Effects of nelfinavir and its M8 metabolite on lymphocyte P-glycoprotein activity during antiretroviral therapy.

PubMed

Donahue, John P; Dowdy, David; Ratnam, Krishna K; Hulgan, Todd; Price, James; Unutmaz, Derya; Nicotera, Janet; Raffanti, Steven; Becker, Mark; Haas, David W

2003-01-01

The efflux pump P-glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P-glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus-negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus-positive patients receiving nelfinavir. The 50% P-glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 micromol/L and 29.5 micromol/L, respectively, for CD4(+) T cells and 19.3 micromol/L and >48 micromol/L, respectively, for CD8(+) T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4(+) (rho = 0.85, P <.0001) and CD8(+) (rho = 0.83, P <.0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus-positive patients, we believe it is likely that CD4(+) and CD8(+) lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P-glycoprotein inhibited by plasma concentrations of nelfinavir.

A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

PubMed

Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

2013-05-01

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application.

PubMed

Chen, Feng; Hu, Zhe-Yi; Parker, Robert B; Laizure, S Casey

2017-06-01

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of exposure multiples of human metabolites for MIST assessment in preclinical safety species without using reference standards or radiolabeled compounds.

PubMed

Ma, Shuguang; Li, Zhiling; Lee, Keun-Joong; Chowdhury, Swapan K

2010-12-20

A simple, reliable, and accurate method was developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volumes of human plasma with blank plasma of animal species and vice versa followed by an analysis using high-resolution full-scan accurate mass spectrometry. This approach provided comparable results (within (±15%) to those obtained from regulated bioanalysis and did not require synthetic standards or radiolabeled compounds. In addition, both qualitative and quantitative data were obtained from a single LC-MS analysis on all metabolites and, therefore, the coverage of any metabolite of interest can be obtained.

Metabolic Signature of Remote Ischemic Preconditioning Involving a Cocktail of Amino Acids and Biogenic Amines.

PubMed

Chao de la Barca, Juan Manuel; Bakhta, Oussama; Kalakech, Hussein; Simard, Gilles; Tamareille, Sophie; Catros, Véronique; Callebert, Jacques; Gadras, Cédric; Tessier, Lydie; Reynier, Pascal; Prunier, Fabrice; Mirebeau-Prunier, Delphine

2016-09-24

Remote ischemic preconditioning (RIPC) is an attractive therapeutic procedure for protecting the heart against ischemia/reperfusion injury. Despite evidence of humoral mediators transported through the circulation playing a critical role, their actual identities so far remain unknown. We sought to identify plasmatic RIPC-induced metabolites that may play a role. Rat plasma samples from RIPC and control groups were analyzed using a targeted metabolomic approach aimed at measuring 188 metabolites. Principal component analysis and orthogonal partial least-squares discriminant analysis were used to identify the metabolites that discriminated between groups. Plasma samples from 50 patients subjected to RIPC were secondarily explored to confirm the results obtained in rats. Finally, a combination of the metabolites that were significantly increased in both rat and human plasma was injected prior to myocardial ischemia/reperfusion in rats. In the rat samples, 124 molecules were accurately quantified. Six metabolites (ornithine, glycine, kynurenine, spermine, carnosine, and serotonin) were the most significant variables for marked differentiation between the RIPC and control groups. In human plasma, analysis confirmed ornithine decrease and kynurenine and glycine increase following RIPC. Injection of the glycine and kynurenine alone or in combination replicated the protective effects of RIPC seen in rats. We have hereby reported significant variations in a cocktail of amino acids and biogenic amines after remote ischemic preconditioning in both rat and human plasma. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01390129. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Recommendations for Improving Identification and Quantification in Non-Targeted, GC-MS-Based Metabolomic Profiling of Human Plasma

PubMed Central

Wang, Hanghang; Muehlbauer, Michael J.; O’Neal, Sara K.; Newgard, Christopher B.; Hauser, Elizabeth R.; Shah, Svati H.

2017-01-01

The field of metabolomics as applied to human disease and health is rapidly expanding. In recent efforts of metabolomics research, greater emphasis has been placed on quality control and method validation. In this study, we report an experience with quality control and a practical application of method validation. Specifically, we sought to identify and modify steps in gas chromatography-mass spectrometry (GC-MS)-based, non-targeted metabolomic profiling of human plasma that could influence metabolite identification and quantification. Our experimental design included two studies: (1) a limiting-dilution study, which investigated the effects of dilution on analyte identification and quantification; and (2) a concentration-specific study, which compared the optimal plasma extract volume established in the first study with the volume used in the current institutional protocol. We confirmed that contaminants, concentration, repeatability and intermediate precision are major factors influencing metabolite identification and quantification. In addition, we established methods for improved metabolite identification and quantification, which were summarized to provide recommendations for experimental design of GC-MS-based non-targeted profiling of human plasma. PMID:28841195

Effects of dynamic exercise on plasma arachidonic acid epoxides and diols in human volunteers

USDA-ARS?s Scientific Manuscript database

Metabolites of the cytochrome P450 pathway may contribute to vasodilation of the vasculature of skeletal muscle during exercise. We determined effects of exercise intensity and duration on plasma concentrations of specific metabolites in the epoxyeicosatrienoic acid family. This allowed us to dete...

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography.

PubMed

Davies, Benjamin J; Herbert, Megan K; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

2006-11-07

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.

UHPLC-(ESI)QTOF MS/MS profiling of quercetin metabolites in human plasma postconsumption of applesauce enriched with apple peel and onion.

PubMed

Lee, Jihyun; Ebeler, Susan E; Zweigenbaum, Jerry A; Mitchell, Alyson E

2012-08-29

An ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-(ESI)QTOF MS/MS) method was developed for measuring individual quercetin metabolites in human plasma with high sensitivity and high selectivity. Quercetin (3,3',4',5,7-pentahydroxyflavone) occurs as glycosides in foods. The composition of glycosides is species and cultivar specific. In humans, quercetin undergoes extensive biotransformation, resulting in a range of metabolites. The bioactivity of quercetin metabolites will depend on the type and position of the conjugates. Herein, individual quercetin metabolites (i.e., sulfate, glucuronide or methyl conjugates) were identified by accurate mass MS in human plasma (females = 8 and males = 8) over 24 h after consumption of applesauce enriched with either micronized apple peel (AP) or onion powder (OP). The AP and OP contained ~180 μmol of quercetin glycosides. The relative amounts of quercetin metabolites were quantified in plasma. The complement of identified quercetin metabolites was similar after consumption of AP and OP. Primary metabolites included the following: quercetin sulfate, quercetin glucuronide, and quercetin diglucuronide. A quercetin glutathione adduct was identified in negative ion mode but not apparent in positive ion mode. The pharmacokinetic parameters for AUC0-24 h and Cmax were significantly different for AP and OP. For example, consumption of the AP resulted in Cmax of quercetin sulfate, 4.6 ng/mL; quercetin glucuronide, 15.5 ng/mL; quercetin diglucuronide, 9.3 ng/mL; quercetin glucuronide sulfate, 1.3 ng/mL; methyl quercetin glucuronide, 7.5 ng/mL; and methyl quercetin diglucuronide, 3.6 ng/mL, whereas the OP resulted in Cmax of quercetin sulfate, 37.3 ng/mL; quercetin glucuronide, 212.8 ng/mL; quercetin diglucuronide, 168.8 ng/mL; quercetin glucuronide sulfate, 43.0 ng/mL; methyl quercetin glucuronide, 90.1 ng/mL; methyl quercetin diglucuronide, 65.4 ng/mL. Gender-related differences in the AUC0-24 h for quercetin sulfate and quercetin sulfate glucuronide metabolites were also observed.

Plasma and serum from nonfasting men and women differ in their lipidomic profiles.

PubMed

Ishikawa, Masaki; Tajima, Yoko; Murayama, Mayumi; Senoo, Yuya; Maekawa, Keiko; Saito, Yoshiro

2013-01-01

Biomarkers will play important roles in disease diagnosis, drug development, and the proper use of drugs. Blood is considered the best biofluid for biomarker research because it is easy to access and a wealth of data are available. However, previous studies revealed that several ionic metabolites showed different levels (including presence or absence) in plasma and serum. Thus, attention should be paid to selecting the best biofluid for biomarker exploration. Many lipid molecules have biological significance and thus would be candidate biomarkers. However, no comprehensive study revealing differences in lipid metabolite levels between plasma and serum has been undertaken. Furthermore, gender differences have not been reported. To clarify the difference in the levels of lipid metabolites between human plasma and serum from both genders, we performed lipid metabolomic analysis using liquid chromatography-mass spectrometry-based systems for phospholipids (PLs), lysoPLs, sphingomyelins, ceramides and oxidative fatty acids. Our results revealed that most of the lipid metabolites were present at similar levels in plasma and serum and in males and females. However, several oxidative fatty acid metabolites showed differences. Of the metabolites related to clotting processes, three showed higher levels in serum than in plasma, and three were detected only in serum. Furthermore, four metabolites were present at different levels between males and females, and two were detected only in males. Thus, attention should be paid to the selection of plasma or serum when utilizing these lipid metabolites as biomarkers.

Is N,N-dimethylglycine N-oxide a choline and betaine metabolite?

PubMed

Lever, Michael; McEntyre, Christopher J; George, Peter M; Chambers, Stephen T

2017-06-27

Choline metabolism is by oxidation to betaine, which is demethylated to N,N-dimethylglycine; dimethylglycine is oxidatively demethylated to sarcosine. This pathway is important for osmoregulation and as a source of methyl groups. We asked whether another metabolite was involved. We synthesized the N-oxide of dimethylglycine (DMGO) by oxidizing dimethylglycine with peracetic acid, and measured DMGO in human plasma and urine by HPLC-MS/MS with positive ion detection, using two chromatography procedures, based on ion exchange and HILIC separations. The molecular ion DMGOH+ (m/z=120) yielded four significant fragments (m/z=103, 102, 58 and 42). The suspected DMGO peak in human body fluids showed all these fragments, and co-chromatographed with added standard DMGO in both HPLC systems. Typical plasma concentrations of DMGO are under 1 μmol/l. They may be lower in metabolic syndrome patients. Urine concentrations are higher, and DMGO has a higher fractional clearance than dimethylglycine, betaine and choline. It was present in all of over 80 human urine and plasma samples assayed. Plasma DMGO concentrations correlate with plasma DMG concentrations, with betaine and choline concentrations, with the osmolyte myo-inositol, and strongly with urinary DMGO excretion. We conclude that DMGO is probably a normal human metabolite.

The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age

PubMed Central

Al-Salameh, Abdallah; Croixmarie, Vincent; Masson, Perrine; Corruble, Emmanuelle; Fève, Bruno; Colle, Romain; Ripoll, Laurent; Walther, Bernard; Boursier-Neyret, Claire; Werner, Erwan; Becquemont, Laurent; Chanson, Philippe

2017-01-01

Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the “normal” metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the “normal” metabolome and its main sources of variation. PMID:28278231

The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age.

PubMed

Trabado, Séverine; Al-Salameh, Abdallah; Croixmarie, Vincent; Masson, Perrine; Corruble, Emmanuelle; Fève, Bruno; Colle, Romain; Ripoll, Laurent; Walther, Bernard; Boursier-Neyret, Claire; Werner, Erwan; Becquemont, Laurent; Chanson, Philippe

2017-01-01

Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the "normal" metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the "normal" metabolome and its main sources of variation.

Disposition, profiling and identification of emixustat and its metabolites in humans.

PubMed

Fitzsimmons, Michael E; Sun, Gang; Kuksa, Vladimir; Reid, Michael J

2018-06-01

1. Emixustat is a small molecule that potently inhibits retinal pigment epithelium 65 isomerohydrolase. Emixustat is in clinical development for the treatment of various retinopathies (i.e. Stargardt disease and diabetic retinopathy). 2. A human absorption, distribution, metabolism, and excretion (ADME) study was conducted with a single dose of [ 14 C]-emixustat in healthy male subjects. Total 14 C content in plasma, urine, and faeces was determined using accelerator mass spectrometry (AMS), and metabolic profiles in pooled plasma and urine were investigated by both HPLC-AMS and 2D LC-MS/MS. 3. After a single, oral 40-mg dose of [ 14 C]-emixustat, recovery of total 14 C was nearly complete within 24 h. Urine was the major route of 14 C elimination; accounting for > 90% of the administered dose. 4. Biotransformation of emixustat occurred primarily at two structural moieties; oxidation of the cyclohexyl moiety and oxidative deamination of the 3R-hydroxypropylamine, both independently and in combination to produce secondary metabolites. Metabolite profiling in pooled plasma samples identified 3 major metabolites: ACU-5124, ACU-5116 and ACU-5149, accounting for 29.0%, 11.5%, and 10.6% of total 14 C, respectively. Emixustat was metabolized in human hepatocytes with unchanged emixustat accounting for 33.7% of sample radioactivity and predominantly cyclohexanol metabolites observed.

Disposition and metabolism of [14C]-levomilnacipran, a serotonin and norepinephrine reuptake inhibitor, in humans, monkeys, and rats

PubMed Central

Brunner, Valérie; Maynadier, Bernadette; Chen, Laishun; Roques, Louise; Hude, Isabelle; Séguier, Sébastien; Barthe, Laurence; Hermann, Philippe

2015-01-01

Levomilnacipran is approved in the US for the treatment of major depressive disorder in adults. We characterized the metabolic profile of levomilnacipran in humans, monkeys, and rats after oral administration of [14C]-levomilnacipran. In vitro binding of levomilnacipran to human plasma proteins was also studied. Unchanged levomilnacipran was the major circulating compound after dosing in all species. Within 12 hours of dosing in humans, levomilnacipran accounted for 52.9% of total plasma radioactivity; the circulating metabolites N-desethyl levomilnacipran N-carbamoyl glucuronide, N-desethyl levomilnacipran, and levomilnacipran N-carbamoyl glucuronide accounted for 11.3%, 7.5%, and 5.6%, respectively. Similar results were seen in monkeys. N-Desethyl levomilnacipran and p-hydroxy levomilnacipran were the main circulating metabolites in rats. Mass balance results indicated that renal excretion was the major route of elimination with 58.4%, 35.5%, and 40.2% of total radioactivity being excreted as unchanged levomilnacipran in humans, monkeys, and rats, respectively. N-Desethyl levomilnacipran was detected in human, monkey, and rat urine (18.2%, 12.4%, and 7.9% of administered dose, respectively). Human and monkey urine contained measurable quantities of levomilnacipran glucuronide (3.8% and 4.1% of administered dose, respectively) and N-desethyl levomilnacipran glucuronide (3.2% and 2.3% of administered dose, respectively); these metabolites were not detected in rat urine. The metabolites p-hydroxy levomilnacipran and p-hydroxy levomilnacipran glucuronide were detected in human urine (≤1.2% of administered dose), and p-hydroxy levomilnacipran glucuronide was found in rat urine (4% of administered dose). None of the metabolites were pharmacologically active. Levomilnacipran was widely distributed with low plasma protein binding (22%). PMID:26150694

Choline and Choline Metabolite Patterns and Associations in Blood and Milk during Lactation in Dairy Cows

PubMed Central

Artegoitia, Virginia M.; Middleton, Jesse L.; Harte, Federico M.; Campagna, Shawn R.; de Veth, Michael J.

2014-01-01

Milk and dairy products are an important source of choline, a nutrient essential for human health. Infant formula derived from bovine milk contains a number of metabolic forms of choline, all contribute to the growth and development of the newborn. At present, little is known about the factors that influence the concentrations of choline metabolites in milk. The objectives of this study were to characterize and then evaluate associations for choline and its metabolites in blood and milk through the first 37 weeks of lactation in the dairy cow. Milk and blood samples from twelve Holstein cows were collected in early, mid and late lactation and analyzed for acetylcholine, free choline, betaine, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine and sphingomyelin using hydrophilic interaction liquid chromatography-tandem mass spectrometry, and quantified using stable isotope-labeled internal standards. Total choline concentration in plasma, which was almost entirely phosphatidylcholine, increased 10-times from early to late lactation (1305 to 13,535 µmol/L). In milk, phosphocholine was the main metabolite in early lactation (492 µmol/L), which is a similar concentration to that found in human milk, however, phosphocholine concentration decreased exponentially through lactation to 43 µmol/L in late lactation. In contrast, phosphatidylcholine was the main metabolite in mid and late lactation (188 µmol/L and 659 µmol/L, respectively), with the increase through lactation positively correlated with phosphatidylcholine in plasma (R 2 = 0.78). Unlike previously reported with human milk we found no correlation between plasma free choline concentration and milk choline metabolites. The changes in pattern of phosphocholine and phosphatidylcholine in milk through lactation observed in the bovine suggests that it is possible to manufacture infant formula that more closely matches these metabolites profile in human milk. PMID:25157578

Identification, Synthesis, and Biological Evaluation of the Metabolites of 3-Amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36), a Promising Rexinoid Lead Compound for the Development of Cancer Chemotherapeutic and Chemopreventive Agents

PubMed Central

Chen, Lian; Conda-Sheridan, Martin; Narasimha Reddy, P. V.; Morrell, Andrew; Park, Eun-Jung; Kondratyuk, Tamara P.; Pezzuto, John M.; van Breemen, Richard B.; Cushman, Mark

2012-01-01

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, 7 phase I and II metabolites were formed by human hepatocytes, and 5 metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB. PMID:22712432

Identification, synthesis, and biological evaluation of the metabolites of 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36), a promising rexinoid lead compound for the development of cancer chemotherapeutic and chemopreventive agents.

PubMed

Chen, Lian; Conda-Sheridan, Martin; Reddy, P V Narasimha; Morrell, Andrew; Park, Eun-Jung; Kondratyuk, Tamara P; Pezzuto, John M; van Breemen, Richard B; Cushman, Mark

2012-06-28

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.

Systematic analysis of the polyphenol metabolome using the Phenol-Explorer database.

PubMed

Rothwell, Joseph A; Urpi-Sarda, Mireia; Boto-Ordoñez, Maria; Llorach, Rafael; Farran-Codina, Andreu; Barupal, Dinesh Kumar; Neveu, Vanessa; Manach, Claudine; Andres-Lacueva, Cristina; Scalbert, Augustin

2016-01-01

The Phenol-Explorer web database details 383 polyphenol metabolites identified in human and animal biofluids from 221 publications. Here, we exploit these data to characterize and visualize the polyphenol metabolome, the set of all metabolites derived from phenolic food components. Qualitative and quantitative data on 383 polyphenol metabolites as described in 424 human and animal intervention studies were systematically analyzed. Of these metabolites, 301 were identified without prior enzymatic hydrolysis of biofluids, and included glucuronide and sulfate esters, glycosides, aglycones, and O-methyl ethers. Around one-third of these compounds are also known as food constituents and corresponded to polyphenols absorbed without further metabolism. Many ring-cleavage metabolites formed by gut microbiota were noted, mostly derived from hydroxycinnamates, flavanols, and flavonols. Median maximum plasma concentrations (C(max)) of all human metabolites were 0.09 and 0.32 μM when consumed from foods or dietary supplements, respectively. Median time to reach maximum plasma concentration in humans (T(max)) was 2.18 h. These data show the complexity of the polyphenol metabolome and the need to take into account biotransformations to understand in vivo bioactivities and the role of dietary polyphenols in health and disease. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioavailability and pharmacokinetic profile of grape pomace phenolic compounds in humans.

PubMed

Castello, Fabio; Costabile, Giuseppina; Bresciani, Letizia; Tassotti, Michele; Naviglio, Daniele; Luongo, Delia; Ciciola, Paola; Vitale, Marilena; Vetrani, Claudia; Galaverna, Gianni; Brighenti, Furio; Giacco, Rosalba; Del Rio, Daniele; Mena, Pedro

2018-05-15

Grape pomace, the major byproduct of the wine and juice industry, is a relevant source of bioactive phenolic compounds. However, polyphenol bioavailability in humans is not well understood, and the inter-individual variability in the production of phenolic metabolites has not been comprehensively assessed to date. The pharmacokinetic and excretive profiles of phenolic metabolites after the acute administration of a drink made from red grape pomace was here investigated in ten volunteers. A total of 35 and 28 phenolic metabolites were quantified in urine and plasma, respectively. The main circulating metabolites included phenyl-γ-valerolactones, hydroxybenzoic acids, simple phenols, hydroxyphenylpropionic acids, hydroxycinnamates, and (epi)catechin phase II conjugates. A high inter-individual variability was shown both in urine and plasma samples, and different patterns of circulating metabolites were unravelled by applying unsupervised multivariate analysis. Besides the huge variability in the production of microbial metabolites of colonic origin, an important variability was observed due to phase II conjugates. These results are of interest to further understand the potential health benefits of phenolic metabolites on individual basis. Copyright © 2018 Elsevier Inc. All rights reserved.

Resveratrol Metabolism in a Non-Human Primate, the Grey Mouse Lemur (Microcebus murinus), Using Ultra-High-Performance Liquid Chromatography–Quadrupole Time of Flight

PubMed Central

Menet, Marie-Claude; Marchal, Julia; Dal-Pan, Alexandre; Taghi, Méryam; Nivet-Antoine, Valérie; Dargère, Delphine; Laprévote, Olivier; Beaudeux, Jean-Louis; Aujard, Fabienne; Epelbaum, Jacques; Cottart, Charles-Henry

2014-01-01

The grey mouse lemur (Microcebus murinus) is a non-human primate used to study the ageing process. Resveratrol is a polyphenol that may increase lifespan by delaying age-associated pathologies. However, no information about resveratrol absorption and metabolism is available for this primate. Resveratrol and its metabolites were qualitatively and quantitatively analyzed in male mouse-lemur plasma (after 200 mg.kg−1 of oral resveratrol) by ultra-high performance liquid chromatography (UHPLC), coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer used in full-scan mode. Data analyses showed, in MSE mode, an ion common to resveratrol and all its metabolites: m/z 227.072, and an ion common to dihydro-resveratrol metabolites: m/z 229.08. A semi-targeted study enabled us to identify six hydrophilic resveratrol metabolites (one diglucurono-conjugated, two monoglucurono-conjugated, one monosulfo-conjugated and two both sulfo- and glucurono-conjugated derivatives) and three hydrophilic metabolites of dihydro-resveratrol (one monoglucurono-conjugated, one monosulfo-conjugated, and one both sulfo- and glucurono-conjugated derivatives). The presence of such metabolites has been already detected in the mouse, rat, pig, and humans. Free resveratrol was measurable for several hours in mouse-lemur plasma, and its two main metabolites were trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate. Free dihydro-resveratrol was not measurable whatever the time of plasma collection, while its hydrophilic metabolites were present at 24 h after intake. These data will help us interpret the effect of resveratrol in mouse lemurs and provide further information on the inter-species characteristics of resveratrol metabolism. PMID:24663435

Determination of the sulfate and glucuronide conjugates of levornidazole in human plasma and urine, and levornidazole and its five metabolites in human feces by high performance liquid chromatography-tandem mass spectrometry.

PubMed

He, Gaoli; Guo, Beining; Zhang, Jing; Li, Yi; Wu, Xiaojie; Fan, Yaxin; Chen, Yuancheng; Cao, Guoying; Yu, Jicheng

2018-04-01

Levornidazole is a novel third-generation nitroimidazoles antibiotic which metabolism and disposition in human are not well known. We have previously developed two methods to quantify levornidazole and its phase I metabolites, Ml (Hydroxylation metabolite), M2 (N-dealkylation metabolite) and M4 (Oxidative dechlorination metabolite), in human plasma and urine. In this study, we developed three novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods and analyzed its phase II metabolites, sulfate conjugate (M6) and glucuronide conjugate (M16), in human plasma and urine, and the parent drug and above-mentioned five metabolites in human feces samples. Analytes and internal standard (IS) in human plasma were extracted by a solid-phase extraction procedure and separated on an ACQUITY UPLC CSH C18 column in gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase. The pretreatment procedures for urine and feces homogenate samples involved a protein precipitation followed by liquid-liquid extraction, and chromatographic separations were performed on the Atlantis T3 columns of different lengths and particle sizes (2.1 × 50 mm, 3 μm and 2.1 × 150 mm, 5 μm), respectively. The mobile phases consisted of formic acid and acetonitrile-methanol solution (v/v, 50:50) in gradient elution. The MS/MS analysis was conducted on TSQ Quantum triple quadrupole mass spectrometer using electrospray ionization with selected reaction monitoring (SRM) in the positive ion mode. The calibration curves for all analytes were linear and the validation ranges were as follows: 0.005-0.500 μg/mL for M6 and 0.005-2.500 μg/mL for M16 in plasma; 0.010-10.000 μg/mL for M6 and M16 in urine; 0.005-1.000 μg/mL for levornidazole, M2, M4 and M16, and 0.010-2.000 μg/mL for M1 and M6 in human feces homogenate. Across these matrices, mean intra- and inter- batch accuracy values were in the ranges of 80.0%-120.0%, and intra- and inter-batch precision values did not exceed 20%. It was fully validated including selectivity, linearity, matrix effect, extraction recovery, stability, dilution integrity, carryover and incurred sample analysis (ISR). These newly developed methods were successfully applied in pharmacokinetics, metabolism and disposition study of levornidazole in 12 healthy Chinese subjects. Copyright © 2018 Elsevier B.V. All rights reserved.

Rice Protein Matrix Enhances Circulating Levels of Xanthohumol Following Acute Oral Intake of Spent Hops in Humans.

PubMed

O'Connor, Annalouise; Konda, Veera; Reed, Ralph L; Christensen, J Mark; Stevens, Jan F; Contractor, Nikhat

2018-03-01

Xanthohumol (XN), a prenylated flavonoid found in hops, exhibits anti-inflammatory and antioxidant properties. However, poor bioavailability may limit therapeutic applications. As food components are known to modulate polyphenol absorption, the objective is to determine whether a protein matrix could enhance the bioavailability of XN post oral consumption in humans. This is a randomized, double-blind, crossover study in healthy participants (n = 6) evaluating XN and its major metabolites (isoxanthohumol [IX], 6- and 8-prenylnaringenin [6-PN, 8-PN]) for 6 h following consumption of 12.4 mg of XN delivered via a spent hops-rice protein matrix preparation or a control spent hops preparation. Plasma XN and metabolites are measured by LC-MS/MS. C max , T max , and area-under-the-curve (AUC) values were determined. Circulating XN and metabolite response to each treatment was not bioequivalent. Plasma concentrations of XN and XN + metabolites (AUC) are greater with consumption of the spent hops-rice protein matrix preparation. Compared to a standard spent hops powder, a protein-rich spent hops matrix demonstrates enhanced plasma levels of XN and metabolites following acute oral intake. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC-MS/MS and its application to a pharmacokinetic study.

PubMed

Bae, Jung-Woo; Choi, Chang-Ik; Jang, Choon-Gon; Lee, Seok-Yong

2011-11-01

A simple and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed and validated for the determination of sibutramine and its N-desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t-butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse-phase Luna C18 column with a mobile phase of acetonitrile-10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI-MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05-20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra- and inter-day validation for sibutramine, M1 and M2 were acceptable. This LC-MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.

Identification of human plasma metabolites exhibiting time-of-day variation using an untargeted liquid chromatography-mass spectrometry metabolomic approach.

PubMed

Ang, Joo Ern; Revell, Victoria; Mann, Anuska; Mäntele, Simone; Otway, Daniella T; Johnston, Jonathan D; Thumser, Alfred E; Skene, Debra J; Raynaud, Florence

2012-08-01

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49-81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies.

Profiling primaquine metabolites in primary human hepatocytes by UPLC-QTOF-MS with 13c stable isotope labeling

USDA-ARS?s Scientific Manuscript database

Primaquine (PQ) is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. However, hemolytic anemia is a dose-limiting side effect of primaquine therapy that limits its widespread use. The major plasma metabolite identified in humans and animals, car...

Experiment requirements: Vitamin D metabolites and bone demineralization, Spacelab 2, experiment no. 1

NASA Technical Reports Server (NTRS)

Schnoes, H. K.; Holton, E. M.; Thirolf, R. G.

1978-01-01

As a contribution toward an understanding of the molecular basis of bone loss, mineral imbalance, and increasing fecal calcium under conditions of prolonged space flight, the blood levels of biologically active vitamin D metabolites of flight crew members will be quantitatively measured. Prior to the mission, the refinement of existing and the development of new techniques for the assay of all vitamin D metabolites will provide an arsenal of methods suitable for a wide range of metabolite levels. In terms of practical application, the analysis of human and animal plasma samples, Spacelab crew plasma samples, and flight hardware are envisioned.

In matrix derivatization of trichloroethylene metabolites in human plasma with methyl chloroformate and their determination by solid-phase microextraction-gas chromatography-electron capture detector.

PubMed

Mudiam, Mohana Krishna Reddy; Jain, Rajeev; Varshney, Meenu; Ch, Ratnasekhar; Chauhan, Abhishek; Goyal, Sudhir Kumar; Khan, Haider A; Murthy, R C

2013-04-15

Trichloroethylene (TCE) is a common industrial chemical that has been widely used as metal degreaser and for many industrial purposes. In humans, TCE is metabolized into dichloroacetic acid (DCA), trichloroacetic acid (TCA) and trichloroethanol (TCOH). A simple and rapid method has been developed for the quantitative determination of TCE metabolites. The procedure involves the in situ derivatization of TCE metabolites with methyl chloroformate (MCF) directly in diluted plasma samples followed by extraction and analysis with solid-phase microextraction (SPME) coupled to gas chromatography-electron capture detector (GC-ECD). Factors which can influence the efficiency of derivatization such as amount of MCF and pyridine (PYR), ratio of water/methanol were optimized. The factors which can affect the extraction efficiencies of SPME were screened using 2(7-4) Placket-Burman Design (PBD). A central composite design (CCD) was then applied to further optimize the most significant factors for optimum SPME extraction. The optimum factors for the SPME extraction were found to be 562.5mg of NaCl, pH at 1 and an extraction time of 22 min. Recoveries and detection limits of all three analytes in plasma were found to be in the range of 92.69-97.55% and 0.036-0.068 μg mL(-1) of plasma, respectively. The correlation coefficients were found to be in the range of 0.990-0.995. The intra- and inter-day precisions for TCE metabolites were found to be in the range of 2.37-4.81% and 5.13-7.61%, respectively. The major advantage of this method is that MCF derivatization allows conversion of TCE metabolites into their methyl esters in very short time (≤30 s) at room temperature directly in the plasma samples, thus makes it a solventless analysis. The method developed was successfully applied to the plasma samples of humans exposed to TCE. Copyright © 2013 Elsevier B.V. All rights reserved.

Choline intake and genetic polymorphisms influence choline metabolite concentrations in human breast milk and plasma123

PubMed Central

Fischer, Leslie M; da Costa, Kerry Ann; Galanko, Joseph; Sha, Wei; Stephenson, Brigitte; Vick, Julie; Zeisel, Steven H

2010-01-01

Background: Choline is essential for infant nutrition, and breast milk is a rich source of this nutrient. Common single nucleotide polymorphisms (SNPs) change dietary requirements for choline intake. Objective: The aim of this study was to determine whether total choline intake and/or SNPs influence concentrations of choline and its metabolites in human breast milk and plasma. Design: We gave a total of 103 pregnant women supplemental choline or a placebo from 18 wk gestation to 45 d postpartum and genotyped the women for 370 common SNPs. At 45 d postpartum, we measured choline metabolite concentrations in breast milk and plasma and assessed the dietary intake of choline by using a 3-d food record. Results: On average, lactating women in our study ate two-thirds of the recommended intake for choline (Adequate Intake = 550 mg choline/d). Dietary choline intake (no supplement) correlated with breast-milk phosphatidylcholine and plasma choline concentrations. A supplement further increased breast-milk choline, betaine, and phosphocholine concentrations and increased plasma choline and betaine concentrations. We identified 5 SNPs in MTHFR that altered the slope of the intake–metabolite concentration relations, and we identified 2 SNPs in PEMT that shifted these curves upward. Individuals who shared sets of common SNPs were outliers in plots of intake–metabolite concentration curves; we suggest that these SNPs should be further investigated to determine how they alter choline metabolism. Conclusion: Total intake of choline and genotype can influence the concentrations of choline and its metabolites in the breast milk and blood of lactating women and thereby affect the amount of choline available to the developing infant. This study was registered at clinicaltrials.gov as NCT00678925. PMID:20534746

Simulation of human plasma concentration-time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling.

PubMed

Kamimura, Hidetaka; Ito, Satoshi; Chijiwa, Hiroyuki; Okuzono, Takeshi; Ishiguro, Tomohiro; Yamamoto, Yosuke; Nishinoaki, Sho; Ninomiya, Shin-Ichi; Mitsui, Marina; Kalgutkar, Amit S; Yamazaki, Hiroshi; Suemizu, Hiroshi

2017-05-01

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

Testicular distribution and toxicity of a novel LTA4H inhibitor in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, P.D., E-mail: pward4@its.jnj.com; La, D.

JNJ 40929837, a novel leukotriene A4 hydrolase inhibitor in drug development, was reported to induce testicular toxicity in rats. The mechanism of toxicity was considered to be rodent specific and not relevant to humans. To further investigate this finding in rats, the distribution and toxicokinetics of JNJ 40929837 and its two metabolites, M1 and M2, were investigated. A quantitative whole body autoradiography study showed preferential distribution and retention of JNJ 40929837-derived radioactivity in the testes consistent with the observed site of toxicity. Subsequent studies with unlabeled JNJ 40929837 showed different metabolite profiles between the plasma and testes. Following a singlemore » oral 50 mg/kg dose of JNJ 40929837, M2 was the primary metabolite in plasma whereas M1 was the primary metabolite in testes. The exposure of M1 was 386-fold higher in the testes compared to plasma whereas M2 had limited exposure in testes. Furthermore, the T{sub max} of M1 was 48 h in testes suggesting a large accumulation potential of this metabolite in testes compared to plasma. Following six months of repeated daily oral dosing, M1 accumulated approximately five-fold in the testes whereas the parent did not accumulate. These results indicate that the toxicokinetic profiles of JNJ 40929837 and its two metabolites in testes are markedly different compared to plasma and support the importance of understanding the toxicokinetic profiles of compounds and their metabolites in organs/tissues where toxicity is observed. - Highlights: • JNJ 40929837-derived radioactivity preferentially distributed into testes • Primary metabolite flip-flop in plasma and testes • The primary metabolite in testes accumulated 5-fold but not parent.« less

Green tea catechins and their metabolites in human skin before and after exposure to ultraviolet radiation.

PubMed

Clarke, Kayleigh A; Dew, Tristan P; Watson, Rachel E B; Farrar, Mark D; Osman, Joanne E; Nicolaou, Anna; Rhodes, Lesley E; Williamson, Gary

2016-01-01

Dietary flavonoids may protect against sunburn inflammation in skin. Preliminary reports using less complete analysis suggest that certain catechins and their metabolites are found in skin biopsies and blister fluid after consumption of green tea; however, it is not known if they are affected by solar-simulated ultraviolet radiation (UVR) or whether conjugated forms, with consequently altered bioactivity, are present. The present study tested the hypothesis that UVR affects the catechin levels in the skin of healthy volunteers after consumption of green tea and how catechins in the plasma are related to their presence in skin tissue samples. In an open oral intervention study, 11 subjects consumed green tea and vitamin C supplements daily for 3months. Presupplementation and postsupplementation plasma samples, suction blister fluid and skin biopsies were collected; the latter two samples were collected both before and after UVR. A sensitive high-performance liquid chromatography/mass spectrometric assay was used to measure the intact catechin metabolites, conjugates and free forms. Seven green tea catechins and their corresponding metabolites were identified postsupplementation in skin biopsies, 20 in blister fluid and 26 in plasma, with 15 green tea catechin metabolites present in both blister fluid and plasma. The valerolactone, O-methyl-M4-O-sulfate, a gut microbiota metabolite of catechins, was significantly increased 1.6-fold by UVR in blister fluid samples. In conclusion, there were some common catechin metabolites in the plasma and blister fluid, and the concentration was always higher in plasma. The results suggest that green tea catechins and metabolites are bioavailable in skin and provide a novel link between catechin metabolites derived from the skin and gut microbiota. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Green tea catechins and their metabolites in human skin before and after exposure to ultraviolet radiation☆☆☆★

PubMed Central

Clarke, Kayleigh A.; Dew, Tristan P.; Watson, Rachel E.B.; Farrar, Mark D.; Osman, Joanne E.; Nicolaou, Anna; Rhodes, Lesley E.; Williamson, Gary

2016-01-01

Dietary flavonoids may protect against sunburn inflammation in skin. Preliminary reports using less complete analysis suggest that certain catechins and their metabolites are found in skin biopsies and blister fluid after consumption of green tea; however, it is not known if they are affected by solar-simulated ultraviolet radiation (UVR) or whether conjugated forms, with consequently altered bioactivity, are present. The present study tested the hypothesis that UVR affects the catechin levels in the skin of healthy volunteers after consumption of green tea and how catechins in the plasma are related to their presence in skin tissue samples. In an open oral intervention study, 11 subjects consumed green tea and vitamin C supplements daily for 3 months. Presupplementation and postsupplementation plasma samples, suction blister fluid and skin biopsies were collected; the latter two samples were collected both before and after UVR. A sensitive high-performance liquid chromatography/mass spectrometric assay was used to measure the intact catechin metabolites, conjugates and free forms. Seven green tea catechins and their corresponding metabolites were identified postsupplementation in skin biopsies, 20 in blister fluid and 26 in plasma, with 15 green tea catechin metabolites present in both blister fluid and plasma. The valerolactone, O-methyl-M4-O-sulfate, a gut microbiota metabolite of catechins, was significantly increased 1.6-fold by UVR in blister fluid samples. In conclusion, there were some common catechin metabolites in the plasma and blister fluid, and the concentration was always higher in plasma. The results suggest that green tea catechins and metabolites are bioavailable in skin and provide a novel link between catechin metabolites derived from the skin and gut microbiota. PMID:26454512

The impact of red cabbage fermentation on bioavailability of anthocyanins and antioxidant capacity of human plasma.

PubMed

Wiczkowski, Wieslaw; Szawara-Nowak, Dorota; Romaszko, Jerzy

2016-01-01

The effect of red cabbage fermentation on anthocyanin bioavailability and plasma antioxidant capacity in humans was studied. In a randomized crossover study, 13 volunteers consumed fresh and fermented red cabbage. Blood and urine samples were collected before and after consumption. Analyses of anthocyanins by HPLC-MS/MS and plasma antioxidant capacity by photochemiluminescence assay were conducted. Red cabbage products contained 20 different nonacylated and acylated anthocyanins with the main structure of cyanidin triglucosides. The anthocyanins ingested were present in physiological fluids in form of 18 native anthocyanins and 12 metabolites (methylated, glucuronided, sulfated). Among cyanidin metabolites identified, methylated forms were predominant. Bioavailability of anthocyanin from fresh red cabbage was over 10% higher than from fermented red cabbage. Upon fresh cabbage consumption, volunteers plasma showed higher antioxidant capacity than after fermented cabbage intake. The study has shown that fermentation process affects red cabbage anthocyanins bioavailability and human plasma antioxidant capacity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice.

PubMed

Shimizu, Makiko; Suemizu, Hiroshi; Mitsui, Marina; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

2017-10-01

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

Identification of an Epoxide Metabolite of Lycopene in Human Plasma Using 13C-Labeling and QTOF-MS.

PubMed

Cichon, Morgan J; Moran, Nancy E; Riedl, Ken M; Schwartz, Steven J; Clinton, Steven K

2018-03-20

The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with 13 C-labeling to investigate the post-prandial oxidative metabolism of lycopene in human plasma. Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.

Simultaneous determination of TPN729 and its five metabolites in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry.

PubMed

Liu, Yang; Shao, Shuguang; Song, Hanlin; Yao, Xueting; Liu, Jie; Liu, Hongzhong; Song, Ling; Jiang, Ji; Liu, Dongyang; Hu, Pei

2018-03-20

A specific and sensitive method was firstly developed using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify TPN729 and its metabolites (TPN729-D1, TPN729-D2, TPN729M15-3 and TPN729M3) in human plasma and (TPN729-D1, TPN729-D2, TPN729M15-3 and TPN729M14) in human urine. Protein precipitation and direct dilution were used to extract TPN729 and its metabolites from plasma and urine, respectively. Ionization of TPN729, TPN729-D1, TPN729-D2, TPN729M15-3, TPN729M3, TPN729M14 and sildenafil (internal standard, IS) was performed using an electrospray ionization (ESI) source in positive mode and detection was carried out with multiple reaction monitoring (MRM) mode. This assay method for TPN729 and its five metabolites has been fully validated in terms of sensitivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, stability, matrix effect and recovery. The LLOQ of TPN729/TPN729-D1/TPN729-D2/TPN729M15-3/TPN729M3 in human plasma and TPN729/TPN729-D1/TPN729-D2/TPN729M15-3/TPN729M14 in human urine were 0.200/0.500/2.00/0.500/1.00 ng/mL and 4.00/2.50/10.0/2.50/1.00 ng/mL, respectively. Inter- and intra-batch precision of TPN729 and its metabolites were less than 15% and the accuracy was within ±15% for both plasma and urine. The extraction recoveries of all analytes at three concentration levels were consistent. In conclusion, the validation results showed that this method was robust, specific, and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of TPN729 in Chinese healthy subjects. Copyright © 2018 Elsevier B.V. All rights reserved.

Phenolic sulfates as new and highly abundant metabolites in human plasma after ingestion of a mixed berry fruit purée.

PubMed

Pimpão, Rui C; Ventura, M Rita; Ferreira, Ricardo B; Williamson, Gary; Santos, Claudia N

2015-02-14

Bioavailability studies are vital to assess the potential impact of bioactive compounds on human health. Although conjugated phenolic metabolites derived from colonic metabolism have been identified in the urine, the quantification and appearance of these compounds in plasma is less well studied. In this regard, it is important to further assess their potential biological activity in vivo. To address this gap, a cross-over intervention study with a mixed fruit purée (blueberry, blackberry, raspberry, strawberry tree fruit and Portuguese crowberry) and a standard polyphenol-free meal was conducted in thirteen volunteers (ten females and three males), who received each test meal once, and plasma metabolites were identified by HPLC-MS/MS. Sulfated compounds were chemically synthesised and used as standards to facilitate quantification. Gallic and caffeic acid conjugates were absorbed rapidly, reaching a maximum concentration between 1 and 2 h. The concentrations of sulfated metabolites resulting from the colonic degradation of more complex polyphenols increased in plasma from 4 h, and pyrogallol sulfate and catechol sulfate reached concentrations ranging from 5 to 20 μm at 6 h. In conclusion, phenolic sulfates reached high concentrations in plasma, as opposed to their undetected parent compounds. These compounds have potential use as biomarkers of polyphenol intake, and their biological activities need to be considered.

Profile of plasma and urine metabolites after the intake of almond [Prunus dulcis (Mill.) D.A. Webb] polyphenols in humans.

PubMed

Urpi-Sarda, Mireia; Garrido, Ignacio; Monagas, María; Gómez-Cordovés, Carmen; Medina-Remón, Alexander; Andres-Lacueva, Cristina; Bartolomé, Begoña

2009-11-11

Nut skins are considered to be a rich source of polyphenols and may be partially responsible for the numerous health effects associated with nut consumption. However, more bioavailability studies of nut skin polyphenols are needed to understand the health effects derived from nut consumption. The aim of the present study was to determine the profiles of both phase II and microbial-derived phenolic metabolites in plasma and urine samples before and after the intake of almond skin polyphenols by healthy human subjects (n = 2). Glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and glucuronide and sulfate conjugates of isorhamnetin, were detected in plasma and urine samples after consumption of almond skin polyphenols. The main microbial-derived metabolites of flavanols, such as 5-(dihydroxyphenyl)-gamma-valerolactone and 5-(hydroxymethoxyphenyl)-gamma-valerolactone, were also detected in their glucuronide and sulfate forms. In addition, numerous metabolites derived from further microbial degradation of hydroxyphenylvalerolactones, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic, and hydroxyhippuric acids, registered major changes in urine after the consumption of almond skin polyphenols. The urinary excretion of these microbial metabolites was estimated to account for a larger proportion of the total polyphenol ingested than phase II metabolites of (epi)catechin, indicating the important role of intestinal bacteria in the metabolism of highly polymerized almond skin polyphenols. To the authors' knowledge this study constitutes the most complete report of the absorption of almond skin polyphenols in humans.

Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method.

PubMed

Choo, K S; Kim, I W; Jung, J K; Suh, Y G; Chung, S J; Lee, M H; Shim, C K

2001-06-01

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection.

PubMed

Brunnenberg, M; Lindenblatt, H; Gouzoulis-Mayfrank, E; Kovar, K A

1998-11-20

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCI. The maximum plasma concentrations were 235-465 ng/ml (MDE), 67-673 ng/ml (HME) and 7-33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.

In vitro metabolism of pyranocoumarin isomers decursin and decursinol angelate by liver microsomes from man and rodents.

PubMed

Li, Li; Zhang, Jinhui; Xing, Chengguo; Kim, Sung-Hoon; Jiang, Cheng; Lü, Junxuan

2013-11-01

The aim of this study is to investigate and compare the metabolic rate and profiles of pyranocoumarin isomers decursin and decursinol angelate using liver microsomes from humans and rodents, and to characterize the major metabolites of decursin and decursinol angelate in human liver microsomal incubations using LC-MS/MS. First, we conducted liver microsomal incubations of decursin and decursinol angelate in the presence or absence of NADPH. We found that in the absence of NADPH, decursin was efficiently hydrolyzed to decursinol by hepatic esterase(s), but decursinol angelate was not. In contrast, formation of decursinol from decursinol angelate was mediated mainly by cytochrome P450(s). Second, we measured the metabolic rate of decursin and decursinol angelate in liver S9 fractions from mice and humans. We found that human liver S9 fractions metabolized both decursin and decursinol angelate more slowly than those of the mouse. Third, we characterized the major metabolites of decursin and decursinol angelate from human liver microsomes incubations using HPLC-UV and LC-MS/MS methods and assessed the in vivo metabolites in mouse plasma from a one-dose PK study. Decursin and decursinol angelate have different metabolite profiles. Nine metabolites of decursin and nine metabolites of decursinol angelate were identified in human liver microsome incubations besides decursinol using a hybrid triple quadruple linear ion trap LC-MS/MS system, and many of them were later verified to be also present in plasma samples from rodent PK studies. Georg Thieme Verlag KG Stuttgart · New York.

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans.

PubMed

Xing, Jie; Zang, Meitong; Zhang, Haiying; Zhu, Mingshe

2015-10-15

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

A Genome-Wide Association Study of the Human Metabolome in a Community-Based Cohort

PubMed Central

Rhee, Eugene P.; Ho, Jennifer E.; Chen, Ming-Huei; Shen, Dongxiao; Cheng, Susan; Larson, Martin G.; Ghorbani, Anahita; Shi, Xu; Helenius, Iiro T.; O’Donnell, Christopher J.; Souza, Amanda L.; Deik, Amy; Pierce, Kerry A.; Bullock, Kevin; Walford, Geoffrey A.; Vasan, Ramachandran S.; Florez, Jose C.; Clish, Clary; Yeh, J.-R. Joanna; Wang, Thomas J.; Gerszten, Robert E.

2014-01-01

SUMMARY Because metabolites are hypothesized to play key roles as markers and effectors of cardio-metabolic diseases, recent studies have sought to annotate the genetic determinants of circulating metabolite levels. We report a genome-wide association study (GWAS) of 217 plasma metabolites, including >100 not measured in prior GWAS, in 2,076 participants of the Framingham Heart Study. For the majority of analytes, we find that estimated heritability explains >20% of inter-individual variation, and that variation attributable to heritable factors is greater than that attributable to clinical factors. Further, we identify 31 genetic loci associated with plasma metabolites, including 23 that have not previously been reported. Importantly, we include GWAS results for all surveyed metabolites, and demonstrate how this information highlights a role for AGXT2 in cholesterol ester and triacylglycerol metabolism. Thus, our study outlines the relative contributions of inherited and clinical factors on the plasma metabolome and provides a resource for metabolism research. PMID:23823483

Alteration of metabolite profiling by cold atmospheric plasma treatment in human myeloma cells.

PubMed

Xu, Dehui; Xu, Yujing; Ning, Ning; Cui, Qingjie; Liu, Zhijie; Wang, Xiaohua; Liu, Dingxin; Chen, Hailan; Kong, Michael G

2018-01-01

Despite new progress of chemotherapy in multiple myeloma (MM) clinical treatment, MM is still a refractory disease and new technology is needed to improve the outcomes and prolong the survival. Cold atmospheric plasma is a rapidly developed technology in recent years, which has been widely applied in biomedicine. Although plasma could efficiently inactivate various tumor cells, the effects of plasma on tumor cell metabolism have not been studied yet. In this study, we investigated the metabolite profiling of He plasma treatment on myeloma tumor cells by gas-chromatography time-of-flight (GC-TOF) mass-spectrometry. Meanwhile, by bioinformatic analysis such as GO and KEGG analysis we try to figure out the metabolism pathway that was significantly affected by gas plasma treatment. By GC-TOF mass-spectrometry, 573 signals were detected and evaluated using PCA and OPLS-DA. By KEGG analysis we listed all the differential metabolites and further classified into different metabolic pathways. The results showed that beta-alanine metabolism pathway was the most significant change after He gas plasma treatment in myeloma cells. Besides, propanoate metabolism and linoleic acid metabolism should also be concerned during gas plasma treatment of cancer cells. Cold atmospheric plasma treatment could significantly alter the metabolite profiling of myeloma tumor cells, among which, the beta-alanine metabolism pathway is the most susceptible to He gas plasma treatment.

Metabolism of bepridil in laboratory animals and humans.

PubMed

Wu, W N; Hills, J F; Chang, S Y; Ng, K T

1988-01-01

The metabolism of bepridil was studied in the Swiss mouse, Sprague-Dawley rat, New Zealand rabbit, rhesus monkey, and healthy human. After oral administration of bepridil-14C-hydrochloride, recoveries of total radioactivity in urine and feces (7 days) were greater than or equal to 80% of the administered dose in all five species. Bepridil and 25 metabolites have been isolated by HPLC and TLC from representative plasma, urine, and fecal extract pools from all species and identified on the basis of TLC, HPLC, and mass spectrometry. The identified metabolites explained 60-99% of the total radioactivity in each sample for rabbit plasma, in which only 17% of the total radioactivity was characterized. Metabolic pathways involving oxidative reactions at seven sites on the bepridil molecule are proposed for each species. Metabolite formation in the five species is described by four interrelated pathways. The metabolic pathway involving aromatic hydroxylation followed by N-dealkylation, N-debenzylation, and N-acetylation was important in all species. Major metabolites produced by this pathway included 4-hydroxy(at N-phenyl)-bepridil (Ia), N-benzyl-4-amino-phenol (IV), and N-acetyl-4-aminophenol (Vy). Metabolite Ia was isolated in significant amounts (greater than or equal to 5% of sample) in all fecal and urine samples except rat urine. Metabolite IV was a major circulating metabolite in all species and a major urinary metabolite in humans. Metabolite Vy was present in significant quantities in urine in all species except rabbit. Other important pathways involved primary reactions such as iso-butyl hydroxylation, pyrrolidine ring oxidation, and N-debenzylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic studies of an orally active platinum anticancer drug by liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Poon, G K; Raynaud, F I; Mistry, P; Odell, D E; Kelland, L R; Harrap, K R; Barnard, C F; Murrer, B A

1995-09-29

Bis(acetato)amminedichloro(cyclohexylamine) platinum(IV) (JM216) is a new orally administered platinum complex with antitumor properties, and is currently undergoing phase II clinical trials. When JM216 was incubated with human plasma ultrafiltrate, 93% of the platinum species were protein-bound and 7% were unbound. The unbound platinum complexes in the ultrafiltrates of human plasma were analysed using a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method. Apart from the parent drug, four metabolites were identified and characterised. These include JM118 [amminedichloro(cyclohexylamine) platinum(II)], JM383 [bis(acetato)ammine(cyclohexylamine)dihydroxo platinum(IV)] and the two isomers JM559 and JM518 [bis(acetato)amminechloro(cyclohexylamine) hydroxo platinum(IV)]. Their elemental compositions were determined by accurate mass measurement during the LC analysis, to confirm their identities. Quantitation of these metabolites by off-line LC atomic absorption spectroscopy demonstrated that JM118 is the major metabolite in plasma from patients receiving JM216 treatment.

Measurement of the alpha4beta2* nicotinic acetylcholine receptor ligand 2-[(18)F]Fluoro-A-85380 and its metabolites in human blood during PET investigation: a methodological study.

PubMed

Sorger, Dietlind; Becker, Georg A; Patt, Marianne; Schildan, Andreas; Grossmann, Udo; Schliebs, Reinhard; Seese, Anita; Kendziorra, Kai; Kluge, Magnus; Brust, Peter; Mukhin, Alexey G; Sabri, Osama

2007-04-01

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS.

PubMed

Kikura-Hanajiri, Ruri; Kawamura, Maiko; Miyajima, Atsuko; Sunouchi, Momoko; Goda, Yukihiro

2011-04-01

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid-acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.

In Vitro Metabolism of Tamoxifen in Human, Rat, and Fish Microsomes

EPA Science Inventory

Results from an in vivo study comparing biologically-active metabolites in the plasma of Wistar rats and cunner fish (Tautogolabrus adspersus) treated with tamoxifen indicate notable differences in circulating metabolite concentrations between these two species. After a single or...

UHPLC-Q-Orbitrap-HRMS-based global metabolomics reveal metabolome modifications in plasma of young women after cranberry juice consumption.

PubMed

Liu, Haiyan; Garrett, Timothy J; Su, Zhihua; Khoo, Christina; Gu, Liwei

2017-07-01

Plasma metabolome in young women following cranberry juice consumption were investigated using a global UHPLC-Q-Orbitrap-HRMS approach. Seventeen female college students, between 21 and 29 years old, were given either cranberry juice or apple juice for three days using a cross-over design. Plasma samples were collected before and after juice consumption. Plasma metabolomes were analyzed using UHPLC-Q-Orbitrap-HRMS followed by orthogonal partial least squares-discriminant analyses (OPLS-DA). S-plot was used to identify discriminant metabolites. Validated OPLS-DA analyses showed that the plasma metabolome in young women, including both exogenous and endogenous metabolites, were altered following cranberry juice consumption. Cranberry juice caused increases of exogenous metabolites including quinic acid, vanilloloside, catechol sulfate, 3,4-dihydroxyphenyl ethanol sulfate, coumaric acid sulfate, ferulic acid sulfate, 5-(trihydroxphenyl)-gamma-valerolactone, 3-(hydroxyphenyl)proponic acid, hydroxyphenylacetic acid and trihydroxybenzoic acid. In addition, the plasma levels of endogenous metabolites including citramalic acid, aconitic acid, hydroxyoctadecanoic acid, hippuric acid, 2-hydroxyhippuric acid, vanilloylglycine, 4-acetamido-2-aminobutanoic acid, dihydroxyquinoline, and glycerol 3-phosphate were increased in women following cranberry juice consumption. The metabolic differences and discriminant metabolites observed in this study may serve as biomarkers of cranberry juice consumption and explain its health promoting properties in human. Copyright © 2017 Elsevier Inc. All rights reserved.

Plasma metabolomics profiling of maintenance hemodialysis based on capillary electrophoresis - time of flight mass spectrometry.

PubMed

Liu, Shuxin; Wang, Lichao; Hu, Chunxiu; Huang, Xin; Liu, Hong; Xuan, Qiuhui; Lin, Xiaohui; Peng, Xiaojun; Lu, Xin; Chang, Ming; Xu, Guowang

2017-08-15

Uremia has been a rapidly increasing health problem in China. Hemodialysis (HD) is the main renal replacement therapy for uremia. The results of large-scale clinical trials have shown that the HD pattern is crucial for long-term prognosis of maintenance hemodialysis (MHD) in uremic patients. Plasma metabolism is very important for revealing the biological insights linked to the therapeutic effects of the HD pattern on uremia. Alteration of plasma metabolites in uremic patients in response to HD therapy has been reported. However, HD-pattern-dependent changes in plasma metabolites remain poorly understood. To this end, a capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolomics method was performed to systemically study the differences between HD and high flux hemodialysis (HFD) on plasma metabolite changes in patients. Three hundred and one plasma samples from three independent human cohorts (i.e., healthy controls, patients with pre-HD/post-HD, and patients with pre-HFD/post-HFD) were used in this study. Metabolites significantly changed (p < 0.05) after a single HD or HFD process. However, 11 uremic retention solutes could be more efficiently removed by HFD. Our findings indicate that a CE-TOF/MS-based metabolomics approach is promising for providing novel insights into understanding the effects of different dialysis methods on metabolite alterations of uremia.

Simple and cost-effective liquid chromatography-mass spectrometry method to measure dabrafenib quantitatively and six metabolites semi-quantitatively in human plasma.

PubMed

Vikingsson, Svante; Dahlberg, Jan-Olof; Hansson, Johan; Höiom, Veronica; Gréen, Henrik

2017-06-01

Dabrafenib is an inhibitor of BRAF V600E used for treating metastatic melanoma but a majority of patients experience adverse effects. Methods to measure the levels of dabrafenib and major metabolites during treatment are needed to allow development of individualized dosing strategies to reduce the burden of such adverse events. In this study, an LC-MS/MS method capable of measuring dabrafenib quantitatively and six metabolites semi-quantitatively is presented. The method is fully validated with regard to dabrafenib in human plasma in the range 5-5000 ng/mL. The analytes were separated on a C18 column after protein precipitation and detected in positive electrospray ionization mode using a Xevo TQ triple quadrupole mass spectrometer. As no commercial reference standards are available, the calibration curve of dabrafenib was used for semi-quantification of dabrafenib metabolites. Compared to earlier methods the presented method represents a simpler and more cost-effective approach suitable for clinical studies. Graphical abstract Combined multi reaction monitoring transitions of dabrafenib and metabolites in a typical case sample.

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady state.

PubMed

Vikingsson, Svante; Strömqvist, Malin; Svedberg, Anna; Hansson, Johan; Höiom, Veronica; Gréen, Henrik

2016-08-01

A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Unbiased Scanning Method and Data Banking Approach Using Ultra-High Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry for Quantitative Comparison of Metabolite Exposure in Plasma across Species Analyzed at Different Dates.

PubMed

Gao, Hongying; Deng, Shibing; Obach, R Scott

2015-12-01

An unbiased scanning methodology using ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry was used to bank data and plasma samples for comparing the data generated at different dates. This method was applied to bank the data generated earlier in animal samples and then to compare the exposure to metabolites in animal versus human for safety assessment. With neither authentic standards nor prior knowledge of the identities and structures of metabolites, full scans for precursor ions and all ion fragments (AIF) were employed with a generic gradient LC method to analyze plasma samples at positive and negative polarity, respectively. In a total of 22 tested drugs and metabolites, 21 analytes were detected using this unbiased scanning method except that naproxen was not detected due to low sensitivity at negative polarity and interference at positive polarity; and 4'- or 5-hydroxy diclofenac was not separated by a generic UPLC method. Statistical analysis of the peak area ratios of the analytes versus the internal standard in five repetitive analyses over approximately 1 year demonstrated that the analysis variation was significantly different from sample instability. The confidence limits for comparing the exposure using peak area ratio of metabolites in animal plasma versus human plasma measured over approximately 1 year apart were comparable to the analysis undertaken side by side on the same days. These statistical analysis results showed it was feasible to compare data generated at different dates with neither authentic standards nor prior knowledge of the analytes.

KAE609 (Cipargamin), a New Spiroindolone Agent for the Treatment of Malaria: Evaluation of the Absorption, Distribution, Metabolism, and Excretion of a Single Oral 300-mg Dose of [14C]KAE609 in Healthy Male Subjects.

PubMed

Huskey, Su-Er W; Zhu, Chun-qi; Fredenhagen, Andreas; Kühnöl, Jürgen; Luneau, Alexandre; Jian, Zhigang; Yang, Ziping; Miao, Zhuang; Yang, Fan; Jain, Jay P; Sunkara, Gangadhar; Mangold, James B; Stein, Daniel S

2016-05-01

KAE609 [(1'R,3'S)-5,7'-dichloro-6'-fluoro-3'-methyl-2',3',4',9'-tetrahydrospiro[indoline-3,1'-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent in clinical development for the treatment of malaria. This study investigated the absorption, distribution, metabolism, and excretion of KAE609 after oral administration of [(14)C]KAE609 in healthy subjects. After oral administration to human subjects, KAE609 was the major radioactive component (approximately 76% of the total radioactivity in plasma); M23 was the major circulating oxidative metabolite (approximately 12% of the total radioactivity in plasma). Several minor oxidative metabolites (M14, M16, M18, and M23.5B) were also identified, each accounting for approximately 3%-8% of the total radioactivity in plasma. KAE609 was well absorbed and extensively metabolized, such that KAE609 accounted for approximately 32% of the dose in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways. M23 was the major metabolite in feces. Subjects reported semen discoloration after dosing in prior studies; therefore, semen samples were collected once from each subject to further evaluate this clinical observation. Radioactivity excreted in semen was negligible, but the major component in semen was M23, supporting the rationale that this yellow-colored metabolite was the main source of semen discoloration. In this study, a new metabolite, M16, was identified in all biologic matrices albeit at low levels. All 19 recombinant human cytochrome P450 enzymes were capable of catalyzing the hydroxylation of M23 to form M16 even though the extent of turnover was very low. Thus, electrochemistry was used to generate a sufficient quantity of M16 for structural elucidation. Metabolic pathways of KAE609 in humans are summarized herein and M23 is the major metabolite in plasma and excreta. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methyl-11C]WAY-100635, in monkey and human plasma by HPLC: comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET.

PubMed

Osman, S; Lundkvist, C; Pike, V W; Halldin, C; McCarron, J A; Swahn, C G; Ginovart, N; Luthra, S K; Bench, C J; Grasby, P M; Wikström, H; Barf, T; Cliffe, I A; Fletcher, A; Farde, L

1996-07-01

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t1/2 = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-11C]WAY-100635 into humans, radioactivity was found to clear rapidly from blood and plasma. By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-11C]WAY-100635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-11C]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha 1-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-11C]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-11 in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake.

Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

PubMed

Flaminio, L; Ripamonti, M; Ascalone, V

1994-05-13

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

PubMed

Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

2015-03-01

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

PubMed Central

López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

2016-01-01

Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

PubMed

López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

2016-01-01

Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

Pairwise alignment of chromatograms using an extended Fisher-Rao metric.

PubMed

Wallace, W E; Srivastava, A; Telu, K H; Simón-Manso, Y

2014-09-02

A conceptually new approach for aligning chromatograms is introduced and applied to examples of metabolite identification in human blood plasma by liquid chromatography-mass spectrometry (LC-MS). A square-root representation of the chromatogram's derivative coupled with an extended Fisher-Rao metric enables the computation of relative differences between chromatograms. Minimization of these differences using a common dynamic programming algorithm brings the chromatograms into alignment. Application to a complex sample, National Institute of Standards and Technology (NIST) Standard Reference Material 1950, Metabolites in Human Plasma, analyzed by two different LC-MS methods having significantly different ranges of elution time is described. Published by Elsevier B.V.

Micro liquid chromatography-mass spectrometry with direct liquid introduction used for separation and quantitation of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma.

PubMed

Ranalder, U B; Lausecker, B B; Huselton, C

1993-07-23

The separation and quantitation of the pentafluorobenzyl derivatives of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma on micro high-performance liquid chromatographic columns (0.32 mm I.D.) is described. The column outlet was directly coupled to the source of a quadrupole mass spectrometer via a simple SFC-frit interface. Negative ion chemical ionization conditions were obtained by coaxial introduction of ammonia as a reagent gas. A signal-to-noise ratio well above 3 was obtained for 1 pg of each analyte injected. The limit of quantitation calculated from spiked biological plasma extracts was 0.3 ng/ml.

Quantification of phenolic acids and their methylates, glucuronides, sulfates and lactones metabolites in human plasma by LC-MS/MS after oral ingestion of soluble coffee.

PubMed

Marmet, Cynthia; Actis-Goretta, Lucas; Renouf, Mathieu; Giuffrida, Francesca

2014-01-01

Chlorogenic acids and derivatives like phenolic acids are potentially bioactive phenolics, which are commonly found in many foods. Once absorbed, chlorogenic and phenolic acids are highly metabolized by the intestine and the liver, producing glucuronidated and/or sulphated compounds. These metabolites were analyzed in human plasma using a validated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method. After protein precipitation, phenolic acids and their metabolites were extracted by using ethanol and chromatographic separation was achieved by reversed-phase using an Acquity UPLC BEH C18 column combined with a gradient elution system using 1% acetic acid aqueous solution and 1% acetic acid with 100% acetonitrile. The method was able to quantify 56 different compounds including 24 phenolic acids, 4 lactones, 15 sulfates and 13 glucuronides metabolites between 5 and 1000nM in plasma for most of them, except for m-dihydrocoumaric acid, 5-ferulloylquinic-glucuronide, 4-methoxycinnamic acid, 3-phenylpropionic acid, 3-(4-methoxyphenyl)propionic acid (25 to 1000nM) and p-dihydrocoumaric acid (50-1000nM). Values of repeatability and intermediate reproducibility were below 15% of deviation in general, and maximum 20% for the lowest concentrations. The validated method was successfully applied to quantify phenolic acids and their metabolites in plasma obtained after oral ingestion of soluble coffee. In conclusion, the developed and validated method is proved to be very sensitive, accurate and precise for the quantification of these possible dietary phenols. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of cyclophosphamide and carboxyethylphosphoramide mustard enantiomers in human plasma and application to clinical pharmacokinetics.

PubMed

Attié de Castro, Francine; Scatena, Gabriel dos Santos; Cass, Quézia Bezerra; Simões, Belinda Pinto; Lanchote, Vera Lucia

2014-11-15

This study describes for the first time a method for the sequential analysis of the enantiomers of cyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in human plasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and separated on a Chiralpak(®) AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5mL/min. No matrix effect was observed in the analysis of the enantiomers of both analytes and the analytical method was linear in the range of 0.05-25.0μg and 250-1000ng of each enantiomer/mL plasma. The coefficients of variation and relative errors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%. CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, during storage for 4h at room temperature, and after 24h inside the autosampler at 4°C, with deviations less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metabolite CEPM in patients with multiple sclerosis (n=10) who received a CY pretransplant conditioning regimen for hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumulation of the (S)-(-)-CY enantiomer (S/R ratio=1.3) and lack of enantioselective exposure to the CEPM metabolite (S/R ratio=1.0). Copyright © 2014 Elsevier B.V. All rights reserved.

Metabolism of a new positive inotropic agent, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (OPC-8212) in the rat, mouse, dog, monkey and human.

PubMed

Miyamoto, G; Sasabe, H; Tominaga, N; Uegaki, N; Tominaga, M; Shimizu, T

1988-10-01

1. After OPC-8212 was orally given to rats, mice, dogs, monkeys and humans, its metabolites were identified by n.m.r. and mass spectrometry, and their concentrations in the plasma, urine and faeces of these species were measured by high-performance liquid chromatography (h.p.l.c.). 2. Hydrolysis of the amide group, oxidation and cleavage of the piperazine ring, O-demethylation of the methoxy group, and conjugation were proposed as metabolic pathways of OPC-8212. 3. In rats, mice and monkeys given OPC-8212 orally, metabolites M-1 to M-6 were detected in the plasma, urine and faeces, while M-1, -4, -5 and M-6 were detected in dogs, and M-1, M-3, M-4, M-5 and M-6 were detected in humans. 4. Conjugates of metabolites M-6 and M-7, with glucuronic acid and sulphuric acid, were observed in the urine of rats and humans.

The plasma and cerebrospinal fluid pharmacokinetics of erlotinib and its active metabolite (OSI-420) after intravenous administration of erlotinib in non-human primates.

PubMed

Meany, Holly J; Fox, Elizabeth; McCully, Cynthia; Tucker, Chris; Balis, Frank M

2008-08-01

Erlotinib hydrochloride is a small molecule inhibitor of epidermal growth factor receptor (EGFR). EGFR is over-expressed in primary brain tumors and solid tumors that metastasize to the central nervous system. We evaluated the plasma and cerebrospinal fluid (CSF) pharmacokinetics of erlotinib and its active metabolite OSI-420 after an intravenous (IV) dose in a non-human primate model. Erlotinib was administered as a 1 h IV infusion to four adult rhesus monkeys. Serial blood and CSF samples were drawn over 48 h and erlotinib and OSI-420 were quantified with an HPLC/tandem mass spectroscopic assay. Pharmacokinetic parameters were estimated using non-compartmental and compartmental methods. CSF penetration was calculated from the AUC(CSF):AUC(plasma). Erlotinib disappearance from plasma after a short IV infusion was biexponential with a mean terminal half-life of 5.2 h and a mean clearance of 128 ml/min per m(2). OSI-420 exposure (AUC) in plasma was 30% (range 12-59%) of erlotinib, and OSI-420 clearance was more than 5-fold higher than erlotinib. Erlotinib and OSI-420 were detectable in CSF. The CSF penetration (AUC(CSF):AUC(plasma)) of erlotinib and OSI-420 was <5% relative to total plasma concentration, but CSF drug exposure was approximately 30% of plasma free drug exposure, which was calculated from published plasma protein binding values. The IV administration of erlotinib was well tolerated. Erlotinib and its active metabolite OSI-420 are measurable in CSF after an IV dose. The drug exposure (AUC) in the CSF is limited relative to total plasma concentrations but is substantial relative the free drug exposure in plasma.

Metabolite characterization of a novel anti-cancer agent, icotinib, in humans through liquid chromatography/quadrupole time-of-flight tandem mass spectrometry.

PubMed

Liu, Dongyang; Jiang, Ji; Zhang, Li; Tan, Fenlai; Wang, Yingxiang; Hu, Pei

2011-08-15

Icotinib is a novel anti-cancer drug that has shown promising clinical efficacy and safety in patients with non-small-cell lung cancer (NSCLC). At this time, the metabolic fate of icotinib in humans is unknown. In the present study, a liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF MS) method was established to characterize metabolites of icotinib in human plasma, urine and feces. In addition, nuclear magnetic resonance (NMR) detection was utilized to determine the connection between side-chain and quinazoline groups for some complex metabolites. In total, 29 human metabolites (21 isomer metabolites) were characterized, of which 23 metabolites are novel compared to the metabolites in rats. This metabolic study revealed that icotinib was extensively metabolized at the 12-crown-4 ether moiety (ring-opening and further oxidation), carbon 15 (hydroxylation) and an acetylene moiety (oxidation) to yield 19 oxidized metabolites and to further form 10 conjugates with sulfate acid or glucuronic acid. To our knowledge, this is the first report of the human metabolic profile of icotinib. Study results indicated that significant attention should be paid to the metabolic profiles of NSCLC patients during the development of icotinib. Copyright © 2011 John Wiley & Sons, Ltd.

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin.

PubMed

Naganuma, H; Kawahara, Y

1990-09-14

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.

Global metabolomic analysis of human saliva and plasma from healthy and diabetic subjects, with and without periodontal disease.

PubMed

Barnes, Virginia M; Kennedy, Adam D; Panagakos, Fotinos; Devizio, William; Trivedi, Harsh M; Jönsson, Thomas; Guo, Lining; Cervi, Shannon; Scannapieco, Frank A

2014-01-01

Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine), increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate) and ω-6 fatty acid (linoleate and arachidonate) signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic signatures associated with periodontal disease in diabetics. The metabolites identified in this study that discriminated the periodontal groups may be useful for developing diagnostics and therapeutics tailored to the diabetic population.

Global Metabolomic Analysis of Human Saliva and Plasma from Healthy and Diabetic Subjects, with and without Periodontal Disease

PubMed Central

Barnes, Virginia M.; Kennedy, Adam D.; Panagakos, Fotinos; Devizio, William; Trivedi, Harsh M.; Jönsson, Thomas; Guo, Lining; Cervi, Shannon; Scannapieco, Frank A.

2014-01-01

Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine), increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate) and ω-6 fatty acid (linoleate and arachidonate) signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic signatures associated with periodontal disease in diabetics. The metabolites identified in this study that discriminated the periodontal groups may be useful for developing diagnostics and therapeutics tailored to the diabetic population. PMID:25133529

Simultaneous determination of ornidazole and its main metabolites in human plasma by LC-MS/MS: application to a pharmacokinetic study.

PubMed

Du, Jiangbo; Ma, Zhiyu; Zhang, Yifan; Wang, Ting; Chen, Xiaoyan; Zhong, Dafang

2014-09-01

Ornidazole is a 5-nitroimidazole antimicrobial agent used for almost 40 years. A novel LC-MS/MS assay was developed and validated for the simultaneous determination of ornidazole and its main metabolites (M3, M6, M16-1, and M16-2) in human plasma. After extraction from 100 μl of plasma by protein precipitation with acetonitrile, all the analytes were separated on a Capcell PAK MG C18 column (100 × 4.6 mm, 5 μm) within 5.0 min and detected by ESI-MS/MS in the positive mode. The validation results met the acceptance criteria as per the US FDA and EMA guidelines. The validated method was successfully applied to a pharmacokinetic study after oral administration of 1000 mg ornidazole to six healthy Chinese volunteers.

Altered metabolism of orally administered loxoprofen in human subjects after an oral administration of loxoprofen for three consecutive days followed by a seven-day washout.

PubMed

Kim, In-Wha; Chung, Suk-Jae; Shim, Chang-Koo

2002-04-01

The effect of pretreatment (i.e., oral administration of loxoprofen for 3 consecutive days followed by a 7-day washout) on the pharmacokinetics and metabolism of the drug was studied in humans. In a control study, a Loxonin tablet (60 mg as loxoprofen anhydrous) was administered orally to 6 healthy male Korean subjects. In a pretreatment study, a Loxonin tablet was administered orally to the subjects once daily for 3 consecutive days. On the 10(th) day, a Loxonin tablet was administered orally to the subjects, and the concentrations of loxoprofen and the trans- and cis-alcohol metabolites in the plasma and urine were measured as a function of time. Using this pretreatment, the area under the curve (AUC) of the trans-alcohol metabolite of loxoprofen in the plasma, but not those of loxoprofen and the cis-alcohol metabolite, was increased (1.5-fold, p < 0.05), leading to increased contribution of the trans-alcohol metabolite to the total urinary recovery of loxoprofen (1.3-fold, p < 0.05). The urinary recovery of total metabolites, which was largely (> 90%) comprised of conjugate metabolites, was also increased as a result of the pretreatment (1.5-fold, p < 0.05). These results indicate that stereoselective reduction to trans-alcohol metabolites as well as the phase II metabolism of loxoprofen may be increased by such a pretreatment in human subjects. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:973-979, 2002

Phenolic and microbial-targeted metabolomics to discovering and evaluating wine intake biomarkers in human urine and plasma.

PubMed

Urpi-Sarda, Mireia; Boto-Ordóñez, María; Queipo-Ortuño, María Isabel; Tulipani, Sara; Corella, Dolores; Estruch, Ramon; Tinahones, Francisco J; Andres-Lacueva, Cristina

2015-09-01

The discovery of biomarkers of intake in nutritional epidemiological studies is essential in establishing an association between dietary intake (considering their bioavailability) and diet-related risk factors for diseases. The aim is to study urine and plasma phenolic and microbial profile by targeted metabolomics approach in a wine intervention clinical trial for discovering and evaluating food intake biomarkers. High-risk male volunteers (n = 36) were included in a randomized, crossover intervention clinical trial. After a washout period, subjects received red wine or gin, or dealcoholized red wine over four weeks. Fasting plasma and 24-h urine were collected at baseline and after each intervention period. A targeted metabolomic analysis of 70 host and microbial phenolic metabolites was performed using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). Metabolites were subjected to stepwise logistic regression to establish prediction models and received operation curves were performed to evaluate biomarkers. Prediction models based mainly on gallic acid metabolites, obtained sensitivity, specificity and area under the curve (AUC) for the training and validation sets of between 91 and 98% for urine and between 74 and 91% for plasma. Resveratrol, ethylgallate and gallic acid metabolite groups in urine samples also resulted in being good predictors of wine intake (AUC>87%). However, lower values for metabolites were obtained in plasma samples. The highest correlations between fasting plasma and urine were obtained for the prediction model score (r = 0.6, P<0.001), followed by gallic acid metabolites (r = 0.5-0.6, P<0.001). This study provides new insights into the discovery of food biomarkers in different biological samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

GC-MS-Based Metabolome and Metabolite Regulation in Serum-Resistant Streptococcus agalactiae.

PubMed

Wang, Zhe; Li, Min-Yi; Peng, Bo; Cheng, Zhi-Xue; Li, Hui; Peng, Xuan-Xian

2016-07-01

Streptococcus agalactiae causes severe systemic infections in human and fish. In the present study, we established a pathogen-plasma interaction model by which we explored how S. agalactiae evaded serum-mediated killing. We found that S. agalactiae grew faster in the presence of yellow grouper plasma than in the absence of the plasma, indicating S. agalactiae evolved a way of evading the fish immune system. To determine the events underlying this phenotype, we applied GC-MS-based metabolomics approaches to identify differential metabolomes between S. agalactiae cultured with and without yellow grouper plasma. Through bioinformatics analysis, decreased malic acid and increased adenosine were identified as the most crucial metabolites that distinguish the two groups. Meanwhile, they presented with decreased TCA cycle and elevated purine metabolism, respectively. Finally, exogenous malic acid and adenosine were used to reprogram the plasma-resistant metabolome, leading to elevated and decreased susceptibility to the plasma, respectively. Therefore, our findings reveal for the first time that S. agalactiae utilizes a metabolic trick to respond to plasma killing as a result of serum resistance, which may be reverted or enhanced by exogenous malic acid and adenosine, respectively, suggesting that the metabolic trick can be regulated by metabolites.

Plasma pharmacokinetics and metabolism of 13-cis- and all-trans-retinoic acid in the cynomolgus monkey and the identification of 13-cis- and all-trans-retinoyl-beta-glucuronides. A comparison to one human case study with isotretinoin.

PubMed

Kraft, J C; Slikker, W; Bailey, J R; Roberts, L G; Fischer, B; Wittfoht, W; Nau, H

1991-01-01

In order to compare the disposition and metabolism of 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (all-trans-RA) in the nonpregnant female cynomolgus monkey, the plasma concentrations of the parent compound, the oxidized metabolites 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, and the conjugate metabolites 13-cis-retinoyl-beta-glucuronide (13-cis-RAG) and all trans-retinoyl-beta-glucuronide (all-trans-RAG), were determined on day 1 and day 10 after oral dosing of 2 and 10 mg 13-cis- and all-trans-RA/kg/day. Both 13-cis-RAG and all-trans-RAG have been identified as major plasma metabolites in these studies using thermospray/HPLC/mass-spectrometry of the intact conjugates. AUC comparisons from 0-24 hr after administration indicated that 13-cis-RA treatment resulted in primarily cis metabolites and all-trans-RA treatment resulted in primarily trans metabolites, although low levels of isomerization products were observed. Comparison of the two doses (2 and 10 mg/kg, po) revealed that the AUCs were proportional to the dose administered. Although qualitatively similar, elimination of 13-cis-RA in the monkey was more rapid than in the human, and approximately a 10-fold greater dose of 13-cis-RA was required in the monkey to produce the AUC values comparable to the human. The elimination of all-trans-RA in monkey was faster than that of 13-cis-RA and tended to increase with repeated dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Milk decreases urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans.

PubMed

Mullen, William; Borges, Gina; Donovan, Jennifer L; Edwards, Christine A; Serafini, Mauro; Lean, Michael E J; Crozier, Alan

2009-06-01

Cocoa drinks containing flavan-3-ols are associated with many health benefits, and conflicting evidence exists as to whether milk adversely affects the bioavailability of flavan-3-ols. The objective was to determine the effect of milk on the bioavailability of cocoa flavan-3-ol metabolites. Nine human volunteers followed a low-flavonoid diet for 2 d before drinking 250 mL of a cocoa beverage, made with water or milk, that contained 45 micromol (-)-epicatechin and (-)-catechin. Plasma and urine samples were collected for 24 h, and flavan-3-ol metabolites were analyzed by HPLC with photodiode array and mass spectrometric detection. Milk affected neither gastric emptying nor the transit time through the small intestine. Two flavan-3-ol metabolites were detected in plasma and 4 in urine. Milk had only minor effects on the plasma pharmacokinetics of an (epi)catechin-O-sulfate and had no effect on an O-methyl-(epi)catechin-O-sulfate. However, milk significantly lowered the excretion of 4 urinary flavan-3-ol metabolites from 18.3% to 10.5% of the ingested dose (P = 0.016). Studies that showed protective effects of cocoa and those that showed no effect of milk on bioavailability used products that have a much higher flavan-3-ol content than does the commercial cocoa used in the present study. Most studies of the protective effects of cocoa have used drinks with a very high flavan-3-ol content. Whether similar protective effects are associated with the consumption of many commercial chocolate and cocoa products containing substantially lower amounts of flavan-3-ols, especially when absorption at lower doses is obstructed by milk, remains to be determined.

GC-MS and LC-MS analysis of nerve agents in body fluids: intra-laboratory verification test using spiked plasma and urine samples.

PubMed

Koller, Marianne; Becker, Christian; Thiermann, Horst; Worek, Franz

2010-05-15

The purpose of this study was to check the applicability of different analytical methods for the identification of unknown nerve agents in human body fluids. Plasma and urine samples were spiked with nerve agents (plasma) or with their metabolites (urine) or were left blank. Seven random samples (35% of all samples) were selected for the verification test. Plasma was worked up for unchanged nerve agents and for regenerated nerve agents after fluoride-induced reactivation of nerve agent-inhibited butyrylcholinesterase. Both extracts were analysed by GC-MS. Metabolites were extracted from plasma and urine, respectively, and were analysed by LC-MS. The urinary metabolites and two blank samples could be identified without further measurements, plasma metabolites and blanks were identified in six of seven samples. The analysis of unchanged nerve agent provided five agents/blanks and the sixth agent after further investigation. The determination of the regenerated agents also provided only five clear findings during the first screening because of a rather noisy baseline. Therefore, the sample preparation was extended by a size exclusion step performed before addition of fluoride which visibly reduced baseline noise and thus improved identification of the two missing agents. The test clearly showed that verification should be performed by analysing more than one biomarker to ensure identification of the agent(s). Copyright (c) 2010 Elsevier B.V. All rights reserved.

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry.

PubMed

Robinson, P R; Jones, M D; Maddock, J; Rees, L W

1991-03-08

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[ def,p ]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madeen, Erin P.; Ognibene, Ted J.; Corley, Richard A.

Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in non-smokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a micro-dose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novelmore » “moving wire” interface between ultra-performance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself, (Cmax= 18.5 ± 15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/-)-DBC-11,12-diol (Cmax= 2.5 ± 1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [14C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (Cmax= 35.8 ± 23.0 pg/pool, Tmax= 6-12 h pool). [14C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (Cmax= 29.4 ± 11.6 pg/pool, Tmax= 6-12 h pool). Parent [14C]-DBC was not detected in urine. This is the first dataset to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.« less

Human Micro-Dosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry

PubMed Central

Madeen, Erin P.; Ognibene, Ted J.; Corley, Richard A.; McQuistan, Tammie J.; Baird, William M.; Bench, Graham; Turteltaub, Ken W.; Williams, David E.

2017-01-01

Metabolism is a key health risk factor for exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in non-smokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a micro-dose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novel “moving wire” interface between ultra-performance liquid chromatography (UPLC) and the AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself, (Cmax= 18.5 ± 15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/−)-DBC-11,12-diol (Cmax= 2.5 ± 1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Deconjugated and conjugated metabolites were detected in urine with [14C]-(+/−)-DBC-tetraol identified as the major metabolite, 88.7% of which was detected upon enzymatic deconjugation (Cmax= 35.8 ± 23.0 pg/pool, Tmax= 6–12 h pool). [14C]-DBC-11,12-diol, of which 94.4% was conjugated and identified in urine (Cmax= 29.4 ± 11.6 pg/pool, Tmax= 6–12 h pool). Parent [14C]-DBC was not detected in the urine. This is the first dataset to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose laboratory animal models to human translation for environmental health risk assessment. PMID:27494294

Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure

PubMed Central

Cooper, Jason P; Hwang, Kyunghwa; Singh, Hardeep; Wang, Dong; Reynolds, C Patrick; Curley, Robert W; Williams, Simon C; Maurer, Barry J; Kang, Min H

2011-01-01

BACKGROUND AND PURPOSE High plasma levels of fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4-HPR has been limiting its therapeutic applications. This study characterized metabolism of 4-HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4-HPR plasma levels in mice and to increase the low bioavailability of 4-HPR. EXPERIMENTAL APPROACH 4-HPR metabolites were identified by mass spectrometric analysis and levels of 4-HPR and its metabolites [N-(4-methoxyphenyl)retinamide (4-MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR)] were quantified by high-performance liquid chromatography (HPLC). Kinetic analysis of enzyme activities and the effects of enzyme inhibitors were performed in pooled human and pooled mouse liver microsomes, and in human cytochrome P450 (CYP) 3A4 isoenzyme microsomes. In vivo metabolism of 4-HPR was inhibited in mice. KEY RESULTS Six 4-HPR metabolites were identified in the plasma of patients and mice. 4-HPR was oxidized to 4-oxo-4-HPR, at least in part via human CYP3A4. The CYP3A4 inhibitor ketoconazole significantly reduced 4-oxo-4-HPR formation in both human and mouse liver microsomes. In two strains of mice, co-administration of ketoconazole with 4-HPR in vivo significantly increased 4-HPR plasma concentrations by > twofold over 4-HPR alone and also increased 4-oxo-4-HPR levels. CONCLUSIONS AND IMPLICATIONS Mice may serve as an in vivo model of human 4-HPR pharmacokinetics. In vivo data suggest that the co-administration of ketoconazole at normal clinical doses with 4-HPR may increase systemic exposure to 4-HPR in humans. PMID:21391977

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine.

PubMed

Buechler, Jochen; Schwab, Matthias; Mikus, Gerd; Fischer, Beate; Hermle, Leo; Marx, Claudia; Grön, Georg; Spitzer, Manfred; Kovar, Karl Artur

2003-08-15

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.

A novel LC-MS/MS method for the simultaneous quantification of topiramate and its main metabolites in human plasma.

PubMed

Milosheska, Daniela; Roškar, Robert

2017-05-10

The aim of the present report was to develop and validate simple, sensitive and reliable LC-MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid-liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10-20μg/mL for TPM, 0.01-2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001-0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.

Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract.

PubMed

de Bock, Martin; Thorstensen, Eric B; Derraik, José G B; Henderson, Harold V; Hofman, Paul L; Cutfield, Wayne S

2013-11-01

Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxytyrosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest source of olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical taken either as liquid or capsules. To quantify the bioavailability and metabolism of oleuropein and hydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years were randomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, and then the opposite strength (but same formulation) a week later. Plasma and urine samples were collected at fixed intervals for 24 h post-ingestion. Phenolic content was analyzed by LC-ESI-MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasma were greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL; p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated and glucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier with liquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailability of phenolic compounds, with males displaying greater plasma area under the curve for conjugated hydroxytyrosol (11,600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosol metabolites were recovered in the urine within 8 h. There was wide inter-individual variation. OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

PubMed

Harada, Sei; Hirayama, Akiyoshi; Chan, Queenie; Kurihara, Ayako; Fukai, Kota; Iida, Miho; Kato, Suzuka; Sugiyama, Daisuke; Kuwabara, Kazuyo; Takeuchi, Ayano; Akiyama, Miki; Okamura, Tomonori; Ebbels, Timothy M D; Elliott, Paul; Tomita, Masaru; Sato, Asako; Suzuki, Chizuru; Sugimoto, Masahiro; Soga, Tomoyoshi; Takebayashi, Toru

2018-01-01

Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population. Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid. In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

Quantitative monitoring of tamoxifen in human plasma extended to 40 metabolites using liquid-chromatography high-resolution mass spectrometry: new investigation capabilities for clinical pharmacology.

PubMed

Dahmane, Elyes; Boccard, Julien; Csajka, Chantal; Rudaz, Serge; Décosterd, Laurent; Genin, Eric; Duretz, Bénédicte; Bromirski, Maciej; Zaman, Khalil; Testa, Bernard; Rochat, Bertrand

2014-04-01

Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans, a technique of detection that shows comparable selectivity and sensitivity to ion transitions (SRM) performed with triple-quadrupole (TQ)-MS but that allows de facto determination of "all" ions including drug metabolites. This could be of potential utility in in vivo drug metabolism and pharmacovigilance studies in order to have a more comprehensive insight in drug biotransformation profile differences in patients. This simultaneous quantitative and qualitative (Quan/Qual) approach has been tested with 20 patients chronically treated with tamoxifen (TAM). The absolute quantification of TAM and three metabolites in plasma was realized using HR- and TQ-MS and compared. The same LC-HR-MS analysis allowed the identification and relative quantification of 37 additional TAM metabolites. A number of new metabolites were detected in patients' plasma including metabolites identified as didemethyl-trihydroxy-TAM-glucoside and didemethyl-tetrahydroxy-TAM-glucoside conjugates corresponding to TAM with six and seven biotransformation steps, respectively. Multivariate analysis allowed relevant patterns of metabolites and ratios to be associated with TAM administration and CYP2D6 genotype. Two hydroxylated metabolites, α-OH-TAM and 4'-OH-TAM, were newly identified as putative CYP2D6 substrates. The relative quantification was precise (<20 %), and the semiquantitative estimation suggests that metabolite levels are non-negligible. Metabolites could play an important role in drug toxicity, but their impact on drug-related side effects has been partially neglected due to the tremendous effort needed with previous MS technologies. Using present HR-MS, this situation should evolve with the straightforward determination of drug metabolites, enlarging the possibilities in studying inter- and intra-patients drug metabolism variability and related effects.

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

PubMed

Hashiguchi, Takuyu; Kurogi, Katsuhisa; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Yasuda, Shin; Liu, Ming-Cheh; Suiko, Masahito

2011-01-01

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

High-performance liquid chromatographic method for the determination of moclobemide and its two major metabolites in human plasma.

PubMed

Rakic, Anita; Miljkovic, Branislava; Pokrajac, Milena; Vucicevic, Katarina

2007-03-12

A selective, sensitive, and simple high-performance liquid chromatographic (HPLC) method was developed for the determination of moclobemide and its two major metabolites, Ro 12-5637 and Ro 12-8095, in human plasma. Sample preparation (0.5 ml of plasma) involved solid-phase extraction (SPE) using Speedisk H(2)O-Philic DVB columns. Separations were performed on a Waters XTerra RP18 column (5 microm, 150 mm x 4.6 mm). The mobile phase consisted of 10 mM KH(2)PO(4) with 1% triethylamine (pH 3.9) and acetonitrile (83:17, v/v), and a flow-rate was 1.2 ml/min. The total run time was 13 min. UV detection was performed at 240 nm. Mean absolute recoveries were > or =90% and the limit of quantification (LOQ) for all analytes was 0.02 mg/l. Calibration curves were linear (r>0.995) over a wide range of the analyte concentrations in plasma; thus, the method is suitable for different clinical studies when large variations in the drug/metabolites concentrations are observed. During a 5-day assay validation procedure the accuracy and precision were tested and proven (relative errors (RE)< or =13%; intra-day coefficient of variation (CV)< or =7%; inter-day CV< or =13%). Many drugs frequently used in the target patient population were evaluated for potential interference in order method selectivity to be ensured. The assay has been used in a clinical pharmacokinetic study to assess steady-state pharmacokinetics of moclobemide and two metabolites in depressive patients on mono- and combined therapy.

Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302).

PubMed

Watanabe, Tanya A; Geary, Richard S; Levin, Arthur A

2006-01-01

In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.

Pharmacokinetics, Metabolism, and Excretion of the Antiviral Drug Arbidol in Humans

PubMed Central

Deng, Pan; Zhong, Dafang; Yu, Kate; Zhang, Yifan; Wang, Ting

2013-01-01

Arbidol is a broad-spectrum antiviral drug that is used clinically to treat influenza. In this study, the pharmacokinetics, metabolism, and excretion of arbidol were investigated in healthy male Chinese volunteers after a single oral administration of 200 mg of arbidol hydrochloride. A total of 33 arbidol metabolites were identified in human plasma, urine, and feces. The principal biotransformation pathways included sulfoxidation, dimethylamine N-demethylation, glucuronidation, and sulfate conjugation. The major drug-related component in the plasma was sulfinylarbidol (M6-1), followed by unmetabolized arbidol, N-demethylsulfinylarbidol (M5), and sulfonylarbidol (M8). The exposures of M5, M6-1, and M8, as determined by the metabolite-to-parent area under the plasma concentration-time curve from 0 to t (AUC0-t) ratio, were 0.9 ± 0.3, 11.5 ± 3.6, and 0.5 ± 0.2, respectively. In human urine, glucuronide and sulfate conjugates were detected as the major metabolites, accounting for 6.3% of the dose excreted within 0 to 96 h after drug administration. The fecal specimens mainly contained the unchanged arbidol, accounting for 32.4% of the dose. Microsomal incubation experiments demonstrated that the liver and intestines were the major organs that metabolize arbidol in humans. CYP3A4 was the major isoform involved in arbidol metabolism, whereas the other P450s and flavin-containing monooxygenases (FMOs) played minor roles. These results indicated possible drug interactions between arbidol and CYP3A4 inhibitors and inducers. Further investigations are needed to understand the importance of M6-1 in the efficacy and safety of arbidol, because of its high plasma exposure and long elimination half-life (25.0 h). PMID:23357765

New insights into the bioavailability of red raspberry anthocyanins and ellagitannins.

PubMed

Ludwig, Iziar A; Mena, Pedro; Calani, Luca; Borges, Gina; Pereira-Caro, Gema; Bresciani, Letizia; Del Rio, Daniele; Lean, Michael E J; Crozier, Alan

2015-12-01

Red raspberries, containing ellagitannins and cyanidin-based anthocyanins, were fed to volunteers and metabolites appearing in plasma and urine were analysed by UHPLC-MS. Anthocyanins were not absorbed to any extent with sub nmol/L concentrations of cyanidin-3-O-glucoside and a cyanidin-O-glucuronide appearing transiently in plasma. Anthocyanins excreted in urine corresponded to 0.007% of intake. More substantial amounts of phase II metabolites of ferulic acid and isoferulic acid, along with 4'-hydroxyhippuric acid, potentially originating from pH-mediated degradation of cyanidin in the proximal gastrointestinal tract, appeared in urine and also plasma where peak concentrations were attained 1-1.5h after raspberry intake. Excretion of 18 anthocyanin-derived metabolites corresponded to 15.0% of intake, a figure substantially higher than obtained in other anthocyanin feeding studies. Ellagitannins pass from the small to the large intestine where the colonic microbiota mediate their conversion to urolithins A and B which appeared in plasma and were excreted almost exclusively as sulfate and glucuronide metabolites. The urolithin metabolites persisted in the circulatory system and were excreted in urine for much longer periods of time than the anthocyanin metabolites although their overall urinary recovery was lower at 7.0% of intake. It is events originating in the proximal and distal gastrointestinal tract, and subsequent phase II metabolism, that play an important role in the bioavailability of both anthocyanins and ellagitannins and it is their metabolites which appear in the circulatory system, that are key to elucidating the mode of action(s) underlying the protective effects of these compounds on human health. Copyright © 2015 Elsevier Inc. All rights reserved.

The heterozygous N291S mutation in the lipoprotein lipase gene impairs whole-body insulin sensitivity and affects a distinct set of plasma metabolites in humans.

PubMed

Berg, Sofia Mikkelsen; Havelund, Jesper; Hasler-Sheetal, Harald; Kruse, Vibeke; Pedersen, Andreas James Thestrup; Hansen, Aleksander Bill; Nybo, Mads; Beck-Nielsen, Henning; Højlund, Kurt; Færgeman, Nils Joakim

Mutations in the lipoprotein lipase gene causing decreased lipoprotein lipase activity are associated with surrogate markers of insulin resistance and the metabolic syndrome in humans. We investigated the hypothesis that a heterozygous lipoprotein lipase mutation (N291S) induces whole-body insulin resistance and alterations in the plasma metabolome. In 6 carriers of a heterozygous lipoprotein lipase mutation (N291S) and 11 age-matched and weight-matched healthy controls, we examined insulin sensitivity and substrate metabolism by euglycemic-hyperinsulinemic clamps combined with indirect calorimetry. Plasma samples were taken before and after the clamp (4 hours of physiological hyperinsulinemia), and metabolites were measured enzymatically or by gas chromatography-mass spectrometry. Compared with healthy controls, heterozygous carriers of a defective lipoprotein lipase allele had elevated fasting plasma levels triglycerides (P < .006), and markedly impaired insulin-stimulated glucose disposal rates (P < .024) and nonoxidative glucose metabolism (P < .015). Plasma metabolite profiling demonstrated lower circulating levels of pyruvic acid and α-tocopherol in the N291S carriers than in controls both before and after stimulation with insulin (all >1.5-fold change and P < .05). Heterozygous carriers with a defective lipoprotein lipase allele are less insulin sensitive and have increased plasma levels of nonesterified fatty acids and triglycerides. The heterozygous N291S carriers also have a distinct plasma metabolomic signature, which may serve as a diagnostic tool for deficient lipoprotein lipase activity and as a marker of lipid-induced insulin resistance. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis.

PubMed

Sahu, Kapendra; Siddiqui, Anees A; Shaharyar, Mohammad; Ahmad, Niyaz; Anwar, Mohammad; Ahmad, Farhan J

2013-07-01

A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Human metabolic profiles are stably controlled by genetic and environmental variation

PubMed Central

Nicholson, George; Rantalainen, Mattias; Maher, Anthony D; Li, Jia V; Malmodin, Daniel; Ahmadi, Kourosh R; Faber, Johan H; Hallgrímsdóttir, Ingileif B; Barrett, Amy; Toft, Henrik; Krestyaninova, Maria; Viksna, Juris; Neogi, Sudeshna Guha; Dumas, Marc-Emmanuel; Sarkans, Ugis; The MolPAGE Consortium; Silverman, Bernard W; Donnelly, Peter; Nicholson, Jeremy K; Allen, Maxine; Zondervan, Krina T; Lindon, John C; Spector, Tim D; McCarthy, Mark I; Holmes, Elaine; Baunsgaard, Dorrit; Holmes, Chris C

2011-01-01

1H Nuclear Magnetic Resonance spectroscopy (1H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top-down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non-identical twin pairs donated plasma and urine samples longitudinally. We acquired 1H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common-environmental), individual-environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual-environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in 1H NMR-detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker-discovery studies. We provide a power-calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect 1H NMR-based biomarkers quantifying predisposition to disease. PMID:21878913

Validation of a sensitive LC-MS assay for quantification of glyburide and its metabolite 4-transhydroxy glyburide in plasma and urine: an OPRU Network study

PubMed Central

Naraharisetti, Suresh Babu; Kirby, Brian J.; Hebert, Mary F.; Easterling, Thomas R.; Unadkat, Jashvant D.

2009-01-01

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 ml) or urine (0.1 ml). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was < 15% whereas the accuracy (% deviation from expected) ranged from −10.1–14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate. PMID:17980680

Online restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry for the simultaneous determination of vanillin and its vanillic acid metabolite in human plasma.

PubMed

Li, De-Qiang; Zhang, Zhi-Qing; Yang, Xiu-Ling; Zhou, Chun-Hua; Qi, Jin-Long

2016-09-01

An automated online solid-phase extraction with restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry was developed and validated for the simultaneous quantification of vanillin and its vanillic acid metabolite in human plasma. After protein precipitation by methanol, which contained the internal standards, the supernatant of plasma samples was injected to the system, the endogenous large molecules were flushed out, and target analytes were trapped and enriched on the adsorbent, resulting in a minimization of sample complexity and ion suppression effects. Calibration curves were linear over the concentrations of 5-1000 ng/mL for vanillin and 10-5000 ng/mL for vanillic acid with a coefficient of determination >0.999 for the determined compounds. The lower limits of quantification of vanillin and vanillic acid were 5.0 and 10.0 ng/mL, respectively. The intra- and inter-run precisions expressed as the relative standard deviation were 2.6-8.6 and 3.2-10.2%, respectively, and the accuracies expressed as the relative error were in the range of -6.1 to 7.3%. Extraction recoveries of analytes were between 89.5 and 97.4%. There was no notable matrix effect for any analyte concentration. The developed method was proved to be sensitive, repeatable, and accurate for the quantification of vanillin and its vanillic acid metabolite in human plasma. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma amino acid and metabolite signatures tracking diabetes progression in the UCD-T2DM rat model

USDA-ARS?s Scientific Manuscript database

Elevations of plasma concentrations of branched-chain amino acids (BCAAs) are observed in human insulin resistance and type 2 diabetes mellitus (T2DM); however, there has been some controversy with respect to the passive or causative nature of the BCAA phenotype. Using untargeted metabolomics, plasm...

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS.

PubMed

Choong, Eva; Rudaz, Serge; Kottelat, Astrid; Guillarme, Davy; Veuthey, Jean-Luc; Eap, Chin B

2009-12-05

A simple and sensitive LC-MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2-200 ng/mL for DUL, OLA, and STN, 4-200 ng/mL for DSTN, 5-1000 ng/mL for ARI, DARI and finally 2-1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

[Interspecies differences of noopept pharmacokinetics].

PubMed

Boĭko, S S; Korotkov, S A; Zherdev, V P; Gudasheva, T A; Ostrovskaia, R U; Voronina, T A

2004-01-01

Significant interspecific differences in the pharmacokinetics of noopept are manifested by a decrease in the drug elimination rate on the passage from rats to rabbits and humans. Very intensive metabolism of noopept was observed upon intravenous administration in rats. In these animals, presystemic elimination mechanisms lead to the formation of a specific metabolite representing a product of drug biotransformation hydroxylated at the phenyl ring. In rabbits, unchanged noopept circulates in the blood for a longer time upon both intravenous and peroral introduction, biotransformation proceeds at a much slower rate, and no metabolites analogous to that found in rats are detected. The noopept pharmacokinetics in humans differs from that in animals by still slower elimination and considerable individual variability. No drug metabolites are found in the human blood plasma, probably because of a relatively small dose and low concentration.

A rapid and sensitive LC-MS/MS-ESI method for the determination of tolvaptan and its two main metabolites in human plasma.

PubMed

Jiang, Juanjuan; Tian, Lei; Huang, Yiling; Yan, Yan; Li, Yishi

2016-08-01

A liquid chromatography-tandem mass spectrometry (LC-MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150×3.9mm, 5μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449-252 for tolvaptan, m/z 479-252 for metabolite DM-4103, m/z 481-252 for metabolite DM-4107 and m/z 463-266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1ng/mL. The method was validated over a linear range from 1 to 500ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15mg tolvaptan tablet. Copyright © 2016. Published by Elsevier B.V.

Pharmacokinetic study of isocorynoxeine metabolites mediated by cytochrome P450 enzymes in rat and human liver microsomes.

PubMed

Zhao, Lizhu; Zang, Bin; Qi, Wen; Chen, Fangfang; Wang, Haibo; Kano, Yoshihiro; Yuan, Dan

2016-06-01

Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of dose on the bioavailability of coffee chlorogenic acids in humans.

PubMed

Stalmach, Angélique; Williamson, Gary; Crozier, Alan

2014-08-01

Single servings of coffee beverage containing low (412 μmol), medium (635 μmol) and high (795 μmol) amounts of chlorogenic acids were administered to eleven healthy volunteers in a double-blind randomised controlled trial. Analysis of plasma and urine collected for 24 h revealed the presence of 12 metabolites in plasma and 16 metabolites in urine, principally in the form of sulphates, and to a lesser extent glucuronides of caffeic, ferulic, dihydrocaffeic and dihydroferulic acids, as well as intact feruloylquinic and caffeoylquinic acids, and sulphated caffeoylquinic acid lactones. Median values of peak plasma concentrations after increasing doses of chlorogenic acids were 1088, 1526 and 1352 nM. In urine the median amounts of metabolites excreted after 24 h following consumption of the three coffees were 101, 160 and 125 μmol, accounting for 24%, 25% and 16% of the doses ingested. Peak plasma concentration and urinary excretion values showed trends towards a reduced bioavailability of chlorogenic acids associated with the highest dose ingested, when expressed as percentages of intake. Potential biomarkers of coffee intake were identified as feruloylquinic acids and sulphated caffeoylquinic acid lactones in plasma and urine with positive moderate to strong coefficients of determination for peak plasma concentrations (0.60-0.81) and amounts excreted in urine (0.36-0.73) (P < 0.05).

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

PubMed

Prónai, L; Yukinobu, I; Láng, I; Fehér, J

1992-01-01

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.

UPLC-MS/MS method for the determination of tobacco-specific biomarker NNAL, tamoxifen and its main metabolites in rat plasma.

PubMed

Xiong, Wei; Zhao, Jiajia; Wang, Ling; Jiang, Xuehua

2017-06-01

Cigarette smoke is known to interact with tamoxifen-metabolizing enzymes and transporters and potentially affect its treatment outcome. 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) because it is frequently used as a biomarker to assess human smoke exposure. In order to study the potential pharmacokinetic interaction between cigarette smoke and tamoxifen in rats a UPLC-MS/MS method for the simultaneous determination of NNAL and tamoxifen along with its metabolites in rat plasma has been developed and validated. Analytes were extracted with methanol and separated on a HSS T3 column by a gradient elution with the mobile phase consisting of acetonitrile and water. The lower limits of quantitation ranged from 0.05 to 0.62 ng/mL. Precisions showed RSD <15.8% and accuracy in the range 80.6-116.0%. Mean analyte recoveries ranged from 76.9 to 108.4%. The method was successfully applied to study the effects of cigarette smoke condensate (CSC), NNK and benzo(a)pyrene pre-treatment on the pharmacokinetics of tamoxifen and its metabolites in rats. Significant effects of CSC, NNK, benzo(a)pyrene were observed on pharmacokinetics of tamoxifen and its metabolites. We also found that plasma NNAL levels are statistically significant correlated with plasma 4-hydroxy-tamoxifen and endoxifen. Copyright © 2016 John Wiley & Sons, Ltd.

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography-mass spectrometry and ion mobility mass spectrometry.

PubMed

Silvestro, Luigi; Tarcomnicu, Isabela; Dulea, Constanta; Attili, Nageswara Rao B N; Ciuca, Valentin; Peru, Dan; Rizea Savu, Simona

2013-10-01

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Determination of Cloretazine (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS).

PubMed

Bai, Feng; Minkin, Patton; Fraga, Charles H; O'Shaughnessy, Melinda A; Gururangan, Sri; Stewart, Clinton F

2007-06-15

A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry.

PubMed

Chhonker, Yashpal S; Pandey, Chandra P; Chandasana, Hardik; Laxman, Tulsankar Sachin; Prasad, Yarra Durga; Narain, V S; Dikshit, Madhu; Bhatta, Rabi S

2016-03-01

The interest in therapeutic drug monitoring has increased over the last few years. Inter- and intra-patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield(TM) C18 column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)-acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease. Copyright © 2015 John Wiley & Sons, Ltd.

Disposition of the Dietary Mutagen 2-Amino-3,8-dimethylimidazo[4,5- f ]quinoxaline in Healthy and Pancreatic Cancer Compromised Humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malfatti, Michael A.; Kuhn, Edward A.; Turteltaub, Kenneth W.

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Once diagnosed, prognosis is poor with a 5-year survival rate of less than 5%. Exposure to carcinogenic heterocyclic amines (HCAs) derived from cooked meat has been shown to be positively associated with pancreatic cancer risk. To evaluate the processes that determines the carcinogenic potential of HCAs for human pancreas, 14-carbon labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a putative human carcinogenic HCA found in well-done cooked meat, was administered at a dietary relevant dose to human volunteers diagnosed with pancreatic cancer undergoing partial pancreatectomy and healthy control volunteers. After 14C-MeIQx exposure,more » blood and urine was collected for pharmacokinetic and metabolite analysis. MeIQx-DNA adducts levels were quantified by accelerator mass spectrometry from pancreatic tissue excised during surgery from the cancer patient group. Pharmacokinetic analysis of plasma revealed a rapid distribution of MeIQx with a plasma elimination half-life of approximately 3.5 hr in 50% of the cancer patients and all of the control volunteers. In 2 of the 4 cancer patients very low levels of MeIQx were detected in plasma and urine suggesting low absorption from the gut into the plasma. Urinary metabolite analysis revealed five MeIQx metabolites with 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid being the most abundant accounting for 25%–50% of the recovered 14-carbon/ml urine. We found there was no discernable difference in metabolite levels between the cancer patient volunteers and the control group. MeIQx-DNA adduct analysis of pancreas and duodenum tissue revealed adduct levels indistinguishable from background levels. Lastly, although other meat-derived HCA mutagens have been shown to bind DNA in pancreatic tissue, indicating that exposure to HCAs from cooked meat cannot be discounted as a risk factor for pancreatic cancer, the results from this current study show that exposure to a single dietary dose of MeIQx does not readily form measurable DNA adducts under the conditions of the experiment.« less

Disposition of the Dietary Mutagen 2-Amino-3,8-dimethylimidazo[4,5- f ]quinoxaline in Healthy and Pancreatic Cancer Compromised Humans

DOE PAGES

Malfatti, Michael A.; Kuhn, Edward A.; Turteltaub, Kenneth W.; ...

2016-02-26

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Once diagnosed, prognosis is poor with a 5-year survival rate of less than 5%. Exposure to carcinogenic heterocyclic amines (HCAs) derived from cooked meat has been shown to be positively associated with pancreatic cancer risk. To evaluate the processes that determines the carcinogenic potential of HCAs for human pancreas, 14-carbon labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a putative human carcinogenic HCA found in well-done cooked meat, was administered at a dietary relevant dose to human volunteers diagnosed with pancreatic cancer undergoing partial pancreatectomy and healthy control volunteers. After 14C-MeIQx exposure,more » blood and urine was collected for pharmacokinetic and metabolite analysis. MeIQx-DNA adducts levels were quantified by accelerator mass spectrometry from pancreatic tissue excised during surgery from the cancer patient group. Pharmacokinetic analysis of plasma revealed a rapid distribution of MeIQx with a plasma elimination half-life of approximately 3.5 hr in 50% of the cancer patients and all of the control volunteers. In 2 of the 4 cancer patients very low levels of MeIQx were detected in plasma and urine suggesting low absorption from the gut into the plasma. Urinary metabolite analysis revealed five MeIQx metabolites with 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid being the most abundant accounting for 25%–50% of the recovered 14-carbon/ml urine. We found there was no discernable difference in metabolite levels between the cancer patient volunteers and the control group. MeIQx-DNA adduct analysis of pancreas and duodenum tissue revealed adduct levels indistinguishable from background levels. Lastly, although other meat-derived HCA mutagens have been shown to bind DNA in pancreatic tissue, indicating that exposure to HCAs from cooked meat cannot be discounted as a risk factor for pancreatic cancer, the results from this current study show that exposure to a single dietary dose of MeIQx does not readily form measurable DNA adducts under the conditions of the experiment.« less

LC-MS/MS-based approach for obtaining exposure estimates of metabolites in early clinical trials using radioactive metabolites as reference standards.

PubMed

Zhang, Donglu; Raghavan, Nirmala; Chando, Theodore; Gambardella, Janice; Fu, Yunlin; Zhang, Duxi; Unger, Steve E; Humphreys, W Griffith

2007-12-01

An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.

Metabolic Disposition of Osimertinib in Rats, Dogs, and Humans: Insights into a Drug Designed to Bind Covalently to a Cysteine Residue of Epidermal Growth Factor Receptor.

PubMed

Dickinson, Paul A; Cantarini, Mireille V; Collier, Jo; Frewer, Paul; Martin, Scott; Pickup, Kathryn; Ballard, Peter

2016-08-01

Preclinical and clinical studies were conducted to determine the metabolism and pharmacokinetics of osimertinib and key metabolites AZ5104 and AZ7550. Osimertinib was designed to covalently bind to epidermal growth factor receptors, allowing it to achieve nanomolar cellular potency (Finlay et al., 2014). Covalent binding was observed in incubations of radiolabeled osimertinib with human and rat hepatocytes, human and rat plasma, and human serum albumin. Osimertinib, AZ5104, and AZ7550 were predominantly metabolized by CYP3A. Seven metabolites were detected in human hepatocytes, also observed in rat or dog hepatocytes at similar or higher levels. After oral administration of radiolabeled osimertinib to rats, drug-related material was widely distributed, with the highest radioactivity concentrations measured at 6 hours postdose in most tissues; radioactivity was detectable in 42% of tissues 60 days postdose. Concentrations of [(14)C]-radioactivity in blood were lower than in most tissues. After the administration of a single oral dose of 20 mg of radiolabeled osimertinib to healthy male volunteers, ∼19% of the dose was recovered by 3 days postdose. At 84 days postdose, mean total radioactivity recovery was 14.2% and 67.8% of the dose in urine and feces. The most abundant metabolite identified in feces was AZ5104 (∼6% of dose). Osimertinib accounted for ∼1% of total radioactivity in the plasma of non-small cell lung cancer patients after 22 days of 80-mg osimertinib once-daily treatment; the most abundant circulatory metabolites were AZ7550 and AZ5104 (<10% of total osimertinib-related material). Osimertinib is extensively distributed and metabolized in humans and is eliminated primarily via the fecal route. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Maternal-fetal disposition of glyburide in pregnant mice is dependent on gestational age.

PubMed

Shuster, Diana L; Risler, Linda J; Liang, Chao-Kang J; Rice, Kenneth M; Shen, Danny D; Hebert, Mary F; Thummel, Kenneth E; Mao, Qingcheng

2014-08-01

Gestational diabetes mellitus is a major complication of human pregnancy. The oral clearance (CL) of glyburide, an oral antidiabetic drug, increases 2-fold in pregnant women during late gestation versus nonpregnant controls. In this study, we examined gestational age-dependent changes in maternal-fetal pharmacokinetics (PK) of glyburide and metabolites in a pregnant mouse model. Nonpregnant and pregnant FVB mice were given glyburide by retro-orbital injection. Maternal plasma was collected over 240 minutes on gestation days (gd) 0, 7.5, 10, 15, and 19; fetuses were collected on gd 15 and 19. Glyburide and metabolites were quantified using high-performance liquid chromatography-mass spectrometry, and PK analyses were performed using a pooled data bootstrap approach. Maternal CL of glyburide increased approximately 2-fold on gd 10, 15, and 19 compared with nonpregnant controls. Intrinsic CL of glyburide in maternal liver microsomes also increased as gestation progressed. Maternal metabolite/glyburide area under the curve ratios were generally unchanged or slightly decreased throughout gestation. Total fetal exposure to glyburide was <5% of maternal plasma exposure, and was doubled on gd 19 versus gd 15. Fetal metabolite concentrations were below the limit of assay detection. This is the first evidence of gestational age-dependent changes in glyburide PK. Increased maternal glyburide clearance during gestation is attributable to increased hepatic metabolism. Metabolite elimination may also increase during pregnancy. In the mouse model, fetal exposure to glyburide is gestational age-dependent and low compared with maternal plasma exposure. These results indicate that maternal glyburide therapeutic strategies may require adjustments in a gestational age-dependent manner if these same changes occur in humans. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Maternal-Fetal Disposition of Glyburide in Pregnant Mice Is Dependent on Gestational Age

PubMed Central

Shuster, Diana L.; Risler, Linda J.; Liang, Chao-Kang J.; Rice, Kenneth M.; Shen, Danny D.; Hebert, Mary F.; Thummel, Kenneth E.

2014-01-01

Gestational diabetes mellitus is a major complication of human pregnancy. The oral clearance (CL) of glyburide, an oral antidiabetic drug, increases 2-fold in pregnant women during late gestation versus nonpregnant controls. In this study, we examined gestational age–dependent changes in maternal-fetal pharmacokinetics (PK) of glyburide and metabolites in a pregnant mouse model. Nonpregnant and pregnant FVB mice were given glyburide by retro-orbital injection. Maternal plasma was collected over 240 minutes on gestation days (gd) 0, 7.5, 10, 15, and 19; fetuses were collected on gd 15 and 19. Glyburide and metabolites were quantified using high-performance liquid chromatography–mass spectrometry, and PK analyses were performed using a pooled data bootstrap approach. Maternal CL of glyburide increased approximately 2-fold on gd 10, 15, and 19 compared with nonpregnant controls. Intrinsic CL of glyburide in maternal liver microsomes also increased as gestation progressed. Maternal metabolite/glyburide area under the curve ratios were generally unchanged or slightly decreased throughout gestation. Total fetal exposure to glyburide was <5% of maternal plasma exposure, and was doubled on gd 19 versus gd 15. Fetal metabolite concentrations were below the limit of assay detection. This is the first evidence of gestational age–dependent changes in glyburide PK. Increased maternal glyburide clearance during gestation is attributable to increased hepatic metabolism. Metabolite elimination may also increase during pregnancy. In the mouse model, fetal exposure to glyburide is gestational age–dependent and low compared with maternal plasma exposure. These results indicate that maternal glyburide therapeutic strategies may require adjustments in a gestational age–dependent manner if these same changes occur in humans. PMID:24898265

Simultaneous determination of flurbiprofen and its hydroxy metabolite in human plasma by liquid chromatography-tandem mass spectrometry for clinical application.

PubMed

Lee, Hye-In; Choi, Chang-Ik; Byeon, Ji-Yeong; Lee, Jung-Eun; Park, So-Young; Kim, Young-Hoon; Kim, Se-Hyung; Lee, Yun-Jeong; Jang, Choon-Gon; Lee, Seok-Yong

2014-11-15

Flurbiprofen (FLB) is one of the phenylalkanoic acid derivatives of non-steroidal anti-inflammatory drugs used for the management of pain and inflammation in patients with arthritis. We developed and validated a rapid and sensitive high-performance liquid chromatography analytical method utilizing tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of FLB and its major metabolite, 4'-hydroxyflurbiprofen (4'-OH-FLB), in human plasma. Probenecid was used as an internal standard (IS). After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of the two analytes was achieved using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)-methanol (15:85, v/v) and quantified by MS/MS detection in ESI negative ion mode. The flow rate of the mobile phase was 250μl/min and the retention times of FLB, 4'-OH-FLB, and IS were 1.1, 0.8, and 0.9min, respectively. The calibration curves were linear over a range of 0.01-10μg/ml for FLB and 0.01-1μg/ml for 4'-OH-FLB. The lower limit of quantifications using 100μl of human plasma was 0.01μg/ml for both analytes. The mean accuracy and precision for intra- and inter-run validation of FLB and 4'-OH-FLB were all within acceptable limits. The present HPLC-MS/MS method showed improved sensitivity for quantification of the FLB and its major metabolite in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans. Copyright © 2014 Elsevier B.V. All rights reserved.

A Targeted Metabolomics MRM-MS Study on Identifying Potential Hypertension Biomarkers in Human Plasma and Evaluating Acupuncture Effects.

PubMed

Yang, Mingxiao; Yu, Zheng; Deng, Shufang; Chen, Xiaomin; Chen, Liang; Guo, Zhenyu; Zheng, Hui; Chen, Lin; Cai, Dingjun; Wen, Bo; Wu, Qiaofeng; Liang, Fanrong

2016-05-16

The critical role of metabolic abnormality in hypertension is increasingly recognized, but its biomarkers are not clearly identified. In this study, 47 chemical compounds recorded by literature were employed as target metabolites of essential hypertension (EH). We detected their content in the plasma of EH patients and healthy subjects by using the Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS). After screening the most altered compounds, acupuncture was used to treat patients for 3 months and these plasma metabolites were tested again. The results showed that oleic acid (OA) and myoinositol (MI) were the most important differential metabolites between the hypertensive plasma and the healthy plasma. They were also closely correlated with 24-hour blood pressure and nocturnal dipping. Moreover, plasma OA and MI could be restored to normal levels by acupuncture, accompanying with reduction of 24-hour systolic and diastolic blood pressure [from 145.10 ± 9.28 mm Hg to 140.70 ± 9.59 mm Hg (P < 0.0001), and 88.35 ± 7.92 mm Hg to 85.86 ± 7.95 mm Hg (P = 0.0024), respectively] and improvement of circadian blood pressure rhythm. This study demonstrated that plasma OA and MI were potential hypertension biomarkers and they could be used to preliminarily assess the treating effects such as acupuncture.

Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

PubMed

Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

1991-01-01

Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

A sensitive LC-MS/MS method for simultaneous determination of R-bambuterol and its active metabolite R-terbutaline in human plasma and urine with application to a clinical pharmacokinetic study.

PubMed

Zhou, Ting; Zhao, Ting; Cheng, Qing; Liu, Shan; Xu, Ling; Tan, Wen

2014-07-01

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of R-bambuterol and its active metabolite R-terbutaline in human plasma and urine was established. The inhibition for the biotransformation of R-bambuterol in plasma was fully investigated. Plasma samples were prepared on ice and neostigmine metilsulfate added as a cholinesterase inhibitor immediately after sample collection. All samples were extracted with ethyl acetate and separated on a C₁₈ column under gradient elution with a mobile phase consisting of methanol and water containing 5 mm ammonium acetate at a flow rate of 0.6 mL/min. The analytes were detected by an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte in plasma. In urine samples, the LLOQs were 20.00 and 500.0 pg/mL for R-bambuterol and R-terbutaline, respectively. The intra- and inter-day precisions were <12.7 and <8.6% for plasma and urine, respectively. The analytical runtime within 6.0 min per sample made this method suitable for high-throughput determination. The validated method has been successfully applied to the human pharmacokinetic study of R-bambuterol involving 10 healthy volunteers. Copyright © 2013 John Wiley & Sons, Ltd.

¹¹C-ORM-13070, a novel PET ligand for brain α₂C-adrenoceptors: radiometabolism, plasma pharmacokinetics, whole-body distribution and radiation dosimetry in healthy men.

PubMed

Luoto, Pauliina; Suilamo, Sami; Oikonen, Vesa; Arponen, Eveliina; Helin, Semi; Herttuainen, Jukka; Hietamäki, Johanna; Holopainen, Aila; Kailajärvi, Marita; Peltonen, Juha M; Rouru, Juha; Sallinen, Jukka; Scheinin, Mika; Virta, Jere; Virtanen, Kirsi; Volanen, Iina; Roivainen, Anne; Rinne, Juha O

2014-10-01

(11)C-labelled 1-[(S)-1-(2,3-dihydrobenzo[1,2]dioxin-2-yl)methyl]-4-(3-methoxy-methylpyridin-2-yl)-piperazine ((11)C-ORM-13070) is a novel PET tracer for imaging of α2C-adrenoceptors in the human brain. Brain α2C-adrenoceptors may be therapeutic targets in several neuropsychiatric disorders, including depression, schizophrenia and Alzheimer's disease. To validate the use of (11)C-ORM-13070 in humans, we investigated its radiometabolism, pharmacokinetics, whole-body distribution and radiation dose. Radiometabolism was studied in a test-retest setting in six healthy men. After intravenous injection of (11)C-ORM-13070, blood samples were drawn over 60 min. Plasma samples were analysed by radio-HPLC for intact tracer and its radioactive metabolites. Metabolite-corrected plasma time-activity curves were used for calculation of pharmacokinetics. In a separate group of 12 healthy men, the whole-body distribution of (11)C-ORM-13070 and radiation exposure were investigated by dynamic PET/CT imaging without blood sampling. Two radioactive metabolites of (11)C-ORM-13070 were detected in human arterial plasma. The proportion of unchanged (11)C-ORM-13070 decreased from 81 ± 4 % of total radioactivity at 4 min after tracer injection to 23 ± 4 % at 60 min. At least one of the radioactive metabolites penetrated into red blood cells, while the parent tracer remained in plasma. The apparent elimination rate constant and corresponding half-life of unchanged (11)C-ORM-13070 in arterial plasma were 0.0117 ± 0.0056 min(-1) and 73.6 ± 35.8 min, respectively. The organs with the highest absorbed doses were the liver (12 μSv/MBq), gallbladder wall (12 μSv/MBq) and pancreas (9.1 μSv/MBq). The mean effective dose was 3.9 μSv/MBq, with a range of 3.6 - 4.2 μSv/MBq. (11)C-ORM-13070 was rapidly metabolized in human subjects after intravenous injection. The effective radiation dose of (11)C-ORM-13070 was in the same range as that of other (11)C-labelled brain receptor tracers. An injection of 500 MBq of (11)C-ORM-13070 would expose a subject to 2.0 mSv of radiation. This supports the use of (11)C-ORM-13070 in repeated PET scans, for example, in receptor occupancy trials with novel drug candidates.

Quantitation of anacetrapib, stable-isotope labeled-anacetrapib (microdose), and four metabolites in human plasma using liquid chromatography tandem mass spectrometry.

PubMed

Chavez-Eng, C M; Lutz, R W; Li, H; Goykhman, D; Bateman, K P; Woolf, E

2016-02-01

An ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of (4S,5R)-5-[3,5-bis (trifluoromethyl)phenyl]-3-{[4'-fluoro-5'-isopropyl-2'-methoxy-4-(trifluoromethyl)biphenyl-2-yl] methyl}-4-methyl-1,3-oxazolidin-2-one (anacetrapib, I) and [(13)C5(15)N]-anacetrapib, II in human plasma has been developed to support a clinical study to determine the absolute bioavailability of I. The analytes and the stable-isotope labeled internal standard ([(13)C7(15)N(2)H7]-anacetrapib, III) were extracted from 100μL of human plasma by liquid-liquid extraction using 20/80 isopropyl alcohol/hexane (v/v). The chromatographic separation of the analytes was achieved using Waters BEH Shield RP 18 (50×2.1mm×1.7μm) column and mobile phase gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.6mL/min flow rate. The MS/MS detection was performed on AB Sciex 5000 or AB 5500 in positive electrospray ionization mode, operated in selected reaction monitoring mode. The assay was validated in the concentration range 1-2000ng/mL for I; and a lower curve range, 0.025-50ng/mL for II. In addition to the absolute bioavailability determination, it was desired to better elucidate the pharmacokinetic behavior of several hydroxylated metabolites of I. Toward this end, two exploratory assays for the hydroxy metabolites of I were qualified in the concentration range 0.5-500ng/mL. All metabolites were separated on a Supelco Ascentis Express Phenyl-Hexyl (50×2.1mm, 2.7μm) column. Metabolite M4 was analyzed in the negative mode with a mobile phase consisting of a gradient mixture of water (A) and acetonitrile (B). The other three metabolites, M1-M3 were analyzed in the positive mode using a mobile phase gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The assays were utilized to support a clinical study in which a microdosing approach was used to determine the pharmacokinetics of anacetrapib and its metabolites. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolism of oral 9-cis-retinoic acid in the human. Identification of 9-cis-retinoyl-beta-glucuronide and 9-cis-4-oxo-retinoyl-beta-glucuronide as urinary metabolites.

PubMed

Sass, J O; Masgrau, E; Saurat, J H; Nau, H

1995-09-01

Data from a number of investigators suggest that the 9-cis-isomer of RA1 (9-cis-RA) may be a promising agent in chemoprevention and treatment of certain types of cancer. Therefore, clinical studies on this retinoid have been initiated. However, up to now, no information has been published on the metabolism of 9-cis-RA in the human. Herein, we report the first data on retinoid metabolism after multiple administration of 9-cis-RA (20 mg/day po) to human volunteers. After 2 and 12-13 hr, plasma concentrations of 9-cis-RA and its metabolites 9,13-dicis-RA, 13-cis-RA, and all-trans-RA were low. In contrast, dosing with 13-cis-RA yielded much higher plasma retinoid levels. Effects on plasma retinol concentrations did not become obvious after any drug treatment. Several retinoid metabolites were found in the urine of 9-cis-RA-treated individuals, and 9-cis-RAG, as well as 9-cis-4-oxo-RAG, could be identified. After treatment with 9-cis-RA, high concentrations of the administered drug were found in the feces, along with comparably low concentrations of 13-cis-RA, 9,13-dicis-RA, and all-trans-RA. Our report indicates that 9-cis-RA is either eliminated much more rapidly than 13-cis-RA, or it is poorly absorbed, and presents the characterization of two urinary glucuronides.

Documenting the kinetic time course of lambda-cyhalothrin metabolites in orally exposed volunteers for the interpretation of biomonitoring data.

PubMed

Khemiri, Rania; Côté, Jonathan; Fetoui, Hamadi; Bouchard, Michèle

2017-07-05

Lambda-cyhalothrin is a pyrethroid pesticide largely used in agriculture. Exposure assessment can be performed by measuring key urinary metabolites. For a proper use of biomonitoring data, it is however important to gain information on the toxicokinetics of these key biomarkers of exposure. A human volunteer study was performed to document the plasma and urinary time courses of major lambda-cyhalothrin metabolites. Seven volunteers ingested 0.025mgkg -1 body weight of lambda-cyhalothrin. Blood samples were withdrawn prior to dosing and at fixed time periods over the 72 h-period following ingestion and complete urine voids were collected pre-exposure and at pre-established intervals over 84h post-dosing. The cis-3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropanecarboxylic acid (CFMP) and 3-phenoxybenzoic acid (3-PBA) metabolites were quantified in these samples. Plasma concentrations of CFMP and 3-PBA increased rapidly after ingestion, with average peak values at 3.1 and 4.0h post-dosing, respectively; subsequent elimination phase showed a rapid decay with a mean half-life (t ½ ) of ≈5.3 and 6.4h for CFMP and 3-PBA, respectively. Urinary rate time courses displayed a profile similar to the plasma concentration-time curves with corresponding mean t ½ of ≈4.2 and 5.9h. In the 84-h period post-treatment, on average 21% of lambda-cyhalothrin dose were excreted in urine as CFMP as compared to 30% as 3-PBA. Overall, CFMP and 3-PBA metabolites were confirmed to be major metabolites of lambda-cyhalothrin and exhibited similar kinetics with short half-lives; they thus both appear as useful biomarkers of exposure to lambda-cyhalothrin in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

Species differences in the formation of vabicaserin carbamoyl glucuronide.

PubMed

Tong, Zeen; Chandrasekaran, Appavu; DeMaio, William; Jordan, Ronald; Li, Hongshan; Moore, Robin; Poola, Nagaraju; Burghart, Peter; Hultin, Theresa; Scatina, JoAnn

2010-04-01

Vabicaserin is a potent 5-hydroxtryptamine 2C full agonist with therapeutic potential for a wide array of psychiatric disorders. Metabolite profiles indicated that vabicaserin was extensively metabolized via carbamoyl glucuronidation after oral administration in humans. In the present study, the differences in the extent of vabicaserin carbamoyl glucuronide (CG) formation in humans and in animals used for safety assessment were investigated. After oral dosing, the systemic exposure ratios of CG to vabicaserin were approximately 12 and up to 29 in monkeys and humans, respectively, and the ratios of CG to vabicaserin were approximately 1.5 and 1.7 in mice and dogs, respectively. These differences in systemic levels of CG are likely related to species differences in the rate and extent of CG formation and elimination. Whereas CG was the predominant circulating metabolite in humans and a major metabolite in mice, dogs, and monkeys, it was a relatively minor metabolite in rats, in which oxidative metabolism was the major metabolic pathway. Although the CG was not detected in plasma or urine of rats, approximately 5% of the dose was excreted in bile as CG in the 24-h collection postdose, indicating the rat had the metabolic capability of producing the CG. In vitro, in a CO(2)-enriched environment, the CG was the predominant metabolite in dog and human liver microsomes, a major metabolite in monkey and mice, and only a very minor metabolite in rats. Carbamoyl glucuronidation and hydroxylation had similar contributions to vabicaserin metabolism in mouse and monkey liver microsomes. However, only trace amounts of CG were formed in rat liver microsomes, and other metabolites were more prominent than the CG. In conclusion, significant differences in the extent of formation of the CG were observed among the various species examined. The exposure ratios of CG to vabicaserin were highest in humans, followed by monkeys, then mice and dogs, and lowest in rats, and the in vitro metabolite profiles generally correlated well with the in vivo metabolites.

Plasma Metabolites Predict Severity of Depression and Suicidal Ideation in Psychiatric Patients-A Multicenter Pilot Analysis.

PubMed

Setoyama, Daiki; Kato, Takahiro A; Hashimoto, Ryota; Kunugi, Hiroshi; Hattori, Kotaro; Hayakawa, Kohei; Sato-Kasai, Mina; Shimokawa, Norihiro; Kaneko, Sachie; Yoshida, Sumiko; Goto, Yu-Ichi; Yasuda, Yuka; Yamamori, Hidenaga; Ohgidani, Masahiro; Sagata, Noriaki; Miura, Daisuke; Kang, Dongchon; Kanba, Shigenobu

2016-01-01

Evaluating the severity of depression (SOD), especially suicidal ideation (SI), is crucial in the treatment of not only patients with mood disorders but also psychiatric patients in general. SOD has been assessed on interviews such as the Hamilton Rating Scale for Depression (HAMD)-17, and/or self-administered questionnaires such as the Patient Health Questionnaire (PHQ)-9. However, these evaluation systems have relied on a person's subjective information, which sometimes lead to difficulties in clinical settings. To resolve this limitation, a more objective SOD evaluation system is needed. Herein, we collected clinical data including HAMD-17/PHQ-9 and blood plasma of psychiatric patients from three independent clinical centers. We performed metabolome analysis of blood plasma using liquid chromatography mass spectrometry (LC-MS), and 123 metabolites were detected. Interestingly, five plasma metabolites (3-hydroxybutyrate (3HB), betaine, citrate, creatinine, and gamma-aminobutyric acid (GABA)) are commonly associated with SOD in all three independent cohort sets regardless of the presence or absence of medication and diagnostic difference. In addition, we have shown several metabolites are independently associated with sub-symptoms of depression including SI. We successfully created a classification model to discriminate depressive patients with or without SI by machine learning technique. Finally, we produced a pilot algorithm to predict a grade of SI with citrate and kynurenine. The above metabolites may have strongly been associated with the underlying novel biological pathophysiology of SOD. We should explore the biological impact of these metabolites on depressive symptoms by utilizing a cross species study model with human and rodents. The present multicenter pilot study offers a potential utility for measuring blood metabolites as a novel objective tool for not only assessing SOD but also evaluating therapeutic efficacy in clinical practice. In addition, modification of these metabolites by diet and/or medications may be a novel therapeutic target for depression. To clarify these aspects, clinical trials measuring metabolites before/after interventions should be conducted. Larger cohort studies including non-clinical subjects are also warranted to clarify our pilot findings.

Potential antiproliferative activity of polyphenol metabolites against human breast cancer cells and their urine excretion pattern in healthy subjects following acute intake of a polyphenol-rich juice of grumixama (Eugenia brasiliensis Lam.).

PubMed

Teixeira, L L; Costa, G R; Dörr, F A; Ong, T P; Pinto, E; Lajolo, F M; Hassimotto, N M A

2017-06-21

The bioavailability and metabolism of anthocyanins and ellagitannins following acute intake of grumixama fruit, native Brazilian cherry, by humans, and its in vitro antiproliferative activity against breast cancer cells (MDA-MB-231) were investigated. A single dose of grumixama juice was administered to healthy women (n = 10) and polyphenol metabolites were analyzed in urine and plasma samples collected over 24 h. The majority of the metabolites circulating and excreted in urine were phenolic acids and urolithin conjugates, the gut microbiota catabolites of both classes of polyphenols, respectively. According to pharmacokinetic parameters, the subjects were divided into two distinct groups, high and low urinary metabolite excretors. The pool of polyphenol metabolites found in urine samples showed a significant inhibition of cell proliferation and G2/M cell cycle arrest in MDA-MB-231 cells. Our findings demonstrate the large interindividual variability concerning the polyphenol metabolism, which possibly could reflect in health promotion.

A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma.

PubMed

Peer, Cody J; Spencer, Shawn D; VanDenBerg, Dustin A H; Pacanowski, Michael A; Horenstein, Richard B; Figg, William D

2012-01-01

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r(2)=0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range. Published by Elsevier B.V.

A Sensitive and Rapid Ultra HPLC-MS/MS Method for the Simultaneous Detection of Clopidogrel and its Derivatized Active Thiol Metabolite in Human Plasma

PubMed Central

Peer, Cody J.; Spencer, Shawn D.; VanDenBerg, Dustin A. H.; Pacanowski, Michael A.; Horenstein, Richard B.; Figg, William D.

2011-01-01

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix®) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3’-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2µm-C18 column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically-relevant concentration range of 0.01–50 ng/mL for parent clopidogrel and 0.1–150 ng/mL (r2= 0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) < ±12% and precision (%CV) of < ±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range. PMID:22169056

Development and validation of a high performance liquid chromatographic method for the determination of oxcarbazepine and its main metabolites in human plasma and cerebrospinal fluid and its application to pharmacokinetic study.

PubMed

Kimiskidis, Vasilios; Spanakis, Marios; Niopas, Ioannis; Kazis, Dimitrios; Gabrieli, Chrysi; Kanaze, Feras Imad; Divanoglou, Daniil

2007-01-17

An isocratic reversed-phase HPLC-UV procedure for the determination of oxcarbazepine and its main metabolites 10-hydroxy-10,11-dihydrocarbamazepine and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine in human plasma and cerebrospinal fluid has been developed and validated. After addition of bromazepam as internal standard, the analytes were isolated from plasma and cerebrospinal fluid by liquid-liquid extraction. Separation was achieved on a X-TERRA C18 column using a mobile phase composed of 20 mM KH(2)PO(4), acetonitrile, and n-octylamine (76:24:0.05, v/v/v) at 40 degrees C and detected at 237 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery and lower limit of quantification according to the FDA validation guidelines. Calibration curves were linear with a coefficient of variation (r) greater than 0.998. Accuracy ranged from 92.3% to 106.0% and precision was between 2.3% and 8.2%. The method has been applied to plasma and cerebrospinal fluid samples obtained from patients treated with oxcarbazepine, both in monotherapy and adjunctive therapy.

A liquid chromatography with tandem mass spectrometry method for simultaneous determination of UTL-5g and its metabolites in human plasma.

PubMed

Shaw, Jiajiu; Wiegand, Richard; Wu, Jianmei; Bao, Xun; Valeriote, Frederick; Li, Jing

2015-06-01

UTL-5g is a novel small-molecule TNF-α inhibitor under investigation as both a chemoprotective and radioprotective agent. Animal studies showed that pretreatment of UTL-5g protected kidney, liver, and platelets from cisplatin-induced toxicity. In addition, UTL-5g reduced liver and lung injuries induced by radiation in vivo. Although a number of preclinical studies have been conducted, a validated bioanalytical method for UTL-5g in human plasma has not been published. In this work, a sensitive and reproducible reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the determination of UTL-5g and its metabolites, 5-methylisoxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA), in human plasma. The method involves a simple methanol precipitation step followed by injection of the supernatant onto a Waters 2695 HPLC system coupled with a Waters Quattro Micro™ triple quadrupole mass spectrometer. Chromatographic separation was accomplished using a Waters Nova-Pak C18 column maintained at 30°C, running at gradient mode with mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.2mL/min. The analytes were monitored under positive electrospray ionization (ESI). Quantitation of these compounds in plasma was linear from 0.05 to 10μM. The lower limit of quantitation (LLOQ) was 0.05, 0.1, and 0.2μM for UTL-5g, ISOX and DCA, respectively. The accuracy and intra-and inter-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method provides a practical tool to measure and characterize the plasma concentration-time profiles for UTL-5g and its metabolites, ISOX and DCA. Copyright © 2015 Elsevier B.V. All rights reserved.

Mass spectrometry profiling reveals altered plasma levels of monohydroxy fatty acids and related lipids in healthy humans after controlled exposure to biodiesel exhaust.

PubMed

Gouveia-Figueira, Sandra; Karimpour, Masoumeh; Bosson, Jenny A; Blomberg, Anders; Unosson, Jon; Sehlstedt, Maria; Pourazar, Jamshid; Sandström, Thomas; Behndig, Annelie F; Nording, Malin L

2018-08-14

Experimental human exposure studies are an effective tool to study adverse health effects from acute inhalation of particulate matter and other constituents of air pollution. In this randomized and double-blinded crossover study, we investigated the systemic effect on bioactive lipid metabolite levels after controlled biodiesel exhaust exposure of healthy humans and compared it to filtered air at a separate exposure occasion. Eicosanoids and other oxylipins, as well as endocannabinoids and related lipids, were quantified in plasma from 14 healthy volunteers at baseline and at three subsequent time points (2, 6, and 24 h) after 1 h exposure sessions. Protocols based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) methods were developed to detect temporal changes in circulating levels after biodiesel exhaust exposure. The exhaust was generated by a diesel engine fed with an undiluted rapeseed methyl ester fuel. Among the 51 analyzed lipid metabolites, PGF 2α , 9,10-DiHOME, 9-HODE, 5-HETE, 11-HETE, 12-HETE, and DEA displayed significant responsiveness to the biodiesel exhaust exposure as opposed to filtered air. Of these, 9-HODE and 5-HETE at 24 h survived the 10% false discovery rate cutoff (p < 0.003). Hence, the majority of the responsive lipid metabolites were monohydroxy fatty acids. We conclude that it is possible to detect alterations in circulating bioactive lipid metabolites in response to biodiesel exhaust exposure using LC-MS/MS, with emphasis on metabolites with inflammation related properties and implications on cardiovascular health and disease. These observations aid future investigations on air pollution effects, especially with regard to cardiovascular outcomes. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous measurement of proguanil and its metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography, and its preliminary application in relation to genetically determined S-mephenytoin 4'-hydroxylation status.

PubMed

Kusaka, M; Setiabudy, R; Chiba, K; Ishizaki, T

1996-02-01

A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers.

Application of dried blood spot cards to determine olive oil phenols (hydroxytyrosol metabolites) in human blood.

PubMed

de Las Hazas, María Carmen López; Motilva, Maria José; Piñol, Carme; Macià, Alba

2016-10-01

In this study, a fast and simple blood sampling and sample pre-treatment method based on the use of the dried blood spot (DBS) cards and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for the quantification of olive oil phenolic metabolites in human blood was developed and validated. After validation, the method was applied to determine hydroxytyrosol metabolites in human blood samples after the acute intake of an olive oil phenolic extract. Using the FTA DMPK-A DBS card under optimum conditions, with 20µL as the blood solution volume, 100µL of methanol/Milli-Q water (50/50, v/v) as the extraction solvent and 7 disks punched out from the card, the main hydroxytyrosol metabolites (hydroxytyrosol-3-O-sulphate and hydroxytyrosol acetate sulphate) were identified and quantified. The developed methodology allowed detecting and quantifying the generated metabolites at low μM levels. The proposed method is a significant improvement over existing methods to determine phenolic metabolites circulating in blood and plasma samples, thus making blood sampling possible with the volunteer pricking their own finger, and the subsequent storage of the blood in the DBS cards prior to chromatographic analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of drug metabolizing cytochrome P450s by the aromatase inhibitor drug letrozole and its major oxidative metabolite 4,4′-methanol-bisbenzonitrile in vitro

PubMed Central

Jeong, Seongwook; Woo, Margaret M.; Flockhart, David A.

2009-01-01

Purpose To determine the inhibitory potency of letrozole and its main human metabolite, 4,4′-methanol-bisbenzonitrilee, on the activities of eight cytochrome P450 (CYP) enzymes. Methods Letrozole and its metabolite were incubated with human liver microsomes (HLMs) (or expressed CYP isoforms) and NADPH in the absence (control) and presence of the test inhibitor. Results Letrozole was a potent competitive inhibitor of CYP2A6 (Ki 4.6 ± 0.05 μM and 5.0 ± 2.4 μM in HLMs and CYP2A6, respectively) and a weak inhibitor of CYP2C19 (Ki 42.2 μM in HLMs and 33.3 μM in CYP2C19), while its metabolite showed moderate inhibition of CYP2C19 and CYP2B6. Letrozole or its metabolite had negligible effect on other CYPs. Conclusions Based on the in vitro Ki values, letrozole is predicted to be a weak inhibitor of CYP2A6 in vivo. Letrozole and its major human metabolite show inhibitory activity towards other CYPs, but clinically relevant drug interactions seem less likely as the Ki values are above the therapeutic plasma concentrations of letrozole. PMID:19198839

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations.

PubMed

Von Moltke, L L; Greenblatt, D J; Granda, B W; Duan, S X; Grassi, J M; Venkatakrishnan, K; Harmatz, J S; Shader, R I

1999-07-01

To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50 ) of 0.61 microm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by approximately 40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition.

Simultaneous determination of blonanserin and its four metabolites in human plasma using ultra-performance liquid chromatography-tandem mass spectrometry.

PubMed

Zhou, Ying; Liu, Ming; Jiang, Ji; Wang, Hongyun; Hu, Pei

2013-11-15

A sensitive and rapid method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the simultaneous determination of blonanserin, its major active metabolite (N-deethyl form) and other three metabolites (N-oxide form, Ethylenediamine form and Carboxylate form) in human plasma. Plasma samples were pre-purified by solid-phase extraction (SPE) and analyzed using a gradient chromatographic separation over an Acquity UPLC CSH C18 column. The mobile phase consisted of acetonitrile-water containing 5mM ammonium formate and 0.1% formic acid at a flow rate of 0.5mL/min. Positive electrospray ionization was employed as the ionization source in the multiple reaction monitoring (MRM) mode. The analysis time was about 3.5min. The method was fully validated over the concentration range of 0.01-1ng/mL for all analytes. The lower limit of quantification (LLOQ) was 0.01ng/mL. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115%. The mean extraction recoveries of all analytes at two concentration levels were consistent. Selectivity, matrix effect and stability were also validated. The method was applied to the pharmacokinetic study of blonanserin in Chinese healthy subjects. Copyright © 2013 Elsevier B.V. All rights reserved.

Metabolomic Characteristics of Arsenic-Associated Diabetes in a Prospective Cohort in Chihuahua, Mexico

PubMed Central

Martin, Elizabeth; González-Horta, Carmen; Rager, Julia; Bailey, Kathryn A.; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Ishida, María C.; Gutiérrez-Torres, Daniela S.; Hernández Cerón, Roberto; Viniegra Morales, Damián; Baeza Terrazas, Francisco A.; Jesse Saunders, R.; Drobná, Zuzana; Mendez, Michelle A.; Buse, John B.; Loomis, Dana; Jia, Wei; García-Vargas, Gonzalo G.; Del Razo, Luz M.; Stýblo, Miroslav; Fry, Rebecca

2015-01-01

Chronic exposure to inorganic arsenic (iAs) has been linked to an increased risk of diabetes, yet the specific disease phenotype and underlying mechanisms are poorly understood. In the present study we set out to identify iAs exposure-associated metabolites with altered abundance in nondiabetic and diabetic individuals in an effort to understand the relationship between exposure, metabolomic response, and disease status. A nested study design was used to profile metabolomic shifts in urine and plasma collected from 90 diabetic and 86 nondiabetic individuals matched for varying iAs concentrations in drinking water, body mass index, age, and sex. Diabetes diagnosis was based on measures of fasting plasma glucose and 2-h blood glucose. Multivariable models were used to identify metabolites with altered abundance associated with iAs exposure among diabetic and nondiabetic individuals. A total of 132 metabolites were identified to shift in urine or plasma in response to iAs exposure characterized by the sum of iAs metabolites in urine (U-tAs). Although many metabolites were altered in both diabetic and nondiabetic 35 subjects, diabetic individuals displayed a unique response to iAs exposure with 59 altered metabolites including those that play a role in tricarboxylic acid cycle and amino acid metabolism. Taken together, these data highlight the broad impact of iAs exposure on the human metabolome, and demonstrate some specificity of the metabolomic response between diabetic and nondiabetic individuals. These data may provide novel insights into the mechanisms and phenotype of diabetes associated with iAs exposure. PMID:25577196

Simultaneous quantitation of delamanid (OPC-67683) and its eight metabolites in human plasma using UHPLC-MS/MS.

PubMed

Meng, Min; Smith, Benjamin; Johnston, Brad; Carter, Spencer; Brisson, Jerry; Roth, Sharin E

2015-10-01

Delamanid (OPC-67683) is a novel nitro-dihydroimidazo-oxazole derivative that is being developed by Otsuka Pharmaceutical Co., Ltd., Japan (referred to as Otsuka hereafter) for the treatment of tuberculosis (TB). An ultra-high performance liquid chromatographic-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for the determination of OPC-67683 and its eight metabolites, DM-6704, DM-6705, DM-6706, DM-6717, DM-6718, DM-6720, DM-6721 and DM-6722 in human plasma to support regulated clinical development. During method development several technical challenges such as poor chromatography, separation of structural isomers, conversion of the analytes, instability in matrix and long cycle time were encountered and overcome. A protein precipitation extraction (PPE) was used to extract plasma samples (50μL) and the resulting extracts were analyzed using reversed phase UHPLC-MS/MS with a electrospray (ESI) and selected reaction monitoring (SRM). The method was fully validated over the calibration curve range of 1.00-500ng/mL for all nine analytes with linear regression and 1/x(2) weighting according to regulatory guidance for bioanalysis. Based on three inter-day precision and accuracy runs, the between-run % relative standard deviation (RSD) for all nine analytes varied from 0.0 to 11.9% and the accuracy ranged from 92.7% to 102.5% of nominal at all quality controls (QC) concentrations, including the lower limit of quantitation (LLOQ) QC at 1.00ng/mL. The extraction recovery of OPC-67683 and its eight metabolites were above 95%. Various short term and long term solution and matrix stability were established including the stability of OPC-67683 and its eight metabolites in human plasma for 708 days at -70°C. Although this method has been used to support regulated clinic studies during the last decade and over ten thousand samples have been analyzed, this is the first time that the method development process and validation data have been published. Copyright © 2015 Elsevier B.V. All rights reserved.

Sensitive determination of three aconitum alkaloids and their metabolites in human plasma by matrix solid-phase dispersion with vortex-assisted dispersive liquid-liquid microextraction and HPLC with diode array detection.

PubMed

Wang, Xiaozhong; Li, Xuwen; Li, Lanjie; Li, Min; Liu, Ying; Wu, Qian; Li, Peng; Jin, Yongri

2016-05-01

A simple and sensitive method for determination of three aconitum alkaloids and their metabolites in human plasma was developed using matrix solid-phase dispersion combined with vortex-assisted dispersive liquid-liquid microextraction and high-performance liquid chromatography with diode array detection. The plasma sample was directly purified by matrix solid-phase dispersion and the eluate obtained was concentrated and further clarified by vortex-assisted dispersive liquid-liquid microextraction. Some important parameters affecting the extraction efficiency, such as type and amount of dispersing sorbent, type and volume of elution solvent, type and volume of extraction solvent, salt concentration as well as sample solution pH, were investigated in detail. Under optimal conditions, the proposed method has good repeatability and reproducibility with intraday and interday relative standard deviations lower than 5.44 and 5.75%, respectively. The recoveries of the aconitum alkaloids ranged from 73.81 to 101.82%, and the detection limits were achieved within the range of 1.6-2.1 ng/mL. The proposed method offered the advantages of good applicability, sensitivity, simplicity, and feasibility, which makes it suitable for the determination of trace amounts of aconitum alkaloids in human plasma samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples.

PubMed

Peng, G W; Sood, V K; Rykert, U M

1985-03-01

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.

Rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry analysis of the novel anticancer agent PR-104 and its major metabolites in human plasma: Application to a pharmacokinetic study.

PubMed

Gu, Yongchuan; Wilson, William R

2009-10-01

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC-MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50mmx2.1mm, 1.8microm) column and gradient of acetonitrile and 0.01% formic acid with a 6min run time. The method had a linear range of 0.1-50microM for PR-104, PR-104A and PR-104G, 0.05-5microM for PR-104H, 0.025-2.5microM for PR-104M and 0.01-1microM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.

Metabolic fate of strawberry polyphenols after chronic intake in healthy older adults.

PubMed

Sandhu, Amandeep K; Miller, Marshall G; Thangthaeng, Nopporn; Scott, Tammy M; Shukitt-Hale, Barbara; Edirisinghe, Indika; Burton-Freeman, Britt

2018-01-24

Strawberries contain a wide array of nutrients and phytochemicals including polyphenols such as anthocyanins, proanthocyanidins and ellagitannins. These polyphenols are absorbed and metabolized to various phenolic metabolites/conjugates in the body, which may play a role in disease risk reduction. In the present study, we investigated the metabolic fate of strawberry polyphenols after chronic (90 days) supplementation of freeze-dried strawberry (24 g d -1 , equivalent to 2 cups of fresh strawberries) vs. control powder in 19 healthy older adults. Blood samples were collected at two time-points i.e., fasting (t = 0 h) and 2 h after the breakfast meal. On days 45 and 90 breakfast also included a control or strawberry drink consistent with their treatment randomization. A total of 21 polyphenolic metabolites were quantified in plasma consisting of 3 anthocyanins/metabolites, 3 urolithin metabolites and 15 phenolic acid metabolites. Among anthocyanins/metabolite, pelargonidin glucuronide (85.7 ± 9.0 nmol L -1 , t = 2 h, day 90) was present in the highest concentration. Persistent concentrations of anthocyanins/metabolites, urolithins and some phenolic acids were observed in fasting (t = 0 h) plasma samples on day 45 and 90 after strawberry drink consumption suggesting a role of enteric, enterohepatic recycling or upregulation of gut microbial and/or human metabolism of these compounds. Our results suggest that strawberry polyphenols are absorbed and extensively metabolized, and can persist in the circulation.

Association of cultured myotubes and fasting plasma metabolite profiles with mitochondrial dysfunction in type 2 diabetes subjects.

PubMed

Abu Bakar, Mohamad Hafizi; Sarmidi, Mohamad Roji

2017-08-22

Accumulating evidence implicates mitochondrial dysfunction-induced insulin resistance in skeletal muscle as the root cause for the greatest hallmarks of type 2 diabetes (T2D). However, the identification of specific metabolite-based markers linked to mitochondrial dysfunction in T2D has not been adequately addressed. Therefore, we sought to identify the markers-based metabolomics for mitochondrial dysfunction associated with T2D. First, a cellular disease model was established using human myotubes treated with antimycin A, an oxidative phosphorylation inhibitor. Non-targeted metabolomic profiling of intracellular-defined metabolites on the cultured myotubes with mitochondrial dysfunction was then determined. Further, a targeted MS-based metabolic profiling of fasting blood plasma from normal (n = 32) and T2D (n = 37) subjects in a cross-sectional study was verified. Multinomial logical regression analyses for defining the top 5% of the metabolites within a 95% group were employed to determine the differentiating metabolites. The myotubes with mitochondrial dysfunction exhibited insulin resistance, oxidative stress and inflammation with impaired insulin signalling activities. Four metabolic pathways were found to be strongly associated with mitochondrial dysfunction in the cultured myotubes. Metabolites derived from these pathways were validated in an independent pilot investigation of the fasting blood plasma of healthy and diseased subjects. Targeted metabolic analysis of the fasting blood plasma with specific baseline adjustment revealed 245 significant features based on orthogonal partial least square discriminant analysis (PLS-DA) with a p-value < 0.05. Among these features, 20 significant metabolites comprised primarily of branched chain and aromatic amino acids, glutamine, aminobutyric acid, hydroxyisobutyric acid, pyroglutamic acid, acylcarnitine species (acetylcarnitine, propionylcarnitine, dodecenoylcarnitine, tetradecenoylcarnitine hexadecadienoylcarnitine and oleylcarnitine), free fatty acids (palmitate, arachidonate, stearate and linoleate) and sphingomyelin (d18:2/16:0) were identified as predictive markers for mitochondrial dysfunction in T2D subjects. The current study illustrates how cellular metabolites provide potential signatures associated with the biochemical changes in the dysregulated body metabolism of diseased subjects. Our finding yields additional insights into the identification of robust biomarkers for T2D associated with mitochondrial dysfunction in cultured myotubes.

Clinical significance of CYP2C19 polymorphisms on the metabolism and pharmacokinetics of 11β-hydroxysteroid dehydrogenase type-1 inhibitor BMS-823778.

PubMed

Cheng, Yaofeng; Wang, Lifei; Iacono, Lisa; Zhang, Donglu; Chen, Weiqi; Gong, Jiachang; Humphreys, William Griffith; Gan, Jinping

2018-01-01

BMS-823778 is an inhibitor of 11β-hydroxysteroid dehydrogenase type-1, and thus a potential candidate for Type 2 diabetes treatment. Here, we investigated the metabolism and pharmacokinetics of BMS-823778 to understand its pharmacokinetic variations in early clinical trials. The metabolism of BMS-823778 was characterized in multiple in vitro assays. Pharmacokinetics were evaluated in healthy volunteers, prescreened as CYP2C19 extensive metabolizers (EM) or poor metabolizers (PM), with a single oral dose of [ 14 C]BMS-823778 (10 mg, 80 μCi). Three metabolites (<5%) were identified in human hepatocytes and liver microsomes (HLM) incubations, including two hydroxylated metabolites (M1 and M2) and one glucuronide conjugate (M3). As the most abundant metabolite, M1 was formed mainly through CYP2C19. M1 formation was also correlated with CYP2C19 activities in genotyped HLM. In humans, urinary excretion of dosed radioactivity was significantly higher in EM (68.8%; 95% confidence interval 61.3%, 76.3%) than in PM (47.0%; 43.5%, 50.6%); only small portions (<2%) were present in faeces or bile from both genotypes. In plasma, BMS-823778 exposure in PM was significantly (5.3-fold, P = 0.0097) higher than in EM. Furthermore, total radioactivity exposure was significantly higher (P < 0.01) than BMS-823778 exposure in all groups, indicating the presence of metabolites. M1 was the only metabolite observed in plasma, and much lower in PM. In urine, the amount of M1 and its oxidative metabolite in EM was 7-fold of that in PM, while more glucuronide conjugates of BMS-823778 and M1 were excreted in PM. CYP2C19 polymorphisms significantly impacted systemic exposure and metabolism pathways of BMS-823778 in humans. © 2017 The British Pharmacological Society.

Prediction of Relative In Vivo Metabolite Exposure from In Vitro Data Using Two Model Drugs: Dextromethorphan and Omeprazole

PubMed Central

Lutz, Justin D.

2012-01-01

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUCm/AUCp) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUCm/AUCp ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data. PMID:22010218

Development and validation of an analytical method for regorafenib and its metabolites in mouse plasma.

PubMed

Fu, Qiang; Chen, Mingqing; Hu, Shuiying; McElroy, Craig A; Mathijssen, Ron H; Sparreboom, Alex; Baker, Sharyn D

2018-05-05

An analytical method was developed for measuring the effect of OATP1B2 deficiency on plasma levels of the kinase inhibitor regorafenib and its metabolites regorafenib-N-oxide, N-desmethyl-regorafenib-N-oxide, and regorafenib-N-β-glucuronide (RG) in mice. Compounds were separated by liquid chromatography and monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. All calibration curves were linear in the selected concentration range (R 2  ≥ 0.99). The lower limit of quantification was 5 ng/mL for the four analytes. Within-day precisions, between-day precisions, and accuracies were 2.59-6.82%, 3.97-11.3%, and 94.5-111%, respectively. The identification and structure elucidation of RG, isolated from human urine, was performed by NMR. Compared with wild-type mice given regorafenib (10 mg/kg), deficiency of the drug transporter OATP1B2 in vivo had minimal effects on plasma levels of parent drug and the metabolite regorafenib-N-oxide, and N-desmethyl-regorafenib-N-oxide. However, the area under the curve and peak levels of RG were increased by 5.6-fold and 5.1-fold, respectively, in OATP1B2-knockout mice. In conclusion, our analytical method allowed accurate and precise quantitation of regorafenib and its main metabolites in mouse plasma, and is suitable for evaluation of transporter-dependent pharmacokinetic properties of these agents in vivo. Published by Elsevier B.V.

Diet-derived phenols in plasma and tissues and their implications for health.

PubMed

Clifford, M N

2004-12-01

This paper seeks to catalyse a reappraisal of the nature, fate and biological significance in humans of phenols, polyphenols and tannins (PPT) consumed in normal diets, and in particular questions the primacy of PPT radical-scavenging mechanisms for the supposed health benefits of diets rich in fruits and vegetables. PPT are classified by structure and function. Arguments are presented to show that cinnamates and derived polyphenols make significantly larger contributions to the total PPT intake than the flavonols and flavones upon which the vast majority of attention has been focussed previously. Daily intakes of total PPT may range from less than 100 mg to in excess of 2 g, and the critical importance of coffee and black tea as the major dietary sources is shown. Only some 5% of the dietary PPT is absorbed in the duodenum, and of this only some 5%, mainly flavanols, reaches the plasma unchanged, the balance being mammalian conjugates. Over 95% of the intake passes to the colon and is fermented by the gut microflora. A fraction of the resulting microbial metabolites is absorbed and appears in the plasma primarily as mammalian conjugates. Even following high intakes of PPT, the plasma metabolites collectively make a very small (less than 5%) and transient contribution to the total concentration of redox active substances in plasma. This explains the failure of most studies that sought to detect an increase in plasma antioxidant power after consuming a PPT-rich meal or supplement. The powerfully antioxidant PPT aglycones, much used in in vitro studies, do not reach the plasma. The redox potential of those unchanged PPT and PPT metabolites that reach the plasma enables them to scavenge damaging radicals, but the endogenous plasma antioxidants, especially ascorbate, are required for disposal of the resultant phenoxyl radicals. Black tea and coffee, the major sources of PPT, are poor sources of ascorbate. It is suggested that if diets rich in fruits and vegetables are health-promoting, and if these effects are due to PPT, then alternatives to radical-scavenging mechanisms must be sought. Evidence is presented to show that some mammalian metabolites of PPT may indeed be able to protect the vascular endothelium and that diets rich in PPT may in humans at normal dietary levels have the ability to protect against Type II diabetes and the metabolic syndrome through effects on glucose absorption and associated hormones. Such effects are recommended for further investigation.

Gas chromatographic-mass spectrometry method for the detection of busulphan and its metabolites in plasma and urine.

PubMed

El-Serafi, Ibrahim; Terelius, Ylva; Twelkmeyer, Brigitte; Hagbjörk, Ann-Louise; Hassan, Zuzana; Hassan, Moustapha

2013-01-15

Busulphan is an alkylating agent used as conditioning regimen prior to stem cell transplantation. Busulphan is metabolized in the liver and four major metabolites have been identified. The first metabolite is tetrahydrothiophene which is oxidized to tetrahydrothiophene 1-oxide, then sulfolane and finally 3-hydroxy sulfolane. Despite the low molecular weight and wide polarity range of busulphan and its four metabolites, the use of a fused silica non-polar column significantly enhanced the automated gas chromatography-mass spectrometry of their detection in one simple method. The limit of quantification was 0.5μM for busulphan and all its metabolites except 3-OH sulfolane, which was 1.25μM. This method was validated for all the compounds in both human plasma and urine. Lower limits of quantifications (LLOQs) were run in pentaplicate per compound and all results were within 20% of the nominal values. The recovery was determined by comparing the peak area of two quality control (QC) samples, before and after extraction in plasma and urine, in triplicate. Acceptable precision and accuracy have been obtained; at least 3 standard curves have been run for each compound using three different QCs covering the calibration curve in triplicate. The QC values were within 15% (SD) of the nominal values. Selectivity and sensitivity of all compounds have been measured. Compounds were stable up to 50 days after extraction in -20°C and 48h at RT. Moreover, the compounds were stable for three cycles of freezing and thawing. The method was applied in a clinical case where the patient received high dose busulphan; all the compounds have been detected, identified and quantified both in plasma and urine. Copyright © 2012 Elsevier B.V. All rights reserved.

The turnover and transport of vitamin D and of a polar metabolite with the properties of 25-hydroxycholecalciferol in human plasma

PubMed Central

Smith, John Edgar; Goodman, DeWitt S.

1971-01-01

Four normal men were injected intravenously with physiological doses (6 μg) of vitamin D3-1,2-3H. Serial samples of plasma were collected for 50 days. Total lipid extracts were chromatographed on silicic acid columns or thin-layer plates in order to characterize the radioactive components. Labeled vitamin D3 disappeared rapidly from plasma (initial half-life approximately 12 hr); after 7 days unchanged vitamin D3 represented less than 1% of circulating radioactivity. Coincident with vitamin D3 disappearance a more polar labeled metabolite appeared with chromatographic and other properties identical with those of 25-hydroxycholecalciferol. The disappearance of the more polar metabolite was relatively slow with a half-life of 19.6 ±0.6 days. A similar half-life was seen in a fifth subject, injected with 80 μg of vitamin D3-3H. Most (approximately 92%) of the plasma total radioactivity was represented by this component throughout the study. Plasma samples collected at various times were adjusted to density (d) 1.21 and were ultracentrifuged to separate plasma lipoproteins from proteins with d > 1.21. In all samples, almost all (mean 94%) of the radioactivity was found in association with proteins of d > 1.21. This observation was confirmed by bioassay, measuring uptake of 45Ca by intestinal slices. All plasma bioassayable vitamin D was found in association with proteins of d > 1.21; 55% of bioactivity was found in the chromatographic fraction corresponding to 25-hydroxycholecalciferol and 44% in the fractions representing vitamin D3. Since both vitamin D3 and its 25-hydroxy metabolite are lipid-soluble sterol derivatives, the finding that these compounds do not circulate in association with the known plasma lipoproteins provides presumptive evidence for the existence of a specific transport protein of d > 1.21. The transport protein for the polar metabolite has been partly characterized by gel filtration on Sephadex G-200 and by electrophoresis on polyacrylamide gel. The protein has an apparent size slightly smaller than plasma albumin (approximate mol wt 50,000-60,000) and an electrophoretic mobility very slightly greater than that of albumin. Studies are in progress to fractionate further and to characterize the transport protein. Images PMID:4330006

Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency.

PubMed

Tran, C; Yazdanpanah, M; Kyriakopoulou, L; Levandovskiy, V; Zahid, H; Naufer, A; Isbrandt, D; Schulze, A

2014-09-25

To develop an accurate stable isotope dilution assay for simultaneous quantification of creatine metabolites ornithine, arginine, creatine, creatinine, and guanidinoacetate in very small blood sample volumes to study creatine metabolism in mice. Liquid-chromatography (C18) tandem mass spectrometry with butylation was performed in positive ionization mode. Stable isotope dilution assay with external calibration was applied to three different specimen types, plasma, whole blood and dried blood spot (DBS). Analytical separation, sensitivity, accuracy, and linearity of the assay were adequate. The stable isotope dilution assay in plasma revealed no significant bias to gold standard methods for the respective analytes. Compared to plasma, we observed an overestimate of creatine and creatinine (2- to 5-fold and 1.2- to 2-fold, respectively) in whole-blood and DBS, and an underestimate of arginine (2.5-fold) in DBS. Validation of the assay in mouse models of creatine deficiency revealed plasma creatine metabolite pattern in good accordance with those observed in human GAMT and AGAT deficiency. Single dose intraperitoneal application of ornithine in wild-type mice lead to fast ornithine uptake (Tmax ≤ 10 min) and elimination (T1/2=24 min), and a decline of guanidinoacetate. The assay is fast and reliable to study creatine metabolism and pharmacokinetics in mouse models of creatine deficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and validation of an ultra-fast and sensitive microflow liquid chromatography-tandem mass spectrometry (MFLC-MS/MS) method for quantification of LSD and its metabolites in plasma and application to a controlled LSD administration study in humans.

PubMed

Steuer, Andrea E; Poetzsch, Michael; Stock, Lorena; Eisenbeiss, Lisa; Schmid, Yasmin; Liechti, Matthias E; Kraemer, Thomas

2017-05-01

Lysergic acid diethylamide (LSD) is a semi-synthetic hallucinogen that has gained popularity as a recreational drug and has been investigated as an adjunct to psychotherapy. Analysis of LSD represents a major challenge in forensic toxicology due to its instability, low drug concentrations, and short detection windows in biological samples. A new, fast, and sensitive microflow liquid chromatography (MFLC) tandem mass spectrometry method for the validated quantification of LSD, iso-LSD, 2-oxo 3-hydroxy-LSD (oxo-HO-LSD), and N-desmethyl-LSD (nor-LSD) was developed in plasma and applied to a controlled pharmacokinetic (PK) study in humans to test whether LSD metabolites would offer for longer detection windows. Five hundred microlitres of plasma were extracted by solid phase extraction. Analysis was performed on a Sciex Eksigent MFLC system coupled to a Sciex 5500 QTrap. The method was validated according to (inter)-national guidelines. MFLC allowed for separation of the mentioned analytes within 3 minutes and limits of quantification of 0.01 ng/mL. Validation criteria were fulfilled for all analytes. PK data could be calculated for LSD, iso-LSD, and oxo-HO-LSD in all participants. Additionally, hydroxy-LSD (HO-LSD) and HO-LSD glucuronide could be qualitatively detected and PK determined in 11 and 8 subjects, respectively. Nor-LSD was only sporadically detected. Elimination half-lives of iso-LSD (median 12 h) and LSD metabolites (median 9, 7.4, 12, and 11 h for oxo-HO-LSD, HO-LSD, HO-LSD-gluc, and nor-LSD, respectively) exceeded those of LSD (median 4.2 h). However, screening for metabolites to increase detection windows in plasma seems not to be constructive due to their very low concentrations. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetic modeling: Prediction and evaluation of route dependent dosimetry of bisphenol A in monkeys with extrapolation to humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisher, Jeffrey W., E-mail: jeffrey.fisher@fda.hhs.gov; Twaddle, Nathan C.; Vanlandingham, Michelle

A physiologically based pharmacokinetic (PBPK) model was developed for bisphenol A (BPA) in adult rhesus monkeys using intravenous (iv) and oral bolus doses of 100 {mu}g d6-BPA/kg (). This calibrated PBPK adult monkey model for BPA was then evaluated against published monkey kinetic studies with BPA. Using two versions of the adult monkey model based on monkey BPA kinetic data from and , the aglycone BPA pharmacokinetics were simulated for human oral ingestion of 5 mg d16-BPA per person (Voelkel et al., 2002). Voelkel et al. were unable to detect the aglycone BPA in plasma, but were able to detectmore » BPA metabolites. These human model predictions of the aglycone BPA in plasma were then compared to previously published PBPK model predictions obtained by simulating the Voelkel et al. kinetic study. Our BPA human model, using two parameter sets reflecting two adult monkey studies, both predicted lower aglycone levels in human serum than the previous human BPA PBPK model predictions. BPA was metabolized at all ages of monkey (PND 5 to adult) by the gut wall and liver. However, the hepatic metabolism of BPA and systemic clearance of its phase II metabolites appear to be slower in younger monkeys than adults. The use of the current non-human primate BPA model parameters provides more confidence in predicting the aglycone BPA in serum levels in humans after oral ingestion of BPA. -- Highlights: Black-Right-Pointing-Pointer A bisphenol A (BPA) PBPK model for the infant and adult monkey was constructed. Black-Right-Pointing-Pointer The hepatic metabolic rate of BPA increased with age of the monkey. Black-Right-Pointing-Pointer The systemic clearance rate of metabolites increased with age of the monkey. Black-Right-Pointing-Pointer Gut wall metabolism of orally administered BPA was substantial across all ages of monkeys. Black-Right-Pointing-Pointer Aglycone BPA plasma concentrations were predicted in humans orally given oral doses of deuterated BPA.« less

Pharmacokinetics of dextromethorphan and its metabolites in horses following a single oral administration.

PubMed

Corado, Carley R; McKemie, Daniel S; Knych, Heather K

2017-06-01

Dextromethorphan is an N-methyl-D-aspartate (NMDA) non-competitive antagonist commonly used in human medicine as an antitussive. Dextromethorphan is metabolized in humans by cytochrome P450 2D6 into dextrorphan, which is reported to be more potent than the parent compound. The goal of this study is to describe the metabolism of and determine the pharmacokinetics of dextromethorphan and its major metabolites following oral administration to horses. A total of 23 horses received a single oral dose of 2 mg/kg. Blood samples were collected at time 0 and at various times up to 96 h post drug administration. Urine samples were collected from 12 horses up to 120 h post administration. Plasma and urine samples were analyzed using liquid chromatography-mass spectrometry, and the resulting data analyzed using non-compartmental analysis. The C max , T max , and the t 1/2 of dextromethorphan were 519.4 ng/mL, 0.55 h, and 12.4 h respectively. The area under the curve of dextromethorphan, free dextrorphan, and conjugated dextrorphan were 563.8, 2.19, and 6,691 h*ng/mL respectively. In addition to free and glucuronidated dextrorphan, several additional glucuronide metabolites were identified in plasma, including hydroxyl-desmethyl dextrorphan, desmethyl dextrorphan, and three forms of hydroxylated dextrorphan. Dextromethorphan was found to be eliminated from the urine predominately as the O-demethylated metabolite, dextrorphan. Several additional metabolites including several novel hydroxy-dextrorphan metabolites were also detected in the urine in both free and glucuronidated forms. No significant undesirable behavioural effects were noted throughout the duration of the study. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.

PubMed

Simón-Manso, Yamil; Lowenthal, Mark S; Kilpatrick, Lisa E; Sampson, Maureen L; Telu, Kelly H; Rudnick, Paul A; Mallard, W Gary; Bearden, Daniel W; Schock, Tracey B; Tchekhovskoi, Dmitrii V; Blonder, Niksa; Yan, Xinjian; Liang, Yuxue; Zheng, Yufang; Wallace, William E; Neta, Pedatsur; Phinney, Karen W; Remaley, Alan T; Stein, Stephen E

2013-12-17

Recent progress in metabolomics and the development of increasingly sensitive analytical techniques have renewed interest in global profiling, i.e., semiquantitative monitoring of all chemical constituents of biological fluids. In this work, we have performed global profiling of NIST SRM 1950, "Metabolites in Human Plasma", using GC-MS, LC-MS, and NMR. Metabolome coverage, difficulties, and reproducibility of the experiments on each platform are discussed. A total of 353 metabolites have been identified in this material. GC-MS provides 65 unique identifications, and most of the identifications from NMR overlap with the LC-MS identifications, except for some small sugars that are not directly found by LC-MS. Also, repeatability and intermediate precision analyses show that the SRM 1950 profiling is reproducible enough to consider this material as a good choice to distinguish between analytical and biological variability. Clinical laboratory data shows that most results are within the reference ranges for each assay. In-house computational tools have been developed or modified for MS data processing and interactive web display. All data and programs are freely available online at http://peptide.nist.gov/ and http://srmd.nist.gov/ .

Metabolic characterization of (±)-praeruptorin A in vitro and in vivo by high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry.

PubMed

Song, Yue-Lin; Jing, Wang-Hui; Yan, Ru; Wang, Yi-Tao

2014-03-01

(±)-Praeruptorin A (PA) is the major bioactive component in Peucedani Radix (Chinese name: Qian-hu), and exhibits dramatically anti-hypertensive effect typically through acting as a calcium channel blocker. The current study aims on the characterization of the metabolic profiles of PA in vitro and in vivo using high performance liquid chromatography (HPLC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). A total of 12 phase I metabolites (M1-12) in rat liver microsomes (RLMs), 9 phase I metabolites (M1-3, M5-6 and M9-12) in human liver microsomes (HLMs), 2 hydrolyzed products in rat plasma (M11 and M12), none metabolite in human plasma, none metabolite in rat intestinal bacteria, 7 metabolites (M1, M4-7, M13 and M15) in PA-treated rat urine and 6 metabolites (M1, M4-7 and M15) in PA-treated feces were detected and tentatively identified using predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (predictive MRM-IDA-EPI) mode in combination with enhanced mass spectrum-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode in the mass spectrometer domain, respectively, while TOF-MS was adopted to confirm the identification. Further, 2 glucuronidated metabolites (M13-14) in RLMs and none metabolite in HLMs of cis-khellactone (CKL), which was the main actual form of PA in vivo, were generated, while its sulfated product was not observed in either rat liver S9 fractions (RS9) or human liver S9 fractions (HS9). Oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation were demonstrated to be the predominant metabolic types for PA in vitro and in vivo. Judging from the decrement of peak areas, PA was metabolized quickly in both RLMs and HLMs, indicating extensively hepatic first-pass elimination. Taken together, the metabolic fates of (±)-praeruptorin A in vitro and in vivo were elucidated in current study, and Q-trap-MS coupled with LightSight™ software can be adopted as a useful tool for quick detection and identification of metabolites in complex biological matrices. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Identification and quantitation of all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma.

PubMed

Eckhoff, C; Nau, H

1990-08-01

Human plasma was analyzed by high performance liquid chromatography for the presence of retinoic acid and 4-oxoretinoic acid isomers. Peaks that coeluted with the reference compounds all-trans-retinoic acid, 13-cis-retinoic acid, and 13-cis-4-oxoretinoic acid were routinely observed in human plasma. These retinoids were unequivocally identified by the following methods: comigration with reference compounds under several high performance liquid chromatographic conditions; comparison of ultraviolet spectra with those of reference compounds; derivatization with diazomethane and coelution of the methyl esters with reference compounds in a high performance liquid chromatographic system as well as in a gas chromatography system with a mass selective detector. In vitro formation of 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid as artifacts during the analytical procedure was excluded by control experiments. The mean plasma concentrations of the vitamin A metabolites in ten male volunteers were: all-trans-retinoic acid: 1.32 +/- 0.46 ng/ml; 13-cis-retinoic acid: 1.63 +/- 0.85 ng/ml; and 13-cis-4-oxoretinoic acid: 3.68 +/- 0.99 ng/ml. After oral dosing with vitamin A (833 IU/kg body weight) in five male volunteers, mean plasma all-trans-retinoic acid increased to 3.92 +/- 1.40 ng/ml and 13-cis-retinoic acid increased to 9.75 +/- 2.18 ng/ml. Maximal plasma 13-cis-4-oxoretinoic acid concentrations (average 7.60 +/- 1.45 ng/ml) were observed 6 h after dosing which was the last time point in this study. Concentrations of all-trans-4-oxoretinoic acid were low or not detectable. Our findings suggest that, in addition to all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid are present in normal human plasma as metabolites of vitamin A.

The relationship between core body temperature and 3,4-methylenedioxymethamphetamine metabolism in rats: implications for neurotoxicity.

PubMed

Goni-Allo, Beatriz; O Mathúna, Brian; Segura, Mireia; Puerta, Elena; Lasheras, Berta; de la Torre, Rafael; Aguirre, Norberto

2008-04-01

A close relationship appears to exist between 3,4-methylenedioxymethamphetamine (MDMA)-induced changes in core body temperature and long-term serotonin (5-HT) loss. We investigated whether changes in core body temperature affect MDMA metabolism. Male Wistar rats were treated with MDMA at ambient temperatures of 15, 21.5, or 30 degrees C to prevent or exacerbate MDMA-induced hyperthermia. Plasma concentrations of MDMA and its main metabolites were determined for 6 h. Seven days later, animals were killed and brain indole content was measured. The administration of MDMA at 15 degrees C blocked the hyperthermic response and long-term 5-HT depletion found in rats treated at 21.5 degrees C. At 15 degrees C, plasma concentrations of MDMA were significantly increased, whereas those of three of its main metabolites were reduced when compared to rats treated at 21.5 degrees C. By contrast, hyperthermia and indole deficits were exacerbated in rats treated at 30 degrees C. Noteworthy, plasma concentrations of MDMA metabolites were greatly enhanced in these animals. Instrastriatal perfusion of MDMA (100 microM for 5 h at 21 degrees C) did not potentiate the long-term depletion of 5-HT after systemic MDMA. Furthermore, interfering in MDMA metabolism using the catechol-O-methyltransferase inhibitor entacapone potentiated the neurotoxicity of MDMA, indicating that metabolites that are substrates for this enzyme may contribute to neurotoxicity. This is the first report showing a direct relationship between core body temperature and MDMA metabolism. This finding has implications on both the temperature dependence of the mechanism of MDMA neurotoxicity and human use, as hyperthermia is often associated with MDMA use in humans.

Unbiased plasma metabolomics reveal the correlation of metabolic pathways and Prakritis of humans.

PubMed

Shirolkar, Amey; Chakraborty, Sutapa; Mandal, Tusharkanti; Dabur, Rajesh

2017-11-25

Ayurveda, an ancient Indian medicinal system, has categorized human body constitutions in three broad constitutional types (prakritis) i.e. Vata, Pitta and Kapha. Analysis of plasma metabolites and related pathways to classify Prakriti specific dominant marker metabolites and metabolic pathways. 38 healthy male individuals were assessed for dominant Prakritis and their fasting blood samples were collected. The processed plasma samples were subjected to rapid resolution liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (RRLC-ESI-QTOFMS). Mass profiles were aligned and subjected to multivariate analysis. Partial least square discriminant analysis (PLS-DA) model showed 97.87% recognition capability. List of PLS-DA metabolites was subjected to permutative Benjamini-Hochberg false discovery rate (FDR) correction and final list of 76 metabolites with p < 0.05 and fold-change > 2.0 was identified. Pathway analysis using metascape and JEPETTO plugins in Cytoscape revealed that steroidal hormone biosynthesis, amino acid, and arachidonic acid metabolism are major pathways varying with different constitution. Biological Go processes analysis showed that aromatic amino acids, sphingolipids, and pyrimidine nucleotides metabolic processes were dominant in kapha type of body constitution. Fat soluble vitamins, cellular amino acid, and androgen biosynthesis process along with branched chain amino acid and glycerolipid catabolic processes were dominant in pitta type individuals. Vata Prakriti was found to have dominant catecholamine, arachidonic acid and hydrogen peroxide metabolomics processes. The neurotransmission and oxidative stress in vata, BCAA catabolic, androgen, xenobiotics metabolic processes in pitta, and aromatic amino acids, sphingolipid, and pyrimidine metabolic process in kaphaPrakriti were the dominant marker pathways. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.

Simultaneous determination of blonanserin and its metabolite in human plasma and urine by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

PubMed

Wen, Yu-Guan; Ni, Xiao-Jia; Zhang, Ming; Liu, Xia; Shang, De-Wei

2012-08-15

Blonanserin is a novel atypical antipsychotic with highly selective receptor antagonist activity to dopamine D₂ and 5-HT(2A). N-desethyl blonanserin (blonanserin C) is its major active metabolite in human plasma. Herein we report a new highly sensitive, selective, and rapid liquid chromatography-tandem mass spectrometry method to determine blonanserin and blonanserin C simultaneously in human plasma and urine, with N-desethyl-chlor-blonanserin (blonanserin D) as internal standard (IS). Blonanserin and blonanserin C were extracted from aliquots of plasma and urine with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using an Agilent Eclipse Plus C₁₈ column. The mobile phase was composed of: acetonitrile and ammonium formate-formic acid buffer containing 5mM ammonium formate and 0.1% formic acid (87:13, v/v). To quantify blonanserin, blonanserin C, and blonanserin D, respectively, multiple reaction monitoring (MRM) transition of m/z 368.2→297.2, m/z 340.2→297.1, and m/z 356.2→313.3 was performed in positive mode. The analysis time was about 5.5 min. The calibration curve was linear in the concentration range of 0.01-2 ng/ml. The lower limit of quantification reached 0.01 ng/ml. The intra and inter-day precision and relative errors were less than 8.0% and 6.6% for three QC levels in plasma and urine. The current LC-MS/MS method was validated as simple, sensitive, and accurate and has been successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Liquid chromatography/tandem mass spectrometry method for simultaneous determination of cocaine and its metabolite (-)ecgonine methyl ester in human acidified stabilized plasma samples.

PubMed

Liu, Yongzhen; Zheng, Bo; Strafford, Stephanie; Orugunty, Ravi; Sullivan, Michael; Gus, Jeffrey; Heidbreder, Christian; Fudala, Paul J; Nasser, Azmi

2014-06-15

Two simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods (low range and high range) were developed and validated for the quantification of cocaine and its metabolite (-)ecgonine methyl ester (EME) in human acidified stabilized plasma samples. In the low range assay, cocaine and the internal standard, cocaine-D3, were extracted using a single step liquid-liquid extraction from human acidified stabilized plasma. For the high range assay, human acidified stabilized plasma containing cocaine, EME, and the internal standards, cocaine-D3 and EME-D3, was mixed with acetonitrile, and the protein precipitate was separated by centrifugation. Both cocaine and EME extracted from both assays were separated on a HILIC column and detected in positive ion mode using multiple reaction monitoring (MRM). Both methods were validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. The linear range for the low range assay was 0.01-5ng/mL for cocaine; in the high range assay values were 5-1000ng/mL for cocaine and 1-200ng/mL for EME. The correlation coefficient (R(2)) values for both assays were 0.993 or greater. The precision and accuracy for intra-day and inter-day were better than 13.0%. The recovery was above 85% and matrix effects were low with the matrix factor ranging from 0.817 to 1.10 for both analytes in both assays. The validated methods were successfully used to quantify the plasma concentrations of cocaine and EME in clinical pharmacokinetic and pharmacodynamic studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemical UPLC-ESI-MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract.

PubMed

Zhang, Mingjie; Wang, Manman; Liang, Jiajia; Wen, Yongqing; Xiong, Zhili

2018-02-01

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC-ESI-MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty-nine components including twenty-five C19-diterpenoid alkaloids and four C20-diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19-diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3-deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16-β-hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous determination of febuxostat and its three active metabolites in human plasma by liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in Chinese healthy volunteers.

PubMed

Wu, Yingli; Mao, Zhengsheng; Liu, Youping; Wang, Xin; Di, Xin

2015-10-10

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of febuxostat and its three active metabolites in human plasma was developed using a ZORBAX SB-C18 column (50 mm × 4.6 mm, 5 μm) and an optimized gradient mobile phase consisting of acetonitrile, water and formic acid. Plasma samples were spiked with the internal standard losartan and then pre-treated using one-step protein precipitation with methanol. Mass spectrometric detection was performed by selective reaction monitoring mode via electrospray ionization source operating in positive ionization mode. The method exhibited good linearity over the concentration range of 10-20,000 ng/mL for febuxostat, 1.0-270 ng/mL for 67M-1 and 67M-2, and 0.8-250 ng/mL for 67 M-4, respectively. The intra- and inter-day precisions were less than 14.7% and the accuracy ranged from -4.3% to 5.1%. The method was successfully applied to a clinical pharmacokinetic study of febuxostat in humans after oral administration of a single dose of febuxostat at 40, 80 and 120 mg. Copyright © 2015 Elsevier B.V. All rights reserved.

Intestinal disposition of quercetin and its phase-II metabolites after oral administration in healthy volunteers.

PubMed

Chalet, Clément; Rubbens, Jari; Tack, Jan; Duchateau, Guus S; Augustijns, Patrick

2018-05-15

Quercetin is one of the main dietary flavonoids and undergoes a substantial intestinal phase-II metabolism. Quercetin conjugates have been detected in plasma and in urine, but their presence in the small intestine has not been assessed. This study aimed to investigate the intestinal metabolism and metabolite excretion of quercetin by the human small intestinal wall after oral dosing. Six healthy volunteers were given a capsule of 500 mg of quercetin with 240 ml of water. Duodenal fluids were collected using the intraluminal sampling technique for 4 h and analysed by LC-MS/MS. Phase-II metabolites of quercetin were detected and quantified in aspirated intestinal fluids. Metabolites appeared almost immediately after administration, indicating an intestinal metabolism and apical excretion into the lumen. Quercetin-3'-O-glucuronide was found to be the main intestinal metabolite. Our results could not conclude on the enterohepatic recycling of quercetin or its metabolites, although several individual profiles showed distinctive peaks. This study highlights the intestinal metabolism and excretion of quercetin and its conjugates in humans and gives insights into the relevant concentrations which should be used to investigate potential food-drug interactions in vitro. © 2018 Royal Pharmaceutical Society.

Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzyme-alkaline hydrolysis.

PubMed

Aizpurua-Olaizola, Oier; Zarandona, Iratxe; Ortiz, Laura; Navarro, Patricia; Etxebarria, Nestor; Usobiaga, Aresatz

2017-04-01

A high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of Δ9-tetrahydrocannabinol (THC), its two metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), and four additional cannabinoids (cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), and cannabinol (CBN)) in 1 mL of human urine and plasma was developed and validated. The hydrolysis process was studied to ensure complete hydrolysis of glucuronide conjugates and the extraction of a total amount of analytes. Initially, urine and plasma blank samples were spiked with THC-COOH-glucuronide and THC-glucuronide, and four different pretreatment methods were compared: hydrolysis-free method, enzymatic hydrolysis with Escherichia Coli β-glucuronidase, alkaline hydrolysis with 10 M NaOH, and enzyme-alkaline tandem hydrolysis. The last approach assured the maximum efficiencies (close to 100%) for both urine and plasma matrices. Regarding the figures of merit, the limits of detection were below 1 ng/mL for all analytes, the accuracy ranged from 84% to 115%, and both within-day and between-day precision were lower than 12%. Finally, the method was successfully applied to real urine and plasma samples from cannabis users. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Plasma appearance and correlation between coffee and green tea metabolites in human subjects.

PubMed

Renouf, Mathieu; Guy, Philippe; Marmet, Cynthia; Longet, Karin; Fraering, Anne-Lise; Moulin, Julie; Barron, Denis; Dionisi, Fabiola; Cavin, Christophe; Steiling, Heike; Williamson, Gary

2010-12-01

Coffee and green tea are two of the most widely consumed hot beverages in the world. Their respective bioavailability has been studied separately, but absorption of their respective bioactive phenolics has not been compared. In a randomised cross-over design, nine healthy subjects drank instant coffee and green tea. Blood samples were collected over 12 h and at 24 h to assess return to baseline. After green tea consumption, (-)-epigallocatechin (EGC) was the major catechin, appearing rapidly in the plasma; (-)-EGC gallate (EGCg) and (-)-epicatechin (EC) were also present, but (-)-EC gallate and C were not detected. Dihydroferulic acid and dihydrocaffeic acid were the major metabolites that appeared after coffee consumption with a long time needed to reach maximum plasma concentration, suggesting metabolism and absorption in the colon. Other phenolic acid equivalents (caffeic acid (CA), ferulic acid (FA) and isoferulic acid (iFA)) were detected earlier, and they peaked at lower concentrations. Summations of the plasma area under the curves (AUC) for the measured metabolites showed 1.7-fold more coffee-derived phenolic acids than green tea-derived catechins (P = 0.0014). Furthermore, we found a significant correlation between coffee metabolites based on AUC. Inter-individual differences were observed, but individuals with a high level of CA also showed a correspondingly high level of FA. However, no such correlation was observed between the tea catechins and coffee phenolic acids. Correlation between AUC and maximum plasma concentration was also significant for CA, FA and iFA and for EGCg. This implies that the mechanisms of absorption for these two classes of compounds are different, and that a high absorber of phenolic acids is not necessarily a high absorber of catechins.

Plasma metabolomic profiles of breast cancer patients after short-term limonene intervention.

PubMed

Miller, Jessica A; Pappan, Kirk; Thompson, Patricia A; Want, Elizabeth J; Siskos, Alexandros P; Keun, Hector C; Wulff, Jacob; Hu, Chengcheng; Lang, Julie E; Chow, H-H Sherry

2015-01-01

Limonene is a lipophilic monoterpene found in high levels in citrus peel. Limonene demonstrates anticancer properties in preclinical models with effects on multiple cellular targets at varying potency. While of interest as a cancer chemopreventive, the biologic activity of limonene in humans is poorly understood. We conducted metabolite profiling in 39 paired (pre/postintervention) plasma samples from early-stage breast cancer patients receiving limonene treatment (2 g QD) before surgical resection of their tumor. Metabolite profiling was conducted using ultra-performance liquid chromatography coupled to a linear trap quadrupole system and gas chromatography-mass spectrometry. Metabolites were identified by comparison of ion features in samples to a standard reference library. Pathway-based interpretation was conducted using the human metabolome database and the MetaCyc database. Of the 397 named metabolites identified, 72 changed significantly with limonene intervention. Class-based changes included significant decreases in adrenal steroids (P < 0.01), and significant increases in bile acids (P ≤ 0.05) and multiple collagen breakdown products (P < 0.001). The pattern of changes also suggested alterations in glucose metabolism. There were 47 metabolites whose change with intervention was significantly correlated to a decrease in cyclin D1, a cell-cycle regulatory protein, in patient tumor tissues (P ≤ 0.05). Here, oral administration of limonene resulted in significant changes in several metabolic pathways. Furthermore, pathway-based changes were related to the change in tissue level cyclin D1 expression. Future controlled clinical trials with limonene are necessary to determine the potential role and mechanisms of limonene in the breast cancer prevention setting. ©2014 American Association for Cancer Research.

A simplified radiometabolite analysis procedure for PET radioligands using a solid phase extraction with micellar medium.

PubMed

Nakao, Ryuji; Halldin, Christer

2013-07-01

A solid phase extraction method has been developed for simple and high-speed direct determination of PET radioligands in plasma. This methodology makes use of a micellar medium and a solid-phase extraction cartridge for displacement of plasma protein bound radioligand and separation of PET radioligands from their radiometabolites without significant preparation. The plasma samples taken from monkey or human during PET measurements were mixed with a micellar eluent containing an anionic surfactant sodium dodecyl sulphate and loaded onto SPE cartridges. The amount of radioactivity corresponding to parent radioligand (retained on the cartridge) and its radioactive metabolites (eluted with micellar eluent) was measured. Under the optimized conditions, excellent separation of target PET radioligands from their radiometabolites was achieved with a single elution and short run-time of 1 min. This method was successfully applied to study the metabolism for (11)C-labelled radioligands in human or monkey plasma. The amount of parent PET radioligands estimated by micellar solid phase extraction strongly corresponded with that determined by radio-LC. The improved throughput permitted the analysis of a large number of plasma samples (up to 13 samples per one PET study) for accurate estimation of metabolite-corrected input function during quantitative PET imaging studies. Solid phase extraction together with micellar medium is fast, sensitive and easy to use, and therefore it is an attractive alternative method to determine relative composition of PET radioligands in plasma. Copyright © 2013 Elsevier Inc. All rights reserved.

Is enzymatic hydrolysis a reliable analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites in human fluids?

PubMed

Quifer-Rada, Paola; Martínez-Huélamo, Miriam; Lamuela-Raventos, Rosa M

2017-07-19

Phenolic compounds are present in human fluids (plasma and urine) mainly as glucuronidated and sulfated metabolites. Up to now, due to the unavailability of standards, enzymatic hydrolysis has been the method of choice in analytical chemistry to quantify these phase II phenolic metabolites. Enzymatic hydrolysis procedures vary in enzyme concentration, pH and temperature; however, there is a lack of knowledge about the stability of polyphenols in their free form during the process. In this study, we evaluated the stability of 7 phenolic acids, 2 flavonoids and 3 prenylflavanoids in urine during enzymatic hydrolysis to assess the suitability of this analytical procedure, using three different concentrations of β-glucuronidase/sulfatase enzymes from Helix pomatia. The results indicate that enzymatic hydrolysis negatively affected the recovery of the precursor and free-form polyphenols present in the sample. Thus, enzymatic hydrolysis does not seem an ideal analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites.

Metabolomic characteristics of arsenic-associated diabetes in a prospective cohort in Chihuahua, Mexico.

PubMed

Martin, Elizabeth; González-Horta, Carmen; Rager, Julia; Bailey, Kathryn A; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Ishida, María C; Gutiérrez-Torres, Daniela S; Hernández Cerón, Roberto; Viniegra Morales, Damián; Baeza Terrazas, Francisco A; Saunders, R Jesse; Drobná, Zuzana; Mendez, Michelle A; Buse, John B; Loomis, Dana; Jia, Wei; García-Vargas, Gonzalo G; Del Razo, Luz M; Stýblo, Miroslav; Fry, Rebecca

2015-04-01

Chronic exposure to inorganic arsenic (iAs) has been linked to an increased risk of diabetes, yet the specific disease phenotype and underlying mechanisms are poorly understood. In the present study we set out to identify iAs exposure-associated metabolites with altered abundance in nondiabetic and diabetic individuals in an effort to understand the relationship between exposure, metabolomic response, and disease status. A nested study design was used to profile metabolomic shifts in urine and plasma collected from 90 diabetic and 86 nondiabetic individuals matched for varying iAs concentrations in drinking water, body mass index, age, and sex. Diabetes diagnosis was based on measures of fasting plasma glucose and 2-h blood glucose. Multivariable models were used to identify metabolites with altered abundance associated with iAs exposure among diabetic and nondiabetic individuals. A total of 132 metabolites were identified to shift in urine or plasma in response to iAs exposure characterized by the sum of iAs metabolites in urine (U-tAs). Although many metabolites were altered in both diabetic and nondiabetic 35 subjects, diabetic individuals displayed a unique response to iAs exposure with 59 altered metabolites including those that play a role in tricarboxylic acid cycle and amino acid metabolism. Taken together, these data highlight the broad impact of iAs exposure on the human metabolome, and demonstrate some specificity of the metabolomic response between diabetic and nondiabetic individuals. These data may provide novel insights into the mechanisms and phenotype of diabetes associated with iAs exposure. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations

PubMed Central

von Moltke, Lisa L; Greenblatt, David J; Granda, Brian W; Duan, Su Xiang; Grassi, Jeffrey M; Venkatakrishnan, Karthik; Harmatz, Jerold S; Shader, Richard I

1999-01-01

Aims To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. Methods Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Results The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50) of 0.61 μm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by ≈40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. Conclusions The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition. PMID:10383565

Development of a low cost portable fluorometry technology and quantification of cannabinoids in body fluids

DOT National Transportation Integrated Search

1977-05-04

Technology was developed for determining delta sup 9-tetrahydrocannabinol (I) and its major metabolite 11-nor-delta sup 9-tetrahydrocannabinol-9-carboxylic acid (II) in human blood plasma utilizing high pressure liquid chromatography (hplc)-ultraviol...

Development, validation and application of the liquid chromatography tandem mass spectrometry method for simultaneous quantification of azilsartan medoxomil (TAK-491), azilsartan (TAK-536), and its 2 metabolites in human plasma.

PubMed

Kuze, Yoji; Kogame, Akifumi; Jinno, Fumihiro; Kondo, Takahiro; Asahi, Satoru

2015-09-15

Azilsartan medoxomil potassium salt (TAK-491) is an orally administered angiotensin II type 1 receptor blocker for the treatment of hypertension and is an ester-based prodrug that is rapidly hydrolyzed to the pharmacologically active moiety, azilsartan (TAK-536), during absorption. TAK-536 is biotransformed to the 2 metabolites M-I by decarboxylation and M-II by dealkylation. In this study, we developed and validated a LC/MS/MS method which can simultaneously determine 4 analytes, TAK-491, TAK-536, M-I and M-II. The bioanalytical method can be outlined as follows: two structural analogues are used as the internal standards. The analytes and the IS are extracted from human plasma using solid phase extraction. After evaporating, the residue is reconstituted and injected into a LC/MS/MS system with an ESI probe and analyzed in the positive ion mode. Separation is performed through a conventional reversed-phase column with a mobile phase of water/acetonitrile/acetic acid (40:60:0.05, v/v/v) mixture at a flow rate of 0.2mL/min. The total run time is 8.5min. The calibration range is 1-2500ng/mL in human plasma for all the analytes. Instability issues of the prodrug, TAK-491, were overcome and all the validation results met the acceptance criteria in accordance with the regulatory guideline/guidance. As a result of the clinical study, the human PK profiles of TAK-536, M-I and M-II were successfully obtained and also it was confirmed that TAK-491 was below the LLOQ (1ng/mL) in the human plasma samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and Validation of a High Pressure Liquid Chromatography-UV Method for the Determination of Treosulfan and Its Epoxy Metabolites in Human Plasma and Its Application in Pharmacokinetic Studies.

PubMed

Koyyalamudi, Sundar R; Kuzhiumparambil, Unnikrishnan; Nath, Christa E; Byrne, Jennifer A; Fraser, Christopher J; O'Brien, Tracey A; Earl, John W; Shaw, Peter J

2016-03-01

Treosulfan (l-threitol-1,4-di-methanesulfonate) is a prodrug of a bifunctional alkylating agent that is being used increasingly in pediatric bone marrow transplantation regimens. The activation pathway is a complex reaction, which consists of two consecutive reactions leading to epoxybutane derivatives which are responsible for DNA alkylation. A simple, sensitive high performance liquid chromatography method for the determination of the sum of treosulfan and its epoxy metabolites by UV detection after derivatization with sodium diethyldithiocarbamate in human plasma was developed and validated. Plasma samples containing treosulfan and epoxy metabolites were converted into thiocarbamate derivative with 10% sodium diethyldithiocarbamate. Dinitrobiphenyl was used as an internal standard. The analysis was carried out using a reversed phase C18 column with a mobile phase consisting of methanol-water (65:35, v/v) at a flow rate of 1 mL/min. The eluent was monitored at 254 nm. The standard calibration curve was established between 2.5 and 50 µg/mL, with a correlation coefficient of 0.9987. Intra- and interday precision and accuracy of the method was <8% and met the analytical criteria. Pharmacokinetic parameters were determined in six children who received intravenous treosulfan (dose range 12-24 g/m(2)) in combination with fludarabine prior to blood or marrow transplantation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Predictors and correlations of phthalate metabolite concentrations in urine and seminal plasma among reproductive-aged men.

PubMed

Wang, Yi-Xin; Liu, Chong; Chen, Ying-Jun; Chen, Heng-Gui; Yang, Pan; Wang, Peng; Huang, Li-Li; Ai, Song-Hua; Duan, Peng; Pan, An; Zeng, Qiang; Lu, Wen-Qing

2018-02-01

Certain phthalates are suspected to be endocrine disruptors that are adversely associated with male reproductive health. However, the predictors and correlations of phthalate metabolite concentrations in urine and seminal plasma among reproductive-aged men have not been thoroughly studied. To investigate the predictors and correlations of phthalate metabolite concentrations in urine and seminal plasma among adult Chinese males. We measured mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-n-octyl phthalate (MOP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) concentrations in seminal plasma and repeated spot-urine samples from 687 men who visited a reproductive center. Mixed-effect models were used to examine the associations of sociodemographic, lifestyle and medical factors with urinary metabolite concentrations. Linear regression models were used to identify predictors of metabolite concentrations in seminal plasma and correlations between metabolite concentrations in spot urine samples and seminal plasma. Measurements taken from spot urine samples poorly predicted same-day seminal plasma concentrations (all R 2 <0.10). Inverse associations were observed between education level and urinary MBP and MEOHP and between household income and urinary MMP; receiving intravenous infusion therapy was associated with increased urinary MBP, MEHHP and MEOHP, use of facial cleanser/cream was associated with increased MEP, and smoking was associated with increased MEHP. The predictors of metabolite concentrations in seminal plasma differed from those in urine, except for the association of intravenous infusion therapy with MBP. BMI was associated with increased seminal plasma MBP, MEHP and MEOHP, smoking was associated with increased MEP, and contact with plastics was associated with increased MEOHP. Phthalate metabolite concentrations in adult men varied in accordance with sociodemographic variables, lifestyle factors and intravenous therapy. Measures of metabolite levels in urine may not directly reflect the exposure status of the male reproductive system. Copyright © 2017 Elsevier Inc. All rights reserved.

Biotransformation of the platinum drug JM216 following oral administration to cancer patients.

PubMed

Raynaud, F I; Mistry, P; Donaghue, A; Poon, G K; Kelland, L R; Barnard, C F; Murrer, B A; Harrap, K R

1996-01-01

This study evaluates the metabolic profile of JM216 [bis(acetato)ammine-dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n = 2-4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m2). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt = 5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients' plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis-amminedichloro-(cyclohexylamine)platinum(II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis-acetatoammine-chloro(cyclohexylalamine)hydroxoplatin um(IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients' plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzymatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(Il) metabolites in plasma that differ significantly from other systemically applied platinum drugs.

Application of antibody-mediated extraction for the stereoselective determination of the active metabolite of loxoprofen in human and rat plasma.

PubMed

Takasaki, W; Tanaka, Y

1992-01-01

Antibody-mediated extraction followed by chiral high-performance liquid chromatography (HPLC) was applied to stereoselective determination in human and rat plasma of the active metabolite [(2S,1'R,2'S)-trans-alcohol] with three chiral centers of Loxoprofen, a 2-arylpropionic acid antiinflammatory agent after oral administration. Antiserum against the (1'R,2'S)-cyclopentanol moiety was obtained from a rabbit immunized with bovine serum albumin conjugate linked to the propionic acid moiety, in which another chiral center is located. Then, the immunoglobulin G purified by a protein A column was coupled to BrCN-activated Sepharose 4B. Plasma samples were applied to the immobilized antibody column. After washing the column to remove unrequired stereoisomers, a mixture of two diastereomers whose configurations were 1'R,2'S in the cyclopentanol moiety was extracted with 95% methanol. The solvent was evaporated and the residue was derivatized with (+)-(R)-1-(1-naphthyl)ethylamine as a chiral reagent to separate the diastereomers by HPLC. This combined analytical method showed the stereoselective metabolism of Loxoprofen in human, that is, 64% of the total amount of four trans-alcohol stereoisomers was in the 2S,1'R,2'S form, which is the active metabolite. This phenomenon was also observed in rats given Loxoprofen and its (2S)- and (2R)-isomers, and is explained by stereoselective ketone reduction of Loxoprofen to the (1'R,2'S)-trans-alcohol and inversion from 2R to 2S in the propionic acid moiety. Antibody-mediated extraction should be a selective and simple clean-up method for determining haptens with complicated structures, combined with an appropriate analytical method.

Development and validation of amitriptyline and its metabolite in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study.

PubMed

Bhatt, Mitesh; Shah, Sanjay

2010-11-01

A method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of amitriptyline and its main metabolite in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective SPE procedure using hydrophilic-lipophilic balance cartridges. The assay involves a simple solid-phase extraction (SPE) procedure of 0.2 mL of human plasma and analysis was performed on a triple-quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode via electrospray ionization (ESI). The standard calibration curve was linear over the ranges 0.370-95.539 ng/mL for amitriptyline and 0.365-94.374 ng/mL for nortriptyline, expressed by the linear correlation coefficient r², which was better than 0.995 for both. The intra- and inter-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 85.3, 88.4 and 80.7% for amitriptyline, nortriptyline and doxepin respectively. Total run time was 1.2 min only for each sample, which makes it possible to analyze more than 400 samples per day. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2010 John Wiley & Sons, Ltd.

Plasma pharmacokinetics of catechin metabolite 4'-O-Me-EGC in healthy humans.

PubMed

Renouf, Mathieu; Redeuil, Karine; Longet, Karin; Marmet, Cynthia; Dionisi, Fabiola; Kussmann, Martin; Williamson, Gary; Nagy, Kornél

2011-10-01

Tea is an infusion of the leaves of the Camellia sinensis plant and is the most widely consumed beverage in the world after water. Green tea contains significant amounts of polyphenol catechins and represents a promising dietary component to maintain health and well-being. Epidemiological studies indicate that polyphenol intake may have potential health benefits, such as, reducing the incidence of coronary heart disease, diabetes and cancer. While bioavailability of green tea bioactives is fairly well understood, some gaps still remain to be filled, especially the identification and quantification of conjugated metabolites in plasma, such as, sulphated, glucuronidated or methylated compounds. In the present study, we aimed to quantify the appearance of green tea catechins in plasma with particular emphasis on their methylated forms. After feeding 400 mL of green tea, 1.25% infusion to 9 healthy subjects, we found significant amounts of EC, EGC and EGCg in plasma as expected. EGC was the most bioavailable catechin, and its methylated form (4'-O-Me-EGC) was also present in quantifiable amounts. Its kinetics followed that of its parent compound. However, the relative amount of the methylated form of EGC was lower than that of the parent compound, an important aspect which, in the literature, has been controversial so far. The quantitative results presented in our study were confirmed by co-chromatography and accurate mass analysis of the respective standards. We show that the relative abundance of 4'-O-Me-EGC is ~40% compared to the parent EGC. 4'-O-Me-EGC is an important metabolite derived from catechin metabolism. Its presence in significant amounts should not be overlooked when assessing human bioavailability of green tea.

Polyamines and Their Metabolites as Diagnostic Markers of Human Diseases

PubMed Central

Park, Myung Hee; Igarashi, Kazuei

2013-01-01

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke. PMID:24009852

Urinary nandrolone metabolites of endogenous origin in man: a confirmation by output regulation under human chorionic gonadotropin stimulation.

PubMed

Reznik, Y; Dehennin, L; Coffin, C; Mahoudeau, J; Leymarie, P

2001-01-01

19-Nortestosterone (nandrolone) is an anabolic steroid compound widely used as a doping agent by athletes. The analysis of its urinary metabolites, 19-norandrosterone (NA) and 19-noretiocholanolone (NE) glucuronides, allows the detection of surreptitious administration of nandrolone in sport. A threshold concentration at 2 microgram/L urinary nandrolone metabolites is advocated by the International Olympic Committee for the detection of doping, but some controversy concerning the validity of this threshold arose from the demonstration of endogenous production of nandrolone in mammals, including humans. The regulation of human nandrolone production and its contribution in vivo to the process of aromatization remain unknown. In the present study 10 healthy men were successively submitted to insulinic stress and gonadal stimulation by hCG administration. Urinary NA and NE concentrations were quantified by gas chromatography-mass spectrometry. NA was detected in basal urine samples from all subjects, with a mean urinary excretion rate (UER) of 3.17 +/- 0.35 ng/h, whereas NE was detected in 4 of 10 (UER range, 0.8-4.7 ng/h). Insulinic hypoglycemia did not significantly modify mean NA UER despite random intraindividual variations between timed urine collections. After hCG administration, NA UER increased by 250% (P < 0.01) and estradiol (E(2)) UER by 260% (P < 0.001). The maximum NA concentration obtained after stimulation was 0.43 microgram/L. NA UER, plasma E(2), and E(2)/T ratio peaked on day 1 after hCG administration, whereas plasma T peaked later on day 3. NA UER correlated with plasma E(2) (r = 0.61; P < 0.001) and E(2)/T (r = 0.51; P < 0.001), but not with plasma T. In conclusion, insulinic stress did not significantly alter nandrolone metabolism, whereas the effect of hCG was a stimulation of NA excretion in all subjects, which constitutes strong support for the endogenous origin of low basal NA excretion. The comparative kinetics of NA UER, plasma E(2), and E(2)/T ratio suggest a contribution of the aromatase process to nandrolone biosynthesis in man.

Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood.

PubMed

Isokawa, Muneki; Kanamori, Takahiro; Funatsu, Takashi; Tsunoda, Makoto

2014-08-01

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry. Copyright © 2014 Elsevier B.V. All rights reserved.

A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

PubMed Central

Gooden, Felicia C. T.; Tabet, Michael R.; Ball, William J.

2014-01-01

The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine’s AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine’s Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse. PMID:24733787

Absorption kinetics and action profiles of subcutaneously administered insulin analogues (AspB9GluB27, AspB10, AspB28) in healthy subjects.

PubMed

Kang, S; Brange, J; Burch, A; Vølund, A; Owens, D R

1991-11-01

The subcutaneous absorption and resulting changes in plasma insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations after subcutaneous bolus injection of three soluble human insulin analogues (AspB9GluB27, monomeric; AspB28, mixture of monomers and dimers; and AspB10, dimeric) and soluble human insulin were evaluated. Fasting healthy male volunteers (n = 7) were studied on five occasions 1 wk apart randomly receiving 0.6 nmol.kg-1 s.c. 125I-labeled AspB10 or soluble human insulin (Novolin R, Novo, Copenhagen); 1st study and 0.6 nmol.kg-1 s.c. 125I-labeled AspB28, AspB9GluB27 or soluble human insulin (2nd study). Residual radioactivity at the injection site was measured over 8 h with frequent venous sampling for plasma immunoreactive insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations. The three analogues were absorbed 2-3 times faster than human insulin. The mean +/- SE time to 50% residual radioactivity was 94 +/- 6 min for AspB10 compared with 184 +/- 10 min for human insulin (P less than 0.001), 83 +/- 8 min for AspB28 (P less than 0.005), and 63 +/- 9 min for AspB9GluB27 (P less than 0.001) compared with 182 +/- 21 min for human insulin. delta Peak plasma insulin analogue levels were significantly higher after each analogue than after human insulin (P less than 0.005). With all three analogues, the mean hypoglycemic nadir occurred earlier at 61-65 min postinjection compared with 201-210 min for the reference human insulins (P less than 0.005). The magnitude of the hypoglycemic nadir was greater after AspB9GluB27 (P less than 0.05) and AspB28 (P less than 0.001) compared with human insulin. There was a significantly faster onset and offset of responses in C-peptide and intermediary metabolite levels after the analogues than after human insulin (P less than 0.05). The rapid absorption and biological actions of these analogues offer potential therapeutic advantages over the current short-acting neutral soluble insulins.

Metabonomics approach to determine metabolic differences between green tea and black tea consumption.

PubMed

Van Dorsten, Ferdi A; Daykin, Clare A; Mulder, Theo P J; Van Duynhoven, John P M

2006-09-06

The purpose of this study was to compare the effects of black and green tea consumption on human metabolism. Seventeen healthy male volunteers consumed black tea, green tea, or caffeine in a randomized crossover study. Twenty-four-hour urine and blood plasma samples were analyzed by NMR-based metabonomics, that is, high-resolution 1H NMR metabolic profiling combined with multivariate statistics. Green and black tea consumption resulted in similar increases in urinary excretion of hippuric acid and 1,3-dihydroxyphenyl-2-O-sulfate, both of which are end products of tea flavonoid degradation by colonic bacteria. Several unidentified aromatic metabolites were detected in urine specifically after green tea intake. Interestingly, green and black tea intake also had a different impact on endogenous metabolites in urine and plasma. Green tea intake caused a stronger increase in urinary excretion of several citric acid cycle intermediates, which suggests an effect of green tea flavanols on human oxidative energy metabolism and/or biosynthetic pathways.

Maternal Choline Status, but Not Fetal Genotype, Influences Cord Plasma Choline Metabolite Concentrations.

PubMed

Visentin, Carly E; Masih, Shannon; Plumptre, Lesley; Malysheva, Olga; Nielsen, Daiva E; Sohn, Kyoung-Jin; Ly, Anna; Lausman, Andrea Y; Berger, Howard; Croxford, Ruth; El-Sohemy, Ahmed; Caudill, Marie A; O'Connor, Deborah L; Kim, Young-In

2015-07-01

Choline deficiency during pregnancy can lead to adverse birth outcomes, including impaired neurodevelopment and birth defects. Genetic variants of choline and one-carbon metabolism may also influence birth outcomes by altering plasma choline concentrations. The effects of maternal ad libitum choline intake during pregnancy and fetal genetic variants on maternal and cord concentrations of choline and its metabolites are unknown. This prospective study sought to assess the effect of 1) maternal dietary choline intake on maternal and cord plasma concentrations of choline and its metabolites, and 2) fetal genetic polymorphisms on cord plasma concentrations. The dietary choline intake of 368 pregnant Canadian women was assessed in early (0-16 wk) and late (23-37 wk) pregnancy with the use of a food frequency questionnaire. Plasma concentrations of free choline and its metabolites were measured in maternal samples at recruitment and delivery, and in the cord blood. Ten fetal genetic variants in choline and one-carbon metabolism were assessed for their association with cord plasma concentrations of free choline and its metabolites. Mean maternal plasma free choline, dimethylglycine, and trimethylamine N-oxide (TMAO) concentrations increased during pregnancy by 49%, 17%, and 13%, respectively (P < 0.005), whereas betaine concentrations decreased by 21% (P < 0.005). Cord plasma concentrations of free choline, betaine, dimethylglycine, and TMAO were 3.2, 2.0, 1.3, and 0.88 times corresponding maternal concentrations at delivery, respectively (all P < 0.005). Maternal plasma concentrations of betaine, dimethylglycine, and TMAO (r(2) = 0.19-0.51; P < 0.0001) at delivery were moderately strong, whereas maternal concentrations of free choline were not significant (r(2) = 0.12; P = 0.06), predictors of cord plasma concentrations of these metabolites. Neither maternal dietary intake nor fetal genetic variants predicted maternal or cord plasma concentrations of choline and its metabolites. These data collectively indicate that maternal choline status, but not fetal genotype, influences cord plasma concentrations of choline metabolites. This trial was registered at clinicaltrials.gov as NCT02244684. © 2015 American Society for Nutrition.

Effect of the long-term regular intake of virgin olive oil on the phenolic metabolites in human fasting plasma.

PubMed

Valls, Rosa-Maria; Soler, Aranzazu; Girona, Josefa; Heras, Mercedes; Romero, Maria-Paz; Covas, Maria-Isabel; Solà, Rosa; Masana, Lluis; Motilva, Maria-Jose

2010-09-21

The effect of repeated consumption of virgin olive oil on endogenous phenolic metabolites of fasting plasma is unknown. For this reason, we hypothesized that regular long-term virgin olive oil intake could have an indirect protection effect on the endogenous phenols. Thus, the aim of the study was to determine the phenolic profile of human plasma in a fasting state of long-term regular virgin olive oil consumers, using the fasting plasma of non-consumers as a natural control. Forty participants living in the area of Reus (Catalonia, Spain) were selected, 20 life-long regular consumers of virgin olive oil and a natural control of 20 non-consumers, the latter being Rumanians who dislike the taste of olive oil. The diet was obtained from 3-day food records. The results showed similar phenolic composition of fasting plasmas of the two volunteer groups. Of special interest is that more of the compounds quantified showed higher concentration in fasting plasma from habitual virgin olive oil consumers. The compounds were semi-quantified using caffeic acid as the calibration standard. The quantification of fasting consumer's plasma showed higher concentration of a hydroxyflavanone type compound (2.90+/-0.04 microM vs 1.5+/-0.04 microM) and a catecholamine derivative (0.70+/-0.03 microM vs 0.56+/-0.03 microM) than the plasma of non-consumers (P<0.05). The results suggest an indirect protective mechanism of long-term regular virgin olive oil consumption related to the protection of the endogenous antioxidant system. Copyright 2010 Elsevier B.V. All rights reserved.

A Genome-Wide Metabolic QTL Analysis in Europeans Implicates Two Loci Shaped by Recent Positive Selection

PubMed Central

Nicholson, George; Rantalainen, Mattias; Li, Jia V.; Maher, Anthony D.; Malmodin, Daniel; Ahmadi, Kourosh R.; Faber, Johan H.; Barrett, Amy; Min, Josine L.; Rayner, N. William; Toft, Henrik; Krestyaninova, Maria; Viksna, Juris; Neogi, Sudeshna Guha; Dumas, Marc-Emmanuel; Sarkans, Ugis; Donnelly, Peter; Illig, Thomas; Adamski, Jerzy; Suhre, Karsten; Allen, Maxine; Zondervan, Krina T.; Spector, Tim D.; Nicholson, Jeremy K.; Lindon, John C.

2011-01-01

We have performed a metabolite quantitative trait locus (mQTL) study of the 1H nuclear magnetic resonance spectroscopy (1H NMR) metabolome in humans, building on recent targeted knowledge of genetic drivers of metabolic regulation. Urine and plasma samples were collected from two cohorts of individuals of European descent, with one cohort comprised of female twins donating samples longitudinally. Sample metabolite concentrations were quantified by 1H NMR and tested for association with genome-wide single-nucleotide polymorphisms (SNPs). Four metabolites' concentrations exhibited significant, replicable association with SNP variation (8.6×10−11

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

PubMed

Tsutsui, Haruhito; Maeda, Toshio; Min, Jun Zhe; Inagaki, Shinsuke; Higashi, Tatsuya; Kagawa, Yoshiyuki; Toyo'oka, Toshimasa

2011-05-12

The number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study. The low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB. Many metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-L-leucine based upon the MS/MS spectra and the retention time on the chromatograms. N-acetyl-L-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Bioanalysis of alectinib and metabolite M4 in human plasma, cross-validation and impact on PK assessment.

PubMed

Heinig, Katja; Miya, Kazuhiro; Kamei, Tomonori; Guerini, Elena; Fraier, Daniela; Yu, Li; Bansal, Surendra; Morcos, Peter N

2016-07-01

Alectinib is a novel anaplastic lymphoma kinase (ALK) inhibitor for treatment of patients with ALK-positive non-small-cell lung cancer who have progressed on or are intolerant to crizotinib. To support clinical development, concentrations of alectinib and metabolite M4 were determined in plasma from patients and healthy subjects. LC-MS/MS methods were developed and validated in two different laboratories: Chugai used separate assays for alectinib and M4 in a pivotal Phase I/II study while Roche established a simultaneous assay for both analytes for another pivotal study and all other studies. Cross-validation assessment revealed a bias between the two bioanalytical laboratories, which was confirmed with the clinical PK data between both pivotal studies using the different bioanalytical methods.

Metabolomic markers of fertility in bull seminal plasma

PubMed Central

Dinh, Thu; Kaya, Abdullah; Topper, Einko; Moura, Arlindo Alencar

2018-01-01

Metabolites play essential roles in biological systems, but detailed identities and significance of the seminal plasma metabolome related to bull fertility are still unknown. The objectives of this study were to determine the comprehensive metabolome of seminal plasma from Holstein bulls and to ascertain the potential of metabolites as biomarkers of bull fertility. The seminal plasma metabolome from 16 Holstein bulls with two fertility rates were determined by gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses of the data were performed, and the pathways associated with the seminal plasma metabolome were identified using bioinformatics approaches. Sixty-three metabolites were identified in the seminal plasma of all bulls. Fructose was the most abundant metabolite in the seminal fluid, followed for citric acid, lactic acid, urea and phosphoric acid. Androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid represented the least predominant metabolites. Partial-Least Squares Discriminant Analysis (PLSDA) revealed a distinct separation between high and low fertility bulls. The metabolites with the greatest Variable Importance in Projection score (VIP > 2) were 2-oxoglutaric acid and fructose. Heat-map analysis, based on VIP score, and univariate analysis indicated that 2-oxoglutaric acid was less (P = 0.02); whereas fructose was greater (P = 0.02) in high fertility than in low fertility bulls. The current study is the first to describe the metabolome of bull seminal plasma using GC-MS and presented metabolites such as 2-oxoglutaric acid and fructose as potential biomarkers of bull fertility. PMID:29634739

Variation of Metabolite and Hormone Contents in Human Milk.

PubMed

Demmelmair, Hans; Koletzko, Berthold

2017-03-01

Animal studies show that the lactation period contributes to metabolic programming of the offspring and that oral leptin and insulin show bioactivity. Stage of lactation, duration of gestation, maternal body composition, and maternal diet seem to influence the concentrations of small molecules in human milk. Variability of small molecule concentrations seems higher in preterm milk than in term milk. Insulin in human milk shows concentrations similar to plasma. Leptin concentration is lower in milk than in plasma and reflects maternal body mass index. Early in lactation, leptin could contribute to mediating the association between maternal and infant body composition. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Plasma metabolomic profiles of breast cancer patients after short-term limonene intervention

PubMed Central

Miller, Jessica A.; Pappan, Kirk; Thompson, Patricia A.; Want, Elizabeth J.; Siskos, Alexandros; Keun, Hector C.; Wulff, Jacob; Hu, Chengcheng; Lang, Julie E.; Chow, H-H. Sherry

2014-01-01

Limonene is a lipophilic monoterpene found in high levels in citrus peel. Limonene demonstrates anti-cancer properties in preclinical models with effects on multiple cellular targets at varying potency. While of interest as a cancer chemopreventive, the biological activity of limonene in humans is poorly understood. We conducted metabolite profiling in 39 paired (pre/post-intervention) plasma samples from early-stage breast cancer patients receiving limonene treatment (2 g QD) before surgical resection of their tumor. Metabolite profiling was conducted using ultra-performance liquid chromatography (UPLC) coupled to a linear trap quadrupole (LTQ) system and gas chromatography mass spectrometry (GC-MS). Metabolites were identified by comparison of ion features in samples to a standard reference library. Pathway-based interpretation was conducted using the human metabolome database (HMDB) and the MetaCyc database. Of the 397 named metabolites identified, 72 changed significantly with limonene intervention. Class-based changes included significant decreases in adrenal steroids (P’s<0.01), and significant increases in bile acids (P’s≤0.05) and multiple collagen breakdown products (P’s<0.001). The pattern of changes also suggested alterations in glucose metabolism. There were 47 metabolites whose change with intervention was significantly correlated to a decrease in cyclin D1, a cell cycle regulatory protein, in patient tumor tissues (P’s≤0.05). Here, oral administration of limonene resulted in significant changes in several metabolic pathways. Further, pathway-based changes were related to the change in tissue level cyclin D1 expression. Future controlled clinical trials with limonene are necessary to determine the potential role and mechanisms of limonene in the breast cancer prevention setting. PMID:25388013

The application of a novel high-resolution mass spectrometry-based analytical strategy to rapid metabolite profiling of a dual drug combination in humans.

PubMed

Xing, Jie; Zang, Meitong; Liu, Huixiang

2017-11-15

Metabolite profiling of combination drugs in complex matrix is a big challenge. Development of an effective data mining technique for simultaneously extracting metabolites of one parent drug from both background matrix and combined drug-related signals could be a solution. This study presented a novel high resolution mass spectrometry (HRMS)-based data-mining strategy to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was verapamil-irbesartan (VER-IRB), which is widely used in clinic to treat hypertension. First, mass defect filter (MDF), as a targeted data mining tool, worked effectively except for those metabolites with similar MDF values. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, was able to recover all relevant metabolites of VER-IRB from the full-scan MS dataset except for trace metabolites buried in the background noise and/or combined drug-related signals. Third, the novel ring double bond (RDB; valence values of elements in structure) filter, could show rich structural information in more sensitive full-scan MS chromatograms; however, it had a low capability to remove background noise and was difficult to differentiate the metabolites with RDB coverage. Fourth, an integrated strategy, i.e., untargeted BS followed by RDB, was effective for metabolite identification of VER and IRB, which have different RDB values. Majority of matrix signals were firstly removed using BS. Metabolite ions for each parent drug were then isolated from remaining background matrix and combined drug-related signals by imposing of preset RDB values/ranges around the parent drug and selected core substructures. In parallel, MDF was used to recover potential metabolites with similar RDB. As a result, a total of 74 metabolites were found for VER-IRB in human plasma and urine, among which ten metabolites have not been previously reported in human. The results demonstrated that the combination of accurate mass-based multiple data-mining techniques, i.e., untargeted background subtraction followed by ring double bond filtering in parallel with targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drug. Copyright © 2017 Elsevier B.V. All rights reserved.

Sensitivity and proportionality assessment of metabolites from microdose to high dose in rats using LC-MS/MS.

PubMed

Ni, Jinsong; Ouyang, Hui; Seto, Carmai; Sakuma, Takeo; Ellis, Robert; Rowe, Josh; Acheampong, Andrew; Welty, Devin; Szekely-Klepser, Gabriella

2010-03-01

The objective of this study was to evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to characterize metabolites in plasma and urine at microdoses in rats and to investigate proportionality of metabolite exposure from a microdose of 1.67 µg/kg to a high dose of 5000 µg/kg for atorvastatin, ofloxacin, omeprazole and tamoxifen. Only the glucuronide metabolite of ofloxacin, the hydroxylation metabolite of omeprazole and the hydration metabolite of tamoxifen were characterized in rat plasma at microdose by LC-MS/MS. The exposure of detected metabolites of omeprazole and tamoxifen appeared to increase in a nonproportional manner with increasing doses. Exposure of ortho- and para-hydroxyatorvastatin, but not atorvastatin and lactone, increased proportionally with increasing doses. LC-MS/MS has demonstrated its usefulness for detecting and characterizing the major metabolites in plasma and urine at microdosing levels in rats. The exposure of metabolites at microdose could not simply be used to predict their exposure at higher doses.

Pharmacokinetics of [14C]Abacavir, a Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase Inhibitor, Administered in a Single Oral Dose to HIV-1-Infected Adults: a Mass Balance Study

PubMed Central

McDowell, James A.; Chittick, Gregory E.; Ravitch, Joshua R.; Polk, Ronald E.; Kerkering, Thomas M.; Stein, Daniel S.

1999-01-01

Abacavir (1592U89) {(−)-(1S, 4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol} is a 2′-deoxyguanosine analogue with potent activity against human immunodeficiency virus (HIV) type 1. To determine the metabolic profile, routes of elimination, and total recovery of abacavir and metabolites in humans, we undertook a phase I mass balance study in which six HIV-infected male volunteers ingested a single 600-mg oral dose of abacavir including 100 μCi of [14C]abacavir. The metabolic disposition of the drug was determined through analyses of whole-blood, plasma, urine, and stool samples, collected for a period of up to 10 days postdosing, and of cerebrospinal fluid (CSF), collected up to 6 h postdosing. The radioactivity from abacavir and its two major metabolites, a 5′-carboxylate (2269W93) and a 5′-glucuronide (361W94), accounted for the majority (92%) of radioactivity detected in plasma. Virtually all of the administered dose of radioactivity (99%) was recovered, with 83% eliminated in urine and 16% eliminated in feces. Of the 83% radioactivity dose eliminated in the urine, 36% was identified as 361W94, 30% was identified as 2269W93, and 1.2% was identified as abacavir; the remaining 15.8% was attributed to numerous trace metabolites, of which <1% of the administered radioactivity was 1144U88, a minor metabolite. The peak concentration of abacavir in CSF ranged from 0.6 to 1.4 μg/ml, which is 8 to 20 times the mean 50% inhibitory concentration for HIV clinical isolates in vitro (0.07 μg/ml). In conclusion, the main route of elimination for oral abacavir in humans is metabolism, with <2% of a dose recovered in urine as unchanged drug. The main route of metabolite excretion is renal, with 83% of a dose recovered in urine. Two major metabolites, the 5′-carboxylate and the 5′-glucuronide, were identified in urine and, combined, accounted for 66% of the dose. Abacavir showed significant penetration into CSF. PMID:10582871

Metabolic profiling of human lung cancer blood plasma using 1H NMR spectroscopy

NASA Astrophysics Data System (ADS)

Kokova, Daria; Dementeva, Natalia; Kotelnikov, Oleg; Ponomaryova, Anastasia; Cherdyntseva, Nadezhda; Kzhyshkowska, Juliya

2017-11-01

Lung cancer (both small cell and non-small cell) is the second most common cancer in both men and women. The article represents results of evaluating of the plasma metabolic profiles of 100 lung cancer patients and 100 controls to investigate significant metabolites using 400 MHz 1H NMR spectrometer. The results of multivariate statistical analysis show that a medium-field NMR spectrometer can obtain the data which are already sufficient for clinical metabolomics.

Quantification of vosaroxin and its metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine using high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Nijenhuis, C M; Lucas, L; Rosing, H; Jamieson, G; Fox, J A; Schellens, J H M; Beijnen, J H

2016-08-01

Vosaroxin is a first-in-class anticancer quinolone derivative topoisomerase II inhibitor that is currently in development in combination with cytarabine for the treatment of acute myeloid leukemia (AML). To investigate vosaroxin pharmacokinetics (PK) in patients, liquid chromatography tandem mass spectrometry (LC-MS/MS) assays to quantify vosaroxin and the two metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine were developed and validated. Immediately after collection the samples were stored at -80°C. Prior to analysis, the plasma samples were subjected to protein precipitation and the urine samples were diluted. For both assays the reconstituted extracts were injected on a Symmetry Shield RP8 column and gradient elution was applied using 0.1% formic acid in water and acetonitrile-methanol (50:50, v/v). Analyses were performed with a triple quadruple mass spectrometer in positive-ion mode. A deuterated isotope of vosaroxin was used as internal standard for the quantification. The validated assays quantify vosaroxin and N-desmethylvosaroxin in the concentration range of 2-500ng/mL in plasma and urine. For O-desmethylvosaroxin the concentration range of 4-500ng/mL in plasma and urine was validated. Dilution integrity experiments show that samples can be diluted 25 fold in control matrix prior to analysis. The expanded concentration range for plasma and urine for vosaroxin and N-desmethylvosaroxin is therefore from 2 to 15,000ng/mL and in plasma for O-desmethylvosaroxin from 4 to 15,000ng/mL. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous determination of metoprolol and its metabolites, α-hydroxymetoprolol and O-desmethylmetoprolol, in human plasma by liquid chromatography with tandem mass spectrometry: Application to the pharmacokinetics of metoprolol associated with CYP2D6 genotypes.

PubMed

Bae, Soo Hyeon; Lee, Joeng Kee; Cho, Doo-Yeoun; Bae, Soo Kyung

2014-06-01

A rapid and simple LC with MS/MS method for the simultaneous determination of metoprolol and its two CYP2D6-derived metabolites, α-hydroxy- and O-desmethylmetoprolol, in human plasma was established. Metoprolol (MET), its two metabolites, and the internal standard chlorpropamide were extracted from plasma (50 μL) using ethyl acetate. Chromatographic separation was performed on a Luna CN column with an isocratic mobile phase consisting of distilled water and methanol containing 0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The total run time was 3.0 min per sample. Mass spectrometric detection was conducted by ESI in positive ion selected-reaction monitoring mode. The linear ranges of concentration for MET, α-hydroxymetoprolol, and O-desmethylmetoprolol were 2-1000, 2-500, and 2-500 ng/mL, respectively, with a lower limit of quantification of 2 ng/mL for all analytes. The coefficient of variation for the assay's precision was ≤ 13.2%, and the accuracy was 89.1-110%. All analytes were stable under various storage and handling conditions and no relevant cross-talk and matrix effect were observed. Finally, this method was successfully applied to assess the influence of CYP2D6 genotypes on the pharmacokinetics of MET after oral administration of 100 mg to healthy Korean volunteers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative metabolomics in vegans and omnivores reveal constraints on diet-dependent gut microbiota metabolite production

PubMed Central

Wu, Gary D; Compher, Charlene; Chen, Eric Z; Smith, Sarah A; Shah, Rachana D; Bittinger, Kyle; Chehoud, Christel; Albenberg, Lindsey G; Nessel, Lisa; Gilroy, Erin; Star, Julie; Weljie, Aalim M; Flint, Harry J; Metz, David C; Bennett, Michael J; Li, Hongzhe; Bushman, Frederic D; Lewis, James D

2015-01-01

Objective The consumption of an agrarian diet is associated with a reduced risk for many diseases associated with a ‘Westernised’ lifestyle. Studies suggest that diet affects the gut microbiota, which subsequently influences the metabolome, thereby connecting diet, microbiota and health. However, the degree to which diet influences the composition of the gut microbiota is controversial. Murine models and studies comparing the gut microbiota in humans residing in agrarian versus Western societies suggest that the influence is large. To separate global environmental influences from dietary influences, we characterised the gut microbiota and the host metabolome of individuals consuming an agrarian diet in Western society. Design and results Using 16S rRNA-tagged sequencing as well as plasma and urinary metabolomic platforms, we compared measures of dietary intake, gut microbiota composition and the plasma metabolome between healthy human vegans and omnivores, sampled in an urban USA environment. Plasma metabolome of vegans differed markedly from omnivores but the gut microbiota was surprisingly similar. Unlike prior studies of individuals living in agrarian societies, higher consumption of fermentable substrate in vegans was not associated with higher levels of faecal short chain fatty acids, a finding confirmed in a 10-day controlled feeding experiment. Similarly, the proportion of vegans capable of producing equol, a soy-based gut microbiota metabolite, was less than that was reported in Asian societies despite the high consumption of soy-based products. Conclusions Evidently, residence in globally distinct societies helps determine the composition of the gut microbiota that, in turn, influences the production of diet-dependent gut microbial metabolites. PMID:25431456

Development and validation of a bioanalytical method for the simultaneous determination of heroin, its main metabolites, naloxone and naltrexone by LC-MS/MS in human plasma samples: Application to a clinical trial of oral administration of a heroin/naloxone formulation.

PubMed

Moreno-Vicente, Raquel; Fernández-Nieva, Zuriñe; Navarro, Arantza; Gascón-Crespí, Irene; Farré-Albaladejo, Magí; Igartua, Manuela; Hernández, Rosa María; Pedraz, José Luis

2015-10-10

A bioanalytical method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification of heroin, its main metabolites and naloxone. In addition, naltrexone was detected qualitatively. This method was used to analyse human plasma samples from a clinical trial after oral administration of a heroin/naloxone formulation in healthy volunteers. O-methylcodeine was used as an internal standard. Samples were kept in an ice-bath during their processing to minimize the degradation of heroin. A short methodology based on protein precipitation with methanol was used for sample preparation. After protein precipitation, only the addition of a formic acid solution was needed to elute heroin, 6-monoacetylmorphine, morphine, naloxone and naltrexone. Morphine metabolites were evaporated to dryness and reconstituted in a formic acid solution. Chromatographic separation was achieved at 35 °C on an X-Bridge Phenyl column (150 × 4.6 mm, 5 μm) using a gradient elution with a mobile phase of ammonium formate buffer at pH 3.0 and formic acid in acetonitrile. The run time was 8 min. The analytes were monitored using a triple quadrupole mass spectrometer with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The method was found to be linear in a concentration range of 10-2000 ng/mL for M3G and 10-1000 ng/mL for the rest of compounds. Quality controls showed accurate values between -3.6% and 4.0% and intra- and inter-day precisions were below 11.5% for all analytes. The overall recoveries were approximately 100% for all analytes including the internal standard. A rapid, specific, precise and simple method was developed for the determination of heroin, its metabolites, naloxone and naltrexone in human plasma. This method was successfully applied to a clinical trial in 12 healthy volunteers. Copyright © 2015 Elsevier B.V. All rights reserved.

Liquid chromatography-electrospray mass spectrometry determination of carbamazepine, oxcarbazepine and eight of their metabolites in human plasma.

PubMed

Breton, Hélène; Cociglio, Marylène; Bressolle, Françoise; Peyriere, Hélène; Blayac, Jean Pierre; Hillaire-Buys, Dominique

2005-12-15

Carbamazepine (CBZ) and oxcarbazepine (OXCBZ) are both antiepileptic drugs, which are prescribed as first-line drugs for the treatment of partial and generalized tonic-clonic epileptic seizures. In this paper, a specific and sensitive liquid chromatography-electrospray ionization mass spectrometry method was described for the simultaneous determination of carbamazepine (CBZ), oxcarbazepine (OXCBZ) and eight of their metabolites [CBZ-10,11-epoxide (CBZ-EP), 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (DiOH-CBZ), 10-hydroxy-10,11-dihydroCBZ (10-OH-CBZ), 2-hydroxycarbamazepine (2-OH-CBZ), 3-hydroxycarbamazepine (3-OH-CBZ), iminostilbene (IM), acridone (AO) and acridine (AI)] in human plasma. The work-up procedure involved a simple precipitation with acetone. Separation of the analytes was achieved within 50 min using a Zorbax eclipse XD8 C8 analytical column. The mobile phase consisted of a mixture of acetonitrile-formate buffer (2 mM, pH 3). Detection was performed using a quadrupole mass spectrometer fitted with an electrospray ion source. Mass spectrometric data were acquired in single ion recording mode at m/z 237 for CBZ, m/z 180 for CBZ-EP and AI, m/z 236 for OXCBZ, m/z 237 for 10-OH-CBZ, m/z 253 for 2-OH-CBZ, 3-OH-CBZ and DiOH-CBZ, m/z 196 for AO and m/z 194 for IM. For all analytes, the drug/internal standard peak height ratios were linked via a quadratic relationship to plasma concentrations. The extraction recovery averaged 90% for CBZ, 80% for OXCBZ and was 80-105% for the metabolites. The lower limit of quantitation was 0.5mg/l for CBZ, 0.4 mg/l for OXCBZ and ranged from 0.02 to 0.3 mg/l for the metabolites. Precision ranged from 2 to 13% and accuracy was between 86 and 112%. This method was found suitable for the analysis of plasma samples collected during therapeutic drug monitoring of patients treated with CBZ or OXCBZ.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

PubMed

Li, Rui; Bi, Yi-An; Vildhede, Anna; Scialis, Renato J; Mathialagan, Sumathy; Yang, Xin; Marroquin, Lisa D; Lin, Jian; Varma, Manthena V S

2017-07-01

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC 50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers.

PubMed

Vinnakota, Chakravarthy V; Peetha, Naga S; Perrizo, Mitch G; Ferris, David G; Oda, Robert P; Rockwood, Gary A; Logue, Brian A

2012-11-01

Cyanide is highly toxic and is present in many foods, combustion products (e.g. cigarette smoke), industrial processes, and has been used as a terrorist weapon. In this study, cyanide and its major metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), were analyzed from various human biofluids of smokers (low-level chronic cyanide exposure group) and non-smokers to gain insight into the relationship of these biomarkers to cyanide exposure. The concentrations of each biomarker tested were elevated for smokers in each biofluid. Significant differences (p < 0.05) were found for thiocyanate in plasma and urine, and ATCA showed significant differences in plasma and saliva. Additionally, biomarker concentration ratios, correlations between markers of cyanide exposure, and other statistical methods were performed to better understand the relationship between cyanide and its metabolites. Of the markers studied, the results indicate plasma ATCA, in particular, showed excellent promise as a biomarker for chronic low-level cyanide exposure.

Piceatannol and Its Metabolite, Isorhapontigenin, Induce SIRT1 Expression in THP-1 Human Monocytic Cell Line

PubMed Central

Kawakami, Shinpei; Kinoshita, Yosuke; Maruki-Uchida, Hiroko; Yanae, Koji; Sai, Masahiko; Ito, Tatsuhiko

2014-01-01

Piceatannol is a phytochemical that is present in large amounts in passion fruit (Passiflora edulis) seeds, and is an analog of resveratrol. Recently, the absorption and metabolism of piceatannol were investigated in rats, and isorhapontigenin, O-methyl piceatannol, was detected as a piceatannol metabolite in rat plasma. To elucidate the function of piceatannol and its metabolites, we investigated the expression of sirtuin 1 (SIRT1) in THP-1 monocytic cells after treatment with piceatannol and its metabolites, and compared their effects with those of resveratrol and its metabolites. Piceatannol and resveratrol upregulated the expression levels of SIRT1 mRNA and SIRT1 protein. An extract of passion fruit seeds, which contained high levels of piceatannol, also upregulated SIRT1 mRNA expression. As for the metabolites, isorhapontigenin upregulated SIRT1 mRNA expression, whereas resveratrol glucuronides and sulfate did not affect SIRT1 expression. These findings indicate that after intake of piceatannol, not only piceatannol itself, but also its metabolite, isorhapontigenin, contributed to the upregulation of SIRT1 expression. PMID:25360511

Quantification of Acetaminophen and Its Metabolites in Plasma Using UPLC-MS: Doors Open to Therapeutic Drug Monitoring in Special Patient Populations.

PubMed

Flint, Robert B; Mian, Paola; van der Nagel, Bart; Slijkhuis, Nuria; Koch, Birgit C P

2017-04-01

Acetaminophen (APAP, paracetamol) is the most commonly used drug for pain and fever in both the United States and Europe and is considered safe when used at registered dosages. Nevertheless, differences between specific populations lead to remarkable changes in exposure to potentially toxic metabolites. Furthermore, extended knowledge is required on metabolite formation after intoxication, to optimize antidote treatment. Therefore, the authors aimed to develop and validate a quick and easy analytical method for simultaneous quantification of APAP, APAP-glucuronide, APAP-sulfate, APAP-cysteine, APAP-glutathione, APAP-mercapturate, and protein-derived APAP-cysteine in human plasma by ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry. The internal standard was APAP-D4 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity ultraperformance liquid chromatography HSS T3 column with a runtime of only 4.5 minutes per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate, formic acid in Milli-Q ultrapure water or in methanol at flow rate of 0.4 mL/minute. A plasma volume of only 10 μL was required to achieve both adequate accuracy and precision. Calibration curves of all 6 analytes were linear. All analytes were stable for at least 48 hours in the autosampler; the high quality control of APAP-glutathione was stable for 24 hours. The method was validated according to the U.S. Food and Drug Administration guidelines. This method allows quantification of APAP and 6 metabolites, which serves purposes for research, as well as therapeutic drug monitoring. The advantage of this method is the combination of minimal injection volume, a short runtime, an easy sample preparation method, and the ability to quantify APAP and all 6 metabolites.

High-performance metabolic profiling of plasma from seven mammalian species for simultaneous environmental chemical surveillance and bioeffect monitoring.

PubMed

Park, Youngja H; Lee, Kichun; Soltow, Quinlyn A; Strobel, Frederick H; Brigham, Kenneth L; Parker, Richard E; Wilson, Mark E; Sutliff, Roy L; Mansfield, Keith G; Wachtman, Lynn M; Ziegler, Thomas R; Jones, Dean P

2012-05-16

High-performance metabolic profiling (HPMP) by Fourier-transform mass spectrometry coupled to liquid chromatography gives relative quantification of thousands of chemicals in biologic samples but has had little development for use in toxicology research. In principle, the approach could be useful to detect complex metabolic response patterns to toxicologic exposures and to detect unusual abundances or patterns of potentially toxic chemicals. As an initial study to develop these possible uses, we applied HPMP and bioinformatics analysis to plasma of humans, rhesus macaques, marmosets, pigs, sheep, rats and mice to determine: (1) whether more chemicals are detected in humans living in a less controlled environment than captive species and (2) whether a subset of plasma chemicals with similar inter-species and intra-species variation could be identified for use in comparative toxicology. Results show that the number of chemicals detected was similar in humans (3221) and other species (range 2537-3373). Metabolite patterns were most similar within species and separated samples according to family and order. A total of 1485 chemicals were common to all species; 37% of these matched chemicals in human metabolomic databases and included chemicals in 137 out of 146 human metabolic pathways. Probability-based modularity clustering separated 644 chemicals, including many endogenous metabolites, with inter-species variation similar to intra-species variation. The remaining chemicals had greater inter-species variation and included environmental chemicals as well as GSH and methionine. Together, the data suggest that HPMP provides a platform that can be useful within human populations and controlled animal studies to simultaneously evaluate environmental exposures and biological responses to such exposures. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Determination of D- and L-pipecolic acid in food samples including processed foods.

PubMed

Fujita, Toru; Fujita, Manabu; Kodama, Taku; Hada, Toshikazu; Higashino, Kazuya

2003-01-01

Pipecolic acid, a metabolite of lysine, is found in human physiological fluids and is thought to play an important role in the central inhibitory gamma-aminobutyric acid system. However, it is unclear whether plasma D- and L-pipecolic acid originate from oral food intake or intestinal bacterial metabolites. We analyzed the contents of D- and L-pipecolic acid in several processed foods including dairy products (cow's milk, cheese and yogurt), fermented beverages (beer and wine) and heated samples (beef, bovine liver, bread and tofu) to clarify the relationship between plasma D- and L-pipecolic acid and dietary foods. Our study revealed that some of the samples contained high concentrations of total pipecolic acid, and a higher proportion of L- than D-isomers. The other samples also showed high proportions of L-pipecolic acid. It was also shown that there is no significant change in the ratio of the D-isomer before and after heat treatment. The heat treatments could not cause the racemization of pipecolic acid in this study. These findings suggest that plasma pipecolic acid, particularly the D-isomer, does not originate from direct food intake and that D- and L-pipecolic acid can possibly be derived from intestinal bacterial metabolites. Copyright 2003 S. Karger AG, Basel

Highly sensitive quantification for human plasma-targeted metabolomics using an amine derivatization reagent.

PubMed

Arashida, Naoko; Nishimoto, Rumi; Harada, Masashi; Shimbo, Kazutaka; Yamada, Naoyuki

2017-02-15

Amino acids and their related metabolites play important roles in various physiological processes and have consequently become biomarkers for diseases. However, accurate quantification methods have only been established for major compounds, such as amino acids and a limited number of target metabolites. We previously reported a highly sensitive high-throughput method for the simultaneous quantification of amines using 3-aminopyridyl-N-succinimidyl carbamate as a derivatization reagent combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Herein, we report the successful development of a practical and accurate LC-MS/MS method to analyze low concentrations of 40 physiological amines in 19 min. Thirty-five of these amines showed good linearity, limits of quantification, accuracy, precision, and recovery characteristics in plasma, with scheduled selected reaction monitoring acquisitions. Plasma samples from 10 healthy volunteers were evaluated using our newly developed method. The results revealed that 27 amines were detected in one of the samples, and that 24 of these compounds could be quantified. Notably, this new method successfully quantified metabolites with high accuracy across three orders of magnitude, with lowest and highest averaged concentrations of 31.7 nM (for spermine) and 18.3 μM (for α-aminobutyric acid), respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of the cytoprotective agent WR-2721 (Amifostine, Ethyol) and its metabolites in human blood using monobromobimane fluorescent labeling and high-performance liquid chromatography.

PubMed

Souid, A K; Newton, G L; Dubowy, R L; Fahey, R C; Bernstein, M L

1998-01-01

WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] is a chemoprotective agent that is currently in pediatric clinical trials. It is a prodrug that is dephosphorylated by alkaline phosphatase to the active free thiol form, WR-1065 [S-2-(3-aminopropylamino)ethanethiol]. It is likely that adequate and sustained cellular levels of the drug are necessary for optimum cytoprotection. To date, a method to measure both plasma and cellular levels of WR-2721 and its metabolites in clinical samples has not been available. In the study reported here the monobromobimane (mBBr) fluorescent labeling method was used to measure these levels when drug was added in vitro to blood samples from normal volunteers. In addition, we present pharmacokinetic data from a pediatric patient receiving WR-2721 (825 mg/m2 x 2). The results can be summarized as follows: (1) WR-2721 was detected in the patient's plasma with a half-life of about 10 min; (2) the WR-1065 concentration in the blood cellular fraction was similar to that of plasma; (3) both WR-1065 and WR-SS-low molecular weight (WR-SS-LMW) metabolites disappeared from plasma and the cellular fraction by 3.6 h after WR-2721 infusion; (4) a large proportion of WR-1065 was oxidized in plasma to WR-SS protein and WR-SS-LMW; (5) a large proportion of WR-1065 in the cellular fraction was oxidized to WR-SS-protein; (6) the WR-SS-LMW concentration in the cellular fraction was low; and (7) saturation of plasma and cellular protein binding sites was possible. The pharmacokinetic data that were generated with this technique could guide clinical trials using WR-2721.

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme.

PubMed

Wang, L H; Ahmad, S; Benter, I F; Chow, A; Mizutani, S; Ward, P E

1991-01-01

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (less than 1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15-5.91 nmol/min/ml), which sequentially converted SP to SP(3-11) and SP(5-11). In turn, the SP(5-11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2-25.5 nmol/min/ml). The Km values of SP for DAP IV and of SP(5-11) for AmM ranged from 32.7 to 123 microM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76-10.8 nmol/min/ml; Km = 90.7 microM). These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

PubMed Central

Zharikova, Olga L.; Fokina, Valentina M.; Nanovskaya, Tatiana N.; Hill, Ronald A.; Mattison, Donald R.; Hankins, Gary D.V.; Ahmed, Mahmoud S.

2014-01-01

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans- (M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a( 4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo. PMID:19679108

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide.

PubMed

Zharikova, Olga L; Fokina, Valentina M; Nanovskaya, Tatiana N; Hill, Ronald A; Mattison, Donald R; Hankins, Gary D V; Ahmed, Mahmoud S

2009-12-15

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

Metabolism and Disposition of a Novel B-Cell Lymphoma-2 Inhibitor Venetoclax in Humans and Characterization of Its Unusual Metabolites.

PubMed

Liu, Hong; Michmerhuizen, Melissa J; Lao, Yanbin; Wan, Katty; Salem, Ahmed Hamed; Sawicki, James; Serby, Michael; Vaidyanathan, Srirajan; Wong, Shekman L; Agarwal, Suresh; Dunbar, Martin; Sydor, Jens; de Morais, Sonia M; Lee, Anthony J

2017-03-01

Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, and excretion of venetoclax in humans. After a single oral dose of [ 14 C]venetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 [2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide] (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-[(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino[2,1-b][1,3]benzoxazin-2-yl]-N-[3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Integrative Metabolic Signatures for Hepatic Radiation Injury

PubMed Central

Su, Gang; Meng, Fan; Liu, Laibin; Mohney, Robert; Kulkarni, Shilpa; Guha, Chandan

2015-01-01

Background Radiation-induced liver disease (RILD) is a dose-limiting factor in curative radiation therapy (RT) for liver cancers, making early detection of radiation-associated liver injury absolutely essential for medical intervention. A metabolomic approach was used to determine metabolic signatures that could serve as biomarkers for early detection of RILD in mice. Methods Anesthetized C57BL/6 mice received 0, 10 or 50 Gy Whole Liver Irradiation (WLI) and were contrasted to mice, which received 10 Gy whole body irradiation (WBI). Liver and plasma samples were collected at 24 hours after irradiation. The samples were processed using Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry. Results Twenty four hours after WLI, 407 metabolites were detected in liver samples while 347 metabolites were detected in plasma. Plasma metabolites associated with 50 Gy WLI included several amino acids, purine and pyrimidine metabolites, microbial metabolites, and most prominently bradykinin and 3-indoxyl-sulfate. Liver metabolites associated with 50 Gy WLI included pentose phosphate, purine, and pyrimidine metabolites in liver. Plasma biomarkers in common between WLI and WBI were enriched in microbial metabolites such as 3 indoxyl sulfate, indole-3-lactic acid, phenyllactic acid, pipecolic acid, hippuric acid, and markers of DNA damage such as 2-deoxyuridine. Metabolites associated with tryptophan and indoles may reflect radiation-induced gut microbiome effects. Predominant liver biomarkers in common between WBI and WLI were amino acids, sugars, TCA metabolites (fumarate), fatty acids (lineolate, n-hexadecanoic acid) and DNA damage markers (uridine). Conclusions We identified a set of metabolomic markers that may prove useful as plasma biomarkers of RILD and WBI. Pathway analysis also suggested that the unique metabolic changes observed after liver irradiation was an integrative response of the intestine, liver and kidney. PMID:26046990

HPLC-ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma.

PubMed

Mistri, Hiren N; Jangid, Arvind G; Pudage, Ashutosh; Shrivastav, Pranav

2008-03-15

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.

Simultaneous determination of chloroquine and its three metabolites in human plasma, whole blood and urine by ion-pair high-performance liquid chromatography.

PubMed

Houzé, P; de Reynies, A; Baud, F J; Benatar, M F; Pays, M

1992-02-14

A method was developed for the separation and measurement of chloroquine and three metabolites (desethylchloroquine, bisdesethylchloroquine and 4-amino-7-chloroquinoline) in biological samples by ion-pair high-performance liquid chromatography with UV detection. The method uses 2,3-diaminoaphthalene as an internal standard and provides a limit of detection between 1 and 2 ng/ml for chloroquine and its metabolites. The assay was linear in the range 12.5-250 ng/ml and the analytical recovery and reproducibility were sufficient. The assay was applied to the analysis of biological samples from a patient undergoing chloroquine chemoprophylaxis and a patient who had ingested chloroquine in a suicide attempt.

Intestinal microbiota-derived metabolomic blood plasma markers for prior radiation injury.

PubMed

Ó Broin, Pilib; Vaitheesvaran, Bhavapriya; Saha, Subhrajit; Hartil, Kirsten; Chen, Emily I; Goldman, Devorah; Fleming, William Harv; Kurland, Irwin J; Guha, Chandan; Golden, Aaron

2015-02-01

Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative, untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of the concurrent metabolic changes in brain and plasma during insulin-induced moderate hypoglycemia using 1H NMR spectroscopy in juvenile rats.

PubMed

Ennis, Kathleen; Lusczek, Elizabeth; Rao, Raghavendra

2017-07-13

Treatment of hypoglycemia in children is currently based on plasma glucose measurements. This approach may not ensure neuroprotection since plasma glucose does not reflect the dynamic state of cerebral energy metabolism. To determine whether cerebral metabolic changes during hypoglycemia could be better characterized using plasma metabolomic analysis, insulin-induced acute hypoglycemia was induced in 4-week-old rats. Brain tissue and concurrent plasma samples were collected from hypoglycemic (N=7) and control (N=7) rats after focused microwave fixation to prevent post-mortem metabolic changes. The concentration of 29 metabolites in brain and 34 metabolites in plasma were determined using 1 H NMR spectroscopy at 700MHz and examined using partial least squares-discriminant analysis. The sensitivity of plasma glucose for detecting cerebral energy failure was assessed by determining its relationship to brain phosphocreatine. The brain and plasma metabolite profiles of the hypoglycemia group were distinct from the control group (brain: R 2 =0.92, Q 2 =0.31; plasma: R 2 =0.95, Q 2 =0.74). Concentration differences in glucose, ketone bodies and amino acids were responsible for the intergroup separation. There was 45% concordance between the brain and plasma metabolite profiles. Brain phosphocreatine correlated with brain glucose (control group: R 2 =0.86; hypoglycemia group: R 2 =0.59; p<0.05), but not with plasma glucose. The results confirm that plasma glucose is an insensitive biomarker of cerebral energy changes during hypoglycemia and suggest that a plasma metabolite profile is superior for monitoring cerebral metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Three Novel Ring Expansion Metabolites of KAE609, a New Spiroindolone Agent for the Treatment of Malaria, in Rats, Dogs, and Humans.

PubMed

Huskey, Su-Er W; Zhu, Chun-qi; Lin, Melissa M; Forseth, Ry R; Gu, Helen; Simon, Oliver; Eggimann, Fabian K; Kittelmann, Matthias; Luneau, Alexandre; Vargas, Alexandra; Li, Hongmei; Wang, Lai; Einolf, Heidi J; Zhang, Jin; Favara, Sarah; He, Handan; Mangold, James B

2016-05-01

KAE609 [(1'R,3'S)-5,7'-dichloro-6'-fluoro-3'-methyl-2',3',4',9'-tetrahydrospiro[indoline-3,1'-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent being developed for the treatment of malaria. After oral dosing of KAE609 to rats and dogs, the major radioactive component in plasma was KAE609. An oxidative metabolite, M18, was the prominent metabolite in rat and dog plasma. KAE609 was well absorbed and extensively metabolized such that low levels of parent compound (≤11% of the dose) were detected in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways (≥93% of the dose) in the feces of rats and dogs. M37 and M23 were the major metabolites in rat and dog feces, respectively. Among the prominent metabolites of KAE609, the isobaric chemical species, M37, was observed, suggesting the involvement of an isomerization or rearrangement during biotransformation. Subsequent structural elucidation of M37 revealed that KAE609, a spiroindolone, undergoes an unusual C-C bond cleavage, followed by a 1,2-acyl shift to form a ring expansion metabolite M37. The in vitro metabolism of KAE609 in hepatocytes was investigated to understand this novel biotransformation. The metabolism of KAE609 was qualitatively similar across the species studied; thus, further investigation was conducted using human recombinant cytochrome P450 enzymes. The ring expansion reaction was found to be primarily catalyzed by cytochrome P450 (CYP) 3A4 yielding M37. M37 was subsequently oxidized to M18 by CYP3A4 and hydroxylated to M23 primarily by CYP1A2. Interestingly, M37 was colorless, whereas M18 and M23 showed orange yellow color. The source of the color of M18 and M23 was attributed to their extended conjugated system of double bonds in the structures. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Promising Metabolite Profiles in the Plasma and CSF of Early Clinical Parkinson's Disease

PubMed Central

Stoessel, Daniel; Schulte, Claudia; Teixeira dos Santos, Marcia C.; Scheller, Dieter; Rebollo-Mesa, Irene; Deuschle, Christian; Walther, Dirk; Schauer, Nicolas; Berg, Daniela; Nogueira da Costa, Andre; Maetzler, Walter

2018-01-01

Parkinson's disease (PD) shows high heterogeneity with regard to the underlying molecular pathogenesis involving multiple pathways and mechanisms. Diagnosis is still challenging and rests entirely on clinical features. Thus, there is an urgent need for robust diagnostic biofluid markers. Untargeted metabolomics allows establishing low-molecular compound biomarkers in a wide range of complex diseases by the measurement of various molecular classes in biofluids such as blood plasma, serum, and cerebrospinal fluid (CSF). Here, we applied untargeted high-resolution mass spectrometry to determine plasma and CSF metabolite profiles. We semiquantitatively determined small-molecule levels (≤1.5 kDa) in the plasma and CSF from early PD patients (disease duration 0–4 years; n = 80 and 40, respectively), and sex- and age-matched controls (n = 76 and 38, respectively). We performed statistical analyses utilizing partial least square and random forest analysis with a 70/30 training and testing split approach, leading to the identification of 20 promising plasma and 14 CSF metabolites. These metabolites differentiated the test set with an AUC of 0.8 (plasma) and 0.9 (CSF). Characteristics of the metabolites indicate perturbations in the glycerophospholipid, sphingolipid, and amino acid metabolism in PD, which underscores the high power of metabolomic approaches. Further studies will enable to develop a potential metabolite-based biomarker panel specific for PD. PMID:29556190

Lymphatic metabolites of quercetin after intestinal administration of quercetin-3-glucoside and its aglycone in rats.

PubMed

Nakamura, Toshiyuki; Kinjo, Chinatsu; Nakamura, Yoshimasa; Kato, Yoji; Nishikawa, Miyu; Hamada, Masahiro; Nakajima, Noriyuki; Ikushiro, Shinichi; Murota, Kaeko

2018-05-01

Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of the intestinal metabolites like quercetin monoglucuronides were transported directly into the lymph and the hepatic metabolites like the diglucuronides were eventually transferred from the plasma into the lymph. These results indicate that the absorbed Q3G is partly transported into the intestinal lymph as quercetin metabolites. Deglycosylation in the enterocyte is also suggested to affect the subsequent metabolic pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

A highly sensitive LC-MS/MS method for simultaneous determination of glycyrrhizin and its active metabolite glycyrrhetinic acid: Application to a human pharmacokinetic study after oral administration.

PubMed

Suzuki, Tsuneharu; Tsukahara, Michiko; Akasaka, Yuko; Inoue, Hideo

2017-12-01

A highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of glycyrrhizin (GL) and its active metabolite, glycyrrhetinic acid (GA), from human plasma was validated and applied to a human pharmacokinetic study. The analytes were extracted from human plasma using an Oasis MAX cartridge and chromatographic separation was performed on an Inertsil ODS-3 column. The detection was performed using an API 4000 mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m/z 823 → 453 for GL and m/z 471 → 149 for GA were obtained. The response was a linear function of concentration over the ranges of 0.5-200 ng/mL for GL and 2-800 ng/mL for GA (both R 2  > 0.998). Using this method, the pharmacokinetics of GL after single oral administration of a clinical dose (75 mg) to six healthy male Japanese volunteers were evaluated. GL was detected in the plasma of all subjects and the average peak concentration was 24.8 ± 12.0 ng/mL. In contrast, peak concentration of GA was 200.3 ± 60.3 ng/mL, i.e. ~8-fold higher than that of GL. This is the first report clarifying pharmacokinetic profiles of GL and GA simultaneously at a therapeutic oral dose of a GL preparation. Copyright © 2017 John Wiley & Sons, Ltd.

High-sensitive LC-MS/MS method for the simultaneous determination of mirodenafil and its major metabolite, SK-3541, in human plasma: application to microdose clinical trials of mirodenafil.

PubMed

Cho, Doo-Yeoun; Bae, Soo Hyeon; Shon, Ji-Hong; Bae, Soo Kyung

2013-03-01

A high-sensitivity LC/MS/MS method was developed and validated for the simultaneous determination of mirodenafil and its major metabolite, SK-3541, in human plasma. Mirodenafil, SK-3541, and udenafil as an internal standard were extracted from plasma samples with methyl tert-butyl ether. Chromatographic separation was performed on a Luna phenyl-hexyl column (100 × 2.0 mm) with an isocratic mobile phase consisting of 5 mM ammonium formate and ACN (23:77, v/v) at a flow rate of 0.35 mL/min. Detection and quantification were performed using a mass spectrometer in selected reaction monitoring mode with positive ESI at m/z 532.3 → 296.1 for mirodenafil, m/z 488.1 → 296.1 for SK-3541, and m/z 517.3 → 283.2 for udenafil. The calibration curves were linear over a concentration range of 2-500 pg/mL using 0.5 mL plasma for the microdose of mirodenafil (100 μg). Analytical method validation of the clinical dose (100 mg), with a calibration curve range of 2-500 ng/mL using 0.025-mL plasma, was also conducted. The other LC-MS/MS conditions were similar to those used for the microdosing. Each method was applied successfully to pharmacokinetic studies after a microdose or clinical dose of mirodenafil to six healthy Korean male volunteers. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma free versus deconjugated metanephrines for diagnosis of phaeochromocytoma

PubMed Central

Pamporaki, Christina; Därr, Roland; Bursztyn, Michael; Stephan, Glöckner; Bornstein, Stefan R.; Lenders, Jacques W.M.; Pacak, Karel; Krinner, Axel; Eisenhofer, Graeme

2013-01-01

Summary Background Diagnosis of phaeochromocytoma is commonly performed by measurements of plasma free normetanephrine and metanephrine. Plasma deconjugated normetanephrine and metanephrine have been proposed as alternative equivalent, but easier to measure biomarkers. Objective The aim of this study was to compare the diagnostic performances of plasma free versus deconjugated normetanephrine and metanephrine in patients tested for phaeochromocytoma. Methods The study population included a reference group of 262 normotensive and hypertensive volunteers, 198 patients with phaeochromocytoma and 528 patients initially suspected of having the tumour, but with negative investigations after at least 2 years of follow up. Measurements were performed using liquid chromatography with electrochemical detection. Results Median plasma concentrations of free normetanephrine were 17-fold higher in patients with phaeochromocytoma than in the reference population, a 72% larger (p<0.001) difference than that for the 10-fold higher levels of plasma deconjugated normetanephrine. In contrast, relative increases of plasma concentrations of free and deconjugated metanephrine were similar. Using upper cut-offs established in the reference population, measurements of plasma free metabolites provided superior diagnostic performance than deconjugated metabolites according to measures of both sensitivity (97% vs 92%, p=0.002) and specificity (93 vs 89%, p=0.012). The area under the receiver operating characteristic curve for the free metabolites was larger than that for the deconjugated metabolites (0.986 vs 0.965, p<0.001). Conclusion Measurements of plasma free normetanephrine and metanephrine are superior to the deconjugated metabolites for diagnosis of phaeochromocytoma. PMID:23461656

Liquid chromatography/mass spectrometry-based plasma metabolic profiling study of escitalopram in subjects with major depressive disorder.

PubMed

Bandu, Raju; Lee, Hyun Jeong; Lee, Hyeong Min; Ha, Tae Hyon; Lee, Heon-Jeong; Kim, Se Joo; Ha, Kyooseob; Kim, Kwang Pyo

2018-05-01

Liquid chromatography-mass spectrometry (LC-MS) method revealed the plasma metabolite profiles in major depressive disorder patients treated with escitalopram (ECTP) (n = 7). Depression severity was assessed according to the 17-item Hamilton Depression Rating Scale. Metabolic profiles were derived from major depressive disorder subject blood samples collected after ECTP treatment. Blood plasma was separated and processed in order to effectively extract metabolites, which were then analyzed using LC-MS. We identified 19 metabolites and elucidated their structures using LC-tandem MS (LC-MS/MS) combined with elemental compositions derived from accurate mass measurements. We further used online H/D exchange experiments to verify the structural elucidations of each metabolite. Identifying molecular metabolites may provide critical insights into the pharmacological and clinical effects of ECTP treatment and may also provide useful information informing the development of new antidepressant treatments. These detailed plasma metabolite analyses may also be used to identify optimal dose concentrations in psychopharmacotherapeutic treatment through drug monitoring, as well as forming the basis for response predictions in depressed subjects. Copyright © 2018 John Wiley & Sons, Ltd.

Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling.

PubMed

Suarez-Diez, Maria; Adam, Jonathan; Adamski, Jerzy; Chasapi, Styliani A; Luchinat, Claudio; Peters, Annette; Prehn, Cornelia; Santucci, Claudio; Spyridonidis, Alexandros; Spyroulias, Georgios A; Tenori, Leonardo; Wang-Sattler, Rui; Saccenti, Edoardo

2017-07-07

Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA-MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids.

Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling

PubMed Central

2017-01-01

Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA–MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids. PMID:28517934

Comprehensive metabolic profiling of chronic low-grade inflammation among generally healthy individuals.

PubMed

Pietzner, Maik; Kaul, Anne; Henning, Ann-Kristin; Kastenmüller, Gabi; Artati, Anna; Lerch, Markus M; Adamski, Jerzy; Nauck, Matthias; Friedrich, Nele

2017-11-30

Inflammation occurs as an immediate protective response of the immune system to a harmful stimulus, whether locally confined or systemic. In contrast, a persisting, i.e., chronic, inflammatory state, even at a low-grade, is a well-known risk factor in the development of common diseases like diabetes or atherosclerosis. In clinical practice, laboratory markers like high-sensitivity C-reactive protein (hsCRP), white blood cell count (WBC), and fibrinogen, are used to reveal inflammatory processes. In order to gain a deeper insight regarding inflammation-related changes in metabolism, the present study assessed the metabolic patterns associated with alterations in inflammatory markers. Based on mass spectrometry and nuclear magnetic resonance spectroscopy we determined a comprehensive panel of 613 plasma and 587 urine metabolites among 925 apparently healthy individuals. Associations between inflammatory markers, namely hsCRP, WBC, and fibrinogen, and metabolite levels were tested by linear regression analyses controlling for common confounders. Additionally, we tested for a discriminative signature of an advanced inflammatory state using random forest analysis. HsCRP, WBC, and fibrinogen were significantly associated with 71, 20, and 19 plasma and 22, 3, and 16 urine metabolites, respectively. Identified metabolites were related to the bradykinin system, involved in oxidative stress (e.g., glutamine or pipecolate) or linked to the urea cycle (e.g., ornithine or citrulline). In particular, urine 3'-sialyllactose was found as a novel metabolite related to inflammation. Prediction of an advanced inflammatory state based solely on 10 metabolites was well feasible (median AUC: 0.83). Comprehensive metabolic profiling confirmed the far-reaching impact of inflammatory processes on human metabolism. The identified metabolites included not only those already described as immune-modulatory but also completely novel patterns. Moreover, the observed alterations provide molecular links to inflammation-associated diseases like diabetes or cardiovascular disorders.

Evaluation of an on-capillary copper complexation methodology for the investigation of in vitro metabolism of dynorphin A 1–17

PubMed Central

Kuhnline, Courtney D.; Lunte, Susan M.

2013-01-01

Dynorphin A 1–17 is an endogenous neuropeptide implicated in a variety of neurological disorders including Alzheimer’s and Parkinson’s diseases and neuropathic pain. Metabolites of this peptide can exhibit their own unique effects in vivo, and it is possible that one of these metabolites is responsible for the neurotoxicity. In this article, the use of CE for the separation of dynorphin A 1–17 from four of its metabolites is described. Buffer additives were investigated to eliminate peptide adsorption to the capillary wall and to improve resolution between closely related metabolites. On-capillary copper complexation was employed and was shown to improve separation efficiency as compared with the separation of native peptides. The method was then applied to in vitro dynorphin metabolism in human plasma as well as rat brain and rat spinal cord slices. PMID:20658491

Investigation of Metabolomic Blood Biomarkers for Detection of Adenocarcinoma Lung Cancer

PubMed Central

Fahrmann, Johannes F.; Kim, Kyoungmi; DeFelice, Brian C.; Taylor, Sandra L.; Gandara, David R.; Yoneda, Ken Y.; Cooke, David T.; Fiehn, Oliver; Kelly, Karen; Miyamoto, Suzanne

2015-01-01

Background Untargeted metabolomics was utilized in case control studies of adenocarcinoma (ADC) lung cancer in order to develop and test metabolite classifiers in serum and plasma as potential biomarkers for diagnosing lung cancer. Methods Serum and plasma were collected and used in two independent case-control studies (ADC1 and ADC2). Controls were frequency matched for gender, age and smoking history. There were 52 ADC cases and 31 controls in ADC1 and 43 ADC cases and 43 controls in ADC2. Metabolomics was conducted using gas chromatography time-of-flight mass spectrometry. Differential analysis was performed on ADC1 and the top candidates (FDR < 0.05) for serum and plasma used to develop individual and multiplex-classifiers that were then tested on an independent set of serum and plasma samples (ADC2). Results Aspartate provided the best accuracy (81.4%) for an individual metabolite classifier in serum whereas pyrophosphate had the best accuracy (77.9%) in plasma when independently tested. Multiplex classifiers of either 2 or 4 serum metabolites had an accuracy of 72.7% when independently tested. For plasma, a multi-metabolite classifier consisting of 8 metabolites gave an accuracy of 77.3% when independently tested. Comparison of overall diagnostic performance between the two blood matrices yielded similar performances. However, serum is most ideal given higher sensitivity for low abundant metabolites. Conclusion This study shows the potential of metabolite-based diagnostic tests for detection of lung adenocarcinoma. Further validation in a larger pool of samples is warranted. Impact These biomarkers could improve early detection and diagnosis of lung cancer. PMID:26282632

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection.

PubMed

Wyss, R; Bucheli, F

1997-10-24

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.

Metabolic Characterization of the Common Marmoset (Callithrix jacchus)

PubMed Central

Go, Young-Mi; Liang, Yongliang; Uppal, Karan; Soltow, Quinlyn A.; Promislow, Daniel E. L.; Wachtman, Lynn M.; Jones, Dean P.

2015-01-01

High-resolution metabolomics has created opportunity to integrate nutrition and metabolism into genetic studies to improve understanding of the diverse radiation of primate species. At present, however, there is very little information to help guide experimental design for study of wild populations. In a previous non-targeted metabolomics study of common marmosets (Callithrix jacchus), Rhesus macaques, humans, and four non-primate mammalian species, we found that essential amino acids (AA) and other central metabolites had interspecies variation similar to intraspecies variation while non-essential AA, environmental chemicals and catabolic waste products had greater interspecies variation. The present study was designed to test whether 55 plasma metabolites, including both nutritionally essential and non-essential metabolites and catabolic products, differ in concentration in common marmosets and humans. Significant differences were present for more than half of the metabolites analyzed and included AA, vitamins and central lipid metabolites, as well as for catabolic products of AA, nucleotides, energy metabolism and heme. Three environmental chemicals were present at low nanomolar concentrations but did not differ between species. Sex and age differences in marmosets were present for AA and nucleotide metabolism and warrant additional study. Overall, the results suggest that quantitative, targeted metabolomics can provide a useful complement to non-targeted metabolomics for studies of diet and environment interactions in primate evolution. PMID:26581102

Individual variability in the human metabolism of an arsenic-containing carbohydrate, 2',3'-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-D-riboside, a naturally occurring arsenical in seafood.

PubMed

Raml, Reingard; Raber, Georg; Rumpler, Alice; Bauernhofer, Thomas; Goessler, Walter; Francesconi, Kevin A

2009-09-01

We report studies on the variability in human metabolism of an oxo-arsenosugar involving the ingestion of a chemically synthesized arsenosugar and quantitative determination of the arsenic metabolites in urine and serum by HPLC coupled with arsenic-selective mass spectrometric detection (ICPMS, inductively coupled plasma mass spectrometry). The total, four-day, urinary excretion of arsenic for six volunteers ranged widely from ca. 4-95%. The arsenic metabolites present in the urine also showed great variability: high arsenic excretion was accompanied by almost complete biotransformation of the ingested oxo-arsenosugar into a multitude of metabolites (>10), whereas the subjects that excreted low amounts of arsenic produced low quantities of metabolites relative to unchanged oxo-arsenosugar and its thio-analogue. Major arsenic urinary metabolites were dimethylarsinate (DMA) and possible intermediates in the degradation of arsenosugar to DMA, namely, dimethylarsinoylethanol (DMAE) and dimethylarsinoylacetate (DMAA) present both as their oxo- and thio-analogues. Thio-DMAE and thio-DMAA were also found in blood serum indicating that these species were formed in the liver rather than on storage of the urine in the bladder. The large variability in the way individuals metabolize arsenosugars has implications for risk assessment of arsenic intake from seafood.

A rapid and sensitive LC-MS/MS assay for the determination of saxagliptin and its active metabolite 5-hydroxy saxagliptin in human plasma and its application to a pharmacokinetic study.

PubMed

Batta, N; Pilli, N R; Derangula, V R; Vurimindi, H B; Damaramadugu, R; Yejella, R P

2015-03-01

The authors proposed a simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method for the simultaneous determination of saxagliptin and its active metabolite 5-hydroxy saxagliptin in human plasma. The developed method was fully validated as per the US FDA guidelines. The method utilized stable labeled isotopes saxagliptin-15 N d2 (IS1) and 5-hydroxy saxagliptin-15 N-d2 (IS2) as internal standards for the quantification of saxagliptin and 5-hydroxy saxagliptin, respectively. Analytes and the internal standards were extracted from human plasma by a single step solid-phase extraction technique without drying, evaporation and reconstitution steps. The optimized mobile phase was composed of 0.1% acetic acid in 5 mM ammonium acetate and acetonitrile (30:70, v/v) and delivered at a flow rate of 0.85 mL/min. The method exhibits the linear calibration range of 0.05-100 ng/mL for both the analytes. The precision and accuracy results for both the analytes were well within the acceptance limits. The different stability experiments conducted in aqueous samples and in matrix samples are meeting the acceptance criteria. The chromatographic run time was set at 1.8 min; hence more than 400 samples can be analyzed in a single day. © Georg Thieme Verlag KG Stuttgart · New York.

Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

PubMed

Boulet, Lysiane; Faure, Patrice; Flore, Patrice; Montérémal, Julien; Ducros, Véronique

2017-06-01

Tryptophan (Trp) is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological states, such as cardiovascular diseases, cancer, immunomodulation or depression. Hence, the quantification of Trp catabolites, from both KYN and 5HT pathways, might be usefulfor the discovery of novel diagnostic and follow-up biomarkers. We have developed a simple method for quantification of Trp and 8 of its metabolites,involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. We also validated the methodin human plasma samples, according to NF EN ISO 15189 criteria. Our method shows acceptable intra- and inter-day coefficients of variation (CV) (<12% and <16% respectively). The linearity entirelycovers the human plasma range. Stabilities of whole blood and of residues weredetermined, as well as the use of 2 different types of collectiontube, enabling us to adapt our process. Matrix effects and reference values showed good agreement compared to the literature. We propose here a method allowing the simultaneous quantification of a panel of Trp catabolites, never used before to our knowledge. This method, witha quickchromatographic runtime (15min) and simple sample preparation, has beenvalidated according to NF EN ISO 15189 criteria. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

An Atmospheric Pressure Chemical Ionization MS/MS Assay Using Online Extraction for the Analysis of 11 Cannabinoids and Metabolites in Human Plasma and Urine.

PubMed

Klawitter, Jelena; Sempio, Cristina; Mörlein, Sophie; De Bloois, Erik; Klepacki, Jacek; Henthorn, Thomas; Leehey, Maureen A; Hoffenberg, Edward J; Knupp, Kelly; Wang, George S; Hopfer, Christian; Kinney, Greg; Bowler, Russell; Foreman, Nicholas; Galinkin, Jeffrey; Christians, Uwe; Klawitter, Jost

2017-10-01

Although, especially in the United States, there has been a recent surge of legalized cannabis for either recreational or medicinal purposes, surprisingly little is known about clinical dose-response relationships, pharmacodynamic and toxicodynamic effects of cannabinoids such as Δ9-tetrahydrocannabinol (THC). Even less is known about other active cannabinoids. To address this knowledge gap, an online extraction, high-performance liquid chromatography coupled with tandem mass spectrometry method for simultaneous quantification of 11 cannabinoids and metabolites including THC, 11-hydroxy-Δ9-tetrahydrocannabinol, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-C-gluc), cannabinol, cannabidiol, cannabigerol, cannabidivarin, Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCV-COOH) was developed and validated in human urine and plasma. In contrast to atmospheric pressure chemical ionization, electrospray ionization was associated with extensive ion suppression in plasma and urine samples. Thus, the atmospheric pressure chemical ionization assay was validated showing a lower limit of quantification ranging from 0.39 to 3.91 ng/mL depending on study compound and matrix. The upper limit of quantification was 400 ng/mL except for THC-C-gluc with an upper limit of quantification of 2000 ng/mL. The linearity was r > 0.99 for all analyzed calibration curves. Acceptance criteria for intrabatch and interbatch accuracy (85%-115%) and imprecision (<15%) were met for all compounds. In plasma, the only exceptions were THCV (75.3%-121.2% interbatch accuracy) and cannabidivarin (interbatch imprecision, 15.7%-17.2%). In urine, THCV did not meet predefined acceptance criteria for intrabatch accuracy. This assay allows for monitoring not only THC and its major metabolites but also major cannabinoids that are of interest for marijuana research and clinical practice.

Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever

PubMed Central

Näsström, Elin; Vu Thieu, Nga Tran; Dongol, Sabina; Karkey, Abhilasha; Voong Vinh, Phat; Ha Thanh, Tuyen; Johansson, Anders; Arjyal, Amit; Thwaites, Guy; Dolecek, Christiane; Basnyat, Buddha; Baker, Stephen; Antti, Henrik

2014-01-01

The host–pathogen interactions induced by Salmonella Typhi and Salmonella Paratyphi A during enteric fever are poorly understood. This knowledge gap, and the human restricted nature of these bacteria, limit our understanding of the disease and impede the development of new diagnostic approaches. To investigate metabolite signals associated with enteric fever we performed two dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A infections and asymptomatic controls, identifying 695 individual metabolite peaks. Applying supervised pattern recognition, we found highly significant and reproducible metabolite profiles separating S. Typhi cases, S. Paratyphi A cases, and controls, calculating that a combination of six metabolites could accurately define the etiological agent. For the first time we show that reproducible and serovar specific systemic biomarkers can be detected during enteric fever. Our work defines several biologically plausible metabolites that can be used to detect enteric fever, and unlocks the potential of this method in diagnosing other systemic bacterial infections. DOI: http://dx.doi.org/10.7554/eLife.03100.001 PMID:24902583

Metabolomic and Genome-wide Association Studies Reveal Potential Endogenous Biomarkers for OATP1B1.

PubMed

Yee, S W; Giacomini, M M; Hsueh, C-H; Weitz, D; Liang, X; Goswami, S; Kinchen, J M; Coelho, A; Zur, A A; Mertsch, K; Brian, W; Kroetz, D L; Giacomini, K M

2016-11-01

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10 -8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection.

PubMed

Ortiz Boyer, F; Fernández Romero, J M; Luque de Castro, M D; Quesada, J M

1999-03-01

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.

Behavioral Effects and Pharmacokinetics of (±)-3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) after Intragastric Administration to Baboons

PubMed Central

Goodwin, Amy K.; Mueller, Melanie; Shell, Courtney D.; Ricaurte, George A.

2013-01-01

(±)-3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”) is a popular drug of abuse. We aimed to characterize the behavioral effects of intragastric MDMA in a species closely related to humans and to relate behavioral effects to plasma MDMA and metabolite concentrations. Single doses of MDMA (0.32–7.8 mg/kg) were administered via an intragastric catheter to adult male baboons (N = 4). Effects of MDMA on food-maintained responding were assessed over a 20-hour period, whereas untrained behaviors and fine-motor coordination were characterized every 30 minutes until 3 hours postadministration. Levels of MDMA and metabolites in plasma were measured in the same animals (n = 3) after dosing on a separate occasion. MDMA decreased food-maintained responding over the 20-hour period, and systematic behavioral observations revealed increased frequency of bruxism as the dose of MDMA was increased. Drug blood level determinations showed no MDMA after the lower doses of MDMA tested (0.32–1.0 mg/kg) and modest levels after higher MDMA doses (3.2–7.8 mg/kg). High levels of 3,4-dihydroxymethamphetamine (HHMA) were detected after all doses of MDMA, suggesting extensive first-pass metabolism of MDMA in the baboon. The present results demonstrate that MDMA administered via an intragastric catheter produced behavioral effects that have also been reported in humans. Similar to humans, blood levels of MDMA after oral administration may not be predictive of the behavioral effects of MDMA. Metabolites, particularly HHMA, may play a significant role in the behavioral effects of MDMA. PMID:23516331

Behavioral effects and pharmacokinetics of (±)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) after intragastric administration to baboons.

PubMed

Goodwin, Amy K; Mueller, Melanie; Shell, Courtney D; Ricaurte, George A; Ator, Nancy A

2013-06-01

(±)-3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a popular drug of abuse. We aimed to characterize the behavioral effects of intragastric MDMA in a species closely related to humans and to relate behavioral effects to plasma MDMA and metabolite concentrations. Single doses of MDMA (0.32-7.8 mg/kg) were administered via an intragastric catheter to adult male baboons (N = 4). Effects of MDMA on food-maintained responding were assessed over a 20-hour period, whereas untrained behaviors and fine-motor coordination were characterized every 30 minutes until 3 hours postadministration. Levels of MDMA and metabolites in plasma were measured in the same animals (n = 3) after dosing on a separate occasion. MDMA decreased food-maintained responding over the 20-hour period, and systematic behavioral observations revealed increased frequency of bruxism as the dose of MDMA was increased. Drug blood level determinations showed no MDMA after the lower doses of MDMA tested (0.32-1.0 mg/kg) and modest levels after higher MDMA doses (3.2-7.8 mg/kg). High levels of 3,4-dihydroxymethamphetamine (HHMA) were detected after all doses of MDMA, suggesting extensive first-pass metabolism of MDMA in the baboon. The present results demonstrate that MDMA administered via an intragastric catheter produced behavioral effects that have also been reported in humans. Similar to humans, blood levels of MDMA after oral administration may not be predictive of the behavioral effects of MDMA. Metabolites, particularly HHMA, may play a significant role in the behavioral effects of MDMA.

Metabolism by conjugation appears to confer resistance to paracetamol (acetaminophen) hepatotoxicity in the cynomolgus monkey.

PubMed

Yu, Hong; Barrass, Nigel; Gales, Sonya; Lenz, Eva; Parry, Tony; Powell, Helen; Thurman, Dale; Hutchison, Michael; Wilson, Ian D; Bi, Luke; Qiao, Junwen; Qin, Qiuping; Ren, Jin

2015-03-01

1. Paracetamol overdose remains the leading cause of acute liver failure in humans. This study was undertaken in cynomolgus monkeys to study the pharmacokinetics, metabolism and the potential for hepatotoxic insult from paracetamol administration as a possible model for human toxicity. 2. No adverse effects were observed for doses of up to 900 mg/kg/d for 14 d. Only minor sporadic increases in alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase in a number of animals were observed, with no clear dose response. 3. Toxicokinetic analysis showed good plasma exposure, albeit with less than proportional rises in Cmax and AUC, with increasing dose. The Cmax values in monkey were up to 3.5 times those associated with human liver toxicity and the AUC approx. 1000 times those associated with liver enzyme changes in 31-44% of human subjects. 4. Metabolite profiling of urine by (1)H NMR spectroscopy revealed paracetamol and its glucuronide and sulphate metabolites. Glutathione-derived metabolites, e.g. the cysteinyl conjugate, were only present in very low concentrations whilst the mercapturate was not detected. 5. These in vivo observations demonstrated that the cynomolgus monkey is remarkably resistant to paracetamol-induced toxicity and a poor model for investigating paracetamol-related hepatotoxicity in humans.

Combining Chimeric Mice with Humanized Liver, Mass Spectrometry, and Physiologically-Based Pharmacokinetic Modeling in Toxicology.

PubMed

Yamazaki, Hiroshi; Suemizu, Hiroshi; Mitsui, Marina; Shimizu, Makiko; Guengerich, F Peter

2016-12-19

Species differences exist in terms of drug oxidation activities, which are mediated mainly by cytochrome P450 (P450) enzymes. To overcome the problem of species extrapolation, transchromosomic mice containing a human P450 3A cluster or chimeric mice transplanted with human hepatocytes have been introduced into the human toxicology research area. In this review, drug metabolism and disposition mediated by humanized livers in chimeric mice are summarized in terms of biliary/urinary excretions of phthalate and bisphenol A and plasma clearances of the human cocktail probe drugs caffeine, warfarin, omeprazole, metoprolol, and midazolam. Simulation of human plasma concentrations of the teratogen thalidomide and its human metabolites is possible with a simplified physiologically based pharmacokinetic model based on data obtained in chimeric mice, in accordance with reported clinical thalidomide concentrations. In addition, in vivo nonspecific hepatic protein binding parameters of metabolically activated 14 C-drug candidate and hepatotoxic medicines in humanized liver mice can be analyzed by accelerator mass spectrometry and are useful for predictions in humans.

Comparative metabolomics in vegans and omnivores reveal constraints on diet-dependent gut microbiota metabolite production.

PubMed

Wu, Gary D; Compher, Charlene; Chen, Eric Z; Smith, Sarah A; Shah, Rachana D; Bittinger, Kyle; Chehoud, Christel; Albenberg, Lindsey G; Nessel, Lisa; Gilroy, Erin; Star, Julie; Weljie, Aalim M; Flint, Harry J; Metz, David C; Bennett, Michael J; Li, Hongzhe; Bushman, Frederic D; Lewis, James D

2016-01-01

The consumption of an agrarian diet is associated with a reduced risk for many diseases associated with a 'Westernised' lifestyle. Studies suggest that diet affects the gut microbiota, which subsequently influences the metabolome, thereby connecting diet, microbiota and health. However, the degree to which diet influences the composition of the gut microbiota is controversial. Murine models and studies comparing the gut microbiota in humans residing in agrarian versus Western societies suggest that the influence is large. To separate global environmental influences from dietary influences, we characterised the gut microbiota and the host metabolome of individuals consuming an agrarian diet in Western society. Using 16S rRNA-tagged sequencing as well as plasma and urinary metabolomic platforms, we compared measures of dietary intake, gut microbiota composition and the plasma metabolome between healthy human vegans and omnivores, sampled in an urban USA environment. Plasma metabolome of vegans differed markedly from omnivores but the gut microbiota was surprisingly similar. Unlike prior studies of individuals living in agrarian societies, higher consumption of fermentable substrate in vegans was not associated with higher levels of faecal short chain fatty acids, a finding confirmed in a 10-day controlled feeding experiment. Similarly, the proportion of vegans capable of producing equol, a soy-based gut microbiota metabolite, was less than that was reported in Asian societies despite the high consumption of soy-based products. Evidently, residence in globally distinct societies helps determine the composition of the gut microbiota that, in turn, influences the production of diet-dependent gut microbial metabolites. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Chemopreventive efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of familial adenomatous polyposis.

PubMed

Perkins, Sarah; Verschoyle, Richard D; Hill, Kirsti; Parveen, Ifat; Threadgill, Michael D; Sharma, Ricky A; Williams, Marion L; Steward, William P; Gescher, Andreas J

2002-06-01

Curcumin, the major yellow pigment in turmeric, prevents the development of adenomas in the intestinal tract of the C57Bl/6J Min/+ mouse, a model of human familial APC. To aid the rational development of curcumin as a colorectal cancer-preventive agent, we explored the link between its chemopreventive potency in the Min/+ mouse and levels of drug and metabolites in target tissue and plasma. Mice received dietary curcumin for 15 weeks, after which adenomas were enumerated. Levels of curcumin and metabolites were determined by high-performance liquid chromatography in plasma, tissues, and feces of mice after either long-term ingestion of dietary curcumin or a single dose of [(14)C]curcumin (100 mg/kg) via the i.p. route. Whereas curcumin at 0.1% in the diet was without effect, at 0.2 and 0.5%, it reduced adenoma multiplicity by 39 and 40%, respectively, compared with untreated mice. Hematocrit values in untreated Min/+ mice were drastically reduced compared with those in wild-type C57Bl/6J mice. Dietary curcumin partially restored the suppressed hematocrit. Traces of curcumin were detected in the plasma. Its concentration in the small intestinal mucosa, between 39 and 240 nmol/g of tissue, reflects differences in dietary concentration. [(14)C]Curcumin disappeared rapidly from tissues and plasma within 2-8 h after dosing. Curcumin may be useful in the chemoprevention of human intestinal malignancies related to Apc mutations. The comparison of dose, resulting curcumin levels in the intestinal tract, and chemopreventive potency suggests tentatively that a daily dose of 1.6 g of curcumin is required for efficacy in humans. A clear advantage of curcumin over nonsteroidal anti-inflammatory drugs is its ability to decrease intestinal bleeding linked to adenoma maturation.

Determination of aniracetam's main metabolite, N-anisoyl-GABA, in human plasma by LC-MS/MS and its application to a pharmacokinetic study.

PubMed

Cai, Shuang; Wang, Lei

2012-05-15

A simple and rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C₁₈ column at 25 °C. The mobile phase consisted of methanol-ammonium acetate (10 mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC-MS/MS method for ABA with advantages of short analysis time (4.5 min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485-19.4 μg/mL in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (RSD) and accuracy (RE) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings. Copyright © 2012 Elsevier B.V. All rights reserved.

Serum and Plasma Metabolomic Biomarkers for Lung Cancer.

PubMed

Kumar, Nishith; Shahjaman, Md; Mollah, Md Nurul Haque; Islam, S M Shahinul; Hoque, Md Aminul

2017-01-01

In drug invention and early disease prediction of lung cancer, metabolomic biomarker detection is very important. Mortality rate can be decreased, if cancer is predicted at the earlier stage. Recent diagnostic techniques for lung cancer are not prognosis diagnostic techniques. However, if we know the name of the metabolites, whose intensity levels are considerably changing between cancer subject and control subject, then it will be easy to early diagnosis the disease as well as to discover the drug. Therefore, in this paper we have identified the influential plasma and serum blood sample metabolites for lung cancer and also identified the biomarkers that will be helpful for early disease prediction as well as for drug invention. To identify the influential metabolites, we considered a parametric and a nonparametric test namely student׳s t-test as parametric and Kruskal-Wallis test as non-parametric test. We also categorized the up-regulated and down-regulated metabolites by the heatmap plot and identified the biomarkers by support vector machine (SVM) classifier and pathway analysis. From our analysis, we got 27 influential (p-value<0.05) metabolites from plasma sample and 13 influential (p-value<0.05) metabolites from serum sample. According to the importance plot through SVM classifier, pathway analysis and correlation network analysis, we declared 4 metabolites (taurine, aspertic acid, glutamine and pyruvic acid) as plasma biomarker and 3 metabolites (aspartic acid, taurine and inosine) as serum biomarker.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS.

PubMed

Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I; Fokina, Valentina M; Nanovskaya, Tatiana N; Hankins, Gary D V; Ahmed, Mahmoud S

2015-04-01

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS

PubMed Central

Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I.; Fokina, Valentina M.; Nanovskaya, Tatiana N.; Hankins, Gary D.V.; Ahmed, Mahmoud S.

2014-01-01

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3, and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples ranged between 87% and 99%, and 85%–95% for urine samples. The differences in retention times among the analytes, and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL and 4.95 ng/mL for MET. LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 μg/mL for MET. The relative deviation of this method was < 14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy ranged between 86% and 114% in plasma, and 94% to 105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. PMID:25164921

Plasma-free vs deconjugated metanephrines for diagnosis of phaeochromocytoma.

PubMed

Pamporaki, Christina; Därr, Roland; Bursztyn, Michael; Glöckner, Stephan; Bornstein, Stefan R; Lenders, Jacques W M; Pacak, Karel; Krinner, Axel; Eisenhofer, Graeme

2013-10-01

The diagnosis of phaeochromocytoma is commonly performed by the measurements of plasma-free normetanephrine and metanephrine. Plasma-deconjugated normetanephrine and metanephrine have been proposed as alternative, equivalent, but easier to measure biomarkers. The aim of this study was to compare the diagnostic performance of plasma-free vs deconjugated normetanephrine and metanephrine in patients tested for phaeochromocytoma. The study population included a reference group of 262 normotensive and hypertensive volunteers, 198 patients with phaeochromocytoma and 528 patients initially suspected of having the tumour, but with negative investigations after at least 2 years of follow-up. Measurements were performed using liquid chromatography with electrochemical detection. Plasma concentrations of free normetanephrine were 17-fold higher in patients with phaeochromocytoma than in the reference population, a 72% larger (P < 0·001) difference than that for the 10-fold higher levels of plasma-deconjugated normetanephrine. In contrast, relative increases in plasma concentrations of free and deconjugated metanephrine were similar. Using upper cut-offs established in the reference population, measurements of plasma-free metabolites provided superior diagnostic performance than deconjugated metabolites according to measures of both sensitivity (97% vs 92%, P = 0·002) and specificity (93% vs 89%, P = 0·012). The area under the receiver operating characteristic curve for the free metabolites was larger than that for the deconjugated metabolites (0·986 vs 0·965, P < 0·001). Measurements of plasma-free normetanephrine and metanephrine are superior to the deconjugated metabolites for diagnosis of phaeochromocytoma. © 2013 John Wiley & Sons Ltd.

Improved motor and cognitive performance with sodium nitrite supplementation is related to small metabolite signatures: a pilot trial in middle-aged and older adults

PubMed Central

DeVan, Allison E.; Cruickshank-Quinn, Charmion; Reisdorph, Nichole; Bassett, Candace J.; Evans, Trent D.; Brooks, Forrest A.; Bryan, Nathan S.; Chonchol, Michel B.; Giordano, Tony; McQueen, Matthew B.; Seals, Douglas R.

2015-01-01

Advancing age is associated with reductions in nitric oxide bioavailability and changes in metabolic activity, which are implicated in declines in motor and cognitive function. In preclinical models, sodium nitrite supplementation (SN) increases plasma nitrite and improves motor function, whereas other nitric oxide-boosting agents improve cognitive function. This pilot study was designed to translate these findings to middle-aged and older (MA/O) humans to provide proof-of-concept support for larger trials. SN (10 weeks, 80 or 160 mg/day capsules, TheraVasc, Inc.) acutely and chronically increased plasma nitrite and improved performance on measures of motor and cognitive outcomes (all p<0.05 or better) in healthy MA/O adults (62 ± 7 years). Untargeted metabolomics analysis revealed that SN significantly altered 33 (160 mg/day) to 45 (80 mg/day) different metabolites, 13 of which were related to changes in functional outcomes; baseline concentrations of 99 different metabolites predicted functional improvements with SN. This pilot study provides the first evidence that SN improves aspects of motor and cognitive function in healthy MA/O adults, and that these improvements are associated with, and predicted by, the plasma metabolome. Our findings provide the necessary support for larger clinical trials on this promising pharmacological strategy for preserving physiological function with aging. PMID:26626856

Dynamics of progesterone, TNF-a and a metabolite of PGF2a in blood plasma of beef cows following embryo transfer

USDA-ARS?s Scientific Manuscript database

Lactating beef cows received an embryo along with no treatment (control; n = 16), controlled internal drug releasing device (CIDR; n = 16), human chorionic gonadotropin (hCG; n = 15), or gonadotropin releasing hormone (GnRH; n = 15) to assess the effectiveness of these treatments in increasing blood...

Concentrations of isoflavones and their metabolites in the blood of pregnant and non-pregnant heifers fed soy bean.

PubMed

Woclawek-Potocka, Izabela; Piskula, Mariusz Krzysztof; Bah, Mamadou; Siemieniuch, Marta Jolanta; Korzekwa, Anna; Brzezicka, Edyta; Skarzynski, Dariusz Jan

2008-10-01

The present study compared the changes in isoflavones (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cyclic and pregnant heifers after feeding with soy bean. Twelve healthy heifers were divided into three groups: cyclic heifers (days 8-12 of the estrous cycle; control group; n=4), an early pregnancy group (2 months pregnant; n=4) and a late pregnancy group (8 months pregnant; n=4). All heifers were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on an HPLC system. In the blood plasma of the early- and late-pregnant heifers, we found lower concentrations and time-dependent decreases in daidzein and genistein in comparison to cyclic heifers (P<0.05). Moreover, we noticed significant increases of equol and para-ethyl-phenol in the blood plasma of the early-pregnant heifers (P<0.05). In contrast, in the blood plasma of the late-pregnant heifers, we did not find an increase in the isoflavone metabolite concentrations compared with the early-pregnant heifers (P>0.05). In conclusion, physiological status (cyclicity or pregnancy) of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the heifers. The stage of pregnancy affects isoflavone absorption, biotransformation and metabolism differently and results in higher concentrations of active metabolites of isoflavones during early pregnancy in comparison to their lower concentrations during late pregnancy. Therefore, we surmise that cows are more sensitive to active isoflavone metabolite actions during early pregnancy than cyclic heifers and heifers in late pregnancy.

Untargeted metabolomic profiling plasma samples of patients with lung cancer for searching significant metabolites by HPLC-MS method

NASA Astrophysics Data System (ADS)

Dementeva, N.; Ivanova, K.; Kokova, D.; Kurzina, I.; Ponomaryova, A.; Kzhyshkowska, J.

2017-09-01

Lung cancer is one of the most common types of cancer leading to death. Consequently, the search and the identification of the metabolites associated with the risk of developing cancer are very valuable. For the purpose, untargeted metabolic profiling of the plasma samples collected from the patients with lung cancer (n = 100) and the control group (n = 100) was conducted. After sample preparation, the plasma samples were analyzed using LC-MS method. Biostatistics methods were applied to pre-process the data for elicitation of dominating metabolites which responded to the difference between the case and the control groups. At least seven significant metabolites were evaluated and annotated. The most part of identified metabolites are connected with lipid metabolism and their combination could be useful for follow-up studies of lung cancer pathogenesis.

Liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of the irreversible BTK inhibitor ibrutinib and its dihydrodiol-metabolite in plasma and its application in mouse pharmacokinetic studies.

PubMed

Rood, Johannes J M; van Hoppe, Stephanie; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

2016-01-25

A validated simple, fast and sensitive bio-analytical assay for ibrutinib and its dihydrodiol metabolite in human and mouse plasma was set up. Sample preparation was performed by protein precipitation, and addition of the respective deuterated internal standards, followed by LC-MS/MS analysis. Separation was performed on a 3.5 μm particle-size, bridged ethylene hybrid column with gradient elution by 0.1% v/v formic acid and acetonitrile. The full eluate was transferred to an electrospray interface in positive ionization mode, and subsequently analyzed by a triple quadrupole mass spectrometer by selected reaction monitoring. The assay was validated in a 5-5000 ng/ml calibration range. Both ibrutinib and dihydrodiol-ibrutinib were deemed stable under refrigerated or frozen storage conditions. At room temperature, ibrutinib showed a not earlier described instability, and revealed rapid degradation at 37 °C. Finally, the assay was used for a pharmacokinetic study of plasma levels in treated FVB mice. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative plasma disposition of fenbendazole, oxfendazole and albendazole in dogs.

PubMed

Gokbulut, C; Bilgili, A; Hanedan, B; McKellar, Q A

2007-09-30

The plasma disposition of fenbendazole (FBZ), oxfendazole (OFZ) and albendazole (ABZ); and the enantiospecific disposition of OFZ, and ABZSO produced were investigated following an oral administration (50 mg/kg) in dogs. Blood samples were collected from 1 to 120 h post-administration. The plasma samples were analysed by high performance liquid chromatography (HPLC). The plasma concentration of FBZ, OFZ, ABZ and their metabolites were significantly different from each other and depended on the drug administered. The sulphone metabolite (FBZSO2) of FBZ was not detected in any plasma samples and the parent molecule ABZ did not reach quantifiable concentrations following FBZ and ABZ administration, respectively. OFZ and its sulphone metabolite attained a significantly higher plasma concentration and remained much longer in plasma compared with FBZ and ABZ and their respective metabolites. The maximum plasma concentrations (Cmax), area under the concentration time curve (AUC) and mean residence time (MRT) of parent OFZ were more than 30, 68 and 2 times those of FBZ, respectively. The same parameters for ABZSO were also significantly greater than those of FBZSO. The ratio for total AUCs of both the parent drug and the metabolites were 1:42:7 for following FBZ, OFZ and ABZ administration, respectively. The enantiomers were never in racemic proportions and (+) enantiomers of both OFZ and ABZSO were predominant in plasma. The AUC of (+) enantiomers of OFZ and ABZSO was, respectively more than three and seven times larger than that of (-) enantiomers of both molecules. It is concluded that the plasma concentration of OFZ was substantially greater compared with FBZ and ABZ. The data on the pharmacokinetic profile of OFZ presented here may contribute to evaluate its potential as an anthelmintic drug for parasite control in dogs.

Development of analyses by high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry of bilberry (Vaccinium myrtilus) anthocyanins in human plasma and urine.

PubMed

Cooke, Darren N; Thomasset, Sarah; Boocock, David J; Schwarz, Michael; Winterhalter, Peter; Steward, William P; Gescher, Andreas J; Marczylo, Timothy H

2006-09-20

Anthocyanins are potent antioxidants that may possess chronic disease preventive properties. Here, rapid, reliable, and reproducible solid-phase extraction, high-performance liquid chromatography (HPLC), and mass spectrometry techniques are described for the isolation, separation, and identification of anthocyanins in human plasma and urine. Recoveries of cyanidin-3-glucoside (C3G) were 91% from water, 71% from plasma, and 81% from urine. Intra- and interday variations for C3G extraction were 9 and 9.1% in plasma and 7.1 and 9.1% in urine and were less than 15% for all anthocyanins from a standardized bilberry extract (mirtoselect). Analysis of mirtoselect by HPLC with UV detection produced spectra with 15 peaks compatible with anthocyanin components found in mirtoselect within a total run time of 15 min. Chromatographic analysis of human urine obtained after an oral dose of mirtoselect yielded 19 anthocyanin peaks. Mass spectrometric analysis employing multiple reaction monitoring suggests the presence of unchanged anthocyanins and anthocyanidin glucuronide metabolites.

Intestinal Microbiota-Derived Metabolomic Blood Plasma Markers for Prior Radiation Injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ó Broin, Pilib; Department of Mathematical Sciences, Yeshiva University, New York, New York; Vaitheesvaran, Bhavapriya

2015-02-01

Purpose: Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. Methods and Materials: Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative,more » untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. Results: We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. Conclusions: Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma.« less

A novel double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]tofogliflozin after oral administration and concomitant intravenous microdose administration of [13C]tofogliflozin in humans.

PubMed

Schwab, Dietmar; Portron, Agnes; Backholer, Zoe; Lausecker, Berthold; Kawashima, Kosuke

2013-06-01

Human mass balance studies and the assessment of absolute oral bioavailability (F) are usually assessed in separate studies. Intravenous microdose administration of an isotope tracer concomitant to an unlabeled oral dose is an emerging technique to assess F. We report a novel double-tracer approach implemented for tofogliflozin combining oral administration of a radiolabel tracer with concomitant intravenous administration of a stable isotope tracer. Tofogliflozin is a potent and selective sodium/glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus currently in clinical development. The objectives of the present study were to assess the systemic exposure of major circulating metabolites, excretion balance, F and contribution of renal clearance (CLR) to total clearance (CL) of tofogliflozin in healthy subjects within one study applying a novel double-tracer technique. Six healthy male subjects received 20 mg [(12)C/(14)C]tofogliflozin (3.73 MBq) orally and a concomitant microdose of 0.1 mg [(13)C]tofogliflozin intravenously. Pharmacokinetics of tofogliflozin were determined for the oral and intravenous route; the pharmacokinetics of the metabolites M1 and M5 were determined for the oral route. Quantification of [(12)C]tofogliflozin in plasma and urine and [(13)C]tofogliflozin in plasma was performed by selective LC-MS/MS methods. For the pre-selected metabolites of tofogliflozin, M1 and M5, a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to plasma and urine samples. Total radioactivity was assessed in plasma, urine and feces. Pharmacokinetic analysis was conducted by non-compartmental methods. The pharmacokinetics of tofogliflozin in healthy subjects were characterized by an F of 97.5 ± 12.3 %, CL of 10.0 ± 1.3 l/h and volume of distribution at steady-state (V(ss)) of 50.6 ± 6.7 l. The main route of elimination of total drug-related material was by excretion into urine (77.0 ± 4.1 % of the dose). The observed CL(R) of 25.7 ± 5.0 ml/min was higher than the product of the estimated glomerular filtration rate (eGFR) and fraction unbound in plasma (f(u)) (eGFR × f(u) 15 ml/min), indicating the presence of net active tubular secretion in the renal elimination of tofogliflozin. However, CLR contributed only 15.5 % to the CL of tofogliflozin, suggesting that reductions in CLR by renal impairment won't significantly affect systemic exposure to tofogliflozin. Tofogliflozin and its metabolite M1 were the only major circulating entities accounting for 46 ± 8.6 and 50 ± 8.2 %, respectively, of total circulating drug-related material, while the metabolite M5 was a minor circulating metabolite accounting for 3.0 ± 0.3 % of total circulating drug-related material. Both the M1 and M5 metabolites were excreted into urine and the major metabolite M1 did not exhibit active tubular secretion. These results demonstrate the utility of the double-tracer approach to provide essential pharmacokinetic data and excretion data for drug-related material in one study at the same dosing occasion. The data obtained allowed the characterization of absorption, distribution, metabolism and excretion of tofogliflozin. Tofogliflozin exhibited highly favorable pharmacokinetic properties as demonstrated by its high F, low CL and a low V(ss. The presence of only one major circulating metabolite of tofogliflozin was unambiguously demonstrated. As a drug targeting the kidney, luminal exposure of the kidney is achieved by renal filtration and active tubular secretion.

Altered metabolites in the plasma of autism spectrum disorder: a capillary electrophoresis time-of-flight mass spectroscopy study.

PubMed

Kuwabara, Hitoshi; Yamasue, Hidenori; Koike, Shinsuke; Inoue, Hideyuki; Kawakubo, Yuki; Kuroda, Miho; Takano, Yosuke; Iwashiro, Norichika; Natsubori, Tatsunobu; Aoki, Yuta; Kano, Yukiko; Kasai, Kiyoto

2013-01-01

Clinical diagnosis and severity of autism spectrum disorders (ASD) are determined by trained clinicians based on clinical evaluations of observed behaviors. As such, this approach is inevitably dependent on the expertise and subjective assessment of those administering the clinical evaluations. There is a need to identify objective biological markers associated with diagnosis or clinical severity of the disorder. To identify novel candidate metabolites as potential biomarkers for ASD, the current study applied capillary electrophoresis time-of-flight mass spectroscopy (CE-TOFMS) for high-throughput profiling of metabolite levels in the plasma of 25 psychotropic-naïve adult males with high-functioning ASD and 28 age-matched typically-developed control subjects. Ten ASD participants and ten age-matched controls were assigned in the first exploration set, while 15 ASD participants and 18 controls were included in the second replication set. By CE-TOFMS analysis, a total of 143 metabolites were detected in the plasma of the first set. Of these, 17 metabolites showed significantly different relative areas between the ASD participants and the controls (p<0.05). Of the 17 metabolites, we consistently found that the ASD participants had significantly high plasma levels of arginine (p = 0.024) and taurine (p = 0.018), and significantly low levels of 5-oxoproline (p<0.001) and lactic acid (p = 0.031) compared with the controls in the second sample set. Further confirmatory analysis using quantification of absolute metabolite concentrations supported the robustness of high arginine (p = 0.001) and low lactic acid (p = 0.003) in the combined sample (n = 53). The present study identified deviated plasma metabolite levels associated with oxidative stress and mitochondrial dysfunction in individuals with ASD.

Altered Metabolites in the Plasma of Autism Spectrum Disorder: A Capillary Electrophoresis Time-of-Flight Mass Spectroscopy Study

PubMed Central

Kuwabara, Hitoshi; Yamasue, Hidenori; Koike, Shinsuke; Inoue, Hideyuki; Kawakubo, Yuki; Kuroda, Miho; Takano, Yosuke; Iwashiro, Norichika; Natsubori, Tatsunobu; Aoki, Yuta; Kano, Yukiko; Kasai, Kiyoto

2013-01-01

Clinical diagnosis and severity of autism spectrum disorders (ASD) are determined by trained clinicians based on clinical evaluations of observed behaviors. As such, this approach is inevitably dependent on the expertise and subjective assessment of those administering the clinical evaluations. There is a need to identify objective biological markers associated with diagnosis or clinical severity of the disorder. To identify novel candidate metabolites as potential biomarkers for ASD, the current study applied capillary electrophoresis time-of-flight mass spectroscopy (CE-TOFMS) for high-throughput profiling of metabolite levels in the plasma of 25 psychotropic-naïve adult males with high-functioning ASD and 28 age-matched typically-developed control subjects. Ten ASD participants and ten age-matched controls were assigned in the first exploration set, while 15 ASD participants and 18 controls were included in the second replication set. By CE-TOFMS analysis, a total of 143 metabolites were detected in the plasma of the first set. Of these, 17 metabolites showed significantly different relative areas between the ASD participants and the controls (p<0.05). Of the 17 metabolites, we consistently found that the ASD participants had significantly high plasma levels of arginine (p = 0.024) and taurine (p = 0.018), and significantly low levels of 5-oxoproline (p<0.001) and lactic acid (p = 0.031) compared with the controls in the second sample set. Further confirmatory analysis using quantification of absolute metabolite concentrations supported the robustness of high arginine (p = 0.001) and low lactic acid (p = 0.003) in the combined sample (n = 53). The present study identified deviated plasma metabolite levels associated with oxidative stress and mitochondrial dysfunction in individuals with ASD. PMID:24058493

Urine and plasma metabolites predict the development of diabetic nephropathy in individuals with Type 2 diabetes mellitus.

PubMed

Pena, M J; Lambers Heerspink, H J; Hellemons, M E; Friedrich, T; Dallmann, G; Lajer, M; Bakker, S J L; Gansevoort, R T; Rossing, P; de Zeeuw, D; Roscioni, S S

2014-09-01

Early detection of individuals with Type 2 diabetes mellitus or hypertension at risk for micro- or macroalbuminuria may facilitate prevention and treatment of renal disease. We aimed to discover plasma and urine metabolites that predict the development of micro- or macroalbuminuria. Patients with Type 2 diabetes (n = 90) and hypertension (n = 150) were selected from the community-cohort 'Prevention of REnal and Vascular End-stage Disease' (PREVEND) and the Steno Diabetes Center for this case-control study. Cases transitioned in albuminuria stage (from normo- to microalbuminuria or micro- to macroalbuminuria). Controls, matched for age, gender, and baseline albuminuria stage, remained in normo- or microalbuminuria stage during follow-up. Median follow-up was 2.9 years. Metabolomics were performed on plasma and urine. The predictive performance of a metabolite for albuminuria transition was assessed by the integrated discrimination index. In patients with Type 2 diabetes with normoalbuminuria, no metabolites discriminated cases from controls. In patients with Type 2 diabetes with microalbuminuria, plasma histidine was lower (fold change = 0.87, P = 0.02) and butenoylcarnitine was higher (fold change = 1.17, P = 0.007) in cases vs. controls. In urine, hexose, glutamine and tyrosine were lower in cases vs. controls (fold change = 0.20, P < 0.001; 0.32, P < 0.001; 0.51, P = 0.006, respectively). Adding the metabolites to a model of baseline albuminuria and estimated glomerular filtration rate metabolites improved risk prediction for macroalbuminuria transition (plasma integrated discrimination index = 0.28, P < 0.001; urine integrated discrimination index = 0.43, P < 0.001). These metabolites did not differ between hypertensive cases and controls without Type 2 diabetes. Type 2 diabetes-specific plasma and urine metabolites were discovered that predict the development of macroalbuminuria beyond established renal risk markers. These results should be confirmed in a large, prospective cohort. © 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.

Buprenorphine metabolites, buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide, are biologically active

PubMed Central

Brown, Sarah M.; Holtzman, Michael; Kim, Thomas; Kharasch, Evan D.

2012-01-01

Background The long-lasting high affinity opioid buprenorphine has complex pharmacology including ceiling effects with respect to analgesia and respiratory depression. Plasma concentrations of the major buprenorphine metabolites norbuprenorphine, buprenorphine-3-glucuronide, and norbuprenorphine-3-glucuronide approximate or exceed those of the parent drug. Buprenorphine glucuronide metabolites pharmacology is undefined. This investigation determined binding and pharmacological activity of the two glucuronide metabolites, and in comparison with buprenorphine and norbuprenorphine. Methods Competitive inhibition of radioligand binding to human mu, kappa, delta opioid and nociceptin receptors was used to determine glucuronide binding affinities for these receptors. Common opiate effects were assessed in vivo in Swiss Webster mice. Antinociception was assessed using a tail-flick assay, respiratory effects were measured using unrestrained whole-body plethysmography, and sedation was assessed by inhibition of locomotion measured by open-field testing. Results Buprenorphine-3-glucuronide had high affinity for human mu (Ki = 4.9±2.7 pM), delta (Ki = 270±0.4 nM), and nociceptin (Ki = 36±0.3 μM) but not kappa receptors. Norbuprenorphine-3-glucuronide had affinity for human kappa (Ki = 300±0.5 nM) and nociceptin (Ki= 18±0.2 μM) but not mu or delta receptors. At the dose tested, buprenorphine-3-glucuronide had a small antinociceptive effect. Neither glucuronide had significant effects on respiratory rate, but norbuprenorphine-3-glucuronide decreased tidal volume. Norbuprenorphine-3-glucuronide also caused sedation. Conclusions Both glucuronide metabolites of buprenorphine are biologically active at doses relevant to metabolite exposures which occur after buprenorphine. Activity of the glucuronides may contribute to the overall pharmacology of buprenorphine. PMID:22037640

The PET radioligand [carbonyl-(11)C]desmethyl-WAY-100635 binds to 5-HT(1A) receptors and provides a higher radioactive signal than [carbonyl-(11)C]WAY-100635 in the human brain.

PubMed

Andrée, Bengt; Halldin, Christer; Pike, Victor W; Gunn, Roger N; Olsson, Hans; Farde, Lars

2002-03-01

5-Hydroxytryptamine (serotonin)-1A (5-HT(1A)) receptors are of key interest in research on the pathophysiology and treatment of psychiatric disorders. The PET radioligand [carbonyl-(11)C]WAY-100635 ((11)C-WAY), where WAY-100635 is (3)H-(N-(2-(1-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) cyclohexane-carboxamide, is commonly used for quantitation of 5-HT(1A) receptors in the human brain. The aim of this PET study was to compare (11)C-WAY with the putative metabolite and selective radioligand [carbonyl-(11)C]desmethyl-WAY-100635 ((11)C-DWAY). A PET examination was performed on each of 5 healthy male volunteers after intravenous injection of (11)C-WAY and (11)C-DWAY on separate occasions. Radioactive metabolites in plasma were determined with high-performance liquid chromatography. The plasma metabolite--corrected input function was used in a kinetic compartment analysis. The simplified reference tissue model and peak equilibrium method, using the cerebellum as reference region, was applied for comparison of data. For both radioligands, the highest radioactivity was observed in the neocortex and the raphe nuclei, whereas radioactivity was low in the cerebellum. The regional binding potentials were similar for the 2 radioligands. The brain uptake was more than 2-fold higher for (11)C-DWAY than for (11)C-WAY, in part because of higher delivery (first-order rate constant K(1), 0.38 vs. 0.16). The time--activity curves were well described by a 3-compartment model for all regions, whereas uptake in the cerebellum could not be described by a 2-compartment model, supporting the existence of kinetically distinguishable nonspecific binding in the cerebellum or radioactive metabolites in the brain for both radioligands. Both radioligands were rapidly metabolized, and <10% of the radioactivity in plasma represented unchanged (11)C-WAY or (11)C-DWAY at 10 min after injection. The metabolic pattern was similar for both radioligands, with the formation of radiolabeled cyclohexanecarboxylic acid and more polar components. For (11)C-WAY, small amounts of an additional labeled metabolite comigrated with reference desmethyl-WAY-100635. The advantages of (11)C-DWAY over (11)C-WAY for research on central 5-HT(1A) receptors is supported by a significantly higher radioactivity signal at equipotent doses, providing improved imaging statistics and advantages in biomathematic modeling and the preclusion of (11)C-DWAY as a metabolite interfering with PET measurements.

Simultaneous quantification of acetaminophen and five acetaminophen metabolites in human plasma and urine by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: Method validation and application to a neonatal pharmacokinetic study.

PubMed

Cook, Sarah F; King, Amber D; van den Anker, John N; Wilkins, Diana G

2015-12-15

Drug metabolism plays a key role in acetaminophen (paracetamol)-induced hepatotoxicity, and quantification of acetaminophen metabolites provides critical information about factors influencing susceptibility to acetaminophen-induced hepatotoxicity in clinical and experimental settings. The aims of this study were to develop, validate, and apply high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) methods for simultaneous quantification of acetaminophen, acetaminophen-glucuronide, acetaminophen-sulfate, acetaminophen-glutathione, acetaminophen-cysteine, and acetaminophen-N-acetylcysteine in small volumes of human plasma and urine. In the reported procedures, acetaminophen-d4 and acetaminophen-d3-sulfate were utilized as internal standards (IS). Analytes and IS were recovered from human plasma (10μL) by protein precipitation with acetonitrile. Human urine (10μL) was prepared by fortification with IS followed only by sample dilution. Calibration concentration ranges were tailored to literature values for each analyte in each biological matrix. Prepared samples from plasma and urine were analyzed under the same HPLC-ESI-MS/MS conditions, and chromatographic separation was achieved through use of an Agilent Poroshell 120 EC-C18 column with a 20-min run time per injected sample. The analytes could be accurately and precisely quantified over 2.0-3.5 orders of magnitude. Across both matrices, mean intra- and inter-assay accuracies ranged from 85% to 112%, and intra- and inter-assay imprecision did not exceed 15%. Validation experiments included tests for specificity, recovery and ionization efficiency, inter-individual variability in matrix effects, stock solution stability, and sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw, and post-preparative). The utility and suitability of the reported procedures were illustrated by analysis of pharmacokinetic samples collected from neonates receiving intravenous acetaminophen. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of dehydroepiandrosterone and its biologically active oxygenated metabolites in human plasma evinces a hormonal imbalance during HIV-TB coinfection.

PubMed

Vecchione, María Belén; Eiras, Javier; Suarez, Guadalupe Verónica; Angerami, Matías Tomás; Marquez, Cecilia; Sued, Omar; Ben, Graciela; Pérez, Héctor Miguel; Gonzalez, Diego; Maidana, Patricia; Mesch, Viviana; Quiroga, María Florencia; Bruttomesso, Andrea Claudia

2018-04-27

An estimated one third of the world's population is affected by latent tuberculosis (TB), which once active represents a leading cause of death among infectious diseases. Human immunodeficiency virus (HIV) infection is a main predisposing factor to TB reactivation. Individuals HIV-TB co-infected develop a chronic state of inflammation associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. This results in a hormonal imbalance, disturbing the physiological levels of cortisol and dehydroepiandrosterone (DHEA). DHEA and its oxygenated metabolites androstenediol (AED), androstenetriol (AET) and 7-oxo-DHEA are immunomodulatory compounds that may regulate physiopathology in HIV-TB co-infection. In order to study possible changes in plasma levels of these hormones, we developed an approach based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). To our knowledge, this represents the first report of their simultaneous measurement in HIV-TB individuals and the comparison with healthy donors, obtaining statistically higher plasma levels of DHEA, AET and 7-oxo-DHEA in patients. Moreover, we found that concentrations of 7-oxo-DHEA positively correlated with absolute CD4+ T cell counts, nadir CD4+ T cell values and with individuals who presented TB restricted to the lungs. This research contributes to understanding the role of these hormones in HIV-TB and emphasizes the importance of deepening their study in this context.

2-Aminoadipic acid is a biomarker for diabetes risk

PubMed Central

Wang, Thomas J.; Ngo, Debby; Psychogios, Nikolaos; Dejam, Andre; Larson, Martin G.; Vasan, Ramachandran S.; Ghorbani, Anahita; O’Sullivan, John; Cheng, Susan; Rhee, Eugene P.; Sinha, Sumita; McCabe, Elizabeth; Fox, Caroline S.; O’Donnell, Christopher J.; Ho, Jennifer E.; Florez, Jose C.; Magnusson, Martin; Pierce, Kerry A.; Souza, Amanda L.; Yu, Yi; Carter, Christian; Light, Peter E.; Melander, Olle; Clish, Clary B.; Gerszten, Robert E.

2013-01-01

Improvements in metabolite-profiling techniques are providing increased breadth of coverage of the human metabolome and may highlight biomarkers and pathways in common diseases such as diabetes. Using a metabolomics platform that analyzes intermediary organic acids, purines, pyrimidines, and other compounds, we performed a nested case-control study of 188 individuals who developed diabetes and 188 propensity-matched controls from 2,422 normoglycemic participants followed for 12 years in the Framingham Heart Study. The metabolite 2-aminoadipic acid (2-AAA) was most strongly associated with the risk of developing diabetes. Individuals with 2-AAA concentrations in the top quartile had greater than a 4-fold risk of developing diabetes. Levels of 2-AAA were not well correlated with other metabolite biomarkers of diabetes, such as branched chain amino acids and aromatic amino acids, suggesting they report on a distinct pathophysiological pathway. In experimental studies, administration of 2-AAA lowered fasting plasma glucose levels in mice fed both standard chow and high-fat diets. Further, 2-AAA treatment enhanced insulin secretion from a pancreatic β cell line as well as murine and human islets. These data highlight a metabolite not previously associated with diabetes risk that is increased up to 12 years before the onset of overt disease. Our findings suggest that 2-AAA is a marker of diabetes risk and a potential modulator of glucose homeostasis in humans. PMID:24091325

2-Aminoadipic acid is a biomarker for diabetes risk.

PubMed

Wang, Thomas J; Ngo, Debby; Psychogios, Nikolaos; Dejam, Andre; Larson, Martin G; Vasan, Ramachandran S; Ghorbani, Anahita; O'Sullivan, John; Cheng, Susan; Rhee, Eugene P; Sinha, Sumita; McCabe, Elizabeth; Fox, Caroline S; O'Donnell, Christopher J; Ho, Jennifer E; Florez, Jose C; Magnusson, Martin; Pierce, Kerry A; Souza, Amanda L; Yu, Yi; Carter, Christian; Light, Peter E; Melander, Olle; Clish, Clary B; Gerszten, Robert E

2013-10-01

Improvements in metabolite-profiling techniques are providing increased breadth of coverage of the human metabolome and may highlight biomarkers and pathways in common diseases such as diabetes. Using a metabolomics platform that analyzes intermediary organic acids, purines, pyrimidines, and other compounds, we performed a nested case-control study of 188 individuals who developed diabetes and 188 propensity-matched controls from 2,422 normoglycemic participants followed for 12 years in the Framingham Heart Study. The metabolite 2-aminoadipic acid (2-AAA) was most strongly associated with the risk of developing diabetes. Individuals with 2-AAA concentrations in the top quartile had greater than a 4-fold risk of developing diabetes. Levels of 2-AAA were not well correlated with other metabolite biomarkers of diabetes, such as branched chain amino acids and aromatic amino acids, suggesting they report on a distinct pathophysiological pathway. In experimental studies, administration of 2-AAA lowered fasting plasma glucose levels in mice fed both standard chow and high-fat diets. Further, 2-AAA treatment enhanced insulin secretion from a pancreatic β cell line as well as murine and human islets. These data highlight a metabolite not previously associated with diabetes risk that is increased up to 12 years before the onset of overt disease. Our findings suggest that 2-AAA is a marker of diabetes risk and a potential modulator of glucose homeostasis in humans.

Toxico-pathological effects in rats induced by lambda-cyhalothrin.

PubMed

Dahamna, S; Harzallah, D; Guemache, A; Sekfali, N

2009-01-01

Pesticides are widely used chemicals making human exposure to pesticides a realistic possibility. Biomonitoring is a common and useful tool for assessing human exposure to pesticides. Pyrethroids are effective insecticides that are often used in household sprays, aerosol bombs, insect repellents, pet shampoos, and lice treatments. Using products containing these compounds will expose people to these chemicals. Since these compounds frequently are used on crOPs, they are often detected in fruits and vegetables. Biomonitoring of exposure is a useful tool for assessing exposure to pesticides. Biomonitoring involves the measurement of the parent pesticide, its metabolite or reaction product in biological media, typically blood or urine, to determine if an exposure has occurred and the extent of that exposure. Although not without its limitations, biomonitoring has great utility in integrating all routes of exposure allowing for one exposure measurement. Pesticides have much shorter environmental half-lives and tend not to bioaccumulate. In fact, from humans within 24 hr as the parent pesticide, a mercapturic acid detoxification product, oxidative or dealkylation metabolites, and/or glucuronide or sulphate-bound metabolites. However, because of the heavy agricultural and residential use of these chemicals, humans are continually exposed to many of these chemicals. The objective of the present study was to explore modification in toxico-pathological responses of rats treated with lambda-cyhalothrin (commercially called karate). Rats (250 g weight), were gavaged by 1/100 LD50 for 4 weeks (one dose every week). Blood was collected before dosing and after 48 hours from the treatment. Enzyme activities were assayed in the plasma samples obtained. Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate transaminase (GPT), Alkaline phosphatase (ALPH) and Glucose. The results showed a decrease in RBC; WBC and Hb. This probably explained by the effect of lambda cyhalothrin on the erythropoiesis and the destruction of cells. An increase of plasma enzyme activities in GOT and GPT were recorded, explain a high energy-generating product. The histopathological results showed alteration on the target organs such as liver and kidney, these biochemical and histological modifications are probably due to the effect of lambda cyhalothrin or their metabolites.

Sex-dependent metabolism of nevirapine in rats: impact on plasma levels, pharmacokinetics and interaction with nortriptyline.

PubMed

Usach, Iris; Compañ, Pablo; Peris, José-Esteban

2018-05-01

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in the treatment of human immunodeficiency virus type 1 (HIV-1) and is the first-choice NNRTI during pregnancy. NVP shows a sex dimorphic profile in humans with sex differences in bioavailability, biotransformation and toxicity. In this study, sex differences in NVP metabolism and inhibition of NVP metabolism by the antidepressant nortriptyline (NT) were evaluated using rats as experimental animals. NVP was administered orally to male and female rats and sex differences in plasma levels and pharmacokinetic parameters were analysed. NVP plasma levels were higher in female compared with male rats, and pharmacokinetic parameters such as maximum plasma concentration (C max ), time to C max (T max ), half-life (t 1/2 ) and area under the plasma concentration-time curve from the time of dosing to the last measurable concentration (AUC last ) showed ca. 4-, 5-, 7- and 22-fold higher values in female rats. In vitro experiments carried out with hepatic microsomes confirmed slower NVP metabolism in female rats, with a maximum velocity (V max ) 2-fold lower than in male hepatic microsomes. The major metabolite in both sexes was 12-hydroxynevirapine (12-OH-NVP), with the V max for this metabolite being 15-fold lower in female compared with male rat hepatic microsomes. Inhibition of NVP metabolism by NT was similar in both sexes, with statistically non-significant differences in 50% inhibitory concentration (IC 50 ) values. In summary, NVP is metabolised more slowly in female compared with male rats, but the inhibitory effect of NT is similar in both sexes. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.

PubMed

Fang, Mingliang; Ivanisevic, Julijana; Benton, H Paul; Johnson, Caroline H; Patti, Gary J; Hoang, Linh T; Uritboonthai, Winnie; Kurczy, Michael E; Siuzdak, Gary

2015-11-03

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

PHARMACOKINETICS OF TRAMADOL HYDROCHLORIDE AND ITS METABOLITE O-DESMETHYLTRAMADOL FOLLOWING A SINGLE, ORALLY ADMINISTERED DOSE IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

PubMed

Boonstra, Jennifer L; Barbosa, Lorraine; Van Bonn, William G; Johnson, Shawn P; Gulland, Frances M D; Cox, Sherry K; Martin-Jimenez, Tomas

2015-09-01

Tramadol is a synthetic, centrally acting, opiate-like analgesic that is structurally related to codeine and morphine. The objective of this study was to determine the pharmacokinetics of tramadol hydrochloride and its major active metabolite O-desmethyltramadol (M1) in the California sea lion (Zalophus californianus). A single dose of tramadol was administered orally in fish at 2 mg/kg to a total of 15 wild California sea lions admitted for rehabilitation. Twenty-four total blood samples were collected post drug administration at 10, 20, 30, and 45 min and at 1, 3, 5, 6, 8, 12, and 24 hr. Blood plasma was separated and stored at -80°C until analysis with high-performance liquid chromatography was performed to determine levels of tramadol and M1, the major active metabolite. The results indicate that the plasma levels of parent tramadol are low or negligible during the first 30-45 min and then reach the predicted mean maximum plasma concentration of 358 ng/ml at 1.52 hr. The M1 metabolite was not detectable in 21 of 24 plasma samples, below the level of quantification of 5 ng/ml in one sample, and detectable at 11 and 17 ng/ml in two of the samples. This study suggests that a 2 mg/kg dose would need to be administered every 6-8 hr to maintain concentrations of tramadol above the minimum human analgesic level for mild to moderate pain. Based on dosing simulations, a dose of 4 mg/kg q8 hr or q12 hr, on average, may represent an adequate compromise, but further studies are needed using a larger sample size. Pharmacodynamic studies are warranted to determine if tramadol provides analgesic effects in this species. The potential for tramadol toxicosis at any dose also has not been determined in this species.

Method development for the determination of 24S-hydroxycholesterol in human plasma without derivatization by high-performance liquid chromatography with tandem mass spectrometry in atmospheric pressure chemical ionization mode.

PubMed

Sugimoto, Hiroshi; Kakehi, Masaaki; Satomi, Yoshinori; Kamiguchi, Hidenori; Jinno, Fumihiro

2015-10-01

We developed a highly sensitive and specific high-performance liquid chromatography with tandem mass spectrometry method with an atmospheric pressure chemical ionization interface to determine 24S-hydroxycholesterol, a major metabolite of cholesterol formed by cytochrome P450 family 46A1, in human plasma without any derivatization step. Phosphate buffered saline including 1% Tween 80 was used as the surrogate matrix for preparation of calibration curves and quality control samples. The saponification process to convert esterified 24S-hydroxycholesterol to free sterols was optimized, followed by liquid-liquid extraction using hexane. Chromatographic separation of 24S-hydroxycholesterol from other isobaric endogenous oxysterols was successfully achieved with gradient mobile phase comprised of 0.1% propionic acid and acetonitrile using L-column2 ODS (2 μm, 2.1 mm id × 150 mm). This assay was capable of determining 24S-hydroxycholesterol in human plasma (200 μL) ranging from 1 to 100 ng/mL with acceptable intra- and inter-day precision and accuracy. The potential risk of in vitro formation of 24S-hydroxycholesterol by oxidation from endogenous cholesterol in human plasma was found to be negligible. The stability of 24S-hydroxycholesterol in relevant solvents and human plasma was confirmed. This method was successfully applied to quantify the plasma concentrations of 24S-hydroxycholesterol in male and female volunteers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efavirenz Primary and Secondary Metabolism In Vitro and In Vivo: Identification of Novel Metabolic Pathways and Cytochrome P450 2A6 as the Principal Catalyst of Efavirenz 7-Hydroxylation

PubMed Central

Ogburn, Evan T.; Jones, David R.; Masters, Andrea R.; Xu, Cong; Guo, Yingying

2010-01-01

Efavirenz primary and secondary metabolism was investigated in vitro and in vivo. In human liver microsome (HLM) samples, 7- and 8-hydroxyefavirenz accounted for 22.5 and 77.5% of the overall efavirenz metabolism, respectively. Kinetic, inhibition, and correlation analyses in HLM samples and experiments in expressed cytochrome P450 show that CYP2A6 is the principal catalyst of efavirenz 7-hydroxylation. Although CYP2B6 was the main enzyme catalyzing efavirenz 8-hydroxylation, CYP2A6 also seems to contribute. Both 7- and 8-hydroxyefavirenz were further oxidized to novel dihydroxylated metabolite(s) primarily by CYP2B6. These dihydroxylated metabolite(s) were not the same as 8,14-dihydroxyefavirenz, a metabolite that has been suggested to be directly formed via 14-hydroxylation of 8-hydroxyefavirenz, because 8,14-dihydroxyefavirenz was not detected in vitro when efavirenz, 7-, or 8-hydroxyefavirenz were used as substrates. Efavirenz and its primary and secondary metabolites that were identified in vitro were quantified in plasma samples obtained from subjects taking a single 600-mg oral dose of efavirenz. 8,14-Dihydroxyefavirenz was detected and quantified in these plasma samples, suggesting that the glucuronide or the sulfate of 8-hydroxyefavirenz might undergo 14-hydroxylation in vivo. In conclusion, efavirenz metabolism is complex, involving unique and novel secondary metabolism. Although efavirenz 8-hydroxylation by CYP2B6 remains the major clearance mechanism of efavirenz, CYP2A6-mediated 7-hydroxylation (and to some extent 8-hydroxylation) may also contribute. Efavirenz may be a valuable dual phenotyping tool to study CYP2B6 and CYP2A6, and this should be further tested in vivo. PMID:20335270

Non-targeted analyses of animal plasma: betaine and choline represent the nutritional and metabolic status.

PubMed

Katayama, K; Sato, T; Arai, T; Amao, H; Ohta, Y; Ozawa, T; Kenyon, P R; Hickson, R E; Tazaki, H

2013-02-01

Simple liquid chromatography-mass spectrometry (LC-MS) was applied to non-targeted metabolic analyses to discover new metabolic markers in animal plasma. Principle component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) were used to analyse LC-MS multivariate data. PCA clearly generated two separate clusters for artificially induced diabetic mice and healthy control mice. PLS-DA of time-course changes in plasma metabolites of chicks after feeding generated three clusters (pre- and immediately after feeding, 0.5-3 h after feeding and 4 h after feeding). Two separate clusters were also generated for plasma metabolites of pregnant Angus heifers with differing live-weight change profiles (gaining or losing). The accompanying PLS-DA loading plot detailed the metabolites that contribute the most to the cluster separation. In each case, the same highly hydrophilic metabolite was strongly correlated to the group separation. The metabolite was identified as betaine by LC-MS/MS. This result indicates that betaine and its metabolic precursor, choline, may be useful biomarkers to evaluate the nutritional and metabolic status of animals. © 2011 Blackwell Verlag GmbH.

Three plasma metabolite signatures for diagnosing high altitude pulmonary edema

NASA Astrophysics Data System (ADS)

Guo, Li; Tan, Guangguo; Liu, Ping; Li, Huijie; Tang, Lulu; Huang, Lan; Ren, Qian

2015-10-01

High-altitude pulmonary edema (HAPE) is a potentially fatal condition, occurring at altitudes greater than 3,000 m and affecting rapidly ascending, non-acclimatized healthy individuals. However, the lack of biomarkers for this disease still constitutes a bottleneck in the clinical diagnosis. Here, ultra-high performance liquid chromatography coupled with Q-TOF mass spectrometry was applied to study plasma metabolite profiling from 57 HAPE and 57 control subjects. 14 differential plasma metabolites responsible for the discrimination between the two groups from discovery set (35 HAPE subjects and 35 healthy controls) were identified. Furthermore, 3 of the 14 metabolites (C8-ceramide, sphingosine and glutamine) were selected as candidate diagnostic biomarkers for HAPE using metabolic pathway impact analysis. The feasibility of using the combination of these three biomarkers for HAPE was evaluated, where the area under the receiver operating characteristic curve (AUC) was 0.981 and 0.942 in the discovery set and the validation set (22 HAPE subjects and 22 healthy controls), respectively. Taken together, these results suggested that this composite plasma metabolite signature may be used in HAPE diagnosis, especially after further investigation and verification with larger samples.

Plasma choline metabolites associate with metabolic stress among young overweight men in a genotype-specific manner

PubMed Central

Yan, J; Winter, L B; Burns-Whitmore, B; Vermeylen, F; Caudill, M A

2012-01-01

OBJECTIVES: We aimed to test the hypotheses that (i) plasma choline metabolites differ between normal (body mass index (BMI)<25 kg m−2) and overweight (BMI ⩾25 kg m−2) men, and (ii) an elevated BMI alters associations between plasma choline metabolites and indicators of metabolic stress. DESIGN: This was a cross-sectional study. A one-time fasting blood sample was obtained for measurements of the choline metabolites and metabolic stress indicators (that is, serum alanine aminotransferase (ALT), glucose, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides and homocysteine), and for genotype determination. SUBJECTS: The analysis was conducted with 237 Mexican American men with a median age of 22 years. RESULTS: Compared with men with a normal BMI (n=98), those with an elevated BMI (n=139) had 6% lower (P=0.049) plasma betaine and an 11% lower (P=0.002) plasma betaine to choline ratio. Among men with an elevated BMI, plasma betaine and the plasma betaine to choline ratio positively associated (P⩽0.044) with a favorable serum cholesterol profile, and inversely associated (P=0.001) with serum ALT, a marker of liver dysfunction. The phosphatidylethanolamine N-methyltransferase (PEMT) 5465G→A (rs7946) genotype interacted (P⩽0.007) with the plasma betaine to choline ratio to modulate indicators of metabolic stress with stronger inverse associations observed among overweight men with the PEMT 5465GG genotype. CONCLUSIONS: Plasma choline metabolites predict metabolic stress among overweight men often in a genotype-specific manner. The diminished betaine among overweight men coupled with the inverse association between betaine and metabolic stress suggest that betaine supplementation may be effective in mitigating some of the metabolic insults arising from lipid overload. PMID:23169489

Carbamazepine and its 10,11-epoxide metabolite in acute mania: clinical and pharmacokinetic correlates.

PubMed

Petit, P; Lonjon, R; Cociglio, M; Sluzewska, A; Blayac, J P; Hue, B; Alric, R; Pouget, R

1991-01-01

The study was designed to investigate the antimanic profile of carbamazepine as a first-line drug in affective or schizoaffective disorders, to correlate the clinical efficacy with the plasma level of carbamazepine and its 10,11-epoxide metabolite, and to test the potential value of monitoring the salivary level. It was administered alone for 3 weeks to 21 acute manic inpatients. During the first week, the dosage was rapidly increased to 800 mg/day in order to produce steady-state plasma levels of carbamazepine on Day 7. The individual dose was then adjusted to maintain the therapeutic range of 8-12 mg/l. Plasma and saliva levels of the drug and its metabolite, as well as clinical status were assessed weekly. Overall, there was 62% globally improved patients and 77% in affective disorders. The improvement of manic symptoms was significantly lower in schizoaffective than in affective disorders, whereas the dropout rate and the need for antipsychotic medication was higher in the former group. The antimanic response was significantly correlated with the plasma levels both of carbamazepine and its epoxide metabolite, with a time-lag consistent with a delayed drug effect. Drug and metabolite concentrations in saliva were close to their plasma free fraction and were strongly correlated with their plasma levels, suggesting the potential value of salivary drug monitoring.

Metabolic fate of poly-(lactic-co-glycolic acid)-based curcumin nanoparticles following oral administration.

PubMed

Harigae, Takahiro; Nakagawa, Kiyotaka; Miyazawa, Taiki; Inoue, Nao; Kimura, Fumiko; Ikeda, Ikuo; Miyazawa, Teruo

2016-01-01

Curcumin (CUR), the main polyphenol in turmeric, is poorly absorbed and rapidly metabolized following oral administration, which severely curtails its bioavailability. Poly-(lactic-co-glycolic acid)-based CUR nanoparticles (CUR-NP) have recently been suggested to improve CUR bioavailability, but this has not been fully verified. Specifically, no data are available about curcumin glucuronide (CURG), the major metabolite of CUR found in the plasma following oral administration of CUR-NP. Herein, we investigated the absorption and metabolism of CUR-NP and evaluated whether CUR-NP improves CUR bioavailability. Following oral administration of CUR-NP in rats, we analyzed the plasma and organ distribution of CUR and its metabolites using high-performance liquid chromatography-tandem mass spectrometry. To elucidate the mechanism of increased intestinal absorption of CUR-NP, we prepared mixed micelles comprised of phosphatidylcholine and bile salts and examined the micellar solubility of CUR-NP. Additionally, we investigated the cellular incorporation of the resultant micelles into differentiated Caco-2 human intestinal cells. Following in vivo administration of CUR-NP, CUR was effectively absorbed and present mainly as CURG in the plasma which contained significant amounts of the metabolite compared with other organs. Thus, CUR-NP increased intestinal absorption of CUR rather than decreasing metabolic degradation and conversion to other metabolites. In vitro, CUR encapsulated in CUR-NP was solubilized in mixed micelles; however, whether the micelles contained CUR or CUR-NP had little influence on cellular uptake efficiency. Therefore, we suggest that the high solubilization capacity of CUR-NP in mixed micelles, rather than cellular uptake efficiency, explains the high intestinal absorption of CUR-NP in vivo. These findings provide a better understanding of the bioavailability of CUR and CUR-NP following oral administration. To improve the bioavailability of CUR, future studies should focus on enhancing the resistance to metabolic degradation and conversion of CUR to other metabolites, which may lead to novel discoveries regarding food function and disease prevention.

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry.

PubMed

Kwok, Wai Him; Ho, Emmie N M; Lau, Ming Yip; Leung, Gary N W; Wong, April S Y; Wan, Terence S M

2013-03-01

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.

Moclobemide excretion in human breast milk.

PubMed Central

Pons, G; Schoerlin, M P; Tam, Y K; Moran, C; Pfefen, J P; Francoual, C; Pedarriosse, A M; Chavinie, J; Olive, G

1990-01-01

1. Six lactating white women, aged 24-36 years, received a single oral dose of 300 mg moclobemide, between 09.00 h and 11.00 h, 3 to 5 days after the delivery of a full term neonate. 2. Complete milk collections were obtained before, 3, 6, 9, 12 and 24 h after drug administration by means of a breast pump. Venous blood samples were drawn before, and 0.5, 1, 3, 4.5, 6, 9, 12, 24 h post-dosing. 3. Moclobemide, and its major metabolite (Ro 12-8095) were measured in milk and plasma samples using h.p.l.c. The active metabolite (Ro 12-5637) could only be detected in plasma. 4. Moclobemide and its metabolites were not detectable in 24 h plasma samples. Cmax, tmax and t1/2 for moclobemide were (mean +/- s.d.) 2.70 +/- 1.24 mg l-1, 2.03 +/- 1.19 h and 2.26 +/- 0.26 h, respectively. 5. The concentrations of moclobemide and Ro 12-8095 in milk were highest at 3 h after drug administration and the drug and metabolite were not detectable after 12 h. Ro 12-5637 was not detected in any milk sample. The percentages of the dose excreted as moclobemide and Ro 12-8095 were (mean +/- s.d.) 0.057 +/- 0.020% and 0.031 +/- 0.011%, respectively. An average 3.5 kg breast-fed neonate would therefore be exposed to only a 0.05 mg kg-1 moclobemide dose (approximately 1% of the maternal dose on the mg kg-1 basis). The low amount of moclobemide excreted into breast milk is unlikely to be hazardous to suckling infants. PMID:2297459

Metabolic profiles of biological aging in American Indians: the Strong Heart Family Study.

PubMed

Zhao, Jinying; Zhu, Yun; Uppal, Karan; Tran, ViLinh T; Yu, Tianwei; Lin, Jue; Matsuguchi, Tet; Blackburn, Elizabeth; Jones, Dean; Lee, Elisa T; Howard, Barbara V

2014-03-01

Short telomere length, a marker of biological aging, has been associated with age-related metabolic disorders. Telomere attrition induces profound metabolic dysfunction in animal models, but no study has examined the metabolome of telomeric aging in human. Here we studied 423 apparently healthy American Indians participating in the Strong Family Heart Study. Leukocyte telomere length (LTL) was measured by qPCR. Metabolites in fasting plasma were detected by untargeted LC/MS. Associations of LTL with each metabolite and their combined effects were examined using generalized estimating equation adjusting for chronological age and other aging-related factors. Multiple testing was corrected using the q-value method (q<0.05). Of the 1,364 distinct m/z features detected, nineteen metabolites in the classes of glycerophosphoethanolamines, glycerophosphocholines, glycerolipids, bile acids, isoprenoids, fatty amides, or L-carnitine ester were significantly associated with LTL, independent of chronological age and other aging-related factors. Participants with longer (top tertile) and shorter (bottom tertile) LTL were clearly separated into distinct groups using a multi-marker score comprising of all these metabolites, suggesting that these newly detected metabolites could be novel metabolic markers of biological aging. This is the first study to interrogate the human metabolome of telomeric aging. Our results provide initial evidence for a metabolic control of LTL and may reveal previously undescribed new roles of various lipids in the aging process.

Lower Concentrations of Circulating Medium and Long-Chain Acylcarnitines Characterize Insulin Resistance in Persons with HIV.

PubMed

Bailin, Samuel S; Jenkins, Cathy A; Petucci, Christopher; Culver, Jeffrey A; Shepherd, Bryan E; Fessel, Joshua P; Hulgan, Todd; Koethe, John R

2018-05-02

In human immunodeficiency virus (HIV)-negative individuals, a plasma metabolite profile, characterized by higher levels of branched-chain amino acids (BCAA), aromatic amino acids, and C3/C5 acylcarnitines, is associated with insulin resistance and increased risk of diabetes. We sought to characterize the metabolite profile accompanying insulin resistance in HIV-positive persons to assess whether the same or different bioenergetics pathways might be implicated. We performed an observational cohort study of 70 nondiabetic, HIV-positive individuals (50% with body mass index ≥30 kg/m 2 ) on efavirenz, tenofovir, and emtricitabine with suppressed HIV-1 RNA levels (<50 copies/mL) for at least 2 years and a CD4 + count over 350 cells/μL. We measured fasting insulin resistance using the homeostatic model assessment 2, plasma free fatty acids (FFA) using gas chromatography, and amino acids, acylcarnitines, and organic acids using liquid chromatography/mass spectrometry. We assessed the relationship of plasma metabolites with insulin resistance using multivariable linear regression. The median age was 45 years, median CD4 + count was 701 cells/μL, and median hemoglobin A1c was 5.2%. Insulin resistance was associated with higher plasma C3 acylcarnitines (p = .01), but not BCAA or C5 acylcarnitines. However, insulin resistance was associated with lower plasma levels of C18, C16, C12, and C2 acylcarnitines (p ≤ .03 for all), and lower C18 and C16 acylcarnitine:FFA ratios (p = .002, and p = .03, respectively). In HIV-positive persons, lower levels of plasma acylcarnitines, including the C2 product of complete fatty acid oxidation, are a more prominent feature of insulin resistance than changes in BCAA, suggesting impaired fatty acid uptake and/or mitochondrial oxidation is a central aspect of glucose intolerance in this population.

Clozapine response and plasma catecholamines and their metabolites.

PubMed

Green, A I; Alam, M Y; Sobieraj, J T; Pappalardo, K M; Waternaux, C; Salzman, C; Schatzberg, A F; Schildkraut, J J

1993-02-01

The atypical neuroleptic clozapine has an unusual profile of clinical effects and a distinctive spectrum of pharmacological actions. Plasma measures of catecholamines and their metabolites have been used in the past to study the action of typical neuroleptics. We obtained longitudinal assessments of plasma measures of dopamine (pDA), norepinephrine (pNE), and their metabolites, homovanillic acid (pHVA) and 3-methoxy-4-hydroxyphenylglycol (pMHPG), in eight treatment-resistant or treatment-intolerant schizophrenic patients who were treated with clozapine for 12 weeks following a prolonged drug-washout period. Our findings from the study of these eight patients suggest the following: Plasma levels of HVA and possibly NE derived from the neuroleptic-free baseline period may predict response to clozapine; plasma levels of HVA and MHPG decrease during the initial weeks of treatment in responders but not in nonresponders; and plasma levels of DA and NE increase in both responders and nonresponders to clozapine.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

PubMed

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Disposition and metabolism of semagacestat, a {gamma}-secretase inhibitor, in humans.

PubMed

Yi, Ping; Hadden, Chad; Kulanthaivel, Palaniappan; Calvert, Nathan; Annes, William; Brown, Thomas; Barbuch, Robert J; Chaudhary, Archana; Ayan-Oshodi, Mosun A; Ring, Barbara J

2010-04-01

Semagacestat is a functional gamma-secretase inhibitor that has been shown to reduce the rate of formation of amyloid-beta in vitro and in vivo. This study was conducted to characterize the disposition of semagacestat in humans. After a single 140-mg dose of [(14)C]semagacestat administered as an oral solution to six healthy male subjects, semagacestat was rapidly absorbed (T(max) approximately 0.5 h) and eliminated from the systemic circulation (terminal t(1/2) approximately 2.4 h). The major circulating metabolites of semagacestat, M2 (hydrolysis of the amide bond proximal to the benzazepine ring) and M3 (benzylic hydroxylation of the benzazepine ring), accounted for approximately 27 and 10% of total radioactivity exposure, respectively, as calculated from relative area under the plasma concentration versus time curve from 0 to 24 h derived from the plasma radiochromatograms. The radioactive dose was almost completely recovered after 7 days postdose, with 87% of the dose in urine and 8% in feces. Unchanged [(14)C]semagacestat in urine accounted for approximately 44% of the dose, which indicates that renal excretion played an important role in elimination. Metabolites M2 and M3, with their related secondary metabolites, each accounted for approximately 20% of the dose in excreta. In vitro data indicate the formation of M3 is primarily mediated by CYP3A, with cDNA-expressed CYP3A5 approximately 2 times more efficient than CYP3A4 in forming M3. Thus, the relative content of CYP3A4 and CYP3A5 in humans will likely determine the formation clearance of M3 after exposure to semagacestat.

A longitudinal analysis of the effects of age on the blood plasma metabolome in the common marmoset, Callithrix jacchus

PubMed Central

Hoffman, Jessica M.; Tran, ViLinh; Wachtman, Lynn M.; Green, Cara L.; Jones, Dean P.; Promislow, Daniel E.L.

2016-01-01

Primates tend to be long-lived for their size with humans being the longest lived of all primates. There are compelling reasons to understand the underlying age-related processes that shape human lifespan. But the very fact of our long lifespan that makes it so compelling, also makes it especially difficult to study. Thus, in studies of aging, researchers have turned to non-human primate models, including chimpanzees, baboons, and rhesus macaques. More recently, the common marmoset, Callithrix jacchus, has been recognized as a particularly valuable model in studies of aging, given its small size, ease of housing in captivity, and relatively short lifespan. However, little is known about the physiological changes that occur as marmosets age. To begin to fill in this gap, we utilized high sensitivity metabolomics to define the longitudinal biochemical changes associated with age in the common marmoset. We measured 2104 metabolites from blood plasma at three separate time points over a 17-month period, and we completed both a cross-sectional and longitudinal analysis of the metabolome. We discovered hundreds of metabolites associated with age and body weight in both male and female animals. Our longitudinal analysis identified age-associated metabolic pathways that were not found in our cross-sectional analysis. Pathways enriched for age-associated metabolites included tryptophan, nucleotide, and xenobiotic metabolism, suggesting these biochemical pathways might play an important role in the basic mechanisms of aging in primates. Moreover, we found that many metabolic pathways associated with age were sex specific. Our work illustrates the power of longitudinal approaches, even in a short time frame, to discover novel biochemical changes that occur with age. PMID:26805607

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

PubMed

Bowden, John A; Heckert, Alan; Ulmer, Candice Z; Jones, Christina M; Koelmel, Jeremy P; Abdullah, Laila; Ahonen, Linda; Alnouti, Yazen; Armando, Aaron M; Asara, John M; Bamba, Takeshi; Barr, John R; Bergquist, Jonas; Borchers, Christoph H; Brandsma, Joost; Breitkopf, Susanne B; Cajka, Tomas; Cazenave-Gassiot, Amaury; Checa, Antonio; Cinel, Michelle A; Colas, Romain A; Cremers, Serge; Dennis, Edward A; Evans, James E; Fauland, Alexander; Fiehn, Oliver; Gardner, Michael S; Garrett, Timothy J; Gotlinger, Katherine H; Han, Jun; Huang, Yingying; Neo, Aveline Huipeng; Hyötyläinen, Tuulia; Izumi, Yoshihiro; Jiang, Hongfeng; Jiang, Houli; Jiang, Jiang; Kachman, Maureen; Kiyonami, Reiko; Klavins, Kristaps; Klose, Christian; Köfeler, Harald C; Kolmert, Johan; Koal, Therese; Koster, Grielof; Kuklenyik, Zsuzsanna; Kurland, Irwin J; Leadley, Michael; Lin, Karen; Maddipati, Krishna Rao; McDougall, Danielle; Meikle, Peter J; Mellett, Natalie A; Monnin, Cian; Moseley, M Arthur; Nandakumar, Renu; Oresic, Matej; Patterson, Rainey; Peake, David; Pierce, Jason S; Post, Martin; Postle, Anthony D; Pugh, Rebecca; Qiu, Yunping; Quehenberger, Oswald; Ramrup, Parsram; Rees, Jon; Rembiesa, Barbara; Reynaud, Denis; Roth, Mary R; Sales, Susanne; Schuhmann, Kai; Schwartzman, Michal Laniado; Serhan, Charles N; Shevchenko, Andrej; Somerville, Stephen E; St John-Williams, Lisa; Surma, Michal A; Takeda, Hiroaki; Thakare, Rhishikesh; Thompson, J Will; Torta, Federico; Triebl, Alexander; Trötzmüller, Martin; Ubhayasekera, S J Kumari; Vuckovic, Dajana; Weir, Jacquelyn M; Welti, Ruth; Wenk, Markus R; Wheelock, Craig E; Yao, Libin; Yuan, Min; Zhao, Xueqing Heather; Zhou, Senlin

2017-12-01

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Determination of metabolite of nicergoline in human plasma by high-performance liquid chromatography and its application in pharmacokinetic studies.

PubMed

Zheng, Rong; Wu, Yi-Hong; Jiang, De-Xi; Zhang, Dan

2012-02-01

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of 10 α -methoxy-6-methyl ergoline-8 β -methanol (MDL, a main metabolite of nicergoline) in human plasma. One-step liquid-liquid extraction (LLE) with diethyl ether was employed as the sample preparation method. Tizanidine hydrochloride was selected as the internal standard (IS). Analysis was carried out on a Diamonsil ODS column (150 mm×4.6 mm, 5 μm) using acetonitrile-ammonium acetate (0.1 mol/L) (15/85, v/v) as mobile phase at detection wavelength of 224 nm. The calibration curves were linear over the range of 2.288-73.2 ng/mL with a lower limit of quantitation (LLOQ) of 2.288 ng/mL. The intra- and inter-day precision values were below 13% and the recoveries were from 74.47% to 83.20% at three quality control levels. The method herein described was successfully applied in a randomized crossover bioequivalence study of two different nicergoline preparations after administration of 30 mg in 20 healthy volunteers.

Determination of metabolite of nicergoline in human plasma by high-performance liquid chromatography and its application in pharmacokinetic studies

PubMed Central

Zheng, Rong; Wu, Yi-Hong; Jiang, De-Xi; Zhang, Dan

2012-01-01

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of 10α-methoxy-6-methyl ergoline-8β-methanol (MDL, a main metabolite of nicergoline) in human plasma. One-step liquid–liquid extraction (LLE) with diethyl ether was employed as the sample preparation method. Tizanidine hydrochloride was selected as the internal standard (IS). Analysis was carried out on a Diamonsil ODS column (150 mm×4.6 mm, 5 μm) using acetonitrile–ammonium acetate (0.1 mol/L) (15/85, v/v) as mobile phase at detection wavelength of 224 nm. The calibration curves were linear over the range of 2.288–73.2 ng/mL with a lower limit of quantitation (LLOQ) of 2.288 ng/mL. The intra- and inter-day precision values were below 13% and the recoveries were from 74.47% to 83.20% at three quality control levels. The method herein described was successfully applied in a randomized crossover bioequivalence study of two different nicergoline preparations after administration of 30 mg in 20 healthy volunteers. PMID:29403722

Identification of Metabolism and Excretion Differences of Procymidone between Rats and Humans Using Chimeric Mice: Implications for Differential Developmental Toxicity.

PubMed

Abe, Jun; Tomigahara, Yoshitaka; Tarui, Hirokazu; Omori, Rie; Kawamura, Satoshi

2018-02-28

A metabolite of procymidone, hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of hydroxylated-PCM was less, resulting in a much lower exposure. The main excretion route of hydroxylated-PCM-glucuronide was bile (the point that hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone's potential for causing teratogenicity in humans must be low compared to that in rats.

Dexamethasone decreases plasma levels of the prochiral fenbendazole and its chiral and achiral metabolites in sheep.

PubMed

Sánchez, S; Small, J; Jones, D G; McKellar, Q A

2003-07-01

1. The effect of co-administration of either short- or long-acting formulations of DXM on hepatic function and the plasma pharmacokinetic behaviour of prochiral fenbendazole (FBZ) and its metabolites was evaluated in sheep. 2. Neither DXM treatment markedly affected any of the biochemical markers of hepatic function tested. In contrast, both formulations significantly modified the plasma pharmacokinetic behaviour of FBZ and its metabolites. 3. Plasma FBZ concentrations and the associated area under the time-concentration curves were significantly lower, although the plasma detection period was longer (72 versus 48 h) in the DXM pretreated animals compared with those given FBZ alone. 4. DXM also appeared to alter the pattern of FBZ absorption, possibly through effects on abomasal pH. The shape of the plasma concentration-time curves for oxfendazole (OFZ) and fenbendazole sulphone (FBZSO(2)) were similar to FBZ, raising the possibility that DXM treatment may have altered the liver biotransformation of the parent drug. 5. The concentrations of the (+) chiral metabolite of OFZ were significantly lower in DXM pretreated animals compared with those given FBZ alone. The trend was similar for the (-) antipode, although the differences between DXM pretreated and non-pretreated animals were not statistically significant.

Human kidney methoxyflurane and sevoflurane metabolism. Intrarenal fluoride production as a possible mechanism of methoxyflurane nephrotoxicity.

PubMed

Kharasch, E D; Hankins, D C; Thummel, K E

1995-03-01

Methoxyflurane nephrotoxicity is mediated by cytochrome P450-catalyzed metabolism to toxic metabolites. It is historically accepted that one of the metabolites, fluoride, is the nephrotoxin, and that methoxyflurane nephrotoxicity is caused by plasma fluoride concentrations in excess of 50 microM. Sevoflurane also is metabolized to fluoride ion, and plasma concentrations may exceed 50 microM, yet sevoflurane nephrotoxicity has not been observed. It is possible that in situ renal metabolism of methoxyflurane, rather than hepatic metabolism, is a critical event leading to nephrotoxicity. We tested whether there was a metabolic basis for this hypothesis by examining the relative rates of methoxyflurane and sevoflurane defluorination by human kidney microsomes. Microsomes and cytosol were prepared from kidneys of organ donors. Methoxyflurane and sevoflurane metabolism were measured with a fluoride-selective electrode. Human cytochrome P450 isoforms contributing to renal anesthetic metabolism were identified by using isoform-selective inhibitors and by Western blot analysis of renal P450s in conjunction with metabolism by individual P450s expressed from a human hepatic complementary deoxyribonucleic acid library. Sevoflurane and methoxyflurane did undergo defluorination by human kidney microsomes. Fluoride production was dependent on time, reduced nicotinamide adenine dinucleotide phosphate, protein concentration, and anesthetic concentration. In seven human kidneys studied, enzymatic sevoflurane defluorination was minima, whereas methoxyflurane defluorination rates were substantially greater and exhibited large interindividual variability. Kidney cytosol did not catalyze anesthetic defluorination. Chemical inhibitors of the P450 isoforms 2E1, 2A6, and 3A diminished methoxyflurane and sevoflurane defluorination. Complementary deoxyribonucleic acid-expressed P450s 2E1, 2A6, and 3A4 catalyzed methoxyflurane and sevoflurane metabolism, in diminishing order of activity. These three P450s catalyzed the defluorination of methoxyflurane three to ten times faster than they did that of sevoflurane. Expressed P450 2B6 also catalyzed methoxyflurane defluorination, but 2B6 appeared not to contribute to renal microsomal methoxyflurane defluorination because the P450 2B6-selective inhibitor had no effect. Human kidney microsomes metabolize methoxyflurane, and to a much lesser extent sevoflurane, to fluoride ion. P450s 2E1 and/or 2A6 and P450 3A are implicated in the defluorination. If intrarenally generated fluoride or other metabolites are nephrotoxic, then renal metabolism may contribute to methoxyflurane nephrotoxicity. The relative paucity of renal sevoflurane defluorination may explain the absence of clinical sevoflurane nephrotoxicity to date, despite plasma fluoride concentrations that may exceed 50 microM.

Analysis of Human Plasma Metabolites across Different Liquid Chromatography - Mass Spectrometry Platforms: Cross-platform Transferable Chemical Signatures

PubMed Central

Telu, Kelly H.; Yan, Xinjian; Wallace, William E.; Stein, Stephen E.; Simón-Manso, Yamil

2016-01-01

RATIONALE The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different LC-MS platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. METHODS Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC-MS platforms using reversed phase chromatography and different chromatographic scales (nano, conventional and UHPLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). RESULTS Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (RSD < 2%); however, substantial differences were found in the LC-MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. CONLUSIONS Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable. PMID:26842580

Discovery and Validation of Pyridoxic Acid and Homovanillic Acid as Novel Endogenous Plasma Biomarkers of Organic Anion Transporter (OAT) 1 and OAT3 in Cynomolgus Monkeys.

PubMed

Shen, Hong; Nelson, David M; Oliveira, Regina V; Zhang, Yueping; Mcnaney, Colleen A; Gu, Xiaomei; Chen, Weiqi; Su, Ching; Reily, Michael D; Shipkova, Petia A; Gan, Jinping; Lai, Yurong; Marathe, Punit; Humphreys, W Griffith

2018-02-01

Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Thawing as a critical pre-analytical step in the lipidomic profiling of plasma samples: New standardized protocol.

PubMed

Pizarro, Consuelo; Arenzana-Rámila, Irene; Pérez-del-Notario, Nuria; Pérez-Matute, Patricia; González-Sáiz, José María

2016-03-17

Lipid profiling is a promising tool for the discovery and subsequent identification of biomarkers associated with various diseases. However, data quality is quite dependent on the pre-analytical methods employed. To date, potential confounding factors that may affect lipid metabolite levels after the thawing of plasma for biomarker exploration studies have not been thoroughly evaluated. In this study, by means of experimental design methodology, we performed the first in-depth examination of the ways in which thawing conditions affect lipid metabolite levels. After the optimization stage, we concluded that temperature, sample volume and the thawing method were the determining factors that had to be exhaustively controlled in the thawing process to ensure the quality of biomarker discovery. Best thawing conditions were found to be: 4 °C, with 0.25 mL of human plasma and ultrasound (US) thawing. The new US proposed thawing method was quicker than the other methods we studied, allowed more features to be identified and increased the signal of the lipids. In view of its speed, efficiency and detectability, the US thawing method appears to be a simple, economical method for the thawing of plasma samples, which could easily be applied in clinical laboratories before lipid profiling studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultra-sensitive LC-MS/MS method for the quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma for a microdose clinical trial.

PubMed

van Nuland, M; Hillebrand, M J X; Rosing, H; Burgers, J A; Schellens, J H M; Beijnen, J H

2018-03-20

In microdose clinical trials a maximum of 100 μg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC-MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC-MS/MS method for quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5-500 pg/mL for gemcitabine and 250-50,000 pg/mL for dFdU were linear, with a correlation coefficient (r 2 ) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioavailability of anthocyanins and colonic polyphenol metabolites following consumption of aronia berry extract.

PubMed

Xie, Liyang; Lee, Sang Gil; Vance, Terrence M; Wang, Ying; Kim, Bohkyung; Lee, Ji-Young; Chun, Ock K; Bolling, Bradley W

2016-11-15

A single-dose pharmacokinetic trial was conducted in 6 adults to evaluate the bioavailability of anthocyanins and colonic polyphenol metabolites after consumption of 500mg aronia berry extract. UHPLC-MS methods were developed to quantitate aronia berry polyphenols and their metabolites in plasma and urine. While anthocyanins were bioavailable, microbial phenolic catabolites increased ∼10-fold more than anthocyanins in plasma and urine. Among the anthocyanins, cyanidin-3-O-galactoside was rapidly metabolized to peonidin-3-O-galactoside. Aronia polyphenols were absorbed and extensively metabolized with tmax of anthocyanins and other polyphenol catabolites from 1.0h to 6.33h in plasma and urine. Despite significant inter-individual variation in pharmacokinetic parameters, concentrations of polyphenol metabolites in plasma and urine at 24h were positively correlated with total AUC in plasma and urine (r=0.93, and r=0.98, respectively). This suggests that fasting blood and urine collections could be used to estimate polyphenol bioavailability and metabolism after aronia polyphenol consumption. Copyright © 2016 Elsevier Ltd. All rights reserved.

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

PubMed Central

Moaddel, Ruin; Venkata, Swarajya Lakshmi Vattem; Tanga, Mary J.; Bupp, James E.; Green, Carol E.; Iyer, Lalitha; Furimsky, Anna; Goldberg, Michael E.; Torjman, Marc C.; Wainer, Irving W.

2010-01-01

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in Complex Regional Pain Syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)-and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C18 stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary, metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients. PMID:20875593

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome.

PubMed

Moaddel, Ruin; Venkata, Swarajya Lakshmi Vattem; Tanga, Mary J; Bupp, James E; Green, Carol E; Iyer, Lalitha; Furimsky, Anna; Goldberg, Michael E; Torjman, Marc C; Wainer, Irving W

2010-10-15

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in complex regional pain syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)- and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C(18) stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients. Published by Elsevier B.V.

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans.

PubMed

Bartolomé, Begoña; Monagas, María; Garrido, Ignacio; Gómez-Cordovés, Carmen; Martín-Alvarez, Pedro J; Lebrón-Aguilar, Rosa; Urpí-Sardà, Mireia; Llorach, Rafael; Andrés-Lacueva, Cristina

2010-09-01

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism. 2010 Elsevier Inc. All rights reserved.

Clinical features and hormonal profiles of cloprostenol-induced early abortions in heifers monitored by ultrasonography

PubMed Central

Lobago, Fikre; Gustafsson, Hans; Bekana, Merga; Beckers, Jean-François; Kindahl, Hans

2006-01-01

Background The present study describes the clinical features and plasma profiles of bovine pregnancy-associated glycoprotein 1 (bPAG1), the main metabolite of prostaglandin F2α (PG metabolite) and progesterone (P4) in heifers in which early abortions were induced. Methods Early abortions were induced in four heifers with cloprostenol and monitored by ultrasonography. Blood samples were collected and the plasma were analyzed for bPAG 1, P4 and PG metabolite. Results The foetal heartbeat rates varied from 170–186 beats per minute for all foetuses up to the date of cloprostenol treatment. Foetal death was confirmed within two days after cloprostenol treatment. Prior to cloprostenol injection, blood plasma concentrations of bPAG1, PG metabolite and P4 varied from 8.4 – 40.0 ng/mL, 158 – 275 pmol/L and 20.7 – 46.9 nmol/L, respectively. After the foetus expelled, the plasma level of bPAG1 began to decrease but the decrease was small and gradual. The estimated half-life of bPAG1 was 1.8 – 6.6 days. The plasma level of the PG metabolite started to have short lasting peaks (above 300 pmol/L) within three hours after cloprostenol treatment. The plasma concentrations of P4 dropped sharply to less than 4 nmol/L after 24 hours of cloprostenol injection. Conclusion The current findings indicated that after early closprostenol-induced foetal death, the plasma concentration of bPAG1 decreased gradually and showed a tendency of variation with the stages of pregnancy. PMID:17121683

Omega-3 Long-Chain Fatty Acids in the Heart, Kidney, Liver and Plasma Metabolite Profiles of Australian Prime Lambs Supplemented with Pelleted Canola and Flaxseed Oils.

PubMed

Nguyen, Don V; Le, Van H; Nguyen, Quang V; Malau-Aduli, Bunmi S; Nichols, Peter D; Malau-Aduli, Aduli E O

2017-08-17

The objective of the study was to ascertain whether human health beneficial omega-3 long-chain (≥C 20 ) polyunsaturated fatty acid ( n -3 LC-PUFA) content in heart, kidney and liver can be enhanced by supplementing prime lambs with graded levels of canola and flaxseed oil. Health status of the lambs, as a consequence of the supplementation, was also investigated by examining their plasma metabolites. Sixty purebred and first-cross lambs were allocated to one of five treatments of lucerne hay basal diet supplemented with isocaloric and isonitrogenous wheat-based pellets without oil inclusion (Control) or graded levels of canola oil at 2.5% (2.5C), 5% (5C), flaxseed oil at 2.5% (2.5F) and 5% (5F) in a completely randomised design. Pre-slaughter blood, post-slaughter kidney, liver and heart samples were analysed for plasma metabolite and fatty acid profiles. Summations of docosapentaenoic acid and docosahexaenoic acid, and total n -3 LC-PUFA were enhanced in the liver and kidney of 5F supplemented lambs with a marked decrease in n -6/ n -3 ratio and significant breed differences detected. There were generally no deleterious impacts on animal health status. A combination of 5% oil supplementation and lamb genetics is an effective and strategic management tool for enhancing n -3 LC-PUFA contents of heart, kidney and liver without compromising lamb health.

Quantitative metabolomics by H-NMR and LC-MS/MS confirms altered metabolic pathways in diabetes.

PubMed

Lanza, Ian R; Zhang, Shucha; Ward, Lawrence E; Karakelides, Helen; Raftery, Daniel; Nair, K Sreekumaran

2010-05-10

Insulin is as a major postprandial hormone with profound effects on carbohydrate, fat, and protein metabolism. In the absence of exogenous insulin, patients with type 1 diabetes exhibit a variety of metabolic abnormalities including hyperglycemia, glycosurea, accelerated ketogenesis, and muscle wasting due to increased proteolysis. We analyzed plasma from type 1 diabetic (T1D) humans during insulin treatment (I+) and acute insulin deprivation (I-) and non-diabetic participants (ND) by (1)H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. The aim was to determine if this combination of analytical methods could provide information on metabolic pathways known to be altered by insulin deficiency. Multivariate statistics differentiated proton spectra from I- and I+ based on several derived plasma metabolites that were elevated during insulin deprivation (lactate, acetate, allantoin, ketones). Mass spectrometry revealed significant perturbations in levels of plasma amino acids and amino acid metabolites during insulin deprivation. Further analysis of metabolite levels measured by the two analytical techniques indicates several known metabolic pathways that are perturbed in T1D (I-) (protein synthesis and breakdown, gluconeogenesis, ketogenesis, amino acid oxidation, mitochondrial bioenergetics, and oxidative stress). This work demonstrates the promise of combining multiple analytical methods with advanced statistical methods in quantitative metabolomics research, which we have applied to the clinical situation of acute insulin deprivation in T1D to reflect the numerous metabolic pathways known to be affected by insulin deficiency.

Omega–3 Long-Chain Fatty Acids in the Heart, Kidney, Liver and Plasma Metabolite Profiles of Australian Prime Lambs Supplemented with Pelleted Canola and Flaxseed Oils

PubMed Central

Nguyen, Don V.; Le, Van H.; Nguyen, Quang V.; Malau-Aduli, Bunmi S.; Nichols, Peter D.

2017-01-01

The objective of the study was to ascertain whether human health beneficial omega–3 long-chain (≥C20) polyunsaturated fatty acid (n-3 LC-PUFA) content in heart, kidney and liver can be enhanced by supplementing prime lambs with graded levels of canola and flaxseed oil. Health status of the lambs, as a consequence of the supplementation, was also investigated by examining their plasma metabolites. Sixty purebred and first-cross lambs were allocated to one of five treatments of lucerne hay basal diet supplemented with isocaloric and isonitrogenous wheat-based pellets without oil inclusion (Control) or graded levels of canola oil at 2.5% (2.5C), 5% (5C), flaxseed oil at 2.5% (2.5F) and 5% (5F) in a completely randomised design. Pre-slaughter blood, post-slaughter kidney, liver and heart samples were analysed for plasma metabolite and fatty acid profiles. Summations of docosapentaenoic acid and docosahexaenoic acid, and total n-3 LC-PUFA were enhanced in the liver and kidney of 5F supplemented lambs with a marked decrease in n-6/n-3 ratio and significant breed differences detected. There were generally no deleterious impacts on animal health status. A combination of 5% oil supplementation and lamb genetics is an effective and strategic management tool for enhancing n-3 LC-PUFA contents of heart, kidney and liver without compromising lamb health. PMID:28817082

Plasma urolithin metabolites correlate with improvements in endothelial function after red raspberry consumption: A double-blind randomized controlled trial.

PubMed

Istas, Geoffrey; Feliciano, Rodrigo P; Weber, Timon; Garcia-Villalba, Rocio; Tomas-Barberan, Francisco; Heiss, Christian; Rodriguez-Mateos, Ana

2018-05-24

Raspberries are a rich source of ellagitannins and anthocyanins. The aim of this work was to investigate whether raspberry consumption can improve vascular function and to understand which phenolic metabolites may be responsible for the effects. A 3 arm double-blind randomized controlled crossover human intervention trial was conducted in 10 healthy males. Flow-mediated dilation (FMD) was measured at baseline, 2 h, and 24 h post-consumption of 200 g and 400 g of red raspberries containing 201 or 403 mg of total (poly)phenols, or a matched control drink. Raspberry (poly)phenol metabolites were analyzed in plasma and urine by UPLC-QTOF mass spectrometry using authentic standards. Significant improvements in FMD were observed at 2 h (1.6% (95%CI 1.2, 1.9) and 1.2% (95% CI 0.8, 1.5)) and 24 h (1.0% (95% CI 0.6, 1.2) and 0.7% (95%CI 0.2, 0.9)) post-consumption of the 200 and 400 g raspberry drinks as compared to control, respectively. Plasma ellagic acid, urolithin A-3-glucuronide and urolithin A-sulfate correlated with the improvements in FMD at 2 and 24 h post consumption, respectively. Consumption of dietary achievable amounts of red raspberries acutely improves endothelial function up to 24 h and ellagitannins may be responsible for the observed effect. Copyright © 2018 Elsevier Inc. All rights reserved.

Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry.

PubMed

Jaremko, Malgorzata; Kasai, Yumi; Barginear, Myra F; Raptis, George; Desnick, Robert J; Yu, Chunli

2010-12-15

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.

Effects of fenfluramine on plasma homovanillic acid in healthy subjects.

PubMed

Hollander, E; Stein, D J; Saoud, J B; DeCaria, C M; Cooper, T B; Islam, M N; Liebowitz, M R; Stanley, M

1992-01-01

The specificity of fenfluramine as a pharmacological probe of the serotonin system has been questioned, since animal studies with high dose l-fenfluramine show increases in striatal levels of the dopamine metabolite homovanillic acid. To test the specificity of fenfluramine in humans with clinical doses, we compared plasma homovanillic acid (pHVA) concentration in healthy volunteers after administration of fenfluramine (60 mg) and placebo. There were no significant effects on pHVA, which supports previous findings that at doses used in pharmacological challenge paradigms, the effect of fenfluramine on the dopamine system is insufficient to alter measures of its change.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

PubMed

Dobrinas, Maria; Choong, Eva; Noetzli, Muriel; Cornuz, Jacques; Ansermot, Nicolas; Eap, Chin B

2011-11-15

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5 mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100 mm, 1.7 μm). A gradient program was used, with a 10 mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4 mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500 ng/mL for nicotine, 1-1000 ng/mL for cotinine, 2-1000 ng/mL for trans-3'-hydroxycotinine and 1-500 ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation. Copyright © 2011 Elsevier B.V. All rights reserved.

Vitamin C-lipid metabolites: uptake and retention and effect on plasma C-reactive protein and oxidized LDL levels in healthy volunteers.

PubMed

Pancorbo, Dario; Vazquez, Carlos; Fletcher, Mary Ann

2008-11-01

Previously, a novel formulation of vitamin C-lipid metabolites (PureWay-C) was shown to be more rapidly taken-up by human T-lymphocytes and more rapidly stimulate neurite outgrowth, fibroblast adhesion and inhibition of xenobiotic-induced T-cell hyperactivation. Here, PureWay-C serum levels were measured in healthy volunteers after oral supplementation. Plasma C-reactive protein and oxidized low density lipoprotein levels (LDL) were also measured. Healthy volunteers maintained a low vitamin C diet for 14 days and, following an overnight fast, received a single oral dose of (vitamin C) 1000 mg of either ascorbic acid (AA), calcium ascorbate (CaA), vitamin C-lipid metabolites (PureWay-C), or calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C). Blood samples were collected immediately prior to the oral dose administration and at various times post ingestion. Twenty-four-hour urine collections were saved for oxalate and uric acid assays. PureWay-C supplementation leads to the highest absolute serum vitamin C levels when compared to AA, CaA and Ester-C. PureWay-C provides a statistically significant greater serum level than calcium ascorbate at 1, 2, 4, and 6 hours post oral supplementation whereas Ester-C shows a less but slightly statistically significant increase at only 1 and 4 hours. Oral supplementation with PureWay-C also led to a greater reduction in plasma C-reactive protein and oxidized LDL levels compared to the other vitamin C formulations. PureWay-C is more rapidly absorbed and leads to higher serum vitamin C levels and greater reduction of plasma levels of inflammatory and oxidative stress markers than other forms of vitamin C, including Ester-C.

Determination of N-methylsuccinimide and 2-hydroxy-N-methylsuccinimide in human urine and plasma.

PubMed

Jönsson, B A; Akesson, B

1997-12-19

A method for determination of N-methylsuccinimide (MSI) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine and of MSI in human plasma was developed. MSI and 2-HMSI are metabolites of the widely used organic solvent N-methyl-2-pyrrolidone (NMP). MSI and 2-HMSI were purified from urine and plasma by C8 solid-phase extraction and analysed by gas chromatography-mass spectrometry in the negative-ion chemical ionisation mode. The intra-day precisions in urine were 2-6% for MSI (50 and 400 ng/ml) and 3-5% for 2-HMSI (1000 and 8000 ng/ml). For MSI in plasma it was 2% (60 and 1200 ng/ml). The between-day precisions in urine were 3-4% for MSI (100 and 1000 ng/ml) and 2-4% for 2-HMSI (10,000 and 18,000 ng/ml) and 3-4% for MSI in plasma (100 and 900 ng/ml). The recoveries from urine were 109-117% for MSI (50 and 400 ng/ml) and 81-89% for 2-HMSI (1000 and 8000 ng/ml). The recovery of MSI from plasma was 91-101% (50 and 500 ng/ml). The detection limits for MSI were 3 ng/ml in urine and 1 ng/ml in plasma and that of 2-HMSI in urine was 200 ng/ml. The method is applicable for analysis of urine and plasma samples from workers exposed to NMP.

Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys.

PubMed

Gokbulut, Cengiz; Akar, Ferda; McKellar, Quintin A

2006-07-01

Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO(2)) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstrating that ABZ was completely metabolised by first-pass mechanisms in donkeys. Maximum plasma concentrations (C(max)) of FBZSO (0.49microg/mL) and FBZSO(2) (0.60microg/mL) were detected at (t(max)) 5.67 and 8.00h, respectively, following administration of FBZSO. The area under the curve (AUC) of the sulphone metabolite (10.33microg h/mL) was significantly higher than that of the parent drug FBZSO (5.17microg h/mL). C(max) of albendazole sulphoxide (ABZSO) (0.08g/mL) and albendazole sulphone (ABZSO(2)) (0.04microg/mL) were obtained at 5.71 and 8.00h, respectively, following ABZ administration. The AUC of the sulphoxide metabolite (0.84microg h/mL) of ABZ was significantly higher than that of the sulphone metabolite (0.50microg h/mL). The highest dry-faecal concentrations of parent molecules were detected at 32, 34 and 30h for FBZSO, FBZ and ABZ, respectively. The sulphide metabolite was significantly higher than the parent molecule after FBZSO administration. The parent molecule was predominant in the faecal samples following FBZ administration. After ABZ administration, the parent molecule was significantly metabolised, probably by gastrointestinal microflora, to its sulphoxide metabolite (ABZSO) that showed a similar excretion profile to the parent molecule in the faecal samples. The AUC of the parent FBZ was significantly higher than that of FBZSO and ABZ in faeces. It is concluded that the plasma concentration of FBZSO was significantly higher than that of FBZ and ABZ. Although ABZ is not licensed for use in Equidae, its metabolites presented a greater plasma kinetic profile than FBZ which is licensed for use in horses. A higher metabolic capacity, first-pass effects and lower absorption of benzimidazoles in donkeys decrease bioavailability and efficacy compared to ruminants.

Absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract

PubMed Central

Atwell, Lauren L.; Hsu, Anna; Wong, Carmen P.; Stevens, Jan F.; Bella, Deborah; Yu, Tian-Wei; Pereira, Clifford B.; Löhr, Christiane V.; Christensen, John Mark; Dashwood, Roderick H.; Williams, David E.; Shannon, Jackilen; Ho, Emily

2015-01-01

Scope Sulforaphane (SFN), an isothiocyanate derived from crucifers, has numerous health benefits. SFN bioavailability from dietary sources is a critical determinant of its efficacy in humans. A key factor in SFN absorption is the release of SFN from its glucosinolate precursor, glucoraphanin, by myrosinase. Dietary supplements are used in clinical trials to deliver consistent SFN doses, but myrosinase is often inactivated in available supplements. We evaluated SFN absorption from a myrosinase-treated broccoli sprout extract (BSE) and are the first to report effects of twice daily, oral dosing on SFN exposure in healthy adults. Methods and results Subjects consumed fresh broccoli sprouts or the BSE, each providing 200 μmol SFN daily, as a single dose and as two 100-μmol doses taken 12 h apart. Using HPLC-MS/MS, we detected ~3 x higher SFN metabolite levels in plasma and urine of sprout consumers, indicating enhanced SFN absorption from sprouts. Twelve-hour dosing retained higher plasma SFN metabolite levels at later time points than 24-hour dosing. No dose responses were observed for molecular targets of SFN (i.e. heme oxygenase-1, histone deacetylase activity, p21). Conclusion We conclude that the dietary form and dosing schedule of SFN may impact SFN absorption and efficacy in human trials. PMID:25522265

Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and neonates.

PubMed

Page-Sharp, Madhu; Strunk, Tobias; Salman, Sam; Hibbert, Julie; Patole, Sanjay K; Manning, Laurens; Batty, Kevin T

2017-11-30

Advances in bioanalytical methods are facilitating micro-volume and dried blood spot (DBS) analysis of drugs in biological matrices for pharmacokinetic studies in children and neonates. We sought to develop a UPLC-MS/MS assay for simultaneous measurement of caffeine, pentoxifylline (PTX) and three metabolites of PTX in both plasma and DBS. Caffeine, PTX, the metabolites M1 (lisofylline), M4 and M5, and the internal standards (caffeine-d 9 and PTX-d 6 ) were separated using a Waters Aquity T3 UPLC C 18 column and gradient mobile phase (water-methanol-formic acid). Retention times for caffeine, M5, M4, PTX and M1 were 1.6, 1.7, 1.9, 2.0 and 2.1min, respectively, with a run time of 5min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1-50mg/L for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were the limits of quantification (100μg/L for caffeine and 10μg/L for PTX, M1, M4 and M5). Caffeine, PTX and the metabolites were stable in DBS for >34days at room and refrigerated temperatures. Plasma and DBS samples were obtained from 24 preterm infants recruited into a clinical pharmacokinetic study of PTX. Paired analysis indicated that DBS concentrations were 9% lower than concurrent plasma concentrations for caffeine, 7% lower for PTX (consistent with the blood:plasma ratio) and 13% lower for M1 (lisofylline). The validated UPLC-MS/MS method is suitable for micro-volume plasma and DBS analysis of caffeine, PTX and its metabolites for pharmacokinetic studies in paediatric patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Prediction of Losartan-Active Carboxylic Acid Metabolite Exposure Following Losartan Administration Using Static and Physiologically Based Pharmacokinetic Models.

PubMed

Nguyen, Hoa Q; Lin, Jian; Kimoto, Emi; Callegari, Ernesto; Tse, Susanna; Obach, R Scott

2017-09-01

The aim of this study was to evaluate a strategy based on static and dynamic physiologically based pharmacokinetic (PBPK) modeling for the prediction of metabolite and parent drug area under the time-concentration curve ratio (AUC m /AUC p ) and their PK profiles in humans using in vitro data when active transport processes are involved in disposition. The strategy was applied to losartan and its pharmacologically active metabolite carboxylosartan as test compounds. Hepatobiliary transport including transport-mediated uptake, canilicular and basolateral efflux, and metabolic clearance estimates were obtained from in vitro studies using human liver microsomes and sandwich-cultured hepatocytes. Human renal clearance of carboxylosartan was estimated from dog renal clearance using allometric scaling approach. All clearance mechanisms were mechanistically incorporated in a static model to predict the relative exposure of carboxylosartan versus losartan (AUC m /AUC p ). The predicted AUC m /AUC p were consistent with the observed data following intravenous and oral administration of losartan. Moreover, the in vitro parameters were used as initial parameters in PBPK permeability-limited disposition models to predict the concentration-time profiles for both parent and its active metabolite after oral administration of losartan. The PBPK model was able to recover the plasma profiles of both losartan and carboxylosartan, further substantiating the validity of this approach. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Plasma trimethylamine N-oxide concentration is associated with choline, phospholipids, and methyl metabolism.

PubMed

Obeid, Rima; Awwad, Hussain M; Rabagny, Yannick; Graeber, Stefan; Herrmann, Wolfgang; Geisel, Juergen

2016-03-01

Elevated plasma concentrations of the gut bacteria choline metabolite trimethylamine N-oxide (TMAO) are associated with atherosclerosis. However, the determinants of TMAO in humans require additional assessment. We examined cardiometabolic risk factors and pathways associated with TMAO concentrations in humans. A total of 283 individuals (mean ± SD age: 66.7 ± 9.0 y) were included in this observational study. Plasma concentrations of trimethylamine, TMAO, choline, lipids, phospholipids, and methyl metabolites were measured. Study participants were divided into 4 groups by median concentrations of TMAO and choline (4.36 and 9.7 μmol/L, respectively). Compared with the group with TMAO and choline concentrations that were less than the median (n = 82), the group with TMAO and choline concentrations that were at least the median (n = 83) was older and had lower high-density lipoprotein (HDL) cholesterol, phospholipids, and methylation potential, higher creatinine, betaine, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM), and higher percentages of men and subjects with diabetes. The difference in plasma TMAO concentrations between men and women (7.3 ± 10.0 compared with 5.4 ± 5.6 μmol/L, respectively) was NS after adjustment for age and creatinine (P = 0.455). The TMAO:trimethylamine ratio was higher in men (P < 0.001). Diabetes was associated with significantly higher plasma TMAO concentration (8.6 ± 12.2 compared with 5.4 ± 5.2 μmol/L) even after adjustments. Sex and diabetes showed an interactive effect on trimethylamine concentrations (P = 0.010) but not on TMAO concentrations (P = 0.950). Positive determinants of TMAO in a stepwise regression model that applied to the whole group were SAH, trimethylamine, choline, and female sex, whereas plasma phosphatidylcholine was a negative determinant. High TMAO and choline concentrations are associated with an advanced cardiometabolic risk profile. Diabetes is related to higher plasma TMAO concentrations but also to alterations in interrelated pathways such as lipids, phospholipids, and methylation. Elevated plasma TMAO concentrations likely reflect a specific metabolic pattern characterized by low HDL and phospholipids in addition to hypomethylation. This trial was registered at clinicaltrials.gov as NCT02586181 and NCT02588898. © 2016 American Society for Nutrition.

Mapping of the circulating metabolome reveals α-ketoglutarate as a predictor of morbid obesity-associated non-alcoholic fatty liver disease.

PubMed

Rodríguez-Gallego, E; Guirro, M; Riera-Borrull, M; Hernández-Aguilera, A; Mariné-Casadó, R; Fernández-Arroyo, S; Beltrán-Debón, R; Sabench, F; Hernández, M; del Castillo, D; Menendez, J A; Camps, J; Ras, R; Arola, L; Joven, J

2015-02-01

Obesity severely affects human health, and the accompanying non-alcoholic fatty liver disease (NAFLD) is associated with high morbidity and mortality. Rapid and non-invasive methods to detect this condition may substantially improve clinical care. We used liquid and gas chromatography-quadruple time-of-flight-mass spectrometry (LC/GC-QTOF-MS) analysis in a non-targeted metabolomics approach on the plasma from morbidly obese patients undergoing bariatric surgery to gain a comprehensive measure of metabolite levels. On the basis of these findings, we developed a method (GC-QTOF-MS) for the accurate quantification of plasma α-ketoglutarate to explore its potential as a novel biomarker for the detection of NAFLD. Plasma biochemical differences were observed between patients with and without NAFLD indicating that the accumulation of lipids in hepatocytes decreased β-oxidation energy production, reduced liver function and altered glucose metabolism. The results obtained from the plasma analysis suggest pathophysiological insights that link lipid and glucose disturbances with α-ketoglutarate. Plasma α-ketoglutarate levels are significantly increased in obese patients compared with lean controls. Among obese patients, the measurement of this metabolite differentiates between those with or without NAFLD. Data from the liver were consistent with data from plasma. Clinical utility was assessed, and the results revealed that plasma α-ketoglutarate is a fair-to-good biomarker in patients (n=230). Other common laboratory liver tests used in routine application did not favourably compare. Plasma α-ketoglutarate is superior to common liver function tests in obese patients as a surrogate biomarker of NAFLD. The measurement of this biomarker may potentiate the search for a therapeutic approach, may decrease the need for liver biopsy and may be useful in the assessment of disease progression.

Classification of LC columns based on the QSRR method and selectivity toward moclobemide and its metabolites.

PubMed

Plenis, Alina; Olędzka, Ilona; Bączek, Tomasz

2013-05-05

This paper focuses on a comparative study of the column classification system based on the quantitative structure-retention relationships (QSRR method) and column performance in real biomedical analysis. The assay was carried out for the LC separation of moclobemide and its metabolites in human plasma, using a set of 24 stationary phases. The QSRR models established for the studied stationary phases were compared with the column test performance results under two chemometric techniques - the principal component analysis (PCA) and the hierarchical clustering analysis (HCA). The study confirmed that the stationary phase classes found closely related by the QSRR approach yielded comparable separation for moclobemide and its metabolites. Therefore, the QSRR method could be considered supportive in the selection of a suitable column for the biomedical analysis offering the selection of similar or dissimilar columns with a relatively higher certainty. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of Silymarin, Glycyrrhizin, and Oxymatrine on the Pharmacokinetics of Ribavirin and Its Major Metabolite in Rats.

PubMed

Liao, Sha; Jin, Xueyuan; Li, Jinglai; Zhang, Tianhong; Zhang, Wenpeng; Shi, Weiguo; Fan, Shiyong; Wang, Xiaoying; Wang, Juan; Zhong, Bohua; Zhang, Zhenqing

2016-04-01

The herb-derived compounds silymarin, glycyrrhizin, and oxymatrine are widely used to treat chronic hepatitis C virus infections in China. They are often prescribed in combination with ribavirin, which has a narrow therapeutic index. We investigated the influence of these compounds on ribavirin pharmacokinetics following concurrent administration at the human dose in rats. Pharmacokinetic parameters were determined in rats following oral (p.o.) administration of ribavirin (30 mg/kg) with or without silymarin (40 mg/kg, p.o.), glycyrrhizin (15 mg/kg, intraperitoneal [i.p.]), or oxymatrine (60 mg/kg, p.o.). Compared with the animals in ribavirin group, silymarin significantly decreased the area under the plasma concentration-time curve (AUC0-t ) and the peak plasma concentration (Cmax ) of ribavirin and ribavirin base by 31.2-44.5% and 48.9-50.0%, respectively. Glycyrrhizin significantly decreased the Cmax and AUC0-t of both ribavirin and its metabolite by 35.3-37.6% and 38.6-39.8%, respectively. However, silymarin or glycyrrhizin did not change the ribavirin metabolite/parent ratios of the AUC and Cmax . Oxymatrine did not induce significant changes in ribavirin concentration, but it significantly decreased the Cmax (26.6%) and AUC (21.8%) of the metabolite. This study indicates that the therapeutic efficacy of ribavirin may be compromised by the concurrent administration of herbal medicines/dietary supplements containing silymarin, glycyrrhizin, or oxymatrine. Copyright © 2016 John Wiley & Sons, Ltd.

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

PubMed Central

2016-01-01

Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as β-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) enabled the identification and quantification of multiple types of Alb xenobiotic adducts in animals and humans during the past three decades. In this perspective, we highlight the history of Alb as a target protein for adduction to environmental and dietary genotoxicants, pesticides, and herbicides, common classes of medicinal drugs, and endogenous electrophiles, and the emerging analytical mass spectrometry technologies to identify Alb-toxicant adducts in humans. PMID:27989119

Pharmacokinetics of aniracetam and its metabolites in rats.

PubMed

Ogiso, T; Iwaki, M; Tanino, T; Ikeda, K; Paku, T; Horibe, Y; Suzuki, H

1998-05-01

The pharmacokinetics of aniracetam (AP), a new cognitive performance enhancer, and its main metabolites was investigated after intravenous (iv) and oral administrations to rat. The plasma levels of AP, 4-p-anisamidobutyric acid (ABA), and p-anisic acid (AA) were determined simultaneously by the HPLC method. The plasma concentrations of the parent drug and ABA quickly declined in a biexponential manner, with rapid terminal decay and a small mean residence time. However, AA yielded nonlinearly high levels at the initial times and the plasma concentrations of 2-pyrrolidinone (PD) were sustained over a relatively long time. When AA was administered intravenously, nonlinearity of the plasma concentrations was also found at higher doses. To describe the time course of the plasma levels of AP and its metabolites after iv administration, a pharmacokinetic model with seven compartments was applied, which included 10 first-order rate constants and one Michaelis-Menten constant. An approximate fit was obtained between the observed and calculated curves based on the model, except for the plasma concentrations of ABA. The plasma concentration-time profiles of AP and its metabolites following oral administration of AP (50 and 100 mg/kg) were similar to those after iv dosing, with the exception of PD, which showed much lower plasma levels than those after iv administration. Elimination of AP and ABA was rapid after oral dosing, and the bioavailability of AP was extremely small (11.4 and 8.6%). As a result, AP was largely metabolized to ABA, AA, and PD in rat.

Morphological characterisation of the cell-growth inhibitory activity of rooperol and pharmacokinetic aspects of hypoxoside as an oral prodrug for cancer therapy.

PubMed

Albrecht, C F; Theron, E J; Kruger, P B

1995-09-01

Hypoxoside is the major diglucoside isolated from the corms of the plant family Hypoxidaceae. It contains an unusual E-pent-1-en-4-yne 5-carbon bridging unit with two distal catechol groups to which the glucose moieties are attached. It is non-toxic for BL6 mouse melanoma cells in tissue culture on condition that the fetal calf serum in the medium is heat-inactivated for 1 hour at 56 degrees C in order to destroy endogenous beta-glucosidase activity. The latter catalyses hypoxoside conversion to its cytotoxic aglucone, rooperol, which, when tested as a pure chemical, caused 50% inhibition of BL6 melanoma cell growth at 10 micrograms/ml. Light and electron microscopy revealed that the cytotoxic effect of rooperol manifested as vacuolisation of the cytoplasm and formation of pores in the plasma membrane. Indications of apoptosis were also found. Pharmacokinetic studies on mice dosed intragastrically with hypoxoside showed that it was deconjugated by bacterial beta-glucosidase to form rooperol in the colon. Surprisingly, no hypoxoside or rooperol was detectable in the serum. Only phase II biotransformation products (sulphates and glucuronides) were present in the portal blood and bile. In contrast, however, in human serum after oral ingestion of hypoxoside, the metabolites can reach relatively high concentrations. Rooperol metabolites isolated from human urine were non-toxic for BL6 melanoma cells in culture up to a concentration of 200 micrograms/ml. In the presence of beta-glucuronidase, which released rooperol from the metabolites, 50% growth inhibition was achieved at a 75 micrograms/ml metabolite concentration. The supernatant of a human melanoma homogenate could also cause deconjugation of the metabolites to form rooperol.(ABSTRACT TRUNCATED AT 250 WORDS)

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies.

PubMed

Plenis, Alina; Chmielewska, Aleksandra; Konieczna, Lucyna; Lamparczyk, Henryk

2007-09-01

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers. Copyright (c) 2007 John Wiley & Sons, Ltd.

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows.

PubMed

Kowalczyk-Zieba, I; Woclawek-Potocka, I; Piskula, M K; Piotrowska-Tomala, K K; Boruszewska, D; Bah, M M; Siemieniuch, M J; Skarzynski, D J

2011-12-01

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows. Copyright © 2011. Published by Elsevier Inc.

Inhibitors of alprazolam metabolism in vitro: effect of serotonin-reuptake-inhibitor antidepressants, ketoconazole and quinidine.

PubMed Central

von Moltke, L L; Greenblatt, D J; Cotreau-Bibbo, M M; Harmatz, J S; Shader, R I

1994-01-01

1. The biotransformation of the triazolobenzodiazepine alprazolam (ALP) to its hydroxylated metabolites (4-OH-ALP and alpha-OH-ALP) was evaluated in human, monkey, rat, and mouse liver microsomes. 2. In all species 4-OH-ALP was the principal metabolite, accounting for 84% of clearance in human microsomes compared with 16% for alpha-OH-ALP. 3. Among the serotonin-specific reuptake inhibitors fluoxetine (FLU) and sertraline (SERT), and their respective demethylated metabolites norfluoxetine (NOR) and desmethylsertraline (DES), NOR was the most potent inhibitor (mean Ki for 4-OH-ALP formation in humans: 11 microM), FLU the weakest (Ki = 83 microM), with SERT and DES falling in between (Ki = 24 and 20 microM). 4. The in vitro data predict 29% inhibition of ALP clearance at mean FLU and NOR plasma concentrations of 77 ng ml-1 and 72 ng ml-1, respectively, after correction for liver:water partition ratios in the range of 12-14. The observed mean degree of inhibition in a previous in vivo study was 21%. 5. Ketoconazole was a potent inhibitor of ALP metabolism in vitro (Ki = 0.046 microM), suggesting that ALP hydroxylation is mediated by the cytochrome P450-3A sub-family. Quinidine was a weak inhibitor (Ki = 626 microM). PMID:7946933

Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma

PubMed Central

Teitelbaum, Aaron M.; Flaker, Alicia M.; Kharasch, Evan D.

2016-01-01

A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-minute run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1 ng/mL, respectively. All analytes were stable following freeze thaw cycles at −80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma. PMID:26963497

Novel sulphur-containing imatinib metabolites found by untargeted LC-HRMS analysis.

PubMed

Vrobel, Ivo; Friedecký, David; Faber, Edgar; Najdekr, Lukáš; Mičová, Kateřina; Karlíková, Radana; Adam, Tomáš

2017-06-15

Untargeted metabolite profiling using high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS), followed by data analysis with the Compound Discoverer 2.0™ software, was used to study the metabolism of imatinib in humans with chronic myeloid leukemia. Plasma samples from control (drug-free) and patient (treated with imatinib) groups were analyzed in full-scan mode and the unknown ions occurring only in the patient group were then, as potential imatinib metabolites, subjected to multi-stage fragmentation in order to elucidate their structure. The application of an untargeted approach, as described in this study, enabled the detection of 24 novel structurally unexpected metabolites. Several sulphur-containing compounds, probably originating after the reaction of reactive intermediates of imatinib with endogenous glutathione, were found and annotated as cysteine and cystine adducts. In the proposed mechanism, the cysteine adducts were formed after the rearrangement of piperazine moiety to imidazoline. On the contrary, in vivo S-N exchange occurred in the case of the cystine adducts. In addition, N-O exchange was observed in the collision cell in the course of the fragmentation of the cystine adducts. The presence of sulphur in the cysteine and cystine conjugates was proved by means of ultra-high resolution measurements using Orbitrap Elite. The detection of metabolites derived from glutathione might improve knowledge about the disposition of imatinib towards bioactivation and help to improve understanding of the mechanism of its hepatotoxicity or nephrotoxicity in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

The effects of diet and physical activity on plasma homovanillic acid in normal human subjects.

PubMed

Kendler, K S; Mohs, R C; Davis, K L

1983-03-01

This study examines the effect of diet and moderate physical activity on plasma levels of the dopamine metabolite homovanillic acid (HVA) in healthy young males. At weekly intervals, subjects were fed four isocaloric meals: polycose (pure carbohydrate), sustecal, low monoamine, and high monoamine. Moderate physical activity consisted of 30 minutes of exercise on a bicycle ergometer. The effect of diet on plasma HVA (pHVA) was highly significant. Compared to the polycose meal, the high monoamine meal significantly increased pHVA. Moderate physical activity also significantly increased pHVA. Future clinical studies using pHVA in man as an index of brain dopamine function should control for the effects of both diet and physical activity.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells.

PubMed

Hawwa, Ahmed F; Millership, Jeff S; Collier, Paul S; McElnay, James C

2009-02-20

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats.

PubMed

Lysne, Vegard; Bjørndal, Bodil; Vik, Rita; Nordrehaug, Jan Erik; Skorve, Jon; Nygård, Ottar; Berge, Rolf K

2015-06-04

The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP), with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.

A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats

PubMed Central

Lysne, Vegard; Bjørndal, Bodil; Vik, Rita; Nordrehaug, Jan Erik; Skorve, Jon; Nygård, Ottar; Berge, Rolf K.

2015-01-01

The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP), with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP. PMID:26053618

Comprehensive Plasma Metabolomic Analyses of Atherosclerotic Progression Reveal Alterations in Glycerophospholipid and Sphingolipid Metabolism in Apolipoprotein E-deficient Mice

PubMed Central

Dang, Vi T.; Huang, Aric; Zhong, Lexy H.; Shi, Yuanyuan; Werstuck, Geoff H.

2016-01-01

Atherosclerosis is the major underlying cause of most cardiovascular diseases. Despite recent advances, the molecular mechanisms underlying the pathophysiology of atherogenesis are not clear. In this study, comprehensive plasma metabolomics were used to investigate early-stage atherosclerotic development and progression in chow-fed apolipoprotein E-deficient mice at 5, 10 and 15 weeks of age. Comprehensive plasma metabolomic profiles, based on 4365 detected metabolite features, differentiate atherosclerosis-prone from atherosclerosis-resistant models. Metabolites in the sphingomyelin pathway were significantly altered prior to detectable lesion formation and at all subsequent time-points. The cytidine diphosphate-diacylglycerol pathway was up-regulated during stage I of atherosclerosis, while metabolites in the phosphatidylethanolamine and glycosphingolipid pathways were augmented in mice with stage II lesions. These pathways, involving glycerophospholipid and sphingolipid metabolism, were also significantly affected during the course of atherosclerotic progression. Our findings suggest that distinct plasma metabolomic profiles can differentiate the different stages of atherosclerotic progression. This study reveals that alteration of specific, previously unreported pathways of glycerophospholipid and sphingolipid metabolism are associated with atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. PMID:27721472

Exposure and food web transfer of pharmaceuticals in ospreys (Pandion haliaetus): Predictive model and empirical data

USGS Publications Warehouse

Lazarus, Rebecca S.; Rattner, Barnett A.; Du, Bowen; McGowan, Peter C.; Blazer, Vicki S.; Ottinger, Mary Ann

2015-01-01

The osprey (Pandion haliaetus) is a well-known sentinel of environmental contamination, yet no studies have traced pharmaceuticals through the water–fish–osprey food web. A screening-level exposure assessment was used to evaluate the bioaccumulation potential of 113 pharmaceuticals and metabolites, and an artificial sweetener in this food web. Hypothetical concentrations in water reflecting “wastewater effluent dominated” or “dilution dominated” scenarios were combined with pH-specific bioconcentration factors (BCFs) to predict uptake in fish. Residues in fish and osprey food intake rate were used to calculate the daily intake (DI) of compounds by an adult female osprey. Fourteen pharmaceuticals and a drug metabolite with a BCF greater than 100 and a DI greater than 20 µg/kg were identified as being most likely to exceed the adult human therapeutic dose (HTD). These 15 compounds were also evaluated in a 40 day cumulative dose exposure scenario using first-order kinetics to account for uptake and elimination. Assuming comparable absorption to humans, the half-lives (t1/2) for an adult osprey to reach the HTD within 40 days were calculated. For 3 of these pharmaceuticals, the estimated t1/2 in ospreys was less than that for humans, and thus an osprey might theoretically reach or exceed the HTD in 3 to 7 days. To complement the exposure model, 24 compounds were quantified in water, fish plasma, and osprey nestling plasma from 7 potentially impaired locations in Chesapeake Bay. Of the 18 analytes detected in water, 8 were found in fish plasma, but only 1 in osprey plasma (the antihypertensive diltiazem). Compared to diltiazem detection rate and concentrations in water (10/12 detects,

Simultaneous determination of pirfenidone and its metabolite in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

PubMed

Wen, Yu-Guan; Liu, Xia; He, Xiu-Ling; Shang, De-Wei; Ni, Xiao-Jia; Zhang, Ming; Wang, Zhan-Zhang; Hu, Jin-Qing; Qiu, Chang

2014-01-01

A simple and rapid analytical method for the simultaneous determination of pirfenidone and its metabolite, 5-carboxy-pirfenidone, in human plasma using liquid chromatography-tandem mass spectrometry has been developed and validated. Aliquots of plasma (0.1 mL) containing pirfenidone and 5-carboxy-pirfenidone, as well as deuterium-labeled internal standards (ISs), were deproteinized using acetonitrile. An Agilent Zorbax Plus C18 column was used for the chromatography, with isocratic elution. The mobile phase was a mixture of acetonitrile and aqueous ammonium formate solution (5 mM) containing 0.1% formic acid (60 : 40, v/v). Using multiple reaction monitoring in positive ionization mode, transitions m/z 186.1 → 65.1, m/z 216.0 → 77.0, m/z 191.1 → 65.1 and m/z 221.0 → 81.0 were chosen to quantify pirfenidone, 5-carboxy-pirfenidone and the two ISs, respectively. The time of analysis was <3 min. The calibration curve was linear over the concentration ranges 0.005-25 μg/mL for pirfenidone, and 0.005-15 μg/mL for 5-carboxy-pirfenidone. The lower limit of quantification for both analytes was 0.005 μg/mL. The intra- and interday precision and relative errors in quality control samples were between -11.7 and 1.3% for pirfenidone and between -5.6 and 2.5% for 5-carboxy-pirfenidone, with mean recoveries ≥90%. The method that has been developed is easy to carry out, sensitive and rapid, and has been successfully used to investigate the pharmacokinetics of pirfenidone in healthy human volunteers. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA) and Its Phase I and II Metabolites in Humans.

PubMed

Steuer, Andrea E; Schmidhauser, Corina; Tingelhoff, Eva H; Schmid, Yasmin; Rickli, Anna; Kraemer, Thomas; Liechti, Matthias E

2016-01-01

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from 0 to 24 h (AUC24) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the Cmax and AUC24 of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism.

Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA) and Its Phase I and II Metabolites in Humans

PubMed Central

Steuer, Andrea E.; Schmidhauser, Corina; Tingelhoff, Eva H.; Schmid, Yasmin; Rickli, Anna; Kraemer, Thomas; Liechti, Matthias E.

2016-01-01

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from 0 to 24 h (AUC24) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the Cmax and AUC24 of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism. PMID:26967321

Enantioselectivity in the Metabolism of Cyclophosphamide in Patients With Multiple or Systemic Sclerosis.

PubMed

de Castro, Francine Attié; Simões, Belinda Pinto; Coelho, Eduardo Barbosa; Lanchote, Vera Lucia

2017-06-01

The aim of this study was to evaluate the enantioselective pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard in patients with systemic or multiple sclerosis. Patients with systemic sclerosis (n = 10) or multiple sclerosis (n = 10), genotyped for the allelic variants of CYP2C9*2 and CYP2C9*3 and of the CYP2B6 G516T polymorphism, were treated with 50 mg cyclophosphamide/kg daily for 4 days. Serial blood samples were collected up to 24 hours after administration of the last cyclophosphamide dose. Cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide enantiomers were analyzed in plasma samples using liquid chromatography-tandem mass spectrometry coupled to chiral column Chiralcel OD-R or Chiralpak AD-RH. Cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-17, TNF-α, and INT-δ in the plasma samples collected before cyclophosphamide infusion were analyzed by Milliplex MAP human cytokine/chemokine. Pharmacokinetic parameters showed higher plasma concentrations of (S)-(-)-cyclophosphamide (AUC 215.0 vs 186.2 μg·h/mL for multiple sclerosis patients and 219.1 vs 179.2 μg·h/mL for systemic sclerosis patients) and (R)-4-hydroxycyclophosphamide (AUC 5.6 vs 3.7 μg·h/mL for multiple sclerosis patients and 6.3 vs 5.6 μg·h/mL for systemic sclerosis patients) when compared to their enantiomers in both groups of patients, whereas the pharmacokinetics of the carboxyethylphosphoramide metabolite was not enantioselective. Cytokines' plasma concentrations were similar between multiple and systemic sclerosis groups. The pharmacokinetics of cyclophosphamide is enantioselective in patients with systemic sclerosis and multiple sclerosis, with higher plasma concentrations of the (S)-(-)-cyclophosphamide enantiomer due to the preferential formation of the (R)-4-hydroxycyclophosphamide metabolite. © 2017, The American College of Clinical Pharmacology.

Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knobloch, M.; Portier, C.J.; Levionnois, O.L.

2006-11-01

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 {mu}g/ml S-ketamine over 120 min under isoflurane anesthesia was performed inmore » Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses)« less

Detection of Baicalin Metabolites Baicalein and Oroxylin-A in Mouse Pancreas and Pancreatic Xenografts

PubMed Central

Lu, Qing-Yi; Zhang, Lifeng; Moro, Aune; Chen, Monica C.; Harris, Diane M.; Eibl, Guido; Go, Vay-Liang W.

2011-01-01

Objectives Scutellaria baicalensis has been a subject of research interests due to its potential multiple therapeutic benefits. This study was to examine the distribution of baicalein, wogonin, oroxylin A and their glucuronide/sulfate conjugated metabolites in plasma, colon, small intestine, lung, liver, pancreas, kidney, and prostate tissues and in pancreatic tumor in a xenograft animal model. In addition, we examined metabolic stability of baicalin in these tissues. Methods A mouse xenograft model was prepared by injection of 3×106 human pancreatic cancer MiaPaCa-2 cells subcutaneously into nude mice. Mice were randomly allocated to control diet (AIN76A) and 1% SB diet (n=8 per group) for 13 weeks. Levels of baicalein, wogonin, oroxylin A, and their conjugates in mouce tissues were measured by high-pressure liquid chromatography following enzymatic hydrolysis and then extraction. Results A substantial amount of baicalin (34–63%) was methylated to oroxylin A and its conjugates in various organs during absorption. While plasma contained predominantly conjugates of baicalein, wogonin, and oroxylin A, both aglycones and conjugates were found in all other tissues investigated and in tumor. Conclusions Substantial accumulation of bioactive metabolites are found in target tissues, suggesting strong potential for SB use as a preventive or adjuvant supplement for pancreatic cancer. PMID:22158070

Different effects of clopidogrel and clarithromycin on the enantioselective pharmacokinetics of sibutramine and its active metabolites in healthy subjects.

PubMed

Shinde, Dhananjay D; Kim, Ho-Sook; Choi, Jae-Seok; Pan, Wei; Bae, Soo Kyung; Yeo, Chang-Woo; Shon, Ji-Hong; Kim, Dong-Hyun; Shin, Jae Gook

2013-05-01

In this study, we assessed the effects of clopidogrel and clarithromycin, known CYP2B6 and CYP3A inhibitors, respectively, on the enantioselective disposition of racemic sibutramine in conjunction with CYP2B6 polymorphisms in humans. Sibutramine showed enantioselective plasma profiles with consistently higher concentrations of R-enantiomers. Clopidogrel and clarithromycin significantly increased the sibutramine plasma concentration, but their effects differed between enantiomers; a 2.2-fold versus 4.1-fold increase in the AUC in S-enantiomer and 1.8-fold versus 2.0-fold for the R-enantiomer, respectively. The AUCs of S- and R-desmethyl metabolites changed significantly during the clopidogrel phase (P < .001 and P < .001, respectively) but not during the clarithromycin phase (P = .099 and P = .090, respectively). Exposure to sibutramine was higher in subjects with the CYP2B6*6/*6 genotype, but no statistical difference was observed among the CYP2B6 genotypes. These results suggest that the enantioselective disposition of sibutramine and its active metabolites are influenced by the altered genetic and environmental factors of CYP2B6 and CYP3A activity in vivo. © The Author(s) 2013.

Pharmacokinetic comparison of different flubendazole formulations in pigs: A further contribution to its development as a macrofilaricide molecule

PubMed Central

Ceballos, L.; Alvarez, L.; Mackenzie, C.; Geary, T.; Lanusse, C.

2015-01-01

Despite the well established ivermectin activity against microfilaria, the success of human filariasis control programmes requires the use of a macrofilaricide compound. Different in vivo trials suggest that flubendazole (FLBZ), an anthelmintic benzimidazole compound, is a highly efficacious and potent macrofilaricide. However, since serious injection site reactions were reported in humans after the subcutaneous FLBZ administration, the search for alternative pharmaceutical strategies to improve the systemic availability of FLBZ has acquired special relevance both in human and veterinary medicine. The goal of the current experimental work was to compare the pharmacokinetic plasma behavior of FLBZ, and its metabolites, formulated as either an aqueous hydroxypropyl- β -cyclodextrin-solution (HPBCD), an aqueous carboxymethyl cellulose-suspension (CMC) or a Tween 80-based formulation, in pigs. Animals were allocated into three groups and treated (2 mg/kg) with FLBZ formulated as either a HPBCD-solution (oral), CMC-suspension (oral) or Tween 80-based formulation (subcutaneous). Only trace amounts of FLBZ parent drug and its reduced metabolite were measured after administration of the different FLBZ formulations in pigs. The hydrolyzed FLBZ (H-FLBZ) metabolite was the main analyte recovered in the bloodstream in pigs treated with the three experimental FLBZ formulations. The oral administration of the HPBCD-solution accounted for significantly higher (P < 0.05) Cmax and AUC (23.1 ± 4.4 μg h/mL) values for the main metabolite (H-FLBZ), compared with those observed for the oral CMC-suspension (AUC = 3.5 ± 1.0 μg h/mL) and injectable Tween 80-based formulation (AUC: 7.5 ± 1.7 μg h/mL). The oral administration of the HPBCD-solution significantly improved the poor absorption pattern (indirectly assessed as the H-FLBZ plasma concentrations) observed after the oral administration of the FLBZ-CMC suspension or the subcutaneous injection of the Tween 80 FLBZ formulation to pigs. Overall, the work reported here indicates that FLBZ pharmacokinetic behavior can be markedly changed by the pharmaceutical formulation. PMID:27120064

Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma

PubMed Central

Sonawane, Poonam; Cho, Hwang Eui; Tagde, Ashujit; Verlekar, Dattesh; Yu, Alice L; Reynolds, C Patrick; Kang, Min H

2014-01-01

Background and Purpose Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA. Experimental Approach Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples. Key Results Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels. Conclusions and Implications We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma. PMID:25039756

Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma.

PubMed

Sonawane, Poonam; Cho, Hwang Eui; Tagde, Ashujit; Verlekar, Dattesh; Yu, Alice L; Reynolds, C Patrick; Kang, Min H

2014-12-01

Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA. Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples. Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels. We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma. © 2014 The British Pharmacological Society.

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

PubMed

Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

2014-11-01

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

Analysis of human plasma metabolites across different liquid chromatography/mass spectrometry platforms: Cross-platform transferable chemical signatures.

PubMed

Telu, Kelly H; Yan, Xinjian; Wallace, William E; Stein, Stephen E; Simón-Manso, Yamil

2016-03-15

The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different liquid chromatography/mass spectrometry (LC/MS) platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC/MS platforms using reversed-phase chromatography and different chromatographic scales (conventional HPLC, UHPLC and nanoLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (relative standard deviation (RSD) <2%); however, substantial differences were found in the LC/MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Associations between Repeated Measures of Maternal Urinary Phthalate Metabolites and Thyroid Hormone Parameters during Pregnancy

PubMed Central

Johns, Lauren E.; Ferguson, Kelly K.; McElrath, Thomas F.; Mukherjee, Bhramar; Meeker, John D.

2016-01-01

Background: Maintaining thyroid homeostasis during pregnancy is essential for normal fetal growth and development. Growing evidence suggests that phthalates interfere with normal thyroid function. Few human studies have investigated the degree to which phthalates may affect thyroid hormone levels in particularly susceptible populations such as pregnant women. Objectives: We examined the associations between repeated measures of urinary phthalate metabolites and plasma thyroid hormone levels in samples collected at up to four time points per subject in pregnancy. Additionally, we investigated the potential windows of susceptibility to thyroid hormone disturbances related to study visit of sample collection. Methods: Data were obtained from pregnant women (n = 439) participating in a nested case–control study of preterm birth with 116 cases and 323 controls. We measured 9 phthalate metabolite concentrations in urine samples collected at up to four study visits per subject during pregnancy (median = 10, 18, 26, and 35 weeks of gestation, respectively). We also measured a panel of thyroid function markers in plasma collected at the same four time points per subject during pregnancy. Results: Although our results were generally null, in repeated measures analyses we observed that phthalate metabolites were largely inversely associated with thyrotropin and positively associated with free and total thyroid hormones. Cross-sectional analyses by study visit revealed that the magnitude and/or direction of these relationships varied by timing of exposure during gestation. Conclusions: These results support previous reports showing the potential for environmental phthalate exposure to alter circulating levels of thyroid hormones in pregnant women. Citation: Johns LE, Ferguson KK, McElrath TF, Mukherjee B, Meeker JD. 2016. Associations between repeated measures of maternal urinary phthalate metabolites and thyroid hormone parameters during pregnancy. Environ Health Perspect 124:1808–1815; http://dx.doi.org/10.1289/EHP170 PMID:27152641

Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation.

PubMed

Sadeque, Abu J M; Palamar, Safet; Usmani, Khawja A; Chen, Chuan; Cerny, Matthew A; Chen, Weichao G

2016-04-01

Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT(2C) receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management. Lorcaserin is primarily cleared by metabolism, which involves multiple enzyme systems with various metabolic pathways in humans. The major circulating metabolite is lorcaserin N-sulfamate. Both human liver and renal cytosols catalyze the formation of lorcaserin N-sulfamate, where the liver cytosol showed a higher catalytic efficiency than renal cytosol. Human sulfotransferases (SULTs) SULT1A1, SULT1A2, SULT1E1, and SULT2A1 are involved in the formation of lorcaserin N-sulfamate. The catalytic efficiency of these SULTs for lorcaserin N-sulfamate formation is widely variable, and among the SULT isoforms SULT1A1 was the most efficient. The order of intrinsic clearance for lorcaserin N-sulfamate is SULT1A1 > SULT2A1 > SULT1A2 > SULT1E1. Inhibitory effects of lorcaserin N-sulfamate on major human cytochrome P450 (P450) enzymes were not observed or minimal. Lorcaserin N-sulfamate binds to human plasma protein with high affinity (i.e., >99%). Thus, despite being the major circulating metabolite, the level of free lorcaserin N-sulfamate would be minimal at a lorcaserin therapeutic dose and unlikely be sufficient to cause drug-drug interactions. Considering its formation kinetic parameters, high plasma protein binding affinity, minimal P450 inhibition or induction potential, and stability, the potential for metabolic drug-drug interaction or toxicological effects of lorcaserin N-sulfamate is remote in a normal patient population. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Determination of the Nicotine Metabolites Cotinine and Trans-3′-Hydroxycotinine in Biologic fluids of Smokers and Non-Smokers using Liquid Chromatography - Tandem Mass Spectrometry: Biomarkers for Tobacco Smoke Exposure and for Phenotyping Cytochrome P450 2A6 Activity

PubMed Central

Jacob, Peyton; Yu, Lisa; Duan, Minjiang; Ramos, Lita; Yturralde, Olivia; Benowitz, Neal L.

2011-01-01

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3′-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by Cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography – tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3′-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1 ng/ mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented. PMID:21208832

Reproducible diagnostic metabolites in plasma from typhoid fever patients in Asia and Africa.

PubMed

Näsström, Elin; Parry, Christopher M; Vu Thieu, Nga Tran; Maude, Rapeephan R; de Jong, Hanna K; Fukushima, Masako; Rzhepishevska, Olena; Marks, Florian; Panzner, Ursula; Im, Justin; Jeon, Hyonjin; Park, Seeun; Chaudhury, Zabeen; Ghose, Aniruddha; Samad, Rasheda; Van, Tan Trinh; Johansson, Anders; Dondorp, Arjen M; Thwaites, Guy E; Faiz, Abul; Antti, Henrik; Baker, Stephen

2017-05-09

Salmonella Typhi is the causative agent of typhoid. Typhoid is diagnosed by blood culture, a method that lacks sensitivity, portability and speed. We have previously shown that specific metabolomic profiles can be detected in the blood of typhoid patients from Nepal (Näsström et al., 2014). Here, we performed mass spectrometry on plasma from Bangladeshi and Senegalese patients with culture confirmed typhoid fever, clinically suspected typhoid, and other febrile diseases including malaria. After applying supervised pattern recognition modelling, we could significantly distinguish metabolite profiles in plasma from the culture confirmed typhoid patients. After comparing the direction of change and degree of multivariate significance, we identified 24 metabolites that were consistently up- or down regulated in a further Bangladeshi/Senegalese validation cohort, and the Nepali cohort from our previous work. We have identified and validated a metabolite panel that can distinguish typhoid from other febrile diseases, providing a new approach for typhoid diagnostics.

Disposition of lasofoxifene, a next-generation selective estrogen receptor modulator, in healthy male subjects.

PubMed

Prakash, Chandra; Johnson, Kim A; Gardner, Mark J

2008-07-01

Disposition of lasofoxifene, a next-generation selective estrogen receptor modulator, was investigated in male volunteers after p.o. administration of a single 20-mg dose of [(14)C]lasofoxifene. Approximately 72% of the administered dose was recovered from the urine and feces, with majority of dose excreted in the feces, probably via bile. The absorption of lasofoxifene in humans was slow with T(max) values typically exceeding 6 h. The C(max) and area under plasma concentration-time profile from time 0 to the last quantifiable time point values of lasofoxifene were lower than those determined for total radioactivity, indicating presence of circulating metabolites. The primary clearance mechanisms for lasofoxifene in humans were direct conjugation (glucuronide and sulfate conjugates) and phase I oxidation, each accounting for about half of the metabolism. Several oxidative metabolites were identified by liquid chromatography/tandem mass spectrometry. The primary phase I metabolites were the result of hydroxylations on the tetraline moiety and the phenyl rings attached to the tetraline, and oxidation on the pyrrolidine moiety. Considering the numerous metabolites seen in vivo, additional in vitro studies using human liver and intestinal microsomes, recombinant cytochromes P450 (P450s), and UDP glucuronosyltransferases (UGTs) were performed. The turnover of lasofoxifene was very slow in liver microsomes, and only two metabolites were identified as two regioisomers of the catechol metabolite. The results from in vitro experiments with recombinant isoforms and P450 isoform-selective inhibitors suggested that the oxidative metabolism of lasofoxifene is catalyzed primarily by CYP3A and CYP2D6. In addition, its glucuronidation is catalyzed by UGTs that are expressed in both the liver (UGT1A1, UGT1A3, UGT1A6, and UGT1A9) and the intestine (UGT1A8 and UGT1A10).

Development and validation of a method using supported liquid extraction for the simultaneous determination of midazolam and 1'-hydroxy-midazolam in human plasma by liquid chromatography with tandem mass spectrometry detection.

PubMed

Svanström, Camilla; Hansson, Gunnar P; Svensson, Leif D; Sennbro, Carl Johan

2012-01-25

The metabolic conversion of midazolam (MDZ) to its main metabolite 1'-hydroxy-midazolam (1-OH-MDZ) can be used as a probe drug for cytochrome P450 3A (CYP3A) activity. A sensitive method for the simultaneous determination of MDZ and its metabolite 1-OH-MDZ in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection was developed and validated. Plasma samples (100 μL) were diluted with 0.5M NH(3) (aq) containing deuterated internal standards. The samples were extracted with ethyl acetate on a 96-well SLE-plate. Separation was performed on a Symmetry Shield RP18 column using an acidic gradient running from 2% to 95% methanol in 3 min. Detection was performed using a triple quadrupole mass spectrometer running in positive electrospray selected reaction monitoring (SRM) mode. The validated dynamic range was 0.2-100 nmol/L for both analytes. In the concentration range 0.6-75 nmol/L the extraction recoveries were in the ranges 91.2-98.6% and 94.5-98.3% for MDZ and 1-OH-MDZ, respectively. Matrix effects were more pronounced for MDZ than for 1-OH-MDZ but the response was still 75.4% or higher compared to a reference. The overall repeatability was within 2.2-7.6% for both analytes, the overall reproducibility was within 3.1-10.2% for both analytes and the overall accuracy bias was within -1.1 to 7.5% for both analytes. The method was successfully applied to determine the plasma concentrations of MDZ and 1-OH-MDZ in 14 healthy volunteers up to 24h after administration of a single oral dose of 2mg MDZ. The SLE technology was found to be convenient and suitable for sample preparation, and the developed method was found to be rapid, selective and reproducible for the simultaneous determination of MDZ and 1-OH-MDZ in human plasma. Copyright © 2011 Elsevier B.V. All rights reserved.

Hair analysis for drugs of abuse. XVI. Disposition of fenethylline and its metabolite into hair and discrimination between fenethylline use and amphetamine use by hair analysis.

PubMed

Kikura, R; Nakahara, Y

1997-01-01

The incorporation tendency of fenethylline (FNT) and its metabolite into rat hair and the discrimination between FNT use and amphetamine (AP) use by hair analysis using gas chromatography-mass spectrometry with selected ion monitoring are described. After the intraperitoneal administrations of FNT to pigmented hairy rats (5 mg/kg/day, 10 days, n = 3), concentrations of FNT and its metabolite, AP, in the rat hair newly grown over 4 weeks were compared with area under the concentration versus time curves (AUCs) of the drugs in the rat plasma. The hair concentrations of FNT and AP were 52 +/- 1.4 and 4.9 +/- 0.6 ng/mg, whereas those of plasma AUCs were 55.9 +/- 23.1 and 22.3 +/- 4.9 micrograms.min/mL, respectively. The ratios of the hair concentrations to the AUCs of FNT tends to be highly incorporated into hair from suggests that FNT tends to be highly incorporated into hair from blood. The analytical method was applied to the determination of the metabolites in scalp hair of humans who were given FNT orally in multiple doses (50 mg/day, 3 days, n = 5) or in a single dose (50 mg/day, 1 day, n = 1). FNT and AP were detected at 0.51 +/- 0.23 and 0.35 +/- 0.12 ng/mg, respectively, in the proximal 1-cm hair segments from subjects given FNT orally for 3 days and 0.25 and 0.11 ng/mg, respectively, in the single-dose sample. In addition, it was found that the concentrations of FNT were 1.2 to 2.7 times greater than those of AP in the human hair samples, except for one sample, although FNT rapidly disappeared from the urine compared with AP. It was concluded that hair would be a good specimen for disclosure of drug history of FNT and for discrimination between FNT use and AP abuse.

Azithromycin metabolite identification in plasma, bile, and tissues of the ball python (Python regius).

PubMed

Hunter, R P; Koch, D E; Coke, R L; Goatley, M A; Isaza, R

2003-04-01

Azithromycin is the first of a class of antibiotics classified as azalides. Six ball pythons (Python regius) were given a single dose of azithromycin at 10 mg/kg p.o. and i.v. in a crossover design. Serial blood samples were collected for unchanged azithromycin and to determine, if possible, the structure and number of circulating azithromycin metabolites. After a 4-month wash-out period, the snakes were given azithromycin p.o. as a single dose of 10 mg/kg for the study of azithromycin metabolism and metabolite tissue distribution. Bile, liver, lung, kidney, and skin samples were analyzed for the metabolites identified from the first experiment. Unchanged azithromycin accounted for 80, 68, and 60% of the total material at 12, 24, and 48 h postadministration in plasma, independent of route of administration. At both 24 and 72 h postadministration, azithromycin accounted for 70% of total azithromycin- associated material in bile. In liver and kidney, unchanged azithromycin accounted for 40% of the total azithromycin-associated material; this doubled in lung and skin. Fifteen metabolites were positively or tentatively identified in plasma, bile, or tissues of all snakes. Four of these possible metabolites: 3'-desamine-3-ene-azithromycin, descladinose dehydroxy-2-ene-azithromycin, 3'-desamine-3-ene descladinose-azithromycin, and 3'-N-nitroso,9a-N-desmethyl-azithromycin are unique to this species. Descladinose-azithromycin, 3'-N-desmethyl,9a-N-desmethyl-azithromycin, and 3'-N-desmethyl, 3'-O-desmethyl-azithromycin were the only metabolites identified in skin. Kidney tissue contained a greater number of metabolites than liver tissue, with 3'-N-didesmethyl-azithromycin being identified only in the kidney. Compared with the dog and cat, a greater number of metabolites were identified in ball python plasma. The percentage of unchanged azithromycin in bile is not different between the three species.

[Simultaneous determination of sivelestat and its metabolite XW-IMP-A in human plasma using HPLC-MS/MS].

PubMed

Wang, Jing; Dai, Xiao-jian; Zhang, Yi-fan; Zhong, Da-fang; Wu, Yu-lin; Chen, Xiao-yan

2015-10-01

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.

An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

PubMed

Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

2016-09-02

Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Simultaneous determination of phosphatidylcholine-derived quaternary ammonium compounds by a LC-MS/MS method in human blood plasma, serum and urine samples.

PubMed

Steuer, Christian; Schütz, Philipp; Bernasconi, Luca; Huber, Andreas R

2016-01-01

The determination of circulating trimethylamine-N-oxide (TMAO), choline, betaine, l-carnitine and O-acetyl-l-carnitine concentration in different human matrices is of great clinical interest. Recent results highlighted the prognostic value of TMAO and quaternary ammonium containing metabolites in the field of cardiovascular and kidney diseases. Herein, we report a method for the rapid and simultaneous measurement of closely related phosphatidylcholine-derived metabolites in three different biological matrices by stable isotope dilution assay. Plasma, serum and urine samples were simply deproteinized and separated by HILIC-chromatography. Detection and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed covering more than the full reference range. Assay performance metrics include intra- and interday imprecision were below 10% for all analytes. To exclude matrix effects standard addition methods were applied for all matrices. It was shown that calibration standards and quality control prepared in water can be used instead of matrix-matched calibration and controls. The LC/MS/MS-based assay described in this article may improve future clinical studies evaluating TMAO and related substances as prognostic markers for cardiovascular risk and all-cause mortality in different patient populations. Copyright © 2015 Elsevier B.V. All rights reserved.

The use of D-penicillamine in patients with rheumatoid arthritis undergoing hemodialysis.

PubMed

Matthey, F; Perrett, D; Greenwood, R N; Baker, L R

1986-05-01

D-penicillamine and its major metabolites cysteine-penicillamine disulphide (CP) and penicillamine disulphide (P2) concentrations were measured in plasma from a hemodialysis patient with rheumatoid arthritis. CP and P2 alone were measured in plasma and a plasma ultrafiltrate from a second patient. On penicillamine 250 mg thrice weekly taken after dialysis pre-dialysis penicillamine concentrations were in the range 5.9-9.9 mumol/l. CP and P2 concentrations remained stable (range 139-197 mumol/l and 10-20 mumol/l) over 5 weeks and were of the same order as previously found in patients with normal renal function on higher doses of the drug. On penicillamine 250 mg daily concentrations of metabolites CP and P2 reach 193 mumol/l and 59.2 mumol/l after 2 and 3 weeks respectively. Concentration of metabolites fell by about half and of penicillamine by about a third after dialysis. Concentration of metabolites in ultrafiltrate were on average 75% lower than in plasma. Penicillamine 250 mg thrice weekly given after dialysis appears to be an appropriate dose for hemodialysis patients with rheumatoid arthritis.

Prediction of Gestational Diabetes through NMR Metabolomics of Maternal Blood.

PubMed

Pinto, Joana; Almeida, Lara M; Martins, Ana S; Duarte, Daniela; Barros, António S; Galhano, Eulália; Pita, Cristina; Almeida, Maria do Céu; Carreira, Isabel M; Gil, Ana M

2015-06-05

Metabolic biomarkers of pre- and postdiagnosis gestational diabetes mellitus (GDM) were sought, using nuclear magnetic resonance (NMR) metabolomics of maternal plasma and corresponding lipid extracts. Metabolite differences between controls and disease were identified through multivariate analysis of variable selected (1)H NMR spectra. For postdiagnosis GDM, partial least squares regression identified metabolites with higher dependence on normal gestational age evolution. Variable selection of NMR spectra produced good classification models for both pre- and postdiagnostic GDM. Prediagnosis GDM was accompanied by cholesterol increase and minor increases in lipoproteins (plasma), fatty acids, and triglycerides (extracts). Small metabolite changes comprised variations in glucose (up regulated), amino acids, betaine, urea, creatine, and metabolites related to gut microflora. Most changes were enhanced upon GDM diagnosis, in addition to newly observed changes in low-Mw compounds. GDM prediction seems possible exploiting multivariate profile changes rather than a set of univariate changes. Postdiagnosis GDM is successfully classified using a 26-resonance plasma biomarker. Plasma and extracts display comparable classification performance, the former enabling direct and more rapid analysis. Results and putative biochemical hypotheses require further confirmation in larger cohorts of distinct ethnicities.

The Human Urine Metabolome

PubMed Central

Bouatra, Souhaila; Aziat, Farid; Mandal, Rupasri; Guo, An Chi; Wilson, Michael R.; Knox, Craig; Bjorndahl, Trent C.; Krishnamurthy, Ramanarayan; Saleem, Fozia; Liu, Philip; Dame, Zerihun T.; Poelzer, Jenna; Huynh, Jessica; Yallou, Faizath S.; Psychogios, Nick; Dong, Edison; Bogumil, Ralf; Roehring, Cornelia; Wishart, David S.

2013-01-01

Urine has long been a “favored” biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca. PMID:24023812

Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS.

PubMed

Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte; Stürup, Stefan; Misra, Sougat; Björnstedt, Mikael; Gammelgaard, Bente

2016-03-01

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.

In vivo metabolism and partitioning of 6-[18F]fluoro-L-meta-tyrosine in whole blood: a unified compartment model

NASA Astrophysics Data System (ADS)

Asselin, Marie-Claude; Wahl, Lindi M.; Cunningham, Vincent J.; Amano, Shigeko; Nahmias, Claude

2002-06-01

Physiological quantification of dynamic PET data requires the determination of an input function, preferably from plasma. A compartmental model relating a parent radiotracer, its radiolabelled metabolites and their exchange between plasma and erythrocytes is presented. This model allows for the time course of radioactivity measured in whole blood to be transformed into the time course of the radiotracer in plasma. The utility of this approach is illustrated with blood data collected on 30 human subjects injected with 6-[18F]fluoro-L-meta-tyrosine (FmT), a pre-synaptic dopaminergic radiotracer. A three-compartment four-parameter model is shown to yield significantly better fits to the blood data than related lower and higher order models. This model is found to be robust to measurement noise, and yet sensitive to metabolic changes induced by pretreatment with carbidopa. For FmT, the between-subject variations are shown to be small enough to warrant the use of a population-based correction;; tissue time-activity curves were simulated to verify that this correction does not significantly affect the precision and accuracy of the derived rate constants. The unified blood model can be adapted for radiotracers other than FmT as long as the blood partition ratio of the parent radiotracer differs from that of its metabolites and/or the rate at which they equilibrate between plasma and erythrocytes is different.

Gemfibrozil markedly increases the plasma concentrations of montelukast: a previously unrecognized role for CYP2C8 in the metabolism of montelukast.

PubMed

Karonen, T; Filppula, A; Laitila, J; Niemi, M; Neuvonen, P J; Backman, J T

2010-08-01

According to available information, montelukast is metabolized by cytochrome P450 (CYP) 3A4 and 2C9. In order to study the significance of CYP2C8 in the pharmacokinetics of montelukast, 10 healthy subjects were administered gemfibrozil 600 mg or placebo twice daily for 3 days, and 10 mg montelukast on day 3, in a randomized, crossover study. Gemfibrozil increased the mean area under the plasma concentration-time curve (AUC)(0-infinity), peak plasma concentration (C(max)), and elimination half-life (t(1/2)) of montelukast 4.5-fold, 1.5-fold, and 3.0-fold, respectively (P < 0.001). After administration of gemfibrozil, the time to reach C(max) (t(max)) of the montelukast metabolite M6 was prolonged threefold (P = 0.005), its AUC(0-7) was reduced by 40% (P = 0.027), and the AUC(0-24) of the secondary metabolite M4 was reduced by >90% (P < 0.001). In human liver microsomes, gemfibrozil 1-O-beta glucuronide inhibited the formation of M6 (but not of M5) from montelukast 35-fold more potently than did gemfibrozil (half-maximal inhibitory concentration (IC(50)) 3.0 and 107 micromol/l, respectively). In conclusion, gemfibrozil markedly increases the plasma concentrations of montelukast, indicating that CYP2C8 is crucial in the elimination of montelukast.

Vitamin C metabolites, independent of smoking status, significantly enhance leukocyte, but not plasma ascorbate concentrations.

PubMed

Moyad, Mark A; Combs, Maile A; Vrablic, Angelica S; Velasquez, Janet; Turner, Benilda; Bernal, Samuel

2008-10-01

The objective of this study was to test the effects of acute doses of vitamin C alone, calcium ascorbate with vitamin C metabolites, and placebo, on total plasma and leukocyte vitamin C concentrations over 24 hours. A double-blind, placebo-controlled, four-way crossover study was performed consisting of four separate phases lasting 24 hours each and utilizing one of four oral 1000-mg preparations within each phase (one of vitamin C alone, two separate vitamin C formulations of calcium ascorbate with vitamin C metabolites, and placebo). There was a 7-day washout between phases, and blood draws at seven time points within each phase of the study for a total of 28 serologic measurements per subject and 420 total measurements for the entire clinical trial. Vitamin C concentration in plasma and leukocytes were measured by high-performance liquid chromatography at baseline and at six sequential time periods over 24 hours. Fifteen healthy males were enrolled, aged 18-39 years; nine were had never smoked and six were chronic smokers. No significant difference in plasma vitamin C levels was observed when comparing the different preparations. However, at 24 hours, calcium ascorbate with metabolites resulted in significantly higher concentrations of vitamin C in leukocytes (P<0.0001) compared with vitamin C alone. These results were similar for both metabolite formulations, and independent of smoking status. Regardless of smoking status, vitamin C metabolites may enhance leukocyte utilization of vitamin C itself, despite no consistent difference in plasma levels among the different preparations. A larger clinical investigation is warranted to confirm these preliminary findings, and to determine the clinical relevance of this impact on overall immune function.

New approaches to evaluate sympathoadrenal system activity in experiments on Earth and in space

NASA Astrophysics Data System (ADS)

Kvetnansky, R.; Noskov, V. B.; Blazicek, P.; Macho, L.; Grigoriev, A. I.; Goldstein, D. S.; Kopin, I. J.

In previous studies the activity of the sympathoadrenal system (SAS) in cosmonauts during space flights was evaluated by measuring plasma catecholamines (CA) levels and urinary CA and their metabolites concentrations. Plasma CA levels are accepted indicators of SAS activity, however, they are determined by the plasma clearances as well as the rates of CA release (spillover-SO) into the bloodstream. Nowadays methods are available which evaluate not only plasma levels of CA but also their release, spillover, uptake, reuptake, degradation and also CA synthesis in vivo measured by plasma levels of dihydroxyphenylalanine (DOPA). Plasma concentrations of DOPA, the CA noradrenaline (NE), adrenaline (ADR), and dopamine (DA), the deaminated catechol metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), and the O-methylated metabolites methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured during immobilization stress (IMO) in conscious rats. Radiotracer methods were used to measure NE SO. IMO markedly increased arterial NE levels but NE SO was less elevated bacause the NE clearance was slightly reduced in IMO rats. Simultaneous measurements of plasma CA and their metabolites provide another means to obtain information about SAS function. For instance, dissociation between changes of plasma DHPG and NE levels can indicate changes in neuronal reuptake of NE. We found marked parallel increases in plasma NE and DHPG levels during acute IMO; however after repeated IMO, plasma NE levels were increased but DHPG responses were less pronounced suggesting a reduced NE reuptake. DOPA, the CA precursor, circulates in plasma at a concentration higher than NE. During stress, increased sympathoneural outflow stimulates DOPA synthesis and release into the circulation supporting the view that changes in plasma DOPA levels during stress reflect in vivo changes in the rate of CA synthesis. We propose to measure the new plasma indicators of SAS activity in cosmonauts and/or in animals before, during and after space flights.

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring.

PubMed

Labat, Laurence; Goncalves, Antonio; Marques, Ana Rita; Duretz, Bénédicte; Granger, Bernard; Declèves, Xavier

2017-08-01

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC-HR-MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full-scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 μL plasma giving a wide polynomial dynamic range (R 2  > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra- and inter-day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples. Copyright © 2017 John Wiley & Sons, Ltd.

Pharmacokinetics, distribution, metabolism, and excretion of the dual reuptake inhibitor [(14)C]-nefopam in rats.

PubMed

Yu, Jian; Solon, Eric; Shen, Helen; Modi, Nishit B; Mittur, Aravind

2016-11-01

1. This study examined the pharmacokinetics, distribution, metabolism, and excretion of [(14)C] nefopam in rats after a single oral administration. Blood, plasma, and excreta were analyzed for total radioactivity, nefopam, and metabolites. Metabolites were profiled and identified. Radioactivity distribution was determined by quantitative whole-body autoradiography. 2. The pharmacokinetic profiles of total radioactivity and nefopam were similar in male and female rats. Radioactivity partitioned approximately equally between plasma and red blood cells. A majority of the radioactivity was excreted in urine within 24 hours and mass balance was achieved within 7 days. 3. Intact nefopam was a minor component in plasma and excreta. Numerous metabolites were identified in plasma and urine generated by multiple pathways including: hydroxylation/oxidation metabolites (M11, M22a and M22b, M16, M20), some of which were further glucuronidated (M6a to M6c, M7a to M7c, M8a and M8b, M3a to M3d); N-demethylation of nefopam to metabolite M21, which additionally undergoes single or multiple hydroxylations or sulfation (M9, M14, M23), with some of the hydroxylated metabolites further glucuronidated (M2a to M2d). 4. Total radioactivity rapidly distributed with highest concentrations found in the urinary bladder, stomach, liver, kidney medulla, small intestine, uveal tract, and kidney cortex without significant accumulation or persistence. Radioactivity reversibly associated with melanin-containing tissues.

Plasma lipid metabolites and refueling performance of Semi palmated Sandpipers at migratory stopovers

USGS Publications Warehouse

Lyons, J.E.; Collazo, J.A.; Guglielmo, C.

2005-01-01

Assessing stopover habitat quality and refueling performance of individual birds is crucial to the conservation and management of migratory shorebirds. Plasma lipid metabolites indicate the trajectory of mass change in individuals and may be a more accurate measure of refueling performance at a particular site than static measures such as nutrient reserves. We measured lipid metabolites of Semipalmated Sandpipers at 4 coastal stopover sites during northward migration: Merritt Island, FL; Georgetown, SC; Pea Island, NC; and Delaware Bay, NJ. We described spatial and temporal variation in metabolic profiles among the 4 stopovers and evaluated the effects of body mass, age, and date on metabolite concentrations. Triglyceride concentration, an indicator of fat deposition, declined during the migration, whereas B-OH-Butyrate, a measure of fasting, increased. Triglyceride concentration correlated with phospholipids and inversely related to B-OH-butyrate, but was not related to body mass or age. Triglyceride levels and estimated percent fat were greater at Delaware Bay than at any stopovers to the south. Plasma metabolite profiles accurately reflected stopover refueling performance and provide an important new technique for assessing stopover habitat quality for migratory shorebirds.

Quantitation of silibinin, a putative cancer chemopreventive agent derived from milk thistle (Silybum marianum), in human plasma by high-performance liquid chromatography and identification of possible metabolites.

PubMed

Hoh, Carmen S L; Boocock, David J; Marczylo, Timothy H; Brown, V A; Cai, Hong; Steward, William P; Berry, David P; Gescher, Andreas J

2007-04-04

Silibinin has recently received attention as a potential cancer chemopreventive agent because of its antiproliferative and anticarcinogenic effects. A simple and specific reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of silibinin in human plasma. Sample preparation involved simple protein precipitation, and separation was achieved on a Waters Atlantis C18 column with flow rate of 1.0 mL/min at 40 degrees C and UV detection at 290 nm. Silibinin was detected as two peaks corresponding to trans-diastereoisomers. The peak area was linear over the investigated concentration range (0-5000 ng/mL). The limits of detection were 2 and 1 ng/mL for the two diastereoisomers (d1 and d2), with a recovery of 53-58%. This method was utilized to detect silibinin in plasma of colorectal patients after 7 days of treatment with silipide (silibinin formulated with phosphatidyl choline).

Application of LC-MS analysis to the characterisation of the in vitro and in vivo metabolite profiles of RGH-1756 in the rat.

PubMed

Gémesi, L I; Kapás, M; Szeberényi, S

2001-03-01

RGH-1756, 1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazol[2,1-b] thiazolyl)-phenoxy]-butyl-4-(14)C)-piperazine dimethane is a novel atypical antipsychotic drug candidate of Gedeon Richter Ltd. The metabolic pathways of the compound have been investigated by profiling the metabolites present in plasma, bile, and faeces samples of rats treated with (14)C-RGH-1756. The metabolites formed in vitro by rat liver microsomes have also been analysed. Good separation of the compounds has been achieved by gradient HPLC method on Zorbax/Bonus RP-C18 column. Radiometry and mass spectrometry have been applied to detect and characterise the metabolites. The metabolite formed by oxidative cleavage of the chain at the carbon atom adjacent to the piperazine nitrogen has been identified as the major plasma metabolite. Glucuronide conjugate of hydroxy-RGH-1756 has been found as one of the main metabolites excreted in the bile where the unchanged compound has not been detected.

Flavanol plasma bioavailability is affected by metabolic syndrome in rats.

PubMed

Margalef, Maria; Pons, Zara; Iglesias-Carres, Lisard; Bravo, Francisca Isabel; Muguerza, Begoña; Arola-Arnal, Anna

2017-09-15

Flavanols, which exert several health benefits, are metabolized after ingestion. Factors such as the host physiological condition could affect the metabolism and bioavailability of flavanols, influencing their bioactivities. This study aimed to qualitatively evaluate whether a pathological state influenced flavanol plasma bioavailability. Standard and cafeteria (CAF) diet fed rats, a robust model of metabolic syndrome (MeS), were administered 1000mg/kg of flavanol enriched grape seed polyphenol extract (GSPE). Flavanols and their metabolites were quantified by HPLC-MS/MS in plasma before and at 2, 4, 7, 24, and 48h after GSPE ingestion. Results showed that in CAF administered rats the maximum time of plasma flavanol concentration was delayed and these animals presented higher levels of plasma phase-II metabolites as well as altered microbial metabolites. In conclusion, this study demonstrated that MeS pathological state modified flavanol bioavailability, supporting the hypothesis that flavanol metabolism, and therefore flavanol functionality, depend on the organism's state of health. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lower plasma choline levels are associated with sleepiness symptoms.

PubMed

Pak, Victoria M; Dai, Feng; Keenan, Brendan T; Gooneratne, Nalaka S; Pack, Allan I

2018-04-01

Sleepiness and cardiovascular disease share common molecular pathways; thus, metabolic risk factors for sleepiness may also predict cardiovascular disease risk. Daytime sleepiness predicts mortality and cardiovascular disease, although the mechanism is unidentified. This study explored the associations between subjective sleepiness and metabolite concentrations in human blood plasma within the oxidative and inflammatory pathways, in order to identify mechanisms that may contribute to sleepiness and cardiovascular disease risk. An exploratory case-control sample of 36 subjects, categorized based on the Epworth Sleepiness Scale (ESS) questionnaire as sleepy (ESS ≥ 10) or non-sleepy (ESS < 10), was recruited among subjects undergoing an overnight sleep study for suspected sleep apnea at the University of Pennsylvania Sleep Center. The average age was 42.4 ± 10.5 years, the mean body mass index (BMI) was 40.0 ± 9.36 kg/m 2 , median Apnea Hypopnea Index (AHI) was 8.2 (IQR: 2.5-26.5), and 52% were male. Fasting morning blood plasma samples were collected after an overnight sleep study. Biomarkers were explored in subjects with sleepiness versus those without using the multiple linear regression adjusting for age, BMI, smoking, Apnea Hypopnea Index (sleep apnea severity), study cohort, and hypertension. The level of choline is significantly lower (P = 0.003) in sleepy subjects (N = 18; mean plasma choline concentration of 8.19 ± 2.62 μmol/L) compared with non-sleepy subjects (N = 18; mean plasma choline concentration of 9.14 ± 2.25 μmol/L). Other markers with suggestive differences (P < 0.1) include isovalerylcarnitine, Alpha-Amino apidipic acid, Spingosine 1 Phosphate, Aspartic Acid, Propionylcarnitine, and Ceramides (fatty acids; C14-C16 and C-18). This pilot study is the first to show that lower levels of plasma choline metabolites are associated with sleepiness. Further exploration of choline and other noted metabolites and their associations with sleepiness will guide targeted symptom management. Copyright © 2017 Elsevier B.V. All rights reserved.

An assessment of 25-hydroxyvitamin D measurements in comparability studies conducted by the Vitamin D Metabolites Quality Assurance Program.

PubMed

Bedner, Mary; Lippa, Katrice A; Tai, Susan S-C

2013-11-15

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements, established the first accuracy-based program for improving the comparability of vitamin D metabolite measurements, the Vitamin D Metabolites Quality Assurance Program. The study samples were human serum or plasma Standard Reference Materials (SRMs) with 25-hydroxyvitamin D values that were determined at NIST. Participants evaluated the materials using immunoassay (IA), liquid chromatography (LC) with mass spectrometric detection, and LC with ultraviolet absorbance detection. NIST evaluated the results for concordance within the participant community as well as trueness relative to the NIST value. For the study materials that contain mostly 25-hydroxyvitamin D3 (25(OH)D3),the coefficient of variation (CV) for the participant results was consistently in the range from 7% to 19%, and the median values were biased high relative to the NIST values. However, for materials that contain significant concentrations of both 25-hydroxyvitamin D2 (25(OH)D2) and 25(OH)D3, the median IA results were biased lower than both the LC and the NIST values, and the CV was as high as 28%. The first interlaboratory comparison results for SRM 972a Vitamin D Metabolites in Human Serum are also reported. Relatively large within-lab and between-lab variability hinders conclusive assessments of bias and accuracy. © 2013.

Simultaneous quantification and semi-quantification of ginkgolic acids and their metabolites in rat plasma by UHPLC-LTQ-Orbitrap-MS and its application to pharmacokinetics study.

PubMed

Qian, Yiyun; Zhu, Zhenhua; Duan, Jin-Ao; Guo, Sheng; Shang, Erxin; Tao, Jinhua; Su, Shulan; Guo, Jianming

2017-01-15

A highly sensitive method using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated for the simultaneous identification and quantification of ginkgolic acids and semi-quantification of their metabolites in rat plasma. For the five selected ginkgolic acids, the method was found to be with good linearities (r>0.9991), good intra- and inter-day precisions (RSD<15%), and good accuracies (RE, from -10.33% to 4.92%) as well. Extraction recoveries, matrix effects and stabilities for rat plasm samples were within the required limits. The validated method was successfully applied to investigate the pharmacokinetics of the five ginkgolic acids in rat plasma after oral administration of 3 dosage groups (900mg/kg, 300mg/kg and 100mg/kg). Meanwhile, six metabolites of GA (15:1) and GA (17:1) were identified by comparison of MS data with reported values. The results of validation in terms of linear ranges, precisions and stabilities were established for semi-quantification of metabolites. The curves of relative changes of these metabolites during the metabolic process were constructed by plotting the peak area ratios of metabolites to salicylic acid (internal standard, IS), respectively. Double peaks were observed in all 3 dose groups. Different type of metabolites and different dosage of each metabolite both resulted in different T max . Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of fasting on the urinary excretion of water-soluble vitamins in humans and rats.

PubMed

Fukuwatari, Tsutomu; Yoshida, Erina; Takahashi, Kei; Shibata, Katsumi

2010-01-01

Recent studies showed that the urinary excretion of the water-soluble vitamins can be useful as a nutritional index. To determine how fasting affects urinary excretion of water-soluble vitamins, a human study and an animal experiment were conducted. In the human study, the 24-h urinary excretion of water-soluble vitamins in 12 healthy Japanese adults fasting for a day was measured. One-day fasting drastically decreased urinary thiamin content to 30%, and increased urinary riboflavin content by 3-fold. Other water-soluble vitamin contents did not show significant change by fasting. To further investigate the alterations of water-soluble vitamin status by starvation, rats were starved for 3 d, and water-soluble vitamin contents in the liver, blood and urine were measured during starvation. Urinary excretion of thiamin, riboflavin, vitamin B(6) metabolite 4-pyridoxic acid, nicotinamide metabolites and folate decreased during starvation, but that of vitamin B(12), pantothenic acid and biotin did not. As for blood vitamin levels, only blood vitamin B(1), plasma PLP and plasma folate levels decreased with starvation. All water-soluble vitamin contents in the liver decreased during starvation, whereas vitamin concentrations in the liver did not decrease. Starvation decreased only concentrations of vitamin B(12) and folate in the skeletal muscle. These results suggest that water-soluble vitamins were released from the liver, and supplied to the peripheral tissues to maintain vitamin nutrition. Our human study also suggested that the effect of fasting should be taken into consideration for subjects showing low urinary thiamin and high urinary riboflavin.

Pharmacokinetic evaluation of tissue distribution of pirfenidone and its metabolites for idiopathic pulmonary fibrosis therapy.

PubMed

Togami, Kohei; Kanehira, Yukimune; Tada, Hitoshi

2015-05-01

Pirfenidone is the first and only clinically used anti-fibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). It was reported previously that pirfenidone metabolites (5-hydroxypirfenidone and 5-carboxypirfenidone) also have anti-fibrotic effects. The present study evaluated the distribution of pirfenidone and its metabolites in the lung, liver and kidney tissues in rats. The time course for the different concentrations of pirfenidone, 5-hydroxypirfenidone and 5-carboxypirfenidone in the lung tissue following oral administration (30 mg/kg) to rats was lower than that in plasma, and the area under the drug concentration-time curve (AUC) ratios of lung/plasma for pirfenidone, 5-hydroxypirfenidone and 5-carboxypirfenidone were 0.52, 0.40 and 0.61, respectively. In in vitro transport experiments, the basolateral-to-apical transport of pirfenidone and its metabolites through the model of lung epithelial cell (Calu-3) monolayers was not significantly different from their apical-to-basolateral transport. In binding experiments, the binding rate of these drugs to the lung tissue was lower than that to the plasma protein. These findings suggest that the low distribution of pirfenidone and its metabolites in the lungs was based on their low affinities with lung tissue and not the transport characteristics of lung epithelial cells. On the other hand, the AUC ratios of liver/plasma for pirfenidone and 5-carboxypirfenidone were 2.3 and 6.5 and the AUC ratios of kidney/plasma were 1.5 and 20, respectively. The binding rates to the liver and kidney tissues were higher than those to the plasma protein. These results suggest that high concentrations of these drugs were found in the liver and kidney tissues. Copyright © 2014 John Wiley & Sons, Ltd.

Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis.

PubMed

Heijne, Wilbert H M; Lamers, Robert-Jan A N; van Bladeren, Peter J; Groten, John P; van Nesselrooij, Joop H J; van Ommen, Ben

2005-01-01

This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Association of platelet responsiveness with clopidogrel metabolism: role of compliance in the assessment of "resistance".

PubMed

Serebruany, Victor; Cherala, Ganesh; Williams, Craig; Surigin, Serge; Booze, Christopher; Kuliczkowski, Wiktor; Atar, Dan

2009-12-01

Noncompliance is probably the major cause of clopidogrel "resistance." However, noncompliance is difficult to prove without confirming that the drug has been administered. Therefore, detection of plasma clopidogrel and/or metabolite(s) as the reliable objective method to confirm compliance is important. We sought to correlate the inhibition of platelet aggregation (IPA) with plasma levels of unchanged clopidogrel (UC), active thiol metabolite (ATM), and inactive carboxyl metabolite (ICM) in a large cohort of patients with coronary artery disease and ischemic stroke treated with clopidogrel. We conducted secondary post-hoc analyses of IPA and plasma UC, ATM, and ICM in a dataset consisting of presumably compliant patients with coronary disease (n = 422) and post-stroke (n = 209). Overall noncompliance rate was 22% (n = 138), while such risks were significantly higher in stroke survivors (n = 79, or 38%) when compared to patients with coronary disease (14%; n = 59; P = .001). Only ICM (19,154 +/- 7,228 ng/ml) was suitable for detecting compliance, while UC (15.2 +/- 9.4 ng/ml), and ATM (8.1 +/- 3.7 ng/ml) in most cases are barely detectable, and diminish over time in the stored samples. The best correlation with IPA (r2 = 0.847) was observed for active metabolite, followed by unchanged clopidogrel (r2 = 0.602), and finally inactive metabolite (r2 = 0.529). The predictive value for noncompliance was also high for inactive metabolite (c-statistic = 0.911). Therapy with clopidogrel is associated with double-digit underestimated risks for noncompliance, especially in stroke survivors, supporting the hypothesis that lack of IPA, and clopidogrel "resistance" are attributed to hidden noncompliance. Plasma ICM, but not UC, or ATM is a useful marker to monitor compliance to clopidogrel in registries and clinical trials.

Metabolite profiling of ginsenosides in rat plasma, urine and feces by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract.

PubMed

Dong, Wei-Wei; Han, Xiong-Zhe; Zhao, Jinhua; Zhong, Fei-Liang; Ma, Rui; Wu, Songquan; Li, Donghao; Quan, Lin-Hu; Jiang, Jun

2018-03-01

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune-modulatory, anti-tumor, anti-aging and anti-inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC-MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng. Copyright © 2017 John Wiley & Sons, Ltd.

Organic Anion-Transporting Polypeptide and Efflux Transporter-Mediated Hepatic Uptake and Biliary Excretion of Cilostazol and Its Metabolites in Rats and Humans.

PubMed

Wang, Chong; Huo, Xiaokui; Wang, Changyuan; Meng, Qiang; Liu, Zhihao; Sun, Pengyuan; Cang, Jian; Sun, Huijun; Liu, Kexin

2017-09-01

Cilostazol undergoes extensive liver metabolism. However, the transporter-mediated hepatic disposition of cilostazol remains unknown. The present study was performed to investigate the hepatic uptake and biliary excretion of cilostazol and its metabolites (OPC-13015 and OPC-13213) using rat liver and human transporter-transfected cells in vitro. Cilostazol uptake by rat liver slices and isolated rat hepatocytes exhibited time-, concentration-, and temperature dependency and was decreased by Oatp inhibitors, which suggested that Oatp was involved in the hepatic uptake of cilostazol. Cilostazol uptake in rat hepatocytes, OATP1B1-, and OATP1B3-HEK293 cells indicated a saturable process with K m values of 2.7 μM, 17.7 μM, and 2.7 μM, respectively. Epigallocatechin gallate, cyclosporin A, rifampicin, and telmisartan inhibited cilostazol uptake in OATP1B1/1B3-HEK293 cells with K i values close to their clinical plasma concentration, which suggested possible drug-drug interactions in humans via OATP1B1/1B3. Moreover, the cumulative biliary excretion of cilostazol and OPC-13015 was significantly decreased by quinidine, bilirubin, and novobiocin in perfused rat liver, but OPC-13213 biliary excretion was only inhibited by novobiocin, which suggested that the efflux transporters Mrp2, Bcrp, and P-gp were involved in the biliary excretion of cilostazol and its metabolites. Our findings indicated that multiple transporters were involved in the hepatic disposition of cilostazol and its metabolites. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Pharmacokinetics and safety of vitamin E δ-tocotrienol after single and multiple doses in healthy subjects with measurement of vitamin E metabolites.

PubMed

Mahipal, Amit; Klapman, Jason; Vignesh, Shivakumar; Yang, Chung S; Neuger, Anthony; Chen, Dung-Tsa; Malafa, Mokenge P

2016-07-01

Vitamin E delta-tocotrienol (VEDT) has demonstrated chemopreventive and antineoplastic activity in preclinical models. The aim of our study was to determine the safety and pharmacokinetics of VEDT and its metabolites after single- and multiple-dose administrations in healthy subjects. Thirty-six subjects received from 100 to 1600 mg of oral VEDT as a single dose or twice daily for 14 consecutive days. A 3 + 3 dose escalation design was utilized. Pharmacokinetic data were derived from high-performance liquid chromatography (HPLC) assays. Serial blood and urine samples were collected before and during VEDT administration, with serum and urine metabolites assessed using HPLC. No drug-related adverse events were observed. Pharmacokinetic parameters for single and multiple doses were, respectively, as follows (shown as range): time to maximum concentration of 4-9.3 and 4.7-7.3 h, maximum concentration of 795.6-3742.6 and 493.3-3746 ng/mL, half-life of 1.7-5.9 and 2.3-6.9 h, and 0-12 h area under the curve of 4518.7-20,781.4 and 1987.7-22,171.2 ng h/mL. Plasma tocotrienols were significantly increased after VEDT administration, indicating oral bioavailability of VEDT in humans. Plasma and urine levels of metabolites, δ-carboxyethyl hydroxychroman, and δ-carboxymethylbutyl hydroxychroman were elevated after VEDT administration in a dose-dependent manner and were 30-60 times significantly higher than δ-tocotrienol levels. VEDT can be safely administered at doses up to 1600 mg twice daily. Plasma VEDT concentrations were comparable to those obtained in VEDT-treated mice in which tumor growth was delayed. Our results suggest that VEDT can be safely consumed by healthy subjects and achieve bioactive levels, supporting the investigation of VEDT for chemoprevention.

Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

PubMed

Abdallah, Inas A; Huang, Peng; Liu, Jing; Lee, David Y; Liu-Chen, Lee-Yuan; Hassan, Hazem E

2017-04-01

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C 18 column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains

PubMed Central

Bennett, Brian J.; Davis, Richard C.; Civelek, Mete; Orozco, Luz; Wu, Judy; Qi, Hannah; Pan, Calvin; Packard, René R. Sevag; Eskin, Eleazar; Yan, Mujing; Kirchgessner, Todd; Wang, Zeneng; Li, Xinmin; Gregory, Jill C.; Hazen, Stanley L.; Gargalovic, Peter S.; Lusis, Aldons J.

2015-01-01

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis. PMID:26694027

Determination of homovanillic acid (HVA) in human plasma by HPLC with coulometric detection and a new SPE procedure.

PubMed

Saracino, M A; Mandrioli, R; Mercolini, L; Ferranti, A; Zaimovic, A; Leonardi, C; Raggi, M A

2006-09-11

A sensitive high-performance liquid chromatographic method has been developed for the determination of homovanillic acid (HVA), the main metabolite of dopamine, in human plasma. Analyses were carried out on a reversed-phase column (C8, 250 mm x 4.6 mm i.d., 5 microm) using a mobile phase composed of 10% methanol and 90% aqueous citrate buffer, containing octanesulfonic acid and EDTA at pH 4.8. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at -0.200 V and the second analytical cell at +0.500 V. A careful solid-phase extraction procedure, based on strong anion exchange (SAX) cartridges (100 mg, 1 mL), was implemented for the pre-treatment of plasma samples. Extraction yield was satisfactory, being the mean value 98.0%. The calibration curve was linear over the concentration range of 0.2-25.0 ng mL(-1) of homovanillic acid. The limit of quantitation (LOQ) was 0.2 ng mL(-1) and the limit of detection (LOD) was 0.1 ng mL(-1). The method was successfully applied to plasma samples from former alcohol, cocaine and heroin addicts. Results were satisfactory in terms of precision and accuracy. Hence, the method is suitable for the determination of homovanillic acid in human plasma.

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo

2012-04-20

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChEmore » polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.« less

Activity of Nitazoxanide and Tizoxanide against Mycobacterium tuberculosis in vitro and in whole blood culture

PubMed Central

Harausz, Elizabeth P.; Chervenak, Keith A.; Good, Caryn E.; Jacobs, Michael R.; Wallis, Robert S.; Sanchez-Felix, Manuel; Boom, W. Henry

2016-01-01

Nitazoxanide (NTZ) and its metabolite tizoxanide (TIZ) were studied as antimycobacterial agents in vitro (in mycobacterial growth indicator tube [MGIT] cultures) and in a whole blood bactericidal assay. Both NTZ and TIZ show high protein binding. In MGIT cultures (albumin concentration=78 µM), inhibition of Mycobacterium tuberculosis growth occurred at total drug concentrations of ≥16 µg/ml, whereas in whole blood cultures (albumin concentration=350 µM), ≥128 µg/ml was required. Free drug fractions at these two conditions were estimated to be 69% and 2%, respectively. Co-incubation of NTZ and TIZ in human plasma for 72 hours nearly completely eliminated their ability to inhibit mycobacterial growth in MGIT. Interactions with plasma proteins may limit the potential of NTZ and TIZ as drugs for human tuberculosis. PMID:27156623

Systematic evaluation of serum and plasma collection on the endogenous metabolome.

PubMed

Zhou, Zhi; Chen, Yanhua; He, Jiuming; Xu, Jing; Zhang, Ruiping; Mao, Yan; Abliz, Zeper

2017-02-01

In metabolomics research, the use of different blood collection methods may influence endogenous metabolites. Ultra HPLC coupled with MS/MS was applied together with multivariate statistics to investigate metabolomics differences in serum and plasma samples handled by different anticoagulants. A total of 135 known representative metabolites were assessed for comprehensive evaluation of the effects of anticoagulants. Exogenous factors, including separation gel ingredients from the serum collection tubes and the anticoagulants, affected mass spectrometer detection. Heparin plasma yielded the best detection of different functional groups and is therefore the optimal blood specimen for metabolomics research, followed by potassium oxalate plasma.

Human urine and plasma concentrations of bisphenol A extrapolated from pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and semi-physiological pharmacokinetic modeling.

PubMed

Miyaguchi, Takamori; Suemizu, Hiroshi; Shimizu, Makiko; Shida, Satomi; Nishiyama, Sayako; Takano, Ryohji; Murayama, Norie; Yamazaki, Hiroshi

2015-06-01

The aim of this study was to extrapolate to humans the pharmacokinetics of estrogen analog bisphenol A determined in chimeric mice transplanted with human hepatocytes. Higher plasma concentrations and urinary excretions of bisphenol A glucuronide (a primary metabolite of bisphenol A) were observed in chimeric mice than in control mice after oral administrations, presumably because of enterohepatic circulation of bisphenol A glucuronide in control mice. Bisphenol A glucuronidation was faster in mouse liver microsomes than in human liver microsomes. These findings suggest a predominantly urinary excretion route of bisphenol A glucuronide in chimeric mice with humanized liver. Reported human plasma and urine data for bisphenol A glucuronide after single oral administration of 0.1mg/kg bisphenol A were reasonably estimated using the current semi-physiological pharmacokinetic model extrapolated from humanized mice data using algometric scaling. The reported geometric mean urinary bisphenol A concentration in the U.S. population of 2.64μg/L underwent reverse dosimetry modeling with the current human semi-physiological pharmacokinetic model. This yielded an estimated exposure of 0.024μg/kg/day, which was less than the daily tolerable intake of bisphenol A (50μg/kg/day), implying little risk to humans. Semi-physiological pharmacokinetic modeling will likely prove useful for determining the species-dependent toxicological risk of bisphenol A. Copyright © 2015 Elsevier Inc. All rights reserved.

The Karlsruhe Metabolomics and Nutrition (KarMeN) Study: Protocol and Methods of a Cross-Sectional Study to Characterize the Metabolome of Healthy Men and Women

PubMed Central

Kriebel, Anita; Dörr, Claudia; Bandt, Susanne; Rist, Manuela; Roth, Alexander; Hummel, Eva; Kulling, Sabine; Hoffmann, Ingrid; Watzl, Bernhard

2016-01-01

Background The human metabolome is influenced by various intrinsic and extrinsic factors. A precondition to identify such biomarkers is the comprehensive understanding of the composition and variability of the metabolome of healthy humans. Sample handling aspects have an important impact on the composition of the metabolome; therefore, it is crucial for any metabolomics study to standardize protocols on sample collection, preanalytical sample handling, storage, and analytics to keep the nonbiological variability as low as possible. Objective The main objective of the KarMeN study is to analyze the human metabolome in blood and urine by targeted and untargeted metabolite profiling (gas chromatography-mass spectrometry [GC-MS], GC×GC-MS, liquid chromatography-mass spectrometry [LC-MS/MS], and1H nuclear magnetic resonance [NMR] spectroscopy) and to determine the impact of sex, age, body composition, diet, and physical activity on metabolite profiles of healthy women and men. Here, we report the outline of the study protocol with special regard to all aspects that should be considered in studies applying metabolomics. Methods Healthy men and women, aged 18 years or older, were recruited. In addition to a number of anthropometric (height, weight, body mass index, waist circumference, body composition), clinical (blood pressure, electrocardiogram, blood and urine clinical chemistry) and functional parameters (lung function, arterial stiffness), resting metabolic rate, physical activity, fitness, and dietary intake were assessed, and 24-hour urine, fasting spot urine, and plasma samples were collected. Standard operating procedures were established for all steps of the study design. Using different analytical techniques (LC-MS, GC×GC-MS,1H NMR spectroscopy), metabolite profiles of urine and plasma were determined. Data will be analyzed using univariate and multivariate as well as predictive modeling methods. Results The project was funded in 2011 and enrollment was carried out between March 2012 and July 2013. A total of 301 volunteers were eligible to participate in the study. Metabolite profiling of plasma and urine samples has been completed and data analysis is currently underway. Conclusions We established the KarMeN study applying a broad set of clinical and physiological examinations with a high degree of standardization. Our experimental approach of combining scheduled timing of examinations and sampling with the multiplatform approach (GC×GC-MS, GC-MS, LC-MS/MS, and1H NMR spectroscopy) will enable us to differentiate between current and long-term effects of diet and physical activity on metabolite profiles, while enabling us at the same time to consider confounders such as age and sex in the KarMeN study. Trial Registration German Clinical Trials Register DRKS00004890; https://drks-neu.uniklinik-freiburg.de/drks_web/navigate.do? navigationId=trial.HTML&TRIAL_ID=DRKS00004890 (Archived by WebCite at http://www.webcitation.org/6iyM8dMtx) PMID:27421387

Analysis of fenretinide and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry and its application to clinical pharmacokinetics.

PubMed

Cho, Hwang Eui; Min, H Kang

2017-01-05

A simple and accurate high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) and N-(4-methoxyphenyl)retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5μm, 50×2.1mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH* 2.4) at a flow rate of 0.5mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2-50ng/mL with a lower limit of quantification of 0.2ng/mL. The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than 90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of the pharmacokinetic study for patients treated with 4-HPR in a clinical trial. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative determination of regorafenib and its two major metabolites in human plasma with high-performance liquid chromatography and ultraviolet detection.

PubMed

Fujita, Kazuma; Miura, Masatomo; Shibata, Hiroyuki

2016-10-01

A simple, highly sensitive and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N-oxidemetabolite (M-2) and the desmethyl N-oxide metabolite (M-5) in human plasma. Regorafenib, M-2, M-5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH2 PO4 (pH 3.5)-acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M-2 and M-5 were 10 ng/mL for each analyte. A procedure using solid-phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra- and inter-day assays were <12.2% for regorafenib, <12.3% for M-2 and <15.1% for M-5. Accuracies for intra- and inter-day assays were <9.4% for regorafenib, <8.0% for M-2 and <12.8% for M-5 over a linear range from 10 to 10,000 ng/mL. This HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

High-Throughput HPLC-MS/MS Method for Quantification of Ibuprofen Enantiomers in Human Plasma: Focus on Investigation of Metabolite Interference.

PubMed

Nakov, Natalija; Bogdanovska, Liljana; Acevska, Jelena; Tonic-Ribarska, Jasmina; Petkovska, Rumenka; Dimitrovska, Aneta; Kasabova, Lilia; Svinarov, Dobrin

2016-11-01

In this research, as a part of the development of fast and reliable HPLC-MS/MS method for quantification of ibuprofen (IBP) enantiomers in human plasma, the possibility of IBP acylglucoronide (IBP-Glu) back-conversion was assessed. This involved investigation of in source and in vitro back-conversion. The separation of IBP enantiomers, its metabolite and rac-IBP-d3 (internal standard), was achieved within 6 min using Chiracel OJ-RH chromatographic column (150 × 2.1 mm, 5 μm). The followed selected reaction monitoring transitions for IBP-Glu (m/z 381.4 → 205.4, m/z 381.4 → 161.4 and m/z 205.4 → 161.4) implied that under the optimized electrospray ionization parameters, in source back-conversion of IBP-Glu was insignificant. The results obtained after liquid-liquid extraction of plasma samples spiked with IBP-Glu revealed that the amount of IBP enantiomers generated by IBP-Glu back-conversion was far <20% of lower limit of quantification sample. These results indicate that the presence of IBP-Glu in real samples will not affect the quantification of the IBP enantiomers; thereby reliability of the method was improved. Additional advantage of the method is the short analysis time making it suitable for the large number of samples. The method was fully validated according to the EMA guideline and was shown to meet all requirements to be applied in a pharmacokinetic study. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Real Patient and its Virtual Twin: Application of Quantitative Systems Toxicology Modelling in the Cardiac Safety Assessment of Citalopram.

PubMed

Patel, Nikunjkumar; Wiśniowska, Barbara; Jamei, Masoud; Polak, Sebastian

2017-11-27

A quantitative systems toxicology (QST) model for citalopram was established to simulate, in silico, a 'virtual twin' of a real patient to predict the occurrence of cardiotoxic events previously reported in patients under various clinical conditions. The QST model considers the effects of citalopram and its most notable electrophysiologically active primary (desmethylcitalopram) and secondary (didesmethylcitalopram) metabolites, on cardiac electrophysiology. The in vitro cardiac ion channel current inhibition data was coupled with the biophysically detailed model of human cardiac electrophysiology to investigate the impact of (i) the inhibition of multiple ion currents (I Kr , I Ks , I CaL ); (ii) the inclusion of metabolites in the QST model; and (iii) unbound or total plasma as the operating drug concentration, in predicting clinically observed QT prolongation. The inclusion of multiple ion channel current inhibition and metabolites in the simulation with unbound plasma citalopram concentration provided the lowest prediction error. The predictive performance of the model was verified with three additional therapeutic and supra-therapeutic drug exposure clinical cases. The results indicate that considering only the hERG ion channel inhibition of only the parent drug is potentially misleading, and the inclusion of active metabolite data and the influence of other ion channel currents should be considered to improve the prediction of potential cardiac toxicity. Mechanistic modelling can help bridge the gaps existing in the quantitative translation from preclinical cardiac safety assessment to clinical toxicology. Moreover, this study shows that the QST models, in combination with appropriate drug and systems parameters, can pave the way towards personalised safety assessment.

Fluoride Ion Regeneration of Cyclosarin (Gf) from Minipig Tissue and Fluids Following Whole Body GF Vapor Exposure

DTIC Science & Technology

2006-11-01

Quantitation of organophosphorus nerve agent metabolites in human urine using isotope dilution gas chromatography- tandem mass spectrometry. J. Anal...Recent developments to improve nerve agent biomarker techniques include methods for measuring fluoride regenerated Sarin (GB) in blood and tissue...Our efforts extend the fluoride ion regeneration method to be able to determine cyclosarin (GF) in red blood cells, plasma, and tissue of minipig

Central activation, metabolites, and calcium handling during fatigue with repeated maximal isometric contractions in human muscle.

PubMed

Cairns, Simeon P; Inman, Luke A G; MacManus, Caroline P; van de Port, Ingrid G L; Ruell, Patricia A; Thom, Jeanette M; Thompson, Martin W

2017-08-01

To determine the roles of calcium (Ca 2+ ) handling by sarcoplasmic reticulum (SR) and central activation impairment (i.e., central fatigue) during fatigue with repeated maximal voluntary isometric contractions (MVC) in human muscles. Contractile performance was assessed during 3 min of repeated MVCs (7-s contraction, 3-s rest, n = 17). In ten participants, in vitro SR Ca 2+ -handling, metabolites, and fibre-type composition were quantified in biopsy samples from quadriceps muscle, along with plasma venous [K + ]. In 11 participants, central fatigue was compared using tetanic stimulation superimposed on MVC in quadriceps and adductor pollicis muscles. The decline of peak MVC force with fatigue was similar for both muscles. Fatigue resistance correlated directly with % type I fibre area in quadriceps (r = 0.77, P = 0.009). The maximal rate of ryanodine-induced Ca 2+ -release and Ca 2+ -uptake fell by 31 ± 26 and 28 ± 13%, respectively. The tetanic force depression was correlated with the combined reduction of ATP and PCr, and increase of lactate (r = 0.77, P = 0.009). Plasma venous [K + ] increased from 4.0 ± 0.3 to 5.4 ± 0.8 mM over 1-3-min exercise. Central fatigue occurred during the early contractions in the quadriceps in 7 out of 17 participants (central activation ratio fell from 0.98 ± 0.05 to 0.86 ± 0.11 at 1 min), but dwindled at exercise cessation. Central fatigue was seldom apparent in adductor pollicis. Fatigue with repeated MVC in human limb muscles mainly involves peripheral aspects which include impaired SR Ca 2+ -handling and we speculate that anaerobic metabolite changes are involved. A faster early force loss in quadriceps muscle with some participants is attributed to central fatigue.

Simultaneous determination of apatinib and its four major metabolites in human plasma using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

PubMed

Ding, Juefang; Chen, Xiaoyan; Dai, Xiaojian; Zhong, Dafang

2012-05-01

Apatinib, also known as YN968D1, is a novel antiangiogenic agent that selectively inhibits vascular endothelial growth factor receptor-2. Currently, apatinib is undergoing phase II/III clinical trials in China for the treatment of solid tumors. Apatinib is extensively metabolized in humans, and its major metabolites in circulation include cis-3-hydroxy-apatinib (M1-1), trans-3-hydroxy-apatinib (M1-2), apatinib-25-N-oxide (M1-6), and cis-3-hydroxy-apatinib-O-glucuronide (M9-2). To investigate the pharmacokinetics of apatinib and its four major metabolites in patients with advanced colorectal cancer, a sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of apatinib, M1-1, M1-2, M1-6, and M9-2 in human plasma. After a simple protein precipitation using acetonitrile as the precipitation solvent, all the analytes and the internal standard vatalanib were separated on a Zorbax Eclipse XDB C(18) column (50 mm × 4.6 mm, 1.8 μm, Agilent) using acetonitrile: 5 mmol/L ammonium acetate with 0.1% formic acid as the mobile phase with gradient elution. A chromatographic total run time of 9 min was achieved. Mass spectrometry detection was conducted through electrospray ionization in positive ion multiple reaction monitoring modes. The method was linear over the concentration range of 3.00-2000 ng/mL for each analyte. The lower limit of quantification for each analyte was 3.00 ng/mL. The intra-assay precision for all the analytes was less than 11.3%, the inter-assay precision was less than 13.8%, and the accuracy was between -5.8% and 3.3%. The validated method was successfully applied to a clinical pharmacokinetic study following oral administration of 500 mg apatinib mesylate in patients with advanced colorectal cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo.

PubMed

Edison, Erica E; Brosnan, Margaret E; Meyer, Christian; Brosnan, John T

2007-12-01

A fraction of the body's creatine and creatine phosphate spontaneously degrades to creatinine, which is excreted by the kidneys. In humans, this amounts to approximately 1-2 g/day and demands a comparable rate of de novo creatine synthesis. This is a two-step process in which l-arginine:glycine amidinotransferase (AGAT) catalyzes the conversion of glycine and arginine to ornithine and guanidinoacetate (GAA); guanidinoacetate methyltransferase (GAMT) then catalyzes the S-adenosylmethionine-dependent methylation of GAA to creatine. AGAT is found in the kidney and GAMT in the liver, which implies an interorgan movement of GAA from the kidney to the liver. We studied the renal production of this metabolite in both rats and humans. In control rats, [GAA] was 5.9 microM in arterial plasma and 10.9 microM in renal venous plasma for a renal arteriovenous (A-V) difference of -5.0 microM. In the rat, infusion of arginine or citrulline markedly increased renal GAA production but infusion of glycine did not. Rats fed 0.4% creatine in their diet had decreased renal AGAT activity and mRNA, an arterial plasma [GAA] of 1.5 microM, and a decreased renal A-V difference for GAA of -0.9 microM. In humans, [GAA] was 2.4 microM in arterial plasma, with a renal A-V difference of -1.1 microM. These studies show, for the first time, that GAA is produced by both rat and human kidneys in vivo.

Evaluation of metabolism, plasma protein binding and other biological parameters after administration of (-)-[(18)F]Flubatine in humans.

PubMed

Patt, Marianne; Becker, Georg A; Grossmann, Udo; Habermann, Bernd; Schildan, Andreas; Wilke, Stephan; Deuther-Conrad, Winnie; Graef, Susanne; Fischer, Steffen; Smits, René; Hoepping, Alexander; Wagenknecht, Gudrun; Steinbach, Jörg; Gertz, Hermann-Josef; Hesse, Swen; Schönknecht, Peter; Brust, Peter; Sabri, Osama

2014-07-01

(-)-[(18)F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α4β2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound+metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90min. A fraction of 15%±2% of (-)-[(18)F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (-)-[(18)F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82±0.03 at 3min p.i. to 0.87±0.03 at 270min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (-)-[(18)F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. (-)-[(18)F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment. Copyright © 2014 Elsevier Inc. All rights reserved.

Clinical and Physiological Correlates of Caffeine and Caffeine Metabolites in Primary Insomnia

PubMed Central

Youngberg, Mark R.; Karpov, Irina O.; Begley, Amy; Pollock, Bruce G.; Buysse, Daniel J.

2011-01-01

Objectives: To explore the relationship between plasma concentrations of caffeine and subjective and polysomnographic measures of sleep in both good sleeper controls (GSC) and individuals with primary insomnia (PI), following the consumption of low-moderate quantities of caffeine in the home environment. Methods: 65 PI and 29 GSC, each consuming < 4 four coffee cup equivalents of caffeine daily, were recruited. Subjects completed a diary detailing sleep habits and caffeine consumption, one night of polysomnography, and a blood sample for measurement of plasma caffeine and its metabolites at bedtime. Plasma concentrations of caffeine, its primary metabolite, paraxanthine, and other metabolites were determined for each subject and correlated with self-report and polysomnographic measures. Results: No statistically significant differences were found between GSC and PI with respect to number of caffeinated beverages consumed (p = 0.91), estimated absolute caffeine ingestion (p = 0.48), time of caffeine consumption (p = 0.22), or plasma concentrations of caffeine (p = 0.92) or paraxanthine (p = 0.88). Significant correlations were found between plasma concentrations of caffeine/paraxanthine and endorsed caffeine intake (r = 0.58, p < 0.05) and estimated absolute caffeine ingestion (r = 0.57, p < 0.05). Plasma caffeine/paraxanthine was significantly correlated with percent stage 1 sleep (r = 0.32, p < 0.05). However, plasma concentrations of caffeine/paraxanthine were not significantly correlated with other subjective or polysomnographic measures of sleep disturbance in either GSC or PI. Conclusions: These data suggest that low-moderate amounts of caffeine consumed in the home environment, and mostly during morning hours, have little effect on subjective or polysomnographic measures of sleep in GSC or PI. Citation: Youngberg MR; Karpov IO; Begley A; Pollock BG; Buysse DJ. Clinical and physiological correlates of caffeine and caffeine metabolites in primary insomnia. J Clin Sleep Med 2011;7(2):196-203. PMID:21509336

Studies on distribution and metabolism of para-methoxymethamphetamine (PMMA) in rats after subcutaneous administration.

PubMed

Rohanova, Miroslava; Balikova, Marie

2009-05-02

p-Methoxymethamphetamine (PMMA) is an illegal psychedelic drug of abuse derived from an amphetamine structure with a risk to health and reports of several cases of intoxications and fatalities caused by its ingestion. However, its pharmacokinetics based on a controlled study is unknown and only partial information on its biotransformation in animal models is available. Our experimental design aimed to study the disposition and kinetic profile of PMMA and its metabolites in rat plasma and selected tissues after the bolus subcutaneous dose of 40mg/kg, using a GC-MS method. Prior to this, we performed a qualitative verification of its metabolites appearing in excreted urine fractions. PMMA maximum plasma concentration of 4014+/-1122ng/mL was reached 30min after dosing, whereas the appearance of metabolites was rather delayed. The disposition of PMMA was characterized by its approximate half-life of 1.0h, volume of distribution of 6.4L/kg and plasma clearance of 4.4L/h. PMMA tissue concentration exceeded plasma and the highest one was found in the lungs (c(max) 42,988+/-10,223ng/g). Penetration through the blood/brain barrier was more efficient considering PMMA and its N-desmethylated metabolite PMA (para-methoxyamphetamine) than hydroxylated metabolites. The maximum brain/plasma ratio value of PMMA (15.8) and PMA (11.8) was reached after 8h of observation. The experimental results ascertained could be useful for subsequent evaluation of the psychotropic or neurotoxic effects of PMMA and for diagnostic concern of intoxication.

Urinary composition and postprandial blood changes in H-secoisolariciresinol diglycoside (SDG) metabolites in rats do not differ between acute and chronic SDG treatments.

PubMed

Rickard, S E; Thompson, L U

2000-09-01

Although chronic exposure to secoisolariciresinol diglycoside (SDG) was shown to alter (3)H-SDG metabolite disposition in rats, the proportion of measured radioactivity attributed to known or unknown SDG metabolites was not determined. Using HPLC and GC-MS, two experiments were conducted to determine the effect of acute (1 d) vs. chronic (10 d) SDG treatment on major urinary metabolites of (3)H-SDG in female, Sprague-Dawley rats (70-72-d-old) over a 48-h period and if new urinary metabolites were detectable in rats fed nonradioactive flaxseed or SDG. A third experiment was conducted to determine changes in postprandial blood levels of (3)H-SDG metabolites over a 24-h period with acute or chronic SDG treatment. Regardless of treatment, enterodiol, enterolactone and secoisolariciresinol accounted for 75-80% of urine radioactivity. Four potential new lignan metabolites, two of which were detected in the urine of rats fed nonradioactive flaxseed or SDG, were found. Type of treatment had no effect on levels of individual urinary metabolites of (3)H-SDG. As observed for plasma lignans in women fed flaxseed, blood radioactivity peaked at 9 h and remained high until 24 h in both treatment groups, suggesting that blood lignan kinetics might be similar with flaxseed or SDG consumption and that they were comparable between humans and rats. In conclusion, the main urinary lignan metabolites were enterodiol, enterolactone and secoisolariciresinol. Urinary composition or blood levels of radioactive lignans were not affected by the duration of SDG exposure. Thus, while chronic SDG exposure alters lignan disposition in rats, it does not change the metabolite profile.

Biotransformation and mass balance of the SGLT2 inhibitor empagliflozin in healthy volunteers.

PubMed

Chen, Lin-Zhi; Jungnik, Arvid; Mao, Yanping; Philip, Elsy; Sharp, Dale; Unseld, Anna; Seman, Leo; Woerle, Hans-Jürgen; Macha, Sreeraj

2015-01-01

1. The absorption, biotransformation and excretion of empagliflozin, an SGLT2 inhibitor, were evaluated in eight healthy subjects following a single 50 mg oral dose of empagliflozin containing ∼100 µCi [(14)C]-empagliflozin. 2. Radioactivity was rapidly absorbed, with plasma levels peaking 1 h post-dose. Total exposure was lower in blood versus plasma, consistent with moderate (28.6-36.8%) red blood cell partitioning. Protein binding was 80.3-86.2%. 3. Most of the radioactive dose was recovered in urine (54.4%) and faeces (41.1%). Unchanged empagliflozin was the most abundant drug-related component in plasma, representing 75.5-77.4% of plasma radioactivity and 79.6% plasma radioactivity AUC0-12 h. Unchanged empagliflozin was the most abundant drug-related component in urine and faeces, representing 43.5% (23.7% of dose) and 82.9% (34.1% of dose) of radioactivity in urine and faeces, respectively. Six metabolites were identified in plasma: three glucuronide conjugates representing 4.7-7.1% of AUC0-12 h and three less abundant metabolites (<0.2-1.9% AUC0-12 h). The most abundant metabolites in urine were two glucuronide conjugates (7.8-13.2% of dose) and in faeces was a tetrahydrofuran ring-opened carboxylic acid metabolite (1.9% of dose). 4. To conclude, empagliflozin was rapidly absorbed and excreted primarily unchanged in urine and faeces. Unchanged parent was the major drug-related component in plasma. Metabolism was primarily via glucuronide conjugation.

Reproducible diagnostic metabolites in plasma from typhoid fever patients in Asia and Africa

PubMed Central

Näsström, Elin; Parry, Christopher M; Vu Thieu, Nga Tran; Maude, Rapeephan R; de Jong, Hanna K; Fukushima, Masako; Rzhepishevska, Olena; Marks, Florian; Panzner, Ursula; Im, Justin; Jeon, Hyonjin; Park, Seeun; Chaudhury, Zabeen; Ghose, Aniruddha; Samad, Rasheda; Van, Tan Trinh; Johansson, Anders; Dondorp, Arjen M; Thwaites, Guy E; Faiz, Abul; Antti, Henrik; Baker, Stephen

2017-01-01

Salmonella Typhi is the causative agent of typhoid. Typhoid is diagnosed by blood culture, a method that lacks sensitivity, portability and speed. We have previously shown that specific metabolomic profiles can be detected in the blood of typhoid patients from Nepal (Näsström et al., 2014). Here, we performed mass spectrometry on plasma from Bangladeshi and Senegalese patients with culture confirmed typhoid fever, clinically suspected typhoid, and other febrile diseases including malaria. After applying supervised pattern recognition modelling, we could significantly distinguish metabolite profiles in plasma from the culture confirmed typhoid patients. After comparing the direction of change and degree of multivariate significance, we identified 24 metabolites that were consistently up- or down regulated in a further Bangladeshi/Senegalese validation cohort, and the Nepali cohort from our previous work. We have identified and validated a metabolite panel that can distinguish typhoid from other febrile diseases, providing a new approach for typhoid diagnostics. DOI: http://dx.doi.org/10.7554/eLife.15651.001 PMID:28483042

Pharmacokinetics, tissue distribution, and metabolism of nitrofurantoin in the channel catfish (Ictalurus punctatus)

USGS Publications Warehouse

Stehly, G.R.; Plakas, S.M.

1993-01-01

The pharmacokinetics, tissue distribution, and metabolism of the drug nitrofurantoin were examined in the channel catfish (Ictalurus punctatus) after intravascular or oral dosing. Mean plasma concentrations of nitrofurantoin after intravascular administration at 1 and 10 mg/kg of body weight were best fit to two- and three-compartment pharmacokinetic models, respectively. Nitrofurantoin was rapidly eliminated from the plasma after intravascular dosing; at 1 and 10 mg/kg, the terminal half-lives were 23 and 46 min, respectively. After oral dosing at 1 mg/kg, peak plasma concentrations (0.06 mu g/ml) occurred at 2 h; the bioavailability was 17%. Residues of nitrofurantoin and its metabolites in the tissues were initially eliminated rapidly but persisted at the later sampling times. Residue concentrations were highest in the plasma and excretory tissues. Approximately 21% and 4% of the oral dose were eliminated in the urine and bile, respectively. Parent nitrofurantoin was the major radiolabelled compound found in the urine; however, the percentage of total residues composed of metabolites increased with time. Biliary residues consisted mostly of nitrofurantoin metabolites. High-performance liquid chromatography revealed the presence of at least five metabolites in the urine and bile.

Bioavailability of cyanidin glycosides from natural chokeberry (Aronia melanocarpa) juice with dietary-relevant dose of anthocyanins in humans.

PubMed

Wiczkowski, Wieslaw; Romaszko, Ewa; Piskula, Mariusz K

2010-12-08

The aim of this study was to investigate the bioavailability of anthocyanins from chokeberry juice with a dietary-relevant dose of anthocyanins. Thirteen healthy volunteers consumed chokeberry juice providing 0.8 mg of anthocyanins/kg of body weight. Before and after juice consumption, blood and urine were collected. Concentration of anthocyanins was measured with HPLC-PDA-MS-ESI. Cyanidin-3-galactoside comprised 66% of total chokeberry anthocyanins. Eight cyanidin derivatives were found in blood and urine after juice consumption. The maximum plasma anthocyanin concentration of 32.7 ± 2.9 nmol/L was reached at 1.3 ± 0.1 h after juice consumption. The anthocyanins' urine excretion rate (62.9 ± 5.0 nmol/h) was the highest within the first 2 h. In total, 0.25 ± 0.02% of the ingested anthocyanins was excreted by the renal route during 24 h, mainly as metabolites of cyanidin. According to these observations, after consumption of a dietary-relevant dose of anthocyanins as natural chokeberry juice, anthocyanins and their metabolites were present in plasma and urine of volunteers.

Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the Myc-Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2-thioxothiazolidin-4-one, in mice.

PubMed

Guo, Jianxia; Parise, Robert A; Joseph, Erin; Egorin, Merrill J; Lazo, John S; Prochownik, Edward V; Eiseman, Julie L

2009-03-01

c-Myc is commonly activated in many human tumors and is functionally important in cellular proliferation, differentiation, apoptosis and cell cycle progression. The activity of c-Myc requires noncovalent interaction with its client protein Max. In vitro studies indicate the thioxothiazolidinone, 10058-F4, inhibits c-Myc/Max dimerization. In this study, we report the efficacy, pharmacokinetics and metabolism of this novel protein-protein disruptor in mice. SCID mice bearing DU145 or PC-3 human prostate cancer xenografts were treated with either 20 or 30 mg/kg 10058-F4 on a qdx5 schedule for 2 weeks for efficacy studies. For pharmacokinetics and metabolism studies, mice bearing PC-3 or DU145 xenografts were treated with 20 mg/kg of 10058-F4 i.v. Plasma and tissues were collected 5-1440 min after dosing. The concentration of 10058-F4 in plasma and tissues was determined by HPLC, and metabolites were characterized by LC-MS/MS. Following a single iv dose, peak plasma 10058-F4 concentrations of approximately 300 muM were seen at 5 min and declined to below the detection limit at 360 min. Plasma concentration versus time data were best approximated by a two-compartment, open, linear model. The highest tissue concentrations of 10058-F4 were found in fat, lung, liver, and kidney. Peak tumor concentrations of 10058-F4 were at least tenfold lower than peak plasma concentrations. Eight metabolites of 10058-F4 were identified in plasma, liver, and kidney. The terminal half-life of 10058-F4 was approximately 1 h, and the volume of distribution was >200 ml/kg. No significant inhibition of tumor growth was seen after i.v. treatment of mice with either 20 or 30 mg/kg 10058-F4. The lack of significant antitumor activity of 10058-F4 in tumor-bearing mice may have resulted from its rapid metabolism and low concentration in tumors.

Study of the plasma proteome of Atlantic cod (Gadus morhua): Effect of exposure to two PAHs and their corresponding diols.

PubMed

Skogland Enerstvedt, Karianne; Sydnes, Magne O; Pampanin, Daniela M

2017-09-01

Occurrence of polycyclic aromatic hydrocarbon (PAH) contamination in the marine environment represents a risk to marine life and humans. In this study, plasma samples from Atlantic cod (Gadus morhua) were analysed by shotgun mass spectrometry to investigate the plasma proteome in response to exposure to single PAHs (naphthalene or chrysene) and their corresponding metabolites (dihydrodiols). In total, 369 proteins were identified and ranked according to their relative abundance. The levels of 12 proteins were found significantly altered in PAH exposed fish and are proposed as new biomarker candidates. Eleven proteins were upregulated, primarily immunoglobulin components, and one protein was downregulated (antifreeze protein type IV.) The uniformity of the upregulated proteins suggests a triggered immune response in the exposed fish. Overall, the results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers for environmental monitoring of PAH related exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anthocyanins and their gut metabolites reduce the adhesion of monocyte to TNFα-activated endothelial cells at physiologically relevant concentrations.

PubMed

Krga, Irena; Monfoulet, Laurent-Emmanuel; Konic-Ristic, Aleksandra; Mercier, Sylvie; Glibetic, Maria; Morand, Christine; Milenkovic, Dragan

2016-06-01

An increasing number of evidence suggests a protective role of dietary anthocyanins against cardiovascular diseases. Anthocyanins' extensive metabolism indicates that their metabolites could be responsible for the protective effects associated with consumption of anthocyanin-rich foods. The aim of this work was to investigate the effect of plasma anthocyanins and their metabolites on the adhesion of monocytes to TNFα-activated endothelial cells and on the expression of genes encoding cell adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were exposed to circulating anthocyanins: cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, anthocyanin degradation product: 4-hydroxybenzaldehyde, or to their gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid, at physiologically-relevant concentrations (0.1-2 μM) and time of exposure. Both anthocyanins and gut metabolites decreased the adhesion of monocytes to HUVECs, with a magnitude ranging from 18.1% to 47%. The mixture of anthocyanins and that of gut metabolites also reduced monocyte adhesion. However, no significant effect on the expression of genes encoding E-selectin, ICAM1 and VCAM1 was observed, suggesting that other molecular targets are involved in the observed effect. In conclusion, this study showed the potency of anthocyanins and their gut metabolites to modulate the adhesion of monocytes to endothelial cells, the initial step in atherosclerosis development, under physiologically-relevant conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

PubMed

Wang, Zhican; Lin, Yvonne S; Dickmann, Leslie J; Poulton, Emma-Jane; Eaton, David L; Lampe, Johanna W; Shen, Danny D; Davis, Connie L; Shuhart, Margaret C; Thummel, Kenneth E

2013-05-01

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia. Copyright © 2013 American Society for Bone and Mineral Research.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug-induced osteomalacia

PubMed Central

Wang, Zhican; Lin, Yvonne S.; Dickmann, Leslie J.; Poulton, Emma-Jane; Eaton, David L.; Lampe, Johanna W.; Shen, Danny D.; Davis, Connie L.; Shuhart, Margaret C.; Thummel, Kenneth E.

2012-01-01

Long-term therapy with certain drugs, especially P450 inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4 and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25OHD3 were evaluated following exposure to P450 inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine and rifampin significantly increased the levels of CYP3A4 but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6′,7′-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1 or CYP27B1 expression. 24R,25(OH)2D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (−10%; p = 0.03), and a non-significant change in 24R,25(OH)2D3 (−8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia. PMID:23212742

An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4'-hydroxylated metabolites in breast cancer patients.

PubMed

Dahmane, E; Mercier, T; Zanolari, B; Cruchon, S; Guignard, N; Buclin, T; Leyvraz, S; Zaman, K; Csajka, C; Decosterd, L A

2010-12-15

There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure. Copyright © 2010 Elsevier B.V. All rights reserved.

Sprague-Dawley rats display metabolism-mediated sex differences in the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fonsart, Julien; Menet, Marie-Claude; Decleves, Xavier

The use of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has been associated with unexplained deaths. Male humans and rodents are more sensitive to acute toxicity than are females, including a potentially lethal hyperthermia. MDMA is highly metabolized to five main metabolites, by the enzymes CYP1A2 and CYP2D. The major metabolite in rats, 3,4-methylenedioxyamphetamine (MDA), also causes hyperthermia. We postulated that the reported sex difference in rats is due to a sexual dimorphism(s). We therefore determined (1) the LD50 of MDMA and MDA, (2) their hyperthermic effects, (3) the activities of liver CYP1A2 and CYP2D, (4) the liver microsomal metabolism ofmore » MDMA and MDA, (5) and the plasma concentrations of MDMA and its metabolites 3 h after giving male and female Sprague-Dawley (SD) rats MDMA (5 mg.kg{sup -1} sc). The LD50 of MDMA was 2.4-times lower in males than in females. MDMA induced greater hyperthermia (0.9 deg. C) in males. The plasma MDA concentration was 1.3-fold higher in males, as were CYP1A2 activity (twice) and N-demethylation to MDA (3.3-fold), but the plasma MDMA concentration (1.4-fold) and CYP2D activity (1.3-fold) were higher in females. These results suggest that male SD rats are more sensitive to MDMA acute toxicity than are females, probably because their CYP1A2 is more active, leading to higher N-demethylation and plasma MDA concentration. This metabolic pathway could be responsible for the lethality of MDMA, as the LD50 of MDA is the same in both sexes. These data strongly suggest that the toxicity of amphetamine-related drugs largely depends on metabolic differences.« less

Pharmacokinetics and tolerability of DA-8031, a novel selective serotonin reuptake inhibitor for premature ejaculation in healthy male subjects.

PubMed

Shin, Dongseong; Lee, SeungHwan; Yi, Sojeong; Yoon, Seo Hyun; Cho, Joo-Youn; Bahng, Mi Young; Jang, In-Jin; Yu, Kyung-Sang

2017-01-01

DA-8031 is a selective serotonin reuptake inhibitor under development for the treatment of premature ejaculation. This is the first-in-human study aimed at evaluating the pharmacokinetics and tolerability of DA-8031 and its metabolites (M1, M2, M4, and M5) in the plasma and urine after administration of a single oral dose in healthy male subjects. A dose block-randomized, double-blind, placebo-controlled, single ascending dose study was conducted. Subjects received either placebo or a single dose of DA-8031 at 5, 10, 20, 40, 60, 80, or 120 mg. DA-8031 and its four metabolites were analyzed in the plasma and urine for pharmacokinetic evaluation. The effect of genetic polymorphisms of cytochrome-P450 (CYP) enzymes on the pharmacokinetics of DA-8031 was evaluated. After a single dose, plasma DA-8031 reached the maximum concentration at a median of 2-3 h and was eliminated with terminal elimination half-life of 17.9-28.7 h. The mean renal clearance was 3.7-5.6 L/h. Dose-proportional pharmacokinetics was observed over the dose range of 20-80 mg. Among the metabolites, M4 had the greatest plasma concentration, followed by M5 and M1. Subjects with CYP2D6 intermediate metabolizer had significantly greater dose-normalized C max and AUC 0- t of DA-8031 as well as smaller metabolic ratios than those subjects with CYP2D6 extensive metabolizer. The most common adverse events were nausea, dizziness, and headache, and no serious adverse events were reported. In conclusion, the systemic exposure of DA-8031 was increased proportionally to the dose within 20-80 mg. Genetic polymorphisms of CYP2D6 had an effect on the systemic exposure of DA-8031. DA-8031 was well tolerated after single doses of 80 mg or less.

Development and validation of a sensitive assay for analysis of midazolam, free and conjugated 1-hydroxymidazolam and 4-hydroxymidazolam in pediatric plasma: Application to Pediatric Pharmacokinetic Study.

PubMed

Moorthy, Ganesh S; Jogiraju, Harini; Vedar, Christina; Zuppa, Athena F

2017-11-01

Pharmacokinetic, pharmacodynamic and pharmacogenomic studies of midazolam are currently being performed in critically ill children to find suitable dose regimens. Sensitive assays using small volumes of plasma are necessary to determine the concentrations of midazolam and its respective metabolites in pediatric studies. Midazolam is metabolized to hydroxylated midazolam isomers, which are present as free as well as the corresponding glucuronide conjugates. A high-performance liquid chromatographic method with tandem mass spectrometry has been developed and validated for the quantification of midazolam, and free and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites in small volumes of plasma. Cleanup consisted of 96-well μ-elution solid phase extraction (SPE). The analytes were separated by gradient elution using a C 18 analytical column with a total run time of 5min. Multiple reaction monitoring was employed using precursor to product ion transitions of m/z 326.2→291.3 for midazolam, m/z 342.1→203.0 for 1-hydroxymidazolam, m/z 342.1→325.1 for 4-hydroxymidazolam and m/z 330.2→295.3 for 2 H 4 -midazolam (internal standard). Since authentic hydroxymidazolamglucuronide standards are not available, samples were hydrolyzed with β-glucuronidase under optimized conditions. Assay conditions were modified and optimized to provide appropriate recovery and stability because 4-hydroxymidazolam was very acid sensitive. Standard curves were linear from 0.5 to 1000ng/mL for all three analytes. Intra- and inter day accuracy and precision for quality control samples (2, 20, 200 and 800ng/mL) were within 85-115% and 15% (coefficient of variation), respectively. Stability in plasma and extracts were sufficient under assay conditions. Plasma samples were processed and analyzed for midazolam, and free 1-hydroxymidazolam and 4-hydroxymidazolam metabolites. Plasma samples that were hydrolyzed with β-glucuronidase were processed and analyzed for midazolam, and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites under the same assay conditions. The difference in concentration between the total and free hydroxymidazolam metabolites provided an estimate of conjugated hydroxymidazolam metabolites. The combination of 96-well μ-elution SPE and LC-MS/MS allows reliable quantification of midazolam and its metabolites in small volumes of plasma for pediatric patients. This assay is currently being successfully utilized for analysis of samples from ongoing clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of concentrations of whole blood levels of choline, betaine, and dimethylglycine and their relations to plasma levels.

PubMed

Awwad, Hussain Mohamad; Kirsch, Susanne H; Geisel, Juergen; Obeid, Rima

2014-04-15

We aimed at developing a method for the measurement of choline and its metabolites in whole blood (WB). After an extraction step, quantification of choline, betaine, and dimethylglycine (DMG) was performed using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Plasma and WB metabolites were evaluated in a group of 61 elderly people. The calibration curves were linear (r(2)>0.997) for all compounds. The inter- and intra-assay coefficients of variation for all analytes were <10%. The recoveries were >90% and the relative matrix effect were ≤4.0%. The median concentrations of choline, betaine, and DMG were 11.3, 27.8, and 5.9μmol/L in plasma and 66.6, 165, and 13.7μmol/L in WB, respectively. There were positive correlations between WB and plasma markers; for choline (r=0.42), betaine (r=0.61), and DMG (r=0.56) (all p≤0.001). The concentrations of betaine in WB and plasma were significantly higher in men than in women. The concentrations of WB choline and DMG did not differ significantly according to sex. In conclusion, we have established a reliable method for measuring choline metabolites in WB. The concentrations of WB choline, betaine, and DMG seem to reflect intracellular concentrations of these metabolites. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of the effects of curcumin and curcumin glucuronide in human hepatocellular carcinoma HepG2 cells.

PubMed

Shoji, Motomu; Nakagawa, Kiyotaka; Watanabe, Akio; Tsuduki, Tsuyoshi; Yamada, Teiko; Kuwahara, Shigefumi; Kimura, Fumiko; Miyazawa, Teruo

2014-05-15

Curcumin is a yellow pigment found in turmeric (Curcuma Longa L.), and is reported, in recent studies, to have several pharmacological effects, including anti-oxidant, anti-inflammatory, anti-tumour and lipid-lowering properties. However, as most curcumin is conjugated when absorbed through the intestine, free curcumin is present at extremely low levels inside the body. Therefore, curcumin metabolites have been presumed to be responsible for the curcumin bioactivity. In this study, we first confirmed that curcumin glucuronide is the major metabolite of curcumin found in the plasma after oral administration of curcumin in rats. Next, we synthesised curcumin glucuronide and compared the effects of curcumin and curcumin glucuronide on gene expression in a human hepatoma cell line (HepG2). We found that the effects of curcumin glucuronide are weaker than those of curcumin and that this difference is related to relative absorption rates of curcumin and curcumin glucuronide into HepG2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of dietary lysine levels on the concentrations of selected nutrient metabolites in blood plasma of late-stage finishing pigs.

PubMed

Regmi, N; Wang, T; Crenshaw, M A; Rude, B J; Liao, S F

2018-04-01

Lysine is the first-limiting amino acid (AA) in typical swine diets and plays very important roles in promoting growth performance of pigs. This research was conducted to study the effects of dietary lysine on blood plasma concentrations of protein, carbohydrate, and lipid metabolites of pigs. Eighteen crossbred finishing pigs (nine barrows and nine gilts; initial BW 92.3 ± 6.9 kg) were individually penned in an environment controlled barn. Pigs were assigned to three dietary treatments according to a randomized complete block design with gender as block and pig as experimental unit (6 pigs/treatment). Three corn and soybean meal-based diets were formulated to contain total lysine at 0.43%, 0.71%, and 0.98% (as-fed basis) for Diets I (lysine deficient), II (lysine adequate), and III (lysine excess) respectively. After 4 weeks on trial, jugular vein blood was collected and plasma was separated. The plasma concentrations of total protein, albumin, urea nitrogen (UN), triglyceride, total cholesterol, and glucose were determined using an ACE Clinical Chemistry System (Alfa Wassermann, Inc., West Caldwell, NJ, USA). Data were analysed using the GLM Procedure with PDIFF (adjust = T) option of SAS. No differences (p > 0.10) were found between barrows and gilts for any of the metabolites measured. While there were no differences (p > 0.10) between pigs fed Diets II and III in plasma concentrations of UN, albumin, and total cholesterol, the concentration of albumin in these pigs was higher (p < .05) than that of pigs fed Diet I, and the concentrations of UN and total cholesterol in these pigs were lower (p < .05) than that of pigs fed Diet I. There were no differences (p > 0.10) among the three dietary treatments in plasma concentrations of total protein, triglycerides, and glucose. These findings indicated that the plasma metabolite profile can be affected by changing dietary lysine content only. Thorough understanding how the plasma metabolite profile is alternated by dietary lysine will facilitate nutrient management for more sustainable swine production. © 2017 Blackwell Verlag GmbH.

Elevated circulating LDL phenol levels in men who consumed virgin rather than refined olive oil are associated with less oxidation of plasma LDL.

PubMed

de la Torre-Carbot, Karina; Chávez-Servín, Jorge L; Jaúregui, Olga; Castellote, Ana I; Lamuela-Raventós, Rosa M; Nurmi, Tarja; Poulsen, Henrik E; Gaddi, Antonio V; Kaikkonen, Jari; Zunft, Hans-Franz; Kiesewetter, Holger; Fitó, Montserrat; Covas, María-Isabel; López-Sabater, M Carmen

2010-03-01

In human LDL, the bioactivity of olive oil phenols is determined by the in vivo disposition of the biological metabolites of these compounds. Here, we examined how the ingestion of 2 similar olive oils affected the content of the metabolic forms of olive oil phenols in LDL in men. The oils differed in phenol concentrations as follows: high (629 mg/L) for virgin olive oil (VOO) and null (0 mg/L) for refined olive oil (ROO). The study population consisted of a subsample from the EUROLIVE study and a randomized controlled, crossover design was used. Intervention periods lasted 3 wk and were preceded by a 2-wk washout period. The levels of LDL hydroxytyrosol monosulfate and homovanillic acid sulfate, but not of tyrosol sulfate, increased after VOO ingestion (P < 0.05), whereas the concentrations of circulating oxidation markers, including oxidized LDL (oxLDL), conjugated dienes, and hydroxy fatty acids, decreased (P < 0.05). The levels of LDL phenols and oxidation markers were not affected by ROO consumption. The relative increase in the 3 LDL phenols was greater when men consumed VOO than when they consumed ROO (P < 0.05), as was the relative decrease in plasma oxLDL (P = 0.001) and hydroxy fatty acids (P < 0.001). Plasma oxLDL concentrations were negatively correlated with the LDL phenol levels (r = -0.296; P = 0.013). Phenols in LDL were not associated with other oxidation markers. In summary, the phenol concentration of olive oil modulates the phenolic metabolite content in LDL after sustained, daily consumption. The inverse relationship of these metabolites with the degree of LDL oxidation supports the in vivo antioxidant role of olive oil phenolics compounds.

4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

PubMed

Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

2011-06-01

We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

Neuropharmacology of 3,4-Methylenedioxypyrovalerone (MDPV), Its Metabolites, and Related Analogs

PubMed Central

Baumann, Michael H.; Bukhari, Mohammad O.; Lehner, Kurt R.; Anizan, Sebastien; Rice, Kenner C.; Concheiro, Marta; Huestis, Marilyn A.

2017-01-01

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive component of so-called bath salts products that has caused serious medical consequences in humans. In this chapter, we review the neuropharmacology of MDPV and related analogs, and supplement the discussion with new results from our preclinical experiments. MDPV acts as a potent uptake inhibitor at plasma membrane transporters for dopamine (DAT) and norepinephrine (NET) in nervous tissue. The MDPV formulation in bath salts is a racemic mixture, and the S isomer is much more potent than the R isomer at blocking DAT and producing abuse-related effects. Elevations in brain extracellular dopamine produced by MDPV are likely to underlie its locomotor stimulant and addictive properties. MDPV displays rapid pharmacokinetics when injected into rats (0.5–2.0 mg/kg), with peak plasma concentrations achieved by 10–20 min and declining quickly thereafter. MDPV is metabolized to 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-OH-3-MeO-PV) in vivo, but motor activation produced by the drug is positively correlated with plasma concentrations of parent drug and not its metabolites. 3,4-Catechol-PV is a potent uptake blocker at DAT in vitro but has little activity after administration in vivo. 4-OH-3-MeO-PV is the main MDPV metabolite but is weak at DAT and NET. MDPV analogs, such as α-pyrrolidinovalerophenone (α-PVP), display similar ability to inhibit DAT and increase extracellular dopamine concentrations. Taken together, these findings demonstrate that MDPV and its analogs represent a unique class of transporter inhibitors with a high propensity for abuse and addiction. PMID:27830575

Determination of N,N-dimethyltryptamine and beta-carboline alkaloids in human plasma following oral administration of Ayahuasca.

PubMed

Yritia, Mercedes; Riba, Jordi; Ortuño, Jordi; Ramirez, Ariel; Castillo, Araceli; Alfaro, Yolanda; de la Torre, Rafael; Barbanoj, Manel J

2002-11-05

Ayahuasca is a South American psychotropic beverage prepared from plants native to the Amazon River Basin. It combines the hallucinogenic agent and 5-HT(2A/2C) agonist N,N-dimethyltryptamine (DMT) with beta-carboline alkaloids showing monoamine oxidase-inhibiting properties. In the present paper, an analytical methodology for the plasma quantification of the four main alkaloids present in ayahuasca plus two major metabolites is described. DMT was extracted by liquid-liquid extraction with n-pentane and quantified by gas chromatography with nitrogen-phosphorus detection. Recovery was 74%, and precision and accuracy were better than 9.9%. The limit of quantification (LOQ) was 1.6 ng/ml. Harmine, harmaline, and tetrahydroharmine (THH), the three main beta-carbolines present in ayahuasca, and harmol and harmalol (O-demethylation metabolites of harmine and harmaline, respectively) were measured in plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Sample preparation was accomplished by solid-phase extraction, which facilitated the automation of the process. All five beta-carbolines were measured using a single detector by switching wavelengths. Separation of harmol and harmalol required only slight changes in the chromatographic conditions. Method validation demonstrated good recoveries, above 87%, and accuracy and precision better than 13.4%. The LOQ was 0.5 ng/ml for harmine, 0.3 ng/ml for harmaline, 1.0 ng/ml for THH, and 0.3 ng/ml for harmol and harmalol. Good linearity was observed in the concentration ranges evaluated for DMT (2.5-50 ng/ml) and the beta-carbolines (0.3-100 ng/ml). The gas chromatography and HPLC methods described allowed adequate characterization of the pharmacokinetics of the four main alkaloids present in ayahuasca, and also of two major beta-carboline metabolites not previously described in the literature.

Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: Effect of sex and arsenic exposure

PubMed Central

Huang, Madelyn C.; Douillet, Christelle; Su, Mingming; Zhou, Kejun; Wu, Tao; Chen, Wenlian; Galanko, Joseph A.; Drobná, Zuzana; Saunders, R. Jesse; Martin, Elizabeth; Fry, Rebecca C.; Jia, Wei; Stýblo, Miroslav

2016-01-01

Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex-specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation. PMID:26883664

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

NASA Astrophysics Data System (ADS)

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-12-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Metabolomics as a promising tool for early osteoarthritis diagnosis.

PubMed

de Sousa, E B; Dos Santos, G C; Duarte, M E L; Moura, V; Aguiar, D P

2017-09-21

Osteoarthritis (OA) is the main cause of disability worldwide, due to progressive articular cartilage loss and degeneration. According to recent research, OA is more than just a degenerative disease due to some metabolic components associated to its pathogenesis. However, no biomarker has been identified to detect this disease at early stages or to track its development. Metabolomics is an emerging field and has the potential to detect many metabolites in a single spectrum using high resolution nuclear magnetic resonance (NMR) techniques or mass spectrometry (MS). NMR is a reproducible and reliable non-destructive analytical method. On the other hand, MS has a lower detection limit and is more destructive, but it is more sensitive. NMR and MS are useful for biological fluids, such as urine, blood plasma, serum, or synovial fluid, and have been used for metabolic profiling in dogs, mice, sheep, and humans. Thus, many metabolites have been listed as possibly associated to OA pathogenesis. The goal of this review is to provide an overview of the studies in animal models and humans, regarding the use of metabolomics as a tool for early osteoarthritis diagnosis. The concept of osteoarthritis as a metabolic disease and the importance of detecting a biomarker for its early diagnosis are highlighted. Then, some studies in plasma and synovial tissues are shown, and finally the application of metabolomics in the evaluation of synovial fluid is described.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

PubMed Central

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-01-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics. PMID:28000704

Quantitative Cross-Species Extrapolation between Humans and Fish: The Case of the Anti-Depressant Fluoxetine

PubMed Central

Margiotta-Casaluci, Luigi; Owen, Stewart F.; Cumming, Rob I.; de Polo, Anna; Winter, Matthew J.; Panter, Grace H.; Rand-Weaver, Mariann; Sumpter, John P.

2014-01-01

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation. PMID:25338069

Plasma amino acid and metabolite signatures tracking diabetes progression in the UCD-T2DM rat model.

PubMed

Piccolo, Brian D; Graham, James L; Stanhope, Kimber L; Fiehn, Oliver; Havel, Peter J; Adams, Sean H

2016-06-01

Elevations of plasma concentrations of branched-chain amino acids (BCAAs) are observed in human insulin resistance and type 2 diabetes mellitus (T2DM); however, there has been some controversy with respect to the passive or causative nature of the BCAA phenotype. Using untargeted metabolomics, plasma BCAA and other metabolites were assessed in lean control Sprague-Dawley rats (LC) and temporally during diabetes development in the UCD-T2DM rat model, i.e., prediabetic (PD) and 2 wk (D2W), 3 mo (D3M), and 6 mo (D6M) post-onset of diabetes. Plasma leucine, isoleucine, and valine concentrations were elevated only in D6M rats compared with D2W rats (by 28, 29, and 30%, respectively). This was in contrast to decreased plasma concentrations of several other amino acids in D3M and/or D6M relative to LC rats (Ala, Arg, Glu, Gln, Met, Ser, Thr, and Trp). BCAAs were positively correlated with fasting glucose and negatively correlated with plasma insulin, total body weight, total adipose tissue weight, and gastrocnemius muscle weight in the D3M and D6M groups. Multivariate analysis revealed that D3M and D6M UCD-T2DM rats had lower concentrations of amino acids, amino acid derivatives, 1,5-anhydroglucitol, and conduritol-β-opoxide and higher concentrations of uronic acids, pantothenic acids, aconitate, benzoic acid, lactate, and monopalmitin-2-glyceride relative to PD and D2W UCD-T2DM rats. The UCD-T2DM rat does not display elevated plasma BCAA concentrations until 6 mo post-onset of diabetes. With the acknowledgement that this is a rodent model of T2DM, the results indicate that elevated plasma BCAA concentrations are not necessary or sufficient to elicit an insulin resistance or T2DM onset. Copyright © 2016 the American Physiological Society.

Plasma amino acid and metabolite signatures tracking diabetes progression in the UCD-T2DM rat model

PubMed Central

Piccolo, Brian D.; Graham, James L.; Stanhope, Kimber L.; Fiehn, Oliver; Havel, Peter J.

2016-01-01

Elevations of plasma concentrations of branched-chain amino acids (BCAAs) are observed in human insulin resistance and type 2 diabetes mellitus (T2DM); however, there has been some controversy with respect to the passive or causative nature of the BCAA phenotype. Using untargeted metabolomics, plasma BCAA and other metabolites were assessed in lean control Sprague-Dawley rats (LC) and temporally during diabetes development in the UCD-T2DM rat model, i.e., prediabetic (PD) and 2 wk (D2W), 3 mo (D3M), and 6 mo (D6M) post-onset of diabetes. Plasma leucine, isoleucine, and valine concentrations were elevated only in D6M rats compared with D2W rats (by 28, 29, and 30%, respectively). This was in contrast to decreased plasma concentrations of several other amino acids in D3M and/or D6M relative to LC rats (Ala, Arg, Glu, Gln, Met, Ser, Thr, and Trp). BCAAs were positively correlated with fasting glucose and negatively correlated with plasma insulin, total body weight, total adipose tissue weight, and gastrocnemius muscle weight in the D3M and D6M groups. Multivariate analysis revealed that D3M and D6M UCD-T2DM rats had lower concentrations of amino acids, amino acid derivatives, 1,5-anhydroglucitol, and conduritol-β-opoxide and higher concentrations of uronic acids, pantothenic acids, aconitate, benzoic acid, lactate, and monopalmitin-2-glyceride relative to PD and D2W UCD-T2DM rats. The UCD-T2DM rat does not display elevated plasma BCAA concentrations until 6 mo post-onset of diabetes. With the acknowledgement that this is a rodent model of T2DM, the results indicate that elevated plasma BCAA concentrations are not necessary or sufficient to elicit an insulin resistance or T2DM onset. PMID:27094034

Cross-species pharmacokinetic comparison from mouse to man of a second-generation antisense oligonucleotide, ISIS 301012, targeting human apolipoprotein B-100.

PubMed

Yu, Rosie Z; Kim, Tae-Won; Hong, An; Watanabe, Tanya A; Gaus, Hans J; Geary, Richard S

2007-03-01

The pharmacokinetics of a 2'-O-(2-methoxyethyl)-modified oligonucleotide, ISIS 301012 [targeting human apolipoprotein B-100 (apoB-100)], was characterized in mouse, rat, monkey, and human. Plasma pharmacokinetics following parental administration was similar across species, exhibiting a rapid distribution phase with t(1/2alpha) of several hours and a prolonged elimination phase with t(1/2beta) of days. The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following s.c. injection, with bioavailability ranging from 80 to 100% in monkeys. Plasma clearance scaled well across species as a function of body weight alone, and this correlation was improved when corrected for plasma protein binding. In all of the animal models studied, the highest tissue concentrations of ISIS 301012 were observed in kidney and liver. Urinary excretion was less than 3% in monkeys and human in the first 24 h. ISIS 301012 is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. However, following 25 mg/kg s.c. administration in mouse and 5-mg/kg i.v. bolus administration in rat, plasma concentrations of ISIS 301012 exceeded their respective protein binding capacity. Thus, urinary excretion increased to 16% or greater within the first 24 h. Albeit slow, urinary excretion of ISIS 301012 and its shortened metabolites is the ultimate elimination pathway of this compound, as demonstrated by 32% of dose recovered in total excreta by 14 days in a rat mass balance study. The pharmacokinetics of ISIS 301012 in human is predictable from the pharmacokinetics measured in animals. The pharmacokinetic properties of ISIS 301012 provide guidance for clinical development and support infrequent dose administration.

Investigation of metabolites for estimating blood deposition time.

PubMed

Lech, Karolina; Liu, Fan; Davies, Sarah K; Ackermann, Katrin; Ang, Joo Ern; Middleton, Benita; Revell, Victoria L; Raynaud, Florence J; Hoveijn, Igor; Hut, Roelof A; Skene, Debra J; Kayser, Manfred

2018-01-01

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor's alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

Cranberry (poly)phenol metabolites correlate with improvements in vascular function: A double-blind, randomized, controlled, dose-response, crossover study.

PubMed

Rodriguez-Mateos, Ana; Feliciano, Rodrigo P; Boeres, Albert; Weber, Timon; Dos Santos, Claudia Nunes; Ventura, M Rita; Heiss, Christian

2016-10-01

Cranberries are rich in potentially bioactive (poly)phenols. The aim of this paper was to investigate whether cranberry juice intake can improve vascular function in healthy men in a dose- and time-dependent manner, and to understand which of the circulating (poly)phenol metabolites correlate with vascular effects. A double-blind randomized controlled crossover trial was conducted in ten healthy males. Flow-mediated dilation (FMD), blood pressure, pulse wave velocity and augmentation index were investigated at baseline, 1, 2, 4, 6, and 8 h post-consumption of cranberry juices containing 409, 787, 1238, 1534, and 1910 mg of total cranberry (poly)phenols (TP), and a control drink. Plasma (poly)phenol metabolites were analyzed by UPLC-Q-TOF MS using authentic standards. We observed dose-dependent increases in FMD at 1, 2, 4, 6, and 8 h with a peak at 4 h and maximal effects with juice containing 1238 mg TP. A total of 60 metabolites were quantified in plasma after cranberry consumption. Twelve (poly)phenol metabolites significantly correlated with the increases in FMD, including ferulic and caffeic acid sulfates, quercetin-3-O-ß-D-glucuronide and a γ-valerolactone sulfate. (Poly)phenols in cranberry juice can improve vascular function in healthy males and this is linked to the presence of specific newly identified plasma metabolites. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma.

PubMed

Orsatti, Laura; Speziale, Roberto; Orsale, Maria Vittoria; Caretti, Fulvia; Veneziano, Maria; Zini, Matteo; Monteagudo, Edith; Lyons, Kathryn; Beconi, Maria; Chan, Kelvin; Herbst, Todd; Toledo-Sherman, Leticia; Munoz-Sanjuan, Ignacio; Bonelli, Fabio; Dominguez, Celia

2015-03-25

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/ml for 3HK, 9.8-20,000 ng/ml for KYN, 0.49-1000 ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%). Copyright © 2015 Elsevier B.V. All rights reserved.

Quantity and source of dietary protein influence metabolite production by gut microbiota and rectal mucosa gene expression: a randomized, parallel, double-blind trial in overweight humans.

PubMed

Beaumont, Martin; Portune, Kevin Joseph; Steuer, Nils; Lan, Annaïg; Cerrudo, Victor; Audebert, Marc; Dumont, Florent; Mancano, Giulia; Khodorova, Nadezda; Andriamihaja, Mireille; Airinei, Gheorghe; Tomé, Daniel; Benamouzig, Robert; Davila, Anne-Marie; Claus, Sandrine Paule; Sanz, Yolanda; Blachier, François

2017-10-01

Background: Although high-protein diets (HPDs) are frequently consumed for body-weight control, little is known about the consequences for gut microbiota composition and metabolic activity and for large intestine mucosal homeostasis. Moreover, the effects of HPDs according to the source of protein need to be considered in this context. Objective: The objective of this study was to evaluate the effects of the quantity and source of dietary protein on microbiota composition, bacterial metabolite production, and consequences for the large intestinal mucosa in humans. Design: A randomized, double-blind, parallel-design trial was conducted in 38 overweight individuals who received a 3-wk isocaloric supplementation with casein, soy protein, or maltodextrin as a control. Fecal and rectal biopsy-associated microbiota composition was analyzed by 16S ribosomal DNA sequencing. Fecal, urinary, and plasma metabolomes were assessed by 1 H-nuclear magnetic resonance. Mucosal transcriptome in rectal biopsies was determined with the use of microarrays. Results: HPDs did not alter the microbiota composition, but induced a shift in bacterial metabolism toward amino acid degradation with different metabolite profiles according to the protein source. Correlation analysis identified new potential bacterial taxa involved in amino acid degradation. Fecal water cytotoxicity was not modified by HPDs, but was associated with a specific microbiota and bacterial metabolite profile. Casein and soy protein HPDs did not induce inflammation, but differentially modified the expression of genes playing key roles in homeostatic processes in rectal mucosa, such as cell cycle or cell death. Conclusions: This human intervention study shows that the quantity and source of dietary proteins act as regulators of gut microbiota metabolite production and host gene expression in the rectal mucosa, raising new questions on the impact of HPDs on the large intestine mucosa homeostasis. This trial was registered at clinicaltrials.gov as NCT02351297. © 2017 American Society for Nutrition.

An Assessment of 25-Hydroxyvitamin D Measurements in Comparability Studies Conducted by the Vitamin D Metabolites Quality Assurance Program

PubMed Central

Bedner, Mary; Lippa, Katrice A.; Tai, Susan S.-C.

2013-01-01

Background The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements, established the first accuracy-based program for improving the comparability of vitamin D metabolite measurements, the Vitamin D Metabolites Quality Assurance Program. Methods Study samples were comprised of human serum or plasma Standard Reference Materials (SRMs) with 25-hydroxyvitamin D values that were determined at NIST. Participants evaluated the materials using immunoassay (IA), liquid chromatography (LC) with mass spectrometric detection, and LC with ultraviolet absorbance detection. NIST evaluated the results for concordance within the participant community as well as trueness relative to the NIST value. Results For the study materials that contain mostly 25-hydroxyvitamin D3 (25(OH)D3), the coefficient of variation (CV) for the participant results was consistently in the range from 7% to 19%, and the median values were biased high relative to the NIST values. However, for materials that contain significant concentrations of both 25-hydroxyvitamin D2 (25(OH)D2) and 25(OH)D3, the median IA results were biased lower than both the LC and the NIST values, and the CV was as high as 28%. The first interlaboratory comparison results for SRM 972a Vitamin D Metabolites in Human Serum are also reported. Conclusions Relatively large within-lab and between-lab variability hinders conclusive assessments of bias and accuracy. PMID:23978484

In vitro biosynthesis, isolation, and identification of predominant metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (RVX-208).

PubMed

Khmelnitsky, Yuri L; Mozhaev, Vadim V; Cotterill, Ian C; Michels, Peter C; Boudjabi, Sihem; Khlebnikov, Vladimir; Madhava Reddy, M; Wagner, Gregory S; Hansen, Henrik C

2013-06-01

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (∼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the β-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

A diet rich in high-glucoraphanin broccoli interacts with genotype to reduce discordance in plasma metabolite profiles by modulating mitochondrial function123

PubMed Central

Armah, Charlotte N; Traka, Maria H; Dainty, Jack R; Defernez, Marianne; Janssens, Astrid; Leung, Wing; Doleman, Joanne F; Potter, John F

2013-01-01

Background: Observational and experimental studies suggest that diets rich in cruciferous vegetables and glucosinolates may reduce the risk of cancer and cardiovascular disease (CVD). Objective: We tested the hypothesis that a 12-wk dietary intervention with high-glucoraphanin (HG) broccoli would modify biomarkers of CVD risk and plasma metabolite profiles to a greater extent than interventions with standard broccoli or peas. Design: Subjects were randomly assigned to consume 400 g standard broccoli, 400 g HG broccoli, or 400 g peas each week for 12 wk, with no other dietary restrictions. Biomarkers of CVD risk and 347 plasma metabolites were quantified before and after the intervention. Results: No significant differences in the effects of the diets on biomarkers of CVD risk were found. Multivariate analyses of plasma metabolites identified 2 discrete phenotypic responses to diet in individuals within the HG broccoli arm, differentiated by single nucleotide polymorphisms associated with the PAPOLG gene. Univariate analysis showed effects of sex (P < 0.001), PAPOLG genotype (P < 0.001), and PAPOLG genotype × diet (P < 0.001) on the plasma metabolic profile. In the HG broccoli arm, the consequence of the intervention was to reduce variation in lipid and amino acid metabolites, tricarboxylic acid (TCA) cycle intermediates, and acylcarnitines between the 2 PAPOLG genotypes. Conclusions: The metabolic changes observed with the HG broccoli diet are consistent with a rebalancing of anaplerotic and cataplerotic reactions and enhanced integration of fatty acid β-oxidation with TCA cycle activity. These modifications may contribute to the reduction in cancer risk associated with diets that are rich in cruciferous vegetables. This trial was registered at clinicaltrials.gov as NCT01114399. PMID:23964055

Phenolic metabolites in plasma and tissues of rats fed with a grape pomace extract as assessed by liquid chromatography-tandem mass spectrometry.

PubMed

Rodriguez Lanzi, Cecilia; Perdicaro, Diahann J; Antoniolli, Andrea; Piccoli, Patricia; Vazquez Prieto, Marcela A; Fontana, Ariel

2018-05-31

Grape pomace extract (GPE) is a rich and relatively low-cost source of phenolic compounds. However, little is known about the main GPE metabolites in mammals, which could help explain the observed health-promoting effects. This study investigated the presence of parent compounds from flavanol, flavonol and stilbene families and their metabolites in rat plasma and tissues after an acute intake of GPE in doses of 300 and 600 mg kg/body weight. The measurement of free compounds and their metabolites was performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results showed the presence of epicatechin, epicatechin methyl-glucuronide, epicatechin methyl-sulphate, catechin, catechin-glucuronide, quercetin methyl-glucuronide, resveratrol-3-glucuronide, resveratrol-4-glucuronide and resveratrol-3-sulphate in plasma, which was dose dependent. The most abundant measured compound in plasma was epicatechin-glucuronide. The presence of glucuronidated and methyl-glucuronidated forms of catechin were observed in the liver at both doses, while epicatechin-glucuronide and methyl-glucuronide were detected only upon intake of 600 mg GPE/kg body weight. At this dose epicatechin-glucuronide and methyl-glucuronide were also detected in muscle, and catechin methyl-glucuronide in adipose tissue. Results show the main GPE metabolites present in rat tissues after oral consumption, contributing to better understand the health benefits of GPE and its potential utilization as a functional ingredient. Copyright © 2018. Published by Elsevier Inc.

Determination of piperphentonamine and metabolites M1 and M6 in human plasma and urine by LC/MS/MS and its application in a pharmacokinetics study in Chinese healthy volunteers.

PubMed

Shi, Aixin; Hu, Xin; Li, Kexin; Li, Jian; Han, Liping; Wan, Huayin; Li, Rubing

2012-11-01

Piperphentonamine hydrochloride (PPTA) is a new calcium sensitizer. A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of piperphentonamine and its metabolites M1 and M6 was developed for the first time and applied to a pharmacokinetics study. Protein precipitation was used for pre-treatment of plasma samples, and solid phase extraction method was used for pre-treatment of urine samples. The chromatographic separation was achieved on a C(18) column using gradient elution in this study: A: 1% acetic acid aqueous solution, and B: acetonitrile. The whole analysis lasted for 10.5min and the gradient flow rate was 0.25mL/min constantly. The detection was performed of a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via a positive electrospray ionization source. The results were that the m/z ratios of monitored precursor ions and product ions of PPTA, M1 and M6 were 354.0→191.8, 356.0→148.7 and 358.0→148.7, respectively. From the standard curve, the concentration ranges of both PPTA and M1 in blood and urine samples were 0.1-500ng/mL and 0.1-200ng/mL, respectively; the concentration ranges of M6 in blood sample and urine sample were 0.2-500ng/mL and 0.2-200ng/mL, respectively; and the correlation coefficient of standard curve was r>0.99. A total of 31 healthy Chinese subjects participated in the pharmacokinetic study of single bolus intravenous injection of piperphentonamine hydrochloride. They were divided into three dosage groups and given 0.2, 0.4 and 0.6mg/kg of PPTA. After drug administration, concentrations of PPTA, M1 and M6 in human plasma and urine samples were determined to evaluation the pharmacokinetic characteristics of PPTA and its metabolites M1 and M6. Copyright © 2012 Elsevier B.V. All rights reserved.

Associations among plasma metabolite levels and short-term exposure to PM2.5 and ozone in a cardiac catheterization cohort.

PubMed

Breitner, Susanne; Schneider, Alexandra; Devlin, Robert B; Ward-Caviness, Cavin K; Diaz-Sanchez, David; Neas, Lucas M; Cascio, Wayne E; Peters, Annette; Hauser, Elizabeth R; Shah, Svati H; Kraus, William E

2016-12-01

Exposure to ambient particulate matter (PM) and ozone has been associated with cardiovascular disease (CVD). However, the mechanisms linking PM and ozone exposure to CVD remain poorly understood. This study explored associations between short-term exposures to PM with a diameter <2.5μm (PM 2.5 ) and ozone with plasma metabolite concentrations. We used cross-sectional data from a cardiac catheterization cohort at Duke University, North Carolina (NC), USA, accumulated between 2001 and 2007. Amino acids, acylcarnitines, ketones and total non-esterified fatty acid plasma concentrations were determined in fasting samples. Daily concentrations of PM 2.5 and ozone were obtained from a Bayesian space-time hierarchical model, matched to each patient's residential address. Ten metabolites were selected for the analysis based on quality criteria and cluster analysis. Associations between metabolites and PM 2.5 or ozone were analyzed using linear regression models adjusting for long-term trend and seasonality, calendar effects, meteorological parameters, and participant characteristics. We found delayed associations between PM 2.5 or ozone and changes in metabolite levels of the glycine-ornithine-arginine metabolic axis and incomplete fatty acid oxidation associated with mitochondrial dysfunction. The strongest association was seen for an increase of 8.1μg/m 3 in PM 2.5 with a lag of one day and decreased mean glycine concentrations (-2.5% [95% confidence interval: -3.8%; -1.2%]). Short-term exposures to ambient PM 2.5 and ozone is associated with changes in plasma concentrations of metabolites in a cohort of cardiac catheterization patients. Our findings might help to understand the link between air pollution and cardiovascular disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

PubMed

Kurko, Johanna; Tringham, Maaria; Tanner, Laura; Näntö-Salonen, Kirsti; Vähä-Mäkilä, Mari; Nygren, Heli; Pöhö, Päivi; Lietzen, Niina; Mattila, Ismo; Olkku, Anu; Hyötyläinen, Tuulia; Orešič, Matej; Simell, Olli; Niinikoski, Harri; Mykkänen, Juha

2016-09-01

Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity. Copyright © 2016 Elsevier Inc. All rights reserved.

Extended Duration Nocturnal Hemodialysis and Changes in Plasma Metabolite Profiles.

PubMed

Kalim, Sahir; Wald, Ron; Yan, Andrew T; Goldstein, Marc B; Kiaii, Mercedeh; Xu, Dihua; Berg, Anders H; Clish, Clary; Thadhani, Ravi; Rhee, Eugene P; Perl, Jeffrey

2018-03-07

In-center, extended duration nocturnal hemodialysis has been associated with variable clinical benefits, but the effect of extended duration hemodialysis on many established uremic solutes and other components of the metabolome is unknown. We determined the magnitude of change in metabolite profiles for patients on extended duration nocturnal hemodialysis. In a 52-week prospective, observational study, we followed 33 patients receiving conventional thrice weekly hemodialysis who converted to nocturnal hemodialysis (7-8 hours per session, three times per week). A separate group of 20 patients who remained on conventional hemodialysis (3-4 hours per session, three times per week) served as a control group. For both groups, we applied liquid chromatography-mass spectrometry-based metabolite profiling on stored plasma samples collected from all participants at baseline and after 1 year. We examined longitudinal changes in 164 metabolites among those who remained on conventional hemodialysis and those who converted to nocturnal hemodialysis using Wilcoxon rank sum tests adjusted for multiple comparisons (false discovery rate <0.05). On average, the nocturnal group had 9.6 hours more dialysis per week than the conventional group. Among 164 metabolites, none changed significantly from baseline to study end in the conventional group. Twenty-nine metabolites changed in the nocturnal group, 21 of which increased from baseline to study end (including all branched-chain amino acids). Eight metabolites decreased after conversion to nocturnal dialysis, including l-carnitine and acetylcarnitine. By contrast, several established uremic retention solutes, including p -cresol sulfate, indoxyl sulfate, and trimethylamine N -oxide, did not change with extended dialysis. Across a wide array of metabolites examined, extended duration hemodialysis was associated with modest changes in the plasma metabolome, with most differences relating to metabolite increases, despite increased dialysis time. Few metabolites showed reduction with more dialysis, and no change in several established uremic toxins was observed. Copyright © 2018 by the American Society of Nephrology.

[Human bioaging acceleration as Chernobyl radiation consequence].

PubMed

Neĭfakh, E A; Liuman, G K

2013-01-01

To monitor human bioaging as a health integral index by blood plasma markers as a molar ratio for biochemically coupled monomers of intracellular lipofuscin, an intracellular polymeric aging pigment with free-radical crossed shifts, has been developed. Lipofuscin includes cell debris with catabolites of lipoperoxic cascade and lipid antioxidants. The latter were detected in the plasma samples of normal adults and children, as well as in Chernobyl clean-up workers (24-62 years old by 1990) with external total gamma-doses of 0.9-145 cSv for 4.2 years. Dynamics for bioaging markers as the molar ratio of blood levels of lipoperoxic catabolites to their antioxidants reflected normal physiologic peculiarities for the studied age periods: oxygen stress for newborns, adaptation during childhood, stability for the middle age and an increased lipoperoxidation (mainly for aging men) due to the age weakening of the antioxidant control. The ratio for the fractions of ma- lone dialdehyde (MDA), a lipoperoxic final catabolite, showed the increase of its binding by plasma proteins in proportions to calendar ages for the norm, as it is the case for lipofuscin; The graph of the age normal molar ratio of protein-bound MDA to the free one was pre-set for calibrations into the developed computer Program to calculate Relative Aging Velocities (Wrel) by bioage increments during the period of human exposure to radiation from the CAPS damage. Wrel were increasing logarithmically to the obtained doses if the total radiation exceeded 4 cSv and exceeded their normal velocities at 50 cSv 10 times or more. Slowing down of Wrel in relation to the calendar age increment was found if the sum doses were lower than 4 cSv. Levels of the studied plasma metabolites as their bioage Moles/Moles markers relative to their norms are dynamically stationary in contrast to the lipofuscin intracellular irreversible accumulation. Earlier it was shown that the decreased vitamin E and A levels with the increased lipoperoxic metabolite blood levels that indicate health consequences for the irradiated CAPS personell with related cytogenetic deviations, as well as for the adult population and children from radio-polluted regions, were restored to norms or corrected by adequate peroral therapy with bioantioxidants.

Simultaneous determination of dextromethorphan, dextrorphan, and guaifenesin in human plasma using semi-automated liquid/liquid extraction and gradient liquid chromatography tandem mass spectrometry.

PubMed

Eichhold, Thomas H; McCauley-Myers, David L; Khambe, Deepa A; Thompson, Gary A; Hoke, Steven H

2007-01-17

A method for the simultaneous determination of dextromethorphan (DEX), dextrorphan (DET), and guaifenesin (GG) in human plasma was developed, validated, and applied to determine plasma concentrations of these compounds in samples from six clinical pharmacokinetic (PK) studies. Semi-automated liquid handling systems were used to perform the majority of the sample manipulation including liquid/liquid extraction (LLE) of the analytes from human plasma. Stable-isotope-labeled analogues were utilized as internal standards (ISTDs) for each analyte to facilitate accurate and precise quantification. Extracts were analyzed using gradient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Use of semi-automated LLE with LC-MS/MS proved to be a very rugged and reliable approach for analysis of more than 6200 clinical study samples. The lower limit of quantification was validated at 0.010, 0.010, and 1.0 ng/mL of plasma for DEX, DET, and GG, respectively. Accuracy and precision of quality control (QC) samples for all three analytes met FDA Guidance criteria of +/-15% for average QC accuracy with coefficients of variation less than 15%. Data from the thorough evaluation of the method during development, validation, and application are presented to characterize selectivity, linearity, over-range sample analysis, accuracy, precision, autosampler carry-over, ruggedness, extraction efficiency, ionization suppression, and stability. Pharmacokinetic data are also provided to illustrate improvements in systemic drug and metabolite concentration-time profiles that were achieved by formulation optimization.

Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease.

PubMed

Sunny, Nishanth E; Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J; Nautiyal, Manisha; Mathew, Justin T; Williams, Caroline M; Cusi, Kenneth

2015-08-15

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.

Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease

PubMed Central

Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J.; Nautiyal, Manisha; Mathew, Justin T.; Williams, Caroline M.; Cusi, Kenneth

2015-01-01

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD. PMID:26058864

Stability of thiopental and pentobarbital in human plasma determined with a new easy and specific gas chromatography-mass spectrometry assay.

PubMed

Martens-Lobenhoffer, J

1999-08-01

A gas chromatographic-mass spectrometric (GC-MS) assay for the determination of thiopental and its main metabolite pentobarbital in human plasma is presented in this study. The sample preparation consists only in the addition of the internal standard barbital and an acidic extraction with ethyl acetate. Analytical separation is accomplished on a RTX-1 15 m x 0.25 mm capillary column with a film thickness of 0.5 micron. The effluent is observed by a mass selective detector operating in the single ion monitoring mode. The limits of detection are 5 ng/ml for pentobarbital and 10 ng/ml for thiopental, the intra-day variabilities are 2.2% and 4.0% and the inter-day variabilities are 3.3% and 7.1% at concentrations of 5 micrograms/ml, respectively. Applying this assay, the stability of thiopental and pentobarbital in human plasma was tested at concentrations of 5 micrograms/ml each. Thiopental is stable in human plasma at least over 41 days stored at -20 degrees C and 5 degrees C, respectively. A decay of about 2%/day is observed under storage at ambient temperature (19-20 degrees C). Pentobarbital is stable under all storage conditions. Methanolic solutions of thiopental are stable for 83 days under storage at 5 degrees C. Aqueous solutions of thiopental-sodium are stable for at least 23 days under storage at 5 degrees C or ambient temperature.

Altered asparagine and glutamate homeostasis precede coronary artery disease and type-2 diabetes.

PubMed

Ottosson, Filip; Smith, Einar; Melander, Olle; Fernandez, Céline

2018-05-16

Type 2 diabetes (T2DM) is accompanied by an increased risk of coronary artery disease (CAD), but the overlapping metabolic disturbances preceding both diseases are insufficiently described. We hypothesized that alterations in metabolism occur years before clinical manifestation of T2DM and CAD and that these alterations are reflected in the plasma metabolome. We thus aimed to identify plasma metabolites that predict future T2DM and CAD. Using targeted liquid chromatography-mass spectrometry (LC-MS), 35 plasma metabolites (amino acid metabolites and acylcarnitines) were quantified in 1049 individuals, free from CAD and diabetes, drawn from a population sample of 5386 in the Malmö Preventive Project (mean age 69.5y, 31% women). The sample included 204 individuals who developed T2DM, 384 who developed CAD and 496 who remained T2DM and CAD free, during a mean follow-up of 6.1 years. In total, 16 metabolites were significantly associated with risk of developing T2DM using logistic regression models. Glutamate (OR=1.96, p=5.4e-12) was the most strongly associated metabolite, followed by increased levels of branched-chain amino acids. Incident CAD was predicted by three metabolites, glutamate (OR=1.28, p=6.6e-4), histidine (OR=0.76, p=5.1e-4) and asparagine (OR=0.80, p=2.2e-3). Glutamate (OR=1.48, p=1.6e-8) and asparagine (OR=0.75, p=1.8e-5) were both associated with a composite endpoint of developing either T2DM or CAD. We identified several plasma metabolites that associated with incidence of T2DM and CAD, where elevated glutamate and reduced asparagine levels associated with both diseases. We thus discovered novel associations that might help shed additional light on why T2DM and CAD commonly co-occur.

Colonic Absorption of Low-Molecular-Weight Metabolites Influenced by the Intestinal Microbiome: A Pilot Study.

PubMed

Matsumoto, Mitsuharu; Ooga, Takushi; Kibe, Ryoko; Aiba, Yuji; Koga, Yasuhiro; Benno, Yoshimi

2017-01-01

Low-molecular-weight metabolites produced by the intestinal microbiome play a direct role in health and disease. However, little is known about the ability of the colon to absorb these metabolites. It is also unclear whether these metabolites are bioavailable. Here, metabolomics techniques (capillary electrophoresis with time-of-flight mass spectrometry, CE-TOFMS), germ-free (GF) mice, and colonized (Ex-GF) mice were used to identify the colonic luminal metabolites transported to colonic tissue and/or blood. We focused on the differences in each metabolite between GF and Ex-GF mice to determine the identities of metabolites that are transported to the colon and/or blood. CE-TOFMS identified 170, 246, 166, and 193 metabolites in the colonic feces, colonic tissue, portal plasma, and cardiac plasma, respectively. We classified the metabolites according to the following influencing factors: (i) the membrane transport system of the colonocytes, (ii) metabolism during transcellular transport, and (iii) hepatic metabolism based on the similarity in the ratio of each metabolite between GF and Ex-GF mice and found 62 and 22 metabolites that appeared to be absorbed from the colonic lumen to colonocytes and blood, respectively. For example, 11 basic amino acids were transported to the systemic circulation from the colonic lumen. Furthermore, many low-molecular-weight metabolites influenced by the intestinal microbiome are bioavailable. The present study is the first to report the transportation of metabolites from the colonic lumen to colonocytes and somatic blood in vivo, and the present findings are critical for clarifying host-intestinal bacterial interactions.

Chromatographic and spectroscopic characterization of urolithins for their determination in biological samples after the intake of foods containing ellagitannins and ellagic acid.

PubMed

García-Villalba, Rocío; Espín, Juan Carlos; Tomás-Barberán, Francisco A

2016-01-08

Ellagitannins and ellagic acid (EA) are metabolized by the gut microbiota to produce urolithins that could be responsible for the health effects attributed to ellagitannin-containing food products. Several urolithin aglycones could be present in fecal samples while glucuronide and sulphate conjugates are mainly found in plasma and urine. So far, the lack of available standards has made difficult their correct identification and quantification. In the present study, UV and MS spectra characteristics of urolithins and their phase II metabolites have been determined using different systems based on liquid chromatography (LC) coupled with diode-array or mass spectrometer detectors with different analyzers (triple quadrupole (QqQ) and quadrupole time-of-flight (QTOF)). Chromatographic separation was achieved on a reversed-phase Poroshell C18 column (3×100mm, 2.7μm). Elution order, characteristic UV spectra, and relative response factors (RRFs) with respect to their parental compound (EA) and the most common metabolite urolithin A (Uro-A) were determined. This contribution, along with the most important mass spectra characteristics (MRM transitions, qualifier/quantifier ratio, accurate mass and fragmentation pattern) will allow the determination of urolithin metabolites in different biological samples and their quantification even if not all metabolites are commercially available. The methods developed in the three systems have been fully validated in terms of linearity, sensitivity, precision, recovery, matrix effect, selectivity and stability. After that, they were successfully applied to complex biological matrices (urine, feces and plasma) from two human studies in which volunteers consumed ellagitannin-containing foods, such as walnuts and pomegranate extracts. Copyright © 2015 Elsevier B.V. All rights reserved.

Pharmacokinetics of intravenous pan-class I phosphatidylinositol 3-kinase (PI3K) inhibitor [14C]copanlisib (BAY 80-6946) in a mass balance study in healthy male volunteers.

PubMed

Gerisch, Michael; Schwarz, Thomas; Lang, Dieter; Rohde, Gabriele; Reif, Stefanie; Genvresse, Isabelle; Reschke, Susanne; van der Mey, Dorina; Granvil, Camille

2017-09-01

To determine the pharmacokinetics of radiolabeled copanlisib (BAY 80-6946) in healthy male volunteers and to investigate the disposition and biotransformation of copanlisib. A single dose of 12 mg copanlisib containing 2.76 MBq [ 14 C]copanlisib was administered as a 1-h intravenous infusion to 6 volunteers with subsequent sampling up to 34 days. Blood, plasma, urine and feces were collected to monitor total radioactivity, parent compound and metabolites. Copanlisib treatment was well tolerated. Copanlisib was rapidly distributed throughout the body with a volume distribution of 1870 L and an elimination half-life of 52.1-h (range 40.4-67.5-h). Copanlisib was the predominant component in human plasma (84% of total radioactivity AUC) and the morpholinone metabolite M1 was the only circulating metabolite (about 5%). Excretion of drug-derived radioactivity based on all 6 subjects was 86% of the dose within a collection interval of 20-34 days with 64% excreted into feces as major route of elimination and 22% into urine. Unchanged copanlisib was the main component excreted into urine (15% of dose) and feces (30% of dose). Excreted metabolites (41% of dose) of copanlisib resulted from oxidative biotransformation. Copanlisib was eliminated predominantly in the feces compared to urine as well as by hepatic biotransformation, suggesting that the clearance of copanlisib would more likely be affected by hepatic impairment than by renal dysfunction. The dual mode of elimination via unchanged excretion of copanlisib and oxidative metabolism decreases the risk of clinically relevant PK-related drug-drug interactions.

Metabolic profiles of male meat eaters, fish eaters, vegetarians, and vegans from the EPIC-Oxford cohort12

PubMed Central

Schmidt, Julie A; Rinaldi, Sabina; Ferrari, Pietro; Carayol, Marion; Achaintre, David; Scalbert, Augustin; Cross, Amanda J; Gunter, Marc J; Fensom, Georgina K; Appleby, Paul N; Key, Timothy J; Travis, Ruth C

2015-01-01

Background: Human metabolism is influenced by dietary factors and lifestyle, environmental, and genetic factors; thus, men who exclude some or all animal products from their diet might have different metabolic profiles than meat eaters. Objective: We aimed to investigate differences in concentrations of 118 circulating metabolites, including acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexose, and sphingolipids related to lipid, protein, and carbohydrate metabolism between male meat eaters, fish eaters, vegetarians, and vegans from the Oxford arm of the European Prospective Investigation into Cancer and Nutrition. Design: In this cross-sectional study, concentrations of metabolites were measured by mass spectrometry in plasma from 379 men categorized according to their diet group. Differences in mean metabolite concentrations across diet groups were tested by using ANOVA, and a false discovery rate–controlling procedure was used to account for multiple testing. Principal component analysis was used to investigate patterns in metabolic profiles. Results: Concentrations of 79% of metabolites differed significantly by diet group. In the vast majority of these cases, vegans had the lowest concentration, whereas meat eaters most often had the highest concentrations of the acylcarnitines, glycerophospholipids, and sphingolipids, and fish eaters or vegetarians most often had the highest concentrations of the amino acids and a biogenic amine. A clear separation between patterns in the metabolic profiles of the 4 diet groups was seen, with vegans being noticeably different from the other groups because of lower concentrations of some glycerophospholipids and sphingolipids. Conclusions: Metabolic profiles in plasma could effectively differentiate between men from different habitual diet groups, especially vegan men compared with men who consume animal products. The difference in metabolic profiles was mainly explained by the lower concentrations of glycerophospholipids and sphingolipids in vegans. PMID:26511225

Metabolic profiles of male meat eaters, fish eaters, vegetarians, and vegans from the EPIC-Oxford cohort.

PubMed

Schmidt, Julie A; Rinaldi, Sabina; Ferrari, Pietro; Carayol, Marion; Achaintre, David; Scalbert, Augustin; Cross, Amanda J; Gunter, Marc J; Fensom, Georgina K; Appleby, Paul N; Key, Timothy J; Travis, Ruth C

2015-12-01

Human metabolism is influenced by dietary factors and lifestyle, environmental, and genetic factors; thus, men who exclude some or all animal products from their diet might have different metabolic profiles than meat eaters. We aimed to investigate differences in concentrations of 118 circulating metabolites, including acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexose, and sphingolipids related to lipid, protein, and carbohydrate metabolism between male meat eaters, fish eaters, vegetarians, and vegans from the Oxford arm of the European Prospective Investigation into Cancer and Nutrition. In this cross-sectional study, concentrations of metabolites were measured by mass spectrometry in plasma from 379 men categorized according to their diet group. Differences in mean metabolite concentrations across diet groups were tested by using ANOVA, and a false discovery rate-controlling procedure was used to account for multiple testing. Principal component analysis was used to investigate patterns in metabolic profiles. Concentrations of 79% of metabolites differed significantly by diet group. In the vast majority of these cases, vegans had the lowest concentration, whereas meat eaters most often had the highest concentrations of the acylcarnitines, glycerophospholipids, and sphingolipids, and fish eaters or vegetarians most often had the highest concentrations of the amino acids and a biogenic amine. A clear separation between patterns in the metabolic profiles of the 4 diet groups was seen, with vegans being noticeably different from the other groups because of lower concentrations of some glycerophospholipids and sphingolipids. Metabolic profiles in plasma could effectively differentiate between men from different habitual diet groups, especially vegan men compared with men who consume animal products. The difference in metabolic profiles was mainly explained by the lower concentrations of glycerophospholipids and sphingolipids in vegans.

Candidate Proteins, Metabolites and Transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

PubMed Central

Finkel, Richard S.; Crawford, Thomas O.; Swoboda, Kathryn J.; Kaufmann, Petra; Juhasz, Peter; Li, Xiaohong; Guo, Yu; Li, Rebecca H.; Trachtenberg, Felicia; Forrest, Suzanne J.; Kobayashi, Dione T.; Chen, Karen S.; Joyce, Cynthia L.; Plasterer, Thomas

2012-01-01

Background Spinal Muscular Atrophy (SMA) is a neurodegenerative motor neuron disorder resulting from a homozygous mutation of the survival of motor neuron 1 (SMN1) gene. The gene product, SMN protein, functions in RNA biosynthesis in all tissues. In humans, a nearly identical gene, SMN2, rescues an otherwise lethal phenotype by producing a small amount of full-length SMN protein. SMN2 copy number inversely correlates with disease severity. Identifying other novel biomarkers could inform clinical trial design and identify novel therapeutic targets. Objective: To identify novel candidate biomarkers associated with disease severity in SMA using unbiased proteomic, metabolomic and transcriptomic approaches. Materials and Methods: A cross-sectional single evaluation was performed in 108 children with genetically confirmed SMA, aged 2–12 years, manifesting a broad range of disease severity and selected to distinguish factors associated with SMA type and present functional ability independent of age. Blood and urine specimens from these and 22 age-matched healthy controls were interrogated using proteomic, metabolomic and transcriptomic discovery platforms. Analyte associations were evaluated against a primary measure of disease severity, the Modified Hammersmith Functional Motor Scale (MHFMS) and to a number of secondary clinical measures. Results A total of 200 candidate biomarkers correlate with MHFMS scores: 97 plasma proteins, 59 plasma metabolites (9 amino acids, 10 free fatty acids, 12 lipids and 28 GC/MS metabolites) and 44 urine metabolites. No transcripts correlated with MHFMS. Discussion In this cross-sectional study, “BforSMA” (Biomarkers for SMA), candidate protein and metabolite markers were identified. No transcript biomarker candidates were identified. Additional mining of this rich dataset may yield important insights into relevant SMA-related pathophysiology and biological network associations. Additional prospective studies are needed to confirm these findings, demonstrate sensitivity to change with disease progression, and assess potential impact on clinical trial design. Trial Registry Clinicaltrials.gov NCT00756821. PMID:22558154

Concordance of Changes in Metabolic Pathways Based on Plasma Metabolomics and Skeletal Muscle Transcriptomics in Type 1 Diabetes

PubMed Central

Dutta, Tumpa; Chai, High Seng; Ward, Lawrence E.; Ghosh, Aditya; Persson, Xuan-Mai T.; Ford, G. Charles; Kudva, Yogish C.; Sun, Zhifu; Asmann, Yan W.; Kocher, Jean-Pierre A.; Nair, K. Sreekumaran

2012-01-01

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry–based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome–based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment. PMID:22415876

Effect of a herbal extract containing curcumin and piperine on midazolam, flurbiprofen and paracetamol (acetaminophen) pharmacokinetics in healthy volunteers

PubMed Central

Volak, Laurie P; Hanley, Michael J; Masse, Gina; Hazarika, Suwagmani; Harmatz, Jerold S; Badmaev, Vladimir; Majeed, Muhammed; Greenblatt, David J; Court, Michael H

2013-01-01

Aims Turmeric extract derived curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) are currently being evaluated for the treatment of cancer and Alzheimer's dementia. Previous in vitro studies indicate that curcuminoids and piperine (a black pepper derivative that enhances curcuminoid bioavailability) could inhibit human CYP3A, CYP2C9, UGT and SULT dependent drug metabolism. The aim of this study was to determine whether a commercially available curcuminoid/piperine extract alters the pharmacokinetic disposition of probe drugs for these enzymes in human volunteers. Methods A randomized placebo-controlled six way crossover study was conducted in eight healthy volunteers. A standardized curcuminoid/piperine preparation (4 g curcuminoids plus 24 mg piperine) or matched placebo was given orally four times over 2 days before oral administration of midazolam (CYP3A probe), flurbiprofen (CYP2C9 probe) or paracetamol (acetaminophen) (dual UGT and SULT probe). Plasma and urine concentrations of drugs, metabolites and herbals were measured by HPLC. Subject sedation and electroencephalograph effects were also measured following midazolam dosing. Results Compared with placebo, the curcuminoid/piperine treatment produced no meaningful changes in plasma Cmax, AUC, clearance, elimination half-life or metabolite levels of midazolam, flurbiprofen or paracetamol (α = 0.05, paired t-tests). There was also no effect of curcuminoid/piperine treatment on the pharmacodynamics of midazolam. Although curcuminoid and piperine concentrations were readily measured in plasma following glucuronidase/sulfatase treatment, unconjugated concentrations were consistently below the assay thresholds (0.05–0.08 μm and 0.6 μm, respectively). Conclusion The results indicate that short term use of this piperine-enhanced curcuminoid preparation is unlikely to result in a clinically significant interaction involving CYP3A, CYP2C9 or the paracetamol conjugation enzymes. PMID:22725836

Development and validation of a sensitive UHPLC-MS/MS method for the simultaneous analysis of tramadol, dextromethorphan chlorpheniramine and their major metabolites in human plasma in forensic context: application to pharmacokinetics.

PubMed

Heneedak, Hala M; Salama, Ismail; Mostafa, Samia; El-Kady, Ehab; El-Sadek, Mohamed

2015-07-01

The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to European Union guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. In the present study, a fast, sensitive and robust method to quantify tramadol, chlorpheniramine, dextromethorphan and their major metabolites, O-desmethyltramadol, dsmethyl-chlorpheniramine and dextrophan, respectively, in human plasma using ibuprofen as internal standard (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction method using ethyl acetate-diethyl-ether (1:1). Extracted samples were analyzed by ultra-high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase containing acetonitrile, water and formic acid (89.2:11.7:0.1) for 2.0 min at a flow rate of 0.25 μL/min into a Hypersil-Gold C18 column, 20 × 2.0 mm (1.9 µm) from Thermoscientific, New York, USA. The calibration curve was linear for the six analytes. The intraday precision (RSD) and accuracy (RE) of the method were 3-9.8 and -1.7-4.5%, respectively. The analytical procedure herein described was used to assess the pharmacokinetics of the analytes in 24 healthy volunteers after a single oral dose containing 50 mg of tramadol hydrochloride, 3 mg chlorpheniramine maleate and 15 mg of dextromethorphan hydrobromide. Copyright © 2014 John Wiley & Sons, Ltd.

Estimates of the pharmacokinetics of famciclovir and its active metabolite penciclovir in young Asian elephants (Elephas maximus).

PubMed

Brock, A Paige; Isaza, Ramiro; Hunter, Robert P; Richman, Laura K; Montali, Richard J; Schmitt, Dennis L; Koch, David E; Lindsay, William A

2012-12-01

To determine plasma pharmacokinetics of penciclovir following oral and rectal administration of famciclovir to young Asian elephants (Elephas maximus). 6 healthy Asian elephants (5 females and 1 male), 4.5 to 9 years old and weighing 1,646 to 2,438 kg. Famciclovir was administered orally or rectally in accordance with an incomplete crossover design. Three treatment groups, each comprising 4 elephants, received single doses of famciclovir (5 mg/kg, PO, or 5 or 15 mg/kg, rectally); there was a minimum 12-week washout period between subsequent famciclovir administrations. Serial blood samples were collected after each administration. Samples were analyzed for famciclovir and penciclovir with a validated liquid chromatography-mass spectroscopy assay. Famciclovir was tolerated well for both routes of administration and underwent complete biotransformation to the active metabolite, penciclovir. Mean maximum plasma concentration of penciclovir was 1.3 μg/mL at 1.1 hours after oral administration of 5 mg/kg. Similar results were detected after rectal administration of 5 mg/kg. Mean maximum plasma concentration was 3.6 μg/mL at 0.66 hours after rectal administration of 15 mg/kg; this concentration was similar to results reported for humans receiving 7 mg/kg orally. Juvenile Asian elephants are susceptible to elephant endotheliotropic herpesvirus. Although most infections are fatal, case reports indicate administration of famciclovir has been associated with survival of 3 elephants. In Asian elephants, a dose of 8 to 15 mg of famciclovir/kg given orally or rectally at least every 8 hours may result in penciclovir concentrations that are considered therapeutic in humans.

Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma.

PubMed

Johnston, Jeffrey S; Phelps, Mitch A; Blum, Kristie A; Blum, William; Grever, Michael R; Farley, Katherine L; Dalton, James T

2008-08-01

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 microL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 microL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3>541.3, 544.2>501.2, and 615.3>572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5-3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 microL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.

High-performance liquid chromatographic determination of certain salicylates and their major metabolites in plasma following topical administration of a liniment to healthy subjects.

PubMed

Dadgar, D; Climax, J; Lambe, R; Darragh, A

1985-08-09

The liniment used is a topical analgesic and anti-inflammatory preparation containing two active constituents, 3-phenylpropylsalicylate and ethyl-5-methoxysalicylate, in solution in isobutyl decanoate. It is known that 3-phenylpropylsalicylate is metabolised to salicylic acid and salicyluric acid and ethyl-5-methoxysalicylate is metabolised to 5-methoxysalicylic acid and gentisic acid. In the present study the separation of the salicylates and their metabolites was carried out on a Waters mu Bondapak C18 column using two different mobile phases, methanol-water (80:20) for the parent drugs and methanol-5% aqueous acetic acid (27:73) for their metabolites. The salicylates and their metabolites were detected by absorption at 310 nm. The limits of detection for parent drugs and metabolites were respectively 0.2 and 0.1 microgram/ml in plasma, using a 1-ml plasma sample and a 20-microliter injection from a reconstituted volume of 250 microliter. Mean percentage coefficients of variation for intra-assay and inter-assay precision were between 3.3 +/- 1.9% to 9.1 +/- 3.7% and 6.8 +/- 2.2% to 15.7 +/- 10.1%, respectively. Linearity, as measured by the correlation coefficient of intra-assay linear regression curves, was better than 0.998 in all cases.

Dietary (Poly)phenolics in Human Health: Structures, Bioavailability, and Evidence of Protective Effects Against Chronic Diseases

PubMed Central

Del Rio, Daniele; Rodriguez-Mateos, Ana; Spencer, Jeremy P.E.; Tognolini, Massimiliano; Borges, Gina

2013-01-01

Abstract Human intervention trials have provided evidence for protective effects of various (poly)phenol-rich foods against chronic disease, including cardiovascular disease, neurodegeneration, and cancer. While there are considerable data suggesting benefits of (poly)phenol intake, conclusions regarding their preventive potential remain unresolved due to several limitations in existing studies. Bioactivity investigations using cell lines have made an extensive use of both (poly)phenolic aglycones and sugar conjugates, these being the typical forms that exist in planta, at concentrations in the low-μM-to-mM range. However, after ingestion, dietary (poly)phenolics appear in the circulatory system not as the parent compounds, but as phase II metabolites, and their presence in plasma after dietary intake rarely exceeds nM concentrations. Substantial quantities of both the parent compounds and their metabolites pass to the colon where they are degraded by the action of the local microbiota, giving rise principally to small phenolic acid and aromatic catabolites that are absorbed into the circulatory system. This comprehensive review describes the different groups of compounds that have been reported to be involved in human nutrition, their fate in the body as they pass through the gastrointestinal tract and are absorbed into the circulatory system, the evidence of their impact on human chronic diseases, and the possible mechanisms of action through which (poly)phenol metabolites and catabolites may exert these protective actions. It is concluded that better performed in vivo intervention and in vitro mechanistic studies are needed to fully understand how these molecules interact with human physiological and pathological processes. Antioxid. Redox Signal. 18, 1818–1892. PMID:22794138

Untargeted Plasma Metabolomics Identifies Endogenous Metabolite with Drug-like Properties in Chronic Animal Model of Multiple Sclerosis*

PubMed Central

Poisson, Laila M.; Suhail, Hamid; Singh, Jaspreet; Datta, Indrani; Denic, Aleksandar; Labuzek, Krzysztof; Hoda, Md Nasrul; Shankar, Ashray; Kumar, Ashok; Cerghet, Mirela; Elias, Stanton; Mohney, Robert P.; Rodriguez, Moses; Rattan, Ramandeep; Mangalam, Ashutosh K.; Giri, Shailendra

2015-01-01

We performed untargeted metabolomics in plasma of B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation, and membrane stability were observed to be significantly altered between EAE and control (p < 0.05, false discovery rate <0.10). Bioinformatics analysis revealed six metabolic pathways being impacted and altered in EAE, including α-linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including ω-3 and ω-6 fatty acids, are commonly decreased in mouse models of multiple sclerosis (MS) and in patients with MS. Daily oral administration of resolvin D1, a downstream metabolite of ω-3, decreased disease progression by suppressing autoreactive T cells and inducing an M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of metabolomics to identify an endogenous metabolite(s) possessing drug-like properties, which is assessed for therapy in preclinical mouse models of MS. PMID:26546682

Oral administration of D-aspartate, but not L-aspartate, depresses rectal temperature and alters plasma metabolites in chicks.

PubMed

Erwan, Edi; Chowdhury, Vishwajit Sur; Nagasawa, Mao; Goda, Ryosei; Otsuka, Tsuyoshi; Yasuo, Shinobu; Furuse, Mitsuhiro

2014-07-25

L-Aspartate (L-Asp) and D-aspartate (D-Asp) are physiologically important amino acids in mammals and birds. However, the functions of these amino acids have not yet been fully understood. In this study, we therefore examined the effects of L-Asp and D-Asp in terms of regulating body temperature, plasma metabolites and catecholamines in chicks. Chicks were first orally administered with different doses (0, 3.75, 7.5 and 15 mmol/kg body weight) of L- or D-Asp to monitor the effects of these amino acids on rectal temperature during 120 min of the experimental period. Oral administration of D-Asp, but not of L-Asp, linearly decreased the rectal temperature in chicks. Importantly, orally administered D-Asp led to a significant reduction in body temperature in chicks even under high ambient temperature (HT) conditions. However, centrally administered D-Asp did not significantly influence the body temperature in chicks. As for plasma metabolites and catecholamines, orally administered D-Asp led to decreased triacylglycerol and uric acid concentrations and increased glucose and chlorine concentrations but did not alter plasma catecholamines. These results suggest that oral administration of D-Asp may play a potent role in reducing body temperature under both normal and HT conditions. The alteration of plasma metabolites further indicates that D-Asp may contribute to the regulation of metabolic activity in chicks. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolomic spectral libraries for data-independent SWATH liquid chromatography mass spectrometry acquisition.

PubMed

Bruderer, Tobias; Varesio, Emmanuel; Hidasi, Anita O; Duchoslav, Eva; Burton, Lyle; Bonner, Ron; Hopfgartner, Gérard

2018-03-01

High-quality mass spectral libraries have become crucial in mass spectrometry-based metabolomics. Here, we investigate a workflow to generate accurate mass discrete and composite spectral libraries for metabolite identification and for SWATH mass spectrometry data processing. Discrete collision energy (5-100 eV) accurate mass spectra were collected for 532 metabolites from the human metabolome database (HMDB) by flow injection analysis and compiled into composite spectra over a large collision energy range (e.g., 10-70 eV). Full scan response factors were also calculated. Software tools based on accurate mass and predictive fragmentation were specially developed and found to be essential for construction and quality control of the spectral library. First, elemental compositions constrained by the elemental composition of the precursor ion were calculated for all fragments. Secondly, all possible fragments were generated from the compound structure and were filtered based on their elemental compositions. From the discrete spectra, it was possible to analyze the specific fragment form at each collision energy and it was found that a relatively large collision energy range (10-70 eV) gives informative MS/MS spectra for library searches. From the composite spectra, it was possible to characterize specific neutral losses as radical losses using in silico fragmentation. Radical losses (generating radical cations) were found to be more prominent than expected. From 532 metabolites, 489 provided a signal in positive mode [M+H] + and 483 in negative mode [M-H] - . MS/MS spectra were obtained for 399 compounds in positive mode and for 462 in negative mode; 329 metabolites generated suitable spectra in both modes. Using the spectral library, LC retention time, response factors to analyze data-independent LC-SWATH-MS data allowed the identification of 39 (positive mode) and 72 (negative mode) metabolites in a plasma pool sample (total 92 metabolites) where 81 previously were reported in HMDB to be found in plasma. Graphical abstract Library generation workflow for LC-SWATH MS, using collision energy spread, accurate mass, and fragment annotation.

A snapshot of plasma metabolites in first-episode schizophrenia: a capillary electrophoresis time-of-flight mass spectrometry study.

PubMed

Koike, S; Bundo, M; Iwamoto, K; Suga, M; Kuwabara, H; Ohashi, Y; Shinoda, K; Takano, Y; Iwashiro, N; Satomura, Y; Nagai, T; Natsubori, T; Tada, M; Yamasue, H; Kasai, K

2014-04-08

Few biomarkers have been known that can easily measure clinical conditions in mental illnesses such as schizophrenia. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) is a new method that can measure ionized and low-molecular-weight metabolites. To explore global metabolomic alterations that characterize the onset of schizophrenia and identify biomarkers, we profiled the relative and absolute concentrations of the plasma metabolites from 30 patients with first-episode schizophrenia (FESZ, four drug-naïve samples), 38 healthy controls and 15 individuals with autism spectrum disorders using CE-TOFMS. Five metabolites had robust changes (increased creatine and decreased betaine, nonanoic acid, benzoic acid and perillic acid) in two independent sample sets. Altered levels of these metabolites are consistent with well-known hypotheses regarding abnormalities of the homocysteine metabolism, creatine kinase-emia and oxidative stress. Although it should be considered that most patients with FESZ received medication, these metabolites are candidate biomarkers to improve the determination of diagnosis, severity and clinical stages, especially for FESZ.

Estrogenic activities of diuron metabolites in female Nile tilapia (Oreochromis niloticus).

PubMed

Pereira, Thiago Scremin Boscolo; Boscolo, Camila Nomura Pereira; Felício, Andreia Arantes; Batlouni, Sergio Ricardo; Schlenk, Daniel; de Almeida, Eduardo Alves

2016-03-01

Some endocrine disrupting chemicals (EDCs) can alter the estrogenic activities of the organism by directly interacting with estrogen receptors (ER) or indirectly through the hypothalamus-pituitary-gonadal axis. Recent studies in male Nile tilapia (Oreochromis niloticus) indicated that diuron may have anti-androgenic activity augmented by biotransformation. In this study, the effects of diuron and three of its metabolites were evaluated in female tilapia. Sexually mature female fish were exposed for 25 days to diuron, as well as to its metabolites 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU), at concentrations of 100 ng/L. Diuron metabolites caused increases in E2 plasma levels, gonadosomatic indices and in the percentage of final vitellogenic oocytes. Moreover, diuron and its metabolites caused a decrease in germinative cells. Significant differences in plasma concentrations of the estrogen precursor and gonadal regulator17α-hydroxyprogesterone (17α-OHP) were not observed. These results show that diuron metabolites had estrogenic effects potentially mediated through enhanced estradiol biosynthesis and accelerated the ovarian development of O. niloticus females. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantitation of plasma thiamine, related metabolites and plasma protein oxidative damage markers in children with autism spectrum disorder and healthy controls.

PubMed

Anwar, Attia; Marini, Marina; Abruzzo, Provvidenza Maria; Bolotta, Alessandra; Ghezzo, Alessandro; Visconti, Paola; Thornalley, Paul J; Rabbani, Naila

2016-11-01

To assess thiamine and related metabolite status by analysis of plasma and urine in autistic children and healthy controls, correlations to clinical characteristics and link to plasma protein markers of oxidative damage. 27 children with autism (21 males and 6 females) and 21 (15 males and 6 females) age-matched healthy control children were recruited. The concentration of thiamine and related phosphorylated metabolites in plasma and urine and plasma protein content of dityrosine, N-formylkynurenine and 3-nitrotyrosine was determined. Plasma thiamine and thiamine monophosphate concentrations were similar in both study groups (median [lower-upper quartile]): autistic children - 6.60 nM (4.48-8.91) and 7.00 nM (5.51-8.55), and healthy controls - 6.82 nM (4.47-7.02) and 6.82 nM (5.84-8.91), respectively. Thiamine pyrophosphate (TPP) was decreased 24% in autistic children compared to healthy controls: 6.82 nM (5.81-8.52) versus 9.00 nM (8.41-10.71), p < .01. Urinary excretion of thiamine and fractional renal clearance of thiamine did not change between the groups. No correlation was observed between clinical markers and the plasma and urine thiamine concentration. Plasma protein dityrosine content was increased 88% in ASD. Other oxidative markers were unchanged. Autistic children had normal plasma and urinary thiamine levels whereas plasma TPP concentration was decreased. The latter may be linked to abnormal tissue handling and/or absorption from gut microbiota of TPP which warrants further investigation. Increased plasma protein dityrosine may reflect increased dual oxidase activity in response to change in mucosal immunity and host-microbe homeostasis.

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes.

PubMed

Staack, Roland F; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

2004-01-15

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices.

PubMed

Zhang, Limin; Ye, Yangfang; An, Yanpeng; Tian, Yuan; Wang, Yulan; Tang, Huiru

2011-02-04

Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using (1)H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycerophosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats.

Identification of Altered Metabolomic Profiles Following a Panchakarma-based Ayurvedic Intervention in Healthy Subjects: The Self-Directed Biological Transformation Initiative (SBTI).

PubMed

Peterson, Christine Tara; Lucas, Joseph; John-Williams, Lisa St; Thompson, J Will; Moseley, M Arthur; Patel, Sheila; Peterson, Scott N; Porter, Valencia; Schadt, Eric E; Mills, Paul J; Tanzi, Rudolph E; Doraiswamy, P Murali; Chopra, Deepak

2016-09-09

The effects of integrative medicine practices such as meditation and Ayurveda on human physiology are not fully understood. The aim of this study was to identify altered metabolomic profiles following an Ayurveda-based intervention. In the experimental group, 65 healthy male and female subjects participated in a 6-day Panchakarma-based Ayurvedic intervention which included herbs, vegetarian diet, meditation, yoga, and massage. A set of 12 plasma phosphatidylcholines decreased (adjusted p < 0.01) post-intervention in the experimental (n = 65) compared to control group (n = 54) after Bonferroni correction for multiple testing; within these compounds, the phosphatidylcholine with the greatest decrease in abundance was PC ae C36:4 (delta = -0.34). Application of a 10% FDR revealed an additional 57 metabolites that were differentially abundant between groups. Pathway analysis suggests that the intervention results in changes in metabolites across many pathways such as phospholipid biosynthesis, choline metabolism, and lipoprotein metabolism. The observed plasma metabolomic alterations may reflect a Panchakarma-induced modulation of metabotypes. Panchakarma promoted statistically significant changes in plasma levels of phosphatidylcholines, sphingomyelins and others in just 6 days. Forthcoming studies that integrate metabolomics with genomic, microbiome and physiological parameters may facilitate a broader systems-level understanding and mechanistic insights into these integrative practices that are employed to promote health and well-being.

Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.

PubMed

Tang, W H Wilson; Wang, Zeneng; Levison, Bruce S; Koeth, Robert A; Britt, Earl B; Fu, Xiaoming; Wu, Yuping; Hazen, Stanley L

2013-04-25

Recent studies in animals have shown a mechanistic link between intestinal microbial metabolism of the choline moiety in dietary phosphatidylcholine (lecithin) and coronary artery disease through the production of a proatherosclerotic metabolite, trimethylamine-N-oxide (TMAO). We investigated the relationship among intestinal microbiota-dependent metabolism of dietary phosphatidylcholine, TMAO levels, and adverse cardiovascular events in humans. We quantified plasma and urinary levels of TMAO and plasma choline and betaine levels by means of liquid chromatography and online tandem mass spectrometry after a phosphatidylcholine challenge (ingestion of two hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine) in healthy participants before and after the suppression of intestinal microbiota with oral broad-spectrum antibiotics. We further examined the relationship between fasting plasma levels of TMAO and incident major adverse cardiovascular events (death, myocardial infarction, or stroke) during 3 years of follow-up in 4007 patients undergoing elective coronary angiography. Time-dependent increases in levels of both TMAO and its d9 isotopologue, as well as other choline metabolites, were detected after the phosphatidylcholine challenge. Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics. Increased plasma levels of TMAO were associated with an increased risk of a major adverse cardiovascular event (hazard ratio for highest vs. lowest TMAO quartile, 2.54; 95% confidence interval, 1.96 to 3.28; P<0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P<0.001), as well as in lower-risk subgroups. The production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota. Increased TMAO levels are associated with an increased risk of incident major adverse cardiovascular events. (Funded by the National Institutes of Health and others.).

Diets high in resistant starch increase plasma levels of trimethylamine-N-oxide, a gut microbiome metabolite associated with CVD risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergeron, Nathalie; Williams, Paul T.; Lamendella, Regina

Production of trimethylamine-N-oxide (TMAO), a biomarker of CVD risk, is dependent on intestinal microbiota, but little is known of dietary conditions promoting changes in gut microbial communities. Resistant starches (RS) alter the human microbiota. We sought to determine whether diets varying in RS and carbohydrate (CHO) content affect plasma TMAO levels. We also assessed postprandial glucose and insulin responses and plasma lipid changes to diets high and low in RS. In a cross-over trial, fifty-two men and women consumed a 2-week baseline diet (41 percentage of energy (%E) CHO, 40 % fat, 19 % protein), followed by 2-week high- andmore » low-RS diets separated by 2-week washouts. RS diets were assigned at random within the context of higher (51–53 %E)v. lower CHO (39–40 %E) intake. Measurements were obtained in the fasting state and, for glucose and insulin, during a meal test matching the composition of the assigned diet. With lower CHO intake, plasma TMAO, carnitine, betaine andγ-butyrobetaine concentrations were higher after the high-v. low-RS diet (P<0·01 each). These metabolites were not differentially affected by highv. low RS when CHO intake was high. Although the high-RS meal reduced postprandial insulin and glucose responses when CHO intake was low (P<0·01 each), RS did not affect fasting lipids, lipoproteins, glucose or insulin irrespective of dietary CHO content. In conclusion, a lower-CHO diet high in RS was associated with higher plasma TMAO levels. These findings, together with the absence of change in fasting lipids, suggest that short-term high-RS diets do not improve markers of cardiometabolic health.« less

Diets high in resistant starch increase plasma levels of trimethylamine-N-oxide, a gut microbiome metabolite associated with CVD risk.

PubMed

Bergeron, Nathalie; Williams, Paul T; Lamendella, Regina; Faghihnia, Nastaran; Grube, Alyssa; Li, Xinmin; Wang, Zeneng; Knight, Rob; Jansson, Janet K; Hazen, Stanley L; Krauss, Ronald M

2016-12-01

Production of trimethylamine-N-oxide (TMAO), a biomarker of CVD risk, is dependent on intestinal microbiota, but little is known of dietary conditions promoting changes in gut microbial communities. Resistant starches (RS) alter the human microbiota. We sought to determine whether diets varying in RS and carbohydrate (CHO) content affect plasma TMAO levels. We also assessed postprandial glucose and insulin responses and plasma lipid changes to diets high and low in RS. In a cross-over trial, fifty-two men and women consumed a 2-week baseline diet (41 percentage of energy (%E) CHO, 40 % fat, 19 % protein), followed by 2-week high- and low-RS diets separated by 2-week washouts. RS diets were assigned at random within the context of higher (51-53 %E) v. lower CHO (39-40 %E) intake. Measurements were obtained in the fasting state and, for glucose and insulin, during a meal test matching the composition of the assigned diet. With lower CHO intake, plasma TMAO, carnitine, betaine and γ-butyrobetaine concentrations were higher after the high- v. low-RS diet (P<0·01 each). These metabolites were not differentially affected by high v. low RS when CHO intake was high. Although the high-RS meal reduced postprandial insulin and glucose responses when CHO intake was low (P<0·01 each), RS did not affect fasting lipids, lipoproteins, glucose or insulin irrespective of dietary CHO content. In conclusion, a lower-CHO diet high in RS was associated with higher plasma TMAO levels. These findings, together with the absence of change in fasting lipids, suggest that short-term high-RS diets do not improve markers of cardiometabolic health.

Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk

PubMed Central

Tang, W.H. Wilson; Wang, Zeneng; Levison, Bruce S.; Koeth, Robert A.; Britt, Earl B.; Fu, Xiaoming; Wu, Yuping; Hazen, Stanley L.

2013-01-01

BACKGROUND Recent studies in animals have shown a mechanistic link between intestinal microbial metabolism of the choline moiety in dietary phosphatidylcholine (lecithin) and coronary artery disease through the production of a proatherosclerotic metabolite, trimethylamine-N-oxide (TMAO). We investigated the relationship among intestinal microbiota-dependent metabolism of dietary phosphatidylcholine, TMAO levels, and adverse cardiovascular events in humans. METHODS We quantified plasma and urinary levels of TMAO and plasma choline and betaine levels by means of liquid chromatography and online tandem mass spectrometry after a phosphatidylcholine challenge (ingestion of two hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine) in healthy participants before and after the suppression of intestinal microbiota with oral broad-spectrum antibiotics. We further examined the relationship between fasting plasma levels of TMAO and incident major adverse cardiovascular events (death, myocardial infarction, or stroke) during 3 years of follow-up in 4007 patients undergoing elective coronary angiography. RESULTS Time-dependent increases in levels of both TMAO and its d9 isotopologue, as well as other choline metabolites, were detected after the phosphatidylcholine challenge. Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics. Increased plasma levels of TMAO were associated with an increased risk of a major adverse cardiovascular event (hazard ratio for highest vs. lowest TMAO quartile, 2.54; 95% confidence interval, 1.96 to 3.28; P<0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P<0.001), as well as in lower-risk subgroups. CONCLUSIONS The production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota. Increased TMAO levels are associated with an increased risk of incident major adverse cardiovascular events. (Funded by the National Institutes of Health and others.) PMID:23614584

Pyranocoumarin Tissue Distribution, Plasma Metabolome and Prostate Transcriptome Impacts of Sub-Chronic Exposure to Korean Angelica Supplement in Mice.

PubMed

Zhang, Jinhui; Li, Li; Tang, Suni; Zhang, Yong; Markiewski, Maciej; Xing, Chengguo; Jiang, Cheng; Lü, Junxuan

2016-01-01

Herbal products containing Korean Angelica gigas Nakai (AGN) root extract are marketed as dietary supplements for memory enhancement, pain killing, and female menopausal symptom relief. We have shown the anticancer activities of AGN supplements in mouse models. To facilitate human anticancer translational research, we characterized the tissue distribution of AGN marker pyranocoumarin compounds decursin (D) and decursinol angelate (DA) ([Formula: see text]% in AGN) and their metabolite decursinol (DOH), assessed the safety of sub-chronic AGN dietary exposure in mice, and explored its impact on plasma aqueous metabolites and the prostate transcriptome. The data show that after a gavage dose, plasma contained readily detectable DOH, but little D and DA, mirroring patterns in the liver. Extra-hepatic tissues retained greater levels of DA and D than the liver did. For sub-chronic exposures, male mice were provided ad libitum AIN93M-pellet diets with 0.5 and 1% AGN for six weeks. No adverse effects were observed on the plasma biochemistry markers of liver and kidney integrity in spite of their enlargement. Histopathological examinations of the liver, kidney and other visceral organs did not reveal tissue abnormalities. Metabolomic assessment of plasma from mice fed the 1%-AGN diet suggested metabolic shifts of key amino acids especially in the methionine-cysteine cycle, purine cycle, and glycolysis-citrate cycle. Prostate transcriptomic profiling identified gene signature changes in the metabolisms of drugs, lipids and cellular energetics, neuro-muscular features, immunity and inflammation, and tumor suppressor/oncogene balance. The safety profile was corroborated with a daily [Formula: see text] injection of AGN extract (100-300[Formula: see text]mg/kg) for four weeks, which resulted in much greater systemic pyranocoumarin exposure than the dietary route did.

Pyranocoumarin tissue distribution, and plasma metabolome and prostate transcriptome impacts of sub-chronic exposure to Korean Angelica supplement in mice

PubMed Central

ZHANG, Jinhui; LI, Li; TANG, Suni; ZHANG, Yong; MARKIEWSKI, Maciej; XING, Chengguo; JIANG, Cheng; LÜ, Junxuan

2016-01-01

Herbal products containing Korean Angelica gigas Nakai (AGN) root extract are marketed as dietary supplements for memory enhancement, pain killing, and female menopausal symptom relief. We have shown anti-cancer activity of AGN supplement in mouse models. To facilitate human anti-cancer translational research, we characterized the tissue distribution of AGN marker pyranocoumarin compounds decursin (D) and decursinol angelate (DA) (~50% in AGN) and their metabolite decursinol (DOH), assessed safety of sub-chronic AGN dietary exposure in mice, and explored the impacts on the plasma aqueous metabolites and prostate transcriptome. The data show that after a gavage dose, plasma contained readily detectable DOH, but little D and DA, mirroring patterns in the liver. Extra-hepatic tissues retained greater level of DA and D than liver. For sub-chronic exposures, male mice were provided ad libitum AIN93M-pellet diet with 0.5 and 1% AGN for 6 weeks. No adverse effect was observed on plasma biochemistry markers of liver and kidney integrity in spite of their enlargement. Histopathological examination of liver, kidney and other visceral organs did not reveal tissue abnormalities. Metabolomic assessment of plasma from the mice fed 1%-AGN diet suggested metabolic shifts of key amino acids especially methionine-cysteine cycle, purine cycle and glycolysis-citrate cycle. Prostate transcriptomic profiling identified gene signature changes of metabolisms of drugs, lipids and cellular energetics, neuro-muscular features, immunity and inflammation, and tumor suppressor/oncogene balance. The safety profile was corroborated with daily i.p. injection of AGN extract (200 mg/kg) for 4 weeks, which resulted in much greater systemic pyranocoumarin exposure than dietary route. PMID:27080944

Bergamottin contribution to the grapefruit juice-felodipine interaction and disposition in humans.

PubMed

Goosen, Theunis C; Cillié, Doré; Bailey, David G; Yu, Chongwoo; He, Kan; Hollenberg, Paul F; Woster, Patrick M; Cohen, Lucinda; Williams, J Andrew; Rheeders, Malie; Dijkstra, H Paul

2004-12-01

Our objectives were to evaluate the contribution of bergamottin to the grapefruit juice-felodipine interaction and to characterize bergamottin disposition. In this study 250 mL grapefruit juice; 2-, 6-, or 12-mg capsules of bergamottin plus water; or water was administered with 5 mg extended-release felodipine to 11 volunteers in a partially randomized, 5-way crossover study. Plasma concentrations of felodipine, its primary metabolite (dehydrofelodipine), bergamottin, and 6',7'-dihydroxybergamottin were determined. Grapefruit juice (containing 1.7 mg bergamottin) increased peak plasma concentration (C max ) and area under the plasma concentration-time curve (AUC) of felodipine by 89% (P < .025) and 54% (P < .025), respectively, compared with water. With 2 mg bergamottin, felodipine C max increased by 33% (P < .05). The increase by bergamottin was markedly variable among individuals (range, -33% to 125%). With 6 mg bergamottin, felodipine C max was enhanced by 35% (P < .025), and with 12 mg bergamottin, felodipine C max increased by 40% (P < .05) and AUC increased by 37% (P < .05) compared with water. Bergamottin measured in plasma after administration of 6 and 12 mg produced C max values of 2.1 and 5.9 ng/mL, respectively, and times to reach C max of 0.8 and 1.1 hours, respectively. The bergamottin metabolite 6',7'-dihydroxybergamottin was detected in plasma of some subjects after bergamottin administration. Bergamottin enhanced the oral bioavailability of felodipine and may cause a clinically relevant drug interaction in susceptible individuals. Grapefruit juice-drug interactions likely also involve other furanocoumarins, possibly acting in combination by additive or synergistic mechanisms. Bergamottin has systemic availability and is metabolized in vivo to 6',7'-dihydroxybergamottin.

Associations between five-factor model of the Positive and Negative Syndrome Scale and plasma levels of monoamine metabolite in patients with schizophrenia.

PubMed

Watanabe, Kenya; Miura, Itaru; Kanno-Nozaki, Keiko; Horikoshi, Sho; Mashiko, Hirobumi; Niwa, Shin-Ichi; Yabe, Hirooki

2015-12-15

The five-factor model of the Positive and Negative Syndrome Scale (PANSS) for schizophrenia symptoms is the most common multiple-factor model used in analyses; its use may improve evaluation of symptoms in schizophrenia patients. Plasma monoamine metabolite levels are possible indicators of clinical symptoms or response to antipsychotics in schizophrenia. We investigated the association between five-factor model components and plasma monoamine metabolites levels to explore the model's biological basis. Plasma levels of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography in 65 Japanese patients with schizophrenia. Significant negative correlation between plasma 5-HIAA levels and the depression/anxiety component was found. Furthermore, significant positive correlation was found between plasma MHPG levels and the excitement component. Plasma HVA levels were not correlated with any five-factor model component. These results suggest that the five-factor model of the PANSS may have a biological basis, and may be useful for elucidating the psychopathology of schizophrenia. Assessment using the five-factor model may enable understanding of monoaminergic dysfunction, possibly allowing more appropriate medication selection. Further studies of a larger number of first-episode schizophrenia patients are needed to confirm and extend these results. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Plasma Metabolomic Changes following PI3K Inhibition as Pharmacodynamic Biomarkers: Preclinical Discovery to Phase I Trial Evaluation.

PubMed

Ang, Joo Ern; Pandher, Rupinder; Ang, Joo Chew; Asad, Yasmin J; Henley, Alan T; Valenti, Melanie; Box, Gary; de Haven Brandon, Alexis; Baird, Richard D; Friedman, Lori; Derynck, Mika; Vanhaesebroeck, Bart; Eccles, Suzanne A; Kaye, Stan B; Workman, Paul; de Bono, Johann S; Raynaud, Florence I

2016-06-01

PI3K plays a key role in cellular metabolism and cancer. Using a mass spectrometry-based metabolomics platform, we discovered that plasma concentrations of 26 metabolites, including amino acids, acylcarnitines, and phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors compared with non-tumor-bearing controls and in addition were increased following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These candidate metabolomics biomarkers were evaluated in a phase I dose-escalation clinical trial of pictilisib. Time- and dose-dependent effects were observed in patients for 22 plasma metabolites. The changes exceeded baseline variability, resolved after drug washout, and were recapitulated on continuous dosing. Our study provides a link between modulation of the PI3K pathway and changes in the plasma metabolome and demonstrates that plasma metabolomics is a feasible and promising strategy for biomarker evaluation. Also, our findings provide additional support for an association between insulin resistance, branched-chain amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther; 15(6); 1412-24. ©2016 AACR. ©2016 American Association for Cancer Research.

Identification of metabolites of Si-Ni-San, a traditional Chinese medicine formula, in rat plasma and urine using liquid chromatography/diode array detection/triple-quadrupole spectrometry.

PubMed

Yan, Zhixiang; Chen, Ying; Li, Tianxue; Zhang, Jie; Yang, Xinghao

2012-02-15

Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple-quadrupole spectrometry (LC-DAD-MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC-Q-TOF/MS/MS.

PubMed

Wang, Nannan; Zhao, Xiaoning; Li, Yiran; Cheng, Congcong; Huai, Jiaxin; Bi, Kaishun; Dai, Ronghua

2018-06-01

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid-liquid extraction and separated on a Shim-pack XR-ODS C 18 column (75 × 3.0 mm, 2.2 μm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone-related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid-related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid-related metabolites. It is concluded the developed UHPLC-Q-TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD. Copyright © 2018 John Wiley & Sons, Ltd.

Pharmacokinetics of tramadol and its major metabolites following rectal and intravenous administration in dogs.

PubMed

Giorgi, M; Del Carlo, S; Saccomanni, G; Łebkowska-Wieruszewska, B; Kowalski, C J

2009-06-01

To compare the rectal and I/V administration of tramadol in dogs, to assess both its pharmacokinetic properties and absolute bioavailability. After rectal administration via suppositories and I/V injection of tramadol (4 mg/kg), the concentration of tramadol and its main metabolites, O-desmethyl-tramadol (M1), N-desmethyl-tramadol (M2) and N,O-didesmethyl-tramadol (M5), were determined in plasma, using high-performance liquid chromatography (HPLC). A balanced cross-over study was used, involving six male Beagle dogs. Plasma concentrations after rectal and I/V administration were fitted on the basis of mono- and bi-compartmental models, respectively. Following rectal administration tramadol was detected from 5 minutes up to 10 hours, in lesser amounts than M5 and M2, while M1 was detected in negligible amounts. Following I/V administration tramadol was detected up to 10 hours, M2 and M5 were detected at similar concentrations, and M1 was present at low concentrations. The area under the curve (AUC) of the three metabolites did not differ significantly after either route of administration of tramadol. The absolute bioavailability of tramadol via rectal administration was 10 (SD 4)%. After rectal administration of tramadol suppositories, absorption of the active ingredient was rapid, but its metabolism quickly transformed the parent drug to high levels of M2 and M5. In the dog, rectal pharmaceutical formulation of tramadol would have a different pharmacokinetic behaviour than in humans.

Associations of beta-endorphin with HVA and MHPG in the plasma of prepubertal boys: effects of familial drug abuse and antisocial personality disorder liability.

PubMed

Moss, H B; Yao, J K

1996-06-01

It is well-established that the secretion of the opioid neuropeptide beta-endorphin is perturbed by the administration of various drugs of abuse. Several investigators have speculated that variations in beta-endorphin secretory regulation may precede the development of a substance use disorder, and thus be a component of the liability for substance abuse. In order to test this hypothesis, we examined fasting, morning plasma concentrations of beta-endorphin and two catecholamine metabolites in prepubertal boys naive to drugs of abuse and at elevated familial risk for a substance use disorder (SA+), and in controls (SA-). Specifically, the dopaminergic metabolite homovanillic acid (pHVA), and the noradrenergic metabolite, 3-methoxy-4-hydroxy-phenylglycol (pMHPG) were measured. Between-group differences were not found for beta-endorphin, pHVA, or pMHPG. Similarly, such differences did not differentiate sons of fathers with Antisocial Personality Disorder and controls. However, regression analysis revealed that although both pHVA and pMHPG predicted beta-endorphin concentrations to similar degrees, the directions of influence were the opposite. pHVA was found to be positively associated with beta-endorphin while pMHPG was found to be negatively associated with beta-endorphin. No between-group differences in these relationships were found. The results suggest an opponent process in catecholaminergic regulation of beta-endorphin in humans, and are consistent with observations in the central nervous system of animal models.