Direct detection of glucuronide metabolites of lidocaine in sheep urine.

PubMed

Doran, Gregory S; Smith, Alistair K; Rothwell, Jim T; Edwards, Scott H

2018-02-15

The anaesthetic lidocaine is metabolised quickly to produce a series of metabolites, including several hydroxylated metabolites, which are further metabolised by addition of a glucuronic acid moiety. Analysis of these glucuronide metabolites in urine is performed indirectly by cleaving the glucuronic acid group using β-glucuronidase. However, direct analysis of intact glucuronide conjugates is a more straightforward approach as it negates the need for long hydrolysis incubations, and minimises the oxidation of sensitive hydrolysis products, while also distinguishing between the two forms of hydroxylated metabolites. A method was developed to identify three intact glucuronides of lidocaine in sheep urine using LC-MS/MS, which was further confirmed by the synthesis of glucuronide derivatives of 3OH-MEGX and 4OH-LIDO. Direct analysis of urine allowed the detection of the glucuronide metabolites of hydroxylidocaine (OH-LIDO), hydroxyl-monoethylglycinexylidide (OH-MEGX), and hydroxy-2,6-xylidine (OH-XYL). Analysis of urine before and after β-glucuronidase digestion showed that the efficiency of hydrolysis of these glucuronide metabolites may be underestimated in some studies. Analysis of urine in the current study from three different sheep with similar glucuronide metabolite concentrations resulted in different hydrolysis efficiencies, which may have been a result of different levels of substrate binding by matrix components, preventing enzyme cleavage. The use of direct analysis of intact glucuronides has the benefit of being less influenced by these matrix effects, while also allowing analysis of unstable metabolites like 4OH-XYL, which rapidly oxidises after hydrolysis. Additionally, direct analysis is less expensive and less time consuming, while providing more information about the status of hydroxylated metabolites in urine. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Microbial carbonylation and hydroxylation of 20(R)-panaxadiol by Aspergillus niger.

PubMed

Yan, Bin; Chen, Zhihua; Zhai, Xuguang; Yin, Guibo; Ai, Yafei; Chen, Guangtong

2018-04-01

20(R)-panaxadiol (PD) was metabolised by the fungus Aspergillus niger AS 3.3926 to its C-3 carbonylated metabolite and five other hydroxylated metabolites (1-6). Their structures were elucidated as 3-oxo-20(R)-panaxadiol (1), 3-oxo-7β-hydroxyl- 20(R)-panaxadiol (2), 3-oxo-7β,23α-dihydroxyl-20(R)-panaxadiol (3), 3,12-dioxo- 7β,23β-dihydroxyl-20(R)-panaxadiol (4), 3-oxo-1α,7β-dihydroxyl-20(R)-panaxadiol (5) and 3-oxo-7β,15β-dihydroxyl-20(R)-panaxadiol (6) by spectroscopic analysis. Among them, compounds 2-6 were new compounds. Pharmacological studies revealed that compound 6 exhibited significant anti-hepatic fibrosis activity.

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.

Metabolism of diclofenac in plants--hydroxylation is followed by glucose conjugation.

PubMed

Huber, Christian; Bartha, Bernadett; Schröder, Peter

2012-12-01

Pharmaceuticals from human or veterinary medication form a new class of micropollutants that poses a serious threat to our aquatic environment and its organisms. The intensively used nonsteroidal anti-inflammatory drug diclofenac is found in the environment worldwide due to its poor elimination during waste water treatment processes. In order to test phytoremediation as a tool for the removal of this drug from waste water, the uptake of the compound into plant tissues and its metabolic pathway was addressed using Hordeum vulgare (barley) and a hairy root cell culture of Armoracia rusticana (horse radish) as model species. Diclofenac is taken up by plants and undergoes rapid metabolization; already after 3h of exposure the drug and its metabolites could be detected in the plant tissues. Similar to its fate in mammalian cells the drug is activated in a phase I reaction resulting in the hydroxylated metabolite 4'OH-diclofenac which is conjugated subsequently in phase II to a glucopyranoside, a typical plant specific metabolite. After exposure to 10 and 100 μM diclofenac a concentration dependent formation of the hydroxylated metabolite was observed, while the formation of the phase II metabolite OH-diclofenac glucopyranoside was not positively affected by the higher concentration. To our knowledge this is the first time these two human painkiller metabolites are shown to occur in plant tissues. Copyright © 2012 Elsevier B.V. All rights reserved.

Inducible hydroxylation and demethylation of the herbicide isoproturon by Cunninghamella elegans.

PubMed

Hangler, Martin; Jensen, Bo; Rønhede, Stig; Sørensen, Sebastian R

2007-03-01

A screening of 27 fungal strains for degradation of the phenylurea herbicide isoproturon was performed and yielded 15 strains capable of converting the herbicide to polar metabolites. The zygomycete fungus Cunninghamella elegans strain JS/2 isolated from an agricultural soil converted isoproturon to several known hydroxylated metabolites. In addition, unknown metabolites were produced in minor amounts. Inducible degradation was indicated by comparing resting cells pregrown with or without isoproturon. This shows that strain JS/2 is capable of partially degrading isoproturon and that one or more of the enzymes involved are inducible upon isoproturon exposure.

Induction of cytochrome P450 1A1 in MCF-7 human breast cancer cells by 4-chlorobiphenyl (PCB3) and the effects of its hydroxylated metabolites on cellular apoptosis

PubMed Central

Ptak, Anna; Ludewig, Gabriele; Rak, Agnieszka; Nadolna, Weronika; Bochenek, Michał; Gregoraszczuk, Ewa L

2010-01-01

Several studies suggest an involvement of PCBs in breast cancer formation, but the results are ambiguous and the mechanisms not clear. We propose that local activation of cytochrome P450 enzymes, CYP1A1 and CYP1B1 by PCB3, may generate active metabolites which affect apoptosis and thereby promote mammary carcinogenesis. To test this hypothesis MCF-7 human breast cancer cells were exposed to 300 nM PCB3 and its hydroxylated metabolites, 4OH-PCB and 3,4diOH-PCB3. The enzyme activity for CYP1A1 was assayed using the EROD assay, and CYP1A1 and CYP1B1 protein expression by western blotting. PCB3 increased CYP1A1 activity (~1.5fold) and protein levels within 6 hrs after exposure. No effect on CYP1B1 protein expression was observed. The effects of PCB3 and both its metabolites on staurosporine-induced apoptosis were determined by measuring DNA fragmentation using ELISA and TUNEL assays, and by measuring caspase-8 and caspase-9 activity. We found that PCB3 and both of its hydroxylated metabolites had no effect on caspase-8 and caspase-9 activity when cells were grown in medium deprived of estrogen, but reduced caspase-9 activity when cells were grown in medium supplemented with serum containing estradiol. Interestingly, a decrease of DNA fragmentation was observed upon treatment with 3,4diOH-PCB3 in both culture conditions, suggesting that 3,4diOH-PCB3 affects a caspase-independent pathway of cell death. In summary, interactions of PCB3 and its metabolites with estradiol by yet unknown mechanisms inhibit caspase 9-related apoptosis and additional, other death pathways are affected by the catechol metabolite 3,4diOH-PCB3. These anti-apoptotic effects and the change in metabolic activity may contribute to the carcinogenic effect of PCBs. PMID:19604582

Alpha-hydroxytamoxifen, a genotoxic metabolite of tamoxifen in the rat: identification and quantification in vivo and in vitro.

PubMed

Boocock, D J; Maggs, J L; White, I N; Park, B K

1999-01-01

The metabolic formation of a-hydroxytamoxifen, a reactive metabolite of tamoxifen in rat liver, was characterized and quantified in vitro (hepatic microsomal incubations) and in vivo (bile-duct cannulated animals). This minor metabolite was identified by chromatographic and mass spectral comparisons with the authentic compound. The rates of formation of alpha-hydroxytamoxifen in incubations (30 min) of tamoxifen (25 microM) with liver microsomal preparations from women (pool of six), female CD1 mice or female Sprague-Dawley rats, as quantified by liquid chromatography-mass spectrometry (LC-MS), were 1.15+/-0.03, 0.30+/-0.05 and 2.70+/-0.35 pmol/min/mg protein, respectively. Selective inhibition of microsomal P450 indicated that alpha-hydroxylation was catalysed predominantly by CYP3A in humans. Bile-duct cannulated and anaesthetized female rats and mice given [14C]tamoxifen (43 micromol/kg, i.v.) excreted, respectively, 24 and 21% of the administered radioactivity in bile over 5 and 3.5 h. The major radiolabelled biliary metabolite in rats, characterized by LC-MS after enzymic hydrolysis of conjugates, was the glucuronide of 4-hydroxytamoxifen (10% of dose) and only 0.1% of the dose was recovered as alpha-hydroxytamoxifen. After administration of alpha-hydroxytamoxifen (43 micromol/kg, i.v.) to rats, only 1.19% of the administered compound was recovered from a glucuronide metabolite in bile, indicating a possible 0.84% alpha-hydroxylation of tamoxifen in vivo. There was, however, no indication of the presence in bile of either O-sulphonate or glutathione conjugates derived from alpha-hydroxytamoxifen. This study shows for the first time that alpha-hydroxytamoxifen can be glucuronylated in rat liver. Whereas sulphonation results in electrophilic genotoxic intermediates, glucuronidation may represent a means of detoxifying alpha-hydroxytamoxifen.

RATE AND CAPACITY OF HEPATIC MICROSOMAL RING HYDROXYLATION OF PHENOL TO HYDROQUINONE AND CATECHOL IN RAINBOW TROUT

EPA Science Inventory

Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol PH) by directly measuring the production of hydroquinone (HQ), the primary metabolite, and catechol (CAT), a secondary metabolite. An HPLC method with integrated ultra...

Metabolite proving fungal cleavage of the aromatic core part of a fluoroquinolone antibiotic

PubMed Central

2012-01-01

Liquid cultures of the basidiomycetous fungus Gloeophyllum striatum were employed to study the biodegradation of pradofloxacin, a new veterinary fluoroquinolone antibiotic carrying a CN group at position C-8. After 16 days of incubation, metabolites were purified by micro-preparative high-performance liquid chromatography. Four metabolites could be identified by co-chromatography with chemically synthesized standards. The chemical structures of three compounds were resolved by 1H-nuclear magnetic resonance spectroscopy plus infrared spectroscopy in one case. All metabolites were confirmed by high resolution mass spectrometry-derived molecular formulae. They comprised compounds in which the carboxyl group or the fluorine atom had been exchanged for a hydroxyl group. Furthermore, replacement of the CN group and the intact amine moiety by a hydroxyl group as well as degradation of the amine substituent were observed. The chemical structure of a catechol-type fluoroquinolone metabolite (F-5) could be fully defined for the first time. The latter initiated a hypothetical degradation sequence providing a unique metabolite, F-13, which consisted of the cyclopropyl-substituted pyridone ring still carrying C-7 and C-8 of pradofloxacin, now linked by a double bond and substituted by a hydroxyl and the CN group, respectively. Most likely, all reactions were hydroxyl radical-driven. Metabolite F-13 proves fungal cleavage of the aromatic fluoroquinolone core for the first time. Hence, two decades after the emergence of the notion of the non-biodegradability of fluoroquinolones, fungal degradation of all key structural elements has been proven. PMID:22214407

Methemoglobinemia Hemotoxicity of Some Antimalarial 8-Aminoquinoline Analogues and Their Hydroxylated Derivatives: Density Functional Theory Computation of Ionization Potentials.

PubMed

Ding, Yuanqing; Liu, Haining; Tekwani, Babu L; Nanayakkara, N P Dhammika; Khan, Ikhlas A; Walker, Larry A; Doerksen, Robert J

2016-07-18

The administration of primaquine (PQ), an essential drug for the treatment and radical cure of malaria, can lead to methemoglobin formation and life-threatening hemolysis for glucose-6-phosphate dehydrogenase deficient patients. The ionization potential (IP, a quantitative measure of the ability to lose an electron) of the metabolites generated by antimalarial 8-aminoquinoline (8-AQ) drugs like PQ has been believed to be correlated in part to this methemoglobinemia hemotoxicity: the lower the IP of an 8-AQ derivative, the higher the concentration of methemoglobin generated. In this work, demethoxylated primaquine (AQ02) was employed as a model, by intensive computation at the B3LYP-SCRF(PCM)/6-311++G**//B3LYP/6-31G** level in water, to study the effects of hydroxylation at various positions on the ionization potential. Compared to the parent AQ02, the IPs of AQ02's metabolites hydroxylated at N1', C5, and C7 were lower by 61, 30, and 19 kJ/mol, respectively, while differences in the IP relative to PQ were small for hydroxylation at all other positions. The C6 position, at which the IP of the hydroxylated metabolite was greater than that of AQ02, by 2 kJ/mol, was found to be unique. Several literature and proposed 8-AQ analogues were studied to evaluate substituent effects on their potential to generate methemoglobin, with the finding that hydroxylations at N1' and C5 contribute the most to the potential hemotoxicity of PQ-based antimalarials, whereas hydroxylation at C7 has little effect. Phenoxylation at C5 in PQ-based 8-AQs can block the hydroxylation at C5 and reduce the potential for methemoglobin generation, while -CF3 and chlorines attached to the phenolic ring can further reduce the risk. The H-shift at N1' during the cationization of hydroxylated metabolites of 8-AQs sharply decreased their IPs, but this effect can be significantly reduced by the introduction of an electron-withdrawing group to the quinoline core. The results and this approach may be

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast.

PubMed

Bermejo, Clara; Ewald, Jennifer C; Lanquar, Viviane; Jones, Alexander M; Frommer, Wolf B

2011-08-15

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation. © The Authors Journal compilation © 2011 Biochemical Society

Diclofenac and metabolite pharmacokinetics in children.

PubMed

van der Marel, Caroline D; Anderson, Brian J; Rømsing, Janne; Jacqz-Aigrain, Evelyne; Tibboel, Dick

2004-06-01

Data concerning metabolism of diclofenac in children are limited to intravenous and enteric coated oral formulations. There are no data examining diclofenac or its hydroxyl metabolite pharmacokinetics after rectal administration in children. Infants (n = 26) undergoing tonsillectomy were given diclofenac 2 mg.kg(-1) followed by 1 mg.kg(-1) 8 h as suppository formulation for postoperative analgesia. Serum was assayed for diclofenac, 4'-hydroxydiclofenac and 5'-hydroxydiclofenac concentrations during the procedure and 1, 2 and 4 h postoperatively. The formation clearances of diclofenac to hydroxyl metabolites were estimated using nonlinear mixed effects models. A single compartment, first order absorption and first order elimination model was used to describe diclofenac pharmacokinetics. Published data from 11 children given enteric-coated diclofenac tablets were used to assess relative bioavailability. Mean (sd) age and weight of the patients were 4.5 (1.5) years and 20.5 (4.1) kg. The formation clearance to 4'-hydroxydiclofenac (% CV) and to 5'-hydroxydiclofenac were 8.41 (8.1) and 3.41 (113) l.h(-1) respectively, standardized to a 70 kg person using allometric '1/4 power' models. Clearance by other routes contributed 33.0 (64) l.h(-1) 70 kg(-1). Elimination clearance of hydroxyl metabolites was fixed at 27.5 l.h(-1) 70 kg(-1). The volumes of distribution of parent diclofenac and its hydroxyl metabolite were 22.8 (19.0) and 45.3 (l.70) kg(-1). The suppository formulation had an absorption half-life of 0.613 (33.2) h with a lag time of 0.188 (24.9) h. Interoccasion variability of formation clearance to 4'-hydroxydiclofenac, diclofenac volume of distribution, absorption half-time and lag time for the suppository was 36%, 55%, 14% and 119%, respectively. The relative bioavailability of the suppository compared with an enteric-coated tablet was 1.26. The formation clearance of the active metabolite 4'-hydroxydiclofenac contributed 19% of total clearance (44.82 l.h(-1

RATE AND CAPACITY OF HEPATIC MICROSOMAL RING HYDROXYLATION OF PHENOL TO HYDROQUINONE AND CATECHOL IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

EPA Science Inventory

Rainbow trout liver microsomes were used to study the rate of ring-hydroxylation of phenol (PH) by directly measuring the production of hydroquinone (HQ), the primary metabolite, and catechol (CAT), a secondary metabolite. An HPLC method with integrated ultroviolet (UV) and elect...

ipso-Hydroxylation and Subsequent Fragmentation: a Novel Microbial Strategy To Eliminate Sulfonamide Antibiotics

PubMed Central

Ricken, Benjamin; Cichocka, Danuta; Parisi, Martina; Lenz, Markus; Wyss, Dominik; Martínez-Lavanchy, Paula M.; Müller, Jochen A.; Shahgaldian, Patrick; Tulli, Ludovico G.; Kohler, Hans-Peter E.; Kolvenbach, Boris A.

2013-01-01

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us. PMID:23835177

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry.

PubMed

Aceña, Jaume; Pérez, Sandra; Eichhorn, Peter; Solé, Montserrat; Barceló, Damià

2017-09-01

The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment. Graphical abstract Studies of metabolism of carbamazepine and ibuprofen in fish.

Organohalogens and their hydroxylated metabolites in the blood of pigs from an open waste dumping site in south India: association with hepatic cytochrome P450.

PubMed

Mizukawa, Hazuki; Nomiyama, Kei; Kunisue, Tatsuya; Watanabe, Michio X; Subramanian, Annamalai; Iwata, Hisato; Ishizuka, Mayumi; Tanabe, Shinsuke

2015-04-01

The concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and their hydroxylated metabolites (OH-PCBs and OH-PBDEs) were measured in the blood of Eurasian wild pigs (Sus scrofa) from a municipal waste open dumping site (DS) and a reference site (RS) in South India. We showed that contamination with OH-PCBs was higher in female pigs from the DS than in all other adult pigs. The highest OH-PCB concentrations were found in piglets from the DS. Moreover, the hepatic expression levels of CYP1A and CYP2B were higher in piglets than in their dam, implying metabolism of PCBs by cytochrome P450 (CYP) enzymes. The OH-PCB congener profiles differed according to sex and collection sites, possibly because of variations in the expression levels of phase I and phase II enzymes among individual pigs, differences in the exposure sources, and maternal transfer of parent PCBs. The hepatic CYP1A expression levels were positively correlated with the blood concentrations of 4OH-CB107, 4OH-CB162, and 4OH-CB187, implying CYP1A-dependent formation of these OH-PCBs in the pig liver. We found no significant correlations between the blood concentrations of OH-PCBs and thyroid hormones (THs); however, the thyroxin (T4) levels were lower in pigs from the DS than in pigs from the RS. Our limited dataset suggest that induced CYP enzymes accelerate the metabolism of xenobiotics and endogenous molecules in pigs. Thus, besides parental compounds, the risk of hydroxylated metabolites entering wildlife and humans living in and around municipal open waste dumping sites should be considered. Copyright © 2015 Elsevier Inc. All rights reserved.

Ecotoxicity of quinoline and hydroxylated derivatives and their occurrence in groundwater of a tar-contaminated field site.

PubMed

Neuwoehner, Judith; Reineke, Anne-Kirsten; Hollender, Juliane; Eisentraeger, Adolf

2009-03-01

In the groundwater of a timber impregnation site higher concentrations of hydroxylated quinolines compared to their parent compounds quinoline and isoquinoline were found. Studying the toxicity of parent compounds and metabolites, genotoxicity was found with metabolic activation in the SOS-Chromotest and Ames fluctuation test only for quinoline. An adverse effect on algae was observed only for the parent compounds quinoline and isoquinoline, while in the Daphnia magna immobilization assay most hydroxylated quinoline derivatives showed toxicity. The highest ecotoxic potential was observed in the Vibrio fischeri luminescence-inhibition assay. Comparing experimental EC50-values with QSAR predicted ones, for all compounds apart from isoquinoline and 2(1H)-quinolinone in the V. fischeri test baseline toxicity or polar nacrosis is indicated. In conclusion, the hydroxylation of quinoline leads to a detoxification of the genotoxic potential, while taken additive mixture toxicity and a safety factor into account parent compounds and metabolites are found of ecotoxicological relevance in the groundwater.

Metabolism and metabolites of polychlorinated biphenyls (PCBs)

PubMed Central

Grimm, FA; Hu, D; Kania-Korwel, I; Lehmler, HJ; Ludewig, G; Hornbuckle, KC; Duffel, MW; Bergman, A; Robertson, LW

2015-01-01

The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity and thereby assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general and in humans in particular. The aim of this document is to provide an overview of PCB metabolism and to identify metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed. PMID:25629923

Study of tamoxifen urinary metabolites in rat by ultra-high-performance liquid chromatography time-of-flight mass spectrometry.

PubMed

Domínguez-Romero, Juan C; García-Reyes, Juan F; Beneito-Cambra, Miriam; Martínez-Romero, Rubén; Martinez-Lara, Esther; Del Moral-Leal, María L; Molina-Díaz, Antonio

2015-08-01

Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra-high-performance liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC-TOFMS analyses, a solid-phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC-QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N-oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O -H2 )], hydroxylation of the ring accompanied by N-desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N-desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC-TOFMS. Copyright © 2015 John Wiley & Sons, Ltd.

Microsomal Oxidation of 2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Results in Species-Dependent Chiral Signatures of the Hydroxylated Metabolites

PubMed Central

2015-01-01

Chiral polychlorinated biphenyls (PCBs) display variable atropisomeric enrichment in wildlife and animal models, especially at higher trophic levels. These differences in PCBs’ chiral signatures are, at least in part, due to species-dependent oxidation of PCBs to hydroxylated PCB metabolites (OH-PCBs). Here, we investigate the hypothesis that the cytochrome P450 (P450) enzyme-mediated oxidation of chiral PCBs results in species-dependent differences in the chiral signatures of OH-PCBs (i.e., the direction and extent of OH-PCBs’ atropisomeric enrichment). To investigate this hypothesis, we incubated PCB 136, a representative chiral PCB, with pooled human liver microsomes (HLMs) or liver microsomes from male guinea pig, hamster, monkey, mouse, and rabbit or female dog and determined average profiles and chiral signatures of the OH-PCBs. 2,2′,3,3′,6,6′-Hexachlorobiphenyl-4-ol (4–136) was the major metabolite in incubations with HLMs and monkey and rabbit microsomes. 2,2′,3,3′,6,6′-Hexachlorobiphenyl-5-ol (5–136) was the major metabolite formed by microsomes from all other species. Both 4–136 and 5–136 were formed atropselectively in all microsomal incubations; however, the direction and extent of the atropisomeric enrichment of both OH-PCB metabolites showed considerable differences across microsomal preparations obtained from different species. These differences in OH-PCBs’ atropisomeric enrichment may not only be toxicologically relevant but may also be useful to study sources and transport of OH-PCBs in the environment. PMID:24467194

Sulfate Metabolites of 4-Monochlorobiphenyl in Whole Poplar Plants

PubMed Central

Zhai, Guangshu; Lehmler, Hans-Joachim; Schnoor, Jerald L.

2013-01-01

4-Monochlorobiphenyl (PCB3) has been proven to be transformed into hydroxylated metabolites of PCB3 (OH-PCB3s) in whole poplar plants in our previous work. However, hydroxylated metabolites of PCBs, including OH-PCB3s, as the substrates of sulfotransferases have not been studied in many organisms including plants in vivo. Poplar (Populus deltoides × nigra, DN34) was used to investigate the further metabolism from OH-PCB3s to PCB3 sulfates because it is a model plant and one that is frequently utilized in phytoremediation. Results showed poplar plants could metabolize PCB3 into PCB3 sulfates during 25 day exposures. Three sulfate metabolites, including 2′-PCB3 sulfate, 3′-PCB3 sulfate and 4′-PCB3 sulfate, were identified in poplar roots and their concentrations increased in the roots from day 10 to day 25. The major products were 2′-PCB3 sulfate and 4′-PCB3 sulfate. However, the concentrations of PCB3 sulfates were much lower than those of OH-PCB3s in the roots, suggesting the sequential transformation of these hydroxylated PCB3 metabolites into PCB3 sulfates in whole poplars. In addition, 2′-PCB3 sulfate or 4′-PCB3 sulfate was also found in the bottom wood samples indicating some translocation or metabolism in woody tissue. Results suggested that OH-PCB3s were the substrates of sulfotransferases which catalyzed the formation of PCB3 sulfates in the metabolic pathway of PCB3. PMID:23215248

Stereoselective and regiospecific hydroxylation of ketamine and norketamine.

PubMed

Desta, Zeruesenay; Moaddel, Ruin; Ogburn, Evan T; Xu, Cong; Ramamoorthy, Anuradha; Venkata, Swarajya Lakshmi Vattem; Sanghvi, Mitesh; Goldberg, Michael E; Torjman, Marc C; Wainer, Irving W

2012-11-01

The objective was to determine the cytochrome P450s (CYPs) responsible for the stereoselective and regiospecific hydroxylation of ketamine [(R,S)-Ket] to diastereomeric hydroxyketamines, (2S,6S;2R,6R)-HK (5a) and (2S,6R;2R,6S)-HK (5b) and norketamine [(R,S)-norKet] to hydroxynorketamines, (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,5S;2R,5R)-HNK (4c), (2S,4S;2R,4R)-HNK (4d), (2S,4R;2R,4S)-HNK (4e), (2S,5R;2R,5S)-HNK (4f). The enantiomers of Ket and norKet were incubated with characterized human liver microsomes (HLMs) and expressed CYPs. Metabolites were identified and quantified using LC/MS/MS and apparent kinetic constants estimated using single-site Michaelis-Menten, Hill or substrate inhibition equation. 5a was predominantly formed from (S)-Ket by CYP2A6 and N-demethylated to 4a by CYP2B6. 5b was formed from (R)- and (S)-Ket by CYP3A4/3A5 and N-demethylated to 4b by multiple enzymes. norKet incubation produced 4a, 4c and 4f and minor amounts of 4d and 4e. CYP2A6 and CYP2B6 were the major enzymes responsible for the formation of 4a, 4d and 4f, and CYP3A4/3A5 for the formation of 4e. The 4b metabolite was not detected in the norKet incubates. 5a and 4b were detected in plasma samples from patients receiving (R,S)-Ket, indicating that 5a and 5b are significant Ket metabolites. Large variations in HNK concentrations were observed suggesting that pharmacogenetics and/or metabolic drug interactions may play a role in therapeutic response.

Hydroxylated PBDEs induce developmental arrest in zebrafish

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

Usenko, Crystal Y., E-mail: Crystal_usenko@baylor.edu; Hopkins, David C.; Trumble, Stephen J., E-mail: Stephen_trumble@baylor.edu

The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was notmore » observed. In short-term exposures (24–28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis. -- Highlights: ► OH-PBDEs induce developmental arrest in a concentration-dependent manner. ► Hydroxyl group location influences biological interaction. ► OH-PBDEs induce oxidative stress. ► Thyroid hormone gene regulation was disrupted following exposure. ► To our knowledge, this is the first whole organism study of OH-PBDE toxicity.« less

Biosynthesis of human diazepam and clonazepam metabolites.

PubMed

de Paula, Núbia C; Araujo Cordeiro, Kelly C F; de Melo Souza, Paula L; Nogueira, Diogo F; da Silva e Sousa, Diego B; Costa, Maísa B; Noël, François; de Oliveira, Valéria

2015-03-01

A screening of fungal and microbial strains allowed to select the best microorganisms to produce in high yields some of the human metabolites of two benzodiazepine drugs, diazepam and clonazepam, in order to study new pharmacological activities and for chemical standard proposes. Among the microorganisms tested, Cunninghamella echinulata ATCC 9244 and Rhizopus arrhizus ATCC 11145 strains, were the most active producers of the mains metabolites of diazepam which included demethylated, hydroxylated derivatives. Beauveria bassiana ATCC 7159 and Chaetomium indicum LCP 984200 produced the 7 amino-clonazepam metabolite and a product of acid hydrolysis of this benzodiazepine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Syntheses and properties of the major hydroxy metabolites in humans of blonanserin AD-5423, a novel antipsychotic agent.

PubMed

Ochi, Takeshi; Sakamoto, Masato; Minamida, Akira; Suzuki, Kenji; Ueda, Tomohiko; Une, Teruaki; Toda, Hiroshi; Matsumoto, Kazuya; Terauchi, Yoshiaki

2005-02-15

Two major metabolites in humans of blonanserin, 2-(4-ethyl-1-piperazinyl)-4-(4-fluorophenyl)-5,6,7,8,9,10-hexahydrocycloocta-[b]pyridine (code name AD-5423), were synthesized. The first, 7-hydroxylated AD-5423, was synthesized through a four-step process starting from 4-fluorobenzoylacetonitrile (1), and the second, 8-hydroxylated AD-5423, a nine-step process also from 1. The optical resolution, structures, and receptor binding properties of the metabolites were documented.

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism.

PubMed

Beste, Lisa; Nahar, Nurun; Dalman, Kerstin; Fujioka, Shozo; Jonsson, Lisbeth; Dutta, Paresh C; Sitbon, Folke

2011-09-01

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Effect of pregnancy on the disposition of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) atropisomers and their hydroxylated metabolites in female mice.

PubMed

Kania-Korwel, Izabela; Barnhart, Christopher D; Lein, Pamela J; Lehmler, Hans-Joachim

2015-09-21

Chiral PCBs, such as PCB 95, are developmental neurotoxicants that undergo atropisomeric enrichment in nonpregnant adult mice. Because pregnancy is associated with changes in hepatic cytochrome P450 enzyme activity as well as lipid disposition and metabolism, this study investigates the effect of pregnancy on the maternal disposition of chiral PCBs. Female C57BL/6 mice (8 weeks old) were dosed daily beginning 2 weeks prior to conception and continuing throughout gestation and lactation (56 days total) with racemic PCB 95 (0, 0.1, 1.0, or 6.0 mg/kg body wt/day) in peanut butter. Levels and chiral signatures of PCB 95 and its hydroxylated metabolites (OH-PCBs) were determined in adipose, blood, brain, and liver. Tissue levels of PCB 95 increased 4- to 12-fold with increasing dose, with considerable enrichment of the second eluting atropisomer in all tissues (EF range 0.11 to 0.26). OH-PCBs displayed atropisomeric enrichment in blood and liver but were not detected in adipose and brain. Levels of PCB 95 and its metabolites were 2- to 11-fold lower in pregnant dams relative to those previously reported in nonpregnant age-matched female mice; however, PCB 95 and OH-PCB profiles and chiral signatures were similar between both studies. In contrast, human brain samples contained racemic PCB 95 residues (EF = 0.50). These results demonstrate that changes in cytochrome P450 enzyme activity and lipid disposition during pregnancy reduce the PCB body burden in dams but do not affect metabolite profiles or chiral signatures. The differences in chiral signatures between mice and humans suggest species-specific differences in atropisomeric disposition, the toxicological significance of which remains to be determined.

Preparation of human drug metabolites using fungal peroxygenases

Treesearch

Marzena Poraj-Kobielska; Matthias Kinne; René Ullrich; Katrin Scheibner; Gernot Kayser; Kenneth E. Hammel; Martin Hofrichter

2011-01-01

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus...

The metabolites of cyclohexylamine in man and certain animals

PubMed Central

Renwick, A. G.; Williams, R. T.

1972-01-01

1. [1-14C]Cyclohexylamine hydrochloride was synthesized and given orally or intraperitoneally to rats, rabbits and guinea pigs (dose 50–500mg/kg) and orally to humans (dose 25 or 200mg/person). The 14C is excreted mainly in the urine, most of the excretion occurring in the first day after dosing. Only small amounts (1–7%) are found in the faeces. 2. In the rat, guinea pig and man, the amine is largely excreted unchanged, only 4–5% of the dose being metabolized in 24h in the rat and guinea pig and 1–2% in man. In the rabbit about two-thirds of the dose is excreted unchanged and about 30% is metabolized. 3. In the rat, five minor metabolites were found, namely cyclohexanol (0.05%), trans-3- (2.2%), cis-4- (1.7%), trans-4- (0.5%) and cis-3-aminocyclohexanol (0.1% of the dose in 24h). 4. In the rabbit, eight metabolites were identified, namely cyclohexanol (9.3%), trans-cyclohexane-1,2-diol (4.7%), cyclohexanone (0.2%), cyclohexylhydroxylamine (0.2%) and trans-3- (11.3%), cis-3- (0.6%), trans-4- (0.4%) and cis-4-aminocyclohexanol (0.2%). 5. In the guinea pig, six minor metabolites were found, namely cyclohexanol (0.5%), trans-cyclohexane-1,2-diol (2.5%) and trans-3- (1.2%), cis-3- (0.2%), trans-4- (0.2%) and cis-4-aminocyclohexanol (0.2%). 6. In man only two metabolites were definitely identified, namely cyclohexanol (0.2%) and trans-cyclohexane-1,2-diol (1.4% of the dose), but man had been given a smaller dose (3mg/kg) than the other species (50mg/kg). 7. The hydroxylated metabolites of cyclohexylamine were excreted in the urine in both free and conjugated forms. 8. Although cyclohexylamine is metabolized to only a minor extent, in rats the metabolism was mainly through hydroxylation of the cyclohexane ring, in man by deamination and in guinea pigs and rabbits by ring hydroxylation and deamination. PMID:4655821

Microsomal metabolism of trenbolone acetate metabolites ...

EPA Pesticide Factsheets

Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

A comparison of octanol-water partitioning between organic chemicals and their metabolites in mammals.

PubMed

Pirovano, Alessandra; Borile, Nicolò; Jan Hendriks, A

2012-08-01

Bioaccumulation models take various elimination and uptake processes into account, estimating rates from chemical lipophilicity, expressed as the octanol-water partition ratio (K(ow)). Here, we focussed on metabolism, which transforms parent compounds into usually more polar metabolites, thus enhancing elimination. The aim of this study was to quantify the change in lipophilicity of relevant organic pollutants undergoing various biotransformation reactions in mammals. We considered oxidation reactions catalyzed by three enzyme groups: cytochrome P450 (CYP), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH). Estimated logK(ow) values of a selected dataset of parent compounds were compared with the logK(ow) of their first metabolites. The logK(ow) decreased by a factor that varies between 0 and -2, depending on the metabolic pathway. For reactions mediated by CYP, the decrease in K(ow) was one order of magnitude for hydroxylated and epoxidated compounds and two orders of magnitude for dihydroxylated and sulphoxidated xenobiotics. On the other hand, no significant change in lipophilicity was observed for compounds N-hydroxylated by CYP and for alcohols and aldehydes metabolized by ADH and ALDH. These trends could be anticipated by the calculus method of logK(ow). Yet, they were validated using experimental logK(ow) values, when available. These relationships estimate the extent to which the elimination of pollutants is increased by biotransformation. Thus, the quantification of the K(ow) reduction can be considered as a first necessary step in an alternative approach to anticipate biotransformation rates, which are hard to estimate with existing methods. Copyright © 2012 Elsevier Ltd. All rights reserved.

[Influence of corynebacteria metabolites on antagonistic activity of H2O2 producing lactobacilli].

PubMed

Bukharin, O V; Sgibnev, A V

2012-01-01

Study combined influence of Corynebacterium genus bacteria metabolites and H2O2 producing lactobacilli on survival rate of Staphylococcus aureus, Escherichia coli and Lactobacillus acidophilus. The ability to inhibit catalase of the test strains used and to reduce bactericidal effect of hydroxyl radical were determined in corynebacteria. H2O2 containing metabolites were obtained by cultivating lactobacilli in mineral medium, the amount of H2O2 was determined by oxidation of TMB by peroxidase. Bactericidal effect of lactobacilli metabolites for test strains treated by corynebacteria metabolites was evaluated by seeding results. Results. Inhibitio by corynebacteria metabolites of S. aureus catalase activity by 30-40% and E. coli catalase activ ity by 40-70% was shown. A reduction of bactericidal effect of hydroxyl radicals by corynebacteria metabolites by 30-35% for S. aureus, 38-42% for E. coli and 70-73% for L. acidophilus was noted. The enchantment of bactericidal effect of lactobacilli after treatment of the test strain by corynebacteria metabolites against S. aureus and E. coli manifested by reduction of the numbe of viable cells by 2-3 lg CFU. For L. acidophilus the bactericidal effect oflactobacilli metabolite in the same conditions reduced, and that led to the increase ofviability by 2-4 lg PFU. A conclusion on the possibility of regulation by associative bacteria the manifestations of antagonistic activity of H2O2 producing dominant microorganisms is made based on the data obtained.

Metabolites of hirsuteine and hirsutine, the major indole alkaloids of Uncaria rhynchophylla, in rats.

PubMed

Nakazawa, Takahiro; Banba, Koh-ichi; Hata, Kazumasa; Nihei, Yutaka; Hoshikawa, Ayumi; Ohsawa, Keisuke

2006-08-01

The metabolic fate of hirsuteine (HT) and hirsutine (HS), the major indole alkaloids of Uncaria rhynchophylla, was investigated using rats. On HPLC analysis, urine from rats orally administered HT were found to contain two metabolites (HT1 and HT2) together with unchanged HT. Similarly HS also was metabolized to two compounds (HS1 and HS2). Metabolite structures were determined to be 11-hydroxyhirsuteine-11-O-beta-D-glucuronide (HT1), 11-hydroxyhirsuteine (HT2), 11-hydroxyhirsutine-11-O-beta-D-glucuronide (HS1) and 11-hydroxyhirsutine (HS2), based on spectroscopic and chemical data. HT1 and HS1 were also detected in bile from rats administered HT and HS, respectively. Total cumulative urinary excretion within 72 h of oral administration was approximately 14% and 26% of the HT and HS doses, respectively, while total cumulative biliary excretion was 35% and 46%, respectively. HT and HS 11-hydroxylation were catalyzed by rat liver microsomes. This 11-hydroxylation activity was inhibited by addition of SKF-525A (a nonselective CYP inhibitor) or cimetidine (a CYP2C inhibitor). These results indicate that orally administered HT and HS are converted to 11-hydroxy metabolites in rats, and that the metabolites are predominantly excreted in bile rather than urine following glucuronidation. Furthermore, the results suggest that CYP2C enzymes are involved, at least in part, in the specific 11-hydroxylation of HT and HS.

Hydroxylative activity of Aspergillus niger towards androst-4-ene and androst-5-ene steroids.

PubMed

Świzdor, Alina; Panek, Anna; Milecka-Tronina, Natalia

2017-10-01

Aspergillus niger, one of fungal species most frequently used for experimental and industrial-scale biotransformations of various organic compounds, is generally known to transform steroids at 16β position. In this work, application of the strain A. niger KCH910 to bioconversion of dehydroepiandrosterone (DHEA), androstenediol and testosterone is described, with emphasis on the metabolic steps leading to the products. Evidence from this study indicated that incubated 5-ene steroids underwent bioconversion within two metabolic pathways: oxidation by the action of 3β-HSD (3β-hydroxysteroid dehydrogenase) to 4-ene steroids, and minor allylic hydroxylation to epimeric 7-alcohols. Further transformation of the 3-oxo-4-ene metabolites resulted in non-selective 16-hydroxylation. It is the first report on an A. niger strain able to introduce not only 16β- but also 16α-hydroxyl function into steroids. Copyright © 2017. Published by Elsevier Inc.

[Pharmacokinetics of (-)-clausenamide and its major metabolite 6-hydroxyl-clausenamide in beagle dogs by HPLC/MS].

PubMed

Song, Min; Qian, Wen; Hang, Tai-Jun; Zhang, Zheng-Xing

2005-10-01

To establish a sensitive and accurate method to study the pharmacokinetics of (-)-clausenamide [(-)-clau] and its major metabolite 6-hydroxyl-clausenamide (6-OH-clau) in the plasma of the Beagle dog. (-)-Clau was orally administered to six Beagle dogs at the dose of 30 mg x kg(-1), venous blood from front leg was sampled and plasma was separated for analysis. After extraction with ethyl acetate, the plasma samples were analyzed by HPLC/MS and the mobile phase was a mixture of methanol-water-acetic acid (60: 40: 0. 8) at the flow rate of 1.0 mL x min(-1). The API-ES positive ion SIM detection was carried out for the detection of both (-)-clau ([M + H] (+), m/z 298 ) and 6-OH-clau ([M + H - H2 O](+), m/z 296) with glipzide (glip) ([M + H](+), m/z 446) as internal standard. The pharmacokinetic parameters were calculated by 3P97 software. There was good linear relationship ( r > 0. 999) between the SIM responses and the concentrations for (-)-clau and 6-OH-clau at the range from 1.0 to 200 ng x mL(-1) and 0.2 to 40.0 ng x mL(-1), respectively. The absolute recovery was greater than 85%. The plasma concentration-time curves of (-)-clau and 6-OH-clau were both best fitted to a two-compartment model. The C(max) of (-)-clau and 6-OH-clau were (21 +/- 10) ng x mL(-1) and (3.9 +/- 2.2) ng x mL(-1), T(max) were (0.8 +/- 0.5) h and (1.3 +/- 0.5) h, T 1/2 alpha were (0.9 +/- 0.6) hand (1.4 +/- 0.6) h, T 1/2 beta were (19 +/- 23) hand (13 +/- 12) h, AUC(0-24 h) were (69 +/- 14) h x ng x mL(-1) and (12 +/- 7) h x ng x mL(-1) respectively. The established HPLC/MS method was sensitive and specific for the determination of (-)-clau. It was shown that the absorption and first phase elimination of (-)-clau were very quick in Beagle dogs, but the terminal elimination was very slow. The plasma concentration profile of its major metabolite 6-OH-clau was similar to (-)-clau and the AUC was relatively small in comparison with (-)-clau.

Metabolites of the phenylurea herbicides chlorotoluron, diuron, isoproturon and linuron produced by the soil fungus Mortierella sp.

PubMed

Badawi, Nora; Rønhede, Stig; Olsson, Stefan; Kragelund, Birthe B; Johnsen, Anders H; Jacobsen, Ole Stig; Aamand, Jens

2009-10-01

Phenylurea herbicides are used worldwide, and often pollute surface- and groundwater in concentrations exceeding the limit value for drinking water (0.1 microg l(-1)). Bacteria degrade phenylurea herbicides by successive N-dealkylation to substituted aniline products. Little is known about the corresponding fungal pathways, however. We here report degradation of chlorotoluron, diuron, isoproturon and linuron by the soil fungus Mortierella sp. Gr4. Degradation was fastest with linuron and resulted in successively dealkylated metabolites and 3,4-dichloroaniline. A major new metabolite was detected that has not yet been fully identified. Thin layer chromatography and nuclear magnetic resonance spectroscopy indicate that it is a non-aromatic diol. Degradation of isoproturon, chlorotoluron and diuron involved successive N-demethylation and, in the case of isoproturon and chlorotoluron, additional hydroxylation. A new hydroxylated isoproturon metabolite was detected. The study thus shows that the fungal pathways differ from the bacterial pathways and yield new metabolites of possible environmental concern.

Detection and characterization of urinary metabolites of boldione by LC-MS/MS. Part I: Phase I metabolites excreted free, as glucuronide and sulfate conjugates, and released after alkaline treatment of the urine.

PubMed

Gómez, C; Pozo, O J; Fabregat, A; Marcos, J; Deventer, K; Van Eenoo, P; Segura, J; Ventura, R

2012-10-01

Boldione (1,4-androstadien-3,17-dione) is included in the list of prohibited substances, issued by the World Anti-Doping Agency (WADA). Endogenous production of low concentrations of boldione has also been reported. The objective of this study was to assess boldione metabolism in humans. Detection of boldione metabolites was accomplished by analysis by liquid chromatography coupled to tandem mass spectrometry of urine samples obtained after administration of the drug and subjected to different sample preparation procedures to analyze the different metabolic fractions (free, glucuronides, sulpfates and released in basic media). In addition to boldione, eight metabolites were detected in the free fraction. Four of them were identified by comparison with standards: 6β-hydroxy-boldenone (M3), androsta-1,4,6-triene-3,17-dione (M5), (5α)-1-androstenedione (M6) and (5α)-1-testosterone (M8). Metabolite M7 was identified as the 5β-isomer of 1-androstenedione, and metabolites M1, M2 and M4 were hydroxylated metabolites and tentative structures were proposed based on mass spectrometric data. After β-glucuronidase hydrolysis, five additional metabolites excreted only as conjugates with glucuronic acid were detected: boldenone, (5β)-1-testosterone (M9), and three metabolites resulting from reduction of the 3-keto group. Boldenone, epiboldenone, and hydroxylated metabolites of boldione, boldenone and 1-testosterone were detected as conjugates with sulfate. In addition, boldione and seven metabolites (boldenone, M2, M3, M4, M5, M7 and M9) increased their concentration in urine after treatment of the urine in alkaline conditions. In summary, 15 boldione metabolites were detected in all fractions. The longer detection time was observed for metabolite M4 after alkaline treatment of the urine, which was detected up to 5 days after boldione administration. Copyright © 2012 John Wiley & Sons, Ltd.

Hydroxylated and methyl sulfone PCB metabolites in adipose and whole blood of polar bear (Ursus maritimus) from East Greenland.

PubMed

Sandala, G M; Sonne-Hansen, C; Dietz, R; Muir, D C G; Valters, K; Bennett, E R; Born, E W; Letcher, R J

2004-09-20

Persistent methyl sulfone (MeSO2-) and hydroxylated (HO-) polychlorinated biphenyl (PCB) metabolites have emerged as important classes of environmental contaminants in vertebrate, aquatic biota and humans. In the present study, PCB, MeSO2-PCB and HO-PCB concentrations and congener patterns were determined in the whole blood and adipose tissue of male (n = 7) and female (n = 12) polar bears (Ursus maritimus) of random age (3-25 years of age), and collected in 1999-2001 from the Ittoqqortoormiit/Scoresby Sound area in central East Greenland. There was no significant difference (P < 0.05) between males and females with respect to PCB or PCB metabolite concentrations in either tissue. The mean sum (Sigma) PCB concentrations were 7020+/-3366 ng/g lipid weight (lw) (range 2708-18148 ng/g lw) and 46.1+/-44.6 ng/g wet weight (ww) (range 12.6-204.2 ng/g ww) in adipose and blood, respectively. The mean Sigma-HO-PCB concentration in whole blood was 182.3+/-72.1 ng/g ww (range 93.8-382.1 ng/g ww). The mean Sigma-HO-PCB to Sigma-PCB concentration ratios in whole blood were 4.59+/-3.58 (range 1.03-11.88) and 8.30+/-5.56 (range 2.16-19.47) in females and males, respectively, which are the highest ratios reported so far for polar bears from any population, or for any free-ranging animal. Sigma-HO-PCB concentrations were greater than all other major classes of organochlorines (i.e. Sigma-PCBs, Sigma-MeSO2-PCBs, Sigma-chlordanes (CHLs), Sigma-hexachlorocyclohexanes (HCHs) and Sigma-chlorobenzenes (CBzs). The mean Sigma-MeSO2-PCB concentrations were 699+/-836 ng/g lw (range 127-3920 ng/g lw) and 10.9+/-9.6 ng/g ww (range 4.3-52.1 ng/g ww) in the adipose and blood, respectively. Regardless of age and sex, in both adipose and whole blood the MeSO2-PCB congener pattern was dominated by 3'- and 4'-MeSO2-CB101 and -CB87, and 4-MeSO2-CB149 (approx. 70% of the Sigma-MeSO2-PCBs). Minor differences in the MeSO2-PCB congener pattern were observed between blood and adipose, which suggests the

EFFECT OF PREGNANCY ON THE DISPOSITION OF 2,2′,3,5′,6-PENTACHLOROBIPHENYL (PCB 95) ATROPISOMERS AND THEIR HYDROXYLATED METABOLITES IN FEMALE MICE

PubMed Central

Kania-Korwel, Izabela; Barnhart, Christopher D.; Lein, Pamela J.; Lehmler, Hans-Joachim

2015-01-01

Chiral PCBs, such as PCB 95, are developmental neurotoxicants that undergo atropisomeric enrichment in non-pregnant, adult mice. Because pregnancy is associated with changes in hepatic cytochrome P450 enzyme activity as well as lipid disposition and metabolism, this study investigates the effect of pregnancy on the maternal disposition of chiral PCBs. Female C57Bl/6 mice (8 weeks old) were dosed daily beginning 2 weeks prior to conception and continuing throughout gestation and lactation (56 days total) with racemic PCB 95 (0, 0.1, 1.0 or 6.0 mg/kg body wt/day) in peanut butter. Levels and chiral signatures of PCB 95 and its hydroxylated metabolites (OH-PCBs) were determined in adipose, blood, brain and liver. Tissue levels of PCB 95 increased 4 to 12 fold with increasing dose, with considerable enrichment of the second eluting atropisomer in all tissues (EF range 0.11 to 0.26). OH-PCBs displayed atropisomeric enrichment in blood and liver, but were not detected in adipose and brain. Levels of PCB 95 and its metabolites were 2 to 11 fold lower in pregnant dams relative to those previously reported in non-pregnant age-matched female mice; however, PCB 95 and OH-PCB profiles and chiral signatures were similar between both studies. In contrast, human brain samples contained racemic PCB 95 residues (EF=0.50). These results demonstrate that changes in cytochrome P450 enzyme activity and lipid disposition during pregnancy reduce the PCB body burden in dams, but do not affect metabolite profiles or chiral signatures. The differences in chiral signatures between mice and humans suggest species-specific differences in atropisomeric disposition, the toxicological significance of which remain to be determined. PMID:26271003

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

PubMed Central

Moserová, Michaela; Kotrbová, Věra; Aimová, Dagmar; Šulc, Miroslav; Frei, Eva; Stiborová, Marie

2009-01-01

A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP. PMID:21217860

Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism1[C][W][OA

PubMed Central

Beste, Lisa; Nahar, Nurun; Dalman, Kerstin; Fujioka, Shozo; Jonsson, Lisbeth; Dutta, Paresh C.; Sitbon, Folke

2011-01-01

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels. PMID:21746809

Effect of Acid on Surface Hydroxyl Groups on Kaolinite and Montmorillonite

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

Sihvonen, Sarah K.; Murphy, Kelly A.; Washton, Nancy M.

Mineral dust aerosol participates in heterogeneous chemistry in the atmosphere. In particular, the hydroxyl groups on the surface of aluminosilicate clay minerals are important for heterogeneous atmospheric processes. These functional groups may be altered by acidic processing during atmospheric transport. In this study, we exposed kaolinite (KGa-1b) and montmorillonite (STx-1b) to aqueous sulfuric acid and then rinsed the soluble reactants and products off in order to explore changes to functional groups on the mineral surface. To quantify the changes due to acid treatment of edge hydroxyl groups, we use 19F magic angle spinning nuclear magnetic resonance spectroscopy and a probemore » molecule, 3,3,3-trifluoropropyldimethylchlorosilane. We find that the edge hydroxyl groups (OH) increase in both number and density with acid treatment. Chemical reactions in the atmosphere may be impacted by the increase in OH at the mineral edge.« less

[Gas chromatographic/mass spectrometric analysis of boldenone urinary metabolites in man].

PubMed

Zhang, J; Liu, C S; Zhou, T H

1991-01-01

The metabolism of boldenone (17 beta-hydroxy-1,4-androstem-3-one) in man has been investigated by gas chromatography/mass spectrometry. After oral administration of a 20 mg dose to man, six metabolites were detected in the conjugated fraction of the urinary samples. Boldenone, the major compound excreted in urine, was detected within 34 h after administration. In addition, several metabolites, resulting from the hydroxylation of boldenone and the reduction of the unsaturated carbon bonds of boldenone, were detected in the urine samples varying from 9 to 83 h. Extraction and fractionation of these metabolites were achieved by using XAD-2 column and gas chromatography. The recovery of the whole procedure was studied. Furthermore, the mass spectra of the metabolites are presented and major fragment pathways are discussed.

The Unexpected and Exceptionally Facile Chemical Modification of the Phenolic Hydroxyl Group of Tyrosine by Polyhalogenated Quinones under Physiological Conditions.

PubMed

Qu, Na; Li, Feng; Shao, Bo; Shao, Jie; Zhai, Guijin; Wang, Fuyi; Zhu, Ben-Zhan

2016-10-17

The phenolic hydroxyl group of tyrosine residue plays a crucial role in the structure and function of many proteins. However, little study has been reported about its modification by chemical agents under physiological conditions. In this study, we found, unexpectedly, that the phenolic hydroxyl group of tyrosine can be rapidly and efficiently modified by tetrafluoro-1,4-benzoquinone and other polyhalogenated quinones, which are the major genotoxic and carcinogenic quinoid metabolites of polyhalogenated aromatic compounds. The modification was found to be mainly due to the formation of a variety of fluoroquinone-O-tyrosine conjugates and their hydroxylated derivatives via nucleophilic substitution pathway. Analogous modifications were observed for tyrosine-containing peptides. Further studies showed that the blockade of the reactive phenolic hydroxyl group of tyrosine in the substrate peptide, even by very low concentration of tetrafluoro-1,4-benzoquinone, can prevent the kinase catalyzed tyrosine phosphorylation. This is the first report showing the exceptionally facile chemical modification of the phenolic hydroxyl group of tyrosine by polyhalogenated quinones under normal physiological conditions, which may have potential biological and toxicological implications.

Identification of BVT.2938 metabolites by LC/MS and LC/MS/MS after in vitro incubations with liver microsomes and hepatocytes.

PubMed

Edlund, Per Olof; Baranczewski, Pawel

2004-03-10

The metabolism of the 5HT2c agonist BVT.2938, 1-(3-[2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy]-2-pyrazinyl)-2(R)-methylpiperazine, was studied in vitro by incubation with rat, monkey and human liver microsomes as well as cryopreserved hepatocytes, followed by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis on a quadrupole-time of flight mass spectrometer for structural elucidation. Deuterium exchange on column was used to differentiate between hydroxylation and N-oxidation. Liver microsomes were incubated in two different buffer systems with optimum conditions for cytochrome P450 activity or UDP-glucuronosyltransferase activity. The major phase I metabolites of BVT.2938 originated from O-deethylation of the pyridine ring, O-dealkylation of the ethylene bridge, pyrazine ring hydroxylation, hydroxylation of pyridine ring and piperazine ring N-hydroxylation. When a hydrogen carbonate buffer system was supplemented with UDPGA, the piperazine carbamoyl-glucuronide from the parent compound was identified together with several glucuronides of the phase I metabolites. The metabolite pattern in hepatocytes was similar to microsomes except that the sulphate at the N-position of the piperazine ring of BVT.2938 was identified, while the carbamoyl-glucuronide was missing. Excellent correlation was obtained between radioactivity detection and the chemiluminescent nitrogen detector when the nitrogen content of the analytes was taken into account.

Detection and measurement of the agonistic activities of PCBs and mono-hydroxylated PCBs to the constitutive androstane receptor using a recombinant yeast assay.

PubMed

Kamata, Ryo; Shiraishi, Fujio; Kageyama, Shiho; Nakajima, Daisuke

2015-10-01

Polychlorinated biphenyls (PCBs) are thought to exert their toxicities mainly by binding to the aryl hydrocarbon receptor and by stimulating transcription of various genes, notably metabolizing enzymes including the cytochrome P450 (CYP) 1 family. However, PCBs and their metabolites could have potential to activate other nuclear receptors and subsequent events. We focused on the constitutive androstane receptor (CAR) inducing CYP2B and measured the agonistic activity of PCBs and mono-hydroxylated PCBs (OH-PCBs) to the CAR using yeast cells transduced with the human CAR and its response pathway. Twenty-nine of 34 tested PCBs and 72 of 91 OH-PCBs exhibited CAR agonistic effects. Of 41 OH-PCBs that had the same chlorination patterns as the tested PCBs, 9 had activities more than twice those of their non-hydroxylated analogs. In particular, 2',4',6'-trichlorobiphenyl-4-ol and 2,2',4',6'-tetrachlorobiphenyl-4-ol were 332- and 22-fold more potent than their analogs and were 15 times and 2.8 times, respectively, as active as a reference substance, 4-tert-octylphenol. The activities of 17 of the OH-PCBs were reduced to less than half those of their non-hydroxylated analogs. Four OH-PCBs derived from 3 active PCBs were inactive. However, a consistent relationship between hydroxyl substituent position and activity could not be discerned. Comprehensive evaluation of the toxic potential of PCBs and their hydroxylated metabolites and their concentrations in the environment are required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hydroxylation increases the neurotoxic potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells.

PubMed

Dingemans, Milou M L; de Groot, Aart; van Kleef, Regina G D M; Bergman, Ake; van den Berg, Martin; Vijverberg, Henk P M; Westerink, Remco H S

2008-05-01

Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants. Our objective was to investigate the effects of a hydroxylated metabolite of 2,2',4,4'-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells. We measured vesicular catecholamine release and [Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively. Acute exposure of PC12 cells to 6-OH-BDE-47 (5 microM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 microM). An additional late increase in [Ca2+]i was often observed at > or =1 microM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 microM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria. The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca2+]i cannot be ruled out.

Iron and its complexation by phenolic cellular metabolites

PubMed Central

Chobot, Vladimir

2010-01-01

Iron is a transition metal that forms chelates and complexes with various organic compounds, also with phenolic plant secondary metabolites. The ligands of iron affect the redox potential of iron. Electrons may be transferred either to hydroxyl radicals, hydrogen peroxide or molecular oxygen. In the first case, oxidative stress is decreased, in the latter two cases, oxidative stress is increased. This milieu-dependent mode of action may explain the non-linear mode of action of juglone and other secondary metabolites. Attention to this phenomenon may help to explain idiosyncratic and often nonlinear effects that result in biological assays. Current chemical assays are discussed that help to explore these aspects of redox chemistry. PMID:20592800

Quantifying fenbendazole and its metabolites in self-medicating wild red grouse Lagopus lagopus scoticus using an HPLC-MS-MS approach.

PubMed

Adam, Aileen; Webster, Lucy M I; Mullen, William; Keller, Lukas F; Johnson, Paul C D

2011-05-11

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC-MS-MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control. Copyright © 2011 Elsevier B.V. All rights reserved.

Phosphate-Linked Silibinin Dimers (PLSd): New Promising Modified Metabolites.

PubMed

Romanucci, Valeria; Gravante, Raffaele; Cimafonte, Martina; Marino, Cinzia Di; Mailhot, Gilles; Brigante, Marcello; Zarrelli, Armando; Fabio, Giovanni Di

2017-08-11

By exploiting the regioselective protection of the hydroxyl groups of silibinin along with the well-known phosphoramidite chemistry, we have developed an efficient strategy for the synthesis of new silibinin-modified species, which we have named Phosphate-Linked Silibinin Dimers (PLSd), in which the monomer units are linked by phosphodiester bonds. The antioxidant abilities of the new PLSd were estimated on HepG2 cells using DPPH free radical scavenging and xanthine/xanthine oxidase assays. The new phosphate-metabolites showed a higher anti-oxidant activity than the silibinin, as well as very low toxicity. The ability to scavenge reactive oxygen species (ROS) such as singlet oxygen () and hydroxyl radical () reveals that the two dimers are able to scavenge about two times more effectively than silibinin. Finally, solubility studies have shown that the PLSd present good water solubility (more than 20 mg·L -1 ) under circumneutral pH values, whereas the silibinin was found to be very poorly soluble (less than 0.4 mg·L -1 ) and not stable under alkaline conditions. Together, the above promising results warrant further investigation of the future potential of the PLSd as anti-oxidant metabolites within the large synthetic polyphenols field.

Mucor hiemalis mediated 14α-hydroxylation on steroids: in vivo and in vitro investigations of 14α-hydroxylase activity.

PubMed

Kolet, Swati P; Haldar, Saikat; Niloferjahan, Siddiqui; Thulasiram, Hirekodathakallu V

2014-07-01

Transformation of testosterone and progesterone into synthetically challenging 14α-hydroxy derivatives was achieved by using fungal strain Mucor hiemalis. Prolonged incubation led to the formation of corresponding 6β/7α,14α-dihydroxy metabolites. The position and stereochemistry of newly introduced hydroxyl group was determined by detailed spectroscopic analyses. The time course experiment indicated that fungal strain initiated transformation by hydroxylation at 14α-position followed by at 6β- or 7α-positions. Studies using cell-free extracts suggest that the 14α-hydroxylase activity is NADPH dependent and belongs to the cytochrome P450 family. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems.

PubMed

Biondi, R; Xia, Y; Rossi, R; Paolocci, N; Ambrosio, G; Zweier, J L

2001-03-01

Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.

Identification and mechanism of formation of potentially genotoxic metabolites of tamoxifen: study by LC-MS/MS.

PubMed

Lim, C K; Yuan, Z X; Jones, R M; White, I N; Smith, L L

1997-06-01

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI MS) and tandem mass spectrometry (MS/MS) have been applied to the study of tamoxifen metabolism in liver microsomes and to the identification of potentially genotoxic metabolites. The results showed that the hydroxylated derivatives, including 4-hydroxytamoxifen and alpha-hydroxytamoxifen are detoxication metabolites, while arene oxides, their free radical precursors or metabolic intermediates, are the most probable species involved in DNA-adduct formation.

Quasi-targeted analysis of hydroxylation-related metabolites of polycyclic aromatic hydrocarbons in human urine by liquid chromatography-mass spectrometry.

PubMed

Tang, Caiming; Tan, Jianhua; Fan, Ruifang; Zhao, Bo; Tang, Caixing; Ou, Weihui; Jin, Jiabin; Peng, Xianzhi

2016-08-26

Metabolite identification is crucial for revealing metabolic pathways and comprehensive potential toxicities of polycyclic aromatic hydrocarbons (PAHs) in human body. In this work, a quasi-targeted analysis strategy was proposed for metabolite identification of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in human urine using liquid chromatography triple quadruple mass spectrometry (LC-QqQ-MS/MS) combined with liquid chromatography high resolution mass spectrometry (LC-HRMS). Potential metabolites of OH-PAHs were preliminarily screened out by LC-QqQ-MS/MS in association with filtering in a self-constructed information list of possible metabolites, followed by further identification and confirmation with LC-HRMS. The developed method can provide more reliable and systematic results compared with traditional untargeted analysis using LC-HRMS. In addition, data processing for LC-HRMS analysis were greatly simplified. This quasi-targeted analysis method was successfully applied to identifying phase I and phase II metabolites of OH-PAHs in human urine. Five metabolites of hydroxynaphthalene, seven of hydroxyfluorene, four of hydroxyphenanthrene, and three of hydroxypyrene were tentatively identified. Metabolic pathways of PAHs in human body were putatively revealed based on the identified metabolites. The experimental results will be valuable for investigating the metabolic processes of PAHs in human body, and the quasi-targeted analysis strategy can be expanded to the metabolite identification and profiling of other compounds in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Biotransformation of the citrus flavone tangeretin in rats. Identification of metabolites with intact flavane nucleus.

PubMed

Nielsen, S E; Breinholt, V; Cornett, C; Dragsted, L O

2000-09-01

The present study was carried out in order to investigate the in vivo biotransformation and excretion of the flavone, tangeretin, found in citrus fruits, by analysing urine and faeces samples from rats after repeated administration of 100 mg/kg body weight/day tangeretin. The formed metabolites were separated and identified by HPLC and the structures elucidated by LC/MS and 1H NMR. Ten new, major metabolites with intact flavonoid structure were identified. The metabolites identified were either demethylated or hydroxylated derivatives of the parent compound and metabolic changes were found primarily to occur in the 4' position of the B-ring. The total urinary excretion of tangeretin metabolites with intact flavan nucleus was about 11% of the administered daily dose.

Profiling and Distribution of Metabolites of Procyanidin B2 in Mice by UPLC-DAD-ESI-IT-TOF-MSn Technique

PubMed Central

Xiao, Ying; Hu, Zhongzhi; Yin, Zhiting; Zhou, Yiming; Liu, Taiyi; Zhou, Xiaoli; Chang, Dawei

2017-01-01

The metabolite profiles and distributions of procyanidin B2 were qualitatively described using UPLC-DAD-ESI-IT-TOF-MSn without help of reference standards, and a possible metabolic pathway was proposed in the present study. Summarily, 53 metabolites (24 new metabolites) were detected as metabolites of procyanidin B2, and 45 of them were tentatively identified. Twenty seven metabolites were assigned as similar metabolites of (−)-epicatechin by scission of the flavanol interflavanic bond C4–C8, including 16 aromatic metabolites, 5 conjugated metabolites, 3 ring-cleavage metabolites, and 2 phenylvalerolactone metabolites. Additionally, 14 metabolites were conjugates of free procyanidin B2, comprising 9 methylation metabolites, 8 sulfation metabolites, 5 hydration metabolites, 2 hydroxylation metabolites, 1 hydrogenation metabolites, and 1 glucuronidation metabolites. The results of metabolite distributions in organs indicated that the conjugated reaction of free procyanidin B2 mainly occurred in liver and diversified metabolites forms were observed in small intestine. The metabolic components of procyanidin B2 identified in mice provided useful information for further study of the bioactivity and mechanism of its action. PMID:28522973

Metabolite Depletion Affects Flux Profiling of Cell Lines.

PubMed

Nilsson, A; Haanstra, J R; Teusink, B; Nielsen, J

2018-06-01

Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hepatocyte or serum albumin protein carbonylation by oxidized fructose metabolites: Glyceraldehyde or glycolaldehyde as endogenous toxins?

PubMed

Dong, Qiang; Yang, Kai; Wong, Stephanie M; O'Brien, Peter J

2010-10-06

Excessive sugar intake in animal models may cause tissue damage associated with oxidative and carbonyl stress cytotoxicity as well as inflammation. Fructose became a 100-fold more cytotoxic if hepatocytes were exposed to a non-toxic infusion of H(2)O(2) so as to simulate H(2)O(2) released by Kupffer cells or infiltrating immune cells. In order to determine the molecular mechanisms involved, protein carbonylation of fructose and its metabolites were determined using the 2,4-dinitrophenylhydrazine method. In a cell-free system, fructose was found to carbonylate bovine serum albumin (BSA) only if low concentrations of FeII/H(2)O(2) were added. Protein carbonylation by the fructose metabolites glyceraldehyde or glycolaldehyde was also markedly increased by FeII/H(2)O(2). The protein carbonylation may be attributed to glyoxal formation by hydroxyl radicals as the glyoxal trapping agent aminoguanidine or hydroxyl radical scavengers prevented protein carbonylation. Glyoxal was also much more effective than other carbonyls at causing protein carbonylation. When BSA was replaced by isolated rat hepatocytes, fructose metabolite glyceraldehyde in the presence of non-toxic 2 microM FeII:8-hydroxyquinoline (HQ) and a H(2)O(2) generating system (glucose/glucose oxidase) markedly increased cytotoxicity, protein carbonylation and reactive oxygen species (ROS)/H(2)O(2) formation. Furthermore this was prevented by hydroxyl radical scavengers or aminoguanidine, a glyoxal scavenger. CuII: 8-hydroxyquinoline increased H(2)O(2) induced hepatocyte protein carbonylation less but was prevented by aminoguanidine. However, cytotoxicity and protein carbonylation induced by glyceraldehyde/CuII:HQ/H(2)O(2) were not affected by hydroxyl radical scavengers. Although fatty liver induced by an excessive sugar diet in animal models has been proposed as the first hit for non-alcoholic steatohepatitis (NASH) we propose that oxidative stress induced by the oxidation of fructose or fructose metabolites

Application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry in the rapid characterization of quinocetone metabolites formed in vitro.

PubMed

Liu, Zhao-Ying; Huang, Ling-Li; Chen, Dong-Mei; Dai, Meng-Hong; Tao, Yan-Fei; Wang, Yu-Lian; Yuan, Zong-Hui

2010-02-01

The application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography (LC/MS-IT-TOF) in the rapid characterization of in vitro metabolites of quinocetone was developed. Metabolites formed in rat liver microsomes were separated using a VP-ODS column with gradient elution. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The elemental compositions of all fragment ions of quinocetone and its metabolites could be rapidly assigned based upon the known compositional elements of protonated molecules. The structure of metabolites were elucidated based on the combination of three techniques: agreement between their proposed structure, the accurate masses, and the elemental composition of ions in their mass spectra; comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite; and the elemental compositions of lost mass numbers in proposed fragmentation pathways. Twenty-seven phase I metabolites were identified as 11 reduction metabolites, three direct hydroxylation metabolites, and 13 metabolites with a combination of reduction and hydroxylation. All metabolites except the N-oxide reduction metabolite M6 are new metabolites of quinocetone, which were not previously reported. The ability to conduct expected biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurement, all in a single experimental run, is one of the most attractive features of this methodology. The results demonstrate the use of LC/MS-IT-TOF approach appears to be rapid, efficient, and reliable in structural characterization of drug metabolites.

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

Sequential Metabolism of Secondary Alkyl Amines to Metabolic-Intermediate Complexes: Opposing Roles for the Secondary Hydroxylamine and Primary Amine Metabolites of Desipramine, (S)-Fluoxetine, and N-Desmethyldiltiazem

PubMed Central

Hanson, Kelsey L.; VandenBrink, Brooke M.; Babu, Kantipudi N.; Allen, Kyle E.; Nelson, Wendel L.

2010-01-01

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine ≫ primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d3-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation. PMID:20200233

Sequential metabolism of secondary alkyl amines to metabolic-intermediate complexes: opposing roles for the secondary hydroxylamine and primary amine metabolites of desipramine, (s)-fluoxetine, and N-desmethyldiltiazem.

PubMed

Hanson, Kelsey L; VandenBrink, Brooke M; Babu, Kantipudi N; Allen, Kyle E; Nelson, Wendel L; Kunze, Kent L

2010-06-01

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine > primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d(3)-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation.

Characterization of metabolites of larotaxel in rat by liquid chromatography coupled with Q exactive high-resolution benchtop quadrupole orbitrap mass spectrometer.

PubMed

Liu, Zhenzhen; Hou, Pengyi; Liu, Lian; Qian, Feng

2018-03-01

1. Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans for drug metabolism studies. Larotaxel is a taxane analog that has the potential for the treatment of various types of cancer. 2. In this study, the metabolism of larotaxel was evaluated after an intravenous dose of 8 mg/kg via the caudal vein to healthy rats and its metabolites were characterized by high performance liquid chromatography coupled with a Q Exactive high-resolution benchtop quadrupole orbitrap mass spectrometer. Rat bio-samples were separated on a Capcell Pak C 18 column (2.1 i.d. × 100 mm; 2.7 μm) with mobile phase of acetonitrile and water. 3. As a result, a total of 34 metabolites were detected and identified by comparing the molecular masses, retention times and spectral patterns of the analytes with those of the parent drug. Three metabolites were confirmed by comparison with reference substances. 4. The prominent metabolites were mainly hydroxyl, dihydroxyl, trihydroxyl and 10-desacetyl analogs of larotaxel, some of which resulted from oxidation of the tert-butyl groups on the side chain and further oxidation and cyclization of the tert-butyl hydroxylated metabolites.

Contribution of hydroxylated atrazine degradation products to the total atrazine load in midwestern streams

USGS Publications Warehouse

Lerch, R.N.; Blanchard, P.E.; Thurman, E.M.

1998-01-01

The contribution of hydroxylated atrazine degradation products (HADPs) to the total atrazine load (i.e., atrazine plus stable metabolites)in streams needs to be determined in order to fully assess the impact of atrazine contamination on stream ecosystems and human health. The objectives of this study were (1) to determine the contribution of HADPs to the total atrazine load in streams of nine midwestern states and (2) to discuss the mechanisms controlling the concentrations of HADPs in streams. Stream samples were collected from 95 streams in northern Missouri at preplant and postplant of 1994 and 1995, and an additional 46 streams were sampled in eight midwestern states at postplant of 1995. Samples were analyzed for atrazine, deethylatrazine (DEA), deisopropylatrazine (DIA), and three HADPs. Overall, HADP prevalence (i.e., frequency of detection) ranged from 87 to 100% for hydroxyatrazine (HA), 0 to 58% for deethylhydroxyatrazine (DEHA), and 0% for deisopropylhydroxyatrazine (DIHA) with method detection limits of 0.04-0.10 ??g L-1. Atrazine metabolites accounted for nearly 60% of the atrazine load in northern Missouri streams at preplant, with HA the predominant metabolite present. Data presented in this study and a continuous monitoring study are used to support the hypothesis that a combination of desorption from stream sediments and dissolved-phase transport control HADP concentrations in streams.The contribution of hydroxylated atrazine degradation products (HADPs) to the total atrazine load (i.e., atrazine plus stable metabolites) in streams needs to be determined in order to fully assess the impact of atrazine contamination on stream ecosystems and human health. The objectives of this study were (1) to determine the contribution of HADPs to the total atrazine load in streams of nine midwestern states and (2) to discuss the mechanisms controlling the concentrations of HADPs in streams. Stream samples were collected from 95 streams in northern Missouri at

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.

Prenatal exposure to polychlorinated biphenyls and their hydroxylated metabolites is associated with motor development of three-month-old infants.

PubMed

Berghuis, Sietske A; Soechitram, Shalini D; Hitzert, Marrit M; Sauer, Pieter J J; Bos, Arend F

2013-09-01

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that are potentially toxic to the developing brain. Hydroxylated metabolites of PCBs (OH-PCBs) are suggested to be even more toxic. Little is known about their short-term effects on human health. To determine whether prenatal background exposure to PCBs and OH-PCBs was associated with the motor development of three-month-old infants. Ninety-seven mother-infant pairs participated in this Dutch, observational cohort study. We determined the concentrations of PCBs and OH-PCBs in cord blood samples. When the infants were three months old we evaluated their motor development by assessing the presence and performance of spontaneous movement patterns from video recordings. We calculated a Motor Optimality Score (MOS). The score could range from low (5) to high (28) optimality. We explored the correlations between PCB and OH-PCB levels and MOS. Subsequently, we tested whether the levels differed between infants with a low (<26) or high (≥26) MOS and whether the levels associated with detailed aspects of their motor repertoires. We found several associations between PCB and OH-PCB levels and MOS, including detailed aspects of the early motor development. High 4-OH-PCB-107 levels were associated with a low MOS (P=.013). High PCB-187 levels were associated with reduced midline arm and leg movements (P=.047 and P=.043, respectively). High 4'-OH-PCB-172 levels were associated with more manipulation (P=.033). Prenatal exposure to high background levels of most PCBs and 4-OH-PCB-107 seems to impair early motor development, whereas only 4'-OH-PCB-172 showed the opposite. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine, Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers' Urine

PubMed Central

McGuffey, James E.; Wei, Binnian; Bernert, John T.; Morrow, John C.; Xia, Baoyun; Wang, Lanqing; Blount, Benjamin C.

2014-01-01

Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies. PMID:25013964

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.

Cerebrospinal fluid ATP metabolites in multiple sclerosis.

PubMed

Lazzarino, G; Amorini, A M; Eikelenboom, M J; Killestein, J; Belli, A; Di Pietro, V; Tavazzi, B; Barkhof, F; Polman, C H; Uitdehaag, B M J; Petzold, A

2010-05-01

Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline. The Expanded Disability Status Scale, MRI brain imaging measures for brain atrophy (ventricular and parenchymal fractions), and cerebrospinal fluid biomarkers for axonal damage (phosphorylated and hyperphosphorylated neurofilaments) were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 micromol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing-remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

Benzene: a case study in parent chemical and metabolite interactions.

PubMed

Medinsky, M A; Kenyon, E M; Schlosser, P M

1995-12-28

Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia and pancytopenia, and acute myelogenous leukemia. A combination of metabolites (hydroquinone and phenol for example) is apparently necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Since benzene and its hydroxylated metabolites (phenol, hydroquinone and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus, the potential exists for competition among various enzymes for phenol. However, zonal localization of Phase I and Phase II enzymes in various regions of the liver acinus regulates this competition. Biologically-based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.

Potential bile acid metabolites. XV. Synthesis of 4 beta-hydroxylated bile acids; unique bile acids in human fetal bile.

PubMed

Iida, T; Momose, T; Chang, F C; Goto, J; Nambara, T

1989-12-01

The 4 beta-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, and cholic acids were synthesized from their respective parent compounds. The principal reactions employed were 1) beta-face cis-dihydroxylation of delta 3 intermediates with osmium tetroxide-N-methylmorpholine N-oxide, 2) selective cathylation of vicinal 3 beta,4 beta-diols followed by oxidation of the resulting 4 beta-monocathylates, or direct selective oxidation at C-3 of 3 beta,4 beta-diols with pyridinium chlorochromate, and 3) stereoselective reduction of the 3-oxo compounds with tert-butylamine-borane complex. The results of analysis of the prepared 4 beta-hydroxylated bile acids with a diequatorial trans-glycol structure and their 3 beta-epimers by proton and carbon-13 nuclear magnetic resonance spectroscopies are briefly discussed along with the mass spectrometric properties.

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.

Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

PubMed Central

Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

2014-01-01

Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

Comprehensive Analysis of Tiamulin Metabolites in Various Species of Farm Animals Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole/Time-of-Flight.

PubMed

Sun, Feifei; Yang, Shupeng; Zhang, Huiyan; Zhou, Jinhui; Li, Yi; Zhang, Jinzhen; Jin, Yue; Wang, Zhanhui; Li, Yanshen; Shen, Jianzhong; Zhang, Suxia; Cao, Xingyuan

2017-01-11

Tiamulin is an antimicrobial widely used in veterinary practice to treat dysentery and pneumonia in pigs and poultry. However, knowledge about the metabolism of tiamulin is very limited in farm animals. To better understand the biotransformation of tiamulin, in the present study, in vitro and in vivo metabolites of tiamulin in rats, chickens, swine, goats, and cows were identified and elucidated using ultra-high performance liquid chromatography coupled to quadrupole/time-of-flight. As a result, a total of 26 metabolites of tiamulin, identified in vitro and in vivo, and majority of metabolites were revealed for the first time. In all farm animals, tiamulin undergoes phase I metabolic routes of hydroxylation in the mutilin part (the ring system), S-oxidation and N-deethylation on side chain, and no phase II metabolite was detected. Among these, 2β- and 8α-hydroxylation and N-deethylation were the main metabolic pathways of tiamulin in farm animals. In addition, we have put forward that 8a-hydroxy-tiamulin and 8a-hydroxy-N-deethyl-tiamulin could be hydroxylated into 8a-hydroxy-mutilin, the marker residue of tiamulin in swine. Furthermore, a significant interspecies difference was observed on the metabolism of tiamulin among various farm animals. The possible marker residues for tiamulin in swine were 8α-hydroxy-tiamulin, N-deethyl-tiamulin, and 8α-hydroxy-N-deethyl-tiamulin, which were consistent with the hypothesis proposed by the European Agency for the Evaluation of Medicinal Products. However, results in present study indicated that three metabolites (2β-hydroxy-tiamulin, N-deethyl-tiamulin, and 2β-hydroxy-N-deethyl-tiamulin) of tiamulin in chickens had larger yields, which implied that 2β-hydroxy-mutilin or N-deethyl-tiamulin was more likely to be regarded as the potential marker residue of tiamulin in chickens.

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

NASA Astrophysics Data System (ADS)

Campos, Christiane Gonçalves; Veras, Henrique César Teixeira; de Aquino Ribeiro, José Antônio; Costa, Patrícia Pinto Kalil Gonçalves; Araújo, Katiúscia Pereira; Rodrigues, Clenilson Martins; de Almeida, João Ricardo Moreira; Abdelnur, Patrícia Verardi

2017-12-01

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. [Figure not available: see fulltext.

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

Quantification of hydroxyl radical produced during phacoemulsification.

PubMed

Gardner, Jonathan M; Aust, Steven D

2009-12-01

To quantitate hydroxyl radicals produced during phacoemulsification with various irrigating solutions and conditions used in cataract surgery. Chemistry and Biochemistry Department, Utah State University, Logan, Utah, USA. All experiments were performed using an Infiniti Vision System phacoemulsifier with irrigation and aspiration. Hydroxyl radicals were quantitated using electron spin resonance spectroscopy and a spectrophotometric assay for malondialdehyde, which is formed by the oxidation of deoxyribose by the hydroxyl radical. Hydroxyl radical production increased during longitudinal-stroking phacoemulsification as power levels were increased in a nonlinear, nonexponential fashion. The detection of hydroxyl radical was reduced in irrigating solutions containing organic molecules (eg, citrate, acetate, glutathione, dextrose) and further reduced in Navstel, an irrigating solution containing a viscosity-modifying agent, hydroxypropyl methylcellulose. Hydroxyl radicals produced in settings representative of those used in phacoemulsification cataract surgery were quantitated using the deoxyribose method. Hydroxyl radical production was dependent on the level of ultrasound power applied and the irrigating solution used. Oxidative stress on the eye during phacoemulsification may be minimized by using irrigating solutions that contain organic molecules, including the viscosity-modifying agent hydroxypropyl methylcellulose, that can compete for reaction with hydroxyl radicals.

Prolyl hydroxylation in elastin is not random.

PubMed

Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon; Merkher, Yulia; Sivan, Sarit S; Heinz, Andrea

2016-10-01

This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

PubMed

Vanduchova, Alena; Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

2016-12-01

The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

Microbial hydroxylation and glycosidation of oleanolic acid by Circinella muscae and their anti-inflammatory activities.

PubMed

Yan, Sensen; Lin, Haijun; Huang, Huilian; Yang, Min; Xu, Bohui; Chen, Guangtong

2018-05-29

Biotransformation of oleanolic acid (OA) by Circinella muscae AS 3.2695 was investigated. Nine hydroxylated and glycosylated metabolites (1-9) were obtained. Their structures were elucidated as 3β,7β-dihydroxyolean-12-en-28-oic acid (1), 3β,7β,21β-trihydroxyolean-12-en-28-oic acid (2), 3β,7α,21β-trihydroxyolean-12-en- 28-oic acid (3), 3β,7β,15α-trihydroxyolean-12-en-28-oic acid (4), 7β,15α-dihydroxy- 3-oxo-olean-12-en-28-oic acid (5), 7β-hydroxy-3-oxo-olean-12-en-28-oic acid (6), oleanolic acid-28-O-β-D-glucopyranosyl ester (7), 3β,21β-dihydroxyolean-12-en-28- oic acid-28-O-β-D-glucopyranosyl ester (8), and 3β,7β,15α-trihydroxyolean-12-en- 28-oic acid-28-O-β-D-glucopyranosyl ester (9) by spectroscopic analysis. Among them, compounds 4 and 9 were new compounds. In addition, anti-inflammatory activities were assayed and evaluated for the isolated metabolites. Most of the metabolites exhibited significant inhibitory activities on lipopolysaccharides-induced NO production in RAW 264.7 cells.

Bioactive metabolites from biotransformation of paeonol by the white-rot basidiomycete Coriolus versicolor.

PubMed

Li, Xiao-Jun; Shi, Xin-Wei; Shuai, Qi; Gao, Jin-Ming; Zhang, An-Ling

2011-08-01

Biotransformation of paeonol (1) with the white-rot basidiomycete Coriolus versicolor afforded two metabolites, 2,4-dihydroxyacetophenone (2) and 2,5-dihydroxy-4-methoxyacetophenone (3), which were identified by spectroscopic methods. Compound 3 showed higher antioxidative, antibacterial, antifungal activities than 1 or 2. The results demonstrate for the first time that C. versicolor has the capacities to catalyze hydroxylation and demethylation reactions on the aromatic compound.

Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

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

Dreiem, Anne; Norwegian Defence Research Establishment, Department for Protection, Kjeller; Rykken, Sidsel

2009-10-15

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34,more » 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4'-OH PCB 36 < 4'-OH PCB 68 < 4-OH PCB 39 < 4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.« less

Oxidative metabolism of BDE-99 by human liver microsomes: predominant role of CYP2B6.

PubMed

Erratico, Claudio A; Szeitz, András; Bandiera, Stelvio M

2012-10-01

Hydroxylated polybrominated diphenyl ethers (PBDEs) have been found in human serum, suggesting that they are formed by in vivo oxidative metabolism of PBDEs. However, the biotransformation of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), a major PBDE detected in human tissue and environmental samples, is poorly understood. In the present study, the oxidative metabolism of BDE-99 was assessed using pooled and single-donor human liver microsomes, a panel of human recombinant cytochrome P450 (CYP) enzymes, and CYP-specific antibodies. Hydroxylated metabolites were quantified using a liquid chromatography/tandem mass spectrometry-based method. In total, 10 hydroxylated metabolites of BDE-99 were produced by human liver microsomes. Six metabolites were identified as 2,4,5-tribromophenol (2,4,5-TBP), 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, and 2-OH-BDE-123 using authentic standards. Three monohydroxy- and one dihydroxy-pentabrominated metabolites were unidentified. Rates of formation of the three major metabolites (2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101) by human liver microsomes ranged from 24.4 to 44.8 pmol/min/mg protein. Additional experiments demonstrated that the dihydroxylated metabolite was a primary metabolite of BDE-99 and was not produced by hydroxylation of a monohydroxy metabolite. Among the panel of recombinant CYP enzymes tested, formation of all 10 hydroxylated metabolites was catalyzed solely by CYP2B6. A combined approach using antibodies to CYP2B6 and single-donor liver microsomes expressing a wide range of CYP2B6 levels confirmed that CYP2B6 was responsible for the biotransformation of BDE-99. Collectively, the results show that the oxidative metabolism of BDE-99 by human liver microsomes is catalyzed solely by CYP2B6 and is an important determinant of the toxicity and bioaccumulation of BDE-99 in humans.

Ruta graveolens Extracts and Metabolites against Spodoptera frugiperda.

PubMed

Ayil-Gutiérrez, Benjamin A; Villegas-Mendoza, Jesús M; Santes-Hernndez, Zuridai; Paz-González, Alma D; Mireles-Martínez, Maribel; Rosas-García, Ninfa M; Rivera, Gildardo

2015-11-01

The biological activity of Ruta graveolens leaf tissue extracts obtained with different solvents (ethyl acetate, ethanol, and water) and metabolites (psoralen, 2- undecanone and rutin) against Spodoptera frugiperda was evaluated. Metabolites levels in extracts were quantified by HPLC and GC. Ethyl acetate and ethanol extracts showed 94% and 78% mortality, respectively. Additionally, psoralen metabolite showed a high mortality as cypermethrin. Metabolite quantification in extracts shows the presence of 2-undecanone (87.9 µmoles mg(-1) DW), psoralen (3.6 µmoles mg(-1) DW) and rutin (0.001 pmoles mg(-1) DW). We suggest that these concentrations of 2-undecanone and psoralen in R. graveolens leaf tissue extracts could be responsible for S. frugiperda mortality.

Filamentous fungal biofilm for production of human drug metabolites.

PubMed

Amadio, Jessica; Casey, Eoin; Murphy, Cormac D

2013-07-01

In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86% of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76%). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2×72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.

Degradation of Ciprofloxacin by Basidiomycetes and Identification of Metabolites Generated by the Brown Rot Fungus Gloeophyllum striatum

PubMed Central

Wetzstein, Heinz-Georg; Stadler, Marc; Tichy, Hans-Volker; Dalhoff, Axel; Karl, Wolfgang

1999-01-01

Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring 14CO2 production from [14C]CIP in liquid cultures. Sixteen species inhabiting wood, soil, humus, or animal dung produced up to 35% 14CO2 during 8 weeks of incubation. Despite some low rates of 14CO2 formation, all species tested had reduced the antibacterial activity of CIP in supernatants to between 0 and 33% after 13 weeks. Gloeophyllum striatum was used to identify the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10% 14CO2 from C-4 and the piperazinyl moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The structures of 11 metabolites were elucidated by high-performance liquid chromatography combined with electrospray ionization mass spectrometry and 1H nuclear magnetic resonance spectroscopy. They fell into four categories as follows: (i) monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an isatin-type compound, proving elimination of C-2, and (iv) metabolites indicating both elimination and degradation of the piperazinyl moiety. A metabolic scheme previously described for enrofloxacin degradation could be confirmed and extended. A new type of metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory evidence for the proposed network of congeners. This may result from sequential hydroxylation of CIP and its congeners by hydroxyl radicals. Our findings reveal for the first time the widespread potential for CIP degradation among basidiomycetes inhabiting various environments, including agricultural soils and animal dung. PMID:10103250

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Lu, Jianghai; He, Chunji; He, Genye; Wang, Xiaobing; Xu, Youxuan; Wu, Yun; Dong, Ying; Ouyang, Gangfeng

2014-07-01

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H](+) as precursor ion in full-scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N-dimethyl, N-desmethyl and N,N-didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono- and di-hydroxylation, methoxylation, N-desmethylation, N,N-didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

PubMed Central

Hibi, Makoto; Mori, Ryosuke; Miyake, Ryoma; Kawabata, Hiroshi; Kozono, Shoko; Takahashi, Satomi

2016-01-01

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities. PMID:26801577

Identification of phase I and II metabolites of the new designer drug α-pyrrolidinohexiophenone (α-PHP) in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

PubMed

Paul, Michael; Bleicher, Sergej; Guber, Susanne; Ippisch, Josef; Polettini, Aldo; Schultis, Wolfgang

2015-11-01

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as 'new psychoactive substances', with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α-pyrrolidinohexiophenone (α-PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß-keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n-hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß-keto-reduced alcohols. Besides the great number of metabolites detected in this sample, α-PHP is still one of the most abundant ions together with its ß-keto-reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α-PHP. Copyright © 2015 John Wiley & Sons, Ltd.

phzO, a Gene for Biosynthesis of 2-Hydroxylated Phenazine Compounds in Pseudomonas aureofaciens 30-84

PubMed Central

Delaney, Shannon M.; Mavrodi, Dmitri V.; Bonsall, Robert F.; Thomashow, Linda S.

2001-01-01

Certain strains of root-colonizing fluorescent Pseudomonas spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent Pseudomonas spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of Pseudomonas aureofaciens 30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA). P. aureofaciens 30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described. PMID:11114932

The impact of the hydroxyl radical photochemical sources on the rivastigmine drug transformation in mimic and natural waters.

PubMed

Passananti, Monica; Temussi, Fabio; Iesce, Maria Rosaria; Mailhot, Gilles; Brigante, Marcello

2013-09-15

In this paper we investigated the degradation of the rivastigmine drug induced by hydroxyl radical in synthetic and natural waters focusing on both reactivity and photoproducts identification. The hydroxyl radical formation rate was quantified by using terephthalic acid as trapping molecule and it was related with the rivastigmine degradation rate. The second order rate constant between hydroxyl radical and rivastigmine was estimated to be ≈ 5.8 × 10(9) M(-1) s(-1). Irradiation of rivastigmine in three natural waters (rain, lake and river) and comparison with degradation rates observed in synthetic solutions using nitrite, nitrate and hydrogen peroxide suggest that, in addition to hydroxyl radical, also nitroderived radicals (NO/NO2) are responsible for the pollutant degradation in natural media. In fact, the evaluated degradation rates in three natural waters are greatly higher than those estimated considering only the reactivity with photogenerated hydroxyl radical. Using nitrites and nitrates as photochemical OH source, the rivastigmine degradation cannot be described considering only the hydroxyl radical reactivity suggesting that NO and NO2 radicals could play a key role during indirect degradation. Moreover main degradation products have been identified by means of HPLC-MS. Hydroxylation of the aromatic ring as well as carbamate and amino chain oxidation were suggested as main reaction mechanisms, but also nitroderived compounds were characterized. Finally polychromatic irradiations of three rivastigmine doped natural waters (rain, river and lake) underlined the role of the indirect degradation that needs to be considered when direct degradation of selected pollutants is negligible under environmental-like conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of a tryptanthrin metabolite in rat liver microsomes by liquid chromatography/electrospray ionization-tandem mass spectrometry.

PubMed

Lee, Sang Kyu; Kim, Ghee Hwan; Kim, Dong Hyeon; Kim, Dong Hyun; Jahng, Yurngdong; Jeong, Tae Cheon

2007-10-01

Tryptanthrin originally isolated from Isatis tinctoria L. has been characterized to have anti-inflammatory activities through the dual inhibition of cyclooxygenase-2 and 5-lipoxygenase mediated prostaglandin and leukotriene syntheses. To characterize phase I metabolite(s), tryptanthrin was incubated with rat liver microsomes in the presence of NADPH-generating system. One metabolite was identified by liquid chromatography/electrospray ionization-tandem mass spectrometry. M1 could be identified as a metabolite mono-hydroxylated on the aromatic ring of indole moiety from the MS(2) spectra of protonated tryptanthrin and M1. The structure of metabolite was confirmed as 8-hydroxytryptanthrin with a chemically synthesized authentic standard. The formation of M1 was NADPH-dependent and was inhibited by SKF-525A, a general CYP-inhibitor, indicating the cytochrome P450 (CYP)-mediated reaction. In addition, it was proposed that M1 might be formed by CYP 1A in rat liver microsomes from the experiments with enriched rat liver microsomes.

Analysis of multiple vitamin D metabolites by ultra-performance supercritical fluid chromatography-tandem mass spectrometry (UPSFC-MS/MS).

PubMed

Jenkinson, Carl; Taylor, Angela; Storbeck, Karl-Heinz; Hewison, Martin

2018-06-15

In recent years, increased interest in the human health benefits of vitamin D has led to demand for improved analysis of patient vitamin D 'status'. Studies to date have focused primarily on a single vitamin D metabolite, 25-hydroxyvitamin D, despite the existence of a broad range of vitamin D metabolites, referred to as the vitamin D metabolome. This study reports on the development of a rapid UPSFC-MS/MS method for the analysis of nine vitamin D metabolites in human serum. Optimum separation was obtained with a Lux-Cellulose chiral column. We observed an orthogonal elution order when compared with ultra-high performance liquid chromatography (UHPLC). The order of elution was reversed based on hydroxyl- group number, however elution order did not differ between isomeric changes in hydroxyl- group position or epimers. Although UPSFC yielded superior resolution and selectivity over previously developed UHPLC-MS/MS methods, improvements in sensitivity could not be achieved owing to the lower injection volume required for UPSFC relative to UHPLC. Method validation was performed on the developed UPSFC-MS/MS method and found to be within acceptable limits. Applying the method to the analysis of human serum samples showed a significant correlation with serum concentrations of metabolites measured by UHPLC-MS/MS (25OHD3 r = 0.997, P=<0.001, and 3-epi-25OHD3 r = 0.996, P ≤0.001). These data indicate that UPSFC provides an efficient analytical platform for rapid analysis of multiple vitamin D metabolites from serum. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Cobalt(II) ion as a promoter of hydroxyl radical and possible 'crypto-hydroxyl' radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers.

PubMed

Moorhouse, C P; Halliwell, B; Grootveld, M; Gutteridge, J M

1985-12-13

Co(II) ions react with hydrogen peroxide under physiological conditions to form a 'reactive species' that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (.OH), their effectiveness being related to their second-order rate constants for reaction with .OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed 'in free solution' and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, .OH radical is formed in a 'site-specific' manner and is difficult to intercept by .OH scavengers. The relationship of these results to the proposed 'crypto .OH' radical is discussed.

Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1.

PubMed

Charvátová, Olga; Foley, B Lachele; Bern, Marshall W; Sharp, Joshua S; Orlando, Ron; Woods, Robert J

2008-11-01

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS.

PubMed

Masters, Andrea R; McCoy, Michael; Jones, David R; Desta, Zeruesenay

2016-03-15

Bupropion metabolites formed via oxidation and reduction exhibit pharmacological activity, but little is known regarding their stereoselective disposition. A novel stereoselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify enantiomers of bupropion, 4-hydroxybupropion, and erythro- and threo-dihydrobupropion. Liquid-liquid extraction was implemented to extract all analytes from 50 μL human plasma. Acetaminophen (APAP) was used as an internal standard. The analytes were separated on a Lux 3 μ Cellulose-3 250×4.6 mm column by methanol: acetonitrile: ammonium bicarbonate: ammonium hydroxide gradient elution and monitored using an ABSciex 5500 QTRAP triple-quadrupole mass spectrometer equipped with electrospray ionization probe in positive mode. Extraction efficiency for all analytes was ≥70%. The stability at a single non-extracted concentration for over 48 h at ambient temperature resulted in less than 9.8% variability for all analytes. The limit of quantification (LOQ) for enantiomers of bupropion and 4-hydroxybupropion was 0.3 ng/mL, while the LOQ for enantiomers of erythro- and threo-hydrobupropion was 0.15 ng/mL. The intra-day precision and accuracy estimates for enantiomers of bupropion and its metabolites ranged from 3.4% to 15.4% and from 80.6% to 97.8%, respectively, while the inter-day precision and accuracy ranged from 6.1% to 19.9% and from 88.5% to 99.9%, respectively. The current method was successfully implemented to determine the stereoselective pharmacokinetics of bupropion and its metabolites in 3 healthy volunteers administered a single 100mg oral dose of racemic bupropion. This novel, accurate, and precise HPLC-MS/MS method should enhance further research into bupropion stereoselective metabolism and drug interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolism of mequindox and its metabolites identification in chickens using LC-LTQ-Orbitrap mass spectrometry.

PubMed

Shan, Qi; Liu, Yiming; He, Limin; Ding, Huanzhong; Huang, Xianhui; Yang, Fan; Li, Yafei; Zeng, Zhenling

2012-01-15

Mequindox (MEQ), 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial and feed additive. Its toxicities have been reported to be closely related to its metabolism. To understand more clearly the metabolic pathways of MEQ, its metabolism in chickens was studied using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry. The structures of the MEQ metabolites and their product ions were easily and reliably characterized based on the accurate MS-squared spectra and known structure of MEQ. Twenty-four metabolites were detected in chicken plasma, bile, faeces, and tissues, of which 12 were detected in vivo for the first time. The major metabolic pathways reported previously for in vitro metabolism of MEQ in chicken microsomes were confirmed in this study, including N→O group reduction, carbonyl reduction, and methyl mono-hydroxylation. In addition, deacetylation and acetyl-hydroxylation of MEQ were shown to be important metabolic pathways. Collectively, these data contribute to our understanding of the in vivo metabolism of MEQ. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

DNA Binding Hydroxyl Radical Probes.

PubMed

Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

2012-01-01

The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite.

PubMed

Reidy, G F; Murray, M

1990-01-30

Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.

Steroid and sterol 7-hydroxylation: ancient pathways.

PubMed

Lathe, Richard

2002-11-01

B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.

Identification and quantification of metabolites common to 17alpha-methyltestosterone and mestanolone in horse urine.

PubMed

Yamada, Masayuki; Aramaki, Sugako; Okayasu, Toshimasa; Hosoe, Tomoo; Kurosawa, Masahiko; Kijima-Suda, Isao; Saito, Koichi; Nakazawa, Hiroyuki

2007-09-21

Anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, which were developed as oral formulations for therapeutic purposes, have been abused in the field of human sports. These anabolic steroids are also used to enhance racing performance in racehorses. In humans, structurally related 17alpha-methyltestosterone (MTS) and mestanolone (MSL), which are anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, have metabolites in common. The purpose of this study was to determine metabolites common to these two steroids in horses, which may serve as readily available screening targets for the doping test of these steroids in racehorses. Urine sample collected after administering MTS and MSL to horses was treated to obtain unconjugated steroid, glucuronide, and sulfate fractions. The fractions were subjected to gas chromatography/mass spectrometry (GC/MS), and 17alpha-methyl-5alpha-androstan-3beta,17beta-diol, 17alpha-hydroxymethyl-5alpha-androstan-3beta,17beta-diol, 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol, and 17alpha-methyl-5alpha-androstan-3beta,16alpha,17beta-triol were detected as the common metabolites by comparison with synthesized reference standards. The urinary concentrations of these metabolites after dosing were determined by GC/MS. 17Alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol was mainly detected in the sulfate fractions of urine samples after administration. This compound was consistently detected for the longest time in the urine samples after dosing with both steroids. The results suggest that 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol is a very useful screening target for the doping test of MTS and MSL in racehorses.

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

PubMed

Kim, Yoon Hee; Won, Yeong-Seon; Yang, Xue; Kumazoe, Motofumi; Yamashita, Shuya; Hara, Aya; Takagaki, Akiko; Goto, Keiichi; Nanjo, Fumio; Tachibana, Hirofumi

2016-05-11

Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

Catalytic Hydroxylation of Polyethylenes

PubMed Central

2017-01-01

Polyolefins account for 60% of global plastic consumption, but many potential applications of polyolefins require that their properties, such as compatibility with polar polymers, adhesion, gas permeability, and surface wetting, be improved. A strategy to overcome these deficiencies would involve the introduction of polar functionalities onto the polymer chain. Here, we describe the Ni-catalyzed hydroxylation of polyethylenes (LDPE, HDPE, and LLDPE) in the presence of mCPBA as an oxidant. Studies with cycloalkanes and pure, long-chain alkanes were conducted to assess precisely the selectivity of the reaction and the degree to which potential C–C bond cleavage of a radical intermediate occurs. Among the nickel catalysts we tested, [Ni(Me4Phen)3](BPh4)2 (Me4Phen = 3,4,7,8,-tetramethyl-1,10-phenanthroline) reacted with the highest turnover number (TON) for hydroxylation of cyclohexane and the highest selectivity for the formation of cyclohexanol over cyclohexanone (TON, 5560; cyclohexanol/(cyclohexanone + ε-caprolactone) ratio, 10.5). The oxidation of n-octadecane occurred at the secondary C–H bonds with 15.5:1 selectivity for formation of an alcohol over a ketone and 660 TON. Consistent with these data, the hydroxylation of various polyethylene materials by the combination of [Ni(Me4Phen)3](BPh4)2 and mCPBA led to the introduction of 2.0 to 5.5 functional groups (alcohol, ketone, alkyl chloride) per 100 monomer units with up to 88% selectivity for formation of alcohols over ketones or chloride. In contrast to more classical radical functionalizations of polyethylene, this catalytic process occurred without significant modification of the molecular weight of the polymer that would result from chain cleavage or cross-linking. Thus, the resulting materials are new compositions in which hydroxyl groups are located along the main chain of commercial, high molecular weight LDPE, HDPE, and LLDPE materials. These hydroxylated polyethylenes have improved wetting

Hydroxyl radical mediated DNA base modification by manmade mineral fibres.

PubMed Central

Leanderson, P; Söderkvist, P; Tagesson, C

1989-01-01

Manmade mineral fibres (MMMFs) were examined for their ability to hydroxylate 2-deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OH-dG), a reaction that is mediated by hydroxyl radicals. It appeared that (1) catalase and the hydroxyl radical scavengers, dimethylsulphoxide and sodium benzoate, inhibited the hydroxylation, whereas Fe2+ and H2O2 potentiated it; (2) pretreatment of MMMFs with the iron chelator, deferoxamine, or with extensive heat (200-400 degrees C), attenuated the hydroxylation; (3) the hydroxylation obtained by various MMMFs varied considerably; (4) there was no apparent correlation between the hydroxylation and the surface area of different MMMFs, although increasing the surface area of a fibre by crushing it increased its hydroxylating capacity; and (5) there was good correlation between the hydroxylation of dG residues in DNA and the hydroxylation of pure dG in solution for the 16 different MMMFs investigated. These findings indicate that MMMFs cause a hydroxyl radical mediated DNA base modification in vitro and that there is considerable variation in the reactivity of different fibre species. The DNA modifying ability seems to depend on physical or chemical characteristics, or both, of the fibre. PMID:2765416

21 CFR 172.814 - Hydroxylated lecithin.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.814 Hydroxylated lecithin. The food additive hydroxylated lecithin may be safely used as an emulsifier in foods in accordance with the following conditions: (a) The additive is...

One drop chemical derivatization--DESI-MS analysis for metabolite structure identification.

PubMed

Lubin, Arnaud; Cabooter, Deirdre; Augustijns, Patrick; Cuyckens, Filip

2015-07-01

Structural elucidation of metabolites is an important part during the discovery and development process of new pharmaceutical drugs. Liquid Chromatography (LC) in combination with Mass Spectrometry (MS) is usually the technique of choice for structural identification but cannot always provide precise structural identification of the studied metabolite (e.g. site of hydroxylation and site of glucuronidation). In order to identify those metabolites, different approaches are used combined with MS data including nuclear magnetic resonance, hydrogen/deuterium exchange and chemical derivatization followed by LC-MS. Those techniques are often time-consuming and/or require extra sample pre-treatment. In this paper, a fast and easy to set up tool using desorption electrospray ionization-MS for metabolite identification is presented. In the developed method, analytes in solution are simply dried on a glass plate with printed Teflon spots and then a single drop of derivatization mixture is added. Once the spot is dried, the derivatized compound is analyzed. Six classic chemical derivatizations were adjusted to work as a one drop reaction and applied on a list of compounds with relevant functional groups. Subsequently, two successive reactions on a single spot of amoxicillin were tested and the methodology described was successfully applied on an in vitro incubated alprazolam metabolite. All reactions and analyses were performed within an hour and gave useful structural information by derivatizing functional groups, making the method a time-saving and efficient tool for metabolite identification if used in addition or in some cases as an alternative to common methods. Copyright © 2015 John Wiley & Sons, Ltd.

Responses to water stress of gas exchange and metabolites in Eucalyptus and Acacia spp.

PubMed

Warren, Charles R; Aranda, Ismael; Cano, F Javier

2011-10-01

Studies of water stress commonly examine either gas exchange or leaf metabolites, and many fail to quantify the concentration of CO₂ in the chloroplasts (C(c)). We redress these limitations by quantifying C(c) from discrimination against ¹³CO₂ and using gas chromatography-mass spectrometry (GC-MS) for leaf metabolite profiling. Five Eucalyptus and two Acacia species from semi-arid to mesic habitats were subjected to a 2 month water stress treatment (Ψ(pre-dawn) = -1.7 to -2.3 MPa). Carbohydrates dominated the leaf metabolite profiles of species from dry areas, whereas organic acids dominated the metabolite profiles of species from wet areas. Water stress caused large decreases in photosynthesis and C(c), increases in 17-33 metabolites and decreases in 0-9 metabolites. In most species, fructose, glucose and sucrose made major contributions to osmotic adjustment. In Acacia, significant osmotic adjustment was also caused by increases in pinitol, pipecolic acid and trans-4-hydroxypipecolic acid. There were also increases in low-abundance metabolites (e.g. proline and erythritol), and metabolites that are indicative of stress-induced changes in metabolism [e.g. γ-aminobutyric acid (GABA) shunt, photorespiration, phenylpropanoid pathway]. The response of gas exchange to water stress and rewatering is rather consistent among species originating from mesic to semi-arid habitats, and the general response of metabolites to water stress is rather similar, although the specific metabolites involved may vary. © 2011 Blackwell Publishing Ltd.

[Analysis of metabolites of quercitrin in rat intestinal flora by using UPLC-ESI-Q-TOF-MS/MS].

PubMed

Qin, Xiao-Li; Sun, Hui-Yuan; Yang, Wu; Li, Yong-Jun; Zheng, Lin; Liu, Ting; Huang, Yong

2017-01-01

To investigate the metabolism of quercitrin in rat intestinal flora and possible biological pathways, laying the foundation for the metabolic mechanism of traditional Chinese medicine glycosides ingredients. UPLC-Q-TOF-MS/MS method was established to detect the quercitrin and its metabolites with 0.1% formic acid solution(A)-0.1% formic acid acetonitrile(B) as the mobile phase for gradient elution at a flow rate of 0.3 mL•min⁻¹. Electrospray negative ion mode was applied to analyze the metabolites of quercitrin in rat intestinal flora. Metabolite ToolsTM, mass defect filter(MDF) and other technologies were used to screen, analyze the metabolites and infer the chemical formula of the metabolites. The results showed that quercitrin would have degalactoside, deoxygenation and acetylation reactions, and the aglycone quercetin resulted from degalactoside would have further reactions such as hydroxylation, deoxygenation, reduction, and ring opening to achieve deoxygenation metabolite kaempferol, C2-C3 double bonds hydrogenation and reduction product taxifolin, and degalactoside product quercetin. The research results showed that quercitrin can be metabolized by rat intestinal flora, which could increase their hydrophobicity and chemical diversity. Copyright© by the Chinese Pharmaceutical Association.

Solar Insolation Effect on the Local Distribution of Lunar Hydroxyl

NASA Astrophysics Data System (ADS)

Kim, Suyeon; Yi, Yu; Hong, Ik-Seon; Sohn, Jongdae

2018-03-01

Moon mineralogy mapper (M3)'s work proved that the moon is not completely dry but has some hydroxyl/water. M3's data confirmed that the amount of hydroxyl on the lunar surface is inversely related to the measured signal brightness, suggesting the lunar surface is sensitive to temperature by solar insolation. We tested the effect of solar insolation on the local distribution of hydroxyl by using M3 data, and we found that most craters had more hydroxyl in shade areas than in sunlit areas. This means that the local distribution of hydroxyl is absolutely influenced by the amount of sunshine. We investigated the factors affecting differences in hydroxyl; we found that the higher the latitude, the larger the difference during daytime. We also measured the pyroxene content and found that pyroxene affects the amount of hydroxyl, but it does not affect the difference in hydroxyl between sunlit and shaded areas. Therefore, we confirmed that solar insolation plays a significant role in the local distribution of hydroxyl, regardless of surface composition.

Characterization of Differential Cocaine Metabolism in Mouse and Rat through Metabolomics-Guided Metabolite Profiling

PubMed Central

Yao, Dan; Shi, Xiaolei; Wang, Lei; Gosnell, Blake A.

2013-01-01

Rodent animal models have been widely used for studying neurologic and toxicological events associated with cocaine abuse. It is known that the mouse is more susceptible to cocaine-induced hepatotoxicity (CIH) than the rat. However, the causes behind this species-dependent sensitivity to cocaine have not been elucidated. In this study, cocaine metabolism in the mouse and rat was characterized through LC-MS-based metabolomic analysis of urine samples and were further compared through calculating the relative abundance of individual cocaine metabolites. The results showed that the levels of benzoylecgonine, a major cocaine metabolite from ester hydrolysis, were comparable in the urine from the mice and rats treated with the same dose of cocaine. However, the levels of the cocaine metabolites from oxidative metabolism, such as N-hydroxybenzoylnorecgonine and hydroxybenzoylecgonine, differed dramatically between the two species, indicating species-dependent cocaine metabolism. Subsequent structural analysis through accurate mass analysis and LC-MS/MS fragmentation revealed that N-oxidation reactions, including N-demethylation and N-hydroxylation, are preferred metabolic routes in the mouse, while extensive aryl hydroxylation reactions occur in the rat. Through stable isotope tracing and in vitro enzyme reactions, a mouse-specific α-glucoside of N-hydroxybenzoylnorecgonine and a group of aryl hydroxy glucuronides high in the rat were identified and structurally elucidated. The differences in the in vivo oxidative metabolism of cocaine between the two rodent species were confirmed by the in vitro microsomal incubations. Chemical inhibition of P450 enzymes further revealed that different P450-mediated oxidative reactions in the ecgonine and benzoic acid moieties of cocaine contribute to the species-dependent biotransformation of cocaine. PMID:23034697

Some reactions of the hydroxyl adduct of adenine

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

Vanhemmen, J.J.

1975-01-01

The chemical reactions of purine derivatives resulting from pulse radiolysis were studied. Some reactions of the hydroxyl adduct of adenine are described and one of these reactions was compared with similar reactions of hydroxyl adducts of other purine derivatives. Evidence is given that in various purines opening of the imidazole ring is due to unimolecular rearrangements of the hydroxyl adducts. (GRA)

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection

PubMed Central

2010-01-01

novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC. PMID:20156336

Pentachlorophenol and hydroxylated polychlorinated biphenyl metabolites in umbilical cord plasma of neonates from coastal populations in Québec.

PubMed Central

Sandau, Courtney D; Ayotte, Pierre; Dewailly, Eric; Duffe, Jason; Norstrom, Ross J

2002-01-01

Concentrations of polychlorinated biphenyls (PCBs), hydroxylated metabolites of PCBs (HO-PCBs) and octachlorostyrene (4-HO-HpCS), and pentachlorophenol (PCP) were determined in umbilical cord plasma samples from three different regions of Québec. The regions studied included two coastal areas where exposure to PCBs is high because of marine-food-based diets--Nunavik (Inuit people) and the Lower North Shore of the Gulf of St. Lawrence (subsistence fishermen)--and a southern Québec urban center where PCB exposure is at background levels (Québec City). The main chlorinated phenolic compound in all regions was PCP. Concentrations of PCP were not significantly different among regions (geometric mean concentration 1,670 pg/g, range 628-7,680 pg/g wet weight in plasma). The ratio of PCP to polychlorinated biphenyl congener number 153 (CB153) concentration ranged from 0.72 to 42.3. Sum HO-PCB (sigma HO-PCBs) concentrations were different among regions, with geometric mean concentrations of 553 (range 238-1,750), 286 (103-788), and 234 (147-464) pg/g wet weight plasma for the Lower North Shore, Nunavik, and the southern Québec groups, respectively. Lower North Shore samples also had the highest geometric mean concentration of sum PCBs (sum of 49 congeners; sigma PCBs), 2,710 (525-7,720) pg/g wet weight plasma. sigma PCB concentrations for Nunavik samples and southern samples were 1,510 (309-6,230) and 843 (290-1,650) pg/g wet weight plasma. Concentrations (log transformed) of sigma HO-PCBs and sigma PCBs were significantly correlated (r = 0.62, p < 0.001), as were concentrations of all major individual HO-PCB congeners and individual PCB congeners. In Nunavik and Lower North Shore samples, free thyroxine (T4) concentrations (log transformed) were negatively correlated with the sum of quantitated chlorinated phenolic compounds (sum PCP and sigma HO-PCBs; r = -0.47, p = 0.01, n = 20) and were not correlated with any PCB congeners or sigma PCBs. This suggests that PCP and

Analysis of hydroxylated metabolites of PCBs (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian inuit.

PubMed Central

Sandau, C D; Ayotte, P; Dewailly, E; Duffe, J; Norstrom, R J

2000-01-01

In this study, we identified the main hydroxylated polychlorinated biphenyls (OH-PCBs) and other chlorinated phenolic compounds and we determined their relative concentrations in whole blood from 13 male and 17 female Inuit from northern Quebec, Canada, and from a pooled whole blood sample from southern Quebec. We also determined concentrations of polychlorinated biphenyls (PCBs). Total OH-PCB concentrations were variable among the Inuit samples, ranging over 2 orders of magnitude (0.117-11.6 ng/g whole blood wet weight). These concentrations were equal to and up to 70 times those found for the southern Quebec pooled whole blood sample. Geometric mean concentrations of total OH-PCBs were 1.73 and 1.01 ng/g whole blood for Inuit men and women, respectively, and 0.161 ng/g whole blood for the southern population pool. There are limited data available for comparison, but the levels of OH-PCBs in Inuit are higher than those previously reported in the literature for other populations. There was a significant correlation (p < 0.005) between OH-PCBs and PCBs (r = 0.84) and both correlated significantly (p < 0.005) with age (r = 0.68 and 0.78, respectively). The ratio of OH-PCBs to PCBs was lower in Inuit (0.11) than in the southern Quebec pool (0.33). There is no apparent explanation for the difference. There was considerable variability in the congener pattern of the identified OH-PCBs. The main metabolite, 4-OH-CB109 (4-OH-2,3,3',4', 5-pentachlorobiphenyl), constituted 12-62% of the total OH-PCBs in the samples. Pentachlorophenol (PCP) was the dominant phenolic compound in blood, constituting 46% (geometric mean) of the total quantitated chlorinated phenolic compounds. PCP concentrations in Inuit blood ranged from 0.558 to 7.77 ng/g on a wet weight basis. All but two Inuit samples had lower concentrations than the southern Quebec pool (6.29 ng/g). The possible role of OH-PCBs in mediating PCB-induced adverse effects needs to be investigated further. Images Figure 1

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

PubMed

Bourgogne, Emmanuel; Culot, Benoit; Dell'Aiera, Sylvie; Chanteux, Hugues; Stockis, Armel; Nicolas, Jean-Marie

2018-06-01

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction. Copyright © 2018 Elsevier B.V. All rights reserved.

Future Directions of Structural Mass Spectrometry using Hydroxyl Radical Footprinting

PubMed Central

Kiselar, Janna G.; Chance, Mark R.

2010-01-01

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposures of proteins to a “white” x-ray beam for milliseconds provide sufficient oxidative modifications to surface amino acid side chains that can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular sub-domains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal to noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis driven

Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry.

PubMed

Holm, Niels Bjerre; Pedersen, Anders Just; Dalsgaard, Petur Weihe; Linnet, Kristian

2015-03-01

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-​(1-Adamant​yl)-​1-​(5-​fluoropentyl)-​1H-​indazole-​3-​carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW-type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high-resolution accurate-mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase-I metabolites of 5F-AKB-48. The modifications included mono-, di-, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N-fluoropentylindazole moiety, dealkylation of the N-fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time-dependent formation of mono-, di-, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F-AKB-48 for use in clinical and forensic screening. Copyright © 2014 John Wiley & Sons, Ltd.

Implications for Metabolite Quantification by Mass Spectrometry in the Absence of Authentic Standards.

PubMed

Hatsis, Panos; Waters, Nigel J; Argikar, Upendra A

2017-05-01

Quantification of metabolites by mass spectrometry in the absence of authentic reference standards or without a radiolabel is often called "semiquantitative," which acknowledges that mass spectrometric responses are not truly quantitative. For many researchers, it is tempting to pursue this practice of semiquantification in early drug discovery and even preclinical development, when radiolabeled absorption, distribution, metabolism, and excretion studies are being deferred to later stages of drug development. The caveats of quantifying metabolites based on parent drug response are explored in this investigation. A set of 71 clinically relevant drugs/metabolites encompassing common biotransformation pathways was subjected to flow injection analysis coupled with electrospray ionization (ESI) mass spectrometry. The results revealed a large variation in ESI response even for structurally similar parent drug/metabolite pairs. The ESI response of each metabolite was normalized to that of the parent drug to generate an ESI relative response factor. Overall, relative response factors ranged from 0.014 (>70-fold lower response than parent) to 8.6 (8.6-fold higher response than parent). Various two-dimensional molecular descriptors were calculated that describe physicochemical, topological, and structural properties for each drug/metabolite. The molecular descriptors, along with the ESI response factors, were used in univariate analyses as well as a principal components analysis to ascertain which molecular descriptors best account for the observed discrepancies in drug/metabolite ESI response. This investigation has shown that the practice of using parent drug response to quantify metabolites should be used with caution. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Comparative metabolites profiles of osthole in normal and osteoporosis rats using liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Wang, Nani; Wang, Xuping; Zhang, Yang; Zhang, Qiaoyan; Xu, Pingcui; Xin, Hailiang; Wu, Renjie; Shou, Dan; Qin, Luping

2018-05-30

Osthole is a derivative of coumnarin, which has been used to treat several diseases, including osteoporosis. To investigate the metabolite profile of osthole in osteoporosis rats was utilized to understand its underlying mechanisms of its anti-osteoporosis effect. In this study, plasma samples were collected from normal and osteoporosis rats after oral administration of osthole and analyzed to identify the metabolites of osthole by high performance liquid chromatography quadrupole time-of-flight mass spectrometry. By comparing the molecular weight and MS fragmentation of the metabolites with those of parent drug and reference standards, a total of 36 metabolites in plasma were identified. Demethylation, hydroxylation, hydroxymethylene loss and reduction, and subsequent glucuronidation, methylation and sulfation were the major metabolic pathways of osthole in both normal and osteoporosis rats. A specific hydration metabolic pathway was found in osteoporosis rats. These results provided a meaningful basis for studying the underlying mechanism of the anti-osteoporosis effect of osthole. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of phase-I metabolites and chronic toxicity study of the Kv1.3 blocker PAP-1 (5-(4-phenoxybutoxy)psoralen) in the rat.

PubMed

Hao, B; Chen, Z-W; Zhou, X-J; Zimin, P I; Miljanich, G P; Wulff, H; Wang, Y-X

2011-03-01

1. PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats. 2. Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds. 3. Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes. 4. Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism. 5. We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.

Oxidative metabolism of BDE-47, BDE-99, and HBCDs by cat liver microsomes: Implications of cats as sentinel species to monitor human exposure to environmental pollutants.

PubMed

Zheng, Xiaobo; Erratico, Claudio; Luo, Xiaojun; Mai, Bixian; Covaci, Adrian

2016-05-01

The in vitro oxidative metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), and individual α-, β- and γ-hexabromocyclododecane (HBCD) isomers catalyzed by cytochrome P450 (CYP) enzymes was screened using cat liver microsomes (CLMs). Six hydroxylated metabolites, namely 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47), 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), 4'-hydroxy-2,2',4,5'- tetrabromodiphenyl ether (4'-OH-BDE-49), and 2'-hydroxy-2,3',4,4'-tetrabromodiphenyl ether (2'-OH-BDE-66), were identified and quantified after incubation of BDE-47. A di-OH-tetra-BDE was also found as metabolite of BDE-47 with CLMs. 5-OH-BDE-47 was the major metabolite formed. Five hydroxylated metabolites (3'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (3'-OH-BDE-99), 5'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99), 6-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (6-OH-BDE-99), 6'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99), and 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether (4'-OH-BDE-101) were formed from BDE-99 incubated with CLMs. Concentrations of BDE-99 metabolites were lower than those of BDE-47. Four or more mono-hydroxylated HBCD (OH-HBCDs), four or more di-hydroxylated HBCD (di-OH-HBCDs), five or more mono-hydroxylated pentabromocyclododecanes (OH-PBCDs), and five or more di-hydroxylated pentabromocyclododecenes (di-OH-PBCDs) were detected after incubation of α-, β-, or γ-HBCD with CLMs. No diastereoisomeric or enantiomeric enzymatic isomerisation was observed incubating α-, β- or γ-HBCD with CLMs. Collectively, our data suggest that (i) BDE-47 is metabolized at a faster rate than BDE-99 by CLMs, (ii) OH-HBCDs are the major hydroxylated metabolites of α-, β- and γ-HBCD produced by CLMs, and (iii) the oxidative metabolism of BDE-47 and

Quantitative and qualitative analysis of the novel antitumor 1,3,4-oxadiazole derivative (GLB) and its metabolites using HPLC-UV and UPLC-QTOF-MS

PubMed Central

Li, Pu; Wang, Xin; Li, Jian; Meng, Zhi-Yun; Li, Shu-Chun; Li, Zhong-Jun; Lu, Ying-Yuan; Ren, Hong; Lou, Ya-Qing; Lu, Chuang; Dou, Gui-Fang; Zhang, Guo-Liang

2015-01-01

Fructose-based 3-acetyl-2,3-dihydro-1,3,4-oxadiazole (GLB) is a novel antitumor agent and belongs to glycosylated spiro-heterocyclic oxadiazole scaffold derivative. This research first reported a simple, specific, sensitive and stable high performance liquid chromatography -ultraviolet detector (HPLC-UV) method for the quantitative determination of GLB in plasma. In this method, the chromatographic separation was achieved with a reversed phase C18 column. The calibration curve for GLB was linear at 300 nm. The lower limit of quantification was 10 ng/mL. The precision, accuracy and stability of the method were validated adequately. This method was successfully applied to the pharmacokinetic study in rats for detection of GLB after oral administration. Moreover, the structures of parent compound GLB and its two major metabolites M1 and M2 were identified in plasma using an ultra performance liquid chromatography- electrospray ionization-quadrupole-time of flight- mass spectrometry (UPLC-ESI-QTOF-MS) method. Our results indicated that the di-hydroxylation (M1) and hydroxylation (M2) of GLB are the major metabolites. In conclusion, the present study provided valuable information on an analytical method for the determination of GLB and its metabolites in rats, can be used to support further developing of this antitumor agent. PMID:26148672

Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

PubMed Central

Gorzolka, Karin; Kölling, Jan; Nattkemper, Tim W.; Niehaus, Karsten

2016-01-01

MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest. PMID:26938880

Transformation of hydroxylated and methoxylated 2,2',4,4',5-brominated diphenyl ether (BDE-99) in plants.

PubMed

Pan, Lili; Sun, Jianteng; Wu, Xiaodan; Wei, Zi; Zhu, Lizhong

2016-11-01

The occurrence and fate of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) have received significant attention. However, there is limited information on the metabolism relationship between OH-pentaBDEs and MeO-pentaBDEs that were frequently detected with relatively high concentrations in the environment. In this study, the biotransformation between OH-BDE-99 and MeO-BDE-99 was investigated in rice, wheat, and soybean plants. All the three plants can metabolize OH-BDE-99 to corresponding homologous methoxylated metabolites, while the transformation from MeO-BDE-99 to OH-BDE-99 could only be found in soybean. The conversion of parent compounds was the highest in soybean, followed by wheat and rice. Transformation products were found mainly in the roots, with few metabolites being translocated to the shoots and solution after exposure. The results of this study provide valuable information for a better understanding of the accumulation and transformation of OH-PBDEs and MeO-PBDEs in different plants. Copyright © 2016. Published by Elsevier B.V.

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

PubMed

Lu, Linling; Qian, Dawei; Yang, Jing; Jiang, Shu; Guo, Jianming; Shang, Er-xin; Duan, Jin-ao

2013-04-01

In this paper, ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and the MetaboLynx™ software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC-Q-TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.

Verification of propofol sulfate as a further human propofol metabolite using LC-ESI-QQQ-MS and LC-ESI-QTOF-MS analysis.

PubMed

Maas, Alexandra; Maier, Christoph; Michel-Lauter, Beate; Broecker, Sebastian; Madea, Burkhard; Hess, Cornelius

2017-03-01

Propofol (2,6-diisopropylphenol) is a water-insoluble, intravenous anesthetic that is widely used for the induction and maintenance of anesthesia as well as for endoscopic and pediatric sedation. After admission, propofol undergoes extensive hepatic and extrahepatic metabolism, including direct conjugation to propofol glucuronide and hydroxylation to 2,6-diisopropyl-1,4-quinol. The latter substance subsequently undergoes phase II metabolism, resulting in the formation of further metabolites (1quinolglucuronide, 4quinolglucuronide and 4quinol-sulfate). Further minor phase I propofol metabolites (2-(ω-propanol)-6-isopropylphenol and 2-(ω-propanol)-6-isopropyl-1,4-quinol)) are also described. Due to its chemical structure with the phenolic hydroxyl group, propofol is also an appropriate substrate for sulfation by sulfotransferases. The existence of propofol sulfate was investigated by liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LCESIQQQ-MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LCESI-QTOF-MS). A propofol sulfate reference standard was used for identification and method development, yielding a precursor at m/z 257 (deprotonated propofol sulfate) and product ions at m/z 177 (deprotonated propofol) and m/z 80 ([SO3]-). Propofol sulfate - a further phase II metabolite of propofol - was verified in urine samples by LC-ESI-QQQ-MS and LC-ESI-QTOF-MS. Analyses of urine samples from five volunteers collected before and after propofol-induced sedation verified the presence of propofol sulfate in urine following propofol administration, whereas ascertained concentrations of this metabolite were significantly lower compared with detected propofol glucuronide concentrations. The existence of propofol sulfate as a further phase II propofol metabolite in humans could be verified by two different detection techniques (LCESIQQQ-MS and LC-ESI-QTOFMS) on the basis of a propofol sulfate

Tandem mass spectrometry approach for the investigation of the steroidal metabolism: structure-fragmentation relationship (SFR) in anabolic steroids and their metabolites by ESI-MS/MS analysis.

PubMed

Musharraf, Syed Ghulam; Ali, Arslan; Khan, Naik Tameem; Yousuf, Maria; Choudhary, Muhammad Iqbal; Atta-ur-Rahman

2013-02-01

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the effect of different substitutions introduced during metabolism on fragmentation patterns of four anabolic steroids including methyltestosterone, methandrostenolone, cis-androsterone and adrenosterone, along with their metabolites. Collision-induced dissociation (CID) analysis was performed to correlate the major product ions of 19 steroids with structural features. The analysis is done to portray metabolic alteration, such as incorporation or reduction of double bonds, hydroxylations, and/or oxidation of hydroxyl moieties to keto functional group on steroidal skeleton which leads to drastically changed product ion spectra from the respective classes of steroids, therefore, making them difficult to identify. The comparative ESI-MS/MS study also revealed some characteristic peaks to differentiate different steroidal metabolites and can be useful for the unambiguous identification of anabolic steroids in biological fluid. Moreover, LC-ESI-MS/MS analysis of fermented extract of methyltestosterone, obtained by Macrophomina phaseolina was also investigated. Copyright © 2012 Elsevier Inc. All rights reserved.

Enantioselective supercritical fluid chromatography-tandem mass spectrometry method for simultaneous estimation of risperidone and its 9-hydroxyl metabolites in rat plasma.

PubMed

Prasad, Thatipamula R; Joseph, Siji; Kole, Prashant; Kumar, Anoop; Subramanian, Murali; Rajagopalan, Sudha; Kr, Prabhakar

2017-11-01

Objective of the current work was to develop a 'green chemistry' compliant selective and sensitive supercritical fluid chromatography-tandem mass spectrometry method for simultaneous estimation of risperidone (RIS) and its chiral metabolites in rat plasma. Methodology & results: Agilent 1260 Infinity analytical supercritical fluid chromatography system resolved RIS and its chiral metabolites within runtime of 6 min using a gradient chromatography method. Using a simple protein precipitation sample preparation followed by mass spectrometric detection achieved a sensitivity of 0.92 nM (lower limit of quantification). With linearity over four log units (0.91-7500 nM), the method was found to be selective, accurate, precise and robust. The method was validated and was successfully applied for simultaneous estimation of RIS and 9-hydroxyrisperidone metabolites (R & S individually) after intravenous and per oral administration to rats.

Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on cardiovascular function in conscious rats.

PubMed

Schindler, Charles W; Thorndike, Eric B; Blough, Bruce E; Tella, Srihari R; Goldberg, Steven R; Baumann, Michael H

2014-01-01

The cardiovascular effects produced by 3,4-methylenedioxymethamphetamine (MDMA; 'Ecstasy') contribute to its acute toxicity, but the potential role of its metabolites in these cardiovascular effects is not known. Here we examined the effects of MDMA metabolites on cardiovascular function in rats. Radiotelemetry was employed to evaluate the effects of s.c. administration of racemic MDMA and its phase I metabolites on BP, heart rate (HR) and locomotor activity in conscious male rats. MDMA (1-20 mg·kg(-1)) produced dose-related increases in BP, HR and activity. The peak effects on HR occurred at a lower dose than peak effects on BP or activity. The N-demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), produced effects that mimicked those of MDMA. The metabolite 3,4-dihydroxymethamphetamine (HHMA; 1-10 mg·kg(-1)) increased HR more potently and to a greater extent than MDMA, whereas 3,4-dihydroxyamphetamine (HHA) increased HR, but to a lesser extent than HHMA. Neither dihydroxy metabolite altered motor activity. The metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) did not affect any of the parameters measured. The tachycardia produced by MDMA and HHMA was blocked by the β-adrenoceptor antagonist propranolol. Our results demonstrate that HHMA may contribute significantly to the cardiovascular effects of MDMA in vivo. As such, determining the molecular mechanism of action of HHMA and the other hydroxyl metabolites of MDMA warrants further study. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on cardiovascular function in conscious rats

PubMed Central

Schindler, Charles W; Thorndike, Eric B; Blough, Bruce E; Tella, Srihari R; Goldberg, Steven R; Baumann, Michael H

2014-01-01

BACKGROUND AND PURPOSE The cardiovascular effects produced by 3,4-methylenedioxymethamphetamine (MDMA; ‘Ecstasy’) contribute to its acute toxicity, but the potential role of its metabolites in these cardiovascular effects is not known. Here we examined the effects of MDMA metabolites on cardiovascular function in rats. EXPERIMENTAL APPROACH Radiotelemetry was employed to evaluate the effects of s.c. administration of racemic MDMA and its phase I metabolites on BP, heart rate (HR) and locomotor activity in conscious male rats. KEY RESULTS MDMA (1–20 mg·kg−1) produced dose-related increases in BP, HR and activity. The peak effects on HR occurred at a lower dose than peak effects on BP or activity. The N-demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), produced effects that mimicked those of MDMA. The metabolite 3,4-dihydroxymethamphetamine (HHMA; 1–10 mg·kg−1) increased HR more potently and to a greater extent than MDMA, whereas 3,4-dihydroxyamphetamine (HHA) increased HR, but to a lesser extent than HHMA. Neither dihydroxy metabolite altered motor activity. The metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) did not affect any of the parameters measured. The tachycardia produced by MDMA and HHMA was blocked by the β-adrenoceptor antagonist propranolol. CONCLUSIONS AND IMPLICATIONS Our results demonstrate that HHMA may contribute significantly to the cardiovascular effects of MDMA in vivo. As such, determining the molecular mechanism of action of HHMA and the other hydroxyl metabolites of MDMA warrants further study. PMID:24328722

Quantifying the vitamin D economy.

PubMed

Heaney, Robert P; Armas, Laura A G

2015-01-01

Vitamin D enters the body through multiple routes and in a variety of chemical forms. Utilization varies with input, demand, and genetics. Vitamin D and its metabolites are carried in the blood on a Gc protein that has three principal alleles with differing binding affinities and ethnic prevalences. Three major metabolites are produced, which act via two routes, endocrine and autocrine/paracrine, and in two compartments, extracellular and intracellular. Metabolic consumption is influenced by physiological controls, noxious stimuli, and tissue demand. When administered as a supplement, varying dosing schedules produce major differences in serum metabolite profiles. To understand vitamin D's role in human physiology, it is necessary both to identify the foregoing entities, mechanisms, and pathways and, specifically, to quantify them. This review was performed to delineate the principal entities and transitions involved in the vitamin D economy, summarize the status of present knowledge of the applicable rates and masses, draw inferences about functions that are implicit in these quantifications, and point out implications for the determination of adequacy. © The Author(s) 2014. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

PubMed

Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

2017-03-01

Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

PAH Metabolites in Bile of European Eel (Anguilla anguilla) from Morocco.

PubMed

Wariaghli, Fatima; Kammann, Ulrike; Hanel, Reinhold; Yahyaoui, Ahmed

2015-12-01

Environmental pollution of fish with organic contaminants is a topic of rising attention in Morocco. Polycyclic aromatic hydrocarbons (PAH) are prominent organic contaminants which are rapidly metabolized in fish. Their metabolites are accumulated in the bile fluid and can be used to assess PAH exposure. The two PAH metabolites 1-hydroxypyrene and 1-hydroxyphenanthrene were quantified in European eels (Anguilla anguilla) from two Moroccan river systems by high-performance liquid chromatography with fluorescence detection. Mean values ranged from 52 to 210 ng/mL 1-hydroxypyrene and from 61 to 73 ng/mL 1-hydroxyphenanthrene. The overall concentrations of PAH metabolites in eel from Morocco appeared moderate compared to eel from European rivers and coastal sites. The present study provides first information on concentrations of PAH metabolites in fish from Morocco.

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli.

PubMed

McCloskey, Douglas; Xu, Julia; Schrübbers, Lars; Christensen, Hanne B; Herrgård, Markus J

2018-04-25

Fast metabolite quantification methods are required for high throughput screening of microbial strains obtained by combinatorial or evolutionary engineering approaches. In this study, a rapid RIP-LC-MS/MS (RapidRIP) method for high-throughput quantitative metabolomics was developed and validated that was capable of quantifying 102 metabolites from central, amino acid, energy, nucleotide, and cofactor metabolism in less than 5 minutes. The method was shown to have comparable sensitivity and resolving capability as compared to a full length RIP-LC-MS/MS method (FullRIP). The RapidRIP method was used to quantify the metabolome of seven industrial strains of E. coli revealing significant differences in glycolytic, pentose phosphate, TCA cycle, amino acid, and energy and cofactor metabolites were found. These differences translated to statistically and biologically significant differences in thermodynamics of biochemical reactions between strains that could have implications when choosing a host for bioprocessing. Copyright © 2018. Published by Elsevier Inc.

Identification of two main urinary metabolites of (/sup 14/C)omeprazole in humans

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

Renberg, L.; Simonsson, R.; Hoffmann, K.J.

1989-01-01

The excretion and metabolism of (/sup 14/C)omeprazole given orally as a suspension was studied in 10 healthy male subjects. An average of 79% of the dose was recovered in the urine in 96 hr, with most of the radioactivity (76% of dose) being eliminated in the first 24 hr. Pooled urine (0-2 hr) from five subjects, containing about 47% of the dose, was analyzed by reverse phase gradient elution LC with radioisotope detection. Omeprazole was completely metabolized to at least six metabolites. The two major metabolites were extensively purified by LC and their structures were determined by MS with derivatizationmore » and use of stable isotopes, 1H NMR, and comparison with synthetic references. They were formed by hydroxylation of a methyl group in the pyridine ring, followed by further oxidation of the alcohol to the corresponding carboxylic acid. Both metabolites retained the sulfoxide group of omeprazole, rendering them as unstable as the parent compound at pH less than 7. They accounted for approximately 28% (hydroxyomeprazole) and 23% (omeprazole acid) of the amount excreted in the 0-2-hr collection interval. Based on in vitro studies with the synthetic metabolites in isolated gastric glands, it is unlikely that M1 and M2 will contribute to the pharmacological effect of omeprazole in humans.« less

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.

Metabolism of boldione in humans by mass spectrometric techniques: detection of pseudoendogenous metabolites.

PubMed

de la Torre, Xavier; Curcio, Davide; Colamonici, Cristiana; Molaioni, Francesco; Botrè, Francesco

2013-01-01

Boldione is an anabolic androgenic steroid (AAS) related to boldenone, androstenedione, and testosterone bearing two double bonds in C1 and C4 positions. Boldione is rapidly transformed to the well-known AAS boldenone, being both compounds included in the list of prohibited substances and methods published yearly by the World Anti-Doping Agency (WADA). After the administration of boldione to a male volunteer, the already described urinary metabolites of boldenone produced after reduction in C4, oxydoreduction in C3 and C17, and hydroxylation have been detected. In addition, minor new metabolites have been detected and their structure postulated after mass spectrometric analyses. Finally, the reduction of the double bound in C1 produces metabolites identical to the endogenously produced ones. A method based on gas chromatography coupled to isotope ratio mass spectrometry (GC/C/IRMS) after a urine sample purification by high performance liquid chromatography (HPLC) permitted to confirm the main synthetic like boldione/boldenone metabolite (17β-hydroxy-5β-androst-1-en-3-one) and boldenone at trace levels (< 5 ng/mL) and then to establish its synthetic or endogenous origin, and to determine the exogenous origin of metabolites with the same chemical structure of the endogenous ones. The detection of pseudoendogenous androgens of synthetic origin partially overlapped boldenone and its main metabolite detection, being an additional proof of synthetic steroids misuse. By the use of IRMS, the correct evaluation of the modifications of the steroid profile after the administration of synthetic AAS that could be converted into endogenous like ones is possible. Copyright © 2013 John Wiley & Sons, Ltd.

Analysis of Particulate and Dissolved Metabolite Pools at Station ALOHA

NASA Astrophysics Data System (ADS)

Boysen, A.; Carlson, L.; Hmelo, L.; Ingalls, A. E.

2016-02-01

Metabolomic studies focus on identifying and quantifying the small organic molecules that are the currency by which an organism lives and dies. Metabolite profiles of microorganisms have the potential to elucidate mechanisms of chemically mediated interactions that influence the success of microbial groups living in a complex environment. However, the chemical diversity of metabolites makes resolving a wide range of compounds analytically challenging. As such, metabolomics has lagged behind other genomic analyses. Here we conduct targeted analysis of over 200 primary and secondary metabolites present in the intracellular and extracellular metabolite pools at Station ALOHA using both reverse phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. We selected the metabolites in our method due to their known importance in primary metabolism, secondary metabolism, and interactions between marine microorganisms such as nutrient exchange, growth promotion, and cell signaling. Through these analyses we obtain a snapshot of microbial community status that, blended with other forms of genomic data, can further our understanding of microbial dynamics. We hypothesize that monitoring a large suite of important metabolites across environmental gradients and diurnal cycles can elucidate factors controlling the distribution and activity of important microbial groups.

DNA Adduct Formation from Metabolic 5'-Hydroxylation of the Tobacco-Specific Carcinogen N'-Nitrosonornicotine in Human Enzyme Systems and in Rats.

PubMed

Zarth, Adam T; Upadhyaya, Pramod; Yang, Jing; Hecht, Stephen S

2016-03-21

N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans.

The radiocarbon hydroxyl technique

NASA Technical Reports Server (NTRS)

Campbell, Malcolm J.; Sheppard, John C.

1994-01-01

The Radiocarbon Technique depends upon measuring the rate of oxidation of CO in an essentially unperturbed sample of air. The airborne technique is slightly different. Hydroxyl concentrations can be calculated directly; peroxyl concentrations can be obtained by NO doping.

A Fast and Effective Pyridine-Free Method for the Determination of Hydroxyl Value of Hydroxyl-Terminated Polybutadiene and Other Hydroxy Compounds

NASA Astrophysics Data System (ADS)

Alex, Ancy Smitha; Kumar, Vijendra; Sekkar, V.; Bandyopadhyay, G. G.

2017-07-01

Hydroxyl-terminated polybutadiene (HTPB) is the workhorse propellant binder for launch vehicle and missile applications. Accurate determination of the hydroxyl value (OHV) of HTPB is crucial for tailoring the ultimate mechanical and ballistic properties of the propellant derived. This article describes a fast and effective methodology free of pyridine based on acetic anhydride, N-methyl imidazole, and toluene for the determination of OHV of nonpolar polymers like HTPB and other hydroxyl compounds. This method gives accurate and reproducible results comparable to standard methods and is superior to existing methods in terms of user friendliness, efficiency, and time requirement.

Spin-locking vs. chemical exchange saturation transfer MRI for investigating chemical exchange process between water and labile metabolite protons

PubMed Central

Jin, Tao; Autio, Joonas; Obata, Takayuki; Kim, Seong-Gi

2010-01-01

Chemical exchange saturation transfer (CEST) and spin-locking (SL) experiments were both able to probe the exchange process between protons of non-equivalent chemical environments. To compare the characteristics of the CEST and SL approaches in the study of chemical exchange effects, we performed CEST and SL experiments at varied pH and concentrated metabolites with exchangeable amide, amine, and hydroxyl protons at 9.4 T. Our results show that: i) On-resonance SL is most sensitive to chemical exchanges in the intermediate exchange regime and is able to detect hydroxyl and amine protons on a millimolar concentration scale. Off-resonance SL and CEST approaches are sensitive to slow-exchanging protons when an optimal SL or saturation pulse power matches the exchanging rate, respectively. ii) Offset frequency-dependent SL and CEST spectra are very similar, and can be explained well with an SL model recently developed by Trott and Palmer. iii) The exchange rate and population of metabolite protons can be determined from offset-dependent SL or CEST spectra or from on-resonance SL relaxation dispersion measurements. iv) The asymmetry of the magnetization transfer ratio (MTRasym) is highly dependent on the choice of saturation pulse power. In the intermediate exchange regime, MTRasym becomes complicated and should be interpreted with care. PMID:21500270

Enantioselective N-demethylation and hydroxylation of sibutramine in human liver microsomes and recombinant cytochrome p-450 isoforms.

PubMed

Shinde, Dhananjay D; Kim, Min-Jung; Jeong, Eun-Sook; Kim, Yang-Weon; Lee, Ji-Woo; Shin, Jae-Gook; Kim, Dong-Hyun

2014-01-01

The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Clint) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Clint ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Clint ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.

Concentration-dependent photodegradation kinetics and hydroxyl-radical oxidation of phenicol antibiotics.

PubMed

Li, Kai; Zhang, Peng; Ge, Linke; Ren, Honglei; Yu, Chunyan; Chen, Xiaoyang; Zhao, Yuanfeng

2014-09-01

Thiamphenicol and florfenicol are two phenicol antibiotics widely used in aquaculture and are ubiquitous as micropollutants in surface waters. The present study investigated their photodegradation kinetics, hydroxyl-radical (OH) oxidation reactivities and products. Firstly, the photolytic kinetics of the phenicols in pure water was studied as a function of initial concentrations (C0) under UV-vis irradiation (λ>200nm). It was found that the kinetics was influenced by C0. A linear plot of the pseudo-first-order rate constant vs C0 was observed with a negative slope. Secondly, the reaction between the phenicol antibiotics and OH was examined with a competition kinetic method under simulated solar irradiation (λ>290nm), which quantified their bimolecular reaction rate constants of (2.13±0.02)×10(9)M(-1)s(-1) and (1.82±0.10)×10(9)M(-1)s(-1) for thiamphenicol and florfenicol, respectively. Then the corresponding OH oxidated half-lives in sunlit surface waters were calculated to be 90.5-106.1h. Some main intermediates were formed from the reaction, which suggested that the two phenicols underwent hydroxylation, oxygenation and dehydrogenation when OH existed. These results are of importance to assess the phenicol persistence in wastewater treatment and sunlit surface waters. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Relationship between urinary polycyclic aromatic hydrocarbon metabolite and cell cycle of lymphocyte in coke oven workers].

PubMed

Pan, B L; Zhang, H T; Zhang, H J; Chen, W T; Yang, J

2016-11-20

Objective: To investigate the relationship between urinary polycyclic aromatic hydrocarbon metabolite and cell cycle of lymphocyte in coke oven workers. Methods: 437 coke oven workers and 163 work-ers in water treatment department were recruited in this study. Flow cytometry was used to detect the cell cycle of lymphocyte. For the measurement of urinary metabolites, urine samples were treated with β-glucuronidase and analyzed using HPLC with a fluorescence detector. Results: The concentrations of urinary 2-naphthol, 2-hydroxyfluorene, 9-phenanthrol and 1-hydroxypyrene l in coke oven workers were significantly higher than those in control group ( P <0.01) . The distributions of cell cycle were analyzed in high exposure group (the content of urinary metabolites high than P 75) and low exposure group (the content of urinary metabolites low than P 25) . According to the content of 1-hydroxypyrene, the proportions of S phase in high exposure group were significant-ly higher than those of low exposure group ( Z =-2.496, P =0.013) , but the proportions of G0/G1 phase were sig-nificantly lower than low exposure group ( Z =-2.074, P =0.038) . The similar results were not been found in other hydroxylated metabolites as internal exposure group. Conclusion: Increasing levels of urinary 1-hydroxypyrene might resulting in cell cycle of lymphocyte disorders, mainly for G0/G1 phase shorten and S phase arrest.

Isoprene photo-oxidation products quantify the effect of pollution on hydroxyl radicals over Amazonia

DOE PAGES

Liu, Yingjun; Seco, Roger; Kim, Saewung; ...

2018-04-11

Nitrogen oxides (NO x) emitted from human activities are believed to regulate the atmospheric oxidation capacity of the troposphere. However, observational evidence is limited for the low-to-median NO x concentrations prevalent outside of polluted regions. Directly measuring oxidation capacity, represented primarily by hydroxyl radicals (OH), is challenging, and the span in NO x concentrations at a single observation site is often not wide. Concentrations of isoprene and its photo-oxidation products were used to infer the equivalent noontime OH concentrations. The fetch at an observation site in central Amazonia experienced varied contributions from background regional air, urban pollution, and biomass burning.more » The afternoon concentrations of reactive nitrogen oxides (NO y), indicative of NO x exposure during the preceding few hours, spanned from 0.3 to 3.5 parts per billion. Accompanying the increase of NO y concentration, the inferred equivalent noontime OH concentrations increased by at least 250% from 0.6 × 10 6 to 1.6 × 10 6 cm -3. The conclusion is that, compared to background conditions of low NO x concentrations over the Amazon forest, pollution increased NO x concentrations and amplified OH concentrations, indicating the susceptibility of the atmospheric oxidation capacity over the forest to anthropogenic influence and reinforcing the important role of NO x in sustaining OH concentrations.« less

Isoprene photo-oxidation products quantify the effect of pollution on hydroxyl radicals over Amazonia

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

Liu, Yingjun; Seco, Roger; Kim, Saewung

Nitrogen oxides (NO x) emitted from human activities are believed to regulate the atmospheric oxidation capacity of the troposphere. However, observational evidence is limited for the low-to-median NO x concentrations prevalent outside of polluted regions. Directly measuring oxidation capacity, represented primarily by hydroxyl radicals (OH), is challenging, and the span in NO x concentrations at a single observation site is often not wide. Concentrations of isoprene and its photo-oxidation products were used to infer the equivalent noontime OH concentrations. The fetch at an observation site in central Amazonia experienced varied contributions from background regional air, urban pollution, and biomass burning.more » The afternoon concentrations of reactive nitrogen oxides (NO y), indicative of NO x exposure during the preceding few hours, spanned from 0.3 to 3.5 parts per billion. Accompanying the increase of NO y concentration, the inferred equivalent noontime OH concentrations increased by at least 250% from 0.6 × 10 6 to 1.6 × 10 6 cm -3. The conclusion is that, compared to background conditions of low NO x concentrations over the Amazon forest, pollution increased NO x concentrations and amplified OH concentrations, indicating the susceptibility of the atmospheric oxidation capacity over the forest to anthropogenic influence and reinforcing the important role of NO x in sustaining OH concentrations.« less

Isoprene photo-oxidation products quantify the effect of pollution on hydroxyl radicals over Amazonia.

PubMed

Liu, Yingjun; Seco, Roger; Kim, Saewung; Guenther, Alex B; Goldstein, Allen H; Keutsch, Frank N; Springston, Stephen R; Watson, Thomas B; Artaxo, Paulo; Souza, Rodrigo A F; McKinney, Karena A; Martin, Scot T

2018-04-01

Nitrogen oxides (NO x ) emitted from human activities are believed to regulate the atmospheric oxidation capacity of the troposphere. However, observational evidence is limited for the low-to-median NO x concentrations prevalent outside of polluted regions. Directly measuring oxidation capacity, represented primarily by hydroxyl radicals (OH), is challenging, and the span in NO x concentrations at a single observation site is often not wide. Concentrations of isoprene and its photo-oxidation products were used to infer the equivalent noontime OH concentrations. The fetch at an observation site in central Amazonia experienced varied contributions from background regional air, urban pollution, and biomass burning. The afternoon concentrations of reactive nitrogen oxides (NO y ), indicative of NO x exposure during the preceding few hours, spanned from 0.3 to 3.5 parts per billion. Accompanying the increase of NO y concentration, the inferred equivalent noontime OH concentrations increased by at least 250% from 0.6 × 10 6 to 1.6 × 10 6 cm -3 . The conclusion is that, compared to background conditions of low NO x concentrations over the Amazon forest, pollution increased NO x concentrations and amplified OH concentrations, indicating the susceptibility of the atmospheric oxidation capacity over the forest to anthropogenic influence and reinforcing the important role of NO x in sustaining OH concentrations.

Systematic and comprehensive strategy for metabolite profiling in bioanalysis using software-assisted HPLC-Q-TOF: magnoflorine as an example.

PubMed

Tian, Xiaoting; Zhang, Yucheng; Li, Zhixiong; Hu, Pei; Chen, Mingcang; Sun, Zhaolin; Lin, Yunfei; Pan, Guoyu; Huang, Chenggang

2016-03-01

Metabolite profiling plays a crucial role in drug discovery and development, and HPLC-Q-TOF has evolved into a powerful and effective high-resolution analytical tool for metabolite detection. However, traditional empirical identification is laborious and incomplete. This paper presents a systematic and comprehensive strategy for elucidating metabolite structures using software-assisted HPLC-Q-TOF that takes full advantage of data acquisition, data processing, and data mining technologies, especially for high-throughput metabolite screening. This strategy has been successfully applied in the study of magnoflorine metabolism based on our previous report of its poor bioavailability and drug-drug interactions. In this report, 23 metabolites of magnoflorine were tentatively identified with detailed fragmentation pathways in rat biological samples (urine, feces, plasma, and various organs) after i.p. or i.g. administration, and for most of these metabolites, the metabolic sites were determined. The phase I biotransformations of magnoflorine (M1-M7, M10-M14) consist of demethylation, dehydrogenation, hydroxylation, methylene to ketone transformation, N-ring opening, and dehydroxylation. The phase II biotransformations (M8, M9, and M15-M23) consist of methylation, acetylation, glucuronidation, and N-acetylcysteine conjugation. The results indicate that the extensive metabolism and wide tissue distribution of magnoflorine and its metabolites may partly contribute to its poor bioavailability and drug-drug interaction, and i.p. administration should thus be a suitable approach for isolating magnoflorine metabolites. In summary, this strategy could provide an efficient, rapid, and reliable method for the structural characterization of drug metabolites and may be applicable for general Q-TOF users.

In vitro metabolism of BDE-47, BDE-99, and α-, β-, γ-HBCD isomers by chicken liver microsomes.

PubMed

Zheng, Xiaobo; Erratico, Claudio; Abdallah, Mohamed Abou-Elwafa; Negreira, Noelia; Luo, Xiaojun; Mai, Bixian; Covaci, Adrian

2015-11-01

The in vitro oxidative metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), and the individual α-, β- and γ-hexabromocyclododecane (HBCD) isomers catalyzed by cytochrome P450 (CYP) enzymes was studied using chicken liver microsomes (CLMs). Metabolites were identified using a liquid chromatography-tandem mass spectrometry method and authentic standards for the oxidative metabolites of BDE-47 and BDE-99. Six hydroxylated tetra-BDEs, namely 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47), 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), 4'-hydroxy-2,2',4,5'- tetrabromodiphenyl ether (4'-OH-BDE-49), and 2'-hydroxy-2,3',4,4'-tetrabromodiphenyl ether (2'-OH-BDE-66), were identified and quantified after incubation of BDE-47 with CLMs. 4'-OH-BDE-49 was the major metabolite formed. Three hydroxylated penta-BDEs (5'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99), 6'-hydroxy-2,2',4,4',5- pentabromodiphenyl ether (6'-OH-BDE-99), and 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether, 4'-OH-BDE-101, were formed incubating BDE-99 with CLMs. Concentrations of BDE-99 metabolites were lower than those of BDE-47. More than four mono-hydroxylated HBCD (OH-HBCD), more than four di-hydroxylated HBCD (di-OH-HBCD), more than five mono-hydroxylated pentabromocyclododecenes (OH-PBCD), and more than five di-hydroxylated pentabromocyclododecenes (di-OH-PBCD) were detected when α-, β-, or γ-HBCD were individually incubated with CLMs. Response values (the ratio between the peak areas of the target compound and its internal standard) for OH-HBCD were 1-3 orders of magnitude higher than those for OH-PBCD, di-OH-HBCD, and di-OH-PBCD, suggesting that OH-HBCD might be the major metabolites of α-, β- and γ-HBCD produced by CLMs. No diastereoisomeric or enantiomeric bioisomerisation was

Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometry.

PubMed

Fandiño, Anabel S; Nägele, Edgar; Perkins, Patrick D

2006-02-01

The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques. Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature. Copyright 2006 John Wiley & Sons, Ltd.

Quantification of Water-Soluble Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy.

PubMed

Lo, Yu-Chang; Chien, Shih-Chang; Mishchuk, Darya O; Slupsky, Carolyn M; Mau, Jeng-Leun

2016-01-01

The water-soluble metabolites in 5 mushrooms were identified and quantified using proton nuclear magnetic resonance (NMR) spectroscopy and software for targeted metabolite detection and quantification. In total, 35 compounds were found in Agaricus brasiliensis, 25 in Taiwanofungus camphoratus, 23 in Ganoderma lucidum (Taiwan) and Lentinus edodes, and 16 in G. lucidum (China). Total amounts of all identified metabolites in A. brasiliensis, T. camphoratus, G. lucidum, G. lucidum (China), and L. edodes were 149,950.51, 12,834.18, 9,549.09, 2,788.41, and 111,726.51 mg/kg dry weight, respectively. These metabolites were categorized into 4 groups: free amino acids and derivatives, carbohydrates, carboxylic acids, and nucleosides. Carbohydrates were the most abundant metabolites among all 4 groups, with mannitol having the highest concentration among all analyzed metabolites (848-94,104 mg/kg dry weight). Principal components analysis (PCA) showed obvious distinction among the metabolites of the 5 different kinds of mushrooms analyzed in this study. Thus PCA could provide an optional analytical way of identifying and recognizing the compositions of flavor products. Furthermore, the results of this study demonstrate that NMRbased metabolomics is a powerful tool for differentiating between various medicinal mushrooms.

Regioselective alkane hydroxylation with a mutant AlkB enzyme

DOEpatents

Koch, Daniel J.; Arnold, Frances H.

2012-11-13

AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

Analysis of 10 metabolites of polymethoxyflavones with high sensitivity by electrochemical detection in high-performance liquid chromatography.

PubMed

Zheng, Jinkai; Bi, Jinfeng; Johnson, David; Sun, Yue; Song, Mingyue; Qiu, Peiju; Dong, Ping; Decker, Eric; Xiao, Hang

2015-01-21

Polymethoxyflavones (PMFs) have been known as a type of bioactive flavones that possess various beneficial biological functions. Accumulating evidence demonstrated that the metabolites of PMFs, that is, hydroxyl PMFs (OH-PMFs), had more potent beneficial biological effects than their corresponding parent PMFs. To facilitate the further identification and quantification of OH-PMFs in biological samples, the aim of this study was to develop a methodology for the simultaneous determination of 10 OH-PMFs using high-performance liquid chromatography (HPLC) coupled with electrochemistry detection. The HPLC profiles of these 10 OH-PMFs affected by different chromatographic parameters (different organic composition in mobile phases, the concentration of trifluoroacetic acid, and the concentration of ammonium acetate) are fully discussed in this study. The optimal condition was selected for the following validation studies. The linearity of calibration curves, accuracy, and precision (intra- and interday) at three concentration levels (low, middle, and high concentration range) were verified. The regression equations were linear (r > 0.9992) over the range of 0.005-10 μM. The limit of detection for 10 OH-PMFs was in the range of 0.8-3.7 ng/mL (S/N = 3, 10 μL injection). The recovery rates ranged from 86.6 to 108.7%. The precisions of intraday and interday analyses were less than 7.37 and 8.63% for relative standard deviation, respectively. This validated method was applied for the analysis of a variety of samples containing OH-PMFs. This paper also gives an example of analyzing the metabolites of nobiletin in mouse urine using the developed method. The transformation from nobiletin to traces of 5-hydroxyl metabolites has been discovered by this effective method, and this is the first paper to report such an association.

Chemoselective Hydroxyl Group Transformation: An Elusive Target‡

PubMed Central

Trader, Darci J.; Carlson, Erin E.

2012-01-01

The selective reaction of one functional group in the presence of others is not a trivial task. A noteworthy amount of research has been dedicated to the chemoselective reaction of the hydroxyl moiety. This group is prevalent in many biologically important molecules including natural products and proteins. However, targeting the hydroxyl group is difficult for many reasons including its relatively low nucleophilicity in comparison to other ubiquitous functional groups such as amines and thiols. Additionally, many of the developed chemoselective reactions cannot be used in the presence of water. Despite these complications, chemoselective transformation of the hydroxyl moiety has been utilized in the synthesis of complex natural product derivatives, the reaction of tyrosine residues in proteins, the isolation of natural products and is the mechanism of action of myriad drugs. Here, methods for selective targeting of this group, as well as applications of several devised methods, are described. PMID:22695722

Sphingolipid hydroxylation in mammals, yeast and plants - An integrated view.

PubMed

Marquês, Joaquim Trigo; Susana Marinho, H; de Almeida, Rodrigo Freire Martins

2018-05-07

This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature, with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as they can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants. Copyright © 2017. Published by Elsevier Ltd.

Simultaneous, untargeted metabolic profiling of polar and nonpolar metabolites by LC-Q-TOF mass spectrometry.

PubMed

Kirkwood, Jay S; Maier, Claudia; Stevens, Jan F

2013-05-01

At its most ambitious, untargeted metabolomics aims to characterize and quantify all of the metabolites in a given system. Metabolites are often present at a broad range of concentrations and possess diverse physical properties complicating this task. Performing multiple sample extractions, concentrating sample extracts, and using several separation and detection methods are common strategies to overcome these challenges but require a great amount of resources. This protocol describes the untargeted, metabolic profiling of polar and nonpolar metabolites with a single extraction and using a single analytical platform. © 2013 by John Wiley & Sons, Inc.

Stereoselective synthesis of an active metabolite of the potent PI3 kinase inhibitor PKI-179.

PubMed

Chen, Zecheng; Venkatesan, Aranapakam M; Dos Santos, Osvaldo; Delos Santos, Efren; Dehnhardt, Christoph M; Ayral-Kaloustian, Semiramis; Ashcroft, Joseph; McDonald, Leonard A; Mansour, Tarek S

2010-03-05

The synthesis and stereochemical determination of 1-(4-(4-((1R,5R,6R)-6-hydroxy-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea (2), an active metabolite of the potent PI3 kinase inhibitor PKI-179 (1), is described. Stereospecific hydroboration of the double bond of 2,5-dihydro-1H-pyrrole 8 gave the 2,3-trans alcohol 9 exclusively. The configuration of the 3-hydroxyl group in 9 was inverted by an oxidation and stereoselective reduction sequence to give the corresponding 2,3-cis isomer 23. Both exo (21) and endo (27) isomers of the metabolite 2 were prepared via a practical synthetic route from 9 and 23, respectively, and the stereochemistry of 2 was determined to be endo. The endo isomer (27) was separated into two enantiomers 28 and 29 by chiral HPLC. Compound 2 was found to be enantiomerically pure and identical to the enantiomer 28. The absolute stereochemistry of the enantiomer 28 was determined by Mosher's method, thus establishing the stereochemistry of the active metabolite 2.

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.

Construction of Viable Soil Defined Media Using Quantitative Metabolomics Analysis of Soil Metabolites

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

Jenkins, Stefan; Swenson, Tami L.; Lau, Rebecca

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Thus, environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids,more » nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the < 1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a

Construction of Viable Soil Defined Media Using Quantitative Metabolomics Analysis of Soil Metabolites

PubMed Central

Jenkins, Stefan; Swenson, Tami L.; Lau, Rebecca; Rocha, Andrea M.; Aaring, Alex; Hazen, Terry C.; Chakraborty, Romy; Northen, Trent R.

2017-01-01

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the <1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable

Construction of Viable Soil Defined Media Using Quantitative Metabolomics Analysis of Soil Metabolites

DOE PAGES

Jenkins, Stefan; Swenson, Tami L.; Lau, Rebecca; ...

2017-12-22

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Thus, environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids,more » nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the < 1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

PubMed

Nakada, Naoyuki; Oda, Kazuo

2015-01-01

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

Investigating Hydroxyl at Asteroid 951 Gaspra

NASA Astrophysics Data System (ADS)

Granahan, James C.

2015-11-01

Recent investigations [Granahan, 2011; 2014] of Galileo Near Infrared Mapping Spectrometer (NIMS) observations of asteroid 951 Gaspra have detected an infrared absorption feature near 2.8 micrometers. These were detected in NIMS data acquired by the Galileo spacecraft on October 29, 1991 at wavelengths ranging from 0.7 - 5.2 micrometers [Carlson et al., 1992]. This abstract presents a summary of the investigation to identify the material creating the 2.8 micrometer spectral absorption feature. The current best match for the observed 951 Gaspra feature is the phyllosilicate bound hydroxyl signature present in a thermally desiccated QUE 99038 carbonaceous chondrite as measured by Takir et al. [2013].The 951 Gaspra absorption feature has been compared to a variety of hydroxyl bearing signatures. Many phyllosilicates, hydroxyl bearing minerals, have absorption minima at different positions (2.7 or 2.85 micrometers). It also differs from similar absorptions in a potential R chondrite analog, LAP 04840. The spectra LAP 04840 has a 2.7 micrometer feature due to biotite and a 2.9 micrometer feature due to adsorbed water [Klima et al., 2007]. 2.8 micrometer absorption minima have been found for adsorbed hydroxyl on the Moon [McCord et al., 2011] and various carbonaceous chondrites [Calvin and King, 1997; Takir et al., 2013]. The best match, with a minimum Euclidean distance difference to the 951 Gaspra feature, is found in the spectrum of QUE 99038 [Takir et al., 2013]. This spectrum is the product of an infrared measurement of a sample that had its adsorbed water baked off and removed in a vacuum chamber. The remaining hydroxyl in the sample belongs to a mixture of phyllosilicates dominated by the presence of cronstedtite.References: Calvin, W. M., and T. V. King (1997), Met. Planet. Sci., 32, 693-702. Carlson, R. W., et al. (1992), Bull. American Astro. Soc., 24, 932. Granahan, J. C. (2011), Icarus, 213, 265-272. Granahan, J. C. (2014), 45th LPSC, #1092. Klima, R., C. et

Echo-Planar Imaging-Based, J-Resolved Spectroscopic Imaging for Improved Metabolite Detection in Prostate Cancer

DTIC Science & Technology

2014-10-01

Imaging (EP-JRESI); Citrate, Choline, Creatine , Spermine, 3Tesla MRI scanner, Endo-rectal MR coil, WET Water Suppression, prostate cancer (PCa...spectroscopic imaging are due to the overlap of metabolite resonances, quantifying few metabolites only (citrate (Cit), choline (Ch), creatine (Cr...concentrations of citrate (Cit), creatine (Cr), choline (Ch) and polyamines that are used to detect and diagnose PCa (2). The challenging task in 1D MRS

Urinary corticosterone metabolite responses to capture and captivity in the cane toad (Rhinella marina).

PubMed

Narayan, Edward J; Cockrem, John F; Hero, Jean-Marc

2011-09-01

Urinary corticosterone metabolite responses to capture have recently been shown for the first time in amphibians, and in the present study urinary corticosterone metabolite responses to capture and to confinement in captivity were measured in adult cane toads (Rhinella marina) in Queensland, Australia. An adrenocorticotropic hormone (ACTH) challenge was used to provide a biological validation for urinary corticosterone metabolite concentrations measured by radioimmunoassay (RIA). Urinary corticosterone metabolite increased 1-2 days after ACTH but not saline injection and then returned to initial values, indicating that the RIA could detect changes in corticosterone secretion in toads. Urinary corticosterone metabolite responses to short-term capture and restraint in plastic bags were first apparent 2h after capture of wild toads. Toads held communally in captivity for 5 days had elevated urinary corticosterone metabolite concentrations. Mean corticosterone concentrations declined significantly after a further 7 days in individual housing chambers. There was no sex difference in urinary corticosterone metabolite responses of toads to ACTH challenge, short-term capture or captivity. The relative amount of variation in the mean corticosterone responses was quantified by calculating coefficients of variation (CV) for each mean corticosterone response. Mean corticosterone at 0 min was more variable for captive toads than wild toads. Furthermore, initial corticosterone concentrations (0 min) were more variable than concentrations during the ACTH challenge, short-term capture and captivity. There was little change in the amount of variation of mean corticosterone levels between male and female toads with increasing time in captivity (12-29 days). This study has shown individual corticosterone responses of amphibians for the first-time, and has provided a novel method for quantifying the relative amount of variation in amphibian corticosterone responses. Copyright © 2011

Quantifying global-brain metabolite level changes with whole-head proton MR spectroscopy at 3T.

PubMed

Davitz, Matthew S; Wu, William E; Soher, Brian J; Babb, James S; Kirov, Ivan I; Huang, Jeffrey; Fatterpekar, Girish; Gonen, Oded

2017-01-01

To assess the sensitivity of non-localized, whole-head 1 H-MRS to an individual's serial changes in total-brain NAA, Glx, Cr and Cho concentrations - metabolite metrics often used as surrogate markers in neurological pathologies. In this prospective study, four back-to-back (single imaging session) and three serial (successive sessions) non-localizing, ~3min 1 H-MRS (TE/TR/TI=5/10 4 /940ms) scans were performed on 18 healthy young volunteers: 9 women, 9 men: 29.9±7.6 [mean±standard deviation (SD)] years old. These were analyzed by calculating a within-subject coefficient of variation (CV=SD/mean) to assess intra- and inter-scan repeatability and prediction intervals. This study was Health Insurance Portability and Accountability Act compliant. All subjects gave institutional review board-approved written, informed consent. The intra-scan CVs for the NAA, Glx, Cr and Cho were: 3.9±1.8%, 7.3±4.6%, 4.0±3.4% and 2.5±1.6%, and the corresponding inter-scan (longitudinal) values were: 7.0±3.1%, 10.6±5.6%, 7.6±3.5% and 7.0±3.9%. This method is shown to have 80% power to detect changes of 14%, 27%, 26% and 19% between two serial measurements in a given individual. Subject to the assumption that in neurological disorders NAA, Glx, Cr and Cho changes represent brain-only pathology and not muscles, bone marrow, adipose tissue or epithelial cells, this approach enables us to quantify them, thereby adding specificity to the assessment of the total disease load. This will facilitate monitoring diffuse pathologies with faster measurement, more extensive (~90% of the brain) spatial coverage and sensitivity than localized 1 H-MRS. Copyright © 2016 Elsevier Inc. 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.

Molecular design and synthesis of functional photothermopolymers from hydroxyl benzoic acids

NASA Astrophysics Data System (ADS)

Tong, Xiao; Gu, Jiangnan; Wang, Liyuan; Zou, Yingquan; Yu, Shangxian

2000-06-01

The most applicable hydroxyl benzoic acid monomers were optimized to synthesize the thermolysis-decarboxylation polymers according to the relative results of TG analysis of hydroxyl benzoic acids, their 13C-NMR spectra analyses and their quantum chemistry calculation with AB-INITIO method. On the basis of the empirical rule -- M/A value rule, while phenols with high M/A value and hydroxyl benzoic acids were both cocondensed with formaldehyde at proper ratio, the novolak resin with carboxyl groups used as a thermal imaging material could be obtained. In the presence of an acid catalyst, such as oxalic acid, a hydroxyl benzoic acid could be additionally polymerized with divinyl benzene (DVB) to synthesize another kind of polymer with not only carboxyl groups but also phenolic hydroxyl groups. The thermal imaging mechanisms of these polymers with carboxyl groups were discussed in the paper.

Spin-locking versus chemical exchange saturation transfer MRI for investigating chemical exchange process between water and labile metabolite protons.

PubMed

Jin, Tao; Autio, Joonas; Obata, Takayuki; Kim, Seong-Gi

2011-05-01

Chemical exchange saturation transfer (CEST) and spin-locking (SL) experiments were both able to probe the exchange process between protons of nonequivalent chemical environments. To compare the characteristics of the CEST and SL approaches in the study of chemical exchange effects, we performed CEST and SL experiments at varied pH and concentrated metabolite phantoms with exchangeable amide, amine, and hydroxyl protons at 9.4 T. Our results show that: (i) on-resonance SL is most sensitive to chemical exchanges in the intermediate-exchange regime and is able to detect hydroxyl and amine protons on a millimolar concentration scale. Off-resonance SL and CEST approaches are sensitive to slow-exchanging protons when an optimal SL or saturation pulse power matches the exchanging rate, respectively. (ii) Offset frequency-dependent SL and CEST spectra are very similar and can be explained well with an SL model recently developed by Trott and Palmer (J Magn Reson 2002;154:157-160). (iii) The exchange rate and population of metabolite protons can be determined from offset-dependent SL or CEST spectra or from on-resonance SL relaxation dispersion measurements. (iv) The asymmetry of the magnetization transfer ratio (MTR(asym)) is highly dependent on the choice of saturation pulse power. In the intermediate-exchange regime, MTR(asym) becomes complicated and should be interpreted with care. Copyright © 2010 Wiley-Liss, Inc.

Urinary Estrogen Metabolites in 2 Soy Trials with Premenopausal Women

PubMed Central

Maskarinec, Gertraud; Morimoto, Yukiko; Heak, Sreang; Isaki, Marissa; Steinbrecher, Astrid; Custer, Laurie; Franke, Adrian A.

2012-01-01

Background Soy consumption may protect against breast cancer through modification of estrogen metabolism. Objective We examined the effect of soy foods on urinary estrogens and the 2-hydroxy (OH)/16α-OH estrone (E1) ratio in 2 dietary interventions with premenopausal women. Methods BEAN1 was a 2-year randomized trial and BEAN2 a 13-month randomized crossover study. In both interventions, study participants consumed a high-soy diet with 2 soy food servings/day and a low-soy diet with <3 servings of soy/week. Urine samples were collected at baseline and at the end of the diet periods, analyzed for 9 estrogen metabolites by liquid chromatography mass spectrometry, and adjusted for creatinine levels. For BEAN1, 2 samples for 188 participants and for BEAN2, 3 samples for 79 women were analyzed. We applied mixed-effects regression models with log-transformed values of estrogen metabolites and soy intake as the exposure variable. Results In BEAN1, no effect of the high-soy diet on individual estrogen metabolites or hydroxylation pathways was observed. The median 2-OH/16α-OH E1 ratio decreased non-significantly in the intervention group from 6.2 to 5.2 as compared to 6.8 and 7.2 in the control group (p=0.63). In BEAN2, only 4-OHE1 was significantly lower after the high-soy diet. Interaction terms of the high-soy diet with equol producer status, ethnicity, and weight status revealed no significant effect modification. Conclusions Contrary to our hypothesis and some previous reports, the results from 2 well controlled dietary interventions do not support an effect of a high-soy diet on a panel of urinary estrogen metabolites and the 2-OH/16α-OHE1 ratio. PMID:22713773

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols.

PubMed

Fenoll, L G; Rodríguez-López, J N; Varón, R; García-Ruiz, P A; García-Cánovas, F; Tudela, J

2000-04-01

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding

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.

Microsomal Metabolism of Prochiral Polychlorinated Biphenyls Results in the Enantioselective Formation of Chiral Metabolites

PubMed Central

2016-01-01

Polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substituents and their metabolites exist as stable rotational isomers, or atropisomers, that are nonsuperimposable mirror images of each other. Additionally, the oxidation of certain axially prochiral PCBs, such as 2,2′,4,6′-tetrachlorobiphenyl (PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl (PCB 102), in the meta position of the symmetrically substituted phenyl ring is expected to form axially chiral hydroxylated metabolites (OH-PCBs); however, the formation of chiral OH-PCBs from prochiral PCBs has not been demonstrated experimentally. Here, we investigate if the oxidation of PCB 51 and PCB 102 by different microsomal preparations results in the formation of chiral OH-PCBs. Gas chromatographic analysis revealed that PCB 51 and PCB 102 were metabolized to 2,2′,4,6′-tetrachlorobiphenyl-3′-ol (OH-PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl-3′-ol (OH-PCB 102), respectively, by liver microsomes from male rats pretreated with different inducers; untreated male monkeys, guinea pigs, rabbits, and hamsters; and female dogs. The formation of both metabolites was inducer- and species-dependent. Both OH-PCB 51 and OH-PCB 102 were chiral and formed enantioselectively by all microsomal preparations investigated. These findings demonstrate that axially chiral PCB metabolites are formed from axially prochiral PCB congeners, a fact that should be considered when studying the environmental fate, transport, and toxicity of OH-PCBs. PMID:28038482

The role of 17β-estradiol metabolites in chromium-induced oxidative stress.

PubMed

Sawicka, Ewa; Długosz, Anna

2017-01-01

The increasing incidence of estrogen-dependent breast cancer and the presence in the environment of a large number of factors that interact with estrogen receptors have sparked interest in chemical influences on estrogen-dependent processes. In a previous work, the authors examined the interaction of estradiol with chromium. In the present article the importance of estradiol biotransformation in these interactions is investigated. There is no information in the available literature about the role of metabolites in exposure to chromium. It seems important because estradiol metabolites have various carcinogenic abilities and their formation during biotransformation could be increased or decreased by environmental enzyme inducers or inhibitors. The metabolites could play a detoxifying role or create a toxic synergism in free radical processes induced by chromium VI (CrVI). The aim of this study was to evaluate the influence of 2 17β-estradiol metabolites - 4-hydroxyestradiol (4-OHE2) and 16α-hydroxyestrone (16α-OHE1) - in conditions of oxidative stress caused by CrVI. Human blood, erythrocytes or mitochondria isolated from human placentas after natural deliveries were used in the experiments. The influence of CrVI, 4-OHE2 and 16-OHE1 on thiobarbituric acid reactive substances (TBARS), the hydroxyl radical (•OH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and the interactions of the metabolites exposed to chromium expressed by these factors were examined. 4-OHE2 reduced the level of TBARS induced by CrVI in mitochondria (p < 0.05) and in erythrocytes (p < 0.05), and increased SOD activity (p < 0.05). 16α-OHE1 increased the activity of GST in erythrocytes exposed to CrVI (p < 0.05). The metabolites do not have toxic interactions with CrVI. On the contrary, they exhibited a protective effect. The mechanism of protection varied: 4-OHE2 decreased TBARS and increased SOD activity, while 16α-OHE1 increased GST

Utilizing ion mobility spectrometry and mass spectrometry for the analysis of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers and their metabolites

DOE PAGES

Zheng, Xueyun; Dupuis, Kevin T.; Aly, Noor A.; ...

2018-03-02

Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants originating from incomplete combustion of organic materials and synthetic sources. PAHs, PCBs, and PBDEs have all been shown to have a significant effect on human health with correlations to cancer and other diseases. Therefore, measuring the presence of these xenobiotics in the environment and human body is imperative for assessing their health risks. To date, their analyses require both gas chromatography and liquid chromatography separations in conjunction with mass spectrometry measurements for detection of both the parent molecules and their hydroxylated metabolites, making theirmore » studies extremely time consuming. Here in this work, we characterized PAHs, PCBs, PBDEs and their hydroxylated metabolites using ion mobility spectrometry coupled with mass spectrometry (IMS-MS) and in combination with different ionization methods including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Finally, the collision cross section and m/z trend lines derived from the IMS-MS analyses displayed distinct trends for each molecule type. Additionally, the rapid isomeric and molecular separations possible with IMS-MS showed great promise for quickly distinguishing the parent and metabolized PAH, PCB, and PDBE molecules in complex environmental and biological samples.« less

Utilizing ion mobility spectrometry and mass spectrometry for the analysis of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers and their metabolites

DOE PAGES

Zheng, Xueyun; Dupuis, Kevin T.; Aly, Noor A.; ...

2018-03-02

Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants originating from incomplete combustion of organic materials and synthetic sources. PAHs, PCBs, and PBDEs have all been shown to have a significant effect on human health with correlations to cancer and other diseases. Therefore, measuring the presence of these xenobiotics in the environment and human body is imperative for assessing their health risks. To date, their analyses require both gas chromatography and liquid chromatography separations in conjunction with mass spectrometry measurements for detection of both the parent molecules and their hydroxylated metabolites, making theirmore » studies extremely time consuming. Here in this work, we characterized PAHs, PCBs, PBDEs and their hydroxylated metabolites using ion mobility spectrometry coupled with mass spectrometry (IMS-MS) and in combination with different ionization methods including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). In conclusion, the collision cross section and m/z trend lines derived from the IMS-MS analyses displayed distinct trends for each molecule type. Additionally, the rapid isomeric and molecular separations possible with IMS-MS showed great promise for quickly distinguishing the parent and metabolized PAH, PCB, and PDBE molecules in complex environmental and biological samples.« less

Utilizing ion mobility spectrometry and mass spectrometry for the analysis of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers and their metabolites

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

Zheng, Xueyun; Dupuis, Kevin T.; Aly, Noor A.

Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants originating from incomplete combustion of organic materials and synthetic sources. PAHs, PCBs, and PBDEs have all been shown to have a significant effect on human health with correlations to cancer and other diseases. Therefore, measuring the presence of these xenobiotics in the environment and human body is imperative for assessing their health risks. To date, their analyses require both gas chromatography and liquid chromatography separations in conjunction with mass spectrometry measurements for detection of both the parent molecules and their hydroxylated metabolites, making theirmore » studies extremely time consuming. Here in this work, we characterized PAHs, PCBs, PBDEs and their hydroxylated metabolites using ion mobility spectrometry coupled with mass spectrometry (IMS-MS) and in combination with different ionization methods including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). In conclusion, the collision cross section and m/z trend lines derived from the IMS-MS analyses displayed distinct trends for each molecule type. Additionally, the rapid isomeric and molecular separations possible with IMS-MS showed great promise for quickly distinguishing the parent and metabolized PAH, PCB, and PDBE molecules in complex environmental and biological samples.« less

Time-Resolved Hydroxyl Radical Footprinting of RNA with X-Rays.

PubMed

Hao, Yumeng; Bohon, Jen; Hulscher, Ryan; Rappé, Mollie C; Gupta, Sayan; Adilakshmi, Tadepalli; Woodson, Sarah A

2018-06-01

RNA footprinting by hydroxyl radical cleavage provides 'snapshots' of RNA tertiary structure or protein interactions that bury the RNA backbone. Generation of hydroxyl radicals with a high-flux synchrotron X-ray beam provides analysis on a short timescale (5-100 msec), which enables the structures of folding intermediates or other transient conformational states to be determined in biochemical solutions or cells. This article provides protocols for using synchrotron beamlines for hydroxyl radical footprinting. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Tuning the Slide-Roll Motion Mode of Carbon Nanotubes via Hydroxyl Groups

NASA Astrophysics Data System (ADS)

Li, Rui; Wang, Shiwei; Peng, Qing

2018-05-01

Controlling the motion of carbon nanotubes is critical in manipulating nanodevices, including nanorobots. Herein, we investigate the motion behavior of SWCNT (10,10) on Si substrate utilizing molecular dynamics simulations. We show that hydroxyl groups have sensitive effect on the carbon nanotube's motion mode. When the hydroxyl groups' ratio on carbon nanotube and silicon substrate surfaces is larger than 10 and 20%, respectively, the motion of carbon nanotube transforms from sliding to rolling. When the hydroxyl groups' ratio is smaller, the slide or roll mode can be controlled by the speed of carbon nanotube, which is ultimately determined by the competition between the interface potential energy and kinetic energy. The change of motion mode holds true for different carbon nanotubes with hydroxyl groups. The chirality has little effect on the motion behavior, as opposed to the diameter, attributed to the hydroxyl groups' ratio. Our study suggests a new route to control the motion behavior of carbon nanotube via hydroxyl groups.

Tuning the Slide-Roll Motion Mode of Carbon Nanotubes via Hydroxyl Groups.

PubMed

Li, Rui; Wang, Shiwei; Peng, Qing

2018-05-08

Controlling the motion of carbon nanotubes is critical in manipulating nanodevices, including nanorobots. Herein, we investigate the motion behavior of SWCNT (10,10) on Si substrate utilizing molecular dynamics simulations. We show that hydroxyl groups have sensitive effect on the carbon nanotube's motion mode. When the hydroxyl groups' ratio on carbon nanotube and silicon substrate surfaces is larger than 10 and 20%, respectively, the motion of carbon nanotube transforms from sliding to rolling. When the hydroxyl groups' ratio is smaller, the slide or roll mode can be controlled by the speed of carbon nanotube, which is ultimately determined by the competition between the interface potential energy and kinetic energy. The change of motion mode holds true for different carbon nanotubes with hydroxyl groups. The chirality has little effect on the motion behavior, as opposed to the diameter, attributed to the hydroxyl groups' ratio. Our study suggests a new route to control the motion behavior of carbon nanotube via hydroxyl groups.

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human, Monkey, Dog, Rat, and Mouse Liver Microsomes

PubMed Central

Li, Wei; Liu, Li; Sun, Baoshan; Guo, Zhenghong; Shi, Caihong; Zhao, Yuqing

2014-01-01

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH3-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH3-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH3-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH3-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH3-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH3-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH3-PPD, especially at the C-20 hydroxyl group. PMID:24736630

Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry.

PubMed

Vikingsson, Svante; Josefsson, Martin; Gréen, Henrik

2015-01-01

The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Serum metabolites associate with CT findings following TBI.

PubMed

Dickens, Alex Mountfort; Posti, Jussi P; Takala, Riikka Sk; Ala-Seppälä, Henna Maria; Mattila, Ismo; Coles, Jonathan Coles; Frantzén, Janek; Hutchinson, Peter John; Katila, Ari J; Kyllönen, Anna; Maanpää, Henna-Riikka; Newcombe, Virginia; Outtrim, Joanne; Tallus, Jussi; Carpenter, Keri; Menon, David; Hyotylainen, Tuulia; Tenovuo, Olli; Oresic, Matej

2018-06-27

There is a need to rapidly detect patients with traumatic brain injury (TBI) who require head computed tomography (CT). Given the energy crisis in the brain following TBI, we hypothesized that serum metabolomics would be a useful tool for developing a set of biomarkers to determine the need for CT and to distinguish between different types of injuries observed. Logistic regression models using metabolite data from the discovery cohort (n=144, Turku, Finland) were used to distinguish between patients with traumatic intracranial findings and negative findings on head CT. The resultant models were then tested in the validation cohort (n=66, Cambridge, UK). The levels of glial fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 were also quantified in the serum from the same patients. Despite there being significant differences in the protein biomarkers in patients with TBI, the model that determined the need for a CT scan validated poorly (AUC=0.64: Cambridge patients). However, using a combination of six metabolites (two amino acids, three sugar derivatives and one ketoacid) it was possible to discriminate patients with intracranial abnormalities on CT and patients with a normal CT (AUC=0.77 in Turku patients and AUC=0.73 in Cambridge patients). Furthermore, a combination of three metabolites could distinguish between diffuse brain injuries and mass lesions (AUC=0.87 in Turku patients and AUC=0.68 in Cambridge patients). This study identifies a set of validated serum polar metabolites, which associate with the need for a CT scan. Additionally, serum metabolites can also predict the nature of the brain injury. These metabolite markers may prevent unnecessary CT scans, thus reducing the cost of diagnostics and radiation load.

Hydroxylated polychlorinated biphenyls decrease circulating steroids in female polar bears (Ursus maritimus).

PubMed

Gustavson, Lisa; Ciesielski, Tomasz M; Bytingsvik, Jenny; Styrishave, Bjarne; Hansen, Martin; Lie, Elisabeth; Aars, Jon; Jenssen, Bjørn M

2015-04-01

As a top predator in the Arctic food chain, polar bears (Ursus maritimus) are exposed to high levels of persistent organic pollutants (POPs). Because several of these compounds have been reported to alter endocrine pathways, such as the steroidogenesis, potential disruption of the sex steroid synthesis by POPs may cause implications for reproduction by interfering with ovulation, implantation and fertility. Blood samples were collected from 15 female polar bears in Svalbard (Norway) in April 2008. The concentrations of nine circulating steroid hormones; dehydroepiandrosterone (DHEA), androstenedione (AN), testosterone (TS), dihydrotestosterone (DHT), estrone (E1), 17α-estradiol (αE2), 17β-estradiol (βE2), pregnenolone (PRE) and progesterone (PRO) were determined. The aim of the study was to investigate associations among circulating levels of specific POP compounds and POP-metabolites (hydroxylated PCBs [OH-PCBs] and hydroxylated PBDEs [OH-PBDEs]), steroid hormones, biological and capture variables in female polar bears. Inverse correlations were found between circulating levels of PRE and AN, and circulating levels of OH-PCBs. There were no significant relationships between the steroid concentrations and other analyzed POPs or the variables capture date and capture location (latitude and longitude), lipid content, condition and body mass. Although statistical associations do not necessarily represent direct cause-effect relationships, the present study indicate that OH-PCBs may affect the circulating levels of AN and PRE in female polar bears and that OH-PCBs thus may interfere with the steroid homeostasis. Increase in PRO and a decrease in AN concentrations suggest that the enzyme CYP17 may be a potential target for OH-PCBs. In combination with natural stressors, ongoing climate change and contaminant exposure, it is possible that OH-PCBs may disturb the reproductive potential of polar bears. Copyright © 2015 Elsevier Inc. All rights reserved.

Steroid conversion with CYP106A2 - production of pharmaceutically interesting DHEA metabolites.

PubMed

Schmitz, Daniela; Zapp, Josef; Bernhardt, Rita

2014-06-05

Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes. In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA. In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as

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.

Characterization of major metabolites of polymethoxylated flavonoids in Pericarpium Citri Reticulatae using liver microsomes immobilized on magnetic nanoparticles coupled with UPLC/MS-MS.

PubMed

Lei, Jun; Xue, Ying; Liu, Yi-Ming; Liao, Xun

2017-01-01

The peels of citrus fruits (Pericarpium Citri Reticulatae, PCR) have long been used in traditional Chinese medicines (TCMs). Polymethoxylated flavonoids (PMFs) were found to be the main components present in PCR extracts, but their metabolism remains unclear which restrain the utilization of this TCM. In the present work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) for in vitro metabolic study on the whole PMFs of PCR. LMMNPs were characterized by transmission electron microscope and Fourier-transform infrared spectrum. The relative enzyme binding capacity of LMMNPs was estimated to be about 428 μg/mg from thermogravimetric analysis. Incubation of LMMNPs with PMFs produced demethylated metabolites of PMFs, six of which were identified by ultrahigh pressure liquid chromatography-mass spectrometry (UPLC-MS/MS). The 3'-hydroxylated tangeretin (T3) was detected from the metabolites of tangeretin for the first time, which suggested that 4'-demethylated and 3'-hydroxylated derivative of tangeretin (3'-hydroxy-5,6,7,8,4'-pentamethoxyflavone, T4) was not only derived from 4'-demethylated tangeretin (T2) as previously reported, but also from T3. This is the first investigation of the metabolism of the whole PMFs, which may shed light on the mechanism of action of PCR.

Effect of curcumin against oxidation of biomolecules by hydroxyl radicals.

PubMed

Borra, Sai Krishna; Mahendra, Jaideep; Gurumurthy, Prema; Jayamathi; Iqbal, Shabeer S; Mahendra, Little

2014-10-01

Among various reactive oxygen species, hydroxyl radicals have the strongest chemical activity, which can damage a wide range of essential biomolecules such as lipids, proteins, and DNA. The objective of this study was to investigate the beneficial effects of curcumin on prevention of oxidative damage of biomolecules by hydroxyl radicals generated in in vitro by a Fenton like reaction. We have incubated the serum, plasma and whole blood with H2O2/Cu2+/ Ascorbic acid system for 4 hours at 37 0C and observed the oxidation of biomolecules like albumin, lipids, proteins and DNA. Curcumin at the concentrations of 50,100 and 200 μmoles, prevented the formation of ischemia modified albumin, MDA, protein carbonyls, oxidized DNA and increased the total antioxidant levels and GSH significantly. These observations suggest the hydroxyl radical scavenging potentials of curcumin and protective actions to prevent the oxidation of biomolecules by hydroxyl radicals.

Hydroxyl radical production in plasma electrolysis with KOH electrolyte solution

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

Saksono, Nelson; Febiyanti, Irine Ayu, E-mail: irine.ayu41@ui.ac.id; Utami, Nissa

2015-12-29

Plasma electrolysis is an effective technology for producing hydroxyl radical (•OH). This method can be used for waste degradation process. This study was conducted to obtain the influence of applied voltage, electrolyte concentration, and anode depth in the plasma electrolysis system for producing hydroxyl radical. The materials of anode and cathode, respectively, were made from tungsten and stainless steel. KOH solution was used as the solution. Determination of hydroxyl radical production was done by measuring H{sub 2}O{sub 2} amount formed in plasma system using an iodometric titration method, while the electrical energy consumed was obtained by measuring the electrical currentmore » throughout the process. The highest hydroxyl radical production was 3.51 mmol reached with 237 kJ energy consumption in the power supply voltage 600 V, 0.02 M KOH, and 0.5 cm depth of anode.« less

High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) using cryopreserved human hepatocytes and assessment of metabolic stability with human liver microsomes

PubMed Central

Gandhi, Adarsh S.; Wohlfarth, Ariane; Zhu, Mingshe; Pang, Shaokun; Castaneto, Marisol; Scheidweiler, Karl B.; Huestis, Marilyn A.

2014-01-01

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolic profiles. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine. To characterize the metabolite profile, human hepatocytes were incubated with 10 μmol/L STS-135 for up to 3 h. High-resolution mass spectrometry with software-assisted data mining identified 29 STS-135 metabolites. Less than 25% of STS-135 parent compound remained after 3 h incubation. Primary metabolites were generated by mono-, di- or trihydroxylation with and without ketone formation, dealkylation and oxidative defluorination of N-fluoropentyl side chain or possible oxidation to carboxylic acid, some of them further glucuronidated. Hydroxylations occurred mainly on the aliphatic adamantane ring and less commonly on the N-pentyl side chain. At 1 h phase I metabolites predominated, while at 3 h phase II metabolites were present in higher amounts. The major metabolites were monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21), both hydroxylated on the adamantane system. Moreover, metabolic stability of STS-135 (1 μmol/L) was assessed in human liver microsomes experiments. The in vitro half-life of STS-135 was 7.2±0.6 min and intrinsic clearance (CLint) was 93.6 mL·min−1·kg−1. This is the first report characterizing STS-135 hepatic metabolic pathways. These data provide potential urinary targets to document STS-135 intake in clinical and forensic settings and potential candidates for pharmacological testing. PMID:24827428

The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

PubMed

Neunzig, J; Milhim, M; Schiffer, L; Khatri, Y; Zapp, J; Sánchez-Guijo, A; Hartmann, M F; Wudy, S A; Bernhardt, R

2017-03-01

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased K m and decreased k cat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites

Effect of Curcumin Against Oxidation of Biomolecules by Hydroxyl Radicals

PubMed Central

Mahendra, Jaideep; Gurumurthy, Prema; Jayamathi; Iqbal, Shabeer S; Mahendra, Little

2014-01-01

Background: Among various reactive oxygen species, hydroxyl radicals have the strongest chemical activity, which can damage a wide range of essential biomolecules such as lipids, proteins, and DNA. Objective: The objective of this study was to investigate the beneficial effects of curcumin on prevention of oxidative damage of biomolecules by hydroxyl radicals generated in in vitro by a Fenton like reaction. Materials and Methods: We have incubated the serum, plasma and whole blood with H2O2/Cu2+/ Ascorbic acid system for 4 hours at 37 0C and observed the oxidation of biomolecules like albumin, lipids, proteins and DNA. Results: Curcumin at the concentrations of 50,100 and 200 μmoles, prevented the formation of ischemia modified albumin, MDA, protein carbonyls, oxidized DNA and increased the total antioxidant levels and GSH significantly. Conclusion: These observations suggest the hydroxyl radical scavenging potentials of curcumin and protective actions to prevent the oxidation of biomolecules by hydroxyl radicals. PMID:25478334

Lysine hydroxylation of collagen in a fibroblast cell culture system

NASA Technical Reports Server (NTRS)

Uzawa, Katsuhiro; Yeowell, Heather N.; Yamamoto, Kazushi; Mochida, Yoshiyuki; Tanzawa, Hideki; Yamauchi, Mitsuo

2003-01-01

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

PubMed

Perchat, Nadia; Saaidi, Pierre-Loïc; Darii, Ekaterina; Pellé, Christine; Petit, Jean-Louis; Besnard-Gonnet, Marielle; de Berardinis, Véronique; Dupont, Maeva; Gimbernat, Alexandra; Salanoubat, Marcel; Fischer, Cécile; Perret, Alain

2018-05-08

Trigonelline (TG; N- methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields ( Z )-2-(( N- methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into ( E )-2-(( N- methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

Simultaneous Quantification of 20 Synthetic Cannabinoids and 21 Metabolites, and Semi-quantification of 12 Alkyl Hydroxy Metabolites in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

Scheidweiler, Karl B.; Huestis, Marilyn A.

2014-01-01

Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative efforts, complicating toxicological analysis. No extensive synthetic cannabinoid quantitative urinary methods are reported in the literature. We developed and validated a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for simultaneously quantifying JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM-2201, MAM-2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM-694, RCS-8, XLR-11 and HU-210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxy metabolite isomers were considered semi-quantitative. β-glucuronidase hydrolyzed urine was extracted with 1 ml Biotage SLE+ columns. Specimens were reconstituted in 150 µL mobile phase consisting of 50% A (0.01% formic acid in water) and 50% B (0.01% formic acid in 50:50 methanol:acetonitrile). 4 and 25 µL injections were performed to acquire data in positive and negative ionization modes, respectively. The LC-MS/MS instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5500 Qtrap mass spectrometer with an electrospray source. Gradient chromatographic separation was achieved utilizing a Restek Ultra Biphenyl column with a 0.5 ml/min flow rate and an overall run time of 19.5 and 11.4 min for positive and negative mode methods, respectively. Quantification was by multiple reaction monitoring with CP 47,497 compounds and HU-210 ionized via negative polarity; all other analytes were acquired in positive mode. Lower and upper limits of linearity were 0.1–1.0 and 50–100 µg/l (r2 > 0.994). Validation parameters were evaluated at three concentrations spanning linear dynamic ranges. Inter-day analytical recovery (bias) and imprecision (N=20) were 88.3–112.2% and 4.3–13.5% coefficient of variation, respectively. Extraction efficiencies and matrix effect (N=10) were 44–110 and −73 to 52%, respectively. We present

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

PubMed Central

2014-01-01

Background Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes. Results In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA. Conclusions In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Tamoxifen metabolism in rat liver microsomes: identification of a dimeric metabolite derived from free radical intermediates by liquid chromatography/mass spectrometry.

PubMed

Jones, R M; Yuan, Z X; Lim, C K

1999-01-01

Tamoxifen has been shown to be a potent liver carcinogen in rats, and generates covalent DNA adducts. On-line high performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been used to further study the metabolites of tamoxifen formed by rat liver microsomes in the presence of NADPH with a view to identifying potential reactive metabolites which may be responsible for the formation of DNA adducts, and liver carcinogenesis. A metabolite has been detected with a protonated molecule at m/z 773. The mass of this compound is consistent with a dimer of hydroxylated tamoxifen (m/z 388). Analysis of 4-hydroxytamoxifen incubated with a rat liver microsomal preparation showed the formation of a similar metabolite with an apparent MH+ ion at m/z 773, believed to be a dimer of 4-hydroxytamoxifen formed by a free radical reaction. The retention time for this metabolite from 4-hydroxytamoxifen is identical to that of the tamoxifen metabolite, suggesting that these two compounds are the same. The levels of the dimer were higher when 4-hydroxytamoxifen was used as substrate and, in addition, two isomers were detected. It is proposed that tamoxifen was first converted to arene oxides which react with DNA or to 4-hydroxytamoxifen, either directly or via 3,4-epoxytamoxifen, which then undergoes activation via a free radical reaction to give reactive intermediates which can then react with DNA and protein, or with themselves, to give the dimers (m/z 773).

Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids.

PubMed

Biondi, Roberto; Brancorsini, Stefano; Poli, Giulia; Egidi, Maria Giulia; Capodicasa, Enrico; Bottiglieri, Livio; Gerli, Sandro; Brillo, Eleonora; Renzo, Gian Carlo Di; Cretoiu, Dragos; Micu, Romeo; Suciu, Nicolae

2018-05-01

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids. Copyright © 2018 The

Phthalate Metabolites, Consumer Habits and Health Effects

PubMed Central

Wallner, Peter; Kundi, Michael; Hohenblum, Philipp; Scharf, Sigrid; Hutter, Hans-Peter

2016-01-01

Phthalates are multifunctional chemicals used in a wide variety of consumer products. The aim of this study was to investigate whether levels of urinary phthalate metabolites in urine samples of Austrian mothers and their children were associated with consumer habits and health indicators. Within an Austrian biomonitoring survey, urine samples from 50 mother-child pairs of five communities (two-stage random stratified sampling) were analysed. The concentrations of 14 phthalate metabolites were determined, and a questionnaire was administered. Monoethyl phthalate (MEP), mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono-(5-carboxy-2-ethylpentyl) phthalate (5cx-MEPP), and 3-carboxy-mono-propyl phthalate (3cx-MPP) could be quantified in the majority of samples. Significant correlations were found between the use of hair mousse, hair dye, makeup, chewing gum, polyethylene terephthalate (PET) bottles and the diethyl phthalate (DEP) metabolite MEP. With regard to health effects, significant associations of MEP in urine with headache, repeated coughing, diarrhoea, and hormonal problems were observed. MBzP was associated with repeated coughing and MEHP was associated with itching. PMID:27428989

Phthalate Metabolites, Consumer Habits and Health Effects.

PubMed

Wallner, Peter; Kundi, Michael; Hohenblum, Philipp; Scharf, Sigrid; Hutter, Hans-Peter

2016-07-15

Phthalates are multifunctional chemicals used in a wide variety of consumer products. The aim of this study was to investigate whether levels of urinary phthalate metabolites in urine samples of Austrian mothers and their children were associated with consumer habits and health indicators. Within an Austrian biomonitoring survey, urine samples from 50 mother-child pairs of five communities (two-stage random stratified sampling) were analysed. The concentrations of 14 phthalate metabolites were determined, and a questionnaire was administered. Monoethyl phthalate (MEP), mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono-(5-carboxy-2-ethylpentyl) phthalate (5cx-MEPP), and 3-carboxy-mono-propyl phthalate (3cx-MPP) could be quantified in the majority of samples. Significant correlations were found between the use of hair mousse, hair dye, makeup, chewing gum, polyethylene terephthalate (PET) bottles and the diethyl phthalate (DEP) metabolite MEP. With regard to health effects, significant associations of MEP in urine with headache, repeated coughing, diarrhoea, and hormonal problems were observed. MBzP was associated with repeated coughing and MEHP was associated with itching.

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

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950

PubMed Central

2016-01-01

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC–MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC–MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC–MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety. PMID:27994733

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950.

PubMed

Salla, Manohar; Butler, Mark S; Pelingon, Ruby; Kaeslin, Geraldine; Croker, Daniel E; Reid, Janet C; Baek, Jong Min; Bernhardt, Paul V; Gillam, Elizabeth M J; Cooper, Matthew A; Robertson, Avril A B

2016-12-08

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro- s -indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R -(+)- N -((1-hydroxy-1,2,3,5,6,7-hexahydro- s -indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide ( 2a ). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a , with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

Spectroscopic determination of ecologically relevant plant secondary metabolites

DOE PAGES

Couture, John J.; Singh, Aditya; Rubert-Nason, Kennedy F.; ...

2016-07-23

Spectroscopy has recently emerged as an effective method to accurately characterize leaf biochemistry in living tissue through the application of chemometric approaches to foliar optical data, but this approach has not been widely used for plant secondary metabolites. Here in this paper, we examine the ability of reflectance spectroscopy to quantify specific phenolic compounds in trembling aspen (Populus tremuloides) and paper birch (Betula papyrifera) that play influential roles in ecosystem functioning related to trophic-level interactions and nutrient cycling.

Exploring the limitations of the Hantzsch method used for quantification of hydroxyl radicals in systems of relevance for interfacial radiation chemistry

NASA Astrophysics Data System (ADS)

Yang, Miao; Soroka, Inna; Jonsson, Mats

2017-01-01

In the presence of Tris or methanol, hydroxyl radicals in systems of relevance for interfacial radiation chemistry can be quantified indirectly via the Hantzsch method by determining the amount of the scavenging product formaldehyde formed. In this work, the influence of the presence of H2O2 on the Hantzsch method using acetoacetanilide (AAA) as derivatization reagent is studied. The experiments show that the measured CH2O concentration deviates from the actual concentration in the presence of H2O2 and the deviation increases with increasing [H2O2]0/[CH2O]0. The deviation is negative, i.e., the measured formaldehyde concentration is lower than the actual concentration. This leads to an underestimation of the hydroxyl radical production in systems containing significant amount of H2O2. The main reason for the deviation is found to be three coupled equilibria involving H2O2, CH2O and the derivative produced in the Hantzsch method.

Serum metabolites are associated with all-cause mortality in chronic kidney disease.

PubMed

Hu, Jiun-Ruey; Coresh, Josef; Inker, Lesley A; Levey, Andrew S; Zheng, Zihe; Rebholz, Casey M; Tin, Adrienne; Appel, Lawrence J; Chen, Jingsha; Sarnak, Mark J; Grams, Morgan E

2018-06-02

Chronic kidney disease (CKD) involves significant metabolic abnormalities and has a high mortality rate. Because the levels of serum metabolites in patients with CKD might provide insight into subclinical disease states and risk for future mortality, we determined which serum metabolites reproducibly associate with mortality in CKD using a discovery and replication design. Metabolite levels were quantified via untargeted liquid chromatography and mass spectroscopy from serum samples of 299 patients with CKD in the Modification of Diet in Renal Disease (MDRD) study as a discovery cohort. Six among 622 metabolites were significantly associated with mortality over a median follow-up of 17 years after adjustment for demographic and clinical covariates, including urine protein and measured glomerular filtration rate. We then replicated associations with mortality in 963 patients with CKD from the African American Study of Kidney Disease and Hypertension (AASK) cohort over a median follow-up of ten years. Three of the six metabolites identified in the MDRD cohort replicated in the AASK cohort: fumarate, allantoin, and ribonate, belonging to energy, nucleotide, and carbohydrate pathways, respectively. Point estimates were similar in both studies and in meta-analysis (adjusted hazard ratios 1.63, 1.59, and 1.61, respectively, per doubling of the metabolite). Thus, selected serum metabolites were reproducibly associated with long-term mortality in CKD beyond markers of kidney function in two well characterized cohorts, providing targets for investigation. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

The mutagenicities of safrole, estragole, eugenol, trans-anethole, and some of their known or possible metabolites for Salmonella typhimurium mutants.

PubMed

Swanson, A B; Chambliss, D D; Blomquist, J C; Miller, E C; Miller, J A

1979-04-01

Safrole, estragole, anethole, and eugenol and some of their known or possible metabolites were tested for mutagenic activity for S. typhimurium TA1535, TA100, and TA98. Highly purified 1'-hydroxyestragole and 1'-hydroxysafrole were mutagenic (approximately 15 and 10 revertants/micromole, respectively) for strain TA100 in the absence of fortified liver microsomes; trans-anethole and estragole appeared to have very weak activity. 3'-Hydroxyanethole was too toxic for an adequate test. Supplementation with NADPH-fortified rat-liver microsomes and cytosol converted 3'-hydroxyanethole to a mutagen(s) and increased the mutagenic activities for strain TA100 of 1'-hydroxyestragole, 1'-hydroxysafrole, estragole, and anethole. No mutagenicity was detected for safrole or eugenol with or without added NADPH-fortified liver preparations. The electrophilic 2',3'-oxides of safrole, 1'-hydroxysafrole, 1'-acetoxysafrole, 1'-oxosafrole, estragole, 1'-hydroxyestragole, and eugenol showed dose-dependent mutagenic activities for strain TA1535 in the absence of fortified liver microsomes. These mutagenic activities ranged from about 330 revertants/micromole for 1'-oxosafrole-2',3'-oxide to about 7000 revertants/micromole for safrole-2',3'-oxide. The arylalkenes, their hydroxylated derivatives, or their epoxides did not show mutagenic activity for strain TA98, except for 1'-oxosafrole-2',3'-oxide, which had weak activity. Since the arylalkenes are hydroxylated and/or epoxidized by hepatic microsomes, hydroxy and epoxide derivatives appear to be proximate and ultimate mutagenic metabolites, respectively, of the arylalkenes.

Effect of surface hydroxyl groups on heat capacity of mesoporous silica

NASA Astrophysics Data System (ADS)

Marszewski, Michal; Butts, Danielle; Lan, Esther; Yan, Yan; King, Sophia C.; McNeil, Patricia E.; Galy, Tiphaine; Dunn, Bruce; Tolbert, Sarah H.; Hu, Yongjie; Pilon, Laurent

2018-05-01

This paper quantifies the effect of surface hydroxyl groups on the effective specific and volumetric heat capacities of mesoporous silica. To achieve a wide range of structural diversity, mesoporous silica samples were synthesized by various methods, including (i) polymer-templated nanoparticle-based powders, (ii) polymer-templated sol-gel powders, and (iii) ambigel silica samples dried by solvent exchange at room temperature. Their effective specific heat capacity, specific surface area, and porosity were measured using differential scanning calorimetry and low-temperature nitrogen adsorption-desorption measurements. The experimentally measured specific heat capacity was larger than the conventional weight-fraction-weighted specific heat capacity of the air and silica constituents. The difference was attributed to the presence of OH groups in the large internal surface area. A thermodynamic model was developed based on surface energy considerations to account for the effect of surface OH groups on the specific and volumetric heat capacity. The model predictions fell within the experimental uncertainty.

Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation.

PubMed

Rose, K; Allan, A; Gauldie, S; Stapleton, G; Dobbie, L; Dott, K; Martin, C; Wang, L; Hedlund, E; Seckl, J R; Gustafsson, J A; Lathe, R

2001-06-29

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7alpha-hydroxylation of oxysterols and 3beta-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7alpha-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7alpha, exemplified by prominent 6alpha-hydroxylation of 5alpha-androstane-3beta,17beta-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (beta-galactosidase) under Cyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7alpha-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7alpha-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6alpha-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7b gene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine.

PubMed

Dubbelman, Anne-Charlotte; Jansen, Robert S; Rosing, Hilde; Darwish, Mona; Hellriegel, Edward; Robertson, Philmore; Schellens, Jan H M; Beijnen, Jos H

2012-07-01

Bendamustine is an alkylating agent consisting of a mechlorethamine derivative, a benzimidazole group, and a butyric acid substituent. A human mass balance study showed that bendamustine is extensively metabolized and subsequently excreted in urine. However, limited information is available on the metabolite profile of bendamustine in human urine. The objective of this study was to elucidate the metabolic pathways of bendamustine in humans by identification of its metabolites excreted in urine. Human urine samples were collected up to 168 h after an intravenous infusion of 120 mg/m(2) (80-95 μCi) [(14)C]bendamustine. Metabolites of [(14)C]bendamustine were identified using liquid chromatography (high-resolution)-tandem mass spectrometry with off-line radioactivity detection. Bendamustine and a total of 25 bendamustine-related compounds were detected. Observed metabolic conversions at the benzimidazole and butyric acid moiety were N-demethylation and γ-hydroxylation. In addition, various other combinations of these conversions with modifications at the mechlorethamine moiety were observed, including hydrolysis (the primary metabolic pathway), cysteine conjugation, and subsequent biotransformation to mercapturic acid and thiol derivatives, N-dealkylation, oxidation, and conjugation with phosphate, creatinine, and uric acid. Bendamustine-derived products containing phosphate, creatinine, and uric acid conjugates were also detected in control urine incubated with bendamustine. Metabolites that were excreted up to 168 h after the infusion included products of dihydrolysis and cysteine conjugation of bendamustine and γ-hydroxybendamustine. The range of metabolic reactions is generally consistent with those reported for rat urine and bile, suggesting that the overall processes involved in metabolic elimination are qualitatively the same in rats and humans.

Age Related Changes in Metabolite Concentrations in the Normal Spinal Cord

PubMed Central

Abdel-Aziz, Khaled; Solanky, Bhavana S.; Yiannakas, Marios C.; Altmann, Daniel R.; Wheeler-Kingshott, Claudia A. M.; Thompson, Alan J.; Ciccarelli, Olga

2014-01-01

Magnetic resonance spectroscopy (MRS) studies have previously described metabolite changes associated with aging of the healthy brain and provided insights into normal brain aging that can assist us in differentiating age-related changes from those associated with neurological disease. The present study investigates whether age-related changes in metabolite concentrations occur in the healthy cervical spinal cord. 25 healthy volunteers, aged 23–65 years, underwent conventional imaging and single-voxel MRS of the upper cervical cord using an optimised point resolved spectroscopy sequence on a 3T Achieva system. Metabolite concentrations normalised to unsuppressed water were quantified using LCModel and associations between age and spinal cord metabolite concentrations were examined using multiple regressions. A linear decline in total N-Acetyl-aspartate concentration (0.049 mmol/L lower per additional year of age, p = 0.010) and Glutamate-Glutamine concentration (0.054 mmol/L lower per additional year of age, p = 0.002) was seen within our sample age range, starting in the early twenties. The findings suggest that neuroaxonal loss and/or metabolic neuronal dysfunction, and decline in glutamate-glutamine neurotransmitter pool progress with aging. PMID:25310093

Bioconversion of dieldrin by wood-rotting fungi and metabolite detection.

PubMed

Kamei, Ichiro; Takagi, Kazuhiro; Kondo, Ryuichiro

2010-08-01

Dieldrin is one of the most persistent organochlorine pesticides, listed as one of the 12 persistent organic pollutants in the Stockholm Convention. Although microbial degradation is an effective way to remediate environmental pollutants, reports on aerobic microbial degradation of dieldrin are limited. Wood-rotting fungi can degrade a wide spectrum of recalcitrant organopollutants, and an attempt has been made to select wood-rotting fungi that can degrade dieldrin, and to identify the metabolite. Thirty-four isolates of wood-rotting fungi were investigated for their ability to degrade dieldrin. Strain YK543 degraded 39.1 +/- 8.8% of dieldrin during 30 days of incubation. Phylogenetic analysis demonstrated that strain YK543 was closely related to the fungus Phlebia brevispora Nakasone TMIC33929, which has been reported as a fungus that can degrade chlorinated dioxins and polychlorinated biphenyls. 9-Hydroxydieldrin was detected as a metabolite in the cultures of strain YK543. It is important to select the microorganisms that degrade organic pollutants, and to identify the metabolic pathway for the development of bioremediation methods. Strain YK543 was selected as a fungus capable of degrading dieldrin. The metabolic pathway includes 9-hydroxylation reported in rat's metabolism catalysed by liver microsomal monooxygenase. This is the first report of transformation of dieldrin to 9-hydroxydieldrin by a microorganism. Copyright (c) 2010 Society of Chemical Industry.

Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls.

PubMed

Möller, Ines; Wintermeyer, Annette; Bender, Katja; Jübner, Martin; Thomas, Andreas; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

2011-09-01

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine. Copyright © 2010 John Wiley & Sons, Ltd.

Hydroxyl migration disorders the surface structure of hydroxyapatite nanoparticles

NASA Astrophysics Data System (ADS)

Cheng, Xiajie; Wu, Hong; Zhang, Li; Ma, Xingtao; Zhang, Xingdong; Yang, Mingli

2017-09-01

The surface structure of nano-hydroxyapatite (HAP) was investigated using a combined simulated annealing and molecular dynamics method. The stationary structures of nano-HAP with 4-7 nm in diameter and annealed under different temperatures were analyzed in terms of pair distribution function, structural factor, mean square displacement and atomic coordination number. The particles possess different structures from bulk crystal. A clear radial change in their atomic arrangements was noted. From core to surface the structures change from ordered to disordered. A three-shell model was proposed to describe the structure evolution of nano-HAP. Atoms in the core zone keep their arrangements as in crystal, while atoms in the surface shell are in short-range order and long-range disorder, adopting a typically amorphous structure. Atoms in the middle shell have small displacements and/or deflections but basically retain their original locations as in crystal. The disordered shell is about 1 nm in thickness, in agreement with experimental observations. The disordering mainly stems from hydroxyl migration during which hydroxyls move to the surface and bond with the exposed Ca ions, and their left vacancies bring about a rearrangement of nearby atoms. The disordering is to some extent different for particles unannealed under different temperatures, resulting from fewer number of migrated hydroxyls at lower temperatures. Particles with different sizes have similar surface structures, and their surface energy decreases with increasing size. Moreover, the surface energy is reduced by hydroxyl migration because the exposed Ca ions on the surface are ionically bonded with the migrated hydroxyls. Our calculations proposed a new structure model for nano-HAP, which indicates a surface structure with activities different from those without surface reorganization. This is particularly interesting because most bioactivities of biomaterials are dominated by their surface activity.

Extractive biotransformation for production of metabolites of poorly soluble compounds: synthesis of 32-hydroxy-rifalazil.

PubMed

Mozhaev, Vadim V; Mozhaeva, Lyudmila V; Michels, Peter C; Khmelnitsky, Yuri L

2008-10-01

A novel reaction system was developed for the production of metabolites of poorly water-soluble parent compounds using mammalian liver microsomes. The system includes the selection and use of an appropriate hydrophobic polymeric resin as a reservoir for the hydrophobic parent compounds and its metabolites. The utility of the extractive biotransformation approach was shown for the production of a low-yielding, synthetically challenging 32-hydroxylated metabolite of the antibiotic rifalazil using mouse liver microsomes. To address the low solubility and reactivity of rifalazil in the predominantly aqueous microsomal catalytic system, a variety of strategies were tested for the enhanced delivery of hydrophobic substrates, including the addition of mild detergents, polyvinylpyrrolidone, glycerol, bovine serum albumin, and hydrophobic polymeric resins. The latter strategy was identified as the most suitable for the production of 32-hydroxy-rifalazil, resulting in up to 13-fold enhancement of the volumetric productivity compared with the standard aqueous system operating at the solubility limit of rifalazil. The production process was optimized for a wide range of reaction parameters; the most important for improving volumetric productivity included the type and amount of the polymeric resin, cofactor recycling system, concentrations of the biocatalyst and rifalazil, reaction temperature, and agitation rate. The optimized extractive biotransformation system was used to synthesize 32-hydroxy-rifalazil on a multimilligram scale.

Radiocarbon tracer measurements of atmospheric hydroxyl radical concentrations

NASA Technical Reports Server (NTRS)

Campbell, M. J.; Farmer, J. C.; Fitzner, C. A.; Henry, M. N.; Sheppard, J. C.

1986-01-01

The usefulness of the C-14 tracer in measurements of atmospheric hydroxyl radical concentration is discussed. The apparatus and the experimental conditions of three variations of a radiochemical method of atmosphere analysis are described and analyzed: the Teflon bag static reactor, the flow reactor (used in the Wallops Island tests), and the aircraft OH titration reactor. The procedure for reduction of the aircraft reactor instrument data is outlined. The problems connected with the measurement of hydroxyl radicals are discussed. It is suggested that the gas-phase radioisotope methods have considerable potential in measuring tropospheric impurities present in very low concentrations.

The profiling of the metabolites of hirsutine in rat by ultra-high performance liquid chromatography coupled with linear ion trap Orbitrap mass spectrometry: An improved strategy for the systematic screening and identification of metabolites in multi-samples in vivo.

PubMed

Wang, Jianwei; Qi, Peng; Hou, Jinjun; Shen, Yao; Yang, Min; Bi, Qirui; Deng, Yanping; Shi, Xiaojian; Feng, Ruihong; Feng, Zijin; Wu, Wanying; Guo, Dean

2017-02-05

Drug metabolites identification and construction of metabolic profile are meaningful work for the drug discovery and development. The great challenge during this process is the work of the structural clarification of possible metabolites in the complicated biological matrix, which often resulting in a huge amount data sets, especially in multi-samples in vivo. Analyzing these complex data manually is time-consuming and laborious. The object of this study was to develop a practical strategy for screening and identifying of metabolites from multiple biological samples efficiently. Using hirsutine (HTI), an active components of Uncaria rhynchophylla (Gouteng in Chinese) as a model and its plasma, urine, bile, feces and various tissues were analyzed with data processing software (Metwork), data mining tool (Progenesis QI), and HR-MS n data by ultra-high performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS). A total of 67 metabolites of HTI in rat biological samples were tentatively identified with established library, and to our knowledge most of which were reported for the first time. The possible metabolic pathways were subsequently proposed, hydroxylation, dehydrogenation, oxidation, N-oxidation, hydrolysis, reduction and glucuronide conjugation were mainly involved according to metabolic profile. The result proved application of this improved strategy was efficient, rapid, and reliable for metabolic profiling of components in multiple biological samples and could significantly expand our understanding of metabolic situation of TCM in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of hydroxyl radical reaction with aromatics by dissolved natural organic matter

USGS Publications Warehouse

Lindsey, M.E.; Tarr, M.A.

2000-01-01

Reaction of aromatic compounds with hydroxyl radical is inhibited by dissolved natural organic matter (NOM). The degree of inhibition is significantly greater than that expected based on a simple model in which aromatic compound molecules bound to NOM are considered to be unreactive. In this study, hydroxyl radical was produced at steady-state concentrations using Fenton chemistry (H2O2 + Fe2+ ??? Fe3+ + HO- + HO??). Suwannee River fulvic acid and humic acid were used as NOM. The most likely mechanism for the observed inhibition is that hydroxyl radical formation occurs in microenvironmental sites remote from the aromatic compounds. In addition to changes in kinetics, pyrene hydroxyl radical reaction also exhibited a mechanistic change in the presence of fulvic acid. The mechanism changed from a reaction that was apparently firstorder in pyrene to one that was apparently secondorder in pyrene, indicating that pyrene self-reaction may have become the dominant mechanism in the presence of fulvic acid. Dissolved NOM causes significant changes in the rate and mechanism of hydroxyl radical degradation of aromatic compounds. Consequently, literature rate constants measured in pure water will not be useful for predicting the degradation of pollutants in environmental systems. The kinetic and mechanistic information in this study will be useful for developing improved degradation methods involving Fenton chemistry.Reaction of aromatic compounds with hydroxyl radical is inhibited by dissolved natural organic matter (NOM). The degree of inhibition is significantly greater than that expected based on a simple model in which aromatic compounds molecules bounds to NOM are considered to be unreactive. In this study, hydroxyl radical was produced at steady-state concentrations using Fenton chemistry (H2O2 + Fe2+ ??? Fe3+ + HO- + HO??). Suwannee River fulvic acid and humic acid were used as NOM. The most likely mechanisms for the observed inhibition is that hydroxyl radical

Sulfur Dioxide Capture by Heterogeneous Oxidation on Hydroxylated Manganese Dioxide.

PubMed

Wu, Haodong; Cai, Weimin; Long, Mingce; Wang, Hairui; Wang, Zhiping; Chen, Chen; Hu, Xiaofang; Yu, Xiaojuan

2016-06-07

Here we demonstrate that sulfur dioxide (SO2) is efficiently captured via heterogeneous oxidation into sulfate on the surface of hydroxylated manganese dioxide (MnO2). Lab-scale activity tests in a fluidized bed reactor showed that the removal efficiency for a simulated flue gas containing 5000 mg·Nm(-3) SO2 could reach nearly 100% with a GHSV (gas hourly space velocity) of 10000 h(-1). The mechanism was investigated using a combination of experimental characterizations and theoretical calculations. It was found that formation of surface bound sulfate proceeds via association of SO2 with terminal hydroxyls. Both H2O and O2 are essential for the generation of reactive terminal hydroxyls, and the indirect role of O2 in heterogeneous SO2 oxidation at low temperature was also revealed. We propose that the high reactivity of terminal hydroxyls is attributed to the proper surface configuration of MnO2 to adsorb water with degenerate energies for associative and dissociative states, and maintain rapid proton dynamics. Viability analyses suggest that the desulfurization method that is based on such a direct oxidation reaction at the gas/solid interface represents a promising approach for SO2 capture.

Degradation in soil and water and ecotoxicity of rimsulfuron and its metabolites.

PubMed

Martins, J M; Chevre, N; Spack, L; Tarradellas, J; Mermoud, A

2001-11-01

The degradation and ecotoxicity of sulfonylurea herbicide rimsulfuron and its major metabolites were examined in batch samples of an alluvial sandy loam and in freshwater. An HPLC-DAD method was adapted to simultaneously identify and quantify rimsulfuron and its metabolites, which was successfully validated by GC-MS analysis. In aqueous solutions, pure rimsulfuron was rapidly hydrolyzed into metabolite 1 (N-(4,6-dimethoxypyrimidin-2-yl)-N-(3-(ethylsulfonyl)-2-pyridinylurea)), which itself was transformed into the more stable metabolite 2 (N-((3-(ethylsulfonyl)-2-pyridinyl)-4,6-dimethoxy-2-pyrimidineamine)), with half-life (t(1/2)) values of 2 and 2.5 days, respectively. Hydrolysis was instantaneous under alkaline conditions (pH = 10). In aqueous suspensions of the alluvial soil (pH = 8), formulated rimsulfuron had a half-life of 7 days, whereas that of metabolite 1 was similar to that in water (about 3.5 days). The degradation of the two major metabolites was also studied in soil suspensions with the pure compounds at concentrations ranging from 1 to 10 mg l(-1). The half-life of metabolite 1 ranged from 3.9 to 5 days, close to the previous values. Metabolite 2 was more persistent and its degradation is strongly dependent on the initial concentration (C0): half-life values ranged from 8.1 to 55 days at 2-10 mg l(-1), respectively. These values are higher than those determined from the kinetics of metabolite 1 transformation into metabolite 2 (t(1/2) = 8-19 days). The ecotoxicity of the three chemicals was evaluated through their effect on Daphnia magna and Vibrio fischeri (Microtox bioassay). No effect was observed on D. magna with 24 and 48 h acute toxicity tests. Similarly, no toxic effect was observed with the Microtox test for the three chemicals in the range of concentrations tested that included the field application dose. Thus, being of low persistence and lacking acute toxicity, these chemicals present a low environmental risk. However, chronic effects

Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites.

PubMed

Liu, J Y; Song, Y C; Zhang, Z; Wang, L; Guo, Z J; Zou, W X; Tan, R X

2004-11-09

Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5alpha,8alpha-epidioxy-ergosta-6,22-diene-3beta-ol as well as other four known compounds ergosta-4,22-diene-3beta-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5'-methoxyl-7'-methyl-(1'H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 microg/mL, respectively.

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.

Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

PubMed

Homan, Erica P; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M; Dawson, Brian; Bertin, Terry K; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H L

2014-01-01

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

ALTERATION OF CATECHOLAMINES IN PHOECHROMOCYTOMA (PC12) CELLS IN VITRO BY THE METABOLITES OF CHLOROTRIAZINE HERBICIDE

EPA Science Inventory

The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified following treatme...

Curable liquid hydrocarbon prepolymers containing hydroxyl groups and process for producing same

NASA Technical Reports Server (NTRS)

Rhein, R. A.; Ingham, J. D. (Inventor)

1978-01-01

Production of hydroxyl containing curable liquid hydrocarbon prepolymers by ozonizing a high molecular weight saturated hydrocarbon polymer such as polyisobutylene or ethylene propylene rubber is discussed. The ozonized material is reduced using reducing agents, preferably diisobutyl aluminum hydride, to form the hydroxyl containing liquid prepolymers having a substantially lower molecular weight than the parent polymer. The resulting curable liquid hydroxyl containing prepolymers can be poured into a mold and readily cured, with reactants such as toluene diisocyanate, to produce highly stable elastomers having a variety of uses such as binders for solid propellants.

Non-linear hydroxyl radical formation rate in dispersions containing mixtures of pyrite and chalcopyrite particles

NASA Astrophysics Data System (ADS)

Kaur, Jasmeet; Schoonen, Martin A.

2017-06-01

The formation of hydroxyl radicals was studied in mixed pyrite-chalcopyrite dispersions in water using the conversion rate of adenine as a proxy for hydroxyl radical formation rate. Experiments were conducted as a function of pH, presence of phosphate buffer, surface loading, and pyrite-to-chalcopyrite ratio. The results indicate that hydroxyl radical formation rate in mixed systems is non-linear with respect to the rates in the pure endmember dispersions. The only exception is a set of experiments in which phosphate buffer is used. In the presence of phosphate buffer, the hydroxyl radical formation is suppressed in mixtures and the rate is close to that predicted based on the reaction kinetics of the pure endmembers. The non-linear hydroxyl radical formation in dispersions containing mixtures of pyrite and chalcopyrite is likely the result of two complementary processes. One is the fact that pyrite and chalcopyrite form a galvanic couple. In this arrangement, chalcopyrite oxidation is accelerated, while pyrite passes electrons withdrawn from chalcopyrite to molecular oxygen, the oxidant. The incomplete reduction of molecular oxygen leads to the formation of hydrogen peroxide and hydroxyl radical. The galvanic coupling appears to be augmented by the fact that chalcopyrite generates a significant amount of hydrogen peroxide upon dispersal in water. This hydrogen peroxide is then available for conversion to hydroxyl radical, which appears to be facilitated by pyrite as chalcopyrite itself produces only minor amounts of hydroxyl radical. In essence, pyrite is a ;co-factor; that facilitates the conversion of hydrogen peroxide to hydroxyl radical. This conversion reaction is a surface-mediated reaction. Given that hydroxyl radical is one of the most reactive species in nature, the formation of hydroxyl radicals in aqueous systems containing chalcopyrite and pyrite has implications for the stability of organic molecules, biomolecules, the viability of microbes, and

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

PubMed

Pencík, Aleš; Casanova-Sáez, Rubén; Pilarová, Veronika; Žukauskaite, Asta; Pinto, Rui; Micol, José Luis; Ljung, Karin; Novák, Ondrej

2018-04-27

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

Non-targeted analysis of petroleum metabolites in groundwater using GC×GC-TOFMS.

PubMed

Mohler, Rachel E; O'Reilly, Kirk T; Zemo, Dawn A; Tiwary, Asheesh K; Magaw, Renae I; Synowiec, Karen A

2013-09-17

Groundwater at fuel release sites often contains nonpolar hydrocarbons that originate from both the fuel release and other environmental sources, as well as polar metabolites of petroleum biodegradation. These compounds, along with other polar artifacts, can be quantified as "total petroleum hydrocarbons" using USEPA Methods 3510/8015B, unless a silica gel cleanup step is used to separate nonpolar hydrocarbons from polar compounds prior to analysis. Only a limited number of these metabolites have been identified by traditional GC-MS methods, because they are difficult to resolve using single-column configurations. Additionally, the targeted use of derivatization limits the detection of many potential metabolites of interest. The objective of this research was to develop a nontargeted GC×GC-TOFMS approach to characterize petroleum metabolites in environmental samples gathered from fuel release sites. The method tentatively identified more than 760 unique polar compounds, including acids/esters, alcohols, phenols, ketones, and aldehydes, from 22 groundwater samples collected at five sites. Standards for 28 polar compounds indicate that effective limits of quantitation for most of these compounds in the groundwater samples range from 1 to 11 μg/L.

Moringa oleifera leaf extracts inhibit 6beta-hydroxylation of testosterone by CYP3A4.

PubMed

Monera, Tsitsi G; Wolfe, Alan R; Maponga, Charles C; Benet, Leslie Z; Guglielmo, Joseph

2008-10-01

Moringa oleifera is a tropical tree often used as a herbal medicine, including by people who test positive for HIV. Since herbal constituents may interact with drugs via inhibition of metabolizing enzymes, we investigated the effects of extracts of M. oleifera on the CYP3A4-mediated 6beta-hydroxylation of testosterone. Methanolic and aqueous leaf and root of extracts of M. oleifera with concentrations between 0.01 and 10 mg/ml were incubated with testosterone and mixed-sex human liver microsomes in the presence of NADPH. Metabolite concentrations were determined by HPLC. The cytotoxicity of the extracts was tested with HepG2 cells using the MTT formazan assay. Significant CYP3A4 inhibitory effects were found, with IC50 values of 0.5 and 2.5 mg/ml for leaf-methanol and leaf-water extracts, respectively. Root extracts were less active. Cytotoxicity was observed only with the leaf-water extract (IC50 = 6 mg/ml). Further investigation is warranted to elucidate the potential of M. oleifera for clinically significant interactions with antiretroviral and other drugs.

Measurement of hydroxyl radical production in ultrasonic aqueous solutions by a novel chemiluminescence method.

PubMed

Hu, Yufei; Zhang, Zhujun; Yang, Chunyan

2008-07-01

Measurement methods for ultrasonic fields are important for reasons of safety. The investigation of an ultrasonic field can be performed by detecting the yield of hydroxyl radicals resulting from ultrasonic cavitations. In this paper, a novel method is introduced for detecting hydroxyl radicals by a chemiluminescence (CL) reaction of luminol-hydrogen peroxide (H2O2)-K5[Cu(HIO6)2](DPC). The yield of hydroxyl radicals is calculated directly by the relative CL intensity according to the corresponding concentration of H2O2. This proposed CL method makes it possible to perform an in-line and real-time assay of hydroxyl radicals in an ultrasonic aqueous solution. With flow injection (FI) technology, this novel CL reaction is sensitive enough to detect ultra trace amounts of H2O2 with a limit of detection (3sigma) of 4.1 x 10(-11) mol L(-1). The influences of ultrasonic output power and ultrasonic treatment time on the yield of hydroxyl radicals by an ultrasound generator were also studied. The results indicate that the amount of hydroxyl radicals increases with the increase of ultrasonic output power (< or = 15 W mL(-1)). There is a linear relationship between the time of ultrasonic treatment and the yield of H2O2. The ultrasonic field of an ultrasonic cleaning baths has been measured by calculating the yield of hydroxyl radicals.

Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites.

PubMed

Khan, Naik T; Zafar, Salman; Noreen, Shagufta; Al Majid, Abdullah M; Al Othman, Zeid A; Al-Resayes, Saud Ibrahim; Atta-ur-Rahman; Choudhary, M Iqbal

2014-07-01

Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2-6, while M. phaseolina transformed compound 1 into polar metabolites 7-11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12-14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1-14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC50 values between 49.0 and 84.9 μM. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of the stereoselective biotransformation of permethrin in human liver microsomes: contributions of cytochrome P450 monooxygenases to the formation of estrogenic metabolites.

PubMed

Lavado, Ramon; Li, Jiwen; Rimoldi, John M; Schlenk, Daniel

2014-04-21

Permethrin (PM) is a pyrethroid insecticide that exists as 4 enantiomers. Biotransformation of PM to estrogen receptor agonists (3-phenoxybenzyl alcohol (PBOH) and 3-(4'-hydroxyphenoxy)-benzyl alcohol (3,4 PBOH)) has been shown to be stereoselective in other vertebrate species. This study evaluated the biotransformation of PM enantiomers in human liver microsomes and with recombinant CYP3A4 and CYP2C19. PBOH and 3,4 PBOH were the only metabolites detected from in vitro incubations including each of the 4 enantiomers of PM with 1R-trans PM having the most efficient NADPH-catalyzed biotransformation to both metabolites. Coincubation with the CYP inhibitor ketoconazole and time course experiments with liver microsomes and recombinant CYP2C19 and CYP3A4 indicated CYP-catalyzed stereoselective cleavage of the ester followed by 4-hydoxylation to 3,4' PBOH. These data indicate potential dispositional differences may occur with PM enantiomers and a shift in putative molecular targets. While cleavage of pyrethroid esters lead to detoxification of the acute neurological effects, formation of the benzyl alcohol and hydroxylated metabolite may lead to estrogenic responses, since each of these metabolites are estrogen receptor ligands. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Metabolomics of Pichia pastoris: impact of buffering conditions on the kinetics and nature of metabolite loss during quenching.

PubMed

Mattanovich, Matthias; Russmayer, Hannes; Scharl-Hirsch, Theresa; Puxbaum, Verena; Burgard, Jonas; Mattanovich, Diethard; Hann, Stephan

2017-05-01

Mass spectrometry-based metabolomic profiling is a powerful strategy to quantify the concentrations of numerous primary metabolites in parallel. To avoid distortion of metabolite concentrations, quenching is applied to stop the cellular metabolism instantly. For yeasts, cold methanol quenching is accepted to be the most suitable method to stop metabolism, while keeping the cells intact for separation from the supernatant. During this treatment, metabolite loss may occur while the cells are suspended in the quenching solution. An experiment for measuring the time-dependent loss of selected primary metabolites in differently buffered quenching solutions was conducted to study pH and salt concentration-dependent effects. Molecular properties of the observed metabolites were correlated with the kinetics of loss to gain insight into the mechanisms of metabolite leakage. Size and charge-related properties play a major role in controlling metabolite loss. We found evidence that interaction with the cell wall is the main determinant to retain a molecule inside the cell. Besides suggesting an improved quenching protocol to keep loss at a minimum, we could establish a more general understanding of the process of metabolite loss during quenching, which will allow to predict optimal conditions for hitherto not analysed metabolites. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

α-Tocopherol does not accelerate depletion of γ-tocopherol and tocotrienol or excretion of their metabolites in rats.

PubMed

Uchida, Tomono; Nomura, Saki; Sakuma, Eri; Hanzawa, Fumiaki; Ikeda, Saiko

2013-07-01

From an enzyme kinetic study using rat liver microsomes, α-tocopherol has been suggested to accelerate the other vitamin E catabolism by stimulating vitamin E ω-hydroxylation, the late limiting reaction of the vitamin E catabolic pathway. To test the effect of α-tocopherol on catabolism of the other vitamin E isoforms in vivo, we determined whether α-tocopherol accelerates depletion of γ-tocopherol and tocotrienol and excretion of their metabolites in rats. Male Wistar rats were fed a γ-tocopherol-rich diet for 6 weeks followed by a γ-tocopherol-free diet with or without α-tocopherol for 7 days. Intake of γ-tocopherol-free diets lowered γ-tocopherol concentrations in serum, liver, adrenal gland, small intestine, and heart, but there was no effect of dietary α-tocopherol on γ-tocopherol concentrations. The level of urinary excretion of γ-tocopherol metabolite was not affected by dietary α-tocopherol. Next, the effect of α-tocopherol on tocotrienol depletion was examined using rats fed a tocotrienol-rich diet for 6 weeks. Subsequent intake of a tocotrienol-free diet with or without α-tocopherol for 7 days depleted concentrations of α- and γ-tocotrienol in serum and tissues, which was accompanied by a decrease in the excretion of γ-tocotrienol metabolite. However, neither the tocotrienol concentration nor γ-tocotrienol metabolite excretion was affected by dietary α-tocopherol. These data showed that dietary α-tocopherol did not accelerate the depletion of γ-tocopherol and tocotrienol and their metabolite excretions, suggesting that the positive effect of α-tocopherol on vitamin E ω-hydroxylase is not sufficient to affect the other isoform concentrations in tissues.

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption.

PubMed

Kremer, Jonathan Isaak; Gömpel, Katharina; Bakuradze, Tamara; Eisenbrand, Gerhard; Richling, Elke

2018-04-01

Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet. We performed a 4-day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 μmol nicotinic acid (NA) and 0.58 μmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N 1 -methylnicotinamide (NMNAM), N 1 -methyl-2-pyridone-5-carboxamide (2-Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC-ESI-MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2-Py), with t max values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h -1 of NA, NAM, NMNAM, and 2-Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM. The results indicate regular coffee consumption to be a source of niacin in human diet. © 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HETEROGENOUS PHOTOREACTION OF FORMALDEHYDE WITH HYDROXYL RADICALS

EPA Science Inventory

Atmospheric heterogeneous photoreactions occur between formaldehyde and hydroxyl radicals to produce formic acid. hese photoreactions not only occur in clouds, but also in other tropospheric hydrometeors such as precipitation and dew droplets. xperiments were performed by irradia...

The hydroxyl species and acid sites on diatomite surface: a combined IR and Raman study

NASA Astrophysics Data System (ADS)

Yuan, P.; Wu, D. Q.; He, H. P.; Lin, Z. Y.

2004-04-01

Diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), Raman spectroscopy of adsorbed pyridine molecules (Py-Raman) and in situ Py-IR have been used to investigate the hydroxyl species and acid sites on diatomite surfaces. The Lewis (L) and Brønsted (B) acid sites, and various hydroxyl species, including isolated hydroxyl groups, H-bonded hydroxyl groups and physically adsorbed water, are identified. The L acid sites in diatomite samples are resulted from the clay impurities, and the B acid sites are resulted from some moderate strength H-bonded hydroxyl groups. At room temperature, both of the isolated and H-bonded silanols associate with the physically adsorbed water by hydrogen bond. After calcination treatment, physically adsorbed water will be desorbed from the silanols, and the silanols will condense with the increase of temperature. Generally, the H-bonded silanols condense more easily than the isolated ones. The properties of surface hydroxyl species of diatomaceous silica are more similar to precipitated silica rather than fumed silica.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study

Identification of absorbed constituents and in vivo metabolites in rats after oral administration of Physalis alkekengi var. franchetii by ultra-high-pressure liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Feng, Xinchi; Huo, Xiaoguang; Liu, Hongxia; Chai, Liwei; Ding, Liqin; Qiu, Feng

2018-03-01

The calyces of Physalis alkekengi var. franchetii (Chinese Lantern, JDL) are well-known as traditional Chinese medicine owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of JDL and their metabolites in vivo are still unclear to date. In this paper, an ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) method was established to identify absorbed constituents and in vivo metabolites in rat biological fluids after oral administration of JDL. Based on the proposed strategy, 33 compounds were observed in dosed rat biosamples. Twelve of 33 compounds were indicated as prototype components of JDL, and 21 compounds were predicted to be metabolites of JDL. Finally, the metabolic pathways were proposed, which were glucuronidation, sulfation, methylation and dehydroxylation for flavonoid constituents and sulfonation and hydroxylation for physalin consitituents. This is the first systematic study on the absorbed constituents and metabolic profiling of JDL and will provide a useful template for screening and characterizing the ingredients and metabolites of traditional Chinese medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Characterization of in vivo metabolites in rat urine following an oral dose of masitinib by liquid chromatography tandem mass spectrometry.

PubMed

Kadi, Adnan A; Amer, Sawsan M; Darwish, Hany W; Attwa, Mohamed W

2018-05-15

Masitinib (MST) is an orally administered drug that targets mast cells and macrophages, important cells for immunity, by inhibiting a limited number of tyrosine kinases. It is currently registered in Europe and USA for the treatment of mast cell tumors in dogs. AB Science announced that the European Medicines Agency has accepted a conditional marketing authorization application for MST to treat amyotrophic lateral sclerosis. In our work, we focused on studying in vivo metabolism of MST in Sprague-Dawley rats. Single oral dose of MST (33 mg kg -1 ) was given to Sprague-Dawley rats (kept in metabolic cages) using oral gavage. Urine was collected and filtered at 0, 6, 12, 18, 24, 48, 72 and 96 h from MST dosing. An equal amount of ACN was added to urine samples. Both organic and aqueous layers were injected into liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect in vivo phase I and phase II MST metabolites. The current work reports the identification and characterization of twenty in vivo phase I and four in vivo phase II metabolites of MST by LC-MS/MS. Phase I metabolic pathways were reduction, demethylation, hydroxylation, oxidative deamination, oxidation and N-oxide formation. Phase II metabolic pathways were the direct conjugation of MST, N-demethyl metabolites and oxidative metabolites with glucuronic acid. Part of MST dose was excreted unchanged in urine. The literature review showed no previous articles have been made on in vivo metabolism of MST or detailed structural identification of the formed in vivo phase I and phase II metabolites.

Quantitative Method for Simultaneous Analysis of Acetaminophen and 6 Metabolites.

PubMed

Lammers, Laureen A; Achterbergh, Roos; Pistorius, Marcel C M; Romijn, Johannes A; Mathôt, Ron A A

2017-04-01

Hepatotoxicity after ingestion of high-dose acetaminophen [N-acetyl-para-aminophenol (APAP)] is caused by the metabolites of the drug. To gain more insight into factors influencing susceptibility to APAP hepatotoxicity, quantification of APAP and metabolites is important. A few methods have been developed to simultaneously quantify APAP and its most important metabolites. However, these methods require a comprehensive sample preparation and long run times. The aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of acetaminophen, the main metabolites acetaminophen glucuronide and acetaminophen sulfate, and 4 Cytochrome P450-mediated metabolites by using liquid chromatography with mass spectrometric (LC-MS) detection. The method was developed and validated for the human plasma, and it entailed a single method for sample preparation, enabling quick processing of the samples followed by an LC-MS method with a chromatographic run time of 9 minutes. The method was validated for selectivity, linearity, accuracy, imprecision, dilution integrity, recovery, process efficiency, ionization efficiency, and carryover effect. The method showed good selectivity without matrix interferences. For all analytes, the mean process efficiency was >86%, and the mean ionization efficiency was >94%. Furthermore, the accuracy was between 90.3% and 112% for all analytes, and the within- and between-run imprecision were <20% for the lower limit of quantification and <14.3% for the middle level and upper limit of quantification. The method presented here enables the simultaneous quantification of APAP and 6 of its metabolites. It is less time consuming than previously reported methods because it requires only a single and simple method for the sample preparation followed by an LC-MS method with a short run time. Therefore, this analytical method provides a useful method for both clinical and research purposes.

Metabolic profile of glyburide in human liver microsomes using LC-DAD-Q-TRAP-MS/MS.

PubMed

Ravindran, Selvan; Basu, Sudipta; Gorti, Santosh Kapil Kumar; Surve, Prashant; Sloka, Navya

2013-05-01

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography-diode array detector-quadruple-ion trap-mass spectrometry/mass spectrometry (LC-DAD-Q-TRAP-MS/MS). An enhanced mass scan-enhanced product ion scan with information-dependent acquisition mode in a Q-TRAP-MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.

Structure and Dynamics of Hydroxyl-Functionalized Protic Ammonium Carboxylate Ionic Liquids.

PubMed

Thummuru, Dhileep Nagi Reddy; Mallik, Bhabani S

2017-10-26

We performed classical molecular dynamics simulations to investigate the structure and dynamics of protic ionic liquids, 2-hydroxy ethylammonium acetate, ethylammonium hydroxyacetate, and 2-hydroxyethylammonium hydroxyacetate at ambient conditions. Structural properties such as density, radial distribution functions, spatial distribution functions, and structure factors have been calculated. Dynamic properties such as mean square displacements, as well as residence and hydrogen bond dynamics have also been calculated. Hydrogen bond lifetimes and residence times change with the addition of hydroxyl groups. We observe that when a hydroxyl group is present on the cation, dynamics become very slow and it forms a strong hydrogen bond with carboxylate oxygen atoms of the anion. The hydroxyl functionalized ILs show more dynamic diversity than structurally similar ILs.

Atmospheric Hydroxyl Radical Production from Electronically Excited NO2 and H2O

NASA Astrophysics Data System (ADS)

Li, Shuping; Matthews, Jamie; Sinha, Amitabha

2008-03-01

Hydroxyl radicals are often called the “detergent” of the atmosphere because they control the atmosphere’s capacity to cleanse itself of pollutants. Here, we show that the reaction of electronically excited nitrogen dioxide with water can be an important source of tropospheric hydroxyl radicals. Using measured rate data, along with available solar flux and atmospheric mixing ratios, we demonstrate that the tropospheric hydroxyl contribution from this source can be a substantial fraction (50%) of that from the traditional O(1D) + H2O reaction in the boundary-layer region for high solar zenith angles. Inclusion of this chemistry is expected to affect modeling of urban air quality, where the interactions of sunlight with emitted NOx species, volatile organic compounds, and hydroxyl radicals are central in determining the rate of ozone formation.

Atmospheric hydroxyl radical production from electronically excited NO2 and H2O.

PubMed

Li, Shuping; Matthews, Jamie; Sinha, Amitabha

2008-03-21

Hydroxyl radicals are often called the "detergent" of the atmosphere because they control the atmosphere's capacity to cleanse itself of pollutants. Here, we show that the reaction of electronically excited nitrogen dioxide with water can be an important source of tropospheric hydroxyl radicals. Using measured rate data, along with available solar flux and atmospheric mixing ratios, we demonstrate that the tropospheric hydroxyl contribution from this source can be a substantial fraction (50%) of that from the traditional O(1D) + H2O reaction in the boundary-layer region for high solar zenith angles. Inclusion of this chemistry is expected to affect modeling of urban air quality, where the interactions of sunlight with emitted NOx species, volatile organic compounds, and hydroxyl radicals are central in determining the rate of ozone formation.

Metabolic regulation and overproduction of primary metabolites

PubMed Central

Sanchez, Sergio; Demain, Arnold L.

2008-01-01

Summary Overproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Biosynthesis of enzymes catalysing metabolic reactions in microbial cells is controlled by well‐known positive and negative mechanisms, e.g. induction, nutritional regulation (carbon or nitrogen source regulation), feedback regulation, etc. The microbial production of primary metabolites contributes significantly to the quality of life. Fermentative production of these compounds is still an important goal of modern biotechnology. Through fermentation, microorganisms growing on inexpensive carbon and nitrogen sources produce valuable products such as amino acids, nucleotides, organic acids and vitamins which can be added to food to enhance its flavour, or increase its nutritive values. The contribution of microorganisms goes well beyond the food and health industries with the renewed interest in solvent fermentations. Microorganisms have the potential to provide many petroleum‐derived products as well as the ethanol necessary for liquid fuel. Additional applications of primary metabolites lie in their impact as precursors of many pharmaceutical compounds. The roles of primary metabolites and the microbes which produce them will certainly increase in importance as time goes on. In the early years of fermentation processes, development of producing strains initially depended on classical strain breeding involving repeated random mutations, each followed by screening or selection. More recently, methods of molecular genetics have been used for the overproduction of primary metabolic products. The development of modern tools of molecular biology enabled more rational approaches for strain improvement. Techniques of transcriptome, proteome and metabolome analysis, as well as metabolic flux analysis. have recently been introduced in order to identify new and

Estrogen Metabolites Are Not Associated With Colorectal Cancer Risk In Postmenopausal Women

PubMed Central

Falk, Roni T.; Dallal, Cher M.; Lacey, James V.; Bauer, Douglas C.; Buist, Diana SM; Cauley, Jane A.; Hue, Trisha F.; LaCroix, Andrea; Tice, Jeffrey A.; Pfeiffer, Ruth M.; Xu, Xia; Veenstra, Timothy D.; Brinton, Louise A.

2015-01-01

Background A potential protective role for estrogen in colon carcinogenesis has been suggested based on exogenous hormone use, but it is unclear from previous studies whether endogenous estrogens are related to colorectal cancer (CRC) risk. These few prior studies focused on parent estrogens; none evaluated effects of estrogen metabolism in postmenopausal women. Methods We followed 15,595 women (ages 55–80) enrolled in B~FIT (Breast and Bone Follow-up to the Fracture Intervention Trial (FIT)) who donated blood between 1992 and 1993 for cancer through December 2004. A panel of 15 estrogen metabolites (EM), including estradiol and estrone, were measured in serum from 187 CRC cases and a subcohort of 501 women not using exogenous hormones at blood draw. We examined EM individually, grouped by pathway (hydroxylation at the C-2, C-4, or C-16 position), and by ratios of the groupings using Cox proportional hazards regression models. Results No significant associations were seen for estrone (HRQ4 v Q1=1.15, 95% CI=0.69–1.93, ptrend=0.54), estradiol (HRQ4 v Q1= 0.98, 95% CI=0.58–1.64, ptrend>0.99) or total EM (the sum of all EM; HRQ4 v Q1=1.35. 95% CI=0.81–2.24, ptrend=0.33). Most metabolites in the 2-, 4- or 16-pathway were unrelated to risk, although a borderline trend in risk was associated with high levels of 17-epiestriol. Conclusion Circulating estrogens and their metabolites were generally unrelated to CRC risk in postmenopausal women. Impact Additional studies are needed to understand how exogenous estrogen may prevent CRC PMID:26104910

Follicular fluid and urinary concentrations of phthalate metabolites among infertile women and associations with in vitro fertilization parameters.

PubMed

Du, Yao-Yao; Fang, Yue-Li; Wang, Yi-Xin; Zeng, Qiang; Guo, Na; Zhao, Hua; Li, Yu-Feng

2016-06-01

Evidence from toxicological studies has demonstrated that phthalates can lead to reduced fertility through effects on folliculogenesis, oocyte maturation and embryonic development, but human data are limited. Concentrations of eight phthalate metabolites in 110 follicular fluid (FF) and urine samples collected from 112 women attending an infertility clinic in Wuhan, China were quantified, and correlations between paired matrices were explored. Associations between metabolite concentrations and in vitro fertilization (IVF) parameters were evaluated with multivariable models. Six metabolites were detected in >72.73% of the FF samples. MEHP and MBP were the dominant metabolites with a median level of 2.80 and 2.05ng/mL, respectively. Significant correlations between the two matrices, urine and FF, were found for MEP (rs=0.44), and MBP (rs=0.22). FF and urinary metabolite concentrations were not associated with any IVF parameters. However, given the prevalence of phthalates exposure, further work is needed to elucidate the potential hazard on female reproduction. Copyright © 2016 Elsevier Inc. All rights reserved.

SU-F-T-676: Measurement of Hydroxyl Radicals in Radiolized Water Systems

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

Ouyang, Z; Ngwa, W; Brigham and Women’s Hospital, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA

2016-06-15

Purpose: Hydroxyl radicals can be produced within tissue by radiation therapy, and they are largely responsible for DNA damage and cell killing. Coumarin-3-carboxylic acid (3-CCA) and crystal violet are reported to react with hydroxyl radicals and can be used for fluorescence and absorbance measurements, respectively. This study assesses the ability of hydroxyl measurement for both 3-CCA and crystal violet in radiolized water systems in order to provide dosimetric information in radiation chemistry and radiation biology experiments. Methods: 3-CCA and crystal violet were both dissolved in phosphate buffered saline (PBS, pH 7.4) with final concentrations 0.5 mg/mL and 0.05 mg/mL. 3-CCAmore » and control solutions (PBS only) were loaded in black bottom 96-well plates. Crystal violet and control solutions were loaded in clear bottom 96-well plates. The prepared solutions were irradiated at 2 Gy using a small animal radiation research platform. Fluorescence reading with 360 nm excitation wavelength and 485 nm emission wavelength was done for 3-CCA, and absorbance reading at wavelength 580 nm was done for crystal violet before and after radiation. Results: 3-CCA showed clear difference in fluorescence before and after radiation, which suggested hydroxyl production during radiation. However, crystal violet absorbance at 580 nm was not changed significantly by radiation. Conclusion: The overall conclusion is that 3-CCA can be used for hydroxyl measurement in radiolized water systems, while crystal violet cannot, although crystal violet is reported widely to react with hydroxyl radicals produced in Fenton reactions. Possible reasons could relate to reaction pH.« less

Oxybuprocaine and five metabolites simultaneously determined in urine by gas chromatography and gas chromatography-mass spectrometry after extraction with Extrelut.

PubMed

Kasuya, F; Igarashi, K; Fukui, M

1987-05-01

We describe a gas-liquid chromatographic (GC) method for determination of oxybuprocaine, and a gas chromatographic-mass spectrometric (GC-MS) method for simultaneous determination of four of its nine metabolites in urine. We used an Extrelut column to simply and rapidly extract oxybuprocaine and its metabolites from urine. For the GC-MS analyses, we monitored the characteristic fragment ions at m/z 353, 395, 369, 411, and 235 for 3-butoxy-4-aminobenzoic acid (metabolite 2, M-2), 3-butoxy-4-acetylaminobenzoic acid (M-3), 3-hydroxy-4-aminobenzoic acid (M-4), 3-hydroxy-4-acetylaminobenzoic acid (M-5), and methaqualone (internal standard), respectively. We quantified the glucuronide of M-2 after enzymic treatment. The assay's selectivity and reproducibility (within-day and between-day CVs less than 8% for all metabolites) make it applicable to determine oxybuprocaine and its metabolites in human urine. Mean 9-h urinary excretion of oxybuprocaine and its five metabolites from four healthy volunteers was 89.2% after a 100-mg oral dose.

Simultaneous determination of eight metabolites of organophosphate and pyrethroid pesticides in urine.

PubMed

Guo, Xin Y; Sun, Li S; Huang, Meng Y; Xu, Wei L; Wang, Ying; Wang, Na

2017-01-02

A simultaneous method for quantifying eight metabolites of organophosphate pesticides and pyrethroid pesticides in urine samples has been established. The analytes were extracted using liquid-liquid extraction coupled with WCX solid phase extraction (SPE) cartridges. Eight metabolites were chemically derivatized before analysis using gas chromatography-tandem mass spectrometry (GC-MS-MS). The separation was performed on a HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm) with temperature programming. The detection was performed under electro-spray ionization (ESI) in multiple reaction monitoring (MRM) mode. An internal standard method was used. The extraction solvent, types of SPE cartridges and eluents were optimized by comparing the sample recoveries under different conditions. The results showed that the calibration curves of the five organophosphorus pesticides metabolites were linear in the range of 0.2-200 μg/L (r 2 ≥ 0.992) and that of the three pyrethroid pesticides metabolites were linear in the range of 0.025-250 μg/L (r 2 ≥ 0.991). The limits of detection (LODs, S/N ≥ 3) and the limits of quantification (LOQs, S/N ≥ 10) of the eight metabolites were 0.008-0.833 μg/L and 0.25-2.5 μg/L, respectively. The recoveries of the eight metabolites ranged from 54.08% to 82.49%. This efficient, stable, and cost-effective method is adequate to handle the large number of samples required for surveying the exposure level of organophosphorus and pyrethroid pesticides in the general population.

Effects of surface hydroxylation on adhesion at zinc/silica interfaces.

PubMed

Le, Ha-Linh Thi; Goniakowski, Jacek; Noguera, Claudine; Koltsov, Alexey; Mataigne, Jean-Michel

2018-06-06

The weak interaction between zinc and silica is responsible for the poor performance of anti-corrosive galvanic zinc coatings on modern advanced high-strength steels, which are fundamental in the automotive industry, and important for rail transport, shipbuilding, and aerospace. With the goal of identifying possible methods for its improvement, we report an ab initio study of the effect of surface hydroxylation on the adhesion characteristics of model zinc/β-cristobalite interfaces, representative of various surface hydroxylation/hydrogenation conditions. We show that surface silanols resulting from dissociative water adsorption at the most stable stoichiometric (001) and (111) surfaces prevent strong zinc-silica interactions. However, dehydrogenation of such interfaces produces oxygen-rich zinc/silica contacts with excellent adhesion characteristics. These are due to partial zinc oxidation and the formation of strong iono-covalent Zn-O bonds between zinc atoms and the under-coordinated excess anions, remnant of the hydroxylation layer. Interestingly, these interfaces appear as the most thermodynamically stable in a wide range of realistic oxygen-rich and hydrogen-lean environments. We also point out that the partial oxidation of zinc atoms in direct contact with the oxide substrate may somewhat weaken the cohesion in the zinc deposit itself. This fundamental analysis of the microscopic mechanisms responsible for the improved zinc wetting on pre-hydroxylated silica substrates provides useful guidelines towards practical attempts to improve adhesion.

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.

Effect of the St. John's wort constituent hyperforin on docetaxel metabolism by human hepatocyte cultures.

PubMed

Komoroski, Bernard J; Parise, Robert A; Egorin, Merrill J; Strom, Stephen C; Venkataramanan, Raman

2005-10-01

St. John's wort is a commonly used herbal medication that increases cytochrome P450 3A (CYP3A) activity. Because docetaxel is inactivated by CYP3A, we studied the effects of the St. John's wort constituent hyperforin on docetaxel metabolism in a human hepatocyte model. Hepatocytes, isolated from three donor livers, were exposed to hyperforin (0.1, 0.5, or 1.5 micromol/L) or rifampin (10 micromol/L) for 48 hours. After 48 hours, hyperforin- or rifampin-containing medium was replaced with medium containing 100 micromol/L docetaxel. After 1 hour, docetaxel metabolism was characterized by liquid chromatography-tandem mass spectrometry. Subsequent incubations characterized the specific cytochrome P450s that produced the docetaxel metabolites observed in hepatocyte incubations. Rifampin induced docetaxel metabolism 6.8- to 32-fold above docetaxel metabolism in control cultures. Hyperforin induced docetaxel metabolism in all three hepatocyte preparations. Hyperforin induction was dose-dependent and, at maximum, was 2.6- to 7-fold greater than that in controls. Docetaxel metabolites identified in rifampin- and hyperforin-treated hepatocyte preparations included the previously described tert-butyl-hydroxylated metabolite and two previously unidentified metabolites involving hydroxylation on the baccatin ring. CYP3A4 produced the tert-butyl-hydroxylated metabolite and the two ring-hydroxylated metabolites. CYP2C8 produced one of the newly described ring-hydroxylated metabolites. Exposure to the St. John's wort constituent hyperforin induces docetaxel metabolism in vitro. This implies that subtherapeutic docetaxel concentrations may result when docetaxel is administered to patients using St. John's wort on a chronic basis. The results also show induction of previously undescribed metabolic pathways for docetaxel, one of which may be analogous to the known 6-alpha-hydroxylation of paclitaxel by CYP2C8.

Development of urine standard reference materials for metabolites of organic chemicals including polycyclic aromatic hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds.

PubMed

Schantz, Michele M; Benner, Bruce A; Heckert, N Alan; Sander, Lane C; Sharpless, Katherine E; Vander Pol, Stacy S; Vasquez, Y; Villegas, M; Wise, Stephen A; Alwis, K Udeni; Blount, Benjamin C; Calafat, Antonia M; Li, Zheng; Silva, Manori J; Ye, Xiaoyun; Gaudreau, Éric; Patterson, Donald G; Sjödin, Andreas

2015-04-01

Two new Standard Reference Materials (SRMs), SRM 3672 Organic Contaminants in Smokers' Urine (Frozen) and SRM 3673 Organic Contaminants in Non-Smokers' Urine (Frozen), have been developed in support of studies for assessment of human exposure to select organic environmental contaminants. Collaborations among three organizations resulted in certified values for 11 hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and reference values for 11 phthalate metabolites, 8 environmental phenols and parabens, and 24 volatile organic compound (VOC) metabolites. Reference values are also available for creatinine and the free forms of caffeine, theobromine, ibuprofen, nicotine, cotinine, and 3-hydroxycotinine. These are the first urine Certified Reference Materials characterized for metabolites of organic environmental contaminants. Noteworthy, the mass fractions of the environmental organic contaminants in the two SRMs are within the ranges reported in population survey studies such as the National Health and Nutrition Examination Survey (NHANES) and the Canadian Health Measures Survey (CHMS). These SRMs will be useful as quality control samples for ensuring compatibility of results among population survey studies and will fill a void to assess the accuracy of analytical methods used in studies monitoring human exposure to these organic environmental contaminants.

Development of urine standard reference materials for metabolites of organic chemicals including polycyclic aromatic hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds

PubMed Central

Schantz, Michele M.; Benner, Bruce A.; Heckert, N. Alan; Sander, Lane C.; Sharpless, Katherine E.; Vander Pol, Stacy S.; Vasquez, Y.; Villegas, M.; Wise, Stephen A.; Alwis, K. Udeni; Blount, Benjamin C.; Calafat, Antonia M.; Li, Zheng; Silva, Manori J.; Ye, Xiaoyun; Gaudreau, Éric; Patterson, Donald G.; Sjödin, Andreas

2016-01-01

Two new Standard Reference Materials (SRMs), SRM 3672 Organic Contaminants in Smokers’ Urine (Frozen) and SRM 3673 Organic Contaminants in Non-Smokers’ Urine (Frozen), have been developed in support of studies for assessment of human exposure to select organic environmental contaminants. Collaborations among three organizations resulted in certified values for 11 hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and reference values for 11 phthalate metabolites, 8 environmental phenols and parabens, and 24 volatile organic compound (VOC) metabolites. Reference values are also available for creatinine and the free forms of caffeine, theobromine, ibuprofen, nicotine, cotinine, and 3-hydroxycotinine. These are the first urine Certified Reference Materials characterized for metabolites of organic environmental contaminants. Noteworthy, the mass fractions of the environmental organic contaminants in the two SRMs are within the ranges reported in population survey studies such as the National Health and Nutrition Examination Survey (NHANES) and the Canadian Health Measures Survey (CHMS). These SRMs will be useful as quality control samples for ensuring compatibility of results among population survey studies and will fill a void to assess the accuracy of analytical methods used in studies monitoring human exposure to these organic environmental contaminants. PMID:25651899

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

Individual variation and repeatability in urinary corticosterone metabolite responses to capture in the cane toad (Rhinella marina).

PubMed

Narayan, Edward J; Molinia, Frank C; Cockrem, John F; Hero, Jean-Marc

2012-01-15

Urinary corticosterone metabolite enzyme-immunoassay (EIA) can be used for the non-invasive assessment of baseline levels and corticosterone responses in amphibians. In this study, urinary corticosterone responses of wild male cane toads (Rhinella marina) to confinement and repeated handling were measured to quantify individual variation in corticosterone responses for the first time in an amphibian species. Urine samples were collected at 0 h in the wild, hourly from 2 to 8 h after transfer into captivity, and again at 12 and 24 h in captivity. Toads were then held in captivity and subjected to the same sampling protocol on three occasions at 14 days intervals to quantify variation in corticosterone metabolite responses within and between toads. Baseline and individual corticosterone metabolite responses in male cane toads were generally consistent, with high statistical repeatabilities for 0 h (r=0.630), 6 h (r=0.793), 12 h (r=0.652) and 24 h (r=0.721) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.567, p=0.033; r=0.728, p=0.014 respectively). Urinary corticosterone responses appear to be a stable, repeatable trait within individuals. Corticosterone responses in amphibians can be more readily measured when urine rather than plasma samples are collected, and the protocol established in the current study can now be applied to the study of variation in corticosterone responses in other amphibians. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

Comparative Analysis of Fruit Metabolites and Pungency Candidate Genes Expression between Bhut Jolokia and Other Capsicum Species.

PubMed

M, Sarpras; Gaur, Rashmi; Sharma, Vineet; Chhapekar, Sushil Satish; Das, Jharna; Kumar, Ajay; Yadava, Satish Kumar; Nitin, Mukesh; Brahma, Vijaya; Abraham, Suresh K; Ramchiary, Nirala

2016-01-01

Bhut jolokia, commonly known as Ghost chili, a native Capsicum species found in North East India was recorded as the naturally occurring hottest chili in the world by the Guinness Book of World Records in 2006. Although few studies have reported variation in pungency content of this particular species, no study till date has reported detailed expression analysis of candidate genes involved in capsaicinoids (pungency) biosynthesis pathway and other fruit metabolites. Therefore, the present study was designed to evaluate the diversity of fruit morphology, fruiting habit, capsaicinoids and other metabolite contents in 136 different genotypes mainly collected from North East India. Significant intra and inter-specific variations for fruit morphological traits, fruiting habits and 65 fruit metabolites were observed in the collected Capsicum germplasm belonging to three Capsicum species i.e., Capsicum chinense (Bhut jolokia, 63 accessions), C. frutescens (17 accessions) and C. annuum (56 accessions). The pungency level, measured in Scoville Heat Unit (SHU) and antioxidant activity measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay showed maximum levels in C. chinense accessions followed by C. frutescens accessions, while C. annuum accessions showed the lowest value for both the traits. The number of different fruit metabolites detected did not vary significantly among the different species but the metabolite such as benzoic acid hydroxyl esters identified in large percentage in majority of C. annuum genotypes was totally absent in the C. chinense genotypes and sparingly present in few genotypes of C. frutescens. Significant correlations were observed between fruit metabolites capsaicin, dihydrocapsaicin, hexadecanoic acid, cyclopentane, α-tocopherol and antioxidant activity. Furthermore, comparative expression analysis (through qRT-PCR) of candidate genes involved in capsaicinoid biosynthesis pathway revealed many fold higher expression of

Comparative Analysis of Fruit Metabolites and Pungency Candidate Genes Expression between Bhut Jolokia and Other Capsicum Species

PubMed Central

M, Sarpras; Gaur, Rashmi; Sharma, Vineet; Chhapekar, Sushil Satish; Das, Jharna; Kumar, Ajay; Yadava, Satish Kumar; Nitin, Mukesh; Brahma, Vijaya; Abraham, Suresh K.; Ramchiary, Nirala

2016-01-01

Bhut jolokia, commonly known as Ghost chili, a native Capsicum species found in North East India was recorded as the naturally occurring hottest chili in the world by the Guinness Book of World Records in 2006. Although few studies have reported variation in pungency content of this particular species, no study till date has reported detailed expression analysis of candidate genes involved in capsaicinoids (pungency) biosynthesis pathway and other fruit metabolites. Therefore, the present study was designed to evaluate the diversity of fruit morphology, fruiting habit, capsaicinoids and other metabolite contents in 136 different genotypes mainly collected from North East India. Significant intra and inter-specific variations for fruit morphological traits, fruiting habits and 65 fruit metabolites were observed in the collected Capsicum germplasm belonging to three Capsicum species i.e., Capsicum chinense (Bhut jolokia, 63 accessions), C. frutescens (17 accessions) and C. annuum (56 accessions). The pungency level, measured in Scoville Heat Unit (SHU) and antioxidant activity measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay showed maximum levels in C. chinense accessions followed by C. frutescens accessions, while C. annuum accessions showed the lowest value for both the traits. The number of different fruit metabolites detected did not vary significantly among the different species but the metabolite such as benzoic acid hydroxyl esters identified in large percentage in majority of C. annuum genotypes was totally absent in the C. chinense genotypes and sparingly present in few genotypes of C. frutescens. Significant correlations were observed between fruit metabolites capsaicin, dihydrocapsaicin, hexadecanoic acid, cyclopentane, α-tocopherol and antioxidant activity. Furthermore, comparative expression analysis (through qRT-PCR) of candidate genes involved in capsaicinoid biosynthesis pathway revealed many fold higher expression of

Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors.

PubMed

Mannan, Ahmad A; Liu, Di; Zhang, Fuzhong; Oyarzún, Diego A

2017-10-20

Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.

In vitro and in vivo metabolic investigation of the Palbociclib by UHPLC-Q-TOF/MS/MS and in silico toxicity studies of its metabolites.

PubMed

Chavan, Balasaheb B; Tiwari, Shristy; G, Shankar; Nimbalkar, Rakesh D; Garg, Prabha; R, Srinivas; Talluri, M V N Kumar

2018-05-14

Palbociclib (PAB) is a CDK4/6 inhibitor and U. S Food and Drug Administration (FDA) granted regular approval for the treatment of hormone receptor (HR) positive, metastatic breast cancer in combination with an aromatase inhibitor in postmenopausal women. Metabolite identification is a crucial aspect during drug discovery and development as the drug metabolites may be pharmacologically active or possess toxicological activity. As there are no reports on the metabolism studies of the PAB, the present study focused on investigation of the in vitro and in vivo metabolic fate of the drug. The in vitro metabolism studies were carried out by using microsomes (HLM and RLM) and S9 fractions (Human and rat). The in vivo metabolism of the drug was studied by administration of the PAB orally to the Sprague-Dawley rats followed by analysis of urine, faeces and plasma samples. The sample preparation includes simple protein precipitation (PP) followed by solid phase extraction (SPE). The extracted samples were analyzed by ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry (UHPLC/Q-TOF/MS/MS). A total of 14 metabolites were detected in in vivo matrices. The PAB was metabolized via hydroxylation, oxidation, sulphation, N-dealkylation, acetylation and carbonylation pathways. A few of the metabolites were also detected in in vitro samples. Metabolite identification and characterization were performed by using UHPLC/Q-TOF/MS/MS in combination with HRMS data. To identify the toxicity potential of these metabolites, in silico toxicity assessment was carried out using TOPKAT and DEREK softwares. Copyright © 2018. Published by Elsevier B.V.

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.

Arsenic Species in Chicken Breast: Temporal Variations of Metabolites, Elimination Kinetics, and Residual Concentrations

PubMed Central

Liu, Qingqing; Peng, Hanyong; Lu, Xiufen; Zuidhof, Martin J.; Li, Xing-Fang; Le, X. Chris

2016-01-01

Background: Chicken meat has the highest per capita consumption among all meat types in North America. The practice of feeding 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox) to chickens lasted for more than 60 years. However, the fate of Rox and arsenic metabolites remaining in chicken are poorly understood. Objectives: We aimed to determine the elimination of Rox and metabolites from chickens and quantify the remaining arsenic species in chicken meat, providing necessary information for meaningful exposure assessment. Methods: We have conducted a 35-day feeding experiment involving 1,600 chickens, of which half were control and the other half were fed a Rox-supplemented diet for the first 28 days and then a Rox-free diet for the final 7 days. We quantified the concentrations of individual arsenic species in the breast meat of 229 chickens. Results: Rox, arsenobetaine, arsenite, monomethylarsonic acid, dimethylarsinic acid, and a new arsenic metabolite, were detected in breast meat from chickens fed Rox. The concentrations of arsenic species, except arsenobetaine, were significantly higher in the Rox-fed than in the control chickens. The half-lives of elimination of these arsenic species were 0.4–1 day. Seven days after termination of Rox feeding, the concentrations of arsenite (3.1 μg/kg), Rox (0.4 μg/kg), and a new arsenic metabolite (0.8 μg/kg) were significantly higher in the Rox-fed chickens than in the control. Conclusion: Feeding of Rox to chickens increased the concentrations of five arsenic species in breast meat. Although most arsenic species were excreted rapidly when the feeding of Rox stopped, arsenic species remaining in the Rox-fed chickens were higher than the background levels. Citation: Liu Q, Peng H, Lu X, Zuidhof MJ, Li XF, Le XC. 2016. Arsenic species in chicken breast: temporal variations of metabolites, elimination kinetics, and residual concentrations. Environ Health Perspect 124:1174–1181; http://dx.doi.org/10.1289/ehp

Arsenic Species in Chicken Breast: Temporal Variations of Metabolites, Elimination Kinetics, and Residual Concentrations.

PubMed

Liu, Qingqing; Peng, Hanyong; Lu, Xiufen; Zuidhof, Martin J; Li, Xing-Fang; Le, X Chris

2016-08-01

Chicken meat has the highest per capita consumption among all meat types in North America. The practice of feeding 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox) to chickens lasted for more than 60 years. However, the fate of Rox and arsenic metabolites remaining in chicken are poorly understood. We aimed to determine the elimination of Rox and metabolites from chickens and quantify the remaining arsenic species in chicken meat, providing necessary information for meaningful exposure assessment. We have conducted a 35-day feeding experiment involving 1,600 chickens, of which half were control and the other half were fed a Rox-supplemented diet for the first 28 days and then a Rox-free diet for the final 7 days. We quantified the concentrations of individual arsenic species in the breast meat of 229 chickens. Rox, arsenobetaine, arsenite, monomethylarsonic acid, dimethylarsinic acid, and a new arsenic metabolite, were detected in breast meat from chickens fed Rox. The concentrations of arsenic species, except arsenobetaine, were significantly higher in the Rox-fed than in the control chickens. The half-lives of elimination of these arsenic species were 0.4-1 day. Seven days after termination of Rox feeding, the concentrations of arsenite (3.1 μg/kg), Rox (0.4 μg/kg), and a new arsenic metabolite (0.8 μg/kg) were significantly higher in the Rox-fed chickens than in the control. Feeding of Rox to chickens increased the concentrations of five arsenic species in breast meat. Although most arsenic species were excreted rapidly when the feeding of Rox stopped, arsenic species remaining in the Rox-fed chickens were higher than the background levels. Liu Q, Peng H, Lu X, Zuidhof MJ, Li XF, Le XC. 2016. Arsenic species in chicken breast: temporal variations of metabolites, elimination kinetics, and residual concentrations. Environ Health Perspect 124:1174-1181; http://dx.doi.org/10.1289/ehp.1510530.

Identification and in silico prediction of metabolites of the model compound, tebufenozide by human CYP3A4 and CYP2C19.

PubMed

Shirotani, Naoki; Togawa, Moe; Ikushiro, Shinichi; Sakaki, Toshiyuki; Harada, Toshiyuki; Miyagawa, Hisashi; Matsui, Masayoshi; Nagahori, Hirohisa; Mikata, Kazuki; Nishioka, Kazuhiko; Hirai, Nobuhiro; Akamatsu, Miki

2015-10-15

The metabolites of tebufenozide, a model compound, formed by the yeast-expressed human CYP3A4 and CYP2C19 were identified to clarify the substrate recognition mechanism of the human cytochrome P450 (CYP) isozymes. We then determined whether tebufenozide metabolites may be predicted in silico. Hydrogen abstraction energies were calculated with the density functional theory method B3LYP/6-31G(∗). A docking simulation was performed using FRED software. Several alkyl sites of tebufenozide were hydroxylated by CYP3A4 whereas only one site was modified by CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen abstraction energy estimation, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Occurrence of selected pesticides and their metabolites in near-surface aquifers of the midwestern United States

USGS Publications Warehouse

Kolpin, D.W.; Michael, Thurman E.; Goolsby, D.A.

1996-01-01

The occurrence and distribution of selected pesticides and their metabolites were investigated through the collection of 837 water-quality samples from 303 wells across the Midwest. Results of this study showed that five of the six most frequently detected compounds were pesticide metabolites. Thus, it was common for a metabolite to be found more frequently in groundwater than its parent compound. The metabolite alachlor ethanesulfonic acid (alachlor-ESA; 2-[(2,6-diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfonic acid) was detected almost 10 times as frequently and at much higher concentrations than its parent compound alachlor (2-chloro-2‘,6‘-diethyl-N-(methoxymethyl)acetamide). The median detectable atrazine (2-chloro-4-ethylamino-6- isopropylamino-s-triazine) concentration was almost half that of atrazine residue (atrazine plus the two atrazine metabolites analyzed). Cyanazine amide [2-chloro-4-(1-carbamoyl-1-methylethylamino)-6-ethylamino-s-triazine] was detected almost twice as frequently as cyanazine (2-chloro-4-ethylamino-6-methylpropionitrileamino-s-triazine). Results show that information on pesticide metabolites is necessary to understand the environmental fate of pesticides. Consequently, if pesticide metabolites are not quantified, the effects of chemical use on groundwater quality would be substantially underestimated. Thus, continued research is needed to identify major degradation pathways for all pesticides and to develop analytical methods to determine their concentrations in water and other environmental media.

Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680.

PubMed

Roh, Changhyun; Seo, Su-Hyun; Choi, Kwon-Young; Cha, Minho; Pandey, Bishnu Prasad; Kim, June-Hyung; Park, Jun-Seong; Kim, Duck Hee; Chang, Ih Seop; Kim, Byung-Gee

2009-07-01

Screening of bacterial whole cells was performed for regioselective hydroxylation of daidzein and genistein. Among the strains examined, Streptomyces avermitilis MA-4680 showed high ortho-dihydroxylation activity to produce 3',4',7-trihydroxyisoflavone and 3',4',5,7-tetrahydroxyisoflavone from daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone), respectively. Using 100 mg cells (wet wt.) and 1% (v/v) Triton X100 in 1 ml of total reaction volume, where 100 microl of the substrate solution (0.5 mM in 10% (v/v) mixed solvent of DMSO:MeOH = 3:7) was added to 900 microl of potassium phosphate buffer (100 mM, pH 7.2), a 16% molar conversion yield of 3',4',7-trihydroxyisoflavone was obtained from 0.5 mM daidzein after 24 h of reaction time at 28 degrees C and 200 rpm. Ketoconazole significantly (ca. 90%) inhibited the ortho-hydroxylation activity of daidzein, suggesting that cytochrome P450 enzymes putatively play roles in regiospecific daidzein hydroxylation. The analysis of the reaction products was determined by gas chromatography/mass spectrometry (GC/MS) and (1)H NMR.

Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

PubMed

Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

2012-12-15

Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

Formation of Δ(1) and Δ(6) testosterone metabolites by human hepatocytes.

PubMed

Fabregat, Andreu; Marcos, Josep; Ventura, Rosa; Casals, Gregori; Jimenez, Wladimiro; Reichenbach, Vedrana; Segura, Jordi; Pozo, Oscar J

2015-03-01

The existence of urinary testosterone (T) metabolites conjugated with cysteine has been recently reported. The formation of a ring double bond by a phase I metabolic transformation and the subsequent nucleophilic conjugation with glutathione was proposed as a putative metabolic pathway for the occurrence of these metabolites in urine. The main goal of the present study was to confirm the first step of the postulated pathway. For that purpose, human hepatocyte cells systems were incubated with a pure T standard. The cell culture supernatants were analyzed by liquid chromatography coupled to mass spectrometry using a selected reaction monitoring method. Major T metabolites such as androsterone and 4-androstene-3,17-dione, together with the recently reported Δ(1) and Δ(6) metabolites were simultaneously quantified. The formation of 1,4-androstadien-3,17-dione, 4,6-androstadien-3,17-dione, 17β-hydroxy-4,6-androstadien-3-one and 17β-hydroxy-1,4-androstadien-3-one (boldenone) after incubation of T in hepatocyte cell cultures was demonstrated by comparing the retention times and the ion ratios of the metabolites with those obtained by analysis of commercial standards. Thus, the formation of double bonds Δ(1) and Δ(6) by hepatic phase I metabolism of T was confirmed. Analogously to T, this pathway might also be present in other steroids, opening the possibility of targeting additional biomarkers. Copyright © 2015. Published by Elsevier Inc.

Detection of adsorbed water and hydroxyl on the Moon.

PubMed

Clark, Roger N

2009-10-23

Data from the Visual and Infrared Mapping Spectrometer (VIMS) on Cassini during its flyby of the Moon in 1999 show a broad absorption at 3 micrometers due to adsorbed water and near 2.8 micrometers attributed to hydroxyl in the sunlit surface on the Moon. The amounts of water indicated in the spectra depend on the type of mixing and the grain sizes in the rocks and soils but could be 10 to 1000 parts per million and locally higher. Water in the polar regions may be water that has migrated to the colder environments there. Trace hydroxyl is observed in the anorthositic highlands at lower latitudes.

Hydrogen-bond rich ionic liquids with hydroxyl cationic tails

NASA Astrophysics Data System (ADS)

Deng, Li; Shi, Rui; Wang, Yanting; Ou-Yang, Zhong-Can

2013-02-01

To investigate if the amphiphilic feature exhibited in ionic liquids (ILs) with nonpolar cationic tails still exists in ILs with polar tails, by performing molecular dynamics simulations for 1-(8-hydroxyoctyl)-3-methyl-imidazolium nitrate (COH) and 1-octyl-3-methyl-imidazolium nitrate (C8), we found that, in COH, cationic tail groups can no longer aggregate to form separated nonpolar tail domains, instead hydroxyl groups form a rich number of hydrogen bonds with other groups, indicating that the hydroxyl substituent changes the IL system from an amphiphilic liquid to a polar liquid. Due to the large amount of hydrogen bonds, COH has slower dynamics than C8.

Generation of hydroxyl radicals and singlet oxygen during oxidation of rhododendrol and rhododendrol-catechol.

PubMed

Miyaji, Akimitsu; Gabe, Yu; Kohno, Masahiro; Baba, Toshihide

2017-03-01

The generation of hydroxyl radicals and singlet oxygen during the oxidation of 4-(4-hydroxyphenyl)-2-butanol (rhododendrol) and 4-(3,4-dihydroxyphenyl)-2-butanol (rhododendrol-catechol) with mushroom tyrosinase in a phosphate buffer (pH 7.4) was examined as the model for the reactive oxygen species generation via the two rhododendrol compounds in melanocytes. The reaction was performed in the presence of 5,5-dimethyl-1-pyrroline- N -oxide (DMPO) spin trap reagents for hydroxyl radical or 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen, and their electron spin resonances were measured. An increase in the electron spin resonances signal attributable to the adduct of DMPO reacting with the hydroxyl radical and that of 4-oxo-TEMP reacting with singlet oxygen was observed during the tyrosinase-catalyzed oxidation of rhododendrol and rhododendrol-catechol, indicating the generation of hydroxyl radical and singlet oxygen. Moreover, hydroxyl radical generation was also observed in the autoxidation of rhododendrol-catechol. We show that generation of intermediates during tyrosinase-catalyzed oxidation of rhododendrol enhances oxidative stress in melanocytes.

QSPR prediction of the hydroxyl radical rate constant of water contaminants.

PubMed

Borhani, Tohid Nejad Ghaffar; Saniedanesh, Mohammadhossein; Bagheri, Mehdi; Lim, Jeng Shiun

2016-07-01

In advanced oxidation processes (AOPs), the aqueous hydroxyl radical (HO) acts as a strong oxidant to react with organic contaminants. The hydroxyl radical rate constant (kHO) is important for evaluating and modelling of the AOPs. In this study, quantitative structure-property relationship (QSPR) method is applied to model the hydroxyl radical rate constant for a diverse dataset of 457 water contaminants from 27 various chemical classes. The constricted binary particle swarm optimization and multiple-linear regression (BPSO-MLR) are used to obtain the best model with eight theoretical descriptors. An optimized feed forward neural network (FFNN) is developed to investigate the complex performance of the selected molecular parameters with kHO. Although the FFNN prediction results are more accurate than those obtained using BPSO-MLR, the application of the latter is much more convenient. Various internal and external validation techniques indicate that the obtained models could predict the logarithmic hydroxyl radical rate constants of a large number of water contaminants with less than 4% absolute relative error. Finally, the above-mentioned proposed models are compared to those reported earlier and the structural factors contributing to the AOP degradation efficiency are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Antioxidative activities of two metabolites of cultured marine fungus, Halorosellinia oceanicum 323 in vitro].

PubMed

Luo, Jinghui; Yang, Yingbao; Lin, Yongcheng; Chen, Zhiliang; Jiang, Guangce

2004-03-01

To investigate the antioxidative effects of 323-A and 323-B, two isomers extracted from the metabolites of cultured marine fungus, Halorosellinia oceanicum 323 in vitro. NADH-PMS-NBT system was used to produce superoxide free radical (O2*-), EDTANa2-Fe(II)-H2O2 system to generate hydroxyl free radical (*OH), H2O2 to stimulate oxidative hemolysis of erythrocytes of rats, Cys-Fe2+ to induce malondialdehyde (MDA) production in homogenates, and ferrous-ascorbic acid system to increase the turbidity of mitochondria suspension in the liver of rats. And the antioxidative activities of 323-A and 323-B were studied. 323-A and 323-B not only scavenge O2*- and *OH produced by the experimental systems directly, but also inhibit H2O2 stimulated oxidative hemolysis of erythrocytes of rats, depress MDA production in homogenates induced by Cys-Fe2+ system, and reduce the turbidity of mitochondria suspension in the liver of rats increased by ferrous-ascorbic acid system in vitro. 323-A and 323-B showed comprehensive cleaning actions on free radicals and protective effects on the functions of tissues and cells against oxidative lesion. The results suggested that the marine microorganic metabolites might be a novel and profound source of antioxidative reagents.

A New Paradigm for Known Metabolite Identification in Metabonomics/Metabolomics: Metabolite Identification Efficiency

PubMed Central

Everett, Jeremy R.

2015-01-01

A new paradigm is proposed for assessing confidence in the identification of known metabolites in metabonomics studies using NMR spectroscopy approaches. This new paradigm is based upon the analysis of the amount of metabolite identification information retrieved from NMR spectra relative to the molecular size of the metabolite. Several new indices are proposed including: metabolite identification efficiency (MIE) and metabolite identification carbon efficiency (MICE), both of which can be easily calculated. These indices, together with some guidelines, can be used to provide a better indication of known metabolite identification confidence in metabonomics studies than existing methods. Since known metabolite identification in untargeted metabonomics studies is one of the key bottlenecks facing the science currently, it is hoped that these concepts based on molecular spectroscopic informatics, will find utility in the field. PMID:25750701

A new paradigm for known metabolite identification in metabonomics/metabolomics: metabolite identification efficiency.

PubMed

Everett, Jeremy R

2015-01-01

A new paradigm is proposed for assessing confidence in the identification of known metabolites in metabonomics studies using NMR spectroscopy approaches. This new paradigm is based upon the analysis of the amount of metabolite identification information retrieved from NMR spectra relative to the molecular size of the metabolite. Several new indices are proposed including: metabolite identification efficiency (MIE) and metabolite identification carbon efficiency (MICE), both of which can be easily calculated. These indices, together with some guidelines, can be used to provide a better indication of known metabolite identification confidence in metabonomics studies than existing methods. Since known metabolite identification in untargeted metabonomics studies is one of the key bottlenecks facing the science currently, it is hoped that these concepts based on molecular spectroscopic informatics, will find utility in the field.

Impact of Ti Incorporation on Hydroxylation and Wetting of Fe 3 O 4

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

Stoerzinger, Kelsey A.; Pearce, Carolyn I.; Droubay, Timothy C.

2017-08-24

Understanding the interaction of water with compositionally tuned metal oxides is central to exploiting their unique catalytic and magnetic properties. However, processes such as hydroxylation, wetting, and resulting changes in electronic structure at ambient conditions are challenging to probe in situ. Here, we examine the hydroxylation and wetting of Fe(3-x)TixO4 epitaxial films directly using ambient pressure X-ray photoelectron spectroscopy under controlled relative humidity. Fe2+ formation promoted by Ti4+ substitution for Fe3+ increases with hydroxylation, commensurate with a decrease in the surface work function or change in the surface dipole. The incorporation of small amounts of Ti (x=0.25) as a bulkmore » dopant dramatically impacts hydroxylation, in part due to surface segregation, leading to coverages closer to that of TiO2 than Fe3O4. However, the Fe(3-x)TixO4 compositional series shows a similar affinity for water physisorption, which begins at notably lower relative humidity than on TiO2. The findings suggest that relative humidity rather than surface hydroxyl density controls wettability. Studies of this kind directly relate to rational design of doped magnetite into more active catalysts for UV/Fenton degradation, the adsorption of contaminants, and the development of spin filters.« less

[Study of scavenging activity of sorghum pigment to hydroxyl free radicals by fluorimetry].

PubMed

Zhang, Hai-rong; Wang, Wen-yan

2007-03-01

A natural product, sorghum pigment, consists of a number of important flavonoid derivatives, occurrs on the seed capsules or in the stems of many sorghums, and is widely applied in different fields of food, cosmetic and dyeing industries, It is important for scavenging hydroxyl free radicals and protection of human healthiness. Scavenging capacities of hydroxyl free radicals with sodium nitrite, quercetin and sorghum pigment were comparatively researched by fluorimetry, and the model of hydroxyl free radicals produced is based on the reaction of Cu2+ -catalyzed oxidation of ascorbic acid in the presence of hydrogen peroxide. The hydroxyl radicals react with benzoic acid, forming a fluorescent product, and the fluorescence intensity was determined by the concentration of hydroxybenzoic acid. The experimental results show that the sodium nitrite, quercetin and sorghum pigment have a quantity-effect relationship for scavenging hydroxyl free radicals, and sodium nitrite and quercetin in comparison with sorghum pigment have high antioxidant capacity. Finally, the quenching mechanisms were explored with sodium nitrite, sorghum pigment, and quercetin respectively. The sorghum pigment and sodium nitrite feature a dynamic quenching processes, while quercetin shows a static quenching processes. A reference method was provided for reasonable exploitation and utilization of sorghum pigment.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

PubMed

Siddiqui, Mahwish; Ahmad, Malik Shoaib; Wahab, Atia-Tul-; Yousuf, Sammer; Fatima, Narjis; Naveed Shaikh, Nimra; Rahman, Atta-Ur-; Choudhary, M Iqbal

2017-01-01

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7 �

Stereoselective Formation of Mono- and Di-Hydroxylated Polychlorinated Biphenyls by Rat Cytochrome P450 2B1

PubMed Central

Lehmler, Hans-Joachim; Wong, Charles S.

2013-01-01

Changes in atropisomer composition of chiral polychlorinated biphenyls (PCBs) and their mono- and di- hydroxylated metabolites (OH- and diOH-PCBs) via rat cytochrome P450 2B1 (CYP2B1) mediated biotransformation were investigated in vitro. Rat CYP2B1 could stereoselectively biotransform chiral PCBs to generate meta-OH-PCBs as the major metabolites after 60 min incubations. Non-racemic enantiomer fractions (EFs: concentration ratios of the (+)-atropisomer or the first-eluting atropisomer over the total concentrations of two atropisomers) of 5-OH-PCBs, were 0.17, 0.20, 0.85, 0.77 and 0.41 for incubations with PCBs 91, 95, 132, 136 and 149, respectively. CYP-mediated stereoselective formation of diOH-PCBs from OH-PCBs was observed for the first time. After 60 min stereoselective biotransformation, the EFs of both 4-OH-PCB 95 and 5-OH-PCB 95 changed from racemic (i.e., 0.50) to 0.62 and 0.46, respectively. These transformations generated statistically non-racemic 4,5-diOH-PCB 95, with EFs of 0.53 and 0.58 for 4-OH-PCB 95 and 5-OH-PCB 95 incubations, respectively. Biotransformation of PCBs 91 and 136 also generated 4,5-diOH-PCB 91 and 4,5-diOH-PCB 136, respectively. These in vitro results were consistent with that observed for stereoselective PCB biotransformation by rat liver microsomes and in vivo. Biotransformation interference between two atropisomers of PCB 136 was investigated for the first time in this study. The biotransformation process of (−)-PCB 136 was significantly disrupted by the presence of (+)-PCB 136, but not the other way around. Thus, stereoselective metabolism of chiral PCBs and OH-PCBs by CYPs is a major mechanism for atropisomer composition change of PCBs and their metabolites in the environment, with the degree of composition change dependent, at least in part, on stereoselective interference of atropisomers with each other at the enzyme level. PMID:24060104

Density functional study of the adsorption of aspirin on the hydroxylated (0 0 1) α-quartz surface

NASA Astrophysics Data System (ADS)

Abbasi, A.; Nadimi, E.; Plänitz, P.; Radehaus, C.

2009-08-01

In this study the adsorption geometry of aspirin molecule on a hydroxylated (0 0 1) α-quartz surface has been investigated using DFT calculations. The optimized adsorption geometry indicates that both, adsorbed molecule and substrate are strongly deformed. Strong hydrogen bonding between aspirin and surface hydroxyls, leads to the breaking of the original hydroxyl-hydroxyl hydrogen bonds (Hydrogenbridges) on the surface. In this case new hydrogen bonds on the hydroxylated (0 0 1) α-quartz surface appear which significantly differ from those at the clean surface. The 1.11 eV adsorption energy reveals that the interaction of aspirin with α-quartz is an exothermic chemical interaction.

Markedly Enhanced Surface Hydroxyl Groups of TiO2 Nanoparticles with Superior Water-Dispersibility for Photocatalysis

PubMed Central

Wu, Chung-Yi; Tu, Kuan-Ju; Deng, Jin-Pei; Lo, Yu-Shiu; Wu, Chien-Hou

2017-01-01

The benefits of increasing the number of surface hydroxyls on TiO2 nanoparticles (NPs) are known for environmental and energy applications; however, the roles of the hydroxyl groups have not been characterized and distinguished. Herein, TiO2 NPs with abundant surface hydroxyl groups were prepared using commercial titanium dioxide (ST-01) powder pretreated with alkaline hydrogen peroxide. Through this simple treatment, the pure anatase phase was retained with an average crystallite size of 5 nm and the surface hydroxyl group density was enhanced to 12.0 OH/nm2, estimated by thermogravimetric analysis, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. Especially, this treatment increased the amounts of terminal hydroxyls five- to six-fold, which could raise the isoelectric point and the positive charges on the TiO2 surface in water. The photocatalytic efficiency of the obtained TiO2 NPs was investigated by the photodegradation of sulforhodamine B under visible light irradiation as a function of TiO2 content, pH of solution, and initial dye concentration. The high surface hydroxyl group density of TiO2 NPs can not only enhance water-dispersibility but also promote dye sensitization by generating more hydroxyl radicals. PMID:28772926

Posaconazole (Noxafil, SCH 56592), a new azole antifungal drug, was a discovery based on the isolation and mass spectral characterization of a circulating metabolite of an earlier lead (SCH 51048).

PubMed

Nomeir, Amin A; Pramanik, Birendra N; Heimark, Larry; Bennett, Frank; Veals, John; Bartner, Peter; Hilbert, Maryjane; Saksena, Anil; McNamara, Paul; Girijavallabhan, Viyyoor; Ganguly, Ashit K; Lovey, Raymond; Pike, Russell; Wang, Haiyan; Liu, Yi-Tsung; Kumari, Pramila; Korfmacher, Walter; Lin, Chin-Chung; Cacciapuoti, Anthony; Loebenberg, David; Hare, Roberta; Miller, George; Pickett, Cecil

2008-04-01

Posaconazole (SCH 56592) is a novel triazole antifungal drug that is marketed in Europe and the United States under the trade name 'Noxafil' for prophylaxis against invasive fungal infections. SCH 56592 was discovered as a possible active metabolite of SCH 51048, an earlier lead. Initial studies have shown that serum concentrations determined by a microbiological assay were higher than those determined by HPLC from animals dosed with SCH 51048. Subsequently, several animals species were dosed with (3)H-SCH 51048 and the serum was analyzed for total radioactivity, SCH 51048 concentration and antifungal activity. The antifungal activity was higher than that expected based on SCH 51048 serum concentrations, confirming the presence of active metabolite(s). Metabolite profiling of serum samples at selected time intervals pinpointed the peak that was suspected to be the active metabolite. Consequently, (3)H-SCH 51048 was administered to a large group of mice, the serum was harvested and the metabolite was isolated by extraction and semipreparative HPLC. LC-MS/MS analysis suggested that the active metabolite is a secondary alcohol with the hydroxyl group in the aliphatic side chain of SCH 51048. All corresponding monohydroxylated diastereomeric mixtures were synthesized and characterized. The HPLC retention time and LC-MS/MS spectra of the diastereomeric secondary alcohols of SCH 51048 were similar to those of the isolated active metabolite. Finally, all corresponding individual monohydroxylated diasteriomers were synthesized and evaluated for in vitro and in vivo antifungal potencies, as well as pharmacokinetics. SCH 56592 emerged as the candidate with the best overall profile.

R-Limonene metabolism in humans and metabolite kinetics after oral administration.

PubMed

Schmidt, Lukas; Göen, Thomas

2017-03-01

We studied the R-limonene (LMN) metabolism and elimination kinetics in a human in vivo study. Four volunteers were orally exposed to a single LMN dose of 100-130 µg kg -1 bw. In each case, one pre-exposure and subsequently all 24 h post-exposure urine samples were collected. From two subjects, blood samples were drawn up to 5 h after exposure. The parent compound was analysed in blood using headspace GC-MS. The metabolites cis- and trans-carveol (cCAR), perillyl alcohol (POH), perillic acid (PA), limonene-1,2-diol (LMN-1,2-OH), and limonene-8,9-diol (LMN-8,9-OH) were quantified in both blood and urine using GC-PCI-MS/MS. Moreover, GC-PCI-MS full-scan experiments were applied for identification of unknown metabolites in urine. In both matrices, metabolites reached maximum concentrations 1-2 h post-exposure followed by rapid elimination with half-lives of 0.7-2.5 h. In relation to the other metabolites, LMN-1,2-OH was eliminated slowest. Nonetheless, overall renal metabolite elimination was completed within the 24-h observation period. The metabolite amounts excreted via urine corresponded to 0.2 % (cCAR), 0.2 % (tCAR), <0.1 % (POH), 2.0 % (PA), 4.3 % (LMN-1,2-OH), and 32 % (LMN-8,9-OH) of the orally administered dose. GC-PCI-MS full-scan analyses revealed dihydroperillic acid (DHPA) as an additional LMN metabolite. DHPA was estimated to account for 5 % of the orally administered dose. The study revealed that human LMN metabolism proceeds fast and is characterised by oxidation mainly of the exo-cyclic double bond but also of the endo-cyclic double bond and of the methyl side chain. The study results may support the prediction of the metabolism of other terpenes or comparable chemical structures.

Sensitive determination of endogenous hydroxyl radical in live cell by a BODIPY based fluorescent probe.

PubMed

Lei, Kepeng; Sun, Mingtai; Du, Libo; Zhang, Xiaojie; Yu, Huan; Wang, Suhua; Hayat, Tasawar; Alsaedi, Ahmed

2017-08-01

The sensitive and selective fluorescence probe for hydroxyl radical analysis is of significance because hydroxyl radical plays key roles in many physiological and pathological processes. In this work, a novel organic fluorescence molecular probe OHP for hydroxyl radical is synthesized by a two-step route. The probe employs 4-bora-3a,4a-diaza-s-indacene (difluoroboron dipyrromethene, BODIPY) as the fluorophore and possesses relatively high fluorescence quantum yields (77.14%). Hydroxyl radical can rapidly react with the probe and quench the fluorescence in a good linear relationship (R 2 =0.9967). The limit of detection is determined to be as low as 11nM. In addition, it has been demonstrated that the probe has a good stability against pH and light illumination, low cytotoxicity and high biocompatibility. Cell culture experimental results show that the probe OHP is sensitive and selective for imaging and tracking endogenous hydroxyl radical in live cells. Copyright © 2017 Elsevier B.V. All rights reserved.

A Quantitative Profiling Method of Phytohormones and Other Metabolites Applied to Barley Roots Subjected to Salinity Stress

PubMed Central

Cao, Da; Lutz, Adrian; Hill, Camilla B.; Callahan, Damien L.; Roessner, Ute

2017-01-01

As integral parts of plant signaling networks, phytohormones are involved in the regulation of plant metabolism and growth under adverse environmental conditions, including salinity. Globally, salinity is one of the most severe abiotic stressors with an estimated 800 million hectares of arable land affected. Roots are the first plant organ to sense salinity in the soil, and are the initial site of sodium (Na+) exposure. However, the quantification of phytohormones in roots is challenging, as they are often present at extremely low levels compared to other plant tissues. To overcome this challenge, we developed a high-throughput LC-MS method to quantify ten endogenous phytohormones and their metabolites of diverse chemical classes in roots of barley. This method was validated in a salinity stress experiment with six barley varieties grown hydroponically with and without salinity. In addition to phytohormones, we quantified 52 polar primary metabolites, including some phytohormone precursors, using established GC-MS and LC-MS methods. Phytohormone and metabolite data were correlated with physiological measurements including biomass, plant size and chlorophyll content. Root and leaf elemental analysis was performed to determine Na+ exclusion and K+ retention ability in the studied barley varieties. We identified distinct phytohormone and metabolite signatures as a response to salinity stress in different barley varieties. Abscisic acid increased in the roots of all varieties under salinity stress, and elevated root salicylic acid levels were associated with an increase in leaf chlorophyll content. Furthermore, the landrace Sahara maintained better growth, had lower Na+ levels and maintained high levels of the salinity stress linked metabolite putrescine as well as the phytohormone metabolite cinnamic acid, which has been shown to increase putrescine concentrations in previous studies. This study highlights the importance of root phytohormones under salinity stress and

Comparative oxidative metabolism of BDE-47 and BDE-99 by rat hepatic microsomes.

PubMed

Erratico, Claudio A; Moffatt, Sarah C; Bandiera, Stelvio M

2011-09-01

Polybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that have become ubiquitous environmental pollutants. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are among the most prevalent PBDEs detected in humans, wildlife, and abiotic environmental matrices. The purpose of this study was to investigate the oxidative metabolism of BDE-47 and BDE-99 in rat hepatic microsomes by comparing metabolite formation rates, kinetic parameters associated with metabolite formation, and the effects of prototypical cytochrome P450 (CYP) inducers. The CYP enzymes involved were also identified. Incubation of BDE-47 with hepatic microsomes from phenobarbital-treated rats generated a total of five hydroxylated (OH-BDE) metabolites, among which 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49) and 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47) were the major metabolites, as identified using authentic standards and quantified by liquid chromatography/mass spectrometry. Incubations of BDE-99 with hepatic microsomes from dexamethasone-treated rats produced a total of seven hydroxylated metabolites, among which 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90) and 6'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99) were the major metabolites. Although the overall rate of oxidative metabolism of BDE-99 by hepatic microsomes was greater than that of BDE-47, para-hydroxylation involving a National Institutes of Health shift mechanism represented a major metabolic pathway for both PBDE congeners. Among the rat recombinant CYP enzymes tested, CYP2A2 and CYP3A1 were the most active in BDE-47 and BDE-99 metabolism, respectively. However, CYP1A1 exhibited the highest activity for 4'-OH-BDE-49 and 6'-OH-BDE-99 formation, and CYP3A1 exhibited the highest activity for 3-OH-BDE-47 and 4-OH-BDE-90 formation. Collectively, the results demonstrate that oxidative metabolism of BDE-47 and BDE-99 is

Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

PubMed

Nishshanka, Upul; Chu, Pak-Sin; Evans, Eric; Reimschuessel, Renate; Hasbrouck, Nicholas; Amarasinghe, Kande; Jayasuriya, Hiranthi

2015-06-24

Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

Correlations of Maternal Buprenorphine Dose, Buprenorphine, and Metabolite Concentrations in Meconium with Neonatal Outcomes

PubMed Central

Kacinko, SL; Jones, HE; Johnson, RE; Choo, RE; Huestis, MA

2009-01-01

For the first time, relationships among maternal buprenorphine dose, meconium buprenorphine and metabolite concentrations, and neonatal outcomes are reported. Free and total buprenorphine and norbuprenorphine, nicotine, opiates, cocaine, benzodiazepines, and metabolites were quantified in meconium from 10 infants born to women who had received buprenorphine during pregnancy. Neither cumulative nor total third-trimester maternal buprenorphine dose predicted meconium concentrations or neonatal outcomes. Total buprenorphine meconium concentrations and buprenorphine/norbuprenorphine ratios were significantly related to neonatal abstinence syndrome (NAS ) scores >4. As free buprenorphine concentration and percentage free buprenorphine increased, head circumference decreased. Thrice-weekly urine tests for opiates, cocaine, and benzodiazepines and self-reported smoking data from the mother were compared with data from analysis of the meconium to estimate in utero exposure. Time of last drug use and frequency of use during the third trimester were important factors associated with drug-positive meconium specimens. The results suggest that buprenorphine and metabolite concentrations in the meconium may predict the onset and frequency of NAS. PMID:18701886

Search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism.

PubMed

Martínez-Ramírez, Jorge A; Strien, Juliane; Walther, Grit; Peters, Frank T

2016-05-01

Fungi colonizing cadavers are capable of drug metabolism and may thus change the metabolite pattern or concentration of drugs in forensic postmortem samples. The purpose of this study was to check for the presence of such changes by searching fungi-specific metabolites of four model drugs (amitriptyline, metoprolol, mirtazapine, and zolpidem) in decomposed postmortem blood samples from 33 cases involving these drugs. After isolation and identification of fungal strains present in the samples, each isolate was incubated in Sabouraud medium at 25°C for up to 120h with each model drug. One part of the supernatants was directly analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), another after liquid-liquid extraction with chlorobutane and concentration. From 21 out of 33 decomposed postmortem blood samples (64%) a total of 30 different strains could be isolated, one from the class of Ascomycete and the rest belonging to 15 species from 8 different genera (number of species): Aspergillus (2), Botrytis (1), Candida (8), Fusarium (1), Mucor (1), Penicillium (1), and Rodothorula (1). In the in vitro studies, these microorganisms were found capable of N-demethylation and N-oxidation of amitriptyline and mirtazapine, O-demethylation followed by side chain oxidation of metoprolol as well as hydroxylation of all four-model drugs. In two of the postmortem blood samples, from which the fungi Aspergillus jensenii, Candida parapsilosis. and Mucor circinelloides had been isolated, a fungi-specific hydroxy zolpidem metabolite was detected. The presence of this metabolite in postmortem samples likely indicates postmortem fungal biodegradation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of allocryptopine and protopine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

PubMed

Huang, Ya-Jun; Xiao, Sa; Sun, Zhi-Liang; Zeng, Jian-Guo; Liu, Yi-Song; Liu, Zhao-Ying

2016-07-15

Allocryptopine (AL) and protopine (PR) have been extensively studied because of their anti-parasitic, anti-arrhythmic, anti-thrombotic, anti-inflammatory and anti-bacterial activity. However, limited information on the pharmacokinetics and metabolism of AL and PR has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of AL and PR in rat liver S9 using a rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOFMS) method. The incubation mixture was processed with 15% trichloroacetic acid (TCA). Multiple scans of AL and PR metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the precursor ion or metabolite. Eight and five metabolites of AL and PR were identified in rat liver S9, respectively. Among these metabolites, seven and two metabolites of AL and PR were identified in the first time, respectively. The demethylenation of the 2,3-methylenedioxy, the demethylation of the 9,10-vicinal methoxyl group and the 2,3-methylenedioxy group were the main metabolic pathways of AL and PR in liver S9, respectively. In addition, the cleavage of the methylenedioxy group of the drugs and subsequent methylation or O-demethylation were also the common metabolic pathways of drugs in liver S9. In addition, the hydroxylation reaction was also the metabolic pathway of AL. This was the first investigation of in vitro metabolism of AL and PR in rat liver S9. The detailed structural elucidations of AL and PR metabolites were performed using a rapid and accurate HPLC/QqTOFMS method. The metabolic pathways of AL and PR in rat were tentatively proposed based on these characterized metabolites and early reports. Copyright © 2016 John Wiley

Damage mechanism of hydroxyl radicals toward adenine—thymine base pair

NASA Astrophysics Data System (ADS)

Tan, Rong-Ri; Wang, Dong-Qi; Zhang, Feng-Shou

2014-02-01

The adenine—thymine base pair was studied in the presence of hydroxyl radicals in order to probe the hydrogen bond effect. The results show that the hydrogen bonds have little effect on the hydroxylation and dehydrogenation happened at the sites, which are not involved in a hydrogen bond, while at the sites involved in hydrogen bond formation in the base pair, the reaction becomes more difficult, both in view of the free energy barrier and the exothermicity. With a 6-311++G(d,p) level of description, both B3LYP and MP2 methods confirm that the C8 site of isolated adenine has the highest possibility to form covalent bond with the hydroxyl radicals, though with different energetics: B3LYP predicts a barrierless pathway, while MP2 finds a transition state with an energy of 106.1 kJ/mol. For the dehydrogenation reactions, B3LYP method predicts that the free energy barrier increases in the order of HN9 < HN61 < HN62 < H2 < H8.

Moringa oleifera leaf extracts inhibit 6β-hydroxylation of testosterone by CYP3A4

PubMed Central

Monera, Tsitsi G.; Wolfe, Alan R.; Maponga, Charles C.; Benet, Leslie Z.; Guglielmo, Joseph

2017-01-01

Background Moringa oleifera is a tropical tree often used as a herbal medicine, including by people who test positive for HIV. Since herbal constituents may interact with drugs via inhibition of metabolizing enzymes, we investigated the effects of extracts of M. oleifera on the CYP3A4-mediated 6ß-hydroxylation of testosterone. Methods Methanolic and aqueous leaf and root of extracts of M. oleifera with concentrations between 0.01 and 10 mg/ml were incubated with testosterone and mixed-sex human liver microsomes in the presence of NADPH. Metabolite concentrations were determined by HPLC. The cytotoxicity of the extracts was tested with HepG2 cells using the MTT formazan assay. Results Significant CYP3A4 inhibitory effects were found, with IC50 values of 0.5 and 2.5 mg/ml for leaf-methanol and leaf-water extracts, respectively. Root extracts were less active. Cytotoxicity was observed only with the leaf-water extract (IC50 = 6 mg/ml). Conclusions Further investigation is warranted to elucidate the potential of M. oleifera for clinically significant interactions with antiretroviral and other drugs. PMID:19745507

Simultaneous extraction of proteins and metabolites from cells in culture

PubMed Central

Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

2014-01-01

Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938

Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite.

PubMed

Zheng, H X; Huang, Y; Frassetto, L A; Benet, L Z

2009-01-01

The effects of single doses of intravenous (IV) ciprofloxacin and rifampin and of multiple doses of rifampin on glyburide exposure and blood glucose levels were investigated in nine healthy volunteers. A single IV dose of rifampin significantly increased the area under the concentration-time curve (AUC) of glyburide and its metabolite. Blood glucose levels were significantly lower than those observed after dosing with glyburide alone. Multiple doses of rifampin induced an increase in liver enzyme levels, leading to a marked decrease in glyburide exposure and blood glucose levels. When IV rifampin was administered after multiple doses of rifampin, the inhibition of hepatic uptake transporters masked the induction effect; however, the relative changes in AUC for glyburide and its hydroxyl metabolite were similar to those seen under noninduced conditions. The studies reported here demonstrate how measurements of the levels of both the parent drug and its primary metabolite are useful in unmasking simultaneous drug-drug induction and inhibition effects and in characterizing enzymatic vs. transporter mechanisms.

An Efficient Approach to Sulfate Metabolites of Polychlorinated Biphenyls

PubMed Central

Li, Xueshu; Parkin, Sean; Duffel, Michael W.; Robertson, Larry W.; Lehmler, Hans-Joachim

2009-01-01

Polychlorinated biphenyls (PCBs), a major class of persistent organic pollutants, are metabolized to hydroxylated PCBs. Several hydroxylated PCBs are substrates of cytosolic phase II enzymes, such as phenol and hydroxysteroid (alcohol) sulfotransferases; however, the corresponding sulfation products have not been isolated and characterized. Here we describe a straightforward synthesis of a series of ten PCB sulfate monoesters from the corresponding hydroxylated PCBs. The hydroxylated PCBs were synthesized by coupling chlorinated benzene boronic acids with appropriate brominated (chloro-)anisoles, followed by demethylation with boron tribromide. The hydroxylated PCBs were sulfated with 2,2,2-trichloroethyl chlorosulfate using DMAP as base. Deprotection with zinc powder/ammonium formate yielded the ammonium salts of the desired PCB sulfate monoesters in good yields when the sulfated phenyl ring contained no or one chlorine substituent. However, no PCB sulfate monoesters were isolated when two chlorines were present ortho to the sulfated hydroxyl group. To aid with future quantitative structure activity relationship studies, the structures of two 2,2,2-trichloroethyl-protected PCB sulfates were verified by X-ray diffraction. PMID:19345419

Identification of three new phase II metabolites of a designer drug methylone formed in rats by N-demethylation followed by conjugation with dicarboxylic acids.

PubMed

Židková, Monika; Linhart, Igor; Balíková, Marie; Himl, Michal; Dvořáčková, Veronika; Lhotková, Eva; Páleníček, Tomáš

2018-06-01

1. Methylone (3,4-methylenedioxy-N-methylcathinone, MDMC), which appeared on the illicit drug market in 2004, is a frequently abused synthetic cathinone derivative. Known metabolic pathways of MDMC include N-demethylation to normethylone (3,4-methylenedioxycathinone, MDC), aliphatic chain hydroxylation and oxidative demethylenation followed by monomethylation and conjugation with glucuronic acid and/or sulphate. 2. Three new phase II metabolites, amidic conjugates of MDC with succinic, glutaric and adipic acid, were identified in the urine of rats dosed subcutaneously with MDMC.HCl (20 mg/kg body weight) by LC-ESI-HRMS using synthetic reference standards to support identification. 3. The main portion of administered MDMC was excreted unchanged. Normethylone, was a major urinary metabolite, of which a minor part was conjugated with dicarboxylic acids. 4. Previously identified ring-opened metabolites 4-hydroxy-3-methoxymethcathinone (4-OH-3-MeO-MC), 3-hydroxy-4-methoxymeth-cathinone (3-OH-4-MeO-MC) and 3,4-dihydroxymethcathinone (3,4-di-OH-MC) mostly in conjugated form with glucuronic and/or sulphuric acids were also detected. 5. Also, ring-opened metabolites derived from MDC, namely, 4-hydroxy-3-methoxycathinone (4-OH-3-MeO-C), 3-hydroxy-4-methoxycathinone (3-OH-4-MeO-C) and 3,4-dihydroxycathinone (3,4-di-OH-C) were identified for the first time in vivo.

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL).

PubMed

Chen, P; Tian, Z; Digenis, G A; Tai, H H

1996-06-01

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.

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

Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme

DOEpatents

Koch, Daniel J.; Arnold, Frances H.

2013-01-29

Cytochrome P450 CYP153A6 from Myobacterium sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.

Synthesis and Evaluation of Vitamin D Receptor-Mediated Activities of Cholesterol and Vitamin D Metabolites

PubMed Central

Teske, Kelly A.; Bogart, Jonathan W.; Sanchez, Luis M.; Yu, Olivia B.; Preston, Joshua V.; Cook, James M.; Silvaggi, Nicholas R.; Bikle, Daniel D.; Arnold, Leggy A.

2016-01-01

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer. PMID:26774929

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

PubMed Central

Winkelmann, Traud; Ratjens, Svenja; Bartsch, Melanie; Rode, Christina; Niehaus, Karsten; Bednarz, Hanna

2015-01-01

Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

Elucidating Rifampin’s Inducing and Inhibiting Effects on Glyburide Pharmacokinetics and Blood Glucose in Healthy Volunteers: Unmasking the Differential Effect of Enzyme Induction and Transporter Inhibition for a Drug and Its Primary Metabolite

PubMed Central

Zheng, HX; Huang, Y; Frassetto, LA; Benet, LZ

2013-01-01

The effects of single doses of intravenous ciprofloxacin and rifampin, multiple doses of rifampin, on glyburide exposure and effect on blood glucose levels in 9 healthy volunteers were investigated. The single intravenous dose of rifampin significantly increased the AUCs of glyburide and metabolite. Blood glucose levels dropped significantly in comparison to when glyburide was dosed alone. Multiple doses of rifampin induced liver enzymes leading to a marked decrease in glyburide exposure and in blood glucose measurements. When intravenous rifampin was given after multiple doses of rifampin, the inhibition of hepatic uptake transporters masked the induction effect, however, relative changes in AUC for glyburide and its hydroxyl metabolite were the same as that seen under non-induced conditions. The studies reported here demonstrate how measurements of both the parent drug and its primary metabolite are useful in unmasking simultaneous drug-drug induction and inhibition effects and characterizing enzymatic versus transporter mechanisms. PMID:18843263

Proline Hydroxylation in Cell Wall Proteins: Is It Yet Possible to Define Rules?

PubMed

Duruflé, Harold; Hervé, Vincent; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth

2017-01-01

Cell wall proteins (CWPs) play critical and dynamic roles in plant cell walls by contributing to developmental processes and response to environmental cues. Since the CWPs go through the secretion pathway, most of them undergo post-translational modifications (PTMs) which can modify their biological activity. Glycosylation is one of the major PTMs of CWPs and refers to N -glycosylation, O -glycosylation and glypiation. Each of these PTMs occurs in different amino acid contexts which are not all well defined. This article deals with the hydroxylation of Pro residues which is a prerequisite for O -glycosylation of CWPs on hydroxyproline (Hyp) residues. The location of Hyp residues is well described in several structural CWPs, but yet rarely described in other CWPs. In this article, it is studied in detail in five Arabidopsis thaliana proteins using mass spectrometry data: one of them (At4g38770, AtPRP4) is a structural CWP containing 32.5% of Pro residues arranged in typical motifs, the others are either rich (27-28%, At1g31580 and At2g10940) or poor (6-8%, At1g09750 and At3g08030) in Pro residues. The known rules of Pro hydroxylation allowed a good prediction of Hyp location in AtPRP4. However, they could not be applied to the other proteins whatever their Pro content. In addition, variability of the Pro hydroxylation patterns was observed within some amino acid motifs in all the proteins and new patterns of Pro hydroxylation are described. Altogether, this work shows that Hyp residues are present in more protein families than initially described, and that Pro hydroxylation patterns could be different in each of them. This work paves the way for completing the existing Pro hydroxylation code.

In vivo proton MRS to quantify anesthetic effects of pentobarbital on cerebral metabolism and brain activity in rat.

PubMed

Du, Fei; Zhang, Yi; Iltis, Isabelle; Marjanska, Malgorzata; Zhu, Xiao-Hong; Henry, Pierre-Gilles; Chen, Wei

2009-12-01

To quantitatively investigate the effects of pentobarbital anesthesia on brain activity, brain metabolite concentrations and cerebral metabolic rate of glucose, in vivo proton MR spectra, and electroencephalography were measured in the rat brain with various doses of pentobarbital. The results show that (1) the resonances attributed to propylene glycol, a solvent in pentobarbital injection solution, can be robustly detected and quantified in the brain; (2) the concentration of most brain metabolites remained constant under the isoelectric state (silent electroencephalography) with a high dose of pentobarbital compared to mild isoflurane anesthesia condition, except for a reduction of 61% in the brain glucose level, which was associated with a 37% decrease in cerebral metabolic rate of glucose, suggesting a significant amount of "housekeeping" energy for maintaining brain cellular integrity under the isoelectric state; and (3) electroencephalography and cerebral metabolic activities were tightly coupled to the pentobarbital anesthesia depth and they can be indirectly quantified by the propylene glycol resonance signal at 1.13 ppm. This study indicates that in vivo proton MR spectroscopy can be used to measure changes in cerebral metabolite concentrations and cerebral metabolic rate of glucose under varied pentobarbital anesthesia states; moreover, the propylene glycol signal provides a sensitive biomarker for quantitatively monitoring these changes and anesthesia depth noninvasively. (c) 2009 Wiley-Liss, Inc.

Metabolites identification of Huo Luo Xiao Ling Dan in rat urine by UPLC coupled with electrospray ionization time-of-flight mass spectrometry.

PubMed

Wang, Fenrong; Wu, Yun; Ai, Yu; Bian, Qiaoxia; Ma, Wen; Lee, David Y-W; Dai, Ronghua

2016-03-01

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula, is used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) method was developed for detection and identification of HLXLD metabolites in rat urine at high and normal clinical dosages. The prototype constituents and their metabolites in urine were analyzed. The mass measurements were accurate within 8 ppm, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 85 compounds were detected in high dosages urine samples by a highly sensitive extracted ion chromatograms method, including 31 parent compounds and 54 metabolites. Our results indicated that phase 2 reactions (e.g. glucuronidation, glutathionidation and sulfation) were the main metabolic pathways of lactones, alkaloids and flavones, while phase I reactions (e.g. hydrogenation and hydroxylation) were the major metabolic reaction for coumarins, paeoniflorin and iridoids. This investigation provided important structural information on the metabolism of HLXLD and provided scientific evidence to obtain a more comprehensive metabolic profile. Copyright © 2015 John Wiley & Sons, Ltd.

Novel denture-cleaning system based on hydroxyl radical disinfection.

PubMed

Kanno, Taro; Nakamura, Keisuke; Ikai, Hiroyo; Hayashi, Eisei; Shirato, Midori; Mokudai, Takayuki; Iwasawa, Atsuo; Niwano, Yoshimi; Kohno, Masahiro; Sasaki, Keiichi

2012-01-01

The purpose of this study was to evaluate a new denture-cleaning device using hydroxyl radicals generated from photolysis of hydrogen peroxide (H2O2). Electron spin resonance analysis demonstrated that the yield of hydroxyl radicals increased with the concentration of H2O2 and light irradiation time. Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant S aureus were killed within 10 minutes with a > 5-log reduction when treated with photolysis of 500 mM H2O2; Candida albicans was killed within 30 minutes with a > 4-log reduction with photolysis of 1,000 mM H2O2. The clinical test demonstrated that the device could effectively reduce microorganisms in denture plaque by approximately 7-log order within 20 minutes.

Novel liquid chromatography-electrospray ionization mass spectrometry method for the quantification in human urine of microbial aromatic acid metabolites derived from dietary polyphenols.

PubMed

Gonthier, Marie-Paule; Rios, Laurent Y; Verny, Marie- Anne; Rémésy, Christian; Scalbert, Augustin

2003-06-15

An HPLC-ESI-MS-MS method was developed to quantify in human urine fourteen aromatic acids known as metabolites of dietary polyphenols. These metabolites were determined simultaneously in a single 20-min chromatographic analysis with multiple reaction monitoring detection. The inter- and intra-day precisions, calculated from quality control samples were 8.8 and 5.3%, respectively, and the mean accuracy was 2.3%. The method was tested on urine samples collected from one healthy volunteer who consumed a polyphenol-rich diet for 3 days. Increased levels of several aromatic acid metabolites were observed, demonstrating that the method can be used to detect changes in the excretion of microbial metabolites induced by the consumption of polyphenol-containing foods in humans.

Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting

PubMed Central

Saladino, Jessica; Liu, Mian; Live, David; Sharp, Joshua S.

2009-01-01

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide in order to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues. PMID:19278868

Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms

PubMed Central

Osakabe, Keishi; Tsao, Cheng Chung; Li, Laigeng; Popko, Jacqueline L.; Umezawa, Toshiaki; Carraway, Daniel T.; Smeltzer, Richard H.; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1999-01-01

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, kcat/Km of CAld5H for coniferyl aldehyde was ≈140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (Ki = 0.59 μM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis. PMID:10430877

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms.

PubMed

Bellin, Daniel L; Sakhtah, Hassan; Rosenstein, Jacob K; Levine, Peter M; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E P; Shepard, Kenneth L

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. 'Images' over a 3.25 × 0.9 mm(2) area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

PubMed Central

Bellin, Daniel L.; Sakhtah, Hassan; Rosenstein, Jacob K.; Levine, Peter M.; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E. P.; Shepard, Kenneth L.

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites produced by microbial biofilms, which can drastically affect colony development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. “Images” over a 3.25 × 0.9 mm area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify, and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression. PMID:24510163

Metabolites profiling of Pulsatilla saponin D in rat by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS).

PubMed

Ouyang, Hui; Zhou, Maofu; Guo, Yicheng; He, Mingzhen; Huang, Hesong; Ye, Xide; Feng, Yulin; Zhou, Xin; Yang, Shilin

2014-07-01

Pulsatilla saponin D, an antitumor substance isolated from traditional Chinese herbal medicine Pulsatilla chinensis (Bge.) Regel, is a promising candidate for new drug development. The purpose of the present study is to establish a simple and practical strategy for the metabolite profiling of Pulsatilla saponin D in vivo. A total of 18 metabolites were identified in rat plasma, urine and feces samples based on MS and MS/MS data by using ESI-Q-TOF-MS/MS, and eight of them (M11-M18) were reported for the first time. The results indicated that deglycosylation, dehydrogenation, hydroxylation and sulfation were the major metabolic transformations of Pulsatilla saponin D in vivo. This study has improved our understanding of the metabolic fate of Pulsatilla saponin D in vivo, and the information gained from the current study is relevant to the pharmacological activity of Pulsatilla saponin D. Copyright © 2014 Elsevier B.V. All rights reserved.

Black Hydroxylated Titanium Dioxide Prepared via Ultrasonication with Enhanced Photocatalytic Activity

PubMed Central

Fan, Chenyao; Chen, Chao; Wang, Jia; Fu, Xinxin; Ren, Zhimin; Qian, Guodong; Wang, Zhiyu

2015-01-01

The amorphous TiO2 derived from hydroxylation has become an effective approach for the enhancement of photocatalytic activity of TiO2 since a kind of special black TiO2 was prepared by engineering disordered layers on TiO2 nanocrystals via hydrogenation. In this contribution, we prepared totally amorphous TiO2 with various degrees of blackness by introducing hydroxyls via ultrasonic irradiation, through which can we remarkably enhance the photocatalytic activity of TiO2 with improved light harvesting and narrowed band gap. PMID:26133789

Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes.

PubMed

Shrestha, Riya; Kim, Ju-Hyun; Nam, Wongshik; Lee, Hye Suk; Lee, Jae-Mok; Lee, Sangkyu

2018-04-01

Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb-drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC 50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Comparison of salicylate and D-phenylalanine for detection of hydroxyl radicals in chemical and biological reactions.

PubMed

Bailey, S M; Fauconnet, A L; Reinke, L A

1997-02-01

Hydroxylation of salicylate and D-phenylalanine was measured to test the usefulness of these compounds for hydroxyl radical (HO(•)) detection in chemical and biological systems. When HO(•) were produced by the photolytic decomposition of hydrogen peroxide, nearly equal amounts of 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were produced from salicylate, with catechol as a minor product. In the photolytic reaction, nearly equal concentrations of p-,m-, and o-tyrosine were formed from D-phenylalanine. When salicylate or D-phenylalanine was present with Fenton reagents or in iron(II) autoxidation systems, the relative proportions of hydroxylated products were similar to those observed after photolysis, although less total products were usually detected. In contrast, when similar experiments were conducted with isolated hepatic microsomes and perfused livers, 2,5-DHBA was the primary product from salicylate, and p-tyrosine was the major product from D-phenylalanine. Cytochrome P-450 enzymes can hydroxylate salicylate to produce 2,5-DHBA, and it is likely that phenylalanine hydroxylase produces most of the p-tyrosine detected in hepatic tissues. Thus, although both salicylate and D-phenylalanine are useful probes for hydroxyl radical formation in chemical systems, hydroxylated products formed from enzymatic reactions complicate interpretation of data from both compounds in vivo.

Comparison of concentrations and profiles of polycyclic aromatic hydrocarbon metabolites in bile of fishes from offshore oil platforms and natural reefs along the California coast

USGS Publications Warehouse

Gale, Robert W.; Tanner, Michael J.; Love, Milton S.; Nishimoto, Mary M.; Schroeder, Donna M.

2012-01-01

To determine the environmental consequences of decommissioning offshore oil platforms on local and regional fish populations, contaminant loads in reproducing adults were investigated at seven platform sites and adjacent, natural sites. Specimens of three species (Pacific sanddab, Citharichthys sordidus; kelp rockfish, Sebastes atrovirens; and kelp bass, Paralabrax clathratus) residing at platforms and representing the regional background within the Santa Barbara Channel and within the San Pedro Basin were collected. Some of the most important contaminant classes related to oil operations are polycyclic aromatic hydrocarbons (PAHs) because of their potential toxicity and carcinogenicity. However, acute exposure cannot be related directly to PAH tissue concentrations because of rapid metabolism of the parent chemicals in fish; therefore, PAH metabolites in bile were measured, targeting free hydroxylated PAHs (OH-PAHs) liberated by enzymatic hydrolysis of the bound PAH glucuronides and sulfates. An ion-pairing method was developed for confirmatory analysis that targeted PAH glucuronides and sulfates. Concentrations of hydroxylated PAHs in all samples (76 fish from platforms and 64 fish from natural sites) were low, ranging from less than the limits of detection (5 to 120 nanograms per milliliter bile; 0.03 to 42 nanograms per milligram protein) to a maximum of 320 nanograms per milliliter bile (32 nanograms per milligram protein). A previously proposed dosimeter of PAH exposure in fish, 1-hydroxypyrene, was not detected at any platform site. Low concentrations of 1-hydroxypyrene were detected in 3 of 12 kelp rockfish collected from a natural reef site off Santa Barbara. The most prevalent OH-PAH, 2-hydroxyfluorene, was detected at low concentrations in seven fish of various species; of these, four were from two of the seven platform sites. The greatest concentrations of 2-hydroxyfluorene were found in three fish of various species from Platform Holly and were only

A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites.

PubMed

Vo Duy, S; Besteiro, S; Berry, L; Perigaud, C; Bressolle, F; Vial, H J; Lefebvre-Tournier, I

2012-08-20

Plasmodium falciparum is the causative agent of malaria, a deadly infectious disease for which treatments are scarce and drug-resistant parasites are now increasingly found. A comprehensive method of identifying and quantifying metabolites of this intracellular parasite could expand the arsenal of tools to understand its biology, and be used to develop new treatments against the disease. Here, we present two methods based on liquid chromatography tandem mass spectrometry for reliable measurement of water-soluble metabolites involved in phospholipid biosynthesis, as well as several other metabolites that reflect the metabolic status of the parasite including amino acids, carboxylic acids, energy-related carbohydrates, and nucleotides. A total of 35 compounds was quantified. In the first method, polar compounds were retained by hydrophilic interaction chromatography (amino column) and detected in negative mode using succinic acid-(13)C(4) and fluorovaline as internal standards. In the second method, separations were carried out using reverse phase (C18) ion-pair liquid chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent in positive detection mode, using d(9)-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r(2)>0.99). The intra- and inter-day accuracy and precision as well as the extraction recovery of each compound were determined. The lower limit of quantitation varied from 50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites. Copyright © 2012 Elsevier B.V. All rights reserved.

Electrochemical behavior of triflusal, aspirin and their metabolites at glassy carbon and boron doped diamond electrodes.

PubMed

Enache, Teodor Adrian; Fatibello-Filho, Orlando; Oliveira-Brett, Ana Maria

2010-08-01

The electrochemical behavior of triflusal (TRF) and aspirin (ASA), before and after hydrolysis in water and in alkaline medium using two different electrode surfaces, glassy carbon and boron doped diamond, was study by differential pulse voltammetry over a wide pH range. The hydrolysis products are 2-(hydroxyl)-4-(trifluoromethyl)-benzoic acid (HTB) for triflusal and salicylic acid (SA) for aspirin, which in vivo represent their main metabolites. The hydrolysis processes were also followed by spectrophotometry. The UV results showed complete hydrolysis after one hour for TRF and after two hours for ASA in alkaline solution. The glassy carbon electrode enables only indirect determination of TRF and ASA through the electrochemical detection of their hydrolysis products HTB and SA, respectively. The oxidation processes of HTB and SA are pH dependent and involve different numbers of electrons and protons. Moreover, the difference between the oxidation peak potential of SA and HTB was equal to 100 mV in the studied pH range from 1 to 8 due to the CF3 of the aromatic ring of HTB molecule. Due to its wider oxidation potential range, the boron doped diamond electrode was used to study the direct oxidation of TRF and ASA, as well as of their respective metabolites HTB and SA.

Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer.

PubMed

Kovacic, Peter

2005-01-01

Cocaine is one of the principal drugs of abuse. Although impressive advances have been made, unanswered questions remain concerning mechanism of toxicity and addiction. Discussion of action mode usually centers on receptor binding and enzyme inhibition, with limited attention to events at the molecular level. This review provides extensive evidence in support of the hypothesis that oxidative metabolites play important roles comprising oxidative stress (OS), reactive oxygen species (ROS), and electron transfer (ET). The metabolites include norcocaine and norcocaine derivatives: nitroxide radical, N-hydroxy, nitrosonium, plus cocaine iminium and formaldehyde. Observed formation of ROS is rationalized by redox cycling involving several possible ET agents. Three potential ones are present in the form of oxidative metabolites, namely, nitroxide, nitrosonium, and iminium. Most attention has been devoted to the nitroxide-hydroxylamine couple which has been designated by various investigators as the principal source of ROS. The proximate ester substituent is deemed important for intramolecular stabilization of reactive intermediates. Reduction potential of nitroxide is in accord with plausibility of ET in the biological milieu. Toxicity by cocaine, with evidence for participation of OS, is demonstrated for many body components, including liver, central nervous system, cardiovascular system, reproductive system, kidney, mitochondria, urine, and immune system. Other adverse effects associated with ROS comprise teratogenesis and apoptosis. Examples of ROS generated are lipid peroxides and hydroxyl radical. Often observed were depletion of antioxidant defenses, and protection by added antioxidants, such as, thiol, salicylate, and deferoxamine. Considerable evidence supports the contention that oxidative ET metabolites of cocaine are responsible for much of the observed OS. Quite significantly, the pro-oxidant, toxic effects, including generation of superoxide and lipid peroxyl

Metabolite identification of the antimalarial naphthoquine using liquid chromatography-tandem high-resolution mass spectrometry in combination with multiple data-mining tools.

PubMed

Sun, Yanhong; Wang, Shuqi; Ji, Jianbo; Zhai, Guangxi; Xing, Jie

2018-06-01

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2-4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMS n ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMS n in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2018 John Wiley & Sons, Ltd.

Optimizing Urine Processing Protocols for Protein and Metabolite Detection.

PubMed

Siddiqui, Nazema Y; DuBois, Laura G; St John-Williams, Lisa; Will, Thompson J; Grenier, Carole; Burke, Emily; Fraser, Matthew O; Amundsen, Cindy L; Murphy, Susan K

In urine, factors such as timing of voids, and duration at room temperature (RT) may affect the quality of recovered protein and metabolite data. Additives may aid with detection, but can add more complexity in sample collection or analysis. We aimed to identify the optimal urine processing protocol for clinically-obtained urine samples that allows for the highest protein and metabolite yields with minimal degradation. Healthy women provided multiple urine samples during the same day. Women collected their first morning (1 st AM) void and another "random void". Random voids were aliquotted with: 1) no additive; 2) boric acid (BA); 3) protease inhibitor (PI); or 4) both BA + PI. Of these aliquots, some were immediately stored at 4°C, and some were left at RT for 4 hours. Proteins and individual metabolites were quantified, normalized to creatinine concentrations, and compared across processing conditions. Sample pools corresponding to each processing condition were analyzed using mass spectrometry to assess protein degradation. Ten Caucasian women between 35-65 years of age provided paired 1 st morning and random voided urine samples. Normalized protein concentrations were slightly higher in 1 st AM compared to random "spot" voids. The addition of BA did not significantly change proteins, while PI significantly improved normalized protein concentrations, regardless of whether samples were immediately cooled or left at RT for 4 hours. In pooled samples, there were minimal differences in protein degradation under the various conditions we tested. In metabolite analyses, there were significant differences in individual amino acids based on the timing of the void. For comparative translational research using urine, information about void timing should be collected and standardized. For urine samples processed in the same day, BA does not appear to be necessary while the addition of PI enhances protein yields, regardless of 4°C or RT storage temperature.

Hydroxyl radical generation by photosystem II.

PubMed

Pospísil, Pavel; Arató, András; Krieger-Liszkay, Anja; Rutherford, A William

2004-06-01

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH

Modeling the thermostability of surface functionalisation by oxygen, hydroxyl, and water on nanodiamonds.

PubMed

Lai, Lin; Barnard, Amanda S

2011-06-01

Understanding nanodiamond functionalisation is of great importance for biological and medical applications. Here we examine the stabilities of oxygen, hydroxyl, and water functionalisation of the nanodiamonds using the self-consistent charge density functional tight-binding simulations. We find that the oxygen and hydroxyl termination are thermodynamically favourable and form strong C–O covalent bonds on the nanodiamond surface in an O2 and H2 gas reservoir, which confirms previous experiments. Yet, the thermodynamic stabilities of oxygen and hydroxyl functionalisation decrease dramatically in a water vapour reservoir. In contrast, H2O molecules are found to be physically adsorbed on the nanodiamond surface, and forced chemical adsorption results in decomposition of H2O. Moreover, the functionalisation efficiency is found to be facet dependent. The oxygen functionalisation prefers the {100} facets as opposed to alternative facets in an O2 and H2 gas reservoir. The hydroxyl functionalisation favors the {111} surfaces in an O2 and H2 reservoir and the {100} facets in a water vapour reservoir, respectively. This facet selectivity is found to be largely dependent upon the environmental temperature, chemical reservoir, and morphology of the nanodiamonds.

Determination of spinetoram and its metabolites in amaranth and parsley using QuEChERS-based extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Park, Ki Hun; Choi, Jeong-Heui; Abd El-Aty, A M; Cho, Soon-Kil; Park, Jong-Hyouk; Kim, Bo Mi; Yang, Angel; Na, Tae Woong; Rahman, Musfiqur; Im, Geon-Jae; Shim, Jae-Han

2012-10-15

In this study, a simultaneous method was developed for the determination of spinetoram (XDE-175-J and XDE-175-L) and its demethyl metabolites (N-demethyl-175-J and N-demethyl-175-L) and formyl metabolites (N-formyl-175-J and N-formyl-175-L) in the minor crops; amaranth and parsley. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction. Afterwards, the analytes were quantified and confirmed via liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM). Calibration curves were linear over the calibration ranges for all the analytes tested with r(2)>0.993. Limits of detection and quantitation were 0.01 and 0.03 mg/kg for all the tested analytes in amaranth and parsley, respectively. Recovery values, at spiking levels 0.05 and 0.25 mg/kg, ranged from 71.0% to 115.2% with relative standard deviations <15%, except for N-formyl-175-J in both amaranth and parsley. This method was applied to field-incurred samples and was shown to provide an adequate sensitivity and performance for the simultaneous determination of spinetoram and metabolites. To the best of our knowledge, this is the first time spinetoram and its metabolites were quantified using LC-MS/MS in minor crops. Copyright © 2012 Elsevier Ltd. All rights reserved.

Twilight Intensity Variation of the Infrared Hydroxyl Airglow

NASA Technical Reports Server (NTRS)

Lowe, R. P.; Gilbert, K. L.; Niciejewski, R. J.

1984-01-01

The vibration rotation bands of the hydroxyl radical are the strongest features in the night airglow and are exceeded in intensity in the dayglow only by the infrared atmospheric bands of oxygen. The variation of intensity during evening twilight is discussed. Using a ground-based Fourier Transform Spectrometer (FTS), hydroxyl intensity measurements as early as 3 deg solar depression were made. Models of the twilight behavior show that this should be sufficient to provide measurement of the main portion of the twilight intensity change. The instrument was equipped with a liquid nitrogen-cooled germanium detector whose high sensitivity combined with the efficiency of the FTS technique permits spectra of the region 1.1 to 1.6 microns at high signal-to-noise to be obtained in two minutes. The use of a polarizer at the entrance aperture of the instrument reduces the intensity of scattered sunlight by a factor of at least ten for zenith observations.

APOE genotype modulates proton magnetic resonance spectroscopy metabolites in the aging brain.

PubMed

Gomar, Jesus J; Gordon, Marc L; Dickinson, Dwight; Kingsley, Peter B; Uluğ, Aziz M; Keehlisen, Lynda; Huet, Sarah; Buthorn, Justin J; Koppel, Jeremy; Christen, Erica; Conejero-Goldberg, Concepcion; Davies, Peter; Goldberg, Terry E

2014-05-01

Proton magnetic resonance spectroscopy ((1)H-MRS) studies on healthy aging have reported inconsistent findings and have not systematically taken into account the possible modulatory effect of APOE genotype. We aimed to quantify brain metabolite changes in healthy subjects in relation to age and the presence of the APOE E4 genetic risk factor for Alzheimer's disease. Additionally, we examined these measures in relation to cognition. We studied a cohort of 112 normal adults between 50 and 86 years old who were genotyped for APOE genetic polymorphism. Measurements of (1)H-MRS metabolites were obtained in the posterior cingulate and precuneus region. Measures of general cognitive functioning, memory, executive function, semantic fluency, and speed of processing were also obtained. General linear model analysis demonstrated that older APOE E4 carriers had significantly higher choline/creatine and myo-inositol/creatine ratios than APOE E3 homozygotes. Structural equation modeling resulted in a model with an excellent goodness of fit and in which the APOE × age interaction and APOE status each had a significant effect on (1)H-MRS metabolites (choline/creatine and myo-inositol/creatine). Furthermore, the APOE × age variable modulation of cognition was mediated by (1)H-MRS metabolites. In a healthy aging normal population, choline/creatine and myo-inositol/creatine ratios were significantly increased in APOE E4 carriers, suggesting the presence of neuroinflammatory processes and greater membrane turnover in older carriers. Structural equation modeling analysis confirmed these possible neurodegenerative markers and also indicated the mediator role of these metabolites on cognitive performance among older APOE E4 carriers. Copyright © 2014 Society of Biological Psychiatry. All rights reserved.

APOE Genotype Modulates Proton Magnetic Resonance Spectroscopy Metabolites in the Aging Brain

PubMed Central

Gomar, Jesus J.; Gordon, Marc L.; Dickinson, Dwight; Kingsley, Peter B.; Uluğ, Aziz M.; Keehlisen, Lynda; Huet, Sarah; Buthorn, Justin J.; Koppel, Jeremy; Christen, Erica; Conejero-Goldberg, Concepcion; Davies, Peter; Goldberg, Terry E.

2013-01-01

Background Proton magnetic resonance spectroscopy (1H-MRS) studies on healthy aging have reported inconsistent findings and have not systematically taken into account the possible modulatory effect of APOE genotype. We aimed to quantify brain metabolite changes in healthy subjects in relation to age and the presence of the APOE E4 genetic risk factor for Alzheimer's disease. Additionally, we examined these measures in relation to cognition. Methods We studied a cohort of 112 normal adults between 50 and 86 years old who were genotyped for APOE genetic polymorphism. Measurements of 1H-MRS metabolites were obtained in the posterior cingulate and precuneus region. Measures of general cognitive functioning, memory, executive function, semantic fluency, and speed of processing were also obtained. Results General linear model analysis demonstrated that older APOE E4 carriers had significantly higher choline/creatine and myoinositol/creatine ratios than APOE E3 homozygotes. Structural equation modeling resulted in a model with an excellent goodness of fit and in which the APOE × age interaction and APOE status each had a significant effect on 1H-MRS metabolites (choline/creatine and myo-inositol/creatine). Furthermore, the APOE × age variable modulation of cognition was mediated by 1H-MRS metabolites. Conclusions In a healthy aging normal population, choline/creatine and myo-inositol/creatine ratios were significantly increased in APOE E4 carriers, suggesting the presence of neuroinflammatory processes and greater membrane turnover in older carriers. Structural equation modeling analysis confirmed these possible neurodegenerative markers and also indicated the mediator role of these metabolites on cognitive performance among older APOE E4 carriers. PMID:23831342

40 CFR 721.10108 - Naphthalenedisulfonic acid, hydrozy-[[[(hydroxyl-disulfo-naphthaleneyl)azo]-alkyl(C=1-5...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Naphthalenedisulfonic acid, hydrozy-[[[(hydroxyl-disulfo-naphthaleneyl)azo]-alkyl(C=1-5)-(sulfoalkoxy)cyclic]azo]-substituted azo-, metal salt... Specific Chemical Substances § 721.10108 Naphthalenedisulfonic acid, hydrozy-[[[(hydroxyl-disulfo...

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

PubMed

Shao, Bo; Mao, Li; Qu, Na; Wang, Ya-Fen; Gao, Hui-Ying; Li, Feng; Qin, Li; Shao, Jie; Huang, Chun-Hua; Xu, Dan; Xie, Lin-Na; Shen, Chen; Zhou, Xiang; Zhu, Ben-Zhan

2017-03-01

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H 2 O 2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of macromolecular baseline of human brain using metabolite cycled semi-LASER at 9.4T.

PubMed

Giapitzakis, Ioannis-Angelos; Avdievich, Nikolai; Henning, Anke

2018-08-01

Macromolecular resonances (MM) arise mainly from cytosolic proteins and overlap with metabolites, influencing metabolite quantification. Macromolecules can serve as valuable biomarkers for diseases and pathologies. The objectives of this study were to characterize MM at 9.4T in the human brain (occipital and left parietal lobe) and to describe the RF coil setup used for MM acquisition in the two regions. An adiabatic inversion pulse was optimised for metabolite nulling at 9.4T using double inversion recovery and was combined for the first time with metabolite cycled (MC) semi-LASER and appropriate coil configuration. MM spectra (seven volunteers) from two brain locations were averaged and smoothed creating MM templates, which were then parametrized using simulated Voigt-shaped lines within LCModel. Quantification was performed on individual data sets, including corrections for different tissue composition and the T 1 and T 2 relaxation of water. Our coil configuration method resulted in efficient B1+ (>30 T/√kW) for both brain regions. The 15 MM components were detected and quantified in MM baselines of the two brain areas. No significant differences in concentration levels of MM between different regions were found. Two new MM peaks were reported (M7 & M8). Double inversion, which was combined with MC semi-LASER, enabled the acquisition of high spectral resolution MM spectra for both brain regions at 9.4T. The 15 MM components were detected and quantified. Two new MM peaks were reported for the first time (M7 & M8) and preliminarily assigned to β-methylene protons of aspartyl-groups. Magn Reson Med 80:462-473, 2018. © 2018 International Society for Magnetic Resonance in Medicine. © 2018 International Society for Magnetic Resonance in Medicine.

Quantitative Measurement of JWH-018 and JWH-073 Metabolites Excreted in Human Urine

PubMed Central

Moran, Cindy L.; Le, Vi-Huyen; Chimalakonda, Krishna C.; Smedley, Amy L.; Lackey, Felisia D.; Owen, Suzanne N.; Kennedy, Paul D.; Endres, Gregory W.; Ciske, Fred L.; Kramer, James B.; Kornilov, Andrei M.; Bratton, L. D.; Dobrowolski, Paul J.; Wessinger, William D.; Fantegrossi, William E.; Prather, Paul P.; James, Laura P.; Radominska-Pandya, Anna; Moran, Jeffery H.

2011-01-01

'K2/SPICE' products are commonly laced with aminoalkylindole synthetic cannabinoids (i.e., JWH-018 and JWH-073) and are touted as ‘legal’ marijuana substitutes. Here we validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) methsod for measuring urinary concentrations of JWH-018, JWH-073, and several potential metabolites of each. The analytical procedure has high capacity for sample throughput and does not require solid phase or liquid extraction. Evaluation of human urine specimens collected after the subjects reportedly administered JWH-018 or a mixture of JWH-018 and JWH-073 provides preliminary evidence of clinical utility. Two subjects that consumed JWH-018 primarily excreted glucuronidated conjugates of 5-(3-(1-naphthoyl)-1H-indol-1-yl)-pentanoic acid (> 50 ng/ml) and (1-(5-hydroxypentyl)- 1H -indol-3-yl)(naphthalene-1-yl)-methanone (> 30 ng/ml). Interestingly, oxidized metabolites of both JWH-018 and JWH-073 were detected in these specimens, suggesting either metabolic demethylation of JWH-018 to JWH-073 or a non-reported, previous JWH-073 exposure. Metabolic profiles generated from a subject who consumed a mixture of JWH-018 and JWH-073 were similar to profiles generated from subjects who presumably consumed JWH-018 exclusively. Oxidized metabolites of JWH-018 and JWH-073 were of the same pattern, but JWH-018 metabolites were excreted at lower concentrations. These results begin clinically validating the LC-MS/MS assay for detecting and quantifying aminoalkylindole metabolites. Full validation awaits further testing. PMID:21506519

Associations between Polybrominated Diphenyl Ether (PBDE) Flame Retardants, Phenolic Metabolites, and Thyroid Hormones during Pregnancy

PubMed Central

Eagle, Sarah; Anthopolos, Rebecca; Wolkin, Amy; Miranda, Marie Lynn

2011-01-01

Background: Polybrominated diphenyl ethers (PBDEs) are chemical additives used as flame retardants in commercial products. PBDEs are bioaccumulative and persistent and have been linked to several adverse health outcomes. Objectives: This study leverages an ongoing pregnancy cohort to measure PBDEs and PBDE metabolites in serum collected from an understudied population of pregnant women late in their third trimester. A secondary objective was to determine whether the PBDEs or their metabolites were associated with maternal thyroid hormones. Methods: One hundred forty pregnant women > 34 weeks into their pregnancy were recruited into this study between 2008 and 2010. Blood samples were collected during a routine prenatal clinic visit. Serum was analyzed for a suite of PBDEs, three phenolic metabolites (i.e., containing an –OH moiety), and five thyroid hormones. Results: PBDEs were detected in all samples and ranged from 3.6 to 694 ng/g lipid. Two hydroxylated BDE congeners (4´-OH-BDE 49 and 6-OH-BDE 47) were detected in > 67% of the samples. BDEs 47, 99, and 100 were significantly and positively associated with free and total thyroxine (T4) levels and with total triiodothyronine levels above the normal range. Associations between T4 and PBDEs remained after controlling for smoking status, maternal age, race, gestational age, and parity. Conclusions: PBDEs and OH-BDEs are prevalent in this cohort, and levels are similar to those in the general population. Given their long half-lives, PBDEs may be affecting thyroid regulation throughout pregnancy. Further research is warranted to determine mechanisms through which PBDEs affect thyroid hormone levels in developing fetuses and newborn babies. PMID:21715241

Reduced metabolites of nitroaromatics are distributed in the environment via the food chain.

PubMed

Nisar, Numrah; Cheema, Kausar J; Powell, Glen; Bennett, Mark; Chaudhary, Safee Ullah; Qadri, Rashad; Yang, Yaodong; Azam, Muhammad; Rossiter, John T

2018-05-15

Increased industrial processes have introduced emerging toxic pollutants into the environment. Phytoremediation is considered to be a very useful, economical and ecofriendly way of controlling these pollutants, however, certain pollutants can potentially travel through the food chain and accumulate at hazardous levels. Four isomers of dinitrotoluenes (DNT) were investigated and observed their potential toxicity towards A. thaliana. Two different aphid species (generalist and specialist) were allowed to feed on plants treated with DNTs and toxicity to aphids determined. Reduced metabolites of DNT (in both plant and aphids) were recovered and quantified through GC-MS analyses. 2,6-DNT was observed to be the toxic of the DNTs tested. Complete metabolism of DNTs to their reduced products was never achieved for higher concentrations. Regioselectivity was observed in the case of 2,4-DNT, with 4A2NT as the dominant isomer. Feeding aphids showed a similar toxicity pattern for DNT isomers as host plants. Metabolites were recovered from the body of aphids, demonstrating the potential transport of metabolites through the food chain. Plants show varied toxicity responses towards the DNT isomers. Aphids fed on A. thaliana plants treated with DNTs were shown to have ANTs present, which reflects the propagation of DNT metabolites through the food chain. Copyright © 2018 Elsevier B.V. All rights reserved.

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols.

PubMed

Dettmer, Katja; Nürnberger, Nadine; Kaspar, Hannelore; Gruber, Michael A; Almstetter, Martin F; Oefner, Peter J

2011-01-01

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid-base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid-base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.

Metabolite identification of triptolide by data-dependent accurate mass spectrometric analysis in combination with online hydrogen/deuterium exchange and multiple data-mining techniques.

PubMed

Du, Fuying; Liu, Ting; Liu, Tian; Wang, Yongwei; Wan, Yakun; Xing, Jie

2011-10-30

Triptolide (TP), the primary active component of the herbal medicine Tripterygium wilfordii Hook F, has shown promising antileukemic and anti-inflammatory activity. The pharmacokinetic profile of TP indicates an extensive metabolic elimination in vivo; however, its metabolic data is rarely available partly because of the difficulty in identifying it due to the absence of appropriate ultraviolet chromophores in the structure and the presence of endogenous interferences in biological samples. In the present study, the biotransformation of TP was investigated by improved data-dependent accurate mass spectrometric analysis, using an LTQ/Orbitrap hybrid mass spectrometer in conjunction with the online hydrogen (H)/deuterium (D) exchange technique for rapid structural characterization. Accurate full-scan MS and MS/MS data were processed with multiple post-acquisition data-mining techniques, which were complementary and effective in detecting both common and uncommon metabolites from biological matrices. As a result, 38 phase I, 9 phase II and 8 N-acetylcysteine (NAC) metabolites of TP were found in rat urine. Accurate MS/MS data were used to support assignments of metabolite structures, and online H/D exchange experiments provided additional evidence for exchangeable hydrogen atoms in the structure. The results showed the main phase I metabolic pathways of TP are hydroxylation, hydrolysis and desaturation, and the resulting metabolites subsequently undergo phase II processes. The presence of NAC conjugates indicated the capability of TP to form reactive intermediate species. This study also demonstrated the effectiveness of LC/HR-MS(n) in combination with multiple post-acquisition data-mining methods and the online H/D exchange technique for the rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.

Structure, electronic properties, and aggregation behavior of hydroxylated carbon nanotubes.

PubMed

López-Oyama, A B; Silva-Molina, R A; Ruíz-García, J; Gámez-Corrales, R; Guirado-López, R A

2014-11-07

We present a combined experimental and theoretical study to analyze the structure, electronic properties, and aggregation behavior of hydroxylated multiwalled carbon nanotubes (OH-MWCNT). Our MWCNTs have average diameters of ~2 nm, lengths of approximately 100-300 nm, and a hydroxyl surface coverage θ~0.1. When deposited on the air/water interface the OH-MWCNTs are partially soluble and the floating units interact and link with each other forming extended foam-like carbon networks. Surface pressure-area isotherms of the nanotube films are performed using the Langmuir balance method at different equilibration times. The films are transferred into a mica substrate and atomic force microscopy images show that the foam like structure is preserved and reveals fine details of their microstructure. Density functional theory calculations performed on model hydroxylated carbon nanotubes show that low energy atomic configurations are found when the OH groups form molecular islands on the nanotube's surface. This patchy behavior for the OH species is expected to produce nanotubes having reduced wettabilities, in line with experimental observations. OH doping yields nanotubes having small HOMO-LUMO energy gaps and generates a nanotube → OH direction for the charge transfer leading to the existence of more hole carriers in the structures. Our synthesized OH-MWCNTs might have promising applications.

An UPLC-MS/MS method for separation and accurate quantification of tamoxifen and its metabolites isomers.

PubMed

Arellano, Cécile; Allal, Ben; Goubaa, Anwar; Roché, Henri; Chatelut, Etienne

2014-11-01

A selective and accurate analytical method is needed to quantify tamoxifen and its phase I metabolites in a prospective clinical protocol, for evaluation of pharmacokinetic parameters of tamoxifen and its metabolites in adjuvant treatment of breast cancer. The selectivity of the analytical method is a fundamental criteria to allow the quantification of the main active metabolites (Z)-isomers from (Z)'-isomers. An UPLC-MS/MS method was developed and validated for the quantification of (Z)-tamoxifen, (Z)-endoxifen, (E)-endoxifen, Z'-endoxifen, (Z)'-endoxifen, (Z)-4-hydroxytamoxifen, (Z)-4'-hydroxytamoxifen, N-desmethyl tamoxifen, and tamoxifen-N-oxide. The validation range was set between 0.5ng/mL and 125ng/mL for 4-hydroxytamoxifen and endoxifen isomers, and between 12.5ng/mL and 300ng/mL for tamoxifen, tamoxifen N-desmethyl and tamoxifen-N-oxide. The application to patient plasma samples was performed. Copyright © 2014 Elsevier B.V. All rights reserved.

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase*

PubMed Central

Liu, Li-Kai; Abdelwahab, Heba; Martin Del Campo, Julia S.; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J.

2016-01-01

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2′-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. PMID:27557658

CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine.

PubMed

Pybus, Brandon S; Sousa, Jason C; Jin, Xiannu; Ferguson, James A; Christian, Robert E; Barnhart, Rebecca; Vuong, Chau; Sciotti, Richard J; Reichard, Gregory A; Kozar, Michael P; Walker, Larry A; Ohrt, Colin; Melendez, Victor

2012-08-02

The 8-aminoquinoline (8AQ) drug primaquine (PQ) is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ's haemotoxic and anti-malarial properties are not fully understood. In the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP) and mono-amine oxidase (MAO) families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements. Relative activity factor (RAF)-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species. As a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy.

PubMed

Brockerman, Jacob A; Okon, Mark; McIntosh, Lawrence P

2014-01-01

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

Synthesis and evaluation of hydroxylated polyamine analogues as antiproliferatives.

PubMed

Bergeron, R J; Müller, R; Huang, G; McManis, J S; Algee, S E; Yao, H; Weimar, W R; Wiegand, J

2001-07-19

A new means of accessing N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPMENSPM) and the first synthesis of (2R,10S)-N(1)-cyclopropylmethyl-2,10-dihydroxy-N(11)-ethylnorspermine [(2R,10S)-(HO)(2)CPMENSPM] are described. Both of these polyamine analogues are shown to be more active against L1210 murine leukemia cell growth than either N(1),N(11)-diethylnorspermine (DENSPM) or (2R,10R)-N(1),N(11)-diethyl-2,10-dihydroxynorspermine [(2R,10R)-(HO)(2)DENSPM] after 96 h of treatment; the activity was comparable to that of (2S,10S)-N(1),N(11)-diethyl-2,10-dihydroxynorspermine [(2S,10S)-(HO)(2)DENSPM] at 96 h. Both cyclopropyl compounds reduced putrescine and spermidine pools, but less effectively than did DENSPM and its derivatives. Only CPMENSPM, and not (2R,10S)-(HO)(2)CPMENSPM, lowered spermine pools. As with DENSPM and (2R,10R)-(HO)(2)DENSPM, both cyclopropyl analogues diminished ornithine decarboxylase and S-adenosylmethionine decarboxylase activity. Unlike the hydroxylated DENSPM compounds, both cyclopropyl norspermines substantially upregulated spermidine/spermine N(1)-acetyltransferase. The most interesting effect of hydroxylating CPMENSPM is the profound reduction in toxicity compared with that of the parent drug. The same phenomenon had been observed for the DENSPM/(2R,10R)-(HO)(2)DENSPM pair. Thus, hydroxylation of norspermine analogues appears to be a way to maintain the compounds' antiproliferative activity while reducing their toxicity.

Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals.

PubMed

Zhou, Juan; Li, Peng; Cheng, Ni; Gao, Hui; Wang, Bini; Wei, Yahui; Cao, Wei

2012-08-01

To understand the antioxidant properties of buckwheat honeys, we investigated their antioxidant effects on hydroxyl radical-induced DNA breaks in the non-site-specific and site-specific systems, the physicochemical properties, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity, chelating, and reducing power assays), total phenolic content and individual phenolic acids were also determined. Total phenolic content of buckwheat honeys ranged from 774 to 1694 mg PA/kg, and p-hydroxybenzoic and p-coumaric acids proved to be the main components in buckwheat honeys. All the buckwheat honey samples possess stronger capability to protect DNA in the non-site-specific systems than in the site-specific systems from being damaged by hydroxyl radicals. In the non-site-specific and site-specific system, buckwheat honeys samples prevented ()OH-induced DNA breaks by 21-78% and 5-31% over control value, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

New Methodology for Known Metabolite Identification in Metabonomics/Metabolomics: Topological Metabolite Identification Carbon Efficiency (tMICE).

PubMed

Sanchon-Lopez, Beatriz; Everett, Jeremy R

2016-09-02

A new, simple-to-implement and quantitative approach to assessing the confidence in NMR-based identification of known metabolites is introduced. The approach is based on a topological analysis of metabolite identification information available from NMR spectroscopy studies and is a development of the metabolite identification carbon efficiency (MICE) method. New topological metabolite identification indices are introduced, analyzed, and proposed for general use, including topological metabolite identification carbon efficiency (tMICE). Because known metabolite identification is one of the key bottlenecks in either NMR-spectroscopy- or mass spectrometry-based metabonomics/metabolomics studies, and given the fact that there is no current consensus on how to assess metabolite identification confidence, it is hoped that these new approaches and the topological indices will find utility.

Enantioselectivity of debrisoquine 4-hydroxylation in Brazilian Caucasian hypertensive patients phenotyped as extensive metabolizers.

PubMed

Cerqueira, P M; Mateus, F H; Cesarino, E J; Bonato, P S; Lanchote, V L

2000-12-01

Debrisoquine (D), an antihypertensive drug metabolized to 4-hydroxydebrisoquine (4-OHD) by CYP2D6, is commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as beta-adrenergic blockers, tricyclic antidepressants, and class 1C antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-OHD or S-(+) and R-(-)-4-OHD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-(-)- and S-(+)-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-OHD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-OHD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-OHD/% of dose excreted as D+4-OHD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5-acetonitrile (9:1, v/v) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-OHD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-(+)-4-OHD and R-(-)-4-OHD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5-acetonitrile-methanol (85:12:3, v/v/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter

[Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

PubMed

Orlova, T I; Bulgakova, V G; Polin, A N

2015-01-01

Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

Hydrogen-assisted versus hydroxyl-assisted CO dissociation over Co-doped Cu(111): A DFT study

NASA Astrophysics Data System (ADS)

Zha, Hao; Dong, Xiuqin; Yu, Yingzhe; Zhang, Minhua

2018-03-01

First principle based density functional theory (DFT) was used to calculate the step-by-step hydrogenation and dissociation reaction network of carbon monoxide (CO) over Co-doped Cu(111) surface as a model for understanding the lateral interaction of surface hydroxyl species (OH) on these reactions. We discussed the Csbnd O bond length and the adsorption energy changes of reaction intermediates under different adsorption circumstances for purpose of making out the effect of surface hydroxyl on the reaction selectivity. Reaction intermediates co-adsorbed with H atom and hydroxyl could undergo H-assisted or OH-assisted routes. The calculations show that the OH-assisted route prefers with the formation of COH, CHOH and CH2OH while general H-assisted route prefers with the formation of HCO, CH2O and CH3O. Considering the rather low activation barrier of COH, CHOH and CH2OH to form CHX, the existence of hydroxyl on the surface is in favor of boosting the CHX and suppressing the methanol.

Engineering Microbial Metabolite Dynamics and Heterogeneity.

PubMed

Schmitz, Alexander C; Hartline, Christopher J; Zhang, Fuzhong

2017-10-01

As yields for biological chemical production in microorganisms approach their theoretical maximum, metabolic engineering requires new tools, and approaches for improvements beyond what traditional strategies can achieve. Engineering metabolite dynamics and metabolite heterogeneity is necessary to achieve further improvements in product titers, productivities, and yields. Metabolite dynamics, the ensemble change in metabolite concentration over time, arise from the need for microbes to adapt their metabolism in response to the extracellular environment and are important for controlling growth and productivity in industrial fermentations. Metabolite heterogeneity, the cell-to-cell variation in a metabolite concentration in an isoclonal population, has a significant impact on ensemble productivity. Recent advances in single cell analysis enable a more complete understanding of the processes driving metabolite heterogeneity and reveal metabolic engineering targets. The authors present an overview of the mechanistic origins of metabolite dynamics and heterogeneity, why they are important, their potential effects in chemical production processes, and tools and strategies for engineering metabolite dynamics and heterogeneity. The authors emphasize that the ability to control metabolite dynamics and heterogeneity will bring new avenues of engineering to increase productivity of microbial strains. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Salt permeation and exclusion in hydroxylated and functionalized silica pores.

PubMed

Leung, Kevin; Rempe, Susan B; Lorenz, Christian D

2006-03-10

We use combined ab initio molecular dynamics (AIMD), grand canonical Monte Carlo, and molecular dynamics techniques to study the effect of pore surface chemistry and confinement on the permeation of salt into silica nanopore arrays filled with water. AIMD shows that 11.6 A diameter hydroxylated silica pores are relatively stable in water, whereas amine groups on functionalized pore surfaces abstract silanol protons, turning into NH3+. Free energy calculations using an ab initio parametrized force field show that the hydroxylated pores strongly attract Na+ and repel Cl- ions. Pores lined with NH3+ have the reverse surface charge polarity. Finally, studies of ions in carbon nanotubes suggest that hydration of Cl- is more strongly frustrated by pure confinement effects than Na+.

Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.

PubMed

Babot, Esteban D; Del Río, José C; Cañellas, Marina; Sancho, Ferran; Lucas, Fátima; Guallar, Víctor; Kalum, Lisbeth; Lund, Henrik; Gröbe, Glenn; Scheibner, Katrin; Ullrich, René; Hofrichter, Martin; Martínez, Angel T; Gutiérrez, Ana

2015-06-15

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106-118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

PubMed Central

Babot, Esteban D.; del Río, José C.; Cañellas, Marina; Sancho, Ferran; Lucas, Fátima; Guallar, Víctor; Kalum, Lisbeth; Lund, Henrik; Gröbe, Glenn; Scheibner, Katrin; Ullrich, René; Hofrichter, Martin; Martínez, Angel T.

2015-01-01

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed. PMID:25862224

Hydroxylation of Benzene via CH Activation Using Bimetallic ...

EPA Pesticide Factsheets

A photoactive bimetallic CuAg@g-C3N4 catalyst system has been designed and synthesized by impregnating copper and silver nanoparticles over the graphitic carbon nitride surface. Its application has been demonstrated in the hydroxylation of benzene under visible light. Prepared for submission to American Chemical Society (ACS) journal, ACS Sustainable Chemistry & Engineering.

Spatial variability in secondary metabolites of the indo-pacific sponge Stylissa massa.

PubMed

Rohde, Sven; Gochfeld, Deborah J; Ankisetty, Sridevi; Avula, Bharathi; Schupp, Peter J; Slattery, Marc

2012-05-01

Chemical diversity represents a measure of selective pressures acting on genotypic variability. In order to understand patterns of chemical ecology and biodiversity in the environment, it is necessary to enhance our knowledge of chemical diversity within and among species. Many sponges produce variable levels of secondary metabolites in response to diverse biotic and abiotic environmental factors. This study evaluated intra-specific variability in secondary metabolites in the common Indo-Pacific sponge Stylissa massa over various geographic scales, from local to ocean basin. Several major metabolites were quantified in extracts from sponges collected in American Samoa, Pohnpei, Saipan, and at several sites and depths in Guam. Concentrations of several of these metabolites varied geographically across the Pacific basin, with American Samoa and Pohnpei exhibiting the greatest differences, and Guam and Saipan more similar to each other. There were also significant differences in concentrations among different sites and depths within Guam. The crude extract of S. massa exhibited feeding deterrence against the omnivorous pufferfish Canthigaster solandri at natural concentrations, however, none of the isolated compounds was deterrent at the maximum natural concentrations observed, nor were mixtures of these compounds, thus emphasizing the need for bioassay-guided isolation to characterize specific chemical defenses. Antibacterial activity against a panel of ecologically relevant pathogens was minimal. Depth transplants, predator exclusion, and UV protection experiments were performed, but although temporal variability in compound concentrations was observed, there was no evidence that secondary metabolite concentration in S. massa was induced by any of these factors. Although the reasons behind the variability observed in the chemical constituents of S. massa are still in question, all sponges are not created equal from a chemical standpoint, and these studies provide

Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol.

PubMed

Zhu, Yan; Hassan, Yousef I; Shao, Suqin; Zhou, Ting

2018-06-29

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly detected in grains infested with Fusarium species. The maximum tolerated levels of DON in the majority of world's countries are restricted to 0.75 mg kg -1 within the human food chain and to less than 1-5 mg kg -1 in animal feed depending on the feed material and/or animal species due to DON's short and long-term adverse effects on human health and animal productivity. The ability to accurately analyze DON and some of its fungal/bacterial metabolites is increasingly gaining a paramount importance in food/feed analysis and research. In this study, we used the immuno-affinity approach to enrich and detect DON and three of its bacterial metabolites, namely 3-epi-DON, 3-keto-DON, and deepoxy-DON (DOM-1). The optimized enrichment step coupled with high performance liquid chromatography can accurately and reproducibly quantify the aforementioned metabolites in feed matrixes (silage extract as an example in this case). It minimizes any background interface and provides a fast and easy-to-operate protocol for the analytical determination of such metabolites. More importantly, the presented data demonstrates the ability of the utilized monoclonal antibody, generated originally to capture DON in Enzyme-Linked Immunosorbent Assays (ELISA), to cross react with three less/non-toxic DON metabolites. This raises the concerns about the genuine need to account for such cross-reactivity when DON contamination is assessed through an immuno-affinity based analyses using the investigated antibody. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

LC-QTOF MS screening of more than 1,000 licit and illicit drugs and their metabolites in wastewater and surface waters from the area of Bogotá, Colombia.

PubMed

Hernández, Félix; Ibáñez, María; Botero-Coy, Ana-María; Bade, Richard; Bustos-López, Martha Cristina; Rincón, Javier; Moncayo, Alejandro; Bijlsma, Lubertus

2015-08-01

A large screening of around 1,000 emerging contaminants, focused on licit and illicit drugs and their metabolites, has been made in urban wastewaters (both influent and effluent) and surface waters from the area of Bogotá, Colombia. After a simple generic solid-phase extraction (SPE) step with Oasis hydrophilic-lipophilic balanced (HLB) cartridges, analyses were made by ultra high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) under MS(E) mode (sequential acquisition of mass spectra at low energy (LE) and high collision energy (HE)). Accurate mass measurements and the information provided by MS(E) on the presence of the (de)protonated molecule and fragment ions allowed the reliable identification of the compounds detected, even without reference standards being available in some cases (tentative identification). The compounds most frequently found were acetaminophen/paracetamol, carbamazepine and its dihydro-dihydroxylated metabolite, clarithromycin, diclofenac, ibuprofen, gemfibrozil, lincomycin, losartan, valsartan, the two metabolites of metamizole (4-acetamido-antipyrine and 4-formylamino-antipyrine), sucralose, and cocaine and its main metabolite benzoylecgonine. Caffeine, the sweetener saccharin, and two hydroxylated metabolites of losartan were tentatively identified in almost all samples analyzed. Pharmaceutical lidocaine was tentatively identified and subsequently confirmed with reference standard. For the first time, a general overview of the occurrence of drugs and their metabolites in the aquatic environment of Colombia has been reported. In the near future, target methodologies, typically based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), will need to be set up for accurate and sensitive quantification of the contaminants selected on the basis on the information provided in the present paper.

Formation of Hydroxylamine from Ammonia and Hydroxyl Radicals

NASA Astrophysics Data System (ADS)

Krim, Lahouari; Zins, Emilie-Laure

2014-06-01

In the interstellar medium, as well as in icy comets, ammonia may be a crucial species in the first step toward the formation of amino-acids and other prebiotic molecules such as hydroxylamine (NH2OH). It is worth to notice that the NH3/H2 ratio in the ISM is 3 10-5 compared the H2O/H2 one which is only 7 10-5. Using either electron-UV irradiations of water-ammonia ices or successive hydrogenation of solid nitric oxide, laboratory experiments have already shown the feasibility of reactions that may take place on the surface of ice grains in molecular clouds, and may lead to the formation of this precursor. Herein is proposed a new reaction pathway involving ammonia and hydroxyl radicals generated in a microwave discharge. Experimental studies, at 3 and 10 K, in solid phase as well as in neon matrix have shown that this reaction proceed via a hydrogen abstraction, leading to the formation of NH2 radical, that further recombine with hydroxyl radical to form hydroxylamine, under non-energetic conditions.

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites.

PubMed

Wei, Cong; Grace, James E; Zvyaga, Tatyana A; Drexler, Dieter M

2012-08-01

The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode. Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5´-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference. The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.

Inhibition of endocannabinoid metabolism by the metabolites of ibuprofen and flurbiprofen.

PubMed

Karlsson, Jessica; Fowler, Christopher J

2014-01-01

In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen. COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4'-hydroxyflurbiprofen and possibly also 3'-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds. It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.

Inhibition of Endocannabinoid Metabolism by the Metabolites of Ibuprofen and Flurbiprofen

PubMed Central

Karlsson, Jessica; Fowler, Christopher J.

2014-01-01

Background In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen. Methodology/Principal Findings COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds. Conclusions/Significance It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo. PMID:25061885

Structure, electronic properties, and aggregation behavior of hydroxylated carbon nanotubes

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

López-Oyama, A. B.; Silva-Molina, R. A.; Ruíz-García, J.

2014-11-07

We present a combined experimental and theoretical study to analyze the structure, electronic properties, and aggregation behavior of hydroxylated multiwalled carbon nanotubes (OH–MWCNT). Our MWCNTs have average diameters of ∼2 nm, lengths of approximately 100–300 nm, and a hydroxyl surface coverage θ∼0.1. When deposited on the air/water interface the OH–MWCNTs are partially soluble and the floating units interact and link with each other forming extended foam-like carbon networks. Surface pressure-area isotherms of the nanotube films are performed using the Langmuir balance method at different equilibration times. The films are transferred into a mica substrate and atomic force microscopy images showmore » that the foam like structure is preserved and reveals fine details of their microstructure. Density functional theory calculations performed on model hydroxylated carbon nanotubes show that low energy atomic configurations are found when the OH groups form molecular islands on the nanotube's surface. This patchy behavior for the OH species is expected to produce nanotubes having reduced wettabilities, in line with experimental observations. OH doping yields nanotubes having small HOMO–LUMO energy gaps and generates a nanotube → OH direction for the charge transfer leading to the existence of more hole carriers in the structures. Our synthesized OH–MWCNTs might have promising applications.« less

Synthesis and properties evaluation of sulfobetaine surfactant with double hydroxyl

NASA Astrophysics Data System (ADS)

Zhou, Ming; Luo, Gang; Zhang, Ze; Li, Sisi; Wang, Chengwen

2017-09-01

A series of sulfobetaine surfactants {N-[(3-alkoxy-2-hydroxyl)propoxy] ethyl-N,N-dimethyl-N-(2-hydroxyl)propyl sulfonate} ammonium chloride were synthesized with raw materials containing linear saturated alcohol, N,N-dimethylethanolamine, sodium 3-chloro-2-hydroxyl propane sulfonic acid and epichlorohydrin. The molecule structures of sulfobetaine surfactants were characterized by FTIR, 1HNMR and elemental analysis. Surface tension measurements can provide us information about the surface tension at the CMC (γCMC), pC20, Γmax and Amin. The pC20 values of sulfobetaine surfactants increase with the hydrophobic chain length increasing. Amin values of the surfactants decrease with increasing hydrophobic chain length from 10 to 14. The critical micelle concentration (CMC) and surface tension (γCMC) values of the sulfobetaine surfactants decrease with increasing hydrophobic chain length from 10 to 16. The lipophilicity of surfactant was enhanced with the increase of the carbon chain, however, the ability of anti-hard water was weakened. The minimum oil/water interfacial tension of four kinds of sulfobetaine surfactants is 10-2-10-3 mN/m magnitude, which indicates that the synthesized bis-hydroxy sulfobetaine surfactants have a great ability to reduce interfacial tension in the surfactant flooding system. The surface tension (γCMC) values of synthesized surfactants were lower compared with conventional anionic surfactant sodium dodecyl sulfonate.

Quantitative metabolite profiling of edible onion species by NMR and HPLC-MS.

PubMed

Soininen, Tuula H; Jukarainen, Niko; Auriola, Seppo O K; Julkunen-Tiitto, Riitta; Karjalainen, Reijo; Vepsäläinen, Jouko J

2014-12-15

Allium genus is a treasure trove of valuable bioactive compounds with potentially therapeutically important properties. This work utilises HPLC-MS and a constrained total-line-shape (CTLS) approach applied to (1)H NMR spectra to quantify metabolites present in onion species to reveal important inter-species differences. Extensive differences were detected between the sugar concentrations in onion species. Yellow onion contained the highest and red onion the lowest amounts of amino acids. The main flavonol-glucosides were quercetin 3,4'-diglucoside and quercetin 4'-glucoside. In general, the levels of flavonols were, higher in yellow onions than in red onions, and garlic and leek contained a lower amount of flavonols than the other Allium species. Our results highlight how (1)H NMR together with HPLC-MS can be useful in the quantification and the identification of the most abundant metabolites, representing an efficient means to pinpoint important functional food ingredients from Allium species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification, quantification, spatiotemporal distribution and genetic variation of major latex secondary metabolites in the common dandelion (Taraxacum officinale agg.).

PubMed

Huber, Meret; Triebwasser-Freese, Daniella; Reichelt, Michael; Heiling, Sven; Paetz, Christian; Chandran, Jima N; Bartram, Stefan; Schneider, Bernd; Gershenzon, Jonathan; Erb, Matthias

2015-07-01

The secondary metabolites in the roots, leaves and flowers of the common dandelion (Taraxacum officinale agg.) have been studied in detail. However, little is known about the specific constituents of the plant's highly specialized laticifer cells. Using a combination of liquid and gas chromatography, mass spectrometry and nuclear magnetic resonance spectrometry, we identified and quantified the major secondary metabolites in the latex of different organs across different growth stages in three genotypes, and tested the activity of the metabolites against the generalist root herbivore Diabrotica balteata. We found that common dandelion latex is dominated by three classes of secondary metabolites: phenolic inositol esters (PIEs), triterpene acetates (TritAc) and the sesquiterpene lactone taraxinic acid β-D-glucopyranosyl ester (TA-G). Purification and absolute quantification revealed concentrations in the upper mgg(-1) range for all compound classes with up to 6% PIEs, 5% TritAc and 7% TA-G per gram latex fresh weight. Contrary to typical secondary metabolite patterns, concentrations of all three classes increased with plant age. The highest concentrations were measured in the main root. PIE profiles differed both quantitatively and qualitatively between plant genotypes, whereas TritAc and TA-G differed only quantitatively. Metabolite concentrations were positively correlated within and between the different compound classes, indicating tight biosynthetic co-regulation. Latex metabolite extracts strongly repelled D. balteata larvae, suggesting that the latex constituents are biologically active. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree

PubMed Central

Rangiah, Kannan; Mahesh, HB; Rajamani, Anantharamanan; Shirke, Meghana D.; Russiachand, Heikham; Loganathan, Ramya Malarini; Shankara Lingu, Chandana; Siddappa, Shilpa; Ramamurthy, Aishwarya; Sathyanarayana, BN

2015-01-01

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC–600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways. PMID:26290780

Circulating metabolite predictors of glycemia in middle-aged men and women.

PubMed

Würtz, Peter; Tiainen, Mika; Mäkinen, Ville-Petteri; Kangas, Antti J; Soininen, Pasi; Saltevo, Juha; Keinänen-Kiukaanniemi, Sirkka; Mäntyselkä, Pekka; Lehtimäki, Terho; Laakso, Markku; Jula, Antti; Kähönen, Mika; Vanhala, Mauno; Ala-Korpela, Mika

2012-08-01

Metabolite predictors of deteriorating glucose tolerance may elucidate the pathogenesis of type 2 diabetes. We investigated associations of circulating metabolites from high-throughput profiling with fasting and postload glycemia cross-sectionally and prospectively on the population level. Oral glucose tolerance was assessed in two Finnish, population-based studies consisting of 1,873 individuals (mean age 52 years, 58% women) and reexamined after 6.5 years for 618 individuals in one of the cohorts. Metabolites were quantified by nuclear magnetic resonance spectroscopy from fasting serum samples. Associations were studied by linear regression models adjusted for established risk factors. Nineteen circulating metabolites, including amino acids, gluconeogenic substrates, and fatty acid measures, were cross-sectionally associated with fasting and/or postload glucose (P < 0.001). Among these metabolic intermediates, branched-chain amino acids, phenylalanine, and α1-acid glycoprotein were predictors of both fasting and 2-h glucose at 6.5-year follow-up (P < 0.05), whereas alanine, lactate, pyruvate, and tyrosine were uniquely associated with 6.5-year postload glucose (P = 0.003-0.04). None of the fatty acid measures were prospectively associated with glycemia. Changes in fatty acid concentrations were associated with changes in fasting and postload glycemia during follow-up; however, changes in branched-chain amino acids did not follow glucose dynamics, and gluconeogenic substrates only paralleled changes in fasting glucose. Alterations in branched-chain and aromatic amino acid metabolism precede hyperglycemia in the general population. Further, alanine, lactate, and pyruvate were predictive of postchallenge glucose exclusively. These gluconeogenic precursors are potential markers of long-term impaired insulin sensitivity that may relate to attenuated glucose tolerance later in life.

Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas

PubMed Central

Isono, Takahiro; Chano, Tokuhiro; Yoshida, Tetsuya; Kageyama, Susumu; Kawauchi, Akihiro; Suzaki, Masafumi; Yuasa, Takeshi

2016-01-01

Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs. PMID:27822416

Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation.

PubMed

González-Pérez, Vanessa; Connolly, Elizabeth A; Bridges, Arlene S; Wienkers, Larry C; Paine, Mary F

2012-11-01

(S)-Warfarin 7-hydroxylation and midazolam 1'-hydroxylation are among the preferred probe substrate reactions for CYP2C9 and CYP3A4/5, respectively. The impact of solvents on enzyme activity, kinetic parameters, and predicted in vivo hepatic clearance (Cl(H)) associated with each reaction has not been evaluated. The effects of increasing concentrations [0.1-2% (v/v)] of six organic solvents (acetonitrile, methanol, ethanol, dimethyl sulfoxide, acetone, isopropanol) were first tested on each reaction using human liver microsomes (HLMs), human intestinal microsomes (midazolam 1'-hydroxylation only), and recombinant enzymes. Across enzyme sources, relative to water, acetonitrile and methanol had the least inhibitory effect on (S)-warfarin 7-hydroxylation (0-58 and 9-96%, respectively); acetonitrile, methanol, and ethanol had the least inhibitory effect on midazolam 1'-hydroxylation (0-29, 0-22, and 0-20%, respectively). Using HLMs, both acetonitrile and methanol (0.1-2%) decreased the V(max) (32-60 and 24-65%, respectively) whereas methanol (2%) increased the K(m) (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin Cl(H) was underpredicted by 21-65% (acetonitrile) and 13-84% (methanol). Acetonitrile, methanol, and ethanol had minimal to modest impact on both the kinetics of midazolam 1'-hydroxylation (10-24%) and predicted midazolam Cl(H) (2-20%). In conclusion, either acetonitrile or methanol at ≤0.1% is recommended as the primary organic solvent for the (S)-warfarin 7-hydroxylation reaction; acetonitrile is preferred if higher solvent concentrations are required. Acetonitrile, methanol, and ethanol at ≤2% are recommended as primary organic solvents for the midazolam 1'-hydroxylation reaction. This information should facilitate optimization of experimental conditions and improve the interpretation and accuracy of in vitro-in vivo predictions involving these two preferred cytochrome P450 probe substrate reactions.

Hydroxyl radical induced transformation of phenylurea herbicides: A theoretical study

NASA Astrophysics Data System (ADS)

Mile, Viktória; Harsányi, Ildikó; Kovács, Krisztina; Földes, Tamás; Takács, Erzsébet; Wojnárovits, László

2017-03-01

Aromatic ring hydroxylation reactions occurring during radiolysis of aqueous solutions are studied on the example of phenylurea herbicides by Density Functional Theory calculations. The effect of the aqueous media is taken into account by using the Solvation Model Based on Density model. Hydroxyl radical adds to the ring because the activation free energies (0.4-47.2 kJ mol-1) are low and also the Gibbs free energies have high negative values ((-27.4) to (-5.9) kJ mol-1). According to the calculations in most of cases the ortho- and para-addition is preferred in agreement with the experimental results. In these reactions hydroxycyclohexadienyl type radicals form. In a second type reaction, when loss of chlorine atom takes place, OH/Cl substitution occurs without cyclohexadienyl type intermediate.

Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly enantioselective separation based on supercritical-fluid chromatography (SFC) and single quadrupole MS detection.

PubMed

Fassauer, Georg M; Hofstetter, Robert; Hasan, Mahmoud; Oswald, Stefan; Modeß, Christina; Siegmund, Werner; Link, Andreas

2017-11-30

Increasing evidence accumulates that metabolites of the dissociative anesthetic ketamine contribute considerably to the biological effects of this drug and could be developed as next generation antidepressants, especially for acute treatment of patients with therapy-refractory major depression. Analytical methods for the simultaneous determination of the plethora of hydroxylated, dehydrogenated and/or demethylated compounds formed after administration of ketamine hydrochloride are a prerequisite for future clinical investigations and a deeper understanding of the individual role of the isomers of these metabolites. In this study, we present development and validation of a method based on supercritical-fluid chromatography (SFC) coupled to single quadrupole MS detection that allows the separation of ketamine as well as all of its relevant metabolites detected in urine of healthy volunteers. Inherently to SFC methods, the run times of the novel protocol are four times shorter than in a comparable HPLC method, the use of organic solvents is reduced and we were able to demonstrate and validate the successful enantioselective separation and quantification of R- and S-ketamine, R- and S-norketamine, R- and S-dehydronorketamine and (2R,6R)- and (2S,6S)-hydroxynorketamine isomers differing in either constitution, stereochemistry, or both, in one run. The developed method may be useful in investigating the antidepressant efficacy of ketamine in clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Promotional effect of surface hydroxyls on electrochemical reduction of CO 2 over SnO x/Sn electrode

DOE PAGES

Cui, Chaonan; Han, Jinyu; Zhu, Xinli; ...

2016-01-16

In this study, tin oxide (SnO x) formation on tin-based electrode surfaces during CO 2 electrochemical reduction can have a significant impact on the activity and selectivity of the reaction. In the present study, density functional theory (DFT) calculations have been performed to understand the role of SnO x in CO 2 reduction using a SnO monolayer on the Sn(112) surface as a model for SnO x. Water molecules have been treated explicitly and considered actively participating in the reaction. The results showed that H 2O dissociates on the perfect SnO monolayer into two hydroxyl groups symmetrically on the surface.more » CO 2 energetically prefers to react with the hydroxyl, forming a bicarbonate (HCO 3(t)*) intermediate, which can then be reduced to either formate (HCOO*) by hydrogenating the carbon atom or carboxyl (COOH*) by protonating the oxygen atom. Both steps involve a simultaneous Csingle bondO bond breaking. Further reduction of HCOO* species leads to the formation of formic acid in the acidic solution at pH < 4, while the COOH* will decompose to CO and H 2O via protonation. Whereas the oxygen vacancy (VO) in the oxide monolayer maybe formed by the reduction, it can be recovered by H 2O dissociation, resulting in two embedded hydroxyl groups. The results show that the hydroxylated surface with two symmetric hydroxyls is energetically more favorable for CO 2 reduction than the hydroxylated VO surface with two embedded hydroxyls. The reduction potential for the former has a limiting-potential of –0.20 V (RHE), lower than that for the latter (–0.74 V (RHE)). Compared to the pure Sn electrode, the formation of SnO x monolayer on the electrode under the operating conditions promotes CO 2 reduction more effectively by forming surface hydroxyls, thereby providing a new channel via COOH* to the CO formation, although formic acid is still the major reduction product.« less

The genetic control of phenformin 4-hydroxylation.

PubMed Central

Shah, R R; Evans, D A; Oates, N S; Idle, J R; Smith, R L

1985-01-01

Previously published results of phenformin 4-hydroxylation in 195 unrelated white British volunteers and 87 family members of 27 randomly selected probands have been subjected to genetic analysis. The results clearly show that about 9% of this population has a genetically determined defect in carrying out this oxidation reaction. The character for the defect is inherited in a Mendelian autosomal recessive fashion. The polymorphism shows a substantial degree of dominance. PMID:4078865

Virtual quantification of metabolites by capillary electrophoresis-electrospray ionization-mass spectrometry: predicting ionization efficiency without chemical standards.

PubMed

Chalcraft, Kenneth R; Lee, Richard; Mills, Casandra; Britz-McKibbin, Philip

2009-04-01

A major obstacle in metabolomics remains the identification and quantification of a large fraction of unknown metabolites in complex biological samples when purified standards are unavailable. Herein we introduce a multivariate strategy for de novo quantification of cationic/zwitterionic metabolites using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) based on fundamental molecular, thermodynamic, and electrokinetic properties of an ion. Multivariate calibration was used to derive a quantitative relationship between the measured relative response factor (RRF) of polar metabolites with respect to four physicochemical properties associated with ion evaporation in ESI-MS, namely, molecular volume (MV), octanol-water distribution coefficient (log D), absolute mobility (mu(o)), and effective charge (z(eff)). Our studies revealed that a limited set of intrinsic solute properties can be used to predict the RRF of various classes of metabolites (e.g., amino acids, amines, peptides, acylcarnitines, nucleosides, etc.) with reasonable accuracy and robustness provided that an appropriate training set is validated and ion responses are normalized to an internal standard(s). The applicability of the multivariate model to quantify micromolar levels of metabolites spiked in red blood cell (RBC) lysates was also examined by CE-ESI-MS without significant matrix effects caused by involatile salts and/or major co-ion interferences. This work demonstrates the feasibility for virtual quantification of low-abundance metabolites and their isomers in real-world samples using physicochemical properties estimated by computer modeling, while providing deeper insight into the wide disparity of solute responses in ESI-MS. New strategies for predicting ionization efficiency in silico allow for rapid and semiquantitative analysis of newly discovered biomarkers and/or drug metabolites in metabolomics research when chemical standards do not exist.

Aromatic Hydroxylation of Salicylic Acid and Aspirin by Human Cytochromes P450

PubMed Central

Bojić, Mirza; Sedgeman, Carl A.; Nagy, Leslie D.; Guengerich, F. Peter

2015-01-01

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1. PMID:25840124

Direct deposition of silver nanoplates on quartz surface by sequence pre-treatment hydroxylation and silanisation.

PubMed

Abu Bakar, Norhayati; Mat Salleh, Muhamad; Ali Umar, Akrajas; Shapter, Joseph George

2017-01-01

Silver nanoparticles deposited on quartz substrates are widely used as SERS substrates. The nanoparticles can be deposited directly from colloidal solution by dipping technique. However, the adhesion of the particles on the quartz surface is very poor. Normally the substrate is pre-treated with hydroxylation or silanisation process. In this paper, we have demonstrated that the application of the sequence pre-treatment hydroxylation and silanisation have improved the density of silver nanoplates desposited on the quartz surface. •Sequence hydroxylation and silanisation pre-treatment assists the deposition of the nanoplate on the surface.•Various immersion times of the quartz surface into the colloidal nanoplates determined size distributions and density surface of the nanoplates on the surface.

Solvent Separating Secondary Metabolites Directly from Biosynthetic Tissue for Surface-Assisted Laser Desorption Ionisation Mass Spectrometry

PubMed Central

Rudd, David; Benkendorff, Kirsten; Voelcker, Nicolas H.

2015-01-01

Marine bioactive metabolites are often heterogeneously expressed in tissues both spatially and over time. Therefore, traditional solvent extraction methods benefit from an understanding of the in situ sites of biosynthesis and storage to deal with heterogeneity and maximize yield. Recently, surface-assisted mass spectrometry (MS) methods namely nanostructure-assisted laser desorption ionisation (NALDI) and desorption ionisation on porous silicon (DIOS) surfaces have been developed to enable the direct detection of low molecular weight metabolites. Since direct tissue NALDI-MS or DIOS-MS produce complex spectra due to the wide variety of other metabolites and fragments present in the low mass range, we report here the use of “on surface” solvent separation directly from mollusc tissue onto nanostructured surfaces for MS analysis, as a mechanism for simplifying data annotation and detecting possible artefacts from compound delocalization during the preparative steps. Water, ethanol, chloroform and hexane selectively extracted a range of choline esters, brominated indoles and lipids from Dicathais orbita hypobranchial tissue imprints. These compounds could be quantified on the nanostructured surfaces by comparison to standard curves generated from the pure compounds. Surface-assisted MS could have broad utility for detecting a broad range of secondary metabolites in complex marine tissue samples. PMID:25786067

Metabolomics for secondary metabolite research.

PubMed

Breitling, Rainer; Ceniceros, Ana; Jankevics, Andris; Takano, Eriko

2013-11-11

Metabolomics, the global characterization of metabolite profiles, is becoming an increasingly powerful tool for research on secondary metabolite discovery and production. In this review we discuss examples of recent technological advances and biological applications of metabolomics in the search for chemical novelty and the engineered production of bioactive secondary metabolites.

Concentrations of dimethylaniline and other metabolites in milk and tissues of dairy cows treated with lidocaine.

PubMed

Hoogenboom, Ron L A P; Zuidema, Tina; Essers, Martien; van Vuuren, Ad M; van Wikselaar, Piet G; van Eijkeren, Jan C H; Mengelers, Marcel J B; Zeilmaker, Marco J; Bulder, Astrid S

2015-01-01

Lidocaine is a topical anaesthetic drug used in dairy cows for laparotomy (caesarean section, abomasal displacement). Because there are no registered drugs for this indication, it can be applied under the so-called Cascade rules (off-label use), with the restriction that the off-label withdrawal periods of 7 days for milk and 28 days for meat are taken into account. In animals, lidocaine is rapidly metabolised into various metabolites, one being 2,6-dimethylaniline (DMA) which is reported to possess carcinogenic and mutagenic properties and detected also in milk. To investigate whether the off-label withdrawal periods are long enough to exclude the presence of lidocaine and DMA, and potential other metabolites, in edible products, a study was performed with eight dairy cows treated with lidocaine by injection in the abdominal muscles. At various time points blood samples, milk and urine were collected. Four animals were slaughtered 3.5 h after treatment, the other four after 48.5 h. The injection site, meat, liver and kidney were analysed for levels of lidocaine, DMA, monoethylglycinexylidide (MEGX) and 3-OH-lidocaine. It was shown that DMA is an important metabolite in dairy cows and can be detected in both meat and milk. In addition, also MEGX, 3-OH-lidocaine and three other metabolites were identified and to some extent quantified. These metabolites were 4-OH-lidocaine, lidocaine-N-oxide and 4-hydroxy-DMA. The latter compound was the most important metabolite in urine. However, levels in milk and meat decreased rapidly after the application. Overall, it can be concluded that the off-label withdrawal times of 7 and 28 days for milk and meat, respectively, guarantee the absence of detectable levels of lidocaine and metabolites.

Conventional liquid chromatography/triple quadrupole mass spectrometer-based metabolite identification and semi-quantitative estimation approach in the investigation of dabigatran etexilate in vitro metabolism

PubMed Central

Hu, Zhe-Yi; Parker, Robert B.; Herring, Vanessa L.; Laizure, S. Casey

2012-01-01

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometer (LC-MS/MS)-based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor-product ion pairs were predicted, to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects of tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using ‘metabolite standards’ generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 convert DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 (or M2) was further metabolized to DAB by CES2 (or CES1). The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples. PMID:23239178

Molecular modeling and molecular dynamics simulation studies on the interactions of hydroxylated polychlorinated biphenyls with estrogen receptor-β.

PubMed

Li, Xiaolin; Ye, Li; Wang, Xiaoxiang; Shi, Wei; Qian, XiangPing; Zhu, YongLiang; Yu, HongXia

2013-10-01

Endocrine-disrupting chemicals have attracted great concern. As major metabolites of polychlorinated biphenyls (PCBs), hydroxylated polychlorinated biphenyls (HO-PCBs) may disrupt estrogen hormone status because of their structural similarity to estrogen endogenous compounds. However, interactions between HO-PCBs and estrogen receptors (ERs) are not fully understood. In the present work, a molecular modeling study combining molecular docking, molecular dynamics simulations, and binding free energy calculations was performed to characterize the interactions of three HO-PCBs (4'-HO-PCB50, 2'-HO-PCB65, and 4'-HO-PCB69) having much different estrogenic activities with ERβ. Docking results showed that binding between ligands and ERβ was stabilized by hydrogen bond and hydrophobic interactions. The binding free energies of three ligands with ERβ were calculated, and further binding free energy decomposition analysis indicated that the dominating driving force of the binding between the ligands and ERβ was the van der Waals interaction. Some key residues, such as Leu298, Phe356, Gly472, His475, and Leu476, played important roles in ligand-receptor interactions by forming hydrophobic and hydrogen bond interactions with ligands. The results may be beneficial to increase understanding of the interactions between HO-PCBs and ERβ.

Herbal medicine yin zhi huang induces CYP3A4-mediated sulfoxidation and CYP2C19-dependent hydroxylation of omeprazole.

PubMed

Fan, Lan; Wang, Guo; Wang, Lian-Sheng; Chen, Yao; Zhang, Wei; Huang, Yuan-Fei; Huang, Rui-Xue; Hu, Dong-Li; Wang, Dan; Zhou, Hong-Hao

2007-10-01

To explore the potential interactions between yin zhi huang (YZH) and omeprazole, a substrate of CYP3A4 and CYP2C19. Eighteen healthy volunteers, including 6 CYP2C19*1/*1, 6 CYP2C19*1/*2 or *3 and 6 CYP2C19*2/*2 were enrolled in a 2-phase, randomized, crossover clinical trial. In each phase, the volunteers received either placebo or 10 mL YZH oral liquid, 3 times daily for 14 d. Then all the patients took a 20 mg omeprazole capsule orally. Blood samples were collected up to 12 h after omeprazole administration. Plasma concentrations of omeprazole and its metabolites were quantified by HPLC with UV detection. After 14 d of treatment of YZH, plasma omeprazole significantly decreased and those of omeprazole sulfone and 5-hydroxyomeprazole significantly increased. The ratios of the area under the plasma concentration-time curves from time 0 to infinity (AUC(0-infinity) of omeprazole to 5-hydroxyomprazole and those of omeprazole to omeprazole sulfone decreased by 64.80%+/-12.51% (P=0.001) and 63.31%+/-18.45% (P=0.004) in CYP2C19*1/*1, 57.98%+/-14.80% (P=0.002) and 54.87%+/-18.42% (P=0.003) in CYP2C19*1/*2 or *3, and 37.74%+/-16.07% (P=0.004) and 45.16%+/-15.54% (P=0.003) in CYP2C19*2/*2, respectively. The decrease of the AUC(0-infinity) ratio of omeprazole to 5-hydroxyomprazole in CYP2C19*1/*1 and CYP2C19*1/*2 or *3 was greater than those in CYP2C19*2/*2 (P=0.047 and P=0.009). YZH induces both CYP3A4-catalyzed sulfoxidation and CYP2C19-dependent hydroxylation of omeprazole leading to decreases in plasma omeprazole concentrations.

Hydroxylation and hydrolysis: two main metabolic ways of spiramycin I in anaerobic digestion.

PubMed

Zhu, Pei; Chen, Daijie; Liu, Wenbin; Zhang, Jianbin; Shao, Lei; Li, Ji-an; Chu, Ju

2014-02-01

The anaerobic degradation behaviors of five macrolides including spiramycin I, II, III, midecamycin and josamycin by sludge were investigated. Within 32days, 95% of spiramycin I, II or III was degraded, while the remove rate of midecamycin or josamycin was 75%. SPM I degradation was much higher in nutrition supplementation than that just in sludge. The degradation products and processes of spiramycin I were further characterized. Three molecules, designated P-1, P-2 and P-3 according to their order of occurrence, were obtained and purified. Structural determination was then performed by nuclear magnetic resonance and MS/MS spectra, and data indicated that hydroxylation and hydrolysis were main reactions during the anaerobic digestion of spiramycin I. P-1 is the intermediate of hydroxylation, and P-2 is the intermediate of hydrolysis. P-3 is the final product of the both reaction. This study revealed a hydroxylation and hydrolysis mechanism of macrolide in anaerobic digestion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biotransformation of fluorophenyl pyridine carboxylic acids by the model fungus Cunninghamella elegans.

PubMed

Palmer-Brown, William; Dunne, Brian; Ortin, Yannick; Fox, Mark A; Sandford, Graham; Murphy, Cormac D

2017-09-01

1. Fluorine plays a key role in the design of new drugs and recent FDA approvals included two fluorinated drugs, tedizolid phosphate and vorapaxar, both of which contain the fluorophenyl pyridyl moiety. 2. To investigate the likely phase-I (oxidative) metabolic fate of this group, various fluorinated phenyl pyridine carboxylic acids were incubated with the fungus Cunninghamella elegans, which is an established model of mammalian drug metabolism. 3.  19 F NMR spectroscopy established the degree of biotransformation, which varied depending on the position of fluorine substitution, and gas chromatography-mass spectrometry (GC-MS) identified alcohols and hydroxylated carboxylic acids as metabolites. The hydroxylated metabolites were further structurally characterised by nuclear magnetic resonance spectroscopy (NMR), which demonstrated that hydroxylation occurred on the 4' position; fluorine in that position blocked the hydroxylation. 4. The fluorophenyl pyridine carboxylic acids were not biotransformed by rat liver microsomes and this was a consequence of inhibitory action, and thus, the fungal model was crucial in obtaining metabolites to establish the mechanism of catabolism.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank

2001-01-01

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants.

Secondary metabolites from marine microorganisms.

PubMed

Kelecom, Alphonse

2002-03-01

After 40 years of intensive research, chemistry of marine natural products has become a mature field. Since 1995, there are signals of decreased interest in the search of new metabolites from traditional sources such as macroalgae and octocorals, and the number of annual reports on marine sponges stabilized. On the contrary, metabolites from microorganisms is a rapidly growing field, due, at least in part, to the suspicion that a number of metabolites obtained from algae and invertebrates may be produced by associated microorganisms. Studies are concerned with bacteria and fungi, isolated from seawater, sediments, algae, fish and mainly from marine invertebrates such as sponges, mollusks, tunicates, coelenterates and crustaceans. Although it is still to early to define tendencies, it may be stated that the metabolites from microorganisms are in most cases quite different from those produced by the invertebrate hosts. Nitrogenated metabolites predominate over acetate derivatives, and terpenes are uncommon. Among the latter, sesquiterpenes, diterpenes and carotenes have been isolated; among nitrogenated metabolites, amides, cyclic peptides and indole alkaloids predominate.

The gene road to royalty--differential expression of hydroxylating genes in the mandibular glands of the honeybee.

PubMed

Malka, Osnat; Karunker, Iris; Yeheskel, Adva; Morin, Shai; Hefetz, Abraham

2009-10-01

The advances in honeybee sociogenomics have paved the way for the study of social communication processes at the gene level, in particular the expression of caste-specific pheromones. The queen honeybee mandibular pheromone provides an excellent model system, in that biosynthesis of the hydroxylating fatty acid caste-specific pheromone appears to be reduced to a single chemical hydroxylation step of stearic acid. Queens are typified by omega-1-hydroxylation, as opposed to the worker-typical omega-hydroxylation. We hypothesized that this bifurcation is the consequence of differential expression of caste-specific genes that code for fatty acid-hydroxylating enzymes from the cytochrome P450 (CYP) family. Bioinformatics studies disclosed two candidate proteins CYP4AA1 and CYP18A1. We thus investigated the expression of these genes in the mandibular glands of queens, and of queenright (QR) and queenless (QL) workers. The real-time PCR results revealed that CYP4AA1 (omega-hydroxylation) was expressed at high levels in both QR and QL workers, whereas in queens its expression was negligible. The expression of CYP18A1 (omega-1-hydroxylation), on the other hand, was high in the queen's glands and negligible in those of QR workers. In QL workers, however, the expression of CYP18A1 was considerably elevated and significantly greater than in QR workers. Three-dimensional structural models constructed for these enzymes demonstrate differences in the active site between CYP18A1 and CYP4AA1, in line with their differential catalytic specificity. The fact that queen pheromone plasticity can be tracked all the way to gene expression provides a new insight into the process of caste differentiation and the accompanying social communication.

Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes.

PubMed Central

Sanderson, J T; Letcher, R J; Heneweer, M; Giesy, J P; van den Berg, M

2001-01-01

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro. PMID:11675267

Identification of drug metabolites in human plasma or serum integrating metabolite prediction, LC-HRMS and untargeted data processing.

PubMed

Jacobs, Peter L; Ridder, Lars; Ruijken, Marco; Rosing, Hilde; Jager, Nynke Gl; Beijnen, Jos H; Bas, Richard R; van Dongen, William D

2013-09-01

Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

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

Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach.

PubMed

Floegel, Anna; Stefan, Norbert; Yu, Zhonghao; Mühlenbruch, Kristin; Drogan, Dagmar; Joost, Hans-Georg; Fritsche, Andreas; Häring, Hans-Ulrich; Hrabě de Angelis, Martin; Peters, Annette; Roden, Michael; Prehn, Cornelia; Wang-Sattler, Rui; Illig, Thomas; Schulze, Matthias B; Adamski, Jerzy; Boeing, Heiner; Pischon, Tobias

2013-02-01

Metabolomic discovery of biomarkers of type 2 diabetes (T2D) risk may reveal etiological pathways and help to identify individuals at risk for disease. We prospectively investigated the association between serum metabolites measured by targeted metabolomics and risk of T2D in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) among all incident cases of T2D (n = 800, mean follow-up 7 years) and a randomly drawn subcohort (n = 2,282). Flow injection analysis tandem mass spectrometry was used to quantify 163 metabolites, including acylcarnitines, amino acids, hexose, and phospholipids, in baseline serum samples. Serum hexose; phenylalanine; and diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5 were independently associated with increased risk of T2D and serum glycine; sphingomyelin C16:1; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2 with decreased risk. Variance of the metabolites was largely explained by two metabolite factors with opposing risk associations (factor 1 relative risk in extreme quintiles 0.31 [95% CI 0.21-0.44], factor 2 3.82 [2.64-5.52]). The metabolites significantly improved T2D prediction compared with established risk factors. They were further linked to insulin sensitivity and secretion in the Tübingen Family study and were partly replicated in the independent KORA (Cooperative Health Research in the Region of Augsburg) cohort. The data indicate that metabolic alterations, including sugar metabolites, amino acids, and choline-containing phospholipids, are associated early on with a higher risk of T2D.

Occurrence of multiple mycotoxins and other fungal metabolites in animal feed and maize samples from Egypt using LC-MS/MS.

PubMed

Abdallah, Mohamed F; Girgin, Gözde; Baydar, Terken; Krska, Rudolf; Sulyok, Michael

2017-10-01

The present study aimed to investigate the occurrence of multiple toxic fungal and bacterial metabolites in 156 animal feed (n = 77) and maize (n = 79) samples collected from three regions in Upper Egypt. The target analytes were quantified using the 'dilute and shoot' approach, followed by a liquid chromatography tandem mass spectrometry analysis. In total, 115 fungal and bacterial metabolites were detected in both matrices, including the regulated mycotoxins in the European Union, in addition to the modified forms such as deoxynivalenol-3-glucosid. Furthermore, some Fusarium, Alternaria, Aspergillus and Penicillum metabolites beside other fungal and bacterial metabolites were detected for the first time in Egypt. All of the samples were contaminated with at least four toxins. On average, 26 different metabolites were detected per sample with a trend of more metabolites in feed than in maize. The maximum number of analytes observed per samples was 54 analytes at maximum concentrations ranging from 0.04 µg kg -1 for tentoxin to 25 040 µg kg -1 for kojic acid. According to the international standards, the contamination rates in the investigated regions were not alarming, except for AFB1 in maize. The necessity of further and continuous monitoring is highly recommended to establish a database for mycotoxin occurrence. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

PubMed Central

Ingelman-Sundberg, M; Kaur, H; Terelius, Y; Persson, J O; Halliwell, B

1991-01-01

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation. PMID:2064611

Assessment of serum pharmacokinetics and urinary excretion of albendazole and its metabolites in human volunteers.

PubMed

Ceballos, Laura; Krolewiecki, Alejandro; Juárez, Marisa; Moreno, Laura; Schaer, Fabian; Alvarez, Luis I; Cimino, Rubén; Walson, Judd; Lanusse, Carlos E

2018-01-01

Soil Transmitted Helminth (STH) infections negatively impact physical and mental development in human populations. Current WHO guidelines recommend morbidity control of these infections through mass drug administration (MDA) using albendazole (ABZ) or mebendazole. Despite major reductions in STH associated morbidity globally, not all programs have demonstrated the expected impact on prevalence of parasite infections. These therapeutic failures may be related to poor programmatic coverage, suboptimal adherence or the exposure of parasites to sub-therapeutic drug concentrations. As part of the DeWorm3 project, we sought to characterize the serum disposition kinetics and pattern of urinary excretion of ABZ and its main metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) in humans, and the assessment of the duration and optimal time point where ABZ and/or its metabolites can be measured in urine as an indirect assessment of an individual's adherence to treatment. Consecutive venous blood and urine samples were collected from eight (8) human volunteers up to 72 h post-ABZ oral administration. ABZ/metabolites were quantified by HPLC. The ABZSO metabolite was the main analyte recovered both in serum and urine. ABZSO Cmax in serum was 1.20 ± 0.44 μg/mL, reached at 4.75 h post-treatment. In urine, ABZSO Cmax was 3.24 ± 1.51 μg/mL reached at 6.50 h post-ABZ administration. Pharmacokinetic data obtained for ABZ metabolites in serum and urine, including the recovery of the ABZ sulphoxide derivative up to 72 h in both matrixes and the recovery of the amino-ABZ sulphone metabolite in urine samples, are suggesting the possibility of developing a urine based method to assess compliance to ABZ treatment. Such an assay may be useful to optimize ABZ use in human patients. ClinicalTrials.gov NCT03192449.

Global tropospheric hydroxyl distribution, budget and reactivity

NASA Astrophysics Data System (ADS)

Lelieveld, Jos; Gromov, Sergey; Pozzer, Andrea; Taraborrelli, Domenico

2016-10-01

The self-cleaning or oxidation capacity of the atmosphere is principally controlled by hydroxyl (OH) radicals in the troposphere. Hydroxyl has primary (P) and secondary (S) sources, the former mainly through the photodissociation of ozone, the latter through OH recycling in radical reaction chains. We used the recent Mainz Organics Mechanism (MOM) to advance volatile organic carbon (VOC) chemistry in the general circulation model EMAC (ECHAM/MESSy Atmospheric Chemistry) and show that S is larger than previously assumed. By including emissions of a large number of primary VOC, and accounting for their complete breakdown and intermediate products, MOM is mass-conserving and calculates substantially higher OH reactivity from VOC oxidation compared to predecessor models. Whereas previously P and S were found to be of similar magnitude, the present work indicates that S may be twice as large, mostly due to OH recycling in the free troposphere. Further, we find that nighttime OH formation may be significant in the polluted subtropical boundary layer in summer. With a mean OH recycling probability of about 67 %, global OH is buffered and not sensitive to perturbations by natural or anthropogenic emission changes. Complementary primary and secondary OH formation mechanisms in pristine and polluted environments in the continental and marine troposphere, connected through long-range transport of O3, can maintain stable global OH levels.

Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry.

PubMed

Krämer, Lisa; Jäger, Christian; Trezzi, Jean-Pierre; Jacobs, Doris M; Hiller, Karsten

2018-02-14

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13 C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13 C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13 C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13 C-labeled bread and quantified 13 C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated.

Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry

PubMed Central

Krämer, Lisa; Jäger, Christian; Jacobs, Doris M.; Hiller, Karsten

2018-01-01

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated. PMID:29443915

Suppression of LPS-induced inflammatory responses by the hydroxyl groups of dexamethasone

PubMed Central

Chuang, Ting-Yun; Cheng, An-Jie; Chen, I-Ting; Lan, Tien-Yun; Huang, I-Hsuan; Shiau, Chung-Wai; Hsu, Chia-Lin; Liu, Ya-Wen; Chang, Zee-Fen; Tseng, Ping-Hui; Kuo, Jean-Cheng

2017-01-01

The innate immune response is a central process that is activated during pathogenic infection in order to maintain physiological homeostasis. It is well known that dexamethasone (Dex), a synthetic glucocorticoid, is a potent immunosuppressant that inhibits the cytokine production induced by bacterial lipopolysaccharides (LPS). Nevertheless, the extent to which the functional groups of Dex control the excessive activation of inflammatory reactions remains unknown. Furthermore, importantly, the role of Dex in the innate immune response remains unclear. Here we explore the mechanism of LPS-induced TNF-α secretion and reveal p38 MAPK signaling as a target of Dex that is involved in control of tumor necrosis factor-α (TNF-α)-converting enzyme (TACE) activity; that later mediates the shedding of TNF-α that allows its secretion. We further demonstrate that the 11-hydroxyl and 21-hydroxyl groups of Dex are the main groups that are involved in reducing LPS-induced TNF-α secretion by activated macrophages. Blockage of the hydroxyl groups of Dex inhibits immunosuppressant effect of Dex during LPS-induced TNF-α secretion and mouse mortality. Our findings demonstrate Dex signaling is involved in the control of innate immunity. PMID:28537905

Measurement of hydroxyl radical density generated from the atmospheric pressure bioplasma jet

NASA Astrophysics Data System (ADS)

Hong, Y. J.; Nam, C. J.; Song, K. B.; Cho, G. S.; Uhm, H. S.; Choi, D. I.; Choi, E. H.

2012-03-01

Atmospheric pressure bioplasmas are being used in a variety of bio-medical and material processing applications, surface modifications of polymers. This plasma can generate the various kinds of radicals when it contacs with the water. Especially, hydroxyl radical species have very important role in the biological and chemical decontamination of media in this situation. It is very important to investigate the hydroxyl radical density in needle-typed plasma jet since it plays a crucial role in interaction between the living body and plasma. We have generated the needle-typed plasma jet bombarding the water surface by using an Ar gas flow and investigated the emission lines by OES (optical emission spectroscopy). It is noted that the electron temperature and plasma density are measured to be about 1.7 eV and 3.4 × 1012 cm-3, respectively, under Ar gas flow ranged from 80 to 300 sccm (standard cubic centimeter per minute) in this experiment. The hydroxyl radical density has also been investigated and measured to be maximum value of 2.6 × 1015 cm-3 for the gas flow rate of 150 sccm in the needle-typed plasma jet by the ultraviolet optical absorption spectroscopy.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

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.

Microbial diversity and metabolite composition of Belgian red-brown acidic ales.

PubMed

Snauwaert, Isabel; Roels, Sanne P; Van Nieuwerburg, Filip; Van Landschoot, Anita; De Vuyst, Luc; Vandamme, Peter

2016-03-16

Belgian red-brown acidic ales are sour and alcoholic fermented beers, which are produced by mixed-culture fermentation and blending. The brews are aged in oak barrels for about two years, after which mature beer is blended with young, non-aged beer to obtain the end-products. The present study evaluated the microbial community diversity of Belgian red-brown acidic ales at the end of the maturation phase of three subsequent brews of three different breweries. The microbial diversity was compared with the metabolite composition of the brews at the end of the maturation phase. Therefore, mature brew samples were subjected to 454 pyrosequencing of the 16S rRNA gene (bacteria) and the internal transcribed spacer region (yeasts) and a broad range of metabolites was quantified. The most important microbial species present in the Belgian red-brown acidic ales investigated were Pediococcus damnosus, Dekkera bruxellensis, and Acetobacter pasteurianus. In addition, this culture-independent analysis revealed operational taxonomic units that were assigned to an unclassified fungal community member, Candida, and Lactobacillus. The main metabolites present in the brew samples were L-lactic acid, D-lactic acid, and ethanol, whereas acetic acid was produced in lower quantities. The most prevailing aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, which might be of impact on the aroma of the end-products. Copyright © 2016 Elsevier B.V. All rights reserved.

LC determination of moclobemide and three metabolites in plasma.

PubMed

Misztal, Genowefa; Skibiński, Robert; Olajossy, Marcin; Paw, Beata

2002-11-07

Moclobemide and three metabolites were quantified using 1 ml of plasma and solid-phase extraction with Bakerbond CN column after the addition of nadolol as the internal standard (I.S.). Separation and detection the analysed substances were achieved isocratically with acetonitrile-methanol-0.067 M phosphate buffer pH 2.65-0.4% triethylamine (12.7:1.9:85:0.4, v/v/v/v), a Nova-pak C(8) column and UV detection at 230 nm. The lower limits of quantitation for moclobemide was 10 ng ml(-1), for M1 (Ro 16-3177)-8 ng ml(-1), for M2 (Ro 12-5637)-10 ng ml(-1) and for M3 (Ro 12-8095)-15 ng ml(-1) (as the metabolites). Accuracies calculated of three concentrations in each of three separate runs were between 84.55 and 93.68 for moclobemide, 83.28 and 92.30 for M1, 86.31 and 92.66 for M2 and 88.42 and 93.40 for M3. Precision data within day were between 5.71 and 7.50 for moclobemide, 2.91 and 6.58 for M1, 4.98 and 6.40 for M2 and 0.94 and 4.73 for M3.

Effects of metformin on metabolite profiles and LDL cholesterol in patients with type 2 diabetes.

PubMed

Xu, Tao; Brandmaier, Stefan; Messias, Ana C; Herder, Christian; Draisma, Harmen H M; Demirkan, Ayse; Yu, Zhonghao; Ried, Janina S; Haller, Toomas; Heier, Margit; Campillos, Monica; Fobo, Gisela; Stark, Renee; Holzapfel, Christina; Adam, Jonathan; Chi, Shen; Rotter, Markus; Panni, Tommaso; Quante, Anne S; He, Ying; Prehn, Cornelia; Roemisch-Margl, Werner; Kastenmüller, Gabi; Willemsen, Gonneke; Pool, René; Kasa, Katarina; van Dijk, Ko Willems; Hankemeier, Thomas; Meisinger, Christa; Thorand, Barbara; Ruepp, Andreas; Hrabé de Angelis, Martin; Li, Yixue; Wichmann, H-Erich; Stratmann, Bernd; Strauch, Konstantin; Metspalu, Andres; Gieger, Christian; Suhre, Karsten; Adamski, Jerzy; Illig, Thomas; Rathmann, Wolfgang; Roden, Michael; Peters, Annette; van Duijn, Cornelia M; Boomsma, Dorret I; Meitinger, Thomas; Wang-Sattler, Rui

2015-10-01

Metformin is used as a first-line oral treatment for type 2 diabetes (T2D). However, the underlying mechanism is not fully understood. Here, we aimed to comprehensively investigate the pleiotropic effects of metformin. We analyzed both metabolomic and genomic data of the population-based KORA cohort. To evaluate the effect of metformin treatment on metabolite concentrations, we quantified 131 metabolites in fasting serum samples and used multivariable linear regression models in three independent cross-sectional studies (n = 151 patients with T2D treated with metformin [mt-T2D]). Additionally, we used linear mixed-effect models to study the longitudinal KORA samples (n = 912) and performed mediation analyses to investigate the effects of metformin intake on blood lipid profiles. We combined genotyping data with the identified metformin-associated metabolites in KORA individuals (n = 1,809) and explored the underlying pathways. We found significantly lower (P < 5.0E-06) concentrations of three metabolites (acyl-alkyl phosphatidylcholines [PCs]) when comparing mt-T2D with four control groups who were not using glucose-lowering oral medication. These findings were controlled for conventional risk factors of T2D and replicated in two independent studies. Furthermore, we observed that the levels of these metabolites decreased significantly in patients after they started metformin treatment during 7 years' follow-up. The reduction of these metabolites was also associated with a lowered blood level of LDL cholesterol (LDL-C). Variations of these three metabolites were significantly associated with 17 genes (including FADS1 and FADS2) and controlled by AMPK, a metformin target. Our results indicate that metformin intake activates AMPK and consequently suppresses FADS, which leads to reduced levels of the three acyl-alkyl PCs and LDL-C. Our findings suggest potential beneficial effects of metformin in the prevention of cardiovascular disease. © 2015 by the American

New insights into the adsorption of 3-(trimethoxysilyl)propylmethacrylate on hydroxylated ZnO nanopowders.

PubMed

Bressy, Christine; Ngo, Van Giang; Ziarelli, Fabio; Margaillan, André

2012-02-14

Functionalization of zinc oxide (ZnO) nano-objects by silane grafting is an attractive method to provide nanostructured materials with a variety of surface properties. Active hydroxyl groups on the oxide surface are one of the causes governing the interfacial bond strength in nanohybrid particles. Here, "as-prepared" and commercially available zinc oxide nanopowders with a wide range of surface hydroxyl density were functionalized by a well-known polymerizable silane coupling agent, i.e., 3-(trimethoxysilyl)propylmethacrylate (MPS). Fourier transform infrared (FTIR) and solid-state (13)C and (29)Si nuclear magnetic resonance (NMR) spectroscopic investigations demonstrated that the silane coupling agent was fully hydrolyzed and linked to the hydroxyl groups already present on the particle surface through covalent and hydrogen bonds. Due to a basic catalyzed condensation of MPS with water, a siloxane layer was shown to be anchored to the nanoparticles through mono- and tridentate structures. Quantitative investigations were performed by thermogravimetric (TGA) and elemental analyses. The amount of silane linked to ZnO particles was shown to be affected by the amount of isolated hydroxyl groups available to react on the particle surface. For as-prepared ZnO nanoparticles, the number of isolated and available hydroxyl groups per square nanometer was up to 3 times higher than the one found on commercially available ZnO nanoparticles, leading to higher amounts of polymerizable silane agent linked to the surface. The MPS molecules were shown to be mainly oriented perpendicular to the oxide surface for all the as-prepared ZnO nanoparticles, whereas a parallel orientation was found for the preheated commercially ZnO nanopowders. In addition, ZnO nanoparticles were shown to be hydrophobized by the MPS treatment with water contact angles higher than 60°.

Comparison of enzyme kinetics of warfarin analyzed by LC-MS/MS QTrap and differential mobility spectrometry.

PubMed

Shaik, Abdul Naveed; Grater, Richard; Lulla, Mukesh; Williams, David A; Gan, Lawrence L; Bohnert, Tonika; LeDuc, Barbara W

2016-01-01

Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3'-hydroxy, 4'-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC-MS/MS QTrap and LC-MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3'-OH, 4'-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (Km and Vmax) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1-3nM using an LC-MS/MS QTrap method which had a run time of 22min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100nM with a run time of 2.8min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific inhibitors of CYP2C9 and CYP3A4 respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

PubMed

Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

2016-08-01

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods.

PubMed

Berisha, Arton; Dold, Sebastian; Guenther, Sabine; Desbenoit, Nicolas; Takats, Zoltan; Spengler, Bernhard; Römpp, Andreas

2014-08-30

An ideal method for bioanalytical applications would deliver spatially resolved quantitative information in real time and without sample preparation. In reality these requirements can typically not be met by a single analytical technique. Therefore, we combine different mass spectrometry approaches: chromatographic separation, ambient ionization and imaging techniques, in order to obtain comprehensive information about metabolites in complex biological samples. Samples were analyzed by laser desorption followed by electrospray ionization (LD-ESI) as an ambient ionization technique, by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging for spatial distribution analysis and by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) for quantitation and validation of compound identification. All MS data were acquired with high mass resolution and accurate mass (using orbital trapping and ion cyclotron resonance mass spectrometers). Grape berries were analyzed and evaluated in detail, whereas wheat seeds and mouse brain tissue were analyzed in proof-of-concept experiments. In situ measurements by LD-ESI without any sample preparation allowed for fast screening of plant metabolites on the grape surface. MALDI imaging of grape cross sections at 20 µm pixel size revealed the detailed distribution of metabolites which were in accordance with their biological function. HPLC/ESI-MS was used to quantify 13 anthocyanin species as well as to separate and identify isomeric compounds. A total of 41 metabolites (amino acids, carbohydrates, anthocyanins) were identified with all three approaches. Mass accuracy for all MS measurements was better than 2 ppm (root mean square error). The combined approach provides fast screening capabilities, spatial distribution information and the possibility to quantify metabolites. Accurate mass measurements proved to be critical in order to reliably combine data from different MS

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

PubMed

Heyno, Eiri; Mary, Véronique; Schopfer, Peter; Krieger-Liszkay, Anja

2011-07-01

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

Characterization of Urinary Phthalate Metabolites Among Custodians

PubMed Central

Cavallari, Jennifer M.; Simcox, Nancy J.; Wakai, Sara; Lu, Chensheng; Garza, Jennifer L.; Cherniack, Martin

2015-01-01

Phthalates, a ubiquitous class of chemicals found in consumer, personal care, and cleaning products, have been linked to adverse health effects. Our goal was to characterize urinary phthalate metabolite concentrations and to identify work and nonwork sources among custodians using traditional cleaning chemicals and ‘green’ or environmentally preferable products (EPP). Sixty-eight custodians provided four urine samples on a workday (first void, before shift, end of shift, and before bedtime) and trained observers recorded cleaning tasks and types of products used (traditional, EPP, or disinfectant) hourly over the work shifts. Questionnaires were used to assess personal care product use. Four different phthalate metabolites [monoethyl phthalate (MEP), monomethyl phthalate (MMP), mono (2-ethylhexyl) phthalate (MEHP), and monobenzyl phthalate (MBzP)] were quantified using liquid chromatography mass spectrometry. Geometric means (GM) and 95% confidence intervals (95% CI) were calculated for creatinine-adjusted urinary phthalate concentrations. Mixed effects univariate and multivariate modeling, using a random intercept for each individual, was performed to identify predictors of phthalate metabolites including demographics, workplace factors, and personal care product use. Creatinine-adjusted urinary concentrations [GM (95% CI)] of MEP, MMP, MEHP, and MBzP were 107 (91.0–126), 2.69 (2.18–3.30), 6.93 (6.00–7.99), 8.79 (7.84–9.86) µg g−1, respectively. An increasing trend in phthalate concentrations from before to after shift was not observed. Creatinine-adjusted urinary MEP was significantly associated with frequency of traditional cleaning chemical intensity in the multivariate model after adjusting for potential confounding by demographics, workplace factors, and personal care product use. While numerous demographics, workplace factors, and personal care products were statistically significant univariate predictors of MMP, MEHP, and MBzP, few

Surface structure and stability of partially hydroxylated silica surfaces

DOE PAGES

Rimsza, J. M.; Jones, R. E.; Criscenti, L. J.

2017-04-04

Surface energies of silicates influence crack propagation during brittle fracture and decrease with surface relaxation caused by annealing and hydroxylation. Molecular-level simulations are particularly suited for the investigation of surface processes. In this work, classical MD simulations of silica surfaces are performed with two force fields (ClayFF and ReaxFF) to investigate the effect of force field reactivity on surface structure and energy as a function of surface hydroxylation. An unhydroxylated fracture surface energy of 5.1 J/m 2 is calculated with the ClayFF force field, and 2.0 J/m 2 is calculated for the ReaxFF force field. The ClayFF surface energies aremore » consistent with the experimental results from double cantilever beam fracture tests (4.5 J/m 2), whereas ReaxFF underestimated these surface energies. Surface relaxation via annealing and hydroxylation was performed by creating a low-energy equilibrium surface. Annealing condensed neighboring siloxane bonds increased the surface connectivity, and decreased the surface energies by 0.2 J/m 2 for ClayFF and 0.8 J/m 2 for ReaxFF. Posthydroxylation surface energies decreased further to 4.6 J/m 2 with the ClayFF force field and to 0.2 J/m 2 with the ReaxFF force field. Experimental equilibrium surface energies are ~0.35 J/m 2, consistent with the ReaxFF force field. Although neither force field was capable of replicating both the fracture and equilibrium surface energies reported from experiment, each was consistent with one of these conditions. Furthermore, future computational investigations that rely on accurate surface energy values should consider the surface state of the system and select the appropriate force field.« less

Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: A combined synthesis, binding and docking study

PubMed Central

McCullough, Christopher; Neumann, Terrence S.; Gone, Jayapal Reddy; He, Zhengjie; Herrild, Christian; Wondergem, Julie; Pandey, Rajesh K.; Donaldson, William A.; Sem, Daniel S.

2014-01-01

Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxyl group interacting with His524 in ERα. One nonsteroid-based dihdroxyl compound was 1000-fold specific for ERβ over ERα, and was also 25-fold specific for agonist ERβ versus antagonist activity. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERβ, versus with Thr299 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERα with high affinity, via hydroxyl hydrogen bonding interactions with the ERα Arg394/Glu353/water triad, and van der Waals interactions with the rest of the molecule. PMID:24315190

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method.

PubMed

Bektaşoğlu, Burcu; Esin Celik, Saliha; Ozyürek, Mustafa; Güçlü, Kubilay; Apak, Reşat

2006-07-07

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more

New Pioglitazone Metabolites and Absence of Opened-Ring Metabolites in New N-Substituted Thiazolidinedione.

PubMed

Campos, Michel Leandro; Cerqueira, Letícia Bonancio; Silva, Bruna Cristina Ulian; Franchin, Taísa Busaranho; Galdino-Pitta, Marina Rocha; Pitta, Ivan Rocha; Peccinini, Rosângela Gonçalves; Pontarolo, Roberto

2018-06-01

Thiazolidinediones (TZDs) are drugs used to treat type 2 diabetes mellitus; however, several safety concerns remain regarding the available drugs in this class. Therefore, the search for new TZD candidates is ongoing; metabolism studies play a crucial step in the development of new candidates. Pioglitazone, one of the most commonly used TZDs, and GQ-11, a new N -substituted TZD, were investigated in terms of their metabolic activity in rat and human liver microsomes to assess their metabolic stability and investigate their metabolites. Methods for preparation of samples were based on liquid-liquid extraction and protein precipitation. Quantitation was performed using liquid chromatography (LC)-tandem mass spectrometry, and the metabolite investigation was performed using ultraperformance LC coupled to a hybrid quadrupole-time of flight mass spectrometer. The predicted intrinsic clearance of GQ-11 was 70.3 and 46.1 ml/kg per minute for rats and humans, respectively. The predicted intrinsic clearance of pioglitazone was 24.1 and 15.9 ml/kg per minute for rats and humans, respectively. The pioglitazone metabolite investigation revealed two unpublished metabolites (M-D and M-A). M-A is a hydration product and may be related to the mechanism of ring opening and the toxicity of pioglitazone. The metabolites of GQ-11 are products of oxidation; no ring-opening metabolite was observed for GQ-11. In conclusion, under the same experimental conditions, a ring-opening metabolite was observed only for pioglitazone. The resistance of GQ-11 to the ring opening is probably related to N -substitution in the TZD ring. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Lung toxicity and tumor promotion by hydroxylated derivatives of 2,6-di-tert-butyl-4-methylphenol (BHT) and 2-tert-butyl-4-methyl-6-iso-propylphenol: correlation with quinone methide reactivity.

PubMed

Kupfer, Rene; Dwyer-Nield, Lori D; Malkinson, Alvin M; Thompson, John A

2002-08-01

Acute pulmonary toxicity and tumor promotion by the food additive 2,6-di-tert-butyl-4-methylphenol (BHT) in mice are well documented. These effects have been attributed to either of two quinone methides, 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) formed through direct oxidation of BHT by pulmonary cytochrome P450 or a quinone methide formed by hydroxylation of a tert-butyl group of BHT (to form BHTOH) followed by oxidation of this metabolite to BHTOH-QM. BHTOH-QM is a more reactive electrophile compared to BHT-QM due to intramolecular interactions of the side-chain hydroxyl with the carbonyl oxygen. To further examine this bioactivation pathway, an analogue of BHTOH was prepared, 2-tert-butyl-6-(1'-hydroxy-1'-methyl)ethyl-4-methylphenol (BPPOH), that is structurally very similar to BHTOH but forms a quinone methide (BPPOH-QM) capable of more efficient intramolecular hydrogen bonding and, therefore, higher electrophilicity than BHTOH-QM. BPPOH-QM was synthesized and its reactivity with water, methanol, and glutathione determined to be >10-fold higher than that of BHTOH-QM. The conversions of BPPOH and BHTOH to quinone methides in lung microsomes from male BALB/cByJ mice were quantitatively similar, but in vivo the former was pneumotoxic at one-half of the dose required for the latter and one-eighth of the dose required for BHT, as determined by increased lung weight:body weight ratios following a single i.p. injection. Similar differences were found in the doses of BHT, BHTOH, or BPPOH required for tumor promotion after a single initiating dose of 3-methylcholanthrene followed by three weekly injections of the phenol. The downregulaton of calpain II, previously shown to accompany lung tumor promotion by BHT and BHTOH, also occurred with BPPOH. The correlation between biologic activities of these phenols and the reactivities of their corresponding quinone methides provides additional support for the role of BHTOH-QM as the principal metabolite

Synthesis and radical scavenging activity of 6-hydroxyl-4-methylcoumarin and its derivatives

NASA Astrophysics Data System (ADS)

Jumal, Juliana; Ayomide, Adetunji Fridaos

2018-06-01

Four compounds of coumarin derivatives namely 6-hydroxyl-4-methylcoumarin (I), 6-hydroxyl-4-methyl-5-(p-nitrophenyl azocoumarin) (II), 6-hydroxyl-4-methyl-5,7-(bis-p-nitrophenyl azocoumarin) (III) and 6-hydroxyl-4-methyl-5,7-(bis-p-chlorophenyl azocoumarin) (IV) were successfully synthesized. These compounds were prepared by reacting hydroquinone with ethylacetoacetate and selected anilines which are chloro and nitro aniline. All synthesized compounds were characterized by CHN micro-elemental analysis, 1H Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FTIR) spectroscopic methods. The infrared spectra of these compounds exhibited five important stretching vibrations: ʋ(-OH), ʋ(C=O), ʋ(C=C), ʋ(C-O) and ʋ(C-N) at 3441-3359 cm-1, 1604-1632 cm-1, 1581-1496 cm-1, 1331-1225 cm-1, 1251-1109 cm-1, respectively. 1H NMR spectra of these compounds show the presence of proton aromatic, proton methyl and proton pyrone ring with the chemical shift at δH 7.00-8.70 ppm, δH 2.20-2.50 ppm and δH 6.10-6.90 ppm, respectively. CHN analysis results of all compounds are in good agreement with the calculated values. All the synthesized compounds were evaluated for their antioxidant activity using DPPH method and ascorbic acid used as the standard. UV-Vis spectroscopic technique was used to investigate the absorbance of these compounds. Compound (II) shows high antioxidant activities compared to compound (I), (III) and (IV) which show moderate to low activities.

Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals

USDA-ARS?s Scientific Manuscript database

Bacteria rely mainly on enzymes, glutathione and other low-molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and tri...

Urinary Concentrations of Polycyclic Aromatic Hydrocarbon Metabolites in Maté Drinkers in Rio Grande do Sul, Brazil

PubMed Central

Lopes, Antonio Barros; Metzdorf, Marcela; Metzdorf, Luiza; Ramalho, Marcos Paulo; Kavalco, Caroline; Etemadi, Arash; Pritchett, Natalie R.; Murphy, Gwen; Calafat, Antonia M.; Abnet, Christian C.; Dawsey, Sanford M.; Fagundes, Renato Borges

2017-01-01

Background Consumption of maté, an infusion of the herb Ilex paraguariensis (yerba maté), is associated with increased risk of esophageal squamous cell carcinoma (ESCC), but the carcinogenic mechanism is unclear. Commercial brands of yerba maté contain high levels of carcinogenic polycyclic aromatic hydrocarbons (PAHs), which are acquired during the traditional drying process. The purpose of this study was to characterize exposure to PAHs in maté drinkers over a wide range of maté consumption. Methods We recruited 244 adults who answered a questionnaire and collected a fasting spot urine specimen. We quantified urinary concentrations of seven PAH metabolites, and assessed associations between self-reported recent maté consumption and urinary PAH metabolites by multivariate regression. Results Recent maté consumption showed a significant dose-response association with 6 of 7 PAH metabolites in unadjusted models (p-for-trend <0.05). After adjustment for creatinine and potential confounders, concentrations of 2-naphthol, 1-hydroxyphenanthrene, and the sum of 2- and 3-hydroxyphenanthrene remained significantly associated with recent maté intake. The sum of the urinary concentrations of the phenanthrene metabolites was similar or higher among maté drinkers who did not smoke than among smokers who did not drink maté. Conclusions Urinary concentrations of PAH metabolites were significantly associated with self-reported amount of recent maté intake, and drinking maté increased urinary concentrations of some PAH metabolites as much as smoking cigarettes. Impact Drinking maté is a source of exposure to potentially carcinogenic PAHs, consistent with the hypothesis that the PAH content of maté may contribute to the increased risk of ESCC in maté drinkers. PMID:29263183

Effect of thyme/cumin essential oils and butylated hydroxyl anisole/butylated hydroxyl toluene on physicochemical properties and oxidative/microbial stability of chicken patties.

PubMed

Sariçoban, Cemalettin; Yilmaz, Mustafa Tahsin

2014-02-01

In this study, effects of thyme/cumin essential oils (EO) and butylated hydroxyl anisole (BHA)/butylated hydroxyl toluene (BHT) on physicochemical properties and storage stability of chicken patties were compared in different storage periods (0, 3, 7, 14, 21, and 28 d). It was found that there were significant (P < 0.05) differences between physicochemical properties of patty samples treated with EO and the synthetic antioxidants. The EO showed similar performance to those of BHA and BHT in limiting TBARS values of chicken patty samples. Similarity in performance was also the case for microbial stability (total aerobic mesophilic, psychrotrophic, lactic acid, and coliform bacteria as well as molds and yeasts); namely, their effects were significant (P < 0.05). Effect of thyme EO was significant (P < 0.05) and remarkable, not allowing any coliform bacteria to grow in the samples. Given that EO were obtained from natural sources, the data suggested that the EO might be more useful than their synthetic counterparts, BHA and BHT, as additives for chicken patties to maintain oxidative/microbial stability and increase shelf life.

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon.

PubMed

Akashi, Kinya; Nishimura, Noriyuki; Ishida, Yoshinori; Yokota, Akiho

2004-10-08

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions. Copyright 2004 Elsevier Inc.

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.

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 of Serum Metabolites Associated With Risk of Type 2 Diabetes Using a Targeted Metabolomic Approach

PubMed Central

Floegel, Anna; Stefan, Norbert; Yu, Zhonghao; Mühlenbruch, Kristin; Drogan, Dagmar; Joost, Hans-Georg; Fritsche, Andreas; Häring, Hans-Ulrich; Hrabě de Angelis, Martin; Peters, Annette; Roden, Michael; Prehn, Cornelia; Wang-Sattler, Rui; Illig, Thomas; Schulze, Matthias B.; Adamski, Jerzy; Boeing, Heiner; Pischon, Tobias

2013-01-01

Metabolomic discovery of biomarkers of type 2 diabetes (T2D) risk may reveal etiological pathways and help to identify individuals at risk for disease. We prospectively investigated the association between serum metabolites measured by targeted metabolomics and risk of T2D in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) among all incident cases of T2D (n = 800, mean follow-up 7 years) and a randomly drawn subcohort (n = 2,282). Flow injection analysis tandem mass spectrometry was used to quantify 163 metabolites, including acylcarnitines, amino acids, hexose, and phospholipids, in baseline serum samples. Serum hexose; phenylalanine; and diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5 were independently associated with increased risk of T2D and serum glycine; sphingomyelin C16:1; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2 with decreased risk. Variance of the metabolites was largely explained by two metabolite factors with opposing risk associations (factor 1 relative risk in extreme quintiles 0.31 [95% CI 0.21–0.44], factor 2 3.82 [2.64–5.52]). The metabolites significantly improved T2D prediction compared with established risk factors. They were further linked to insulin sensitivity and secretion in the Tübingen Family study and were partly replicated in the independent KORA (Cooperative Health Research in the Region of Augsburg) cohort. The data indicate that metabolic alterations, including sugar metabolites, amino acids, and choline-containing phospholipids, are associated early on with a higher risk of T2D. PMID:23043162

Comparison of chemiluminescence methods for analysis of hydrogen peroxide and hydroxyl radicals

NASA Astrophysics Data System (ADS)

Pehrman, R.; Amme, M.; Cachoir, C.

2006-01-01

Assessment of alpha radiolysis influence on the chemistry of geologically disposed spent fuel demands analytical methods for radiolytic product determination at trace levels. Several chemiluminescence methods for the detection of radiolytic oxidants hydrogen peroxide and hydroxyl radicals are tested. Two of hydrogen peroxide methods use luminol, catalyzed by either μ-peroxidase or hemin, one uses 10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate and one potassium periodate. All recipes are tested as batch systems in basic conditions. For hydroxyl radical detection luminophores selected are 3-hydroxyphthalic hydrazide and rutin. Both methods are tested as batch systems. The results are compared and the applicability of the methods for near-field dissolution studies is discussed.

Properties of the Nafion membrane impregnated with hydroxyl ammonium based ionic liquids

NASA Astrophysics Data System (ADS)

Garaev, Valeriy; Kleperis, Janis; Pavlovica, Sanita; Vaivars, Guntars

2012-08-01

In this work, the Nafion 112 membrane impregnated with nine various hydroxyl ammonium based ionic liquids have been investigated. The used ionic liquids were combined from hydroxyl ammonium cations (2-hydroxyethylammonium/HEA, bis(2- hydroxyethyl)ammonium/BHEA, tris(2-hydroxyethyl)ammonium/THEA) and carboxylate anions (formate, acetate, lactate). The membranes are characterized by conductivity and thermal stability measurements. It was found, that almost all composites have 10 times higher ion conductivity than a pure Nafion 112 at 90 °C in ambient environment due to the higher thermal stability. The thermal stability of Nafion membrane was increased by all studied nine ionic liquids. In this work, only biodegradable ionic liquids were used for composite preparation.

Prediction of biotransformation products of the fungicide fluopyram by electrochemistry coupled online to liquid chromatography-mass spectrometry and comparison with in vitro microsomal assays.

PubMed

Mekonnen, Tessema F; Panne, Ulrich; Koch, Matthias

2018-04-01

Biotransformation processes of fluopyram (FLP), a new succinate dehydrogenase inhibitor (SDHI) fungicide, were investigated by electrochemistry (EC) coupled online to liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Oxidative phase I metabolite production was achieved using an electrochemical flow-through cell equipped with a boron-doped diamond (BDD) electrode. Structural elucidation and prediction of oxidative metabolism pathways were assured by retention time, isotopic patterns, fragmentation, and accurate mass measurements using EC/LC/MS, LC-MS/MS, and/or high-resolution mass spectrometry (HRMS). The results obtained by EC were compared with conventional in vitro studies by incubating FLP with rat and human liver microsomes (RLM, HLM). Known phase I metabolites of FLP (benzamide, benzoic acid, 7-hydroxyl, 8-hydroxyl, 7,8-dihydroxyl FLP, lactam FLP, pyridyl acetic acid, and Z/E-olefin FLP) were successfully simulated by EC/LC/MS. New metabolites including an imide, hydroxyl lactam, and 7-hydroxyl pyridyl acetic acid oxidative metabolites were predicted for the first time in our study using EC/LC/MS and liver microsomes. We found oxidation by dechlorination to be one of the major metabolism mechanisms of FLP. Thus, our results revealed that EC/LC/MS-based metabolic elucidation was more advantageous on time and cost of analysis and enabled matrix-free detection with valuable information about the mechanisms and intermediates of metabolism processes. Graphical abstract Oxidative metabolism of fluopyram.

Quantitative determination of five metabolites of aspirin by UHPLC-MS/MS coupled with enzymatic reaction and its application to evaluate the effects of aspirin dosage on the metabolic profile.