Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization.

PubMed

Manini, P; Andreoli, R; Mutti, A; Bergamaschi, E; Niessen, W M

1998-01-01

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).

Glucuronidation and its impact on the bioactivity of [6]-shogaol.

PubMed

Wang, Pei; Zhao, Yantao; Zhu, Yingdong; Sang, Shengmin

2017-09-01

-shogaol (6S) from ginger has been reported to have diverse bioactivities and can be widely metabolized in animals and humans; however, the impact of glucuronidation on its bioactivity is still largely unknown. This study investigates the glucuronidation of 6S and its effect on cell cytotoxicity and Nrf2-inducing activities of 6S. The glucuronidated metabolite of 6S, 4-O-monoglucuronide 6S (6S-G), was synthesized and characterized for the first time. Glucuronidation of 6S in humans was studied using microsomes of the liver and intestine and recombinant UDP-glucuronosyltransferase (UGTs). The kinetics of 6S glucuronidation by human liver and intestinal microsomes followed the substrate inhibition kinetics model. The intrinsic glucuronidation clearance (CL int ) of 6S in human liver microsomes was higher than that in human intestine microsomes. Among the recombinant UGTs examined, UGT1A1, 1A3, 1A6, 1A8, 1A10, 2B7, 2B15, and 2B17 exhibited glucuronidation activity toward 6S, with UGT2B7 being the most potent one. Compared with 6S, the glucuronidation of 6S largely eliminated its cell cytotoxicity against human colon cancer cell lines HT-116 and HT-29, and its Nrf2-inducing activity. The findings from current study provide foundations for understanding the role of glucuronidation in biotransformation and biological activities of 6S. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of the β-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases (UGTs)

PubMed Central

Oleson, Lauren; Court, Michael H.

2009-01-01

Glucuronidation studies using microsomes and recombinant UDP-glucuronosyltransferases (rUGTs) can be complicated by the presence of endogenous β-glucuronidases leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used β-glucuronidase inhibitor, although as of yet it is not clear whether this reagent should be routinely added to glucuronidation incubations. Here we determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and rUGTs. Despite the use of buffered incubation solutions it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM failed to show enhancement of any of the glucuronidation activities evaluated that could be considered consistent with inhibition of β-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for 5-hydroxytryptamine and estradiol glucuronidation by pHLMs with 35% decrease at 20 mM saccharolactone concentration. Endogenous β-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes, and insect-cell expressed rUGTs, but not for kidney or intestinal microsomes, or HEK293 microsomes. However, the extent of hydrolysis was relatively small representing only 9 to 19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations and, if used, saccharolactone concentrations should be titrated to achieve activity enhancement without inhibition. PMID:18718121

Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation

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

Zhu, Liangliang; Xiao, Ling; Xia, Yangliu

This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In themore » presence of DES (0–6.25 μM), K{sub m} values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while V{sub max} values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate V{sub max} from 0.016 to 0.81 nmol/min/mg, while lifting K{sub m} in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with K{sub A}, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4. - Highlights: • E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES. • E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES. • Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the activity of UGT1A4.« less

Triterpenes and flavonol glucuronides from Oenothera cheiranthifolia.

PubMed

Nakanishi, Tsutomu; Inatomi, Yuka; Murata, Hiroko; Ishida, Syun-Suke; Fujino, Yuri; Miura, Kanako; Yasuno, Yoshito; Inada, Akira; Lang, Frank A; Murata, Jin

2007-02-01

A new ursane-type triterpene, named as cheiranthic acid (1), was isolated from the MeOH extract of whole plants of Oenothera cheiranthifolia (Onagraceae) along with an isomeric pair of known oleanane- and ursane-type triterpenes (arjunolic acid and asiatic acid) and three flavonol glucuronide analogues (quercetin 3-O-glucuronide, its n-butyl ester, and myricetin 3-O-glucuronide). Their structures were elucidated based on spectroscopic evidence.

Characterization of raloxifene glucuronidation. Potential role of UGT1A8 genotype on raloxifene metabolism in vivo

PubMed Central

Sun, Dongxiao; Jones, Nathan R; Manni, Andrea; Lazarus, Philip

2014-01-01

Raloxifene is a 2nd-generation selective estrogen receptor modulator used for the prevention and treatment of osteoporosis and the prevention of breast cancer in postmenopausal women. Raloxifene is extensively metabolized by glucuronidation to form raloxifene-6-glucuronide (ral-6-Gluc) and raloxifene-4′-glucuronide (ral-4′-Gluc). The goal of the present study was to determine whether functional polymorphisms in active UGTs could play a role in altered raloxifene glucuronidation in vivo. Using homogenates from HEK293 UGT-overexpressing cell lines, raloxifene was shown to be glucuronidated primarily by the hepatic UGTs 1A1 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10; no detectable raloxifene glucuronidation activity was found for UGT2B enzymes. Functional UGT1A1 transcriptional promoter genotypes were significantly (ptrend=0.005) associated with ral-6-Gluc formation in human liver microsomes, and, consistent with the decreased raloxifene glucuronidation activities observed in vitro with cell line over-expressing UGT1A8 variants, the UGT1A8*2 variant was significantly (p=0.023) correlated with total raloxifene glucuronide formation in human jejunum homogenates. While ral-4′-Gluc exhibited 1/100th the anti-estrogenic activity of raloxifene itself as measured by binding to the estrogen receptor, raloxifene glucuronides comprised ∼99% of the circulating raloxifene dose in raloxifene-treated subjects, with ral-4′-Gluc comprising ∼70% of raloxifene glucuronides. Plasma ral-6-Gluc (ptrend=0.0025), ral-4′-Gluc (ptrend=0.001), and total raloxifene glucuronides (ptrend=0.001) were increased in raloxifene-treated subjects who were predicted slow metabolizers [UGT1A8 (*1/*3)] vs intermediate metabolizers [UGT1A8 (*1/*1) or UGT1A8 (*1/*2)] vs fast metabolizers [UGT1A8 (*2/*2). These data suggest that raloxifene metabolism may be dependent on UGT1A8 genotype and that UGT1A8 genotype may play an important role in overall response to raloxifene. PMID:23682072

Hepatic disposition of the acyl glucuronide 1-O-gemfibrozil-beta-D-glucuronide: effects of clofibric acid, acetaminophen, and acetaminophen glucuronide.

PubMed

Sabordo, L; Sallustio, B C; Evans, A M; Nation, R L

2000-10-01

Glucuronidation of carboxylic acid compounds results in the formation of electrophilic acyl glucuronides. Because of their polarity, carrier-mediated hepatic transport systems play an important role in determining both intra- and extrahepatic exposure to these reactive conjugates. We have previously shown that the hepatic membrane transport of 1-O-gemfibrozil-beta-D-glucuronide (GG) is carrier-mediated and inhibited by the organic anion dibromosulfophthalein. In this study, we examined the influence of 200 microM acetaminophen, acetaminophen glucuronide, and clofibric acid on the disposition of GG (3 microM) in the recirculating isolated perfused rat liver preparation. GG was taken up by the liver, excreted into bile, and hydrolyzed within the liver to gemfibrozil, which appeared in perfusate but not in bile. Mean +/- S. D. hepatic clearance, apparent intrinsic clearance, hepatic extraction ratio, and biliary excretion half-life of GG were 10.4 +/- 1.4 ml/min, 94.1 +/- 17.9 ml/min, 0.346 +/- 0.046, and 30.9 +/- 4.9 min, respectively, and approximately 73% of GG was excreted into bile. At the termination of the experiment (t = 90 min), the ratio of GG concentrations in perfusate, liver, and bile was 1:35:3136. Acetaminophen and acetaminophen glucuronide had no effect on the hepatic disposition of GG, suggesting relatively low affinities of acetaminophen conjugates for hepatic transport systems or the involvement of multiple transport systems for glucuronide conjugates. In contrast, clofibric acid increased the hepatic clearance, extraction ratio, and apparent intrinsic clearance of GG (P <.05) while decreasing its biliary excretion half-life (P <.05), suggesting an interaction between GG and hepatically generated clofibric acid glucuronide at the level of hepatic transport. However, the transporter protein(s) involved remains to be identified.

Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin

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

Liu, Wei; Feng, Qian; Li, Ye

2012-12-15

Emodin is a poorly bioavailable but promising plant-derived anticancer drug candidate. The low oral bioavailability of emodin is due to its extensive glucuronidation in the intestine and liver. Caco-2 cell culture model was used to investigate the interplay between UDP-glucuronosyltransferases (UGTs) and efflux transporters in the intestinal disposition of emodin. Bidirectional transport assays of emodin at different concentrations were performed in the Caco-2 monolayers with or without multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP) efflux transporter chemical inhibitors. The bidirectional permeability of emodin and its glucuronide in the Caco-2 monolayers was determined. Emodin was rapidly metabolized tomore » emodin glucuronide in Caco-2 cells. LTC4, a potent inhibitor of MRP2, decreased the efflux of emodin glucuronide and also substantially increased the intracellular glucuronide level in the basolateral-to-apical (B–A) direction. MK-571, chemical inhibitor of MRP2, MRP3, and MRP4, significantly reduced the efflux of glucuronide in the apical-to-basolateral (A–B) and B–A directions in a dose-dependent manner. However, dipyridamole, a BCRP chemical inhibitor demonstrated no effect on formation and efflux of emodin glucuronide in Caco-2 cells. In conclusion, UGT is a main metabolic pathway for emodin in the intestine, and the MRP family is composed of major efflux transporters responsible for the excretion of emodin glucuronide in the intestine. The coupling of UGTs and MRP efflux transporters causes the extensive metabolism, excretion, and low bioavailability of emodin. -- Highlights: ► Glucuronidation is the main reason for the poor oral bioavailability of emodin. ► Efflux transporters are involved in the excretion of emodin glucuronide. ► The intestine is the main organ for metabolism of emodin.« less

Repaglinide-gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism

PubMed Central

Gan, Jinping; Chen, Weiqi; Shen, Hong; Gao, Ling; Hong, Yang; Tian, Yuan; Li, Wenying; Zhang, Yueping; Tang, Yuwei; Zhang, Hongjian; Humphreys, William Griffith; Rodrigues, A David

2010-01-01

AIM To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. METHODS Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. RESULTS The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [3H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. CONCLUSIONS Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil. PMID:21175442

Repaglinide-gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism.

PubMed

Gan, Jinping; Chen, Weiqi; Shen, Hong; Gao, Ling; Hong, Yang; Tian, Yuan; Li, Wenying; Zhang, Yueping; Tang, Yuwei; Zhang, Hongjian; Humphreys, William Griffith; Rodrigues, A David

2010-12-01

To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil. © 2010 The Authors. British Journal of Clinical Pharmacology © 2010 The British Pharmacological Society.

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms.

PubMed

Street, Christina M; Zhu, Zhaohui; Finel, Moshe; Court, Michael H

2017-01-01

1. Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast. 2. Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes >80% of activity at bisphenol-A concentrations under 5 μM, while UGT1A9 contributes up to 50% of activity at higher concentrations. 3. Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p = 0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes. 4. Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p = 0.006). 5. UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.

Identification of Phase II Metabolites of Thiol-conjugated [6]-Shogaol in Mouse Urine Using High-Performance Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Chen, Huadong; Sang, Shengmin

2012-01-01

Ginger is frequently consumed as a spice and has numerous medicinal properties. Extensive research has characterized the anti-inflammatory, antioxidant, and antitumor activities of ginger. Previously, we reported the mercapturic acid pathway as a major metabolic route of [6]-shogaol in mice and the thiol conjugates of [6]-shogaol existed in the glucuronidated and sulfated forms in mouse urine. However, their structures are still unknown. In the present study, we further investigated the phase II metabolism of thiol-conjugated [6]-shogaol in mouse urine, in which we identified sixteen phase II metabolites of thiol-conjugated [6]-shogaol: 5-cysteinyl-[6]-shogaol glucuronide (9), 5-N-acetylcysteinyl-[6]-shogaol glucuronide (10), 5-cysteinylglycinyl-[6]-shogaol glucuronide (11), 5-methylthio-[6]-shogaol glucuronide (12), 5-cysteinyl-M6 glucuronide (13 and 14), 5-cysteinyl-M6 sulfate (15 and 16), 5-N-acetylcysteinyl-M6 glucuronide (17 and 18), 5-cysteinylglycinyl-M6 glucuronide (19 and 20), 5-cysteinylglycinyl-M6 sulfate (21 and 22), and 5-methylthio-M6 glucuronide (23 and 24) using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing their MSn (n =1! 4) spectra as well as comparing with the tandem mass spectra of authentic standards. To our knowledge, this is the first report involving identification of phase II urinary metabolites of [6]-shogaol in mice. PMID:23031413

Identification of phase II metabolites of thiol-conjugated [6]-shogaol in mouse urine using high-performance liquid chromatography tandem mass spectrometry.

PubMed

Chen, Huadong; Sang, Shengmin

2012-10-15

Ginger is frequently consumed as a spice and has numerous medicinal properties. Extensive research has characterized the anti-inflammatory, antioxidant, and antitumor activities of ginger. Previously, we reported the mercapturic acid pathway as a major metabolic route of [6]-shogaol in mice and the thiol conjugates of [6]-shogaol existed in the glucuronidated and sulfated forms in mouse urine. However, their structures are still unknown. In the present study, we further investigated the phase II metabolism of thiol-conjugated [6]-shogaol in mouse urine, in which we identified sixteen phase II metabolites of thiol-conjugated [6]-shogaol: 5-cysteinyl-[6]-shogaol glucuronide (9), 5-N-acetylcysteinyl-[6]-shogaol glucuronide (10), 5-cysteinylglycinyl-[6]-shogaol glucuronide (11), 5-methylthio-[6]-shogaol glucuronide (12), 5-cysteinyl-M6 glucuronide (13 and 14), 5-cysteinyl-M6 sulfate (15 and 16), 5-N-acetylcysteinyl-M6 glucuronide (17 and 18), 5-cysteinylglycinyl-M6 glucuronide (19 and 20), 5-cysteinylglycinyl-M6 sulfate (21 and 22), and 5-methylthio-M6 glucuronide (23 and 24) using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing their MS(n) (n=1-4) spectra as well as comparing with the tandem mass spectra of authentic standards. To the best of our knowledge, this is the first report involving identification of phase II urinary metabolites of [6]-shogaol in mice. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro and in vivo metabolism of verproside in rats.

PubMed

Kim, Min Gi; Hwang, Deok-Kyu; Jeong, Hyeon-Uk; Ji, Hye Young; Oh, Sei-Ryang; Lee, Yongnam; Yoo, Ji Seok; Shin, Dae Hee; Lee, Hye Suk

2012-10-12

Verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a biologically active compound with anti-inflammatory, antinociceptic, antioxidant, and anti-asthmatic properties. Twenty-one metabolites were identified in bile and urine samples obtained after intravenous administration of verproside in rats using liquid chromatography-quadrupole Orbitrap mass spectrometry. Verproside was metabolized by O-methylation, glucuronidation, sulfation, and hydrolysis to verproside glucuronides (M1 and M2), verproside sulfates (M3 and M4), picroside II (M5), M5 glucuronide (M7), M5 sulfate (M9), isovanilloylcatalpol (M6), M6 glucuronide (M8), M6 sulfate (M10), 3,4-dihydroxybenzoic acid (M11), M11 glucuronide (M12), M11 sulfates (M13 and M14), 3-methyoxy-4-hydroxybenzoic acid (M15), M15 glucuronides (M17 and M18), M15 sulfate (M20), 3-hydroxy-4-methoxybenzoic acid (M16), M16 glucuronide (M19), and M16 sulfate (M21). Incubation of verproside with rat hepatocytes resulted in thirteen metabolites (M1-M11, M13, and M14). Verproside sulfate, M4 was a major metabolite in rat hepatocytes. After intravenous administration of verproside, the drug was recovered in bile (0.77% of dose) and urine (4.48% of dose), and O-methylation of verproside to picroside II (M5) and isovanilloylcatalpol (M6) followed by glucuronidation and sulfation was identified as major metabolic pathways compared to glucuronidation and sulfation of verproside in rats.

Establishment and Use of New MDCK II Cells Overexpressing Both UGT1A1 and MRP2 to Characterize Flavonoid Metabolism via the Glucuronidation Pathway

PubMed Central

Wang, Meifang; Yang, Guangyi; He, Yu; Xu, Beibei; Zeng, Min; Yin, Taijun; Gao, Song; Hu, Ming

2017-01-01

Scope The purpose of this study is to characterize how overexpression of an efflux transporter and an UDP-glucuronosyltransferase (UGT) affects the cellular kinetics of glucuronidation processes. Methods and Results A new MDCK II cell line overexpressing both MRP2 and UGT1A1 (MDCKII-UGT1A1/MRP2 cells) was developed and used to determine how overexpression of an efflux transporter affects the kinetics of cellular flavonoid glucuronide production. The results showed that most model flavonoids (from a total of 13) were mainly metabolized into glucuronides in the MDCKII-UGT1A1/MRP2 cells and the glucuronides were rapidly excreted. Flavonoids with three or fewer hydroxyl group at 7, 3′ or 6 hydroxyl group were also metabolized into sulfates. Mechanistic studies using 7-hydroxylflavone showed that its glucuronide was mainly (90%) effluxed by BCRP with a small (10%) but significant contribution from MRP2. Maximal velocity of glucuronide production MDCK-MRP2/UGT1A1 cells showed a fairly good correlation (R2 >0.8) with those derived using UGT1A1 microsomes, but other kinetic parameters (e.g., Km) did not correlate. Conclusion Overexpression of a second efficient efflux transporter did not significantly change the fact that BCRP is the dominant transporter for flavonoid glucuronide nor did it diminish the influence of the efflux transporter as the “gate keeper” of glucuronidation process. PMID:26833852

Flavonol glycosides of sea buckthorn (Hippophaë rhamnoides ssp. sinensis) and lingonberry (Vaccinium vitis-idaea) are bioavailable in humans and monoglucuronidated for excretion.

PubMed

Lehtonen, Henna-Maria; Lehtinen, Outi; Suomela, Jukka-Pekka; Viitanen, Matti; Kallio, Heikki

2010-01-13

Glucuronidation and excretion of sea buckthorn and lingonberry flavonols were investigated in a postprandial trial by analyzing the intact forms of flavonol glycosides as well as glucuronides in plasma, urine, and feces. Four study subjects consumed sea buckthorn (study day 1) and lingonberry (study day 2) breakfasts, and blood, urine, and fecal samples were collected for 8, 24, and 48 h, respectively. Both glycosides and glucuronides of the flavonol quercetin as well as kaempferol glucuronides were detected in urine and plasma samples after the consumption of lingonberries; 14% of flavonols in urine were glycosides, and 86% were glucuronidated forms (wt %). After the consumption of sea buckthorn, 5% of flavonols excreted in urine were detected intact, and 95% as the glucuronides (wt %). Solely glucuronides of flavonols isorhamnetin and quercetin were found in plasma after the consumption of sea buckthorn berries. Only glycosides were detected in the feces after each berry trial. Flavonol glycosides and glucuronides remained in blood and urine quite long, and the peak concentrations appeared slightly later than previously described. The berries seemed to serve as a good flavonol supply, providing steady flavonol input for the body for a relatively long time.

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions.

PubMed

Perreault, Martin; Wunsch, Ewa; Białek, Andrzej; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Poirier, Guy G; Milkiewicz, Piotr; Barbier, Olivier

2018-01-01

Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower K M values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool.

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions

PubMed Central

Perreault, Martin; Białek, Andrzej; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Poirier, Guy G.

2018-01-01

Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower KM values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool. PMID:29850459

Antioxidant activity of phenolic acids and their metabolites: synthesis and antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic acid.

PubMed

Piazzon, A; Vrhovsek, U; Masuero, D; Mattivi, F; Mandoj, F; Nardini, M

2012-12-19

The main metabolites of caffeic and ferulic acids (ferulic acid-4'-O-sulfate, caffeic acid-4'-O-sulfate, and caffeic acid-3'-O-sulfate), the most representative phenolic acids in fruits and vegetables, and the acyl glucuronide of ferulic acid were synthesized, purified, and tested for their antioxidant activity in comparison with those of their parent compounds and other related phenolics. Both the ferric reducing antioxidant power (FRAP) assay and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging method were used. Ferulic acid-4'-O-sulfate and ferulic acid-4'-O-glucuronide exhibited very low antioxidant activity, while the monosulfate derivatives of caffeic acid were 4-fold less efficient as the antioxidant than caffeic acid. The acyl glucuronide of ferulic acid showed strong antioxidant action. The antioxidant activity of caffeic acid-3'-O-glucuronide and caffeic acid-4'-O-glucuronide was also studied. Our results demonstrate that some of the products of phenolic acid metabolism still retain strong antioxidant properties. Moreover, we first demonstrate the ex vivo synthesis of the acyl glucuronide of ferulic acid by mouse liver microsomes, in addition to the phenyl glucuronide.

Studies on the flavonoid substrates of human UDP-glucuronosyl transferase (UGT) 2B7.

PubMed

Xie, Shenggu; You, Linya; Zeng, Su

2007-08-01

Flavonoids are found in fruits, vegetables, nuts, seeds, herbs, spices, stems and flowers, as well as in tea and red wine. They are prominent components of citrus fruits and other food sources, are consumed regularly with the human diet, and have been shown to have many biological functions, including antioxidant and chelating properties. This study suggests features of the flavonoid structure necessary for it to act as a substrate of human UGT2B7. Generally speaking, flavonol has higher glucuronidation activity than flavones and isoflavones. Differences in C3' position have an important effect on UGT2B7 glucuronidation activity, and the various substituents have different influences on glucuronidation activity. For flavonol, the bulky group at C4' can enhance glucuronidation activity. Increasing the number of hydroxyl groups of flavonoids will increase their glucuronidation activity towards UGT2B7, while conjugation of glycon will weaken the activity, and hydroxyl position can also have an important role in activity. The high glucuronidation efficiency observed with many flavonoids suggests that the contribution of UGT2B7 to the metabolism of flavonoids may be significant. The results suggest that we should not only pay attention to glucuronidation activity, but should also attach importance to the regioselectivity of glucuronidation.

[Enantioselectivity in the excretion of glucuronides of carprofen in man, dogs and horses].

PubMed

Delatour, P; Garnier, F; Maire, R

1996-10-01

After administration of the racemic drug, the stereoselective quantification of the enantiomers of free and conjugated carprofen was performed in human plasma and in plasma, urine and bile of dogs and horses. In humans, the plasma profile of free carprofen and its glucuronides is not stereoselective and the glucuronides excreted in urine are close to a racemate. In dogs and horses on the contrary, the R(-) enantiomer of the free drug is predominant in plasma, while urine and/or bile concentrations of the glucuronides are high in comparison to plasma with a strong selectivity for the S(+) enantiomer. Because glucuronidation of carprofen, as a carboxylic compound, is known to be the major metabolic pathway in most species, interspecies discrepancies in the stereoselective disposition of carprofen seem to be mainly related to the stereoselectivity in the excretion of the glucuronides. Finally, the high plasma concentrations of carprofen glucuronides in human in comparison to other animal species suggest that the former could be specifically subjected to immunological side effects in the time course of treatments by this type of compounds.

Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

PubMed Central

Li, Jia; He, Chunyong; Fang, Lianxiang; Yang, Li; Wang, Zhengtao

2016-01-01

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4. PMID:27005621

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans

PubMed Central

Fujiwara, Ryoichi; Yoda, Emiko; Tukey, Robert H.

2018-01-01

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans. PMID:29079228

In Vitro Stability of Free and Glucuronidated Cannabinoids in Blood and Plasma Following Controlled Smoked Cannabis

PubMed Central

Karschner, Erin L.; Desrosiers, Nathalie A.; Gorelick, David A.; Huestis, Marilyn A.

2013-01-01

BACKGROUND Blood and plasma cannabinoid stability is important for test interpretation and is best studied in authentic rather than fortified samples. METHODS Low and high blood and plasma pools were created for each of 10 participants after they smoked a cannabis cigarette. The stabilities of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide, and THCCOOH-glucuronide were determined after 1 week at room temperature; 1, 2, 4, 12, and 26 (±2) weeks at 4 °C; and 1, 2, 4, 12, 26 (±2), and 52 (±4) weeks at −20 °C. Stability was assessed by Friedman test. RESULTS Numbers of THC-glucuronide and CBD-positive blood samples were insufficient to assess stability. In blood, 11-OH-THC and CBN were stable for 1 week at room temperature, whereas THC and THCCOOH-glucuronide decreased and THCCOOH increased. In blood, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, and CBN were stable for 12, 4, 4, 12, and 26 weeks, respectively, at 4 °C and 12, 12, 26, 26, and 52 weeks at −20 °C. In plasma, THC-glucuronide, THC, CBN, and CBD were stable for 1 week at room temperature, whereas THCCOOH-glucuronide and 11-OH-THC decreased and THCCOOH increased. In plasma, THC-glucuronide, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, CBN, and CBD were stable for 26, 26, 2, 2, 26, 12, and 26 weeks, respectively, at 4 °C and 52, 52, 26, 26, 52, 52, and 52 weeks, respectively, at −20 °C. CONCLUSIONS Blood and plasma samples should be stored at −20 °C for no more than 3 and 6 months, respectively, to assure accurate cannabinoid quantitative results. PMID:23519966

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin.

PubMed

Sadeque, Abu J M; Usmani, Khawja A; Palamar, Safet; Cerny, Matthew A; Chen, Weichao G

2012-04-01

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.

Direct radioimmunoassay of urinary estrogen and pregnanediol glucuronides during the menstrual cycle

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

Stanczyk, F.Z.; Miyakawa, I.; Goebelsmann, U.

Assays measuring immunoreactive estrone glucuronide (E/sub 1/G), estradiol-3-glucuronide (E/sub 2/-3G), estradiol-17..beta..-glucuronide (E/sub 2/-17G), estriol-3-glucuronide (E/sub 3/-3G), estriol-16..cap alpha..-glucuronide (E/sub 3/-16G), and pregnanediol-3..cap alpha..-glucuronide (Pd-3G) directly in diluted urine were developed and validated. These estrogen and pregnanediol glucuronide fractions were measured in aliquots of 24-hour and overnight samples of urine collected daily from seven women for one menstrual cycle. Urinary hormone excretion was correlated with daily serum estradiol (E/sub 2/), progesterone (P), and lutenizing hormonee (LH) levels. A sharp midcycle LH peak preceded by a preovulatory rise in serum E/sub 2/ and followed by luteal phase serum P levels were notedmore » in each of the seven apparently ovulatory cycles. Twenty-four-hour and overnight urinary excretion patterns of estrogen glucuronides were similar to those of serum E/sub 2/. Of the five estrogen glucuronide fractions tested, excretion of E/sub 2/-17G exhibited the earliest and steepest ascending slope of the preovulatory estrogen surge and correlated best with serum E/sub 2/ levels. Urinary excretion of E/sub 1/-G, E/sub 2/-3G, and E/sub 3/-16G also showed an early and steep preovulatory rise and preceded that of E/sub 3/-3G, whereas urinary excretion of E/sub 3/-3G exhibited the poorest correlation with serum E/sub 2/ concentrations. The urinary excretion of Pd-3G rose parallel to serum P levels and was markedly elevated 2 to 3 days after the midcycle LH peak in both 24-hour and overnight collections of urine. These results indicate that among the urinary estrogen conjugate fractions tested, E/sub 2/-17G is the one that most suitably predicts ovulation.« less

Metabolism of isotretinoin. Biliary excretion of isotretinoin glucuronide in the rat.

PubMed

Meloche, S; Besner, J G

1986-01-01

The biliary metabolites of isotretinoin were examined after iv administration of 4-20-mg/kg doses to vitamin A-normal bile duct-cannulated rats. Analysis of bile by reverse phase high performance liquid chromatography showed that injection of isotretinoin is followed by a rapid excretion of metabolites in bile. Isotretinoin glucuronide was identified as the major metabolite in bile. A specific high performance liquid chromatography method based on the assay of generated isotretinoin in beta-glucuronidase-treated bile was developed for the determination of isotretinoin glucuronide in bile samples. The excretion rate of isotretinoin glucuronide increased rapidly to reach a maximum 55 min after dosing and then declined exponentially. After 330 min of collection, biliary excretion of isotretinoin glucuronide was almost complete, and the metabolite accounted for 34.8-37.9% of the dose. These results indicate that conjugation with glucuronic acid represents a major pathway for the metabolism of pharmacological doses of isotretinoin. The maximum excretion rate of isotretinoin glucuronide in bile increased in a linear manner with the dose of isotretinoin, and no delay was observed after the larger doses. These data suggest that glucuronidation and biliary excretion are not saturated at high pharmacological doses of isotretinoin.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

PubMed

Smith, Dennis A; Hammond, Timothy; Baillie, Thomas A

2018-06-01

While simple O - (ether-linked) and N -glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Synthesis, physicochemical properties, and biological activity of bile acids 3-glucuronides: Novel insights into bile acid signalling and detoxification.

PubMed

Mostarda, Serena; Passeri, Daniela; Carotti, Andrea; Cerra, Bruno; Colliva, Carolina; Benicchi, Tiziana; Macchiarulo, Antonio; Pellicciari, Roberto; Gioiello, Antimo

2018-01-20

Glucuronidation is considered an important detoxification pathway of bile acids especially in cholestatic conditions. Glucuronides are less toxic than the parent free forms and are more easily excreted in urine. However, the pathophysiological significance of bile acid glucuronidation is still controversial and debated among the scientific community. Progress in this field has been strongly limited by the lack of appropriate methods for the preparation of pure glucuronides in the amount needed for biological and pharmacological studies. In this work, we have developed a new synthesis of bile acid C3-glucuronides enabling the convenient preparation of gram-scale quantities. The synthesized compounds have been characterized in terms of physicochemical properties and abilities to modulate key nuclear receptors including the farnesoid X receptor (FXR). In particular, we found that C3-glucuronides of chenodeoxycholic acid and lithocholic acid, respectively the most abundant and potentially cytotoxic species formed in patients affected by cholestasis, behave as FXR agonists and positively regulate the gene expression of transporter proteins, the function of which is critical in human conditions related to imbalances of bile acid homeostasis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

In vitro Inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea, and Curcuma xanthorrhiza Extracts and Constituents on Human Liver Glucuronidation Activity.

PubMed

Husni, Zulhilmi; Ismail, Sabariah; Zulkiffli, Mohd Halimhilmi; Afandi, Atiqah; Haron, Munirah

2017-07-01

Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza are commonly consumed as herbal medicines. However their effects on human liver glucuronidation activity are not yet evaluated. In this study, we evaluate the inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza extracts and their constituents on human liver glucuronidation activity. Herbal extracts (aqueous, methanolic and ethanolic extracts) and their constituents were incubated with human liver microsomes with the addition of UDPGA to initiate the reaction. Working concentrations of herbal extracts and their constituents ranged from 10 μg/mL to 1000 μg/mL and 10 μM to 300 μM respectively. IC50 was determined by monitoring the decrement of glucuronidation activity with the increment of herbal extracts or phytochemical constituent's concentrations. All herbal extracts inhibited human liver glucuronidation activity in range of 34.69 μg/mL to 398.10 μg/mL whereas for the constituents, only xanthorrhizol and curcumin (constituents of Curcuma xanthorrhiza ) inhibited human liver glucuronidation activity with IC50 of 538.50 and 32.26 μM respectively. In the present study, we have proved the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to interfere with in vitro glucuronidation process in human liver microsomes. This study documented the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to inhibit human liver glucuronidation activity which may affect the metabolism of therapeutic drugs or hazardous toxicants that follow the same glucuronidation pathway. Abbreviations used: UGT: Uridine 5'-diphospho-glucuronosyltransferase; 4-MU: 4-methylumbelliferone; IC50: Half Maximal Inhibitory Concentration; Km: Michaelis constant; Vmax: Maximum velocity.

In vitro Inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea, and Curcuma xanthorrhiza Extracts and Constituents on Human Liver Glucuronidation Activity

PubMed Central

Husni, Zulhilmi; Ismail, Sabariah; Zulkiffli, Mohd Halimhilmi; Afandi, Atiqah; Haron, Munirah

2017-01-01

Background: Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza are commonly consumed as herbal medicines. However their effects on human liver glucuronidation activity are not yet evaluated. Objective: In this study, we evaluate the inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza extracts and their constituents on human liver glucuronidation activity. Materials and Methods: Herbal extracts (aqueous, methanolic and ethanolic extracts) and their constituents were incubated with human liver microsomes with the addition of UDPGA to initiate the reaction. Working concentrations of herbal extracts and their constituents ranged from 10 μg/mL to 1000 μg/mL and 10 μM to 300 μM respectively. IC50 was determined by monitoring the decrement of glucuronidation activity with the increment of herbal extracts or phytochemical constituent's concentrations. Results: All herbal extracts inhibited human liver glucuronidation activity in range of 34.69 μg/mL to 398.10 μg/mL whereas for the constituents, only xanthorrhizol and curcumin (constituents of Curcuma xanthorrhiza) inhibited human liver glucuronidation activity with IC50 of 538.50 and 32.26 μM respectively. Conclusion: In the present study, we have proved the capabilities of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza to interfere with in vitro glucuronidation process in human liver microsomes. SUMMARY This study documented the capabilities of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza to inhibit human liver glucuronidation activity which may affect the metabolism of therapeutic drugs or hazardous toxicants that follow the same glucuronidation pathway. Abbreviations used: UGT: Uridine 5’-diphospho-glucuronosyltransferase; 4-MU: 4-methylumbelliferone; IC50: Half Maximal Inhibitory Concentration; Km: Michaelis constant; Vmax: Maximum velocity. PMID:28808386

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.

Role of UDP-Glucuronosyltransferase (UGT) 2B2 in Metabolism of Triiodothyronine: Effect of Microsomal Enzyme Inducers in Sprague Dawley and UGT2B2-Deficient Fischer 344 Rats

PubMed Central

Richardson, Terrilyn A.; Klaassen, Curtis D.

2010-01-01

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) can impact thyroid hormone homeostasis in rodents. Increased glucuronidation can result in reduction of serum thyroid hormone and a concomitant increase in thyroid-stimulating hormone (TSH). UGT2B2 is thought to glucuronidate triiodothyronine (T3). The purposes of this study were to determine the role of UGT2B2 in T3 glucuronidation and whether increased T3 glucuronidation mediates the increased TSH observed after MEI treatment. Sprague Dawley (SD) and UGT2B2-deficient Fischer 344 (F344) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum thyroxine (T4), T3, and TSH concentrations, hepatic androsterone/T4/T3 glucuronidation, and thyroid follicular cell proliferation were determined. In both SD and F344 rats, MEI treatments decreased serum T4, whereas serum T3 was maintained (except with PCB treatment). Hepatic T4 glucuronidation increased significantly after MEI in both rat strains. Compared with the other MEI, only PCN treatment significantly increased T3 glucuronidation (281 and 497%) in both SD and UGT2B2-deficient F344 rats, respectively, and increased both serum TSH and thyroid follicular cell proliferation. These data demonstrate an association among increases in T3 glucuronidation, TSH, and follicular cell proliferation after PCN treatment, suggesting that T3 is glucuronidated by other PCN-inducible UGTs in addition to UGT2B2. These data also suggest that PCN (rather than 3-MC or PCB) promotes thyroid tumors through excessive TSH stimulation of the thyroid gland. PMID:20421340

5α-Androst-16-en-3α-ol β-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat.

PubMed

Starkenmann, Christian; Mayenzet, Fabienne; Brauchli, Robert; Troccaz, Myriam

2013-12-01

5α-Androst-16-en-3α-ol (α-androstenol) is an important contributor to human axilla sweat odor. It is assumed that α-andostenol is excreted from the apocrine glands via a H2 O-soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H2 O-soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α-androstenol, β-androstenol sulfates, 5α-androsta-5,16-dien-3β-ol (β-androstadienol) sulfate, α-androstenol β-glucuronide, α-androstenol α-glucuronide, β-androstadienol β-glucuronide, and α-androstenol β-glucuronide furanose. The occurrence of α-androstenol β-glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α-androstenol was observed after incubation of the sterile human sweat or α-androstenol β-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β-glucuronidase activities. We demonstrated that if α- and β-androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H2 O-soluble precursor of α-androstenol in apocrine secretion should be a β-glucuronide. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

PubMed

Jančová, Petra; Siller, Michal; Anzenbacherová, Eva; Křen, Vladimír; Anzenbacher, Pavel; Simánek, Vilím

2011-09-01

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-β-D-glucuronides, B-7-O-β-D-glucuronides, A-20-O-β-D-glucuronides and B-20-O-β-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-β-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.

PubMed

Gufford, Brandon T; Graf, Tyler N; Paguigan, Noemi D; Oberlies, Nicholas H; Paine, Mary F

2015-11-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-D-glucuronide (15.7 mg), silybin A-5-O-β-D-glucuronide (1.6 mg), and silybin A-4´´-O-β-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides

PubMed Central

Gufford, Brandon T.; Graf, Tyler N.; Paguigan, Noemi D.; Oberlies, Nicholas H.

2015-01-01

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-d-glucuronide (15.7 mg), silybin A-5-O-β-d-glucuronide (1.6 mg), and silybin A-4´´-O-β-d-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action. PMID:26316643

Identification of hydroxyropivacaine glucuronide in equine urine by ESI+/MS/MS.

PubMed Central

Harkins, J D; Karpiesiuk, W; Tobin, T; Dirikolu, L; Lehner, A F

2000-01-01

Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following beta-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI+/MS/MS, the presence of a [M+H]+ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide +1. The abundance of this ion diminished after glucuronidase treatment with concomitant appearance of a m/z 291 peak, which is consistent with its hydrolysis to hydroxyropivacaine. In further work, the m/z 467 material was fragmented in the MS/MS system, yielding fragments interpretable as hydroxyropivacaine glucuronide. These data are consistent with the presence of a hydroxyropivacaine glucuronide in equine urine and constitute the first direct demonstration of a specific glucuronide metabolite in equine urine. PMID:10935884

Comparison of the effects of curcumin and curcumin glucuronide in human hepatocellular carcinoma HepG2 cells.

PubMed

Shoji, Motomu; Nakagawa, Kiyotaka; Watanabe, Akio; Tsuduki, Tsuyoshi; Yamada, Teiko; Kuwahara, Shigefumi; Kimura, Fumiko; Miyazawa, Teruo

2014-05-15

Curcumin is a yellow pigment found in turmeric (Curcuma Longa L.), and is reported, in recent studies, to have several pharmacological effects, including anti-oxidant, anti-inflammatory, anti-tumour and lipid-lowering properties. However, as most curcumin is conjugated when absorbed through the intestine, free curcumin is present at extremely low levels inside the body. Therefore, curcumin metabolites have been presumed to be responsible for the curcumin bioactivity. In this study, we first confirmed that curcumin glucuronide is the major metabolite of curcumin found in the plasma after oral administration of curcumin in rats. Next, we synthesised curcumin glucuronide and compared the effects of curcumin and curcumin glucuronide on gene expression in a human hepatoma cell line (HepG2). We found that the effects of curcumin glucuronide are weaker than those of curcumin and that this difference is related to relative absorption rates of curcumin and curcumin glucuronide into HepG2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Antimicrobial and demelanizing activity of Ganoderma lucidum extract, p-hydroxybenzoic and cinnamic acids and their synthetic acetylated glucuronide methyl esters.

PubMed

Heleno, Sandrina A; Ferreira, Isabel C F R; Esteves, Ana P; Ćirić, Ana; Glamočlija, Jasmina; Martins, Anabela; Soković, Marina; Queiroz, Maria João R P

2013-08-01

Mushroom extracts or isolated compounds may be useful in the search of new potent antimicrobial agents. Herein, it is described the synthesis of protected (acetylated) glucuronide derivatives of p-hydroxybenzoic and cinnamic acids, two compounds identified in the medicinal mushroom Ganoderma lucidum. Their antimicrobial and demelanizing activities were evaluated and compared to the parent acids and G. lucidum extract. p-Hydroxybenzoic and cinnamic acids, as also their protected glucuronide derivatives revealed high antimicrobial (antibacterial and antifungal) activity, even better than the one showed by commercial standards. Despite the variation in the order of parent acids and the protected glucuronide derivatives, their antimicrobial activity was always higher than the one revealed by the extract. Nevertheless, the extract was the only one with demelanizing activity against Aspergillus niger. The acetylated glucuronide derivatives could be deprotected to obtain glucuronide metabolites, which circulate in the human organism as products of the metabolism of the parent compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regiospecificity of Human UDP-glucuronosyltransferase Isoforms in Chalcone and Flavanone Glucuronidation Determined by Metal Complexation and Tandem Mass Spectrometry

PubMed Central

Niemeyer, Emily D.; Brodbelt, Jennifer S.

2013-01-01

The glucuronidation of a series of chalcones (2'-hydroxychalcone, 2',4'-dihydroxychalcone, 3,2'-dihydroxychalcone, 4,2'-dihydroxychalcone, and cardamonin) and their corresponding cyclized flavanones (7-hydroxyflavanone, 3'-hydroxyflavanone, 4'-hydroxyflavanone, and alpinetin) by nine human UDP-glucuronosyltransferase (UGT) 1A enzymes was evaluated. A post-column metal complexation LC-MS/MS strategy was used successfully to produce characteristic mass spectrometric product ions that were utilized in combination with elution order trends to identify chalcone and flavanone monoglucuronides unambiguously, thus allowing determination of the regioselectivities of the UGT1A isoforms. The presence of hydroxy groups on the A or B-ring had a significant effect on the glucuronide product yield and the site where glucuronidation occurred. For example, for reaction with UGT1A9, formation of the 2'-O-glucuronide was increased for dihydroxychalcones with A-ring hydroxy substituents. In contrast, although UGT1A8 reacted with 3,2'-dihydroxychalcone and 4,2'-dihydroxychalcone to yield 2'-O-glucuronide products, the presence of a B-ring hydroxy group at the 4' position on cardamonin and 2',4'-dihydroxychalcone quenched the reaction at the OH-2' position. Moreover, the A-ring OH-4 group promoted glucuronidation at the 2' position for the reaction of 4,2'-dihydroxychalcone with UGT1A1 and 1A3. For UGT1A7, hydroxy group substituents on the chalcone A-ring also promoted cyclization and formation of the corresponding flavanone glucuronide. PMID:23713759

Regiospecificity of human UDP-glucuronosyltransferase isoforms in chalcone and flavanone glucuronidation determined by metal complexation and tandem mass spectrometry.

PubMed

Niemeyer, Emily D; Brodbelt, Jennifer S

2013-06-28

The glucuronidation of a series of chalcones (2'-hydroxychalcone, 2',4'-dihydroxychalcone, 3,2'-dihydroxychalcone, 4,2'-dihydroxychalcone, and cardamonin) and their corresponding cyclized flavanones (7-hydroxyflavanone, 3'-hydroxyflavanone, 4'-hydroxyflavanone, and alpinetin) by eight human UDP-glucuronosyltransferase (UGT) 1A enzymes was evaluated. A postcolumn metal complexation LC-MS/MS strategy was used successfully to produce characteristic mass spectrometric product ions that were utilized in combination with elution order trends to identify chalcone and flavanone monoglucuronides unambiguously, thus allowing determination of the regioselectivities of the UGT1A isoforms. The presence of hydroxy groups on the A- or B-ring had a significant effect on the glucuronide product yield and the site where glucuronidation occurred. For example, for reaction with UGT1A9, formation of the 2'-O-glucuronide was increased for dihydroxychalcones with A-ring hydroxy substituents. In contrast, although UGT1A8 reacted with 3,2'-dihydroxychalcone and 4,2'-dihydroxychalcone to yield 2'-O-glucuronide products, the presence of a B-ring hydroxy group at the 4' position on cardamonin and 2',4'-dihydroxychalcone quenched the reaction at the OH-2' position. Moreover, the A-ring OH-4 group promoted glucuronidation at the 2' position for the reaction of 4,2'-dihydroxychalcone with UGT1A1 and 1A3. For UGT1A7, hydroxy group substituents on the chalcone A-ring also promoted cyclization and formation of the corresponding flavanone glucuronide.

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans.

PubMed

Fujiwara, Ryoichi; Yoda, Emiko; Tukey, Robert H

2018-02-01

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Biochemistry of metalocenes. The organ distribution of hydroxyacetyl (/sup 103/Ru)ruthenocene and its glucuronide in mice

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

Taylor, A.J.; Macha, J.; Wenzel, M.

1980-01-01

Hydroxyacetyl(/sup 103/Ru)ruthenocene and its o-glucuronide were prepared in vitro by incubation of acetyl(/sup 103/Ru)ruthenocene with rat-liver homogenate, NADPH, and UDP-glucuronate. The factors affecting hydroxylation and glucuronidation in vitro were optimized for acetylruthenocene. Hydroxyacetyl(/sup 103/Ru)ruthenocene glucuronide showed no affinity for the adrenal glands, but after iv administration of hydroxyacetyl(/sup 103/Ru)ruthenocene there was a distinct accumulation of Ru-103 in adrenals, similar to that found after administration of acetyl(/sup 103/Ru)ruthenocene.

Species-Associated Differences in the Inhibition of Propofol Glucuronidation by Magnolol

PubMed Central

Yang, Lu; Zhu, Liangliang; Ge, Guangbo; Xiao, Ling; Wu, Yan; Liang, Sicheng; Cao, Yunfeng; Yang, Ling; Wang, Dong

2014-01-01

Magnolol, a major active constituent in herbal medicine, potently inhibits propofol glucuronidation in human liver microsomes, with inhibition constants in the nanomolar range. This study was conducted to investigate magnolol-induced inhibition of propofol glucuronidation in liver microsomes from Swiss–Hauschka mice, Sprague–Dawley rats, Chinese Bama pigs, and cynomolgus macaques. Results indicated that magnolol (10 μM) inhibited propofol glucuronidation in liver microsomes from Bama pigs and cynomolgus macaques but not in those from mice or rats. Data from liver microsomes from Bama pigs indicated a competitive inhibition mechanism, with a Ki of 1.7 μM. In contrast to that of pig liver microsomes, the inhibition of microsomes from cynomolgus macaques followed a noncompetitive mechanism, with a Ki of 3.4 μM. In summary, this study indicates that magnolol-induced inhibition of propofol glucuronidation varies substantially among species, and the Ki values determined by using liver microsomes from various experimental animal species far exceed that for human liver microsomes. The inhibition of propofol glucuronidation by magnolol in liver microsomes from all animal species tested was significantly lower than the inhibition previously demonstrated in human liver microsomes. Hepatic microsomes from Swiss–Hauschka mice, Sprague–Dawley rats, Chinese Bama pigs, and cynomolgus macaques are not effective models of the inhibition of glucuronidation induced by magnolol in humans. PMID:25199099

Species-associated differences in the inhibition of propofol glucuronidation by magnolol.

PubMed

Yang, Lu; Zhu, Liangliang; Ge, Guangbo; Xiao, Ling; Wu, Yan; Liang, Sicheng; Cao, Yunfeng; Yang, Ling; Wang, Dong

2014-07-01

Magnolol, a major active constituent in herbal medicine, potently inhibits propofol glucuronidation in human liver microsomes, with inhibition constants in the nanomolar range. This study was conducted to investigate magnolol-induced inhibition of propofol glucuronidation in liver microsomes from Swiss-Hauschka mice, Sprague-Dawley rats, Chinese Bama pigs, and cynomolgus macaques. Results indicated that magnolol (10 μM) inhibited propofol glucuronidation in liver microsomes from Bama pigs and cynomolgus macaques but not in those from mice or rats. Data from liver microsomes from Bama pigs indicated a competitive inhibition mechanism, with a Ki of 1.7 μM. In contrast to that of pig liver microsomes, the inhibition of microsomes from cynomolgus macaques followed a noncompetitive mechanism, with a Ki of 3.4 μM. In summary, this study indicates that magnolol-induced inhibition of propofol glucuronidation varies substantially among species, and the Ki values determined by using liver microsomes from various experimental animal species far exceed that for human liver microsomes. The inhibition of propofol glucuronidation by magnolol in liver microsomes from all animal species tested was significantly lower than the inhibition previously demonstrated in human liver microsomes. Hepatic microsomes from Swiss-Hauschka mice, Sprague-Dawley rats, Chinese Bama pigs, and cynomolgus macaques are not effective models of the inhibition of glucuronidation induced by magnolol in humans.

Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes

PubMed Central

Redmon, Joanna M.; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H.

2016-01-01

Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.Significant concentrations of conjugated (but not unconjugated) genistein, daidzein, and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine.β-glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats.Glucuronidation rates of genistein, daidzein, and equol by cat livers were consistently ranked within the lowest three out of 13 species’ livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species.Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species. PMID:26366946

Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes.

PubMed

Redmon, Joanna M; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H

2016-01-01

1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species' differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species. 2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine. 3. β-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats. 4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species' livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species. 5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species.

Perinatal development of conjugative enzyme systems.

PubMed Central

Lucier, G W

1976-01-01

The problems and priorities involved in studying the role of conjugagive enzymes in developmental pharmacology are discussed and evaluated. The relative rates of UDP glucuronyltransferase and beta-glucuronidase were studied during perinatal development in hepatic and extrahepatic tissues to determine the net balance of glucuronidation or deglucuronidation at different developmental stages. In general, deglucuronidation predominated over glucuronidation in fetal tissues whereas the converse was evident in adults. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an extremely toxic contaminant of some organochlorine compounds, was shown to be a potent inducer of some hepatic and extrahepatic drug-metabolizing enzymes. TCDD, administered during gestation, induced the postnatal activities of p-nitrophenol glucuronyltransferase and benzpyrene hydroxylase in rats. Foster mother experiments revealed that the postnatal induction was caused primarily by newborn exposure to TCDD in the mother's milk. Tissue distribution experiments with TCDD-14C confirmed these findings. Although TCDD induced non-steroid glucuronidation, no significant effects were evident on the postnatal development of steroid glucuronidation. The synthetic estrogen diethylstilbestrol (DES) is metabolized primarily by glucuronidation. The postnatal development of DES glucuronidation, like the steroid pathway, was not affected by gestational TCDD treatment. The fetal distribution of DES and DES-glucuronide, at different stages of development, correlated well with the perinatal development of steroid glucuronyltransferase activity. PMID:829487

Glucuronidation of Drugs and Drug-Induced Toxicity in Humanized UDP-Glucuronosyltransferase 1 Mice

PubMed Central

Kutsuno, Yuki; Itoh, Tomoo; Tukey, Robert H.

2014-01-01

UDP-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various drugs. Although experimental rodents are used in preclinical studies to predict glucuronidation and toxicity of drugs in humans, species differences in glucuronidation and drug-induced toxicity have been reported. Humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) were recently developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of humans and regular mice. The kinetics of etodolac, diclofenac, and ibuprofen acyl-glucuronidation in hUGT1 mice were almost comparable to those in humans, rather than in mice. We further investigated the hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. Because ALT levels were increased at 6 hours after dosing in hUGT1 mice and at 24 hours after dosing in regular mice, the onset pattern of ibuprofen-induced liver toxicity in hUGT1 mice was different from that in regular mice. These data suggest that hUGT1 mice can be valuable tools for understanding glucuronidations of drugs and drug-induced toxicity in humans. PMID:24764149

Disposition of Naringenin via Glucuronidation Pathway Is Affected by Compensating Efflux Transporters of Hydrophilic Glucuronides

PubMed Central

Xu, Haiyan; Kulkarni, Kaustubh H.; Singh, Rashim; Yang, Zhen; Wang, Stephen W.J.; Tam, Vincent H.; Hu, Ming

2010-01-01

The purposes of this study were to investigate how efflux transporters and UDP-glucuronosyltransferases (UGT) affect the disposition of naringenin. A rat intestinal perfusion model with bile duct cannulation was used along with rat intestinal and liver microsomes. In the intestinal perfusion model, both absorption and subsequent excretion of naringenin metabolites were rapid and site-dependent (p < 0.05). Naringenin was absorbed the most in colon and its glucuronides were excreted the most in duodenum. In metabolism studies, the intrinsic clearance value of naringenin glucuronidation was the highest in jejunum microsomes, followed by liver, ileal and colonic microsomes. The rapid metabolism in microsomes did not always translate into more efficient excretion in the rat perfusion model, however, because of presence of rate-limiting efflux transporters. When used separately, MK-571 (an inhibitor of multidrug resistance-related protein 2 or Mrp2) or dipyridamole (an inhibitor of breast cancer resistance protein or Bcrp1) did not affect excretion of naringenin glucuronides, but when used together, they significantly (p < 0.05) decreased intestinal and biliary excretion of naringenin glucuronides. In conclusion, efflux transporters Mrp2 and Bcrp1 are shown to compensate for each other and enable the intestinal excretion of flavonoid (i.e., naringenin) glucuronides. PMID:19736994

Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems☆

PubMed Central

Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

2014-01-01

Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR− rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. PMID:24406246

Analysis of intact glucuronides and sulfates of serotonin, dopamine, and their phase I metabolites in rat brain microdialysates by liquid chromatography-tandem mass spectrometry.

PubMed

Uutela, Päivi; Reinilä, Ruut; Harju, Kirsi; Piepponen, Petteri; Ketola, Raimo A; Kostiainen, Risto

2009-10-15

A method for the analysis of intact glucuronides and sulfates of common neurotransmitters serotonin (5-HT) and dopamine (DA) as well as of 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat brain microdialysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Enzyme-assisted synthesis using rat liver microsomes as a biocatalyst was employed for the production of 5-HT-, 5-HIAA-, DOPAC-, and HVA-glucuronides for reference compounds. The sulfate conjugates were synthesized either chemically or enzymatically using a rat liver S9 fraction. The LC-MS/MS method was validated by determining the limits of detection and quantitation, linearity, and repeatability for the quantitative analysis of 5-HT and DA and their glucuronides, as well as of 5-HIAA, DOPAC, and HVA and their sulfate-conjugates. In this study, 5-HT-glucuronide was for the first time detected in rat brain. The concentration of 5-HT-glucuronide (1.0-1.7 nM) was up to 2.5 times higher than that of free 5-HT (0.4-2.1 nM) in rat brain microdialysates, whereas the concentration of DA-glucuronide (1.0-1.4 nM) was at the same level or lower than the free DA (1.2-2.4 nM). The acidic metabolites of neurotransmitters, 5-HIAA, HVA, and DOPAC, were found in free and sulfated form, whereas their glucuronidation was not observed.

Simultaneous Quantification of Free and Glucuronidated Cannabinoids in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

Scheidweiler, Karl B.; Desrosiers, Nathalie A.; Huestis, Marilyn A.

2012-01-01

Background Cannabis is the most commonly abused drug of abuse and is commonly quantified during urine drug testing. We conducted a controlled drug administration studies investigating efficacy of urinary cannabinoid glucuronide metabolites for documenting recency of cannabis intake and for determining stability of urinary cannabinoids. Methods A liquid chromatography tandem mass spectrometry method was developed and validated quantifying Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol, cannabinol, THC-glucuronide and THCCOOH-glucuronide in 0.5 ml human urine via supported-liquid extraction. Chromatography was performed on an Ultra Biphenyl column with a gradient of 10 mmol/l ammonium acetate, pH 6.15 and 15% methanol in acetonitrile at 0. 4ml/min. Analytes were monitored by positive and negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. Results Linear ranges were 0.5–50 ng/ml for THC-glucuronide, 1–100 ng/ml for THCCOOH, 11-OH-THC and cannabidiol, 2–100 ng/ml for THC and cannabinol, and 5–500 ng/ml for THCCOOH-glucuronide (R2>0.99). Mean extraction efficiencies were 34–73% with analytical recovery (bias) 80.5–118.0% and total imprecision 3.0–10.2% coefficient of variation. Conclusion This method simultaneously quantifies urinary cannabinoids and phase II glucuronide metabolites, and enables evaluation of urinary cannabinoid glucuronides for documenting recency of cannabis intake and cannabinoid stability. The assay is applicable for routine urine cannabinoid testing. PMID:22771478

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

PubMed

Zenger, Katharina; Agnolet, Sara; Schneider, Bernd; Kraus, Birgit

2015-07-22

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

Glucuronidation of 6 alpha-hydroxy bile acids by human liver microsomes.

PubMed Central

Radomińska-Pyrek, A; Zimniak, P; Irshaid, Y M; Lester, R; Tephly, T R; St Pyrek, J

1987-01-01

The glucuronidation of 6-hydroxylated bile acids by human liver microsomes has been studied in vitro; for comparison, several major bile acids lacking a 6-hydroxyl group were also investigated. Glucuronidation rates for 6 alpha-hydroxylated bile acids were 10-20 times higher than those of substrates lacking a hydroxyl group in position 6. The highest rates measured were for hyodeoxy- and hyocholic acids, and kinetic analyses were carried out using these substrates. Rigorous product identification by high-field proton nuclear magnetic resonance and by electron impact mass spectrometry of methyl ester/peracetate derivatives revealed that 6-O-beta-D-glucuronides were the exclusive products formed in these enzymatic reactions. These results, together with literature data, indicate that 6 alpha-hydroxylation followed by 6-O-glucuronidation constitutes an alternative route of excretion of toxic hydrophobic bile acids. PMID:3110212

Detection and identification of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide in nitrite adulterated urine specimens containing morphine and its glucuronides.

PubMed

Luong, Susan; Fu, Shanlin

2014-03-01

In vitro urine adulteration is a well-documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine-3-glucuronide and morphine-6-glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography-mass spectrometry (LC-MS) when morphine and morphine-6-glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2-nitro-morphine and 2-nitro-morphine-6-glucuronide, respectively. These reaction products were also formed when an authentic morphine-positive urine specimen was fortified with nitrite. 2-Nitro-morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography-mass spectrometry (GC-MS) after forming a trimethylsilyl derivative. On the contrary, morphine-3-glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC-MS and GC-MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide as markers for the indirect monitoring of morphine and morphine-6-glucuronide in urine specimens adulterated with nitrite. Copyright © 2013 John Wiley & Sons, Ltd.

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

PubMed

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

2013-10-01

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

In Vitro Glucuronidation and Sulfation of ε-Viniferin, a Resveratrol Dimer, in Humans and Rats.

PubMed

Courtois, Arnaud; Jourdes, Michael; Dupin, Adeline; Lapèze, Caroline; Renouf, Elodie; Biais, Benoît; Teissedre, Pierre-Louis; Mérillon, Jean-Michel; Richard, Tristan; Krisa, Stéphanie

2017-05-03

ε-Viniferin is a resveratrol dimer that possesses antioxidant or anti-inflammatory activities. However little is known about the metabolism of this oligostilbene. This study was thus undertaken as a first approach to identify and characterize the metabolites of ε-viniferin and to describe the kinetic profile of their appearance in humans and rats. The glucuronides and sulfates of ε-viniferin were first obtained by chemical hemi-synthesis and were fully characterized by UPLC-MS and NMR spectroscopy. Then, ε-viniferin was incubated with human or rat S9 liver fractions that led to the formation of four glucuronoconjugates and four sulfoconjugates. In both species, ε-viniferin was subjected to an intense metabolism as 70 to 80% of the molecule was converted to glucuronides and sulfates. In humans, the hepatic clearance of ε-viniferin (V max /K m ) for glucuronidation and sulfation were 4.98 and 6.35 µL/min/mg protein, respectively, whereas, in rats, the hepatic clearance for glucuronidation was 20.08 vs. 2.59 µL/min/mg protein for sulfation. In humans, three major metabolites were observed: two glucuronides and one sulfate. By contrast, only one major glucuronide was observed in rats. This strong hepatic clearance of ε-viniferin in human and rat could explain its poor bioavailability and could help to characterize its active metabolites.

Studies on the disturbance of glucuronide formation in infectious hepatitis

PubMed Central

Vest, M. F.; Fritz, E.

1961-01-01

The ability of the liver to form glucuronides was measured in 10 patients with infectious hepatitis. One test was done at the onset and another about four weeks later after the clinical symptoms had disappeared. N-acetyl-p-aminophenol (N.A.P.A.) or acetanilide was administered in doses ranging from 10 to 20 mg. per kg. body weight, either orally or by intravenous injection. N.A.P.A. is conjugated by the liver at the hydroxyl group and excreted in the urine as sulphuric and glucuronic acid conjugates. Total conjugated p-aminophenol, free N.A.P.A., and N.A.P.A. glucuronide were estimated in the urine of our patients. In the blood the disappearance of N.A.P.A. (free form) and the formation of N.A.P.A. glucuronide were traced. During the acute phase of hepatitis the excretion of total conjugated p-aminophenol and of N.A.P.A. glucuronide in the urine is lower than after recovery from the disease. Likewise free N.A.P.A. disappears more slowly from the circulation and the peak concentration of N.A.P.A. glucuronide in the serum remains lower at the onset of hepatitis than after clinical cure. These results indicate that glucuronide formation during the acute stage of infectious hepatitis is depressed, as are other transformation mechanisms, i.e., of hippuric acid. PMID:13925655

Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems.

PubMed

Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

2014-03-01

Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR(-) rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of UDP-Glucuronosyltransferase (UGT) 1A Polymorphism (rs8330 and rs10929303) on Glucuronidation Status of Acetaminophen

PubMed Central

Tahir, Imtiaz Mahmood; Iqbal, Tahira; Saleem, Sadaf; Perveen, Sofia; Farooqi, Aboubakker

2017-01-01

Interindividual variability in polymorphic uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) ascribed to genetic diversity is associated with relative glucuronidation level among individuals. The present research was aimed to study the effect of 2 important single nucleotide polymorphisms (SNPs; rs8330 and rs10929303) of UGT1A1 gene on glucuronidation status of acetaminophen in healthy volunteers (n = 109). Among enrolled volunteers, 54.13% were male (n = 59) and 45.87% were female (n = 50). The in vivo activity of UGT1A1 was investigated by high-performance liquid chromatography-based analysis of glucuronidation status (ie, acetaminophen and acetaminophen glucuronide) in human volunteers after oral intake of a single dose (1000 mg) of acetaminophen. The TaqMan SNP genotyping assay was used for UGT1A1 genotyping. The wild-type genotype (C/C) was observed the most frequent one for both SNPs (rs8330 and rs10929303) and associated with fast glucuronidator phenotypes. The distribution of variant genotype (G/G) for SNP rs8330 was observed in 5% of male and 8% of the female population; however, for SNP rs10929303, the G/G genotype was found in 8% of both genders. A trimodal distribution (fast, intermediate, and slow) based on phenotypes was observed. Among the male participants, the glucuronidation phenotypes were observed as 7% slow, 37% intermediate, and 56% fast glucuronidators; however, these findings for the females were slightly different as 8%, 32%, and 60% respectively. The k-statistics revealed a compelling evidence for good concordance between phenotype and genotype with a k value of 1.00 for SNP rs8330 and 0.966 for SNP rs10929303 in our population. PMID:28932176

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells

PubMed Central

Spigoni, Valentina; Fantuzzi, Federica; Tassotti, Michele; Brighenti, Furio; Bonadonna, Riccardo C.; Dei Cas, Alessandra

2017-01-01

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot (Citrus bergamia), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7-O-neohesperidoside-6″-O-HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7-O-glucuronide, hesperetin-3′-O-glucuronide, naringenin-7-O-glucuronide and naringenin-4′-O-glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4′-O-glucuronide and hesperetin-7-O-glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs. PMID:29211032

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells.

PubMed

Spigoni, Valentina; Mena, Pedro; Fantuzzi, Federica; Tassotti, Michele; Brighenti, Furio; Bonadonna, Riccardo C; Del Rio, Daniele; Dei Cas, Alessandra

2017-12-06

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot ( Citrus bergamia ), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7- O -neohesperidoside-6″- O -HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7- O -glucuronide, hesperetin-3'- O -glucuronide, naringenin-7- O -glucuronide and naringenin-4'- O -glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4'- O -glucuronide and hesperetin-7- O -glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs.

Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1.

PubMed

Zhou, Jin; Tracy, Timothy S; Remmel, Rory P

2010-11-01

Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Glucuronidation of deoxynivalenol (DON) by different animal species: identification of iso-DON glucuronides and iso-deepoxy-DON glucuronides as novel DON metabolites in pigs, rats, mice, and cows.

PubMed

Schwartz-Zimmermann, Heidi E; Hametner, Christian; Nagl, Veronika; Fiby, Iris; Macheiner, Lukas; Winkler, Janine; Dänicke, Sven; Clark, Erica; Pestka, James J; Berthiller, Franz

2017-12-01

The Fusarium mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereal-based food and feed. Mammals metabolize DON by conjugation to glucuronic acid (GlcAc), the extent and regioselectivity of which is species-dependent. So far, only DON-3-glucuronide (DON-3-GlcAc) and DON-15-GlcAc have been unequivocally identified as mammalian DON glucuronides, and DON-7-GlcAc has been proposed as further DON metabolite. In the present work, qualitative HPLC-MS/MS analysis of urine samples of animals treated with DON (rats: 2 mg/kg bw, single bolus, gavage; mice: 1 mg/kg bw, single i.p. injection; pigs: 74 µg/kg bw, single bolus, gavage; cows: 5.2 mg DON/kg dry mass, oral for 13 weeks) revealed additional DON and deepoxy-DON (DOM) glucuronides. To elucidate their structures, DON and DOM were incubated with human (HLM) and rat liver microsomes (RLM). Besides the expected DON/DOM-3- and 15-GlcAc, minor amounts of four DON- and four DOM glucuronides were formed. Isolation and enzymatic hydrolysis of four of these compounds yielded iso-DON and iso-DOM, the identities of which were eventually confirmed by NMR. Incubation of iso-DON and iso-DOM with RLM and HLM yielded two main glucuronides for each parent compound, which were isolated and identified as iso-DON/DOM-3-GlcAc and iso-DON/DOM-8-GlcAc by NMR. Iso-DON-3-GlcAc, most likely misidentified as DON-7-GlcAc in the literature, proved to be a major DON metabolite in rats and a minor metabolite in pigs. In addition, iso-DON-8-GlcAc turned out to be one of the major DON metabolites in mice. DOM-3-GlcAc was the dominant DON metabolite in urine of cows and an important DON metabolite in rat urine. Iso-DOM-3-GlcAc was detected in urine of DON-treated rats and cows. Finally, DON-8,15-hemiketal-8-glucuronide, a previously described by-product of DON-3-GlcAc production by RLM, was identified in urine of DON-exposed mice and rats. The discovery of several novel DON-derived glucuronides in animal urine requires adaptation of the currently used methods for DON-biomarker analysis.

UGT1A and UGT2B genetic variation alters nicotine and nitrosamine glucuronidation in european and african american smokers.

PubMed

Wassenaar, Catherine A; Conti, David V; Das, Soma; Chen, Peixian; Cook, Edwin H; Ratain, Mark J; Benowitz, Neal L; Tyndale, Rachel F

2015-01-01

Identifying sources of variation in the nicotine and nitrosamine metabolic inactivation pathways is important to understanding the relationship between smoking and cancer risk. Numerous UGT1A and UGT2B enzymes are implicated in nicotine and nitrosamine metabolism in vitro; however, little is known about their roles in vivo. Within UGT1A1, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B17, 47 variants were genotyped, including UGT2B10*2 and UGT2B17*2. The association between variation in these UGTs and glucuronidation activity within European and African American current smokers (n = 128), quantified as urinary ratios of the glucuronide over unconjugated compound for nicotine, cotinine, trans-3'-hydroxycotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was investigated in regression models assuming a dominant effect of variant alleles. Correcting for multiple testing, three UGT2B10 variants were associated with cotinine glucuronidation, rs2331559 and rs11726322 in European Americans and rs835309 in African Americans (P ≤ 0.0002). Additional variants predominantly in UGT2B10 were nominally associated with nicotine (P = 0.008-0.04) and cotinine (P = <0.001-0.02) glucuronidation in both ethnicities in addition to UGT2B10*2 in European Americans (P = 0.01, P < 0.001). UGT2B17*2 (P = 0.03) in European Americans and UGT2B7 variants (P = 0.02-0.04) in African Americans were nominally associated with 3HC glucuronidation. UGT1A (P = 0.007-0.01), UGT2B10 (P = 0.02), and UGT2B7 (P = 0.02-0.03) variants in African Americans were nominally associated with NNAL glucuronidation. Findings from this initial in vivo study support a role for multiple UGTs in the glucuronidation of tobacco-related compounds in vivo, in particular UGT2B10 and cotinine glucuronidation. Findings also provide insight into ethnic differences in glucuronidation activity, which could be contributing to ethnic disparities in the risk for smoking-related cancers. Cancer Epidemiol Biomarkers Prev; 24(1); 94-104. ©2014 AACR. ©2014 American Association for Cancer Research.

The Inhibition of Hepatic and Renal Glucuronidation of p-Nitrophenol and 4-Methylumbelliferone by Oil Palm Empty Fruit Bunch Lignin and Its Main Oxidation Compounds

PubMed Central

Salleh, Norliyana Mohamad; Ismail, Sabariah; Ibrahim, Mohamad Nasir Mohamad

2017-01-01

Background: In order to develop oil palm empty fruit bunch (EFB) lignin as a nutraceutical and health supplement, the investigation of its potential in interacting with other drugs via inhibition of drug-metabolizing enzymes (DMEs) would ensure product safety. Objective: The study was aimed to investigate the in vitro effect of oil palm EFB lignin and its main oxidation compounds on phase II DME UDP-glucuronosyltransferases (UGTs) in rat liver and kidney microsomes. Materials and Methods: The p-nitrophenol (p-NP) and 4-methylumbelliferone (4-MU) were employed as probe substrates in glucuronidation assays. The effect of soda oil palm EFB lignin on Vmax, Km, CLint, Ki, and mode of inhibition of 4-MU glucuronidation in RLM was also determined. Results: The inhibitory potency of oil palm EFB lignin for both p-NP and 4-MU glucuronidation in rat liver microsome (RLM) and rat kidneys microsomes (RKM) was found to be in the rank order of soda > kraft > organosolv. However, the inhibitory potency of its main oxidation compounds were in the rank order of vanillin > syringaldehyde > p-hydroxybenzaldehyde. Soda oil palm EFB lignin exhibited mixed-type inhibition against 4-MU glucuronidation in RLM, showing the change in apparent Vmax and with only a minor effect on Km compared with control. Conclusions: The findings showed that effect of oil palm EFB lignin on both p-NP and 4-MU glucuronidation in RLM and RKM was enhanced by the presence of vanillin as well as flavonoids. Kinetic study showed that soda oil palm EFB lignin exhibited strong inhibition on UGT activity in RLM with mixed-type inhibition mode. SUMMARY The inhibitory potential of oil palm EFB lignin extracts for p-NP and 4-MU glucuronidation in RLM and RKM can be listed in the following rank order: soda > kraft > organosolvThe inhibitory potential of oil palm EFB lignin main oxidation compounds for p-NP and 4-MU glucuronidation in RLM and RKM can be listed in the following rank order: vanillin > syringaldehyde > p-hydroxybenzaldehydeResults suggested that the effect of oil palm EFB lignin on p-NP and 4-MU glucuronidation activity in both RLM and RKM was enhanced by the presence of vanillin as well as total flavonoid contentResults also suggested that oil palm EFB lignin may inhibit glucuronidation of substrate by UGT enzymes, especially UGT1A6, particularly in rat liver Abbreviations used: p-NP: p-Nitrophenol, 4-MU: 4-Methylumbelliferone, EFB: Empty fruit bunch, DME: Drug-metabolizing enzymes, UGT: UDPglucuronosyltransferase, Vmax: Maximal reaction velocity, Km: Michaelis-Menten constant, CLint: Intrinsic clearance, Ki: Dissociation constant of an inhibitor enzyme complex, 4-MUG: 4-Methylumbelliferone glucuronide, DMSO: Dimethyl sulfoxide, IC50: Half maximal inhibitory concentration, p-NPG: p-Nitrophenol glucuronide, RKM: Rat kidneys microsomes, RLM: Rat liver microsome, UDPGA: UDPglucuronic acid, TCA: trichloroacetic acid, MPA: mycophenolic acid PMID:28479734

The Inhibition of Hepatic and Renal Glucuronidation of p-Nitrophenol and 4-Methylumbelliferone by Oil Palm Empty Fruit Bunch Lignin and Its Main Oxidation Compounds.

PubMed

Salleh, Norliyana Mohamad; Ismail, Sabariah; Ibrahim, Mohamad Nasir Mohamad

2017-01-01

In order to develop oil palm empty fruit bunch (EFB) lignin as a nutraceutical and health supplement, the investigation of its potential in interacting with other drugs via inhibition of drug-metabolizing enzymes (DMEs) would ensure product safety. The study was aimed to investigate the in vitro effect of oil palm EFB lignin and its main oxidation compounds on phase II DME UDP-glucuronosyltransferases (UGTs) in rat liver and kidney microsomes. The p -nitrophenol ( p -NP) and 4-methylumbelliferone (4-MU) were employed as probe substrates in glucuronidation assays. The effect of soda oil palm EFB lignin on V max , K m , CL int , K i , and mode of inhibition of 4-MU glucuronidation in RLM was also determined. The inhibitory potency of oil palm EFB lignin for both p -NP and 4-MU glucuronidation in rat liver microsome (RLM) and rat kidneys microsomes (RKM) was found to be in the rank order of soda > kraft > organosolv. However, the inhibitory potency of its main oxidation compounds were in the rank order of vanillin > syringaldehyde > p -hydroxybenzaldehyde. Soda oil palm EFB lignin exhibited mixed-type inhibition against 4-MU glucuronidation in RLM, showing the change in apparent V max and with only a minor effect on K m compared with control. The findings showed that effect of oil palm EFB lignin on both p -NP and 4-MU glucuronidation in RLM and RKM was enhanced by the presence of vanillin as well as flavonoids. Kinetic study showed that soda oil palm EFB lignin exhibited strong inhibition on UGT activity in RLM with mixed-type inhibition mode. The inhibitory potential of oil palm EFB lignin extracts for p -NP and 4-MU glucuronidation in RLM and RKM can be listed in the following rank order: soda > kraft > organosolvThe inhibitory potential of oil palm EFB lignin main oxidation compounds for p -NP and 4-MU glucuronidation in RLM and RKM can be listed in the following rank order: vanillin > syringaldehyde > p-hydroxybenzaldehydeResults suggested that the effect of oil palm EFB lignin on p -NP and 4-MU glucuronidation activity in both RLM and RKM was enhanced by the presence of vanillin as well as total flavonoid contentResults also suggested that oil palm EFB lignin may inhibit glucuronidation of substrate by UGT enzymes, especially UGT1A6, particularly in rat liver Abbreviations used: p -NP: p -Nitrophenol, 4-MU: 4-Methylumbelliferone, EFB: Empty fruit bunch, DME: Drug-metabolizing enzymes, UGT: UDPglucuronosyltransferase, V max : Maximal reaction velocity, K m : Michaelis-Menten constant, CLint: Intrinsic clearance, K i : Dissociation constant of an inhibitor enzyme complex, 4-MUG: 4-Methylumbelliferone glucuronide, DMSO: Dimethyl sulfoxide, IC50: Half maximal inhibitory concentration, p -NPG: p -Nitrophenol glucuronide, RKM: Rat kidneys microsomes, RLM: Rat liver microsome, UDPGA: UDPglucuronic acid, TCA: trichloroacetic acid, MPA: mycophenolic acid.

Three-dimensional quantitative structure-activity relationship studies on UGT1A9-mediated 3-O-glucuronidation of natural flavonols using a pharmacophore-based comparative molecular field analysis model.

PubMed

Wu, Baojian; Morrow, John Kenneth; Singh, Rashim; Zhang, Shuxing; Hu, Ming

2011-02-01

Glucuronidation is often recognized as one of the rate-determining factors that limit the bioavailability of flavonols. Hence, design and synthesis of more bioavailable flavonols would benefit from the establishment of predictive models of glucuronidation using kinetic parameters [e.g., K(m), V(max), intrinsic clearance (CL(int)) = V(max)/K(m)] derived for flavonols. This article aims to construct position (3-OH)-specific comparative molecular field analysis (CoMFA) models to describe UDP-glucuronosyltransferase (UGT) 1A9-mediated glucuronidation of flavonols, which can be used to design poor UGT1A9 substrates. The kinetics of recombinant UGT1A9-mediated 3-O-glucuronidation of 30 flavonols was characterized, and kinetic parameters (K(m), V(max), CL(int)) were obtained. The observed K(m), V(max), and CL(int) values of 3-O-glucuronidation ranged from 0.04 to 0.68 μM, 0.04 to 12.95 nmol/mg/min, and 0.06 to 109.60 ml/mg/min, respectively. To model UGT1A9-mediated glucuronidation, 30 flavonols were split into the training (23 compounds) and test (7 compounds) sets. These flavonols were then aligned by mapping the flavonols to specific common feature pharmacophores, which were used to construct CoMFA models of V(max) and CL(int), respectively. The derived CoMFA models possessed good internal and external consistency and showed statistical significance and substantive predictive abilities (V(max) model: q(2) = 0.738, r(2) = 0.976, r(pred)(2) = 0.735; CL(int) model: q(2) = 0.561, r(2) = 0.938, r(pred)(2) = 0.630). The contour maps derived from CoMFA modeling clearly indicate structural characteristics associated with rapid or slow 3-O-glucuronidation. In conclusion, the approach of coupling CoMFA analysis with a pharmacophore-based structural alignment is viable for constructing a predictive model for regiospecific glucuronidation rates of flavonols by UGT1A9.

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

PubMed

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

2012-08-29

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

The UDP-Glucuronosyltransferase (UGT) 1A Polymorphism c.2042C>G (rs8330) Is Associated with Increased Human Liver Acetaminophen Glucuronidation, Increased UGT1A Exon 5a/5b Splice Variant mRNA Ratio, and Decreased Risk of Unintentional Acetaminophen-Induced Acute Liver FailureS⃞

PubMed Central

Freytsis, Marina; Wang, Xueding; Peter, Inga; Guillemette, Chantal; Hazarika, Suwagmani; Duan, Su X.; Greenblatt, David J.; Lee, William M.

2013-01-01

Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury after acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen-glucuronidating enzymes (UGTs 1A1, 1A6, 1A9, and 2B15). Of these, only three linked single nucleotide polymorphisms (SNPs) located in the shared UGT1A-3′UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all the tested concentrations of acetaminophen. Mechanistic studies using luciferase-UGT1A-3′UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (P = 0.027, χ2 test) in patients who had acute liver failure from unintentional acetaminophen overdose compared with patients with acute liver failure from other causes or a race- or ethnicity-matched population. Together, these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation and could affect an individual’s risk for acetaminophen-induced liver injury. PMID:23408116

Influence of substrates on the in vitro kinetics of steviol glucuronidation and interaction between steviol glycosides metabolites and UGT2B7.

PubMed

Chen, Jun-Ming; Xia, Yong-Mei; Zhang, Yan-Dong; Zhang, Tong-Tong; Peng, Qing-Rui; Fang, Yun

2018-06-01

Steviol glycosides, a natural sweetener, may perform bioactivities via steviol, their main metabolite in human digestion. The metabolising kinetics, i.e. glucuronidation kinetics and interaction between steviol glycosides or their metabolites and metabolising enzyme, are important for understanding the bioactivity and cytotoxicity. The present study investigated kinetics of steviol glucuronidation in human liver microsome and a recombinant human UDP-glucuronosyltransferases isomer, UGT2B7, along with molecular docking to analyse interaction between UGT2B7 and steviol or glucose. The active pocket of UGT2B7 is consisted of Arg352, Leu347, Lys343, Phe339, Tyr354, Lys355 and Leu353. The influence of stevioside, rebaudioside A, glucose and some chemotherapy reagents on the glucuronidation was also studied. The predicted hepatic clearence suggested that steviol could be classified as high-clearence drug. The steviol glycosides did not affect the glucuronidation of steviol notably.

Evaluation of abalone β-glucuronidase substitution in current urine hydrolysis procedures.

PubMed

Malik-Wolf, Brittany; Vorce, Shawn; Holler, Justin; Bosy, Thomas

2014-04-01

This study examined the potential of abalone β-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia β-glucuronidase and Escherichia coli β-glucuronidase. Abalone β-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia β-glucuronidase. JWH 018 N-(5-hydroxypentyl) β-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone β-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli β-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone β-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone β-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone β-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids.

Isolation, purification, and structural characterization of flunixin glucuronide in the urine of greyhound dogs.

PubMed

Brady, T C; Kind, A J; Hyde, W H; Favrow, M; Hill, D W

1998-04-01

A urinary metabolite of flunixin in greyhound dogs was isolated and purified by a gradient-elution solid-phase extraction technique. The purified metabolite was shown to be hydrolyzed to free flunixin by strong base and by beta-glucuronidase, suggesting the presence of a C1-beta-glucuronide ester of flunixin. The metabolite was further characterized by positive-ion, tandem MS with electrospray ionization. Mass spectral data showed the presence of a protonated molecular ion (M+1) at m/z 473, which was consistent with the molecular weight of protonated flunixin glucuronide, and a product ion at m/z 297, which was consistent with the molecular weight of protonated flunixin. Collisionally induced dissociation of the m/z 297 product ion showed a fragmentation pattern consistent with that of standard flunixin. These data support the contention that this metabolite of flunixin in greyhound urine is the C1-beta-glucuronide of flunixin. Acyl glucuronide metabolites of some organic acid drugs have been shown to bind covalently to tissue proteins in vitro, in vivo, and ex vivo. The presence of this metabolite may, therefore, have pharmacokinetic and pharmacodynamic implications for flunixin in greyhound dogs, as well as in other animal species in which the acyl glucuronide of flunixin is a metabolite.

Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

PubMed

Jeong, Hyeon-Uk; Kim, Ju-Hyun; Kong, Tae Yeon; Choi, Won Gu; Lee, Hye Suk

2016-04-01

Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

Overestimation of Flavonoid Aglycones as a Result of the ex vivo Deconjugation of Glucuronides by the Tissue β-Glucuronidase

PubMed Central

Lu, Qing-Yi; Zhang, Lifeng; Eibl, Guido; Go, Vay-Liang W.

2013-01-01

Flavonoid glucuronides are the main circulating metabolites of flavonoids in humans and animals. There has been a growing interest in the biological function of glucuronides. In order to differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycone and metabolic conjugate in vivo. Many organs and body fluids of humans and animals exhibit β-glucuronidase against flavonoid glucuronides. Studies have shown that β-glucuronidase within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycone than actual in vivo concentrations. The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone, a β-glucuronidase inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides. Our data demonstrate that in mouse liver tissues and human tumor xenografts levels of quercetin and methylated quercetin aglycones could be over-estimated by 7 fold. The inhibition of deconjugation of quercetin and baicalein glucuronides by saccharo-1,4-lactone is dose-dependent. The amount of saccharo-1,4-lactone used to produce optimal inhibition of the enzyme activity is in the range of 15 – 24 μmol per gram of liver tissue. The use of β-glucuronidase inhibitor blocks the ex vivo deconjugation resulting in an accurate estimation of tissue levels of aglycone and conjugate. Our study described here can be extended to other animal models and human studies with different types of substrates of β-glucuronidase. PMID:24176739

Pharmacokinetics of acetaminophen, codeine, and the codeine metabolites morphine and codeine-6-glucuronide in healthy Greyhound dogs

PubMed Central

KuKanich, Butch

2009-01-01

The purpose of this study was to determine the pharmacokinetics of codeine and the active metabolites morphine and codeine-6-glucuronide after IV codeine administration and the pharmacokinetics of acetaminophen (APAP), codeine, morphine, and codeine-6-glucuronide after oral administration of combination product containing acetaminophen and codeine to dogs. Six healthy Greyhound dogs were administered 0.734 mg/kg codeine IV and acetaminophen (10.46 mg/kg mean dose) with codeine (1.43 mg/kg mean dose) orally. Blood samples were obtained at predetermined time points for the determination of codeine, morphine, and codeine-6-glucuronide plasma concentrations by LC/MS and acetaminophen by HPLC with UV detection. Codeine was rapidly eliminated after IV administration (T½ =1.22 hr; clearance=29.94 mL/min/kg; volume of distribution=3.17 L/kg) with negligible amounts of morphine present, but large amounts of codeine-6-glucuronide (CMAX=735.75 ng/mL) were detected. The oral bioavailability of codeine was 4%, morphine concentrations were negligible, but large amounts of codeine-6-glucuronide (CMAX=1952.86 ng/mL) were detected suggesting substantial first pass metabolism. Acetaminophen was rapidly absorbed (CMAX=6.74 μg/mL; TMAX=0.85 hr) and eliminated (T½=0.96 hr). In conclusion, the pharmacokinetics of codeine were similar to other opioids in dogs with a short half-life, rapid clearance, large volume of distribution, and poor oral bioavailability. High concentrations of codeine-6-glucuronide were detected after IV and oral administration. PMID:20444020

Pharmacokinetics of acetaminophen, codeine, and the codeine metabolites morphine and codeine-6-glucuronide in healthy Greyhound dogs.

PubMed

KuKanich, B

2010-02-01

The purpose of this study was to determine the pharmacokinetics of codeine and the active metabolites morphine and codeine-6-glucuronide after i.v. codeine administration and the pharmacokinetics of acetaminophen (APAP), codeine, morphine, and codeine-6-glucuronide after oral administration of combination product containing acetaminophen and codeine to dogs. Six healthy Greyhound dogs were administered 0.734 mg/kg codeine i.v. and acetaminophen (10.46 mg/kg mean dose) with codeine (1.43 mg/kg mean dose) orally. Blood samples were collected at predetermined time points for the determination of codeine, morphine, and codeine-6-glucuronide plasma concentrations by LC/MS and acetaminophen by HPLC with UV detection. Codeine was rapidly eliminated after i.v. administration (T(1/2) = 1.22 h; clearance = 29.94 mL/min/kg; volume of distribution = 3.17 L/kg) with negligible amounts of morphine present, but large amounts of codeine-6-glucuronide (C(max) = 735.75 ng/mL) were detected. The oral bioavailability of codeine was 4%, morphine concentrations were negligible, but large amounts of codeine-6-glucuronide (C(max) = 1952.86 ng/mL) were detected suggesting substantial first pass metabolism. Acetaminophen was rapidly absorbed (C(max) = 6.74 microg/mL; T(max) = 0.85 h) and eliminated (T(1/2) = 0.96 h). In conclusion, the pharmacokinetics of codeine was similar to other opioids in dogs with a short half-life, rapid clearance, large volume of distribution, and poor oral bioavailability. High concentrations of codeine-6-glucuronide were detected after i.v. and oral administration.

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.

PubMed

Ternes, T A; Kreckel, P; Mueller, J

1999-01-12

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.

Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin.

PubMed

Gill, Katherine L; Houston, J Brian; Galetin, Aleksandra

2012-04-01

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.

Characterization of In Vitro Glucuronidation Clearance of a Range of Drugs in Human Kidney Microsomes: Comparison with Liver and Intestinal Glucuronidation and Impact of Albumin

PubMed Central

Gill, Katherine L.; Houston, J. Brian

2012-01-01

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CLint, UGT) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CLint, UGT on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CLint, UGT in different tissues. Although BSA increased CLint, UGT in all tissues, the extent was tissue- and drug-dependent. Scaled CLint, UGT in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min−1 · g tissue−1 in liver, kidney, and intestinal microsomes. Renal CLint, UGT (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CLint, UGT for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CLint, UGT (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CLint, UGT is particularly important for UGT1A9 substrates. PMID:22275465

Human hydroxylated metabolites of BDE-47 and BDE-99 are glucuronidated and sulfated in vitro.

PubMed

Erratico, Claudio; Zheng, Xiaobo; Ryden, Andreas; Marsh, Goran; Maho, Walid; Covaci, Adrian

2015-07-16

Polybrominated diphenyl ethers (PBDEs) were used worldwide as additive flame retardants and are classified as persistent, bioaccumulable and toxic environmental pollutants. In humans, the hydroxylated metabolites of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) formed in vitro have also been detected in vivo. To further characterize the metabolism of BDE-47 and BDE-99 and to identify candidate markers for monitoring the human exposure to PBDEs using non-invasive approaches, glucuronidation and sulfation of hydroxylated metabolites of BDE-47 and BDE-99 were investigated using human liver microsomes and cytoplasm, respectively. The formed Phase II metabolites were analyzed by liquid chromatography-tandem mass spectrometry using a novel approach to develop analytical methods in absence of authentic standards. All available standards for hydroxylated metabolites of BDE-47 and BDE-99 were glucuronidated and sulfated, showing that glucuronidation and sulfation are part of the metabolism pathway of BDE-47 and BDE-99 in vitro. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-47 were (a) 2,4-DBP-Gluc and 5-Gluc-BDE-47, and (b) 2'-Sulf-BDE-28, 4-Sulf-BDE-42 and 3-Sulf-BDE-47, respectively. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-99 were (a) 2,4,5-TBP-Gluc and 6'-Gluc-BDE-99, and (b) 3'-Sulf-BDE-99 and 5'-Sulf-BDE-99, respectively. Apparent Km values associated with the formation of sulfated metabolites of BDE-47 and BDE-99 were ten times lower than those of the corresponding glucuronidated metabolites, suggesting that sulfated rather than glucuronidated metabolites of OH-PBDEs might be used as markers of human exposure to PBDEs using a non-invasive approach based on urine sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Disposition and metabolism of codeine after single and chronic doses in one poor and seven extensive metabolisers.

PubMed

Chen, Z R; Somogyi, A A; Reynolds, G; Bochner, F

1991-04-01

1. The pharmacokinetics, metabolism and partial clearances of codeine to morphine, norcodeine and codeine-6-glucuronide after single (30 mg) and chronic (30 mg 8 h for seven doses) administration of codeine were studied in eight subjects (seven extensive and one poor metaboliser of dextromethorphan). Codeine, codeine-6-glucuronide, morphine and norcodeine were measured by high performance liquid chromatographic assays. 2. After the single dose, the time to achieve maximum plasma codeine concentrations was 0.97 +/- 0.31 h (mean +/- s.d.) and for codeine-6-glucuronide it was 1.28 +/- 0.49 h. The plasma AUC of codeine-6-glucuronide was 15.8 +/- 4.5 times higher than that of codeine. The AUC of codeine in saliva was 3.4 +/- 1.1 times higher than that in plasma. The elimination half-life of codeine was 3.2 +/- 0.3 h and that of codeine-6-glucuronide was 3.2 +/- 0.9 h. 3. The renal clearance of codeine was 183 +/- 59 ml min-1 and was inversely correlated with urine pH (r = 0.81). These data suggest that codeine undergoes filtration at the glomerulus, tubular secretion and passive reabsorption. The renal clearance of codeine-6-glucuronide was 55 +/- 21 ml min-1, and was not correlated with urine pH. Its binding to human plasma was less than 10%. These data suggest that codeine-6-glucuronide undergoes filtration at the glomerulus and tubular reabsorption. This latter process is unlikely to be passive. 4. After chronic dosing, the pharmacokinetics of codeine and codeine-6-glucuronide were not significantly different from the single dose pharmacokinetics. 5. After the single dose, 86.1 +/- 11.4% of the dose was recovered in urine, of which 59.8 +/- 10.3% was codeine-6-glucuronide, 7.1 +/- 1.1% was total morphine, 6.9 +/- 2.1% was total norcodeine and 11.8 +/- 3.9% was unchanged codeine. These recoveries were not significantly different (P greater than 0.05) after chronic administration. 6. After the single dose, the partial clearance to morphine was 137 +/- 31 ml min-1 in the seven extensive metabolisers and 8 ml min-1 in the poor metaboliser; to norcodeine the values were 103 +/- 33 ml min-1 and 90 ml min-1; to codeine-6-glucuronide the values were 914 +/- 129 ml min-1 and 971 ml min-1; and intrinsic clearance was 1568 +/- 103 ml min-1 and 1450 ml min-1. These values were not significantly (P greater than 0.05) altered by chronic administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry.

PubMed

Pu, Fan; McKinney, Andrew R; Stenhouse, Allen M; Suann, Craig J; McLeod, Malcolm D

2004-12-25

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.

Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients

PubMed Central

Romero-Lorca, Alicia; Novillo, Apolonia; Gaibar, María; Bandrés, Fernando; Fernández-Santander, Ana

2015-01-01

Tamoxifen is used to prevent and treat estrogen-dependent breast cancer. It is described as a prodrug since most of its antiestrogen effects are exerted through its hydroxylated metabolites 4-OH-tamoxifen and endoxifen. In prior work, we correlated optimal plasma levels of these metabolites with certain genotypes of CYP2D6 and SULT1A2. This descriptive study examines correlations between concentrations of tamoxifen's glucuronide metabolites and genotypes UGT1A4 Pro24Thr, UGT1A4 Leu48Val, UGT2B7 His268Tyr, UGT2B15 Asp85YTyr UGT2B15 Lys523Thr and UGT2B17del in 132 patients with estrogen receptor-positive breast cancer under treatment with tamoxifen. Patients were genotyped by real-time and conventional PCR-RFLP. The glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide were isolated from blood plasma and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Individuals who were homozygous for UGT1A448VAL showed significantly lower mean concentrations of both glucuronide metabolites compared to subjects genotyped as wt/wt plus wt/48Val (p=0.037 and p=0.031, respectively). Women homozygous for UGT2B7268Tyr also showed mean substrate/product ratios of 4-OH-tamoxifen/4-OH-tamoxifen-O-glucuronide and 4-OH-tamoxifen/4-OH-tamoxifen-N-glucuronide indicative of reduced glucuronidase activity compared to wt homozygotes or to heterozygotes for the polymorphism (p=0.005 and p=0.003, respectively). In contrast, UGT2B15 Lys523Thr and UGT2B17del were associated with possibly increased enzyme activity. Patients with at least one variant allele UGT2B15523Thr showed significantly higher 4-OH-tamoxifen-O-glucuronide and endoxifen-glucuronide levels (p=0.023 and p=0.025, respectively) indicating a variant gene-dose effect. Higher 4-OH-tamoxifen-N-glucuronide levels observed in UGT2B17del genotypes (p=0.042) could be attributed to a mechanism that compensates for the greater expression of other genes in UGT2B17 del/del individuals. Our observations suggest that patients carrying mutations UGT1A448Val, UGT2B7268Tyr or with wt genotypes for UGT2B17nodel and UGT2B15523Lys could be the best candidates for a good response to tamoxifen therapy in terms of eliciting effective plasma active tamoxifen metabolite levels. However, additional studies examining the effects of UGT genotype on overall patient response to TAM are needed to further examine the role of UGT polymorphisms in the therapeutic efficacy of TAM. PMID:26176234

Overestimation of flavonoid aglycones as a result of the ex vivo deconjugation of glucuronides by the tissue β-glucuronidase.

PubMed

Lu, Qing-Yi; Zhang, Lifeng; Eibl, Guido; Go, Vay Liang W

2014-01-01

Flavonoid glucuronides are the main circulating metabolites of flavonoids in humans and animals. There has been a growing interest in the biological function of glucuronides. In order to differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycone and metabolic conjugate in vivo. Many organs and body fluids of humans and animals exhibit β-glucuronidase against flavonoid glucuronides. Studies have shown that β-glucuronidase within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycone than actual in vivo concentrations. The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone, a β-glucuronidase inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides. Our data demonstrate that in mouse liver tissues and human tumor xenografts levels of quercetin and methylated quercetin aglycones could be over-estimated by 7-fold. The inhibition of deconjugation of quercetin and baicalein glucuronides by saccharo-1,4-lactone is dose-dependent. The amount of saccharo-1,4-lactone used to produce optimal inhibition of the enzyme activity is in the range of 15-24μmol per gram of liver tissue. The use of β-glucuronidase inhibitor blocks the ex vivo deconjugation resulting in an accurate estimation of tissue levels of aglycone and conjugate. Our study described here can be extended to other animal models and human studies with different types of substrates of β-glucuronidase. Copyright © 2013 Elsevier B.V. All rights reserved.

Metabolic patterns of JWH-210, RCS-4, and THC in pig urine elucidated using LC-HR-MS/MS: Do they reflect patterns in humans?

PubMed

Schaefer, Nadine; Helfer, Andreas G; Kettner, Mattias; Laschke, Matthias W; Schlote, Julia; Ewald, Andreas H; Meyer, Markus R; Menger, Michael D; Maurer, Hans H; Schmidt, Peter H

2017-04-01

The knowledge of pharmacokinetic (PK) properties of synthetic cannabinoids (SCs) is important for interpretation of analytical results found for example in intoxicated individuals. In the absence of human data from controlled studies, animal models elucidating SC PK have to be established. Pigs providing large biofluid sample volumes were tested for prediction of human PK data. In this context, the metabolic fate of two model SCs, namely 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4), was elucidated in addition to Δ 9 -tetrahydrocannabinol (THC). After intravenous administration of the compounds, hourly collected pig urine was analyzed by liquid chromatography-high resolution mass spectrometry. The following pathways were observed: for JWH-210, hydroxylation at the ethyl side chain or pentyl chain and combinations of them followed by glucuronidation; for RCS-4, hydroxylation at the methoxyphenyl moiety or pentyl chain followed by glucuronidation as well as O-demethylation followed by glucuronidation or sulfation; for THC, THC glucuronidation, 11-hydroxylation, followed by carboxylation and glucuronidation. For both SCs, parent compounds could not be detected in urine in contrast to THC. These results were consistent with those obtained from human hepatocyte and/or human case studies. Urinary markers for the consumption of JWH-210 were the glucuronide of the N-hydroxypentyl metabolite (detectable for 3-4 h) and of RCS-4 the glucuronides of the N-hydroxypentyl, hydroxy-methoxyphenyl (detectable for at least 6 h), and the O-demethyl-hydroxy metabolites (detectable for 4 h). Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of Recent Cannabis Use: Whole-Blood and Plasma Free and Glucuronidated Cannabinoid Pharmacokinetics following Controlled Smoked Cannabis Administration

PubMed Central

Schwope, David M.; Karschner, Erin L.; Gorelick, David A.; Huestis, Marilyn A.

2013-01-01

BACKGROUND Δ9-Tetrahydrocannabinol (THC) is the most frequently observed illicit drug in investigations of accidents and driving under the influence of drugs. THC-glucuronide has been suggested as a marker of recent cannabis use, but there are no blood data following controlled THC administration to test this hypothesis. Furthermore, there are no studies directly examining whole-blood cannabinoid pharmacokinetics, although this matrix is often the only available specimen. METHODS Participants (9 men, 1 woman) resided on a closed research unit and smoked one 6.8% THC cannabis cigarette ad libitum. We quantified THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide and THCCOOH-glucuronide directly in whole blood and plasma by liquid chromatography/ tandem mass spectrometry within 24 h of collection to obviate stability issues. RESULTS Median whole blood (plasma) observed maximum concentrations (Cmax) were 50 (76), 6.4 (10), 41 (67), 1.3 (2.0), 2.4 (3.6), 89 (190), and 0.7 (1.4) μg/L 0.25 h after starting smoking for THC, 11-OH-THC, THCCOOH, CBD, CBN, and THCCOOH-glucuronide, respectively, and 0.5 h for THC-glucuronide. At observed Cmax, whole-blood (plasma) detection rates were 60% (80%), 80% (90%), and 50% (80%) for CBD, CBN, and THC-glucuronide, respectively. CBD and CBN were not detectable after 1 h in either matrix (LOQ 1.0 μg/L). CONCLUSIONS Human whole-blood cannabinoid data following cannabis smoking will assist whole blood and plasma cannabinoid interpretation, while furthering identification of recent cannabis intake. PMID:21836075

In silico prediction of acyl glucuronide reactivity

NASA Astrophysics Data System (ADS)

Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

2011-11-01

Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

Chemical communication in tilapia: a comparison of Oreochromis mossambicus with O. niloticus.

PubMed

Hubbard, Peter C; Mota, Vasco C; Keller-Costa, Tina; da Silva, José Paulo; Canário, Adelino V M

2014-10-01

In allopatric speciation species differentiation generally results from different selective pressures in different environments, and identifying the traits responsible helps to understand the isolation mechanism(s) involved. Male Mozambique tilapia (Oreochromis mossambicus) use urine to signal dominance; furthermore, 5β-pregnane-3α,17,20β-triol-3α-glucuronide (and its α-epimer, 5β-pregnane-3α,17,20α-triol-3α-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia (Oreochromisniloticus) is a close relative; species divergence probably resulted from geographical separation around 6 million years ago. This raises the question of whether the two species use similar urinary chemical cues during reproduction. The olfactory potency of urine, and crude extracts, from either species was assessed by the electro-olfactogram and the presence of the steroid glucuronides in urine from the Nile tilapia by liquid-chromatography/mass-spectrometry. Both species showed similar olfactory sensitivity to urine and respective extracts from either species, and similar sensitivity to the steroid glucuronides. 5β-Pregnan-3α,17α,20β-triol-3α-glucuronide was present at high concentrations (approaching 0.5mM) in urine from Nile tilapia, with 5β-pregnan-3α,17α,20α-triol-3α-glucuronide present at lower concentrations, similar to the Mozambique tilapia. Both species also had similar olfactory sensitivity to estradiol-3-glucuronide, a putative urinary cue from females. Together, these results support the idea that reproductive chemical cues have not been subjected to differing selective pressure. Whether these chemical cues have the same physiological and behavioural roles in O. niloticus as O. mossambicus remains to be investigated. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of hydrolysis on identifying prenatal cannabis exposure

PubMed Central

Gray, Teresa R.; Barnes, Allan J.

2011-01-01

Identification of prenatal cannabis exposure is important due to potential cognitive and behavioral consequences. A two-dimensional gas chromatography–mass spectrometry method for cannabinol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 8β,11-dihydroxy-THC, and 11-nor-9-carboxy-THC (THCCOOH) quantification in human meconium was developed and validated. Alkaline, enzymatic, and enzyme–alkaline tandem hydrolysis conditions were optimized with THC- and THCCOOH-glucuronide reference standards. Limits of quantification ranged from 10 to 15 ng/g, and calibration curves were linear to 500 ng/g. Bias and intra-day and inter-day imprecision were <12.3%. Hydrolysis efficiencies were analyte-dependent; THC-glucuronide was effectively cleaved by enzyme, but not base. Conversely, THCCOOH-glucuronide was most sensitive to alkaline hydrolysis. Enzyme–alkaline tandem hydrolysis maximized efficiency for both glucuronides. Identification of cannabinoid-positive meconium specimens nearly doubled following alkaline and enzyme–alkaline hydrolysis. Although no 11-OH-THC glucuronide standard is available, enzymatic hydrolysis improved 11-OH-THC detection in authentic specimens. Maximal identification of cannabis-exposed neonates and the widest range of cannabis biomarkers are achieved with enzyme–alkaline tandem hydrolysis. PMID:20517601

Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

PubMed Central

Trottier, Jocelyn; Perreault, Martin; Rudkowska, Iwona; Levy, Cynthia; Dallaire-Theroux, Amélie; Verreault, Mélanie; Caron, Patrick; Staels, Bart; Vohl, Marie-Claude; Straka, Robert J.; Barbier, Olivier

2014-01-01

Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes detoxifies cholestatic bile acids (BAs). We aimed at i) characterizing the circulating BA-glucuronide (-G) pool composition in humans, ii) evaluating how sex and UGT polymorphisms influence this composition, and iii) analyzing the effects of lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and post-fenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of 5 BA-G species, including CDCA-3G, and up-regulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrates that fenofibrate stimulates BA glucuronidation in humans, and thus reduces bile acid toxicity in the liver. PMID:23756370

False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

PubMed

Arndt, Torsten; Grüner, Joachim; Schröfel, Stefanie; Stemmerich, Karsten

2012-11-30

Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented the inhalation of the sanitizer vapor, the formation of propyl glucuronides and thus false-positive DRI(®) EIA EtG screening results, proving that propyl alcohols are almost exclusively taken up by respiration. The widespread use of propanol-containing products such as hand sanitizers may lead to sufficient uptake of propyl alcohols and excretion of significant amounts of propyl glucuronides to yield false-positive DRI(®) EIA EtG screening results. Thus, positive EtG immunoassay results have to be controlled by mass-spectrometry, in clinical cases at least if ethanol intake is denied by the patient. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Oral coadministration of β-glucuronidase to increase exposure of extensively glucuronidated drugs that undergo enterohepatic recirculation.

PubMed

Eichenbaum, Gary; Hsu, C-P; Subrahmanyam, Vangala; Chen, Jing; Scicinski, Jan; Galemmo, Robert A; Tuman, Robert W; Johnson, Dana L

2012-07-01

Extensive first-pass metabolism can significantly limit a drug's oral exposure levels. In this work, we introduce an innovative approach for increasing the oral bioavailability of a drug that undergoes extensive reversible glucuronidation and enterohepatic recirculation through intraduodenal coadministration of the deconjugating enzyme β-glucuronidase. Intraduodenal administration of JNJ-10198409 (10 mg/kg) with β-glucuronidase (34,000-140,000 units/kg) to catheterized rats resulted in a significant increase (p < 0.005) in the mean area under the plasma concentration versus time curve (AUC; approx. threefold) and maximum plasma concentration (C(max); approx. twofold) of JNJ-10198409. The AUC and C(max) were 60 ± 18 ng h/mL and 76 ± 29 ng/mL, respectively, with no enzyme and 177 ± 55 ng h/mL and 129 ± 41 ng/mL, respectively, with β-glucuronidase coadministered. Moreover, the AUC of the primary glucuronide metabolite increased approximately sevenfold from 1173 ± 361 (ng h)/mL with no enzyme coadministered to 8723 ± 2133 ng h/mL with coadministered enzyme. These pharmacokinetic data support the hypothesis that when the primary glucuronide is secreted into the duodenum via the bile duct, the glucuronide is converted by β-glucuronidase back to the parent compound. The parent compound is then reabsorbed and reconjugated, resulting in elevated systemic exposures to both parent and glucuronide. Potential clinical and preclinical applications and considerations for this approach are discussed. Copyright © 2012 Wiley Periodicals, Inc.

Inhibition of UDP-glucuronosyltransferase (UGT)-mediated glycyrrhetinic acid 3-O-glucuronidation by polyphenols and triterpenoids.

PubMed

Koyama, Mayuko; Shirahata, Tatsuya; Hirashima, Rika; Kobayashi, Yoshinori; Itoh, Tomoo; Fujiwara, Ryoichi

2017-08-01

Glycyrrhetinic acid (GA) is an active metabolite of glycyrrhizin, which is a main constituent in licorice (Glycyrrhiza glabra). While GA exhibits a wide variety of pharmacological activities in the body, it is converted to a toxic metabolite GA 3-O-glucuronide by hepatic UDP-glucuronosyltransferases (UGTs). To avoid the development of the toxic metabolite-induced pseudohyperaldosteronism (pseudoaldosteronism), there is a limitation in maximum daily dosage of licorice and in combined usage of other glycyrrhizin-containing natural medicine. In this study, we investigated the inhibitory effects of various polyphenols and triterpenoids on the UGT-mediated GA 3-O-glucuronidation. In human liver microsomes, UGT-mediated GA glucuronidation was significantly inhibited by protopanaxadiol with an IC 50 value of 59.2 μM. Isoliquiritigenin, rosmarinic acid, alisol B, alisol acetate, and catechin moderately inhibited the GA glucuronidation with IC 50 values of 96.4 μM, 125 μM, 160 μM, 163 μM, and 164 μM. Other tested 19 polyphenols and triterpenoids, including liquiritigenin, did not inhibit UGT-mediated GA glucuronidation in human liver microsomes. Our data indicate that relatively higher dosage of licorice can be used without a risk of developing pseudohyperaldosteronism in combination of natural medicine containing protopanaxadiol such as Panax ginseng. Furthermore, supplemental protopanaxadiol and isoliquiritigenin might be useful in preventing licorice-inducing pseudoaldosteronism. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Use of Isoform-Specific UGT Metabolism to Determine and Describe Rates and Profiles of Glucuronidation of Wogonin and Oroxylin A by Human Liver and Intestinal Microsomes

PubMed Central

Zhou, Qiong; Zheng, Zhijie; Xia, Bijun; Tang, Lan; Lv, Chang; Liu, Wei; Liu, Zhongqiu; Hu, Ming

2010-01-01

Purposes Glucuronidation via UDP-glucuronosyltransferases (or UGTs) is a major metabolic pathway. The purposes of this study are to determine the UGT-isoform specific metabolic fingerprint (or GSMF) of wogonin and oroxylin A, and to use isoform-specific metabolism rates and kinetics to determine and describe their glucuronidation behaviors in tissue microsomes. Methods In vitro glucuronidation rates and profiles were measured using expressed UGTs and human intestinal and liver microsomes. Results GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoform-specific metabolism could describe their metabolism rates and profiles in human liver and intestinal microsomes. Conclusion GSMF and isoform-specific metabolism profiles can determine and describe glucuronidation rates and profiles in human tissue microsomes. PMID:20411407

Direct and efficient liquid chromatographic-tandem mass spectrometric method for opiates in urine drug testing - importance of 6-acetylmorphine and reduction of analytes.

PubMed

Andersson, Maria; Stephanson, Nikolai; Ohman, Inger; Terzuoli, Tommy; Lindh, Jonatan D; Beck, Olof

2014-04-01

Opiates comprise a class of abused drugs that is of primary interest in clinical and forensic urine drug testing. Determination of heroin, codeine, or a multi-drug ingestion is complicated since both heroin and codeine can lead to urinary excretion of free and conjugated morphine. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantage over gas chromatography-mass spectrometry by simplifying sample preparation but increases the number of analytes. A method based on direct injection of five-fold diluted urine for confirmation of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide and 6-acetylmorphine was validated using LC-MS/MS in positive electrospray mode monitoring two transitions using selected reaction monitoring. The method was applied for the analysis of 3155 unknown urine samples which were positive for opiates in immunochemical screening. A linear response was observed for all compounds in the calibration curves covering more than three orders of magnitude. Cut off was set to 2 ng/ml for 6-acetylmorphine and 150 ng/ml for the other analytes. 6-Acetylmorphine was found to be effective (sensitivity 82%) in detecting samples as heroin intake. Morphine-3-glucuronide and codeine-6-glucuronide was the predominant components of total morphine and codeine, 84% and 93%, respectively. The authors have validated a robust LC-MS/MS method for rapid qualitative and quantitative analysis of opiates in urine. 6-Acetylmorphine has been demonstrated as a sensitive and important parameter for a heroin intake. A possible interpretation strategy to conclude the source of detected analytes was proposed. The method might be further developed by reducing the number of analytes to morphine-3-glucuronide, codeine-6-glucuronide and 6-acetylmorphine without compromising test performance. Copyright © 2013 John Wiley & Sons, Ltd.

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

PubMed

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Resveratrol-3-O-glucuronide and resveratrol-4′-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin

PubMed Central

Zunino, Susan J.; Storms, David H.

2017-01-01

Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4′-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4′-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with topoisomerase I inhibitors. PMID:28781690

Resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin.

PubMed

Zunino, Susan J; Storms, David H

2017-08-01

Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with topoisomerase I inhibitors.

Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver.

PubMed

Papageorgiou, Ioannis; Freytsis, Marina; Court, Michael H

2016-10-01

Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3'-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3'-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF. Published by Elsevier Inc.

Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO.

PubMed

Rydevik, Axel; Bondesson, Ulf; Thevis, Mario; Hedeland, Mikael

2013-10-01

A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost. Copyright © 2013 Elsevier B.V. All rights reserved.

Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis.

PubMed

Liang, Si-Cheng; Xia, Yang-Liu; Hou, Jie; Ge, Guang-Bo; Zhang, Jiang-Wei; He, Yu-Qi; Wang, Jia-Yue; Qi, Xiao-Yi; Yang, Ling

2016-02-01

Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Validation of a rapid and sensitive LC-MS/MS method for determination of exemestane and its metabolites, 17β-hydroxyexemestane and 17β-hydroxyexemestane-17-O-β-D-glucuronide: application to human pharmacokinetics study.

PubMed

Wang, Ling-Zhi; Goh, Sok-Hwei; Wong, Andrea Li-Ann; Thuya, Win-Lwin; Lau, Jie-Ying Amelia; Wan, Seow-Ching; Lee, Soo-Chin; Ho, Paul C; Goh, Boon-Cher

2015-01-01

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17β-dihydroexemestane (active metabolite) and 17β-dihydroexemestane-17-O-β-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 μm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1% aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17β-dihydroexemestane, 17β-dihydroexemestane-d3, 17β-dihydroexemestane-17-O-β-D-glucuronide, and 17β-dihydroexemestane-17-O-β-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4-40.0 ng/mL, for exemestane; and 0.2-15.0 ng/mL, for 17β-dihydroexemestane and 17β-dihydroexemestane-17-O-β-D-glucuronide, with coefficient of determination (r2) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17β-dihydroexemestane and 92.0 to 103.2% for 17β-dihydroexemestane-17-O-β-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25 mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP.

PubMed

Järvinen, Erkka; Deng, Feng; Kidron, Heidi; Finel, Moshe

2018-04-01

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E 1 -G), estradiol-3- and 17-glucuronides (E 2 -3G and E 2 -17G), as well as estriol-3- and 16-glucuronides (E 3 -3G and E 3 -16G) are found in human plasma and urine. Unlike E 2 -17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E 1 -G, E 2 -3G, E 3 -3G, E 3 -16G and estrone-3-sulfate (E 1 -S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E 1 -S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E 1 -G and E 2 -3G, were still transported by BCRP at 10-fold higher rates than E 1 -S. BCRP also transported E 3 -16G at higher rates than the studied MRPs, while it transported E 3 -3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E 1 -G, E 2 -3G, E 3 -3G and E 3 -16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K m values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at K m values below 20 μM, but lower V max values than other transporters. In the case of E 3 -3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E 3 -16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Autism and Phthalate Metabolite Glucuronidation

ERIC Educational Resources Information Center

Stein, T. Peter; Schluter, Margaret D.; Steer, Robert A.; Ming, Xue

2013-01-01

Exposure to environmental chemicals may precipitate autism spectrum disorders (ASD) in genetically susceptible children. Differences in the efficiency of the glucuronidation process may substantially modulate substrate concentrations and effects. To determine whether the efficiency of this pathway is compromised in children with ASD, we measured…

Developmental changes rather than repeated administration drive paracetamol glucuronidation in neonates and infants.

PubMed

Krekels, Elke H J; van Ham, Saskia; Allegaert, Karel; de Hoon, Jan; Tibboel, Dick; Danhof, Meindert; Knibbe, Catherijne A J

2015-09-01

Based on recovered metabolite ratios in urine, it has been concluded that paracetamol glucuronidation may be up-regulated upon multiple dosing. This study investigates paracetamol clearance in neonates and infants after single and multiple dosing using a population modelling approach. A population pharmacokinetic model was developed in NONMEM VI, based on paracetamol plasma concentrations from 54 preterm and term neonates and infants, and on paracetamol, paracetamol-glucuronide and paracetamol-sulphate amounts in urine from 22 of these patients. Patients received either a single intravenous propacetamol dose or up to 12 repeated doses. Paracetamol and metabolite disposition was best described with one-compartment models. The formation clearance of paracetamol-sulphate was 1.46 mL/min/kg(1.4), which was about 5.5 times higher than the formation clearance of the glucuronide of 0.266 mL/min/kg. The renal excretion rate constants of both metabolites was estimated to be 11.4 times higher than the excretion rate constant of unchanged paracetamol, yielding values of 0.580 mL/min/kg. Developmental changes were best described by bodyweight in linear relationships on the distribution volumes, the formation of paracetamol-glucuronide and the unchanged excretion of paracetamol, and in an exponential relationship on the formation of paracetamol-sulphate. There was no evidence for up-regulation or other time-varying changes in any of the model parameters. Simulations with this model illustrate how paracetamol-glucuronide recovery in urine increases over time due to the slower formation of this metabolite and in the absence of up-regulation. Developmental changes, described by bodyweight-based functions, rather than up-regulation, explain developmental changes in paracetamol disposition in neonates and infants.

In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism.

PubMed

Atsriku, Christian; Hoffmann, Matthew; Moghaddam, Mehran; Kumar, Gondi; Surapaneni, Sekhar

2015-01-01

1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with <10% metabolized in dog and human, <20% metabolized in rabbit and monkey and >75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.

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

PubMed Central

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

2015-01-01

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

Role of UDP-glucuronosyltransferase isoforms in 13-cis retinoic acid metabolism in humans.

PubMed

Rowbotham, Sophie E; Illingworth, Nicola A; Daly, Ann K; Veal, Gareth J; Boddy, Alan V

2010-07-01

13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.

Biotransformation of Bisphenol AF to Its Major Glucuronide Metabolite Reduces Estrogenic Activity

PubMed Central

Yin, Jie; Zhang, Jing; Feng, Yixing; Shao, Bing

2013-01-01

Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and Vmax of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings. PMID:24349450

Methadone inhibits CYP2D6 and UGT2B7/2B4 in vivo: a study using codeine in methadone- and buprenorphine-maintained subjects

PubMed Central

Gelston, Eloise A; Coller, Janet K; Lopatko, Olga V; James, Heather M; Schmidt, Helmut; White, Jason M; Somogyi, Andrew A

2012-01-01

AIMS To compare the O-demethylation (CYP2D6-mediated), N-demethylation (CYP3A4-mediated) and 6-glucuronidation (UGT2B4/7-mediated) metabolism of codeine between methadone- and buprenorphine-maintained CYP2D6 extensive metabolizer subjects. METHODS Ten methadone- and eight buprenorphine-maintained subjects received a single 60 mg dose of codeine phosphate. Blood was collected at 3 h and urine over 6 h and assayed for codeine, norcodeine, morphine, morphine-3- and -6-glucuronides and codeine-6-glucuronide. RESULTS The urinary metabolic ratio for O-demethylation was significantly higher (P = 0.0044) in the subjects taking methadone (mean ± SD, 2.8 ± 3.1) compared with those taking buprenorphine (0.60 ± 0.43), likewise for 6-glucuronide formation (0.31 ± 0.24 vs. 0.053 ± 0.027; P < 0.0002), but there was no significant difference (P = 0.36) in N-demethylation. Similar changes in plasma metabolic ratios were also found. In plasma, compared with those maintained on buprenorphine, the methadone-maintained subjects had increased codeine and norcodeine concentrations (P < 0.004), similar morphine (P = 0.72) and lower morphine-3- and -6- and codeine-6-glucuronide concentrations (P < 0.008). CONCLUSION Methadone is associated with inhibition of CYP2D6 and UGTs 2B4 and 2B7 reactions in vivo, even though it is not a substrate for these enzymes. Plasma morphine was not altered, owing to the opposing effects of inhibition of both formation and elimination; however, morphine-6-glucuronide (analgesically active) concentrations were substantially reduced. Drug interactions with methadone are likely to include drugs metabolized by various UGTs and CYP2D6. PMID:22092298

A novel method for the quantitation of gingerol glucuronides in human plasma or urine based on stable isotope dilution assays.

PubMed

Schoenknecht, Carola; Andersen, Gaby; Schieberle, Peter

2016-11-15

The bio-active compounds of ginger (Zingiber officinale Roscoe), the gingerols, are gaining considerable attention due to their numerous beneficial health effects. In order to elucidate the physiological relevance of the ascribed effects their bioavailability has to be determined taking their metabolization into account. To quantitate in vivo generated [6]-, [8]- and [10]-gingerol glucuronides in human plasma and urine after ginger tea consumption, a simultaneous and direct liquid chromatography-tandem mass spectrometry method based on stable isotope dilution assays was established and validated. The respective references as well as the isotopically labeled substances were synthesized and characterized by mass spectrometry and NMR. Selective isolation of gingerol glucuronides from human plasma and urine by a mixed-phase anion-exchange SPE method led to recovery rates between 80.8 and 98.2%. LC-MS/MS analyses in selected reaction monitoring modus enabled a highly sensitive quantitation of gingerol glucuronides with LoQs between 3.9-9.8nmol/L in plasma and 39.3-161.1nmol/L in urine. The method precision in plasma and urine varied in the range±15%, whereas the intra-day accuracy in plasma and urine showed values between 78 and 122%. The developed method was then applied to a pilot study in which two volunteers consumed one liter ginger tea. Pharmacokinetic parameters like the maximum concentration (c max ), the time to reach c max (t max ), area under the curve (AUC), elimination rate constant (k el ) and elimination half-life (t 1/2 ) were calculated from the concentration-time curve of each gingerol glucuronide. The obtained results will enable more detailed investigation of gingerol glucuronides as bioactives in their physiologically relevant concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

Fate of glucuronide conjugated estradiol in the environment

USDA-ARS?s Scientific Manuscript database

The fate and transport of conjugated reproductive hormones, which are polar compared to parent hormones, are little understood. Laboratory bench-scale soil (Hamar; Sandy, mixed, frigid typic Endoaquolls) sorption studies were conducted using [14C] 17ß-estradiol-3-glucuronide for a range of concentra...

EFFECTS OF BDE-47 ON NUCLEAR RECEPTOR REGULATED GENES AND IMPLICATIONS FOR THYROID HORMONE DISRUPTION.

EPA Science Inventory

Previous studies have shown that exposure to polybrominated diphenyl ethers (PBDEs) can decrease thyroid hormone levels via the induction of hepatic uridinediphosphate-glucoronosyltransferase, (UGTs) which catalyze glucuronidation of T4 resulting in T4-glucuronide excretion. Bas...

Lethal morphine intoxication in a patient with a sickle cell crisis and renal impairment: case report and a review of the literature.

PubMed

Lagas, Jurjen S; Wagenaar, Jiri F P; Huitema, Alwin D R; Hillebrand, Michel J X; Koks, Cornelis H W; Gerdes, Victor E A; Brandjes, Desiderius P M; Beijnen, Jos H

2011-09-01

Morphine-6-glucuronide, the active metabolite of morphine, and to a lesser extent morphine itself are known to accumulate in patients with renal failure. A number of cases on non-lethal morphine toxicity in patients with renal impairment report high plasma concentrations of morphine-6-glucuronide, suggesting that this metabolite achieves sufficiently high brain concentrations to cause long-lasting respiratory depression, despite its poor central nervous system penetration. We report a lethal morphine intoxication in a 61-year-old man with sickle cell disease and renal impairment, and we measured concentrations of morphine and morphine-6-glucuronide in blood, brain and cerebrospinal fluid. There were no measurable concentrations of morphine-6-glucuronide in cerebrospinal fluid or brain tissue, despite high blood concentrations. In contrast, the relatively high morphine concentration in the brain suggests that morphine itself was responsible for the cardiorespiratory arrest in this patient. Given the fatal outcome, we recommend to avoid repeated or continuous morphine administration in renal failure.

Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS

PubMed Central

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

2015-01-01

Background Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. Methods After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. Results and Conclusion More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS - 8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides, hydroxyphenyl RCS-8, as well as the demethyl-hydroxycyclohexyl RCS-8 glucuronide. PMID:24946920

Glucuronidation of OTS167 in Humans Is Catalyzed by UDP-Glucuronosyltransferases UGT1A1, UGT1A3, UGT1A8, and UGT1A10

PubMed Central

Ramírez, Jacqueline; Mirkov, Snezana; House, Larry K.

2015-01-01

OTS167 is a potent maternal embryonic leucine zipper kinase inhibitor undergoing clinical testing as antineoplastic agent. We aimed to identify the UDP-glucuronosyltransferases (UGTs) involved in OTS167 metabolism, study the relationship between UGT genetic polymorphisms and hepatic OTS167 glucuronidation, and investigate the inhibitory potential of OTS167 on UGTs. Formation of a single OTS167-glucuronide (OTS167-G) was observed in pooled human liver (HLM) (Km = 3.4 ± 0.2 µM), intestinal microsomes (HIM) (Km = 1.7 ± 0.1 µM), and UGTs. UGT1A1 (64 µl/min/mg) and UGT1A8 (72 µl/min/mg) exhibited the highest intrinsic clearances (CLint) for OTS167, followed by UGT1A3 (51 µl/min/mg) and UGT1A10 (47 µl/min/mg); UGT1A9 was a minor contributor. OTS167 glucuronidation in HLM was highly correlated with thyroxine glucuronidation (r = 0.91, P < 0.0001), SN-38 glucuronidation (r = 0.79, P < 0.0001), and UGT1A1 mRNA (r = 0.72, P < 0.0001). Nilotinib (UGT1A1 inhibitor) and emodin (UGT1A8 and UGT1A10 inhibitor) exhibited the highest inhibitory effects on OTS167-G formation in HLM (68%) and HIM (47%). We hypothesize that OTS167-G is an N-glucuronide according to mass spectrometry. A significant association was found between rs6706232 and reduced OTS167-G formation (P = 0.03). No or weak UGT inhibition (range: 0–21%) was observed using clinically relevant OTS167 concentrations (0.4–2 µM). We conclude that UGT1A1 and UGT1A3 are the main UGTs responsible for hepatic formation of OTS167-G. Intestinal UGT1A1, UGT1A8, and UGT1A10 may contribute to first-pass OTS167 metabolism after oral administration. PMID:25870101

Glucuronidation of OTS167 in Humans Is Catalyzed by UDP-Glucuronosyltransferases UGT1A1, UGT1A3, UGT1A8, and UGT1A10.

PubMed

Ramírez, Jacqueline; Mirkov, Snezana; House, Larry K; Ratain, Mark J

2015-07-01

OTS167 is a potent maternal embryonic leucine zipper kinase inhibitor undergoing clinical testing as antineoplastic agent. We aimed to identify the UDP-glucuronosyltransferases (UGTs) involved in OTS167 metabolism, study the relationship between UGT genetic polymorphisms and hepatic OTS167 glucuronidation, and investigate the inhibitory potential of OTS167 on UGTs. Formation of a single OTS167-glucuronide (OTS167-G) was observed in pooled human liver (HLM) (Km = 3.4 ± 0.2 µM), intestinal microsomes (HIM) (Km = 1.7 ± 0.1 µM), and UGTs. UGT1A1 (64 µl/min/mg) and UGT1A8 (72 µl/min/mg) exhibited the highest intrinsic clearances (CLint) for OTS167, followed by UGT1A3 (51 µl/min/mg) and UGT1A10 (47 µl/min/mg); UGT1A9 was a minor contributor. OTS167 glucuronidation in HLM was highly correlated with thyroxine glucuronidation (r = 0.91, P < 0.0001), SN-38 glucuronidation (r = 0.79, P < 0.0001), and UGT1A1 mRNA (r = 0.72, P < 0.0001). Nilotinib (UGT1A1 inhibitor) and emodin (UGT1A8 and UGT1A10 inhibitor) exhibited the highest inhibitory effects on OTS167-G formation in HLM (68%) and HIM (47%). We hypothesize that OTS167-G is an N-glucuronide according to mass spectrometry. A significant association was found between rs6706232 and reduced OTS167-G formation (P = 0.03). No or weak UGT inhibition (range: 0-21%) was observed using clinically relevant OTS167 concentrations (0.4-2 µM). We conclude that UGT1A1 and UGT1A3 are the main UGTs responsible for hepatic formation of OTS167-G. Intestinal UGT1A1, UGT1A8, and UGT1A10 may contribute to first-pass OTS167 metabolism after oral administration. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS.

PubMed

Feng, Wan-Yong; Wen, Jenny; Stauber, Kathe

2018-04-18

Recently, it has been an increasing concern on the bioactivation and adverse reactions associated with consumption of herbal and nature products such as coumarin family. 7-ethoxycoumarin is one of coumarin family compounds, but little information is available regarding its potential reactive metabolites. In this study, we investigated its metabolism in cryopreserved male/female mixed human, male Cynomolgus monkey, male Beagle dog and male Sprague Dawley rat hepatocytes. Following the incubation of 7-ethoxylcoumarin in the hepatocytes for 2 hr, 28 metabolites were detected and identified using high resolution LC-Q-Exactive system in the positive ion and negative ion modes. O-deethylation, glucuronidation, sulfation, oxygenation, oxidative ring-opening, hydrogenation, glutathionation, dehydrogenation, cysteination, glucosidation, methylation, and hydrolysis were observed. At least sixteen metabolites were newly identified. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3 (O-deethylation and glucuronidation), M5 (hydrolysis and mono-oxygenation), M14 (Odeethylation), M16 (hydrolysis), M22 (oxidative ring-opening and oxygenation) and M27 (mono-oxygenation) appeared to be major metabolites in human hepatocytes. M3, M5, M8, M13 (mono-oxygenation), M14, M16, M18 (O-deethylation and sulfation), M22 and M27 appeared to be major metabolites in monkey hepatocytes. M14, M16, M18, M20 (glutathionation and dehydrogenation) and M27 appeared to be major metabolites in dog hepatocytes. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3, M5, M13, M14, M16, M17 (cysteination), M18, M20, and M22 appeared to be major metabolites in rat hepatocytes. Species differences in metabolism of 7-ethoxylcoumarin in hepatocytes were observed across humans, monkeys, dogs and rats. The analysis of metabolites suggests that 7-ethoxylcoumarin may undergo 3,4-epoxidation responsible for formation of glutathione and its derived cysteine conjugates, and carboxylic acid and its glucuronides, glucosides and sulfate, besides well-known 7-hydroxycoumarin, coumarin-7-O-glucuronide, and coumarin-7-O-glucuronide. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Regioselectivity of Human UDP-Glucuronosyltransferase Isozymes in Flavonoid Biotransformation by Metal Complexation and Tandem Mass Spectrometry

PubMed Central

Robotham, Scott A.; Brodbelt, Jennifer S.

2011-01-01

Based on reactions with five flavonoids, the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones versus flavonols. PMID:21889496

DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

EPA Science Inventory

Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic The UDP-glucuronosyltransferases of the blood-brain barrier: their role in drug metabolism and detoxication

PubMed Central

Ouzzine, Mohamed; Gulberti, Sandrine; Ramalanjaona, Nick; Magdalou, Jacques; Fournel-Gigleux, Sylvie

2014-01-01

UDP-glucuronosyltransferases (UGTs) form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-D-glucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds) by the linkage of glucuronic acid from the high energy donor, UDP-α-D-glucuronic acid. In brain, UGTs actively participate to the overall protection of the tissue against the intrusion of potentially harmful lipophilic substances that are metabolized as hydrophilic glucuronides. These metabolites are generally inactive, except for important pharmacologically glucuronides such as morphine-6-glucuronide. UGTs are mainly expressed in endothelial cells and astrocytes of the blood brain barrier (BBB). They are also associated to brain interfaces devoid of BBB, such as circumventricular organ, pineal gland, pituitary gland and neuro-olfactory tissues. Beside their key-role as a detoxication barrier, UGTs play a role in the steady-state of endogenous compounds, like steroids or dopamine (DA) that participate to the function of the brain. UGT isoforms of family 1A, 2A, 2B and 3A are expressed in brain tissues to various levels and are known to present distinct but overlapping substrate specificity. The importance of these enzyme species with regard to the formation of toxic, pharmacologically or physiologically relevant glucuronides in the brain will be discussed. PMID:25389387

Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

PubMed Central

Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

2015-01-01

Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

EPA Science Inventory

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

USDA-ARS?s Scientific Manuscript database

The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

Urinary analysis reveals high deoxynivalenol exposure in pregnant women from Croatia.

PubMed

Sarkanj, Bojan; Warth, Benedikt; Uhlig, Silvio; Abia, Wilfred A; Sulyok, Michael; Klapec, Tomislav; Krska, Rudolf; Banjari, Ines

2013-12-01

In this pilot survey the levels of various mycotoxin biomarkers were determined in third trimester pregnant women from eastern Croatia. First void urine samples were collected and analysed using a "dilute and shoot" LC-ESI-MS/MS multi biomarker method. Deoxynivalenol (DON) and its metabolites: deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were detected in 97.5% of the studied samples, partly at exceptionally high levels, while ochratoxin A was found in 10% of the samples. DON exposure was primarily reflected by the presence of deoxynivalenol-15-glucuronide with a mean concentration of 120 μg L(-1), while free DON was detected with a mean concentration of 18.3 μg L(-1). Several highly contaminated urine samples contained a third DON conjugate, tentatively identified as deoxynivalenol-7-glucuronide by MS/MS scans. The levels of urinary DON and its metabolites measured in this study are the highest ever reported, and 48% of subjects were estimated to exceed the provisional maximum tolerable daily intake (1 μg kg(-1) b.w.). Copyright © 2013 Elsevier Ltd. All rights reserved.

Resveratrol increases phagocytosis and lipopolysaccharide-induced interleukin-1β production, but decreases surface expression of Toll-like receptor 2 in THP-1 monocytes.

PubMed

Zunino, Susan J; Hwang, Daniel H; Huang, Shurong; Storms, David H

2018-02-01

THP-1 monocytes were used to evaluate the effects of physiological levels of resveratrol aglycone, resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate on phagocytosis, IL-1β, IL-1α, and IL-18 production, viability, and TLR2 and TLR4 expression. THP-1 cells were treated with 1, 5, 10, and 15μM resveratrol or metabolites. Resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate had no effect on the functional parameters tested. Resveratrol aglycone increased phagocytosis at concentrations of 5, 10, and 15μM and LPS-induced IL-1β production at concentrations of 10 and 15μM. Expression of TLR4 increased slightly after resveratrol treatment, but surface expression of TLR2 was reduced as resveratrol concentrations increased. Our data suggest that resveratrol may be effective in modulating monocyte function in an environment where there is direct exposure to the aglycone, such as at the gut epithelium. Published by Elsevier Ltd.

A budesonide prodrug accelerates treatment of colitis in rats.

PubMed Central

Cui, N; Friend, D R; Fedorak, R N

1994-01-01

Although oral glucocorticoids are the treatment of choice for moderate to severe ulcerative pancolitis, their systemic side effects and adrenal suppression account for considerable morbidity. An oral glucocorticoid-conjugate (prodrug), budesonide-beta-D-glucuronide, which is not absorbed in the small intestine but is hydrolysed by colonic bacterial and mucosal beta-glucuronidase to release free budesonide into the colon was synthesised. The objective of this study was to compare treatment with budesonide-beta-D-glucuronide with treatment with free budesonide by examining: (1) the healing of experimental colitis and (2) the extent of adrenal suppression. Pancolitis was induced with 4% acetic acid. Animals were then randomised to receive oral therapy for 72 hours with (1) budesonide-beta-D-glucuronide, (2) free budesonide, or (3) vehicle. Drug efficacy and colitic healing was determined by measuring gross colonic ulceration, myeloperoxidase activity, and in vivo colonic fluid absorption. Adrenal suppression was determined by measuring plasma adrenocorticotrophic hormone and serum corticosterone. Vehicle-treated colitis animals had gross ulceration, increased myeloperoxidase activity, and net colonic fluid secretion. Treatment with oral budesonide-beta-D-glucuronide accelerated all measures of colitis healing at a fourfold lower dose than did free budesonide. Furthermore, treatment with budesonide-beta-D-glucuronide did not result in adrenal suppression whereas free budesonide treatment did. A newly synthesised orally administered glucocorticoid-conjugate accelerates colitis healing with limited adrenal suppression. Development of an orally administered colon-specific steroid delivery system represents a novel approach to inflammatory bowel disease treatment. PMID:7959202

13-cis-retinoic acid metabolism in vivo. The major tissue metabolites in the rat have the all-trans configuration.

PubMed

McCormick, A M; Kroll, K D; Napoli, J L

1983-08-02

The liver and intestinal metabolites of orally dosed 13-cis-[11-3H]retinoic acid were analyzed in normal and 13-cis-retinoic acid treated rats 3 h after administration of the radiolabeled retinoid. all-trans-Retinoic acid was identified as a liver and intestinal mucosa metabolite in normal rats given physiological doses of 13-cis-[3H]retinoic acid. all-trans-Retinoyl glucuronide was identified as the most abundant radiolabeled metabolite in mucosa and a prominent liver metabolite under the same conditions. Thus, the major 13-cis-retinoic acid metabolites retained in liver and mucosa, two retinoid target tissues, had the all-trans configuration. These data indicate that the isomerization of 13-cis-retinoic acid to all-trans-retinoic acid and the subsequent conversion to all-trans-retinoyl glucuronide are central events in the in vivo metabolism of 13-cis-retinoic acid in the rat. Moreover, the all-trans-retinoic acid detected in vivo could account for a significant fraction of the physiological activity of 13-cis-retinoic acid. The tissue disposition and metabolism of orally dosed 13-cis-[3H]retinoic acid are modulated by retinoid treatment. Chronic 13-cis-retinoic acid treatment apparently increased the intestinal accumulation of all-trans-retinoic acid, all-trans-retinoyl glucuronide, and 13-cis-retinoyl glucuronide. The liver concentrations of tritiated all-trans-retinoic acid and all-trans-retinoyl glucuronide were also elevated in 13-cis-retinoic acid treated rats.

Expression of β-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

PubMed

Cheng, C-M; Chen, F M; Lu, Y-L; Tzou, S-C; Wang, J-Y; Kao, C-H; Liao, K-W; Cheng, T-C; Chuang, C-H; Chen, B-M; Roffler, S; Cheng, T-L

2013-05-01

Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

Physiological barriers to the oral delivery of curcumin.

PubMed

Berginc, K; Trontelj, J; Basnet, N Skalko; Kristl, A

2012-06-01

Curcumin, a principal component from Curcuma longa, with antioxidant and anti-inflammatory activities was proposed as a potential candidate for the preventation and/or treatment of cancer and chronic diseases. However, curcumin could not achieve its expected therapeutic outcome in clinical trials due to its low solubility and poor bioavailability. The actual intestinal physiological barriers limiting curcumin absorption after oral administration have not been fully investigated. To identify the main barriers curtailing its absorption, in vitro permeability of curcumin and flux of its glucuronide were monitored in rat jejunum and Transwell grown Caco-2 cells. Curcumin was more permeable under acidic conditions, but the permeability was substantially below the permeability of highly permeable standards. Its efflux could not be inhibited by specific Pgp and MRP inhibitors. BCRP was found to participate in curcumin transport, but the Organic Anion Transporting Polypeptide (OATP) did not. The permeability of curcumin significantly increased when the structure of mucus was compromised. The inhibitor of curcumin metabolism, piperin, failed to act as a permeability enhancer. Piperin inhibited Pgp and MRP transporters and decreased the amount of glucuronide transported back into the intestine. Inclusion of piperin in curcumin-containing formulations is highly recommended as to inhibit curcumin glucuronidation and to increase the transport of formed glucuronides into the plasma, therefore increasing the probability of glucuronide distribution into target tissue and inter-convertion to curcumin. It would also be beneficial, if curcumin delivery systems could reversibly compromise the mucous integrity to minimize the non-specific binding of curcumin to its constituents.

Bioavailability of trans-resveratrol from red wine in humans.

PubMed

Vitaglione, Paola; Sforza, Stefano; Galaverna, Gianni; Ghidini, Cristiana; Caporaso, Nicola; Vescovi, Pier Paolo; Fogliano, Vincenzo; Marchelli, Rosangela

2005-05-01

Many in vitro studies demonstrated significant biological effects of trans-resveratrol. Thus, understanding the rate of intestinal absorption and metabolization in vivo of trans-resveratrol is the prerequisite to evaluate its potential health impact. Bioavailability studies mainly in animals or in humans using the pure compound at very high doses were performed. In this work, trans-resveratrol bioavailability from a moderate consumption of red wine in 25 healthy humans has been studied by three different experiments. The wine ingestion was associated to three different dietary approaches: fasting, a standard meal, a meal with high and low amount of lipids. Trans-resveratrol 3- and 4'-glucuronides were synthesized, purified, and characterized as pure standards. Bioavailability data were obtained by measuring the concentration of free, 3-glucuronide and 4'-glucuronide trans-resveratrol by high-performance liquid chromatography (HPLC), both with ultraviolet (UV) and mass spectrometry (MS) detection, in serum samples taken at different times after red wine administration. Free trans-resveratrol was found, in trace amounts, only in some serum samples collected 30 min after red wine ingestion while after longer times resveratrol glucuronides predominated. Trans-resveratrol bioavailability was shown to be independent from the meal or its lipid content. The finding in human serum of trans-resveratrol glucuronides, rather than the free form of the compound, with a high interindividual variability, raises some doubts about the health effects of dietary resveratrol consumption and suggests that the benefits associated to red wine consumption could be probably due to the whole antioxidant pool present in red wine.

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.

Human urine and plasma concentrations of bisphenol A extrapolated from pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and semi-physiological pharmacokinetic modeling.

PubMed

Miyaguchi, Takamori; Suemizu, Hiroshi; Shimizu, Makiko; Shida, Satomi; Nishiyama, Sayako; Takano, Ryohji; Murayama, Norie; Yamazaki, Hiroshi

2015-06-01

The aim of this study was to extrapolate to humans the pharmacokinetics of estrogen analog bisphenol A determined in chimeric mice transplanted with human hepatocytes. Higher plasma concentrations and urinary excretions of bisphenol A glucuronide (a primary metabolite of bisphenol A) were observed in chimeric mice than in control mice after oral administrations, presumably because of enterohepatic circulation of bisphenol A glucuronide in control mice. Bisphenol A glucuronidation was faster in mouse liver microsomes than in human liver microsomes. These findings suggest a predominantly urinary excretion route of bisphenol A glucuronide in chimeric mice with humanized liver. Reported human plasma and urine data for bisphenol A glucuronide after single oral administration of 0.1mg/kg bisphenol A were reasonably estimated using the current semi-physiological pharmacokinetic model extrapolated from humanized mice data using algometric scaling. The reported geometric mean urinary bisphenol A concentration in the U.S. population of 2.64μg/L underwent reverse dosimetry modeling with the current human semi-physiological pharmacokinetic model. This yielded an estimated exposure of 0.024μg/kg/day, which was less than the daily tolerable intake of bisphenol A (50μg/kg/day), implying little risk to humans. Semi-physiological pharmacokinetic modeling will likely prove useful for determining the species-dependent toxicological risk of bisphenol A. Copyright © 2015 Elsevier Inc. All rights reserved.

Challenges in the indirect quantitation of acyl-glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards.

PubMed

Srinivasan, Karthik; Nouri, Parya; Kavetskaia, Olga

2010-07-01

This paper describes the quantitation of acyl-glucuronide metabolites (M26 and M5) of a cardiovascular-drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base-treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base-hydrolysis of an acyl-glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether-glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet-chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites. Copyright (c) 2009 John Wiley & Sons, Ltd.

Cremophor EL-based nanoemulsion enhances transcellular permeation of emodin through glucuronidation reduction in UGT1A1-overexpressing MDCKII cells.

PubMed

Zhang, Tianpeng; Dong, Dong; Lu, Danyi; Wang, Shuai; Wu, Baojian

2016-03-30

Oral emodin, a natural anthraquinone and active component of many herbal medicines, is poorly bioavailable because of extensive first-pass glucuronidation. Here we aimed to prepare emodin nanoemulsion (EMO-NE) containing cremophor EL, and to assess its potential for enhancing transcellular absorption of emodin using UGT1A1-overexpressing MDCKII cells (or MDCK1A1 cells). EMO-NE was prepared using a modified emulsification technique and subsequently characterized by particle size, morphology, stability, and drug release. MDCKII cells were stably transfected with UGT1A1 using the lentiviral transfection approach. Emodin transport and metabolism were evaluated in Transwell-cultured MDCK1A1 cells after apical dosing of EMO-NE or control solution. The obtained EMO-NE (116 ± 6.5 nm) was spherical and stable for at least 2 months. Emodin release in vitro was a passive diffusion-driven process. EMO-NE administration increased the apparent permeability of emodin by a 2.3-fold (p<0.001) compared to the pure emodin solution (1.2 × 10(-5) cm/s vs 5.3 × 10(-6) cm/s). Further, both apical and basolateral excretion of emodin glucuronide (EMO-G) were significantly decreased (≥56.5%, p<0.001) in EMO-NE group. This was accompanied by a marked reduction (57.4%, p<0.001) in total emodin glucuronidation. It was found that the reduced glucuronidation was due to inhibition of cellular metabolism by cremophor EL. Cremophor EL inhibited UGT1A1-mediated glucuronidation of emodin using the mixed-type inhibition mechanism. In conclusion, cremophor EL-based nanoemulsion greatly enhanced transcellular permeation of emodin through inhibition of UGT metabolism. This cremophor EL-based nanoformulation may be a promising strategy to improve the oral bioavailability of emodin. Copyright © 2016 Elsevier B.V. All rights reserved.

Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups

PubMed Central

Murphy, Sharon E.; Park, Sung-Shim L.; Thompson, Elizabeth F.; Wilkens, Lynne R.; Patel, Yesha; Stram, Daniel O.; Le Marchand, Loic

2014-01-01

Nicotine metabolism influences smoking behavior and differences in metabolism probably contribute to ethnic variability in lung cancer risk. We report here on the proportion of nicotine metabolism by cytochrome P450 2A6-catalyzed C-oxidation, UDP-glucuronosyl transferase 2B10 (UGT2B10)-catalyzed N-glucuronidation and flavin monooxygenase 3-catalyzed N-oxidation in five ethnic/racial groups and the role of UGT2B10 genotype on the metabolic patterns observed. Nicotine and its metabolites were quantified in urine from African American (AA, n = 364), Native Hawaiian (NH, n = 311), White (n = 437), Latino (LA, n = 453) and Japanese American (JA, n = 674) smokers. Total nicotine equivalents, the sum of nicotine and six metabolites, and nicotine metabolism phenotypes were calculated. The relationship of UGT2B10 genotype to nicotine metabolic pathways was determined for each group; geometric means were computed and adjusted for age, sex, creatinine, and body mass index. Nicotine metabolism patterns were unique across the groups, C-oxidation was lowest in JA and NH (P < 0.0001), and N-glucuronidation lowest in AA (P < 0.0001). There was no difference in C-oxidation among Whites and AA and LA. Nicotine and cotinine glucuronide ratios were 2- and 3-fold lower in AA compared with Whites. Two UGT variants, a missense mutation (Asp67Tyr, rs61750900) and a splice variant (rs116294140) accounted for 33% of the variation in glucuronidation. In AA, the splice variant accounted for the majority of the reduced nicotine glucuronidation. UGT2B10 variant allele carriers had increased levels of C-oxidation (P = 0.0099). Our data indicate that the relative importance of nicotine metabolic pathways varies by ethnicity, and all pathways should be considered when characterizing the role of nicotine metabolism on smoking behavior and cancer risk. PMID:25233931

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

PubMed Central

Koszewski, Nicholas J.; Haynes, Joseph S.; Horst, Ronald L.

2012-01-01

1,25-Dihydroxyvitamin D3 [1,25(OH)2D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)2D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)2D from 1,25-dihydroxyvitamin D3-25-β-glucuronide [β-gluc-1,25(OH)2D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)2D than 1,25(OH)2D, demonstrating targeted delivery of 1,25(OH)2D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)2D or β-gluc-1,25(OH)2D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)2D with 25-hydroxyvitamin D3-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)2D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)2D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)2D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model. PMID:22114117

The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and β-glucuronidase in human colorectal tumours

PubMed Central

Tobin, Peter; Clarke, Stephen; Seale, J Paul; Lee, Soon; Solomon, Michael; Aulds, Sally; Crawford, Michael; Gallagher, James; Eyers, Tony; Rivory, Laurent

2006-01-01

Aims Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by β-glucuronidase may represent a significant pathway of SN-38 formation. Methods The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 µm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). Results The rate of conversion of irinotecan (9.6 µM) was lower in tumour tissue than matched normal colon mucosa samples (0.30 ± 0.14 pmol min−1 mg−1 protein and 0.77 ± 0.59 pmol min−1 mg−1 protein, respectively; P < 0.005). In contrast, no significant difference was observed in β-glucuronidase activity between tumour and matched normal colon samples (4.56 ± 6.9 pmol min−1 mg−1 protein and 3.62 ± 2.95 pmol min−1 mg−1 protein, respectively, using 9.6 µM SN-38 glucuronide; P > 0.05). β-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2 > 0.23, P < 0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of β-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P < 0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. Conclusions Tumour β-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo. PMID:16842384

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

PubMed Central

Miki, Satomi; Shiba, Yuko; Minekawa, Shoko; Nishikawa, Tomomi; Mukai, Rie; Terao, Junji; Kawai, Yoshichika

2013-01-01

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites. PMID:24260490

Cysteine Scanning Mutagenesis of Transmembrane Domain 10 in Organic Anion Transporting Polypeptide 1B1

PubMed Central

2015-01-01

Organic anion transporting polypeptide (OATP) 1B1 is an important drug transporter expressed in human hepatocytes. Previous studies have indicated that transmembrane (TM) domain 2, 6, 8, 9, and in particular 10 might be part of the substrate binding site/translocation pathway. To explore which amino acids in TM10 are important for substrate transport, we mutated 34 amino acids individually to cysteines, expressed them in HEK293 cells, and determined their surface expression. Transport activity of the two model substrates estrone-3-sulfate and estradiol-17β-glucuronide as well as of the drug substrate valsartan for selected mutants was measured. Except for F534C and F537C, all mutants were expressed at the plasma membrane of HEK293 cells. Mutants Q541C and A549C did not transport estradiol-17β-glucuronide and showed negligible estrone-3-sulfate transport. However, A549C showed normal valsartan transport. Pretreatment with the anionic and cell impermeable sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) affected the transport of each substrate differently. Pretreatment of L545C abolished estrone-3-sulfate uptake almost completely, while it stimulated estradiol-17β-glucuronide uptake. Further analyses revealed that mutant L545C in the absence of MTSES showed biphasic kinetics for estrone-3-sulfate that was converted to monophasic kinetics with a decreased apparent affinity, explaining the previously seen inhibition. In contrast, the apparent affinity for estradiol-17β-glucuronide was not changed by MTSES treatment, but the Vmax value was increased about 4-fold, explaining the previously seen stimulation. Maleimide labeling of L545C was affected by preincubation with estrone-3-sulfate but not with estradiol-17β-glucuronide. These results strongly suggest that L545C is part of the estrone-3-sulfate binding site/translocation pathway but is not directly involved in binding/translocation of estradiol-17β-glucuronide. PMID:24673529

Tissue and species differences in the glucuronidation of glabridin with UDP-glucuronosyltransferases.

PubMed

Guo, Bin; Fang, Zhongze; Yang, Lu; Xiao, Ling; Xia, Yangliu; Gonzalez, Frank J; Zhu, Liangliang; Cao, Yunfeng; Ge, Guangbo; Yang, Ling; Sun, Hongzhi

2015-04-25

Glabridin (GA) has gained wide application in the cosmetics and food industry. This study was performed to investigate its metabolic inactivation and elimination by glucuronidation by use of liver and intestine microsomes from humans (HLM and HIM) and rats (RLM and RIM), and liver microsomes from cynomolgus monkeys and beagle dogs (CyLM and DLM). Both hydroxyl groups at the C2 and C4 positions of the B ring are conjugated to generate two mono-glucuronides (M1 and M2). HIM, RIM and RLM showed the most robust activity in catalyzing M2 formation with intrinsic clearance values (Clint) above 2000 μL/min/mg, with little measurable M1 formation activity. DLM displayed considerable activity both in M1 and M2 formation, with Clint values of 71 and 214 μL/min/mg, respectively, while HLM and CyLM exhibited low activities in catalyzing M1 and M2 formation, with Clint values all below 20 μL/min/mg. It is revealed that UGT1A1, 1A3, 1A9, 2B7, 2B15 and extrahepatic UGT1A8 and 1A10 are involved in GA glucuronidation. Nearly all UGTs preferred M2 formation except for UGT1A1. Notably, UGT1A8 displayed the highest activity with a Clint value more than 5-fold higher than the other isoforms. Chemical inhibition studies, using selective inhibitors of UGT1A1, 1A9, 2B7 and 1A8, further revealed that UGT1A8 contributed significantly to intestinal GA glucuronidation in humans. In summary, this in vitro study demonstrated large species differences in GA glucuronidation by liver and intestinal microsomes, and that intestinal UGTs are important for the pathway in humans. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Development and validation of an UPLC-MS/MS method for the quantification of irinotecan, SN-38 and SN-38 glucuronide in plasma, urine, feces, liver and kidney: Application to a pharmacokinetic study of irinotecan in rats.

PubMed

Basu, Sumit; Zeng, Min; Yin, Taijun; Gao, Song; Hu, Ming

2016-03-15

The objective of this research is to develop and validate a sensitive and reproducible UPLC-MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88-10000 nM, 39-5000 nM, 48.8-6250 nM and 48.8-6250 nM, respectively (R(2)>0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25-2000 nM, 4.88-1250 nM, 9.8-1250 nM and 9.8-1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC-MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 μl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples. Published by Elsevier B.V.

Development and validation of an UPLC-MS/MS method for the quantification of irinotecan, SN 38 and SN-38 glucuronide in plasma, urine, feces, liver and kidney: Application to a pharmacokinetic study of irinotecan in rats

PubMed Central

Basu, Sumit; Zeng, Min; Yin, Taijun; Gao, Song; Hu, Ming

2016-01-01

The objective of this research is to develop and validate a sensitive and reproducible UPLC-MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88 –10000 nM, 39 – 5000 nM, 48.8 –6250 nM and 48.8 – 6250 nM, respectively (R2 > 0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25 – 2000 nM, 4.88 – 1250 nM, 9.8 – 1250 nM and 9.8 – 1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC-MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 µl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples. PMID:26894853

Voucher-Based Reinforcement for Alcohol Abstinence Using the Ethyl-Glucuronide Alcohol Biomarker

ERIC Educational Resources Information Center

McDonell, Michael G.; Howell, Donelle N,; McPherson, Sterling; Cameron, Jennifer M.; Srebnik, Debra; Roll, John M.; Ries, Richard K.

2012-01-01

This study assessed the effects of a contingency management (CM) intervention for alcohol consumption in 10 alcohol-dependent participants. An ABCA design was used. Vouchers were provided contingent on results of ethyl glucuronide (EtG) urine tests (an alcohol biomarker with a 2-day detection period) and alcohol breath tests during the C phase.…

RELATIONSHIP BETWEEN HEPATIC MICROSOMAL THYROXINE GLUCURONIDATION AND TOTAL SERUM THYROXINE CONCENTRATIONS IN RATS TREATED WITH PCDDS, PCDFS AND PCBS

EPA Science Inventory

RELATIONSHIP BETWEEN HEPATIC MICROSOMAL THYROXINE GLUCURONIDATION AND TOTAL SERUM THYROXINE CONCENTRATIONS IN RATS TREATED WITH PCDDs, PCDFs AND PCBs. D G Ross, K M Crofton, M J DeVito, NHEERL, ORD, USEPA, RTP, NC.
Many PHAHs decrease thyroxine (T4), possibly due to inducti...

Gender and oral contraceptive steroids as determinants of drug glucuronidation: effects on clofibric acid elimination.

PubMed Central

Miners, J O; Robson, R A; Birkett, D J

1984-01-01

The disposition of clofibric acid, a drug metabolised largely by glucuronidation, was studied in eight males, eight females and eight females receiving oral contraceptive steroids (OCS). Clofibric acid plasma clearance was not significantly different in males compared to the control female group but was 48% greater (P less than 0.01) in women receiving OCS compared to non-pill using females. This difference was due to an alteration in clofibric acid metabolic clearance as there were no differences between the groups in clofibric acid free fraction. Along with previous data the results suggest that induction of drug glucuronidation by OCS may be of clinical importance, although any sex-related differences are unlikely to be of clinical significance. PMID:6487463

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry.

PubMed

Pettersson Bergstrand, Madeleine; Meyer, Markus R; Beck, Olof; Helander, Anders

2018-03-01

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites. Copyright © 2017 John Wiley & Sons, Ltd.

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

PubMed

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

2009-11-11

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

Internal Hydrolysis Indicator for Sample Specific Monitoring of β-Glucuronidase Activity.

PubMed

Taylor, Lacy L; Flint, Noah A; Ma, Vinh; Hill, Brandy M; Clark, Chantry J; Strathmann, Frederick G

2017-06-01

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Resveratrol-3-O-glucuronide and resveratrol-4’-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin

USDA-ARS?s Scientific Manuscript database

Resveratrol has been reported to inhibit or induce DNA damage depending upon the type of cell and experimental conditions. Dietary resveratrol is present in the body mostly as metabolites and little is known about the activities of these metabolic products. We evaluated physiologically obtainable ...

Effect of piperine on the bioavailability and pharmacokinetics of emodin in rats.

PubMed

Di, Xin; Wang, Xin; Di, Xin; Liu, Youping

2015-11-10

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) has been widely used as a traditional medicine and was shown to possess a multitude of health-promoting properties in pre-clinical studies, but its bioavailability was low due to the extensive glucuronidation in liver and intestine, hindering the development of emodin as a feasible chemopreventive agent. In this study, piperine, as a bioenhancer, was used to enhance the bioavailability of emodin by inhibiting its glucuronidation. The pharmacokinetic profiles of emodin after oral administration of emodin (20mg/kg) alone and in combination with piperine (20mg/kg) to rats were investigated via a validated LC/MS/MS method. As the in vivo pharmacokinetic studies had indicated, the AUC and Cmax of emodin were increased significantly after piperine treatment, and the glucuronidation of emodin was markedly inhibited. Our study demonstrated that piperine significantly improved the in vivo bioavailability of emodin and the influence of piperine on the pharmacokinetics of emodin may be attributed to the inhibition of glucuronidation of emodin. Further research is needed to investigate the detailed mechanism of improved bioavailability of emodin via its combination with piperine. Copyright © 2015 Elsevier B.V. All rights reserved.

HPLC-DAD-ESI/MS(n) analysis of phenolic compounds for quality control of Grindelia robusta Nutt. and bioactivities.

PubMed

Ferreres, Federico; Grosso, Clara; Gil-Izquierdo, Angel; Valentão, Patrícia; Azevedo, Carolina; Andrade, Paula B

2014-06-01

The phenolic composition of herbal tea (HT) and hydromethanolic extract (HME) obtained from Grindelia robusta Nutt. was studied by HPLC-DAD-ESI/MS(n). Thirty six flavonoids and hydroxycinnamic acids were detected, from which thirty are described for the first time in this species. Quantification by HPLC-DAD showed that diosmetin-7-O-glucuronide-3'-O-pentoside+apigenin-7-O-glucuronide-4'-O-pentoside, apigenin-7-O-glucuronide+diosmetin-7-O-glucuronide and 3,5-dicaffeoylquinic acid+3,4-dicaffeoylquinic acid were the major compounds. Since the health-promoting effects of natural phenolic compounds against brain disorders is of increasing interest, HT and HME were also tested against oxygen and nitrogen reactive species and against enzymes related with Alzheimer's disease and depression. Extracts displayed strong in vitro scavenging activity and monoamine oxidase-A (MAO-A) inhibitory activity. The anti-MAO-A capacity was observed at non-toxic concentrations for SH-SY5Y human neuroblastoma cell line, reinforcing the benefits of G. robusta HT. However, no protection against hydrogen peroxide treatment was observed. Copyright © 2014 Elsevier B.V. All rights reserved.

Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.

PubMed

Thevis, Mario; Dib, Josef; Thomas, Andreas; Höppner, Sebastian; Lagojda, Andreas; Kuehne, Dirk; Sander, Mark; Opfermann, Georg; Schänzer, Wilhelm

2015-01-01

Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2)  = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer's disease

PubMed Central

Ho, Lap; Ferruzzi, Mario G.; Janle, Elsa M.; Wang, Jun; Gong, Bing; Chen, Tzu-Ying; Lobo, Jessica; Cooper, Bruce; Wu, Qing Li; Talcott, Stephen T.; Percival, Susan S.; Simon, James E.; Pasinetti, Giulio Maria

2013-01-01

Epidemiological and preclinical studies indicate that polyphenol intake from moderate consumption of red wines may lower the relative risk for developing Alzheimer's disease (AD) dementia. There is limited information regarding the specific biological activities and cellular and molecular mechanisms by which wine polyphenolic components might modulate AD. We assessed accumulations of polyphenols in the rat brain following oral dosage with a Cabernet Sauvignon red wine and tested brain-targeted polyphenols for potential beneficial AD disease-modifying activities. We identified accumulations of select polyphenolic metabolites in the brain. We demonstrated that, in comparison to vehicle-control treatment, one of the brain-targeted polyphenol metabolites, quercetin-3-O-glucuronide, significantly reduced the generation of β-amyloid (Aβ) peptides by primary neuron cultures generated from the Tg2576 AD mouse model. Another brain-targeted metabolite, malvidin-3-O-glucoside, had no detectable effect on Aβ generation. Moreover, in an in vitro analysis using the photo-induced cross-linking of unmodified proteins (PICUP) technique, we found that quercetin-3-O-glucuronide is also capable of interfering with the initial protein-protein interaction of Aβ1–40 and Aβ1–42 that is necessary for the formation of neurotoxic oligomeric Aβ species. Lastly, we found that quercetin-3-O-glucuronide treatment, compared to vehicle-control treatment, significantly improved AD-type deficits in hippocampal formation basal synaptic transmission and long-term potentiation, possibly through mechanisms involving the activation of the c-Jun N-terminal kinases and the mitogen-activated protein kinase signaling pathways. Brain-targeted quercetin-3-O-glucuronide may simultaneously modulate multiple independent AD disease-modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.—Ho, L., Ferruzzi, M. G., Janle, E. M., Wang, J., Gong, B., Chen, T.-Y., Lobo, J., Cooper, B., Wu, Q. L., Talcott, S. T., Percival, S. S., Simon, J. E., Pasinetti, G. M. Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer's disease. PMID:23097297

Red wine and component flavonoids inhibit UGT2B17 in vitro

PubMed Central

2012-01-01

Background The metabolism and excretion of the anabolic steroid testosterone occurs by glucuronidation to the conjugate testosterone glucuronide which is then excreted in urine. Alterations in UGT glucuronidation enzyme activity could alter the rate of testosterone excretion and thus its bioavailability. The aim of this study is to investigate if red wine, a common dietary substance, has an inhibitory effect on UGT2B17. Methods Testosterone glucuronidation was assayed using human UGT2B17 supersomes with quantification of unglucuronidated testosterone over time using HPLC with DAD detection. The selected red wine was analyzed using HPLC; and the inhibitory effects of the wine and phenolic components were tested independently in a screening assay. Further analyses were conducted for the strongest inhibitors at physiologically relevant concentrations. Control experiments were conducted to determine the effects of the ethanol on UGT2B17. Results Over the concentration range of 2 to 8%, the red wine sample inhibited the glucuronidation of testosterone by up to 70% over 2 hours. The ethanol content had no significant effect. Three red wine phenolics, identified by HPLC analyses, also inhibited the enzyme by varying amounts in the order of quercetin (72%), caffeic acid (22%) and gallic acid (9%); using a ratio of phenolic:testosterone of 1:2.5. In contrast p-coumaric acid and chlorogenic acid had no effect on the UGT2B17. The most active phenolic was selected for a detailed study at physiologically relevant concentrations, and quercetin maintained inhibitory activity of 20% at 2 μM despite a ten-fold excess of testosterone. Conclusion This study reports that in an in vitro supersome-based assay, the key steroid-metabolizing enzyme UGT2B17 is inhibited by a number of phenolic dietary substances and therefore may reduce the rate of testosterone glucuronidation in vivo. These results highlight the potential interactions of a number of common dietary compounds on testosterone metabolism. Considering the variety of foodstuffs that contain flavonoids, it is feasible that diet can elevate levels of circulating testosterone through reduction in urinary excretion. These results warrant further investigation and extension to a human trial to delineate the health implications. PMID:22958586

Diabetes mellitus reduces activity of human UDP-glucuronosyltransferase 2B7 in liver and kidney leading to decreased formation of mycophenolic acid acyl-glucuronide metabolite.

PubMed

Dostalek, Miroslav; Court, Michael H; Hazarika, Suwagmani; Akhlaghi, Fatemeh

2011-03-01

Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients.

Circulating sex hormones in relation to anthropometric, sociodemographic and behavioural factors in an international dataset of 12,300 men

PubMed Central

Appleby, Paul N.; Albanes, Demetrius; Black, Amanda; Chan, June M.; Chen, Chu; Cirillo, Piera M.; Cohn, Barbara A.; Cook, Michael B.; Donovan, Jenny L.; Ferrucci, Luigi; Garland, Cedric F.; Giles, Graham G.; Goodman, Phyllis J.; Habel, Laurel A.; Haiman, Christopher A.; Holly, Jeff M. P.; Hoover, Robert N.; Kaaks, Rudolf; Knekt, Paul; Kolonel, Laurence N.; Kubo, Tatsuhiko; Le Marchand, Loïc; Luostarinen, Tapio; MacInnis, Robert J.; Mäenpää, Hanna O.; Männistö, Satu; Metter, E. Jeffrey; Milne, Roger L.; Nomura, Abraham M. Y.; Oliver, Steven E.; Parsons, J. Kellogg; Peeters, Petra H.; Platz, Elizabeth A.; Riboli, Elio; Ricceri, Fulvio; Rinaldi, Sabina; Rissanen, Harri; Sawada, Norie; Schaefer, Catherine A.; Schenk, Jeannette M.; Stanczyk, Frank Z.; Stampfer, Meir; Stattin, Pär; Stenman, Ulf-Håkan; Tjønneland, Anne; Trichopoulou, Antonia; Thompson, Ian M.; Tsugane, Shoichiro; Vatten, Lars; Whittemore, Alice S.; Ziegler, Regina G.

2017-01-01

Introduction Sex hormones have been implicated in the etiology of a number of diseases. To better understand disease etiology and the mechanisms of disease-risk factor associations, this analysis aimed to investigate the associations of anthropometric, sociodemographic and behavioural factors with a range of circulating sex hormones and sex hormone-binding globulin. Methods Statistical analyses of individual participant data from 12,330 male controls aged 25–85 years from 25 studies involved in the Endogenous Hormones Nutritional Biomarkers and Prostate Cancer Collaborative Group. Analysis of variance was used to estimate geometric means adjusted for study and relevant covariates. Results Older age was associated with higher concentrations of sex hormone-binding globulin and dihydrotestosterone and lower concentrations of dehydroepiandrosterone sulfate, free testosterone, androstenedione, androstanediol glucuronide and free estradiol. Higher body mass index was associated with higher concentrations of free estradiol, androstanediol glucuronide, estradiol and estrone and lower concentrations of dihydrotestosterone, testosterone, sex hormone-binding globulin, free testosterone, androstenedione and dehydroepiandrosterone sulfate. Taller height was associated with lower concentrations of androstenedione, testosterone, free testosterone and sex hormone-binding globulin and higher concentrations of androstanediol glucuronide. Current smoking was associated with higher concentrations of androstenedione, sex hormone-binding globulin and testosterone. Alcohol consumption was associated with higher concentrations of dehydroepiandrosterone sulfate, androstenedione and androstanediol glucuronide. East Asians had lower concentrations of androstanediol glucuronide and African Americans had higher concentrations of estrogens. Education and marital status were modestly associated with a small number of hormones. Conclusion Circulating sex hormones in men are strongly associated with age and body mass index, and to a lesser extent with smoking status and alcohol consumption. PMID:29281666

Assessing cannabis consumption frequency: Is the combined use of free and glucuronidated THCCOOH blood levels of diagnostic utility?

PubMed

Hädener, Marianne; Martin Fabritius, Marie; König, Stefan; Giroud, Christian; Weinmann, Wolfgang

2017-07-01

Heavy cannabis consumption is considered incompatible with safe driving. In Swiss traffic policy, drivers suspected of regular cannabis use are therefore required to undergo medical assessment of their long-term fitness to drive. A whole blood concentration of the cannabis metabolite 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THCCOOH) of 40 µg/L is currently used by Swiss forensic experts as the decision limit for regular cannabis consumption. The present study aimed to investigate the suitability of THCCOOH-glucuronide blood levels as an additional and/or better marker for the frequency of cannabis use. Whole blood samples collected from 23 heavy (≥10 joints/month) and 25 occasional smokers (≥1 joint/month, but ≤ 1 joint/week) enrolled in a placebo-controlled cannabis smoking study were analyzed for THCCOOH and THCCOOH-glucuronide. Based on receiver operating characteristic (ROC) curve analysis, concentration thresholds could be established for distinguishing between these two groups. Proposed thresholds for heavy use were THCCOOH-glucuronide > 52 µg/L (100% specificity; 41% sensitivity) and/or total THCCOOH > 58 µg/L (100% specificity; 43% sensitivity). Optimum thresholds for occasional use were THCCOOH-glucuronide < 5 µg/L (73% specificity; 97% sensitivity) and/or total THCCOOH < 5 µg/L (62% specificity; 98% sensitivity). Our results indicate that the THCCOOH-glucuronide whole blood concentration is a useful parameter that complements the free THCCOOH level to assess the frequency of cannabis consumption. The consideration of the blood concentrations of both free and glucuronidated THCCOOH improves the identification of heavy users whose fitness to drive has to be carefully assessed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Chocolate Matrix Factors Modulate Pharmacokinetic Behavior of Cocoa Flavan-3-Ol Phase-II Metabolites Following Oral Consumption by Sprague-Dawley Rats

PubMed Central

Neilson, Andrew P.; Sapper, Teryn N.; Janle, Elsa M.; Rudolph, Ralf; Matusheski, Nathan V.; Ferruzzi, Mario G.

2010-01-01

The impact of carbohydrates and milk on the bioavailability of catechin (C) and epicatechin (EC) from chocolate has been previously studied. However, little data exists regarding potential modulation of the phase-II metabolism by these chocolate matrix factors. The objectives of this study were to assess the impact of matrix composition on qualitative and quantitative profiles of circulating catechins and their metabolites following administration of commercially relevant chocolate confections. Sprague-Dawley rats were fed 1.5 g of a confection (reference dark, high sucrose, or milk chocolate) by intragastric gavage, and plasma samples were collected over 8 h. HPLC-MS analysis was performed to quantify C, EC and their metabolites. The predominant metabolites were O-glucuronides (2 metabolites), and O-Me-O-glucuronides (3 metabolites). Plasma concentrations of metabolites were generally the highest for high sucrose treatment and lowest for milk treatment, while reference dark treatment generally resulted in intermediate concentrations. The O-Me-(±)-C/EC-O-β-glucuronide (peak 4) was significantly higher for the high sucrose treatment (2325 nM*h) versus the milk treatment (1300 nM*h). Additionally, CMAX values for (±)-C/EC-O-β-glucuronide (peak 3), and two O-Me-(±)-C/EC-O-β-glucuronides (peaks 4 and 6) were significantly higher for high sucrose treatment (4012, 518, and 2518 nM, respectively) versus the milk treatment (2590, 240, and 1670 nM, respectively). Milk and sucrose appear to modulate both metabolism and plasma pharmacokinetics, and to a lesser extent, the overall bioavailability of catechins from chocolate confections. PMID:20446738

Chocolate matrix factors modulate the pharmacokinetic behavior of cocoa flavan-3-ol phase II metabolites following oral consumption by Sprague-Dawley rats.

PubMed

Neilson, Andrew P; Sapper, Teryn N; Janle, Elsa M; Rudolph, Ralf; Matusheski, Nathan V; Ferruzzi, Mario G

2010-06-09

The impact of carbohydrates and milk on the bioavailability of catechin (C) and epicatechin (EC) from chocolate has been previously studied. However, little data exist regarding potential modulation of the phase II metabolism by these chocolate matrix factors. The objectives of this study were to assess the impact of matrix composition on qualitative and quantitative profiles of circulating catechins and their metabolites following administration of commercially relevant chocolate confections. Sprague-Dawley rats were administered 1.5 g of a confection (reference dark, high sucrose, or milk chocolate) by intragastric gavage, and plasma samples were collected over 8 h. High-performance liquid chromatography-mass spectrometry analysis was performed to quantify C, EC, and their metabolites. The predominant metabolites were O-glucuronides (two metabolites) and O-Me-O-glucuronides (three metabolites). Plasma concentrations of metabolites were generally the highest for high sucrose treatment and lowest for milk treatment, while the reference dark treatment generally resulted in intermediate concentrations. The O-Me-(+/-)-C/EC-O-beta-glucuronide (peak 4) was significantly higher for the high sucrose treatment (2325 nM h) versus the milk treatment (1300 nM h). Additionally, C(MAX) values for (+/-)-C/EC-O-beta-glucuronide (peak 3) and two O-Me-(+/-)-C/EC-O-beta-glucuronides (peaks 4 and 6) were significantly higher for the high sucrose treatment (4012, 518, and 2518 nM, respectively) versus the milk treatment (2590, 240, and 1670 nM, respectively). Milk and sucrose appear to modulate both metabolism and plasma pharmacokinetics and, to a lesser extent, the overall bioavailability of catechins from chocolate confections.

Absorption and pharmacokinetics of grapefruit flavanones in beagles.

PubMed

Mata-Bilbao, Maria de Lourdes; Andrés-Lacueva, Cristina; Roura, Elena; Jáuregui, Olga; Escribano, Elvira; Torre, Celina; Lamuela-Raventós, Rosa Maria

2007-07-01

The present study evaluated the pharmacokinetics of three different grapefruit flavanone forms in dog plasma and demonstrated their absorption after an oral intake of a grapefruit extract; pharmacokinetic parameters of these forms were also determined. Ten healthy beagles were administered 70 mg citrus flavonoids as a grapefruit extract contained in capsules, while two additional dogs were used as controls and given an excipient. The grapefruit flavanone naringin, along with its metabolites naringenin and naringenin glucuronide, was detected in dog plasma. Blood samples were collected between 0 and 24 h after administration of the extract. Naringin reached its maximun plasma concentration at around 80 min, whereas naringenin and naringenin glucuronide reached their maximun plasma concentrations at around 20 and 30 min, respectively. Maximum plasma concentrations of naringin, naringenin and naringenin glucuronide (medians and ranges) were 0.24 (0.05-2.08), 0.021 (0.001-0.3) and 0.09 (0.034-0.12) micromol/l, respectively. The areas under the curves were 23.16 l (14.04-70.62) min x micromol/for nariningin, 1.78 (0.09-4.95) min x micromol/l for naringenin and 22.5 (2.74-99.23) min x micromol/l for naringenin glucuronide. The median and range values for mean residence time were 3.3 (1.5-9.3), 2.8 (0.8-11.2) and 8.0 (2.3-13.1) h for naringin, naringenin and naringenin glucuronide, respectively. The results of the present study demonstrate the absorption of grapefruit flavanones via the presence of their metabolites in plasma, thus making an important contribution to the field since the biological activities ascribed to these compounds rely on their specific forms of absorption.

Circulating sex hormones in relation to anthropometric, sociodemographic and behavioural factors in an international dataset of 12,300 men.

PubMed

Watts, Eleanor L; Appleby, Paul N; Albanes, Demetrius; Black, Amanda; Chan, June M; Chen, Chu; Cirillo, Piera M; Cohn, Barbara A; Cook, Michael B; Donovan, Jenny L; Ferrucci, Luigi; Garland, Cedric F; Giles, Graham G; Goodman, Phyllis J; Habel, Laurel A; Haiman, Christopher A; Holly, Jeff M P; Hoover, Robert N; Kaaks, Rudolf; Knekt, Paul; Kolonel, Laurence N; Kubo, Tatsuhiko; Le Marchand, Loïc; Luostarinen, Tapio; MacInnis, Robert J; Mäenpää, Hanna O; Männistö, Satu; Metter, E Jeffrey; Milne, Roger L; Nomura, Abraham M Y; Oliver, Steven E; Parsons, J Kellogg; Peeters, Petra H; Platz, Elizabeth A; Riboli, Elio; Ricceri, Fulvio; Rinaldi, Sabina; Rissanen, Harri; Sawada, Norie; Schaefer, Catherine A; Schenk, Jeannette M; Stanczyk, Frank Z; Stampfer, Meir; Stattin, Pär; Stenman, Ulf-Håkan; Tjønneland, Anne; Trichopoulou, Antonia; Thompson, Ian M; Tsugane, Shoichiro; Vatten, Lars; Whittemore, Alice S; Ziegler, Regina G; Allen, Naomi E; Key, Timothy J; Travis, Ruth C

2017-01-01

Sex hormones have been implicated in the etiology of a number of diseases. To better understand disease etiology and the mechanisms of disease-risk factor associations, this analysis aimed to investigate the associations of anthropometric, sociodemographic and behavioural factors with a range of circulating sex hormones and sex hormone-binding globulin. Statistical analyses of individual participant data from 12,330 male controls aged 25-85 years from 25 studies involved in the Endogenous Hormones Nutritional Biomarkers and Prostate Cancer Collaborative Group. Analysis of variance was used to estimate geometric means adjusted for study and relevant covariates. Older age was associated with higher concentrations of sex hormone-binding globulin and dihydrotestosterone and lower concentrations of dehydroepiandrosterone sulfate, free testosterone, androstenedione, androstanediol glucuronide and free estradiol. Higher body mass index was associated with higher concentrations of free estradiol, androstanediol glucuronide, estradiol and estrone and lower concentrations of dihydrotestosterone, testosterone, sex hormone-binding globulin, free testosterone, androstenedione and dehydroepiandrosterone sulfate. Taller height was associated with lower concentrations of androstenedione, testosterone, free testosterone and sex hormone-binding globulin and higher concentrations of androstanediol glucuronide. Current smoking was associated with higher concentrations of androstenedione, sex hormone-binding globulin and testosterone. Alcohol consumption was associated with higher concentrations of dehydroepiandrosterone sulfate, androstenedione and androstanediol glucuronide. East Asians had lower concentrations of androstanediol glucuronide and African Americans had higher concentrations of estrogens. Education and marital status were modestly associated with a small number of hormones. Circulating sex hormones in men are strongly associated with age and body mass index, and to a lesser extent with smoking status and alcohol consumption.

Quantification of major compounds from Ixeris dentata, Ixeris dentata Var. albiflora, and Ixeris sonchifolia and their comparative anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 cells.

PubMed

Karki, Subash; Park, Hee-Juhn; Nugroho, Agung; Kim, Eon Ji; Jung, Hyun Ah; Choi, Jae Sue

2015-01-01

The aim of the present study was to evaluate the comparative anti-inflammatory activities of Ixeris dentata (ID), Ixeris dentata var. albiflora (IDA), and Ixeris sonchifolia (IS) and to identify the main compounds present in extracts. The anti-inflammatory activity was evaluated through lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 murine macrophages. Five main compounds consisting of chlorogenic acid, caffeic acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, and luteolin were used for simultaneous high-performance liquid chromatography quantification. The total phenolic content present in ID (30 mg/g GAE), IDA (35.33 mg/g GAE), and IS (43.79 mg/g GAE) was correlated to the corresponding LPS-induced NO production inhibitory effect in RAW 264.7 cells as expressed with IC(50) values 26.19, 21.43, and 7.59 μg/mL, respectively. Luteolin 7-O-glucoside was found as the major compound in ID (8.76 mg/g dry weight) and IDA (10.35 mg/g dry weight) and luteolin 7-O-glucuronide was the major compound in IS (34.66 mg/g dry weight). Luteolin 7-O-glucoside and luteolin 7-O-glucuronide inhibited LPS-induced NO production with IC(50) values of 30 and 4.5 μM, respectively. Furthermore, luteolin, luteolin 7-O-glucoside, and luteolin 7-O-glucuronide suppressed the expression of iNOS and COX-2, and t-BHP-induced ROS generation in LPS-stimulated RAW 264.7 cells. These results clearly showed that the anti-inflammatory potential of ID, IDA, and IS extract are primarily due to their contents of luteolin 7-O-glucoside and luteolin 7-O-glucuronide, respectively.

Variable Isoflavone Contents of Red Clover Products Affect Intestinal Disposition of Biochanin A, Formononetin, Genistein and Daidzein

PubMed Central

Wang, Stephen W.J.; Chen, Yan; Joseph, Tiby; Hu, Ming

2009-01-01

Marketed red clover products use a wide variety of labels and the isoflavone contents from the lable is ambiguous. In the present study, we analyzed the content of various isoflavone products, and determined a) the content and b) how sample matrix of red clover products affects intestinal disposition of main isoflavones within it using the human intestinal Caco-2 cell model. Analysis using high and ultra-performance liquid chromatography indicates that the isoflavone content varied significantly (p<0.05) between the chosen products. Consequently, rates of isoflavone absorption across the Caco-2 cell monolayers varied (p<0.05) greatly. Unexpectedly, permeabilities of biochanin A and formononetin (two key biomarkers) were found to be significantly affected (p<0.05) by the product matrix. As expected, biochanin A was the only isoflavone with noticeable metabolite peaks in both apical and basolateral sides. Interestingly, rates of metabolism and the polarity of the glucuronidated biochanin A excretion were also significantly altered (p<0.05) by product matrix. Studies using breast cancer resistance protein inhibitor dipyridamole showed that both the apical and basolateral excretion of biochanin A glucuronides were significantly (P<0.05) reduced (7.5 and 9.4-fold, respectively) when dipyridamole is present. This provides evidence that BCRP is the main transporter responsible for the apical efflux of isoflavone glucuronides. In conclusion, the isoflavone contents of the marketed red clover products are highly variable, and product matrix significantly affected intestinal disposition of red clover isoflavones by altering their absorption rates, permeabilities, biochanin A glucuronide excretion rates, and the polarity of biochanin A glucuronide excretion. This research provides scientific evidence to support the standardization effort so that consumers can make intelligent product choices. PMID:18370585

Maternal Buprenorphine Dose, Placenta Buprenorphine and Metabolite Concentrations and Neonatal Outcomes

PubMed Central

Concheiro, Marta; Jones, Hendreé E.; Johnson, Rolley E.; Choo, Robin; Shakleya, Diaa M.; Huestis, Marilyn A.

2010-01-01

Buprenorphine is approved as pharmacotherapy for opioid dependence in non-pregnant patients in multiple countries, and is currently under investigation for pregnant women in the US and Europe. This research evaluates the disposition of buprenorphine, opiates, cocaine, and metabolites in 5 term placentas from a US cohort. Placenta and matched meconium concentrations were compared, and relationships between maternal buprenorphine dose, placenta concentrations, and neonatal outcomes following controlled administration during gestation were investigated. Buprenorphine and/or metabolites were detected in all placenta specimens and were uniformly distributed across this tissue (CV<27.5%, 4 locations), except for buprenorphine in 3 placentas. In 2 of these, buprenorphine was not detected in some locations and, in the 3rd placenta, was totally absent. Median (range) concentrations were buprenorphine 1.6ng/g (not detected to 3.2), norbuprenorphine 14.9ng/g (6.2 to 24.2), buprenorphine-glucuronide 3ng/g (1.3 to 5.0) and norbuprenorphine-glucuronide 14.7ng/g (11.4 to 25.8). Placenta is a potential alternative matrix for detecting in utero buprenorphine exposure, but at lower concentrations (15–70 fold) than in meconium. Statistically significant correlations were observed for mean maternal daily dose from enrollment to delivery and placenta buprenorphine-glucuronide concentration, and for norbuprenorphine-glucuronide concentrations and time to neonatal abstinence syndrome (NAS) onset and duration, and for norbuprenorphine/norbuprenorphine-glucuronide ratio and maximum NAS score, and newborn length. Analysis of buprenorphine and metabolites in this alternative matrix, an abundant waste product available at the time of delivery, may be valuable for prediction of neonatal outcomes for clinicians treating newborns of buprenorphine-exposed women. PMID:20216119

Development of a fast screening and confirmatory method by liquid chromatography-quadrupole-time-of-flight mass spectrometry for glucuronide-conjugated methyltestosterone metabolite in tilapia.

PubMed

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

2012-05-23

This paper describes the development of a fast method to screen and confirm methyltestosterone 17-O-glucuronide (MT-glu) in tilapia bile. The method consists of solid-phase extraction (SPE) followed by high-performance liquid chromatography-mass spectrometry. The system used was an Agilent 6530 Q-TOF with an Agilent Jet stream electrospray ionization interface. The glucuronide detected in the bile was characterized as MT-glu by comparison with a chemically synthesized standard. MT-glu was detected in bile for up to 7 days after dosing. Semiquantification was done with matrix-matched calibration curves, because MT-glu showed signal suppression due to matrix effects. This method provides a suitable tool to monitor the illegal use of methyltestosterone in tilapia culture.

Effects of kale ingestion on pharmacokinetics of acetaminophen in rats.

PubMed

Yamasaki, Izumi; Uotsu, Nobuo; Yamaguchi, Kohji; Takayanagi, Risa; Yamada, Yasuhiko

2011-12-01

Kale is a cruciferous vegetable (Brassicaceae) that contains a large amount of health-promoting phytochemicals. The chronic ingestion of cabbage of the same family is known to accelerate conjugating acetaminophen (AA) and decrease the plasma AA level. Therefore, we examined to clarify the effects of kale on the pharmacokinetics of AA, its glucuronide (AA-G) and sulfate (AA-S). AA was orally administered to rats pre-treated with kale or cabbage (2000 mg/kg/day) for one week. Blood samples were collected from the jugular vein, and the concentrations of AA, AA-G and AA-S were determined. In results, kale ingestion induced an increase in the area under the concentration-time curve (AUC) and a decrease in the clearance of AA, whereas cabbage had almost no influence. In addition, there were significant differences in the AUC of AA-G between the control and kale groups. mRNA expression levels of UDP-glucuronosyltransferases, the enzymes involved in glucuronidation, in the kale group were significantly higher than those in the control group. In conclusion, kale ingestion increased the plasma concentrations of both AA and AA-G. The results suggest that kale ingestion accelerates the glucuronidation of AA, but an increase of plasma AA levels has a different cause than the cause of glucuronidation.

Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

PubMed Central

Suominen, Tina; Uutela, Päivi; Ketola, Raimo A.; Bergquist, Jonas; Hillered, Lars; Finel, Moshe; Zhang, Hongbo; Laakso, Aki; Kostiainen, Risto

2013-01-01

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain. PMID:23826355

High pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

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

Sabourin, P.J.; Bechtold, W.E.; Henderson, R.F.

1988-05-01

The glucuronide and sulfate conjugates of benzene metabolite as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl (/sup 14/C)glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount ofmore » metabolite present in urine following exposure to (/sup 3/H)benzene was determined using p-nitrophenyl (/sup 14/C)glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm (/sup 3/H)benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.« less

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry.

PubMed

Kakimoto, Kensaku; Toriba, Akira; Ohno, Takanori; Ueno, Mariko; Kameda, Takayuki; Tang, Ning; Hayakawa, Kazuichi

2008-05-15

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.

Elucidation of markers for monitoring morphine and its analogs in urine adulterated with pyridinium chlorochromate.

PubMed

Luong, Susan; Kuzhiumparambil, Unnikrishnan; Fu, Shanlin

2015-09-17

Currently, procedures that identify the drugs 'destroyed' in adulterated urine specimens are very limited. This study aimed to determine the effect of pyridinium chlorochromate (PCC) on routine opiate assays and identify reaction products formed. Results/methodology: Opiate-positive urines adulterated with PCC (20 and 100 mM) were analyzed using CEDIA ® immunoassay and GC-MS. Urine and water samples spiked with 6-monoacetylmorphine, morphine and its glucuronides (10 µg/ml) and PCC (0.02-100 mM) were monitored with LC-MS, and the products characterized. PCC significantly decreased the abundance of morphine, codeine and IS. Adulterated water and urine samples containing 6-monoacetylmorphine, morphine and morphine-3-glucuronide yielded morphinone-3-glucuronide, 7,14-dihydroxy-6-monoacetylmorphine, 7,8-diketo-6-monoacetylmorphine and 7,8-diketo-morphine (tentative assignment). Reaction pathways may be different in the two matrices.

Favipiravir inhibits acetaminophen sulfate formation but minimally affects systemic pharmacokinetics of acetaminophen

PubMed Central

Zhao, Yanli; Harmatz, Jerold S; Epstein, Carol R; Nakagawa, Yukako; Kurosaki, Chie; Nakamura, Tetsuro; Kadota, Takumi; Giesing, Dennis; Court, Michael H; Greenblatt, David J

2015-01-01

Aims The antiviral agent favipiravir is likely to be co-prescribed with acetaminophen (paracetamol). The present study evaluated the possiblility of a pharmacokinetic interaction between favipiravir and acetaminophen, in vitro and in vivo. Methods The effect of favipivir on the transformation of acetaminophen to its glucuronide and sulfate metabolites was studied using a pooled human hepatic S9 fraction in vitro. The effect of acute and extended adminstration of favipiravir on the pharmacokinetics of acetaminophen and metabolites was evaluated in human volunteers. Results Favipiravir inhibited the in vitro formation of acetaminophen sulfate, but not acetaminophen glucuronide. In human volunteers, both acute (1 day) and extended (6 days) administration of favipiravir slightly but significantly increased (by about 20 %) systemic exposure to acetaminophen (total AUC), whereas Cmax was not significantly changed. AUC for acetaminophen glucuronide was increased by 23 to 35 % above control by favipiravir, while AUC for acetaminophen sulfate was reduced by about 20 % compared to control. Urinary excretion of acetaminophen sulfate was likewise reduced to 44 to 65 % of control values during favipiravir co-administration, while excretion of acetaminophen glucuronide increased to 17 to 32 % above control. Conclusion Favipiravir inhibits acetaminophen sulfate formation in vitro and in vivo. However the increase in systemic exposure to acetaminophen due to favipiravir co-administration, though statistically significant, is small in magnitude and unlikely to be of clinical importance. PMID:25808818

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

PubMed

Algeelani, Sara; Alam, Novera; Hossain, Md Amin; Mikus, Gerd; Greenblatt, David J

2018-08-01

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC 50 ) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC 50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC 50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

Quantification of conjugated metabolites of drugs in biological matrices after the hydrolysis with β-glucuronidase and sufatase: a review of bio-analytical methods.

PubMed

Ding, Yue; Peng, Ming; Zhang, Tong; Tao, Jian-Sheng; Cai, Zhen-Zhen; Zhang, Yong

2013-10-01

Glucuronidation and sulfation represent two major pathways in phase II drug metabolism in humans and other mammalian species. The great majority of drugs, for example, polyphenols, flavonoids and anthraquinones, could be transformed into sulfated and glucuronidated conjugates simultaneously and extensively in vivo. The pharmacological activities of drug conjugations are normally decreased compared with those of their free forms. However, some drug conjugates may either bear biological activities themselves or serve as excellent sources of biologically active compounds. As the bioactivities of drugs are thought to be relevant to the kinetics of their conjugates, it is essential to study the pharmacokinetic behaviors of the conjugates in more detail. Unfortunately, the free forms of drugs cannot be detected directly in most cases if their glucuronides and sulfates are the predominant forms in biological samples. Nevertheless, an initial enzymatic hydrolysis step using β-glucuronidase and/or sulfatase is usually performed to convert the glucuronidated and/or sulfated conjugates to their free forms prior to the extraction, purification and other subsequent analysis steps in the literature. This review provides fundamental information on drug metabolism pathways, the bio-analytical strategies for the quantification of various drug conjugates, and the applications of the analytical methods to pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.

Determination of bisphenol A, triclosan and their metabolites in human urine using isotope-dilution liquid chromatography-tandem mass spectrometry.

PubMed

Provencher, Gilles; Bérubé, René; Dumas, Pierre; Bienvenu, Jean-François; Gaudreau, Eric; Bélanger, Patrick; Ayotte, Pierre

2014-06-27

Bisphenol A (BPA) and triclosan (TCS) are ubiquitous environmental phenols exhibiting endocrine disrupting activities that may be involved in various health disorders in humans. There is a need to measure separately free forms and conjugated metabolites because only the former are biologically active. We have developed sensitive methods using isotope-dilution liquid chromatography-tandem mass spectrometry for individual measurements of free BPA and TCS as well as their metabolites, BPA glucuronide (BPAG), BPA monosulfate (BPAS), BPA disulfate (BPADS), TCS glucuronide (TCSG) and TCS sulfate (TCSS) in urine. Comparative analyses of urine samples from 46 volunteers living in the Quebec City area using the new methods and a GC-MS/MS method previously used in our laboratory revealed very strong correlations for total BPA (Spearman's rs=0.862, p<0.0001) and total TCS concentrations (rs=0.942, p<0.0001). Glucuronide metabolites were the most abundant BPA and TCS species in urine samples (>94% of total urinary concentrations). Unconjugated TCS concentrations represented a small proportion of total TCS species (median=1.6%) but its concentration was likely underestimated due to losses by adsorption to the surface of polypropylene tubes used for sample storage. To our knowledge, we are the first to report levels of free, sulfated and glucuronidated TCS levels in human urine. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation.

PubMed

Griffeth, L K; Rosen, G M; Rauckman, E J

1985-01-01

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.

Inhibition of intestinal microflora beta-glucuronidase modifies the distribution of the active metabolite of the antitumor agent, irinotecan hydrochloride (CPT-11) in rats.

PubMed

Takasuna, K; Hagiwara, T; Hirohashi, M; Kato, M; Nomura, M; Nagai, E; Yokoi, T; Kamataki, T

1998-01-01

SN-38, a metabolite of irinotecan hydrochloride (CPT-11), is considered to play a key role in the development of diarrhea as well as in the antitumor activity of CPT-11. We have previously found that the inhibition of beta-glucuronidase, which hydrolyzes detoxified SN-38 (SN-38 glucuronide) to reform SN-38, in the lumen by eliminating the intestinal microflora with antibiotics, markedly ameliorates the intestinal toxicity of CPT-11 in rats. In this study we compared the disposition of CPT-11 and its metabolites in rats treated with and without antibiotics. Rats were given drinking water containing 1 mg/ml penicillin and 2 mg/ml streptomycin from 5 days before the administration of CPT-11 (60 mg/kg i.v.) and throughout the experiment. CPT-11, SN-38 glucuronide and SN-38 concentrations in the blood, intestinal tissues and intestinal luminal contents were determined by HPLC. Antibiotics had little or no effect on the pharmacokinetics of CPT-11, SN-38 glucuronide or SN-38 in the blood, or in the tissues or contents of the small intestine, which has less beta-glucuronidase activity in its luminal contents. In contrast, antibiotics markedly reduced the AUC1-24 h of SN-38 (by about 85%) in the large intestine tissue without changing that of CPT-11, and this was accompanied by a complete inhibition of the deconjugation of SN-38 glucuronide in the luminal contents. These results suggest that SN-38, which results from the hydrolysis of SN-38 glucuronide by beta-glucuronidase in the intestinal microflora, contributes considerably to the distribution of SN-38 in the large intestine tissue, and that inhibition of the beta-glucuronidase activity by antibiotics results in decreased accumulation of SN-38 in the large intestine.

Quantification of Cannabinoids and their Free and Glucuronide Metabolites in Whole Blood by Disposable Pipette Extraction and Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Scheidweiler, Karl B.; Newmeyer, Matthew N.; Barnes, Allan J.; Huestis, Marilyn A.

2016-01-01

Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1 h for cannabidiol (CBD) and cannabinol (CBN) and THC-glucuronide in whole blood after smoking, suggesting their applicability for identifying recent intake. However, whole blood collection may not occur for up to 4 h during driving under the influence of drugs investigations, making a recent-use marker with a 6-8 h detection window helpful for improving whole blood cannabinoid interpretation. Other minor cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and its metabolite 11-nor-9-carboxy-THCV (THCVCOOH) might also be useful. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry method for quantification of THC, its phase I and glucuronide phase II metabolites, and 5 five minor cannabinoids. Cannabinoids were extracted from 200 μL whole blood via disposable pipette extraction, separated on a C18 column, and detected via electrospray ionization in negative mode with scheduled multiple reaction mass spectrometric monitoring. Linear ranges were 0.5-100 μg/L for THC and THCCOOH; 0.5-50 μg/L for 11-OH-THC, CBD, CBN, and THC-glucuronide; 1-50 μg/L for CBG, THCV, and THCVCOOH; and 5-500 μg/L for THCCOOH-glucuronide. Inter-day accuracy and precision at low, mid and high quality control (QC) concentrations were 95.1-113% and 2.4-8.5%, respectively (n=25). Extraction recoveries and matrix effects at low and high QC concentrations were 54.0-84.4% and −25.8-30.6%, respectively. By simultaneously monitoring multiple cannabinoids and metabolites, identification of recent cannabis administration or discrimination between licit medicinal and illicit recreational cannabis use can be improved. PMID:27236483

In vitro inhibitory effects of major bioactive constituents of Andrographis paniculata, Curcuma longa and Silybum marianum on human liver microsomal morphine glucuronidation: A prediction of potential herb-drug interactions arising from andrographolide, curcumin and silybin inhibition in humans.

PubMed

Uchaipichat, Verawan

2018-02-01

This study aimed to investigate the liver microsomal inhibitory effects of silybin, silychristin, andrographolide, and curcumin by using morphine as an in vitro UGT2B7 probe substrate, and predict the magnitude of the herb-drug interaction arising from these herbal constituents' inhibition in vivo. Studies were performed in the incubation with and without bovine serum albumin (BSA). Andrographolide and curcumin showed a marked inhibition on morphine 3- and 6-glucuronidation with IC 50 of 50&87 and 96&111 μM, respectively. In the presence of 2%BSA, andrographolide also showed a strong inhibition on morphine 3- and 6-glucuronidation (IC 50 4.4&21.6 μM) whereas curcumin showed moderate inhibition (IC 50 338&333 μM). In the absence and presence of 2%BSA, morphine 3- and 6-glucuronidation was moderately inhibited by silybin (IC 50 583&862 and 1252&1421 μM, respectively), however was weakly inhibited by silychristin (IC 50 3527&3504 and 1124&1530 μM, respectively). The K i of andrographolide, curcumin and silybin on morphine 3- and 6-glucuronidation were 7.1&9.5, 72.7&65.2, and 224.5&159.7 μM, respectively, while the respective values generated from the system containing 2%BSA were 2.4&3.1, 96.4&108.8, and 366.3&394.5 μM. Using the in vitro and in vivo extrapolation approach, andrographolide was herbal component that may have had a potential interaction in vivo when it was co-administered with morphine. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Absorption of N-phenylpropenoyl-L-amino acids in healthy humans by oral administration of cocoa (Theobroma cacao).

PubMed

Stark, Timo; Lang, Roman; Keller, Daniela; Hensel, Andreas; Hofmann, Thomas

2008-10-01

Besides flavan-3-ols, a family of N-phenylpropenoyl-L-amino acids (NPAs) has been recently identified as polyphenol/amino acid conjugates in the seeds of Theobroma cacao as well as in a variety of herbal drugs. Stimulated by reports on their biological activity, the purpose of this study was to investigate if these amides are absorbed by healthy volunteers after administration of a cocoa drink. For the first time, 12 NPAs were quantified in human urine by means of a stable isotope dilution analysis with LC-MS/MS (MRM) detection. A maximum amount was found in the urine taken 2 h after the cocoa consumption. The highest absolute amount of NPAs excreted with the urine was found for N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid (5), but the highest recovery rate (57.3 and 22.8%), that means the percentage amount of ingested amides excreted with the urine, were determined for N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid (6) and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine (13). In order to gain first insights into the NPA metabolism in vivo, urine samples were analyzed by LC-MS/MS before and after beta-glucuronidase/sulfatase treatment. As independent of the enzyme treatment the same NPA amounts were found in urine, there is strong evidence that these amides are metabolized neither via their O-glucuronides nor their O-sulfates. In order to screen for caffeic acid O-glucuronides as potential NPA metabolites, urine samples were screened by means of LC-MS/MS for caffeic acid 3-O-beta-D-glucuronide and 4-O-beta-D-glucuronide. But not even trace amounts of one of these glucuronides were detectable, thus excluding them as major NPA metabolites and underlining the importance of future investigations on a potential O-methylation or reduction of the N-phenylpropenoyl moiety in NPAs.

Quantification of cannabinoids and their free and glucuronide metabolites in whole blood by disposable pipette extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Scheidweiler, Karl B; Newmeyer, Matthew N; Barnes, Allan J; Huestis, Marilyn A

2016-07-01

Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1h for cannabidiol (CBD) and cannabinol (CBN) and Δ(9)-tetrahydrocannabinol (THC)-glucuronide in whole blood after smoking, suggesting their applicability for identifying recent intake. However, whole blood collection may not occur for up to 4h during driving under the influence of drugs investigations, making a recent-use marker with a 6-8h detection window helpful for improving whole blood cannabinoid interpretation. Other minor cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and its metabolite 11-nor-9-carboxy-THCV (THCVCOOH) might also be useful. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry method for quantification of THC, its phase I and glucuronide phase II metabolites, and 5 five minor cannabinoids. Cannabinoids were extracted from 200μL whole blood via disposable pipette extraction, separated on a C18 column, and detected via electrospray ionization in negative mode with scheduled multiple reaction mass spectrometric monitoring. Linear ranges were 0.5-100μg/L for THC and 11-nor-9-carboxy-THC (THCCOOH); 0.5-50μg/L for 11-hydroxy-THC (11-OH-THC), CBD, CBN, and THC-glucuronide; 1-50μg/L for CBG, THCV, and THCVCOOH; and 5-500μg/L for THCCOOH-glucuronide. Inter-day accuracy and precision at low, mid and high quality control (QC) concentrations were 95.1-113% and 2.4-8.5%, respectively (n=25). Extraction recoveries and matrix effects at low and high QC concentrations were 54.0-84.4% and -25.8-30.6%, respectively. By simultaneously monitoring multiple cannabinoids and metabolites, identification of recent cannabis administration or discrimination between licit medicinal and illicit recreational cannabis use can be improved. Published by Elsevier B.V.

Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection.

PubMed

Wang, Caihong; Wu, Caisheng; Zhang, Jinlan; Jin, Ying

2015-04-15

Prenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites. This study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWH-derived prenylflavonoid monomers in rats. The study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers. Major plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma. The pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for ∼99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)(0-96 h) values for total icaritin (24.1 vs. 32.0 mg/L h). Icaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates. Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.

Identification and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-COOH-glu) in hair by ultra-performance liquid chromatography tandem mass spectrometry as a potential hair biomarker of cannabis use.

PubMed

Pichini, Simona; Marchei, Emilia; Martello, Simona; Gottardi, Massimo; Pellegrini, Manuela; Svaizer, Fiorenza; Lotti, Andrea; Chiarotti, Marcello; Pacifici, Roberta

2015-04-01

We developed and validated an ultra-high-pressure liquid chromatography-tandem mass spectrometry method to identify and quantify 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide in hair of cannabis consumers. After hair washing with methyl alcohol and diethyl ether and subsequent addition of amiodarone as internal standard hair samples were treated with 500 μl VMA-T M3 buffer reagent for 1 h at 100 °C. After cooling, 10 μl VMA-T M3 extract were injected into chromatographic system. Chromatographic separation was carried out on a reversed phase column using a linear gradient elution with two solvents: 5 mM ammonium formate pH 3.0 (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The flow rate was kept constant at 0.4 ml/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode via positive electrospray ionization. Linear calibration curves were obtained for 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide with correlation coefficients (r(2)) of 0.99 and a limit of quantification of 0.25 pg/mg hair. Analytical recovery was between 79.6% and 100.7% and intra- and inter-assay imprecision and inaccuracy were always lower than 15%. Ultra-high-pressure liquid chromatography-tandem mass spectrometry analysis of 20 different hair samples of cannabis consumers disclosed the presence of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide in the range of 0.5-8.6 pg/mg hair. These data provided a good start to consider 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide as alternative hair biomarker of cannabis consumption. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A two-step ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry with mass defect filtering method for rapid identification of analogues from known components of different chemical structure types in Fructus Gardeniae-Fructus Forsythiae herb pair extract and in rat's blood.

PubMed

Zhou, Wei; Shan, Jinjun; Meng, Minxin

2018-08-17

Fructus Gardeniae-Fructus Forsythiae herb pair is an herbal formula used extensively to treat inflammation and fever, but few systematic identification studies of the bioactive components have been reported. Herein, the unknown analogues in the first-step screening were rapidly identified from representative compounds in different structure types (geniposide as iridoid type, crocetin as crocetin type, jasminoside B as monocyclic monoterpene type, oleanolic acid as saponin type, 3-caffeoylquinic acid as organic acid type, forsythoside A as phenylethanoid type, phillyrin as lignan type and quercetin 3-rutinoside as flavonoid type) by UPLC-Q-Tof/MS combined with mass defect filtering (MDF), and further confirmed with reference standards and published literatures. Similarly, in the second step, other unknown components were rapidly discovered from the compounds identified in the first step by MDF. Using the two-step screening method, a total of 58 components were characterized in Fructus Gardeniae-Fructus Forsythiae (FG-FF) decoction. In rat's blood, 36 compounds in extract and 16 metabolites were unambiguously or tentatively identified. Besides, we found the principal metabolites were glucuronide conjugates, with the glucuronide conjugates of caffeic acid, quercetin and kaempferol confirmed as caffeic acid 3-glucuronide, quercetin 3-glucuronide and kaempferol 3-glucuronide by reference standards, respectively. Additionally, most of them bound more strongly to human serum albumin than their respective prototypes, predicted by Molecular Docking and Simulation, indicating that they had lower blood clearance in vivo and possibly more contribution to pharmacological effects. This study developed a novel two-step screening method in addressing how to comprehensively screen components in herbal medicine by UPLC-Q-Tof/MS with MDF. Copyright © 2018 Elsevier B.V. All rights reserved.

Chemotaxonomic features associated with flavonoids of cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) in relation to hops (Humulus lupulus L.).

PubMed

Vanhoenacker, Gerd; Van Rompaey, Philippe; De Keukeleire, Denis; Sandra, Pat

2002-02-01

The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-beta-D-glucuronide (3), and apigenin-7-O-beta-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.

Olive oil phenolics are dose-dependently absorbed in humans.

PubMed

Visioli, F; Galli, C; Bornet, F; Mattei, A; Patelli, R; Galli, G; Caruso, D

2000-02-25

Olive oil phenolic constituents have been shown, in vitro, to be endowed with potent biological activities including, but not limited to, an antioxidant action. To date, there is no information on the absorption and disposition of such compounds in humans. We report that olive oil phenolics, namely tyrosol and hydroxytyrosol, are dose-dependently absorbed in humans after ingestion and that they are excreted in the urine as glucuronide conjugates. Furthermore, an increase in the dose of phenolics administered increased the proportion of conjugation with glucuronide.

Novel Strip Test for Circulating Hormones

DTIC Science & Technology

1996-10-01

estrone- 3 -glucuronide was combined with 1 equivalent of 4-amino-benzo-15-crown-5 and 1.2 equivalents of 1 -ethyl- 3 -( 3 - dimethylaminopropyl )carbodiimide...solution containing 5 mg/ml of El-g and 10 mg/ml of 1 -ethyl- 3 -( 3 -dimethylamino-propyl)carbodiimide (EDC) in water at pH 4.2. Fibers are then rinsed with... hydrochloride in water at pH 4.0. The mixture was stirred at room temperature for 48 hours and the resulting estrone- 3 -glucuronide-4-amino-benzo-15-crown

Impact of trans-resveratrol-sulfates and -glucuronides on endothelial nitric oxide synthase activity, nitric oxide release and intracellular reactive oxygen species.

PubMed

Ladurner, Angela; Schachner, Daniel; Schueller, Katharina; Pignitter, Marc; Heiss, Elke H; Somoza, Veronika; Dirsch, Verena M

2014-10-17

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings.

Increased synthesis of a new oleanane-type saponin in hairy roots of marigold (Calendula officinalis) after treatment with jasmonic acid.

PubMed

Markowski, Michał; Długosz, Marek; Szakiel, Anna; Durli, Mathieu; Poinsignon, Sophie; Bouguet-Bonnet, Sabine; Vernex-Loset, Lionel; Krier, Gabriel; Henry, Max

2018-04-18

Native plant of marigold (Calendula officinalis L.) synthesizes oleanolic acid saponins classified as glucosides or glucuronides according to the first residue in sugar chain bound to C-3 hydroxyl group. Hairy root culture, obtained by transformation with Agrobacterium rhizogenes strain 15834, exhibit a potent ability of synthesis of oleanolic acid glycosides. The HPLC profile of saponin fraction obtained from C. officinalis hairy roots treated with plant stress hormone, jasmonic acid, showed the 10-times increase of the content of one particular compound, determined by NMR and MALDI TOF as a new bisdesmoside saponin, 3-O-β-d-glucuronopyranosyl-28-O-β-d-galactopyranosyl-oleanolic acid. Such a diglycoside does not occur in native C. officinalis plant. It is a glucuronide, whereas in the native plant glucuronides are mainly accumulated in flowers, while glucosides are the most abundant saponins in roots. Thus, our results revealed that the pathways of saponin biosynthesis, particularly reactions of glycosylation, are altered in C. officinalis hairy root culture.

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 Skin-Absorbed Resveratrol into Its Glucuronized Form in Mouse Skin

PubMed Central

Pluskal, Tomáš; Ito, Ken; Hori, Kousuke; Ebe, Masahiro; Yanagida, Mitsuhiro; Kondoh, Hiroshi

2014-01-01

Resveratrol (RESV) is a plant polyphenol, which is thought to have beneficial metabolic effects in laboratory animals as well as in humans. Following oral administration, RESV is immediately catabolized, resulting in low bioavailability. This study compared RESV metabolites and their tissue distribution after oral uptake and skin absorption. Metabolomic analysis of various mouse tissues revealed that RESV can be absorbed and metabolized through skin. We detected sulfated and glucuronidated RESV metabolites, as well as dihydroresveratrol. These metabolites are thought to have lower pharmacological activity than RESV. Similar quantities of most RESV metabolites were observed 4 h after oral or skin administration, except that glucuronidated RESV metabolites were more abundant in skin after topical RESV application than after oral administration. This result is consistent with our finding of glucuronidated RESV metabolites in cultured skin cells. RESV applied to mouse ears significantly suppressed inflammation in the TPA inflammation model. The skin absorption route could be a complementary, potent way to achieve therapeutic effects with RESV. PMID:25506824

Evaluation of the Potential Pharmacokinetic Interaction between Atomoxetine and Fluvoxamine in Healthy Volunteers.

PubMed

Todor, Ioana; Popa, Adina; Neag, Maria; Muntean, Dana; Bocsan, Corina; Buzoianu, Anca; Vlase, Laurian; Gheldiu, Ana-Maria; Briciu, Corina

2017-01-01

Attention deficit hyperactivity disorder (ADHD) is frequently associated with other psychiatric pathologies. Therefore, the present study investigated a possible pharmacokinetic interaction between atomoxetine (ATX), a treatment option for ADHD, and an antidepressant, namely, fluvoxamine (FVX). Designed as an open-label, non-randomized clinical trial, the study included 2 periods. In period 1 (reference), each subject received ATX 25 mg (single-dose), whereas in period 2 (test), all subjects were given a combination of ATX 25 mg + FVX 100 mg, following a 6-day pretreatment regimen with the enzymatic inhibitor. Non-compartmental methods were employed to determine the pharmacokinetic parameters of ATX and its main active metabolite (glucuronidated form), 4-hydroxyatomoxetine-O-glucuronide. The results revealed significant differences between the study periods for Cmax, AUC0-t and AUC0-∞ values corresponding to ATX and its metabolite. Small, but statistically significant increases in AUC values were reported for both parent drug (1,583.05 ± 1,040.29 vs. 2,111.55 ± 1,411.59 ng*h/ml) and 4-hydroxyatomoxetine-O-glucuronide (5,754.71 ± 1,235.5 vs. 6,293.17 ± 1,219.34 ng*h/ml) after combined treatment of ATX and the enzymatic inhibitor. FVX had a modest effect on the pharmacokinetics of ATX and 4-hydroxyatomoxetine-O-glucuronide. The presence or absence of any clinical consequences associated with this pharmacokinetic drug-drug interaction needs to be established in future studies. © 2016 S. Karger AG, Basel.

Conceptual biotransformation of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide in rat whole embryo culture.

PubMed

Creech Kraft, J; Juchau, M R

1992-05-28

In cultured rat conceptuses, intraamniotic microinjections of 2500 ng/mL of 4-oxo-13-cis-retinoic acid, 600 ng/mL 4-oxo-all-trans-retinoic acid or 4000 ng/mL all-trans-retinoyl-beta-glucuronide, produce qualitatively and quantitatively similar patterns of dysmorphogenesis as those reported after the intraamniotic microinjection of 250 ng/mL all-trans-retinoic acid [Lee et al., Teratology 44: 313-323, 1991; Creech Kraft et al., Teratology 45: 259-270, 1992]. In the present study, we utilized HPLC techniques to analyze retinoid levels in cultured rat conceptuses, 1.5 hr after intraamniotic microinjections of 4-oxo-13-cis-retinoic acid (2500 ng/mL), 4-oxo-all-trans-retinoic acid (600 ng/mL) or all-trans-retinoyl-beta-glucuronide (4000 ng/mL). Our findings show that, after the microinjections of 4-oxo-all-trans-retinoic acid or 4-oxo-13-cis-retinoic acid (at these selected concentrations), 4-oxo-all-trans-retinoic acid was predominant in the embryos proper at concentrations of about 200 nM. This was roughly equivalent to the levels of all-trans-retinoic acid assayed after microinjections of all-trans-retinoyl-beta-glucuronide (4000 ng/mL). We conclude from these studies that both 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid behave as ultimate or proximate dysmorphogens.

An activity-based near-infrared glucuronide trapping probe for imaging β-glucuronidase expression in deep tissues.

PubMed

Cheng, Ta-Chun; Roffler, Steve R; Tzou, Shey-Cherng; Chuang, Kuo-Hsiang; Su, Yu-Cheng; Chuang, Chih-Hung; Kao, Chien-Han; Chen, Chien-Shu; Harn, I-Hong; Liu, Kuan-Yi; Cheng, Tian-Lu; Leu, Yu-Ling

2012-02-15

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.

Role of gemfibrozil as an inhibitor of CYP2C8 and membrane transporters.

PubMed

Tornio, Aleksi; Neuvonen, Pertti J; Niemi, Mikko; Backman, Janne T

2017-01-01

Cytochrome P450 (CYP) 2C8 is a drug metabolizing enzyme of major importance. The lipid-lowering drug gemfibrozil has been identified as a strong inhibitor of CYP2C8 in vivo. This effect is due to mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide. In vivo, gemfibrozil is a fairly selective CYP2C8 inhibitor, which lacks significant inhibitory effect on other CYP enzymes. Gemfibrozil can, however, have a smaller but clinically meaningful inhibitory effect on membrane transporters, such as organic anion transporting polypeptide 1B1 and organic anion transporter 3. Areas covered: This review describes the inhibitory effects of gemfibrozil on CYP enzymes and membrane transporters. The clinical drug interactions caused by gemfibrozil and the different mechanisms contributing to the interactions are reviewed in detail. Expert opinion: Gemfibrozil is a useful probe inhibitor of CYP2C8 in vivo, but its effect on membrane transporters has to be taken into account in study design and interpretation. Moreover, gemfibrozil could be used to boost the pharmacokinetics of CYP2C8 substrate drugs. Identification of gemfibrozil 1-O-β-glucuronide as a potent mechanism-based inhibitor of CYP2C8 has led to recognition of glucuronide metabolites as perpetrators of drug-drug interactions. Recently, also acyl glucuronide metabolites of clopidogrel and deleobuvir have been shown to strongly inhibit CYP2C8.

HSCCC-based strategy for preparative separation of in vivo metabolites after administration of an herbal medicine: Saussurea laniceps, a case study.

PubMed

Yi, Tao; Zhu, Lin; Zhu, Guo-Yuan; Tang, Yi-Na; Xu, Jun; Fan, Jia-Yi; Zhao, Zhong-Zhen; Chen, Hu-Biao

2016-09-13

This paper reports a novel strategy based on high-speed counter-current chromatography (HSCCC) technique to separate in vivo metabolites from refined extract of urine after administration of an herbal medicine. Saussurea laniceps (SL) was chosen as a model herbal medicine to be used to test the feasibility of our proposed strategy. This strategy succeeded in the case of separating four in vivo metabolites of SL from the urine of rats. Briefly, after oral administration of SL extract to three rats for ten days (2.0 g/kg/d), 269.1 mg of umbelliferone glucuronide (M1, purity, 92.5%), 432.5 mg of scopoletin glucuronide (M2, purity, 93.2%), 221.4 mg of scopoletin glucuronide (M3, purity, 92.9%) and 319.0 mg of scopoletin glucuronide (M4, purity, 90.4%) were separated from 420 mL of the rat urine by HSCCC using a two-phase solvent system composed of methyl tert-butyl ether-n-butanol-acetonitrile-water (MTBE-n-BuOH-ACN-H2O) at a volume ratio of 10:30:11:49. The chemical structures of the four metabolites, M1 to M4, were confirmed by MS and (1)H, (13)C NMR. As far as we know, this is the first report of the successful separation of in vivo metabolites by HSCCC after administration of an herbal medicine.

PET imaging of β-glucuronidase activity by an activity-based 124I-trapping probe for the personalized glucuronide prodrug targeted therapy.

PubMed

Su, Yu-Cheng; Cheng, Ta-Chun; Leu, Yu-Ling; Roffler, Steve R; Wang, Jaw-Yuan; Chuang, Chih-Hung; Kao, Chien-Han; Chen, Kai-Chuan; Wang, Hsin-Ell; Cheng, Tian-Lu

2014-12-01

Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity-based trapping probe for positron emission tomography (PET). We generated a (124)I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form (124)I-TrapG that could be selectively activated by βG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of (124)I-TrapG. βG targeting of (124)I-TrapG in vivo was examined by micro-PET. The biodistribution of (131)I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at βG-expressing CT26 (CT26/mβG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. (124)I-TrapG preferentially accumulated in CT26/mβG but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that (124)I-TrapG signals in CT26/mβG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous βG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. (124)I-TrapG exhibited low cytotoxicity allowing long-term monitoring of βG activity in vivo to aid in the optimization of prodrug targeted therapy. ©2014 American Association for Cancer Research.

Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of cannabinoids and phase I and II metabolites in meconium.

PubMed

Prego-Meleiro, Pablo; Lendoiro, Elena; Concheiro, Marta; Cruz, Angelines; López-Rivadulla, Manuel; de Castro, Ana

2017-05-12

A liquid chromatography-tandem mass spectrometry (LC-MSMS) method was developed and fully validated for the determination of Δ 9 -tetrahydrocannabinol (THC), 11-hydroxyTHC (OHTHC), 11-nor-9-carboxyTHC (THCCOOH), 8-β-11-dihydroxyTHC (diOHTHC), cannabinol, cannabidiol, and THC and THCCOOH glucuronides in 0.25±0.02g meconium. Samples were homogenized in methanol and subjected to cation exchange solid-phase extraction. Chromatographic separation was performed on a Kinetex C18 column (50 mm×2.1mm, 2.6μm) at 35°C, with a gradient of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min; total run time was 10min. Two transitions per analyte were monitored in MRM mode. The method was specific and sensitive; LOD was from 1 to 2ng/g, and LOQ from 4 to 10ng/g; linearity ranged from 4 to 400 ng/g for all the analytes, except for THC glucuronide (10-400ng/g); intra-assay, inter-assay and total imprecision were <11.2%, <13.45% and <15.6%, respectively; accuracy ranged from 93.9% to 109.0% of the target concentration; matrix effect, extraction and process efficiency ranged from -26.4% to -71.4%, 49.9% to 69.5% and 14.3% to 45.0%, respectively. The inclusion of THC and THCCOOH glucuronides avoided the need for the hydrolysis process, thus facilitating sample pretreatment. Application of the method to 19 authentic meconium specimens from uncontrolled pregnancies or women suspicious of drug consumption revealed fetal cannabis exposure in 4 newborns. THCCOOH (24.1-288.8ng/g), diOHTHC (53.2-332.4ng/g), THC (4.2-7.7ng/g), CBN (30.7-93.3ng/g) and CBD (7.1-251.5ng/g) were detected in all cases; THCCOOH glucuronide (190.2-306.8ng/g) in 3 cases; and OHTHC (11.9ng/g) in the remaining one; however, THC glucuronide was not identified in any specimen. Copyright © 2017 Elsevier B.V. All rights reserved.

Race, Gender, and Genetic Polymorphism Contribute to Variability in Acetaminophen Pharmacokinetics, Metabolism, and Protein-Adduct Concentrations in Healthy African-American and European-American Volunteers.

PubMed

Court, Michael H; Zhu, Zhaohui; Masse, Gina; Duan, Su X; James, Laura P; Harmatz, Jerold S; Greenblatt, David J

2017-09-01

Over 30 years ago, black Africans from Kenya and Ghana were shown to metabolize acetaminophen faster by glucuronidation and slower by oxidation compared with white Scottish Europeans. The objectives of this study were to determine whether similar differences exist between African-Americans and European-Americans, and to identify genetic polymorphisms that could explain these potential differences. Acetaminophen plasma pharmacokinetics and partial urinary metabolite clearances via glucuronidation, sulfation, and oxidation were determined in healthy African-Americans (18 men, 23 women) and European-Americans (34 men, 20 women) following a 1-g oral dose. There were no differences in acetaminophen total plasma, glucuronidation, or sulfation clearance values between African-Americans and European-Americans. However, median oxidation clearance was 37% lower in African-Americans versus European-Americans (0.57 versus 0.90 ml/min per kilogram; P = 0.0001). Although acetaminophen total or metabolite clearance values were not different between genders, shorter plasma half-life values (by 11-14%; P < 0.01) were observed for acetaminophen, acetaminophen glucuronide, and acetaminophen sulfate in women versus men. The UGT2B15*2 polymorphism was associated with variant-allele-number proportional reductions in acetaminophen total clearance (by 15-27%; P < 0.001) and glucuronidation partial clearance (by 23-48%; P < 0.001). UGT2B15 *2/*2 genotype subjects also showed higher acetaminophen protein-adduct concentrations than *1/*2 (by 42%; P = 0.003) and *1/*1 (by 41%; P = 0.003) individuals. Finally, CYP2E1 *1D/*1D genotype African-Americans had lower oxidation clearance than *1C/*1D (by 42%; P = 0.041) and *1C/*1C (by 44%; P = 0.048) African-Americans. Consequently, African-Americans oxidize acetaminophen more slowly than European-Americans, which may be partially explained by the CYP2E1*1D polymorphism. UGT2B15*2 influences acetaminophen pharmacokinetics in both African-Americans and European-Americans. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

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

PubMed

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

2014-01-01

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

Assessment of human deoxynivalenol exposure using an LC-MS/MS based biomarker method.

PubMed

Warth, Benedikt; Sulyok, Michael; Fruhmann, Philipp; Berthiller, Franz; Schuhmacher, Rainer; Hametner, Christian; Adam, Gerhard; Fröhlich, Johannes; Krska, Rudolf

2012-05-20

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcA's), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcA's) was estimated to be 20.4±2.4 μg L⁻¹ (max. 63 μg L⁻¹). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcA's were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcA's in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of quercetin and quercetin 3-glucuronide on the expression of bone sialoprotein gene.

PubMed

Kim, Dong-Soon; Takai, Hideki; Arai, Masato; Araki, Shouta; Mezawa, Masaru; Kawai, Yoshichika; Murota, Kaeko; Terao, Junji; Ogata, Yorimasa

2007-06-01

Quercetin is a typical flavonol-type flavonoid and is present in a variety of vegetables, and their antioxidant effect implies their possible role in the prevention of oxidative stress related chronic diseases. Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite crystals. Previously, we reported that isoflavone (genistein) activated BSP gene transcription is mediated through an inverted CCAAT box in the proximal BSP gene promoter. The present study investigates the regulation of BSP transcription in a rat osteoblast-like cell line, ROS 17/2.8 cells, by quercetin and its conjugated metabolite quercetin 3-glucuronide. Quercetin and quercetin 3-glucuronide (5 microM) increased the BSP mRNA levels at 12 h and quercetin upregulated the Cbfa1/Runx2 mRNA expression at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, quercetin increased the luciferase activity of the construct (pLUC3), including the promoter sequence nucleotides -116 to -43. Transcriptional stimulations by quercetin were almost completely abrogated in the constructs that included 2 bp mutations in the inverted CCAAT and FRE elements whereas the CCAAT-protein complex did not change after stimulation by quercetin according to gel shift assays. Quercetin increased the nuclear protein binding to the FRE and 3'-FRE. These data suggest that quercetin and quercetin 3-glucuronide increased the BSP mRNA expression, and that the inverted CCAAT and FRE elements in the promoter of the BSP gene are required for quercetin induced BSP transcription.

Paracetamol pharmacokinetics and metabolism in young women.

PubMed

Allegaert, Karel; Peeters, Mariska Y; Beleyn, Bjorn; Smits, Anne; Kulo, Aida; van Calsteren, Kristel; Deprest, Jan; de Hoon, Jan; Knibbe, Catherijne A J

2015-11-13

There is relevant between individual variability in paracetamol clearance in young women. In this pooled study, we focused on the population pharmacokinetic profile of intravenous paracetamol metabolism and its covariates in young women. Population PK parameters using non-linear mixed effect modelling were estimated in a pooled dataset of plasma and urine PK studies in 69 young women [47 at delivery, 8/47 again 10-15 weeks after delivery (early postpartum), and 7/8 again 1 year after delivery (late postpartum), 22 healthy female volunteers with or without oral contraceptives]. Population PK parameters were estimated based on 815 plasma samples and 101 urine collections. Compared to healthy female volunteers (reference group) not on oral contraceptives, being at delivery was the most significant covariate for clearance to paracetamol glucuronide (Factor = 2.03), while women in early postpartum had decreased paracetamol glucuronidation clearance (Factor = 0.55). Women on contraceptives showed increased paracetamol glucuronidation clearance (Factor = 1.46). The oestradiol level did not further affect this model. Being at delivery did not prove significant for clearance to paracetamol sulphate, but was higher in pregnant women who delivered preterm (<37 weeks, Factor = 1.34) compared to term delivery and non-pregnant women. Finally, clearance of unchanged paracetamol was dependent on urine flow rate. Compared to healthy female volunteers not on oral contraceptives, urine paracetamol glucuronidation elimination in young women is affected by pregnancy (higher), early postpartum (lower) or exposure to oral contraceptives (higher), resulting in at least a two fold variability in paracetamol clearance in young women.

The pharmacokinetics and disposition of MK-0524, a Prosglandin D2 Receptor 1 antagonist, in rats, dogs and monkeys.

PubMed

Chang, S W; Reddy, V; Pereira, T; Dean, B J; Xia, Y-Q; Seto, C; Franklin, R B; Karanam, B V

2007-05-01

MK-0524 is a potent, selective and orally active Prosglandin D(2) Receptor 1 (DP(1)) antagonist currently under clinical development for the treatment of niacin-induced flushing. Experiments to study the pharmacokinetics, metabolism and excretion of MK-0524 were conducted in rats, dogs and monkeys. MK-0524 displayed linear kinetics and rapid absorption following an oral dose. Following intravenous (i.v.) administration of MK-0524 to rats and dogs (1 and 5 mg/kg), the mean Cl(p) was approximately 2 and approximately 6 ml/min/kg, the T(1/2) was approximately 7 and approximately 13 h and the Vd(ss) was approximately 1 and approximately 5 L/kg, respectively. In monkeys dosed i.v. at 3 mg/kg, the corresponding values were 8 ml/min/kg, 3 h and 1 L/kg, respectively. Following oral dosing of MK-0524 to rats (5, 25 and 100 mg/kg), dogs (5 mg/kg) and monkeys (3 mg/kg), the absorption was rapid with the mean C(max) occurring between 1 and 4 h. Absolute oral bioavailability values in rats, dogs and monkeys were 50, 70 and 8%, respectively. The major circulating metabolite was the acyl glucuronide of MK-0524 (M2), with ratios of glucuronide to the parent aglycone being highest in the monkey followed by dog and rat. In bile duct-cannulated rats and dogs, MK-0524 was eliminated primarily via acyl glucuronidation followed by biliary excretion of the acyl glucuronide, M2, the major drug-related entity in bile.

Glucuronidated Quercetin Lowers Blood Pressure in Spontaneously Hypertensive Rats via Deconjugation

PubMed Central

Galindo, Pilar; Rodriguez-Gómez, Isabel; González-Manzano, Susana; Dueñas, Montserrat; Jiménez, Rosario; Menéndez, Carmen; Vargas, Félix; Tamargo, Juan; Santos-Buelga, Celestino; Pérez-Vizcaíno, Francisco; Duarte, Juan

2012-01-01

Background Chronic oral quercetin reduces blood pressure and restores endothelial dysfunction in hypertensive animals. However, quercetin (aglycone) is usually not present in plasma, because it is rapidly metabolized into conjugated, mostly inactive, metabolites. The aim of the study is to analyze whether deconjugation of these metabolites is involved in the blood pressure lowering effect of quercetin. Methodology/Principal Findings We have analyzed the effects on blood pressure and vascular function in vitro of the conjugated metabolites of quercetin (quercetin-3-glucuronide, Q3GA; isorhamnetin-3-glucuronide, I3GA; and quercetin-3′-sulfate, Q3'S) in spontaneously hypertensive rats (SHR). Q3GA and I3GA (1 mg/kg i.v.), but not Q3'S, progressively reduced mean blood pressure (MBP), measured in conscious SHR. The hypotensive effect of Q3GA was abolished in SHR treated with the specific inhibitor of β-glucuronidase, saccharic acid 1,4-lactone (SAL, 10 mg/ml). In mesenteric arteries, unlike quercetin, Q3GA had no inhibitory effect in the contractile response to phenylephrine after 30 min of incubation. However, after 1 hour of incubation Q3GA strongly reduced this contractile response and this effect was prevented by SAL. Oral administration of quercetin (10 mg/Kg) induced a progressive decrease in MBP, which was also suppressed by SAL. Conclusions Conjugated metabolites are involved in the in vivo antihypertensive effect of quercetin, acting as molecules for the plasmatic transport of quercetin to the target tissues. Quercetin released from its glucuronidated metabolites could be responsible for its vasorelaxant and hypotensive effect. PMID:22427863

Impact of Trans-Resveratrol-Sulfates and -Glucuronides on Endothelial Nitric Oxide Synthase Activity, Nitric Oxide Release and Intracellular Reactive Oxygen Species

PubMed Central

Ladurner, Angela; Schachner, Daniel; Schueller, Katharina; Pignitter, Marc; Heiss, Elke H.; Somoza, Veronika; Dirsch, Verena M.

2015-01-01

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings. PMID:25329867

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans.

PubMed

Bartolomé, Begoña; Monagas, María; Garrido, Ignacio; Gómez-Cordovés, Carmen; Martín-Alvarez, Pedro J; Lebrón-Aguilar, Rosa; Urpí-Sardà, Mireia; Llorach, Rafael; Andrés-Lacueva, Cristina

2010-09-01

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism. 2010 Elsevier Inc. All rights reserved.

Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice.

PubMed

Medina, S; Ferreres, F; García-Viguera, C; Horcajada, M N; Orduna, J; Savirón, M; Zurek, G; Martínez-Sanz, J M; Gil, J I; Gil-Izquierdo, A

2013-01-15

Citrus juice intake has been highlighted because of its health-promoting effects. LC-MS based metabolomics approaches are applied to obtain a better knowledge on changes in the concentration of metabolites due to its dietary intake and allow a better understanding of involved metabolic pathways. Eight volunteers daily consumed 400 mL of juice for four consecutive days and urine samples were collected before intake and 24h after each citrus juice intake. Urine samples were analysed by nanoHPLC-q-TOF, followed by principal component analysis (PCA) and Student's t-test (p<0.05). PCA showed a separation between two groups (before and after citrus juice consumption). This approach allowed the identification of four endocrine compounds (tetrahydroaldosterone-3-glucuronide, cortolone-3-glucuronide, testosterone-glucuronide and 17-hydroxyprogesterone), which belonged to the steroid biosynthesis pathway as significant metabolites upregulated by citrus juice intake. Additionally, these results confirmed the importance of using the non-targeted metabolomics technique to identify new endogenous metabolites, up- or down-regulated as a consequence of food intake. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlations between conceptal concentrations of all-trans-retinoic acid and dysmorphogenesis after microinjections of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, or retinol in cultured whole rat embryos.

PubMed

Kraft, J C; Juchau, M R

1992-01-01

Retinol (4,000 ng/ml), all-trans-retinoyl-beta-glucuronide (4,000 ng/ml), and 13-cis-retinoic acid (1,500 ng/ml) each produced dysmorphogenic effects qualitatively similar to those elicited by 250 ng/ml of all-trans-retinoic acid after microinjections of the respective individual retinoids into the amniotic cavities of cultured whole rat embryos. Subsequent HPLC analyses of the cultured whole conceptuses, embryos proper, yolk sacs, and culture media (24 hr after microinjections) indicated that conceptal biotransformation of each of the retinoids had occurred during the culture period. All-trans-retinoic acid was present in the embryos proper at quantitatively similar concentrations (20-100 nM) after microinjections of the selected quantities of each of the microinjected retinoids: retinol, all-trans-retinoyl-beta-glucuronide, 13-cis-retinoic acid, or all-trans-retinoic acid. The results suggested that all-trans-retinoic acid acted as an ultimate dysmorphogen for the retinoids tested with respect to the anomalies monitored in the embryo culture system.

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype.

PubMed

Mantione, K; Zhu, W; Rialas, C; Casares, F; Cadet, P; Franklin, A L; Tonnesen, J; Stefano, G B

2002-03-01

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [3H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present.

The metabolism of 2-trifluormethylaniline and its acetanilide in the rat by 19F NMR monitored enzyme hydrolysis and 1H/19F HPLC-NMR spectroscopy.

PubMed

Tugnait, M; Lenz, E M; Hofmann, M; Spraul, M; Wilson, I D; Lindon, J C; Nicholson, J K

2003-01-01

The urinary excretion profile and identity of the metabolites of 2-trifluoromethyl aniline (2-TFMA) and 2-trifluoromethyl acetanilide (2-TFMAc), following i.p. administration to the rat at 50 mg kg(-1), were determined using a combination of 19F NMR monitored enzyme hydrolysis, SPEC-MS and 19F/1H HPLC-NMR. A total recovery of approximately 96.4% of the dose was excreted into the urine as seven metabolites. The major routes of metabolism were N-conjugation (glucuronidation), and ring-hydroxylation followed by sulphation (and to a lesser extent glucuronidation). The major metabolites excreted into the urine for both compounds were a labile N-conjugated metabolite (a postulated N-glucuronide) and a sulphated ring-hydroxylated metabolite (a postulated 4-amino-5-trifluoromethylphenyl sulphate) following dosing of 2-TFMA. These accounted for approximately 53.0 and 31.5% of the dose, respectively. This study identifies problems on sample component instability in the preparation and analysis procedures.

A HPLC-Q-TOF-MS-based urinary metabolomic approach to identification of potential biomarkers of metabolic syndrome.

PubMed

Yu, Zhi-rui; Ning, Yu; Yu, Hao; Tang, Nai-jun

2014-04-01

Metabolic syndrome (MetS) is a serious threat to public health worldwide with an increased risk of developing type 2 diabetes, cardiovascular diseases and all-cause morbidity and mortality. In this study, a urinary metabolomic approach was performed on high performance liquid chromatography quadrupole time-of-flight mass spectrometry to discriminate 36 male MetS patients and 36 sex and age matched healthy controls. Pattern recognition analyses (principal component analysis and orthogonal projections to latent structures discriminate analysis) commonly demonstrated the difference between MetS patients and no-MetS subjects. This study found 8 metabolites that showed significant changes in patients with MetS, including branch-chain and aromatic amino acids (leucine, tyrosine, phenylalanine and tryptophan), short-chain acylcanitine (tiglylcarnitine), tricarboxylic acid (TCA) cycle intermediate (cis-aconitic acid) and glucuronidated products (cortolone-3-glucuronide and tetrahydroaldosterone-3-glucuronide). The candidate biomarkers revealed in this study could be useful in providing clues for further research focusing on the in-depth investigation of the cause of and cure for MetS.

Two new constituents from Erigeron breviscapus.

PubMed

Li, Jing; Yu, De-Quan

2013-09-01

Two novel constituents, named erigeronones A (1) and B (2), together with apigenin-7-O-β-galacturonide (3), quercetin-7-O-β-glucuronide (4), quercetin-3-O-β-galacturonide (5), and eriodictyol-7-O-β-glucuronide (6), were isolated from the whole grass of Erigeron breviscapus (Vant) Hand.-Mazz (Compositae). Their structures were established on the basis of spectral analyses and comparison with the literature data. Both new compounds 1 and 2 possess a γ-pyrone moiety that is rare in nature. Compound 1 showed significant protective effect on H2O2-injured human umbilical vein endothelial cells.

Neonatal Maturation of Paracetamol (Acetaminophen) Glucuronidation, Sulfation, and Oxidation Based on a Parent-Metabolite Population Pharmacokinetic Model

PubMed Central

Cook, Sarah F.; Stockmann, Chris; Samiee-Zafarghandy, Samira; King, Amber D.; Deutsch, Nina; Williams, Elaine F.; Wilkins, Diana G.; van den Anker, John N.

2017-01-01

Objectives This study aimed to model the population pharmacokinetics of intravenous paracetamol and its major metabolites in neonates and to identify influential patient characteristics, especially those affecting the formation clearance (CLformation) of oxidative pathway metabolites. Methods Neonates with a clinical indication for intravenous analgesia received five 15-mg/kg doses of paracetamol at 12-h intervals (<28 weeks’ gestation) or seven 15-mg/kg doses at 8-h intervals (≥28 weeks’ gestation). Plasma and urine were sampled throughout the 72-h study period. Concentration-time data for paracetamol, paracetamol-glucuronide, paracetamol-sulfate, and the combined oxidative pathway metabolites (paracetamol-cysteine and paracetamol-N-acetylcysteine) were simultaneously modeled in NONMEM 7.2. Results The model incorporated 259 plasma and 350 urine samples from 35 neonates with a mean gestational age of 33.6 weeks (standard deviation 6.6). CLformation for all metabolites increased with weight; CLformation for glucuronidation and oxidation also increased with postnatal age. At the mean weight (2.3 kg) and postnatal age (7.5 days), CLformation estimates (bootstrap 95% confidence interval; between-subject variability) were 0.049 L/h (0.038–0.062; 62 %) for glucuronidation, 0.21 L/h (0.17–0.24; 33 %) for sulfation, and 0.058 L/h (0.044–0.078; 72 %) for oxidation. Expression of individual oxidation CLformation as a fraction of total individual paracetamol clearance showed that, on average, fractional oxidation CLformation increased <15 % when plotted against weight or postnatal age. Conclusions The parent-metabolite model successfully characterized the pharmacokinetics of intravenous paracetamol and its metabolites in neonates. Maturational changes in the fraction of paracetamol undergoing oxidation were small relative to between-subject variability. PMID:27209292

Neonatal Maturation of Paracetamol (Acetaminophen) Glucuronidation, Sulfation, and Oxidation Based on a Parent-Metabolite Population Pharmacokinetic Model.

PubMed

Cook, Sarah F; Stockmann, Chris; Samiee-Zafarghandy, Samira; King, Amber D; Deutsch, Nina; Williams, Elaine F; Wilkins, Diana G; Sherwin, Catherine M T; van den Anker, John N

2016-11-01

This study aimed to model the population pharmacokinetics of intravenous paracetamol and its major metabolites in neonates and to identify influential patient characteristics, especially those affecting the formation clearance (CL formation ) of oxidative pathway metabolites. Neonates with a clinical indication for intravenous analgesia received five 15-mg/kg doses of paracetamol at 12-h intervals (<28 weeks' gestation) or seven 15-mg/kg doses at 8-h intervals (≥28 weeks' gestation). Plasma and urine were sampled throughout the 72-h study period. Concentration-time data for paracetamol, paracetamol-glucuronide, paracetamol-sulfate, and the combined oxidative pathway metabolites (paracetamol-cysteine and paracetamol-N-acetylcysteine) were simultaneously modeled in NONMEM 7.2. The model incorporated 259 plasma and 350 urine samples from 35 neonates with a mean gestational age of 33.6 weeks (standard deviation 6.6). CL formation for all metabolites increased with weight; CL formation for glucuronidation and oxidation also increased with postnatal age. At the mean weight (2.3 kg) and postnatal age (7.5 days), CL formation estimates (bootstrap 95% confidence interval; between-subject variability) were 0.049 L/h (0.038-0.062; 62 %) for glucuronidation, 0.21 L/h (0.17-0.24; 33 %) for sulfation, and 0.058 L/h (0.044-0.078; 72 %) for oxidation. Expression of individual oxidation CL formation as a fraction of total individual paracetamol clearance showed that, on average, fractional oxidation CL formation increased <15 % when plotted against weight or postnatal age. The parent-metabolite model successfully characterized the pharmacokinetics of intravenous paracetamol and its metabolites in neonates. Maturational changes in the fraction of paracetamol undergoing oxidation were small relative to between-subject variability.

Evaluation of a Potential Metabolism-Mediated Drug-Drug Interaction Between Atomoxetine and Bupropion in Healthy Volunteers.

PubMed

Todor, Ioana; Popa, Adina; Neag, Maria; Muntean, Dana; Bocsan, Corina; Buzoianu, Anca; Vlase, Laurian; Gheldiu, Ana-Maria; Briciu, Corina

2016-01-01

To evaluate the impact of bupropion on the pharmacokinetic profile of atomoxetine and its main active metabolite (glucuronidated form), 4-hydroxyatomoxetine-O-glucuronide, in healthy volunteers. An open-label, non-randomized, two-period, sequential clinical trial was conducted as follows: during Period I (Reference), each volunteer received a single oral dose of 25 mg atomoxetine, whilst during Period II (Test), a combination of 25 mg atomoxetine and 300 mg bupropion was administered to all volunteers, after a pretreatment regimen with bupropion for 7 days. Next, after determining atomoxetine and 4-hydroxyatomoxetine-O-glucuronide plasma concentrations, their pharmacokinetic parameters were calculated using a noncompartmental method and subsequently compared to determine any statistically significant differences between the two periods. Bupropion intake influenced all the pharmacokinetic parameters of both atomoxetine and its metabolite. For atomoxetine, Cmax increased from 226±96.1 to 386±137 ng/mL and more importantly, AUC0-∞ was significantly increasedfrom 1580±1040 to 8060±4160 ng*h/mL, while the mean t1/2 was prolonged after bupropion pretreatment. For 4-hydroxyatomoxetine-O-glucuronide, Cmax and AUC0-∞  were decreased from 707±269 to 212±145 ng/mL and from 5750±1240 to 3860±1220 ng*h/mL, respectively. These results demonstrated that the effect of bupropion on CYP2D6 activity was responsible for an increased systemic exposure to atomoxetine (5.1-fold) and also for a decreased exposure to its main metabolite (1.5-fold). Additional studies are required in order to evaluate the clinical relevance of this pharmacokinetic drug interaction.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Free and Glucuronide Whole Blood Cannabinoids' Pharmacokinetics after Controlled Smoked, Vaporized, and Oral Cannabis Administration in Frequent and Occasional Cannabis Users: Identification of Recent Cannabis Intake.

PubMed

Newmeyer, Matthew N; Swortwood, Madeleine J; Barnes, Allan J; Abulseoud, Osama A; Scheidweiler, Karl B; Huestis, Marilyn A

2016-12-01

There is increasing interest in markers of recent cannabis use because following frequent cannabis intake, Δ 9 -tetrahydrocannabinol (THC) may be detected in blood for up to 30 days. The minor cannabinoids cannabidiol, cannabinol (CBN), and THC-glucuronide were previously detected for ≤2.1 h in frequent and occasional smokers' blood after cannabis smoking. Cannabigerol (CBG), Δ 9 -tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-THCV might also be recent use markers, but their blood pharmacokinetics have not been investigated. Additionally, while smoking is the most common administration route, vaporization and edibles are frequently used. We characterized blood pharmacokinetics of THC, its phase I and phase II glucuronide metabolites, and minor cannabinoids in occasional and frequent cannabis smokers for 54 (occasional) and 72 (frequent) hours after controlled smoked, vaporized, and oral cannabis administration. Few differences were observed between smoked and vaporized blood cannabinoid pharmacokinetics, while significantly greater 11-nor-9-carboxy-THC (THCCOOH) and THCCOOH-glucuronide concentrations occurred following oral cannabis. CBG and CBN were frequently identified after inhalation routes with short detection windows, but not detected following oral dosing. Implementation of a combined THC ≥5 μg/L plus THCCOOH/11-hydroxy-THC ratio <20 cutoff produced detection windows <8 h after all routes for frequent smokers; no occasional smoker was positive 1.5 h or 12 h following inhaled or oral cannabis, respectively. Vaporization and smoking provide comparable cannabinoid delivery. CBG and CBN are recent-use cannabis markers after cannabis inhalation, but their absence does not exclude recent use. Multiple, complimentary criteria should be implemented in conjunction with impairment observations to improve interpretation of cannabinoid tests. Clinicaltrials.gov Identifier: NCT02177513. © 2016 American Association for Clinical Chemistry.

Evaluation of equine urine reactivity towards phase II metabolites of 17-hydroxy steroids by liquid chromatography/tandem mass spectrometry.

PubMed

Fidani, M; Gamberini, M C; Pasello, E; Palazzoli, F; De Iuliis, P; Montana, M; Arioli, F

2009-01-01

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (-18 degrees C, 4 degrees C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17beta- and 17alpha-glucuronide steroids were exposed to hydrolysis leading to non-conjugated steroids. Only 17beta-hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17alpha-hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4 degrees C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at -18 degrees C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination. (c) 2008 John Wiley & Sons, Ltd.

Analysis of the repaglinide concentration increase produced by gemfibrozil and itraconazole based on the inhibition of the hepatic uptake transporter and metabolic enzymes.

PubMed

Kudo, Toshiyuki; Hisaka, Akihiro; Sugiyama, Yuichi; Ito, Kiyomi

2013-02-01

The plasma concentration of repaglinide is reported to increase greatly when given after repeated oral administration of itraconazole and gemfibrozil. The present study analyzed this interaction based on a physiologically based pharmacokinetic (PBPK) model incorporating inhibition of the hepatic uptake transporter and metabolic enzymes involved in repaglinide disposition. Firstly, the plasma concentration profiles of inhibitors (itraconazole, gemfibrozil, and gemfibrozil glucuronide) were reproduced by a PBPK model to obtain their pharmacokinetic parameters. The plasma concentration profiles of repaglinide were then analyzed by a PBPK model, together with those of the inhibitors, assuming a competitive inhibition of CYP3A4 by itraconazole, mechanism-based inhibition of CYP2C8 by gemfibrozil glucuronide, and inhibition of organic anion transporting polypeptide (OATP) 1B1 by gemfibrozil and its glucuronide. The plasma concentration profiles of repaglinide were well reproduced by the PBPK model based on the above assumptions, and the optimized values for the inhibition constants (0.0676 nM for itraconazole against CYP3A4; 14.2 μM for gemfibrozil against OATP1B1; and 5.48 μM for gemfibrozil glucuronide against OATP1B1) and the fraction of repaglinide metabolized by CYP2C8 (0.801) were consistent with the reported values. The validity of the obtained parameters was further confirmed by sensitivity analyses and by reproducing the repaglinide concentration increase produced by concomitant gemfibrozil administration at various timings/doses. The present findings suggested that the reported concentration increase of repaglinide, suggestive of synergistic effects of the coadministered inhibitors, can be quantitatively explained by the simultaneous inhibition of the multiple clearance pathways of repaglinide.

17alpha- and 17beta-boldenone 17-glucuronides: synthesis and complete characterization by 1H and 13C NMR.

PubMed

Casati, Silvana; Ottria, Roberta; Ciuffreda, Pierangela

2009-02-01

Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. 17alpha- and 17beta-boldenone 17-glucuronides were synthesized, purified and characterized in order to provide suitable standards for the identification and quantification of these metabolites.

Hydroxytyrosol in functional hydroxytyrosol-enriched biscuits is highly bioavailable and decreases oxidised low density lipoprotein levels in humans.

PubMed

Mateos, Raquel; Martínez-López, Sara; Baeza Arévalo, Gema; Amigo-Benavent, Miryam; Sarriá, Beatriz; Bravo-Clemente, Laura

2016-08-15

Hydroxytyrosol (HT) and its derivatives in olive oil protect low-density lipoproteins (LDL) against oxidation. Biscuits could be a convenient alternative to broaden consumers' choice of HT-rich foods, although the biscuit matrix could affect HT bioavailability. We performed a crossover, randomized, double-blind study to evaluate HT bioavailability in HT-enriched biscuits (HT-B) versus non-enriched biscuits (C-B), and the effects on oxidative postprandial status. On two separate days, 13 subjects consumed 30 g of C-B or HT-B (5.25mg HT) after overnight-fasting. Blood and urine were collected at different intervals and analysed by LC-MS-QToF. After HT-B consumption, plasma metabolites peaked between 0.5 and 1h and were extensively excreted in urine. HT-sulphate and 3,4-dihydroxyphenylacetic acid (DOPAC)-sulphate were the main metabolites, followed by DOPAC and homovanillic acid (HVA). HT-glucuronide, DOPAC-glucuronide, HVA-glucuronide and HVA-sulphate were also detected. Postprandial oxidised-LDL concentrations decreased with HT-B. HT is a promising functional ingredient and, in biscuits, it is highly bioavailable and lowers postprandial oxidised-LDL levels. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemical conversion of corticosteroids to 3 alpha,5 alpha-tetrahydro derivatives. Synthesis of 5 alpha-cortol 3-glucuronides and 5 alpha-cortolone 3-glucuronides.

PubMed

Hosoda, H; Osanai, K; Nambara, T

1991-12-01

The synthesis of the 3-glucuronides of 5 alpha-cortol-20 alpha, 5 alpha-cortolone-20 alpha and their 20 beta-epimers is described. The 5 alpha-cortol 20,21-diacetates (12, 17) and 5 alpha-cortolone 20,21-diacetates (14, 19) were the key intermediates. Sodium borohydride reduction of the carbonyl group at C-20 in 5 alpha-tetrahydrocortisol 3-tert-butyldimethylsilyl ether 17,21-acetonide (8) gave the 20 alpha-hydroxy-acetonide (9). Selective removal of the acetonide ring was successful when the 20 alpha-acetoxy-17 alpha,21-acetonide (10) was treated with 50% acetic acid. Subsequent acetylation with acetic anhydride in pyridine, followed by removal of the protecting group at C-3 in the silyl ether-acetate (11) gave the desired 20 alpha-intermediate (12). The 11-ketone (14) was prepared from 11 by oxidation with pyridinium chlorochromate, followed by desilylation. The 20 beta-acetates (17, 19) were synthesized from 21-acetoxy-3 alpha,11 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one 3-tert-butyldimethylsilyl ether (15). Introduction of the glucuronyl residue at C-3 was carried out by means of the Koenigs-Knorr reaction.

Phenolic constituents from Alchemilla vulgaris L. and Alchemilla mollis (Buser) Rothm. at different dates of harvest.

PubMed

Duckstein, Sarina M; Lotter, Eva M; Meyer, Ulrich; Lindequist, Ulrike; Stintzing, Florian C

2012-01-01

Acetone/water extracts from the leaves, including stalks, of Alchemilla vulgaris L. and A. mollis (Buser) Rothm. were investigated for their phenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 24 and 27 compounds were detected for A. vulgaris and A. mollis, respectively. Pedunculagin and agrimoniin, as described in earlier reports for A. vulgaris, as well as other monomeric and oligomeric ellagitannins such as sanguiin H-10, castalagin/vescalagin, and galloyl-bis-hexahydroxydiphenoyl (HHDP) hexose constituted the major phenolic fraction of both plant species. Also, gallic and chlorogenic acids were found in both extracts. Interestingly, catechin and a procyanidin trimer were detected only in A. mollis. The flavonoid fraction comprised quercetin glucuronide as major compound in addition to several other quercetin glycosides. Most interestingly, a tentatively identified kaempferol glucuronide and a methylated quercetin glucuronide were exclusively found in A. mollis. Finally, the overall phenolic fingerprints of both Alchemilla species, harvested in May and August, i.e. at the beginning and the end of the flowering period, were compared. A general accumulation of phenolic constituents was observed later in the year, especially with regard to the ellagitannins.

Phenolic constituents from Alchemilla vulgaris L. and Alchemilla mollis (Buser) Rothm. at different dates of harvest.

PubMed

Duckstein, Sarina M; Lotter, Eva M; Meyer, Ulrich; Lindequist, Ulrike; Stintzing, Florian C

2013-01-01

Acetone/water extracts from the leaves, including stalks, of Alchemilla vulgaris L. and A. mollis (Buser) Rothm. were investigated for their phenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 24 and 27 compounds were detected for A. vulgaris and A. mollis, respectively. Pedunculagin and agrimoniin, as described in earlier reports for A. vulgaris, as well as other monomeric and oligomeric ellagitannins such as sanguiin H-10, castalagin/vescalagin, and galloyl-bis-hexahydroxydiphenoyl (HHDP) hexose constituted the major phenolic fraction of both plant species. Also, gallic and chlorogenic acids were found in both extracts. Interestingly, catechin and a procyanidin trimer were detected only in A. mollis. The flavonoid fraction comprised quercetin glucuronide as major compound in addition to several other quercetin glycosides. Most interestingly, a tentatively identified kaempferol glucuronide and a methylated quercetin glucuronide were exclusively found in A. mollis. Finally, the overall phenolic fingerprints of both Alchemilla species, harvested in May and August, i.e. at the beginning and the end of the flowering period, were compared. A general accumulation of phenolic constituents was observed later in the year, especially with regard to the ellagitannins.

Metabolism of isorhynchophylline in rats detected by LC-MS.

PubMed

Wang, Wei; Ma, Chao-Mei; Hattori, Masao

2010-01-01

This paper investigates the metabolic fate of isorhynchophylline (ISOR) as a main bioactive oxindole alkaloid in the traditional Chinese medicine. After oral administration of ISOR to rats, plasma, bile, urine and feces were analyzed by LC-MS. Hydroxylation of ISOR and successive glucuronidation proceeded in vitro by incubation with rat liver microsomes. ISOR was identified in plasma, 11-hydroxyisorhynchophylline 11-O--D-glucuronide (MI1) and 10-hydroxyisorhynchophylline 10-O--D-glucuronide (MI2) in bile, and free 11-hydroxyisorhynchophylline (MI3) and 10-hydroxyisorhynchophylline (MI4) in urine and feces. Within 24 h, 71.6% of ISOR was excreted into the feces (in 20.0 g) and 13.8% into the urine (in 20.0 ml) of rats after oral administration of 37.5 mg/kg. Monitoring by LC-MS showed that 8.5% of ISOR was metabolized to MI3 and MI4 in a ratio of ca. 1:1. Specific inhibition of CYP isozymes indicated that CYP2D, CYP1A1/2 and CYP2C participate in ISOR hydroxylation. ISOR was involved in the circulatory system after oral administration. Cytochrome P450 (CYP) in rat liver microsomes played a key role in ISOR hydroxylation.

Attempts to develop a new nuclear measurement technique of β-glucuronidase levels in biological samples

NASA Astrophysics Data System (ADS)

Ünak, T.; Avcibasi, U.; Yildirim, Y.; Çetinkaya, B.

2003-01-01

β-Glucuronidase is one of the most important hydrolytic enzymes in living systems and plays an essential role in the detoxification pathway of toxic materials incorporated into the metabolism. Some organs, especially liver and some tumour tissues, have high level of β-glucuronidase activity. As a result the enzymatic activity of some kind of tumour cells, the radiolabelled glucuronide conjugates of cytotoxic, as well as radiotoxic compounds have potentially very valuable diagnostic and therapeutic applications in cancer research. For this reason, a sensitive measurement of β-glucuronidase levels in normal and tumour tissues is a very important step for these kinds of applications. According to the classical measurement method of β-glucuronidase activity, in general, the quantity of phenolphthalein liberated from its glucuronide conjugate, i.e. phenolphthalein-glucuronide, by β-glucuronidase has been measured by use of the spectrophotometric technique. The lower detection limit of phenolphthalein by the spectrophotometric technique is about 1-3 μg. This means that the β-glucuronidase levels could not be detected in biological samples having lower levels of β-glucuronidase activity and therefore the applications of the spectrophotometric technique in cancer research are very seriously limited. Starting from this consideration, we recently attempted to develop a new nuclear technique to measure much lower concentrations of β-glucuronidase in biological samples. To improve the detection limit, phenolphthalein-glucuronide and also phenyl-N-glucuronide were radioiodinated with 131I and their radioactivity was measured by use of the counting technique. Therefore, the quantity of phenolphthalein or aniline radioiodinated with 131I and liberated by the deglucuronidation reactivity of β-glucuronidase was used in an attempt to measure levels lower than the spectrophotometric measurement technique. The results obtained clearly verified that 0.01 pg level of phenolphthalein or aniline could easily be detected at least 106 times more sensitively. The preliminary results obtained on some biological samples have shown that β-glucuronidase levels could reasonably be measured by use of the nuclear technique. In addition, our results also indicate the potential application of the radiolabelling technique to measure very low β-glucuronidase levels in different biological samples in cancer research and other related fields. The objective of our study is to demonstrate the potential application of the nuclear measurement technique in different biological samples.

Application of dried spot cards as a rapid sample treatment method for determining hydroxytyrosol metabolites in human urine samples. Comparison with microelution solid-phase extraction.

PubMed

Serra, Aida; Rubió, Laura; Macià, Alba; Valls, Rosa-M; Catalán, Úrsula; de la Torre, Rafael; Motilva, Maria-José

2013-11-01

Two different rapid sample pretreatment strategies, dried spot cards, and microelution solid-phase extraction plates (μSPE), with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) have been developed and validated for the determination of hydroxytyrosol and its metabolites in spiked human urine samples. Hydroxytyrosol, hydroxytyrosol-3'-O-glucuronide, hydroxytyrosol-4'-O-glucuronide, hydroxytyrosol-3-O-sulphate, and homovanillic alcohol-4'-O-glucuronide were used as the target compounds. Using the FTA DMPK-A dried urine spot card under optimum conditions, with 5 μL of preconcentrated urine volume and 100 μL of methanol/water (50/50, v/v) as the elution solvent, the extraction recovery (%R) of the compounds studied was higher than 80%, and the matrix effect (%ME) was less than 8%. The stability of these cards and punching at the centre or side of the card were also studied, obtaining an excellent stability after 7 days of storage and complete homogeneity across the surface of the dried drop. The different μSPE parameters that affect the efficiency were also studied, and under optimum conditions, the %R and the %ME were higher than 70% and lower than 17%, respectively. The linearity range in dried urine spot cards was 2.5-20 μM for all the metabolites, with the exception of hydroxytyrosol-3-O-sulphate and hydroxytyrosol, which were 0.3-70 μM and 2.5-50 μM respectively. With regards to μSPE, the linearity range was 0.5-5 μM for all the studied compounds, except for hydroxytyrosol-3-O-sulphate, which was 0.08-5 μM. The quantification limits (LOQs) were 0.3-2.5 μM and 0.08-0.5 μM in dried spot cards and in μSPE, respectively. The two developed methods were then applied and compared for determining hydroxytyrosol and its metabolites in human 24 h-urine samples after a sustained consumption (21 days) of a phenol-enriched virgin olive oil. The metabolites identified were hydroxytyrosol in its glucuronide and sulphate forms, homovanillic alcohol in its glucuronide and sulphate forms, homovanillic acid sulphate and hydroxytyrosol acetate sulphate.

Novel Resveratrol-Based Substrates for Human Hepatic, Renal, and Intestinal UDP-Glucuronosyltransferases

PubMed Central

2015-01-01

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by β-glucuronidase hydrolysis and LC–MS/MS analysis. NI-12a was glucuronidated at both the −COOH and −OH functions, NI-ST-05 formed a novel N–O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation. PMID:24571610

Determination of sulfates and glucuronides of endogenic steroids in biofluids by high-performance liquid chromatography/orbitrap mass spectrometry

NASA Astrophysics Data System (ADS)

Semenistaya, E. N.; Virus, E. D.; Rodchenkov, G. M.

2009-04-01

the possibility of selective determination of testosterone and epitestosterone glucuronides in urine by high-performance liquid chromatography/high-resolution mass spectrometry using solid phase microextraction on a meps cartridge was studied. the effect of the biological matrix on the spectra of conjugated steroids can be taken into account by using the spectra of conjugates recorded for urine samples after hydrolysis as reference spectra. the conditions of fragmentation in the ion source were optimized for separate analytes. this method was used for analyzing real samples with different testosterone/epitestosterone ratios. variations in conjugate contents and qualitative changes in the steroid profile of endogenic compounds were observed.

Effect of piperine on the bioavailability and pharmacokinetics of rosmarinic acid in rat plasma using UPLC-MS/MS.

PubMed

Yang, Jun-Hui; Mao, Kun-Jun; Huang, Ping; Ye, Yin-Jun; Guo, Hua-Shan; Cai, Bao-Chang

2018-02-01

1. The purpose of the present study was to investigate the effect of piperine (PP) on the pharmacokinetics of rosmarinic acid (RA) in rat plasma and to determine whether PP could enhance the oral bioavailability of RA via inhibition of its glucuronidation. 2. The pharmacokinetic profiles of RA between oral administration of RA (50 mg/kg) alone and in combination with different oral dose PP (20, 40, 60, and 80 mg/kg) to rats were investigated via a validated UPLC/MS/MS method. 3. The AUC and C max of RA were significantly increased in combination with different dose PP dose dependently, especially in the presence of 60 and 80 mg/kg PP (p < 0.01). The relative bioavailability of RA in the presence of 20, 40, 60, and 80 mg/kg PP was 1.24-, 1.32-, 2.02-, and 2.26-folds higher, respectively, compared with the control group given RA alone. Compared with RA, the pharmacokinetic modulations of RA glucuronide were even more apparent, and the glucuronidation of RA was remarkedly inhibited. 4. This study demonstrated that PP significantly improved the in vivo bioavailability of RA partly attributing to the inhibition of gut and hepatic metabolism enzymes of RA.

Influence of the Time of Intravenous Administration of Paracetamol on its Pharmacokinetics and Ocular Disposition in Rabbits.

PubMed

Karbownik, Agnieszka; Bienert, Agnieszka; Płotek, Włodzimierz; Grabowski, Tomasz; Cerbin-Koczorowska, Magdalena; Wolc, Anna; Grześkowiak, Edmund

2017-06-01

Paracetamol is one of the most common analgesics and antipyretics applied in health care. The aim of the study was to investigate the influence of the time-of-day administration on the paracetamol pharmacokinetics and its penetration into aqueous humour (AH). Rabbits were divided into three groups: I-receiving paracetamol at 08.00 h, II-receiving paracetamol at 16.00 h, and III-receiving paracetamol at 24.00 h. Paracetamol was administered intravenously at a single dose of 35 mg/kg. The concentrations of paracetamol and its metabolite (paracetamol glucuronide) in the plasma, as well as in AH were measured with the validated HPLC-UV method. No significant differences in the pharmacokinetic parameters of paracetamol was observed. When the drug was administered at 24.00 h,  elimination half-life (t 1/2kel ) of paracetamol glucuronide was longer than when the drug was administered 08.00 h (P = 0.0193). In addition, a statistically significant increase in the paracetamol glucuronide/paracetamol ratio was observed when the drug was administered at 08.00 vs. 16.00 h (P ≤ 0.0001) and 24.00 h (P ≤ 0.0001). There was no chronobiological effect on the pharmacokinetic parameters of paracetamol.

Detection of ethyl glucuronide in blood spotted on different surfaces.

PubMed

Winkler, M; Kaufmann, E; Thoma, D; Thierauf, A; Weinmann, W; Skopp, G; Alt, A

2011-07-15

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Associations Between Genetic Ancestries and Nicotine Metabolism Biomarkers in the Multiethnic Cohort Study

PubMed Central

Wang, Hansong; Park, Sungshim L.; Stram, Daniel O.; Haiman, Christopher A.; Wilkens, Lynne R.; Hecht, Stephen S.; Kolonel, Laurence N.; Murphy, Sharon E.; Le Marchand, Loïc

2015-01-01

Differences in internal dose of nicotine and tobacco-derived carcinogens among ethnic/racial groups have been observed. In this study, we explicitly examined the relationships between genetic ancestries (genome-wide average) and 19 tobacco-derived biomarkers in smokers from 3 admixed groups in the Multiethnic Cohort Study (1993–present), namely, African ancestry in African Americans (n = 362), Amerindian ancestry in Latinos (n = 437), and Asian and Native Hawaiian ancestries in Native Hawaiians (n = 300). After multiple comparison adjustment, both African and Asian ancestries were significantly related to a greater level of free cotinine; African ancestry was also significantly related to lower cotinine glucuronidation (P's < 0.00156). The predicted decrease in cotinine glucuronidation was 8.6% (P = 4.5 × 10−6) per a 20% increase in African ancestry. Follow-up admixture mapping revealed that African ancestry in a 12-Mb region on chromosome 4q was related to lower cotinine glucuronidation (P's < 2.7 × 10−7, smallest P = 1.5 × 10−9), although this is the same region reported in our previous genome-wide association study. Our results implicate a genetic ancestral component in the observed ethnic/racial variation in nicotine metabolism. Further studies are needed to identify the underlying genetic variation that could potentially be ethnic/racial specific. PMID:26568573

Alamethicin for using in bioavailability studies? - Re-evaluation of its effect.

PubMed

Vollmer, Maren; Klingebiel, Mirko; Rohn, Sascha; Maul, Ronald

2017-03-01

A major pathway for the elimination of drugs is the biliary and renal excretion following the formation of more hydrophilic secondary metabolites such as glucuronides. For in vitro investigations of the phase II metabolism, hepatic microsomes are commonly used in the combination with the pore-forming peptide alamethicin, also to give estimates for the in vivo situation. Thus, alamethicin may represent a neglected parameter in the characterization of microsomal in vitro assays. In the present study, the influence of varying alamethicin concentrations on glucuronide formation of selected phenolic compounds was investigated systematically. A correlation between the alamethicin impact and the lipophilicity of the investigated substrates was analyzed as well. Lipophilicity was determined by the logarithm of the octanol-water partition coefficient. For every substrate, a distinct alamethicin concentration could be detected leading to a maximal glucuronidation activity. Further increase of the alamethicin application led to negative effects. The differences between the maximum depletion rates with and without alamethicin addition varied between 2.7% and 18.2% depending on the substrate. A dependence on the lipophilicity could not be confirmed. Calculation of the apparent intrinsic clearance led to a more than 2-fold increase using the most effective alamethicin concentration compared to the alamethicin free control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-β-Glucuronide.

PubMed

Kimoto, Emi; Li, Rui; Scialis, Renato J; Lai, Yurong; Varma, Manthena V S

2015-11-02

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

Development of an on-line solid phase extraction ultra-high-performance liquid chromatography technique coupled to tandem mass spectrometry for quantification of bisphenol S and bisphenol S glucuronide: Applicability to toxicokinetic investigations.

PubMed

Grandin, Flore; Picard-Hagen, Nicole; Gayrard, Véronique; Puel, Sylvie; Viguié, Catherine; Toutain, Pierre-Louis; Debrauwer, Laurent; Lacroix, Marlène Z

2017-12-01

Regulatory measures and public concerns regarding bisphenol A (BPA) have led to its replacement by structural analogues, such as Bisphenol S (BPS), in consumer products. At present, no toxicokinetic investigations have been conducted to assess the factors determining human internal exposure to BPS for subsequent risk assessment. Toxicokinetic studies require reliable analytical methods to measure the plasma concentrations of BPS and its main conjugated metabolite, BPS-glucuronide (BPS-G). An efficient on-line SPE-UPLC-MS/MS method for the simultaneous quantification of BPS and BPS-G in ovine plasma was therefore developed and validated in accordance with the European Medicines Agency guidelines for bioanalytical method validation. This method has a limit of quantification of 3ngmL -1 for BPS and 10ngmL -1 for BPS-G, an analytical capacity of 200 samples per day, and is particularly well suited to toxicokinetic studies. Use of this method in toxicokinetic studies in sheep showed that BPS, like BPA, is efficiently metabolized into its glucuronide form. However, the clearances and distributions of BPS and BPS-G were lower than those of the corresponding unconjugated and glucuroconjugated forms of BPA. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolism and pharmacokinetics of rhynchophylline in rats.

PubMed

Wang, Wei; Ma, Chao-Mei; Hattori, Masao

2010-01-01

The alkaloid, rhynchophylline (RHY), from the stems and hooks of Uncaria rhynchophylla was revealed in recent years to have protective effect on neuronal damage. The present research was carried out to investigate the in vivo metabolism of this bioactive alkaloid. After administering RHY to rats, LC-MS detected RHY in plasma, bile, brain, urine and feces, the glucuronides, 11-hydroxyrhynchophylline 11-O-beta-D-glucuronide (M1) and 10-hydroxyrhynchophylline 10-O-beta-D-glucuronide (M2) in bile, and 11-hydroxyrhynchophylline (M3) and 10-hydroxyrhynchophylline (M4) in urine and feces. Within 24 h, 78.0% of RHY was excreted into the feces and 12.6% into the urine of rats after oral administration of 37.5 mg/kg. Monitoring by LC-MS showed that 9.4% of RHY was metabolized to M3 and M4 in a ratio of about 1 : 1. RHY was also detected in the brain (0.650 ng/g) at 3 h after oral administration of the same dose. Cytochrome P450 (CYP) in rat liver microsomes played a key role in RHY hydroxylation. Specific inhibition of CYP isozymes indicated that CYP2D, CYP1A1/2 and CYP2C participated in RHY hydroxylation, but not CYP3A.

Kinetic disposition of lorazepam with focus on the glucuronidation capacity, transplacental transfer in parturients and racemization in biological samples.

PubMed

Papini, Olga; da Cunha, Sergio Pereira; da Silva Mathes, Angelo do Carmo; Bertucci, Carlo; Moisés, Elaine Christine Dantas; de Barros Duarte, Luciana; de Carvalho Cavalli, Ricardo; Lanchote, Vera Lucia

2006-02-13

The present study investigates the kinetic disposition with focus on the racemization, glucuronidation capacity and the transplacental transfer of lorazepam in term parturients during labor. The study was conducted on 10 healthy parturients aged 18-37 years with a gestational age of 36-40.1 weeks, treated with a single oral dose of 2 mg racemic lorazepam 2-9 h before delivery. Maternal venous blood and urine samples were obtained over a 0-48 h interval and the umbilical cord sample was obtained immediately after clamping. Lorazepam enantiomers were determined in plasma and urine samples by LC-MS/MS using a Chiralcel OD-R column. In vitro racemization of lorazepam required the calculation of the pharmacokinetic parameters as isomeric mixtures. The data were fitted to two-compartment model and the pharmacokinetic parameters are reported as means (95% CI): t(1/2a) 3.2h (2.6-3.7 h), K(a) 0.23 h(-1) (0.19-0.28 h(-1)), t(1/2) 10.4h (9.4-11.3h), beta 0.068 h(-1) (0.061-0.075h(-1)), AUC(0-infinity) 175.3(ngh)/ml (145.7-204.8(ngh)/ml), Cl/F 2.6 ml/(minkg) (2.3-2.9 ml/(minkg)), Vd/F178.8l (146.5-211.1l), Fel 0.3% (0.1-0.5%), and Cl(R) 0.010 ml/(minkg) (0.005-0.015 ml/(minkg)). Placental transfer of lorazepam evaluated as the ratio of vein umbilical/maternal vein plasma concentrations, obtained as an isomeric mixture, was 0.73 (0.52-0.94). Pregnancy changes the pharmacokinetics of lorazepam, with an increase in the apparent distribution volume, an increase in apparent oral clearance, and a reduction of elimination half-life. The increase in oral clearance may indicate an increase in glucuronidation capacity, with a possible reduction in the plasma concentrations of drugs depending on glucuronidation capacity as the major metabolic pathway.

Metabolism of 1-fluoro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 1,1,1-trifluoro-2-chloroethane.

PubMed

Yin, H; Jones, J P; Anders, M W

1995-03-01

1-Fluoro-1,1,2-trichloroethane (HCFC-131a), 1,2-dichloro-1,1-difluoroethane (HCFC-132b), and 1,1,1-trifluoro-2-chloroethane (HCFC-133a) were chosen as models for comparative metabolism studies on 1,1,1,2-tetrahaloethanes, which are under consideration as replacements for ozone-depleting chlorofluorocarbons (CFCs). Male Fischer 344 rats were given 10 mmol/kg ip HCFC-131a or HCFC-132b or exposed by inhalation to 1% HCFC-133a for 2 h. Urine collected in the first 24 h after exposure was analyzed by 19F NMR and GC/MS and with a fluoride-selective ion electrode for the formation of fluorine-containing metabolites. Metabolites of HCFC-131a included 2,2-dichloro-2-fluoroethyl glucuronide, 2,2-dichloro-2-fluoroethyl sulfate, dichlorofluoroacetic acid, and inorganic fluoride. Metabolites of HCFC-132b were characterized as 2-chloro-2,2-difluoroethyl glucuronide, 2-chloro-2,2-difluoroethyl sulfate, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, chlorodifluoroacetaldehyde-urea adduct, and inorganic fluoride. HCFC-133a was metabolized to 2,2,2-trifluoroethyl glucuronide, trifluoroacetic acid, trifluoroacetaldehyde hydrate, trifluoroacetaldehyde-urea adduct, inorganic fluoride, and a minor, unidentified metabolite. With HCFC-131a and HCFC-132b, glucuronide conjugates of 2,2,2-trihaloethanols were the major urinary metabolites, whereas with HCFC-133a, a trifluoroacetaldehyde-urea adduct was the major urinary metabolite. Analysis of metabolite distribution in vivo indicated that aldehydic metabolites increased as fluorine substitution increased in the order HCFC-131a < HCFC-132b < HCFC-133a. With NADPH-fortified rat liver microsomes, HCFC-133a and HCFC-132b were biotransformed to trifluoroacetaldehyde and chlorodifluoroacetaldehyde, respectively, whereas HCFC-131a was converted to dichlorofluoroacetic acid. No covalently bound metabolites were detected by 19F NMR spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary Metabolites of the Dietary Carcinogen PhIP are Predictive of Colon DNA Adducts After a Low Dose Exposure in Humans

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

Malfatti, M; Dingley, K; Nowell, S

2006-04-28

Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation in humans, the most mass abundant HA, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers were administered a dietary relevant dose of [{sup 14}C]PhIP 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosingmore » for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N{sup 2}-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide, had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.« less

Monitoring of ovarian activity by measurement of urinary excretion rates of estrone glucuronide and pregnanediol glucuronide using the Ovarian Monitor, Part II: reliability of home testing.

PubMed

Blackwell, Leonard F; Vigil, Pilar; Gross, Barbara; d'Arcangues, Catherine; Cooke, Delwyn G; Brown, James B

2012-02-01

The UNDP/WHO/World Bank/Special Programme of Research, Development and Research Training in Human Reproduction (Geneva) set up a study to determine whether it is feasible for women to monitor their ovarian activity reliably by home testing. Daily self-monitoring of urinary hormone metabolites for menstrual cycle assessment was evaluated by comparison of results obtained with the Home Ovarian Monitor by untrained users both at home and in study centres. Women collected daily data for urinary estrone glucuronide (E1G) and pregnanediol glucuronide (PdG) for two cycles, then the procedure was repeated in the women's local centre (in Chile, Australia or New Zealand) giving a total of 113 duplicate cycles. The tests were performed without the benefit of replicates or quality controls. The home and centre cycles were normalized and compared to identify assay errors, and the resulting home and centre menstrual cycle profiles were averaged. Reliable mean cycle profiles were obtained with the home and centre excretion rates agreeing to within 36 ± 21 nmol/24 h for E1G and 0.77 ± 0.28 µmol/24 h for baseline PdG values (1-5 µmol/24 h). The cycles had a mean length of 28.1 ± 3.1 days (n = 112; 5th and 95th percentiles: 24 and 35 days, respectively), a mean follicular phase of 14.8 ± 3.1 days (n = 107; 5th and 95th percentiles: 11 and 21 days) and a mean luteal phase length of 13.3 ± 1.5 days (n = 106; 5th and 95th percentiles: 11 and 17 days), calculated from the day of the LH peak. The study confirmed that the Ovarian Monitor pre-coated assay tubes worked well even in the hands of lay users, without standard curves, quality controls or replicates. Point-of-care monitoring to give reliable fertility data is feasible.

Pharmacokinetics and Metabolism of (R,R)-Methoxyfenoterol in Rat

PubMed Central

Siluk, Danuta; Mager, Donald E.; Kim, Hee Seung; Wang, Yan; Furimsky, Anna M.; Ta, Amy; Iyer, Lalitha V.; Green, Carol E.; Wainer, Irving W.

2010-01-01

(R,R)-Fenoterol (Fen), a β2-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results of the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen.The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3 min × nmol ml-1), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146 ml min-1 kg-1, the T1/2 was significantly longer, 152.9 versus 108.9 min and the area under the curve (AUC) was significantly increased, 300 versus 119 min × nmol ml-1.(R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6% while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation.The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G. PMID:20039779

Pharmacokinetics and metabolism of (R,R)-methoxyfenoterol in rat.

PubMed

Siluk, D; Mager, D E; Kim, H S; Wang, Y; Furimsky, A M; Ta, A; Iyer, L V; Green, C E; Wainer, I W

2010-03-01

(R,R)-fenoterol (Fen), a beta(2)-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results from the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen. The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3 min x nmol ml(-1)), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146 ml min(-1) kg(-1), the T(1/2) was significantly longer, 152.9 versus 108.9 min, and the area under the curve (AUC) was significantly increased, 300 versus 119 min x nmol ml(-1). (R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6%, while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation. The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G.

Bioactive Androgens and Glucuronidated Androgen Metabolites are Associated with Subcutaneous and Ectopic Skeletal Muscle Adiposity among Older Black Men

PubMed Central

Miljkovic, Iva; Cauley, Jane A; Dressen, Amy S; Gordon, Christopher L; Goodpaster, Bret H; Kuller, Lewis H; Bunker, Clareann H; Patrick, Alan L; Wheeler, Victor W; Orwoll, Eric S; Zmuda, Joseph M

2011-01-01

Aging is associated with declining serum levels of androgenic hormones and with increased skeletal muscle fat infiltration, an emerging risk factor for type 2 diabetes mellitus (T2DM). Androgens regulate fat mass and glucose homeostasis, but the effect of androgenic hormones on skeletal muscle fat infiltration is largely unknown. Thus, the aim of the current study was to examine the association of serum androgens and their precursors and metabolites with skeletal muscle fat infiltration and T2DM in a black male population group at high risk of T2DM. Serum androgens, estrogens, and androgen precursors and metabolites were measured using mass spectrometry, and calf skeletal muscle fat distribution [subcutaneous and intermuscular fat; skeletal muscle density] were measured using quantitative computed tomography in 472 Afro-Caribbean men aged 65 and older. Bioactive androgens, testosterone, free testosterone and dihydrotestosterone, were associated with less skeletal muscle fat infiltration (r=−0.14 to −0.18, P<0.05) and increased skeletal muscle density (r=0.10 to 0.14, P<0.05), independent of total adiposity. Additionally, glucuronidated androgen metabolites were associated with less subcutaneous fat (r=−0.11 to −0.15, P<0.05). Multivariate logistic regression analysis identified an increased level of 3α-diol-3 glucuronide (OR=1.38, P<0.01) and a decreased level of dihydrotestosterone (OR=0.66, P<0.01) to be significantly associated with T2DM. Our findings suggest that in elderly black men, independent of total adiposity, bioactive androgens and glucuronidated androgen metabolites may play previously unrecognized role in skeletal muscle fat distribution. Longitudinal studies are needed to further evaluate the relationship between androgens and androgen metabolites with changes in skeletal muscle fat distribution with aging and the incidence of T2DM. PMID:21353258

Impediments to enhancement of CPT-11 anticancer activity by E. coli directed beta-glucuronidase therapy.

PubMed

Hsieh, Yuan-Ting; Chen, Kai-Chuan; Cheng, Chiu-Min; Cheng, Tian-Lu; Tao, Mi-Hua; Roffler, Steve R

2015-01-01

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.

Species differences in the formation of vabicaserin carbamoyl glucuronide.

PubMed

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

2010-04-01

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

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction

PubMed Central

Gufford, Brandon T.; Chen, Gang; Vergara, Ana G.; Lazarus, Philip; Oberlies, Nicholas H.

2015-01-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4′- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27–66 µM; UGT1A1, 3.2–8.3 µM; UGT1A8, 19–73 µM; and UGT1A10, 65–120 µM) encompassed reported intestinal tissue concentrations (20–310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product–drug interactions in the context of cancer prevention. PMID:26070840

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

PubMed

Gufford, Brandon T; Chen, Gang; Vergara, Ana G; Lazarus, Philip; Oberlies, Nicholas H; Paine, Mary F

2015-09-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Competing pathways in drug metabolism. I. Effect of input concentration on the conjugation of gentisamide in the once-through in situ perfused rat liver preparation

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

Morris, M.E.; Yuen, V.; Tang, B.K.

1988-05-01

Sulfation and glucuronidation are two parallel pathways for the metabolism of phenolic substrates. Gentisamide (GAM) was used as a model compound to examine the effects of parallel competing pathways on drug disappearance and metabolite formation in the once-through perfused rat liver preparation. GAM was found to form one glucuronide (GAM-5G) and two sulfate (GAM-2S and GAM-5S) conjugates. These GAM conjugates were biosynthesized in recirculating rat liver preparations, and were isolated by preparative high-performance liquid chromatography. Specific incorporation of 35S-sodium sulfate and (14C)glucose into GAM sulfate and glucuronide conjugates revealed corresponding elution patterns as labeled GAM metabolites. Their identities were characterizedmore » by enzymatic and acid hydrolyses and by NMR spectroscopy. Gentisamide-5-sulfate (GAM-5S) and gentisamide-5-glucuronide (GAM-5G) are major metabolites, and gentisamide-2-sulfate (GAM-2S) is a minor metabolite. Single-pass rat liver perfusions were used to examine the effect of stepwise increases/decreases of input GAM concentration (CIn) on the extraction ratio (E) of GAM and formation of metabolites. The E of GAM remained constant (about 0.89) at input concentrations from 0.9 to 120 microM and decreased at CIn greater than 120 microM. Metabolite patterns, however, changed with GAM CIn, even when E was constant at CIn up to 120 microM. GAM-5S was present as the major metabolite of GAM at all GAM CInS in most liver preparations but the proportions of GAM-5S and GAM-2S decreased at increasing CIn; the proportion of GAM-5G, a minor metabolite at low CIn, increased with increasing CIn. Biliary excretion rates at steady state accounted for 5.3 +/- 2.7% (mean +/- S.D.) of the input rate: GAM-5G was the predominant metabolite found.« less

Impediments to Enhancement of CPT-11 Anticancer Activity by E. coli Directed Beta-Glucuronidase Therapy

PubMed Central

Hsieh, Yuan-Ting; Chen, Kai-Chuan; Cheng, Chiu-Min; Cheng, Tian-Lu; Tao, Mi-Hua; Roffler, Steve R.

2015-01-01

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity. PMID:25688562

Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

PubMed

Hanioka, Nobumitsu; Isobe, Takashi; Kinashi, Yu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

2016-07-01

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

PubMed

Mino, Yasuaki; Naito, Takafumi; Shimoyama, Kumiko; Ogawa, Noriyoshi; Kawakami, Junichi

2017-07-01

Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7  µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R 2  = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase activity may be determined by mycophenolic acid exposure and complement fraction C3 in systemic lupus erythematosus patients.

Direct measurement of Bisphenol A (BPA), BPA glucuronide and BPA sulfate in a diverse and low-income population of pregnant women reveals high exposure, with potential implications for previous exposure estimates: a cross-sectional study.

PubMed

Gerona, Roy R; Pan, Janet; Zota, Ami R; Schwartz, Jackie M; Friesen, Matthew; Taylor, Julia A; Hunt, Patricia A; Woodruff, Tracey J

2016-04-12

Bisphenol A (BPA) is a ubiquitous, endocrine-disrupting environmental contaminant that increases risk of some adverse developmental effects. Thus, it is important to characterize BPA levels, metabolic fate and sources of exposure in pregnant women. We used an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytic method to directly and simultaneously measure unconjugated BPA (uBPA), BPA glucuronide and BPA sulfate in the urine of a population of ethnically and racially diverse, and predominately low-income pregnant women (n = 112) in their second trimester. We also administered a questionnaire on dietary and non-dietary sources of exposure to BPA. We found universal and high exposure to uBPA and its metabolites: median concentrations were 0.25, 4.67, and 0.31 μg/g creatinine for uBPA, BPA glucuronide, and BPA sulfate, respectively. The median Total BPA (uBPA + BPA in glucuronide and sulfate forms) level was more than twice that measured in U.S. pregnant women in NHANES 2005-2006, while 30 % of the women had Total BPA levels above the 95th percentile. On average, Total BPA consisted of 71 % BPA in glucuronide form, 15 % BPA in sulfate form and 14 % uBPA, however the proportion of BPA in sulfate form increased and the proportion of uBPA decreased with Total BPA levels. Occupational and non-occupational contact with paper receipts was positively associated with BPA in conjugated (glucuronidated + sulfated) form after adjustment for demographic characteristics. Recent consumption of foods and beverages likely to be contaminated with BPA was infrequent among participants and we did not observe any positive associations with BPA analyte levels. The high levels of BPA analytes found in our study population may be attributable to the low-income status of the majority of participants and/or our direct analytic method, which yields a more complete evaluation of BPA exposure. We observed near-universal exposure to BPA among pregnant women, as well as substantial variability in BPA metabolic clearance, raising additional concerns for effects on fetal development. Our results are consistent with studies showing thermal paper receipts to be an important source of exposure, point to the difficulty pregnant women have avoiding BPA exposure on an individual level, and therefore underscore the need for changes in BPA regulation and commerce.

An investigation of the stability of free and glucuronidated 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in authentic urine samples.

PubMed

Skopp, Gisela; Pötsch, Lucia

2004-01-01

Preanalytical stability of a drug and its major metabolites is an important consideration in pharmacokinetic studies or whenever the analyte pattern is used to estimate drug habits. Firstly, the stability of free and glucuronidated 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH, THCCOOglu) in authentic urine samples was investigated. Random urine samples of cannabis users (n = 38) were stored at -20, 4, and 20 degrees C up to 15 days and up to 5 days at 40 degrees C, and alterations of the analyte pattern during storage were followed by liquid chromatography-tandem mass spectrometry. Secondly, the influence of pH (range 5.0-8.0) on the stability of the analytes was studied using spiked urine to elucidate the results obtained from authentic samples. In authentic urine samples, the initial pH ranged from 5.1 to 8.8. The glucuronide was found to be highly labile at a storage temperature of 4 degrees C and above. Initially, 18 urine samples tested positive for THCCOOH. After 2 days storage at 20 degrees C, THCCOOH was detectable in a further 4 samples, and 7 more samples tested positive for THCCOOH (5-81 ng/mL) after 15 days. Depending on time and temperature, the glucuronide concentration decreased, resulting in an increase of THCCOOH concentration. However, a loss in mean total THCCOOH concentration was found, which was significantly higher in deteriorated samples than in samples without signs of deterioration after 15 days of storage at 20 degrees C. In the drug-free urine sample separately spiked with THCCOOglu or THCCOOH, the investigations on the stability of the target analytes at various pH values revealed that THCCOOH was stable at pH 5.0. At higher pH values, its concentration slightly decreased with time, and about 69% of the initial THCCOOH concentration was still present at pH 8.0 on day 5. THCCOOglu concentrations rapidly decreased with increasing pH value. For example, only 72% of the initial THCCOOglu concentration could be detected at pH 5.0 on day 1. Degradation of the glucuronide resulted in formation of THCCOOH, which was observed even at pH 5.0. In light of the present findings, advanced forensic interpretations based on the presence of THCCOOH or the pattern of THCCOOH and THCCOOglu in stored urine samples seems questionable.

Early neuropsychological adverse events after switching from PI/r to dolutegravir could be related to hyperthyroidism in patients under levothyroxine.

PubMed

Berger, Jean-Luc; Nguyen, Yohan; Lebrun, Delphine; Migault, Caroline; Hentzien, Maxime; Marty, Hélène; Bani-Sadr, Firouzé

2017-01-01

We report two patients who had taken levothyroxine at the same dose for several years and who had stable thyroid stimulating hormone (TSH) levels, and who developed clinical and biological hyperthyroidism following switch from ritonavir-boosted protease inhibitors (PIs) to dolutegravir-based HAART. Levothyroxine is metabolized by deiodination and glucuronidation and the induction of glucuronidation by ritonavir leads to an increased elimination of levothyroxine and a necessity of higher daily doses. Patients who switch from ritonavir-boosted PIs to antiretroviral drugs-based HAART with minimal drug-interaction such as dolutegravir, may require an adjustment in their dose of levothyroxine in order to prevent hyperthyroidism due to impaired elimination of levothyroxine without ritonavir.

Cyprotuoside C and Cyprotuoside D, Two New Cycloartane Glycosides from the Rhizomes of Cyperus rotundus.

PubMed

Lin, San-Qing; Zhou, Zhong-Liu; Li, Chun-Yan

2018-01-01

Cyprotuoside C (1) and cyprotuoside D (2), two new cycloartane glycosides were isolated from the ethanol extract of the rhizomes of Cyperus rotundus. Their structures were identified as 24R-9,10-seco-cycloartan-1(10),9(11)-dien-3β,7β,24,25-tetraol 3-O-β-D-xylopyranosyl-(1→4)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl-25-O-β-D-glucuronide (1) and 9,10-seco-cycloartan-1(10),9(11),23(24)-trien-3β,7β,25-triol 3-O-β-D-xylopyranosyl-(1→4)-{α-L-arabinopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]}-β-D-glucopyranosyl-25-O-β-D-glucuronide (2) by spectroscopic methods.

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.

Transformation of codeine and codeine-6-glucuronide to opioid analogues by urine adulteration with pyridinium chlorochromate: potential issue for urine drug testing.

PubMed

Luong, Susan; Ung, Alison T; Kalman, John; Fu, Shanlin

2014-07-30

Pyridinium chlorochromate (PCC) is the active ingredient of 'Urine Luck', a commercially available in vitro adulterating agent used to conceal the presence of drugs in a urine specimen. The exposure of codeine and its major glucuronide metabolite codeine-6-glucuronide (C6G) to PCC was investigated to determine whether PCC is an effective masking agent for these opiate compounds. Following the addition of PCC to both spiked and authentic codeine and C6G-positive urine specimens, the samples were monitored using liquid chromatography/mass spectrometry (LC/MS). Stable reaction products were identified and characterized using high-resolution MS analysis and, where possible, nuclear magnetic resonance (NMR) analysis. It was determined that PCC effectively oxidizes codeine and C6G, thus altering the original codeine-to-C6G ratio in the urine specimen. Four reaction products were identified for codeine: codeinone, 14-hydroxycodeinone, 6-O-methylcodeine and 8-hydroxy-7,8-dihydrocodeinone. Similarly, three reaction products were identified for C6G: codeinone, codeine and a lactone of C6G (tentative assignment). Besides addressing the complications added to interpretation, more investigation is warranted to further determine their potential for use as markers for monitoring the presence of codeine and C6G in urine specimens adulterated with PCC. Copyright © 2014 John Wiley & Sons, Ltd.

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.

Enhancing the bioavailability of resveratrol by combining it with piperine

PubMed Central

Johnson, Jeremy J.; Nihal, Minakshi; Siddiqui, Imtiaz A.; Scarlett, Cameron O.; Bailey, Howard H.; Mukhtar, Hasan; Ahmad, Nihal

2012-01-01

Scope Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin shown to possess a multitude of health-promoting properties in pre-clinical studies. However, the poor in vivo bioavailability of resveratrol due to its rapid metabolism is being considered as a major obstacle in translating its effects in humans. In this study, we examined the hypothesis that piperine will enhance the pharmacokinetic parameters of resveratrol via inhibiting its glucuronidation, thereby slowing its elimination. Methods and results Employing a standardized LC/MS assay, we determined the effect of piperine co-administration with resveratrol on serum levels resveratrol and resveratrol-3-O-β-d-glucuronide in C57BL mice. Mice were administered resveratrol (100 mg/kg; oral gavage) or resveratrol (100 mg/kg; oral gavage) + piperine (10 mg/kg; oral gavage), and the serum levels of resveratrol and resveratrol-3-O-β-d-glucuronide were analyzed at different times. We found that the degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serum concentration (Cmax) was increased to 1544% with the addition of piperine. Conclusion Our study demonstrated that piperine significantly improves the in vivo bioavailability of resveratrol. However, further detailed research is needed to study the mechanism of improved bioavailability of resveratrol via its combination with piperine as well as its effect on resveratrol metabolism. PMID:21714124

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

PubMed

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

2017-04-01

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

Species and gender differences in the metabolism and distribution of tertiary amyl methyl ether in male and female rats and mice after inhalation exposure or gavage administration.

PubMed

Sumner, Susan C J; Janszen, Derek B; Asgharian, Bahman; Moore, Timothy A; Parkinson, Horace D; Fennell, Timothy R

2003-01-01

Tertiary amyl methyl ether (TAME) is a gasoline fuel additive used to reduce emissions. Understanding the metabolism and distribution of TAME is needed to assess potential human health issues. The effect of dose level, duration of exposure and route of administration on the metabolism and distribution of TAME were investigated in male and female F344 rats and CD-1 mice following inhalation or gavage administration. By 48 h after exposure, >96% of the administered radioactivity was expired in air (16-71%) or eliminated in urine and feces (28-72%). Following inhalation exposure, mice had a two- to threefold greater relative uptake of [14C]TAME compared with rats. Metabolites were excreted in urine of rats and mice that are formed by glucuronide conjugation of tertiary amyl alcohol (TAA), oxidation of TAA to 2,3-dihydroxy-2-methylbutane and glucuronide conjugation of 2,3-dihydroxy-2-methylbutane. A saturation in the uptake and metabolism of TAME with increased exposure concentration was indicated by a decreased relative uptake of total [14C]TAME equivalents and an increase in the percentage expired as volatiles. A saturation of P-450 oxidation of TAA was indicated by a disproportional decrease of 2,3-dihydroxy-2-methylbutane and its glucuronide conjugate with increased exposure concentration. Copyright 2003 John Wiley & Sons, Ltd.

Hepatotoxicity Induced by Sophora flavescens and Hepatic Accumulation of Kurarinone, a Major Hepatotoxic Constituent of Sophora flavescens in Rats.

PubMed

Jiang, Peng; Zhang, Xiuwen; Huang, Yutong; Cheng, Nengneng; Ma, Yueming

2017-10-25

Our previous study showed that kurarinone was the main hepatotoxic ingredient of Sophora flavescens , accumulating in the liver. This study characterized the mechanism of Sophora flavescens extract (ESF) hepatotoxicity and hepatic accumulation of kurarinone. ESF impaired hepatic function and caused fat accumulation in the liver after oral administration (1.25 and 2.5 g/kg for 14 days in rats). Serum metabolomics evaluation based on high-resolution mass spectrometry was conducted and real-time PCR was used to determine the expression levels of CPT-1, CPT-2, PPAR-α, and LCAD genes. Effects of kurarinone on triglyceride levels were evaluated in HL-7702 cells. Tissue distribution of kurarinone and kurarinone glucuronides was analyzed in rats receiving ESF (2.5 g/kg). Active uptake of kurarinone and kurarinone glucuronides was studied in OAT2-, OATP1B1-, OATP2B1-, and OATP1B3-transfected HEK293 cells. Our results revealed that after oral administration of ESF in rats, kurarinone glucuronides were actively transported into hepatocytes by OATP1B3 and hydrolyzed into kurarinone, which inhibited fatty acid β-oxidation through the reduction of l-carnitine and the inhibition of PPAR-α pathway, ultimately leading to lipid accumulation and liver injury. These findings contribute to understanding hepatotoxicity of kurarinone after oral administration of ESF.

Bisphenol A glucuronidation in patients with Parkinson's disease.

PubMed

Landolfi, Annamaria; Troisi, Jacopo; Savanelli, Maria Cristina; Vitale, Carmine; Barone, Paolo; Amboni, Marianna

2017-12-01

Bisphenol A (BPA) is a widely distributed estrogen-mimetic molecule, with well-established effects on the dopaminergic system. It can be found in canned food, dental sealants, thermal paper, etc. BPA undergoes liver conjugation with glucuronic acid and is subsequently excreted in the urine. In the present study we quantified the concentration of free and conjugated Bisphenol A in blood of patients affected by Parkinson Disease, using their spouses as controls. An interview was performed to determine possible confounders in BPA exposure. Free and conjugated BPA were quantified by gas chromatography coupled with mass spectrometry. Parkinson's Disease patients carried a statistically significant lower amount of conjugated Bisphenol A compared to controls. The two populations were mostly homogeneous in terms of exposure to possible Bisphenol A sources. The only exceptions were exposure to canned tuna and canned tomatoes PD patients consumed significantly more of both (p<0.05). Moreover, no difference in Bisphenol A glucuronidation was found after stratification by typology of anti-Parkinson's drug taken and after conversion to the Levodopa Equivalent Daily Dose. BPA glucuronidation was decreased in patients with Parkinson disease. The possible unique mechanisms underlying Bisphenol A metabolism in PD patients deserve further elucidation. Moreover, further study is needed to assess a possible BPA role in Parkinson's Disease pathogenesis, due to its documented dopaminergic toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous determination of ezetimibe and its glucuronide metabolite in human plasma by solid phase extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Guo, Lin; Wang, Meng-meng; He, Min; Qiu, Fu-rong; Jiang, Jian

2015-04-01

A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed to quantify ezetimibe (EZM) and its major glucuronide (ezetimibe glucuronide, EZM-G) in human plasma simultaneously. The analytes were purified by solid phase extraction (SPE) without hydrolysis. Separation of the analytes was achieved using acetonitrile-water (0.08% formic acid) (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min on an Agilent Extend C18 column. The analytes were detected by LC-MS/MS using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to detect EZM and EZM-G, respectively. The analytical method was linear over the concentration range of 0.1-20 ng/mL for EZM and 0.5-200 ng/mL for EZM-G. Within- and between-run precision for EZM was no more than 8.6% and 12.8%; and for EZM-G was no more than 9.0% and 8.7%, respectively. This method was reproducible and reliable, and was successfully used to analyze human plasma samples for application in a bioequivalence study. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of pentachlorophenol and its glucuronide and sulfate conjugates in fish bile and exposure water

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

Stehly, G.R.; Hayton, W.L.

1988-08-01

The glucuronide and sulfate conjugates of pentachlorophenol (PCP) that were present in the bile and exposure water of goldfish (Carassius auratus) were used to develop methodology to quantify PCP and its metabolites. Reverse phase HPLC with radioactivity detection separated PCP and its metabolites, and was used to verify a method of quantification that used differential extraction and scintillation counting. Extractions of aqueous phase at pH 2 or 8, with butanol, ethyl acetate, or ether indicated that ether at pH 8 best separated PCP from its metabolites. The sulfate conjugate of PCP was the major metabolite produced when goldfish were exposedmore » to 125 micrograms UC-PCP/l. It was present primarily in the exposure water, but also appeared in the bile.« less

Residues of the lampricides 3-trifluoromethyl-4-nitrophenol and niclosamide in muscle tissue of rainbow trout

USGS Publications Warehouse

Hubert, T.D.; Bernardy, J.A.; Vue, C.; Dawson, V.K.; Boogaard, M.A.; Schreier, Theresa M.; Gingerich, W.H.

2005-01-01

Rainbow trout (Oncorhyncus mykiss) were exposed to the 14C- labeled lampricide 3-trifluoromethyl-4-nitrophenol (TFM) (2.1 mg/L) or niclosamide (0.055 mg/L) in an aerated static water bath for 24 h. Fish were sacrificed immediately after exposure. Subsamples of skin-on muscle tissue were analyzed for residues of the lampricides. The primary residues in muscle tissue from fish exposed to TFM were parent TFM (1.08 ?? 0.82 nmol/g) and TFM-glucuronide (0.44 ?? 0.24 nmol/g). Muscle tissue from fish exposed to niclosamide contained niclosamide (1.42 ?? 0.51 nmol/g), niclosamide-glucuronide (0.0644 ?? 0.0276 nmol/g), and a metabolite not previously reported, niclosamide sulfate ester (1.12 ?? 0.33 nmol/g).

Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine.

PubMed

Arndt, Torsten; Schröfel, Stefanie; Güssregen, Brunhilde; Stemmerich, Karsten

2014-04-01

Ethyl glucuronide (EtG) in urine is considered a specific marker of recent ethanol consumption. There is an ongoing debate about whether inhalation or transdermal resorption of sanitizer ethanol is the underlying cause for positive EtG findings after hand disinfection. Desderman(®) pure (Schülke & Mayr GmbH, Norderstedt) with 78.2g 96% (v/v) ethanol/100g and approx. 10% 2-propanol was used for multiple hand disinfection without and under an exhauster. Simulating a common working day in a clinic, 5 co-workers of our lab used the sanitizer 32 fold within 8h and 2 persons were merely exposed to the sanitizer vapor but without any dermal sanitizer contact. Any additional ethanol intake or exposition was reliably excluded. Spot urine was collected at baseline, after 1, 2, 4, 6 … 14, and finally 24h after the first sanitizer use. A validated LC-MS/MS was used for MRM and MS(3) of EtG and qualitative analyses of ethyl sulfate and 2-propyl glucuronide. Multiple hand disinfection caused positive EtG findings of up to 2.1mg/L or 1.7mg/g creatinine in 4 out of 5 test persons and even of 0.6mg/L or 0.8mg/g for 2 controls which were merely exposed to the sanitizer vapor but without any sanitizer contact. EtG results between the clinical (0.5mg/g) and the forensic (0.1mg/g) cut-off were obtained even 6h after the last sanitizer exposition. An exhauster prevented the sanitizer vapor inhalation and reduced the EtG excretion to mostly below the detection limit of 0.02mg/g. The maximum value was 0.09mg/g. Ethyl sulfate and 2-propyl glucuronide (2-PpG) were detectable only in the EtG positive samples. 2-PpG is a metabolite of 2-propanol, which is quite frequently used in disinfectants. Thus, the detection of this substance can be used in cases of odd EtG results as an indicator of (unintended) sanitizer exposition. Ethanol from hand sanitizers is predominantly incorporated by the respiratory tract but not via the skin. It can cause a distinct ethyl glucuronide excretion and thus analytically true-positive but forensically false-positive EtG findings in the urine of ethanol abstaining persons. Since accidental ethanol inhalation can occur quite frequently in the working place or even private household, such a situation should always be considered when EtG is used as a marker of recent ethanol consumption. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Influence of hepatic impairment on the pharmacokinetics and safety profile of dapagliflozin: an open-label, parallel-group, single-dose study.

PubMed

Kasichayanula, Sreeneeranj; Liu, Xiaoni; Zhang, Weijiang; Pfister, Marc; LaCreta, Frank P; Boulton, David W

2011-11-01

Dapagliflozin, a selective inhibitor of renal sodium glucose co-transporter 2, is under development for the treatment of type 2 diabetes mellitus. Dapagliflozin elimination is primarily via glucuronidation to an inactive metabolite, dapagliflozin 3-O-glucuronide. Pharmacokinetic studies are recommended in subjects with impaired hepatic function if hepatic metabolism accounts for a substantial portion of the absorbed drug. The purpose of our study was to compare the pharmacokinetics of dapagliflozin in patients with mild, moderate, or severe hepatic impairment (HI) with healthy subjects. This was an open-label, parallel-group study in male or female patients with mild, moderate, or severe HI (6 per group according to Child-Pugh classification) and in 6 healthy control subjects. The control subjects were matched to the combined HI group for age (±10 years), weight (±20%), sex, and smoking status, with no deviations from normal in medical history, physical examination, ECG, or laboratory determinations. All participants received a single 10-mg oral dose of dapagliflozin, and the pharmacokinetics of dapagliflozin and dapagliflozin 3-O-glucuronide were characterized. Dapagliflozin tolerability was also assessed throughout the study. Demographic characteristics and baseline physical measurements (weight, height, and body mass index) were similar among the 18 patients in the HI groups (58-126 kg; 151.2-190.0 cm, and 31.5-37.7 kg/m(2), respectively) and the healthy subject group (65.0-102.6 kg; 166.0-184.0 cm, and 23.3-34.3 kg/m(2), respectively). In those with mild, moderate, or severe HI, dapagliflozin mean C(max) values were 12% lower and 12% and 40% higher than healthy subjects, respectively. Mean dapagliflozin AUC(0-∞) values were 3%, 36%, and 67% higher compared with healthy subjects, respectively. Dapagliflozin 3-O-glucuronide mean C(max) values were 4% and 58% higher and 14% lower in those with mild, moderate, or severe HI compared with healthy subjects, respectively, and mean dapagliflozin 3-O-glucuronide AUC(0-∞) values were 6%, 100%, and 30% higher compared with healthy subjects, respectively. These values were highly dependent on the calculated creatinine clearance of each group. All adverse events were mild or moderate, with no imbalance in frequency between groups. Compared with healthy subjects, systemic exposure to dapagliflozin in subjects with HI was correlated with the degree of HI. Single 10-mg doses of dapagliflozin were generally well tolerated by participants in this study. Due to the higher dapagliflozin exposures in patients with severe HI, the benefit:risk ratio should be individually assessed because the long-term safety profile and efficacy of dapagliflozin have not been specifically studied in this population. Copyright © 2011 Elsevier HS Journals, Inc. All rights reserved.

Metabolism of 1-nitropyrene in germ-free and conventional rats.

PubMed

Kinouchi, T; Morotomi, M; Mutai, M; Fifer, E K; Beland, F A; Ohnishi, Y

1986-04-01

The distribution, covalent binding and metabolism of radioactive 1-nitropyrene (1-NP) were examined following its oral administration to conventional and germ-free male Wistar rats. With both groups of animals, the liver, kidney, bladder, adipose tissue and gastrointestinal tract had the highest specific radioactivity. However, the maximum concentration of radioactivity occurred at 12 hr in conventional rats as compared to 24 hr in germ-free animals. This difference may be due to the faster transit time of the intestinal contents through conventional rats. At 48 hr after treatment, the covalent binding of 1-NP metabolites was greatest in liver and kidney of conventional rats, while in germ-free rats, substantial binding was also found in the gastrointestinal tract. The mutagenic activity in Salmonella typhimurium TA98 of fecal extracts and urine from conventional rats was greater in the presence of an S9 mix, whereas similar extracts from germ-free animals were more mutagenic in the absence of S9. The major fecal metabolites in germ-free rats were (in order of decreasing concentration): 3-nitropyrenol greater than 1-NP greater than 4,5-dihydroxy-4,5-dihydro-1-NP greater than 6-nitropyrenol greater than 8-nitropyrenol. With the exception of 1-NP, similar metabolites were found in the urine as their glucuronide conjugates. In the feces from conventional rats, substantial nitro reduction and N-acetylation occurred with the major metabolites being: 1-NP greater than 1-aminopyrene greater than 8-acetylaminopyrenol greater than 6-acetylaminopyrenol greater than 3-acetylaminopyrenol. The major metabolites identified in the urine from conventional rats were glucuronide conjugates of 6- and 8-acetylaminopyrenol, while the major biliary conjugates identified were glucuronide conjugates of 4,5-dihydroxy-4,5-dihydro-1-NP and 3-, 6-, and 8-nitropyrenol, although the relative proportion of glucuronide conjugates of 6- and 8-aminopyrenol and 6- and 8-acetylaminopyrenol increased in later stages of the biliary excretion. The polar and beta-glucuronidase-refractory metabolites, which may be sulfate and glutathione conjugates, remain to be identified.

UDP-Glucuronosyltransferase 1A Compromises Intracellular Accumulation and Anti-Cancer Effect of Tanshinone IIA in Human Colon Cancer Cells

PubMed Central

Liu, Miao; Wang, Qiong; Liu, Fang; Cheng, Xuefang; Wu, Xiaolan; Wang, Hong; Wu, Mengqiu; Ma, Ying; Wang, Guangji; Hao, Haiping

2013-01-01

Background and Purpose NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA. Experimental Approach We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity. Key Results Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect. Conclusions and Implications UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs’ substrates. PMID:24244442

Inhibitory Effects of Commonly Used Herbal Extracts on UDP-Glucuronosyltransferase 1A4, 1A6, and 1A9 Enzyme Activities

PubMed Central

Mohamed, Mohamed-Eslam F.

2011-01-01

The aim of this study was to investigate the effect of commonly used botanicals on UDP-glucuronosyltransferase (UGT) 1A4, UGT1A6, and UGT1A9 activities in human liver microsomes. The extracts screened were black cohosh, cranberry, echinacea, garlic, ginkgo, ginseng, milk thistle, saw palmetto, and valerian in addition to the green tea catechin epigallocatechin gallate (EGCG). Formation of trifluoperazine glucuronide, serotonin glucuronide, and mycophenolic acid phenolic glucuronide was used as an index reaction for UGT1A4, UGT1A6, and UGT1A9 activities, respectively, in human liver microsomes. Inhibition potency was expressed as the concentration of the inhibitor at 50% activity (IC50) and the volume in which the dose could be diluted to generate an IC50-equivalent concentration [volume/dose index (VDI)]. Potential inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. EGCG inhibited UGT1A4 with an IC50 value of (mean ± S.E.) 33.8 ± 3.1 μg/ml. Milk thistle inhibited both UGT1A6 and UGT1A9 with IC50 values of 59.5 ± 3.6 and 33.6 ± 3.1 μg/ml, respectively. Saw palmetto and cranberry weakly inhibited UGT1A6 and UGT1A9, respectively, with IC50 values >100 μg/ml. For each inhibition, VDI was calculated to determine the potential of achieving IC50-equivalent concentrations in vivo. VDI values for inhibitors indicate a potential for inhibition of first-pass glucuronidation of UGT1A4, UGT1A6, and UGT1A9 substrates. These results highlight the possibility of herb-drug interactions through modulation of UGT enzyme activities. Further clinical studies are warranted to investigate the in vivo extent of the observed interactions. PMID:21632963

Polychlorinated Biphenyl Congeners that Increase the Glucuronidation and Biliary Excretion of Thyroxine Are Distinct from the Congeners that Enhance the Serum Disappearance of Thyroxine

PubMed Central

Martin, L. A.; Wilson, D. T.; Reuhl, K. R.; Gallo, M. A.

2012-01-01

Polychlorinated biphenyl (PCB) congeners differentially reduce serum thyroxine (T4) in rats, but little is known about their ability to affect biliary excretion of T4. Thus, male Sprague-Dawley rats were orally administered Aroclor-1254, Aroclor-1242 (32 mg/kg per day), PCB-95, PCB-99, PCB-118 (16 mg/kg per day), PCB-126 (40 μg/kg per day), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (3.9 μg/kg per day), or corn oil for 7 days. Twenty-four hours after the last dose, [125I]T4 was administered intravenously, and blood, bile, and urine samples were collected for quantifying [125I]T4 and in bile [125I]T4 metabolites. Serum T4 concentrations were reduced by all treatments, but dramatic reductions occurred in response to Aroclor-1254, PCB-99 [phenobarbital (PB)-type congener], and PCB-118 (mixed-type congener). None of the treatments increased urinary excretion of [125I]T4. Aroclor-1254, PCB-118, TCDD, and PCB-126 (TCDD-type congener) increased biliary excretion of T4-glucuronide by 850, 756, 710, and 573%, respectively, corresponding to marked induction of hepatic UDP-glucuronosyltransferase (UGT) activity toward T4. PCB-95 and PCB-99 did not induce UGT activity; therefore, the increased biliary excretion of T4-glucuronide was related to the affinity of congeners for the aryl hydrocarbon receptor. The disappearance of [125I]T4 from serum was rapid (within 15-min) and was increased by Aroclor-1254, PCB-99 and PCB-118. Thus, reductions in serum T4 in response to PCBs did not always correspond with UGT activity toward T4 or with increased biliary excretion of T4-glucuronide. The rapid disappearance of [125I]T4 from the serum of rats treated with PB-like PCBs suggests that increased tissue uptake of T4 is an additional mechanism by which PCBs may reduce serum T4. PMID:22187485

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

Mendizabal, A.F.; Quiroga, S.; Farinati, Z.

The usefulness of the direct 4-hour radioimmunoassay of estriol-16-glucuronide (E/sub 3/G) and pregnanediol-3-glucuronide (P/sub 2/G) in first morning urine (FMU) for establishing a prognosis of the early pregnancy outcome was evaluated in 106 patients that became pregnant. Microaliquots of FMU were serially assayed from day 3 of the conception cycle until day 80 of pregnancy. The E/sub 3/G and P/sub 2/G profiles of 19 pregnancies which terminated in spontaneous abortion with either a diagnosis of the blighted ovum syndrome (n = 11) or presumption of a corpus luteum/trophoblast failure (n = 8) have been compared with those of clinically normalmore » pregnancies (n = 87). Normal pregnancies displayed typical patterns of E/sub 3/G and P/sub 2/G development, while variations were observed in abortive events that reflected changes of the fetoplacental unit.« less

Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities.

PubMed

Kim, D H; Hong, S W; Kim, B T; Bae, E A; Park, H Y; Han, M J

2000-04-01

The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated. By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product. The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22. Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL. GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori. GAMG, the minor metabolite of GL, was the sweetest.

Physicochemical and Pharmacological Characterization of Permanently Charged Opioids.

PubMed

Mazak, Karoly; Noszal, Bela; Hosztafi, Sandor

2017-01-01

The main aim of synthesizing permanently charged opioids is to ensure that they do not enter the central nervous system. Such drugs can provide analgesic activity with reduced sedation and other side effects on the central nervous system. We undertook a search of bibliographic databases for peer-reviewed research literature and also summarized our published results in this field. The present review focuses on the characterization of permanently charged opioids by various physicochemical methods, and in vitro as well as in vivo tests. The basicity and lipophilicity of opioid alkaloids are discussed at the microscopic, speciesspecific level. Glucuronide conjugates of opioids are also reviewed. Whereas the primary metabolite morphine-3-glucuronide does not bind to opioid receptors with high affinity, morphine-6-glucuronide is a potent analgesic, at least, partly due to its unexpectedly high lipophilicity. We discuss the quaternary ammonium opioid derivatives of a permanent positive charge, detailing their antinociceptive activity and effects on gastrointestinal motility in various in vivo animal tests and in vitro studies. Compounds with antagonistic activity are also reviewed. The last part of our study concentrates on sulfate conjugates of morphine derivatives that display unique pharmacological properties because they carry a negative charge at any pH value in the human body. In conclusion, the findings of this review confirm the importance of permanently charged opioids in the investigated fields of pharmacology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Bisphenol-A (BPA), BPA glucuronide, and BPA sulfate in mid-gestation umbilical cord serum in a Northern and Central California population

PubMed Central

Gerona, Roy R.; Woodruff, Tracey J.; Dickenson, Carrie A.; Pan, Janet; Schwartz, Jackie M.; Sen, Saunak; Friesen, Matthew M.; Fujimoto, Victor Y.; Hunt, Patricia A.

2013-01-01

Bisphenol-A (BPA) is an endocrine disrupting chemical used in numerous consumer products, resulting in universal exposure in the United States. Prenatal exposure to BPA is associated with numerous reproductive and developmental effects in animals. However, little is known about human fetal exposure or metabolism of BPA during mid-gestation. In the present study, we present a new liquid chromatography-tandem mass spectrometry method to directly measure concentrations of BPA and two predominant metabolic conjugates – BPA glucuronide and BPA sulfate – in umbilical cord serum collected from elective 2nd trimester pregnancy terminations. We detected at least one form of BPA in all umbilical cord serum samples: BPA (GM 0.16; range

Human Metabolite Lamotrigine-N(2)-glucuronide Is the Principal Source of Lamotrigine-Derived Compounds in Wastewater Treatment Plants and Surface Water.

PubMed

Zonja, Bozo; Pérez, Sandra; Barceló, Damià

2016-01-05

Wastewater and surface water samples, extracted with four solid-phase extraction cartridges of different chemistries, were suspect-screened for the anticonvulsant lamotrigine (LMG), its metabolites, and related compounds. LMG, three human metabolites, and a LMG synthetic impurity (OXO-LMG) were detected. Preliminary results showed significantly higher concentrations of OXO-LMG in wastewater effluent, suggesting its formation in the wastewater treatment plants (WWTPs). However, biodegradation experiments with activated sludge demonstrated that LMG is resistant to degradation and that its human metabolite lamotrigine-N(2)-glucuronide (LMG-N2-G) is the actual source of OXO-LMG in WWTPs. In batch reactors, LMG-N2-G was transformed, following pseudo-first-order kinetics to OXO-LMG and LMG, but kinetic experiments suggested an incomplete mass balance. A fragment ion search applied to batch-reactor and environmental samples revealed another transformation product (TP), formed by LMG-N2-G oxidation, which was identified by high-resolution mass spectrometry. Accounting for all TPs detected, a total mass balance at two concentration levels in batch reactors was closed at 86% and 102%, respectively. In three WWTPs, the total mass balance of LMG-N2-G ranged from 71 to 102%. Finally, LMG-N2-G and its TPs were detected in surface water samples with median concentration ranges of 23-139 ng L(-1). The results of this study suggest that glucuronides of pharmaceuticals might also be sources of yet undiscovered, but environmentally relevant, transformation products.

Systematic Analysis of Main Constituents in Rat Biological Samples after Oral Administration of the Methanol Extract of Fructus Aurantii by HPLC-ESI-MS/MS.

PubMed

Zhang, Jingze; Gao, Wenyuan; Liu, Zhen; Zhang, Zhidan; Liu, Changxiao

2014-01-01

High performance liquid chromatography (HPLC) with diode array detection (DAD) and electrospray ionization tandem mass spectrometry (ESI/MS/MS) was used to analyze the main components in the methanol extract of Fructus Aurantii (FA) and the metabolites in rat biological samples after oral administration of the methanol extract of FA. There were 31 constituents identified in the extract of FA including 2 alkaloids, 1 coumarin, 10 flavonoid glycosides and 18 ploymethoxylated flavones. According to the UV spectrum and MS fragment character of main components in the methanol extract of FA, 18 parent constituents and 11 metabolites were tentatively identified in rat biological samples. Three groups of components in biological samples detected included flavonoid glycosides, their glucuronides and ploymethoxylated flavones. It was interested that flavonoid glycosides, their glucuronides and ploymethoxylated flavones can be investigated in rat plasma and urine, while in rat feces samples only flavonoid glycosides were detected. Triglycosyl, naringenin, neoeriocitrin, neoeriocitrin narirutin and hesperidin were the main components in rat feces which were found either in the plasma or in urine samples. However, naringin and neohesperidin were the main flavonoid glycosides which absorbed after oral administration. Except flavonoid glycosides and their glucuronides, ploymethoxylated flavones also the constituents absorbed because it was investigated mainly in rat plasma and urine but not in feces samples. The identification and elucidation of parent and metabolism components analyzed in biological samples provided the data for further pharmacological and clinical research on FA.

High Resolution Mass Spectrometric Analysis of Secoiridoids and Metabolites as Biomarkers of Acute Olive Oil Intake-An Approach to Study Interindividual Variability in Humans.

PubMed

Silva, Sandra; Garcia-Aloy, Mar; Figueira, Maria Eduardo; Combet, Emilie; Mullen, William; Bronze, Maria Rosário

2018-01-01

Phenolic compounds are minor components of extra virgin olive oil (EVOO). Secoiridoids are the major components contributing to the phenolic content of EVOO. Information is lacking regarding their potential as biomarkers for EVOO intake. Healthy volunteers (n = 9) ingested 50 mL of EVOO in a single dose containing 322 mg kg -1 total phenolic content (caffeic acid equivalents) and 6 mg 20 g -1 hydroxytyrosol and its derivatives. Plasma is collected before (0 h) and at 0.5, 1, 2, 4, and 6 h after ingestion. Urine samples are collected prior to ingestion (0 h) and at 0-4, 4-8, 8-15, and 15-24 h. Samples are analyzed by UPLC coupled with an Exactive Orbitrap MS. Partial least squares discriminant analysis with orthogonal signal correction is applied to screen for metabolites that allow sample discrimination. Plasma biomarkers and urine biomarkers are selected although individual variability is observed among volunteers. Results are in accordance with in vitro experiments performed (in vitro digestion and hepatic microsomal activity assays). Plasma (elenolic acid + H 2 ; p-HPEA-EA + H 2 + glucuronide) and urinary (3,4-DHPEA-EA, 3,4-DHPEA-EA + H 2 +glucuronide, methyl 3,4-DHPEA-EA + H 2 +glucuronide) secoiridoid compounds are selected as biomarkers to monitor EVOO intake showing good predictive ability according to multivariate analysis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

PubMed

Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

2010-03-01

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

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

PubMed

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

1995-09-01

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

Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis.

PubMed

Morris, Ayodele A; Chester, Scot A; Strickland, Erin C; McIntire, Gregory L

2014-10-01

Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

CYP2C8-mediated interaction between repaglinide and steviol acyl glucuronide: In vitro investigations using rat and human matrices and in vivo pharmacokinetic evaluation in rats.

PubMed

Xu, Yunting; Zhou, Dandan; Wang, Yedong; Li, Jiajun; Wang, Meiyu; Lu, Jia; Zhang, Hongjian

2016-08-01

CYP2C8 is involved in the metabolic clearance of several important drugs and recent reports have shown that acyl glucuronides of gemfibrozil and clopidogrel are potent time-dependent inhibitors of CYP2C8 activity. In the present study, the inhibitory effect of steviol acyl glucuronide (SVAG), a circulating metabolite formed after the ingestion of rebaudioside A, was investigated using in vitro and in vivo systems. Results indicated that SVAG was a reversible but not a time-dependent inhibitor of CYP2C8-mediated paclitaxel 6α-hydroxylation. SVAG was also capable of inhibiting CYP2C8-mediated repaglinide 3'-hydroxylation in human liver microsomes and recombinant human CYP2C8, with Ki values of 15.8 μM and 11.6 μM, respectively. In contrast, SVAG did not exhibit inhibitory effect on CYP2C8 activity in rat liver microsomes. In addition, co-administration of rebaudioside A with repaglinide in rats did not lead to AUC and Cmax changes of repaglinide. Although mathematic prediction using a simplified mechanistic model revealed a moderate interaction potential between repaglinide and SVAG, cautions should be given to patients with hypoglycemia if repaglinide and rebaudioside A are used in combination for the blood sugar control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasma metabolomic biomarkers of mixed nuts exposure inversely correlate with severity of metabolic syndrome.

PubMed

Mora-Cubillos, Ximena; Tulipani, Sara; Garcia-Aloy, Mar; Bulló, Mònica; Tinahones, Francisco J; Andres-Lacueva, Cristina

2015-12-01

To identify the most discriminant dietary biomarkers of nuts exposure in subjects with metabolic syndrome (MetS), and investigate the potential association between exposure and the severity of the MetS diagnostic traits. We applied the untargeted LC-ESI-qToF-MS-driven metabolomic workflow to explore the changes occurring in the plasma metabolome of MetS subjects following 12-wk intake of mixed nuts (30 g/d; nuts versus control groups). Urolithin A glucuronide was the most discriminative biomarker of nuts exposure, showing the highest predictive capacity (area under the ROC curve = 89.6% [80.8-98.4]) despite the interindividual variation expected for a host-microbial cometabolite. Furthermore, the detection of urolithin A glucuronide in plasma showed significant inverse correlation with basal abdominal adiposity (waist circumference: r = -0.550, p < 0.01; waist-hip ratio: r = -0.409, p < 0.05) and impaired glycemic control (fasting insulin: r = -0.414, p < 0.05; HOMA-IR: r = -0.417, p < 0.05). Significant changes in medium-chain dicarboxylic acids, recognized as alternative energy substrates that are particularly relevant in the case of glycemic control impairment, were also associated with nut consumption. Higher levels of utolithin A glucuronide are reported in subjects with less severe MetS traits, especially in females. We believe that this inverse correlation may be related with profile of gut microbial dysbiosis, recently associated to subjects with MetS. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

PubMed

Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

2016-09-01

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Hair testing of GHB: an everlasting issue in forensic toxicology.

PubMed

Busardò, Francesco Paolo; Pichini, Simona; Zaami, Simona; Pacifici, Roberta; Kintz, Pascal

2018-01-26

In this paper, the authors present a critical review of different studies regarding hair testing of endogenous γ-hydroxybutyrate (GHB), concentrations in chronic users, and values measured after a single GHB exposure in drug facilitated sexual assault (DFSA) cases together with the role of a recently identified GHB metabolite, GHB-glucuronide. The following databases (up to March 2017) PubMed, Scopus and Web of Science were used, searching the following key words: γ-hydroxybutyrate, GHB, GHB glucuronide, hair. The main key words "GHB" and "γ-hydroxybutyrate" were searched singularly and then associated individually to each of the other keywords. Of the 2304 sources found, only 20 were considered appropriate for the purpose of this paper. Summing up all the studies investigating endogenous GHB concentration in hair, a very broad concentration range from 0 to 12 ng/mg was found. In order to detect a single GHB dose in hair it is necessary to commonly wait 1 month for collecting hair and a segmental analysis of 3 or 5 mm fragments and the calculation of a ratio between the targeted segment and the others represent a reliable method to detect a single GHB intake considering that the ratios presently proposed vary from 3 and 10. The only two studies so far performed, investigating GHB-Glucuronide in hair, show that the latter does not seem to provide any diagnostic information regarding GHB exposure. A practical operative protocol is proposed to be applied in all suspected cases of GHB-facilitated sexual assault (GHB-FSA).

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.

Genetics Home Reference: Crigler-Najjar syndrome

MedlinePlus

... from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form ... the body. The bilirubin-UGT enzyme performs a chemical reaction called glucuronidation. During this reaction, the enzyme transfers ...

Pharmacokinetics and Biliary Excretion of Fisetin in Rats.

PubMed

Huang, Miao-Chan; Hsueh, Thomas Y; Cheng, Yung-Yi; Lin, Lie-Chwen; Tsai, Tung-Hu

2018-06-14

The hypothesis of this study is that fisetin and phase II conjugated forms of fisetin may partly undergo biliary excretion. To investigate this hypothesis, male Sprague-Dawley rats were used for the experiment, and their bile ducts were cannulated with polyethylene tubes for bile sampling. The pharmacokinetic results demonstrated that the average area-under-the-curve (AUC) ratios ( k (%) = AUC conjugate /AUC free-form ) of fisetin, its glucuronides, and its sulfates were 1:6:21 in plasma and 1:4:75 in bile, respectively. Particularly, the sulfated metabolites were the main forms that underwent biliary excretion. The biliary excretion rate ( k BE (%) = AUC bile /AUC plasma ) indicates the amount of fisetin eliminated by biliary excretion. The biliary excretion rates of fisetin, its glucuronide conjugates, and its sulfate conjugates were approximately 144, 109, and 823%, respectively, after fisetin administration (30 mg/kg, iv). Furthermore, biliary excretion of fisetin is mediated by P-glycoprotein.

The mechanism of hydrothermal hydrolysis for glycyrrhizic acid into glycyrrhetinic acid and glycyrrhetinic acid 3-O-mono-β-D-glucuronide in subcritical water.

PubMed

Fan, Rui; Li, Nan; Xu, Honggao; Xiang, Jun; Wang, Lei; Gao, Yanxiang

2016-01-01

To improve the bioactivity and sweetness properties of glycyrrhizic acid (GL), the hydrothermal hydrolysis of GL into glycyrrhetinic acid (GA) and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) in subcritical water was investigated. The effects of temperature, time and their interaction on the conversion ratios were analyzed and the reactions were elaborated with kinetics and thermodynamics. The results showed that GL hydrothermal hydrolysis was significantly (P < 0.05) affected by reaction time and temperature, as well as their interaction, and could be fitted into first-order kinetics. The thermodynamic analysis indicated that the hydrolysis of GL was endergonic and non-spontaneous. The hydrolytic pathways were composed of complex consecutive and parallel reactions. It was concluded that subcritical water may be a potential medium for producing GAMG and GA. Copyright © 2015 Elsevier Ltd. All rights reserved.

Determination of the binding properties of p-cresyl glucuronide to human serum albumin.

PubMed

Yi, Dan; Monteiro, Elisa Bernardes; Chambert, Stéphane; Soula, Hédi A; Daleprane, Julio B; Soulage, Christophe O

2018-04-26

p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×10 3  M -1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin. Copyright © 2018. Published by Elsevier B.V.

Bisphenol A and its analogs: Do their metabolites have endocrine activity?

PubMed

Gramec Skledar, Darja; Peterlin Mašič, Lucija

2016-10-01

Structural analogs of bisphenol A are commonly used as its alternatives in industrial and commercial applications. Nevertheless, the question arises whether the use of other bisphenols is justified as replacements for bisphenol A in mass production of plastic materials. To evaluate the influence of metabolic reactions on endocrine activities of bisphenols, we conducted a systematic review of the literature. Knowledge about the metabolic pathways and enzymes involved in metabolic biotransformations is essential for understanding and predicting mechanisms of toxicity. Bisphenols are metabolized predominantly by the glucuronidation reaction, which is considered their most important detoxification pathway, as based on current knowledge, glucuronides do not have activity on endocrine receptors. In contrast, several oxidative metabolites of bisphenols with enhanced endocrine activities are presented, and these findings indicate that oxidative metabolites of bisphenols can still have endocrine activities in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

PubMed Central

Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

2015-01-01

The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

Flower color changes in three Japanese hibiscus species: further quantitative variation of anthocyanin and flavonols.

PubMed

Shimokawa, Satoshi; Iwashina, Tsukasa; Murakami, Noriaki

2015-03-01

One anthocyanin and four flavonols were detected from the petals of Hibiscus hamabo, H. tiliaceus and H. glaber. They were identified as cyanidin 3-0- sambubioside, gossypetin 3-O-glucuronide-8-O-glucoside, quercetin 7-O-rutinoside, gossypetin 3-O-glucoside and gossypetin 8-O-glucuronide by UV spectra, LC-MS, acid hydrolysis and HPLC. The flavonoid composition was essentially the same among the petals ofH. hamabo, H. tiliaceus and H. glaber, and there was little quantitative variation, except for cyanidin 3-O-sambubioside, the content of which in the petals ofH. tiliaceus and H. glaber was much higher than in that of H. hamabo. Flower colors of H. tiliaceus and H. glaber change from yellow to red, and that of H. hamabo changes from yellow to orange. These changes were caused by contents of anthocyanin and flavonols, which increased after flowering of H. hamabo, H. tiliaceus and H. glaber.

Biotransformation and mass balance of the SGLT2 inhibitor empagliflozin in healthy volunteers.

PubMed

Chen, Lin-Zhi; Jungnik, Arvid; Mao, Yanping; Philip, Elsy; Sharp, Dale; Unseld, Anna; Seman, Leo; Woerle, Hans-Jürgen; Macha, Sreeraj

2015-01-01

1. The absorption, biotransformation and excretion of empagliflozin, an SGLT2 inhibitor, were evaluated in eight healthy subjects following a single 50 mg oral dose of empagliflozin containing ∼100 µCi [(14)C]-empagliflozin. 2. Radioactivity was rapidly absorbed, with plasma levels peaking 1 h post-dose. Total exposure was lower in blood versus plasma, consistent with moderate (28.6-36.8%) red blood cell partitioning. Protein binding was 80.3-86.2%. 3. Most of the radioactive dose was recovered in urine (54.4%) and faeces (41.1%). Unchanged empagliflozin was the most abundant drug-related component in plasma, representing 75.5-77.4% of plasma radioactivity and 79.6% plasma radioactivity AUC0-12 h. Unchanged empagliflozin was the most abundant drug-related component in urine and faeces, representing 43.5% (23.7% of dose) and 82.9% (34.1% of dose) of radioactivity in urine and faeces, respectively. Six metabolites were identified in plasma: three glucuronide conjugates representing 4.7-7.1% of AUC0-12 h and three less abundant metabolites (<0.2-1.9% AUC0-12 h). The most abundant metabolites in urine were two glucuronide conjugates (7.8-13.2% of dose) and in faeces was a tetrahydrofuran ring-opened carboxylic acid metabolite (1.9% of dose). 4. To conclude, empagliflozin was rapidly absorbed and excreted primarily unchanged in urine and faeces. Unchanged parent was the major drug-related component in plasma. Metabolism was primarily via glucuronide conjugation.

In vitro metabolism of [14C]-benalaxyl in hepatocytes of rats, dogs and humans.

PubMed

Nallani, Gopinath C; ElNaggar, Shaaban F; Shen, Li; Chandrasekaran, Appavu

2017-03-01

The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [ 14 C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [ 14 C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacokinetics of Curcumin Diethyl Disuccinate, a Prodrug of Curcumin, in Wistar Rats.

PubMed

Bangphumi, Kunan; Kittiviriyakul, Chuleeporn; Towiwat, Pasarapa; Rojsitthisak, Pornchai; Khemawoot, Phisit

2016-12-01

Curcumin is the major bioactive component of turmeric, but has poor oral bioavailability that limits its clinical applications. To improve the in vitro solubility and alkaline stability, we developed a prodrug of curcumin by succinylation to obtain curcumin diethyl disuccinate, with the goal of improving the oral bioavailability of curcumin. The in vivo pharmacokinetic profile of curcumin diethyl disuccinate was compared with that of curcumin in male Wistar rats. Doses of curcumin 20 mg/kg intravenous or 40 mg/kg oral were used as standard regimens for comparison with the prodrug at equivalent doses in healthy adult rats. Blood, tissues, urine, and faeces were collected from time zero to 48 h after dosing to determine the prodrug level, curcumin level and a major metabolite by liquid chromatography-tandem spectrometry. The absolute oral bioavailability of curcumin diethyl disuccinate was not significantly improved compared with curcumin, with both compounds having oral bioavailability of curcumin less than 1 %. The major metabolic pathway of the prodrug was rapid hydrolysis to obtain curcumin, followed by glucuronidation. Interestingly, curcumin diethyl disuccinate gave superior tissue distribution with higher tissue to plasma ratio of curcumin and curcumin glucuronide in several organs after intravenous dosing at 1 and 4 h. The primary elimination route of curcumin glucuronide occurred via biliary and faecal excretion, with evidence of an entry into the enterohepatic circulation. Curcumin diethyl disuccinate did not significantly improve the oral bioavailability of curcumin due to first pass metabolism in the gastrointestinal tract. Further studies on reduction of first pass metabolism are required to optimise delivery of curcumin using a prodrug approach.

Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry.

PubMed

Krüger, Ralf; Vogeser, Michael; Burghardt, Stephan; Vogelsberger, Rita; Lackner, Karl J

2010-12-01

Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time <3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole.

Determination of a novel nonfluorinated quinolone, nemonoxacin, in human feces and its glucuronide conjugate in human urine and feces by high-performance liquid chromatography-triple quadrupole mass spectrometry.

PubMed

He, Gaoli; Guo, Beining; Yu, Jicheng; Zhang, Jing; Wu, Xiaojie; Cao, Guoying; Shi, Yaoguo; Tsai, Cheng-Yuan

2015-05-01

Three methods were developed and validated for determination of nemonoxacin in human feces and its major metabolite, nemonoxacin acyl-β- d-glucuronide, in human urine and feces. Nemonoxacin was extracted by liquid-liquid extraction in feces homogenate samples and nemonoxacin acyl-β- d-glucuronide by a solid-phase extraction procedure for pretreatment of both urine and feces homogenate sample. Separation was performed on a C18 reversed-phase column under isocratic elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. Both analytes were determined by liquid chromatography-tandem mass spectrometry with positive electrospray ionization in selected reaction monitoring mode and gatifloxacin as the internal standard. The lower limit of quantitation (LLOQ) of nemonoxacin in feces was 0.12 µg/g and the calibration curve was linear in the concentration range of 0.12-48.00 µg/g. The LLOQ of the metabolite was 0.0010 µg/mL and 0.03 µg/g in urine and feces matrices, while the linear range was 0.0010-0.2000 µg/mL and 0.03-3.00 µg/g, respectively. Validation included selectivity, accuracy, precision, linearity, recovery, matrix effect, carryover, dilution integrity and stability, indicating that the methods can quantify the corresponding analytes with excellent reliability. The validated methods were successfully applied to an absolute bioavailability clinical study of nemonoxacin malate capsule. Copyright © 2014 John Wiley & Sons, Ltd.

Involvement of UDP-Glucuronosyltransferases and Sulfotransferases in the Excretion and Tissue Distribution of Resveratrol in Mice

PubMed Central

Böhmdorfer, Michaela; Szakmary, Akos; Schiestl, Robert H.; Vaquero, Javier; Riha, Juliane; Brenner, Stefan; Thalhammer, Theresia; Szekeres, Thomas; Jäger, Walter

2017-01-01

Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4′-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol. PMID:29231856

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30.

PubMed

Sakurama, Haruko; Kishino, Shigenobu; Uchibori, Yoshie; Yonejima, Yasunori; Ashida, Hisashi; Kita, Keiko; Takahashi, Satomi; Ogawa, Jun

2014-05-01

Baicalin (baicalein 7-O-β-D-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-D-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.

Inhibitory effect of ciprofloxacin on β-glucuronidase-mediated deconjugation of mycophenolic acid glucuronide.

PubMed

Kodawara, Takaaki; Masuda, Satohiro; Yano, Yoshitaka; Matsubara, Kazuo; Nakamura, Toshiaki; Masada, Mikio

2014-07-01

The interaction between mycophenolate (MPA) and quinolone antibiotics such as ciprofloxacin is considered to reduce the enterohepatic recycling of MPA, which is biotransformed in the intestine from MPA glucuronide (MPAG) conjugate excreted via the biliary system; however, the molecular mechanism underlying this biotransformation of MPA is still unclear. In this study, an in vitro system was established to evaluate β-glucuronidase-mediated deconjugation and to examine the influence of ciprofloxacin on the enzymatic deconjugation of MPAG and MPA resynthesis. Resynthesis of MPA via deconjugation of MPAG increased in a time-dependent manner from 5 to 60 min in the presence of β-glucuronidase. Ciprofloxacin and phenolphthalein-β-d-glucuronide (PhePG), a typical β-glucuronidase substrate, significantly decreased the production of MPA from MPAG in the β-glucuronidase-mediated deconjugation system. In addition, enoxacin significantly inhibited the production of MPA from MPAG, while levofloxacin and ofloxacin had no inhibitory effect on MPA synthesis. Pharmacokinetic analysis revealed that ciprofloxacin showed a dose-dependent inhibitory effect on MPA production from MPAG via β-glucuronidase with a half-maximal inhibitory concentration (IC50 ) value of 30.4 µm. While PhePG inhibited the β-glucuronidase-mediated production of MPA from MPAG in a competitive manner, ciprofloxacin inhibited MPA synthesis via noncompetitive inhibition. These findings suggest that the reduction in the serum MPA concentration during the co-administration of ciprofloxacin is at least in part due to the decreased enterohepatic circulation of MPA because of noncompetitive inhibition of deconjugation of MPAG by intestinal β-glucuronidase. Copyright © 2014 John Wiley & Sons, Ltd.

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn's Disease Patients and Unaffected First-Degree Relatives.

PubMed

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn's disease.

Metabolism and disposition of bisphenol A in female rats.

PubMed

Snyder, R W; Maness, S C; Gaido, K W; Welsch, F; Sumner, S C; Fennell, T R

2000-11-01

Bisphenol A (BPA), which is used in the manufacture of polycarbonates, elicits weak estrogenic activity in in vitro and in vivo test systems. The objectives of this study were to compare the patterns of disposition of radioactivity in adult female F-344 and CD rats after oral administration of (14)C BPA (100 mg/kg), to isolate the glucuronide of BPA and to assess its estrogenic activity in vitro, and to evaluate the transfer of radioactivity to pups from lactating dams administered (14)C BPA. Over 6 days, F-344 rats excreted more radioactivity in urine than CD rats. The major metabolite in urine was identified as bisphenol A glucuronide (BPA gluc) by incubation with beta-glucuronidase and (1)H and (13)C NMR spectroscopy. In lactating CD rats administered (14)C BPA (100 mg/kg) by gavage, only a small fraction of the label was found in milk, with 0.95 +/- 0.66, 0.63 +/- 0.13, and 0.26 +/- 0.10 microg equiv/ml (mean +/- SD) from dams collected 1, 8, and 26 h after dosing, respectively. Radioactivity in pup carcasses indicated exposure in the range of microgram equivalents per kilogram; those values ranged from 44.3 +/- 24.4 for pups separated from their lactating dams at 2 h to 78.4 +/- 10.9 at 24 h. BPA gluc was the prominent metabolite in milk and plasma. In test systems for activation of in vitro estrogen receptors alpha and beta, BPA gluc did not show appreciable efficacy at concentrations up to 0.03 mM, indicating that metabolism via glucuronidation is a detoxication reaction. Copyright 2000 Academic Press.

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

PubMed

Wang, Lai-Xi; Heredia, Alonso; Song, Haijing; Zhang, Zhaojun; Yu, Biao; Davis, Charles; Redfield, Robert

2004-10-01

Resveratrol is a natural product with diverse biological activities. We have previously reported that resveratrol possesses potent synergistic inhibitory activity against human immunodeficiency virus (HIV)-1 infection in combination with nucleoside analogs (Heredia et al. 2000. J Acquir Immune Defic Syndr 25:246-255). As a part of our program in developing resveratrol as a component for anti-HIV chemotherapy, we describe in this article the characterization, chemical synthesis, and biological effects of the human metabolites of resveratrol. We found that resveratrol was metabolized in humans into two metabolites, which were characterized as resveratrol-3-O- and 4'-O-glucuronides. For further biological studies, we reported two simple, alternative methods for the synthesis of the metabolites. The cytotoxic and antiviral activities of resveratrol and its metabolites were compared in cell culture experiments using human peripheral blood mononuclear cells. Whereas resveratrol was cytotoxic at > or =30 microM, no cytotoxicity was observed for the metabolites at concentrations as high as 300 microM. However, resveratrol showed strong synergistic anti-HIV activity with didanosine at 10 microM, but no synergistic effects were observed for either of the metabolites at up to 300 microM. Nevertheless, the in vitro activity of the metabolites (resveratrol glucuronides) may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human beta-glucuronidase could convert the metabolites back to resveratrol locally or systematically in vivo. The present studies have implications for future development of resveratrol and/or its derivatives as a chemotherapeutic agent. Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association

A Pharmacokinetic/Pharmacodynamic Model of Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Patients Treated with Flavopiridol

PubMed Central

Ji, Jia; Mould, Diane R.; Blum, Kristie A.; Ruppert, Amy S.; Poi, Ming; Zhao, Yuan; Johnson, Amy J.; Byrd, John C.; Grever, Michael R.; Phelps, Mitch A.

2013-01-01

Purpose Flavopiridol, the first clinically evaluated cyclin dependent kinase inhibitor, demonstrates activity in patients with refractory chronic lymphocytic leukemia, but prevalent and unpredictable tumor lysis syndrome (TLS) presents a major barrier to its broad clinical use. The purpose of this study was to investigate the relationships between pretreatment risk factors, drug pharmacokinetics, and TLS. Experimental Design A population pharmacokinetic/pharmacodynamic model linking drug exposure and TLS was developed. Plasma data of flavopiridol and its glucuronide metabolite (flavo-G) were obtained from 111 patients treated in early phase trials with frequent sampling following initial and/or escalated doses. TLS grading was modeled with logistic regression as a pharmacodynamic endpoint. Demographics, baseline disease status, and blood chemistry variables were evaluated as covariates. Results Gender was the most significant pharmacokinetic covariate, with females displaying higher flavo-G exposure than males. Glucuronide metabolite exposure was predictive of TLS occurrence, and bulky lymphadenopathy was identified as a significant covariate on TLS probability. The estimated probability of TLS occurrence in patients with baseline bulky lymphadenopathy < 10 cm or > 10 cm during the first two treatments was 0.111 (SE% 13.0%) and 0.265, (SE% 17.9%) respectively, when flavo-G area under the plasma concentration vs. time curve was at its median value in whole patient group. Conclusions This is the first population pharmacokinetic/pharmacodynamic model of TLS. Further work is needed to explore potential mechanisms and to determine if the associations between TLS, gender and glucuronide metabolites are relevant in CLL patients treated with other cyclin dependent kinase inhibitors. PMID:23300276

Pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract.

PubMed

Wen, Zhiming; Dumas, Todd E; Schrieber, Sarah J; Hawke, Roy L; Fried, Michael W; Smith, Philip C

2008-01-01

Silymarin, a mixture of polyphenolic flavonoids extracted from milk thistle (Silybum marianum), is composed mainly of silychristin, silydianin, silybin A, silybin B (SB(B)), isosilybin A (ISB(A)), and isosilybin B. In this study, the plasma concentrations of free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans were measured using liquid chromatography-electrospray ionization-mass spectrometry, after a single oral dose of 600 mg of standardized milk thistle extracts to three healthy volunteers. Pharmacokinetic analysis indicated that silymarin flavonolignans were rapidly eliminated with short half-lives (1-3 and 3-8 h for free and conjugated, respectively). The AUC(0-->infinity) values of the conjugated silymarin flavonolignans were 4- to 30-fold higher than those of their free fractions, with SB(B) (mean AUC(0-->infinity) = 51 and 597 microg x h/l for free and conjugated, respectively) and ISB(A) (mean AUC(0-->infinity) = 30 and 734 microg x h/l for free and conjugated, respectively) exhibiting higher AUC(0-->infinity) values in comparison with other flavonolignans. Near the plasma peak times (1-3 h), the free, sulfated, and glucuronidated flavonolignans represented approximately 17, 28, and 55% of the total silymarin, respectively. In addition, the individual silymarin flavonolignans exhibited quite different plasma profiles for both the free and conjugated fractions. These data suggest that, after oral administration, silymarin flavonolignans are quickly metabolized to their conjugates, primarily forming glucuronides, and the conjugates are primary components present in human plasma.

Dose-dependent interaction between gemfibrozil and repaglinide in humans: strong inhibition of CYP2C8 with subtherapeutic gemfibrozil doses.

PubMed

Honkalammi, Johanna; Niemi, Mikko; Neuvonen, Pertti J; Backman, Janne T

2011-10-01

Gemfibrozil 1-O-β-glucuronide inactivates CYP2C8 irreversibly. We investigated the effect of gemfibrozil dose on CYP2C8 activity in humans using repaglinide as a probe drug. In a randomized, five-phase crossover study, 10 healthy volunteers ingested 0.25 mg of repaglinide 1 h after different doses of gemfibrozil or placebo. Concentrations of plasma repaglinide, gemfibrozil, their metabolites, and blood glucose were measured. A single gemfibrozil dose of 30, 100, 300, and 900 mg increased the area under the concentration-time curve of repaglinide 1.8-, 4.5-, 6.7-, and 8.3-fold (P < 0.001), and its peak concentration 1.4-, 1.7-, 2.1-, and 2.4-fold (P < 0.05), compared with placebo, respectively. Gemfibrozil pharmacokinetics was characterized by a slightly more than dose-proportional increase in the area under the curve of gemfibrozil and its glucuronide. The gemfibrozil-repaglinide interaction could be mainly explained by gemfibrozil 1-O-β-glucuronide concentration-dependent, mechanism-based inhibition of CYP2C8, with a minor contribution by competitive inhibition of organic anion-transporting polypeptide 1B1 at the highest gemfibrozil dose. The findings are consistent with ∼50% inhibition of CYP2C8 already with a single 30-mg dose of gemfibrozil and >95% inhibition with 900 mg. In clinical drug-drug interaction studies, a single 900-mg dose of gemfibrozil can be used to achieve nearly complete inactivation of CYP2C8.

Curcumin Affects Phase II Disposition of Resveratrol Through Inhibiting Efflux Transporters MRP2 and BCRP

PubMed Central

Ge, Shufan; Yin, Taijun; Xu, Beibei; Gao, Song; Hu, Ming

2015-01-01

Purpose To evaluate the impact of curcumin on the disposition of resveratrol phase II metabolites in vivo, and explain the observations by performing in vitro studies in transporter-overexpressed cells. Methods Pharmacokinetic studies of resveratrol with and without the co-administration of curcumin were performed in both FVB wild-type and Bcrp1 (−/−) mice. Human UGT1A9-overexpressing HeLa cells and human MRP2-overexpressing MDCK II-UGT1A1 cells were used as in vitro tools to further determine the impact of curcumin as a transporter inhibitor on resveratrol metabolites. Results We observed higher exposure of resveratrol conjugates in Bcrp1 (−/−) mice compared to wild-type mice. In wild-type mice, curcumin increased the AUC of resveratrol glucuronide by 4-fold compared to the mice treated without curcumin. The plasma levels of resveratrol and its sulfate conjugate also increased moderately. In Bcrp1 (−/−) mice, there was a further increase (6-fold increase) in AUC of resveratrol glucuronide observed when curcumin was co-administered compared to AUC values obtained in wild-type mice without curcumin treatment. In the presence of 50nM curcumin, the clearance of resveratrol-3-O-glucuronide and resveratrol-3-O-sulfate reduced in both MRP2-overexpressing MDCKII-UGT1A1 cells and Human UGT1A9-overexpressing HeLa cells. Conclusions These results suggest that curcumin alters the phase II distribution of resveratrol through inhibiting efflux transporters including MRP2 and BCRP. PMID:26502886

Urinary Excretion of Buprenorphine, Norbuprenorphine, Buprenorphine-Glucuronide, and Norbuprenorphine-Glucuronide in Pregnant Women Receiving Buprenorphine Maintenance Treatment

PubMed Central

Kacinko, Sherri L.; Jones, Hendree E.; Johnson, Rolley E.; Choo, Robin E.; Concheiro-Guisan, Marta; Huestis, Marilyn A.

2011-01-01

BACKGROUND Buprenorphine (BUP) is under investigation as a medication therapy for opioid-dependent pregnant women. We investigated BUP and metabolite disposition in urine from women maintained on BUP during the second and third trimesters of pregnancy and postpartum. METHODS We measured BUP, norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and NBUP-Gluc concentrations in 515 urine specimens collected thrice weekly from 9 women during pregnancy and postpartum. Specimens were analyzed using a fully validated liquid chromatography-mass spectrometry method with limits of quantification of 5 µg/L for BUP and BUP-Gluc and 25 µg/L for NBUP and its conjugated metabolite. We examined ratios of metabolites across trimesters and postpartum to identify possible changes in metabolism during pregnancy. RESULTS NBUP-Gluc was the primary metabolite identified in urine and exceeded BUP-Gluc concentrations in 99% of specimens. Whereas BUP-Gluc was identified in more specimens than NBUP, NBUP exceeded BUP-Gluc concentrations in 77.9% of specimens that contained both analytes. Among all participants, the mean BUP-Gluc:NBUP-Gluc ratio was significantly higher in the second trimester compared to the third trimester, and there were significant intrasubject differences between trimesters in 71% of participants. In 3 women, the percent daily dose excreted was higher during pregnancy than postpregnancy, consistent with other data indicating increased renal elimination of drugs during pregnancy. CONCLUSIONS These data are the first to evaluate urinary disposition of BUP and metabolites in a cohort of pregnant women. Variable BUP excretion during pregnancy may indicate metabolic changes requiring dose adjustment during later stages of gestation. PMID:19325013

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids

PubMed Central

Morrish, Glynn A; Foster, David J R; Somogyi, Andrew A

2006-01-01

Aims To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids. Methods M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 µm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70–500 µm) using three microsomal samples. Results Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean ± SD) was 4.8-fold greater than M6G Vmax (555 ± 110 vs. 115 ± 19 nmol mg−1 protein h−1; P = 0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg−1 protein h−1). Km values for M3G and M6G formation were not significantly different (1.12 ± 0.37 vs. 1.11 ± 0.31 mm; P = 0.89, 0.02; −0.29, 0.32 mm). M3G and M6G formation was inhibited (P < 0.01) with a significant increase in the M3G/M6G ratio (P < 0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320 ± 42 µm and 192 ± 12 µm for M3G and M6G, respectively, and (S)-methadone Ki of 226 ± 30 µm and 152 ± 20 µm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P = 0.017, 128; 55, 202 µm) and (S)-methadone (P = 0.026, 75; 22, 128 µm). Conclusions Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G. PMID:16487227

Bioavailability of orange juice (poly)phenols: the impact of short-term cessation of training by male endurance athletes.

PubMed

Pereira-Caro, Gema; Polyviou, Thelma; Ludwig, Iziar A; Nastase, Ana-Maria; Moreno-Rojas, José Manuel; Garcia, Ada L; Malkova, Dalia; Crozier, Alan

2017-09-01

Background: Physical exercise has been reported to increase the bioavailability of citrus flavanones. Objective: We investigated the bioavailability of orange juice (OJ) (poly)phenols in endurance-trained males before and after cessation of training for 7 d. Design: Ten fit, endurance-trained males, with a mean ± SD maximal oxygen consumption of 58.2 ± 5.3 mL · kg -1 · min -1 , followed a low (poly)phenol diet for 2 d before drinking 500 mL of OJ containing 398 μmol of (poly)phenols, of which 330 μmol was flavanones. After the volunteers stopped training for 7 d the feeding study was repeated. Urine samples were collected 12 h pre- and 24 h post-OJ consumption. Bioavailability was assessed by the quantitative analysis of urinary flavanone metabolites and (poly)phenol catabolites with the use of high-pressure liquid chromatography-high resolution mass spectrometry. Results: During training, 0-24-h urinary excretion of flavanone metabolites, mainly hesperetin-3'- O -glucuronide, hesperetin-3'-sulfate, naringenin-4'- O -glucuronide, naringenin-7- O -glucuronide, was equivalent to 4.2% of OJ flavanone intake. This increased significantly to 5.2% when OJ was consumed after the volunteers stopped training for 7 d. Overall, this trend, although not significant, was also observed with OJ-derived colonic catabolites, which, after supplementation in the trained state, were excreted in amounts equivalent to 51% of intake compared with 59% after cessation of training. However, urinary excretion of 3 colonic catabolites of bacterial origin, most notably, 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid, did increase significantly when OJ was consumed postcessation compared with precessation of training. Data were also obtained on interindividual variations in flavanone bioavailability. Conclusions: A 7-d cessation of endurance training enhanced, rather than reduced, the bioavailability of OJ flavanones. The biological significance of these differences and whether they extend to the bioavailability of other dietary (poly)phenols remain to be determined. Hesperetin-3'- O -glucuronide and the colonic microbiota-derived catabolite 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid are key biomarkers of the consumption of hesperetin- O -glycoside-containing OJ and other citrus products. This trial was registered at clinicaltrials.gov as NCT02627547. © 2017 American Society for Nutrition.

Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography-time-of-flight mass spectrometry.

PubMed

Saleh, Aljona; Stephanson, Niclas Nikolai; Granelli, Ingrid; Villén, Tomas; Beck, Olof

2012-11-15

In this study a rapid liquid chromatography-time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8 μm particles. Total analysis time was 4 min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100 ng/mL for amphetamines and opiates, and 5 ng/mL for buprenorphines, with lower limits of quantification were 2.8-41 ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97-0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography-time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC-MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20 mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography-time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells

PubMed Central

Fahrmayr, C; König, J; Auge, D; Mieth, M; Fromm, MF

2012-01-01

BACKGROUND AND PURPOSE The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACH Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTS Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONS Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition. PMID:21923755

IN VITRO METABOLISM OF THYROID HORMONES BY RECOMBINANT HUMAN UDP-GLUCORONOSYLTRANSFERASES AND SULFOTRANSFERASES

EPA Science Inventory

Endocrine disruptors can decrease thyroid hormone levels via the induction of hepatic uridinediphosphate-glucoronosyltransferases (UGTs) and sulfotransferases (SULTs). Due to their ability to catalyze glucuronidation and sulfation of hormones and xenobiotics, UGTs and SULTs play ...

Effects of a Model Inducer, Phenobarbital, on Thyroid Hormone Glucuronidation in Rat Hepatocytes

EPA Science Inventory

In vivo, hepatic enzyme inducers such as phenobarbital (PB) decrease circulating thyroid hormone (TH) concentrations. This decrease in circulating TH occurs in part through extrathyroidal mechanisms. Specifically, through the induction of hepatic xenobiotic metabolizing enzymes...

IN VIVO RATE OF PHENOL GLUCURONIDATION BY RAINBOW TROUT

EPA Science Inventory

Induction of vitellogenin (VTG) in male fish has become an accepted biomarker for xenoestrogenicity. This study utilized the male rainbow trout liver slice model to determine the estrogenicity of parent compound, methoxychlor (MXC) and metabolites, di-hydroxy methoxychlor (HPTE) ...

Alternate pathways of thyroid hormone metabolism.

PubMed

Wu, Sing-Yung; Green, William L; Huang, Wen-Sheng; Hays, Marguerite T; Chopra, Inder J

2005-08-01

The major thyroid hormone (TH) secreted by the thyroid gland is thyroxine (T(4)). Triiodothyronine (T(3)), formed chiefly by deiodination of T(4), is the active hormone at the nuclear receptor, and it is generally accepted that deiodination is the major pathway regulating T(3) bioavailability in mammalian tissues. The alternate pathways, sulfation and glucuronidation of the phenolic hydroxyl group of iodothyronines, the oxidative deamination and decarboxylation of the alanine side chain to form iodothyroacetic acids, and ether link cleavage provide additional mechanisms for regulating the supply of active hormone. Sulfation may play a general role in regulation of iodothyronine metabolism, since sulfation of T(4) and T(3) markedly accelerates deiodination to the inactive metabolites, reverse triiodothyronine (rT(3)) and T(2). Sulfoconjugation is prominent during intrauterine development, particularly in the precocial species in the last trimester including humans and sheep, where it may serve both to regulate the supply of T(3), via sulfation followed by deiodination, and to facilitate maternal-fetal exchange of sulfated iodothyronines (e.g., 3,3'-diiodothyronine sulfate [T(2)S]). The resulting low serum T(3) may be important for normal fetal development in the late gestation. The possibility that T(2)S or its derivative, transferred from the fetus and appearing in maternal serum or urine, can serve as a marker of fetal thyroid function is being studied. Glucuronidation of TH often precedes biliary-fecal excretion of hormone. In rats, stimulation of glucuronidation by various drugs and toxins may lead to lower T(4) and T(3) levels, provocation of thyrotropin (TSH) secretion, and goiter. In man, drug induced stimulation of glucuronidation is limited to T(4), and does not usually compromise normal thyroid function. However, in hypothyroid subjects, higher doses of TH may be required to maintain euthyroidism when these drugs are given. In addition, glucuronidates and sulfated iodothyronines can be hydrolyzed to their precursors in gastrointestinal tract and various tissues. Thus, these conjugates can serve as a reservoir for biologically active iodothyronines (e.g., T(4), T(3), or T(2)). The acetic acid derivatives of T(4), tetrac and triac, are minor products in normal thyroid physiology. However, triac has a different pattern of receptor affinity than T(3), binding preferentially to the beta receptor. This makes it useful in the treatment of the syndrome of resistance to thyroid hormone action, where the typical mutation affects only the beta receptor. Thus, adequate binding to certain mutated beta receptors can be achieved without excessive stimulation of alpha receptors, which predominate in the heart. Ether link cleavage of TH is also a minor pathway in normal subjects. However, this pathway may become important during infections, when augmented TH breakdown by ether-link cleavage (ELC) may assist in bactericidal activity. There is a recent claim that decarboxylated derivates of thyronines, that is, monoiodothyronamine (T(1)am) and thyronamine (T(0)am), may be biologically important and have actions different from those of TH. Further information on these interesting derivatives is awaited.

75 FR 50891 - N-alkyl (C8-C18) Primary Amines and Acetate Salts; Exemption from the Requirement of a Tolerance

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-18

... alcohols and hydrocarbons; surfactants such as polyoxyethylene polymers and fatty acids; carriers such as... NAPAAS primary amines and primary amine acetate salt may also be conjugated, whether by glucuronidation...

The concomitant use of lapatinib and paracetamol - the risk of interaction.

PubMed

Karbownik, Agnieszka; Szałek, Edyta; Sobańska, Katarzyna; Grabowski, Tomasz; Klupczynska, Agnieszka; Plewa, Szymon; Wolc, Anna; Magiera, Magdalena; Porażka, Joanna; Kokot, Zenon J; Grześkowiak, Edmund

2018-02-20

Lapatinib is a tyrosine kinase inhibitor used for the treatment of breast cancer. Paracetamol is an analgesic commonly applied to patients with mild or moderate pain and fever. Cancer patients are polymedicated, which involves high risk of drug interactions during therapy. The aim of the study was to assess the interaction between lapatinib and paracetamol in rats. The rats were divided into three groups of eight animals in each. One group received lapatinib + paracetamol (I L + PA ), another group received lapatinib (II L ), whereas the last group received paracetamol (III PA ). A single dose of lapatinib (100 mg/kg b.w.) and paracetamol (100 mg/kg b.w.) was administered orally. Plasma concentrations of lapatinib, paracetamol and its metabolites - glucuronide and sulphate, were measured with the validated HPLC-MS/MS method and HPLC-UV method, respectively. The pharmacokinetic parameters of both drugs were calculated using non-compartmental methods. The co-administration of lapatinib and paracetamol increased the area under the plasma concentration-time curve (AUC) and the maximum concentration (C max ) of lapatinib by 239.6% (p = 0.0030) and 184% (p = 0.0011), respectively. Lapatinib decreased the paracetamol AUC 0-∞ by 48.8% and C max by 55.7%. In the I L + PA group the C max of paracetamol glucuronide was reduced, whereas the C max of paracetamol sulphate was higher than in the III PA group. Paracetamol significantly affected the enhanced plasma exposure of lapatinib. Additionally, lapatinib reduced the concentrations of paracetamol. The co-administration of lapatinib decreased the paracetamol glucuronidation but increased the sulphation. The findings of this study may be of clinical relevance to patients requiring analgesic therapy.

An evaluation of the DRI-ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post-mortem urine.

PubMed

Turfus, Sophie C; Vo, Tu; Niehaus, Nadia; Gerostamoulos, Dimitri; Beyer, Jochen

2013-06-01

A commercial enzyme immunoassay for the qualitative and semi-quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post-mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut-off of 0.5 µg/ml and LC-MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post-mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut-off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post-mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade-offs between sensitivity and specificity for LC-MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut-offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC-MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi-quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.

Glucuronidation and Sulfation Kinetics of Diflunisal in Man.

NASA Astrophysics Data System (ADS)

Loewen, Gordon Rapheal

Diflunisal is a nonsteroidal anti-inflammatory drug used in the treatment of arthritis and musculoskeletal pain. Diflunisal exhibits concentration- and dose-dependent kinetics, the mechanism of which has not been determined. The purpose of this study was to determine the mechanism(s) responsible for non-linear disposition of diflunisal and to examine environmental factors which may affect the elimination of diflunisal. The metabolites of diflunisal, including a new metabolite, the sulphate conjugate, were purified by column and semi-preparative high pressure liquid chromatography. Assays for the quantitation of diflunisal and conjugates in urine and diflunisal in plasma were developed. Plasma protein binding of diflunisal in blank plasma and in plasma obtained following multiple doses of diflunisal was determined by equilibrium dialysis. Total body clearance of diflunisal decreased when dose increased from 100 to 750 mg. Total clearance increased when dose increased from 750 to 1000 mg. The percent of recovered dose eliminated as the acyl glucuronide decreased and the percent eliminated as the sulphate increased with increasing dose of diflunisal. Plasma protein binding of diflunisal was concentration dependent over a range of diflunisal plasma concentrations of 3 to 257 mug/ml. Total clearance, and to a lesser degree, unbound clearance of diflunisal were decreased following multiple dose administration of 250 and 500 mg diflunisal. Percent of recovered dose eliminated as the acyl glucuronide decreased and percent eliminated as the sulphate conjugate increased following multiple dosing. Plasma protein binding of diflunisal was similar in blank plasma and plasma obtained at steady state. Unbound clearance of diflunisal exceeded liver plasma flow. Frequency distributions of the elimination of the conjugates of diflunisal were normally distributed. Sex, smoking, and use of vitamins or oral contraceptives were identified as factors which may affect the elimination of diflunisal.

Validated LC-MS/MS method for the determination of 3-hydroxflavone and its glucuronide in blood and bioequivalent buffers: application to pharmacokinetic, absorption, and metabolism studies.

PubMed

Xu, Beibei; Yang, Guanyi; Ge, Shufan; Yin, Taijun; Hu, Ming; Gao, Song

2013-11-01

The purpose of this study is to develop an UPLC-MS/MS method to quantify 3-hydroxyflavone (3-HF) and its metabolite, 3-hydroxyflavone-glucuronide (3-HFG) from biological samples. A Waters BEH C8 column was used with acetonitrile/0.1% formic acid in water as mobile phases. The mass analysis was performed in an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by acetonitrile was used to extract the analytes from blood. The results showed that the linear response range was 0.61-2500.00 nM for 3-HF and 0.31-2500.00 nM for 3-HFG. The intra-day variance is less than 16.5% and accuracy is in 77.7-90.6% for 3-HF and variance less than 15.9%, accuracy in 85.1-114.7% for 3-HFG. The inter-day variance is less than 20.2%, accuracy is in 110.6-114.2% for 3-HF and variance less than 15.6%, accuracy in 83.0-89.4% for 3-HFG. The analysis was done within 4.0 min. Only 10 μl of blood is needed due to the high sensitivity of this method. The validated method was successfully used to pharmacokinetic study in A/J mouse, transport study in the Caco-2 cell culture model, and glucuronidation study using mice liver and intestine microsomes. The applications revealed that this method can be used for 3-HF and 3-HFG analysis in blood as well as in bioequivalent buffers such HBSS and KPI. Copyright © 2013 Elsevier B.V. All rights reserved.

Pharmacokinetic drug-drug interaction between erlotinib and paracetamol: A potential risk for clinical practice.

PubMed

Karbownik, Agnieszka; Szałek, Edyta; Sobańska, Katarzyna; Grabowski, Tomasz; Wolc, Anna; Grześkowiak, Edmund

2017-05-01

Erlotinib is a tyrosine kinase inhibitor available for the treatment of non-small cell lung cancer. Paracetamol is an analgesic agent, commonly used in cancer patients. Because these drugs are often co-administered, there is an increasing issue of interaction between them. The aim of the study was to investigate the effect of paracetamol on the pharmacokinetic parameters of erlotinib, as well as the influence of erlotinib on the pharmacokinetics of paracetamol. The rabbits were divided into three groups: the rabbits receiving erlotinib (I ER ), the group receiving paracetamol (II PR ), and the rabbits receiving erlotinib+paracetamol (III ER+PR ). A single dose of erlotinib was administered orally (25mg) and was administered intravenously (35mg/kg). Plasma concentrations of erlotinib, its metabolite (OSI420), paracetamol and its metabolites - glucuronide and sulphate were measured with the validated method. During paracetamol co-administration we observed increased erlotinib maximum concentration (C max ) and area under the plasma concentration-time curve from time zero to infinity (AUC 0-∞ ) by 87.7% and 31.1%, respectively. In turn, erlotinib lead to decreased paracetamol AUC 0-∞ by 35.5% and C max by 18.9%. The mean values of paracetamol glucuronide/paracetamol ratios for C max were 32.2% higher, whereas paracetamol sulphate/paracetamol ratios for C max and AUC 0-∞ were 37.1% and 57.1% lower in the II PR group, when compared to the III ER+PR group. Paracetamol had significant effect on the enhanced plasma exposure of erlotinib. Additionally, erlotinib contributed to the lower concentrations of paracetamol. Decreased glucuronidation and increased sulphation of paracetamol after co-administration of erlotinib were also observed. Copyright © 2017. Published by Elsevier B.V.

Pharmacokinetics of paracetamol in patients with chronic pancreatitis.

PubMed

Siepsiak, Magdalena; Szałek, Edyta; Karbownik, Agnieszka; Grabowski, Tomasz; Mziray, Marzanna; Adrych, Krystian; Grześkowiak, Edmund

2016-08-01

Chronic pancreatitis (CP) is a progressive, irreversible disease causing damage of the gland. Abdominal pains are a typical symptom of pancreatitis both in the chronic and acute form. Paracetamol is one of analgesics used for treating mild or moderate pain. Functional and anatomical changes in the gastrointestinal tract caused by pancreatitis may influence on the pharmacokinetics of administered drugs. In the present study we analysed the pharmacokinetics of paracetamol after oral and intravenous administration to patients with CP. The patients were allocated to one of the two groups of the drug under study: I iv, intravenous administration of paracetamol 1000mg (n=17; mean [SD] age, 46.18 [13.78] years; and BMI, 22.03 [2.62]kg/m(2)) and II po, oral administration of paracetamol 1000mg (n=17; mean [SD] age, 48.29 [10.08] years; and BMI, 22.50 [2.92]kg/m(2). The plasma concentrations of paracetamol and its metabolite (glucuronide) were measured with the validated high-pressure liquid chromatography (HPLC) method with ultraviolet (UV) detection. The main pharmacokinetic parameters for paracetamol after iv and po administration to patients with CP were as follows: Cmax, 19.00 [4.50] and Cmax, 9.26 [3.35]μg/ml; AUC0-t, 42.37 [13.92] and 36.68 [11.7]μg×h/mL, respectively. After iv and po administration the AUC ratio between the metabolite (glucuronide) and paracetamol was enhanced. The research findings revealed that patients with chronic pancreatitis had lower concentrations of paracetamol. Therefore, it may be necessary to apply additional analgesic therapy. Moreover, we observed enhanced glucuronidation in our patients. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Validated LC-MS/MS method for the determination of 3-Hydroxflavone and its glucuronide in blood and bioequivalent buffers: Application to pharmacokinetic, absorption, and metabolism studies

PubMed Central

Xu, Beibei; Yang, Guanyi; Ge, Shufan; Yin, Taijun; Hu, Ming; Gao, Song

2015-01-01

The purpose of this study is to develop an UPLC-MS/MS method to quantify 3-hydroxy-flavone (3-HF) and its metabolite, 3-hydroxyflavone-glucuronide (3-HFG) from biological samples. A Waters BEH C8 column was used with acetonitrile/0.1 % formic acid in water as mobile phases. The mass analysis was performed in an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by acetonitrile was used to extract the analytes from plasma. The results showed that the linear response range was 0.61– 2,500.00 nM for 3-HF and 0.31– 2,500.00 nM for 3-HFG. The intra-day variance is less 16.48 % and accuracy is in 77.70–90.64 % for 3-HF and variance less than 15.86%, accuracy in 85.08–114.70 % for 3-HFG. The inter-day variance is less than 20.23 %, accuracy is in 110.58–114.2 % for 3-HF and variance less than 15.59 %, accuracy in 83.00–89.40% for 3-HFG. The analysis was done within 4.0 min. Only 10 μL of blood is needed due to the high sensitivity of this method. The validated method was successfully used to pharmacokinetic study in A/J mouse, transport study in the Caco-2 cell culture model, and glucuronidation study using mice liver and intestine microsomes. The applications revealed that this method can be used for 3-HF and 3-HFG analysis in blood as well as in bioequivalent buffers such HBSS and KPI. PMID:23973631

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.

The fate of benzoic acid in various species

PubMed Central

Bridges, J. W.; French, M. R.; Smith, R. L.; Williams, R. T.

1970-01-01

1. The urinary excretion of orally administered [14C]benzoic acid in man and 20 other species of animal was examined. 2. At a dose of 50mg/kg, benzoic acid was excreted by the rodents (rat, mouse, guinea pig, golden hamster, steppe lemming and gerbil), the rabbit, the cat and the capuchin monkey almost entirely as hippuric acid (95–100% of 24h excretion). 3. In man at a dose of 1mg/kg and the rhesus monkey at 20mg/kg benzoic acid was excreted entirely as hippuric acid. 4. At 50mg/kg benzoic acid was excreted as hippuric acid to the extent of about 80% of the 24h excretion in the squirrel monkey, pig, dog, ferret, hedgehog and pigeon, the other 20% being found as benzoyl glucuronide and benzoic acid, the latter possibly arising by decomposition of the former. 5. On increasing the dose of benzoic acid to 200mg/kg in the ferret, the proportion of benzoyl glucuronide excreted increased and that of hippuric acid decreased. This did not occur in the rabbit, which excreted 200mg/kg almost entirely as hippuric acid. It appears that the hedgehog and ferret are like the dog in respect to their metabolism of benzoic acid. 6. The Indian fruit bat produced only traces of hippuric acid and possibly has a defect in the glycine conjugation of benzoic acid. The main metabolite in this animal (dose 50mg/kg) was benzoyl glucuronide. 7. The chicken, side-necked turtle and gecko converted benzoic acid mainly into ornithuric acid, but all three species also excreted smaller amounts of hippuric acid. PMID:4990586

Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT-751

PubMed Central

Innocenti, Federico; Ramírez, Jacqueline; Obel, Jennifer; Xiong, Julia; Mirkov, Snezana; Chiu, Yi-Lin; Katz, David A.; Carr, Robert A.; Zhang, Wei; Das, Soma; Adjei, Araba; Moyer, Ann M.; Chen, Pei Xian; Krivoshik, Andrew; Medina, Diane; Gordon, Gary B.; Ratain, Mark J.; Sahelijo, Leonardo; Weinshilboum, Richard M.; Fleming, Gini F.; Bhathena, Anahita

2013-01-01

Objective ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751. Methods UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes. Results UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (> 2) was associated with an average 34% increase in ABT-751 clearance (P= 0.044), an 18% reduction in ABT-751 AUC (P = 0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P =0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 Ctrough (P = 0.009). Conclusion These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability. PMID:23670235

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.

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

PubMed Central

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-01-01

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. PMID:28346357

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

PubMed

Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

2017-03-25

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Pharmacokinetics, oral bioavailability, and metabolic profile of resveratrol and its dimethylether analog, pterostilbene, in rats

PubMed Central

Kapetanovic, Izet M.; Huang, Zhihua; Thompson, Thomas N.; McCormick, David L.

2011-01-01

Purpose Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a naturally occurring polyphenol with a broad range of possible health benefits, including anti-cancer activity. However, the biological activity of resveratrol may be limited by poor absorption and first-pass metabolism: only low plasma concentrations of resveratrol are seen following oral administration, and metabolism to glucuronide and sulfate conjugates is rapid. Methylated polyphenol analogs (such as pterostilbene [3,5-dimethoxy-4′-hydroxy-trans-stilbene], the dimethylether analog of resveratrol) may overcome these limitations to pharmacologic efficacy. The present study was designed to compare the bioavailability, pharmacokinetics, and metabolism of resveratrol and pterostilbene following equimolar oral dosing in rats. Methods The agents were administered orally via gavage for 14 consecutive days at 50 or 150 mg/kg/day for resveratrol and 56 or 168 mg/kg/day for pterostilbene. Two additional groups were dosed once intravenously with 10 and 11.2 mg/kg for resveratrol and pterostilbene, respectively. Plasma concentrations of agents and metabolites were measured using a high-pressure liquid chromatograph-tandem mass spectrometer system. Noncompartmental analysis was used to derive pharmacokinetic parameters. Results Resveratrol and pterostilbene were approximately 20 and 80% bioavailable, respectively. Following oral dosing, plasma levels of pterostilbene and pterostilbene sulfate were markedly greater than were plasma levels of resveratrol and resveratrol sulfate. Although plasma levels of resveratrol glucuronide exceeded those of pterostilbene glucuronide, those differences were smaller than those of the parent drugs and sulfate metabolites. Conclusions When administered orally, pterostilbene demonstrates greater bioavailability and total plasma levels of both the parent compound and metabolites than does resveratrol. These differences in agent pharmacokinetics suggest that the in vivo biological activity of equimolar doses of pterostilbene may be greater than that of resveratrol. PMID:21116625

Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated LC-MS/MS analyses.

PubMed

Maas, Alexandra; Maier, Christoph; Iwersen-Bergmann, Stefanie; Madea, Burkhard; Hess, Cornelius

2017-11-30

Besides its clinical application, the anaesthetic agent propofol is being increasingly misused, mostly by healthcare professionals, and its abuse potential gained worldwide attention after the tragic death of Michael Jackson in 2009. Due to the short duration of its narcotic effects, propofol abuse is especially easy to hide compared with the use of other recreational drugs. However, propofol possesses a very narrow therapeutic window between the desired effect and potentially fatal toxicity, making abuse of the drug extremely dangerous even in experienced physicians. Consequently, it is important that forensic laboratories possess a sensitive and specific method for the detection of chronic propofol abuse. We present a simple, fast and reliable method to simultaneously extract propofol and its main metabolite propofol glucuronide from hair, followed by sensitive LC-MS/MS analyses, allowing to determine a chronic propofol abuse. Difficulties regarding the detection of propofol using LC-MS/MS were solved by using a derivatization reaction with 2-fluoro-1-methylpyridinium-p-toluene-sulfonate and triethylamine. Reliability of extraction method and subsequent LC-MS/MS analyses was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, processed sample stability, matrix effects and recovery. Appropriate quantification (LLOQ=10pg/mg hair) and detection limits (3.6pg/mg hair for propofol and 7.8 pg/mg hair for propofol glucuronide) could be achieved, enabling to detect even small amounts of both analytes. Applicability of the method was confirmed by analysis of three human hair samples from deceased with suspicion of chronic propofol abuse. Copyright © 2017 Elsevier B.V. All rights reserved.

Utilising an LC-MS/MS-based multi-biomarker approach to assess mycotoxin exposure in the Bangkok metropolitan area and surrounding provinces.

PubMed

Warth, Benedikt; Petchkongkaew, Awanwee; Sulyok, Michael; Krska, Rudolf

2014-01-01

Human exposures to mycotoxins through dietary intake are a major health hazard and may result in various pathophysiological effects. Although Thailand is a country at increased risk due to its climatic conditions, no comprehensive dataset is available to perform proper exposure assessment of its population with regard to mycotoxins. Therefore, this pilot study was conducted to investigate and evaluate the exposure levels of major mycotoxins (aflatoxin B₁, ochratoxin A, fumonisins, zearalenone and trichothecenes). Sixty first-morning urine samples were collected from healthy volunteers who live in the Bangkok metropolitan area and surrounding provinces (Pathumthani, Nonthaburi, Samutprakarn and Samutsakorn). Urine samples were analysed by a LC-MS/MS-based multi-biomarker method following a so-called 'dilute and shoot' approach. Results generally indicated low mycotoxin exposures in most individuals through the determination of the four biomarkers that were detected in urine samples, i.e. aflatoxin M₁, ochratoxin A (OTA), as well as the deoxynivalenol (DON) metabolites DON-3-glucuronide and DON-15-glucuronide in 10 of 60 individuals. The maximum concentrations were used to estimate the daily intake confirming that none of the individuals exceeded the tolerable daily intake (TDI) of DON (maximum 26% of TDI) or OTA (maximum 22% of TDI). However, the maximum exposure of aflatoxin B₁, estimated to be 0.91 µg (kg bw)⁻¹ day⁻¹, should raise some concerns and suggests further studies utilising a more sensitive method. Low exposure to Fusarium toxins was also confirmed by the absence of zearalenone, α-zearalanol, β-zearalanol and zearalenone-14-glucuronide as well as T-2 toxin, HT-2 toxin, nivalenol and free DON. This is the first multi-mycotoxin biomarker study performed in Southeast Asia.

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

PubMed

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

1991-01-01

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

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn’s Disease Patients and Unaffected First-Degree Relatives

PubMed Central

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn’s disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn’s disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn’s disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn’s disease. PMID:26824357

Toxicokinetics of 1,2-diethylbenzene in male Sprague-Dawley rats-part 1: excretion and metabolism of [(14)C]1,2-diethylbenzene.

PubMed

Payan, J P; Beydon, D; Cossec, B; Ensminger, A; Fabry, J P; Ferrari, E

1999-12-01

The excretion and metabolism of neurotoxic 1,2-diethylbenzene (1, 2-DEB) was studied in male Sprague-Dawley rats after i.v. (1 mg/kg) or oral (1 or 100 mg/kg) administration of 1,2-diethyl[U-(14)C]benzene ([(14)C]1,2-DEB). Whatever the treatment, radioactivity was mainly excreted in urine (65-76% of the dose) and to a lower extent in feces (15-23% of the dose), or via exhaled air (3-5% of the dose). However, experiments with rats fitted with a biliary cannula demonstrated that about 52 to 64% of the administered doses (1 or 100 mg/kg) were initially excreted in bile. Biliary metabolites were extensively reabsorbed from the gut and ultimately excreted in urine after several enterohepatic circulations. Insignificant amounts of unchanged 1,2-DEB were recovered in the different excreta (urine, bile, and feces). As reported previously, presence of 1-(2'-ethylphenyl)ethanol (EPE) was confirmed in urine and demonstrated in bile and feces. The two main [(14)C]1,2-DEB metabolites accounted for 57 to 79% of urinary and biliary radioactivity, respectively. Beta-Glucuronidase hydrolysis and electron impact mass spectra results strongly supported their glucuronide structure. Additionally, these two main metabolites were thought to be the glucuronide conjugates of the two potential enantiomers of EPE. The results indicate that the main initial conversion step of the primary metabolic pathway of 1,2-DEB appears to be the hydroxylation of the alpha-carbon atom of the side chain. The presence of two glucuronide conjugates of EPE in the urine in a ratio different from one suggests that the metabolic conversion of 1, 2-DEB is under stereochemical control.

A comparison of direct and indirect analytical approaches to measuring total nicotine equivalents in urine.

PubMed

Taghavi, Taraneh; Novalen, Maria; Lerman, Caryn; George, Tony P; Tyndale, Rachel F

2018-05-31

Total nicotine equivalents (TNE), the sum of nicotine and metabolites in urine, is a valuable tool for evaluating nicotine exposure. Most methods for measuring TNE involve two-step enzymatic hydrolysis for indirect quantification of glucuronide metabolites. Here, we describe a rapid, low-cost direct liquid chromatography - tandem mass spectrometry (LCMS) assay. In 139 smokers' urine samples, Bland-Altman, correlation, and regression analyses were used to investigate differences in quantification of nicotine and metabolites, TNE, and nicotine metabolite ratio (NMR) between direct and indirect LCMS methods. DNA from a subset (n=97 smokers) was genotyped for UGT2B10*2 and UGT2B17*2 and the known impact of these variants was evaluated using urinary ratios determined by the direct versus indirect method. The direct method showed high accuracy (0-9% bias) and precision (3-14% coefficient of variation) with similar distribution of nicotine metabolites to literary estimates and good agreement between the direct and indirect methods for nicotine, cotinine, and 3-hydroxycotinine (ratios 0.99-1.07), but less agreement for their respective glucuronides (ratios 1.16-4.17). The direct method identified urinary 3HC+3HC-GLUC/COT as having the highest concordance with plasma NMR and provided substantially better estimations of the established genetic impact of glucuronidation variants compared to the indirect method. Direct quantification of nicotine and metabolites is less time-consuming and less costly, and provides accurate estimates of nicotine intake, metabolism rate and the impact of genetic variation in smokers. Lower cost and maintenance combined with high accuracy and reproducibility make the direct method ideal for smoking biomarker, NMR and pharmacogenomics studies. Copyright ©2018, American Association for Cancer Research.

Nutritional Status Differentially Alters Cytochrome P450 3A4 (CYP3A4) and Uridine 5'-Diphospho-Glucuronosyltransferase (UGT) Mediated Drug Metabolism: Effect of Short-Term Fasting and High Fat Diet on Midazolam Metabolism.

PubMed

Lammers, Laureen A; Achterbergh, Roos; Romijn, Johannes A; Mathôt, Ron A A

2018-06-06

Previous studies have shown that nutritional status can alter drug metabolism which may result in treatment failure or untoward side effects. This study assesses the effect of two nutritional conditions, short-term fasting, and a short-term high fat diet (HFD) on cytochrome P450 3A4 (CYP3A4) and uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated drug metabolism by studying the pharmacokinetics of midazolam and its main metabolites. In a randomized-controlled cross-over trial, nine healthy subjects received a single intravenous administration of 0.015 mg/kg midazolam after: (1) an overnight fast (control); (2) 36 h of fasting; and (3) an overnight fast after 3 days of a HFD consisting of 500 ml of cream supplemented to their regular diet. Pharmacokinetic parameters were analyzed simultaneously using non-linear mixed-effects modeling. Short-term fasting increased CYP3A4-mediated midazolam clearance by 12% (p < 0.01) and decreased UGT-mediated metabolism apparent 1-OH-midazolam clearance by 13% (p < 0.01) by decreasing the ratio of clearance and the fraction metabolite formed (ΔCL 1-OH-MDZ /f 1-OH-MDZ ). Furthermore, short-term fasting decreased apparent clearance of 1-OH-midazolam-O-glucuronide (CL 1-OH-MDZ-glucuronide /(f 1-OH-MDZ-glucuronide  × f 1-OH-MDZ )) by 20% (p < 0.01). The HFD did not affect systemic clearance of midazolam or metabolites. Short-term fasting differentially alters midazolam metabolism by increasing CYP3A4-mediated metabolism but by decreasing UGT-mediated metabolism. In contrast, a short-term HFD did not affect systemic clearance of midazolam.

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

PubMed

Fang, Zhong-Ze; Wang, Haina; Cao, Yun-Feng; Sun, Dong-Xue; Wang, Li-Xuan; Hong, Mo; Huang, Ting; Chen, Jian-Xing; Zeng, Jia

2015-03-01

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism. © 2014 Wiley Periodicals, Inc.

Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Ayala, Daniel C.; Morin, Dexter; Buckpitt, Alan R.

2015-01-01

Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up. PMID:25853821

Herb–drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7

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

Ma, Hai-Ying, E-mail: cmu4h-mhy@126.com; Sun, Dong-Xue; Cao, Yun-Feng

2014-05-15

Herb–drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (K{sub i}) were determined for themore » compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (K{sub i}) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb–drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7. - Highlights: • Specific inhibition of andrographolide derivatives towards UGT2B7. • Herb-drug interaction related withAndrographis paniculata. • Guidance for design of UGT2B7 specific inhibitors.« less

Development of a new multi-analyte assay for the simultaneous detection of opioids in serum and other body fluids using liquid chromatography-tandem mass spectrometry.

PubMed

Eckart, K; Röhrich, J; Breitmeier, D; Ferner, M; Laufenberg-Feldmann, R; Urban, R

2015-09-15

A liquid chromatography-tandem mass spectrometry method using electrospray ionization in positive ionization mode was developed for the simultaneous detection of multiple opioid-type drugs in plasma. The presented assay allows the quantitative determination of alfentanil, buprenorphine, codeine, desomorphine, dextromethorphan, dextrorphan, dihydrocodeine, dihydromorphine, ethylmorphine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, naloxone, naltrexone, oxycodone, oxymorphone, pentazocine, pethidine, pholcodine, piritramide, remifentanil, sufentanil, and tramadol as well as the metabolites 6-monoacetylmorphine, bisnortilidine, morphine-3-glucuronide, morphine-6-glucuronide, naltrexol, norbuprenorphine, norfentanyl, norpethidine, nortilidine, and O-desmethyltramadol. Serum and blood samples were purified by solid-phase extraction. The analytes were separated on a phenyl-hexyl (100mm) column by formic acid/acetonitrile gradient elution using an UPLC 1290 Infinity coupled with a 6490 Triple Quadrupole mass spectrometer. The limits of detection ranged from 0.02 to 0.6ng/mL and the lower limits of quantification ranged from 0.1 to 2.0ng/mL. The calibration curves were linear between Calibration Levels 1-6 for all 35 substances. Recovery rates ranged between 51 and 88% for all compounds except alfentanil, bisnortilidine, pethidine, and morphine-3-glucuronide. The matrix effect ranged from 86% for ethylmorphine to 105% for desomorphine. Using the validation procedure proposed by the German Society of Toxicological and Forensic Chemistry, acceptable precision and accuracy data for almost all analytes were obtained. The method was successfully applied to 206 authentic serum samples provided by the palliative and intensive care units of the University Medical Center and the police authorities. Furthermore, a suspected fatal intoxication is demonstrated by an analysis of the sufentanil in post mortem body fluids and tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative Rationalization of Gemfibrozil Drug Interactions: Consideration of Transporters-Enzyme Interplay and the Role of Circulating Metabolite Gemfibrozil 1-O-β-Glucuronide.

PubMed

Varma, Manthena V S; Lin, Jian; Bi, Yi-an; Kimoto, Emi; Rodrigues, A David

2015-07-01

Gemfibrozil has been suggested as a sensitive cytochrome P450 2C8 (CYP2C8) inhibitor for clinical investigation by the U.S. Food and Drug Administration and the European Medicines Agency. However, gemfibrozil drug-drug interactions (DDIs) are complex; its major circulating metabolite, gemfibrozil 1-O-β-glucuronide (Gem-Glu), exhibits time-dependent inhibition of CYP2C8, and both parent and metabolite also behave as moderate inhibitors of organic anion transporting polypeptide 1B1 (OATP1B1) in vitro. Additionally, parent and metabolite also inhibit renal transport mediated by OAT3. Here, in vitro inhibition data for gemfibrozil and Gem-Glu were used to assess their impact on the pharmacokinetics of several victim drugs (including rosiglitazone, pioglitazone, cerivastatin, and repaglinide) by employing both static mechanistic and dynamic physiologically based pharmacokinetic (PBPK) models. Of the 48 cases evaluated using the static models, about 75% and 98% of the DDIs were predicted within 1.5- and 2-fold of the observed values, respectively, when incorporating the interaction potential of both gemfibrozil and its 1-O-β-glucuronide. Moreover, the PBPK model was able to recover the plasma profiles of rosiglitazone, pioglitazone, cerivastatin, and repaglinide under control and gemfibrozil treatment conditions. Analyses suggest that Gem-Glu is the major contributor to the DDIs, and its exposure needed to bring about complete inactivation of CYP2C8 is only a fraction of that achieved in the clinic after a therapeutic gemfibrozil dose. Overall, the complex interactions of gemfibrozil can be quantitatively rationalized, and the learnings from this analysis can be applied in support of future predictions of gemfibrozil DDIs. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Catechol-O-Methyltransferase and UDP-Glucuronosyltransferases in the Metabolism of Baicalein in Different Species.

PubMed

Zhang, Ruiya; Cui, Yonglei; Wang, Yan; Tian, Xiangge; Zheng, Lu; Cong, HaiJian; Wu, Bin; Huo, Xiaokui; Wang, Chao; Zhang, BaoJing; Wang, Xiaobo; Yu, Zhonghui

2017-12-01

Baicalein is the major bioactive flavonoid in some herb medicines and dietary plants; however, the detailed metabolism pathway of its major metabolite oroxylin A-7-O-β-D-glucuronide in human was not clear. It was important to illustrate the major metabolic enzymes that participate in its elimination for the clinic use of baicalein. We first revealed a two-step metabolism profile for baicalein and illustrated the combination of catechol-O-methyltransferase (COMT) and uridine diphosphate-glucuronosyltransferases (UGTs) in drug metabolism, further evaluated its bioactivity variation during drug metabolism. The metabolism profiles were systematically characterized in different human biology preparations; after then, the anti-inflammatory activities of metabolites were evaluated in LPS-induced RAW264.7 cell. The first-step metabolite of baicalein was isolated and identified as oroxylin A; soluble-bound COMT (S-COMT) was the major enzyme responsible for its biotransformation. Specially, position 108 mutation of S-COMT significantly decreases the elimination. Meantime, oroxylin A was rapidly metabolized by UGTs, UGT1A1, -1A3, -1A6, -1A7, -1A8, -1A9, and -1A10 which were involved in the glucuronidation. Considerable species differences were observed with 1060-fold K m (3.05 ± 1.86-3234 ± 475 μM) and 330-fold CL int (5.93-1973 μL/min/mg) variations for baicalein metabolism. Finally, the middle metabolite oroxylin A exhibited a potent anti-inflammatory activity with the IC 50 value of 28 μM. The detailed kinetic parameters indicated that COMT provide convenience for the next glucuronidation; monkey would be a preferred animal model for the preclinical investigation of baicalein. Importantly, oroxylin A should be reconsidered in evaluating baicalein efficacy against inflammatory diseases.

Altered thyroid status in lake trout (Salvelinus namaycush) exposed to co-planar 3,3',4,4',5-pentachlorobiphenyl.

PubMed

Brown, Scott B; Evans, Robert E; Vandenbyllardt, Lenore; Finnson, Ken W; Palace, Vince P; Kane, Andrew S; Yarechewski, Alvin Y; Muir, Derek C G

2004-03-30

Recent studies indicate that co-planar 3,3',4,4',5-pentachlorobiphenyl (PCB) congeners or their metabolites may disrupt thyroid function in fishes. Although co-planar PCB have been detected at microgram per kilogram levels in fish from contaminated areas, few studies have examined mechanisms whereby, co-planar PCBs may alter thyroid function in fish. We treated immature lake trout by intraperitoneal (i.p.)-injection or dietary gavage with vehicle containing 0, 0.7, 1.2, 25 or 40 microg 3,3',4,4',5-pentachlorobiphenyl (PCB 126) per kgBW. Blood and tissue samples were collected at various times up to 61 weeks following exposure. The treatments produced sustained dose-dependent elevations of tissue (PCB 126) concentrations. Thyroid epithelial cell height (TECH), plasma thyroxine (T4) and 3,3',5-triiodo-l-thyronine (T3) concentrations, hepatic 5'-monodeiodinase, hepatic glucuronidation of T4 and T3, as well as plasma T4 kinetics and fish growth were analyzed. Exposure to the highest doses of PCB 126 caused increased TECH, plasma T4 dynamics and T4-glucuronidation (T4-G). PCB 126 did not affect 5'-monodeiodinase and T3-glucuronidation (T3-G) and there were no effects on fish growth or condition. Because T3 status and growth were unaffected, the thyroid system was able to compensate for the alterations caused by the PCB 126 exposure. It is clear that concentrations of co-planar PCBs similar to those found in predatory fish from contaminated areas in the Great Lakes are capable of enhancing metabolism of T4. These changes may be of significance when T4 requirements are high for other reasons (e.g. periods of rapid growth, warm temperatures, metamorphosis, and parr-smolt transformation).

Interaction between rhein acyl glucuronide and methotrexate based on human organic anion transporters.

PubMed

Yuan, Yuan; Yang, Hua; Kong, Linghua; Li, Yuan; Li, Ping; Zhang, Hongjian; Ruan, Jianqing

2017-11-01

Rhein, a major bioactive compound of many medicinal herbs and the prodrug of diacerein, is often used with low dose of methotrexate as drug combination to treat rheumatoid arthritis. In this study, potential drug-drug interaction between methotrexate and rhein was investigated based on organic anion transporters (OAT). Our study demonstrated that rhein acyl glucuronide (RAG), the major metabolite of rhein in the human blood circulation, significantly inhibited the uptake of p-aminohippurate in hOAT1 transfected cells with IC 50 value of 691 nM and estrone sulfate uptake in hOAT3 transfected cells with IC 50 value of 78.5 nM. As the substrate of both hOAT1 and hOAT3, the methotrexate transport was significantly inhibited by RAG in hOAT1 transfected cells at 50 μM and hOAT3 transfected cells at 1 μM by 69% and 87%, respectively. Further in vivo study showed that after co-administrated with RAG in rats the AUC 0-24 values of methotrexate increased from 3109 to 5370 ng/mL*hr and the t 1/2 was prolonged by 40.5% (from 7.4 to 10.4 h), demonstrating the inhibitory effect of RAG on methotrexate excretion. In conclusion, rhein acyl glucuronide could significantly decrease the transport of methotrexate by both hOAT1 and hOAT3. The combination use of rhein, diacerein or other rhein-containing herbs with methotrexate may cause obvious drug-drug interaction and require close monitoring for potential drug interaction in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

DETERMINATION OF KOW VALUES FOR A SERIES OF ARYL GLUCURONIDES

EPA Science Inventory

An important perameter in toxicokinetic modeling is the octanol/water partition coefficient (Kow). This parameter has often been used to predict the accumulation of contaminants from water to fish (Klamer and Beekman 1995); however, few Kow values are available for modeling the b...

A preliminary investigation on the distribution of cannabinoids in man.

PubMed

Gronewold, Antonia; Skopp, Gisela

2011-07-15

An LC/MS/MS procedure to determine THC along with its major metabolites 11-OH-THC, THC-COOH and its glucuronide as well as the cannabinoids CBD and CBN was applied to 5 post mortem cases to study their distribution into some less commonly studied matrices. Analytes were determined in fluids and tissue homogenates following protein precipitation and liquid-liquid extraction. Gall bladder fluid exhibited maximum concentrations of all analytes except THC, which was detectable in high concentrations in muscle tissue along with CBD. THC was also present in lung specimens, whereas its concentration in liver samples was low or not detectable at all. Liver und kidney specimens contained appreciable amounts of THC-COOglu. Findings from bile support extensive enterohepatic recirculation of the glucuronide. Muscle tissue seems an interesting specimen to detect multiple cannabis use, and brain may serve as an alternative specimen for blood; nevertheless, the present findings should be substantiated by further investigations. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Metabolic comparison of radiolabeled aniline- and phenol-phthaleins with (131)I.

PubMed

Avcibaşi, Uğur; Avcibaşi, Nesibe; Unak, Turan; Unak, Perihan; Müftüler, Fazilet Zümrüt; Yildirim, Yeliz; Dinçalp, Haluk; Gümüşer, Fikriye Gül; Dursun, Ebru Rükşen

2008-05-01

The metabolic comparison of aniline- and phenol-phthaleins radiolabeled with (131)I ((131)I-APH and (131)I-PPH, respectively) has been investigated in this study. To compare the metabolic behavior of these phthaleins and their glucuronide conjugates radiolabeled with (131)I, scintigraphic and biodistributional techniques were applied using male Albino rabbits. The results obtained have shown that these compounds were successfully radioiodinated with a radioiodination yield of about 100%. Maximum uptakes of (131)I-APH and (131)I-PPH, which were metabolized as N- and O-glucuronides, were observed within 2 h in the bladder and in the small intestine, respectively. In the case of verification of considerably up taking of these compounds also by tumors developed in the small intestine and in the bladder tissues, these results can be expected to be encouraging to test these compounds, which will be radiolabeled with other radioiodines such as (125)I, (123)I and (124)I as imaging and therapeutic agents in nuclear medical applications.

NMR-based phytochemical analysis of Vitis vinifera cv Falanghina leaves. Characterization of a previously undescribed biflavonoid with antiproliferative activity.

PubMed

Tartaglione, Luciana; Gambuti, Angelita; De Cicco, Paola; Ercolano, Giuseppe; Ianaro, Angela; Taglialatela-Scafati, Orazio; Moio, Luigi; Forino, Martino

2018-03-01

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3-O-β-d-glucopyranoside, quercetin-3-O-β-d-glucuronide, kaempferol-3-O-β-d-glucopyranoside and kaempferol-3-O-β-d-glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimation of phenolic conjugation by colonic mucosa.

PubMed Central

Ramakrishna, B S; Gee, D; Weiss, A; Pannall, P; Roberts-Thomson, I C; Roediger, W E

1989-01-01

Conjugation of phenol by the colonic mucosa was assessed in vivo using dialysis tubing containing 1.5 ml of 1 mmol/l acetaminophen (paracetamol) and 10 mmol/l butyrate. These were allowed to equilibrate in the rectum for one hour. The glucuronidated and sulphated conjugates of acetaminophen were measured by high pressure liquid chromatography and bicarbonate concentrations by gas analysis. In 21 subjects without colonic disease sulphate conjugation was observed in all cases, with a mean (SE) of 3.86 (0.66) nmol/hour, while glucuronide conjugation was found in seven of 21 cases. Mean (SE) bicarbonate output of 42.9 (3.9) mumol/hour (n = 21) indicated healthy colonic mucosal metabolism and phenolic sulphation in dialysate and agreed with published sulphation rates obtained with cultured cells of colonic epithelium. Acetaminophen sulphation suggests that the colonic mucosa has an important role in the conjugation of phenols, and the method reported here would be useful in assessing the detoxification capacity of the colonic mucosa in diseases of the rectal mucosa. PMID:2738167

Metabolism of benoxinate in humans.

PubMed

Kasuya, F; Igarashi, K; Fukui, M

1987-04-01

The metabolism of benoxinate hydrochloride [2-(diethylamino)ethyl 4-amino-3-butoxybenzoate monohydrochloride; oxybuprocaine] was examined in humans after administration of a single oral dose. The drug was almost completely absorbed and was rapidly excreted in the urine (92.1% of dose in 9 h). Nine metabolites and unchanged drug were isolated from the urine and identified by comparison of TLC, GC, and GC-MS with authentic compounds. Any metabolites reflecting initial loss of the butyl side chain of benoxinate could not be detected. This suggests that the ester portion is metabolized more rapidly than the O-butyl side chain. 3-Butoxy-4-aminobenzoic acid, the hydrolyzed product of benoxinate, was primarily excreted (70-90% of dose) as the glucuronide together with a trace of the glycine conjugate (0.35% of dose). In addition, 3-butoxy-4-acetylaminobenzoic acid, 3-hydroxy-4-aminobenzoic acid, and 3-hydroxy-4-acetylaminobenzoic acid were identified, the latter two being detected partly as the glucuronides (1.20 and 1.43% of dose, respectively).

Infusions of artichoke and milk thistle represent a good source of phenolic acids and flavonoids.

PubMed

Pereira, Carla; Barros, Lillian; Carvalho, Ana Maria; Santos-Buelga, Celestino; Ferreira, Isabel C F R

2015-01-01

Cynara scolymus L. (artichoke) and Silybum marianum (L.) Gaertn (milk thistle) are two herbs well-known for their efficiency in the prevention/treatment of liver injuries, among other chronic diseases. Therefore, the aim of this work was to characterize specific bioactive components, phenolic compounds, in hydromethanolic extracts but also in infusions (the most commonly used preparations) obtained from the whole plant of milk thistle and artichoke. The phenolic profiles were accessed using HPLC-DAD-MS/ESI. Infusions of both species presented higher phenolic contents than the hydromethanolic extracts. Milk thistle presented a similar phenolic composition between the two preparations, revealing only differences in the quantities obtained. Nevertheless, artichoke revealed a slightly different profile considering infusion and hydromethanolic extracts. Apigenin-7-O-glucuronide was the major flavonoid found in milk thistle, while luteolin-7-O-glucuronide was the most abundant in artichoke. Therefore, infusions of both artichoke and milk thistle represent a good source of bioactive compounds, especially phenolic acids and flavonoids.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia.

PubMed

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H

2015-11-15

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Absorption and metabolism of milk thistle flavanolignans in humans.

PubMed

Calani, Luca; Brighenti, Furio; Bruni, Renato; Del Rio, Daniele

2012-12-15

This study evaluated the absorption and metabolism of milk thistle flavonolignans silychristin, silydianin, silybin and isosilybin isomers (all together known as silymarin) in humans. Fourteen volunteers consumed an extract of milk thistle and urine was collected up to 48 h after consumption. Thirty-one metabolites were identified in urine by means of HPLC-MS/MS, monoglucuronides being the most common excreted form, followed by sulphate-glucuronides and diglucuronides, respectively. The excretion of monoglucuronides peaked 2 h after consumption, whereas sulphate-glucuronide and diglucuronide excretion peaked at 8 h. The bioavailability of milk thistle flavanolignans was 0.45±0.28% (mean±SD). In conclusion, milk thistle flavonolignans are extensively modified after ingestion and recovered in urine as sulpho- and glucuronyl-conjugates, indicating a strong affinity for hepatic phase II enzymes. All future studies (in vitro and in vivo) dealing with the effects of milk thistle should start by considering the modification of its flavonolignans after ingestion by humans. Copyright © 2012 Elsevier GmbH. All rights reserved.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: advances in understanding breast milk-induced neonatal hyperbilirubinemia

PubMed Central

Fujiwara, Ryoichi; Maruo, Yoshihiro; Chen, Shujuan; Tukey, Robert H.

2015-01-01

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. PMID:26342858

Identification of Metabolism and Excretion Differences of Procymidone between Rats and Humans Using Chimeric Mice: Implications for Differential Developmental Toxicity.

PubMed

Abe, Jun; Tomigahara, Yoshitaka; Tarui, Hirokazu; Omori, Rie; Kawamura, Satoshi

2018-02-28

A metabolite of procymidone, hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of hydroxylated-PCM was less, resulting in a much lower exposure. The main excretion route of hydroxylated-PCM-glucuronide was bile (the point that hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone's potential for causing teratogenicity in humans must be low compared to that in rats.

Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

USDA-ARS?s Scientific Manuscript database

UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

USDA-ARS?s Scientific Manuscript database

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavono...

Differences Between Human and Rat Intestinal and Hepatic Bisphenol-A Glucuronidation and the Influence of Alamethicin on In vitro Kinetic Measurements

EPA Science Inventory

The extent to which membrane disrupting agents, such as alamethicin, may alter cofactor transport and influence in vitro kinetic measurements of glucurondiation is a major concern regarding the characterization and extrapolation of inter-and intra-species pharmacokinetics of bisp...

The fetal programming of dietary fructose and saturated fat on hepatic quercetin glucuronidation in rats

USDA-ARS?s Scientific Manuscript database

Objective Phase II biotransformation of flavonoids generates bioactive metabolites in vivo. However, data on the effect of environmental and physiological factors and fetal programming on phase II pathways toward flavonoids are limited. We examined the effect of parental exposure to a diet high in s...

Simultaneous LC-MS/MS quantitation of acetaminophen and its glucuronide and sulfate metabolites in human dried blood spot samples collected by subjects in a pilot clinical study.

PubMed

Li, Wenkui; Doherty, John P; Kulmatycki, Kenneth; Smith, Harold T; Tse, Francis Ls

2012-06-01

In support of a pilot clinical trial using acetaminophen as the model compound to assess dried blood spot (DBS) sampling as the method for clinical pharmacokinetic sample collection, a novel sensitive LC-MS/MS method was developed and validated for the simultaneous determination of acetaminophen and its major metabolites, acetaminophen glucuronide and sulfate, in human DBS samples collected by subjects via fingerprick. The validated assay dynamic range was from 50.0 to 5000 ng/ml for each compound using a 1/8´´ (3-mm) disc punched from a DBS sample. Baseline separation of the three analytes was achieved to eliminate the possible impact of insource fragmentation of the conjugated metabolites on the analysis of the parent. The overall extraction efficiency was from 61.3 to 78.8% for the three analytes by direct extraction using methanol. The validated method was successfully implemented in the pilot clinical study with the obtained pharmacokinetic parameters in agreement with the values reported in literature.

Metabolic profile of salidroside in rats using high-performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry.

PubMed

Han, Fei; Li, Yan-ting; Mao, Xin-juan; Zhang, Xiao-shu; Guan, Jiao; Song, Ai-hua; Yin, Ran

2016-03-01

A high-performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR MS) method was developed to study the in vivo metabolism of salidroside for the first time. Plasma, urine, bile, and feces samples were collected from male rats after a single intragastric gavage of salidroside at a dose of 50 mg/kg. Besides the parent drug, a total of seven metabolites (three phase I and four phase II metabolites) were detected and tentatively identified by comparing their mass spectrometry profiles with those of salidroside. Results indicated that metabolic pathways of salidroside in male rats included hydroxylation, dehydrogenation, glucuronidation, and sulfate conjugation. Among them, glucuronidation and sulfate conjugation were the major metabolic reactions. And most important, the detection of the sulfation metabolite of p-tyrosol provides a clue for whether the deglycosylation of salidroside occurs in vivo after intragastric gavage. In summary, results obtained in this study may contribute to the better understanding of the safety and mechanism of action of salidroside.

Randomised, double blind trial of two loading dose regimens of diamorphine in ventilated newborn infants.

PubMed

Barker, D P; Simpson, J; Pawula, M; Barrett, D A; Shaw, P N; Rutter, N

1995-07-01

To compare the safety and efficacy of two loading doses of diamorphine in 27 ventilated newborn infants in a randomised double blind trial. Fifty or 200 mcg/kg were infused intravenously over 30 minutes, followed by a 15 mcg/kg/hour continuous infusion. Serial measurements were made of physiology, behaviour, and stress hormones. Both loading doses produced small but significant falls in blood pressure. The 200 mcg/kg dose produced greater respiratory depression, and two infants deteriorated clinically, requiring resuscitation. Loading reduced respiratory effort in most of the infants, but had little effect on behavioural activity. Stress hormone concentrations were reduced at six hours in both dosage groups; differences between loading doses were not significant. Morphine, morphine-3-glucuronide, and morphine-6-glucuronide were detected in the plasma of all patients. No significant differences in concentrations between loading doses were found. Diamorphine reduces the stress response in ventilated newborn infants. A high loading dose confers no benefit, and may produce undesirable physiological effects. A 50 mcg/kg loading dose seems to be safe and effective.

Randomised, double blind trial of two loading dose regimens of diamorphine in ventilated newborn infants.

PubMed Central

Barker, D. P.; Simpson, J.; Pawula, M.; Barrett, D. A.; Shaw, P. N.; Rutter, N.

1995-01-01

AIMS--To compare the safety and efficacy of two loading doses of diamorphine in 27 ventilated newborn infants in a randomised double blind trial. METHODS--Fifty or 200 mcg/kg were infused intravenously over 30 minutes, followed by a 15 mcg/kg/hour continuous infusion. Serial measurements were made of physiology, behaviour, and stress hormones. RESULTS--Both loading doses produced small but significant falls in blood pressure. The 200 mcg/kg dose produced greater respiratory depression, and two infants deteriorated clinically, requiring resuscitation. Loading reduced respiratory effort in most of the infants, but had little effect on behavioural activity. Stress hormone concentrations were reduced at six hours in both dosage groups; differences between loading doses were not significant. Morphine, morphine-3-glucuronide, and morphine-6-glucuronide were detected in the plasma of all patients. No significant differences in concentrations between loading doses were found. CONCLUSIONS--Diamorphine reduces the stress response in ventilated newborn infants. A high loading dose confers no benefit, and may produce undesirable physiological effects. A 50 mcg/kg loading dose seems to be safe and effective. PMID:7552591

Antibacterial and antiparasitic activity of oleanolic acid and its glycosides isolated from marigold (Calendula officinalis).

PubMed

Szakiel, Anna; Ruszkowski, Dariusz; Grudniak, Anna; Kurek, Anna; Wolska, Krystyna I; Doligalska, Maria; Janiszowska, Wirginia

2008-11-01

The antibacterial and antiparasitic activities of free oleanolic acid and its glucosides and glucuronides isolated from marigold (Calendula officinalis) were investigated. The MIC of oleanolic acid and the effect on bacterial growth were estimated by A600 measurements. Oleanolic acid's influence on bacterial survival and the ability to induce autolysis were measured by counting the number of cfu. Cell morphology and the presence of endospores were observed under electron and light microscopy, respectively. Oleanolic acid inhibited bacterial growth and survival, influenced cell morphology and enhanced the autolysis of Gram-positive bacteria suggesting that bacterial envelopes are the target of its activity. On the other hand, glycosides of oleanolic acid inhibited the development of L3 Heligmosomoides polygyrus larvae, the infective stage of this intestinal parasitic nematode. In addition, both oleanolic acid and its glycosides reduced the rate of L3 survival during prolonged storage, but only oleanolic acid glucuronides affected nematode infectivity. The presented results suggest that oleanolic acid and its glycosides can be considered as potential therapeutic agents.

Bioavailability of curcumin and curcumin glucuronide in the central nervous system of mice after oral delivery of nano-curcumin.

PubMed

Szymusiak, Magdalena; Hu, Xiaoyu; Leon Plata, Paola A; Ciupinski, Paulina; Wang, Zaijie Jim; Liu, Ying

2016-09-10

Curcumin is a bioactive molecule extracted from Turmeric roots that has been recognized to possess a wide variety of important biological activities. Despite its great pharmacological activities, curcumin is highly hydrophobic, which results in poor bioavailability. We have formulated this hydrophobic compound into stable polymeric nanoparticles (nano-curcumin) to enhance its oral absorption. Pharmacokinetic analysis after oral delivery of nano-curcumin in mice demonstrated approximately 20-fold reduction in dose requirement when compared to unformulated curcumin to achieve comparable plasma and central nervous system (CNS) tissue concentrations. This investigation corroborated our previous study of curcumin functionality of attenuating opioid tolerance and dependence, which shows equivalent efficacy of low-dose (20mg/kg) nano-curcumin and high-dose (400mg/kg) pure curcumin in mice. Furthermore, the highly selective and validated liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify curcumin glucuronide, the major metabolite of curcumin. The results suggest that the presence of curcumin in the CNS is essential for prevention and reversal of opioid tolerance and dependence. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

PubMed Central

Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

2009-01-01

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

Urinary sex steroid hormone and placental leucine aminopeptidase concentration differences between live births and stillbirth of Bornean orangutans (Pongo pygmaeus).

PubMed

Kinoshita, Kodzue; Sano, Yusuke; Takai, Akira; Shimizu, Mika; Kobayashi, Toshio; Ouchi, Akihiro; Kuze, Noko; Inoue-Murayama, Miho; Idani, Gen'ichi; Okamoto, Munehiro; Ozaki, Yasuhiko

2017-02-01

Under the environment of pregnancy, the placenta assumes an important steroidogenic role in the maintenance of pregnancy. Urinary placental leucine aminopeptidase (PLAP), estrone-3-glucuronide (E 1 G), and pregnanediol-3-glucuronide (PdG) concentrations were compared among five pregnancies (four live births and one stillbirth) in four orangutans. The gestation period of the stillbirth (223 days) was shorter than that of the live births (239-254 days). In females who gave a live birth, average PLAP and E 1 G concentrations increased until the delivery. Conversely, in the female who gave a stillbirth, PLAP concentration failed to increase, and E 1 G concentration was significantly low in late pregnancy period. Regarding PdG concentrations, there was no significant difference among all pregnancies. This is the first study reporting a change in urinary PLAP, E 1 G, and PdG concentrations during orangutan stillbirth and live birth pregnancies. The findings will assist in developing pregnancy screening tests. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Physiologically Based Pharmacokinetic Modeling Suggests Limited Drug–Drug Interaction Between Clopidogrel and Dasabuvir

PubMed Central

Fu, W; Badri, P; Bow, DAJ; Fischer, V

2017-01-01

Dasabuvir, a nonnucleoside NS5B polymerase inhibitor, is a sensitive substrate of cytochrome P450 (CYP) 2C8 with a potential for drug–drug interaction (DDI) with clopidogrel. A physiologically based pharmacokinetic (PBPK) model was developed for dasabuvir to evaluate the DDI potential with clopidogrel, the acyl‐β‐D glucuronide metabolite of which has been reported as a strong mechanism‐based inhibitor of CYP2C8 based on an interaction with repaglinide. In addition, the PBPK model for clopidogrel and its metabolite were updated with additional in vitro data. Sensitivity analyses using these PBPK models suggested that CYP2C8 inhibition by clopidogrel acyl‐β‐D glucuronide may not be as potent as previously suggested. The dasabuvir and updated clopidogrel PBPK models predict a moderate increase of 1.5–1.9‐fold for Cmax and 1.9–2.8‐fold for AUC of dasabuvir when coadministered with clopidogrel. While the PBPK results suggest there is a potential for DDI between dasabuvir and clopidogrel, the magnitude is not expected to be clinically relevant. PMID:28411400

Metabolism of nitazoxanide in rats, pigs, and chickens: Application of liquid chromatography coupled to hybrid linear ion trap/Orbitrap mass spectrometer.

PubMed

Huang, Xianhui; Guo, Chunna; Chen, Zhangliu; Liu, Yahong; He, Limin; Zeng, Zhenling; Yan, Chaoqun; Pan, Guangfang; Li, Shuaipeng

2015-09-01

Nitazoxanide (NTZ) is a nitrothiazole benzamide compound with a broad activity spectrum against parasites, Gram-positive and Gram-negative anaerobic bacteria, and viruses. In this study, hybrid linear ion trap/Orbitrap mass spectrometer providing a high mass resolution and accuracy was used to investigate the metabolism of NTZ in rats, pigs, and chickens. The results revealed that acetylation and glucuronidation were the main metabolic pathways in rats and pigs, whereas acetylation and sulfation were the major metabolic pathways in chickens, which indicated interspecies variations in drug metabolism and elimination. With the accurate mass data and the characteristic MS(n) product ions, we identified six metabolites in which tizoxanide and hydroxylated tizoxanide were phase I metabolites and tizoxanide glucuronide, tizoxanide glucose, tizoxanide sulfate and hydroxyl tizoxanide sulfate were phase II metabolites. Hydroxylated tizoxanide and tizoxanide glucose were identified for the first time. All the comprehensive data were provided to make out the metabolism of NTZ in rats, pigs and chickens more clearly. Copyright © 2015 Elsevier B.V. All rights reserved.

Metabolic Profile of Zearalenone in Liver Microsomes from Different Species and Its in Vivo Metabolism in Rats and Chickens Using Ultra High-Pressure Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry.

PubMed

Yang, Shupeng; Zhang, Huiyan; Sun, Feifei; De Ruyck, Karl; Zhang, Jinzhen; Jin, Yue; Li, Yanshen; Wang, Zhanhui; Zhang, Suxia; De Saeger, Sarah; Zhou, Jinhui; Li, Yi; De Boevre, Marthe

2017-12-27

To explore differences of zearalenone (ZEN) metabolism between various species, phase I and II metabolism by liver microsomes of animals and human were investigated using ultra high-pressure liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF MS). A total of 24 metabolites were identified, among which 12 were reported for the first time. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways of ZEN, and significant differences in various species were also observed. Reduction was the main reaction in swine and human, whereas hydroxylation was predominant in rats, chickens, goats, and cows in in vitro systems. Furthemore, in vivo metabolism of ZEN in rats and chickens was investigated, and 23 and 6 metabolites were identified in each species, respectively. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways in rats, while reduction and sulfation predominated in chickens. These results further enrich the biotransformation profile of ZEN, providing a helpful reference for assessing the risks to animals and humans.

Transport of the soy isoflavone daidzein and its conjugative metabolites by the carriers SOAT, NTCP, OAT4, and OATP2B1.

PubMed

Grosser, Gary; Döring, Barbara; Ugele, Bernhard; Geyer, Joachim; Kulling, Sabine E; Soukup, Sebastian T

2015-12-01

Soy isoflavones (IF) are phytoestrogens, which interact with estrogen receptors. They are extensively metabolized by glucuronosyltransferases and sulfotransferases, leading to the modulation of their estrogenic activity. It can be assumed that this biotransformation also has a crucial impact on the uptake of IF by active or passive cellular transport mechanisms, but little is known about the transport of IF phase II metabolites into the cell. Therefore, transport assays for phase II metabolites of daidzein (DAI) were carried out using HEK293 cell lines transfected with five human candidate carriers, i.e., organic anion transporter OAT4, sodium-dependent organic anion transporter (SOAT), Na(+)-taurocholate cotransporting polypeptide (NTCP), apical sodium-dependent bile acid transporter ASBT, and organic anion transporting polypeptide OATP2B1. Cellular uptake was monitored by UHPLC-DAD. DAI monosulfates were transported by the carriers NTCP and SOAT in a sodium-dependent manner, while OAT4-HEK293 cells revealed a partly sodium-dependent transport for these compounds. In contrast, DAI-7,4'-disulfate was only taken up by NTCP-HEK293 cells. DAI-7-glucuronide, but not DAI-4'-glucuronide, was transported exclusively by OATP2B1 in a sodium-independent manner. DAI-7-glucuronide-4'-sulfate, DAI-7-glucoside, and DAI were no substrate of any of the tested carriers. In addition, the inhibitory potency of the DAI metabolites toward estrone-sulfate (E1S) uptake of the above-mentioned carriers was determined. In conclusion, human SOAT, NTCP, OATP2B1, and OAT4 were identified as carriers for the DAI metabolites. Several metabolites were able to inhibit carrier-dependent E1S uptake. These findings might contribute to a better understanding of the bioactivity of IF especially in case of hormone-related cancers.

Inhibition of human UDP-glucuronosyltransferase enzymes by lapatinib, pazopanib, regorafenib and sorafenib: Implications for hyperbilirubinemia.

PubMed

Miners, John O; Chau, Nuy; Rowland, Andrew; Burns, Kushari; McKinnon, Ross A; Mackenzie, Peter I; Tucker, Geoffrey T; Knights, Kathleen M; Kichenadasse, Ganessan

2017-04-01

Kinase inhibitors (KIs) are a rapidly expanding class of drugs used primarily for the treatment of cancer. Data relating to the inhibition of UDP-glucuronosyltransferase (UGT) enzymes by KIs is sparse. However, lapatinib (LAP), pazopanib (PAZ), regorafenib (REG) and sorafenib (SOR) have been implicated in the development of hyperbilirubinemia in patients. This study aimed to characterise the role of UGT1A1 inhibition in hyperbilirubinemia and assess the broader potential of these drugs to perpetrate drug-drug interactions arising from UGT enzyme inhibition. Twelve recombinant human UGTs from subfamilies 1A and 2B were screened for inhibition by LAP, PAZ, REG and SOR. IC 50 values for the inhibition of all UGT1A enzymes, except UGT1A3 and UGT1A4, by the four KIs were <10μM. LAP, PAZ, REG and SOR inhibited UGT1A1-catalysed bilirubin glucuronidation with mean IC 50 values ranging from 34nM (REG) to 3734nM (PAZ). Subsequent kinetic experiments confirmed that REG and SOR were very potent inhibitors of human liver microsomal β-estradiol glucuronidation, an established surrogate for bilirubin glucuronidation, with mean K i values of 20 and 33nM, respectively. K i values for LAP and PAZ were approximately 1- and 2-orders of magnitude higher than those for REG and SOR. REG and SOR were equipotent inhibitors of human liver microsomal UGT1A9 (mean K i 678nM). REG and SOR are the most potent inhibitors of a human UGT enzyme identified to date. In vitro-in vivo extrapolation indicates that inhibition of UGT1A1 contributes significantly to the hyperbilirubinemia observed in patients treated with REG and SOR, but not with LAP and PAZ. Inhibition of other UGT1A1 substrates in vivo is likely. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and validation of a rapid multi-biomarker liquid chromatography/tandem mass spectrometry method to assess human exposure to mycotoxins.

PubMed

Warth, Benedikt; Sulyok, Michael; Fruhmann, Philipp; Mikula, Hannes; Berthiller, Franz; Schuhmacher, Rainer; Hametner, Christian; Abia, Wilfred Angie; Adam, Gerhard; Fröhlich, Johannes; Krska, Rudolf

2012-07-15

Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers. This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach. Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously. We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins. Copyright © 2012 John Wiley & Sons, Ltd.

Role of UDP-Glucuronosyltransferase 1A1 in the Metabolism and Pharmacokinetics of Silymarin Flavonolignans in Patients with HCV and NAFLD.

PubMed

Xie, Ying; Miranda, Sonia R; Hoskins, Janelle M; Hawke, Roy L

2017-01-15

Silymarin is the most commonly used herbal medicine by patients with chronic liver disease. Silymarin flavonolignans undergo rapid first-pass metabolism primarily by glucuronidation. The aims of this investigation were: (1) to determine the association of UGT1A1*28 polymorphism with the area under the plasma concentration-time curves (AUCs) for silybin A (SA) and silybin B (SB); (2) to evaluate the effect of UGT1A1*28 polymorphism on the profile of flavonolignan glucuronide conjugates found in the plasma; and (3) to investigate the role of UGT1A1 enzyme kinetics on the pharmacokinetics of SA and SB. AUCs and metabolic ratios for thirty-three patients with chronic liver disease administered oral doses of silymarin were compared between different UGT1A1*28 genotypes. The AUCs, metabolic ratios, and the profiles of major SA and SB glucuronides did not differ significantly among the three UGT1A1 genotypes. In contrast, an increase in the proportion of sulfated flavonolignan conjugates in plasma was observed in subjects with UGT1A1*28/*28 genotype compared to subjects carrying wild type alleles. Differences in SA and SB in vitro intrinsic clearance estimates for UGTIA1 correlated inversely with SA and SB exposures observed in vivo indicating a major role for UGT1A1 in silymarin metabolism. In addition, a significant difference in the metabolic ratio observed between patients with NAFLD and HCV suggests that any effect of UGT1A1 polymorphism may be obscured by a greater effect of liver disease on the pharmacokinetics of silymarin. Taken together, these results suggest the presence of the UGT1A1*28 allele does not contribute significantly to a large inter-subject variability in the pharmacokinetics of silybin A and silybin B which may obscure the ability to detect beneficial effects of silymarin in patients with liver disease.

Detection of in utero cannabis exposure by umbilical cord analysis.

PubMed

Kim, Jiyoung; de Castro, Ana; Lendoiro, Elena; Cruz-Landeira, Angelines; López-Rivadulla, Manuel; Concheiro, Marta

2018-04-01

According to the 2014 National Survey on Drug Use and Health, 5.3% of pregnant women smoked marijuana in the past month. This prevalence is expected to increase as a growing number of states and countries are now considering legalization. Although the umbilical cord is becoming a useful objective tool to detect in utero drug exposure, currently data about analytical methods and its utility to detect cannabis exposure are scarce. The objective of this work was to develop a method for the determination of Δ 9 -tetrahydrocannabinol (THC), 11-hydroxyTHC (THC-OH), 11-nor-9-carboxy-THC (THCCOOH), 8-β-11-dihydroxyTHC (THC-diOH), THC and THCCOOH glucuronides, and cannabidiol (CBD) in the umbilical cord by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with dual ionization source. Umbilical cord samples (0.5 g) were homogenized in methanol and extracted by solid-phase extraction. Reversed-phase chromatographic separation was performed in 14 minutes, and 2 transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1-10 to 20-200 ng/g), precision (4.1%-23.4%), accuracy (87.5%-111.4%), matrix effect (-54.8% to -5.8%), extraction efficiency (25%-45.6%), limits of detection and quantification (1-10 ng/g), and endogenous (n = 5) or exogenous interferences (not detected). The method was applied to 13 authentic samples from cannabis-exposed newborns, which meconium samples had tested positive for cannabis. Twelve cord specimens tested positive for THCCOOH-glucuronide (1.6-19.1 ng/g). We developed and validated a specific and sensitive method for the simultaneous determination of THC, its metabolites, including THC and THCCOOH glucuronides, and CBD in umbilical cord samples by LC-MS/MS. The analysis of authentic samples showed the usefulness of umbilical cord to detect cannabis in utero exposure. Copyright © 2017 John Wiley & Sons, Ltd.

The influence of paroxetine on the pharmacokinetics of atomoxetine and its main metabolite.

PubMed

Todor, Ioana; Popa, Adina; Neag, Maria; Muntean, Dana; Bocsan, Corina; Buzoianu, Anca; Vlase, Laurian; Gheldiu, Ana-Maria; Chira, Ruxandra; Briciu, Corina

2015-01-01

To evaluate the effects of paroxetine on the pharmacokinetics of atomoxetine and its main metabolite, 4-hydroxyatomoxetine-O-glucuronide, after coadministration of atomoxetine and paroxetine in healthy volunteers. 22 healthy volunteers, extensive metabolizers, took part in this open-label, non-randomized, clinical trial. The study consisted of two periods: Reference, when a single oral dose of 25 mg atomoxetine was administrated to each subject and Test, when 25 mg atomoxetine and 20 mg paroxetine were coadministered. Between the two periods, the volunteers received an oral daily dose of 20-40 mg paroxetine, for 6 days. Atomoxetine and 4-hydroxyatomoxetine-O-glucuronide plasma concentrations were determined within the first 48 hours following drug administration. The pharmacokinetic parameters of both compounds were assessed using a non-compartmental method and the analysis of variance aimed at identifying any statistical significant differences between the pharmacokinetic parameters of atomoxetine and its main metabolite, corresponding to each study period. Paroxetine modified the pharmacokinetic parameters of atomoxetine. Cmax increased from 221.26±94.93 to 372.53±128.28 ng/mL, while AUC0-t and AUC0-∞ also increased from 1151.19±686.52 to 6452.37±3388.76 ng*h/mL, and from 1229.15±751.04 to 7111.74±4195.17 ng*h/mL respectively. The main metabolite pharmacokinetics was also influenced by paroxetine intake, namely Cmax, AUC0-t and AUC0-∞ decreased from 688.76±270.27 to 131.01±100.43 ng*h/mL, and from 4810.93±845.06 to 2606.04±923.88 and from 4928.55±853.25 to 3029.82 ±941.84 respectively. Multiple-dose paroxetine intake significantly influenced atomoxetine and its active metabolite pharmacokinetics, causing a 5.8-fold increased exposure to atomoxetine and 1.6-fold reduced exposure to 4-hydroxyatomoxetine-O-glucuronide.

Effects of clofibric acid on the biliary excretion of benoxaprofen glucuronide and taurine conjugate in rats.

PubMed

Okada, K; Kanoh, H; Mohri, K

2011-10-01

Benoxaprofen (BOP) is a 2-methyl propionic acid derivative with anti-inflammatory activity. BOP has an asymmetric carbon, and receives chiral inversion from R to S in vivo. BOP is metabolized to glucuronide (BOP-G) and taurine conjugate (BOP-T). The configuration of BOP-G is mainly S, and that of BOP-T is R. Chiral inversion of R to S of the propionic acid moiety and amino acid conjugation of carboxyl compounds proceed via an acyl CoA intermediate. It is known that fibrates, used in hyperlipidemia, induce acyl CoA synthetase and increase CoA concentration. We administered racemic BOP (10 mg/kg body weight) to rats (CFA+) pre-administered clofibric acid (CFA, 280 mg/kg/day), and studied BOP, BOP-G, and BOP-T enantiomer concentrations in plasma and bile up to 12 h after administration. The findings were compared with those in rats (CFA-) that had not received CFA. Furthermore, we studied the amounts of BOP-G enantiomer produced by glucuronidation in vitro using microsomes pretreated with CFA. The amounts of (S)-BOP-G in CFA+ rats were 2.7-fold larger than that in CFA- rats. Although (R)-BOP-T was excreted in CFA- rats, BOP-T could not be detected in CFA+ rats. Plasma clearance values of racemic BOP and (S)-BOP in CFA+ rats were 5-fold and 6-fold larger than those in CFA- rats, respectively. (S)-BOP-G formation activities were higher than (R)-BOP-G formation activities in both CFA+and CFA- microsomes. These findings suggest that CFA increases biliary excretion of (S)-BOP-G and facilitates plasma elimination of BOP, and further suggests that CFA predominantly induces chiral inversion to S rather than metabolic reaction to (R)-BOP-T, resulting in an increase of (S)-BOP-G.

Pseudoendogenous presence of β-boldenone sulphate and glucuronide in untreated young bulls from the food chain.

PubMed

Chiesa, Luca; Pasquale, Elisa; Panseri, Sara; Cannizzo, Francesca T; Biolatti, Bartolomeo; Pavlovic, Radmila; Arioli, Francesco

2015-01-01

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is considered fully compliant up to 2 ng ml(-1). Greater attention has been placed on β-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and β-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of β-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of β-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that β-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a frequency of 78% and 46%, respectively, showing the endogenous origin of boldenone.

Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers' Urine by High Throughput Liquid Chromatography-Mass Spectrometry.

PubMed

von Weymarn, Linda B; Thomson, Nicole M; Donny, Eric C; Hatsukami, Dorothy K; Murphy, Sharon E

2016-03-21

Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after β-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.

Elucidation of arctigenin pharmacokinetics after intravenous and oral administrations in rats: integration of in vitro and in vivo findings via semi-mechanistic pharmacokinetic modeling.

PubMed

Gao, Qiong; Zhang, Yufeng; Wo, Siukwan; Zuo, Zhong

2014-11-01

Although arctigenin (AR) has attracted substantial research interests due to its promising and diverse therapeutic effects, studies regarding its biotransformation were limited. The current study aims to provide information regarding the pharmacokinetic properties of AR via various in vitro and in vivo experiments as well as semi-mechanistic pharmacokinetic modeling. Our in vitro rat microsome incubation studies revealed that glucuronidation was the main intestinal and liver metabolic pathway of AR, which occurred with V max, K m, and Clint of 47.5 ± 3.4 nmol/min/mg, 204 ± 22 μM, and 233 ± 9 μl/min/mg with intestinal microsomes and 2.92 ± 0.07 nmol/min/mg, 22.7 ± 1.2 μM, and 129 ± 4 μl/min/mg with liver microsomes, respectively. In addition, demethylation and hydrolysis of AR occurred with liver microsomes but not with intestinal microsomes. In vitro incubation of AR and its metabolites in intestinal content demonstrated that glucuronides of AR excreted in bile could be further hydrolyzed back to the parent compound, suggesting its potential enterohepatic circulation. Furthermore, rapid formation followed by fast elimination of arctigenic acid (AA) and arctigenin-4'-O-glucuronide (AG) was observed after both intravenous (IV) and oral administrations of AR in rats. Linear pharmacokinetics was observed at three different doses for AR, AA, and AG after IV administration of AR (0.48-2.4 mg/kg, r (2) > 0.99). Finally, an integrated semi-mechanistic pharmacokinetic model using in vitro enzyme kinetic and in vivo pharmacokinetic parameters was successfully developed to describe plasma concentrations of AR, AA, and AG after both IV and oral administration of AR at all tested doses.

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates.

PubMed

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

2015-09-07

E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

Toxicokinetics and metabolism of 1,2-diethylbenzene in male Sprague Dawley rats--part 2: evidence for in vitro and in vivo stereoselectivity of 1,2-diethylbenzene metabolism.

PubMed

Payan, J P; Cossec, B; Beydon, D; Fabry, J P; Ferrari, E

2001-06-01

In a previous study, it was shown that the neurotoxic compound 1,2-diethylbenzene (1,2-DEB) is mainly hydroxylated in the alkyl chain to give 1-(2'-ethylphenyl)ethanol (1,2-EPE) and excreted in urine of rats as two glucuronide compounds (GA1 and GA2). Some findings have suggested that the two enantiomers of 1,2-EPE are formed in vivo. In the present study, a chiral high-performance liquid chromatography method was developed to separate the two enantiomers of 1,2-EPE from a synthesized racemic mixture. Absolute configuration of both enantiomers was determined after esterification with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid and analysis of their (1)H NMR spectra in CCl(4) added with Eu (fod)(3). The two main urinary metabolites, GA1 and GA2, from [(14)C]1,2-DEB-treated Sprague-Dawley rats (80 mg/kg, i.p.) were identified, after hydrolysis with beta-glucuronidase from Escherichia coli, as (R) and (S) glucuronide conjugates of 1,2-EPE, respectively. In vitro hydroxylation of 1,2-DEB and glucuroconjugation of 1,2-EPE were under stereoselective control in S9 fraction or microsomes from male Sprague-Dawley rat liver. The V(max) and K(m) constants for (R)1,2-EPE enantiomer formation determined in S9 fraction were greater than those for the (S) enantiomer. In the plasma of bile duct-cannulated rats, the ratio was 1.2 +/- 0.02 over the 1- to 4-h period after oral administration of [(14)C]1,2-DEB (100 mg/kg). In contrast, the glucuroconjugation rate of (S)1,2-DEB enantiomer was 4 times that of (R)1,2-EPE glucuroconjugation. A similar ratio of (R) to (S)1,2-EPE glucuronide conjugates was obtained in the plasma of bile duct-cannulated rats.

Higher urine 1-hydroxy pyrene glucuronide (1-OHPG) is associated with tobacco smoke exposure and drinking maté in healthy subjects from Rio Grande do Sul, Brazil

PubMed Central

Fagundes, Renato B; Abnet, Christian C; Strickland, Paul T; Kamangar, Farin; Roth, Mark J; Taylor, Philip R; Dawsey, Sanford M

2006-01-01

Background The highest rates of esophageal squamous cell carcinoma (ESCC) in Brazil occur in Rio Grande do Sul, the most southern state, which has incidence rates of 20.4/100,000/year for men and 6.5/100,000/year for women. Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) through tobacco smoke and other sources may increase the risk of ESCC. The aims of the current study were to investigate the degree and sources of PAH exposure of the inhabitants of this region of southern Brazil. Methods Two hundred healthy adults (half smokers, half non smokers, half male and half female) were recruited, given a standardized questionnaire, and asked to provide a urine sample for measurement of 1-hydroxypyrene glucuronide (1-OHPG), a PAH metabolite). Urine 1-OHPG concentrations were measured using immunoaffinity chromatography and synchronous fluorescence spectroscopy and urine cotinine was measured using a dipstick test. We examined factors associated with 1-OHPG concentration using Wilcoxon tests and multiple linear regression. Results Urine 1-hydroxypyrene glucuronide (1-OHPG) was successfully measured on 199 subjects. The median (interquartile range) of urine 1-OHPG in the 199 participants was 2.09 pmol/mL (0.51, 5.84). Tobacco smoke exposure and maté drinking were statistically significantly associated with higher urine 1-OHPG concentrations in the multivariate linear regression model. Conclusion Tobacco smoke and maté both contribute to high levels of benzo[a]pyrene exposure in the people of southern Brazil. This high PAH exposure may contribute to the high rates of ESCC observed in this population. The increased urine 1-OHPG concentrations associated with maté suggest that contaminants, not just thermal injury, may help explain the increased risk of ESCC previously reported for maté consumption. PMID:16729889

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

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

Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase Imore » metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 µM), and higher intrinsic clearance at lower substrate concentrations (<0.07 µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.« less

Introduction of sample tubes with sodium azide as a preservative for ethyl glucuronide in urine.

PubMed

Luginbühl, Marc; Weinmann, Wolfgang; Al-Ahmad, Ali

2017-09-01

Ethyl glucuronide (EtG) is a direct alcohol marker, which is widely used for clinical and forensic applications, mainly for abstinence control. However, the instability of EtG in urine against bacterial degradation or the post-collectional synthesis of EtG in contaminated samples may cause false interpretation of EtG results in urine samples. This study evaluates the potential of sodium azide in tubes used for urine collection to hinder degradation of ethyl glucuronide by bacterial metabolism taking place during growth of bacterial colonies. The tubes are part of a commercial oral fluid collection device. The sampling system was tested with different gram-positive and gram-negative bacterial species previously observed in urinary tract infections, such as Escherichia coli, Staphylococcus aureus, Enterecoccus faecalis, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Inhibition of bacterial growth by sodium azide, resulting in lower numbers of colony forming units compared to control samples, was observed for all tested bacterial species. To test the prevention of EtG degradation by the predominant pathogen in urinary tract infection, sterile-filtered urine and deficient medium were spiked with EtG, and inoculated with E. coli prior to incubation for 4 days at 37 °C in tubes with and without sodium azide. Samples were collected every 24 hours, during four consecutive days, whereby the colony forming units (CFU) were counted on Columbia blood agar plates, and EtG was analyzed by LC-MS/MS. As expected, EtG degradation was observed when standard polypropylene tubes were used for the storage of contaminated samples. However, urine specimens collected in sodium azide tubes showed no or very limited bacterial growth and no EtG degradation. As a conclusion, sodium azide is useful to reduce bacterial growth of gram-negative and gram-positive bacteria. It inhibits the degradation of EtG by E. coli and can be used for the stabilization of EtG in urine samples.

Pharmacokinetic evaluation of a sprinkle-dose regimen of a once-daily, extended-release morphine formulation.

PubMed

Eliot, Lise; Butler, Jackie; Devane, John; Loewen, Gordon

2002-02-01

Morphine sulfate extended-release (MSER) uses a drug-delivery technology that allows once-daily dosing. It is possible to open the MSER capsule and sprinkle the contents on soft food, a potentially useful alternative to the intact capsule in patients who have difficulty swallowing. This study compared the bioavailability of MSER and its metabolites morphine-3-glucuronide and morphine-6-glucuronide after administration of MSER in a sprinkle-dose regimen relative to an intact capsule swallowed whole. This was a randomized, open-label, single-dose, crossover study, with a 7-day washout period between the 2 dosing days. Healthy volunteers were randomized to receive an intact 60-mg MSER capsule swallowed whole or the contents of a 60-mg MSER capsule sprinkled on applesauce. Blood samples were collected and analyzed for concentrations of morphine and its active glucuronide metabolites. Pharmacokinetic (PK) parameters were calculated and bioequivalence assessed. Bioequivalence was concluded if the 90% CIs of the ratio of log-transformed values for maximum concentration (Cmax) and area under the plasma concentration-time curve (AUC) were within 80% to 125%. Of 30 subjects enrolled, 28 completed the study and were eligible for PK evaluation. Two subjects were withdrawn for reasons unrelated to study treatment. The plasma concentration-time profiles of morphine and its metabolites were superimposable after administration of the 2 regimens. Cmax and total systemic exposure-based on AUC from time 0 to the last quantifiable concentration (AUC(last)) and AUC from time 0 to infinity (AUC(infinity))-were comparable between treatments. The 90% CIs for morphine AUC(last), AUC, and Cmax ratios were 98 to 109, 96 to 106, and 95 to 117, respectively. Similar 90% CIs were obtained for the morphine metabolites. In this study in healthy volunteers, sprinkling the entire contents of an MSER capsule onto applesauce and swallowing without chewing was bioequivalent to swallowing an intact MSER capsule.

Metabolism of 1-Fluro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 1,1,1-trifluoro-2-chloroethane

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

Yin, H.; Jones, J.P.; Anders, M.W.

1995-03-01

1-Fluoro-1,1,2-trichloroethane (HCFC-131a), 1,2-dichloro-1,1-difluoroethane (HCFC-132b), and 1,1,1-trifluoro-2-chloroethane (HCFC-133a) were chosen as models for comparative metabolism studies on 1,1,1,2-tetrahaloethanes, which are under consideration as replacements for ozone-depleting chlorofluorocarbons (CFCs). Male Fischer 344 rats were given 10 mmol/kg ip HCFC-131a or HCFC-132b or exposed by inhalation to 1% HCFC-133a for 2 h. Urine collected in the first 24 h after exposure was analyzed by {sup 19}F NMR and GC/MS and with a fluoride-selective ion electrode for the formation of fluorine-containing metabolites. Metabolites of HCFC-131a included 2,2-dichloro-2-fluoroethyl glucuronide, 2,2-dichloro-2-fluorethyl sulfate, dichlorofluoroacetic acid, and inorganic fluoride. Metabolites of HCFC-132b were characterized as 2-chloro-2,2-difluoroethyl glucuronide, 2-chloro-2,2-difluoroethylmore » sulfate, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, chlorodifluoroacetaldehyde-urea adduct, inorganic fluoride, and a minor, unidentified metabolite. With HCFC-131a and HCFC-132b, glucuronide conjugates of 2,2,2-trihaloethanols were the major urinary metabolites, whereas with HCFC-133a, a trifluroacetaldehyde-urea adduct was the major urinary metabolite. Analysis of metabolite distribution in vivo indicated that aldehydic metabolites increased as fluorine substitution increased in the order HCFC-131a < HCFC-132b < HCFC-133a. With NADPH-fortified rat liver microsomes, HCFC-133a and HCFC-133a and HCFC-132b were biotransformed to trifluoroacetaldehyde and chlorodifluoroacetaldehyde, respectively, whereas HCFC-131a was converted to dichlorofluoroacetic acid. No covalently bound metabolites of HCFC-131a and HCFC-133a metabolites were detected by {sup 19}F NMR spectroscopy. 18 refs., 2 figs., 3 tabs.« less

Effects of in vivo hepatic ischemia-reperfusion injury on the hepatobiliary disposition of rhodamine 123 and its metabolites in isolated perfused rat livers.

PubMed

Parasrampuria, Ridhi; Shaik, Imam H; Mehvar, Reza

2012-01-01

A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury.

Characteristics of Men Who Report Persistent Sexual Symptoms After Finasteride Use for Hair Loss.

PubMed

Basaria, Shehzad; Jasuja, Ravi; Huang, Grace; Wharton, Whitney; Pan, Hong; Pencina, Karol; Li, Zhuoying; Travison, Thomas G; Bhawan, Jag; Gonthier, Renaud; Labrie, Fernand; Dury, Alain Y; Serra, Carlo; Papazian, Allen; O'Leary, Michael; Amr, Sami; Storer, Thomas W; Stern, Emily; Bhasin, Shalender

2016-12-01

Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Academic medical center. Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression.

Importance of 5α-reductase gene polymorphisms on circulating and intraprostatic androgens in prostate cancer.

PubMed

Lévesque, Éric; Laverdière, Isabelle; Lacombe, Louis; Caron, Patrick; Rouleau, Mélanie; Turcotte, Véronique; Têtu, Bernard; Fradet, Yves; Guillemette, Chantal

2014-02-01

Polymorphisms in the genes SRD5A1 and SRD5A2 encoding androgen biosynthetic 5α-reductase enzymes have been associated with an altered risk of biochemical recurrence after radical prostatectomy in localized prostate cancer. To gain potential insights into SRD5A biologic effects, we examined the relationship between SRD5A prognostic markers and endogenous sex-steroid levels measured by mass spectrometry in plasma samples and corresponding prostatic tissues of patients with prostate cancer. We report that five of the seven SRD5A markers differentially affect sex-steroid profiles of dihydrotestosterone and its metabolites in both the circulation and prostatic tissues of patients with prostate cancer. Remarkably, a 32% increase in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A rs2208532 polymorphism. Moreover, SRD5A2 markers were associated predominantly with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 protective allele was associated with high circulating androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) levels as opposed to lower levels of both 3α-diol-17G and androsterone-glucuronide observed with the rs2208532 SRD5A2 risk allele. Moreover, SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, were associated with higher circulating levels of 3α-diol-3G. The SRD5A2 rs676033 variant further correlated with enhanced intraprostatic exposure to 5α-reduced steroids (dihydrotestosterone and its metabolite 3β-diol). Similarly, the SRD5A1 rs166050C risk variant was associated with greater prostatic exposure to androsterone, whereas no association was noted with circulating steroids. Our data support the association of 5α-reductase germline polymorphisms with the hormonal milieu in patients with prostate cancer. Further studies are needed to evaluate if these variants influence 5α-reductase inhibitor efficacy. ©2013 AACR.

Plasma Pharmacokinetic Determination of Canagliflozin and Its Metabolites in a Type 2 Diabetic Rat Model by UPLC-MS/MS.

PubMed

Dong, Song-Tao; Niu, Hui-Min; Wu, Yin; Jiang, Jia-Lei; Li, Ying; Jiang, Kun-Yu; Wang, Xin; Zhang, Mao-Fan; Han, Ming-Feng; Meng, Sheng-Nan

2018-05-20

Canagliflozin is a novel, orally selective inhibitor of sodium-dependent glucose co-transporter-2 (SGLT2) for the treatment of patients with type 2 diabetes mellitus. In this study, a sensitive and efficient UPLC-MS/MS method for the quantification of canagliflozin and its metabolites in rat plasma was established and applied to pharmacokinetics in a type 2 diabetic rat model. We firstly investigated the pharmacokinetic changes of canagliflozin and its metabolites in type 2 diabetic rats in order to use canagliflozin more safely, reasonably and effectively. We identified three types of O-glucuronide metabolites (M5, M7 and M17), two kinds of oxidation metabolites (M8 and M9) and one oxidation and glucuronide metabolite (M16) using API 5600 triple-TOF-MS/MS. Following liquid⁻liquid extraction by tert-butyl methyl ether, chromatographic separation of canagliflozin and its metabolites were performed on a Waters XBridge BEH C18 column (100 × 2.1 mm, 2.5 μm) using 0.1% acetonitrile⁻formic acid (75:15, v / v ) as the mobile phase at a flow rate of 0.7 mL/min. Selected ion monitoring transitions of m / z 462.00→191.10, 451.20→153.10, 638.10→191.10 and 478.00→267.00 were chosen to quantify canagliflozin, empagliflozin (IS), O-glucuronide metabolites (M5, M7 and M17), and oxidation metabolites (M9) using an API 5500-triple-MS/MS in the positive electrospray ionization mode. The validation of the method was found to be of sufficient specificity, accuracy and precision. The pathological condition of diabetes could result in altered pharmacokinetic behaviors of canagliflozin and its metabolites. The pharmacokinetic parameters (AUC 0⁻t , AUC 0⁻∞ , CL z /F, and V z /F) of canagliflozin were significantly different between the CTRL and DM group rats ( p < 0.05 or p < 0.01), which may subsequently cause different therapeutic effects.

Characteristics of Men Who Report Persistent Sexual Symptoms After Finasteride Use for Hair Loss

PubMed Central

Basaria, Shehzad; Jasuja, Ravi; Huang, Grace; Wharton, Whitney; Pan, Hong; Pencina, Karol; Li, Zhuoying; Travison, Thomas G.; Bhawan, Jag; Gonthier, Renaud; Labrie, Fernand; Dury, Alain Y.; Serra, Carlo; Papazian, Allen; O'Leary, Michael; Amr, Sami; Storer, Thomas W.; Stern, Emily

2016-01-01

Context: Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. Objective: To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Participants: Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Outcomes: Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Setting: Academic medical center. Results: Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. Conclusions: We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression. PMID:27662439

Low Cotinine Glucuronidation Results in Higher Serum and Saliva Cotinine in African American Compared to White Smokers.

PubMed

Murphy, Sharon E; Sipe, Christopher J; Choi, Kwangsoo; Raddatz, Leah M; Koopmeiners, Joseph S; Donny, Eric C; Hatsukami, Dorothy K

2017-07-01

Background: Tobacco exposure is often quantified by serum or saliva concentrations of the primary nicotine metabolite, cotinine. However, average cotinine concentrations are higher in African Americans (AA) compared with Whites with similar smoking levels. Cotinine is metabolized by UGT2B10 and CYP2A6, and low UGT2B10 activity is common in AA, due to the prevalence of a UGT2B10 splice variant. Methods: UGT2B10 activity was phenotyped in 1,446 smokers (34% AA) by measuring the percentage of cotinine excreted as a glucuronide. Urinary total nicotine equivalents (TNE), the sum of nicotine and 6 metabolites, were determined to quantify smoking dose, and cotinine and 3'-hydroxycotinine were quantified in saliva (study 1) or serum (study 2). Results: Ninety-seven smokers (78% AA) were null for UGT2B10 activity, and the saliva and serum cotinine levels, after adjustment for TNE and cigarettes per day (CPD), were 68% and 48% higher in these smokers compared with nonnull smokers ( P < 0.001). After adjustment for TNE and CPD, salivary cotinine was 35% higher, and serum cotinine 24% higher in AA versus White smokers, but with additional adjustment for UGT2B10 activity, there were no significant differences in saliva and serum cotinine concentrations between these two groups. Conclusions: UGT2B10 activity significantly influences plasma cotinine levels, and higher cotinine concentrations in AA versus White smokers (after adjustment for smoking dose) result from lower levels of UGT2B10-catalyzed cotinine glucuronidation by AA. Impact: UGT2B10 activity or genotype should be considered when using cotinine as a tobacco exposure biomarker, particularly in populations such as AA with high frequencies of UGT2B10 nonfunctional variants. Cancer Epidemiol Biomarkers Prev; 26(7); 1093-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Benzylic oxidation of gemfibrozil-1-O-beta-glucuronide by P450 2C8 leads to heme alkylation and irreversible inhibition.

PubMed

Baer, Brian R; DeLisle, Robert Kirk; Allen, Andrew

2009-07-01

Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker

PubMed Central

Berger, Lisa; Fendrich, Michael; Jones, Joseph; Fuhrmann, Daniel; Plate, Charles; Lewis, Douglas

2014-01-01

Aims This study aimed to evaluate the performance of ethyl glucuronide (EtG) in hair and fingernails as a long-term alcohol biomarker. Design Cross-sectional survey with probability sampling. Setting Midwestern United States. Participants Participants were 606 undergraduate college students between the ages of 18 and 25 years at the time of selection for potential study participation. Measurements EtG concentrations in hair and fingernails were measured by liquid chromatography-tandem mass spectrometry at three thresholds [30 picograms (pg) per milligram (mg); 20 pg/mg; and 8 pg/mg]. Any weekly alcohol use, increasing-risk drinking and high-risk drinking on average during the past 12 weeks was assessed by participant interview using the time-line follow-back method. Findings In both hair and fingernails at all three EtG thresholds, sensitivity was greatest for the high-risk drinking group [hair: 0.43, confidence interval (CI) = 0.17, 0.69 at 30 pg/mg, 0.71, CI = 0.47, 0.95 at 20 pg/mg; 0.93, CI = 0.79, 1.00 at 8 pg/mg; fingernails: 1.00, CI = 1.00–1.00 at 30, 20 and 8 pg/mg] and specificity was greatest for any alcohol use (hair: 1.00, CI = 1.00, 1.00 at 30 and 20 pg/mg; 0.97, CI = 0.92–0.99 at 8 pg/mg; fingernails: 1.00, CI = 1.00–1.00 at 30, 20 and 8 pg/mg). Areas under the receiver operating characteristic curves were significantly higher for EtG concentration in fingernails than hair for any weekly alcohol use (P = 0.02, DeLong test, two-tailed) and increasing-risk drinking (P = 0.02, DeLong test, two-tailed). Conclusions Ethyl glucuronide, especially in fingernails, may have potential as a quantitative indicator of alcohol use. PMID:24524319

CHEMICAL MODIFICATION MODULATES ESTROGENIC ACTIVITY, OXIDATIVE REACTIVITY, & METABOLIC STABILITY IN 4′F-DMA, A NEW BENZOTHIOPHENE SELECTIVE ESTROGEN RECEPTOR MODULATOR

PubMed Central

Liu, Hong; Bolton, Judy L.; Thatcher, Gregory R. J.

2008-01-01

The benzothiophene SERMs raloxifene and arzoxifene, in the clinic or clinical trials for treatment of breast cancer and postmenopausal symptoms, are highly susceptible to oxidative metabolism and formation of electrophilic metabolites. 4′F-DMA, fluoro-substituted desmethyl arzoxifene (DMA), showed attenuated oxidation to quinoids in incubation with rat hepatocytes as well as in rat and human liver microsomes. Incubations of 4′F-DMA with hepatocytes yielded only one glucuronide conjugate and no GSH conjugates; whereas DMA underwent greater metabolism giving two glucuronide conjugates, one sulfate conjugate, and two GSH conjugates. Phase I and phase II metabolism was further evaluated in human small intestine microsomes and in human intestinal Caco-2 cells. In comparison to DMA, 4′F-DMA formed significantly less glucuronide and sulfate conjugates. The formation of quinoids was futher explored in hepatocytes in which DMA was observed to give concentration and time dependent depletion of GSH accompanied by damage to DNA which showed inverse dependence on GSH; in contrast, GSH depletion and DNA damage were almost completely abrogated in incubations with 4′F-DMA. 4′F-DMA shows ligand binding affinity to ERα and ERβ with similarity to both raloxifene and to DMA. ER-mediated biological activity was measured with the ERE-luciferase reporter system in transfected MCF-7 cells and Ishikawa cells, and in MCF-7 cells proliferation was measured. In all systems, 4′F-DMA exhibited anitestrogenic acitivty of comparable potency to raloxifene, but did not manifest estrogenic properties, mirroring previous results on inhibition of estradiol-mediated induction of alkaline phosphatase activity in Ishikawa cells. These results suggest that 4′F-DMA might be an improved benzothiophene SERM with similar antiestrogenic activity to raloxifene, but improved metabolic stability and attenuated toxicity; showing that simple chemical modification can abrogate oxidative bioactivation to potentially toxic metabolites without loss of activity. PMID:16780356

[Induction of uridine 5'-diphosphate-glucuronosyltransferase gene expression by sulforaphane and its mechanism: experimental study in human colon cancel cells].

PubMed

Wang, Min; Li, Yan-Qing; Zhong, Ning; Chen, Jian; Xu, Xiao-Qun; Yuan, Meng-Biao

2005-03-30

To study the induction of expression of uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A in colon cancer cells by sulforaphane (SFN) and its possible mechanism. Human colon cancer cells of the line Caco-2 were cultured and added with SFN of different terminal concentrations, all below the concentration of IC(50). RT-PCR was used to examine the expression of UGT1A mRNA induced by SFN. Western blotting was used to detect the expression of UGT1A protein. The glucuronidation rate of N-hydroxy-PhIP was measured by high performance liquid chromatography (HPLC). The nuclear localization of transcription factor Nrf2 was observed by confocal laser microscopy. (1) Expression of UGT1A mRNA was observed in the Cac0-2 cells induced by SFN of the concentrations of 10 micromol/L approximately 35 micromol/L in a dose-independent manner (P < 0.05). Sulforaphane of the concentration of 25 micromol/L induced the UGT1A mRNA expression time-dependently. The levels of UGT1A1, UGT1A8, and UGT1A10 mRNA expression were significantly increased in the cells treated with 25 micromol/L sulforaphane compared to that in the controls (P = 0.006, P = 0.017, and P = 0.008 respectively). (2) The UGT1A protein band intensity increased significantly in the Coco-2 cells treated with sulforaphane of the concentrations 10 micromol/L approximately 30 micromol/L for 24 h in comparison with the control cells. (3) When the microsomes from the untreated Caco-2 cells were incubated with N-hydroxy-PhIP there was a minor HPLC peak at the expected retention time for N-hydroxy-PhIP-N2-glucuronide. This peak was dramatically increased in the sulforaphane-treated cells, suggesting higher activities of glucuronidation of N-hydroxy-PhIP. (4) Cytoplasmic labeling of NF-E2-related factor 2 (Nrf2), a transcription factor, with no nuclear staining was observed in the non-stimulated cells, whereas an intense nuclear labeling was observed in the sulforaphane-treated cells, indicating the induction of nuclear translocation of Nrf2 by sulforaphane. (1) Low dose sulforaphane induces the expression of UGT1A, UGT1A1, UGT1A A8, and UGT1A A10 mRNA significantly. These changes are accompanied by an increase in UGT1A1 protein and increase in heterocyclic aromatic amine glucuronidation. (2) The induction of the phase II enzyme activity by SFN occurs at the transcriptional level and is regulated by Nrf2.

Overexpression of Aldo-Keto Reductase 1C3 (AKR1C3) in LNCaP Cells Diverts Androgen Metabolism towards Testosterone Resulting in Resistance to the 5α-Reductase Inhibitor Finasteride

PubMed Central

Byrns, Michael C.; Mindnich, Rebekka; Duan, Ling; Penning, Trevor M.

2012-01-01

Type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ4-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signalling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ4-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17β-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17β-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17β-glucuronide was significantly decreased. Δ4-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ4-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ4-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to testosterone that results in proliferation in androgen sensitive prostate cancer. This effect is seen despite high levels of uridine glucuronosyl transferases suggesting that AKR1C3 activity can surmount the effects of this elimination pathway. Treatment options in prostate cancer that target 5α-reductase where AKR1C3 co-exists may be less effective due to the diversion of Δ4-Adione to testosterone. PMID:22265960

Quantification of Phenol, Phenyl Glucuronide, and Phenyl Sulfate in Blood of Unanesthetized Rainbow Trout by On-line Microdialysis Sampling

EPA Science Inventory

In this study we have developed a novel method to estimate in vivo rates of metabolism in unanesthetized fish. This method provides a basis for evaluating the accuracy of in vitro-in vivo metabolism extrapolations. As such, this research will lead to improved risk assessments f...

NEW MEDIUM FOR THE SIMULTANEOUS DETECTION OF TOTAL COLIFORMS AND ESCHERICHIA COLI IN WATER (PUBLISHED ERRATUM APPEARS IN APP ENVIRON MICROBIOL 1993 DEC;59(12):4378)

EPA Science Inventory

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the b...

EFFECTS OF LOW DOSE MIXTURES OF PCB126 AND PERCHLORATE ON THE HYPTHALAMIC-PITUITARY-THYROID (HPT) AXIS IN THE MALE RAT.

EPA Science Inventory

Perchlorate (ClO4) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) are environmental contaminants known to disturb thyroid hormone homeostasis by well defined modes of action that lead to hypothyroidism in the rat. PCB126 increases phase II conjugation of T4 (T4-glucuronide) by indu...

Cholesterol-lowering effects and mechanisms in view of bile acid pathway of resveratrol and resveratrol-glucuronides

USDA-ARS?s Scientific Manuscript database

Resveratrol (Res) was previously reported to be capable of lowering plasma TC and LDL-C. The mechanism behind Res is not clearly understood, although it is presumed to have an effect on bile acid metabolism in the liver: a significant way in eliminating cholesterol from the body. As one of the major...

Two novel creatinine adducts of andrographolide in human urine.

PubMed

Qiu, Feng; Cui, Liang; Chen, Lixia; Sun, Jiawen; Yao, Xinsheng

2012-09-01

Andrographolide is a major labdane diterpenoid of the traditional Chinese and Ayurvedic medicine. Andrographis paniculate (Burm) Nees, is used in clinical situations in China mainly to treat fever, cold, and inflammation. In our previous study, fifteen metabolites of andrographolide were identified in human urine. However, there are still two other unknown metabolites. The aim of this study was to elucidate the structures of these two metabolites. 3. The two metabolites which are probably epimers were identified as creatinine adducts, and their structures were determined to be 14-deoxy-12-(creatinine-5-yl)-andrographolide-19-O-β-D-glucuronide A (Metabolite 1) and 14-deoxy-12-(creatinine-5-yl)-andrographolide-19-O-β-D-glucuronide B (Metabolite 2) by means of spectroscopic evidences. 4. It is for the first time that the formation of creatinine adducts as a novel metabolic pathway is reported. The mechanism was presumed that β-carbon (C-12) of α, β-unsaturated carbonyl was attacked by a 5-anion intermediate of creatinine formed through elimination of a proton, followed by the double bond migration from 12(13) to 13(14) and elimination of the hydroxyl group at C-14.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

PubMed Central

Iverson, Sonya V.; Eriksson, Sofi; Xu, Jianqiang; Prigge, Justin R.; Talago, Emily A.; Meade, Tesia A.; Meade, Erin S.; Capecchi, Mario R.; Arnér, Elias S.J.; Schmidt, Edward E.

2013-01-01

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug-metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however their constitutive metabolic state supported more robust GSH biosynthesis-, glutathionylation-, and glucuronidation-systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage. PMID:23743293

Intravenous paracetamol as an antipyretic and analgesic medication: the significance of drug metabolism.

PubMed

Giamarellos-Bourboulis, Evangelos J; Spyridaki, Aikaterini; Savva, Athina; Georgitsi, Marianna; Tsaganos, Thomas; Mouktaroudi, Maria; Raftogiannis, Maria; Antonopoulou, Anastasia; Papaziogas, Vassilios; Baziaka, Fotini; Sereti, Kalliopi; Christopoulos, Petros; Marioli, Androniki; Kanni, Theodora; Maravitsa, Panagiota; Pantelidou, Ilianna; Leventogiannis, Konstantinos; Tsiaoussis, Panagiotis; Lymberopoulou, Korina; Koutelidakis, Ioannis M

2014-01-01

One prospective, open-label, non-randomized study was conducted in 100 patients to define the antipyretic and analgesic effect of a new intravenous formulation of 1 g of paracetamol; 71 received paracetamol for the management of fever and 29 received paracetamol for pain relief after abdominal surgery or for neoplastic pain. Serial follow-up measurements of core temperature and of pain intensity were done for 6 h. Additional rescue medications were recorded for 5 days. Blood was sampled for the measurement of free paracetamol (APAP) and of glucuronide-APAP and N-sulfate-APAP by an HPLC assay. Defervescence, defined as core temperature below or equal to 37.1°C, was achieved in 52 patients (73.2%) within a median time of 3 h. Patients failing to become afebrile with the first dose of paracetamol became afebrile when administered other agents as rescue medications. Analgesia was achieved in 25 patients (86.4%) within a median time of 2 h. Serum levels of glucuronide-APAP were greater among non-responders to paracetamol. The presented results suggest that the intravenous formulation of paracetamol is clinically effective depending on drug metabolism.

Determination of ethyl glucuronide in hair: a rapid sample pretreatment involving simultaneous milling and extraction.

PubMed

Mönch, Bettina; Becker, Roland; Nehls, Irene

2014-01-01

A combination of simultaneous milling and extraction known as micropulverized extraction was developed for the quantification of the alcohol marker ethyl glucuronide (EtG) in hair samples using a homogeneous reference material and a mixer mill. Best extraction results from 50 mg of hair were obtained with 2-mL plastic tubes containing two steel balls (∅ = 5 mm), 0.5 mL of water and with an oscillating frequency of 30 s(-1) over a period of 30 min. EtG was quantified employing a validated GC-MS procedure involving derivatization with pentafluoropropionic acid anhydride. This micropulverization procedure was compared with dry milling followed by separate aqueous extraction and with aqueous extraction after manual cutting to millimeter-size snippets. Micropulverization yielded 28.0 ± 1.70 pg/mg and was seen to be superior to manually cutting (23.0 ± 0.83 pg/mg) and equivalent to dry grinding (27.7 ± 1.71 pg/mg) with regard to completeness of EtG extraction. The option to process up to 20 samples simultaneously makes micropulverization especially valuable for the high throughput of urgent samples.

The bioactivity of dietary anthocyanins is likely to be mediated by their degradation products.

PubMed

Kay, Colin D; Kroon, Paul A; Cassidy, Aedin

2009-05-01

To date the in vitro mechanistic bioactivity of anthocyanins has been exclusively explored using aglycones and glycoside conjugates, despite a lack of evidence establishing these as the biologically available forms. We conducted intestinal epithelial cell (Caco-2 cells) culture experiments, which indicated that after a 4 h incubation of anthocyanins in cell-free culture media (DMEM), 57% of the initial cyanidin-3-glucoside (C3G) and 96% of cyanidin had degraded. The level of degradation was not statistically different from that of cultured cell incubations, suggesting that degradation was spontaneous. Degradation products were identified as protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), and were confirmed in two other buffer matrices (phosphate and Hank's buffers). In cultured cell media the degradation products PCA and PGA were metabolised to glucuronide and sulphate conjugates, as indicated by both enzyme hydrolysis (sulphatase and glucuronidase treatments) and MS (PCA and PGA m/z = 155; sulphate = 235; glucuronide = 331). These data suggest a significant proportion of intestinal metabolites of anthocyanins are likely to be conjugates of their degradation products. Future efforts to establish the biological activities of anthocyanins should therefore include the investigation of phenolic acid and aldehyde products of degradation, along with their respective metabolites.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

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

Ohara, Kazuaki, E-mail: Kazuaki_Ohara@kirin.co.jp; Wakabayashi, Hideyuki; Taniguchi, Yoshimasa

Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found thatmore » quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.« less

Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium for detection of alcohol abuse during pregnancy: Correlation study between both biomarkers.

PubMed

Cabarcos, Pamela; Tabernero, María Jesús; Otero, José Luís; Míguez, Martha; Bermejo, Ana María; Martello, Simona; De Giovanni, Nadia; Chiarotti, Marcello

2014-11-01

This article presents results from 47 meconium samples, which were analyzed for fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for detection of gestational alcohol consumption. A validated microwave assisted extraction (MAE) method in combination with GC-MS developed in the Institute of Forensic Science (Santiago de Compostela) was used for FAEE and the cumulative concentration of ethyl myristate, ethyl palmitate and ethyl stearate with a cut-off of 600ng/g was applied for interpretation. A simple method for identification and quantification of EtG has been evaluated by ultrasonication followed solid phase extraction (SPE). Successful validation parameters were obtained for both biochemical markers of alcohol intake. FAEE and EtG concentrations in meconium ranged between values lower than LOD and 32,892ng/g or 218ng/g respectively. We have analyzed FAEE and EtG in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. Certain agreement between the two biomarkers was found as they are both a very specific alcohol markers, making it a useful analysis for confirmation. Copyright © 2014 Elsevier B.V. All rights reserved.

Abacavir Pharmacokinetics During Chronic Therapy in HIV-1-Infected Adolescents and Young Adults

PubMed Central

Sleasman, JW; Robbins, BL; Cross, SJ; Lindsey, JC; Kraimer, JM; Heckman, BE; Sprenger, HL; Tustin, NB; Rose, CH; Poston, PA; Neal, EF; Pakes, GE; Nikanjam, M; Capparelli, EV

2009-01-01

The pharmacokinetics of abacavir and its metabolites were investigated in 30 human immunodeficiency virus (HIV)-infected adolescents and young adults 13-25 years of age, equally divided into two groups: <18 years of age and ≥18 years of age. All the subjects received the recommended adult dose of 300 mg twice daily. The area under the plasma concentration-time curve (AUC) and half-life of abacavir did not differ significantly between the age groups or by gender or race, and there were only modest associations of age with apparent abacavir clearance and with volume of distribution. There were no significant correlations of carboxylate or glucuronide metabolite levels with age or gender, although glucuronide AUC was higher in Hispanic subjects than in African-American subjects. Zidovudine and lamivudine concentration profiles were also similar in the two age groups. A novel aspect of the study included an assessment of intracellular carbovir, zidovudine, and lamivudine triphosphate levels, and these were found to be similar in the two age-based groups. Overall, these findings suggest that current recommendations relating to adult dosages are appropriate for adolescents and young adults. PMID:19118380

Deposition of ethyl glucuronide in WHP rat hair after chronic ethanol intake.

PubMed

Małkowska, Anna; Szutowski, Mirosław; Dyr, Wanda

2012-01-01

This study investigated the relationship between ethanol intake in rats and the resulting level of ethyl glucuronide (EtG) in rat hair. Rats (n = 50) consumed a 10% ethanol solution for 4 weeks, then EtG was extracted from samples of their hair using a novel extraction procedure involving freezing and thawing. The EtG concentration was measured using gas chromatography and mass spectrometry. The animals voluntarily drank ethanol, with daily consumption in most rats exceeding 5 g/kg b.w. The silylated EtG was stable for at least 28 h. The limit of detection was 0.03 ng/mg, and the limit of quantification was 0.1 ng/mg. Hair samples from rats that consumed ethanol had EtG levels ranging from 0.17-20.72 ng/mg in female rats and 0.15-13.72 ng/mg in males. There was a correlation between the amount of alcohol consumed and the EtG levels in hair from female (p < 0.01), but not male, rats. The method presented allows detection and quantification of EtG in rat hair. We also observed differences in EtG deposition in male and female rats.

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

PubMed

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

2017-10-01

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

Fomiroid A, a Novel Compound from the Mushroom Fomitopsis nigra, Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

PubMed Central

Chiba, Tomohiro; Sakurada, Tsuyoshi; Watanabe, Rie; Yamaguchi, Kohji; Kimura, Yasuhisa; Kioka, Noriyuki; Kawagishi, Hirokazu; Matsuo, Michinori; Ueda, Kazumitsu

2014-01-01

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C30H48O3. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe. PMID:25551765

Recovery of Escherichia coli from Soil after Addition of Sterile Organic Wastes

PubMed Central

Unc, Adrian; Gardner, Julie; Springthorpe, Susan

2006-01-01

Laboratory batch tests indicate that addition of sterile municipal sewage biosolids to clay soil from four depths increases the numbers of Escherichia coli isolates recoverable in EC-MUG broth (EC broth with 4-methylumbelliferyl-β-glucuronide). This effect was most marked for the deeper soil layers, with increases of about 2.6 orders of magnitude in E. coli most probable number. PMID:16517690

Fate of estrogens in a pilot-scale step-feed anoxic/oxic wastewater treatment system controlling by nitrogen and phosphorus removal.

PubMed

Chen, Qingcai; Li, Zebing; Hua, Xiaoyu

2018-05-01

The control measures for estrogens in the aquatic environment are topics of growing concern. It is a meaningful issue to finding optimal process parameters for efficient removal of estrogens with the purpose of efficient total nitrogen (TN) or total phosphorus (TP) removal in sewage treatment plants. The present paper is concerned with the relationships between the estrogen removal and TN or TP removal in a pilot-scale three-stage anoxic/oxic (A/O) system treating real municipal wastewater. The total removal efficiency for estrone (E1) and 17β-estradiol (E2) and their sulfate and glucuronide conjugates were on average 87% in the pilot-scale system. The concentrations of the sulfate and glucuronide conjugates of estrogens (E1 and E2) in the system were much lower than the estrogens, which might be caused by the rapid degradation of conjugates in the pilot-scale system. The average removal efficiencies of E1 and E2 and their sulfate and glucuronide conjugates were significantly lower under high TP removal conditions than those under high TN removal conditions that suggested that the ammonia oxidation promotes estrogen degradation. When the system achieved efficient TN removal, the concentrations of both E1 and E2 were generally lower in the aerobic zones than those in the anoxic zones. Instead, when the system achieved efficient TP removal conditions, the estrogen concentrations were higher in the aerobic zones than in the anoxic zones. However, it was thought that the variation of the concentrations of the estrogen conjugates had weak influence on concentrations of the free estrogens. The increase of the free estrogens in the aerobic zones could be attributed to the release of the estrogens adsorbed on the sludge. The variation of estrogens in a three-stage A/O system can be properly estimated and measured by a binary linear regression model with the variables of TP and TON (NO 2 - -N and NO 3 - -N), which is probably the important information for the improvement and optimization of wastewater treatment processes to obtain higher removal efficiency for estrogens.

Decrease in lean body mass in men with prostate cancer receiving androgen deprivation therapy: mechanism and biomarkers.

PubMed

Hara, Noboru; Ishizaki, Fumio; Saito, Toshihiro; Nishiyama, Tsutomu; Kawasaki, Takashi; Takahashi, Kota

2013-02-01

To elucidate the mechanism of the androgen deprivation therapy (ADT)-related decrease in lean body mass (LBM). The LBM and blood samples were studied before and after 6 months of ADT in 72 patients with localized prostate cancer. The LBM was assessed using a foot-to-foot bioelectrical impedance analyzer. Before ADT, the LBM correlated with none of the serum sex steroid levels; however, it correlated closely with serum 5α-androstane-3α,17β-diol glucuronide (Spearman's rank correlation coefficient = 0.409, P = .001) and insulin-like growth factor-1 (IGF-I, Spearman's rank correlation coefficient = 0.329, P = .005). After ADT, the LBM decreased by 0.9% (P = .036), and the serum testosterone and dihydrotestosterone had decreased by 96.8% and 94.3%, respectively (P <.001 for both), and the IGF-I had increased by 11.6% (from 19.9 to 22.2 nmol/L, P = .001). The serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D] levels decreased after ADT by 9.8% (from 66.2 to 59.7 pg/mL, P = .008), and the post-treatment LBM correlated inversely with 1,25(OH)2D (Spearman's rank correlation coefficient = -0.343, P = .003). The post-treatment LBM was dissociated with 5α-androstane-3α,17β-diol glucuronide and IGF-I. The pretreatment and post-treatment LBMs both correlated inversely with serum sex hormone-binding globulin (P = .024 and P = .016, respectively). The deficiency in androgen levels was suggested to be a link to the ADT-related decrease in LBM; the androgen metabolite 5α-androstane-3α,17β-diol glucuronide has a potential value for assessing the LBM in untreated men. IGF-I also promotes muscle building and is positively regulated during ADT. Sex hormone-binding globulin possibly accelerates the ADT-related decrease in LBM. Although the mechanism for the decrease in 1,25(OH)2D and its inverse correlation with LBM during ADT is unclear, 1,25(OH)2D might be a biomarker reflecting the ADT-related decrease in LBM. Copyright © 2013 Elsevier Inc. All rights reserved.

Metabolic disposition of 14C-bromfenac in healthy male volunteers.

PubMed

Osman, M; Chandrasekaran, A; Chan, K; Scatina, J; Ermer, J; Cevallos, W; Sisenwine, S F

1998-08-01

The metabolic disposition of 14C-bromfenac, an orally active, potent, nonsteroidal, nonnarcotic, analgesic agent was investigated in six healthy male subjects after a single oral 50-mg dose. The absorption of radioactivity was rapid, producing a mean maximum plasma concentration (Cmax) of 4.9 +/- 1.8 microg x equiv/mL, which was reached 1.0 +/- 0.5 hours after administration. Unchanged drug was the major component found in plasma, and no major metabolites were detected in the plasma. Total radioactivity recovered over a 4-day period from four of the six subjects averaged 82.5% and 13.2% of the dose in the urine and feces, respectively. Excretion into urine was rapid; most of the radioactivity was excreted during the first 8 hours. Five radioactive chromatographic peaks, a cyclic amide and four polar metabolites, were detected in 0- to 24-hour urine samples. Similarity of metabolite profiles between humans and cynomolgus monkeys permitted use of this animal model to generate samples after a high dose for structure elucidation. Liquid chromatography/mass spectrometry (LC/MS) analysis of monkey urine samples indicated that the four polar metabolites were two pairs of diastereoisomeric glucuronides whose molecular weight differed by two daltons. Enzyme hydrolysis, cochromatography, and LC/MS experiments resulted in the identification of a hydroxylated cyclic amide as one of the aglycones, which formed a pair of diastereoisomeric glucuronides after conjugation. Data also suggested that a dihydroxycyclic amide formed by the reduction of the ketone group that joins the phenyl rings formed the second pair of diastereoisomeric glucuronides. Further, incubation of various reference standards in control (blank) urine and buffer with and without creatinine indicated that the hydroxy cyclic amide released from enzyme hydrolysis can undergo ex vivo transformations to a condensation product between creatinine and an alpha-keto acid derivative of the hydroxy cyclic amide that is formed by oxidation and ring opening. Further experiments with a dihydroxylated cyclic amide after reduction of the keto function indicated that it too can form a creatinine conjugate.

Sequential Activation of Classic PKC and Estrogen Receptor α Is Involved in Estradiol 17ß-D-Glucuronide-Induced Cholestasis

PubMed Central

Barosso, Ismael R.; Zucchetti, Andrés E.; Boaglio, Andrea C.; Larocca, M. Cecilia; Taborda, Diego R.; Luquita, Marcelo G.; Roma, Marcelo G.; Crocenzi, Fernando A.; Sánchez Pozzi, Enrique J.

2012-01-01

Estradiol 17ß-d-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα. Conclusion: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC. PMID:23209816

Serum steroid concentrations remain within normal postmenopausal values in women receiving daily 6.5mg intravaginal prasterone for 12 weeks.

PubMed

Martel, Céline; Labrie, Fernand; Archer, David F; Ke, Yuyong; Gonthier, Renaud; Simard, Jean-Nicolas; Lavoie, Lyne; Vaillancourt, Mario; Montesino, Marlene; Balser, John; Moyneur, Érick

2016-05-01

This study integrates all data obtained in women aged 40-80years enrolled with moderate to severe symptoms of vulvovaginal atrophy (VVA) who received daily intravaginal administration of 0.50% (6.5mg) dehydroepiandrosterone (DHEA; prasterone) for 12weeks (n=723; ITT-S population) as compared with placebo (n=266; ITT-S population). To this end, serum steroid levels (DHEA, DHEA-sulfate (DHEA-S), androst-5-ene-3β, 17β-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17β-diol 17-glucuronide (3α-diol-17G)) were measured at Day 1 and Week 12 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following validation performed according to the FDA guidelines [1-6]. In agreement with the mechanisms of intracrinology where DHEA is exclusively transformed intracellularly into active sex steroids which act and are inactivated locally before being released as glucuronided or sulfated metabolites for elimination by the kidneys and liver, all sex steroids remained well within normal postmenopausal values following administration of intravaginal DHEA. Serum estradiol, the most relevant sex steroid, was measured after 12weeks of treatment at 3.36pg/ml (cITT-S population) or 19% below the normal postmenopausal value of 4.17pg/ml. On the other hand, serum E1-S, the best recognized marker of global estrogenic activity, shows an average value of 209pg/ml at 12 weeks compared to 220pg/ml in normal postmenopausal women. Moreover, serum ADT-G, the main metabolite of androgens, also remains well within normal postmenopausal values. The present data shows that a low daily intravaginal dose (6.5mg) of DHEA (prasterone) which is efficacious on the symptoms and signs of VVA, permits to achieve the desired local efficacy without systemic exposure, in agreement with the stringent mechanisms of menopause established after 500 million years of evolution where each cell in each tissue is the master of its sex steroid exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabolism and pharmacokinetics of novel selective vascular endothelial growth factor receptor-2 inhibitor apatinib in humans.

PubMed

Ding, Juefang; Chen, Xiaoyan; Gao, Zhiwei; Dai, Xiaojian; Li, Liang; Xie, Cen; Jiang, Haoyuan; Zhang, Lijia; Zhong, Dafang

2013-06-01

Apatinib is a new oral antiangiogenic molecule that inhibits vascular endothelial growth factor receptor-2. The present study aimed to determine the metabolism, pharmacokinetics, and excretion of apatinib in humans and to identify the enzymes responsible for its metabolism. The primary routes of apatinib biotransformation included E- and Z-cyclopentyl-3-hydroxylation, N-dealkylation, pyridyl-25-N-oxidation, 16-hydroxylation, dioxygenation, and O-glucuronidation after 3-hydroxylation. Nine major metabolites were confirmed by comparison with reference standards. The total recovery of the administered dose was 76.8% within 96 hours postdose, with 69.8 and 7.02% of the administered dose excreted in feces and urine, respectively. About 59.0% of the administered dose was excreted unchanged via feces. Unchanged apatinib was detected in negligible quantities in urine, indicating that systemically available apatinib was extensively metabolized. The major circulating metabolite was the pharmacologically inactive E-3-hydroxy-apatinib-O-glucuronide (M9-2), the steady-state exposure of which was 125% that of the apatinib. The steady-state exposures of E-3-hydroxy-apatinib (M1-1), Z-3-hydroxy-apatinib (M1-2), and apatinib-25-N-oxide (M1-6) were 56, 22, and 32% of parent drug exposure, respectively. Calculated as pharmacological activity index values, the contribution of M1-1 to the pharmacology of the drug was 5.42 to 19.3% that of the parent drug. The contribution of M1-2 and M1-6 to the pharmacology of the drug was less than 1%. Therefore, apatinib was a major contributor to the overall pharmacological activity in humans. Apatinib was metabolized primarily by CYP3A4/5 and, to a lesser extent, by CYP2D6, CYP2C9, and CYP2E1. UGT2B7 was the main enzyme responsible for M9-2 formation. Both UGT1A4 and UGT2B7 were responsible for Z-3-hydroxy-apatinib-O-glucuronide (M9-1) formation.

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

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

Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase Imore » metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in this paper in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 μM), and higher intrinsic clearance at lower substrate concentrations (<0.07 μM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Finally, kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.« less

Morphine Glucuronidation and Elimination in Intensive Care Patients: A Comparison with Healthy Volunteers.

PubMed

Ahlers, Sabine J G M; Välitalo, Pyry A J; Peeters, Mariska Y M; Gulik, Laura van; van Dongen, Eric P A; Dahan, Albert; Tibboel, Dick; Knibbe, Catherijne A J

2015-11-01

Although morphine is used frequently to treat pain in the intensive care unit, its pharmacokinetics has not been adequately quantified in critically ill patients. We evaluated the glucuronidation and elimination clearance of morphine in intensive care patients compared with healthy volunteers based on the morphine and morphine-3-glucuronide (M3G) concentrations. A population pharmacokinetic model with covariate analysis was developed with the nonlinear mixed-effects modeling software (NONMEM 7.3). The analysis included 3012 morphine and M3G concentrations from 135 intensive care patients (117 cardiothoracic surgery patients and 18 critically ill patients), who received continuous morphine infusions adapted to individual pain levels, and 622 morphine and M3G concentrations from a previously published study of 20 healthy volunteers, who received an IV bolus of morphine followed by a 1-hour infusion. For morphine, a 3-compartment model best described the data, whereas for M3G, a 1-compartment model fits best. In intensive care patients with a normal creatinine concentration, a decrease of 76% was estimated in M3G clearance compared with healthy subjects, conditional on the M3G volume of distribution being the same in intensive care patients and healthy volunteers. Furthermore, serum creatinine concentration was identified as a covariate for both elimination clearance of M3G in intensive care patients and unchanged morphine clearance in all patients and healthy volunteers. Under the assumptions in the model, M3G elimination was significantly decreased in intensive care patients when compared with healthy volunteers, which resulted in substantially increased M3G concentrations. Increased M3G levels were even more pronounced in patients with increased serum creatinine levels. Model-based simulations show that, because of the reduction in morphine clearance in intensive care patients with renal failure, a 33% reduction in the maintenance dose would result in morphine serum concentrations equal to those in healthy volunteers and intensive care patients with normal renal function, although M3G concentrations remain increased. Future pharmacodynamic investigations are needed to identify target concentrations in this population, after which final dosing recommendations can be made.

In Vitro Effects of Bisphenol A β-D-Glucuronide (BPA-G) on Adipogenesis in Human and Murine Preadipocytes

PubMed Central

Boucher, Jonathan G.; Boudreau, Adèle; Ahmed, Shaimaa

2015-01-01

Background Exposure to common environmental substances, such as bisphenol A (BPA), has been associated with a number of negative health outcomes. In vivo, BPA is rapidly converted to its predominant metabolite, BPA-glucuronide (BPA-G), which has long been believed to be biologically inactive because it lacks estrogenic activity. However, the effects of BPA-G on cellular metabolism have not been characterized. In the present study we examined the effect of BPA-G on adipogenesis. Methods The effect of BPA-G on the differentiation of human and 3T3L1 murine preadipocytes was evaluated in vitro by quantifying lipid accumulation and the expression of adipogenic markers. Results Treatment of 3T3L1 preadipocytes with 10 μM BPA-G induced a significant increase in lipid accumulation, mRNA expression of the adipogenic markers sterol regulatory element binding factor 1 (SREBF1) and lipoprotein lipase (LPL), and protein levels of LPL, aP2, and adipsin. Treatment of primary human preadipocytes with BPA-G also induced adipogenesis as determined by aP2 levels. Co-treatment of cells with the estrogen receptor (ER) antagonist fulvestrant (ICI) significantly inhibited the BPA-G–induced increase in LPL and aP2 levels, whereas treatment with ICI alone had no effect. Moreover, BPA-G did not display any significant estrogenic activity. Conclusions To our knowledge, this study is the first to report that BPA-G induces adipocyte differentiation and is not simply an inactive metabolite. The fact that BPA-G induced adipogenesis and was inhibited by an ER antagonist yet showed no estrogenic activity suggests that it has no classical ER transcriptional activation function and acts through a pathway that remains to be determined. Citation Boucher JG, Boudreau A, Ahmed S, Atlas E. 2015. In vitro effects of bisphenol A β-D-glucuronide (BPA-G) on adipogenesis in human and murine preadipocytes. Environ Health Perspect 123:1287–1293; http://dx.doi.org/10.1289/ehp.1409143 PMID:26018136

Dihydrocodeine Overdoses in a Neonate and in a 14-year-old Girl Who Were Both Genotyped as Cytochrome P450 2D6*1/*10-*36: Comparing Developmental Ages and Drug Monitoring Data With the Results of Pharmacokinetic Modeling.

PubMed

Shimizu, Makiko; Kondo, Tatsuki; Fukuoka, Tetsuya; Tanaka, Toshihiro; Yamazaki, Hiroshi

2018-04-01

A high activity of cytochrome P450 2D6 (CYP2D6) reportedly leads to toxicity of dihydrocodeine/codeine by increasing toxic potential of their metabolite dihydromorphine/morphine, which are further metabolized to highly active dihydromorphine 6-O-glucuronide and the less active morphine 3-O-glucorinide but rapidly excreted into urine as water-soluble forms. A case of acute respiratory depression after administration of prescribed dihydrocodeine phosphate (2.0 mg/d divided twice a day for 2 days) to a 1-month-old baby boy genotyped as CYP2D6*1/*10-*36 is described. The case is compared with that of a 14-year-old girl, also genotyped as CYP2D6*1/*10-*36, presenting in an agitated state after an overdose (37 mg) of dihydrocodeine phosphate taken as simultaneous ingestion of multiple over-the-counter tablets. In contrast to the rapid clearance of dihydrocodeine from blood in the 14-year-old girl (apparent half-life of 3 hours), the 1-month-old baby boy still had high serum concentrations of dihydrocodeine (400 nmol/L) and dihydromorphine (1.9 nmol/L) 21 hours after the last oral administration of dihydrocodeine-containing cough mixture. The rapid clearance in the 14-year-old girl was mainly attributed to dihydrocodeine glucuronidation and partly attributed to dihydromorphine formation, as determined by liquid chromatography-tandem mass spectrometry analyses. However, the conjugation ratios of dihydrocodeine and dihydromorphine in the neonate were low in comparison with those in the 14-year-old girl and with those measured in 3-, 6-, and 13-year-old control subjects, resulting from the poorly developed glucuronidation potential of the neonate. The current observations suggest that the CYP2D6*1/*10-*36 genotype seen in the 2 Japanese patients may not significantly contribute to the likelihood of dihydrocodeine overdose but highlight the importance of considering age when prescribing dihydrocodeine.

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

DOE PAGES

Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree; ...

2017-01-21

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase Imore » metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in this paper in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 μM), and higher intrinsic clearance at lower substrate concentrations (<0.07 μM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Finally, kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.« less

Pharmacokinetics of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol and conjugate metabolites in healthy human subjects.

PubMed

Zick, Suzanna M; Djuric, Zora; Ruffin, Mack T; Litzinger, Amie J; Normolle, Daniel P; Alrawi, Sara; Feng, Meihua Rose; Brenner, Dean E

2008-08-01

Ginger shows promising anticancer properties. No research has examined the pharmacokinetics of the ginger constituents 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol in humans. We conducted a clinical trial with 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol, examining the pharmacokinetics and tolerability of these analytes and their conjugate metabolites. Human volunteers were given ginger at doses from 100 mg to 2.0 g (N = 27), and blood samples were obtained at 15 minutes to 72 hours after a single p.o. dose. The participants were allocated in a dose-escalation manner starting with 100 mg. There was a total of three participants at each dose except for 1.0 g (N = 6) and 2.0 g (N = 9). No participant had detectable free 6-gingerol, 8-gingerol, 10-gingerol, or 6-shogaol, but 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol glucuronides were detected. The 6-gingerol sulfate conjugate was detected above the 1.0-g dose, but there were no detectable 10-gingerol or 6-shogaol sulfates except for one participant with detectable 8-gingerol sulfate. The C(max) and area under the curve values (mean +/- SE) estimated for the 2.0-g dose are 0.85 +/- 0.43, 0.23 +/- 0.16, 0.53 +/- 0.40, and 0.15 +/- 0.12 microg/mL; and 65.6.33 +/- 44.4, 18.1 +/- 20.3, 50.1 +/- 49.3, and 10.9 +/- 13.0 microg x hr/mL for 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol. The corresponding t(max) values are 65.6 +/- 44.4, 73.1 +/- 29.4, 75.0 +/- 27.8, and 65.6 +/- 22.6 minutes, and the analytes had elimination half-lives <2 hours. The 8-gingerol, 10-gingerol, and 6-shogaol conjugates were present as either glucuronide or sulfate conjugates, not as mixed conjugates, although 6-gingerol and 10-gingerol were an exception. Six-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol are absorbed after p.o. dosing and can be detected as glucuronide and sulfate conjugates.

Pharmacokinetics of 6-, 8-, 10-Gingerols and 6-Shogaol and Conjugate Metabolites in Healthy Human Subjects

PubMed Central

Zick, Suzanna M.; Djuric, Zora; Ruffin, Mack T.; Litzinger, Amie J.; Normolle, Daniel P.; Feng, Meihua Rose; Brenner, Dean E.

2009-01-01

Background Ginger demonstrates promising anticancer properties. No research has examined the pharmacokinetics of the ginger constituents 6-, 8-, 10-gingerol and 6-shogaol in humans. We conducted a clinical trial with 6-, 8-, 10-gingerol and 6-shogaol examining the pharmacokinetics and tolerability of these analytes and their conjugate metabolites Methods Human volunteers were given ginger at doses from 100 mg, to 2.0 g (N=27), and blood samples were obtained at 15 minutes to 72 hours after a single oral dose. Participants were allocated in a dose-escalation manner starting with 100 mg. There was a total of three participants at each dose except for 1.0 g (N=6) and 2.0 g (N=9). Results No participant had detectable free 6-, 8-, 10-gingerol or 6-shogaol, but 6-, 8-, 10-gingerol and 6-shogaol glucuronides were detected. The 6-gingerol sulfate conjugate was detected above the 1.0 g dose but there were no detectable 10-gingerol or 6-shogaol sulfates except for one participant with detectable 8-gingerol sulfate. The Cmax and AUC values (Mean±SE) estimated for the 2.0 g dose are 0.85±0.43, 0.23±0.16, 0.53±0.40, and 0.15±0.12 μg/mL ; and 65.6.33±44.4, 18.1±20.3, 50.1±49.3, and 10.9±13.0 μg·hr/mL for 6-, 8-, 10-gingerol, and 6-shogaol. The corresponding tmax values are 65.6±44.4, 73.1±29.4, 75.0±27.8, and 65.6±22.6 minutes and the analytes had elimination half-lives < 2hr. The 8-, 10-gingerol and 6-shogaol conjugates were present as either glucuronide or sulfate conjugates, not as mixed conjugates, although 6-, 10-gingerol were an exception. Conclusion Six-, 8-, 10-gingerol and 6-shogaol is absorbed after oral dosing and can be detected as glucuronide and sulfate conjugates. PMID:18708382

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.

PubMed

Li, Jian-Ping; Guo, Jian-Ming; Shang, Er-Xin; Zhu, Zhen-Hua; Liu, Yang; Zhao, Bu-Chang; Zhao, Jing; Tang, Zhi-Shu; Duan, Jin-Ao

2017-05-10

Acetylsalicylic acid (Aspirin, ASA) is a famous drug for cardiovascular diseases in recent years. Effects of ASA dosage on the metabolic profile have not been fully understood. The purpose of our study is to establish a rapid and reliable method to quantify ASA metabolites in biological matrices, especially for glucuronide metabolites whose standards are not commercially available. Then we applied this method to evaluate the effects of ASA dosage on the metabolic and excretion profile of ASA metabolites in rat urine. Salicylic acid (SA), gentisic acid (GA) and salicyluric acid (SUA) were determined directly by UHPLC-MS/MS, while salicyl phenolic glucuronide (SAPG) and salicyluric acid phenolic glucuronide (SUAPG) were quantified indirectly by measuring the released SA and SUA from SAPG and SUAPG after β-glucuronidase digestion. SUA and SUAPG were the major metabolites of ASA in rat urine 24h after ASA administration, which accounted for 50% (SUA) and 26% (SUAPG). When ASA dosage was increased, the contributions dropped to 32% and 18%, respectively. The excretion of other three metabolites (GA, SA and SAPG) however showed remarkable increases by 16%, 6% and 4%, respectively. In addition, SUA and SUAPG were mainly excreted in the time period of 12-24h, while GA was excreted in the earlier time periods (0-4h and 4-8h). SA was mainly excreted in the time period of 0-4h and 12-24h. And the excretion of SAPG was equally distributed in the four time periods. We went further to show that the excretion of five metabolites in rat urine was delayed when ASA dosage was increased. In conclusion, we have developed a rapid and sensitive method to determine the five ASA metabolites (SA, GA, SUA, SAPG and SUAPG) in rat urine. We showed that ASA dosage could significantly influence the metabolic and excretion profile of ASA metabolites in rat urine. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor

PubMed Central

Van Roy, F. P.; Heirwegh, K. P. M.

1968-01-01

1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied. PMID:5660631

Echinacea sanguinea and Echinacea pallida extracts stimulate glucuronidation and basolateral transfer of Bauer alkamids 8 and 10 and ketone 24 and inhibit p-glycoprotein transporter in Caco-2 cells

USDA-ARS?s Scientific Manuscript database

The use of Echinacea as a medicinal herb is prominent in the United States, and many studies have assessed the effectiveness of Echinacea as an immunomodulator. We hypothesized that Bauer alkamides 8, 10 and 11 and ketone 24 were absorbed similarly either as pure compounds or from Echinacea sanguin...

[Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes].

PubMed

Wang, Qi; Dai, Zhong; Zhang, Yu-Jie; Ma, Shuang-Cheng

2016-12-01

To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant Ki was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with Ki=5.400±0.956(P<0.05) in HLM system, Ki =10.020±0.611(P<0.05) in RLM system, Ki=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines. Copyright© by the Chinese Pharmaceutical Association.

A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat.

PubMed

Selvam, Anjan Panneer; Muthukumar, Sriram; Kamakoti, Vikramshankar; Prasad, Shalini

2016-03-21

We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001-100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia

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

Fujiwara, Ryoichi, E-mail: fujiwarar@pharm.kitasato-u.ac.jp; Maruo, Yoshihiro; Chen, Shujuan

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepaticmore » tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression. - Highlights: • Breast-feeding can be a factor for the development of neonatal hyperbilirubinemia. • UDP-glucuronosyltransferase (UGT) 1A1 is the sole bilirubin-metabolizing enzyme. • Extrahepatic UGT1A1 plays an important role in bilirubin metabolism. • We discuss the potential mechanism of breast milk-induced neonatal jaundice.« less

Liquid Microjunction Surface Sampling Coupled with High-Pressure Liquid Chromatography-Electrospray Ionization-Mass Spectrometry for Analysis of Drugs and Metabolites in Whole-Body Thin Tissue Sections

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

Kertesz, Vilmos; Van Berkel, Gary J

2010-01-01

In this work, a commercially available autosampler was adapted to perform direct liquid microjunction (LMJ) surface sampling followed by a high-pressure liquid chromatography (HPLC) separation of the extract components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the utility of coupling a separation with this direct liquid extraction based surface sampling approach, four different organs (brain, lung, kidney, and liver) from whole-body thin tissue sections of propranolol dosed and control mice were examined. The parent drug was observed in the chromatograms of the surface sampling extracts from all the organs of the dosed mouse examined. In addition, twomore » isomeric phase II metabolites of propranolol (an aliphatic and an aromatic hydroxypropranolol glucuronide) were observed in the chromatograms of the extracts from lung, kidney, and liver. Confirming the presence of one or the other or both of these glucuronides in the extract from the various organs was not possible without the separation. These drug and metabolite data obtained using the LMJ surface sampling/HPLC-MS method and the results achieved by analyzing similar samples by conventional extraction of the tissues and subsequent HPLC-MS analysis were consistent.« less

Anti-inflammatory effects, nuclear magnetic resonance identification, and high-performance liquid chromatography isolation of the total flavonoids from Artemisia frigida.

PubMed

Wang, Qinghu; Jin, Jinmei; Dai, Nayintai; Han, Narenchaoketu; Han, Jingjing; Bao, Baiyinmuqier

2016-04-01

The aerial parts of Artemisia frigida Willd. are used to treat joint swelling, renal heat, abnormal menstruation, and sore carbuncle. The anti-inflammatory effects of A. frigida have been well-known in folk medicine, suggesting that components extracted from A. frigida could potentially treat inflammatory disease. With the aim of discovering bioactive compounds, in this study, we extracted total flavonoids from the aerial parts of A. frigida and investigated their anti-inflammatory effects against inflammation induced by carrageenan and egg albumin in rats. At the doses studied, total flavonoids (100 mg/kg, 200 mg/kg, and 400 mg/kg) and some isolated compounds (30 mg/kg) showed significant and dose-dependent anti-inflammatory effects. According to the high-performance liquid chromatography analysis of the total flavonoids from A. frigida, there are five major compounds, namely, 5-hydroxy-3',4'-dimethoxy-7-O-β-d-glucuronide (F1), 5-hydroxy-3',4',5'-trimethoxy-7-O-β-d-glucuronide (F2), 5,7,3'-trihydroxy-6,4'-dimethoxyflavone (F3), 5,3'-dihydroxy-6,7,4'-trimethoxyflavone (F4), and 5,3'-dihydroxy-3,6,7,4'-tetramethoxyflavone (F5), which may explain the anti-inflammatory activity. Copyright © 2016. Published by Elsevier B.V.

Simultaneous quantification of multiple components in rat plasma by UPLC-MS/MS and pharmacokinetic study after oral administration of Huangqi decoction.

PubMed

Zeng, Jia-Kai; Li, Yuan-Yuan; Wang, Tian-Ming; Zhong, Jie; Wu, Jia-Sheng; Liu, Ping; Zhang, Hua; Ma, Yue-Ming

2018-05-01

A rapid, sensitive and accurate UPLC-MS/MS method was developed for the simultaneous quantification of components of Huangqi decoction (HQD), such as calycosin-7-O-β-d-glucoside, calycosin-glucuronide, liquiritin, formononetin-glucuronide, isoliquiritin, liquiritigenin, ononin, calycosin, isoliquiritigenin, formononetin, glycyrrhizic acid, astragaloside IV, cycloastragenol, and glycyrrhetinic acid, in rat plasma. After plasma samples were extracted by protein precipitation, chromatographic separation was performed with a C 18 column, using a gradient of methanol and 0.05% acetic acid containing 4mm ammonium acetate as the mobile phase. Multiple reaction monitoring scanning was performed to quantify the analytes, and the electrospray ion source polarity was switched between positive and negative modes in a single run of 10 min. Method validation showed that specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability for 14 components met the requirements for their quantitation in biological samples. The established method was successfully applied to the pharmacokinetic study of multiple components in rats after intragastric administration of HQD. The results clarified the pharmacokinetic characteristics of multiple components found in HQD. This research provides useful information for understanding the relation between the chemical components of HQD and their therapeutic effects. Copyright © 2017 John Wiley & Sons, Ltd.

LC-MS-MS analysis of 2,4-dinitrophenol and its phase I and II metabolites in a case of fatal poisoning.

PubMed

Politi, Lucia; Vignali, Claudia; Polettini, Aldo

2007-01-01

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of biological fluids (blood, urine, gastric content, and bile) collected at autopsy in a case of suspected 2,4-dinitrophenol (DNP) fatal poisoning allowed the determination of DNP and its known metabolites (2-amino-4-nitrophenol and nitro-4-aminophenol). The tentative identification of three conjugated metabolites (DNP glucuronide, DNP sulfate, and 2-amino-4-nitrophenol glucuronide) could be made on the basis of their pseudomolecular ion, isotopic and fragmentation patterns, and retention characteristics. Another DNP metabolite reported in the literature, 2,4-diaminophenol, was not detected in the samples. Postmortem blood concentrations were 48.4 mg/L for DNP and 1.2 mg/L for 2-amino-4-nitrophenol. Gas chromatography-MS screening and quantification in postmortem blood revealed the presence of toxic concentrations of citalopram and its desmethylated metabolite (0.58 and 0.40 mg/L, respectively) and therapeutic or lower than therapeutic levels of olanzapine (0.04 mg/L), desalkylflurazepam (0.02 mg/L), and nordazepam (0.01 mg/L). Based on LC-MS-MS results and on available literature data on DNP poisonings, it was concluded that DNP poisoning played a contributing role, together with citalopram, in the cause of death.

Urinary steroid hormone analysis of ovarian cycles and pregnancy in mandrills (Mandrillus sphinx) indicate that menses, copulatory behavior, sexual swellings and reproductive condition are associated with changing estrone conjugates (E(1)C) and pregnanediol-3-glucuronide (PdG).

PubMed

Phillips, Rebecca Sellin; Wheaton, Catharine J

2008-07-01

The objective of this study was to determine if sexual swellings in mandrills (Mandrillus sphinx) are a reflection of reproductive endocrine state. Urine samples were assayed using an enzyme immunoassay measuring pregnanediol-3-glucuronide (PdG) and estrone conjugates (E(1)C). Hormone patterns of ovarian cycles, pregnancy and lactation were characterized and compared with sexual swellings and copulations relative to menses and peak E(1)C. Cycle lengths averaging 28.7 days and pregnancy length of 181 days determined by hormonal and sexual swelling measures were similar to those reported in other Old World primate species. First day of copulation was observed during rising E(1)C concentrations and preceded observations of peak swelling by 1-2 days. Observations of peak sexual swellings occurred at or on the day after peak E(1)C and decreased following the ovulatory increase in PdG. Observations of menses and sexual swellings are a useful method to track mandrill ovarian cycles and can assist zoos in determining the reproductive state of females in their collections. Zoo Biol 27:320-330, 2008. (c) 2008 Wiley-Liss, Inc.

Pharmacokinetic study of nobiletin and tangeretin in rat serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry.

PubMed

Manthey, John A; Cesar, Thais B; Jackson, Erin; Mertens-Talcott, Susanne

2011-01-12

Nobiletin (NOB) and tangeretin (TAN), two of the main polymethoxylated flavones (PMFs) in citrus, influence a number of key biological pathways in mammalian cells. Although the impacts of NOB and TAN on glucose homeostasis and cholesterol regulation have been investigated in human clinical trials, much information is still lacking about the metabolism and oral bioavailability of these compounds in animals. In this study, NOB and TAN were administered to rats by gavage and intraperitoneal (ip) injection, and the blood serum concentrations of these compounds and their main metabolites were monitored by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). In addition to the administered compounds, two metabolites of TAN and eight metabolites of NOB were detected and measured over 24 h. With identical oral doses, nearly 10-fold higher absorption of NOB occurred compared to TAN. For both compounds, maximum levels of glucuronidated metabolites occurred in the blood serum at later time points (∼5-8 h) compared to the earlier T(max) values for NOB and TAN. In most cases the glucuronides occurred at substantially higher concentrations than the aglycone metabolites. Low levels of NOB and TAN and their metabolites were detectable in rat blood serum even at 24 h after treatment.

Sex Is a Determinant for Deoxynivalenol Metabolism and Elimination in the Mouse

PubMed Central

Pestka, James J.; Clark, Erica S.; Schwartz-Zimmermann, Heidi E.

2017-01-01

Based on prior observations that deoxynivalenol (DON) toxicity is sex-dependent, we compared metabolism and clearance of this toxin in male and female mice. Following intraperitoneal challenge with 1 mg/kg bw DON, the dose used in the aforementioned toxicity study, ELISA and LC–MS/MS analyses revealed that by 24 h, most DON and DON metabolites were excreted via urine (49–86%) as compared to feces (1.2–8.3%). Females excreted DON and its principal metabolites (DON-3-, DON-8,15 hemiketal-8-, and iso-DON-8-glucuronides) in urine more rapidly than males. Metabolite concentrations were typically 2 to 4 times higher in the livers and kidneys of males than females from 1 to 4 h after dosing. Trace levels of DON-3-sulfate and DON-15-sulfate were found in urine, liver and kidneys from females but not males. Fecal excretion of DON and DON sulfonates was approximately 2-fold greater in males than females. Finally, decreased DON clearance rates in males could not be explained by glucuronidation activities in liver and kidney microsomes. To summarize, increased sensitivity of male mice to DON’s toxic effects as compared to females corresponds to decreased ability to clear the toxin via urine but did not appear to result from differences in toxin metabolism. PMID:28777306

Disposition, metabolism and mass balance of [14C]apremilast following oral administration

PubMed Central

Hoffmann, Matthew; Kumar, Gondi; Schafer, Peter; Cedzik, Dorota; Capone, Lori; Kei-Fong, Lai; Gu, Zheming; Heller, Dennis; Feng, Hao; Surapaneni, Sekhar; Laskin, Oscar; Wu, Anfan

2011-01-01

Apremilast is a novel, orally available small molecule that specifically inhibits PDE4and thus modulates multiple pro- and anti-inflammatory mediators, and is currently under clinical development for the treatment of psoriasis and psoriatic arthritis.The pharmacokinetics and disposition of [14C]apremilastwas investigated following a single oral dose (20 mg, 100 uCi) to healthy male subjects. Approximately 58% of the radioactive dose was excreted in urine, while faeces contained 39%. Mean Cmax, AUC0 and tmax values for apremilast in plasma were 333 ng/mL, 1970 ng*h/mL and 1.5 h. Apremilast was extensively metabolized via multiple pathways, with unchanged drug representing 45% of the circulating radioactivity and <7% of the excreted radioactivity. The predominant metabolite was O-desmethyl apremilast glucuronide, representing 39% of plasma radioactivity and 34% of excreted radioactivity. The only other radioactive components that represented >4%of the excreted radioactivity were O-demethylated apremilast and its hydrolysis product. Additional minor circulating and excreted compounds were formed via O-demethylation, O-deethylation, N-deacetylation, hydroxylation, glucuronidation and/or hydrolysis. The major metabolites were at least 50-fold less pharmacologically active than apremilast. Metabolic clearance of apremilast was the major route of elimination, while non-enzymatic hydrolysis and excretion of unchanged drug were involved to a lesser extent. PMID:21859393

Repression by sustained-release. beta. -glucuronidase inhibitors of chemical carcinogen-mediated induction of a marker oncofetal protein in rodents

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

Walaszek, Z.; Hanausek-Walaszek, M.; Webb, T.E.

1988-01-01

The degree of induction of an oncofetal protein marker in rodents by selected chemical carcinogens has been correlated with changes in carcinogenicity induced by dietary D-glucaro-1,4-lactone (GL) based anticarcinogens. These potent anticarcinogens may act to increase the clearance of carcinogens as glucuronides through the inhibition of ..beta..-glucuronidase. The sustained-release forms are particularly effective, 1.5 mmol/kg of GL maintaining serum ..beta..-glucuronidase activity at or below 50% for only 1 h, while an equivalent amount of calcium glucarate (CGT) maintained this level of inhibition for over 5 h. CGT or other sustained-release inhibitors, when fed to rodents during administration of carcinogens thatmore » undergo glucuronidation, caused a marked reduction in the induction of the marker protein. For those systems where other markers of carcinogenesis were also assessed, it was determined the inhibition of marker-protein induction was quantitatively similar to both the inhibition of binding of the carcinogen to DNA and the subsequent induction of tumors in target organs. The following carcinogens were administered intraperitoneally: benzo(a)pryene; 7,12-demethylbenz(a)anthracene; 3-methylcholanthrene; 2-acetylaminofluorene; 2-naphthylamine; N-nitroso-N,N-dibutylamine; aflatoxin B1; 1-nitropyrene.« less

Chirality Influence of Zaltoprofen Towards UDP-Glucuronosyltransferases (UGTs) Inhibition Potential.

PubMed

Jia, Lin; Hu, Cuimin; Wang, Haina; Liu, Yongzhe; Liu, Xin; Zhang, Yan-Yan; Li, Wei; Wang, Li-Xuan; Cao, Yun-Feng; Fang, Zhong-Ze

2015-06-01

Zaltoprofen (ZLT) is a nonsteroidal antiinflammation drug, and has been clinically employed to treat rheumatoid arthritis, osteoarthritis, and other chronic inflammatory pain conditions. The present study aims to investigate the chirality influence of zaltoprofen towards the inhibition potential towards UDP-glucuronosyltransferases (UGTs) isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation system was employed to investigate the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT isoforms. The inhibition difference capability was observed for the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT1A8 and UGT2B7, but not for other tested UGT isoforms. (R)-zaltoprofen exhibited noncompetitive inhibition towards UGT1A8 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters were calculated to be 35.3 μM and 19.2 μM for UGT1A8 and UGT2B7. (R)-zaltoprofen and (S)-zaltoprofen exhibited a different inhibition type towards UGT1A7. Based on the reported maximum plasma concentration of (R)-zaltoprofen in vivo, a high drug-drug interaction between (R)-zaltoprofen and the drugs mainly undergoing UGT1A7, UGT1A8, and UGT2B7-catalyzed glucuronidation was indicated. © 2015 Wiley Periodicals, Inc.

Lipid changes in hepatic microsomes and its relationship to P-nitrophenol glucuronidation in an experimental model of portal hypertension.

PubMed

Ghanem, C; Ghisolfi, C; Marabotto, L; Ouviña, G; Rubio, M; Perazzo, J; Lemberg, A; Bengochea, L

1997-10-01

The liver is responsible for the most important metabolic pathway of non polar compounds. The aim of the present work was to study the p-nitrophenol glucuronidation and its relationship with lipidic composition of microsomal membrane in a model of hepatic portal hypertension and hepatocellular damage induced by monocrotaline. A global increment in liver microsomal phospholipids as well as changes in the phospholipid pattern (phosphatidylethanolamine and sphingomyelin increased up to 156 +/- 13 and 195 +/- 14% respectively) were detected in monocrotaline intoxicated rats when it were compared to control rats. The microsomal cholesterol content showed a decrease in monocrotaline intoxicated rats. (4.1 +/- 0.7 against 6.6 +/- 1.5 micrograms/mg of microsomal protein, in control rats). When p-nitrophenol activity was measured, Km from monocrotaline intoxicated rats was 0.137 mM, and Vmax was 2.9 nmol of p-nitrophenol/mg microsomal protein since in control group Km was 0.322 mM, and Vmax was 4.5 nmol of p-nitrophenol/mg microsomal protein. It is concluded that monocrotaline intoxicated rats showed a different behavior in the kinetics of p-nitrophenol UDP-glucuronyltransferase, as well as a different microsomal lipidic profile, when compared to control group.

Biouptake, toxicity and biotransformation of triclosan in diatom Cymbella sp. and the influence of humic acid.

PubMed

Ding, Tengda; Lin, Kunde; Bao, Lianjun; Yang, Mengting; Li, Juying; Yang, Bo; Gan, Jay

2018-03-01

Triclosan is one of the most frequently detected emerging contaminants in aquatic environment. In this study, we investigated the biouptake, toxicity and biotransformation of triclosan in freshwater algae Cymbella sp. The influence of humic acid, as a representative of dissolved organic matter, was also explored. Results from this study showed that triclosan was toxic to Cymbella sp. with 72 h EC 50 of 324.9 μg L -1 . Humic acid significantly reduced the toxicity and accumulation of triclosan in Cymbella sp. SEM analysis showed that Cymbella sp. were enormously damaged under 1 mg L -1 triclosan exposure and repaired after the addition of 20 mg L -1 humic acid. Triclosan can be significantly taken up by Cymbella sp. The toxicity of triclosan is related to bioaccumulated triclosan as the algal cell numbers decreased when intracellular triclosan increased. A total of 11 metabolites were identified in diatom cells and degradation pathways are proposed. Hydroxylation, methylation, dechlorination, amino acids conjunction and glucuronidation contributed to the transformative reactions of triclosan in Cymbella sp., producing biologically active products (e.g., methyl triclosan) and conjugation products (e.g., glucuronide or oxaloacetic acid conjugated triclosan), which may be included in the detoxification mechanism of triclosan. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hplc-nmr identification of the human urinary metabolites of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV).

PubMed

Shockcor, J P; Wurm, R M; Frick, L W; Sanderson, P N; Farrant, R D; Sweatman, B C; Lindon, J C

1996-02-01

1. Human urine samples from a clinical trial of the anti-HIV compound (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-cyto sin e (BW524W91) have been analysed using 19F-nmr and 1H-hplc-nmr spectroscopy. 2. The identities and relative levels of the xenobiotic species in the urine have been determined by 470-MHz 19F-nmr spectroscopy and by directly coupled 600-MHz 1H-hplc-nmr in the stop-flow mode with confirmation of the metabolite identities being made by comparison with nmr spectra of synthetic standard compounds. 3. The principal urinary xenobiotic was the unchanged drug, but the glucuronide ether conjugate at the 5' position of BW524W91, one of the two diastereomeric sulphoxides and the deaminated metabolite were also characterized. 4. The detection limit of directly coupled hplc-600-MHz 1H-nmr spectroscopy was evaluated by measuring two-dimensional nmr spectra of the glucuronide conjugate of BW524W91 and shown to be approximately 1 microgram material for 1H-1H-TOCSY and 20 micrograms metabolite for 1H-13C-HMQC spectra for overnight (16 h) acquisition.

Differential effects of quercetin and its two derivatives (isorhamnetin and isorhamnetin-3- glucuronide) in inhibiting proliferation of human breast cancer MCF-7 cells.

PubMed

Wu, Qiu; Kroon, Paul A; Shao, Hongjun; Needs, Paul W; Yang, Xingbin

2018-06-15

Quercetin (Que) has consistently been reported to be useful cytotoxic compound in vivo and in vitro, but little is known on its metabolites. Here we examined and compared cytotoxic effect of Que and its water-soluble metabolites, isorhamnetin (IS) and isorhamnetin-3-glucuronide (I3G) in human breast cancer MCF-7 cells, and explain their tumor-inhibitory mechanism and structure-function relationship. The results showed that Que, IS and I3G could dose-dependently inhibit the growth of MCF-7 cells, and the cytotoxic effect was ranked as Que > IS > I3G. Furthermore, Que, IS and I3G mediated the cell-cycle arrest principally in S phase, followed by the decrease in the number of G0/G1 and G2/M, and 70.8%, 68.9% and 49.8% MCF-7 tumor cells entered early phase apotosis when treated with 100 µM Que, IS and I3G for 48 h, respectively. Moreover, induction of apoptosis by Que, IS and I3G were accompanied with the marginal generation of intracellular ROS. Given these results, Que, IS and I3G possess strong cytotoxic effect through a ROS-dependent apoptosis pathway in MCF-7 cells.

The effect of gemfibrozil on repaglinide pharmacokinetics persists for at least 12 h after the dose: evidence for mechanism-based inhibition of CYP2C8 in vivo.

PubMed

Tornio, A; Niemi, M; Neuvonen, M; Laitila, J; Kalliokoski, A; Neuvonen, P J; Backman, J T

2008-09-01

Repaglinide is metabolized by cytochrome P450 (CYP) 2C8 and 3A4. Gemfibrozil has the effect of increasing the area under the concentration-time curve (AUC) of repaglinide eightfold. We studied the effect of dosing interval on the extent of the gemfibrozil-repaglinide interaction. In a randomized five-phase crossover study, 10 healthy volunteers ingested 0.25 mg repaglinide, with or without gemfibrozil pretreatment. Plasma repaglinide, gemfibrozil, their metabolites, and blood glucose were measured. When the last dose of 600 mg gemfibrozil was ingested simultaneously with repaglinide, or 3, 6, or 12 h before, it increased the AUC(0-infinity) of repaglinide 7.0-, 6.5-, 6.2- and 5.0-fold, respectively (P < 0.001). The peak repaglinide concentration increased approximately twofold (P < 0.001), and the half-life was prolonged from 1.2 h to 2-3 h (P < 0.001) during all the gemfibrozil phases. The drug interaction effects persisted at least 12 h after gemfibrozil was administered, although plasma gemfibrozil and gemfibrozil 1-O-beta-glucuronide concentrations were only 5 and 10% of their peak values, respectively. The long-lasting interaction is likely caused by mechanism-based inhibition of CYP2C8 by gemfibrozil glucuronide.

Gemfibrozil is a strong inactivator of CYP2C8 in very small multiple doses.

PubMed

Honkalammi, J; Niemi, M; Neuvonen, P J; Backman, J T

2012-05-01

Therapeutic doses of gemfibrozil cause mechanism-based inactivation of CYP2C8 via formation of gemfibrozil 1-O-β-glucuronide. We investigated the extent of CYP2C8 inactivation caused by three different doses of gemfibrozil twice dailyfor 5 days, using repaglinide as a probe drug, in 10 healthy volunteers. At the end of this 5-day regimen, there were dose-dependent increases in the area under the plasma concentration–time curve from 0 to infinity (AUC0–∞) of repaglinide by3.4-, 5.5-, and 7.0-fold corresponding to 30, 100, and 600 mg of gemfibrozil, respectively, as compared with the control phase (P < 0.001). On the basis of a mechanism-based inactivation model involving gemfibrozil 1-O-β-glucuronide, a gemfibrozil dose of 30 mg twice daily was estimated to inhibit CYP2C8 by >70% and 100 mg twice daily was estimated to inhibit it by >90%. Hence, gemfibrozil is a strong inactivator of CYP2C8 even in very small, subtherapeutic, multiple doses. Administration of small gemfibrozil doses may be useful in optimizing the pharmacokinetics of CYP2C8 substrate drugs and in reducing the formation of their potentially toxic metabolites via CYP2C8.

Isolation of phenolic compounds from iceberg lettuce and impact on enzymatic browning.

PubMed

Mai, Franziska; Glomb, Marcus A

2013-03-20

Enzymatic browning is generally reported as the reaction between phenolic substances and enzymes. The quality of iceberg lettuce is directly linked to this discoloration. In particular, the color change of lettuce stems considerably reduces consumer acceptance and thus decreases sales revenue of iceberg lettuce. Ten phenolic compounds (caffeic acid, chlorogenic acid, phaseolic acid, chicoric acid, isochlorogenic acid, luteolin-7-O-glucuronide, quercetin-3-O-glucuronide, quercetin-3-O-galactoside, quercetin-3-O-glucoside, and quercetin-3-O-(6″-malonyl)-glucoside) were isolated from Lactuca sativa var. capitata by multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC). In addition, syringin was identified for the first time in iceberg lettuce. This polyphenolic ingredient was previously not mentioned for the family of Cichorieae in general. The purity and identity of isolated compounds were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS techniques. Furthermore, the relationship between discoloration of iceberg lettuce and enzymatic browning was thoroughly investigated. Unexpectedly, the total concentration of phenolic compounds and the activity of polyphenol oxidase were not directly related to the browning processes. Results of model incubation experiments of plant extract solutions led to the conclusion that in addition to the typical enzymatic browning induced by polyphenol oxidases, further mechanisms must be involved to explain total browning of lettuce.

Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates.

PubMed

Sorich, Michael J; Smith, Paul A; Miners, John O; Mackenzie, Peter I; McKinnon, Ross A

2008-01-01

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

Preliminary buprenorphine sublingual tablet pharmacokinetic data in plasma, oral fluid and sweat during treatment of opioid-dependent pregnant women

PubMed Central

Concheiro, Marta; Jones, Hendreé E.; Johnson, Rolley E.; Choo, Robin; Huestis, Marilyn A.

2011-01-01

Background Buprenorphine is currently under investigation as a pharmacotherapy to treat pregnant women for opioid dependence. This research evaluates buprenorphine (BUP), norbuprenophine (NBUP), buprenorphine-glucuronide (BUP-Gluc) and norbuprenorphine-glucuronide (NBUP-Gluc) pharmacokinetics after high dose (14–20 mg) BUP sublingual tablet administration in three opioid-dependent pregnant women. Methods Oral fluid and sweat specimens were collected in addition to plasma specimens for 24 h during gestation weeks 28 or 29 and 34, and 2 months after delivery. Tmax was not affected by pregnancy; however, BUP and NBUP Cmax and AUC0–24h tended to be lower during pregnancy compared to postpartum levels. Results Statistically significant but weak positive correlations were found for BUP plasma and OF concentrations, and BUP/NBUP ratios in plasma and OF. Conclusion Statistically significant negative correlations were observed for times of specimen collection and BUP and NBUP OF/plasma ratios. BUP-Gluc and NBUP-Gluc were detected in only 5% of OF specimens. In sweat, BUP and NBUP were detected in only 4 of 25 (12 or 24 h) specimens in low concentrations (<2.4 ng/patch). These preliminary data describe BUP and metabolite pharmacokinetics in pregnant women and suggest that, like methadone, upward dose adjustments may be needed with advancing gestation. PMID:21860340

Trichothecenes Mycotoxin Studies

DTIC Science & Technology

1986-02-01

15-monoacetoxyscirpenol with C-UDP-glucuronic acid (data not shown). We were unable to hydrolyze this metabolite by incu- bation with either bovine ...then, this metabolite appears to be non- toxic. This coupled with our findings that this glucuronide is not hydrolyzed by E. coli or bovine liver a...leakage or violation of the skin patches by two of the animals toward the end of these early experiments. Because of their small size and higher level of

Orange juice (poly)phenols are highly bioavailable in humans.

PubMed

Pereira-Caro, Gema; Borges, Gina; van der Hooft, Justin; Clifford, Michael N; Del Rio, Daniele; Lean, Michael E J; Roberts, Susan A; Kellerhals, Michele B; Crozier, Alan

2014-11-01

We assessed the bioavailability of orange juice (poly)phenols by monitoring urinary flavanone metabolites and ring fission catabolites produced by the action of the colonic microbiota. Our objective was to identify and quantify metabolites and catabolites excreted in urine 0-24 h after the acute ingestion of a (poly)phenol-rich orange juice by 12 volunteers. Twelve volunteers [6 men and 6 women; body mass index (in kg/m(2)): 23.9-37.2] consumed a low (poly)phenol diet for 2 d before first drinking 250 mL pulp-enriched orange juice, which contained 584 μmol (poly)phenols of which 537 μmol were flavanones, and after a 2-wk washout, the procedure was repeated, and a placebo drink was consumed. Urine collected for a 24-h period was analyzed qualitatively and quantitatively by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). A total of 14 metabolites were identified and quantified in urine by using HPLC-MS after orange juice intake. Hesperetin-O-glucuronides, naringenin-O-glucuronides, and hesperetin-3'-O-sulfate were the main metabolites. The overall urinary excretion of flavanone metabolites corresponded to 16% of the intake of 584 μmol (poly)phenols. The GC-MS analysis revealed that 8 urinary catabolites were also excreted in significantly higher quantities after orange juice consumption. These catabolites were 3-(3'-methoxy-4'-hydroxyphenyl)propionic acid, 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 3'-methoxy-4'-hydroxyphenylacetic acid, hippuric acid, 3'-hydroxyhippuric acid, and 4'-hydroxyhippuric acid. These aromatic acids originated from the colonic microbiota-mediated breakdown of orange juice (poly)phenols and were excreted in amounts equivalent to 88% of (poly)phenol intake. When combined with the 16% excretion of metabolites, this percentage raised the overall urinary excretion to ∼ 100% of intake. When colon-derived phenolic catabolites are included with flavanone glucuronide and sulfate metabolites, orange juice (poly)phenols are much-more bioavailable than previously envisaged. In vitro and ex vivo studies on mechanisms underlying the potential protective effects of orange juice consumption should use in vivo metabolites and catabolites detected in this investigation at physiologic concentrations. The trial was registered at BioMed Central Ltd (www.controlledtrials.com) as ISRCTN04271658. © 2014 American Society for Nutrition.

Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MSn, and LC-HR-MS/MS.

PubMed

Michely, Julian A; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2017-02-01

Derivatives of N,N-diallyltryptamine (DALT) can be classified as new psychoactive substances. Biotransformation and detectability of 5-fluoro-DALT (5-F-DALT), 7-methyl-DALT (7-Me-DALT), and 5,6-methylenedioxy-DALT (5,6-MD-DALT) are described here. Their metabolites detected in rat urine and pooled human liver microsomes were identified by liquid chromatography (LC)-high resolution (HR)-tandem mass spectrometry (MS/MS). In addition, the human cytochrome-P450 (CYP) isoenzymes involved in the main metabolic steps were identified and detectability tested in urine by the authors' urine screening approaches using GC-MS, LC-MS n , or LC-HR-MS/MS. Aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations could be proposed for all compounds as main pathways. Carboxylation after initial hydroxylation of the methyl group could also be detected for 7-Me-DALT and O-demethylenation was observed for 5,6-MD-DALT. All phase I metabolites were extensively glucuronidated or sulfated. Initial phase I reactions were catalyzed by CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5. Rat urine samples were analyzed following two different low-dose administrations. GC-MS was not able to monitor consumption reliably, but all three compounds are predicted to be detectable in cases of overdose. The LC-MS n and LC-HR-MS/MS approaches were suitable for detecting an intake of all three compounds mainly via their metabolites. However, after the lowest dose, a reliable monitoring could only be achieved for 5-F-DALT via LC-MS n and LC-HR-MS/MS and for 7-Me-DALT via LC-HR-MS/MS. The most abundant targets in both LC-MS screenings were one of two hydroxy-aryl metabolites and both corresponding glucuronides for 5-F-DALT, one N-deallyl hydroxy-aryl, the carboxy, and one dihydroxy-aryl metabolite for 7-Me-DALT, and the demethylenyl metabolite, its oxo metabolite, and glucuronide for 5,6-MD-DALT.

Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs)

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

Fang, Zhong-Ze; Joint Center for Translational Medicine, Dalian Institute of Chemical Physics Chinese Academy of Sciences and The first Affiliated Hospital of Liaoning Medical University, No.457, Zhongshan Road, Dalian 116023; Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892

The wide utilization of ginseng provides the high risk of herb–drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb–drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employedmore » as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb–drug interaction induced by this kind of inhibition, the ginsenoside Rg{sub 3} was selected as an example, and the inhibition kinetic type and parameters (K{sub i}) were determined. Rg{sub 3} competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (K{sub i} values) were calculated to be 22.6, 7.9, 1.9, and 2.0 μM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg{sub 3} (400 ng/ml (0.5 μM)) after intramuscular injection of 60 mg Rg{sub 3}, the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng–drug interaction. - Highlights: ► Structure-dependent inhibition of ginsenoside towards UDP-glucuronosyltransferases. ► Rg{sub 3}′ inhibition towards UGT isoforms can induce in vivo drug–drug interaction. ► Broadening knowledge on ginsenosides' inhibition towards drug-metabolizing enzymes.« less

Hormones and diet: low insulin-like growth factor-I but normal bioavailable androgens in vegan men

PubMed Central

Allen, N E; Appleby, P N; Davey, G K; Key, T J

2000-01-01

Mean serum insulin-like growth factor-I was 9% lower in 233 vegan men than in 226 meat-eaters and 237 vegetarians (P = 0.002). Vegans had higher testosterone levels than vegetarians and meat-eaters, but this was offset by higher sex hormone binding globulin, and there were no differences between diet groups in free testosterone, androstanediol glucuronide or luteinizing hormone. © 2000 Cancer Research Campaign PMID:10883675

Testosterone and androstanediol glucuronide among men in NHANES III.

PubMed

Duan, Chuan Wei; Xu, Lin

2018-03-09

Most of the androgen replacement therapies were based on serum testosterone and without measurements of total androgen activities. Whether those with low testosterone also have low levels of androgen activity is largely unknown. We hence examined the association between testosterone and androstanediol glucuronide (AG), a reliable measure of androgen activity, in a nationally representative sample of US men. Cross-sectional analysis was based on 1493 men from the Third National Health and Nutrition examination Survey (NHANES III) conducted from 1988 to 1991. Serum testosterone and AG were measured by immunoassay. Kernel density was used to estimate the average density of serum AG concentrations by quartiles of testosterone. Testosterone was weakly and positively correlated with AG (correlation coefficient = 0.18). The kernel density estimates show that the distributions are quite similar between the quartiles of testosterone. After adjustment for age, the distributions of AG in quartiles of testosterone did not change. The correlation between testosterone and AG was stronger in men with younger age, lower body mass index, non-smoking and good self-rated health and health status. Serum testosterone is weakly correlated with total androgen activities, and the correlation is even weaker for those with poor self-rated health. Our results suggest that measurement of total androgen activity in addition to testosterone is necessary in clinical practice, especially before administration of androgen replacement therapy.

Isolation and identification of metabolites of osthole in rats.

PubMed

Lv, X; Wang, C-Y; Hou, J; Zhang, B-J; Deng, S; Tian, Y; Huang, S-S; Zhang, H-L; Shu, X-H; Zhen, Y-H; Liu, K-X; Yao, J-H; Ma, X-C

2012-11-01

Osthole (Ost), one of the major components of Cnidium monnieri (L.) Cusson, is had the structure of an isopentenoxy-coumarin with a range of pharmacological activities. In the present study, the metabolism of Ost in male Sprague-Dawley rats was investigated by identifying Ost metabolites excreted in rat urine. Following an oral dose of 40 mg/kg Ost, 10 phase I and 3 phase II metabolites were isolated from the urine of rats, and their structures identified on the basis of a range of spectroscopic data, including 2D-NMR techniques. These metabolites were fully characterized as 5'-hydroxyl-osthole (M-1), osthenol (M-2), 4'-hydroxyl-osthole (M-3), 3, 5'-dihydroxyl-osthole (M-4), 5'-hydroxyl-osthenol (M-5), 4'-hydroxyl-2', 3'-dihydro-osthenol (M-6), 4'-hydroxyl-osthenol (M-7), 3, 4'-dihydroxyl-osthole (M-8), 2', 3'-dihydroxyl-osthole (M-9), 5'-hydroxyl-2', 3'-dihydroosthole (M-10), osthenol-7-O-β-D-glucuronide (M-11), osthole-4'-O-β-D-glucuronide (M-12) and osthole-5'-O-β-D-glycuronate (M-13). This is the first identification of M-1, M-3 to M-13 in vivo. On the basis of the metabolites profile, a possible metabolic pathway for Ost metabolism in rats has been proposed. This is the first systematic study on the phases I and II metabolites of 8-isopentenoxy-coumarin derivative.

A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat

PubMed Central

Panneer Selvam, Anjan; Muthukumar, Sriram; Kamakoti, Vikramshankar; Prasad, Shalini

2016-01-01

We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001–100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours. PMID:26996103

StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight.

PubMed

Yogendra, Kalenahalli N; Dhokane, Dhananjay; Kushalappa, Ajjamada C; Sarmiento, Felipe; Rodriguez, Ernesto; Mosquera, Teresa

2017-03-01

The resistance to late blight is either qualitative or quantitative in nature. Quantitative resistance is durable, but challenging due to polygenic inheritance. In the present study, the diploid potato genotypes resistant and susceptible to late blight, were profiled for metabolites. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to pathogen infection revealed increased accumulation of morphinone, codeine-6-glucuronide and morphine-3-glucuronides. These BIAs are antimicrobial compounds and possibly involved in cell wall reinforcement, especially through cross-linking cell wall pectins. Quantitative reverse transcription-PCR studies revealed higher expressions of TyDC, NCS, COR-2 and StWRKY8 transcription factor genes, in resistant genotypes than in susceptible genotype, following pathogen inoculation. A luciferase transient expression assay confirmed the binding of the StWRKY8 TF to promoters of downstream genes, elucidating a direct regulatory role on BIAs biosynthetic genes. Sequence analysis of StWRKY8 in potato genotypes revealed polymorphism in the WRKY DNA binding domain in the susceptible genotype, which is important for the regulatory function of this gene. A complementation assay of StWRKY8 in Arabidopsis wrky33 mutant background was associated with decreased fungal biomass. In conclusion, StWRKY8 regulates the biosynthesis of BIAs that are both antimicrobial and reinforce cell walls to contain the pathogen to initial infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous qualitative and quantitative analysis of flavonoids and alkaloids from the leaves of Nelumbo nucifera Gaertn. using high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

PubMed

Guo, Yujie; Chen, Xi; Qi, Jin; Yu, Boyang

2016-07-01

A reliable method, combining qualitative analysis by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and quantitative assessment by high-performance liquid chromatography with photodiode array detection, has been developed to simultaneously analyze flavonoids and alkaloids in lotus leaf extracts. In the qualitative analysis, a total of 30 compounds, including 12 flavonoids, 16 alkaloids, and two proanthocyanidins, were identified. The fragmentation behaviors of four types of flavone glycoside and three types of alkaloid are summarized. The mass spectra of four representative components, quercetin 3-O-glucuronide, norcoclaurine, nuciferine, and neferine, are shown to illustrate their fragmentation pathways. Five pairs of isomers were detected and three of them were distinguished by comparing the elution order with reference substances and the mass spectrometry data with reported data. In the quantitative analysis, 30 lotus leaf samples from different regions were analyzed to investigate the proportion of eight representative compounds. Quercetin 3-O-glucuronide was found to be the predominant constituent of lotus leaf extracts. For further discrimination among the samples, hierarchical cluster analysis, and principal component analysis, based on the areas of the eight quantitative peaks, were carried out. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolism of para-aminophenol by rat hepatocytes.

PubMed

Yan, Z; Nikelly, J G; Killmer, L; Tarloff, J B

2000-08-01

Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

METABOLISM AND DISPOSITION OF ACETAMINOPHEN: RECENT ADVANCES IN RELATION TO HEPATOTOXICITY AND DIAGNOSIS

PubMed Central

McGill, Mitchell R.; Jaeschke, Hartmut

2013-01-01

Acetaminophen (APAP) is one of the most widely used drugs. Though safe at therapeutic doses, overdose causes mitochondrial dysfunction and centrilobular necrosis in the liver. The first studies of APAP metabolism and activation were published more than forty years ago. Most of the drug is eliminated by glucuronidation and sulfation. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively. However, some is converted by CYP2E1 and other cytochrome P450 enzymes to a reactive intermediate that can bind to sulfhydryl groups. The metabolite can deplete liver glutathione (GSH) and modify cellular proteins. GSH binding occurs spontaneously, but may also involve GSH-S-transferases. Protein binding leads to oxidative stress and mitochondrial damage. The glucuronide, sulfate, and GSH conjugates are excreted by transporters in the canalicular (Mrp2 and Bcrp) and basolateral (Mrp3 and Mrp4) hepatocyte membranes. Conditions that interfere with metabolism and metabolic activation can alter the hepatotoxicity of the drug. Recent data providing novel insights into these processes, particularly in humans, are reviewed in the context of earlier work, and the effects of altered metabolism and reactive metabolite formation are discussed. Recent advances in the diagnostic use of serum adducts are covered. PMID:23462933

Metabolic interactions between acetaminophen (paracetamol) and two flavonoids, luteolin and quercetin, through in-vitro inhibition studies.

PubMed

Cao, Lei; Kwara, Awewura; Greenblatt, David J

2017-12-01

Excessive exposure to acetaminophen (APAP, paracetamol) can cause liver injury through formation of a reactive metabolite that depletes hepatic glutathione and causes hepatocellular oxidative stress and damage. Generation of this metabolite is mediated by Cytochrome-P450 (CYP) isoforms, mainly CYP2E1. A number of naturally occurring flavonoids can mitigate APAP-induced hepatotoxicity in experimental animal models. Our objective was to determine the mechanism of these protective effects and to evaluate possible human applicability. Two flavonoids, luteolin and quercetin, were evaluated as potential inhibitors of eight human CYP isoforms, of six UDP-glucuronosyltransferase (UGT) isoforms and of APAP glucuronidation and sulfation. The experimental model was based on in-vitro metabolism by human liver microsomes, using isoform-specific substrates. Luteolin and quercetin inhibited human CYP isoforms to varying degrees, with greatest potency towards CYP1A2 and CYP2C8. However, 50% inhibitory concentrations (IC 50 values) were generally in the micromolar range. UGT isoforms were minimally inhibited. Both luteolin and quercetin inhibited APAP sulfation but not glucuronidation. Inhibition of human CYP activity by luteolin and quercetin occurred with IC 50 values exceeding customary in-vivo human exposure with tolerable supplemental doses of these compounds. The findings indicate that luteolin and quercetin are not likely to be of clinical value for preventing or treating APAP-induced hepatotoxicity. © 2017 Royal Pharmaceutical Society.

Short term impact of Tribulus terrestris intake on doping control analysis of endogenous steroids.

PubMed

Saudan, Christophe; Baume, Norbert; Emery, Caroline; Strahm, Emmanuel; Saugy, Martial

2008-06-10

Tribulus terrestris is a nutritional supplement highly debated regarding its physiological and actual effects on the organism. The main claimed effect is an increase of testosterone anabolic and androgenic action through the activation of endogenous testosterone production. Even if this biological pathway is not entirely proven, T. terrestris is regularly used by athletes. Recently, the analysis of two female urine samples by GC/C/IRMS (gas chromatography/combustion/isotope-ratio-mass-spectrometry) conclusively revealed the administration of exogenous testosterone or its precursors, even if the testosterone glucuronide/epitestosterone glucuronide (T/E) ratio and steroid marker concentrations were below the cut-off values defined by World Anti-Doping Agency (WADA). To argue against this adverse analytical finding, the athletes recognized having used T. terrestris in their diet. In order to test this hypothesis, two female volunteers ingested 500 mg of T. terrestris, three times a day and for two consecutive days. All spot urines were collected during 48 h after the first intake. The (13)C/(12)C ratio of ketosteroids was determined by GC/C/IRMS, the T/E ratio and DHEA concentrations were measured by GC/MS and LH concentrations by radioimmunoassay. None of these parameters revealed a significant variation or increased above the WADA cut-off limits. Hence, the short-term treatment with T. terrestris showed no impact on the endogenous testosterone metabolism of the two subjects.

Metabolism and disposition of ABT-894, a novel α4β2 neuronal acetylcholine receptor agonist, in mice and monkeys.

PubMed

Liu, Hong; Fu, Wentao; Wetter, Jill; Xu, Hongyu; Guan, Zhiwen; Stuart, Patricia

2014-06-01

1.  Metabolism and disposition of ABT-894 was investigated in hepatocytes, in mice and monkeys receiving [(14)C]ABT-894. 2.  In hepatocytes, turnover rate of ABT-894 was slow in all species with more than 90% of parent remaining. M3 (carbamoyl glucuronide) and M6 (mono-oxidation) were detected across species. 3.  ABT-894 showed species-specific disposition profiles. ABT-894 was primarily eliminated by renal secretion in mice. Whereas, monkey mainly cleared ABT-894 metabolically. 4.  ABT-894 underwent two primary routes of metabolism in monkeys: N-carbamoyl glucuronidation to form M3 and oxidation product M1. M3 was the major metabolite in monkey excreta. M3 was observed in mice urine. Circulating levels of M3 in terms of M3/ABT-894 ratios were essentially absent in mice, but were high in monkeys. 5.  Understanding the species difference in the clearance mechanism is the key to the accurate projection of the human clearance and preclinical safety assessment. Lack of species difference in the metabolism of ABT-894 in hepatocytes certainly creates a challenge in predicting its metabolism and pharmacokinetics in human. Based on available metabolism and pharmacokinetic data of ABT-894 in human, monkey is the preferred species in predicting human clearance since it presents a similar clearance mechanism from that observed in human.

Structure-activity relationships of flavonoids as natural inhibitors against E. coli β-glucuronidase.

PubMed

Weng, Zi-Miao; Wang, Ping; Ge, Guang-Bo; Dai, Zi-Ru; Wu, Da-Chang; Zou, Li-Wei; Dou, Tong-Yi; Zhang, Tong-Yan; Yang, Ling; Hou, Jie

2017-11-01

Bacterial β-glucuronidases play key roles in the deconjugation of a variety of endogenous and drug glucuronides, thus have been recognized as important targets to modulate the enterohepatic circulation of various glucuronides. In this study, more than 30 natural flavonoids were collected and their inhibitory effects against E. coli β-glucuronidase (EcGUS) were assayed. The results demonstrated that some flavonoids including scutellarein, luteolin, baicalein, quercetin and scutellarin displayed strong to moderate inhibitory effects against EcGUS, with the IC 50 values ranging from 5.76 μM to 29.64 μM, while isoflavones and dihydroflavones displayed weak inhibitory effects against EcGUS. Further investigation on inhibition kinetics revealed that scutellarein and luteolin functioned as potent competitive inhibitors against EcGUS-mediated PNPG hydrolysis, with the K i values less than 3.0 μM. Molecular docking simulations demonstrated that scutellarein and luteolin could be well-docked into the catalytic site of EcGUS, while the binding areas of these two natural inhibitors on EcGUS were highly overlapped with that of PNPG on EcGUS. Additionally, the structure-inhibition relationships of natural flavonoids against EcGUS are also summarized, which will be very helpful for the medicinal chemists to design and develop more potent flavonoid-type inhibitors against EcGUS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of proflavine metabolites in rainbow trout.

PubMed

Yu, Z; Hayton, W L; Chan, K K

1997-04-01

Proflavine (3,6-diaminoacridine) has potential for use as an antiinfective in fish, and its metabolism by rainbow trout was therefore studied. Fourteen hours after intraarterial bolus administration of 10 mg/kg of proflavine, three metabolites were found in liver and bile, and one metabolite was found in plasma using reversed-phase HPLC with UV detection at 262 nm. Treatment with hydrochloric acid converted the three metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with beta-glucuronidase and saccharic acid 1,4-lactone, a specific beta-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination of UV-VIS absorption and mass spectra, HPLC-purified metabolites were isolated from liver. Data from these experiments suggested that the proflavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuronosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. When synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix-assisted, laser desorption ionization, time-of-flight MS. In addition, they coeluted on HPLC under different mobile phase conditions. Finally, the in vitro incubation with liver subcellular preparations confirmed this characterization and provided the evidence that APG can be formed by glucuronidation of AP or acetylation of PG.

A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat

NASA Astrophysics Data System (ADS)

Panneer Selvam, Anjan; Muthukumar, Sriram; Kamakoti, Vikramshankar; Prasad, Shalini

2016-03-01

We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001-100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours.

Urinary pregnandiol-3-glucuronide and estrone conjugates to creatinine ratios in early pregnancies complicated by vaginal bleeding.

PubMed

Davidson, B J

1986-10-01

There is no simple and rapid test available to predict the outcome of an early pregnancy complicated by vaginal bleeding. In this prospective study, 15 women with normal pregnancies collected a weekly urine sample between 6 and 13 weeks' gestation. A single random urine sample was obtained from 15 women with bleeding who continued to carry their child and 50 women who proceeded to have a spontaneous abortion (SAB). Pregnandiol-3-glucuronide (PDG) was determined with the use of enzyme-multiplied immunoassay technique (EMIT) and estrone conjugates (E1C) were measured by radioimmunoassay (RIA). The ratios of these metabolites to creatinine (C) were calculated. PDG/C ratios in normal women rose gradually from 6 weeks on. All women with bleeding during a normal pregnancy had ratios in the normal range, but 94% of women with a SAB had ratios below the normal range. The E1C/C ratio remained unchanged from 6 to 11 weeks and then rose rapidly. Until 11 weeks, there was no clear separation between the E1C/C ratios of the women with a SAB and the women with bleeding who continued their pregnancies. The prognosis of threatened abortion can be made by a urinary PDG/C ratio but not by an E1C/C ratio. EMIT is simple and quick and uses technology present in many laboratories.