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PubMed1983

Periportal localization of lorazepam glucuronidation in the isolated perfused rat liver.

The relative effects of pretreatment with allyl alcohol and carbon tetrachloride on oxidative and glucuronide metabolism of lorazepam have been compared in the isolated perfused rat liver. Livers from rats pretreated for 24 hr with allyl alcohol (1.8 ml/kg, 1:50 solution, to induce pericentral hepatic necrosis), carbon tetrachloride (0.8 mg/kg in corn oil, to induce perivenular hepatic necrosis), or vehicle were perfused with 20% rat blood, 80% Krebs bicarbonate buffer at 20 ml/min. After 300 micrograms of lorazepam had been added to the reservoir, perfusate concentrations of lorazepam were measured in the perfusate at timed intervals. After 180 min, lorazepam and lorazepam glucuronide were measured in perfusate, bile, and liver homogenate. Allyl alcohol and carbon tetrachloride lowered lorazepam clearance by 47% and 77%, respectively. Recovery of lorazepam glucuronide after 180 min was lowered by 35% by treatment with allyl alcohol and increased 73% by treatment with carbon tetrachloride. Glucuronide recovery permitted estimation of fractional glucuronide vs. nonglucuronide clearance. In control rats, glucuronide clearance accounted for 25% of total clearance. Allyl alcohol caused a 64% reduction in glucuronide clearance but only a 39% reduction in nonglucuronide clearance. In contrast, carbon tetrachloride caused a 60% reduction in glucuronide clearance but an 83% reduction in nonglucuronide clearance. The differences in ratios of the changes in glucuronide and nonglucuronide clearance provide further circumstantial evidence that is consistent with the hypothesis of predominant periportal localization of glucuronidation and pericentral localization of oxidative metabolism of lorazepam.

Allyl Compounds↗
PubMed1995

Characterization of the in vitro glucuronidation of flurbiprofen enantiomers.

To investigate the glucuronidation of the R- and S-enantiomers of the nonsteroidal antiinflammatory drug, flurbiprofen, by liver microsomes of several mammals, including humans, a new and reliable HPLC method for the separation and quantification of the corresponding diastereoisomeric glucuronides has been developed. Interspecies comparison revealed that the glucuronidation of flurbiprofen was highly efficient with liver microsomes of humans, monkeys, rats, and guinea pigs (in decreasing ranking order). Gunn rats, which present a genetic defect in the bilirubin UDP-glucuronosyltransferase (UGT) isoforms, were still able to glucuronidate the drug. The R-enantiomer was glucuronidated faster than the S-form by liver microsomes of rats and humans. Although the KM of glucuronidation of R- and S-enantiomers by rat liver UGT were in same order of magnitude (apparent KM 0.52 and 0.57 mM, respectively), the apparent Vmax's were significantly different (9.34 and 5.48 nmol/min.mg of protein). Regardless of the enantiomer considered, the glucuronidation of flurbiprofen was strongly increased up to 5-fold by treatment of rats with phenobarbital and, at a lower extent, by 3-methylcholanthrene. In contrast, the treatment of rats with ciprofibrate markedly decreased the activity. Glucuronidation of R-flurbiprofen was more enhanced by phenobarbital than that of the S-antipode. Each flurbiprofen enantiomer could weakly inhibit the glucuronidation of its antipode in a noncompetitive way. The apparent Ki was 0.51 mM with R-flurbiprofen as a substrate, and 0.37 mM with S-enantiomer. On the other hand, the rat liver UGT2B1 isoform, stably expressed in V79 cells, could glucuronidate flurbiprofen in an appreciable amount.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Formation of a beta-glucuronidase-resistant glucuronide conjugate of digitoxin by dog liver microsomes.

The aim of these studies was to characterize the glucuronide conjugates of digitoxin and digitoxigenin monodigitoxoside (DMD) produced by liver microsomes from the dog with respect to hydrolysis by beta-glucuronidase and to behavior on HPLC. These results have been compared with studies of conjugates produced by liver microsomes from the rat. Glucuronidation was similar with both substrates with dog microsomes, whereas rat microsomes formed the glucuronide with DMD but not with digitoxin. The DMD glucuronide from both species was completely hydrolyzed by beta-glucuronidase, but no hydrolysis of digitoxin glucuronide was detected. The digitoxin glucuronide was hydrolyzed by a buffer at pH 1.5 but not at pH 10. After acid hydrolysis, the major products appear to be digitoxigenin and DMD glucuronide. These results suggest that glucuronidation of certain drugs by the dog is quite different from that of other species and that the dog may be the only species that possesses a glucuronosyltransferase capable of forming a glucuronide conjugate with digitoxin. The dog also has a glucuronosyltransferase, similar to that in the rat, which is responsible for glucuronidation of DMD. Whether this represents a single glucuronosyltransferase or two different enzymes remains to be elucidated.

Animals↗
PubMed1997

Comparison of stably expressed rat UGT1.1 and UGT2B1 in the glucuronidation of opioid compounds.

Opioids are important drugs used as analgesics, antitussives, antidiarrheals, and in the therapy of myocardial infarctions, and as antagonists of opioid intoxication. The glucuronidation of these compounds, catalyzed by UDP-glucuronosyltransferases (UGTs), is well known to be a primary step in their metabolism to hydrophilic products and in their ultimate excretion. The present study was designed to compare the reactivity and relative glucuronidation efficiencies of opioid agonists, antagonists, and partial agonists with two rat UGT isoforms; UGT1.1, which is generally considered the "bilirubin UGT," and UGT2B1, which has previously been shown to catalyze the glucuronidation of testosterone, chloramphenicol, and (-)-morphine. Rat UGT2B1, stably expressed in HK293 cells, exhibited high glucuronidation rates and catalytic efficiencies for many opioids, although values for (-)-morphine and nalorphine were the highest. In contrast, these compounds were very poor substrates for expressed rat UGT1.1. Comparably high glucuronidation rates and efficiencies were found for buprenorphine and diprenorphine with both UGT isoforms. These results suggest that opioids with morphinan-based chemical structures similar to (-)-morphine interact with UGTs differently than those with oripavine-based chemical structures similar to buprenorphine. To investigate the contribution of rat UGT1.1 and UGT2B1 in the overall rate of glucuronidation of buprenorphine in the rat liver, hepatic microsomes from Gunn rats (where UGT1.1 activity is absent) and Wistar rats (where UGT1.1 activity is present) were studied. Buprenorphine glucuronidation activity in Gunn rat liver microsomes exhibit approximately 25% of rates observed in Wistar rat liver microsomes, whereas (-)-morphine, naloxone, and naltrexone glucuronidation rates were not significantly different in microsomal preparations from Gunn and Wistar rats. These data suggest that UGT2B1 is the major hepatic enzyme involved in the glucuronidation of (-)-morphine and naloxone in livers from untreated rats, whereas buprenorphine glucuronidation is preferentially catalyzed by rat UGT1.1.

Analgesics, Opioid↗
PubMed2003

Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids.

Glucuronidation of carboxylic acids, primarily catalyzed by hepatic UDP-glucuronosyltransferases, is an important phase II metabolic pathway functioning in detoxification. Acyl glucuronides of 2-aryl propionates, however, can form covalently bound protein adducts, which may generate hypersensitive reactions. We previously identified and quantified R- and S-flurbiprofen acyl glucuronides in human urine following the oral administration of flurbiprofen by liquid chromatography/electrospray ionization mass spectrometry. Recent studies also demonstrated the inhibitory effect of bile acids and their metabolites toward rat hepatic bile acid acyl glucuronidation, which may also be the target of the flurbiprofen isoenzyme. We therefore performed a kinetic analysis of rat hepatic flurbiprofen UDP-glucuronosyltransferase using bisubstrate kinetic analysis and inhibition studies. The results indicated that both bile acid and its metabolites clearly inhibited flurbiprofen acyl glucuronidation. The inhibitory effect on flurbiprofen was more efficient than the effect seen on bile acid acyl glucuronidation. Unconjugated, glycine- and taurine-conjugated chenodeoxycholic acids inhibited glucuronidation using a noncompetitive mechanism, whereas the inhibition by chenodeoxycholic acid 24-acyl glucuronide occurred according to a mixed type mechanism. The inhibition by bile acids and their metabolites may be responsible for the suppression of the toxicity of carboxy-linked glucuronides.

Animals↗
PubMed2006

Identification of human UDP-glucuronosyltransferase responsible for the glucuronidation of niflumic acid in human liver.

PURPOSE: To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of niflumic acid in human liver. METHODS: The glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). RESULTS: Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a Km value of 16 microM, which is similar to that found with recombinant UGT1A1. The glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3beta-glucuronidation activity (r=0.78, p<0.05). Beta-estradiol inhibited niflumic acid glucuronidation with an IC50 of 25 microM in HLMs, comparable to that for UGT1A1. CONCLUSIONS: These findings indicate that UGT1A1 is the main isozyme involved in the glucuronidation of niflumic acid in the human liver.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2006

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the C-glucuronidation of phenylbutazone.

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics and endogeneous compounds. There have been many studies on the formation of O-, N- or S-glucuronides and identification of the UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of these glucuronides. However, there is no information available on which UGT isoform(s) catalyzes C-glucuronidation. In the present study, 16 human UGTs (UGTs 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) were cloned and expressed in baculovirus-infected insect cells and investigated to determine their C-glucuronidating activity toward phenylbutazone (PB). Among the UGT isoforms investigated, only UGT1A9 catalyzed PB C-glucuronidation. Human liver and kidney microsomes, which are well known to express UGT1A9, had C-glucuronidating activity toward PB. However, the jejunum, which did not express UGT1A9, had no C-glucuronidating activity. These results demonstrate for the first time that PB C-glucuronidation is catalyzed by only UGT1A9.

Cells, Cultured↗
PubMed2004

Glucuronidation of aliphatic alcohols in human liver microsomes in vitro.

The glucuronidation of several short-chained aliphatic alcohols in vitro, with the use of human liver microsomes (HLM) as catalyst, was performed, and the kinetics were studied. The concentrations of the glucuronides were determined by gas chromatography-mass spectrometry after derivatization to the trimethylsilyl compounds. Alcohols from ethanol to pentanols were found to couple with activated glucuronic acid in HLM to a widely varying amount. For analytic reasons the glucuronide of methanol, which is formed after methanol consumption in human beings in vivo, could not be determined. The length of the alkyl chain played the decisive role in the maximum turnover rate of the glucuronidation. The affinity of the alcohols to UDP-glucuronosyltransferase (UDPGT), which catalyzes the glucuronidation reaction, was shown to depend strongly on their structure. Alcohols with a very short alkyl chain and secondary alcohols were glucuronidated much more slowly in comparison with findings for the longer chain primary alcohols and showed less affinity to UDPGT. The alcohols mutually inhibited glucuronidation. However, ethanol inhibited the glucuronidation of isopentanol or n-pentanol only in high concentrations, whereas the two pentanols inhibited each other to a high degree. The glucuronidation of aliphatic alcohols is probably catalyzed by only one of several very similar enzymes of the UDPGT. This finding was indicated by the fact that the Michaelis-Menten constants of the alcohols--with the use of different lots of the HLM from different liver donors--had nearly the same values.

Alcohols↗
PubMed2006

Quaternary ammonium-linked glucuronidation of trans-4-hydroxytamoxifen, an active metabolite of tamoxifen, by human liver microsomes and UDP-glucuronosyltransferase 1A4.

Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. Trans-4-hydroxy-TAM (trans-4-HO-TAM), one of the TAM metabolites in humans, has been considered to be an active metabolite of TAM because of its higher affinity toward estrogen receptors (ERs) than the parent drug and other side-chain metabolites. In the present study, we found a new potential metabolic pathway of trans-4-HO-TAM and its geometrical isomer, cis-4-HO-TAM, via N-linked glucuronic acid conjugation for excretion in humans. N+-Glucuronides of 4-HO-TAM isomers were isolated along with O-glucuronides from a reaction mixture consisting of trans- or cis-4-HO-TAM and human liver microsomes fortified with UDP-glucuronic acid and identified with their respective synthetic specimens by high performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Although N- and O-glucuronidating activities of human liver microsomes toward trans-4-HO-TAM were nearly comparable, O-glucuronidation was predominant for cis-4-HO-TAM conjugation. Only UGT1A4 catalyzed the N-linked glucuronidation of 4-HO-TAM among recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. In contrast, all UGT isoforms, except for UGT1A3 and UGT1A4, catalyzed O-glucuronidation of 4-HO-TAM. Although O-glucuronidation of 4-HO-TAM greatly decreased binding affinity for human ERs, 4-HO-TAM N+-glucuronide still had binding affinity similar to 4-HO-TAM itself, suggesting that N+-glucuronide might contribute to the biological activity of TAM in vivo.

Animals↗
PubMed2001

Flavonoid glucuronides are substrates for human liver beta-glucuronidase.

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.

Chromatography, High Pressure Liquid↗
PubMed2000

Glucuronidation of apomorphine.

Apomorphine, a dopaminergic receptor agonist, is largely used in the therapy of Parkinson's disease. In this study, we characterized the glucuronidation of apomorphine and other catechols in rat liver and brain microsomes, using UDP-[U-14C]glucuronic acid and separation of the glucuronides formed by a thin layer chromatographic method. rat liver microsomes glucuronidate apomorphine at a significant rate, that was increased in the presence of dithiothreitol. Two apomorphine glucuronides were separated by high pressure liquid chromatography. We showed by electrospray mass spectrometry that both products were monoglucuronides. Other catechols were also glucuronidated in liver microsomes at various rates, and among them, 4-nitrocatechol was the most efficiently conjugated. in rat brain microsomes, only 4-nitrocatechol was significantly glucuronidated, suggesting that in the liver, several uridine-diphosphate glucuronosyltransferase (UGT) isoforms participate to the conjugation of catechols. To determine which isoforms catalyze apomorphine glucuronidation, two recombinant enzymes expressed in V79 cells were used. The isoform UGT1A6 was unable to glucuronidate apomorphine, but we observed a significant activity catalyzed by the isoform UGT2B1. These results provide, to our knowledge, the first demonstration of apomorphine conjugation by recombinant UGT2B1, and the first evidence of the lack of apomorphine glucuronidation in the rat brain.

Animals↗
PubMed2001

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Chromatography, High Pressure Liquid↗
PubMed2001

HPLC/1H NMR spectroscopic studies of the reactive alpha-1-O-acyl isomer formed during acyl migration of S-naproxen beta-1-O-acyl glucuronide.

A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

Acylation↗
PubMed2006

Kinetics of acetaminophen glucuronidation by UDP-glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential implications in acetaminophen-induced hepatotoxicity.

The importance of uridine 5'-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway.

Acetaminophen↗
PubMed2003

Glucuronidation of 1'-hydroxyestragole (1'-HE) by human UDP-glucuronosyltransferases UGT2B7 and UGT1A9.

Estragole (4-allyl-1-methoxybenzene) is a naturally occurring food flavoring agent found in basil, fennel, bay leaves, and other spices. Estragole and its metabolite, 1'-hydroxyestragole (1'-HE), are hepatocarcinogens in rodent models. Recent studies from our laboratory have shown that glucuronidation of 1'-HE is a major detoxification pathway for estragole and 1'-HE, accounting for as much as 30% of urinary metabolites of estragole in rodents. Therefore, this study was designed to investigate the glucuronidation of 1'-HE in human liver microsomes in vitro and identify the specific uridine diphosphate glucuronosyltransferase (UGT) isoforms responsible for 1'-HE glucuronidation. The formation of the glucuronide of 1'-HE (1'-HEG) followed atypical kinetics, and the data best fit to a Hill equation, resulting in apparent kinetic parameters of Km = 1.45 mM, Vmax = 164.5 pmoles/min/mg protein, and n = 1.4. There was a significant intersubject variation in 1'-HE glucuronidation in 27 human liver samples, with a CV of 42%. A screen of cDNA expressed UGT isoforms indicated that UGT2B7 (83.94 +/- 0.188 pmols/min/mg), UGT1A9 (51.36 +/- 0.72 pmoles/min/mg), and UGT2B15 (8.18 +/- 0.037 pmoles/min/mg) were responsible for 1'-HEG formation. Glucuronidation of 1'-HE was not detected in cells expressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, and UGT1A10. 1'-HE glucuronidation in 27 individual human liver samples significantly (p < 0.05) correlated with the glucuronidation of other UGT2B7 substrates (morphine and ibuprofen). These results imply that concomitant chronic intake of therapeutic drugs and dietary components that are UGT2B7 and/or UGT1A9 substrates may interfere with estragole metabolism. Our results also have toxicogenetic significance, as UGT2B7 is polymorphic and could potentially result in genetic differences in glucuronidation of 1'-HE and, hence, toxicity of estragole.

Analgesics, Opioid↗
PubMed2004

Bisphenol a glucuronidation and excretion in liver of pregnant and nonpregnant female rats.

In male rats challenged with the environmental estrogen bisphenol A, the compound is highly glucuronidated in the liver and is excreted largely into the bile. Given that in pregnancy the microsomal glucuronidation toward bisphenol A is attenuated, we hypothesized that elimination of bisphenol A from the liver may be reduced in pregnancy. This study was conducted to trace the elimination of bisphenol A in female rats, especially in pregnancy. In Sprague-Dawley rats, 1.5 mumol of bisphenol A was perfused into the liver via the portal vein. In both the male and the nonpregnant female, the infused bisphenol A was glucuronidated, then the resultant glucuronide was excreted mainly into the bile. In pregnant rats, however, bilious excretion of bisphenol A glucuronide was 60% of that observed in nonpregnant rats, and venous excretion increased reciprocally. During 1-h perfusion, total excretion of the glucuronide from the liver of male, nonpregnant female, and pregnant rats was 889.5 +/- 69.6, 1256.7 +/- 54.8, and 1038.8 +/- 33.3 nmoles, respectively. In Eisai hyperbilirubinemic rats (EHBR), perfusion of the liver with bisphenol A enabled us to determine that multidrug resistance-associated protein (MRP)2-mediating transport is the mechanism behind excretion of the glucuronide into the bile. The expression of MRP2 has been reported to be noticeably reduced in pregnancy. These results suggest that bisphenol A elimination by hepatic glucuronidation is slightly less in pregnancy than in non-pregnancy and that in pregnancy, more bisphenol A glucuronide is eliminated to the vein because of reduced MRP2 expression.

Animals↗
PubMed2005

Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions.

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Alamethicin↗
PubMed2002

Involvement of multiple UDP-glucuronosyltransferase 1A isoforms in glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin in human liver microsomes.

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide. Phenytoin is oxidized to 4'-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4'-HPPH is metabolized to 4'-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4'-HPPH O-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4'-HPPH, and UDP-glucuronic acid was identified as 4'-HPPH O-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4'-HPPH O-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 microM of 4'-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4'-HPPH O-glucuronide. The 4'-HPPH O-glucuronidation in pooled human liver microsomes was inhibited by beta-estradiol as a typical substrate for UGT1A1 (IC(50) = 21.1 microM) and imipramine as a typical substrate for UGT1A4 (IC(50) = 57.7 microM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC(50) = 167.1 microM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC(50) = 287.6 microM) were not prominent. The interindividual difference in the 4'-HPPH O-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4'-HPPH O-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4'-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.

Biotransformation↗