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PubMed2001

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin.

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.

Animals↗
PubMed2005

Isoflavones modulate the glucuronidation of estradiol in human liver microsomes.

Soy food has been associated with a reduced incidence of hormonal cancer in Asian countries, and the soy isoflavones daidzein and genistein are believed to protect against tumors induced by the endogenous hormone 17beta-estradiol (E2). In the present study, we have examined if daidzein and genistein as well as several structurally related isoflavones are able to modulate the in vitro glucuronidation of E2 in human hepatic microsomes. It is known that different isoforms of UDP-glucuronosyltransferase (UGT) are involved in E2 glucuronidation: UGT1A1 leads exclusively to the 3-glucuronide and is stimulated by E2 via homotropic kinetics, whereas UGT2B7 gives rise to the 17-glucuronide of E2 following Michaelis-Menten kinetics. In our study, daidzein markedly stimulated the 3-glucuronidation, thereby enhancing the metabolic clearance of E2. In contrast, genistein inhibited the 3-glucuronidation. The 17-glucuronidation of E2 was not affected by either compound. Formononetin and the daidzein metabolites equol, 3'-hydroxy-daidzein, 6-hydroxy-daidzein and glycitein behaved similar to daidzein, whereas biochanin A resembled genistein. The effect of daidzein on the 3-glucuronidation of E2 in human hepatic microsomes was also obtained with human recombinant UGT1A1. Since the only other compound known to stimulate E2 glucuronidation via allosteric kinetics is 17alpha-ethynylestradiol, our study is the first report of the heterotropic stimulation of a UGT by a non-steroidal and naturally occurring compound. An enhanced rate of glucuronidation of E2 by daidzein and its metabolites may contribute to the putative protection of soy against hormonal cancer.

Allosteric Regulation↗
PubMed2004

Characterization of afloqualone N-glucuronidation: species differences and identification of human UDP-glucuronosyltransferase isoform(s).

Afloqualone (AFQ) is one of the centrally acting muscle relaxants. AFQ N-glucuronide is the most abundant metabolite in human urine when administered orally, whereas it was not detected in the urine when administered to rats, dogs, and monkeys. Species differences in AFQ N-glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. The kinetics of AFQ N-glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K(m) and V(max) values accounted for 2019 +/- 85.9 muM and 871.2 +/- 17.9 pmol/min/mg protein, respectively. The V(max) and intrinsic clearance (CL(int)) values of AFQ N-glucuronidation in human liver were approximately 4- to 10-fold and 2- to 4-fold higher than those in rat, dog, and monkey, respectively. Among 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms, both UGT1A4 and UGT1A3 exhibited high AFQ N-glucuronosyltransferase activities. The K(m) value of AFQ N-glucuronidation in recombinant UGT1A4 microsomes was very close to that in human liver microsomes. The formation of AFQ N-glucuronidation by human liver, jejunum, and recombinant UGT1A4 microsomes was effectively inhibited by trifluoperazine, a known specific substrate for UGT1A4. The AFQ N-glucuronidation activities in seven human liver microsomes were significantly correlated with trifluoperazine N-glucuronidation activities (r(2) = 0.798, p < 0.01). In contrast, the K(m) value of AFQ N-glucuronidation in recombinant UGT1A3 microsomes was relatively close to that in human jejunum microsomes. These results demonstrate that AFQ N-glucuronidation in human is mainly catalyzed by UGT1A4 in the liver and by UGT1A3, as well as UGT1A4 in the intestine.

Adolescent↗
PubMed2006

Structural characterization of anti-HIV drug candidate PA-457 [3-O-(3',3'-dimethylsuccinyl)-betulinic acid] and its acyl glucuronides in rat bile and evaluation of in vitro stability in human and animal liver microsomes and plasma.

PA-457 [3-O-(3',3'-dimethylsuccinyl)-betulinic acid] represents a new class of anti-HIV drug candidates termed maturation inhibitors. After oral administration to rats, PA-457 was metabolized to several glucuronide conjugates and mainly eliminated into rat bile. Liquid chromatography-electrospray ionization-mass spectrometry analysis showed that the glucuronidation products of PA-457 were acyl glucuronides including one di-glucuronide, di-PA-457G, and two mono-glucuronides, referred to as mono-PA-457G (I) and mono-PA-457G (II), respectively. In-source fragmentation of MS spectra supported the conclusion that mono-PA-457G (I) was glucuronidated at the C-28 carboxyl of PA-457, whereas mono-PA-457G (II) was conjugated at the dimethylsuccinic acid side chain of the C-3 position. Quantification demonstrated that the predominant glucuronide of PA-457 in rat bile was mono-PA-457G (I) with lower amounts of mono-PA-457G (II) and di-PA-457G. In vitro stability indicated that the mono-acyl glucuronides of PA-457 were not degraded after incubation with 0.1 M phosphate buffer (pH 4, 7.4 and 9), plasma (human, rat, and mouse), and UDP-glucuronosyltransferase reaction media (without uridine 5'-diphosphoglucuronic acid) with microsomes (human, rat, and mouse liver microsomes), respectively, whereas the minor diglucuronide was unstable in rodent liver microsomes. All glucuronides of PA-457 could be hydrolyzed both by beta-glucuronidase and alkaline (1 M NaOH). Minor putative acyl migration products were slowly formed at pH 9, suggesting that the acyl glucuronides of PA-457 have relatively high in vitro stability.

Animals↗
PubMed2006

Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.

Catalysis↗
PubMed2002

In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance.

The glucuronidation of a number of commonly used hepatic uridine diphosphate glucuronosyltransferase drug substrates has been studied in human tissue microsomes. Prediction of in vivo hepatic drug glucuronidation from liver microsomal data yielded a consistent 10-fold under-prediction. Consideration of protein binding was observed to be pivotal when predicting in vivo glucuronidation for acid substrates. Studies using human intestinal microsomes demonstrated the majority of drugs to be extensively glucuronidated such that the intrinsic clearance (CL(int)) of ethinylestradiol (CL(int) = 1.3 microl/min/mg) was twice that obtained using human liver microsomes (CL(int) = 0.7 microl/min/mg). The potential extrahepatic in vivo glucuronidation was calculated for a range of drug substrates from human microsomal data. These results indicate the contribution of intestinal drug glucuronidation to systemic drug clearance to be much less than either hepatic or renal glucuronidation. Therefore, data obtained with intestinal microsomes may be misleading in the assessment of the contribution of this organ to systemic glucuronidation. The use of hepatocytes to assess metabolic stability for drugs predominantly metabolized by glucuronidation was also investigated. Metabolic clearances for a range of drugs obtained using fresh preparations of human hepatocytes predicted accurately hepatic clearance reported in vivo. The use of cryopreserved hepatocytes as an in vitro tool to predict in vivo metabolism was also assessed with an excellent correlation obtained for a number of extensively glucuronidated drugs (R(2) = 0.80, p < 0.001).

Albumins↗
PubMed1999

Conjugation of the enantiomers of ketotifen to four isomeric quaternary ammonium glucuronides in humans in vivo and in liver microsomes.

The antiallergic drug ketotifen is chiral due to a nonplanar seven-membered ring containing a keto group. Earlier studies have revealed glucuronidation at the tertiary amino group as a major metabolic pathway in humans. Chemical synthesis of glucuronides from racemic ketotifen now led to four isomers separable by HPLC of which two each could be ascribed to (R)-(+)- and (S)-(-)-ketotifen by synthesis from the enantiomers. According to (1)H NMR analysis of the (S)-ketotifen N-glucuronides, the conformation of the piperidylidene ring differs between the two isomers. Enzymatic hydrolysis with Escherichia coli beta-glucuronidase proceeded at a lower rate with the slower eluting (S)-ketotifen glucuronide than with the three other isomers. On incubation of the ketotifen enantiomers (0.5-200 microM) with human liver microsomes in the presence of UDP-glucuronic acid and Triton X-100, the N-glucuronides of (R)-ketotifen were produced with an apparent K(M) 15 microM and V(max) 470 pmol/min/mg protein. The two (S)-ketotifen glucuronides were formed by two-enzyme kinetics with K(M1) 1.3 microM and K(M2) 92 microM and V(max) values of 60 and 440 pmol/min/mg protein. After ingestion of 1 mg of racemic ketotifen, 10 healthy subjects excreted in urine 17 +/- 5% of the dose in the form of N-glucuronides. The (R)-ketotifen glucuronide isomers contributed one-sixth only, whereas the remainder consisted primarily of the (S)-ketotifen glucuronide isomer, which eluted last. Differential hydrolysis or membrane transport may be responsible for the discrepancy between N-glucuronide isomer ratios in vitro and in vivo.

Anti-Allergic Agents↗
PubMed2000

In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin.

The UDP-glucuronosyltransferases (UGTs) are a superfamily of membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to attain maximal enzyme activity. The goals of the current work were to develop a universal method to glucuronidate xenobiotic substrates using microsomes, and to apply this method to sequential oxidation-glucuronidation reactions. Three assays of UGT catalytic activity estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and morphine-3-glucuronidation, which are relatively selective probes for human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment of microsomes with the pore-forming peptide alamethicin (50 microg/mg protein) resulted in conjugation rates 2 to 3 times the rates observed with untreated microsomes. Addition of physiological concentrations of Mg(2+) to the alamethicin-treated microsomes yielded rates that were 4 to 7 times the rates with untreated microsomes. Optimized assay conditions were found not to detrimentally affect cytochrome P450 activity as determined by effects on testosterone 6beta-hydroxylation and 7-ethoxycoumarin deethylation. Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of K(m)(app) = 0.017 mM, V(max)(app) = 0.4 nmol/mg/min, and n = 1.8. Formation of acetaminophen-O-glucuronide also best fit the Hill equation, with K(m)(app) = 4 mM, V(max)(app) = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics, with K(m)(app) = 2 mM and V(max)(app) = 2. 5 nmol/mg/min. Finally, alamethicin treatment of microsomes was found to be effective in facilitating the sequential oxidation-glucuronidation of 7-ethoxycoumarin.

Acetaminophen↗
PubMed2005

Stereoselectivity and interaction between the glucuronidation of S-(-)- and R-(+)-propranolol in rat hepatic microsomes pretreated with different inducers.

Phase II glucuronidation metabolism of side-chain propranolol was studied using microsomes from rats treated with the inducers beta-naphthoflavone (BNF) or dexamethasone (Dex). The glucuronide concentrations of propranolol enantiomers were assayed by RP-HPLC. The kinetic constants of glucuronidation, Km, Vmax and Clint were determined. There are significant differences between the R- and S-enantiomeric glucuronide in Km, Vmax and Clint P < 0.05, P < 0.01 and P < 0.05 in control microsome. There are significant differences in Km and Clint (P < 0.01 or P < 0.001) but no significant differences in Vmax (P > 0.05) between R and S-enantiomeric glucuronide in the microsomes induced with Dex and BNF. The formation of S-(-)-propranolol glucuronide was inhibited by R-(+)-propranolol from the rat microsomes pretreated with BNF and Dex. The glucuronidation metabolism of propranolol enantiomers exhibited the stereoselectivity in rat hepatic microsomes induced with BNF or Dex. Multiple UGT1A and 2B may be involved in stereoselective O-glucuronidation of propranolol enantiomers in rat liver microsomes. The glucuronides produced were in favor of the R-enantiomer. There is an interaction between the glucuronidation of R- and S-enantiomer.

Adrenergic beta-Antagonists↗
PubMed1995

In vitro glucuronidation of peroxisomal proliferators: 2-ethylhexanoic acid enantiomers and their structural analogs.

In order to investigate the glucuronidation of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, by liver microsomes of several mammalian species including man, a gas chromatography method for the quantification of the corresponding glucuronides was developed. The variation coefficients for intra- and interassay repeatability were less than 3 and 7%, respectively. The rat liver UDP-glucuronosyl-transferase (UGT) presented similar Km and Vmax toward the two enantiomers. The glucuronidation of the racemate 2-EHA or its enantiomers was strongly increased up to six times by treatment of the rats with phenobarbital and, to a lesser extent, by 3-methylcholanthrene. In contrast, the treatment of the rats clofibrate did not modify the activity. The induction was not stereoselective. The Gunn rats, which present a genetic defect in the bilirubin UGT isoforms, were able to glucuronidate the drug as well as the congenic strain. Moreover, the UGT-2B1 isoform, stably expressed in V79 cells, glucuronidated 2-EHA in an appreciable amount. Interspecies comparison indicated that the most active glucuronidation of 2-EHA occurred in the dog and the rat. The lowest activities were observed in the man and the rabbit. In all species considered, except rabbit and guinea pig which glucuronidated the R isomer faster, the R and S enantiomers were glucuronidated to a similar extent. The glucuronidation activity toward compounds chemically related to 2-EHA increased as a function of molecular weight, but was not affected by the position of the methyl or the ethyl moiety on the hydrocarbon chain. A correlation between the glucuronidation rate of 2-EHA and analogs and the activity of PCoA oxidase was observed.

Animals↗
PubMed1984

Glucuronidation in rat intestinal epithelial cells. Dependence of glucose supply and resistance to inhibition by ethanol and fasting.

Glucuronidation of 1-naphthol was studied in mucosal cells isolated from the rat intestine. Glucuronidation was directly dependent on the supply of extracellular carbohydrates. Basal glucuronidation (ca. 0.3 nmoles/min X mg cell protein) was increased 2- to 3-fold by adding glucose or fructose to the incubation medium. Saturation of glucuronidation was achieved by adding 0.3 mM glucose, while saturation by fructose was not reached at concentrations below 2 mM. No carbohydrate reserves able to support glucuronidation appear to be present in intestinal cells, since no difference in glucuronidation was observed between cells prepared from fasted (18 or 42 h) and control rats. Glucuronidation was decreased by adding D-galactosamine to the incubation medium, but only when extracellular glucose was present. Various chemicals which are known to inhibit glucuronidation in hepatocytes (ethanol, diethyl ether, sorbitol) did not influence the glucuronidation of 1-naphthol in isolated intestinal cells. Only when ethanol was added to mucosal cells in the absence of extracellular glucose was a small decrease in glucuronidation observed.

Animals↗
PubMed2000

Mycophenolic acid glucuronidation and its inhibition by non-steroidal anti-inflammatory drugs in human liver and kidney.

OBJECTIVE: The aims of this investigation were to study the glucuronidation of mycophenolic acid (MPA) in human liver and kidney and to search for a compound that inhibits MPA glucuronidation among the non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: A sensitive and reproducible radiometric assay was developed to measure the rate of MPA glucuronidation in human liver and kidney microsomes. The assay employed uridine 5'-diphosphate-[U-14C]-glucuronic acid (UDPGA) and MPA-glucuronide was isolated by TLC. The final concentrations of UDPGA and MPA necessary were 1 mM (liver), and MPA concentration was 0.5 mM (kidney). The inhibition of MPA glucuronidation was studied with 18 NSAIDs and tacrolimus. RESULTS: Glucuronosyl transferase activity followed Michaelis-Menten kinetics and the Km (mean +/- SD; mM) was 0.31+/-0.06 (liver; n = 5) and 0.28+/-0.07 (kidney; n = 5; P = 0.555); the Vmax (mean SD; nmol/mg per minute) was 5.2+/-1.4 (liver; n = 5) and 10.5+/-1.2 (kidney; n = 5; P = 0.0005). The MPA glucuronidation rates (mean +/- SD; nmol/min/mg) were 3.3+/-0.9 (liver; n = 10) and 7.8+/-1.5 (kidney; n = 10; P = 0.0002). The rate of MPA glucuronidation ranged between 2.0 and 5.1 nmol/ mg per minute with a 2.5-fold variation (liver) and between 5.7 and 9.8 nmol/mg per minute with a 1.7-fold variation (kidney). The inhibition study was performed in liver and revealed that the percentage of control ranged from 8%+/-3% (niflumic acid) to 119%+/-16% (Ketoralac). The inhibition curves for MPA glucuronidation rate were determined with the four most effective inhibitors: niflumic acid, flufenamic acid, mefenamic acid and diflunisal. Their IC50 estimates (microM) were 8+/-1, 19+/-9, 63+/-8 and 109+/-15, respectively (liver), and 8+/-2, 13+/-2, 49+/-4 and 122+/-18, respectively (kidney). The IC50 estimate for niflumic acid was eightfold lower than the peak plasma levels after a single oral dose of 250 mg of this drug. CONCLUSION: The human liver and kidney are important sites of MPA glucuronidation. MPA glucuronidation was inhibited to various extents by different NSAIDs and the four most effective inhibitors were niflumic acid, flufenamic acid, mefenamic acid and diflunisal. These drugs have similar molecular structures consisting of two aromatic rings bearing a carboxylic group.

Aged↗
PubMed1989

The enantioselective glucuronidation of morphine in rats and humans. Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme.

The formation of morphine glucuronides is enantio- and regioselective in rats and humans. In rat liver microsomes, natural (-)-morphine formed only the 3-O-glucuronide, whereas the unnatural (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions, with the 6-O-glucuronide being the principal product. In human liver microsomes, both the 3-OH-and 6-OH positions were glucuronidated with each of the enantiomers, with the 3-O-glucuronide being the major product with (-)-morphine, and the 6-OH position preferred with the (+)-enantiomer. By using a series of biochemical and biological situations such as induction by xenobiotics, ontogeny, selective inhibition and genetic deficiencies, which are considered to be diagnostic of UDP-glucuronosyltransferase heterogeneity, we determined that two UDP-glucuronosyltransferase isoenzymes were responsible for the glucuronidation of morphine in rat liver. One isoenzyme (the so-called "morphine UDP-glucuronosyltransferase") was responsible for the glucoronidation at the (-)-3-OH and (+)-6-OH positions of morphine, whereas the other formed only the (+)-morphine-3-glucuronide. Evidence from enzyme induction and the genetically deficient deficient Gunn rat suggested that bilirubin UDPGT may be responsible for the (+)-morphine-3-UDP-glucuronosyltransferase activity. In human kidney, glucuronidation of both (-)- and (+)-enantiomers at the 6-OH position was deficient, whereas the activity at the 3-OH positions was still present, which indicated the involvement of two UDP-glucuronosyltransferases in the glucuronidation of morphine in man, as well as rats.

Age Factors↗
PubMed1996

Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites. pH stability and synthesis by human and dog liver.

Glucuronide conjugates of arylamines are thought to be important in the carcinogenic process. This study investigated the pH stability and synthesis of glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites by human and dog liver. Both dog and human liver slices incubated with 0.06 mM [3H]-4-aminobiphenyl produced the N-glucuronide of 4-aminobiphenyl as the major product. After 2 hr of incubation, the N-glucuronide of 4-aminobiphenyl represented 52 and 27% of the total radioactivity recovered by HPLC in dog and human, respectively. When 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, or N-hydroxy-N-acetyl-4-aminobiphenyl was added to human microsomes containing [14C]UDP-glucuronic acid, a new product peak was detected by HPLC. At 0.5 mM, the rate of glucuronidation was N-hydroxy-N-acetyl-4-aminobiphenyl > N-hydroxy-4-aminobiphenyl > 4-aminobiphenyl. The rate of formation of the N-glucuronide of 4-aminobiphenyl was similar to that observed with benzidine and N-acetylbenzidine. The glucuronides of 4-aminobiphenyl and N-hydroxy-4-aminobiphenyl were both acid labile with T1/2 values of 10.5 and 32 min, respectively, at pH 5.5. The glucuronide of N-hydroxy-N-acetyl-4-aminobiphenyl was not acid labile with T1/2 values at pH 5.5 and 7.4 of 55 and 68 min, respectively. The glucuronide of 4-aminobiphenyl was the most acid labile conjugate examined. Thus, the glucuronide of 4-aminobiphenyl is a major product of dog and human liver slice metabolism and likely to play an important role in the carcinogenic process.

Aminobiphenyl Compounds↗
PubMed1995

Stereoselective pharmacokinetics of oxprenolol and its glucuronides in humans.

OBJECTIVE: To study the pharmacokinetics of R(+)- and S(-)-oxprenolol and their corresponding glucuronide conjugates in healthy subjects. METHODS: An oral dose of 80 mg racemic oxprenolol was given to eight male volunteers. Venous blood samples and urine were collected as a function of time. Oxprenolol enantiomers in plasma and urine were determined by an enantiospecific HPLC method. Oxyprenolol glucuronides in plasma and urine were measured as oxprenolol equivalents after enzymatic hydrolysis. RESULTS: For R-oxprenolol the area under the plasma concentration-time curve was slightly higher (R/S ratio, 1.19) and the oral clearance slightly lower (R/S ratio, 0.84) than those parameters for S-oxprenolol. The free fraction of R-oxprenolol in plasma was 4% higher than that of S-oxprenolol. The intrinsic clearance of S-oxprenolol was 1.5 times larger than that of R-oxprenolol, and a maximum of 3% of the dose was excreted as unchanged enantiomers in the urine. The plasma concentrations of S-oxprenolol glucuronide were more than three times higher than those of R-oxprenolol glucuronide. Twenty-five percent of the dose of the R-enantiomer was excreted in the urine as R-oxprenolol glucuronide; 29% of the S-enantiomer dose was excreted as S-oxprenolol glucuronide. The renal clearance of R-oxprenolol glucuronide was, on average, 172 ml/min, suggesting active tubular secretion. In contrast, the renal clearance of S-oxprenolol glucuronide was only 49 ml/min, which can be explained by the plasma binding of the compound. CONCLUSIONS: Our results show small differences in disposition between R- and S-oxprenolol but a marked difference in disposition between the glucuronides. The difference in plasma concentrations between the oxprenolol glucuronides is mainly attributable to the stereoselectivity of the renal excretion.

Adult↗
PubMed1990

Multiplicity of in vitro glucuronidation of 2-hydroxyestriol.

In vitro glucuronidation of 2-hydroxyestriol has been investigated by means of HPLC with dual-electrode coulometric detection. When incubated with rat or dog liver microsomal preparation in the presence of UDPGA, 2-hydroxyestriol was transformed into the 2-glucuronide together with a small amount of 16- and/or 17-glucuronides. In contrast, incubation of 2-hydroxyestriol with guinea-pig liver microsomal preparation yielded the 3-glucuronide and a trace amount of the 2-glucuronide, but no ring D glucuronides. Upon pretreatment with 3-methylcholanthrene male rat liver exhibited a marked increase in both 2- and 3-glucuronidation activities, whereas female rat liver showed an elevation only in 2-glucuronidation. On the other hand, in male and female rats pretreatment with phenobarbital caused a relatively small increase in the glucuronidation activity of the liver. In the male guinea-pig, glucuronidation was not affected by pretreatment with either of the two compounds. The present result demonstrates the multiplicity of hepatic 2-hydroxyestriol UDP-glucuronyl-transferase in the rat, guinea-pig and dog.

Animals↗
PubMed1995

Determination of the glucurono-conjugated position in bile alcohol glucuronides present in a patient with cerebrotendinous xanthomatosis.

The determination of the glucurono-conjugated position in three bile alcohol glucuronides secreted in bile of a patient with cerebrotendinous xanthomatosis was carried out by a nuclear magnetic resonance study. The bile sample was extracted with ethanol and chromatographed on an ion-exchange column, a reverse-phase partition column and a silica gel column to isolate glucurono-conjugates of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol, 5 beta-cholestane-3 alpha, 7 alpha 12 alpha, 23-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol. Proton and 13C nuclear magnetic resonance spectra of the two biliary bile alcohol glucuronides, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol glucuronide and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol glucuronide were identical with those of the synthetic glucuronide 7 alpha, 12 alpha, 25-trihydroxy-5 beta-cholestane-3 alpha-O beta-D-glucopyranosyluronic acid and the isolated glucuronide 3 alpha, 7 alpha, 12 alpha, 25-tetrahydroxy-5 beta-cholestane-23-O-beta-D-glucopyranosyluronic acid from urine of a patient with cerebrotendinous xanthomatosis, respectively. Hence, the glucurono-conjugated positions of the biliary 25-tetrol glucuronide and the biliary 23,25-pentol glucuronide were C-3 and C-23, respectively. By comparison of the 13C chemical shift data with that of the unconjugated bile alcohol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23-tetrol, the glucurono-conjugated position of the natural 23-tetrol glucuronide was determined to be C-23. Thus, the natural 23-tetrol glucuronide can be formulated as 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane-23-O-beta-D-glucopyranosyluronic acid.

Bile↗
PubMed1991

Cytotoxic effect and uptake mechanism by isolated rat hepatocytes of lithocholate and its glucuronide and sulfate.

The hepatotoxicity and uptake mechanism of lithocholate and its glucuronide and sulfate were studied using isolated rat hepatocytes. Cytotoxicity was in the order of lithocholate greater than lithocholate-glucuronide greater than lithocholate-sulfate; their 50% cytotoxic concentrations on hepatocytes were 50, 150 and 700 microM, respectively. Thus, glucuronidation as well as sulfation acted to detoxify lithocholate, not relating to the previously reported higher cholestatic effect of lithocholate-glucuronide than lithocholate. Lithocholate uptake was linear up to 50 microM, whereas the uptakes of lithocholate-glucuronide and sulfate were saturable with an apparent Km and Vmax of 32 microM and 6.4 nmol/min per 10(6) cells for lithocholate-glucuronide and 26 microM and 11.8 nmol/min per 10(6) cells for lithocholate-sulfate. Na+ replacement by choline+ had no effect on the uptake of lithocholate and lithocholate-glucuronide, whereas it slightly inhibited lithocholate-sulfate uptake. Lithocholate-glucuronide uptake was inhibited by lithocholate-sulfate and sulfobromophthalein, whereas lithocholate-glucuronide and sulfobromophthalein had no effect on lithocholate-sulfate uptake. These data indicate that hepatic lithocholate uptake is mediated by simple diffusion, and that hepatic uptake of lithocholate-glucuronide and sulfate is mainly mediated by a Na(+)-independent carrier.

Animals↗