Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by (19)F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid-liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C(18)). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5'-deoxy-5-fluorocytidine (5'-DFCR). Treatment with beta-glucuronidase from three sources showed that it was a glucuroconjugate of 5'-DFCR. HPLC-TIS-MS-MS and (1)H NMR allowed identification of the unknown metabolite as 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.
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PMID:Isolation of an unknown metabolite of capecitabine, an oral 5-fluorouracil prodrug, and its identification by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry as a glucuroconjugate of 5'-deoxy-5-fluorocytidine, namely 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine. 1286 40

After intravenous administration of (-)-epicatechin gallate to Wistar male rats, its biliary metabolites were examined. Deconjugated forms of (-)-epicatechin gallate metabolites were prepared by beta-glucuronidase/sulfatase treatment and purified by HPLC. Five compounds were subjected to FAB-MS and NMR analyses. These metabolites were shown to be (-)-epicatechin gallate, 3'-O-methyl-(-)-epicatechin gallate, 4'-O-methyl-(-)-epicatechin gallate, 4' '-O-methyl-(-)-epicatechin gallate, and 3',4' '-di-O-methyl-(-)-epicatechin gallate. After oral administration, five major metabolites excreted in rat urine were purified in their deconjugated forms and their chemical structures identified. They were degradation products from (-)-epicatechin gallate, pyrogallol, 5-(3,4-dihydroxyphenyl)-gamma-valerolactone, 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid, 3-(3-hydroxyphenyl)propionic acid, and m-coumaric acid. Time course analysis of the identified (-)-epicatechin gallate metabolites showed that (-)-epicatechin gallate and its conjugate appeared in the plasma with their highest levels 0.5 h after oral administration; their levels rapidly decreased, and then they disappeared by 6 h. The degradation products, mainly in their conjugated forms, emerged at 6 h, peaked at 24 h, and disappeared by 48 h. In urine samples, (-)-epicatechin gallate and its methylated metabolites were hardly detected and the degradation products began to be excreted in the 6-24 h period, peaked in the 24-48 h period, and then began to disappear. The most abundant metabolite in both the plasma and the urine was found to be the conjugated form of pyrogallol. On the basis of these results, a possible metabolic route of (-)-epicatechin gallate orally administered to the rat is proposed.
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PMID:Identification of metabolites of (-)-epicatechin gallate and their metabolic fate in the rat. 1292 15

Glucuronides of piperazine hydroxylamines are rarely reported in the literature, and even more rarely are their structures unambiguously identified. One major metabolite was detected by liquid chromatography/mass spectrometry-radioactivity in urine from monkeys treated with the aryl piperazine oral hypoglycemic agent 9-[(1S,2R)-2-fluoro-1-methylpropyl]-2-methoxy-6-(1-piperazinyl) purine hydrochloride (1). The mass spectrum of this metabolite indicated that it was both monooxygenated and glucuronidated on the piperazine ring. Possible structures included the N- or O-glucuronic acid conjugates of a carbinolamine, hydroxylamine, or N-oxide. Treatment with beta-glucuronidase gave a monooxygenated derivative of the parent compound. 1H NMR analysis of either the glucuronic acid conjugate or the monooxygenated product provided insufficient evidence to unambiguously determine their structures. Incubation of 1 with pig liver microsomes resulted in formation of the same monooxygenated derivative derived from beta-glucuronidase treatment of the glucuronide metabolite. This in vitro system was used to generate sufficient material for analysis by 13C NMR, and the metabolite was identified as a hydroxylamine derivative 2. Incubation of the hydroxylamine with monkey liver microsomes and uridine diphospho-5'-glucuronic acid gave the same glucuronic acid conjugate as that observed in monkey urine. 13C NMR analysis of this biosynthetic product led to its unequivocal structure assignment as the O-glucuronic acid conjugate of the hydroxylamine 3.
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PMID:Identification of a hydroxylamine glucuronide metabolite of an oral hypoglycemic agent. 1474 39

1: The use of fluorine-19 nuclear magnetic resonance (19F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored. 2: Ten patients were anaesthetized by sevoflurane for 135-660 min at a flow rate of 6 l min(-1). Urine samples were collected before, directly after and 24 h after discontinuation of anaesthesia. 3: 19F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the 19F-NMR spectrum. HFIP concentrations after beta-glucuronidase treatment were quantified by (19)F-nuclear magnetic resonance. Concentrations directly after and 24 h after discontinuation of anaesthesia were 131 +/- 41 (mean +/- SEM) and 61 +/- 19 mol mg(-1) creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure. 4: Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from beta-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24 h. 5: Since 19F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 +/- 0.7 and 1.4 +/- 0.4 mol mg(-1) creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not. 6. The results show that 19F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. 19F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.
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PMID:Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients. 1520 1

The absorption, metabolism, and excretion of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide monomethanesulfonate (1), a GABAA receptor partial agonist potentially useful in treating generalized anxiety disorder, have been evaluated in both Sprague-Dawley rats and cynomolgus monkeys using [14C]1. In both species, mass balance was achieved within 48 h postdose, with the majority of drug-related material excreted within the feces; the clearance of 1 in each species had both metabolic and renal components. In addition to the metabolites produced by aliphatic hydroxylation and/or N-dealkylation of 1, two unique metabolites were detected: a putative carbamic acid (M7) in rat plasma and monkey bile, and an N-carbamoyl glucuronide (M8) in both rat and monkey bile. Metabolite M8 was structurally deciphered by liquid chromatographytandem mass spectrometry and NMR, and was readily generated in vitro upon incubation of [14C]1 with rat liver microsomes fortified with uridine 5'-diphosphoglucuronic acid trisodium salt and alamethicin under a CO2 atmosphere. Treatment of M8 with beta-glucuronidase afforded 1 directly. The presence of M8 in bile and its notable absence from other matrices suggests the enterohepatic cycling of 1 via M8. Although the structure of M7 was not elucidated unequivocally due to its inability to be formed in vitro and its minimal absolute quantities in limited biological matrices, data herein clearly support its structural rationalization. Furthermore, since M7 is the precursor of M8, detection of M8 is indirect evidence of its existence. It is proposed that M7 arises from an equilibrium between 1 and dissolved CO2-equivalents both in vivo and in vitro, similar to carbamino bonds observed in hemoglobin and certain amino acids, respectively.
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PMID:Biotransformation of a GABAA receptor partial agonist in sprague-dawley rats and cynomolgus monkeys: identification of two unique N-carbamoyl metabolites. 1608 72

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.
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PMID:Two exo-beta-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases. 1631 14

The potential cancer therapeutic agent, 6,7-(dimethoxy-2, 4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine (JNJ-10198409), formed three N-glucuronides that were positively identified by liquid chromatography-tandem mass spectrometry and NMR as N-amine-glucuronide (Glu-A), 1-N-pyrazole-glucuronide (Glu-B), and 2-N-pyrazole-glucuronide (Glu-C). All three N-glucuronides were detected in rat liver microsomes, whereas only Glu-A and -B were found in monkey and human liver microsomes. In contrast to common glucuronides, Glu-B was completely resistant to beta-glucuronidase. Kinetic analyses revealed that glucuronidation of JNJ-10198409 in human liver microsomes exhibited atypical kinetics that may be described by a two-site binding model. For the high affinity binding, K(m) values were 1.2 and 5.0 microM, and V(max) values were 2002 and 2,403 nmol min(-1) mg(-1) for Glu-A and Glu-B, respectively. Kinetic constants of low affinity binding were not determined due to low solubility of the drug. Among the human UDP-glucuronosyltransferases (UGTs) tested, UGT1A9, 1A8, 1A7, and 1A4 were the most active isozymes to produce Glu-A; for the formation of Glu-B, UGT1A9 was the most active enzyme, followed by UGT1A3, 1A7, and 1A4. Glucuronidation of JNJ-10198409 by those UGT1A enzymes followed classic Michaelis-Menten kinetics. In contrast, no glucuronides were formed by all UGT2B isozymes tested, including UGT2B4, 2B7, 2B15, and 2B17. Collectively, these results suggested that glucuronidation of JNJ-10198409 in human liver microsomes is catalyzed by multiple UGT1A enzymes. Since UGT1A enzymes are widely expressed in various tissues, it is anticipated that both hepatic and extrahepatic glucuronidation will likely contribute to the elimination of the drug in humans. Additionally, conjugation at the nitrogens of the pyrazole ring represents a new structural moiety for UGT1A-mediated reactions.
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PMID:N-glucuronidation of the platelet-derived growth factor receptor tyrosine kinase inhibitor 6,7-(dimethoxy-2,4-dihydroindeno[1,2-C]pyrazol-3-yl)-(3-fluoro-phenyl)-amine by human UDP-glucuronosyltransferases. 1645 2

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.
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PMID:3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio. 1678 68

Sesamin, the major sesame oil lignan, is recognized for its health-promoting effects, including the lowering of cholesterol and elevation of gamma-tocopherol in rats and humans. However, little is known about the absorption and metabolism of sesamin in humans. In this study, 6 healthy volunteers took a single dose of sesame oil (508 micromol sesamin) and their urine was collected for four 12-h periods. The urine samples were treated with beta-glucuronidase/sulphatase and extracted with chloroform. The major urinary sesamin metabolite in the chloroform extract was collected using HPLC diode array detector and characterized as (1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane using NMR and mass spectroscopy. A quantitative (1)H-NMR technique, based on the methylenedioxyphenyl protons signal (delta 5.91), was used for the quantification of the metabolite in the chloroform extracts of urine. The excretion of the sesamin catechol metabolite ranged from 22.2 to 38.6% (mean +/- SD, 29.3 +/- 5.6) of the ingested dose and happened mainly in the 1st 12 h after ingestion.
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PMID:Quantitative NMR analysis of a sesamin catechol metabolite in human urine. 1737 58

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30min, 1 and 2h) after injection. Samples were treated with beta-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30min and 1h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.
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PMID:Bioavailability of apocynin through its conversion to glycoconjugate but not to diapocynin. 1797 2


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