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)

The influences of paraffin and GMA-embedding on acid phosphatase, esterase and beta-glucuronidase activity of differently fixed or freeze substituted rat livers were studied. 1. Embedding generally causes a reduction of the enzyme activities but improves considerably the quality of the microscopical pictures when compared with appropriate cryostat sections. Embedding therefore may serve as a very useful tool for detail studies on the cytological level. 2. The embedding media act differently on the reactive sites: a. Paraffin causes a heavy denaturation of the enzyme activity in lysosomes but preserves the activities of the ergastoplasmic (= "microsomal") enzymes. The degree of denaturation increases with increasing embedding temperature. b. GMA-embedding delivered opposite effects by preserving lysosomal activities and quenching endoplasmic enzymes. UV-polymerization of GMA causes a general inactivation of enzymes. 3. The histochemical reactivity of substrates such as glycogen was not influenced by the embedding. However, its most natural localization is achieved by freeze drying or isopropanol freeze substitution followed by GMA-embedding.
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PMID:The influence of glycol methacrylate (GMA) and paraffin embedding on freeze substituted and fixed tissues for enzyme histochemistry. 10 5

A rapid and simple high-performance liquid chromatography analytical method is described for the quantitative determination of phenytoin and five of its metabolites--phenytoin dihydrodiol, catechol, methoxycatechol, para-hydroxyphenytoin, and meta-hydroxyphenytoin--in biological materials. Following ethyl acetate extraction and incubation with beta-glucuronidase, samples were passed through a C18 Sep-Pak cartridge. The eluate was chromatographed on a reverse-phase 10 cm C18 Radial Nova-Pak (5 micron) column using a mobile phase of isopropanol:water (20:80) at a flow rate of 1.4 ml/min and monitored at 235 nm. Total chromatographic analysis time was 23 min, with complete baseline resolution of all metabolites. Five different columns and 10 different mobile phase conditions were studied to determine the best system. The 10-cm C18 Radial Nova-Pak (5 micron) gave the best resolution, reproducibility, and durability. This method should prove applicable for clinical use as well as for research purposes.
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PMID:Isocratic separation of phenytoin metabolites by high-performance liquid chromatography from human and animal microsomes and urine. 291 55

We describe a liquid chromatographic screening procedure for the detection of stimulant laxatives in urine. A 2-ml urine sample was incubated with 500 U of beta-glucuronidase for 2 h at 60 degrees C. The sample was acidified with sodium acetate (pH 5.0) and extracted with 5 ml of an isopropanol-chloroform (1:9) mixture. The organic layer was cleaned up further by washing with 5 ml disodium hydrogen-phosphate (pH 7.5) before being transferred to a conical tube and evaporated to dryness. The residue was reconstituted in 100 microliters mobile phase and 3 microliters were injected onto a Hewlett-Packard Hypersil ODS (5 microns) column. The ultraviolet absorbance of the eluent was monitored at 225 nm. Rhein, bisacodyl diphenol, bisoxatin diphenol, phenolphthalein, bisacodyl, bisoxatin and danthron all eluted within 6 min. The screen was evaluated using urine specimens obtained from 19 patients who claimed they had taken one or more of the laxatives under consideration within the past 48 h. Only two patients who claimed to have taken Coloxyl and Danthron showed negative results. Eighteen of twenty laxatives (90%) taken by the patients were detected and their identity verified by plotting post-run ultraviolet spectra. We therefore conclude that the screen is sufficiently reliable to be of help in the early detection of surreptitious abusers of stimulant laxatives.
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PMID:Screening procedure for stimulant laxatives in urine using high-performance liquid chromatography with diode array detection. 323 41

Four unconjugated metabolites, which were produced through the oxidation of the isopropyl chain of 2-isopropylnaphthalene (2-IPN), were isolated from the urine of rabbits receiving 2-IPN orally and identified: 2-(2-naphthyl)propionic acid, 2-(2-naphthyl)-2-propanol, 2-(2-naphthyl)-1,2-propanediol, and 2-(2-naphthyl)-2-hydroxypropionic acid, together with a small amount of the unchanged compound. Further, the unconjugated metabolites, which were produced through the oxidation of the naphthalene ring, were isolated and identified: 2-isopropylnaphthols and 2-isopropyl-5,6 (or 7,8)-dihydronaphthalene-5,6 (or 7,8)-diol. The identification of these metabolites was made by means of TLC, GLC, MS, IR, GC/MS, and FT-NMR. The presence of glucuronides of metabolites B, C, D, F, and H was also suggested by TLC and GLC of the extract obtained after hydrolysis by beta-glucuronidase. In addition, quantitative determination of the metabolites indicated that the total urinary excretion of the metabolites except 2-isopropylnaphthols in 24 hr after administration was about 29% of the dose.
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PMID:Identification and determination of urinary metabolites of 2-isopropylnaphthalene in rabbits. 360 68

This assay method allows a simultaneous determination of imipramine, desipramine, their 2-hydroxylated metabolites, and imipramine-N-oxide in 0.5 ml of plasma or 0.1 ml of urine within 35 min by an ion-paired, reversed phase (C18) high-performance liquid chromatography (HPLC) with electrochemical detection. The analytes are extracted from alkalinized plasma or urine with 5 ml of a 90/10 mixture (by vol) of diethyl either/2-propanol, back-extracted into 0.5 ml of 0.1 mol/L phosphoric acid. Urine samples are enzymatically treated with beta-glucuronidase/arylsulfatase before extraction. The electrochemical detection is performed with a glassy carbon electrode set at +0.85 V against the Ag/AgCl reference electrode. Recoveries for the analytes and the internal standard (propericiazine) from plasma or urine ranged from 66.4 to 105.7% with coefficients of variation (CVs) of < 6.8%. The intra- and interassay CVs for the analytes were < 17.4% in plasma and < 14.2% in urine. The limits of determination (a signal-to-noise ratio of 3) for imipramine, desipramine, 2-hydroxyimipramine, 2-hydroxydesipramine, and imipramine-N-oxide were 0.5, 0.3, 0.02, 0.02, and 1.0 microgram/L, respectively. Only four of the 23 psychotropic drugs, which might be coadministered with imipramine or desipramine, were considered to be the possible sources to interfere with the assay. We evaluated clinical applicability of this method by determining plasma concentration- and urinary excretion-time courses of the respective analytes in an extensive and a poor metabolizer of the debrisoquine/sparteine-type oxidation after a single oral dose of imipramine HCl (25 mg). The present method appears to be suitable not only for the therapeutic drug monitoring of imipramine and its active metabolites but also for studying the pharmacogenetically related metabolism of imipramine or desipramine.
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PMID:Simultaneous high-performance liquid chromatography-electrochemical detection determination of imipramine, desipramine, their 2-hydroxylated metabolites, and imipramine N-oxide in human plasma and urine: preliminary application to oxidation pharmacogenetics. 833 3

Eighty urine specimens collected from drug rehabilitation programs, which had been screened by immunoassay and confirmed positive by gas chromatography-mass spectrometry (GC-MS) for methamphetamine, were further analyzed for alpha-benzyl-N-methylphenethylamine (BNMPA) and its urinary metabolites, N-demethyl-BNMPA, diphenyl-2-propanone (DP2P), diphenyl-2-propanol, p-OH-N-demethyl-BNMPA, and p-OH-BNMPA. BNMPA is an impurity of illicit methamphetamine synthesis. Analysis of BNMPA and its metabolites was performed by quantitative GC-MS following beta-glucuronidase hydrolysis, liquid-liquid extraction, and derivatization with heptafluorobutyric anhydride. Two urine specimens contained detectable amounts of BNMPA and/or its metabolites. One contained trace amounts (greater than the limit of detection but less than the limit of quantitation) of N-demethyl-BNMPA and DP2P, as well as 0.04 mg/L p-OH-N-demethyl-BNMPA. The other contained trace amounts of BNMPA, p-OH-BNMPA, and p-OH-N-demethyl-BNMPA, as well as 0.03 mg/L N-demethyl-BNMPA. Prior to analyzing these urine specimens, pure reference material of p-OH-BNMPA was made available, and analysis confirmed our previous tentative identification of p-OH-BNMPA as a major metabolite of BNMPA. Detection of BNMPA or its metabolites in biological samples may serve as a marker of illicit methamphetamine administration.
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PMID:alpha-benzyl-N-methylphenethylamine (BNMPA), an impurity of illicit methamphetamine synthesis: III. Detection of BNMPA and metabolites in urine of methamphetamine users. 886 98

An automated high-performance liquid chromatographic method, benzodiazepines by REMEDi HS, was used to analyze benzodiazepines and their metabolites after beta-glucuronidase hydrolysis of 1-mL urine specimens from the following: 924 clinic and hospital patients whose specimens had previously been found to be presumptively positive using either EMIT or Triage immunoassay methodologies and 128 individuals whose specimens had screened negative by EMIT d.a.u.TM. REMEDi analyses did not correlate with the immunoassay results in 136 of the positive and three of the negative urine specimens. Gas chromatographic-mass spectrometric (GC-MS) confirmatory analyses were performed on these discordant specimens using 3 mL beta-glucuronidase-hydrolyzed urine followed by extraction with chloroform-isopropanol (9:1) and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. Two benzodiazepines, flunitrazepam and clonazepam, and their 7-amino metabolites were analyzed without prior derivatization. The analyses established 87% concordance between REMEDi and GC-MS versus 13% concordance with immunoassay for the subset. GC-MS analysis of these 142 specimens demonstrated two reasons for the nonconcurrence between REMEDi and EMIT: EMIT had given either false-negative or false-positive results and EMIT had given a positive result even though the determined metabolites were below the 200-ng/mL cutoff for the immunoassay and the 80-ng/mL cutoff for REMEDi. A total of 23 specimens were found to contain only lorazepam by REMEDi and GC-MS, 15 of which had been screened by Triage. A reevaluation of these 23 specimens by EMIT d.a.u. demonstrated that 11 were positive. This finding was in contrast to previous reports that EMIT will not detect lorazepam glucuronide in urine. An unexpected finding was the REMEDi identification and subsequent GC-MS confirmation of 7-aminoflunitrazepam, a urinary metabolite of flunitrazepam that is not available in the United States and that represented illicit use by four patients. A distinct advantage of REMEDi proved to be its capability in identifying demoxepam, a major metabolite of chlordiazepoxide; GC-MS analysis could not detect this metabolite because of its thermal decomposition to nordiazepam. To further evaluate the specificity of REMEDi, we conducted GC-MS analyses in a random fashion on 55 additional nondiscordant urine specimens that were identified as either positive or negative, as well as 22 specimens identified as containing 7-aminoclonazepam by REMEDi. Concurrence was observed between the two methods for all specimens, with the exception of one apparent false positive for alpha-hydroxyalprazolam by REMEDi. The reproducibility of the REMEDi method was found to be excellent; it was assessed by comparing results of 266 specimens that were reprocessed in different batches and for known calibrators and controls also processed with each batch. Study results demonstrated that the automated REMEDi assay for urinary benzodiazepines and their metabolites was comparable with GC-MS but had distinct advantages over GC-MS because of the following reasons: simplicity of the assay, less time required for analyses, and provision of additional information concerning the parent benzodiazepine.
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PMID:Identification of urinary benzodiazepines and their metabolites: comparison of automated HPLC and GC-MS after immunoassay screening of clinical specimens. 888 78

We developed a high-performance liquid chromatography (HPLC) method for quantitating p-hydroxy-N-benzylamphetamine glucuronide (pHBAG) and p-hydroxy-benzphetamine glucuronide (pHBZG), which are urinary metabolites of benzphetamine, in humans. Urine samples were hydrolysed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight and the treated urine was applied to a solid phase extraction column. After washing the column with water, 0.01 mol/L acetic acid and methanol, pHBA and pHBZ were eluted with dichloromethane:isopropanol:28% ammonium hydroxide (78.4:19.6:2.0 v/v). The eluate was evaporated and the residue was dissolved in acetonitrile: 5 mmol/L 1-pentane sulphonic acid (5:95 v/v) and analysed by HPLC with gradient elution. The amounts of urinary pHBAG and pHBZG excreted by two human subjects after oral administration of 10 mg benzphetamine hydrochloride were determined. About 10-15% of benzphetamine was found to be excreted as pHBAG and pHBZG, and almost all of these metabolites were excreted within 24 h. Urine samples should be collected as early as possible after ingestion of benzphetamine to detect pHBAG and pHBZG.
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PMID:Development of a method for the quantitation of benzphetamine metabolites in human urine by high-performance liquid chromatography. 983 92

We developed a method for simultaneous analysis of benzphetamine (BZ) and its metabolites, p-hydroxy-N-benzylamphetamine (pHBA), p-hydroxybenzphetamine (pHBZ), amphetamine (AP), methamphetamine and p-hydroxymethamphetamine by micellar electrokinetic chromatography (MEKC). Urine samples from 0-15 h (3-h intervals) after oral administration of BZ (10 mg) were hydrolyzed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight. The treated urine was applied to a solid phase extraction column Bond Elut Certify. After sequentially washing the column with water, 0.1 mol/l acetic acid and methanol, the samples were eluted with dichloromethane:isopropanol:28% ammonium hydroxide=78.4:19.6:2.0 (v/v %). The eluate was evaporated and the residue dissolved in running buffer was analyzed by MEKC. In urine from 0-3 h, AP, pHBZ and pHBA were detected. After that, only pHBA, which is one of the major metabolites of BZ in human urine, could be detected in the urine by the present method. A method for quantitation of pHBA by MEKC is described here. The effects of acetonitrile and sodium dodecyl sulfate in the running buffer of MEKC on the separation of BZ and its metabolites are also reported.
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PMID:Simultaneous analysis of benzphetamine and its metabolites, and quantitation of urinary p-hydroxy-N-benzylamphetamine by micellar electrokinetic chromatography. 985 14

A sensitive and stereospecific method was developed to determine propafenone (PPF), 5-hydroxypropafenone (5-OHP) as well as their glucuronide and sulfate conjugates in human plasma. Quantitative analyses and preparative isolations of PPF and 5-OHP were performed on a Nucleosil C18 column after liquid-liquid extraction. Afterwards the enantiomeric ratios of PPF and 5-OHP were determined on a Chiral-AGP column with ion trap mass spectrometric detection in the selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The enantiomers of PPF and 5-OHP were separated with different mobile phases. For PPF enantiomers, the mobile phase consisted of 10 mM ammonium acetate buffer (pH 5.96)-1-propanol (100:9, v/v), at a flow-rate of 0.5 ml/min; And for 5-OHP enantiomers, the mobile phase was 10 mM ammonium acetate buffer (pH 4.1)-2-propanol (100:0.9, v/v), at a flow-rate of 0.6 ml/min. The SRM transitions m/z 342 to m/z 324 and m/z 358 to m/z 340 were monitored for detection of enantiomers of PPF and 5-OHP, respectively. Linear calibration curves were obtained in the concentration range of 20-1600 ng/ml for each enantiomer of PPF and 20-500 ng/ml for the 5-OHP enantiomer. The limits of quantification for each enantiomer of PPF and 5-OHP were found to be 20 ng/ml. Precision and accuracy were within 11% over the calibration range for each of the analytes. Incubation of the plasma samples with beta-glucuronidase/arylsulfatase and the use of the specific beta-glucuronidase inhibitor saccharo-1,4-lactone allows the quantitation of both the glucuronide and sulfate conjugates of the enantiomers. The method was applied to human plasma samples from ten Chinese male volunteers after oral administration of 300 mg racemic propafenone.
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PMID:Enantioselective determination of propafenone and its metabolites in human plasma by liquid chromatography-mass spectrometry. 1002 38


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