Simultaneous evaluation of six human glucuronidation activities in liver microsomes using liquid chromatography–tandem mass spectrometry

This article describes the development of a procedure for the simultaneous evaluation of the activity of six different uridine diphosphate (UDP)–glucuronyltransferases (UGTs) in human liver microsomes (HLMs). The method consists of incubations of probe substrates for UGT1A1 (etoposide), UGT1A3 (chen...

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Bibliographic Details
Published in:Analytical biochemistry 2012-08, Vol.427 (1), p.52-59
Main Authors: Gagez, Anne-Laure, Rouguieg-Malki, Koukeb, Sauvage, François-Ludovic, Marquet, Pierre, Picard, Nicolas
Format: Article
Language:English
Subjects:
Chromatography, Liquid
Drug metabolism
Female
Genotype
Glucuronides - chemistry
Glucuronosyltransferase - analysis
Glucuronosyltransferase - genetics
Humans
Inactivation, Metabolic
Liquid chromatography
Liver - enzymology
Male
Microsomes
Microsomes, Liver - enzymology
Phenotype
Substrate Specificity
Tandem Mass Spectrometry
UDP–glucuronosyltransferase
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Summary:This article describes the development of a procedure for the simultaneous evaluation of the activity of six different uridine diphosphate (UDP)–glucuronyltransferases (UGTs) in human liver microsomes (HLMs). The method consists of incubations of probe substrates for UGT1A1 (etoposide), UGT1A3 (chenodeoxycholic acid), UGT1A4 (trifluoperazine), UGT1A6 (serotonin), UGT1A9 (mefenamic acid), and UGT2B7 (azidothymidine) with HLMs. The six substrates were divided into three different incubations (etoposide+mefenamic acid; chenodeoxycholic acid+serotonin+azidothymidine; and trifluoperazine alone), the media of which were pooled before analysis. Glucuronide formation rates were determined in a single run of 20min using a validated liquid chromatography–tandem mass spectrometry method. No significant difference was observed between glucuronidation activities measured using the current procedure and individual incubations of the probes. The method was used successfully for the determination of UGT activities in 44 individual HLM preparations and for the phenotyping of preparations predicted to have altered UGT1A1 and UGT2B7 activities because of known genetic polymorphisms.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.04.031