Dansyl Glutathione as a Trapping Agent for the Quantitative Estimation and Identification of Reactive Metabolites

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• 作者：Jinping Gan ; Timothy W. Harper ; Mei-Mann Hsueh ; Qinling Qu ; and W. Griffith Humphreys
• 刊名：Chemical Research in Toxicology
• 出版年：2005
• 出版时间：May 2005
• 年：2005
• 卷：18
• 期：5
• 页码：896 - 903
• 全文大小：153K
• 年卷期：v.18,no.5(May 2005)
• ISSN：1520-5010

文摘

A sensitive and quantitative method was developed for the estimation of reactive metaboliteformation in vitro. The method utilizes reduced glutathione (GSH) labeled with a fluorescencetag as a trapping agent and fluorescent detection for quantitation. The derivatization of GSHwas accomplished by reaction of oxidized glutathione (GSSG) with dansyl chloride to formdansylated GSSG. Subsequent reduction of the disulfide bond yielded dansylated GSH (dGSH).Test compounds were incubated with human liver microsomes in the presence of dGSH andNADPH, and the resulting mixtures were analyzed by HPLC coupled with a fluorescencedetector and a mass spectrometer for the quantitation and mass determination of the resultingdGSH adducts. The comparative chemical reactivity of dGSH vs GSH was investigated bymonitoring the reaction of each with 1-chloro-2,4-dinitrobenzene or R-(+)-pulegone afterbioactivation. dGSH was found to be equivalent to GSH in chemical reactivity toward boththiol reactive molecules. dGSH did not serve as a cofactor for glutathione S-transferase (GST)-mediated conjugation of 3,4-dichloronitrobenzene in incubations with either human liver S9fractions or a recombinant GST, GSTM1-1. Reference compounds were tested in this assay,including seven compounds that have been reported to form GSH adducts along with sevendrugs that are among the most prescribed in the current U.S. market and have not beenreported to form GSH adducts. dGSH adducts were detected and quantitated in incubationswith all seven positive reference compounds; however, there were no dGSH adducts observedwith any of the widely prescribed drugs. In comparison with existing methods, this method issensitive, quantitative, cost effective, and easy to implement.