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

Abstract

A sensitive and quantitative method was developed for the estimation of reactive metabolite formation in vitro. The method utilizes reduced glutathione (GSH) labeled with a fluorescence tag as a trapping agent and fluorescent detection for quantitation. The derivatization of GSH was accomplished by reaction of oxidized glutathione (GSSG) with dansyl chloride to form dansylated GSSG. Subsequent reduction of the disulfide bond yielded dansylated GSH (dGSH). Test compounds were incubated with human liver microsomes in the presence of dGSH and NADPH, and the resulting mixtures were analyzed by HPLC coupled with a fluorescence detector and a mass spectrometer for the quantitation and mass determination of the resulting dGSH adducts. The comparative chemical reactivity of dGSH vs GSH was investigated by monitoring the reaction of each with 1-chloro-2,4-dinitrobenzene or R-(+)-pulegone after bioactivation. dGSH was found to be equivalent to GSH in chemical reactivity toward both thiol 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 S9 fractions 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 seven drugs that are among the most prescribed in the current U.S. market and have not been reported to form GSH adducts. dGSH adducts were detected and quantitated in incubations with all seven positive reference compounds; however, there were no dGSH adducts observed with any of the widely prescribed drugs. In comparison with existing methods, this method is sensitive, quantitative, cost effective, and easy to implement.