Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites
Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites
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DOI：
10.1021/tx0496791
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发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Humphreys, WG
中科院分区：
文献类型：
--
作者：
Gan, JP;Harper, TW;Humphreys, WG
建立了一种灵敏、定量的体外反应性代谢物生成的检测方法。该方法利用标记有荧光标签的还原型谷胱甘肽（GSH）作为捕获剂，并通过荧光检测进行定量。通过氧化型谷胱甘肽（GSSG）与丹磺酰氯反应形成丹磺酰化GSSG来完成谷胱甘肽的衍生化。随后二硫键的还原产生丹磺酰化GSH（dGSH）。在存在dGSH和NADPH的情况下，将供试化合物与人肝微粒体一起孵育，并通过与荧光检测器和质谱仪偶联的HPLC分析所得混合物，以定量和质量测定所得dGSH加合物。通过监测生物活化后dGSH与GSH各自与1-氯-2，4-二硝基苯或R-（+）-胡薄荷酮的反应来研究dGSH与GSH的比较化学反应性。发现dGSH在对两种硫醇反应性分子的化学反应性方面等同于GSH。在与人肝S9组分或重组GST（GSTM 1 -1）孵育时，dGSH不作为谷胱甘肽S-转移酶（GST）介导的3，4-二氯硝基苯偶联的辅因子。在本试验中检测了参比化合物，包括7种已报告可形成GSH加合物的化合物沿着7种目前美国市场上处方最多但未报告可形成GSH加合物的药物。在与所有7种阳性参比化合物的孵育中检测并定量dGSH加合物;然而，在任何广泛处方的药物中均未观察到dGSH加合物。与现有的方法相比，该方法具有灵敏度高、定量性好、成本低、易于实现等优点。
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.