The determination of hepatic glutathione at tissue and subcellular level

Abstract

IntroductionGlutathione (GSH) through its important function in the antioxidant protection of cells and in the conjugation of drugs and xenobiotics has crucial importance in pharmacology and toxicology. Since GSH is most often measured in liver tissue and different cell organelles it is important to choose the method that best suits for the determination of GSH. MethodsThe GSH content of cell organelles isolated from control and BSO-treated liver tissues was determined by the GSH-NEM-HPLC-UV, monochlorobimane-GSH-HPLC-fluorescence method and DTNB-GSH recycling assay to find the most suitable method for GSH determination from cell organelles. ResultsThe GSH level of organelles could easily be measured by the monochlorobimane-HPLC-fluorescent method. The addition of monochlorobimane to the homogenisation buffer prevented the oxidation of GSH during isolation. The formation of monochlorobimane-GSH adduct was accelerated by the intrinsic GST activity of samples, however the omission of GST from the GSH standards could cause the overestimation of GSH content of biological samples. NEM is an excellent thiol protective agent and the GSH-NEM conjugate can be directly analysed by HPLC-UV, but the relatively high limit of detection made the method unsuitable for the determination of GSH from cell organelles. Although the DTNB-GSH recycling assay is quite simple and rapid the stabilization of GSH and the efficiency of detection lag behind the monochlorobimane-HPLC-fluorescent method. DiscussionThe monochlorobimane-HPLC-fluorescent method can be advised for the determination of GSH from pharmacologically and toxicological relevant cell organelles and liver tissue whilst addition of monochlorobimane to the homogenisation buffer prevented the autoxidation of GSH.