[70] Determination of glutathione and glutathione disulfide in biological samples

Publisher Summary There are a number of procedures, for example, chemical, enzymatic, and chromatographic for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in biological samples. Enzymatic and chromatographic methods for the determination of glutathione in biological samples are described in this chapter. Because GSH readily oxidizes nonenzymatically and because it is a good substrate of γ-glutamyl transpeptidase, the biological samples are acidified quickly to reduce oxidation of GSH to GSSG and to mixed disulfides, and to inactivate γ-glutamyl transpeptidase. Glutathione oxidizes rapidly at pH values greater than 7. Acid treatment inactivates γ-glutamyl transpeptidase, which catalyzes the reactions that decrease the levels of both GSH and GSSG. The optimum method for treating biological samples depends upon the tissue and the experimental system. The discussed 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG reductase recycling assay for total glutathione is a specific, sensitive, rapid, and reliable procedure. However, because the method depends on an accurate standard curve, appropriate standards containing the protein precipitating agent are essential.