Determination of Glutathione and Glutathione Disulfide in Biological Samples Using Acrylonitrile as a Thiol-Blocking Reagent

Abstract

Biological samples containing glutathione (GSH) and glutathione disulfide (GSSG) were deproteinized by HClO₄ or metaphosphoric acid in the presence of bathophenanthroline disulfate. The total glutathione (GSH+2×GSSG) in the sample was determined using 5, 5′-dithiobis(2-nitrobenzoic acid) and glutathione reductase by the standard enzymatic recycles methods. GSH was determined as the difference between the concentration of the total glutathione and GSSG; GSSG was determined after blocking the thiol group of GSH with acrylonitrile. The method presented here is an improved version of that reported by Griffith (Anal. Biochem., 106, 207 (1980)) using 2-vinylpyridine as the modifying reagent. By using the present method, an analysis of GSH and GSSG is possible without exposure to any bad odor or the need to distill 2-vinylpyridine. In addition, this method was improved by a deproteinization procedure. Highly accurate results could be rapidly obtained by this method.