Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.