Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

t his protocol describes a procedure for determining glutathione (G sH) and glutathione disulfide (G ssG) concentrations in blood and other tissues. artifactual oxidation to G ssG of 5–15% of the G sH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of G ssG. t his can be prevented by derivatizing G sH 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. G ssG concentration is then measured by spectrophotometry with the G sH recycling method, on the basis of conversion of GssG to G sH by glutathione reductase and naDpH and reaction with 5,5 ′-dithiobis-(2-nitrobenzoic acid). G sH concentration is instead measured by either of two methods: by analysis of Gs-neM conjugates by H plc in the same sample that is used to measure G ssG or, alternatively, by analysis of G sH by spectrophotometry (G sH recycling method) on one additional sample aliquot that has not been derivatized with neM. t he procedure can assay GsH and GssG in blood and other tissues in 30 min or less.