Improved procedure for determining glutathione in plasma as an index of myocardial oxidative stress

Glutathione plays an important role in the detoxification processes of electrophilic metabolites of xenobiotics and oxygen free radicals, such that release of reduced and oxidized glutathione into the plasma is considered a reliable index of oxidative stress. However, reduced glutathione in plasma undergoes spontaneous autoxidation, with mixed disulfide formation. We developed a new, simple, quick method to overcome this problem by treating the blood, immediately after collection, with thiol reagents. We add 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide to the blood before determination of total and oxidized glutathione, respectively. We find the proposed assay useful for investigating oxidative stress in clinical situations.