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1- Free GSH=DTNB+Sample+Substrate 2- Total GSH=DTNB+Sample+Substrate+ NADPH+glutathione reductase 3- GSSG=Total GSH-Free GSH Another method to measure GSSG separately, is to remove the free GSH from DTNB reaction process by modification of free GSH with N-ethylmaleimide (NEM) or vinylpyridine prior to conducting assay