The low concentration of GSSG (high GSH/GSSG ratio) in tissues coupled with the need to prevent GSH oxidation during sample preparation, are important considerations for the accurate measurement of GSSG and GSH/GSSG ratios. Guntherberg and Rost (2) first reported the use of N?ethylmaleimide (NEM) reacting with GSH to form a stable complex, therefore removing the GSH prior to the quantification of GSSG in tissues. Unfortunately, NEM inhibits GR. To overcome this problem, Griffith (3) employed 2?vinylpyridine (2-VP) to derivatize GSH. Although 2-VP does not significantly inhibit GR, it reacts relatively slowly with GSH and is not very soluble in aqueous solutions. Sample Types: biological fluids. Range: 0.1 - 3.0 μM. Sensitivity: 0.1 μM. Time: 20 minutes ? Reduced glutathione (GSH) is a tripeptide (g-glutamylcysteinylglycine) that contains a free thiol group. GSH is a major tissue antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols and hydrogen peroxide to water. In the GPx catalyzed reaction, the formation of a disulfide bond between two GSH molecules gives rise to oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of ?-nicotinamide adenine dinucleotide phosphate (?-NADPH2). When cells are exposed to increased levels of oxidative stress, GSSG will accumulate and the ratio of GSH to GSSG will decrease. Therefore, the determination of the GSH/GSSG ratio and the quantification of GSSG are useful indicators of oxidative stress in cells and tissues.