Rhonda Moeller: Quantification of reduced Glutathione and Glutathione Disulfide using Glutathione Reductase and 2-Vinylpyridine.   Glutathione is a tripeptide that can exist in either a reduced form (GSH) or as a dimeric oxidized disulfide (GSSG). Reduced glutathione is the most abundant intracellular thiol and low molecular weight tripeptide found in living cells.  In vivo, reduced glutathione plays a role in maintaining the redox status of the cell, and prevents oxidative damage by oxy-radicals.  A deficiency in GSH can result in increased susceptibility to cell lysis due to lipid peroxidation.  A procedure was evaluated that quantifies the amounts of GSH and GSSG spectroscopically via an  enzymatic recycling assay that uses GSH, glutathione reductase and NADPH.  GSSG was selectively determined  by assaying samples in which GSH was derivatized by 2-vinyl-pyridine (2-VP).  This procedure effectively blocks the pre-existing GSH from entering the recycling assay and being quantified.   The results showed that 2-VP masks 98% of the GSH present in a pure GSH sample.  In contrast to other masking agents, such as NEM, 2-VP does not inhibit glutathione reductase and is therefore a better reagent for the derivitization process.  It was demonstrated that, on an equi-molar basis, the rate of reduction of GSH was approximately half that of GSSG, in accordance with the known two to one molar ratio of the dimer to the monomer.