Differential Rates of Glutathione Oxidation for Assessment of Cellular Redox Status and Antioxidant Capacity by Capillary Electrophoresis-Mass Spectrometry: An Elusive Biomarker of Oxidative Stress

Glutathione metabolism plays a fundamental role in maintaining homeostasis and regulating the redox environment of a cell. Despite the widespread interest in quantifying glutathione metabolites in oxidative stress research, conventional techniques are hampered by complicated sample handling procedures to prevent significant oxidation artifacts generated during sample collection, sample pretreatment, and/or chemical analysis. In this report, a simple and validated method for glutathione analysis from filtered red blood cell (RBC) lysates was developed using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with fingerprick microsampling and ultrafiltration. About a 3-fold improvement in precision with nanomolar detection limits was achieved when using online sample preconcentration with CE-ESI-MS via a modified injection sequence, which permitted accurate determination of the intracellular reduced/oxidized glutathione ratio (GSH/GSSG), as well as other glutathione species, including protein-bound glutathione mixed disulfide (PSSG), free glutathione mixed disulfides (GSSR) and glutathione thioether conjugates (GSX). In this work, the redox status of filtered hemolysates was determined by the equilibrium half-cell reduction potential for glutathione (E(GSSG/2GSH)), whereas its intrinsic antioxidant capacity was assessed by the apparent rate of metal-catalyzed oxidation of glutathione. In-vitro incubation studies of intact RBCs with 1-chloro-2,4-dinitrobenzene (CDNB) and N-acetyl-L-cysteine (NAC) were found to significantly alter E(GSSG/2GSH) and/or glutathione oxidation kinetics (e.g., k(GSSG)) relative to normal controls based on their function as a toxic electrophilic compound and a competitive free radical scavenging/reducing agent, respectively. Differential rates of glutathione oxidation (DIRGO) using CE-ESI-MS offers a novel strategy for global assessment of the impact of intrinsic metabolite constituents (i.e., metabolome) and/or extrinsic perturbants on cellular redox status that is relevant to improved understanding of aging and the pathogenesis of acute or chronic disease states.