Analysis of antioxidant molecules is potentially important to understanding the role of oxidative stress (1)(2)(3)(4)(5)(6) in disease, as oxidative damage is accompanied or preceded by their depletion. Reduced glutathione (GSH) is ubiquitous and abundant; it can be oxidized to its disulfide form (GSSG) in response to an oxidative perturbation. Usually, however, this species is rapidly reduced by the action of glutathione reductase (7). If GSSG accumulates within the cell, it can create protein-glutathione adducts via thiol-disulfide exchange reactions. Thus, in addition to the ratio of GSH to GSSG, the content of glutathionylated proteins (GSSPs) can indicate oxidative stress. The analysis of GSSPs has potential advantages over measurements of GSH and GSSG because GSSPs are more stable than GSSG, being less prone to enzymatic reduction by glutathione reductases (8). Because blood can be studied as an indicator of the overall body oxidative status, GSSPs and, particularly, glutathionyl hemoglobin (Hb-SSG) could represent useful markers of oxidative stress. The use of Hb-SSG as a clinical marker has been proposed (9)(10). Significant increases in GSSP concentrations have been found in diabetes mellitus, hyperlipidemia, Friedreich ataxia, and chronic renal failure (10)(11)(12)(13). The technology commonly used for the assays of GSSPs, electrospray ionization mass spectrometry (ESI-MS), is not widely available. In addition, we have recently reported (14) that oxygenated Hb is able to artificially produce large amounts of GSSG and GSSP if not adequately manipulated. We have therefore developed a rapid and sensitive HPLC method to measure GSSPs that could avoid possible pitfalls and artifacts. Monoclonal anti-GSH antibody was obtained from Virogen. Sheep anti-mouse IgG, horseradish peroxidase conjugate, was obtained from Amersham Pharmacia Biotech. All others chemicals were from Sigma. Human blood was obtained by venipuncture, …