A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY METHOD FOR MEASUREMENT OF OXIDIZED GLUTATHIONE IN BIOLOGICAL SAMPLES

A high-performance liquid chromatography method to determine oxidized glutathione (GSSG) in biological samples with ultraviolet-visible dete ction using N-ethylmaleimide to prevent reduced glutathione (GSH) oxid ation is described. Previous methods based on highperformance liquid c hromatography to quantitate GSH and GSSG are unsuitable for determinin g GSSG in biological samples. This is due to GSH oxidation during samp le processing. N-Ethylmaleimide, but not iodacetic acid, prevents this oxidation. Blood GSH oxidation measured by the widely used method of Reed ct al. (Anal. Biochem. 106, 55-62, 1980) can be as high as 24 +/- 6% (n = 6). When blood samples were assayed by our procedure, GSH oxi dation was only 0.13 +/- 0.28% (n = 5). GSH can be determined enzymati cally, i.e., with glutathione-S-transferase, but perchloric acid shoul d not be used to deproteinize samples. Trichloroacetic acid (15% final concentration) may be used. This method allows an accurate calculatio n of the GSH/GSSG ratio, which is important for determining oxidative stress in tissues in various pathophysiological situations. (C) 1994 A cademic Press, Inc.