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 detection using N-ethylmaleimide to prevent reduced glutathione (GSH) oxidation is described. Previous methods based on high-performance liquid chromatography to quantitative GSH and GSSG are unsuitable for determining GSSG in biological samples. This is due to GSH oxidation during sample processing. N-Ethylmaleimide, but not iodacetic acid, prevents this oxidation. Blood GSH oxidation measured by the widely used method of Reed et 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 oxidation was only 0.13 +/- 0.28% (n = 5). GSH can be determined enzymatically, i.e., with glutathione-S-transferase, but perchloric acid should not be used to deproteinize samples. Trichloroacetic acid (15% final concentration) may be used. This method allows an accurate calculation of the GSH/GSSG ratio, which is important for determining oxidative stress in tissues in various pathophysiological situations.
Asensi
M
M
Departamento de Fisiología, Universidad de Valencia, Spain.
Sastre
J
J
Pallardó
F V
FV
García de la Asunción
J
J
Estrela
J M
JM
Viña
J
J
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005033:Ethylmaleimide; D005978:Glutathione / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000378:metabolism; D019803:Glutathione Disulfide; D006454:Hemoglobins / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D010084:Oxidation-Reduction; D010472:Perchlorates / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014238:Trichloroacetic Acid / Q000494:pharmacology
D006454:Hemoglobins; D010472:Perchlorates; D014238:Trichloroacetic Acid; D007501:Iron; D005978:Glutathione; D005033:Ethylmaleimide; D019803:Glutathione Disulfide