The time course of mixed disulfide formation between GSH and proteins in rat blood after oxidative stress with tert-butyl hydroperoxide

Variations in time of GSH, GSSG and glutathione-protein mixed disulfides (GSSP) were studied in rat blood in vitro experiments of oxidative stress with tert-butyl hydroperoxide (t-BOOH, dose range 0.3-2 mM; time range 15 sec-60 min). The aim was to elucidate the potential for GSSG reduction of protein-bound SH groups (PSH). GSSP was estimated by two methods, indirectly from GSHt (GSH + 2 GSSG) variations and directly from precipitated and washed proteins. After treatment with t-BOOH, GSH and GSSG concentrations showed an immediate (15-30 sec) drop and a peak respectively and returned to control levels (time zero values) between 30 and 60 min. A t-BOOH dose-dependent minimum of GSHt and a corresponding GSSP maximum were obtained within 1-6 min and subsequently returned to control values. Basal GSH, GSSG and GSSP levels were similar in aged and fresh blood. In contrast, after treatment with 1 mM t-BOOH substantial differences in kinetic patterns were observed: for intance GSSP concentrations were higher in aged than in fresh blood with no return to the initial values. The pretreatment of aged blood with 10 mM glucose decreased GSSP formation and produced a reversible pattern similar to that observed in fresh blood. The role of glucose in regulating GSSP generation is discussed. © 1994.