In Vitro Studies on Degradation of Gamma-L-Glutamyl-L-Cysteine and Gamma-L-Glutamyl-D-Cysteine in Blood: Implications for Treatment of Stroke.

Treatment for ischemic stroke involves a thrombolytic agent to re-establish blood flow in the brain. However, delayed reperfusion may cause injury to brain capillaries. Previous studies indicate that the antioxidant gamma-L-glutamyl-L-cysteine (γ-Glu-Cys) contributes to reducing reperfusion injury to the cerebral vasculature in rats, when administered intravascularly. To determine the stability of γ-Glu-Cys in blood, the peptide was incubated in rat serum in vitro, and its degradation was quantified by high-pressure liquid chromatography. The half-time (t1/2) for degradation of γ-Glu-Cys was 11 ± 1 minute (mean ± SD, n = 3). A similar pattern of degradation was observed when γ-Glu-Cys was incubated in the presence of human plasma (t1/2 = 17 ± 8 minutes, n = 3). In a second series of experiments, degradation of an analog (γ-Glu-D-Cys) was tested in rat serum and found to be more stable than the native molecule. The initial velocity for degradation of γ-Glu-D-Cys (0.12 ± 0.02 mM/min; mean ± SD, n = 3) was significantly (P = 0.006) less than that of γ-Glu-Cys (0.22 ± 0.03 mM/min; mean ± SD, n = 3). Furthermore, an in vitro assay indicated that the analog has as an oxidative capacity that equals that of the original peptide in the presence of rat serum and human plasma. Finally, both peptides were found to be similarly effective in preventing lysis of intact cells using in vitro assays. These studies show that γ-Glu-Cys remains intact in blood for several minutes, and the analog γ-Glu-D-Cys may be a more stable, but similarly effective antioxidant.