Comparison of HPLC and enzymatic recycling assays for the measurement of oxidized glutathione in rat brain

Glutathione (reduced, GSH and oxidized, GSSG) concentrations were analysed in rat cerebellar homogenate using high performance liquid chromatography with ultraviolet detection (HPLC-UV) or enzymatic recycling assays. GSSG levels found using the HPLC-UV assay were 200-fold higher than those obtained with the enzymatic recycling procedure. Reduction of synthetic GSSG by glutathione reductase showed total conversion to GSH as assessed by HPLC-UV analysis. In contrast, only approximately 50% of the HPLC peak for GSSG could be reduced by glutathione reductase. Increasing the period of incubation with glutathione reductase for longer than 15 min did not alter GSSG levels. These results suggest that another substance present in brain tissue is derivatized and eluted at the same time as GSSG using the HPLC-UV assay, thus contributing to the apparently high GSSG levels found employing this technique.