COMPARISON OF HPLC AND ENZYMATIC RECYCLING ASSAYS FOR THE MEASUREMENTOF OXIDIZED GLUTATHIONE IN RAT-BRAIN

Glutathione (reduced, GSH and oxidized, GSSG) concentrations were anal ysed in rat cerebellar homogenate using high performance liquid chroma tography with ultraviolet detection (HPLC-UV) or enzymatic recycling a ssays. 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 5 0% of the HPLC peak for GSSG could be reduced by glutathione reductase . Increasing the period of incubation with glutathione reductase for l onger than 15 min did not alter GSSG levels. These results suggest tha t 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.