Status of glutathione and other thiols and disulfides in human plasma.

While plasma thiols, including homocysteine (HCys), glutathione (GSH), and cysteine (Cys), are being investigated as potential indicators of disease risk and health status, low levels, poor stability, and the lack of comprehensive methodologies have hampered their accurate assessment. Using our previously described HPLC with electrochemical detection method, our goal was to assess levels, stability, and distribution of biologically relevant thiols and disulfides in human plasma. In fresh plasma, processed immediately after collection, low levels of Cys, cystine, Cys-Gly, and the mixed disulfide Cys-GSH (CSSG) were consistently observed, whereas the levels of GSH and Cys-Gly disulfide were often below the limits of detection. These profiles were a consequence of poor thiol stability, as thiol standards added to human plasma were lost rapidly due to autoxidation or formation of mixed disulfides. A 75% loss of added GSH observed after 30 min was accounted for completely by the formation of GSH disulfide (24%) and CSSG (74%). Similar changes were found with other thiols when added to plasma. Thiols lost to oxidation were recovered quantitatively by reducing samples with potassium borohydride (KBH(4)) prior to analysis. In a study of 106 healthy adults, mean total thiol levels in plasma were: Cys (201 microM) > Cys-Gly (101 microM) > HCys (7 microM) > gamma-Glu-Cys (5 microM) > GSH (4 microM). All together, these results account for the poor stability of thiols in plasma and provide a method for their comprehensive and accurate determination.