Measurement of reduced and oxidized glutathione in cultures of adult rat hepatocytes

A reversed-phase ion-exchange high-performance liquid chroamtographic technique, suitable for the separate measurement of reduced (GSH) and oxidized (GSSG) glutathione in cultures of adult rat hepatocytes, is described. A commercially available Nucleosil 120-7NH₂ column was used. A complete run took ca. 22 min. The retention times for GSH and GSSG were 10.6 and 12.7 min, respectively, providing a resolution coefficient of 1.4. The coefficients of variation for GSH and GSSG were ca. 5 and 25%, respectively, for freshly isolated hepatocytes, and 16 and 15%, respectively, for 24-h cultured hepatocytes. The detector response was linear as a function of GSH and GSSG concentration and the hepatocytes concentration studied. Addition of up to 1.5 mg/ml bovine serum albumin to the culture medium had no effect on the linearity. The recovery for standards, ranging from 0 to 150 nmol of GSH or GSSG per millilitre in the presence of hepatocytes, was 98% for GSH and 80% for GSSG. The detection limit of the method was between 0.5 and 1.0 nmol of GSH and GSSG per millilitre. In cultured rat hepatocytes, the GSH content increased during the first 24 h of culture, followed by a slow decrease. After six days of culture, the GSH content was less than 50% of the value found for freshly isolated hepatocytes. GSSG was present in cultured rat hepatocytes in only small amounts and becomes unmeasurable after four days of culture.