An Improved Simultaneous Measurement of Oxidized and Reduced Glutathione in Biological Samples by High-Performance Liquid Chromatography Following Derivatization with Dansyl Chloride

Abstract

Reduced (GSH) and oxidized glutathione (GSSG) concentrations in biological samples can be determined simultaneously utilizing high-performance liquid chromatography (HPLC) following derivatization with dansyl chloride. In the present study, two points were investigated in detail to improve upon their method : 1) pH in the derivatization reaction and 2) the method of deproteinization prior to dansylation. Dansylation of standard GSH after S-carboxymethylation and of GSSG both gave the highest relative fluorescence at pH 8.5.Degradation of GSSG to GSH was not detected when GSSG was dansylated at pH 8.5 or more acidic pHs but was apparent at pH 9.0 and even more obvious at pH 10.0. The highest fluorescence values for dansylated GSH and GSSG were also obtained in tissue homogenates following the derivatization at pH 8.5. Although the value for dansylated GSH in the homogenates was also high at pH 9.0, this probably was due to degradation of intrinsic GSSG to GSH. Deproteinization with trichloroacetic acid (TCA) was found to give considerably higher relative fluorescence of dansylated GSSG than that with perchloric acid (PCA), whereas the dansylated-GSH concentrations obtained with the two acids were similar. Moreover, dansylated GSSG was more stable in TCA than in PCA, although there was no difference between these acids in relation to stability of dansylated GSH. In the livers and kidneys of rats, the GSH and GSSG concentrations obtained by the present method were consistent with those obtained by the recycling enzymatic method.