Determination of intracellular glutathione in rat hepatocytes after treatment of environmental pollutants by capillary electrophoresis with laser-induced fluorescence detection

Abstract

A method for the determination of rat hepatocellular glutathione (GSH) by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection utilizing helium–cadmium (He–Cd) laser line at 325 nm has been developed. The GSH in hepatocyte cells was tagged with three fluorogenic reagents, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and 4-( N, N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). These reagents were permeable into cells and reacted with GSH to produce highly fluorescent derivatives, but the efficiency was different among the reagents. Judging from fluorescence microscope analysis, ABD-F seems to be suitable for the tagging of hepatocellular GSH, in terms of the permeability to the cells and the reaction selectivity to GSH. The resulting ABD-GSH was effectively resolved by capillary zone electrophoresis (CZE) with a running buffer of 20 mM phosphate (pH 7.5) at 30 kV applied potential. The determination of intracellular GSH in isolated rat hepatocytes before and after treatments with environmental pollutants was carried out with the CE conditions. The GSH concentrations in untreated cells obtained from 8–11 year old rats were calculated as 14–103 fmol per cell. On the other hand, the hepatocellular GSH levels after treatment of bisphenol A and 1,3-dichloropropene were obviously decreased as comparing with control GSH concentration. The degrees of the depletion were essentially similar to results obtained from HPLC-FL method.