Capillary Isotachophoresis Determination of Trace Oxidized Glutathione in Blood

Abstract

A capillary isotachophoresis (CITP) method performed in a column-coupling apparatus has been developed for the simultaneous determination of glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood samples. The determination of GSSG and GSH concentrations in biological samples is important because of their roles in oxidative stress. Different concentrations of a leading ion in the coupled columns (concentration cascade) and a large volume (37 µl) of the injected sample facilitated a GSSG concentration of between 2 and 25 µmol/l. A reaction between iodoacetate and GSH under alkaline conditions was used to prepare the sample in order to avoid oxidation of GSH to GSSG. This step eliminated the main source of systematic errors with regard to the determination of the GSSG concentration. A linear relationship (R2=0.9969) between the zone length of S-(carboxymethyl)glutathione (the product of the reaction between GSH and iodoacetate) and the concentration of GSH (40-120 µmol/l) was obtained. The method was applied to the analysis of bovine blood samples that had been diluted by a factor of ten with satisfactory results.