Improved method for determination of cellular thiols, disulfides and protein mixed disulfides using HPLC with...

The role of glutathione in cellular responses to toxic insult is well established in the literature and is largely based on chemical determinations of GSH. Although these methods have proved to be adequate to demonstrate the relationships between cellular levels of GSH and xenobiotic toxicity, they may be inadequate for careful investigations regarding the regulation of cellular concentrations of GSH, GSSG and GSSProt. Enzymatic and chromatographic methods provide increased specificity and sensitivity, but the problems of variable accuracy or tedious derivitization steps remain. The HPLC-EC method constitutes a significant improvement over previous methods by satisfying the requirements for selectivity, sensitivity, accuracy and convenience. The utility of the HPLC-EC method has been described in two systems of toxicological interest, particularly with regard to the determination of GSSProt. In further studies, the HPLC-EC method has been useful in investigating the mechanism(s) of alternate nucleophiles, such as cysteamine. The capability to determine various thiols simultaneously in a given biological sample greatly facilitates these investigations. In view of its power as an analytical tool, the HPLC-EC method has the potential for widespread application to the study of cellular thiols.