Improved Method for Determination of Cellular Thiols, Disulfides and Protein Mixed Disulfides Using HPLC with Electrochemical Detection

Abstract

The broad interest in GSH^* as a cellular mediator in the defense against toxicological injury has prompted the development of a variety of methods for measuring cellular glutathione, comprised predominantly of GSH and its disulfides, GSSG and GSSProt. As investigations have increasingly focused on the biochemical mechanisms underlying the disposition of glutathione, the need has arisen for methods providing greater analytical capabilities. The earliest methods developed for thiol determination utilized the stoichiometric reaction of thiols with chemical agents to produce a colored (Ellman, 1959) or fluorescent (Hissin and Hilf, 1976) product. These methods were convenient, but they did not provide a specific determination of GSH. While GSH constitutes the major non-protein thiol in most cell types, inclusion of other thiols or non-thiol compounds (Benson et al., 1975) in the measurement led to overestimation of GSH levels. In addition, chemical methods lacked adequate sensitivity to measure low levels of GSH (Jensen et al., 1985). Despite these difficulties, a difference method has been developed (Habeeb, 1973) and utilized (Isaacs & Hinkley, 1977) to measure hepatic GSH, GSSG and GSSProt. Specificity for GSH and sensitivity have been increased by combining enzymatic recycling with a colorimetric determination (Tietze, 1969), although this method did not differentiate between GSH and GSSG. While GSH could be removed from the reaction using N-ethylmaleimide for GSSG determination, this thiol-blocking reagent, as well as protein precipitants and denaturants, can alter the kinetics of the reaction (Griffith, 1980; Brigelius et al., 1983) leading to erroneous results. Recently, rapid chromatographic analysis using HPLC has been applied to GSH determinations (Reed et al., 1980). While specificity was insured based on column retention time and the sensitivity was adequate for the determination of hepatic GSSProt (Brigelius et al., 1983), lengthy pre-column derivitization was required. In the present report, a new method that utilizes HPLC separation and thiol-specific electrochemical detection for thiol determination is demonstrated. This analytical system provides some advantages lacking in previous methods. Specificity for GSH and other thiols is based on column retention time and does not require thiol detection. derivitization. The sensitivity of the method allows quantitation of GSH in the picomole range, which is adequate for the determination of hepatic GSSProt. Finally, the presence of endogenous biological materials or reagents used in sample preparation has been found not to interfere with thiol detection. The applicability of this method to biological systems is illustrated by the determination of the glutathione status in rat liver and erythrocytes.