Measurement of Tissue Sulfhydryls and Disulfides in Tissue Protein and Nonprotein Fractions by High Performance Liquid Chromatography Using Electrochemical Detection

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Abstract
A specific, sensitive and quantitative method for measuring tissue sulfhydryl (SH) and disulfide levels in nonprotein, protein, and protein-bound fraction has been developed by using HPLC with electrochemical detection. Protein and nonprotein fractions are separated through perchloric acid precipitation. The protein fraction is divided into two aliquots: one which undergoes protein hydrolysis in HCl and the other which undergoes sodium borohydride reduction. The nonprotein, protein and protein-bound fractions generated are then separated by HPLC and the various sulfhydryls (e.g., cysteine, glutathione) and disulfides (e.g., cystine, glutathione disulfide) are measured directly by a dual gold mercury electrode thin layer electrochemical cell. The chromatography takes less than 15 min to separate cystine, cysteine, glutathione, and glutathione disulfide. This assay is more specific and as sensitive as other assays employing 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). Furthermore, this procedure allows the simultaneous measurement of SH and disulfides in the nanogram range without complex extractions or derivatizations.