RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain.

The RP-HPLC method for a simultaneous separation and quantitation of the dinitrophenyl derivative of cystathionine (N,N'-di-DNP) in biological samples together with GSH, GSSG, cysteine and cystine, provides a very useful tool for investigation of the transsulfuration pathway in biological samples, at the same providing results which reflect the redox status (GSH/GSSG ratio) and the potential of the generation of H₂S. An application of the method for the study of the process of transsulfuration in various human brain regions shows the presence of cystathionine in all the investigated regions; it also demonstrates that cystathionine levels vary greatly between particular regions. The highest level in the thalamus and the lowest in the cerebellum were associated with respectively a low or high γ-cystathionase activity, and at the same time, a high cysteine and GSH level in the thalamus and a low value in the cerebellum. Based on the above results, one may suggest a regulatory mechanism responsible for inhibition of the CGL activity at high concentration values of cysteine and/or GSH. Simultaneous determinations of GSH and GSSG levels allow for determining the GSH/GSSG ratio, which reflects tissue redox status. The method may be also employed in determining the activity of γ-cystathionase and cystathionine-β synthase.