Purpose. To apply a high performance liquid chromatographic radiotracer method to test a variety of L-cysteine prodrugs and one dipeptide prodrug for their ability to synthesize glutathione in cultured rat lenses. Methods. Rat lenses were incubated for 48 h in a medium containing [14C(U)]-glycine and prodrugs. Following homogenization and derivatization, lens extracts were analyzed to determine the extent of biosynthetic incorporation of this labeled amino acid into [14C]-glutathione using high performance liquid chromatography with radioisotope and ultraviolet absorption detection. All of the thiazolidine prodrugs contained masked sulfhydryl groups to stabilize them against air oxidation. L-buthionine-(S,R)-sulfoximine - an inhibitor of the first step in glutathione biosynthesis - was present in media containing the dipeptide prodrug. Results. In all cases, a large [14C]-labeled peak eluted just prior to [14C]-glutathione. This peak had some characteristics of the mixed disulfide of glutathione and L-cysteine, viz., L-cysteine/glutathione disulfide, but requires further investigation in order to be positively identified. Of the eleven L-cysteine prodrugs investigated, the most effective was 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, which increased the rate of [14C]-glutathione biosynthesis 35% over that of the controls. A number of other L-cysteine prodrugs were somewhat effective, increasing glutathione synthesis 5-30% over the controls, while several L-cysteine prodrugs were totally ineffective. The only dipeptide prodrug investigated, viz., γ-L-glutamyl-L-cysteine ethyl ester, increased the biosynthesis of [14C]-glutathione 18% over control. Biosynthetic rates based on ultraviolet absorption of the derivatized glutathione demonstrated a similar pattern, the compounds most effective in synthesizing [14C]-glutathione generally yielding the highest ultraviolet glutathione concentrations and the ineffective compounds showing the lowest concentrations. Conclusions. 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid and N-acetyl-L-cysteine were the only compounds that were statistically significant in yielding higher levels of both ultraviolet and radioactive glutathione as compared to their respective controls. Thus, these prodrugs have very promising anti-cataract potential.
Bibliographical noteFunding Information:
This research was supported in part by National Institutes of Health research grant EY-01197-21K. W. B. Rathbun is a Research to Prevent Blindness Senior Scientific Investigator and H. T. Nagasawa is a Department of Veterans Affairs Research Career Scientist. The authors wish to thank Geeta Kadambi for conducting the preliminary [35S]-L-cystinei ncubation study and David Peterson and Donald Foss of Beckman Instruments for invaluable aid in various technical phases of HPLC operation and data analysis.