AN HPLC RADIOTRACER METHOD FOR ASSESSING THE ABILITY OF L-CYSTEINE PRODRUGS TO MAINTAIN GLUTATHIONE LEVELS IN THE CULTURED RAT LENS

Purpose. To apply a high performance liquid chromatographic radiotrace r method to test a variety of L-cysteine prodrugs and one dipeptide pr odrug for their ability to synthesize glutathione in cultured rat lens es. Methods. Rat lenses were incubated for 48 h in a medium containing [C-14(U)]-glycine and prodrugs. Following homogenization and derivati zation, lens extracts were analyzed to determine the extent of biosynt hetic incorporation of this labeled amino acid into [C-14]-glutathione using high performance liquid chromatography with radioisotope and ul traviolet absorption detection. All of the thiazolidine prodrugs conta ined masked sulfhydryl groups to stabilize them against air oxidation. L-buthionine-(S,R)-sulfoximine - an inhibitor of the first step in gl utathione biosynthesis - was present in media containing the dipeptide prodrug. Results. In all cases, a large [C-14]-labeled peak eluted ju st prior to [C-14]-glutathione. This peak had some characteristics of the mixed disulfide of glutathione and L-cysteine, viz., L-cysteine/gl utathione 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 aci d, which increased the rate of [C-14]-glutathione biosynthesis 35% ove r that of the controls. A number of other L-cysteine prodrugs were som ewhat effective, increasing glutathione synthesis 5-30% over the contr ols, while several L-cysteine prodrugs were totally ineffective. The o nly dipeptide prodrug investigated, viz., gamma-L-glutamyl-L-cysteine ethyl ester, increased the biosynthesis of [C-14]-glutathione 18% over control. Biosynthetic rates based on ultraviolet absorption of the de rivatized glutathione demonstrated a similar pattern, the compounds mo st effective in synthesizing [C-14]-glutathione generally yielding the highest ultraviolet glutathione concentrations and the ineffective co mpounds showing the lowest concentrations. Conclusions. 2(R,S)-methylt hiazolidine-4(R)-carboxylic acid, 2(R,S)-n-propylthiazolidine-4(R)-car boxylic acid and N-acetyl-L-cysteine were the only compounds that were statistically significant in yielding higher levels of both ultraviol et and radioactive glutathione as compared to their respective control s. Thus, these prodrugs have very promising anti-cataract potential.