High-performance liquid chromatography/fluorescence detection of S-methylglutathione formed by...

Glutathione-S-transferase T1 (GSTT1-1) is a major isoenzyme for the biotransformation of halomethanes. The enzyme activity is located, among other places, in human liver and erythrocytes and is subject to a genetic polymorphism. Metabolism of the halomethanes via GSTT1-1 yields S-methylglutathione (MeSG). A new HPLC assay for the enzymatic formation of MeSG was developed. The glutathione conjugate was derivatized with 9-fluorenylmethyl chloroformate, followed by reverse-phase HPLC with gradient elution and fluorescence detection. The limit of detection was as low as about 39 pmol MeSG on-column. Including derivatization and HPLC analysis, samples could be run at 42-min intervals, thus enabling a high sample throughput. The entire method was validated for analyte recovery (78.2%) and for variations in detector response with replicated injections (11.8%) and with analyses on each of 11 consecutive days (15.2%) with erythrocyte lysate incubations as the matrix. The time-, protein-, and substrate-dependences of the enzymatic catalysis with the model substrates methyl bromide (MeBr) and methyl chloride (MeCl) were studied. Due to its strong electrophilic character, MeBr caused a high level of spontaneous MeSG formation from glutathione in a protein-free medium and a substrate-trapping side reaction in the presence of proteins. Therefore, enzymatic MeSG formation rates may only be determined with MeBr concentrations of at least 3000 ppm in the presence of limited amounts of protein (e.g. 100 microl erythrocyte lysate). In contrast, MeCl showed a lower alkylating potential allowing enzymatic catalysis to be the dominant reaction in incubations with 10,000 ppm MeCl and 2 ml erythrocyte lysate.