High-performance liquid chromatography/fluorescence detection of S-methylglutathione formed by glutathione-S-transferase T1 in vitro

Glutathione-S-transferase T1 (GSTT1-1) is a major isoenzyme for the biotran sformation of halomethanes. The enzyme activity is located, among other pla ces, in human liver and erythrocytes and is subject to a genetic polymorphi sm. 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 chloroform ate, followed by reverse-phase HPLC with gradient elution and fluorescence detection. The limit of detection was as low as about 39 pmol MeSG on-colum n. Including derivatization and HPLC analysis, samples could be run at 42-m in intervals, thus enabling a high sample throughput. The entire method was validated for analyte recovery (78.2%) and for variations in detector resp onse with replicated injections (11.8%) and with analyses on each of 11 con secutive days (15.2%) with erythrocyte lysate incubations as the matrix. Th e time-, protein-, and substrate-dependences of the enzymatic catalysis wit h the model substrates methyl bromide (MeBr) and methyl chloride (MeCl) wer e studied. Due to its strong electrophilic character, MeBr caused a high le vel of spontaneous MeSG formation from glutathione in a protein-free medium and a substrate-trapping side reaction in the presence of proteins. Theref ore, enzymatic MeSG formation rates may only be determined with MeBr concen trations of at least 3000 ppm in the presence of limited amounts of protein (e.g. 100 mul erythrocyte lysate). In contrast, MeCl showed a lower alkyla ting potential allowing enzymatic catalysis to be the dominant reaction in incubations with 10,000 ppm MeCl and 2 ml erythrocyte lysate.