Simultaneous determination of cytosolic glutathione S-transferase and microsomal epoxide hydrolase activity toward benzo[ a]pyrene-4,5-oxide by high-performance liquid chromatography

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Cytosolic glutathione S-transferase (GST) and microsomal epoxide hydrolase (EH) are important detoxification enzymes for many epoxide xenobiotics. We have developed a rapid, simple, and convenient HPLC assay which measures both of these enzyme activities toward benzo[ a]pyrene-4,5-oxide (BaPO) in tissue homogenates. Tissue fractions were incubated at 37°C in the presence of 5 m m glutathione. Reactions were initiated by addition of BaPO and terminated by the addition of ice-cold acetonitrile containing 2-methoxynaphthalene as an internal standard. Samples were analyzed directly on a 15-cm C18 reverse-phase column at room temperature, with a ternary solvent program which utilized 0.01% ammonium phosphate buffer (pH 3.5), acetonitrile, and water. The uv absorbance (260 nm) was monitored. Baseline resolution of BaPO, BaPO-GSH, and BaPo-diol and the internal standard was accomplished in 10 min. In rat hepatic S9, production of both BaPO-GSH and BaPO-diol was linear with time and protein up to 15 min and 500 μg/ml, respectively. Coefficients of variation for replicate analyses were 2.7 and 3.7% for GST and EH activities in S9, respectively. With fluorescence detection (ex, 241; em, 389 nm), this assay was sensitive enough to measure GST and EH activities in mononuclear leukocytes (MNL). GST and EH activities in 109 human MNL samples were 142 ± 74 (mean ± SD; range 21–435) pmol/mg/min and 19 ± 9 (mean ± SD; range 3–59) pmol/mg/min, respectively. These results demonstrate the simplicity, high sensitivity, and applicability of this assay for a broad range of tissues.