High-performance liquid chromatographic assay of cytosolic glutathione S-transferase activity with styrene oxide

A high-performance liquid chromatographic (HPLC) assay for measuring cytosolic glutathione S-transferase activity with styrene oxide is described. After incubating lung or liver cytosol with reduced glutathione and styrene oxide, unreacted styrene oxide is extracted into ethyl acetate. An aliquot of the aqueous phase is evaporated to dryness and reconstituted in the mobile phase for HPLC analysis. The two glutathione conjugates of styrene oxide [S-(1-phenyl-2-hydroxyethyl)glutathione and S-(2-phenyl-2-hydroxyethyl)glutathione] are separated in less than 10 min; quantitation of transferase activity is based on the comparison of the UV absorbance of the two conjugates at 254 nm with synthetic conjugate standards. As little as 1 nmole of either conjugate can be quantitated with good precision. This assay has advantages over previously published methods for measuring styrene oxide glutathione S-transferase activity as it does not depend on the use of relatively unstable and expensive radiolabelled substrates.