NADPH-dependent formation of polar and nonpolar estrogen metabolites following incubations of 17β-estradiol with human liver microsomes

By using a versatile high-pressure liquid chromatography method ( total elution time similar to 135 min) developed in the present study, we detected the formation of some 20 nonpolar radioactive metabolite peaks ( designated as M1 through M20), in addition to a large number of polar hydroxylated or keto metabolites, following incubations of [H-3] 17beta-estradiol with human liver microsomes or cytochrome P450 3A4 in the presence of NADPH as a cofactor. The formation of most of the nonpolar estrogen metabolite peaks ( except M9) was dependent on the presence of human liver microsomal proteins, and could be selectively inhibited by the presence of carbon monoxide. Among the four cofactors (NAD, NADH, NADP, NADPH) tested, NADPH was the optimum cofactor for the metabolic formation of polar and nonpolar estrogen metabolites in vitro, although NADH also had a weak ability to support the reactions. These observations suggest that the formation of most of the nonpolar estrogen metabolite peaks requires the presence of liver microsomal enzymes and NADPH. Chromatographic analyses showed that these nonpolar estrogen metabolites were not the monomethyl ethers of catechol estrogens or the fatty acid esters of 17beta-estradiol. Analyses using liquid chromatography/ mass spectrometry and NMR showed that M15 and M16, two representative major nonpolar estrogen metabolites, are diaryl ether dimers of 17beta-estradiol. The data of our present study suggest a new metabolic pathway for the NADPH-dependent, microsomal enzyme-mediated formation of estrogen diaryl ether dimers, along with other nonpolar estrogen metabolites.