High-performance liquid chromatography separation of hydroxylated estradiol metabolites: formation of estradiol metabolites by liver microsomes from male and female rats.

A high-performance liquid chromatography method has been described for the separation of estradiol (E2), estrone (E1) and 27 hydroxylated and keto derivatives of these estrogens. Chromatography of a mixture of 29 estrogen standards resulted in 20 different peaks. Solvent extraction followed by the chromatographic separation and quantification of radioactive metabolites was used for studies on the metabolism of [4-14C]E2 by liver microsomes from adult male and female rats. Liver microsomes from male rats metabolized [4-14C]E2 more rapidly and to a larger number of metabolites than liver microsomes from female rats. Under conditions in which less than 10% of the substrate was metabolized, major metabolites from liver microsomes of male rats cochromatographed with E1, 2-OH E2, 15 alpha-OH E2 and 16 alpha-OH E2, and major metabolites from liver microsomes of female rats cochromatographed with E1, 2-OH E2 and 16 alpha-OH E2. The identity of the metabolites was confirmed by mass spectrometry. Using liver microsomes from male rats and conditions in which more extensive metabolism of the substrate occurred, more than 15 additional metabolites of [4-14C]E2 were observed. Liver microsomes from male rats were many-fold more active than liver microsomes from female rats at catalyzing the 2-, 15 alpha- and 16 alpha-hydroxylation of E2. Our studies on the metabolism of [4-14C]E2 by rat liver microsomes indicate that the profile of E2 metabolites is dependent on the time of incubation, microsomal protein concentration and substrate concentration.