Clinical analysis on steroids. XXIX. Evidence for 4-hydroxylation of estradiol 17-sulfate by rat liver microsomes.

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Abstract
When estradiol 17-sulfate was incubated with rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system, 4-hydroxyestradiol 17-sulfate was obtained as a minor product accompanying the major metabolite, 2-hydroxyestradiol 17-sulfate. This 4-hydroxylation was shown to occur without cleavage of the conjugate group. At the substrate concentration of 200 μM, which is about twice the Km value of estradiol 17-sulfate 2-hydroxylase, the amounts of 2-and 4-hydroxylated products formed by liver microsomes from male rats were 15.03±1.43 and 0.23±0.01%, respectively. Analogous results were obtained with microsomes from female rats under the same conditions : the yields of 2-and 4-hydroxylated metabolites were 8.75±0.97 and 0.14±0.02%, respectively. Thus, the relative ratios of 2-and 4-hydroxylation of estradiol 17-sulfate were approximately 60 : 1 in both sexes. A synthesis of the authentic conjugate, 4-hydroxyestradiol 17-sulfate, and a method for its assay by high-performance liquid chromatography with an electrochemical detector are also described.