Metabolism of 17α-Ethinylestradiol by Human Liver Microsomesin Vitro: Aromatic Hydroxylation and Irreversible Protein Binding of Metabolites

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Abstract
Incubation of 2,4,6,7-[3H] estradiol and 2,4,6,7-[3H] ethinylestradiol with human liver microsomes and a NADPH-regenerating system resulted in an oxidative attack on tritiated positions of the estrogens. The tritium of the oxidized portions we recovered from the incubations as HTO. Hydroxylation of estradiol and ethinylestradiol at ring B is of minor importance and accounted for only 4–5% as compared to the oxidation of C-2 and C-4 of ring A. Using a substrate concentration of 20 nmol/ml, the initial velocities of hydrogen displacement (X̄ ± SD) from C-2 and C-4 were 119 ± 58 (estradiol) and 237 ± 104 (ethinylestradiol) . The corresponding figures for displacement from the positions 6,7 were 4.2 ± 2.1 (estradiol) and 9.9 ± 4.8 (ethinylestradiol) . The apparent difference between the oxidation of ring A and ring B of estradiol and of ethinylestradiol is explained by the fact that hydroxylation is hindered at C-16 due to the 17α-ethinyl group, and by the competitive nature of 16- and 2-hydroxylation of estrogens. The different rates of aromatic hydroxylation of estradiol and ethinylestradiol caused irreversible binding of metabolites of both estrogens to microsomal protein to different extents. Whereas 342 ± 135 pmoles of estradiol was bound to 1 mg microsomal protein, the corresponding figure for ethinylestradiol amounted to 584 ± 296 pmoles/mg microsomal protein per 30 min incubation. The irreversible binding to protein was diminished by addition of glutathione to the incubation mixture thus trapping the reactive intermediates involved in the binding reaction. The apparent Km values of the oxidation of 2,4,6,7-[3H] estradiol and 2,4,6,7-[3H] ethinylestradiol by human liver microsomes were identical, 5 × 10−5m. The Km of ethinylestradiol obtained for the oxidation of ethinylestradiol with rat liver microsomes was found in the same range (3.6 × 10−5m).

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