Catechol estrogen formation by brain tissue: a comparison of the release of tritium from [2-3H]estradiol with [6,7-3H]2-hydroxyestradiol formation from [6,7-3H]estradiol by rabbit hypothalami in vitro

A detailed comparison was made between the formation of tritiated water (3H2O) and [6,7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6,7-3H-labeled estradiol, respectively. Maximum activity for both functions was associated with the soluble fractions (S2, 17,500 X g supernatant, for tritium release; S3, 100,000 X g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100,000 X g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 mM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 microM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/2 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min