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*

Abstract

In the course of establishing an assay for estrogen-2-hydroxylase activity, 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. The amounts of both ³H₂O and [6,7-³H]2-OHE₂ formed were stimulated severalfold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S₂, 17,500 × g supernatant, for tritium release; S₃, 100,000 × g supernatant, for 2-OHE₂ formation). In contrast, maximal ³H2O formation by rat liver was associated with the microsomal (P₃, 100,000 × 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 HIM) 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 μM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t_½12 of 2-OHE₂ during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using ³H₂O formation from [2-³H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE₂ is inhibited, thereby making it practical to quantify enzyme activity on the basis of theamount of catechol estrogen formed. Evidence is also presented that the release of ³H₂O from [2-³H]estradiol by hypothalamic tissue, unrelated to 2-OHE₂ formation, may be enzymatically mediated.