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

Roscoe M. Hersey, Pamela Gunsalus, Tom Lloyd, Judith Weisz

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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 t1/212 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.

Original languageEnglish (US)
Pages (from-to)1902-1911
Number of pages10
JournalEndocrinology
Volume109
Issue number6
DOIs
StatePublished - Dec 1981

All Science Journal Classification (ASJC) codes

  • Endocrinology

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