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New aromatase assay and its application for inhibitory studies of aminoglutethimide on microsomes of human term placenta

Authors
Journal
Journal of Steroid Biochemistry
0022-4731
Publisher
Elsevier
Publication Date
Volume
17
Issue
3
Identifiers
DOI: 10.1016/0022-4731(82)90204-7

Abstract

Abstract A new, simple, fast and highly practicable aromatase assay and its application is described. This test depends on the release of tritiated water after aromatization of [1β,2β- 3H]- or [1,2- 3H]-androstendione or testosterone. In tests with [1β,2β- 3H]-androstendione, nonradioactivity labeled estrogens are formed whereas in tests using [1,2- 3H]-androstendione as substrate both estrogens and water contain tritium atoms. Tritiated water is determined by a two-phase scintillation technique depending on the limited emulsifying capacity of dioxane-based scintillation solution for water. A small volume (0.1 ml) of water is completely emulsified by the scintillation solution (10ml) and all tritium-labeled substances can be measured. After addition of 2 ml distilled water 95% of the tritiated water is partitioned in the aqueous phase and only (1β,2β- 3H]-androstendione or [1,2- 3H]-androstendione and tritium labeled estrogens can be counted. The amount of tritiated water in each test can be calculated by impulse differences before and after addition of 2ml distilled water. This aromatase assay using [1,2- 3H]-androstendione was compared to a method described by Thompson and Siiteri [1] depending on an extraction of steroids prior to scintillation counting. A good agreement of both methods was found. In tests with human term placenta aromatase in microsomes the apparent K m of androstendione was determined to be 8.9 nM. Aminoglutethimide showed a 50% inhibition of the placental microsomal aromatase at 0.6 μM.

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