New aromatase assay and its application for inhibitory studies of aminoglutethimide on microsomes of human term placenta.
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 [1 beta,2 beta-3H]- or [1,2-3H]-androstendione or testosterone. In tests with [1 beta,2 beta-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 (10 ml) 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 (1 beta,2 beta-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 2 ml distilled water. This aromatase assay using [1,2-3H]-androstendione was compared to a method described by Thompson and Siiteri [1] depending on 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 Km of androstendione was determined to be 8.9 nM. Aminoglutethimide showed a 50% inhibition of the placental microsomal aromatase at 0.6 microM.
D000616:Aminoglutethimide / Q000494:pharmacology*; D000735:Androstenedione / Q000378:metabolism; D001141:Aromatase / Q000032:analysis*; D005260:Female; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D010088:Oxidoreductases / Q000032:analysis*; D010920:Placenta / Q000187:drug effects / Q000201:enzymology*; D011247:Pregnancy
D000616:Aminoglutethimide; D000735:Androstenedione; D010088:Oxidoreductases; D001141:Aromatase