19-hydroxy-4-androstene-3-17-dione and 19-oxo-delta(4)-androstene-3-17-dione

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.

Topics: Androstenedione; Aromatase; Chromatography, High Pressure Liquid; Estrone; Female; Humans; Microsomes; Placenta

19-Oxoandrostenedione, the product of 19-hydroxyandrostenedione by the 19-oxidase activity of the purified P-450(11)beta system of adrenal cortex mitochondria, was further oxidized and demethylated at the 10-position to give the C18-steroids, estrone (aromatase reaction) and 19-norandrostenedione (nonaromatizing 10-demethylase or C10-19 lyase reaction). These reactions, together with the initial hydroxylation of androstenedione at C19, form a sequence of P-450(11)beta-catalyzed C19-steroid 19-monooxygenase reactions. P-450(11)beta is thus similar to placental endoplasmic P-450AROM in some of its substrate specificity, but the two forms of P-450 appear to be different in both physiology and properties.

Topics: Adrenal Cortex; Androstenedione; Animals; Aromatase; Cattle; Chromatography, High Pressure Liquid; Estrone; Gas Chromatography-Mass Spectrometry; Hydroxylation; Kinetics; Mitochondria; Oxidoreductases; Steroid 11-beta-Hydroxylase; Steroid Hydroxylases

Purified bovine adrenal P-45011 beta has been shown to catalyze conversions of cortisol to cortisone (11-oxidase activity), and 19-hydroxyandrostenedione to 19-oxoandrostenedione (19-oxidase activity), in the reconstituted system consisting of NADPH, NADPH:adrenodoxin reductase, and adrenodoxin. The turnover numbers (mol of product formed/min/mol of P-450) were 1.2 for the 11-oxidase activity and 1.4 for the 19-oxidase activity. No reactions took place when any one of the electron-donating components were omitted either in the presence or in the absence of added NADP+. Likewise, rabbit antibody prepared against P-45011 beta immunoprecipitated the 11-oxidase activity with concomitant loss of deoxycorticosterone 11 beta-hydroxylase activity.

Topics: Adrenal Glands; Adrenodoxin; Androstenedione; Animals; Cattle; Cortisone; Cytochrome P-450 Enzyme System; Ferredoxin-NADP Reductase; Gas Chromatography-Mass Spectrometry; Hydrocortisone; Isoenzymes; NADP; Rabbits

Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O(2) and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with (2)H in addition to (18)O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[(18)O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent (16)O substrates were aromatized under (18)O(2), the formic acid from both substrates contained one atom of (18)O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O(2) is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10beta-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10-C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.

Topics: Androstenedione; Chemical Phenomena; Chemistry; Estrenes; Estrogens; Female; Humans; In Vitro Techniques; Kinetics; Microsomes; Models, Biological; Oxygen Isotopes; Placenta; Tritium

Estrogen is believed to be biosynthesized from androstenedione in placental microsomes by a multienzyme pathway in which 19-hydroxyandrostenedione and 19-oxoandrostenedione (or the hydrated form) are obligatory intermediates. However, both 19-hydroxyandrostenedione and 19-oxoandrostenedione competitively inhibited aromatization of androstenedione, and all three steroids were shown to be mutually competitive. 19-Hydroxyandrostenedione and 19-oxoandrostenedione also competed with androstenedione for binding sites in the microsomes at 4 degrees C. In confirmation of the work of Hollander (Hollander, N. (1962), Endocrinology 71, 723-728), and of Osawa and Shibata (Osawa, Y., and Shibata, K., (1973), Abstracts of the 55th Meeting of the Endocrine Society, Abstract 116) when androstenedione and 19-hydroxyandrostenedione were incubated together, both were converted to estrogen, but little androstenedione was converted to 19-hydroxyandrostenedione. Considered together, these results are incompatible with the multienzyme pathway. Rather, these results may be explained by aromatization of androstenedione at a single catalytic site via enzyme-bound transition states. Both proposed intermediates are, according to this view, by-products which can also be aromatized.

Topics: Androstenedione; Binding Sites; Binding, Competitive; Carbon Radioisotopes; Female; Humans; Isotope Labeling; Kinetics; Mathematics; Microsomes; Placenta; Pregnancy; Tritium