Utilization of Oxygen and Reduced Nicotinamide Adenine Dinucleotide Phosphate by Human Placental Microsomes during Aromatization of Androstenedione

Abstract

Abstract The addition of androstenedione and NADPH to human placental microsomes results in a marked increase in the rate of O2 and NADPH utilization concomitant with estrogen formation. Initially, 5 to 12 moles of O2 and an equivalent amount of NADPH are consumed per mole of estrogen formed, the ratio varying from one microsomal preparation to another. In the presence, however, of 10 to 20 mm KCN, 3 moles of O2, and 3 moles of NADPH are consumed per mole of estrogen formed. This ratio was invariant even when a concentration of KCN sufficiently high to inhibit aromatization was employed. Both NADPH and O2 are required for the conversion of 19-hydroxyandrostenedione to 19-oxoandrostenedione, suggesting that a second hydroxylation is responsible for this conversion rather than oxidoreduction of the 19-alcohol. These results are consistent with previous data suggesting three successive hydroxylations catalyzed by a mixed function oxidase(s) are required during C19 steroid aromatization. The rate of appearance of estrogen intermediates was examined in an effort to determine to what extent the accumulation of these compounds might account for the high rate of O2 consumption observed in the absence of KCN. It was determined that essentially all of the O2 consumed could be accounted for by accumulation of 19-hydroxyandrostenedione and 19-oxoandrostenedione and estrogens. In the presence of KCN, the rates of formation of 19-hydroxyandrostenedione and 19-oxoandrostenedione were greatly inhibited under steady state conditions while the rate of aromatization was only slightly decreased. Moreover, under all conditions, the rate of formation of 19-hydroxyandrostenedione was greater than its rate of conversion to estrogen. Also, the rate of formation of 19-oxoandrostenedione was greater than its rate of disappearance in the absence of KCN. These observations suggest that the final hydroxylation is the rate-limiting step in the aromatization of androstenedione.