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To study the serum levels of 19-hydroxyandrostenedione (19-OH-A), known as an obligatory intermediate of estrogen biosynthesis and considered to be one of the hypertensinogens, a method using GC-MS with application of synthesized [7,7-d2]androstenedione (A), [7,7-d2] 19-OH-A and [9,11-d2]estrone(E1) as internal standards was newly developed. Normal pregnant women and pregnant women complicated with hypertension near term were selected for the study. The levels of 19-OH-A and E1 increased significantly as gestation progressed [19-OH-A; 224.7 +/- 72.1 (1st trimester), 655.5 +/- 325.4 (2nd trimester). 1517.8 +/- 543.6 (3rd trimester)pg/ml], and a positive correlation was found between the levels of the two steroids. No apparent change was observed in A levels during the course of pregnancy. The mean levels of 19-OH-A in pregnancy complicated with hypertension at 2nd and 3rd trimester were 761.7 +/- 348.9 and 1473.0 +/- 491.4 pg/ml, which were compatible with those in normal pregnancy. The levels of 19-OH-A at delivery in maternal vein (MV) were 1735.1 +/- 683.9 pg/ml. Significantly higher levels of 19-OH-A were found in umbilical vein (UV) (1977.2 +/- 564.9 pg/ml) than those in umbilical artery (109.7 +/- 49.1 pg/ml). 19-OH-A concentration in term placental tissue was 16.3 ng/g.w.w. tissue. This is the first report to demonstrate the serum 19-OH-A levels measured by GC-MS and also to demonstrate the levels in the cord blood. The results indicate that 19-OH-A may be the product of pregnancy and may be derived from the feto-placental compartment.

We reported on the unusually high isotope effect of non-aromatizing androgen 19-hydroxylase in sheep and dog adrenals and the validity of the [3H] water method using [19-3H3] androgen. We have extended the study to examine whether this 19-hydroxylation is catalyzed by a cytochrome P-450 dependent enzyme. Sheep adrenal homogenate (1.65 mg prot.) was incubated in the presence of NADPH (5.6mM) with [19-3H3, 4-14C]-androstenedione (A) (3.2 microM, 8.24 x 10(4) dpm 3H/micrograms, 3H/14C = 17.2) in a total of 1.2 ml PO4 buffer under air at pH 7.4 for 2, 5 and 10 min. [19-3H2, 4-14C]-19-hydroxy-A (19-OHA) with added carrier was purified through extraction, TLC, acetylation to form 19-AcOA, and further TLC to give 19-hydroxylase activity as assessed by the product isolation method. Simultaneously, the [3H] water was measured by distillation, and with correction by the apparent kinetic isotope effect (KH/KT = 11.8), used for assessment of 19-hydroxylase activity. The effects on the hydroxylation by cofactor (NADPH, NADH), incubation atmosphere (N2, CO/O2), cytochrome P-450 inhibitors (metyrapone, clotrimazole) and heating were measured by both methods. Compared to the complete system (89.6pmol/min/mg as 100%), carbon monoxide suppressed 15.8, 59.3 and 86.4% of the 19-hydroxylation when a CO/O2 ratio of 0.1, 1 and 9 was used, respectively. Replacement to nitrogen atmosphere decreased the activity by 93.8%. Replacement of NADPH with NADH (7.5mM) caused more than a 92.1% decrease in activity. Metyrapone at 50 and 200 microM and and clotrimazole at 2.5 and 10 microM suppressed the activity by 82.8, 90.4, 85.4 and 94.9%, respectively. A larger scale sheep adrenal incubation of A (250 microM) under 18O2 atmosphere and isolation of 19-AcOA were carried out in a similar manner. The gas chromatography-mass spectrometry analysis of the purified product showed 48.5% of the product to be 18O-labeled as [M+ + 2], m/e 346. Thus, the non-aromatizing androgen 19-hydroxylase requires NADPH and molecular oxygen. It is strongly inhibited by carbon monoxide and cytochrome P-450 inhibitors. These results indicate that the enzyme system responsible for non-aromatizing androgen 19-hydroxylase in adrenal is a cytochrome P-450 dependent monooxygenase.

19-hydroxyandrostenedione (19-OH A), known for many years as an obligatory intermediate of estrogen biosynthesis, has recently been found to be an amplifier of aldosterone action and in itself a hypertensinogenic steroid (J. Steroid Biochem., 16; 329, 1982). To search the presence of a non-aromatizing andorgen 19-hydroxylase in fetal organs, metabolism of labeled androstenedione(A) in fetal adrenal, liver, lung, brain and kidney was studied. 19-hydroxylase activity was calculated by the amounts of 19-OH A formed from A. The tissue homogenate was incubated with [4-14C]A and NADPH for different periods of time under air. The product, [4-14C] 19-OH A with added carrier standard [6,7-3H] 19-OH A was separated and purified by TLC, acetylation and TLC to constant 3H/14C ratio. The identity of the product was established by C.C.D. and by GC-MS analysis of purified product 19-AcOA in a larger scale adrenal incubation of non-labeled A. 19-hydroxylase activity was 0.9 pmol/min/mg protein for adrenal, but there was a negligible quantity of 19-OH A in lung, brain and kidney. Estrogen production was also assayed by 3H-water method using [1 beta-3H, 4-14C] A (3H/14C = 69.9). No detectable amount of estrogen was found in incubation of any homogenate except for liver. These results indicate that the non-aromatizing 19-hydroxylase activity in fetal adrenal gland is much higher than that in other organs. This is the first report to demonstrate the presence of 19-hydroxylase in human fetal adrenal tissue. It is suggested that 19-OH A secreted from fetal adrenal may also be associated with serum concentration of fetal circulation.