Clearance and production of dehydroepiandrosterone and its sulfate in female baboons.

Serum concentrations of dehydroepiandrosterone (D) are greater and those of D sulfate (DS) are lower in female baboons than in women, suggesting interspecies differences in relative production and catabolism of these steroids. To examine this possibility, the metabolic clearance rate (MCR), interconversion (rho), and production of D and DS were determined in five adult female baboons by constant intravenous infusion of [3H]DS and [14C]D. MCR-D (mean +/- SE) was greater (407 +/- 72.8 1/day; 23.1 +/- 3.4 1/day.kg, P less than 0.01) than MCR-DS (44 +/- 5.7 1/day; 2.5 +/- 0.3 1/day.kg). rho-D leads to DS (mean % +/- SE) was greater (45.4 +/- 3.0, P less than 0.001) than rhoDS leads to D (3.8 +/- 0.6), indicating that the equilibrium favors DS formation. Calculated D production and secretion rates were similar (4.5 and 4.4 microgram/min, respectively), whereas DS production (4.1 microgram/min) was twice its secretion rate (2.1 microgram/min). The large difference between MCR-D and MCR-DS resembles that in human beings. However, when clearance is expressed per kilogram body weight, MCR-D is similar to that in man, but MCR-DS is approximately 15-fold greater in the baboon. It is concluded that compared to values in human beings, the greater MCR-DS in baboons maintains the lower serum DS concentration, whereas the higher serum D levels probably result from the relatively greater secretion rate of D baboons.

Contribution of dehydroepiandrosterone and its sulfate to estradiol in baboon pregnancy.

The metabolic clearance rates (MCR), conversion ratios (C), interconversion (rho), production rates (PR), secretion rates (SR), and relative contributions of maternal dehydroepiandrosterone (D) and D-sulfate (DS) to serum estradiol (E2) were determined in five pregnant baboons (Papio papio; 154-167 days gestation, term = 184 days) by constant intravenous infusion of [3H]DS and [14C]D. MCR-D (mean +/- SE) was greater (39.2 +/- 3.3 1/day.kg, P less than 0.001) than MCR-DS (3.1 +/- 0.3 1/day.kg). Because C-D leads to DS (5.460 +/- 0.461) exceeded (P less than 0.001) C-DS leads to (0.006 +/- 0.001), rho-DS leads to DS was greater (42.5% +/- 3.0%, P less than 0.001) than rho-DS leads to D (7.8 +/- 1.0%). C-D leads to E2 was greater (0.256 +/- 0.040) than C-DS leads to E2 (0.002 +/- 0.000). Using these values and serum levels of D (2.46 microgram/100 ml) and DS (18.9 microgram/100 ml) reported previously, SR and PR of D and DS were calculated. Of the total D produced (11.5 microgram/min), 98% was secreted, whereas only 32% of DS produced (7.1 microgram/min) was due to secretion. Using the serum D and DS levels and their conversion to E2, it was calculated that 89.7% of serum E2 was formed directly from D, 4.4% from D via DS, 1.8% from DS directly, and 4.1% from DS via D. It is concluded that, in spite of the low serum D level resulting from a high MCR, the large SR-D and more efficient conversion of D to E2 makes D, and not DS, the major circulating estrogen precursor in late baboon pregnancy.

Identification of 7xi-hydroxytetrahydrocortisol and 7xi-hydroxytetrahydrocortisone from urine of newborn baboons (Papio papio).

Two highly polar, unconjugated metabolltes of tetrahydrocortisol and tetrahydrocortisone, which had been shown previously to account for greater than 40% of [14C]cortisol (F) administered to baboon neonates, were isolated from urine of a term newborn animal. Metabolites were extracted with ethyl acetate, purified by sequential paper chromatography and crystallization, and identified by chemical and biochemical procedures. Both metabolites of F are 5beta-pregnanes with a hydroxyl of unknown orientation at C-7, a 3alpha-hydroxyl, and a dihydroxyacetone side chain. The two compounds differ from each other by the presence of either oxo or hydroxyl functions at C-11. The following nomenclature is proposed: unknown 1: 3alpha,7xi,11beta,17,21-pentahydroxy-5beta-pregnan-20-one (7xi-hydroxytetrahydrocortisol); unknown 11: 3alpha,7xi,17,21-tetrahydroxy-5beta-pregnane-11,20-dione (7xi-hydroxytetrahydrocortisone). Production of these compounds compensates quantitatively for the decreased formation of F glucuronoside metabolites by baboon neonates, compared with that in adults. Therefore, we propose that 7-hydroxylation is a major alternative pathway for F metabolism in the perinatal period of the baboon.

Serum dehydroepiandrosterone and dehydroepiandrosterone sulphate in baboon (Papio Papio) pregnancy.

Dehydroepiandrosterone (D) and dehydroepiandrosterone sulphate (DS) concentrations were determined by radioimmunoassay in peripheral sera of non-pregnant, pregnant (55 days to term) and newborn baboons and in umbilical sera of animals delivered by Caesarean section close to term. D concentrations (mean +/- SD, microng/100 ml, N) in non-pregnany animals (1.61 +/- 1.32, 23) were not different (P greater than 0.05) from those during pregnancy (1.80 +/- 1.21, 101). DS concentrations, expressed as unconjugated D, in non-pregnant (13.5 +/- 6.0, 23) and pregnant (15.1 +/- 7.5, 101) animals were also similar (P greater than 0.05). However, both D (P less than 0.01) and DS (P less than 0.005) levels increased with gestational age such that serum D (2.46 +/- 1.39, 23, P less than 0.05) and DS (18.9 +/- 5.7, 23, P less than 0.001) levels between 150 and 180 days gestation were greater than in non-pregnant animals. These increases may be important since oestrogen production rises rapidly during late gestation and both D and DS can serve as oestrogen precursors. In both non-pregnant (P less than 0.005) and pregnant (P less than 0.001) animals D and DS concentrations (ratio 1:8) were correlated. Cord serum D levels (2.4 +/- 1.4, 5) were not different from those of maternal serum (P greater than 0.05), while cord DS levels (40.3 +/- 14.8) were greater (P less than 0.001) than those of maternal serum. This may reflect rapid equilibration of D but not DS between foetal and maternal circulations. In sera from neonates, D (19.4 +/- 14.6, 8) and DS 567 +/- 570, 8) concentrations were greater (P less than 0.001) than those in maternal serum, indicating marked post-partal changes in clearance or production of both compounds. The high levels of D and DS in cord and newborn wera are compatible with the view that the baboon foetus makes appreciable contributions of oestrogen precurosrs in pregnancy.

Oestrogen formation from C19 precursors in human choriocarcinoma in culture.

A cloned cell line of human choriocarcinoma was evaluated as a model of human placental oestrogen production. Oestrone formation from dehydroepiandrosterone (D), D-sulphate (DS) or 4-androstenedione (A) was less than or equal to 5% of oestradiol-17beta (Oe2) formation. Oe2 formation from D and A was similar (100-150 pmole/h/10(7) cells); that from DS was 10 times less. Omitting serum from the medium increased Oe2 yield from DS 4-fold; addition of albumin restored these yields to control values (P greater than 0.05, t-test), presumably by binding DS. N6,O2'-dibutyryl-adenosine 3',5'-cyclic monophosphoric acid and theophylline treatment for 72 h stimulated (P less than 0.01) Oe2 formation from D (36%), DS (66%) and A (183%). In intact cells, sulphatase activity, Oe2 formation from D and Oe2 formation from DS equalled those in homogenates (P greater than 0.05) but Oe2 formation from D was greater than that from DS in both systems (P less than 0.001), indicating a deficiency of sulphatase relative to subsequent enzymes of oestrogen synthesis. Steroids, at concentrations previously shown to inhibit placental sulphatase or 3beta-hydroxysteroid dehydrogenase, did not inhibit choriocarcinoma enzymes. Except for its relative sulphatase deficiency and insusceptibility of oestrogen synthesizing enzymes to steroid inhibitors, choriocarcinoma appears to be a useful model of placental oestrogen synthesis.

Metabolic clearance and production rates of progesterone in non-pregnant and pregnant baboons (Papio papio).

The metabolic clearance rate (MCR) of progesterone was examined in the baboon to determine whether the increased serum progesterone concentrations during gestation result from increased production or decreased clearance. MCR was determined by constant infusion of [3H]progesterone in 6 non-pregnant baboons and 13 pregnant animals, between 58 and 175 days gestation. Progesterone MCR (mean+/-SE) was significantly greater in pregnant (1316+/-105 1/day, p less than 0.025; 87.3 +/- 6.2 1/day/kg, p less than 0.01) than in non-pregnant animals (893 +/- 75 1/day; 53.1 +/- 3.5 1/day/kg). There was no correlation between progesterone MCR and gestational age. Mean (+/- SE) progesterone MCR was similar during the follicular (938+/-119 1/day; 58.8+/-3.8 1/day/kg) and luteal (802+/-78 1/day; 52.4+/-6.8 1/day/kg) phases of the menstrual cycle. Serum progesterone concentrations (mean +/- SE, determined by radioimmunoassay, were 11.3+/-1.2 ng/ml in pregnant animals and 5.9+/-2.2 ng/ml during the luteal phase of the menstrual cycle. The mean (+/- SE) calculated progesterone production rates (MCR X serum progesterone concentration) were 4.57+/-1.74 mg/day during the luteal phase of the menstrual cycle and 15.26+/-2.48 mg/day during the last two-thirds of gestation. Thus, increased serum progesterone concentrations in pregnant baboons reflect enhanced production rather than decreased clearance. This is analogous to humans, but contrasts with rhesus monkeys, in which production is not increased during pregnancy. The elevated baboon progesterone MCR, despite increased serum corticosteroid binding capacity, indicates that other factors such as changes in maternal metabolism may also be important in regulating progesterone clearance.

The metabolism of cortisol by term baboon neonates (Papio papio).

The metabolism of iv administered (4-14C)cortisol (F) was examined in 3 female, spontaneously delivered, term baboons less than 24th old. Sixty and 80% of 14C was recovered in urine within 24 and 68 h,respectively. The distribution of urinary 14C was 44.7% unconjugated, 18.1% glucuronoside, 3.3% sulfate, and 24% unextractable with ethyl acetate. The metabolites were isolated by chromatography and crystallization. Eight per cent of unconjugated and 60% of glucuronoside metabolites were more polar than the cortols, the majority being unknown I (Rf 0.15), and unknown II (Rf 0.35), (csf., 6beta-ol-F), Rf 0.44, ethyl acetate-chloroofrmmethanol-water, 25:75:50:50). Unconjugated cortisol plus cortisone (E) represented less than 1% or urinary 14C and tetrahydrocortisone (THE) glucuronoside represented 1.2%. Excretion of tetrahydrocortisol (THF) and products of side-chain cleavage were negligible. Excretion of 20beta-hydroxy metabolites and 6beta-ol-F was less than or equal to 5% of urinary 14C. The cortisol production rate (mean +/- SE) calculated from the specific activity of THE was 4.95 +/- 1.92 mg/day. The glucuronoside/unconjugated 14C-ratio (0.4) contrasts with those previously reported in nonpregnant (4.0), pregnant (1.0), and postpartum (1.3) animals, indicating that the metabolic pattern in newborns is an exaggeration of that in pregnancy. In neonates, unknown I and II compensate quantitatively for decreased glucuronoside excretion. Unknowns I and II are derivatives of THF and THE, suggesting that increased hydroxylase or deficient glucuronyl transferase, rather than impaired delta4-reductase, is responsible for decreased glucuronoside excretion. The low F production rate, reduced glucuronoside formation, and increase in highly polar compounds relative to nonpregnant adults resemble the situation in humans. However, the reduction in glucuronosides is compensated for, quantitatively, by highly polar metabolites, which are extractable from baboon urine with ethyl acetate but are nonextractable from the urine fo human neonates.

The metabolic clearance rates and interconversion of cortisol and cortisone in pregnant and nonpregnant baboons.

Pregnancy alters the pattern of maternal cortisol (F) metabolism and increases the maternal serum cortisol-binding capacity (CBC) of baboons. To determine whether these changes are associated with alterations in F clearance,the metabolic clearance rate (MCR) and interconversion (p) of F and cortisone (E) were measured by continuous infusion of (3H)F and (14C)E in 9 regularly menstruating and 7 pregnant baboons (Papio papio). In nonpregnant animals, the values (X +/- SE) for MCR-E (488 +/- 48 1/day) were greater (P less than 0.001) than those for MCR-F (214 +/- 22 1/day). The p value for the conversion of E leads to F (62.8 +/- 4.7%) was greater (P less than 0.001) than that for the reaction F leads to E (41.6 +/- 3.7%), indicating that F formation is favored. Consistent with MCR-E greater than MCR-F, the per cent of F bound to proteins other than albumin (75 +/- 2) was greater (P less than 0.001) than the per cent of E bound (52 +/-3). The production rate (MCR x peripheral concentration; mug/min) of F (55.1 +/- 7.9) was greater (P less than 0.001) than that of E (28.5 +/-3.9) with essentially all of the F being secreted directly (secretion rate 51.2 +/- 7.9 mug/min). Essentially all of the E produced was derived from circulating F, vitually none being secreted directly (secretion rate 4.6 +/- 3.9 mug/min). Pregnancy did not alter the MCR-F (190 +/- 23 1/day), MCR-E 525+/- 51 1/day), per cent of F (79 +/- 3), or per cent of E (49 +/-3) bound,or F (57.2 +/- 9.2 mug/min) or E (35.5 +/- 4.9 mug/min) production rates. CBC (mug F/100 ml) was significantly (P less than 0.01) elevated (25.3 +/- 2.3, nonpregnant vs 35.1 +/- u.6, pregnant). In addition, p E leads to F was increased (75.5 +/- 1.8%) as was p F leads to E (54.3 +/- 3.7%; P less than 0.01). We have concluded that the MCR-F during pregnancy is more dependent on alterations in maternal metabolism than on the increased serum CBC characteristic of gestation. We suggest that the latter factor may be important in regulating the physiologic levels of the other steroids which bind to it.

Utilization of dehydroepiandrosterone and its sulphate for oestrogen production by pregnant baboons (Papio papio).

The effect of varying maternal circulating 3beta-hydroxy-5-androsten-17-one (D) and its sulphate (DS) on urinary oestrogen (Oe) excretion by baboons was determined between 65 and 174 days gestation (term = 184 days). Oe (mainly oestrone) was determined by radioimmunoassay after enzymic hydrolysis of conjugates. Increments in Oe were estimated from the difference between Oe during 5 days pre- and post-treatment. Oestradiol-17beta (5 mg in 5.0 ml saline-ethanol, 1:1) administered iv increased (P less than 0.001) Oe by 2.46 +/- 0.71, 7 (mean, mg +/- SD, number of experiments). The response to 100 mg D (3.20 +/- 1.50, 7) was greater (P less than 0.001) than that due to 100 mg DS (0.03 +/- 0.75, 7). The response to DS was not different from that due to saline-ethanol alone (-0.05 +/- 0.27, 5). Betamethasone (9alpha-fluoro-11beta,17,21-trihydroxy-16beta-methyl-1,4-pregnadiene-3,20-dione) administration (3 mg bi-daily, im) caused an approximately 90% reduction in Oe within 4 days. In animals treated chronically with betamethasone the response due to 100 mg D (2.74 +/- 1.04, 5) was again greater (P less than 0.001) that that due to 100 mg DS (0.10 +/- 0.09, 5). ACTH (8 U or 40 U, im, N = 11) produced a variable but not significant mean change in Oe at either dose level. Metyrapone (300 mg every 4 h for 6 doses) increased (P less than 0.005) Oe (0.77 +/- 0.48, 5). It is concluded that in normal pregnant animals and those treated with betamethasone, in which endogenous levels of D and DS are presumably reduced, D is preferentially used over DS for oestrogen produced. Variations in DS availability do not produce marked changes in oestrogen production. This suggest that rapid increases in oestrogen production, as occur for example close to term, may result from increased availability of D rather than DS. The variable responses to ACTH and the increase in Oe following metyrapone administration are compatible with significant foetal contributions of oestrogen precursors, the production of which is under ACTH control.

Inhibition of placental 3beta-hydroxy-steroid dehydrogenase by naturally occurring steroids. A potential mechanism regulating oestrogen synthesis from unconjugated precursors.

We have proposed that inhibition of placental steroid 3-sulphatase by endogenous steroids may regulate oestrogen synthesis during human pregnancy. The possibility that an additional regulatory mechanism, involving the placental 3beta-hydroxy-steroid dehydrogenase (SDH), may also be operative has now been examined. Inhibitory effects of naturally occurring steroids on SDH activity were determined from the reduction in initial rate of conversion of 3H-dehydroepiandrosterone to non-digitonin precipitable products by 10 000 x g supernatant from homogenates of human term placentae. The apparent Km for dehydroepiandrosterone was 0.33 x 10(-6) M. delta4-3-Oxo products of SDH action (4-androstene-3,17-dione, app. Ki=0.60x10(-6) M; progesterone, app. Ki=1.5x10(-6) M) were the most potent inhibitors and appeared to act non-competitively. Delta5-3beta-Hydroxy alternative substrates were less inhibitory and in the case of pregnenolone (app. Ki=4.5x10(-6) M) behaved competitively. 11beta-, 16alpha-, 17alpha- or 21-hydroxylation and epimerization of 3beta- or 17beta-hydroxyl functions of inhibitors decreased their activity. It is concluded that inhibition of both sulphatase and SDH by endogenous steroids may provide complementary methods of regulating placental oestrogen synthesis in vivo. The SDH mechanism may regulate oestrogen synthesis from unconjugated precursors, either formed within the placenta or derived from the circulation. The major potential inhibitors appear to be delta4-3-ketones, acting non-competitively, and formed within the placenta. In the sulphatase mechanism alternative substrates of extraplacenta origin, acting competitively, are the major potential inhibitors controlling utilization of conjugated precursors.

Cortisol metabolism in the baboon during pregnancy and the postpartum period.

The metabolism of iv-administered 14C-cortisol (F) by pregnant baboons (107, 124 and 150 days gestation) was compared with that previously reported for nonpregnant animals and with that of animals examined 6-18 h after spontaneous vaginal delivery (178 plus or minus 4 days). Unconjugated, glucuronoside (beta-glucuronidase) and sulfate (H2SO4-ethyl acetate) fractions were extracted with ethyl acetate from urine containing more than 80% of injected 14C. Metabolites of interest were isolated by paper partition chromatography and purified by crystallization and derivative formation. Compared with nonpregnant animals, the following changes (P less than 0.05) were observed in pregnancy: (1) an increase in the percent urinary 14C in the unconjugated fraction and a decrease in the proportion of 14C appearing in the glucuronoside fraction; (2) an increase in excretion of metabolites more polar than the cortols; (3) a decrease in excretion of metabolites less polar than cortisone in the glucuronoside fraction; (4) an increase in unconjugated F excretion. Production rate of F (11.9 plus or minus 0.7 mg/day) estimated by isotope dilution and percent urinary 14C in tetrahydrocortisol and tetrahydrocortisone from the glucuronoside fraction were as in nonpregnant animals. With the exception of an increase in F production (22.7 plus or minus 0.8 mg/day), presumably the result of the stress of labor, F metabolism in the immediate postpartum period was strikingly similar to timals indicates that changes in the mother alone can account for the altered metabolic disposition of F in pregnancy and suggests that the fetus takes little part in metabolism of maternal circulating F.