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The origin of and metabolic fate of deoxycorticosterone and deoxycorticosterone sulfate in pregnant women and their fetuses
J.
sreroid Biochem. Vol. 20, No. 1, pp. 237-243,1984 Printedin Great Britain.All rightsreserved
0022-4731/84 $3.00+ 0.00 Copyright0 1984PergamonPressLtd
THE ORIGIN OF AND METABOLIC FATE OF DEOXYCORTICOSTERONE AND DEOXYCORTICOSTERONE SULFATE IN PREGNANT WOMEN AND THEIR FETUSES M. LINETTECASEY,ALIREZAGUERAMI,CRAIGA. WINKELand PAULC. MACDONALD Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Department of Biochemistry. The University of Texas Southwestern Medical School, Dallas, Texas 75235. U.S.A.
Summary-DOC and DOC-SO,, which are present in large amounts in the blood of pregnant women, are derived from sources other than maternal adrenal. Other investigators demonstrated that treatment of near-term pregnant women with ACTH or dexamethasone did not cause alterations in the blood levels of DOC. To define the source(s) of DOC and DOC-SO, in plasma of pregnant women, we evaluated the conversion of plasma progesterone (P) to DOC in extraadrenal sites. DOC is formed from plasma P and, provided that the pregnancy is one characterized by the usual large production of estrogen, DOC production in a given woman is proportional to the level of P in plasma. Unlike other steroid conversions or interconversions, however, the fractional conversion of P to DOC among apparently normal persons varied widely 0.011 f 0.003 (mean + SEM, n = 40, range = 0.001 to 0.030). In women pregnant with a normal living fetus, the product of the production rate of P and the fractional conversion of P to DOC is sufficient to account for the majority of DOC produced in the mother. There may be a second source of DOC, i.e. the transfer of DOC from the fetal to the maternal compartment in a manner that involves (a) direct transfer of DOC by way of trophoblast and (b) by desulfurylation of DOC-SO., from fetal umbilical arterial plasma in trophoblast and thence transfer of DOC liberated in trophoblast to the maternal compartment. Presently, it is clear that DOC-SO, in blood of pregnant women is not derived from plasma DOC; and there is little or no evidence in support of the proposition that DOC-SO, (as a sulfoconjugate) is transferred from the fetal to the maternal compartment because of placental hydrolysis to DOC. Among the extraadrenal tissue sites identified as those in which 21-hydroxylation of plasma P could be effected are some also believed to be tissue sites of mineralocorticosteroid action, viz. kidney, aorta. thymus, and spleen. Quantitatively, the origin of DOC in the fetus is not as clear as in the maternal compartment; yet. many tissues of the fetus have been identified in which both steroid 21-hydroxylase and 21hydroxysteroid sulfotransferase activity are present. Thus, in the human fetus, extraadrenal as well as adrenal production of DOC and DOC-SO, are possible.
INTRODUCTION Pregnancy in women is characterized by a series of hormonal excesses that are of unparalleled magnitude in the mammalian kingdom. This generality is applicable to the production of estrogenic hormones, i.e. estradiol-17P and estriol as well as progesterone (P) [cf. 11. This near unbridled production of steroid hormones also is applicable to the fetal adrenal cortex, which may daily produce upwards of 200 mg of steroids, principally As-3P-hydroxysteroids as the sulfate esters of these steroids, such as dehydroisoandrosterone sulfate and pregnenolone sulfate [cf. 21. In a similar manner, there are incredible amounts of biologically active protein hormones produced during human pregnancy, e.g. chorionic gonadotropin (hCG) and placental lactogen (hPL) [cf. 1, 31.
Address correspondence to: M. Linette Casey, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235, U.S.A. .%aZO,l--Q
237
But more than that, there is an equally massive increase in the production of mineralocorticosteroids in women during the latter few weeks and months of pregnancy [4, 51. This enormous production of mineralocorticosteroids in pregnant women constitutes an intriguing biological phenomenon, the control of which many investigators have attempted to define, but also one that has been suggestive, to many, that mineralocorticosteroid excess may be fundamental in the pathophysiology of pregnancy disorders that are characterized by sodium retention, edema formation, and, ultimately, hypertension. It is known that the rate of secretion of aldosterone by the adrenal cortices of pregnant women commonly is 10-20 times that found in men and nonpregnant women [4, 51. It is clear, however, that this large amount of aldosterone secretion in pregnant women is due to increased stimulation of the zona glomerulosa of the maternal adrenal by angiotensin II [6], which is present in plasma of pregnant women in high concentrations [cf. 71. This latter event is mediated by an estrogen-induced increase in biosynthesis of angiotensinogen [8, cf. lo], the precursor of angiotensin I, together with increased amounts of
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M. LINETFECASEY et
rerun that is secreted by the juxtaglomerular apparatus of kidney [ll]. These two phenomena, in concert, bring about an increase in angiotensin I, which thence, in the endothelium of many blood vessels, principally those in lung, is converted to angiotensin II, the trophic agent for the zona glomerulosa of the adrenal and the site of aldosterone biosynthesis. That this is a correct formulation of the mechanisms by which the rates of aldosterone secretion by the adrenals are increased in pregnant women is supported by the findings that there is a decrease in aldosterone secretion in pregnant women after salt intake is increased [12] or after the development of hypertension [5, 131 as in men and nonpregnant women. Conversely, low salt diets [12] and hemorrhage lead to an increase in renin secretion and an increase in aldosterone secretion in pregnant women as in men and nonpregnant women [ 131. Thus, whereas the rates of aldosterone secretion in pregnant women, compared with those in men and nonpregnant women, are massive, the rate of aldosterone secretion in pregnant women is affected by known negative and positive effecters. In addition to increased aldosterone secretion in pregnant women, however, there also is a striking increase in the rate of formation of another mineralocorticosteroid. i.e. deoxycorticosterone [DOC] and its sulfate ester, DOC-.S04. The biosynthetic mechanism(s) responsible for the massive increase in DOC production in pregnant women is unclear; for example, neither ACTH [14-171 nor dexamethasone [14-16, 181 treatment of pregnant women causes a change in the levels of DOC in blood of treated subjects. Angiotensin II administration does not affect blood levels of DOC [ 191. But, it also is known that in men and nonpregnant women, DOC secretion by the adrenal cortex is regulated by the action of ACTH [20]; therefore, most of the DOC in the maternal compartment cannot arise by adrenal secretion. These findings, together with the fact that the levels of DOC and DOC-SO4 in fetal umbilical plasma are considerably greater than those in maternal plasma, led to the suggestion that DOC and DOC-SO4 in maternal plasma could be accounted for by transfer from the fetus [cf. 21, 221. For a variety of reasons, we speculated that an alternative explanation for the origin of DOC in pregnant women may be more tenable; viz., DOC could originate by extraadrenal 21-hydroxylation of plasma P. The rate of secretion of P in normal pregnancy can be massive, i.e. 200-600 mg per day [3]; ergo, a small fractional conversion of plasma P to DOC could account for a massive increase in DOC formation. It should be recalled that the rate of DOC secretion in men and nonpregnant women is only about 50 pg per day [cf. 231. Since rates of secretion of DOC in pregnant women may be greater than 8 mg per day [cf. 231, it seemed unlikely that transfer of this amount of DOC from fetus to mother could be accomplished.
al.
These hypotheses were tested as follows: (1) determination of the fractional conversion of plasma progesterone to DOC ([p]p-g$c) in men and in pregnant, nonpregnant, and adrenalectomized women; (2) determination of the production rate (PR) of P; (3) comparison of the estimated PR-DOC derived from plasma P with the total PR-DOC in the maternal compartment; (4) determination of the levels of the DOC and DOC-SO4 in umbilical arterial and venous sera and in maternal serum; (5) evaluation of steroid 2 1-hydroxylase and 2 1-hydroxysteroid sulfotransferase activities in various maternal and fetal tissues; and (6) an evaluation of the origin of plasma DOC-SO, from a variety of putative circulating C,,-precursors, e.g. plasma DOC, P, and pregnenolone. These studies were conducted, and are continuing to be conducted, on the basis of the proposition that there appear to be no negative effecters (with respect to alterations in ACTH or angiotensin II) for the formation of DOC in pregnant women. Specifically, in the case of aldosterone, negative effecters such as increased salt intake, alterations in body position, and the development of hypertension act to reduce circulating levels of angiotensin II and thence the rate of aldosterone secretion. These negative effecters do not appear to be operative with respect to extraadrenal formation of DOC in near-term pregnant women. Thus, it can be envisioned that in the sequence of events that are involved in the pathophysiology of mineralocorticosteroid excess, viz. sodium retention, edema formation, and hypertension, there is no physiologic means for reducing the rate of formation of DOC in near-term pregnant women, save for a reduction in the rate of placental formation of progesterone, which has not been described short of death of the fetus or delivery of the placenta, and (as described later) decreased secretion of estrogen [24].
MATERIALS AND METHODS
Steroids
Radiolabeled and nonradiolabeled steroids were purified by gradient-elution column chromatography on celite-ethylene glycol, liquid-liquid partition chromatography on cehte, and by thin-layer chromatography (TLC) as previously described in detail [25, 261. Radiolabeled steroids were infused intravenously either as a bolus injection in a 10 ml solution that contained NaCl (0.15 M), glucose (5%, w/v), and ethanol (6%, v/v) ([p]‘-sy’) or else as a continuous infusion for times that varied from 4-16 h in a solution of glucose (5%, w/v) that contained ethanol (6%, V/V) ([p]p-~~c). In cases in which urine collection was conducted, the urine was collected from the commencement of the radiolabeled tracer infusions for the succeeding 72 to 96 h.
DOC and DOC-sulfate
3l.l
I I
1
P
‘4C
4
l- L
DOC
Tetrohydw- DOC (Glucuronosids)
r
Fig. 1. In viva internal standard method for estimation or D”C Urinary tetrahydro-DOC (glucuronoside) is con[P]’ -BU sidered to be a unique metabolite of plasma DOC.
Determination of the [p] p-Bu D°C, /PI
DOGDsoBc_SO,
or
,,DOGDf;-SO,
The transfer constants of conversion of either P to DOC or DOC to DOC-SO, were determined by use of the radiolabeled internal standard in vivo technique that has been described in detail [26, 271. Briefly, tritium-labeled precursor (or product) or carbon-1C labeled precursor (or product) were infused intravenously (Fig. 1). Studies also were conducted by evaluation of the so called “blood to blood” transfer constant of conversion of one steroid to another by the continuous infusion of radiolabeled tracer precursor and product and the sampling of blood at times when equilibrium conditions were believed to have been established. Radiolabeled DOC or DOC-SO, or both were isolated from plasma, and from the 3H:‘4C of the product of the steroid under investigation and that of the radiolabeled tracers infused, the transfer constant(s) of conversion was computed. Also, after the infusion of radiolabeled precursor and product hormones, urine was collected for a time sufficient to ensure complete excretion of all radiolabeled metabolites (Fig. 2). From the urine collected (after hydrolysis of steroid conjugates), steroids were isolated and separated, and unique metabolites of precursor and products were purified by a variety of chro14C
(50)
1
I
DOC-
-
/
SO4
I
1
Tetmhydm - DOC (Glucumnosida)
matographic techniques that included gradientelution column chromatography on ethylene glycolcelite, partition chromatography on celite, multiple thin-layer chromatographic steps, derivative formation, and chromatography of the derivatives, and finally by repeated crystallization to achieve constant 3H:‘4C ratios [25, 261. Alternatively, a portion of the steroids may have been treated by radiolabeled acetic anhydride to establish the specific activity of a particular metabolite(s). These studies were conducted to compute the production rate of the steroid(s) in question. In pregnant women, the blP-EC was computed from the ratios of the concentrations of P:DOC in plasma and [p]p-Bycdetermined post-partum. The values were similar. Determination of steroid 21 -hydroxylase and 21hydroxy suIfotransferase activities in human tissues
By use of differential centrifugation procedures of homogenates of human adult and fetal tissues, microsome-enriched preparations were prepared for assay of steroid 21-hydroxylase activity. Incubations were conducted with [3H]P, in various concentrations, for various times, and with various amounts of tissue protein in the presence of an NADPH-generating system [27]. The assays were linear with times of incubation for 2 h and with protein concentrations up to 1 mg. In all tissues in which steroid 21-hydroxylase activity was demonstrated, the apparent K,,, of the enzyme for P was similar [27-281. In a similar manner, a cytosolic fraction was prepared for the assay of 21-hydroxysteroid sulfotransferase activity. An excess amount of sulfate donor, phosphoadenosine phosphosulfate, was used to optimize activity. Optimum conditions were established for substrate concentration, time of incubation, pH, and protein concentrations. The reaction was linear with time of incubation and with protein concentrations that varied up to 240 kg [29]. Determination activity
of
placental
steroid
21-sulfatase
Trophoblastic tissue, principally free of adventitious and vascular tissue, was prepared. A microsome-enriched preparation was prepared as described [27]. Optimum conditions were established with respect to pH of the incubation mixture and time of incubation and tissue protein concentration. It is notable that the only human tissue in which steroid 21-sulfatase activity is demonstrable is in trophoblastic tissue of the human placenta [30].
1 DOC CS04)
Fig. 2. In viva internal standard method for evaluation of potential interconversion of plasma DOC and DOC-SO+ Urinary DOC(SOJ and urinary tetrahydro-DOC (glucuronoside)
239
in pregnancy
are considered to be unique metabolites of plasma DOC-SO, and plasma DOC, respectively.
RESULTS
In previous studies [25], and in studies that were conducted in the present investigation, we found that
M. LINEITE CASEY et al
240
Steroid 21-sulJh_tase activity LIVER BILE ,NTESTlNE
a
I ‘4C3
50
PLASMA
3H7 PLASMA
P
DOC
DOC-SO,
50
-c < 0.3
+ >I500
I
t
v
DOC-SO4
Fig. 3. Formulation of metabolic disposition of plasma DOC and DOC-SO,. Plasma DOC and DOC-SOI are not interconverted. DOC is sulfurylated in liver. but intrahepatic DOC-SO, does not escape into blood; rather, it is excreted quantitatively into bile wherein it is converted, by way of the action of bacterial enzymes, to progesterone or metabolites thereof.
Intravenously administered radiolabeled DOCSO4 in the human is not hydrolyzed to DOC. except by the action of bacterial enzymes in the intestine [26]. Thus, it can be concluded that in men and in nonpregnant women, steroid 21-sulfatase activity is not a constituent of human tissues in men or nonpregnant women. By way of contrast, steroid 21-sulfatase activity is demonstrable readily in tissue preparations of the human placenta [30]. Since steroid 21-steroid sulfatase activity is present in placenta, the potential exists for the hydrolysis of DOC-SO, (of fetal origin) in placenta and, thence, the transfer of the DOC so formed into the mother [cf. 221. DISCUSSION
[p]p-,D,oc m men
and in nonpregnant, pregnant, and adrenalectomized women is 0.011 + 0.003 (mean + SEM, n = 40). There are several notable features with respect to [p] ’ suD°C. First, the [p]p-BTc varies widely among presumably normal persons, viz. wEC of 0.001 to 0.030 have been found [25, 311. On the other hand, in a given person, [p]‘-t$’ is remarkably constant irrespective of the plasma level of P, i.e. in a given woman, during the follicular and luteal phase of the ovarian cycle [32], with wide variations in the concentrations of circulating progesterone, and in women during pregnancy, the [p]p-~~c is the same in a given woman [25]. There are two notable exceptions to this generality: In women pregnant with either (1) a dead or (2) an anencephalic fetus in whom progesterone levels are high, the [p]p-,D,ocis strikingly reduced [24]. On the other hand, in men and in nonpregnant and pregnant women, the [p]“““~~“““~ uniformly is 0 [26]. Thus, circulating DOC is not converted to DOC-SO, that enters plasma (Fig. 3). The conversion of plasma [3H]P and [3H]pregnenolone to plasma [3H]DOC-S04 also is negligible in nonpregnant persons. the
From the results of the present and previous investigations [24,25,38]. it is clear that DOC in the plasma of near-term pregnant women arises primarily by extraadrenal conversion of plasma P (of placental origin) to DOC in nonadrenal tissue(s). some of which appear to be those in which mineralocorticosteroids are known to exert specific actions, e.g. kidney 1271. aorta [33], and lymphoid tissues [34]. In addition, some DOC in mother may arise by transfer from the fetus by: (a) direct transfer of DOC from fetus to mother [21] and (b) transfer of DOC from fetus to mother [22] after hydrolysis of fetally derived DOC-SO, to DOC in placenta by action of steroid 21-sulfatase activity [30]. In pregnant women, there must be little secretion of DOC or DOC-SO4 by the maternal adrenal cortex [25. 261. This is consistent with the observation that the rates of secretion of other Czl-steroids (except for aldosterone [5]) by the maternal adrenal are either not increased or are reduced [3Y]. The origin of DOC-SO4 in the maternal compartment is unknown. Clearly. plasma DOC is not converted to DOC-SOI that enters plasma (251. The KIDNEY MEN AND NONPREGNANT WOMEN
Extraadrenalsteroid21-hydroxylaseand sulfotransferase activities Steroid
21-hydroxylase
activity
has been
-DOG
demons-
in a number of tissues of the human adult 1271 and fetus [28, 33, 341. Notable among these tissues are those in which mineralocorticosteroids are believed to act, e.g. kidney [28], blood vessels [33], thymus. spleen, and, possibly, urinary bladder [34]. Thus, the potential exists for the formation of a potent mineralocorticosteroid in its tissue site of action. In these studies, we also found considerable 21-hydroxysteroid sulfotransferase activity in human fetal liver, adrenal, kidney, and intestine [35]; on the other hand, there was little 21-hydroxysteroid sulfotransferase activity in tissues of the adult, except in adrenal and liver, as demonstrated by others [36, 371. trated
1 J
Fig. 4. Hypothetical model to explain the origin of DOC and DOC-SO4 in pregnant women. Estrogen. directly or indirectly, stimulates extraadrenal steroid 21-hydroxylase activity and may, as in other animal species. act to stimulate 21-hydroxysteroid sulfotransferase activity in extraadrenal sites of DOC formation.
241
DOC and DOC-sulfate in pregnancy
same is true (in men and nonpregnant women) of plasma P and pregnenolone; namely, these two Gi-steroids also are not converted to DOC-SO4 that enters plasma. There seems to be little likelihood of the transfer of DOC-SO4 as such [22] from fetus to mother since there is steroid 21-sulfatase activity [3O] in the human placenta. Thus, the origin of DOC-SO, in pregnant women is an enigma. The in situ formation of DOC-SO, in an extraadrenal site in pregnant women from a circulating precursor other than DOC or from a precursor yet unidentified is a possibility. Whereas, as we have stated, the formation of DOC in extraadrenal tissues is not affected by alterations in the levels of angiotensin II or ACTH, the [p]p-~~c in women who are pregnant, but hypoestrogenic, is decreased [24]. Namely, in women with a dead or anencephalic fetus, i.e. in women whose pregnancies are characterized by relative hypoestrogenism, there is a decrease in the [p]p-Fcc and [p]‘-@‘. We reached this conclusion since treatment of women with such hypoestrogenic pregnancies with estrogen effected an increase in [p]‘-BsJ”’and an increase in the PR-DOC without a decrease in the MCR-DOC [24]. Yet other factors also appear to be important in the regulation of extraadrenal 21-hydroxylase activity. Recently, it was shown that immunologic challenge of experimental animals (in which 21-hydroxylase activity is identifiable in splenic tissue [40]) causes a striking decrease in the specific activity of steroid 21-hydroxylase in splenic tissue [41]. Since the gene that encodes for steroid 21-hydroxylase activity is within the HLA locus [42], it is intriguing to speculate that immunologic challenge may act to influence the rate of 21-hydroxylation of Czl-steroids. Thus, there may be physiologic and pathophysiologic processes that do act to bring about alterations in the rate of DOC formation from plasma P. Quantitatively, the origin of DOC in the fetus is less clear. It has been demonstrated that both DOC and DOC-SO4 can be formed in the fetal adrenal; but. also, it has been shown that in many fetal tissues, steroid 21-hydroxylase [28, 33, 341 and 21-hydroxysteroid sulfotransferase [29, 351 activities are demonstrable. There is evidence that is suggestive that estrogen acts, at least in many experimental animals, to increase steroid sulfotransferase activity [43]. Thus, it is conceivable that during pregnancy, in both the mother and fetus, steroid 21-hydroxylase and sulfotransferase activities are increased in tissues other than liver and adrenal. Thereby, an explanation could be offered for the dramatic rise in DOC and DOC-SO, levels in both the maternal and the fetal compartments. An explanation also could be offered for the finding that the PR-DOC appears to be proportional to the level of P in plasma provided that the pregnancy is not associated with hypoestrogenism. Based on the blood concentration of P in late pregnancy, as well as the apparent K, of steroid 21-hydroxylase for P in extraadrenal tissues
[28, 291, during late pregnancy, the steroid 21-hydroxylase enzyme in extraadrenal tissues could become saturated. If this were true, then only by virtue of an increase in steroid 21-hydroxylase activity, as a consequence of the hyperestrogenism of pregnancy, could the [p]p-~~c remain constant, i.e. irrespective of P levels. To date, there has been no pathophysiologic alteration in extraadrenal DOC formation that can be ascribed specifically to an entity known to be associated with apparent mineralocorticosteroid excess. Perhaps the one exception to that generalization has been the identification of a higher incidence of pregnancy-induced hypertension (PIH) among women in whom [p]p-~~c is greater than 0.025 [44]. On the other hand, this cannot be the single cause of PIH since PIH occurs in women in whom [p]p-~~c is not greater than that in women who do not develop PIH [44]. Nonetheless, the potential for the unbridled production of a potent mineralocorticosteroid, DOC, in its potential sites of action (such as kidney and blood vessel) remains as a tantalizing possibility to explain heretofore inexplicable pathophysiologic disorders of human pregnancy and even those ascribed to the premenstrual syndrome in ovulatory women in whom DOC levels are high in the mid-luteal phase of the ovarian cycle as a consequence of the extraadrenal conversion of progesterone, which is secreted in large amounts by the corpus luteum. Present avenues of investigation include an evaluation of the origin of the very potent mineralocorticosteroid, 19-nor-DOC, and the role of DOC-fatty acid esters, which now are known to be formed in fetal kidney tissues. To date, it has not been possible to demonstrate, in humans, the conversion of DOC to 19-norDOC [45]. On the other hand, in preliminary studies, we found that, in fetal kidney tissue, DOC is readily esterified with fatty acids to form fatty acid esters of DOC [46]. Thus, the role of DOC-fatty acid ester(s) as potential mineralocorticosteroids is one avenue for research in a continuing exploration of the role of extraadrenal DOC as a mineralocorticosteroid of physiologic or pathophysiologic importance in pregnant or ovulatory women. AcknoM~ledgement-These investigations were supported, in part. by USPHS Grant No. 5-P50-HD11149. REFERENCES
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38.
39.
plasma progesterone to deoxycorticosterone during estrogen treatment of women pregnant with a dead fetus. J. clin. Invest. 69 (1982) 469-478. Winkel C.A., Milewich L., Parker Jr. C.R., Gant N.F., Simpson E.R. and MacDonald P.C.: Conversion of plasma progesterone to deoxycorticosterone in men, nonpregnant and pregnant women, and adrenalectomized subjects: evidence for steroid 21-hydroxylase activity in nonadrenal tissues. J. clin. Invest. 66 (1980) 803-812. Casey M.L. and MacDonald P.C.: Metabolism of deoxycorticosterone and deoxycorticosterone sulfate in men and women. J. clin. Invest. 70 (1982) 312-319. Winkel CA.. Simpson E.R.. Milewich L. and MacDonald P.C.: Deoxycorticosterone biosynthesis in human kidney: potential for the formation of a potent mineralocorticosteroid in its site of action. Proc. natn. Acad. Sci., U.S.A. 77 (1980) 7069-7073. Winkel C.A., Casey M.L., Simpson E.R. and MacDonald P.C.: Deoxycorticosterone biosynthesis from progesterone in kidney tissue of the human fetus. J. clin. Endocr. Metab. 53 (1981) 10-15. Casey M.L., Howell M.L., Winkel C.A., Simpson E.R. and MacDonald P.C.: Deoxycorticosterone sulfate biosynthesis in human fetal kidney. J. clin. Endocr. Metab. 53 (1981) 990-996. Mathis J.M., Johnston J.D., MacDonald P.C. and Casey M.L.: Steroid 21-sulfatase activity in human placenta. J. steroid Biochem. 19 (1983) 575-579. Winkel C.A., Parker Jr. C.R., Milewich L., Simpson E.R. and MacDonald P.C.: The fractional conversion of circulating progesterone (P) to deoxycorticosterone (DOC) and the ratio of the concentration of DOC to P in plasma (DOC:P) of near-term pregnant women. Proc. Sot. Gynecol. Invest., 27th Ann Meet., Denver (1980) p. 218 (Abstract). Winkel CA., Parker Jr. C.R., Simpson E.R. and MacDonald P.C.: Production rate of deoxycorticosterone in women during the follicular and luteal phase of the ovarian cycle: the role of extraadrenal 21hydroxylation of circulating progesterone in deoxycorticosterone production. J. clin. Endocr. Metab. 51 (1980) 13541358. Casey M.L. and MacDonald P.C.: Formation of deoxycorticosterone from progesterone in extraadrenal tissues: demonstration of steroid 21-hydroxylase activity in human aorta. J. clin. Endocr. Metab. 55 (1982) 804-806. Casey M.L.. Winkel CA. and MacDonald P.C.: Conversion of progesterone to deoxycorticosterone in the human fetus: steroid 21-hydroxylase activity in fetal tissues. J. steroid Biochem. 18 (1983) 449-452. Casey M.L. and MacDonald P.C.: Sulfurylation of deoxycorticosterone in human fetal tissues and in tissues of a prepubertal boy. J. steroid Biochem. 19 (1983). Wengle B.: Distribution of some steroid sulphokinases in foetal human tissues. Acta endocr. Copenh. 52 (1966) 607-618. Bostrom H. and Wengle B.: Studies on ester sulphates. 23. Distribution of phenol and steroid sulphokinases in foetal human tissues. Acta endocr., Copenh. 56 (1967) 69 l-704. Parker Jr. C.R., Everett R.B., Quirk J.G., Whalley P.J., Gant N.F. and MacDonald P.C.: Plasma concentrations of deoxycorticosterone throughout pregnancy in normal women and in women who developed pregnancy-induced hypertension. Am. J. Obstet. Gynecol. 138 (1980) 626631. Gibson M. and Tulchinskv D.: The maternal adrenal. In Maternal-Fetal Endocrkzology. (Edited by D. Tulchinsky and K.J. Ryan). W.B. Saunders, Philadelphia (1980) 129.
DOC and DOC-sulfate in pregnancy 40. Wmkel C.A.. Wade C.E., Danley D.L., MacDonald P.C. and Casey M.L.: Conversion of progesterone to deoxycorticosterone in guinea pig spleen: an animal model for the study of steroid 21-hydroxylase activity in extraadrenal sites. f. steroid Biochem. 19 (1983) in press. 41. Winkef C.A. and Danley D.L.: The effect of immunologic challenge on splenic steroid 21-hydroxyiase activit; in the g&tea pig. Proc. Endocr.. Sm. -65th Ann. Meet. San Anronio. TX, D. 329 (Abstract). 42. Levine L.S., Zachman M., New M.I., ‘Prader A.. PollackM.S.,O’NeillG.H.,YangS.Y.,OberfieldS.E. and Dupong B.: Genetic mapping of the 21hydroxylase-deficiency gene within the HLA hnkage group. N. l%gI. J. Med. 299 (1978) 911. 43. Singer S.S. and Sylvester S.: Enzymatic sulfation of steroids: II. The control of hepatic cortisol sulfotransferase activity and of the individual hepatic steroid
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sulfotransferases of rats by gonads and gonadal hormones. Endocrinology 99 (1976) 1346-1352. 44. Winkel CA., Casey M.L., Guerami A., Rawlins SC., Cox, K., MacDonald S.C. and Parker Jr. C.R.: Ratio of plasma deoxycorticosterone (DOC) levels to plasma progesterone (P) levels in pregnant women who did or did not develop pregnancy-induced hypertension. Proc. Sot. Gyrtecol.-Iniest., L&h Ann. Me>;., Washington, DC (1983) p. 185 (Abstract). 45. Casey M.L. and Guerami A.: The origin of 19-nordeoxycorticosterone (19-nor-DOC) in man. Proc. Sac. Gvnecol. Invest., 30th Ann. Meetina. Washineton D.C. ([9X3), p. 134 (Abstract). y 46. Lucas J.A., Casey M.L. and MacDonald P.C.: The formation of a deoxycorticosterone-fatty acid ester in kidney tissue of the human fetus. Proc. Sot. Gynecoi. Invest., 30th Ann. Meeting, Washington D.C. (1983) p. 295 (Abstract).
sreroid Biochem. Vol. 20, No. 1, pp. 237-243,1984 Printedin Great Britain.All rightsreserved
0022-4731/84 $3.00+ 0.00 Copyright0 1984PergamonPressLtd
THE ORIGIN OF AND METABOLIC FATE OF DEOXYCORTICOSTERONE AND DEOXYCORTICOSTERONE SULFATE IN PREGNANT WOMEN AND THEIR FETUSES M. LINETTECASEY,ALIREZAGUERAMI,CRAIGA. WINKELand PAULC. MACDONALD Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Department of Biochemistry. The University of Texas Southwestern Medical School, Dallas, Texas 75235. U.S.A.
Summary-DOC and DOC-SO,, which are present in large amounts in the blood of pregnant women, are derived from sources other than maternal adrenal. Other investigators demonstrated that treatment of near-term pregnant women with ACTH or dexamethasone did not cause alterations in the blood levels of DOC. To define the source(s) of DOC and DOC-SO, in plasma of pregnant women, we evaluated the conversion of plasma progesterone (P) to DOC in extraadrenal sites. DOC is formed from plasma P and, provided that the pregnancy is one characterized by the usual large production of estrogen, DOC production in a given woman is proportional to the level of P in plasma. Unlike other steroid conversions or interconversions, however, the fractional conversion of P to DOC among apparently normal persons varied widely 0.011 f 0.003 (mean + SEM, n = 40, range = 0.001 to 0.030). In women pregnant with a normal living fetus, the product of the production rate of P and the fractional conversion of P to DOC is sufficient to account for the majority of DOC produced in the mother. There may be a second source of DOC, i.e. the transfer of DOC from the fetal to the maternal compartment in a manner that involves (a) direct transfer of DOC by way of trophoblast and (b) by desulfurylation of DOC-SO., from fetal umbilical arterial plasma in trophoblast and thence transfer of DOC liberated in trophoblast to the maternal compartment. Presently, it is clear that DOC-SO, in blood of pregnant women is not derived from plasma DOC; and there is little or no evidence in support of the proposition that DOC-SO, (as a sulfoconjugate) is transferred from the fetal to the maternal compartment because of placental hydrolysis to DOC. Among the extraadrenal tissue sites identified as those in which 21-hydroxylation of plasma P could be effected are some also believed to be tissue sites of mineralocorticosteroid action, viz. kidney, aorta. thymus, and spleen. Quantitatively, the origin of DOC in the fetus is not as clear as in the maternal compartment; yet. many tissues of the fetus have been identified in which both steroid 21-hydroxylase and 21hydroxysteroid sulfotransferase activity are present. Thus, in the human fetus, extraadrenal as well as adrenal production of DOC and DOC-SO, are possible.
INTRODUCTION Pregnancy in women is characterized by a series of hormonal excesses that are of unparalleled magnitude in the mammalian kingdom. This generality is applicable to the production of estrogenic hormones, i.e. estradiol-17P and estriol as well as progesterone (P) [cf. 11. This near unbridled production of steroid hormones also is applicable to the fetal adrenal cortex, which may daily produce upwards of 200 mg of steroids, principally As-3P-hydroxysteroids as the sulfate esters of these steroids, such as dehydroisoandrosterone sulfate and pregnenolone sulfate [cf. 21. In a similar manner, there are incredible amounts of biologically active protein hormones produced during human pregnancy, e.g. chorionic gonadotropin (hCG) and placental lactogen (hPL) [cf. 1, 31.
Address correspondence to: M. Linette Casey, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235, U.S.A. .%aZO,l--Q
237
But more than that, there is an equally massive increase in the production of mineralocorticosteroids in women during the latter few weeks and months of pregnancy [4, 51. This enormous production of mineralocorticosteroids in pregnant women constitutes an intriguing biological phenomenon, the control of which many investigators have attempted to define, but also one that has been suggestive, to many, that mineralocorticosteroid excess may be fundamental in the pathophysiology of pregnancy disorders that are characterized by sodium retention, edema formation, and, ultimately, hypertension. It is known that the rate of secretion of aldosterone by the adrenal cortices of pregnant women commonly is 10-20 times that found in men and nonpregnant women [4, 51. It is clear, however, that this large amount of aldosterone secretion in pregnant women is due to increased stimulation of the zona glomerulosa of the maternal adrenal by angiotensin II [6], which is present in plasma of pregnant women in high concentrations [cf. 71. This latter event is mediated by an estrogen-induced increase in biosynthesis of angiotensinogen [8, cf. lo], the precursor of angiotensin I, together with increased amounts of
238
M. LINETFECASEY et
rerun that is secreted by the juxtaglomerular apparatus of kidney [ll]. These two phenomena, in concert, bring about an increase in angiotensin I, which thence, in the endothelium of many blood vessels, principally those in lung, is converted to angiotensin II, the trophic agent for the zona glomerulosa of the adrenal and the site of aldosterone biosynthesis. That this is a correct formulation of the mechanisms by which the rates of aldosterone secretion by the adrenals are increased in pregnant women is supported by the findings that there is a decrease in aldosterone secretion in pregnant women after salt intake is increased [12] or after the development of hypertension [5, 131 as in men and nonpregnant women. Conversely, low salt diets [12] and hemorrhage lead to an increase in renin secretion and an increase in aldosterone secretion in pregnant women as in men and nonpregnant women [ 131. Thus, whereas the rates of aldosterone secretion in pregnant women, compared with those in men and nonpregnant women, are massive, the rate of aldosterone secretion in pregnant women is affected by known negative and positive effecters. In addition to increased aldosterone secretion in pregnant women, however, there also is a striking increase in the rate of formation of another mineralocorticosteroid. i.e. deoxycorticosterone [DOC] and its sulfate ester, DOC-.S04. The biosynthetic mechanism(s) responsible for the massive increase in DOC production in pregnant women is unclear; for example, neither ACTH [14-171 nor dexamethasone [14-16, 181 treatment of pregnant women causes a change in the levels of DOC in blood of treated subjects. Angiotensin II administration does not affect blood levels of DOC [ 191. But, it also is known that in men and nonpregnant women, DOC secretion by the adrenal cortex is regulated by the action of ACTH [20]; therefore, most of the DOC in the maternal compartment cannot arise by adrenal secretion. These findings, together with the fact that the levels of DOC and DOC-SO4 in fetal umbilical plasma are considerably greater than those in maternal plasma, led to the suggestion that DOC and DOC-SO4 in maternal plasma could be accounted for by transfer from the fetus [cf. 21, 221. For a variety of reasons, we speculated that an alternative explanation for the origin of DOC in pregnant women may be more tenable; viz., DOC could originate by extraadrenal 21-hydroxylation of plasma P. The rate of secretion of P in normal pregnancy can be massive, i.e. 200-600 mg per day [3]; ergo, a small fractional conversion of plasma P to DOC could account for a massive increase in DOC formation. It should be recalled that the rate of DOC secretion in men and nonpregnant women is only about 50 pg per day [cf. 231. Since rates of secretion of DOC in pregnant women may be greater than 8 mg per day [cf. 231, it seemed unlikely that transfer of this amount of DOC from fetus to mother could be accomplished.
al.
These hypotheses were tested as follows: (1) determination of the fractional conversion of plasma progesterone to DOC ([p]p-g$c) in men and in pregnant, nonpregnant, and adrenalectomized women; (2) determination of the production rate (PR) of P; (3) comparison of the estimated PR-DOC derived from plasma P with the total PR-DOC in the maternal compartment; (4) determination of the levels of the DOC and DOC-SO4 in umbilical arterial and venous sera and in maternal serum; (5) evaluation of steroid 2 1-hydroxylase and 2 1-hydroxysteroid sulfotransferase activities in various maternal and fetal tissues; and (6) an evaluation of the origin of plasma DOC-SO, from a variety of putative circulating C,,-precursors, e.g. plasma DOC, P, and pregnenolone. These studies were conducted, and are continuing to be conducted, on the basis of the proposition that there appear to be no negative effecters (with respect to alterations in ACTH or angiotensin II) for the formation of DOC in pregnant women. Specifically, in the case of aldosterone, negative effecters such as increased salt intake, alterations in body position, and the development of hypertension act to reduce circulating levels of angiotensin II and thence the rate of aldosterone secretion. These negative effecters do not appear to be operative with respect to extraadrenal formation of DOC in near-term pregnant women. Thus, it can be envisioned that in the sequence of events that are involved in the pathophysiology of mineralocorticosteroid excess, viz. sodium retention, edema formation, and hypertension, there is no physiologic means for reducing the rate of formation of DOC in near-term pregnant women, save for a reduction in the rate of placental formation of progesterone, which has not been described short of death of the fetus or delivery of the placenta, and (as described later) decreased secretion of estrogen [24].
MATERIALS AND METHODS
Steroids
Radiolabeled and nonradiolabeled steroids were purified by gradient-elution column chromatography on celite-ethylene glycol, liquid-liquid partition chromatography on cehte, and by thin-layer chromatography (TLC) as previously described in detail [25, 261. Radiolabeled steroids were infused intravenously either as a bolus injection in a 10 ml solution that contained NaCl (0.15 M), glucose (5%, w/v), and ethanol (6%, v/v) ([p]‘-sy’) or else as a continuous infusion for times that varied from 4-16 h in a solution of glucose (5%, w/v) that contained ethanol (6%, V/V) ([p]p-~~c). In cases in which urine collection was conducted, the urine was collected from the commencement of the radiolabeled tracer infusions for the succeeding 72 to 96 h.
DOC and DOC-sulfate
3l.l
I I
1
P
‘4C
4
l- L
DOC
Tetrohydw- DOC (Glucuronosids)
r
Fig. 1. In viva internal standard method for estimation or D”C Urinary tetrahydro-DOC (glucuronoside) is con[P]’ -BU sidered to be a unique metabolite of plasma DOC.
Determination of the [p] p-Bu D°C, /PI
DOGDsoBc_SO,
or
,,DOGDf;-SO,
The transfer constants of conversion of either P to DOC or DOC to DOC-SO, were determined by use of the radiolabeled internal standard in vivo technique that has been described in detail [26, 271. Briefly, tritium-labeled precursor (or product) or carbon-1C labeled precursor (or product) were infused intravenously (Fig. 1). Studies also were conducted by evaluation of the so called “blood to blood” transfer constant of conversion of one steroid to another by the continuous infusion of radiolabeled tracer precursor and product and the sampling of blood at times when equilibrium conditions were believed to have been established. Radiolabeled DOC or DOC-SO, or both were isolated from plasma, and from the 3H:‘4C of the product of the steroid under investigation and that of the radiolabeled tracers infused, the transfer constant(s) of conversion was computed. Also, after the infusion of radiolabeled precursor and product hormones, urine was collected for a time sufficient to ensure complete excretion of all radiolabeled metabolites (Fig. 2). From the urine collected (after hydrolysis of steroid conjugates), steroids were isolated and separated, and unique metabolites of precursor and products were purified by a variety of chro14C
(50)
1
I
DOC-
-
/
SO4
I
1
Tetmhydm - DOC (Glucumnosida)
matographic techniques that included gradientelution column chromatography on ethylene glycolcelite, partition chromatography on celite, multiple thin-layer chromatographic steps, derivative formation, and chromatography of the derivatives, and finally by repeated crystallization to achieve constant 3H:‘4C ratios [25, 261. Alternatively, a portion of the steroids may have been treated by radiolabeled acetic anhydride to establish the specific activity of a particular metabolite(s). These studies were conducted to compute the production rate of the steroid(s) in question. In pregnant women, the blP-EC was computed from the ratios of the concentrations of P:DOC in plasma and [p]p-Bycdetermined post-partum. The values were similar. Determination of steroid 21 -hydroxylase and 21hydroxy suIfotransferase activities in human tissues
By use of differential centrifugation procedures of homogenates of human adult and fetal tissues, microsome-enriched preparations were prepared for assay of steroid 21-hydroxylase activity. Incubations were conducted with [3H]P, in various concentrations, for various times, and with various amounts of tissue protein in the presence of an NADPH-generating system [27]. The assays were linear with times of incubation for 2 h and with protein concentrations up to 1 mg. In all tissues in which steroid 21-hydroxylase activity was demonstrated, the apparent K,,, of the enzyme for P was similar [27-281. In a similar manner, a cytosolic fraction was prepared for the assay of 21-hydroxysteroid sulfotransferase activity. An excess amount of sulfate donor, phosphoadenosine phosphosulfate, was used to optimize activity. Optimum conditions were established for substrate concentration, time of incubation, pH, and protein concentrations. The reaction was linear with time of incubation and with protein concentrations that varied up to 240 kg [29]. Determination activity
of
placental
steroid
21-sulfatase
Trophoblastic tissue, principally free of adventitious and vascular tissue, was prepared. A microsome-enriched preparation was prepared as described [27]. Optimum conditions were established with respect to pH of the incubation mixture and time of incubation and tissue protein concentration. It is notable that the only human tissue in which steroid 21-sulfatase activity is demonstrable is in trophoblastic tissue of the human placenta [30].
1 DOC CS04)
Fig. 2. In viva internal standard method for evaluation of potential interconversion of plasma DOC and DOC-SO+ Urinary DOC(SOJ and urinary tetrahydro-DOC (glucuronoside)
239
in pregnancy
are considered to be unique metabolites of plasma DOC-SO, and plasma DOC, respectively.
RESULTS
In previous studies [25], and in studies that were conducted in the present investigation, we found that
M. LINEITE CASEY et al
240
Steroid 21-sulJh_tase activity LIVER BILE ,NTESTlNE
a
I ‘4C3
50
PLASMA
3H7 PLASMA
P
DOC
DOC-SO,
50
-c < 0.3
+ >I500
I
t
v
DOC-SO4
Fig. 3. Formulation of metabolic disposition of plasma DOC and DOC-SO,. Plasma DOC and DOC-SOI are not interconverted. DOC is sulfurylated in liver. but intrahepatic DOC-SO, does not escape into blood; rather, it is excreted quantitatively into bile wherein it is converted, by way of the action of bacterial enzymes, to progesterone or metabolites thereof.
Intravenously administered radiolabeled DOCSO4 in the human is not hydrolyzed to DOC. except by the action of bacterial enzymes in the intestine [26]. Thus, it can be concluded that in men and in nonpregnant women, steroid 21-sulfatase activity is not a constituent of human tissues in men or nonpregnant women. By way of contrast, steroid 21-sulfatase activity is demonstrable readily in tissue preparations of the human placenta [30]. Since steroid 21-steroid sulfatase activity is present in placenta, the potential exists for the hydrolysis of DOC-SO, (of fetal origin) in placenta and, thence, the transfer of the DOC so formed into the mother [cf. 221. DISCUSSION
[p]p-,D,oc m men
and in nonpregnant, pregnant, and adrenalectomized women is 0.011 + 0.003 (mean + SEM, n = 40). There are several notable features with respect to [p] ’ suD°C. First, the [p]p-BTc varies widely among presumably normal persons, viz. wEC of 0.001 to 0.030 have been found [25, 311. On the other hand, in a given person, [p]‘-t$’ is remarkably constant irrespective of the plasma level of P, i.e. in a given woman, during the follicular and luteal phase of the ovarian cycle [32], with wide variations in the concentrations of circulating progesterone, and in women during pregnancy, the [p]p-~~c is the same in a given woman [25]. There are two notable exceptions to this generality: In women pregnant with either (1) a dead or (2) an anencephalic fetus in whom progesterone levels are high, the [p]p-,D,ocis strikingly reduced [24]. On the other hand, in men and in nonpregnant and pregnant women, the [p]“““~~“““~ uniformly is 0 [26]. Thus, circulating DOC is not converted to DOC-SO, that enters plasma (Fig. 3). The conversion of plasma [3H]P and [3H]pregnenolone to plasma [3H]DOC-S04 also is negligible in nonpregnant persons. the
From the results of the present and previous investigations [24,25,38]. it is clear that DOC in the plasma of near-term pregnant women arises primarily by extraadrenal conversion of plasma P (of placental origin) to DOC in nonadrenal tissue(s). some of which appear to be those in which mineralocorticosteroids are known to exert specific actions, e.g. kidney 1271. aorta [33], and lymphoid tissues [34]. In addition, some DOC in mother may arise by transfer from the fetus by: (a) direct transfer of DOC from fetus to mother [21] and (b) transfer of DOC from fetus to mother [22] after hydrolysis of fetally derived DOC-SO, to DOC in placenta by action of steroid 21-sulfatase activity [30]. In pregnant women, there must be little secretion of DOC or DOC-SO4 by the maternal adrenal cortex [25. 261. This is consistent with the observation that the rates of secretion of other Czl-steroids (except for aldosterone [5]) by the maternal adrenal are either not increased or are reduced [3Y]. The origin of DOC-SO4 in the maternal compartment is unknown. Clearly. plasma DOC is not converted to DOC-SOI that enters plasma (251. The KIDNEY MEN AND NONPREGNANT WOMEN
Extraadrenalsteroid21-hydroxylaseand sulfotransferase activities Steroid
21-hydroxylase
activity
has been
-DOG
demons-
in a number of tissues of the human adult 1271 and fetus [28, 33, 341. Notable among these tissues are those in which mineralocorticosteroids are believed to act, e.g. kidney [28], blood vessels [33], thymus. spleen, and, possibly, urinary bladder [34]. Thus, the potential exists for the formation of a potent mineralocorticosteroid in its tissue site of action. In these studies, we also found considerable 21-hydroxysteroid sulfotransferase activity in human fetal liver, adrenal, kidney, and intestine [35]; on the other hand, there was little 21-hydroxysteroid sulfotransferase activity in tissues of the adult, except in adrenal and liver, as demonstrated by others [36, 371. trated
1 J
Fig. 4. Hypothetical model to explain the origin of DOC and DOC-SO4 in pregnant women. Estrogen. directly or indirectly, stimulates extraadrenal steroid 21-hydroxylase activity and may, as in other animal species. act to stimulate 21-hydroxysteroid sulfotransferase activity in extraadrenal sites of DOC formation.
241
DOC and DOC-sulfate in pregnancy
same is true (in men and nonpregnant women) of plasma P and pregnenolone; namely, these two Gi-steroids also are not converted to DOC-SO4 that enters plasma. There seems to be little likelihood of the transfer of DOC-SO4 as such [22] from fetus to mother since there is steroid 21-sulfatase activity [3O] in the human placenta. Thus, the origin of DOC-SO, in pregnant women is an enigma. The in situ formation of DOC-SO, in an extraadrenal site in pregnant women from a circulating precursor other than DOC or from a precursor yet unidentified is a possibility. Whereas, as we have stated, the formation of DOC in extraadrenal tissues is not affected by alterations in the levels of angiotensin II or ACTH, the [p]p-~~c in women who are pregnant, but hypoestrogenic, is decreased [24]. Namely, in women with a dead or anencephalic fetus, i.e. in women whose pregnancies are characterized by relative hypoestrogenism, there is a decrease in the [p]p-Fcc and [p]‘-@‘. We reached this conclusion since treatment of women with such hypoestrogenic pregnancies with estrogen effected an increase in [p]‘-BsJ”’and an increase in the PR-DOC without a decrease in the MCR-DOC [24]. Yet other factors also appear to be important in the regulation of extraadrenal 21-hydroxylase activity. Recently, it was shown that immunologic challenge of experimental animals (in which 21-hydroxylase activity is identifiable in splenic tissue [40]) causes a striking decrease in the specific activity of steroid 21-hydroxylase in splenic tissue [41]. Since the gene that encodes for steroid 21-hydroxylase activity is within the HLA locus [42], it is intriguing to speculate that immunologic challenge may act to influence the rate of 21-hydroxylation of Czl-steroids. Thus, there may be physiologic and pathophysiologic processes that do act to bring about alterations in the rate of DOC formation from plasma P. Quantitatively, the origin of DOC in the fetus is less clear. It has been demonstrated that both DOC and DOC-SO4 can be formed in the fetal adrenal; but. also, it has been shown that in many fetal tissues, steroid 21-hydroxylase [28, 33, 341 and 21-hydroxysteroid sulfotransferase [29, 351 activities are demonstrable. There is evidence that is suggestive that estrogen acts, at least in many experimental animals, to increase steroid sulfotransferase activity [43]. Thus, it is conceivable that during pregnancy, in both the mother and fetus, steroid 21-hydroxylase and sulfotransferase activities are increased in tissues other than liver and adrenal. Thereby, an explanation could be offered for the dramatic rise in DOC and DOC-SO, levels in both the maternal and the fetal compartments. An explanation also could be offered for the finding that the PR-DOC appears to be proportional to the level of P in plasma provided that the pregnancy is not associated with hypoestrogenism. Based on the blood concentration of P in late pregnancy, as well as the apparent K, of steroid 21-hydroxylase for P in extraadrenal tissues
[28, 291, during late pregnancy, the steroid 21-hydroxylase enzyme in extraadrenal tissues could become saturated. If this were true, then only by virtue of an increase in steroid 21-hydroxylase activity, as a consequence of the hyperestrogenism of pregnancy, could the [p]p-~~c remain constant, i.e. irrespective of P levels. To date, there has been no pathophysiologic alteration in extraadrenal DOC formation that can be ascribed specifically to an entity known to be associated with apparent mineralocorticosteroid excess. Perhaps the one exception to that generalization has been the identification of a higher incidence of pregnancy-induced hypertension (PIH) among women in whom [p]p-~~c is greater than 0.025 [44]. On the other hand, this cannot be the single cause of PIH since PIH occurs in women in whom [p]p-~~c is not greater than that in women who do not develop PIH [44]. Nonetheless, the potential for the unbridled production of a potent mineralocorticosteroid, DOC, in its potential sites of action (such as kidney and blood vessel) remains as a tantalizing possibility to explain heretofore inexplicable pathophysiologic disorders of human pregnancy and even those ascribed to the premenstrual syndrome in ovulatory women in whom DOC levels are high in the mid-luteal phase of the ovarian cycle as a consequence of the extraadrenal conversion of progesterone, which is secreted in large amounts by the corpus luteum. Present avenues of investigation include an evaluation of the origin of the very potent mineralocorticosteroid, 19-nor-DOC, and the role of DOC-fatty acid esters, which now are known to be formed in fetal kidney tissues. To date, it has not been possible to demonstrate, in humans, the conversion of DOC to 19-norDOC [45]. On the other hand, in preliminary studies, we found that, in fetal kidney tissue, DOC is readily esterified with fatty acids to form fatty acid esters of DOC [46]. Thus, the role of DOC-fatty acid ester(s) as potential mineralocorticosteroids is one avenue for research in a continuing exploration of the role of extraadrenal DOC as a mineralocorticosteroid of physiologic or pathophysiologic importance in pregnant or ovulatory women. AcknoM~ledgement-These investigations were supported, in part. by USPHS Grant No. 5-P50-HD11149. REFERENCES
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