Clinical Endocrinology (1978) 8,295-303.
PROLACTIN A N D A D R E N A L A N D R O G E N SECRETION A. V E R M E U L E N AND S. A N D 0
Department of Endocrinology and Metabolic Diseases, Medical Clinic, Academic Hospital, State University, Ghent, Belgium (Received I 9 August 1977; revised 5 October 1977; accepted 24 October 1977)
SUMMARY
The aim of this study was t o investigate the role of prolactin in the secretion by the adrenal of dehydroepiandrosterone (DHA) and its sulphate (DHA-S). Therefore prolactin and DHA-S levels were determined in different groups of subjects under various physiological and pathological conditions. In patients with prolactinomas, or with pharmacologically induced hyperprolactinaemia, plasma DHA-S and, t o lesser extend, DHA concentrations were elevated, and the DHA-S blood production rate greatly increased. ACTH stimulation which did not influence DHA-S concentrations in normals, caused a significant increase in patients with prolactinomas; the increase in DHA concentrations was similar in normal subjects and in prolactinoma patients. Bromocriptine treatment of prolactinaemia patients normalized both prolactin and DHA-S concentrations. Acute elevation of prolactin in normal subjects by TRH stimulation or by short term administration of sulpiride, in contrast to long term treatment, did not influence DHA-S levels. During pregnancy, notwithstanding high prolactin concentrations, DHA-S concentrations were lower than during the menstrual cycle. In patients with prolactinomas, given glucocorticoid replacement with cortisol, DHA-S concentrations were low (normal) notwithstanding persistently high prolactin concentrations. It is concluded that only prolonged elevation of prolactin induces increased DHA-S secretion by the adrenal cortex and that normal ACTH secretion is a prerequisite for this effect. The absence of elevated DHA-S concentrations in pregnancy might be explained by the rapid rate of metabolism, as the DHA-S production rate is increased. The intimate mechanism of prolactin and ACTH interaction at the adrenal cortex remains however unknown. Recently, high plasma dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulphate (DHA-S) levels were reported in patients with prolactinomas (Bassi et al., 1977; Vermeulen et al., 1977), while Donabedian et al. (1976) observed high urinary excretion of 17oxosteroids. Moreover, we reported (Vermeulen et al., 1977) that in patients with pharmacologically elevated plasma prolactin concentrations, DHA-S concentrations were similarly Correspondence: Dr A. Vermeulen, Department of Endocrinology and Metabolic Diseases, Medical Clinic, Academic Hospital, State University, Ghent, Belgium. 03004664/78/0400-0295 $02.00 0 1978 Blackwell Scientific Publications Ltd
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increased. We suggested therefore that prolactin might have an androgen secretion stimulating effect. In the view of these results we decided t o study in more detail the influence of prolactin on DHA-S levels in man. M A T E R I A L S AND METHODS Subjects Plasma prolactin and androgen concentrations were studied in several groups of subjects. (A) Control groups: (1) normal post-menopausal women (n = 40), (2) normal elderly males (>50 years) ( n = 20), (3) normal women during the first days ( 2 - 5 ) of the follicular phase of the cycle (n = 20), (4) normal young males (age 18-25) ( n = 20); (B) patients with prolactinomas with intact hypothalamo-pituitary-adrenal axis as shown by a normal insulin tolerance test, and with impaired ACTH secretion on corticoid treatment; (C) five patients with prolactinomas before and after treatment with bromocriptine 2 X 2.5 mg per day for 6 weeks; (D) six normal subjects and four patients with prolactinomas after acute ACTH stimulation (Synacthen 0.25 mg i.v.); (E) six normal men before and after i.v. injection of 200 pg of TRH, blood samples being taken at 0, 20 and 60 min after TRH injection; (F) three normal post-menopausal women before and after 1 0 days and I month of treatment with sulpiride 2 X SO mg day; and (G) twenty normal pregnant women at different stages of pregnancy. Finally DHA-S production rate was determined in two normal women, in three normal males and in two patients with prolactinomas, using the single i.v. injection method. Methods Blood samples for hormone determination were obtained in the fasting state between 08.00 and 10.00 h. Steroid hormones were determined as previously described (Vermeulen & Verdonck, 1976); DHA-S was determined after solvolysis as described by Andre &James (1973). Prolactin was assayed by a double-antibody method using the commercial CEAIRE-SORIN (Belgium) kit and are expressed in ng/ml NIH-1 standard (1 ng = 40 piu MRC 71/282). Plasma cortisol was determined using the method of Mattingly (1963). Measurement of the metabolic clearance rate (MCR) ofDHA-S A total of 5 pCi of 73H-DHA-S, TRK 267 (Amersham), specific activity 2.4 Ci/mmol, dissolved in 5% ethanol in normal saline, were rapidly injected intravenously between 08.00 and 10.00 hours; blood samples were taken hourly for 8 h from the opposite arm in the recumbent patient. The MCR was calculated on the basis of the slow portion of the disappearance curve, i.e. after the first hour, when the rate of decrease in the concentration of the isotopic labelled DHA-S can be represented by a single exponential curve (Gant et aZ., 1971): MCR = k/D X 1440 l/day where k = 0.6931t ?h(min) and D = fraction of administered radioactivity per litre of blood at zero time, obtained by extrapolation (Tait & Burstein, 1964). Isolation of 3H-DHA-Sfrom plasma To 1 ml of plasma were added 20 pg of cold DHA-S as internal standard. After mixing, the plasma free steroids were extracted with 10 ml of diethylether. After adding 2 ml 1 N sulphuric acid saturated with NaCl, the residual plasma was extracted with 20 ml of ethylacetate and kept overnight at 37'C. After evaporation, the residue was chromatographed on
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Whatman 40 paper and developed in the Bush A2 system. The D zone was eluted with methanol chloroform 1:1 v/v. Appropriate aliquots were taken for radioimmunoassay (recovery) and for determination of radioactivity. As plasma DHA-S concentrations do not show important diurnal variations (De Moor & Heyns, 1969; Rosenfield et al., 1975; Korth-Schutz et al., 1976) blood production rate (BPR) of DHA-S was derived using the plasma DS concentration obtained in the fasting blood sample.
RESULTS Normal values in different control groups are given in Table 1 t o contrast with values obtained in different groups of patients with prolactinomas. In nine young (SO years) (n = 20) Women early follicular phase (n = 20) Post-menopausal women (n = 40)
12.1+1.9* 11.8t3.6 9.0k1.2 7.3k2.6 Patients with prolactinomas with intact pituitaryadrenal axis Young males (n = 7) 1301.0t483 Young women (SO years) (n = 3)
DHA (nmol/l)
DHA-S (bmol/l)
23.3+1.4* 6.4k0.6 1 6 - l t1.5 8.6k0.8
5.04+0.39* 2.25t0.29 2.75k0.21 2.14k0.18
20.9k3.6 35.1 k6.1 19.8-33.0**
9.14k1.46 6.93+1.07 2.50-11.61**
*, MeankSEM; **,range of values. groups (Table 1). Similarly in three women of post-menopausal age with prolactinomas, DHA concentrations were increased with respect t o normal post-menopausal women; in two, DHA-S concentrations were also increased whereas in one a value (2.5 pnol/l) at the upper limit of the normal range was observed. In seven male subjects with prolactinomas (prolactin 300-3680 ng/ml) and an intact pituitary-adrenal axis, mean DHA-S was significantly increased (P< 0.02) with respect to normal controls, whereas mean DHA concentration was not significantly different. In six patients with prolactinomas, DHA concentrations could be followed during treatment with bromocriptine. As shown in Table 2, after 6 weeks of treatment prolactin as well as both DHA-S and DHA concentrations decreased significantly associated with resumption of cyclical ovarian activity or activation of Leydig cell secretion (as evidenced by an increase of plasma testosterone from 4.36 to 16.35 nmol/l). In six patients with prolactinomas (four men and two women) treated for several weeks with hydrocortisone 40 mg per day, DHA and DHA-S concentrations were normal or decreased, despite high prolactin concentrations (Table 3). Finally, whereas in normal subjects (n = 6 ) ACTH stimulation (0.25 mg Synacthen i.v.) caused a rapid increase of DHA but not
A . Vermeulen and S. Ando DHA-S (Fig. I), in patients with a prolactinoma (n = 4), ACTH stimulation resulted in a significant increase of both DHA and DHA-S, prolactin remaining unchanged. TRH stimulation (200 pg i.v.) in young males (n = 6) resulted in an increase of prolactin from a basal concentration of 11.0k2.7 (meankS.D.) ng/ml t o a mean of 37.7k7.9 ng/ml (365+103% of basal value) 20 min after injection which had returned t o 19*2 ng/ml Table 2. Influence of bromocriptine 2 X 2.5 mg/day on plasma hormone Pre-treatment
Treatment
Patient
Sex
Age
Prolactin (ng/ml)
DHA-S (Ctmol/l)
DHA (nmol/l)
Prolactin (ng/ml)
DHA-S (pmol/l)
DHA (nmol/l)
D.M.S. M.M. C.L. C.A. V.M.
F F F F M
39 19 39 40 26
1900 183 78 898 490
8.79 4.25 5.43 3.61 14.24
27.6 33.4 16.9 14.0 55.7
60 10 21 40 48
6.53 3.83 3.71 3.01 8.03
22.9 7.1 10.6 8.2 5.5
Table 3. Hormone concentrations in patients with prolactinomas on cortisol treatment 40 mg/day Patient
Sex
Age
Prolactin (ng/ml)
DHA (nmol/l)
DHA-S (Ctmolb)
W.G. M.A. K.F. R.W. V.D.S. G.G.
M M M M F F
43 38 27 41 52 50
220 200 3585 1140 200 1131
0.71 8.32 2.64
0.07 2.64 0.79 5.36 0.21 0.14
-
4.50 1.43
DHA
DHA-S
F
70
‘I 10 9
. -0 E
8
2-
10 -
0.2
I-
5-
0.1
I
I
I
I
I
I
I
l
l
1
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(176+31% of basal value) 60 min after injection. As shown in Fig.2 neither DHA,DHA-S nor
cortisol increased significantly. Neither changes in DHA nor in DHA-S were correlated with changes in prolactin, whereas a highly significant correlation (Y = 0.89, P < 0.001) between changes in cortisol and DHA (but not DHA-S) was observed. Ten days of treatment with sulpiride (2 X 50 mg/day) of three normal post-menopausal women, resulted in greatly increased prolactin concentration, but had no definite influence on DHA-S levels; after 1 month of treatment on the other hand, prolactin concentrations were not further increased, but DHA-S and DHA were now clearly elevated (Table 4). During gestation (weeks 5-38) prolactin concentrations (n = 20) varied between 4 and 167 ng/ml (mean 77+13 ng/ml), DHA concentrations between 3.2 nmol/l and 24.7 nmol/l (mean 11.9k1.2 nmol/l) and DHA-S levels between 0.39 and 5.79 pmol/l (mean 2.71k0.3 pmol/l). No correlation was observed between prolactin and either DHA or DHA-S concentrations. DHA and DHA-S concentrations decreased with duration of pregnancy: during the first trimester mean concentrations were 13.7+2.1 nmol/l and 3.64k0.64 pmol/l respectively,
30k T
Cortisol
20
DHA-S
Time (min)
Fig. 2. Influence of TR 00 fig I.V., on plasma concentrations of prolactin, cortisol, dehydroepiandrosterone (DHA) and its sulphate (DHA-S) in normal subjects (n = 6). Table 4. Influence of sulpiride treatment on plasma prolactin, DHA and DHA-S After 10 days treatment
Before treatment
After 1 month of treatment
Age Prolactin DHA-S DHA Prolactin DHA-S DHA Prolactin DHA-S DHA (ng/ml) (fimol/l) (nmol/l) (ng/ml) (fimol/l) (nmol/l) (ng/ml) (fimol/l) (nmol/l) -~
V.W. Q Da.S. 9 Hc.A. 0
54 76 52
21.0 16.1 10.8
2.15 1.75 2.11
12.5 4.8 8.8
180 167 167
4.64 1.64 1.96
21.4 5.8 9.5
156 132 159
6.00 4.43 4.29
24.3 10.0 13.6
A . Vermeillen and S. Ando
300
during the second trimester 11.OtO.3 nmol/l and 2.61k0.07 pmol/l and during the third trimester 11.3k0.14 nmol/l and 2.07k0.36 pmol, respectively. Prolactin concentrations on the other hand increased with duration of pregnancy from 15.7t2.7 ng/ml during the first trimester, t o 71k14 ng/ml during the second and 1 3 6 t 9 ng during the third trimester. Finally the metabolic clearance rate and blood production rate was measured in three normal men, two normal women and in two patients with prolactinomas. As shown in Table 5, the metabolic clearance rates of DHA-S in the two patients with prolactinomas were within normal limits (Wang et d . , 1967), whereas their blood production rates were greatly increased when compared t o the control subjects. Table 5. Metabolic clearance rate (MCR) and blood production rate (BPR) of DHA-S in normal subjects and in two patients with prolactinomas
Normals L.L.* D.V.M. V.A. T.R.
BPR (rmol)
Sex
Age
ADV (1)
k
MCR (1/24 hours)
Plasma DHA-S (w)
F
46 57 50 54
5.3 7.9 6.5 10.1
0.0959 0.1052 0.071 3 0.0520
12.2 20.1 11.0 12.6
4.50 1.25 3.32 1.06
54.9 25.1 36.5 13.4
28 35
6.4 6.6
0.0984 0.0422
15.1 8.9
8.14 9.1 1
122.9 81.1
M M M Prolactinoma patients V.P.R.** F R.F. M
*, Early follicular phase; **,amenorrhoea for 9 years. ADV = apparent distribution volume. DISCUSSION
It is evident from our results and from data in the literature, that in several clinical conditions, characterized by high circulating prolactin concentrations, increased plasma DHA-S and, t o a lesser extent, plasma DHA levels are found. This was observed in patients of either sex with prolactinomas, in young women with hyperprolactinaemia induced by chronic sulpiride treatment and in post-menopausal women treated chronically with phenothiazines or butyrophenones (Vermeulen et al., 1977). Moreover short term ACTH stimulation, which in accordance with previous observations (Vaitukaitis et d . , 1969; Nieschlag et d . , 1973) did not influence plasma DHA-S within 120 min in normal subjects, caused a significant increase in plasma DHA-S levels in patients with prolactinomas. The relative increase in DHA in both normals and hyperprolactinaemic subjects was similar with higher absolute values in the latter. This suggests a higher reserve in androgen secretion capacity, despite similar cortisol secretion. Bromocriptine treatment of patients with prolactinomas lowered prolactin concentrations, with a simultaneous decrease of DHA-S and DHA concentrations, as well as resumption of cyclical activity of the ovary or activation of Leydig cell function. This emphasizes the role of prolactin in the elevation of DHA-S levels. In patients with prolactinomas treated with cortisol, DHA-S and DHA concentrations were either normal or more often decreased, despite persistence of high prolactin concentrations. This suggests that the long-term effect of prolactin on the adrenal cortex requires the presence of ACTH.
Prolactin and adrenal androgen secretion
30 1
That the increased DHA-S concentrations in patients with prolactinomas were not the consequence of a decreased rate of metabolism is shown by the normal MCR. This was already suggested by the results reported by Donabedian et al. (1976) and by Bassi et al. (1 977) who observed increased urinary 17-oxosteroid and DHA-S excretion, respectively. All these results suggest a stimulating effect of prolactin on DHA-S secretion by the adrenal cortex. As neither we ourselves (Vermeulen et al., 1977) nor Bassi etal. (1977) observed any significant increase in androstenedione levels, this suggests an effect of prolactin on the adrenal A5-30-01-dehydrogenase. Another possibility, as suggested by Bassi et al. (1977), is a direct effect of prolactin on sulphate synthesis by the adrenal cortex. As sulphates secreted by the adrenal cortex are essentially A 5 3 0 hydroxy-steroids, this latter hypothesis does not exclude the former. Several experiments in animal species suggest an effect of prolactin on the adrenal cortex: indeed prolactin receptors have been detected on adrenal membrane preparations from several species (Posner et al., 1975; Marshall et al., 1975, 1976); synergism between ACTH and prolactin in production of corticosterone from isolated rat adrenal cells has been reported (Lis et al., 1974) whereas Witorsch & Kitay (1972) as well as Gustafsson & Steinberg (1975) reported an inhibition of adrenal Sa-reductase activity by prolactin. Although it is hazardous t o extrapolate these findings to man, they strengthen the hypothesis of a direct effect of prolactin on the human adrenal cortex. Another set of experimental data seems however t o conflict with the hypothesis of a stimulatory effect of prolactin on adrenal androgen secretion. TRH stimulation causes an increase in prolactin, but has no influence on either DHA-S or DHA; similarly short term treatment with sulpiride (10 days) causes a highly significant increase in prolactin without significantly affecting DHA-S or DHA. Moreover during pregnancy, with its greatly increased prolactin concentrations, we found in accordance with earlier observations (Candy, 1970; Nieschlag et al., 1974), DHA-S and DHA concentrations to be decreased when compared to the values during the menstrual cycle. However the lack of effect of either TRH or short term sulpiride treatment on DHA-S does not necessarily refute the hypothesis of an adrenal stimulating effect of prolactin: indeed prolonged elevation of prolactin might be necessary for prolactin t o have any effect on the adrenal cortex. A similar prolonged elevation of plasma prolactin concentration is necessaw before it induces amenorrhoea, although shortening of the luteal phase may occur earlier. As far as pregnancy is concerned, it is well known that metabolism of DHA-S is very rapid and despite decreased plasma concentrations, due to the increased MCR (Cant et al., 1971) the blood production rate of DHA-S is likely t o be increased. The lack of effect of bovine prolactin o n adrenocortical or gonadal function in normal men, as reported by Varma et al. (1977) seems also to be against the hypothesis of a stimulatory effect of human prolactin on adrenal DHA-S secretion. These data should be interpreted with caution, as on the one hand bovine prolactin may have effects distinct from human prolactin, and on the other, it was administered for a relatively short period ( 5 days) only. There remains the possibility that it is not prolactin itself but a different substance secreted both by prolactinomas and by the normal pituitary after chronic administration of drugs with central antidopaminergic effects, that is responsible for the stimulation of androgen secretion by the adrenal cortex. Nevertheless, all experimental data available so far are compatible with the hypothesis that prolonged elevation of plasma prolactin stimulates adrenal DHA-S secretion. This stimulatory effect seems t o require the presence of ACTH, since in its absence low DHA-S concentrations are usually observed. The mechanism leading
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to this increased DHA-S secretion remains unknown and whether physiological prolactin levels influence adrenal androgen secretion in man remains t o be proven. ACKNOWLEDGMENT
Part of this work was supported by grant No. 20.477 of the FWGO (Brussels). REFERENCES ANDRE, C.M. & JAMES, V.H.T. (1973) Assay of plasma dehydroepiandrosterone and its sulfate by competitive protein binding. Clinica Chimica Acta, 43,295-303. BASSI, F., GIUSTI, G., BORSI, L., CATTANEO, S., GIANOTTI, P., FORTI, G., PAZZAGLI, M., VIGIANI, C. & SERIO, M. (1977) Plasma androgens in women with hyperprolactinaemic amenorrhoea. Clinical Endocrinology, 6,5-10. DE MOOR, P. & HEYNS, W. (1966) Androgens in Normal and Pathological Conditions (ed. by A. Vermeulen and D. Exley), pp. 54-61, Excerpta Medica ICS 101. DONABEDIAN, R.R., MAY, P.B. & TAN, Y.S. (1976) Abnormal adrenal steroidogenesis in patients with hyperprolactinaemia, galactorrhea or both. 56th Annual Meeting of the Endocrine Society, Abstract 372, p. 243. GANDY, H.M. (1970) Androgens. In: Endocrinology of Pregnancy (ed. by F. Fuchs and A. Klapper), p. 130. Harper and Row, New York. GANT, N.F., HUTCHINSON, H.T., SIITERI, P. & MACDONALD, P. (1971) Study of the metabolic clearance rate of dehydroisoandrosterone sulfate in pregnancy. American Journal o f Obstetrics and Gynecology, 111,555-561. GUSTAFSSON, J.A. & STEINBERG, A. (1975) Influence of prolactin on the metabolism of steroid hormones in rat liver and adrenals. Acta Endocrinologica (Kbh.], 78,545-553. KORTH-SCHUTZ, S., LEVINE, L.S. & NEW, M.I. (1976) Dehydroepiandrosterone sulfate (DS) levels, a rapid test for abnormal adrenal androgen secretion. Journal of Clinical Endocrinology and Metabolism, 42,1005-1023. LIS, M., GILORDEAU, J. & CHRETIEN, M. (1973) Effect of prolactin on corticosterone production by rat adrenals. Clinical Research, 21,1027. MARSHALL, S., GELATO, M. & MEITES, J. (1975) Serum prolactin levels and prolactin binding activity in adrenals and kidneys of male rats after dehydration, salt loading and unilateral nephrectomy. Proceedings of the Society of Experimental Biology and Medicine, 149, 185-188. MARSHALL, S., KLEDZIK, G.S., GELATO, M., CAMPBELL, G.A. & MEITES, J. (1976) Effects of estrogen and testosterone on specific prolactin binding in the kidneys and adrenals of rats. Steroids, 27,187-195. MATTINGLY, D. (1962) A simple fluorimetric method for the estimation of free 11-hydroxycorticoids in human plasma. Journal of Pathology, 15,374-379. NIESCHLAG, E., LORIAUX, D.L., REIDER, H.J., ZUCKER, LR., KIRCHNER, M.A. & LIPSETT, M.B. (1 973) The secretion of dehydroepiandrosterone and dehydroepiandrosterone sulphate in man. Journal of Endocrinology, 57,123-1 34. NIESCHLAG, E., WALK, T. & SCHINDLER, A.E. (1974) Dehydroepiandrosterone (DHA) and DHAsulfate during pregnancy in maternal blood. Hormones and Metabolic Research, 6,170-171. POSNER, B.I., KELLY, P.A., SHIU, R.P.C. & FRIESEN, H.G. (1975) Studies of insulin,growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology, 95, 5 2 1-53 1. ROSENFIELD, R.S., ROSENBERG, B.J., FUKUSHIMA, D.K. & HELLMAN, L. (1975) 24 hour secretory pattern of dehydroisoandrosterone and dehydroisoandrosterone sulfate. Journal of Clinical Endocrinology and Metabolism, 40,850-855. TAIT, J.F. & BURSTEIN, S. (1964) In vivo studies of steroid dynamics in man. The Hormones, Vol. 5 (ed. by J. Pincus, K. V. Thimann and E. B. Astwood), pp. 441-557. Academic Press, New York. VAITUKAITIS, J.L., DALE, S.L. & MELBY, J.C. (1969) Role of ACTH in the secretion of free dehydroepiandrosterone and its sulfate ester in man. Journal of Clinical Endocrinology and Metabolism, 29, 1443-1447.
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VARMA, M.M., HUSEMAN, C.A., JOHANSSON, A.J. & BLIZZARD, R.M. (1977) Effect of prolactin on adrenocortical and gonadal function in normal men. Journal o f Clinical Endocrinology and Metabolism, 44,760-761. VERMEULEN, A. (1976) Plasma androgen levels during the menstrual cycle. American Journal of Obstetrics and Gynecology, 1 2 5 , 4 9 1 4 9 4 . VERMEULEN, A. & VERDONCK, L. (1976) Radioimmunoassay of 17p-hydroxy-5a-androstan-3-one, 4 androstene-3,17 dione, dehydroepiandrosterone, 17P-hydroxy-progesteroneand progesterone and its application to human plasma. Journal of Steroid Biochemistry, 7, 1-10. VERMEULEN, A., SUY, E. & RUBENS, R. (1977) Effect of prolactin on plasma DHEA(S) levels. Journal o f Clinical Endocrinology and Metabolism, 44,1222-1 225. WANG, D.Y., BULBROOK, R.D., SNEDOON, A. & HAMILTON, T. (1967) The metabolic clearance rate of dehydroepiandrosterone, testosterone and their sulphate esters in man, rat and rabbit. Journal of Endocrinology, 38,307-318. WITORSCH, R.J. & KITAY, J.I. (1972) Pituitary hormones affecting adrenal Sa-reductase activity: ACTH, Growth hormone and prolactin. Endocrinology, 91,764-769.
PROLACTIN A N D A D R E N A L A N D R O G E N SECRETION A. V E R M E U L E N AND S. A N D 0
Department of Endocrinology and Metabolic Diseases, Medical Clinic, Academic Hospital, State University, Ghent, Belgium (Received I 9 August 1977; revised 5 October 1977; accepted 24 October 1977)
SUMMARY
The aim of this study was t o investigate the role of prolactin in the secretion by the adrenal of dehydroepiandrosterone (DHA) and its sulphate (DHA-S). Therefore prolactin and DHA-S levels were determined in different groups of subjects under various physiological and pathological conditions. In patients with prolactinomas, or with pharmacologically induced hyperprolactinaemia, plasma DHA-S and, t o lesser extend, DHA concentrations were elevated, and the DHA-S blood production rate greatly increased. ACTH stimulation which did not influence DHA-S concentrations in normals, caused a significant increase in patients with prolactinomas; the increase in DHA concentrations was similar in normal subjects and in prolactinoma patients. Bromocriptine treatment of prolactinaemia patients normalized both prolactin and DHA-S concentrations. Acute elevation of prolactin in normal subjects by TRH stimulation or by short term administration of sulpiride, in contrast to long term treatment, did not influence DHA-S levels. During pregnancy, notwithstanding high prolactin concentrations, DHA-S concentrations were lower than during the menstrual cycle. In patients with prolactinomas, given glucocorticoid replacement with cortisol, DHA-S concentrations were low (normal) notwithstanding persistently high prolactin concentrations. It is concluded that only prolonged elevation of prolactin induces increased DHA-S secretion by the adrenal cortex and that normal ACTH secretion is a prerequisite for this effect. The absence of elevated DHA-S concentrations in pregnancy might be explained by the rapid rate of metabolism, as the DHA-S production rate is increased. The intimate mechanism of prolactin and ACTH interaction at the adrenal cortex remains however unknown. Recently, high plasma dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulphate (DHA-S) levels were reported in patients with prolactinomas (Bassi et al., 1977; Vermeulen et al., 1977), while Donabedian et al. (1976) observed high urinary excretion of 17oxosteroids. Moreover, we reported (Vermeulen et al., 1977) that in patients with pharmacologically elevated plasma prolactin concentrations, DHA-S concentrations were similarly Correspondence: Dr A. Vermeulen, Department of Endocrinology and Metabolic Diseases, Medical Clinic, Academic Hospital, State University, Ghent, Belgium. 03004664/78/0400-0295 $02.00 0 1978 Blackwell Scientific Publications Ltd
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increased. We suggested therefore that prolactin might have an androgen secretion stimulating effect. In the view of these results we decided t o study in more detail the influence of prolactin on DHA-S levels in man. M A T E R I A L S AND METHODS Subjects Plasma prolactin and androgen concentrations were studied in several groups of subjects. (A) Control groups: (1) normal post-menopausal women (n = 40), (2) normal elderly males (>50 years) ( n = 20), (3) normal women during the first days ( 2 - 5 ) of the follicular phase of the cycle (n = 20), (4) normal young males (age 18-25) ( n = 20); (B) patients with prolactinomas with intact hypothalamo-pituitary-adrenal axis as shown by a normal insulin tolerance test, and with impaired ACTH secretion on corticoid treatment; (C) five patients with prolactinomas before and after treatment with bromocriptine 2 X 2.5 mg per day for 6 weeks; (D) six normal subjects and four patients with prolactinomas after acute ACTH stimulation (Synacthen 0.25 mg i.v.); (E) six normal men before and after i.v. injection of 200 pg of TRH, blood samples being taken at 0, 20 and 60 min after TRH injection; (F) three normal post-menopausal women before and after 1 0 days and I month of treatment with sulpiride 2 X SO mg day; and (G) twenty normal pregnant women at different stages of pregnancy. Finally DHA-S production rate was determined in two normal women, in three normal males and in two patients with prolactinomas, using the single i.v. injection method. Methods Blood samples for hormone determination were obtained in the fasting state between 08.00 and 10.00 h. Steroid hormones were determined as previously described (Vermeulen & Verdonck, 1976); DHA-S was determined after solvolysis as described by Andre &James (1973). Prolactin was assayed by a double-antibody method using the commercial CEAIRE-SORIN (Belgium) kit and are expressed in ng/ml NIH-1 standard (1 ng = 40 piu MRC 71/282). Plasma cortisol was determined using the method of Mattingly (1963). Measurement of the metabolic clearance rate (MCR) ofDHA-S A total of 5 pCi of 73H-DHA-S, TRK 267 (Amersham), specific activity 2.4 Ci/mmol, dissolved in 5% ethanol in normal saline, were rapidly injected intravenously between 08.00 and 10.00 hours; blood samples were taken hourly for 8 h from the opposite arm in the recumbent patient. The MCR was calculated on the basis of the slow portion of the disappearance curve, i.e. after the first hour, when the rate of decrease in the concentration of the isotopic labelled DHA-S can be represented by a single exponential curve (Gant et aZ., 1971): MCR = k/D X 1440 l/day where k = 0.6931t ?h(min) and D = fraction of administered radioactivity per litre of blood at zero time, obtained by extrapolation (Tait & Burstein, 1964). Isolation of 3H-DHA-Sfrom plasma To 1 ml of plasma were added 20 pg of cold DHA-S as internal standard. After mixing, the plasma free steroids were extracted with 10 ml of diethylether. After adding 2 ml 1 N sulphuric acid saturated with NaCl, the residual plasma was extracted with 20 ml of ethylacetate and kept overnight at 37'C. After evaporation, the residue was chromatographed on
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297
Whatman 40 paper and developed in the Bush A2 system. The D zone was eluted with methanol chloroform 1:1 v/v. Appropriate aliquots were taken for radioimmunoassay (recovery) and for determination of radioactivity. As plasma DHA-S concentrations do not show important diurnal variations (De Moor & Heyns, 1969; Rosenfield et al., 1975; Korth-Schutz et al., 1976) blood production rate (BPR) of DHA-S was derived using the plasma DS concentration obtained in the fasting blood sample.
RESULTS Normal values in different control groups are given in Table 1 t o contrast with values obtained in different groups of patients with prolactinomas. In nine young (SO years) (n = 20) Women early follicular phase (n = 20) Post-menopausal women (n = 40)
12.1+1.9* 11.8t3.6 9.0k1.2 7.3k2.6 Patients with prolactinomas with intact pituitaryadrenal axis Young males (n = 7) 1301.0t483 Young women (SO years) (n = 3)
DHA (nmol/l)
DHA-S (bmol/l)
23.3+1.4* 6.4k0.6 1 6 - l t1.5 8.6k0.8
5.04+0.39* 2.25t0.29 2.75k0.21 2.14k0.18
20.9k3.6 35.1 k6.1 19.8-33.0**
9.14k1.46 6.93+1.07 2.50-11.61**
*, MeankSEM; **,range of values. groups (Table 1). Similarly in three women of post-menopausal age with prolactinomas, DHA concentrations were increased with respect t o normal post-menopausal women; in two, DHA-S concentrations were also increased whereas in one a value (2.5 pnol/l) at the upper limit of the normal range was observed. In seven male subjects with prolactinomas (prolactin 300-3680 ng/ml) and an intact pituitary-adrenal axis, mean DHA-S was significantly increased (P< 0.02) with respect to normal controls, whereas mean DHA concentration was not significantly different. In six patients with prolactinomas, DHA concentrations could be followed during treatment with bromocriptine. As shown in Table 2, after 6 weeks of treatment prolactin as well as both DHA-S and DHA concentrations decreased significantly associated with resumption of cyclical ovarian activity or activation of Leydig cell secretion (as evidenced by an increase of plasma testosterone from 4.36 to 16.35 nmol/l). In six patients with prolactinomas (four men and two women) treated for several weeks with hydrocortisone 40 mg per day, DHA and DHA-S concentrations were normal or decreased, despite high prolactin concentrations (Table 3). Finally, whereas in normal subjects (n = 6 ) ACTH stimulation (0.25 mg Synacthen i.v.) caused a rapid increase of DHA but not
A . Vermeulen and S. Ando DHA-S (Fig. I), in patients with a prolactinoma (n = 4), ACTH stimulation resulted in a significant increase of both DHA and DHA-S, prolactin remaining unchanged. TRH stimulation (200 pg i.v.) in young males (n = 6) resulted in an increase of prolactin from a basal concentration of 11.0k2.7 (meankS.D.) ng/ml t o a mean of 37.7k7.9 ng/ml (365+103% of basal value) 20 min after injection which had returned t o 19*2 ng/ml Table 2. Influence of bromocriptine 2 X 2.5 mg/day on plasma hormone Pre-treatment
Treatment
Patient
Sex
Age
Prolactin (ng/ml)
DHA-S (Ctmol/l)
DHA (nmol/l)
Prolactin (ng/ml)
DHA-S (pmol/l)
DHA (nmol/l)
D.M.S. M.M. C.L. C.A. V.M.
F F F F M
39 19 39 40 26
1900 183 78 898 490
8.79 4.25 5.43 3.61 14.24
27.6 33.4 16.9 14.0 55.7
60 10 21 40 48
6.53 3.83 3.71 3.01 8.03
22.9 7.1 10.6 8.2 5.5
Table 3. Hormone concentrations in patients with prolactinomas on cortisol treatment 40 mg/day Patient
Sex
Age
Prolactin (ng/ml)
DHA (nmol/l)
DHA-S (Ctmolb)
W.G. M.A. K.F. R.W. V.D.S. G.G.
M M M M F F
43 38 27 41 52 50
220 200 3585 1140 200 1131
0.71 8.32 2.64
0.07 2.64 0.79 5.36 0.21 0.14
-
4.50 1.43
DHA
DHA-S
F
70
‘I 10 9
. -0 E
8
2-
10 -
0.2
I-
5-
0.1
I
I
I
I
I
I
I
l
l
1
Prolactin and adrenal androgen secretion
299
(176+31% of basal value) 60 min after injection. As shown in Fig.2 neither DHA,DHA-S nor
cortisol increased significantly. Neither changes in DHA nor in DHA-S were correlated with changes in prolactin, whereas a highly significant correlation (Y = 0.89, P < 0.001) between changes in cortisol and DHA (but not DHA-S) was observed. Ten days of treatment with sulpiride (2 X 50 mg/day) of three normal post-menopausal women, resulted in greatly increased prolactin concentration, but had no definite influence on DHA-S levels; after 1 month of treatment on the other hand, prolactin concentrations were not further increased, but DHA-S and DHA were now clearly elevated (Table 4). During gestation (weeks 5-38) prolactin concentrations (n = 20) varied between 4 and 167 ng/ml (mean 77+13 ng/ml), DHA concentrations between 3.2 nmol/l and 24.7 nmol/l (mean 11.9k1.2 nmol/l) and DHA-S levels between 0.39 and 5.79 pmol/l (mean 2.71k0.3 pmol/l). No correlation was observed between prolactin and either DHA or DHA-S concentrations. DHA and DHA-S concentrations decreased with duration of pregnancy: during the first trimester mean concentrations were 13.7+2.1 nmol/l and 3.64k0.64 pmol/l respectively,
30k T
Cortisol
20
DHA-S
Time (min)
Fig. 2. Influence of TR 00 fig I.V., on plasma concentrations of prolactin, cortisol, dehydroepiandrosterone (DHA) and its sulphate (DHA-S) in normal subjects (n = 6). Table 4. Influence of sulpiride treatment on plasma prolactin, DHA and DHA-S After 10 days treatment
Before treatment
After 1 month of treatment
Age Prolactin DHA-S DHA Prolactin DHA-S DHA Prolactin DHA-S DHA (ng/ml) (fimol/l) (nmol/l) (ng/ml) (fimol/l) (nmol/l) (ng/ml) (fimol/l) (nmol/l) -~
V.W. Q Da.S. 9 Hc.A. 0
54 76 52
21.0 16.1 10.8
2.15 1.75 2.11
12.5 4.8 8.8
180 167 167
4.64 1.64 1.96
21.4 5.8 9.5
156 132 159
6.00 4.43 4.29
24.3 10.0 13.6
A . Vermeillen and S. Ando
300
during the second trimester 11.OtO.3 nmol/l and 2.61k0.07 pmol/l and during the third trimester 11.3k0.14 nmol/l and 2.07k0.36 pmol, respectively. Prolactin concentrations on the other hand increased with duration of pregnancy from 15.7t2.7 ng/ml during the first trimester, t o 71k14 ng/ml during the second and 1 3 6 t 9 ng during the third trimester. Finally the metabolic clearance rate and blood production rate was measured in three normal men, two normal women and in two patients with prolactinomas. As shown in Table 5, the metabolic clearance rates of DHA-S in the two patients with prolactinomas were within normal limits (Wang et d . , 1967), whereas their blood production rates were greatly increased when compared t o the control subjects. Table 5. Metabolic clearance rate (MCR) and blood production rate (BPR) of DHA-S in normal subjects and in two patients with prolactinomas
Normals L.L.* D.V.M. V.A. T.R.
BPR (rmol)
Sex
Age
ADV (1)
k
MCR (1/24 hours)
Plasma DHA-S (w)
F
46 57 50 54
5.3 7.9 6.5 10.1
0.0959 0.1052 0.071 3 0.0520
12.2 20.1 11.0 12.6
4.50 1.25 3.32 1.06
54.9 25.1 36.5 13.4
28 35
6.4 6.6
0.0984 0.0422
15.1 8.9
8.14 9.1 1
122.9 81.1
M M M Prolactinoma patients V.P.R.** F R.F. M
*, Early follicular phase; **,amenorrhoea for 9 years. ADV = apparent distribution volume. DISCUSSION
It is evident from our results and from data in the literature, that in several clinical conditions, characterized by high circulating prolactin concentrations, increased plasma DHA-S and, t o a lesser extent, plasma DHA levels are found. This was observed in patients of either sex with prolactinomas, in young women with hyperprolactinaemia induced by chronic sulpiride treatment and in post-menopausal women treated chronically with phenothiazines or butyrophenones (Vermeulen et al., 1977). Moreover short term ACTH stimulation, which in accordance with previous observations (Vaitukaitis et d . , 1969; Nieschlag et d . , 1973) did not influence plasma DHA-S within 120 min in normal subjects, caused a significant increase in plasma DHA-S levels in patients with prolactinomas. The relative increase in DHA in both normals and hyperprolactinaemic subjects was similar with higher absolute values in the latter. This suggests a higher reserve in androgen secretion capacity, despite similar cortisol secretion. Bromocriptine treatment of patients with prolactinomas lowered prolactin concentrations, with a simultaneous decrease of DHA-S and DHA concentrations, as well as resumption of cyclical activity of the ovary or activation of Leydig cell function. This emphasizes the role of prolactin in the elevation of DHA-S levels. In patients with prolactinomas treated with cortisol, DHA-S and DHA concentrations were either normal or more often decreased, despite persistence of high prolactin concentrations. This suggests that the long-term effect of prolactin on the adrenal cortex requires the presence of ACTH.
Prolactin and adrenal androgen secretion
30 1
That the increased DHA-S concentrations in patients with prolactinomas were not the consequence of a decreased rate of metabolism is shown by the normal MCR. This was already suggested by the results reported by Donabedian et al. (1976) and by Bassi et al. (1 977) who observed increased urinary 17-oxosteroid and DHA-S excretion, respectively. All these results suggest a stimulating effect of prolactin on DHA-S secretion by the adrenal cortex. As neither we ourselves (Vermeulen et al., 1977) nor Bassi etal. (1977) observed any significant increase in androstenedione levels, this suggests an effect of prolactin on the adrenal A5-30-01-dehydrogenase. Another possibility, as suggested by Bassi et al. (1977), is a direct effect of prolactin on sulphate synthesis by the adrenal cortex. As sulphates secreted by the adrenal cortex are essentially A 5 3 0 hydroxy-steroids, this latter hypothesis does not exclude the former. Several experiments in animal species suggest an effect of prolactin on the adrenal cortex: indeed prolactin receptors have been detected on adrenal membrane preparations from several species (Posner et al., 1975; Marshall et al., 1975, 1976); synergism between ACTH and prolactin in production of corticosterone from isolated rat adrenal cells has been reported (Lis et al., 1974) whereas Witorsch & Kitay (1972) as well as Gustafsson & Steinberg (1975) reported an inhibition of adrenal Sa-reductase activity by prolactin. Although it is hazardous t o extrapolate these findings to man, they strengthen the hypothesis of a direct effect of prolactin on the human adrenal cortex. Another set of experimental data seems however t o conflict with the hypothesis of a stimulatory effect of prolactin on adrenal androgen secretion. TRH stimulation causes an increase in prolactin, but has no influence on either DHA-S or DHA; similarly short term treatment with sulpiride (10 days) causes a highly significant increase in prolactin without significantly affecting DHA-S or DHA. Moreover during pregnancy, with its greatly increased prolactin concentrations, we found in accordance with earlier observations (Candy, 1970; Nieschlag et al., 1974), DHA-S and DHA concentrations to be decreased when compared to the values during the menstrual cycle. However the lack of effect of either TRH or short term sulpiride treatment on DHA-S does not necessarily refute the hypothesis of an adrenal stimulating effect of prolactin: indeed prolonged elevation of prolactin might be necessary for prolactin t o have any effect on the adrenal cortex. A similar prolonged elevation of plasma prolactin concentration is necessaw before it induces amenorrhoea, although shortening of the luteal phase may occur earlier. As far as pregnancy is concerned, it is well known that metabolism of DHA-S is very rapid and despite decreased plasma concentrations, due to the increased MCR (Cant et al., 1971) the blood production rate of DHA-S is likely t o be increased. The lack of effect of bovine prolactin o n adrenocortical or gonadal function in normal men, as reported by Varma et al. (1977) seems also to be against the hypothesis of a stimulatory effect of human prolactin on adrenal DHA-S secretion. These data should be interpreted with caution, as on the one hand bovine prolactin may have effects distinct from human prolactin, and on the other, it was administered for a relatively short period ( 5 days) only. There remains the possibility that it is not prolactin itself but a different substance secreted both by prolactinomas and by the normal pituitary after chronic administration of drugs with central antidopaminergic effects, that is responsible for the stimulation of androgen secretion by the adrenal cortex. Nevertheless, all experimental data available so far are compatible with the hypothesis that prolonged elevation of plasma prolactin stimulates adrenal DHA-S secretion. This stimulatory effect seems t o require the presence of ACTH, since in its absence low DHA-S concentrations are usually observed. The mechanism leading
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to this increased DHA-S secretion remains unknown and whether physiological prolactin levels influence adrenal androgen secretion in man remains t o be proven. ACKNOWLEDGMENT
Part of this work was supported by grant No. 20.477 of the FWGO (Brussels). REFERENCES ANDRE, C.M. & JAMES, V.H.T. (1973) Assay of plasma dehydroepiandrosterone and its sulfate by competitive protein binding. Clinica Chimica Acta, 43,295-303. BASSI, F., GIUSTI, G., BORSI, L., CATTANEO, S., GIANOTTI, P., FORTI, G., PAZZAGLI, M., VIGIANI, C. & SERIO, M. (1977) Plasma androgens in women with hyperprolactinaemic amenorrhoea. Clinical Endocrinology, 6,5-10. DE MOOR, P. & HEYNS, W. (1966) Androgens in Normal and Pathological Conditions (ed. by A. Vermeulen and D. Exley), pp. 54-61, Excerpta Medica ICS 101. DONABEDIAN, R.R., MAY, P.B. & TAN, Y.S. (1976) Abnormal adrenal steroidogenesis in patients with hyperprolactinaemia, galactorrhea or both. 56th Annual Meeting of the Endocrine Society, Abstract 372, p. 243. GANDY, H.M. (1970) Androgens. In: Endocrinology of Pregnancy (ed. by F. Fuchs and A. Klapper), p. 130. Harper and Row, New York. GANT, N.F., HUTCHINSON, H.T., SIITERI, P. & MACDONALD, P. (1971) Study of the metabolic clearance rate of dehydroisoandrosterone sulfate in pregnancy. American Journal o f Obstetrics and Gynecology, 111,555-561. GUSTAFSSON, J.A. & STEINBERG, A. (1975) Influence of prolactin on the metabolism of steroid hormones in rat liver and adrenals. Acta Endocrinologica (Kbh.], 78,545-553. KORTH-SCHUTZ, S., LEVINE, L.S. & NEW, M.I. (1976) Dehydroepiandrosterone sulfate (DS) levels, a rapid test for abnormal adrenal androgen secretion. Journal of Clinical Endocrinology and Metabolism, 42,1005-1023. LIS, M., GILORDEAU, J. & CHRETIEN, M. (1973) Effect of prolactin on corticosterone production by rat adrenals. Clinical Research, 21,1027. MARSHALL, S., GELATO, M. & MEITES, J. (1975) Serum prolactin levels and prolactin binding activity in adrenals and kidneys of male rats after dehydration, salt loading and unilateral nephrectomy. Proceedings of the Society of Experimental Biology and Medicine, 149, 185-188. MARSHALL, S., KLEDZIK, G.S., GELATO, M., CAMPBELL, G.A. & MEITES, J. (1976) Effects of estrogen and testosterone on specific prolactin binding in the kidneys and adrenals of rats. Steroids, 27,187-195. MATTINGLY, D. (1962) A simple fluorimetric method for the estimation of free 11-hydroxycorticoids in human plasma. Journal of Pathology, 15,374-379. NIESCHLAG, E., LORIAUX, D.L., REIDER, H.J., ZUCKER, LR., KIRCHNER, M.A. & LIPSETT, M.B. (1 973) The secretion of dehydroepiandrosterone and dehydroepiandrosterone sulphate in man. Journal of Endocrinology, 57,123-1 34. NIESCHLAG, E., WALK, T. & SCHINDLER, A.E. (1974) Dehydroepiandrosterone (DHA) and DHAsulfate during pregnancy in maternal blood. Hormones and Metabolic Research, 6,170-171. POSNER, B.I., KELLY, P.A., SHIU, R.P.C. & FRIESEN, H.G. (1975) Studies of insulin,growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology, 95, 5 2 1-53 1. ROSENFIELD, R.S., ROSENBERG, B.J., FUKUSHIMA, D.K. & HELLMAN, L. (1975) 24 hour secretory pattern of dehydroisoandrosterone and dehydroisoandrosterone sulfate. Journal of Clinical Endocrinology and Metabolism, 40,850-855. TAIT, J.F. & BURSTEIN, S. (1964) In vivo studies of steroid dynamics in man. The Hormones, Vol. 5 (ed. by J. Pincus, K. V. Thimann and E. B. Astwood), pp. 441-557. Academic Press, New York. VAITUKAITIS, J.L., DALE, S.L. & MELBY, J.C. (1969) Role of ACTH in the secretion of free dehydroepiandrosterone and its sulfate ester in man. Journal of Clinical Endocrinology and Metabolism, 29, 1443-1447.
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VARMA, M.M., HUSEMAN, C.A., JOHANSSON, A.J. & BLIZZARD, R.M. (1977) Effect of prolactin on adrenocortical and gonadal function in normal men. Journal o f Clinical Endocrinology and Metabolism, 44,760-761. VERMEULEN, A. (1976) Plasma androgen levels during the menstrual cycle. American Journal of Obstetrics and Gynecology, 1 2 5 , 4 9 1 4 9 4 . VERMEULEN, A. & VERDONCK, L. (1976) Radioimmunoassay of 17p-hydroxy-5a-androstan-3-one, 4 androstene-3,17 dione, dehydroepiandrosterone, 17P-hydroxy-progesteroneand progesterone and its application to human plasma. Journal of Steroid Biochemistry, 7, 1-10. VERMEULEN, A., SUY, E. & RUBENS, R. (1977) Effect of prolactin on plasma DHEA(S) levels. Journal o f Clinical Endocrinology and Metabolism, 44,1222-1 225. WANG, D.Y., BULBROOK, R.D., SNEDOON, A. & HAMILTON, T. (1967) The metabolic clearance rate of dehydroepiandrosterone, testosterone and their sulphate esters in man, rat and rabbit. Journal of Endocrinology, 38,307-318. WITORSCH, R.J. & KITAY, J.I. (1972) Pituitary hormones affecting adrenal Sa-reductase activity: ACTH, Growth hormone and prolactin. Endocrinology, 91,764-769.
