Effect of Spironolactone on Sex Hormones in Man B. STRIPP, A. A. TAYLOR, F. C. BARTTER, J. R. GILLETTE, D. L. LORIAUX, R. EASLEY, AND R. H. MENARD National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014, Brown University, Providence, Rhode Island prolactin. These findings are consistent with a previously-reported spironolactone-induced destruction of the microsomal enzyme cytochrome P-450, an enzyme necessary for 17-hydroxylase and desmolase activity. The results do not explain the decrease of libido, the impotence, and the gynecomastia frequently associated with spironolactone therapy in males (/ Clin Endocrinol Metab 41: 777, 1975)

ABSTRACT. Administration of spironolactone at a dosage of 400 mg/day to healthy male volunteers for 5 days resulted in a significant rise in plasma progesterone and 17a-hydroxyprogesterone which persisted throughout the study. A transient increase in plasma FSH and LH concentration was observed after the second but not the third or fifth days of drug administration. There was no change in plasma concentration of testosterone, 17/3-estradiol, or


PIRONOLACTONE is a steroid analog which, because of its competitive antagonism to the action of aldosterone, is widely used as a potassium-conserving diuretic. Recently, spironolactone in high dosages has been successfully used as an antihypertensive agent in the treatment of syndromes which manifest a proven or suspected excess of sodium-retaining steroids, syndromes such as primary aldosteronism or "low-renin essential hypertension" (1-4). The drug, however, does cause untoward reactions in as many as 20% of the patients to whom it is given (5). The occurrence of side effects such as decrease of libido, impotence, and gynecomastia in males and breast tenderness and menstrual irregularities in females (1,5-7) suggests that spironolactone may interfere with the normal function of endocrine systems responsible for sexual function. Received April 14, 1975. Presented in part at the meeting of the American Society for Clinical Investigation, Atlantic City, N.J., May 1974, and at the meeting of the Endocrine Society, Atlanta, Ga., June 1974. Trivial and systematic names used in this paper: testosterone = 17j8-hydroxyandrost-4-en-3-one; progesterone = pregn-4-ene-3,20-dione; 17a-estradiol = estra-l,3,5(10)-triene-3,17a-diol; 17a-hydroxyprogesterone = pregn-4-ene-3, 20-dione-17a-ol. Canrenone (aldadiene) = 3-(3-oxo-17/3-hydroxy-4,6androstadien-17ayl) proprionate.

Antiandrogenic properties of spironolactone have been demonstrated in animals. Androgen-dependent hepatic drug metabolism (8) and growth of the seminal vesicles (9) in male rats are inhibited by spironolactone. Moreover, spironolactone has been shown to decrease the biosynthesis of testosterone in the testis of various animal species by decreasing microsomal cytochrome P-450 content (10-13). Recently it was reported that spironolactone also decreases cytochrome P-450 content and inhibits 17o:-hydroxylase activity in guinea pig adrenal gland, and that this inhibition can be competitively antagonized by the addition of progesterone to the incubation medium (14). The purpose of the present study was to investigate spironolactone-induced changes in sex steroids in normal male volunteers, in an attempt to define the biochemical alterations leading to the development of gynecomastia and the decrease of libido and impotence that occur during spironolactone therapy in some male patients. Materials and Methods Six healthy male volunteers, aged 21 to 33, participated in the study. Diet and activity were unrestricted. Blood samples were obtained between 8 AM and 9 AM on days 1, 3, 4, 6, and 8 of the study. All blood was collected from the antecubital vein into polystyrene syringes and the


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JCE & M • 1975 Vol 41 • No 4



plasma separated immediately after centrifugation at 4 C in heparinized glass tubes. Plasma was stored in polystyrene tubes at — 20 C until analyzed. Serum sodium, potassium, chloride, and total carbon dioxide concentration was determined at least every other day throughout the study. Spironolactone (Aldactone1*) was administered immediately after phlebotomy as a single, 400 mg oral daily dose on days 3 through 7 of the study. Plasma testosterone (15), progesterone (16), estradiol (17), 17a-hydroxyprogesterone (16), prolactin (18), follicle stimulating hormone (FSH) (19), and luteinizing hormone (LH) (20), were determined by radioimmunoassay. Daily urinary 17-ketosteroids were measured in 2 subjects by a modification of the method of Zimmermann (21). Serum sodium, potassium, chloride,

and CO2 were determined by standard automated methods. Group and paired data were analyzed statistically by student's t test. Results The only clinical effects of spironolactone were subjective complaints of lethargy and easy fatigability. These were experienced by 4 of the 6 volunteers. No subject developed gynecomastia or experienced impotence during or after drug treatment was stopped. Serum sodium and chloride concentrations had decreased an average of 5 mEq/1 by the fifth day of spironolactone treatment, as compared to pretreatment values. There

TABLE 1. Effect of spironolactone on plasma progesterone, ]L7a-hydroxyprogesterone, 17/3-estradiol, FSH, LH, prolactin, serum electrolytes, and urinary 17-ketosteroids Spironolactone (400 mg/day) Control a

Day 2 b


Day 3 b


Day 5 b

Progesterone (ng/ml)

0.97 ±0.1 (15)

2.5 ± 0.2 (6)

2.7 ± 0.4


17a-hydroxyprogesterone (ng/ml)

1.29 ± .09 (16)

2.04 ± 0.14 (6)

2.04 ± 0.2


Testosterone (ng/dl)

773 ± 8 6 (15)

855 ± 65

17/8-estradiol (pg/ml)

25.6 ± 1.8 (14)

31.3 ± 4.2 (6)

a 2.2 ± 0.4

b (6)

2.16 ±0.17 (6)

760 ± 107 (6)

713 ± 77

30.5 ± 3.4


40.0 ± 9.3 (6)


10.1 ± 1.4




FSH (mlU/ml)

7.8 ± 1.2 (15)

LH (mlU/ml)

9.1 ± 0.6 (15)

11.9 ± 1.1 (6)

10.0 ± 1.4


10.4 ± 1.3 (6)

Prolactin (ng/ml)

17.9 ± 3.4 (15)

22.0 ± 6.5 (6)

20.2 ± 5.4


24.0 ± 3.9 (6)

Serum Na (mEq/L)

141 ± 0.4 (6)

138 ± 0.8 (6)

139 ± 0.9 (6)

135 ± 0.8 (6)

Serum K (mEq/L)

3.9 ±0.2 (6)

3.8 ± 0.2


4.2 ± 0.3 (6)

3.9 ± 0.2 (6)

Serum Cl (mEq/L).

101 ± 1.0 (6)

99 ± 1.1 (6)

99 ± 1.1 (6)

96 ± 0 . 9 (6)

Serum CO2 (mEq/L)

28 ± 0.4 (6)

27 ± 0.8 (6)

28 ± 0.5 (6)

27 ± 1.2


14.2 ± 1.2 (2)

15.5 ± 1.1 (2)

14.5 ± 1.1 (2)

14.5 ± 0.4


Urinary 17-ketosteroids (mg/24 h)

9.1 ± 1.4

9.2 ± 1.4


a = mean ± standard error of mean b = number of determinations

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FIG. 1. The effect of spironolactone in 6 normal male subjects on serum progesterone, 17hydroxyprogesterone, 17/3estradiol, testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. The data are plotted as changes (A) from values of control days. The asterisks (*) indicate those values which differ significantly from the values of the control days (P < .05).


2 3 5 DAY

2 3 5 DAY

2.0 r *




2 3 5 DAY

was no significant change in serum potassium or CO2 during the study. The plasma steroid data are shown in Table 1 and Figure 1. Plasma progesterone increased from 0.95 ± 0.10 ng/ml (mean ± SEM) during the control period to a maximum value of 2.7 ± 0.4 ng/ml by the third day of spironolactone administration. Plasma 17a-hydroxyprogesterone increased from 1.29 ± 0.09 ng/ml during the control period to a value of 2.16 ± 0.17 ng/ml by the fifth day of spironolactone treatment. Spironolactone and its major metabolite, canrenone, do not interfere significantly with the assay of progesterone or ^a-hydroxyprogesterone.1 There were small but statistically significant increases in plasma LH and FSH after 2 days of spironolactone administration. FSH and LH control values of 7.8 ± 1.2 mlU/ml and 9.1 ± 0.6 mlU/ml, respectively, increased to 9.1 ± 1.4 and 11.9 ±1.1 after 2 days of drug administration. 1 A concentration of 10 /zmol spironolactone or canrenone/ml plasma, which approximates the highest concentration seen in patients who have received 400 mg spironolactone (22) caused less than 5% deviation in the measurement of progesterone or 17ahydroxyprogesterone as compared to the measurement of the same plasma samples without spironolactone or canrenone.

2 3 5 DAY

After the third and fifth days of spironolactone, plasma FSH and LH were highly variable and were not significantly different from the pretreatment values. No significant changes in plasma testosterone, 17/3-estradiol, or prolactin in the 6 subjects or in urinary 17-ketosteroids in 2 subjects were observed during treatment with spironolactone. Discussion Direct interference of spironolactone or its major metabolite, canrenone, with the radioimmunoassays for progesterone and 17ahydroxyprogesterone does not account for the increased plasma concentration of these 2 steroids following spironolactone therapy. Since spironolactone increases hepatic drug metabolism in several mammals (8,11,23,24) including man (25), it is unlikely that the increases in plasma progesterone or 17ahydroxyprogesterone resulted from impairment of their metabolism in the liver. It appears more likely that the rise in plasma progesterone and 17a-hydroxyprogesterone occurred as a result of increased or of continued synthesis with a decrease of further metabolism. These observations, however, do not provide information about either the mechanism or the site(s) of increased proges-

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terone or 17a-hydroxyprogesterone synthesis. Menard et al. (11,13) have reported that spironolactone, but not its metabolites canrenone or canrenoate-K, destroys the microsomal enzyme cytochrome P-450 in the testes of several animal species. This destruction in the dog testis is associated with a decrease in 17«-hydroxylase activity and in secretion of testosterone into the spermatic vein (12). Moreover, destruction of cytochrome P-450 and decreased 17ahydroxylase activity was demonstrated in the adrenals of animals that produce predominantly cortisol (14). The finding of increased plasma progesterone in human subjects taking spironolactone is consistent with decreased 17a-hydroxylation in either the testis or adrenal or both. This hypothesis, however, does not explain the increase of plasma 17a-hydroxyprogesterone observed in this study. Two possible explanations are (a) that spironolactone may interfere with enzymes such as 21-hydroxylase, required to convert 17-hydroxyprogesterone to 11 desoxycortisol. This is not an unlikely possibility as 21-hydroxylase, like 17a-hydroxylase, is a cytochrome P450-dependent microsomal enzyme (14). Alternatively, (b) spironolactone may interfere with the desmolase (also a microsomal enzyme) which converts 17 hydroxyprogesterone and 17a-hydroxypregnenolone to androstenedione and dehydroepiandrosterone, respectively (26). Since secreted androstenedione and dehydroepiandrosterone contribute significantly to measurable urinary 17-ketosteroids (26), a decrease in desmolase activity might be reflected in a decrease of urinary 17-ketosteroid excretion. However, no significant change in urinary 17-ketosteroids was seen after 5 days of spironolactone treatment in the 2 subjects in which those steroids were measured. In any event, the postulated inhibition of 21 hydroxylase, or of desmolase, would presumably be relatively greater than the inhibition of 17-hydroxylase to produce the observed increase in plasma 17-hydroxyprogesterone.

i 1975 No 4

The elucidation of spironolactone's inhibition of specific steroid biosynthetic steps in the testis, and the possible clinical implications of these changes, await more detailed investigation. There was a tendency for plasma testosterone to be lower after 5 days of spironolactone treatment than during the pretreatment period, but the changes in plasma testosterone were highly variable from individual to individual, and statistical significance was not achieved. The relatively small dosage of spironolactone (about 5-8 mg/Kg BW) used in these human experiments, as compared to the dosage of 40-200 mg/Kg used in animal experiments (9-14), could explain our failure to measure any decrease in plasma testosterone concentration. Our findings of transient increases in plasma FSH and LH suggest that plasma testosterone may have been transiently depressed, despite our failure to show a significant change.2 In the presence of an intact pituitary-testicular axis this rise in LH and FSH may have restored plasma testosterone to the normal values observed in this study. The potential effects of spironolactone on pituitary, gonadal, and adrenal function, when the drug is given over longer periods of time, is largely unknown and is currently under investigation. These findings fail to elucidate a mechanism by which spironolactone produces gynecomastia, impotence, or decrease of libido. References 1. Spark, R. F., and J. C. Melby. Ann Intern Med 69: 685, 1968. 2. Brown, J. J., D. L. Davies, J. B. Ferriss, R. Fraser, E. Haywood, A. F. Lever, and J. I. S. Robertson. BrMedJ2: 729, 1972. 3. Crane, M. C , J. J. Harris, and V. J. Johns. Am J Med 52: 457, 1972. 4. Carey, R. M., J. G. Douglas, R. Schweikert, and G. W. Liddle. Arch Intern Med 130: 849, 1972. 2

A significant increase in plasma LH in 5 of 7 normal volunteers after 2 weeks of spironolactone treatment was reported by Pentikainen, et al. (27).

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SPIRONOLACTONE ON SEX HORMONES 5. Greenblatt, D. H., and J. Koch-Wesser. JAMA 225: 40, 1973. 6. Clark, E.JAMA 193: 163, 1965. 7. Levitt, J. I. JAMA 211: 2014, 1970. 8. Stripp, B., M. E. Hamrick, N. G. Zampaglione, and J. R. Gillette. J Pharmacol Exp Ther 176: 766, 1971. 9. Hamrick, M. E., N. G. Zampaglione, B. Stripp, and J. R. Gillette. Biochem Pharmacol 22: 293, 1973. 10. Stripp, B., R. Menard, N. E. Zampaglione, M. E. Hamrick, and J. R. Gillette. Drug Metabolism and Disposition 1: 216, 1973. 11. Menard, R. H., B. Stripp, and J. R. Gillette. Endocrinology 94: 1628, 1974. 12. Stripp, B., R. Menard, L. Loriaux, A. Taylor, J. Gillette, and F. C. Bartter../ Clin Invest. 53: 79a, 1974. 13. Menard, R. H., B. Stripp, D. L. Loriaux, F. C. Bartter, and J. R. Gillette, 56th Annual Meeting of the Endocrine Society, 1974 (Abstract No. 98), p. A-104. 14. Menard, R. H., H. F. Martin, B. Stripp, J. Gillette, and F. C. Bartter. Life Sci [II] 15: 1639, 1974. 15. Nieschlag, E., and D. L. Loriaux, J Klin Chem 10: 164,.1972.


16. Stroh, C. A., and M. B. LipsettJ Clin Endocrinol Metab 28: 1426, 1968. 17. Loriaux, D. L., H. J. Ruder, and M. B. Lipsett. Steroids 18: 463, 1971. 18. Auberb, M. L., R. L. Backer, B. B. Saxena, and S. RaitiJ Clin Endocrinol Metab 38: 1115, 1974. 19. Cargille, C. M., and P. O. RayfordJ Lab Clin Med 75: 1030, 1970. 20. Odell, W. P., G. T. Ross, and P. L. RayfordJ Clin Invest 46: 248, 1967. 21. Peterson, R. E., and C. E. Pierce. "Lipids and the Steroid Hormones in Clinical Medicine" 147,1960. 22. Feller, D. R., and M. C. Gerald. Biochem Pharmacol 20: 1991, 1971. 23. Sadee, W., M. Dagcioglu, and R. Schroder. J Pharmacol Exp Ther 185: 686, 1973. 24. Solymoss, B., S. Toth, S. Varga, J. Werringloer, and G. Zsigsmond. Can J Physiol Pharmacol 49: 841, 1971. 25. Huffman, D. H., D. W. Shoeman, P. Pentikainen, and D. L. Azarnoff. Pharmacol. 10: 338, 1974. 26. Peterson, R. E., Chapter 4 In Christy, N.P. (ed.), The Human Adrenal Cortex, Harper and Row, 1971, p. 87. 27. Pentikainen, P., L. A. Pentikainen, D. H. Huffman, and D. L. Azamoff. Clin Res 21: 472, 1973.

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Effect of spironolactone on sex hormones in man.

Administration spironolactone at a dosage of 400 mg/day to healthy male volunteers for 5 days resulted in a significant rise in plasma progesterone an...
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