Gynecomastia and Semen Abnormalities Induced by Spironolactone in Normal Men RAUL CAMINOS-TORRES, LISA MA, AND PETER J. SNYDER Endocrine Section, Department of Medicine, University of Pennsylvania School of Medicine, 522 Johnson Pavilion, 36th and Hamilton Walk, Philadelphia, Pennsylvania 19174 ABSTRACT. The clinical and hormonal effects of spironolactone on the pituitary-testicular axis were evaluated in healthy, young men. One group of nine men took 100 mg daily for 4 weeks, none for 4 weeks, then 400 mg daily for 4 weeks. The dialyzable fraction of testosterone increased by 20% (P < 0.01) during both periods of spironolactone administration. The serum concentrations of FSH, LH, testosterone and estradiol, however, did not change during either period, nor did the FSH and LH responses to synthetic gonadotropin-releasing hormone. Another group of 9 men took 400 mg of spironolactone daily for up to 24 weeks. During this time 6 developed gynecomastia and 2 noted a decrease in libido. Two men had decreases in sperm density and motility that were apparently drug-related, although the mean sperm density of all 9 men did
G
YNECOMASTIA and impotence are recognized side-effects of spironolactone administration to men, especially at doses exceeding 100 mg daily (1-4). These effects suggest that spironolactone affects testosterone secretion, metabolism, or action. Data from several studies indicate that spironolactone has the potential for producing all of these effects. Administration of spironolactone to several animal species causes a pronounced decrease in testicular microsomal P-450 levels and in microsomal 17-hydroxylase (5), the enzyme responsible for the conversion of progesterone to 17hydroxyprogesterone. Administration of spironolactone to men appears to increase the conversion of testosterone to estradiol (6). Both spironolactone (7,8) and canrenone (8), which is the principal circulating metabolite of spironolactone, inhibit the binding of dihydrotestosterone (DHT) to Received October 26, 1976. Supported by U.S.P.H.S. grants HD 08555 and RR 40 and a grant from Searle Laboratories.
not change significantly. No change occurred in the mean serum concentrations of FSH, LH, testosterone or estradiol. In vitro incubation of canrenone, the principal circulating metabolite of spironolactone, in concentrations achieved in vivo, with serum from normal men resulted in a small but significant displacement of testosterone from its binding protein and in a small spurious increase in die serum testosterone concentration. We conclude that spironolactone-induced gynecomastia and occasional semen abnormalities do not appear to be due to changes in the serum concentrations of testosterone or estradiol. We hypothesize that these changes may be related to binding of canrenone to tissue androgen receptors. (/ Clin Endocrinol Metab 45: 255, 1977)
the DHT receptor in rat ventral prostate cytosol. Which of these biochemical effects is, or are, responsible for the observed gynecomastia and decreased libido in men is unclear. Administration of 400 mg of spironolactone to normal men once a day for 5 days produced an elevation of serum progesterone and 17-hydroxyprogesterone concentrations (9), suggesting that spironolactone may inhibit testosterone biosynthesis in man. The serum testosterone concentration in those men, however, did not decrease nor did their serum estradiol concentration increase. The purpose of the present investigation was to determine the clinical and hormonal effects of long-term spironolactone administration on the pituitary-testicular axis in healthy, young men. Men who were healthy and young were chosen to avoid the concomitant effects of age, illness, and medications on the pituitary-testicular axis. One group of men was given 100 mg of spironolactone daily for 4 weeks and, later, 400 mg daily for 4 weeks, in order to assess any
255
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CAMINOS-TORRES, MA AND SNYDER
hormonal effects. A second group of men took 400 mg daily for 24 weeks in order to determine any additional hormonal effects of prolonged administration and to correlate clinical with hormonal effects. Sera from a third group of men, who did not take spironolactone, were used to determine the effects of spironolactone and its metabolites on the steroid assays employed. Materials and Methods Short-term administration of spironolactone to normal men Each subject, both in this shorMerm study and in the long-term study described below, gave informed, written consent to a protocol approved by the University of Pennsylvania Committee on Investigations in Humans. No subject had any illness or was taking any medications suspected of altering the pituitary-testicular axis. In the short-term study, nine healthy young men, ages 18-39 (mean age, 25), each took 100 mg of spironolactone daily (50 mg every 12 h) for 4 weeks, stopped the medication for 4 weeks, then took 400 mg daily (200 mg every 12 h) for 4 weeks. Blood was sampled twice between 0800 and 1000 h on each of 3 consecutive days before beginning each dose of medication and in a similar fashion at the end of 2 and 4 weeks of each dose. The serum LH and FSH responses to a 50 jug bolus iv dose of gonadotropin-releasing hormone (GnRH) were determined in each subject prior to beginning the first dose and at the end of 4 weeks administration of each dose. Blood was sampled for LH and FSH at -30, - 1 5 , 0, 10, 20, 30, 45, 60, 90, 120, 150, and 180 min in relation to each GnRH dose.
In vitro effects of spironolactone, and potassium canrenoate
JCE & M • 1977 Vol 45 • No 2
canrenone
The in vitro effects of spironolactone and its principal metabolites on the immunoassays for testosterone and estradiol and on the dialyzable fraction of testosterone were determined. Each of these three compounds (Searle Laboratories, Chicago, Illinois) was dissolved in 95% ethanol and added to aliquots of serum from 6 normal young men so that the final concentrations of each compound were 16, 80, 400, and 2000 ng/ml. Each aliquot was then assayed for testosterone, estradiol, and dialyzable fraction of testosterone. These concentrations were chosen to approximate the canrenone levels achieved in vivo by the administration of 200 mg of spironolactone every 12 h. The subjects in this study who took that dose had mean serum concentration of canrenone, which is the principal circulating metabolite of spironolactone (10), of 400600 ng/ml 12 h after the previous dose (Table 2). Since the serum canrenone concentration usually reaches a peak 2-3 h after an oral dose, and the concentration falls gradually during the next 12 h (10), it is likely that the serum concentrations of canrenone in the present study subjects were usually higher than those measured 12 h after a dose. Although the serum levels of spironolactone and potassium canrenoate were not measured in the subjects who were administered spironolactone, the serum concentrations of these two compounds in subjects taking spironolactone are generally much less than that of canrenone (10). The concentrations of spironolactone and potassium canrenoate used in this in vitro study, therefore, were probably many times greater than occur in vivo during the oral administration of 400 mg of spironolactone daily. Assays
Long-term administration of spironolactone to normal men Nine healthy men, ages 21-28, consented to take 400 mg of spironolactone daily (200 mg every 12 h) for 24 weeks. Blood was sampled twice between 0800 and 1000 h on each of 3 consecutive days prior to beginning the medication and in a similar fashion every 4 weeks. A semen sample was analyzed every two weeks. Subjects were examined for gynecomastia every 4 weeks.
FSH and LH were determined by immunoassays, as described previously (11), using reagents provided by the National Pituitary Agency. The results are expressed in terms of the 2nd IRP-HMG, where one /xg of LER 907 equals 38 mlU of FSH and 219 mlU of LH. Serum testosterone was determined by immunoassay using antiserum to testosterone-3carboxymethyloxime-BSA and testosteronetyrosine methyl ester-125I (Bio-RIA, Montreal, Canada). Ether extracts of sera were assayed
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SPIRONOLACTONE-INDUCEDGYNECOMASTIA
TABLE 1. Effect of short-term spironolactone administration to normal men on serum concentrations of FSH, LH, testosterone and estradiol and on the dialyzable fraction of testosterone Spironolactone, 400 mg/day
Spironolactone, 100 mg/day 2 weeks
Pre-Rx Basal serum FSH (mlU/ml) Maximum serum FSH* Basal serum LH (mlU/ml) Maximum serum LH (mlU/ml)* Serum testosterone (ng/dl) % Dialyzable testosterone Serum estradiol (pg/ml)
5.1 :t 0.7 6.0 :t 0.9 11.3 :t 1.4 42.5 :t 5.5 447 :t 4 7 3.40:t 0.16 25 :t 2
4 weeks
4.2 ± 0.5
4.3 :t 0.3 5.2 :t 0.5 11.9 :t 1.3 35.4 :t 3.4 608 :t 6 7 4.12:t 0.231 27 :t 1
12.1 ± 1.4 _
498
± 36 —
29
± 2
Pre-Rx
2 weeks
4.2 ± 0.4
4.2 ± 0.3
11.2 ± 1.1
15.7+ 0.2 _ 497 ± 32 _ 30 ± 2
_
516
± 56 _
26 ± 2
4 weeks 4.0 dt 0.3 5.0 1t 0.5 13.5 dt 1.6 38.1 i: 3.7 587 2t 6 6 4.02 i: 0.15f 25 :t 1
Nine healthy young men took 100 mg of spironolactone daily for 4 weeks, stopped the medication for 4 weeks, then took 400 nig daily for 4 weeks. Blood was sampled from each subject six times prior to taking the medication and similarly after 2 and 4 weeks administration. All values are means ± SEM. * Maximum response to a 50 /xg iv bolus dose of synthetic gonadotropin-releasing hormone, t P < 0.01.
phate, pH 7.4 (PBS) and incubated with 3Htestosterone overnight at 4 C. The 5 ml of diluted serum was then dialyzed against 5 ml of PBS in dialysis cells at 37 C for 24 h. The percentage of dialyzable testosterone in the diluted serum was calculated, DPM in dialysate x 100/DPM in serum + dialysate. Multiplication by 0.367 gave the percentage of dialyzable testosterone in the undiluted serum (11). The figure, 0.367, was obtained experimentally as the ratio of the percentage of dialyzable testosterone in an undiluted serum pool (3.2%) to that in the same pool diluted 1:5 (9.4%), corrected for a 10.8% volume change when undiluted serum was used. The serum concentration of canrenone was determined by Dr. Thomas C. Hutsell at Searle Laboratories by fluorimetric analysis (12). Semen was analyzed as described in Paulsen (13). Statistical analyses were performed by the paired and non-paired t tests (14).
without prior chromatography. Cross-reactivity of dihydrostestosterone was 50% that of testosterone. The lower limit of sensitivity of the assay was 20 pg per assay tube; the intraassay and interassay coefficients of variation were 8.9% and 13.3%. Serum estradiol was determined by immunoassay using antiestradiol serum raised to 17/3 estradiol-6-carboxymethyloxime-bovine serum albumin and using estradiol-7-succinyl-125I (Micromedic, Horsham, Pa.). Benzene extracts of sera were assayed without prior chromatography. Cross-reactivity of estrone was < 1 % that of estradiol. The lower limit of sensitivity of the assay was 2 pg per assay tube; the intraassay and interassay coefficients of variation were 12% and 14%. The dialyzable fraction of testosterone was determined by equilibrium dialysis, as previously described (11). One ml of serum was diluted to 5 ml with 0.15M NaCl, 0.01M phos-
TABLE 2. Effect of long-term spironolactone administration to normal men Week 0 11
Gynecomastia (cumulative #) Serum canrenone (ng/ml) Serum testosterone (ng/dl) Serum estradiol (pg/ml) Serum FSH (mlU/ml) Serum LH (mlU/ml) Sperm density (106/ml)
9 0 _ 578 ± 26 ± 8.9 ± 7.5 ± 81 ±
63 4 1.3 1.5 19
4
8
12
16
20
9 0 483 ± 52 629 + 75 25 ± 3 9.4 ± 1.6 8.4 ± 1.9 69 ± 14
9 0 575 ± 57 575 ± 54 27 ± 6 11.1 ± 2.0 8.7 + 2.0 96 ± 30
i) : 606 i: 115 615 it 53 27 ii 5 11.0:t 1.9 9.2 it 2.0 46 2t 12
9 3 417 ± 77 610 ± 58 29 ± 4 9.5 ± 1.6 9.3 ± 2.4 53 ± 15
-1 576 :t 126 562 :t 36 23 :t 3 10.1 :t 2.0 7.8:t 1.5 75 :t 23
24
6 6 493
± 120
549 ± 49 28 ± 2 10.8 ± 1.8 8.4 ± 2.0 82 ± 27
Nine healthy young men took 400 mg of spironolactone daily (200 mg every 12 h) for up to 24 weeks. The number of subjects completing each 4 week period is given as "n." The cumulative number of subjects who developed gynecomastia by the end of each period includes subjects who dropped out prior to that time. Blood and semen were obtained as described in Materials and Methods. All values are means ± SEM. No statistically significant hormonal changes occurred.
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JCE & M • 1977 Vol 45 • No 2
CAMINOS-TORRES, MA AND SNYDER
258
Results Short-term administration of spironolactone to normal men (Table 1) When nine healthy young men took 100 mg or 400 mg of spironolactone daily for 4 weeks, none noted any change in libido, and none developed gynecomastia. No changes occurred in their serum concentrations of FSH, LH, testosterone, or estradiol or in their FSH and LH responses to GnRH during either the 100 or 400 mg doses. The dialyzable fraction of testosterone, however, was approximately 20% higher (P < 0.01) during the administration of both the 100 and 400 mg doses than prior to treatment. Long-term administration of spironolactone to normal men (Table 2) Nine healthy men took 400 mg of spironolactone daily for up to 24 weeks. Six men completed the entire 24 week course, and 3 stopped after 16 weeks, 2 because of hyperkalemia and 1 because of painful gynecomastia. Six of the 9 men eventually developed gynecomastia, 3 by 12 weeks, 1 by 20 weeks, and 2 by 24 weeks. The gynecomastia was bilateral in 5, was always less than 2 cm in diameter by palpitation, and regressed in less than 2 months in 4 and in 7 months in 1. Two men reported a possible decrease in libido. The serum canrenone concentrations in all nine men generally ranged between 200
and 1000 ng/ml, levels consistent with the ingestion of 200 mg of spironolactone 12 h before. The mean serum concentrations of testosterone, estradiol, FSH and LH did not change throughout the 24 weeks. When the data from only the men who developed gynecomastia was considered, still no changes were apparent. The mean sperm density, likewise, showed no significant change, but two of the subjects appeared to have significant decreases in sperm density and motility after beginning spironolactone, persistance of these abnormalities during spironolactone administration, and return of both parameters to normal after spironolactone was discontinued (Table 3). The serum concentrations of PRL, T4, T3, and TSH were also measured throughout both the short-term and long-term studies, and no changes occurred in any of these parameters. In vitro effects of spironolactone, canrenone, and potassium canrenoate (Table 4) Canrenone increased the dialyzable fraction of testosterone at concentrations of 400 and 2000 ng/ml by 14% and 28%, respectively (P < 0.01 for both). Furthermore, the increase in the dialyzable fraction resulting from the addition of 2000 ng/ml of canrenone was significantly greater (P < 0.01) than that resulting from the addition of 400 ng/ml. The addition of 2000 ng/ml of canrenone spuriously increased the value of testosterone by this immunoassay by 35%. Neither
TABLE 3. Semen characteristics of subjects 8 and 9 before, during and after spironolactone administration Week Subject 8 Sperm density (106/ml) Motility
(0-4+)
Subject 9 Sperm density (106/ml) Motility (0-4+)
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
32
44
59
57
35
9
20
12
—
—
23
—
26
18
—
12
—
39
51
3
3
2
3
3
3
—
—
2
—
2
2
—
1
—
3
3
—
101
93
17
6
14
2
6
11
10
—
—
10
—
45
86
—
—
3
2
1
1
1
1
1
1
1
—
—
1
—
2
2
—
Each subject took 400 mg spironolactone daily, subject 8 for 24 weeks and subject 9 for only 16 weeks, because of the development of hyperkalemia.
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SPIRONOLACTONE-INDUCEDGYNECOMASTIA
259
TABLE 4. Effects of adding sprionolactone, canrenone, and potassium canrenoate in vitro to serum on the dialyzable fraction of testosterone and on the immunoassays of testosterone and estradiol Canrenone (ng/ml)
Spironolactone (ng/ml) No Addition Testosterone (ng/dl)
497 ±36
16
80
474 ±59
478 ±52
400 488 ±45
2000 488 ± 54
16
80
498 ±40
558 ±48
400 539 ±58
Potassium canrenoate (ng/ml) 2000 672* ±72
16
80
468 ± 44
499 ±52
400 457 ± 44
2000 440 ±58
% Dialyzable testosterone
3.80 ±0.35
3.49 ±0.31
3.69 ±0.41
3.68 ±0.42
3.87 ±0.40
3.68 ±0.37
3.81 ±0.36
4.23 ± 0.30*
4.86 ± 0.29*
3.98 ±0.36
3.46 ±0.31
3.99 ±0.59
3.89 ±0.41
Estradiol (pg/ml)
30 ± 4
24 ± 1
27 ±3
26 ±2
29 ±3
26 ±4
28 ± 4
22 ±2
31 ±4
29 ±4
26 ±3
27 ±3
27 ±3
Sera from 6 healthy young men were divided into aliquots to which spironolactone, etc. were added to make the concentrations shown above. All aliquots from a single subject were tested in the same dialysis run and the same testosterone and estradiol immunoassay runs. The values above are the means ± SEM for the six subjects. • P < 0.01 by paired t test.
spironolactone nor potassium canrenoate affected any of these assays. Discussion The administration of 400 mg of spironolactone daily for up to 24 weeks to 9 healthy young men produced gynecomastia in 6 of them and apparent decreases in sperm density and motility in 2 of them. Gynecomastia has previously been reported to occur in men who received this dose of spironolactone for primary aldosteronism (2) and for low-renin hypertension (4), but has not previously been reported in normal men. Semen abnormalities have not been previously reported in either patients or normal subjects. Although semen changes occurred in only 2 of the 9 subjects, and although semen quality can change considerably during a six month period in an apparently normal man without the reason being obvious, the pattern of semen changes in these 2 subjects (Table 3) suggests that the changes were drug-related. Sperm density in both subjects was normal prior to spironolactone administration, decreased markedly and to subnormal levels within 4 weeks of initiation of spironolactone, remained subnormal throughout the course of spironolactone, and returned to normal after its discontinuation. Sperm motility was similarly affected. Any hormonal changes that mediate these two effects of spironolactone should therefore have been observable during 24 weeks
of spironolactone treatment. No changes occurred during this time, however, in any of the parameters usually used to assess the pituitary-testicular axis: the serum concentrations of testosterone, FSH, or LH. Spironolactone-induced gynecomastia and occasional impairment of semen quality, therefore, appear not to be related to a decrease in serum testosterone concentration, even though spironolactone can inhibit the activity of testicular 17-hydroxylase activity in animals (5) and, possibly, in man (9). Spironolactone-induced gynecomastia also appears not to be related to an increase in serum estradiol concentration, even though spironolactone may cause an increase in the peripheral conversion of testosterone to estradiol in men (6). The only hormonal parameter that was significantly altered by the administration of spironolactone was the dialyzable fraction of testosterone. When normal men took either 100 or 400 mg of spironolactone for 4 weeks, their dialyzable fraction of testosterone increased by approximately 20%. The reason for this increase appears to be that canrenone, in serum concentrations achieved during the oral administration of 400 mg of spironolactone daily (Table 2), displaced testosterone from its binding protein (Table 4). On the other hand, canrenone spuriously elevated the measured serum testosterone concentration to approximately the same degree, by the method used here. In view of these two phenomena the lack of change of the serum testosterone con-
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CAMINOS-TORRES, MA AND SNYDER
centration during spironolactone administration probably indicates that the actual total testosterone concentration was lower during spironolactone administration than before, but that the absolute free testosterone concentration was unchanged. Although displacement of testosterone from its serum binding protein and from a testosterone antibody by canrenone has not been described previously, similar effects of canrenone and spironolactone have been described with respect to other proteins. Spironolactone and canrenone interfered with immunoassays and competitive protein binding assays for 11-deoxycorticosterone (15), and a metabolite of canrenone interfered with an immunoassay for aldosterone (16). Neither spironolactone nor canrenone interfered significantly with immunoassays for progesterone or 17hydroxyprogesterone (9). Of potentially more relevance to the present study, both spironolactone and canrenone displaced dihydrotestosterone from irs receptor in rat ventral prostate cytosol (8). It is tempting to speculate, therefore, that the spironolactone-induced gymecomostia and semen abnormalities may have been related to the binding of canrenone to tissue androgen receptors. We conclude that the administration of 400 mg of spironolactone daily to normal young men will frequently produce gynecomastia and occasionally impair sperm density and motility in 12-24 weeks. These changes do not appear to be due to alterations in the serum concentrations of testosterone, free testosterone, or estradiol, but may result from the binding of canrenone to tissue androgen receptors.
JCE & M • 1977 Vol 45 • No 2
References 1. Clark, E., Spironolactone therapy and gynecomastiaJAMA 193: 163, 1965. 2. Spark, R. F., and J. C. Melby, Aldosteronism in hypertension. The spironolactone response test, Ann Intern Med 69: 685, 1968. 3. Adlin, E. V., A. D. Marks, and B. J. Channick, Spironolactone and hydrochlorothiazide in essential hypertension, Arch Intern Med 130: 855, 1972. 4. Douglas, J. C , J. W. Hollifield, and G. W. Liddle, Treatment of low-renin essential hypertension. Comparison of spironolactone and a hydrochlorothiazide-triamterene combination, JAMA 227: 518, 1974. 5. Menard, R. H., B. Stripp, and J. R. Gillette, Spironolactone and testicular cytochrome P450: Decreased testosterone formation in several species and changes in hepatic drug metabolism, Endocrinology 94: 1628, 1974.
6. Huffman, D. H., and D. L. Azarnoff, Effect of spironolactone on metabolic conversion of androgens to estrogens, Clin Res 23: 476A, 1975. 7. Corvol, P., A. Michaud, J. Menard, M. Freifeld, and J. Mahoudeau, Antiandrogenic effect of spironolactones: mechanism of action, Endocrinology 97: 52, 1975. 8. Pita, J. C , Jr., M. E. Lippman, E. B. Thompson, and D. L. Loriaux, Interaction of spironolactone and digitalis with the 5a-dihydrotestosterone (DHT) receptor of rat ventral prostate, Endocrinology 97: 1521, 1975. 9. Stripp, B., A. A. Taylor, F. C. Bartter, J. R. Gillette, D. L. Loriaux, R. Easley, and R. H. Menard, Effect of spironolactone on sex hormones in man, J Clin Endocrinol Metab 41: 777, 1975. 10. Sadee, W., M. Dageioglu, and R. Schroder, Pharmacokinetics of spironolactone, canrenone and canrenoate-K in humans, J Pharmacol Exp Ther 185: 686, 1973. 11. Snyder, P. J., J. F. Reitano, and R. D. Utiger, Serum LH and FSH responses to synthetic gonadotropin releasing hormone in normal men, J Clin Endocrinol Metab 41: 938, 1975. 12. Gochman, N., and C. E. Gantt, A fluorimetric method for the determination of a major spironolactone (Aldactone) metabolite in human plasma, J Pharmacol Exp Ther 135: 312, 1962. 13. Paulsen, C. A., In Williams, R. H. (ed.), Textbook of Endocrinology, ed. 5, W. B. Saunders Co., Acknowledgments Philadelphia, 1974, p. 332. We thank Dr. Michael S. Anderson of Searle Labora- 14. Snedecor, G. W., and W. G. Cochran, Statistical Methods, ed. 5, Iowa State University Press, Ames, tories for his advice throughout the course of this Iowa, 1967, p. 59. study. We also thank Ms. Cordelia Shute and the staff of the Clinical Research Center, Hospital of the 15. Tan, S. Y., and P. J. Mulrow, Interference of spironolactone in 11-deoxycorticosterone radioUniversity of Pennsylvania for performing the in vivo assays, J Clin Endocrinol Metab 41: 791, 1975. studies and Ms. Donna Samuel and Ms. Mary Saxon 16. Sadee, W., A. M. Finn, P. Schmiedek, and A. for preparation of the manuscript. Baethmann, Aldosterone plasma interference by a Reagents for the FSH and LH immunoassays were spironolactone metobolite, Steroids 25: 301, 1975. gifts from the NIAMDD.
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G
YNECOMASTIA and impotence are recognized side-effects of spironolactone administration to men, especially at doses exceeding 100 mg daily (1-4). These effects suggest that spironolactone affects testosterone secretion, metabolism, or action. Data from several studies indicate that spironolactone has the potential for producing all of these effects. Administration of spironolactone to several animal species causes a pronounced decrease in testicular microsomal P-450 levels and in microsomal 17-hydroxylase (5), the enzyme responsible for the conversion of progesterone to 17hydroxyprogesterone. Administration of spironolactone to men appears to increase the conversion of testosterone to estradiol (6). Both spironolactone (7,8) and canrenone (8), which is the principal circulating metabolite of spironolactone, inhibit the binding of dihydrotestosterone (DHT) to Received October 26, 1976. Supported by U.S.P.H.S. grants HD 08555 and RR 40 and a grant from Searle Laboratories.
not change significantly. No change occurred in the mean serum concentrations of FSH, LH, testosterone or estradiol. In vitro incubation of canrenone, the principal circulating metabolite of spironolactone, in concentrations achieved in vivo, with serum from normal men resulted in a small but significant displacement of testosterone from its binding protein and in a small spurious increase in die serum testosterone concentration. We conclude that spironolactone-induced gynecomastia and occasional semen abnormalities do not appear to be due to changes in the serum concentrations of testosterone or estradiol. We hypothesize that these changes may be related to binding of canrenone to tissue androgen receptors. (/ Clin Endocrinol Metab 45: 255, 1977)
the DHT receptor in rat ventral prostate cytosol. Which of these biochemical effects is, or are, responsible for the observed gynecomastia and decreased libido in men is unclear. Administration of 400 mg of spironolactone to normal men once a day for 5 days produced an elevation of serum progesterone and 17-hydroxyprogesterone concentrations (9), suggesting that spironolactone may inhibit testosterone biosynthesis in man. The serum testosterone concentration in those men, however, did not decrease nor did their serum estradiol concentration increase. The purpose of the present investigation was to determine the clinical and hormonal effects of long-term spironolactone administration on the pituitary-testicular axis in healthy, young men. Men who were healthy and young were chosen to avoid the concomitant effects of age, illness, and medications on the pituitary-testicular axis. One group of men was given 100 mg of spironolactone daily for 4 weeks and, later, 400 mg daily for 4 weeks, in order to assess any
255
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256
CAMINOS-TORRES, MA AND SNYDER
hormonal effects. A second group of men took 400 mg daily for 24 weeks in order to determine any additional hormonal effects of prolonged administration and to correlate clinical with hormonal effects. Sera from a third group of men, who did not take spironolactone, were used to determine the effects of spironolactone and its metabolites on the steroid assays employed. Materials and Methods Short-term administration of spironolactone to normal men Each subject, both in this shorMerm study and in the long-term study described below, gave informed, written consent to a protocol approved by the University of Pennsylvania Committee on Investigations in Humans. No subject had any illness or was taking any medications suspected of altering the pituitary-testicular axis. In the short-term study, nine healthy young men, ages 18-39 (mean age, 25), each took 100 mg of spironolactone daily (50 mg every 12 h) for 4 weeks, stopped the medication for 4 weeks, then took 400 mg daily (200 mg every 12 h) for 4 weeks. Blood was sampled twice between 0800 and 1000 h on each of 3 consecutive days before beginning each dose of medication and in a similar fashion at the end of 2 and 4 weeks of each dose. The serum LH and FSH responses to a 50 jug bolus iv dose of gonadotropin-releasing hormone (GnRH) were determined in each subject prior to beginning the first dose and at the end of 4 weeks administration of each dose. Blood was sampled for LH and FSH at -30, - 1 5 , 0, 10, 20, 30, 45, 60, 90, 120, 150, and 180 min in relation to each GnRH dose.
In vitro effects of spironolactone, and potassium canrenoate
JCE & M • 1977 Vol 45 • No 2
canrenone
The in vitro effects of spironolactone and its principal metabolites on the immunoassays for testosterone and estradiol and on the dialyzable fraction of testosterone were determined. Each of these three compounds (Searle Laboratories, Chicago, Illinois) was dissolved in 95% ethanol and added to aliquots of serum from 6 normal young men so that the final concentrations of each compound were 16, 80, 400, and 2000 ng/ml. Each aliquot was then assayed for testosterone, estradiol, and dialyzable fraction of testosterone. These concentrations were chosen to approximate the canrenone levels achieved in vivo by the administration of 200 mg of spironolactone every 12 h. The subjects in this study who took that dose had mean serum concentration of canrenone, which is the principal circulating metabolite of spironolactone (10), of 400600 ng/ml 12 h after the previous dose (Table 2). Since the serum canrenone concentration usually reaches a peak 2-3 h after an oral dose, and the concentration falls gradually during the next 12 h (10), it is likely that the serum concentrations of canrenone in the present study subjects were usually higher than those measured 12 h after a dose. Although the serum levels of spironolactone and potassium canrenoate were not measured in the subjects who were administered spironolactone, the serum concentrations of these two compounds in subjects taking spironolactone are generally much less than that of canrenone (10). The concentrations of spironolactone and potassium canrenoate used in this in vitro study, therefore, were probably many times greater than occur in vivo during the oral administration of 400 mg of spironolactone daily. Assays
Long-term administration of spironolactone to normal men Nine healthy men, ages 21-28, consented to take 400 mg of spironolactone daily (200 mg every 12 h) for 24 weeks. Blood was sampled twice between 0800 and 1000 h on each of 3 consecutive days prior to beginning the medication and in a similar fashion every 4 weeks. A semen sample was analyzed every two weeks. Subjects were examined for gynecomastia every 4 weeks.
FSH and LH were determined by immunoassays, as described previously (11), using reagents provided by the National Pituitary Agency. The results are expressed in terms of the 2nd IRP-HMG, where one /xg of LER 907 equals 38 mlU of FSH and 219 mlU of LH. Serum testosterone was determined by immunoassay using antiserum to testosterone-3carboxymethyloxime-BSA and testosteronetyrosine methyl ester-125I (Bio-RIA, Montreal, Canada). Ether extracts of sera were assayed
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257
SPIRONOLACTONE-INDUCEDGYNECOMASTIA
TABLE 1. Effect of short-term spironolactone administration to normal men on serum concentrations of FSH, LH, testosterone and estradiol and on the dialyzable fraction of testosterone Spironolactone, 400 mg/day
Spironolactone, 100 mg/day 2 weeks
Pre-Rx Basal serum FSH (mlU/ml) Maximum serum FSH* Basal serum LH (mlU/ml) Maximum serum LH (mlU/ml)* Serum testosterone (ng/dl) % Dialyzable testosterone Serum estradiol (pg/ml)
5.1 :t 0.7 6.0 :t 0.9 11.3 :t 1.4 42.5 :t 5.5 447 :t 4 7 3.40:t 0.16 25 :t 2
4 weeks
4.2 ± 0.5
4.3 :t 0.3 5.2 :t 0.5 11.9 :t 1.3 35.4 :t 3.4 608 :t 6 7 4.12:t 0.231 27 :t 1
12.1 ± 1.4 _
498
± 36 —
29
± 2
Pre-Rx
2 weeks
4.2 ± 0.4
4.2 ± 0.3
11.2 ± 1.1
15.7+ 0.2 _ 497 ± 32 _ 30 ± 2
_
516
± 56 _
26 ± 2
4 weeks 4.0 dt 0.3 5.0 1t 0.5 13.5 dt 1.6 38.1 i: 3.7 587 2t 6 6 4.02 i: 0.15f 25 :t 1
Nine healthy young men took 100 mg of spironolactone daily for 4 weeks, stopped the medication for 4 weeks, then took 400 nig daily for 4 weeks. Blood was sampled from each subject six times prior to taking the medication and similarly after 2 and 4 weeks administration. All values are means ± SEM. * Maximum response to a 50 /xg iv bolus dose of synthetic gonadotropin-releasing hormone, t P < 0.01.
phate, pH 7.4 (PBS) and incubated with 3Htestosterone overnight at 4 C. The 5 ml of diluted serum was then dialyzed against 5 ml of PBS in dialysis cells at 37 C for 24 h. The percentage of dialyzable testosterone in the diluted serum was calculated, DPM in dialysate x 100/DPM in serum + dialysate. Multiplication by 0.367 gave the percentage of dialyzable testosterone in the undiluted serum (11). The figure, 0.367, was obtained experimentally as the ratio of the percentage of dialyzable testosterone in an undiluted serum pool (3.2%) to that in the same pool diluted 1:5 (9.4%), corrected for a 10.8% volume change when undiluted serum was used. The serum concentration of canrenone was determined by Dr. Thomas C. Hutsell at Searle Laboratories by fluorimetric analysis (12). Semen was analyzed as described in Paulsen (13). Statistical analyses were performed by the paired and non-paired t tests (14).
without prior chromatography. Cross-reactivity of dihydrostestosterone was 50% that of testosterone. The lower limit of sensitivity of the assay was 20 pg per assay tube; the intraassay and interassay coefficients of variation were 8.9% and 13.3%. Serum estradiol was determined by immunoassay using antiestradiol serum raised to 17/3 estradiol-6-carboxymethyloxime-bovine serum albumin and using estradiol-7-succinyl-125I (Micromedic, Horsham, Pa.). Benzene extracts of sera were assayed without prior chromatography. Cross-reactivity of estrone was < 1 % that of estradiol. The lower limit of sensitivity of the assay was 2 pg per assay tube; the intraassay and interassay coefficients of variation were 12% and 14%. The dialyzable fraction of testosterone was determined by equilibrium dialysis, as previously described (11). One ml of serum was diluted to 5 ml with 0.15M NaCl, 0.01M phos-
TABLE 2. Effect of long-term spironolactone administration to normal men Week 0 11
Gynecomastia (cumulative #) Serum canrenone (ng/ml) Serum testosterone (ng/dl) Serum estradiol (pg/ml) Serum FSH (mlU/ml) Serum LH (mlU/ml) Sperm density (106/ml)
9 0 _ 578 ± 26 ± 8.9 ± 7.5 ± 81 ±
63 4 1.3 1.5 19
4
8
12
16
20
9 0 483 ± 52 629 + 75 25 ± 3 9.4 ± 1.6 8.4 ± 1.9 69 ± 14
9 0 575 ± 57 575 ± 54 27 ± 6 11.1 ± 2.0 8.7 + 2.0 96 ± 30
i) : 606 i: 115 615 it 53 27 ii 5 11.0:t 1.9 9.2 it 2.0 46 2t 12
9 3 417 ± 77 610 ± 58 29 ± 4 9.5 ± 1.6 9.3 ± 2.4 53 ± 15
-1 576 :t 126 562 :t 36 23 :t 3 10.1 :t 2.0 7.8:t 1.5 75 :t 23
24
6 6 493
± 120
549 ± 49 28 ± 2 10.8 ± 1.8 8.4 ± 2.0 82 ± 27
Nine healthy young men took 400 mg of spironolactone daily (200 mg every 12 h) for up to 24 weeks. The number of subjects completing each 4 week period is given as "n." The cumulative number of subjects who developed gynecomastia by the end of each period includes subjects who dropped out prior to that time. Blood and semen were obtained as described in Materials and Methods. All values are means ± SEM. No statistically significant hormonal changes occurred.
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JCE & M • 1977 Vol 45 • No 2
CAMINOS-TORRES, MA AND SNYDER
258
Results Short-term administration of spironolactone to normal men (Table 1) When nine healthy young men took 100 mg or 400 mg of spironolactone daily for 4 weeks, none noted any change in libido, and none developed gynecomastia. No changes occurred in their serum concentrations of FSH, LH, testosterone, or estradiol or in their FSH and LH responses to GnRH during either the 100 or 400 mg doses. The dialyzable fraction of testosterone, however, was approximately 20% higher (P < 0.01) during the administration of both the 100 and 400 mg doses than prior to treatment. Long-term administration of spironolactone to normal men (Table 2) Nine healthy men took 400 mg of spironolactone daily for up to 24 weeks. Six men completed the entire 24 week course, and 3 stopped after 16 weeks, 2 because of hyperkalemia and 1 because of painful gynecomastia. Six of the 9 men eventually developed gynecomastia, 3 by 12 weeks, 1 by 20 weeks, and 2 by 24 weeks. The gynecomastia was bilateral in 5, was always less than 2 cm in diameter by palpitation, and regressed in less than 2 months in 4 and in 7 months in 1. Two men reported a possible decrease in libido. The serum canrenone concentrations in all nine men generally ranged between 200
and 1000 ng/ml, levels consistent with the ingestion of 200 mg of spironolactone 12 h before. The mean serum concentrations of testosterone, estradiol, FSH and LH did not change throughout the 24 weeks. When the data from only the men who developed gynecomastia was considered, still no changes were apparent. The mean sperm density, likewise, showed no significant change, but two of the subjects appeared to have significant decreases in sperm density and motility after beginning spironolactone, persistance of these abnormalities during spironolactone administration, and return of both parameters to normal after spironolactone was discontinued (Table 3). The serum concentrations of PRL, T4, T3, and TSH were also measured throughout both the short-term and long-term studies, and no changes occurred in any of these parameters. In vitro effects of spironolactone, canrenone, and potassium canrenoate (Table 4) Canrenone increased the dialyzable fraction of testosterone at concentrations of 400 and 2000 ng/ml by 14% and 28%, respectively (P < 0.01 for both). Furthermore, the increase in the dialyzable fraction resulting from the addition of 2000 ng/ml of canrenone was significantly greater (P < 0.01) than that resulting from the addition of 400 ng/ml. The addition of 2000 ng/ml of canrenone spuriously increased the value of testosterone by this immunoassay by 35%. Neither
TABLE 3. Semen characteristics of subjects 8 and 9 before, during and after spironolactone administration Week Subject 8 Sperm density (106/ml) Motility
(0-4+)
Subject 9 Sperm density (106/ml) Motility (0-4+)
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
32
44
59
57
35
9
20
12
—
—
23
—
26
18
—
12
—
39
51
3
3
2
3
3
3
—
—
2
—
2
2
—
1
—
3
3
—
101
93
17
6
14
2
6
11
10
—
—
10
—
45
86
—
—
3
2
1
1
1
1
1
1
1
—
—
1
—
2
2
—
Each subject took 400 mg spironolactone daily, subject 8 for 24 weeks and subject 9 for only 16 weeks, because of the development of hyperkalemia.
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SPIRONOLACTONE-INDUCEDGYNECOMASTIA
259
TABLE 4. Effects of adding sprionolactone, canrenone, and potassium canrenoate in vitro to serum on the dialyzable fraction of testosterone and on the immunoassays of testosterone and estradiol Canrenone (ng/ml)
Spironolactone (ng/ml) No Addition Testosterone (ng/dl)
497 ±36
16
80
474 ±59
478 ±52
400 488 ±45
2000 488 ± 54
16
80
498 ±40
558 ±48
400 539 ±58
Potassium canrenoate (ng/ml) 2000 672* ±72
16
80
468 ± 44
499 ±52
400 457 ± 44
2000 440 ±58
% Dialyzable testosterone
3.80 ±0.35
3.49 ±0.31
3.69 ±0.41
3.68 ±0.42
3.87 ±0.40
3.68 ±0.37
3.81 ±0.36
4.23 ± 0.30*
4.86 ± 0.29*
3.98 ±0.36
3.46 ±0.31
3.99 ±0.59
3.89 ±0.41
Estradiol (pg/ml)
30 ± 4
24 ± 1
27 ±3
26 ±2
29 ±3
26 ±4
28 ± 4
22 ±2
31 ±4
29 ±4
26 ±3
27 ±3
27 ±3
Sera from 6 healthy young men were divided into aliquots to which spironolactone, etc. were added to make the concentrations shown above. All aliquots from a single subject were tested in the same dialysis run and the same testosterone and estradiol immunoassay runs. The values above are the means ± SEM for the six subjects. • P < 0.01 by paired t test.
spironolactone nor potassium canrenoate affected any of these assays. Discussion The administration of 400 mg of spironolactone daily for up to 24 weeks to 9 healthy young men produced gynecomastia in 6 of them and apparent decreases in sperm density and motility in 2 of them. Gynecomastia has previously been reported to occur in men who received this dose of spironolactone for primary aldosteronism (2) and for low-renin hypertension (4), but has not previously been reported in normal men. Semen abnormalities have not been previously reported in either patients or normal subjects. Although semen changes occurred in only 2 of the 9 subjects, and although semen quality can change considerably during a six month period in an apparently normal man without the reason being obvious, the pattern of semen changes in these 2 subjects (Table 3) suggests that the changes were drug-related. Sperm density in both subjects was normal prior to spironolactone administration, decreased markedly and to subnormal levels within 4 weeks of initiation of spironolactone, remained subnormal throughout the course of spironolactone, and returned to normal after its discontinuation. Sperm motility was similarly affected. Any hormonal changes that mediate these two effects of spironolactone should therefore have been observable during 24 weeks
of spironolactone treatment. No changes occurred during this time, however, in any of the parameters usually used to assess the pituitary-testicular axis: the serum concentrations of testosterone, FSH, or LH. Spironolactone-induced gynecomastia and occasional impairment of semen quality, therefore, appear not to be related to a decrease in serum testosterone concentration, even though spironolactone can inhibit the activity of testicular 17-hydroxylase activity in animals (5) and, possibly, in man (9). Spironolactone-induced gynecomastia also appears not to be related to an increase in serum estradiol concentration, even though spironolactone may cause an increase in the peripheral conversion of testosterone to estradiol in men (6). The only hormonal parameter that was significantly altered by the administration of spironolactone was the dialyzable fraction of testosterone. When normal men took either 100 or 400 mg of spironolactone for 4 weeks, their dialyzable fraction of testosterone increased by approximately 20%. The reason for this increase appears to be that canrenone, in serum concentrations achieved during the oral administration of 400 mg of spironolactone daily (Table 2), displaced testosterone from its binding protein (Table 4). On the other hand, canrenone spuriously elevated the measured serum testosterone concentration to approximately the same degree, by the method used here. In view of these two phenomena the lack of change of the serum testosterone con-
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260
CAMINOS-TORRES, MA AND SNYDER
centration during spironolactone administration probably indicates that the actual total testosterone concentration was lower during spironolactone administration than before, but that the absolute free testosterone concentration was unchanged. Although displacement of testosterone from its serum binding protein and from a testosterone antibody by canrenone has not been described previously, similar effects of canrenone and spironolactone have been described with respect to other proteins. Spironolactone and canrenone interfered with immunoassays and competitive protein binding assays for 11-deoxycorticosterone (15), and a metabolite of canrenone interfered with an immunoassay for aldosterone (16). Neither spironolactone nor canrenone interfered significantly with immunoassays for progesterone or 17hydroxyprogesterone (9). Of potentially more relevance to the present study, both spironolactone and canrenone displaced dihydrotestosterone from irs receptor in rat ventral prostate cytosol (8). It is tempting to speculate, therefore, that the spironolactone-induced gymecomostia and semen abnormalities may have been related to the binding of canrenone to tissue androgen receptors. We conclude that the administration of 400 mg of spironolactone daily to normal young men will frequently produce gynecomastia and occasionally impair sperm density and motility in 12-24 weeks. These changes do not appear to be due to alterations in the serum concentrations of testosterone, free testosterone, or estradiol, but may result from the binding of canrenone to tissue androgen receptors.
JCE & M • 1977 Vol 45 • No 2
References 1. Clark, E., Spironolactone therapy and gynecomastiaJAMA 193: 163, 1965. 2. Spark, R. F., and J. C. Melby, Aldosteronism in hypertension. The spironolactone response test, Ann Intern Med 69: 685, 1968. 3. Adlin, E. V., A. D. Marks, and B. J. Channick, Spironolactone and hydrochlorothiazide in essential hypertension, Arch Intern Med 130: 855, 1972. 4. Douglas, J. C , J. W. Hollifield, and G. W. Liddle, Treatment of low-renin essential hypertension. Comparison of spironolactone and a hydrochlorothiazide-triamterene combination, JAMA 227: 518, 1974. 5. Menard, R. H., B. Stripp, and J. R. Gillette, Spironolactone and testicular cytochrome P450: Decreased testosterone formation in several species and changes in hepatic drug metabolism, Endocrinology 94: 1628, 1974.
6. Huffman, D. H., and D. L. Azarnoff, Effect of spironolactone on metabolic conversion of androgens to estrogens, Clin Res 23: 476A, 1975. 7. Corvol, P., A. Michaud, J. Menard, M. Freifeld, and J. Mahoudeau, Antiandrogenic effect of spironolactones: mechanism of action, Endocrinology 97: 52, 1975. 8. Pita, J. C , Jr., M. E. Lippman, E. B. Thompson, and D. L. Loriaux, Interaction of spironolactone and digitalis with the 5a-dihydrotestosterone (DHT) receptor of rat ventral prostate, Endocrinology 97: 1521, 1975. 9. Stripp, B., A. A. Taylor, F. C. Bartter, J. R. Gillette, D. L. Loriaux, R. Easley, and R. H. Menard, Effect of spironolactone on sex hormones in man, J Clin Endocrinol Metab 41: 777, 1975. 10. Sadee, W., M. Dageioglu, and R. Schroder, Pharmacokinetics of spironolactone, canrenone and canrenoate-K in humans, J Pharmacol Exp Ther 185: 686, 1973. 11. Snyder, P. J., J. F. Reitano, and R. D. Utiger, Serum LH and FSH responses to synthetic gonadotropin releasing hormone in normal men, J Clin Endocrinol Metab 41: 938, 1975. 12. Gochman, N., and C. E. Gantt, A fluorimetric method for the determination of a major spironolactone (Aldactone) metabolite in human plasma, J Pharmacol Exp Ther 135: 312, 1962. 13. Paulsen, C. A., In Williams, R. H. (ed.), Textbook of Endocrinology, ed. 5, W. B. Saunders Co., Acknowledgments Philadelphia, 1974, p. 332. We thank Dr. Michael S. Anderson of Searle Labora- 14. Snedecor, G. W., and W. G. Cochran, Statistical Methods, ed. 5, Iowa State University Press, Ames, tories for his advice throughout the course of this Iowa, 1967, p. 59. study. We also thank Ms. Cordelia Shute and the staff of the Clinical Research Center, Hospital of the 15. Tan, S. Y., and P. J. Mulrow, Interference of spironolactone in 11-deoxycorticosterone radioUniversity of Pennsylvania for performing the in vivo assays, J Clin Endocrinol Metab 41: 791, 1975. studies and Ms. Donna Samuel and Ms. Mary Saxon 16. Sadee, W., A. M. Finn, P. Schmiedek, and A. for preparation of the manuscript. Baethmann, Aldosterone plasma interference by a Reagents for the FSH and LH immunoassays were spironolactone metobolite, Steroids 25: 301, 1975. gifts from the NIAMDD.
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