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Plasma free androgen patterns in hirsute women and their diagnostic implications
Plasma Free Androgen Patterns in Hirsute Women and Their Diagnostic Implications
ROBERT L. ROSENFIELD.
M.D
Chicago, Illinois
From the Department of Pediatrics, University of Chicago, Pritzker School of Medicine. Wyler Children’s Hospital, 5825 S. Maryland’Avcnue. Chicago, Illinois. Studies carried out in the Clinical Research Center of the IJniversity of Chicago Hospital under Grants RR-305 and RR-55 from the General Clinical Research Centers Program of- the Division of Research Resources. National Institutes of Health. The rcsoarch was qupported in part by USPHS Cranf HD-OG308. Requests for reprints should be addressed to Dr. Robert L. Rosenfield. Department of Pediatrics, The University of Chicago, 5825 Maryland Avenue, Chicago, Illinois 60637. Manuscript accFpted Octohcr 27, 1978.
The 24-hour pattern of plasma total and free testosterone and other lirfl-hydrozrysteroids has been characterized in six women with moderately severe male-pattern hirsutism. Four distinct plasma androgen patterns were identified: (1) total and free plasma testosterone levels were elevated 80 per cent or more of the time, (2) !he free plasma testosterone level was con&&y elevated, where-as total testosterone was in the high-normal range, (3) plasma free androgens were undergoing marked &asic fluctuations around the upper lhnits of normal such that sampling blood for multiple andrhgens and/or at multiple intervals was necessaq to disclw the hyperandrogenic state, and (4) plasma free androgens fluctuated within the normal range. The variation of the plasma androgens was suggestive of intermittent and independent episodes of adrenal and ovarian androgen prodetion. Because of these fluctuations, a single plasma testosterone leGe1was tit necessarily representative of the average concentration over a 24-hour period. For example, the 95 per cent confidence limits are such that a single plasma testost rone level may fall within only 38 per cent of the a&hour mean. ThdLs t short-term estimate of the average daily testosterone level is the mean of hourly determinations over a l-hour per@ the 95 per cent confidence l&nits of this figure are within 18 per cent of the 24-hour mean. Nevertheless, this 3-hour sampling protocol would not have detected the subtle hyperandrogenic state present in twq patients unless androgens other than free teskerone (e.g., free lir@-hydroxysteroids) were measured in the same plasma specimens. The patterns of the plasma testosterone in several hirsute women are reported here. Fluctuations in testosterone were sought because the plasma testosterone concentration is increased in only about half the cases in which more extensive studies reveal abnormalities of testosterone binding, precursors, metaboliteslor metabolism [I-J]. One possible explanation for such disparities would be that a single testosterone level is not necessarily representative of the mean over a prolonged period of time. Although it is well established that plasma testosterone fluctuates within a relatively narrow range in normal women [~-II], there is some indication that it may vary relatively widely in hirsute women. On the basis of sampling blood at 2 to 4 hour intervals for 24 hours, Givens et al. [8] concluded that the variability of plasma testosterone was greater than normal in two of 12 hirsute women. On the basis of assaying four blood samples over a two day period, Ismael et al. [12) reported thq/ a single testosterone level was normal in 40 per cent of a group of himute women found to have uniformly abnormal mean testosterone leyels. Neither group focused on
March 1979
The American
Journal of Medicine
Volume
66
417
ANDROGEN PATTERNS IN HIRSUTISM-ROSENFIELD
Clock a
normal
time
daytime
Clock time
range
+I
range
of lrvrlrl600-4OOhr
lgure 1. Three patterns of plasma androgens (see text) in four different hirsute women. Shaded Ueas indicate normal rang&.
the level of free (unbound] testosterone and other 17/3-hydroxysteroids although the plasma concentrations of these free androgens are more closely related to hirsutism than are total plasma testosterone levels [1,2]. Because of these considerations, a systematic. charac: terization of the pattern of plasma free androgens of selected hirsute women has been undertaken and is reported here. These data have important implications as regards the optimal sampling regimen for the detection of the hyperandrogenic state in women. METHODS Six 17 to 32 year old women with moderately severe malepattern hirsutism [13] were selected for these extensive studies after preliminary tests indicated that their androgen patterns would be of particular interest. Four were diagnosed as having an excessive ovarian source of androgen on the basis of their response to dexamethasone suppression [14]: two subjects (Cases BIB and IV] had normal responses to dexamethasone, suggesting a major adrenal component. After obtaining informed consent, the subjects were admitted to the Clinical Research Centers of the University of Chicago Hospitals during a medroxyprogesterone acetate-withdrawal-induced menstrual period. Commencing at 0800the following morning, blood samples were drawn through an indwelling catheter at approximately 4 hour intervals for a 24 hour period. Meanwhile, between 0800 and 1100 hours, blood was taken at 15
418
March 1979 The American Journal of Medicine
minute intervals for 2 to 3 hours from five subjects. Activities were sedentary except for bedrest from 0800 to 1100 hours. Plasma testosterone and dehydroepiandrosterone sulfate (US) were radioimmunoassayed using a modification of the method of Ismael[l5] and the method of Buster and Abraham [16], respectively. Plasma Ii’@-hydroxysteroids and cortisol were measured by competitive protein binding [1,17]. Plasma lY&hydroxysteroids are those ether-extractable substances in plasma which bind to testosterone-estradiol binding globulin (TEBG); two-thirds of these substances in normal women are accounted for by dihydrotestosierpne, androstenediol and androstanediol, as well as testosterone [2,18]. Testosterone binding to plasma proteins was assessed by a charcoal adsorption assay [l], and the plasma free testosterone and apparent free Ii’@-hydroxysteroids levels were expressed in terms of absolute concentrations in whole, 37% plasma [19]. All measurements of each parameter in a subject were performed as a single batch. The precision of each of these assays is between 11 and 14 per cent (coefficient of variation). Statistical analyses were performed on Hewlett-Packard 9810A and Wang programmable calculators. All p values are one-tailed. For computations regarding the relationship between the 24-hour mean and a subset of any particular parameter, the subset under consideration was excluded from the calculation of the 24-hour mean. Subsets considered were means of levels of a hormone at intervals of I or more hours, the intervals being hourly if three or four values were considered. Ninety-five per cent confidence limits were calculated from the distribution of t [2O].
Volume 66
;\Nl)ROGI:N
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PATTERNS
IN HIRSUTISM-ROSENFIELD
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70-
I
I-
ONE
THREE*
Two* NUMBER
FOUR +
OF SAMPLES
*MEAN AT (EOUALI INTERVALS ff
t-4 HOURS
igure 2. The reliability with which the 24-hour mean free plasma tktosterone of hirsute women is predicted from 1 to 4 hourly measurements at 0800 to 1200
hours. Shaded areas show 95 per cent confidence limits. RESULTS Four patterns of plasma androgens were found. The pattern displayed in Case I (Figure 1) was typical of the two cases studied. The plasma total testosterone level is moderately elevated 80 per cent or more of the time in conjunction with a consistent elevation of the plasma free testosterone level. Case II (Figure l] ilhfstrates another category. Here, plasma free testosterone was consistently abnormal, yet the X-hour mean total plasma testosterone was clearly normal (54 ng/dl) with a marginally elevated total testosterone level in only one of 14 samples. This discrepancy between the total and free plasma iestosterone was due to the subnormal TEBG level which is frequently found in hirsute women (11. A third type is comprised of cases in which androgens markedly fluctuate [up to twofold] about the upper limits of normal. In Case IIIA (Figure l), the patient had an elevated free testosterone level in only five of 14 specimens; her mid-morning free plasma testosterone concentration was normal for over 1.5 hours. Her %&hour mean plasma free testosterone level as determined by the present protocol was marginally elevated (11.1 pg/ml; normal %-hour mean f standard deviation from data on five normal women studied on a similar protocol [S] calculated according to (191 = 5.1 f 1.5). It is noteworthy that either her free 17@-hydroxysteroids or her free testosterone were excessive in 93 per cent of the blood samples obtained. Consequently, her free l7/3hydroxysteroid levels were high on $1 but one occasion when her free testosterone level was normal. A more
difficult diagnostic problem in this category is represented by Case IIIB [Figure 1). This patient had a slightly elevated free testosterone level in only three of 18 samplings; her %-hour mean plasma free testosterone level (9.0 pg/ml) was marginally elevated. High plasma levels of free 17/3-hydroxysteroids were observed on seven of the 17 occasions when her free testosterone level was normal; her %-hour mean free l’//!?-hydroxysteroid level was 59 pg/ml (normal 36 f 8.5 [6,19]). An androgenic basis for hirsutism could not be clearly identified by our protocol in Case IV (not shown). The measurement of the adrenal androgen DS was not of any additional diagnostic help in this series; it was increased (613 pg/dl) in only Case II in which plasma free androgens were abnormal. It was possible to discern an intermittent pattern to the plasma androgen levels in some of these hirsute women. Plasma testosterone and other li’&hydroxysteroid levels fell for about 1.5 hours from an 8 A.M. peak and then underwent a small, but definite surge in mid-morning in Cases I and IIIB (Figure 1). These changes occurred in the absence of a significant change in testosterone binding and simultaneously with falling cortisol levels. The variability of the plasma free testosterone of hirsute women was significantly greater (p
March 1979
The American Journal of Medicine
Volume 66
419
ANDROGEN PATTERNS IN HIRSUTISM-ROSENFIELD
plasma concentration of that steroid in that person. The 95 per cent confidence limits are such that a single total plasma testosterone level may fall within only 38 per cent,of the 24-hour mean. Because of this variability a computation was made of the agreement between the mean of a subset of testosterone levels and the 24-hour mean testosterone level for each of these patients. The mean plasma total testosterone of two, three and four morning samples at 1 to 4 hourly intervals may differ (95 per cent confidence limits] 26 per cent, 20 per cent and 18per cent, respectively, from the &hour average. The data for the plasma free testosterone were not significantly different and are depicted in Figure 2. This figure shows that the best short-term estimate of the average 24-hour plasma free testosterone level is obtained from the mean of 4 hourly samples over a 3-hour period: in this situation one has 95 per cent confidence that the value obtained is within 18per cent of the mean for that day. The degree of fluctuation of testosterone differs from that of the other steroids measured. Cortisol varies more widely than testosterone: the mean f 95 per cent confidence limits for single cortisol levels between 0800 and 1200hours was 110 per cent f 114per cent of the 24hour mean; the mean of 4 hourly plasma cortisol determinations was within about 71 per cent of the mean. On the other hand, DS levels were relatively stable, the 95 per cent confidence limits of the mean ranging from 27 per cent for a single sample to 16per cent, 15per cent and 13 per cent for the mean of two to four levels, respectively; these figures are similar to those expected on the basis of assay variability. The comparable figures for plasma V&hydroxysteroid concentrations are intermediate between those for cortisol and DS. They range successively from 52 per cent for a single sample to 34 per cent, 28 per cent and 29 per cent for the mean of two to four samples. V@hydroxysteroid levels followed a diurnal pattern resembling that of cortisol in that levels at 2000to 2400hours were less than the 0800 mean (p
420
March 1979
The American Journal of Medicine
terone levels, but consistent elevation of plasma free testosterone levels. A single, accurate, elevated free plasma testosterone level would settle the diagnosis in patients in both this and the previous group. A third type is comprised of patients whose plasma androgens undergo marked pha8ic fluctuations around the upper limits of normal with unremarkable levels of free plasma testosterone for over an hour at a time. In such cases, measurement of plasma free l’/&hydroxysteroids, OF perhaps other an.drogens such as androstenedione, in a single sample may provide evidence of hyperandrogenism. Although there is an element of nonspecificity in this assay [2,18], the level of these substances correlates with hirsutism as well as the level of testosterone [l]. Unknown is the extent to which the l7& hydroxysteroids are directly androgenic or to which they are markers of testosterone overproduction by virtue of being testosterone precursors dr metabolites arising from an accelerated rate of testosterone turnover [21]. Other patients of this third sort may require multiple sampling with measurement of both free plasma testosterone and other androgens to detect the endocrine abnormality. In yet other hirsute women an endocrinologic basis for the hirsutism cannot clearly be identified by these means. Plasma androgen levels of hirsute women were found to vary in a pattern suggestive of intermittent and independent episodes of adrenal and ovarian secretion. For example,.Figttre 1 shows patients in whom androgen levels tended to parallel the early morning rise and fall of cortisol levels, but in Lvhom a secondary surge of androgens occurred in mid-morning while the cortisol level was still falling. Since there is no reason to suspect a change in androgen clearance, androgen binding and posture not changing significantly during these studies, these changes probably result from,successive adrenal and ovarian secretory surges. Similar changes in the plasma androgen:cortisol ratio occur normally [6]. These data are compatible with the fact that both an ACTH and a luteinizing hormone surge can be expected within any 3-hour period [22,23].The fact that androgen levels are not more clearly pulsatible is probably related to the fact that ovarian and adrenal androgen production are to a great extent indirect, proceeding considerably through secretion of prehormones, and are probably independent [24]. Because free androgen levels fluctuate more Widely in hirsute than in normal women, a single plasma androgen determination is not necessarily representative of the average level in a hirsute women. A single plasma testosterone value may differ as much as 38 per cent from the 24-hour mean, for example. Calculating the average df samples taken at hourly intervals improves the reliability of the estimate of the mean (Figure 2). Sampling at very short intervals, as suggested by Goldzieher et al. [25], does not seem advisable, because we find that androgen production may be quiescent for about 1.5 hours. Because of our observation that an-
Volume 66
ANDROGEN PATTERNS IN HIRSUTISM-ROSENFIELD
drogen production may be intermittent and because of theoretic considerations regarding the timing of tropic hormone surges, sampling hourly for 3 hours would seem to be the optimal means of detecting elevated androgen levels and obtaining a close estimate of the %-hour mean plasma concentration of testosterone and the other 17@-hydroxysteroids in hirsute women who present a difficult diagnostic problem. This sampling
protocol also gives an optimal estimate of the mean testosterone level in males [15]. ACKNOWLEDGMENT This manuscript was written during my tenure as a Fogarty Senior International Fellow with the help of Dr. A. Nimrod of Prof. Hans Lindner’s Department of Hormone Research, Weizmann Institute of Science.
REFERENCES 1.
2. 3.
4. 5.
Rosenfield RL: Plasma testosterone binding globulin and indexes of the concentration of unbound androgens in normal and hirsute subiects. 1 . . Clin Endocrinol Metab 32: 717,1971. Rosenfield RL: Studies of the relation of plasma androgen levels to androgen action in women. J Steroid Biochem 6: 695,1975. Bardin CW, Lipsett MB: Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries. J Clin Invest 46: 891.1967. Abraham GE, Chakmakjian ZH: Plasma steroids in hirsutism. Obstet Gynecol44: 171,1974. Dupon C, Rosenfield RL, Cleary RE: Sequential changes in total and free testosterone and androstenedione in plasma during spontaneous and clomid-induced ovulatory cycles. Am J Obstet Gynecol115:478,1973. Rosenfield RL. Helke JC: Small diurnal and episodic fluctuations of the plasma free testosterone level in normal females. Am J Obstet Gynecoll20: 461,1974. Judd HL, Yen SSC: Serum androstenedione & testosterone levels during the menstrual cvcle. I Clin Endocrinol Metab 36: 475, 1973: Givens JR, Andersen RN, Ragland JB. et al.: Adrenal function :_ l_:.._..r:_- l-x..“__, ,.I.-..,, e “,.“..,...“_ ,I- ,,,,,, “” drostenedione, testosterone, 17-hydroxyprogesterone. cortisol, LH & FSH to dexamethasone & l/2 unit of ACTH. J Clin Endocrinol Metab 40: 988,1975. Dawood MY, Saxena BB: Plasma testosterone & dihydrotestosterone in ovulatory & anovulatory cycles. Am J Obstet Gynecol126: 430,1976. Tyler JPP, Newton JR, Collins WP: Variations in the concentration of testosterone in peripheral plasma from healthy women. Acta Endocrinol80: 542,197s Aedo AR, Nunez M, Landgren B-M, et al.: Studies on the pattern of circulating steroids in the normal menstrual cycle. 3. Circadian variations in the periovulatory period. Acta Endocrinol84: 320,1977. Ismail AAA. Davidson DW. Souka AR, et al.: The evaluation of the role of androgens in hirsutism 8r the use of a new I
9.
10.
11.
12.
13. 14. 15. 16. 17.
18. 19.
I
20. 21.
22. 23. 24. “5.
March 1979
anti-androgen “cyproterone acetate” for therapy. J Clin Endocrinol Metab 39: 8X1974. Thomas PK, Ferriman DG: Variation in facial and pubic hair growth in white women. Am J Phys Anthropol 15: 171, 1957. Kim MH. Rosenfield RL. Dupon C: The effect of dexamethasone on olasma free androgens during the normal menstrual cycle. Am J Obstet G$ecol 126:982, 1976. Rosenfield RL. lones T. Fang VS: The relationship between plasma testosterone and mean LH levels in men. J Clin Endocrinol Metab 45: 30, 1977. Buster JE, Abraham GE: Radioimmunoassay of plasma dehydroepiandrosterone sulfate. Anal Letters 5: 543, 1972. Murphy BEP: Some studies of the protein binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocrinol Metab 27: 973,1967. Andre CM, James VHT: Plasma androgens in idiopathic hirsutism. Steroids 24: 295, 1974. MOB GW Jr., Rosenfield RL Testosterone binding and free plasma androgen concentrations under physiologic conditions. Submitted for publication. Bailev NTI: Statistical Methods in Biologv, Universitv _” English _ Press, 1959. Mahoudeau IA. Bardin CW. Liosett MB: The metabolic clearance rate and origin of plasma dihydrotestosterone in man and its conversion to the 5n-androstanediols. J Clin Invest 50: 1338,1971. Weitzman ED. Fukushima D. Noneire C. et al.: Twentv-four hour pattern of the episodic se&etion of cortisol in normal subjects. J Clin Endocrinol Metab 33: 14,1971. Santen RJ, Bardin EW: Episodic luteinizing hormone secretion in man. J Clin Invest 52: 2617,1973. Rosenfield RL: Relationship of androgens to female hirsutism and infertility. J Reprod Med 11: 87,1973. Goldzieher JW, Dozier TS, Smith KD, et al.: Improving the diagnostic reliability of rapidly fluctuating plasma hormone levels by optimized multiple-sampling techniques. J Clin Endocrinol Metab 43: 824,1976.
The American Journal of Medicine
Volume 66
421
ROBERT L. ROSENFIELD.
M.D
Chicago, Illinois
From the Department of Pediatrics, University of Chicago, Pritzker School of Medicine. Wyler Children’s Hospital, 5825 S. Maryland’Avcnue. Chicago, Illinois. Studies carried out in the Clinical Research Center of the IJniversity of Chicago Hospital under Grants RR-305 and RR-55 from the General Clinical Research Centers Program of- the Division of Research Resources. National Institutes of Health. The rcsoarch was qupported in part by USPHS Cranf HD-OG308. Requests for reprints should be addressed to Dr. Robert L. Rosenfield. Department of Pediatrics, The University of Chicago, 5825 Maryland Avenue, Chicago, Illinois 60637. Manuscript accFpted Octohcr 27, 1978.
The 24-hour pattern of plasma total and free testosterone and other lirfl-hydrozrysteroids has been characterized in six women with moderately severe male-pattern hirsutism. Four distinct plasma androgen patterns were identified: (1) total and free plasma testosterone levels were elevated 80 per cent or more of the time, (2) !he free plasma testosterone level was con&&y elevated, where-as total testosterone was in the high-normal range, (3) plasma free androgens were undergoing marked &asic fluctuations around the upper lhnits of normal such that sampling blood for multiple andrhgens and/or at multiple intervals was necessaq to disclw the hyperandrogenic state, and (4) plasma free androgens fluctuated within the normal range. The variation of the plasma androgens was suggestive of intermittent and independent episodes of adrenal and ovarian androgen prodetion. Because of these fluctuations, a single plasma testosterone leGe1was tit necessarily representative of the average concentration over a 24-hour period. For example, the 95 per cent confidence limits are such that a single plasma testost rone level may fall within only 38 per cent of the a&hour mean. ThdLs t short-term estimate of the average daily testosterone level is the mean of hourly determinations over a l-hour per@ the 95 per cent confidence l&nits of this figure are within 18 per cent of the 24-hour mean. Nevertheless, this 3-hour sampling protocol would not have detected the subtle hyperandrogenic state present in twq patients unless androgens other than free teskerone (e.g., free lir@-hydroxysteroids) were measured in the same plasma specimens. The patterns of the plasma testosterone in several hirsute women are reported here. Fluctuations in testosterone were sought because the plasma testosterone concentration is increased in only about half the cases in which more extensive studies reveal abnormalities of testosterone binding, precursors, metaboliteslor metabolism [I-J]. One possible explanation for such disparities would be that a single testosterone level is not necessarily representative of the mean over a prolonged period of time. Although it is well established that plasma testosterone fluctuates within a relatively narrow range in normal women [~-II], there is some indication that it may vary relatively widely in hirsute women. On the basis of sampling blood at 2 to 4 hour intervals for 24 hours, Givens et al. [8] concluded that the variability of plasma testosterone was greater than normal in two of 12 hirsute women. On the basis of assaying four blood samples over a two day period, Ismael et al. [12) reported thq/ a single testosterone level was normal in 40 per cent of a group of himute women found to have uniformly abnormal mean testosterone leyels. Neither group focused on
March 1979
The American
Journal of Medicine
Volume
66
417
ANDROGEN PATTERNS IN HIRSUTISM-ROSENFIELD
Clock a
normal
time
daytime
Clock time
range
+I
range
of lrvrlrl600-4OOhr
lgure 1. Three patterns of plasma androgens (see text) in four different hirsute women. Shaded Ueas indicate normal rang&.
the level of free (unbound] testosterone and other 17/3-hydroxysteroids although the plasma concentrations of these free androgens are more closely related to hirsutism than are total plasma testosterone levels [1,2]. Because of these considerations, a systematic. charac: terization of the pattern of plasma free androgens of selected hirsute women has been undertaken and is reported here. These data have important implications as regards the optimal sampling regimen for the detection of the hyperandrogenic state in women. METHODS Six 17 to 32 year old women with moderately severe malepattern hirsutism [13] were selected for these extensive studies after preliminary tests indicated that their androgen patterns would be of particular interest. Four were diagnosed as having an excessive ovarian source of androgen on the basis of their response to dexamethasone suppression [14]: two subjects (Cases BIB and IV] had normal responses to dexamethasone, suggesting a major adrenal component. After obtaining informed consent, the subjects were admitted to the Clinical Research Centers of the University of Chicago Hospitals during a medroxyprogesterone acetate-withdrawal-induced menstrual period. Commencing at 0800the following morning, blood samples were drawn through an indwelling catheter at approximately 4 hour intervals for a 24 hour period. Meanwhile, between 0800 and 1100 hours, blood was taken at 15
418
March 1979 The American Journal of Medicine
minute intervals for 2 to 3 hours from five subjects. Activities were sedentary except for bedrest from 0800 to 1100 hours. Plasma testosterone and dehydroepiandrosterone sulfate (US) were radioimmunoassayed using a modification of the method of Ismael[l5] and the method of Buster and Abraham [16], respectively. Plasma Ii’@-hydroxysteroids and cortisol were measured by competitive protein binding [1,17]. Plasma lY&hydroxysteroids are those ether-extractable substances in plasma which bind to testosterone-estradiol binding globulin (TEBG); two-thirds of these substances in normal women are accounted for by dihydrotestosierpne, androstenediol and androstanediol, as well as testosterone [2,18]. Testosterone binding to plasma proteins was assessed by a charcoal adsorption assay [l], and the plasma free testosterone and apparent free Ii’@-hydroxysteroids levels were expressed in terms of absolute concentrations in whole, 37% plasma [19]. All measurements of each parameter in a subject were performed as a single batch. The precision of each of these assays is between 11 and 14 per cent (coefficient of variation). Statistical analyses were performed on Hewlett-Packard 9810A and Wang programmable calculators. All p values are one-tailed. For computations regarding the relationship between the 24-hour mean and a subset of any particular parameter, the subset under consideration was excluded from the calculation of the 24-hour mean. Subsets considered were means of levels of a hormone at intervals of I or more hours, the intervals being hourly if three or four values were considered. Ninety-five per cent confidence limits were calculated from the distribution of t [2O].
Volume 66
;\Nl)ROGI:N
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PATTERNS
IN HIRSUTISM-ROSENFIELD
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70-
I
I-
ONE
THREE*
Two* NUMBER
FOUR +
OF SAMPLES
*MEAN AT (EOUALI INTERVALS ff
t-4 HOURS
igure 2. The reliability with which the 24-hour mean free plasma tktosterone of hirsute women is predicted from 1 to 4 hourly measurements at 0800 to 1200
hours. Shaded areas show 95 per cent confidence limits. RESULTS Four patterns of plasma androgens were found. The pattern displayed in Case I (Figure 1) was typical of the two cases studied. The plasma total testosterone level is moderately elevated 80 per cent or more of the time in conjunction with a consistent elevation of the plasma free testosterone level. Case II (Figure l] ilhfstrates another category. Here, plasma free testosterone was consistently abnormal, yet the X-hour mean total plasma testosterone was clearly normal (54 ng/dl) with a marginally elevated total testosterone level in only one of 14 samples. This discrepancy between the total and free plasma iestosterone was due to the subnormal TEBG level which is frequently found in hirsute women (11. A third type is comprised of cases in which androgens markedly fluctuate [up to twofold] about the upper limits of normal. In Case IIIA (Figure l), the patient had an elevated free testosterone level in only five of 14 specimens; her mid-morning free plasma testosterone concentration was normal for over 1.5 hours. Her %&hour mean plasma free testosterone level as determined by the present protocol was marginally elevated (11.1 pg/ml; normal %-hour mean f standard deviation from data on five normal women studied on a similar protocol [S] calculated according to (191 = 5.1 f 1.5). It is noteworthy that either her free 17@-hydroxysteroids or her free testosterone were excessive in 93 per cent of the blood samples obtained. Consequently, her free l7/3hydroxysteroid levels were high on $1 but one occasion when her free testosterone level was normal. A more
difficult diagnostic problem in this category is represented by Case IIIB [Figure 1). This patient had a slightly elevated free testosterone level in only three of 18 samplings; her %-hour mean plasma free testosterone level (9.0 pg/ml) was marginally elevated. High plasma levels of free 17/3-hydroxysteroids were observed on seven of the 17 occasions when her free testosterone level was normal; her %-hour mean free l’//!?-hydroxysteroid level was 59 pg/ml (normal 36 f 8.5 [6,19]). An androgenic basis for hirsutism could not be clearly identified by our protocol in Case IV (not shown). The measurement of the adrenal androgen DS was not of any additional diagnostic help in this series; it was increased (613 pg/dl) in only Case II in which plasma free androgens were abnormal. It was possible to discern an intermittent pattern to the plasma androgen levels in some of these hirsute women. Plasma testosterone and other li’&hydroxysteroid levels fell for about 1.5 hours from an 8 A.M. peak and then underwent a small, but definite surge in mid-morning in Cases I and IIIB (Figure 1). These changes occurred in the absence of a significant change in testosterone binding and simultaneously with falling cortisol levels. The variability of the plasma free testosterone of hirsute women was significantly greater (p
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plasma concentration of that steroid in that person. The 95 per cent confidence limits are such that a single total plasma testosterone level may fall within only 38 per cent,of the 24-hour mean. Because of this variability a computation was made of the agreement between the mean of a subset of testosterone levels and the 24-hour mean testosterone level for each of these patients. The mean plasma total testosterone of two, three and four morning samples at 1 to 4 hourly intervals may differ (95 per cent confidence limits] 26 per cent, 20 per cent and 18per cent, respectively, from the &hour average. The data for the plasma free testosterone were not significantly different and are depicted in Figure 2. This figure shows that the best short-term estimate of the average 24-hour plasma free testosterone level is obtained from the mean of 4 hourly samples over a 3-hour period: in this situation one has 95 per cent confidence that the value obtained is within 18per cent of the mean for that day. The degree of fluctuation of testosterone differs from that of the other steroids measured. Cortisol varies more widely than testosterone: the mean f 95 per cent confidence limits for single cortisol levels between 0800 and 1200hours was 110 per cent f 114per cent of the 24hour mean; the mean of 4 hourly plasma cortisol determinations was within about 71 per cent of the mean. On the other hand, DS levels were relatively stable, the 95 per cent confidence limits of the mean ranging from 27 per cent for a single sample to 16per cent, 15per cent and 13 per cent for the mean of two to four levels, respectively; these figures are similar to those expected on the basis of assay variability. The comparable figures for plasma V&hydroxysteroid concentrations are intermediate between those for cortisol and DS. They range successively from 52 per cent for a single sample to 34 per cent, 28 per cent and 29 per cent for the mean of two to four samples. V@hydroxysteroid levels followed a diurnal pattern resembling that of cortisol in that levels at 2000to 2400hours were less than the 0800 mean (p
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terone levels, but consistent elevation of plasma free testosterone levels. A single, accurate, elevated free plasma testosterone level would settle the diagnosis in patients in both this and the previous group. A third type is comprised of patients whose plasma androgens undergo marked pha8ic fluctuations around the upper limits of normal with unremarkable levels of free plasma testosterone for over an hour at a time. In such cases, measurement of plasma free l’/&hydroxysteroids, OF perhaps other an.drogens such as androstenedione, in a single sample may provide evidence of hyperandrogenism. Although there is an element of nonspecificity in this assay [2,18], the level of these substances correlates with hirsutism as well as the level of testosterone [l]. Unknown is the extent to which the l7& hydroxysteroids are directly androgenic or to which they are markers of testosterone overproduction by virtue of being testosterone precursors dr metabolites arising from an accelerated rate of testosterone turnover [21]. Other patients of this third sort may require multiple sampling with measurement of both free plasma testosterone and other androgens to detect the endocrine abnormality. In yet other hirsute women an endocrinologic basis for the hirsutism cannot clearly be identified by these means. Plasma androgen levels of hirsute women were found to vary in a pattern suggestive of intermittent and independent episodes of adrenal and ovarian secretion. For example,.Figttre 1 shows patients in whom androgen levels tended to parallel the early morning rise and fall of cortisol levels, but in Lvhom a secondary surge of androgens occurred in mid-morning while the cortisol level was still falling. Since there is no reason to suspect a change in androgen clearance, androgen binding and posture not changing significantly during these studies, these changes probably result from,successive adrenal and ovarian secretory surges. Similar changes in the plasma androgen:cortisol ratio occur normally [6]. These data are compatible with the fact that both an ACTH and a luteinizing hormone surge can be expected within any 3-hour period [22,23].The fact that androgen levels are not more clearly pulsatible is probably related to the fact that ovarian and adrenal androgen production are to a great extent indirect, proceeding considerably through secretion of prehormones, and are probably independent [24]. Because free androgen levels fluctuate more Widely in hirsute than in normal women, a single plasma androgen determination is not necessarily representative of the average level in a hirsute women. A single plasma testosterone value may differ as much as 38 per cent from the 24-hour mean, for example. Calculating the average df samples taken at hourly intervals improves the reliability of the estimate of the mean (Figure 2). Sampling at very short intervals, as suggested by Goldzieher et al. [25], does not seem advisable, because we find that androgen production may be quiescent for about 1.5 hours. Because of our observation that an-
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drogen production may be intermittent and because of theoretic considerations regarding the timing of tropic hormone surges, sampling hourly for 3 hours would seem to be the optimal means of detecting elevated androgen levels and obtaining a close estimate of the %-hour mean plasma concentration of testosterone and the other 17@-hydroxysteroids in hirsute women who present a difficult diagnostic problem. This sampling
protocol also gives an optimal estimate of the mean testosterone level in males [15]. ACKNOWLEDGMENT This manuscript was written during my tenure as a Fogarty Senior International Fellow with the help of Dr. A. Nimrod of Prof. Hans Lindner’s Department of Hormone Research, Weizmann Institute of Science.
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Rosenfield RL: Plasma testosterone binding globulin and indexes of the concentration of unbound androgens in normal and hirsute subiects. 1 . . Clin Endocrinol Metab 32: 717,1971. Rosenfield RL: Studies of the relation of plasma androgen levels to androgen action in women. J Steroid Biochem 6: 695,1975. Bardin CW, Lipsett MB: Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries. J Clin Invest 46: 891.1967. Abraham GE, Chakmakjian ZH: Plasma steroids in hirsutism. Obstet Gynecol44: 171,1974. Dupon C, Rosenfield RL, Cleary RE: Sequential changes in total and free testosterone and androstenedione in plasma during spontaneous and clomid-induced ovulatory cycles. Am J Obstet Gynecol115:478,1973. Rosenfield RL. Helke JC: Small diurnal and episodic fluctuations of the plasma free testosterone level in normal females. Am J Obstet Gynecoll20: 461,1974. Judd HL, Yen SSC: Serum androstenedione & testosterone levels during the menstrual cvcle. I Clin Endocrinol Metab 36: 475, 1973: Givens JR, Andersen RN, Ragland JB. et al.: Adrenal function :_ l_:.._..r:_- l-x..“__, ,.I.-..,, e “,.“..,...“_ ,I- ,,,,,, “” drostenedione, testosterone, 17-hydroxyprogesterone. cortisol, LH & FSH to dexamethasone & l/2 unit of ACTH. J Clin Endocrinol Metab 40: 988,1975. Dawood MY, Saxena BB: Plasma testosterone & dihydrotestosterone in ovulatory & anovulatory cycles. Am J Obstet Gynecol126: 430,1976. Tyler JPP, Newton JR, Collins WP: Variations in the concentration of testosterone in peripheral plasma from healthy women. Acta Endocrinol80: 542,197s Aedo AR, Nunez M, Landgren B-M, et al.: Studies on the pattern of circulating steroids in the normal menstrual cycle. 3. Circadian variations in the periovulatory period. Acta Endocrinol84: 320,1977. Ismail AAA. Davidson DW. Souka AR, et al.: The evaluation of the role of androgens in hirsutism 8r the use of a new I
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anti-androgen “cyproterone acetate” for therapy. J Clin Endocrinol Metab 39: 8X1974. Thomas PK, Ferriman DG: Variation in facial and pubic hair growth in white women. Am J Phys Anthropol 15: 171, 1957. Kim MH. Rosenfield RL. Dupon C: The effect of dexamethasone on olasma free androgens during the normal menstrual cycle. Am J Obstet G$ecol 126:982, 1976. Rosenfield RL. lones T. Fang VS: The relationship between plasma testosterone and mean LH levels in men. J Clin Endocrinol Metab 45: 30, 1977. Buster JE, Abraham GE: Radioimmunoassay of plasma dehydroepiandrosterone sulfate. Anal Letters 5: 543, 1972. Murphy BEP: Some studies of the protein binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocrinol Metab 27: 973,1967. Andre CM, James VHT: Plasma androgens in idiopathic hirsutism. Steroids 24: 295, 1974. MOB GW Jr., Rosenfield RL Testosterone binding and free plasma androgen concentrations under physiologic conditions. Submitted for publication. Bailev NTI: Statistical Methods in Biologv, Universitv _” English _ Press, 1959. Mahoudeau IA. Bardin CW. Liosett MB: The metabolic clearance rate and origin of plasma dihydrotestosterone in man and its conversion to the 5n-androstanediols. J Clin Invest 50: 1338,1971. Weitzman ED. Fukushima D. Noneire C. et al.: Twentv-four hour pattern of the episodic se&etion of cortisol in normal subjects. J Clin Endocrinol Metab 33: 14,1971. Santen RJ, Bardin EW: Episodic luteinizing hormone secretion in man. J Clin Invest 52: 2617,1973. Rosenfield RL: Relationship of androgens to female hirsutism and infertility. J Reprod Med 11: 87,1973. Goldzieher JW, Dozier TS, Smith KD, et al.: Improving the diagnostic reliability of rapidly fluctuating plasma hormone levels by optimized multiple-sampling techniques. J Clin Endocrinol Metab 43: 824,1976.
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