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The Excretion of Free 17-Ketosteroids in Hirsutism
The Excretion of Free 17-Ketosteroids in Hirsutism JAY J. GOLD, M.D., and SARA GOLDBERG, Ph.D."'
IN
ATI'EMPTS to classify women with hirsutism into specific groups for facilitation and direction of proper therapy, numerous approaches have been utilized. It is a difficult problem to differentiate that form of hirsutism which has been clinically related to adrenal, ovarian, andjor thyroid disease from that which is familial in nature or iatrogenic in origin. Furthermore the idiopathic form, by its very name, defies classification. Members of this latter group often display completely normal urinary steroid levels, in terms of urinary 17ketosteroids ( 17-KS) and C-21 deoxysteroids. In the course of studies on normal and hirsute women, 9 • 10 the nature of our work necessitated extensive hydrolysis of urinary steroids. This required separation of urinary steriods into the free components, glucuronides and sulfates. Examination of the various fractions was then performed. It rapidly became apparent, in terms of the free 17-KS, that there were marked differences in the amount of free 17-KS excreted by normal and by hirsute women. Furthermore, there appeared to be differences in the individual17-KS contained in this free fraction. It was then logical to assume that, if certain androgenic 17-KS (even though of relatively low-potency androgenicity) in the hirsute group free fraction were not present in the normal group, these steroids might be at least partially responsible for the hirsutism. This would appear to be particularly important in that group of hirsute patients who exhibit normal to high-normal17-KS excretion with normal fractionation patterns. Few studies on free steroids have been reported specifically and the bulk of these have been related to 17-hydroxycorticosteroid excretion. 1 • 14-16 • 23 The
From Michael Reese Hospital, Chicago, Ill. This study has been supported in part by grants from the United States Public Health Service {Grant A-1670), and the Stacy H. VanValkenburgh Memorial Grant for Cancer Research from the American Cancer Society (P-185). *Present address: 19 Methodella Street, Jerusalem, Israel. We are grateful to Drs. Kenneth Thompson and Harold Upjohn for the Zn-cortrophin and Medrol used in this study and generously supplied by them, respectively. We are also grateful to Dr. Donald Holub and Dr. Fred Katz of Columbia University, College of Physicians and Surgeons for supplying us with fresh frozen urine from the patient with adrenal carcinoma.
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fewer papers found describing studies on free and conjugated 17-KS report them only as part of another studyY· 20• 22 • 25 Two of these13 • 22 described very low to absent free 17-KS in adrenogenitals, which results are quite different from ours. In one of these studies the urine was~:¢6I1ected in the presence of a preservative. We did not take this specific precaution but collected the specimens at low temperatures. Studies to exclude the possibility of spontaneous hydrolysis prior to analysis were performed and are detailed below. METHODS AND MATERIALS
Fresh 24-hour urines were utilized for this study. The urines were extracted for free steroids as soon as feasible after the 24-hour urine collection was completed. This meant a maximal lapse of time of 4 hours between collection and analysis, unless the urines had been frozen immediately upon completion of collection. A 600-cc. aliquot of urine was extracted at neutral pH 3 times with 300 cc. of redistilled ethyl acetate. The residual urine was discarded and the ethyl acetate extracts were combined. This was then washed 3 times with IN NAOH and then twice with distilled water. The washings were discarded and the washed ethyl acetate was dried with sodium sulfate and evaporated under reduced pressure. The dried extract was dissolved in redistilled absolute methanol and 90% of the methanol extract was spotted on a wide strip ( 3 em. wide) of Whatman No.1 filter paper. The remaining 10% was spotted on a narrow strip ( 1 em. wide). The strips were run on the DeCourcy paper chromatographic system5 for 5-7 hours at room temperature. The strips were then dried at room temperature under a stream of air. The narrow strip was dipped into 70% phosphoric acid, heated at 85°C. for 10 min. and then observed for :fluorescent spots6 under a UV light source ( 3660° A). The Rf's, fluorescent color, etc. were recorded and the parallel areas on the wide strip were delineated, cut, and eluted with redistilled absolute methanol. A Zimmerman reaction was run on 10% of the methanol extract of each eluted spot. The remainder was then individually chromatographed on duplicate strips on the Savard system19 (ligroin: propylene glycol). Upon completion of the run, one strip was developed for fluorescence as noted above. Parallel areas of the duplicate strip were then eluted with methanol. Half of this eluate underwent Zimmerman quantitation and the remainder was examined for its sulfuric acid spectrum on a Cary recording spectrophotometer. The total17-KS were also measured11 on each of the 24-hr. urines studied. From this treatment it was possible to determine the total17-KS, free 17-KS, and the components of the free 17-KS fraction. In order to determine whether spontaneous hydrolysis, which would arti-
17 -KS
VoL. 14, No. 1, 1963
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ficially alter our results, had occurred during urine collection or extraction, the following studies were done. In three 24-hr. urine collections from normal women, each voiding was immediately collected and divided in half. One half was extracted for free 17-KS at collection and the other half was placed in a 24-hr. pool that was kept cold ( 4 °C.). The portion added to the 24-hr. pool was measured for free 17-KS at the end of the 24-hr. collection. The sum of the extractions of the separate voiding was then compared to the value obtained from extraction of the total24-hr. collection and no significant difference was noted. In addition three, fresh 24-hr. urines were extracted for free 17-KS and then placed in the refrigerator at 4°C. without preservative for an additional24-48 hr. They were then re-extracted for free 17-KS, and none was found. One can only conclude from these studies that spontaneous hydrolysis, under the conditions of our experiment, did not contribute to or alter in any way the results we obtained. These data are summarized in Table 1. Patients studied included females in the first and second halves of their: TABLE 1. Data from Studies to Determine Presence of Spontaneous. Hydrolysis During Urine Collection and Extraction Patient
B.J.
S.G.
F.B.
II I
Specimen
1st 8 hr. 2nd 8 hr. 3rd 8 hr.
55 0 0
1st 8 hr. 2nd 8 hr. 3rd 8 hr.
0 0 0
1st Voiding 2nd Voiding 3rd Voiding 4th Voiding 5th Voiding TOTAL
I: i I i!
ZimmeTman chTomogen (p.g.)* Delayed emtTaction:t: Immediate emtTactiont
0 33 40 34 0 107
} }
47
0
}
94
94
FRESH URINE
M.S.
FiTit emtmction
Second emtTaction
314 p.g./24 hr.
0 (24 hr. at 4°C.) 0 (48 hr. at 4°C.) 0 (48 hr. at 4°C.)
S.G.
R.B.
270 p.g./24 hr.
*Calculated on basis of dehydroepiandrosterone as a standard. t Half of each voiding. :!:Half of each voiding pooled 24 hr.
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menstrual cycles and in the three trimesters of pregnancy; normal males; and females with hirsutism due to the Stein-Leventhal syndrome and to adrenal virilism, as well as patients with unclassified hirsutism. Finally, several patients with acute and chronic hepatic disease and chronic renal disease were also evaluated. In all, results of studies on 78 patients are included in this report. RESULTS Normals (Fig. 1 )
The normal female ( 6 patients) excreted a mean of 7.5 mg. of total17-KS per day in the first half of the menstrual cycle. Extraction for free 17-KS revealed only Zimmerman chromogen in the amount of 0.12 mg. per day, with .no identifiable 17-KS on fractionation. During the second half of the menstrual cycle the over-all pattern ( 7 patients) remained the same. Androsterone (A) andjor other mildly androgenic 17-KS were not found. Etiocholanolone (E) was found in one of the 7 patients. The normal male excreted more total 17-KS (13.0 mg. per day), as expected, and slightly more free Zimmerman chromogen ( 0.22 mg.) than the normal female. Again, fractionation revealed no identifiable 17-KS. On the other hand, the normal pregnant female excreted a mean of more than 3 times the free 17-KS (0.64 mg. per day-the mean of the three trimesters) excreted by the nonpregnant female. Furthermore, especially in the first trimester, A and E now began to be identified in this free fraction. The highest excretion of free steroid occurred at the end of the first trimester ( 0.92 mg. per day), with a progressive decrease to the third trimester ( 0.45 mg. per day). Hirsutism Patients (Fig. 2)
Stein-Leventhal syndrome.21 The clinical criteria for this group included menstrual disturbances, variable hirsutism, and bilateral polycystic ovaries. Most had normal urinary 17-KS and all had normal urinary pregnanetriol. Patients studied with the Stein-Leventhal syndrome excreted a mean of TOTAL 17-KS MC./DAY
0
NORMAL FEMALE ht HALF OF CYCLE !61 2nd HALF OF CYCLE !71
NORMALMALE-<10>
5
10
1.s + 1.sr 6.9 1.0+~
FREE 17-KS MG./DAY
15
0
1.0
05
1.5
2.0
ANDROST. ETIOCH
= .053'
NONE
NONE
po.J4 = .oza•
NONE
I OF 7
po.zz±.25'
NONE
NONE
po.12
NORMAL PREGNANCY 1st TRIMESTER !51 2nd TRIMESTER !7l 3rd TRIMESTER !91
ALL TRIMESTERS
0.92 ± .57•
10.3+3. 6.4-+- 1.43*
12.6 + 2.43*
9.8
0.56-+- .18*
~0.45±.16*
30F5
30F5
2 OF 7
lOF 7
1 OF9
30F9
0.64
!MEANS I *STANDARD ERROR OF MEAN
Fig. 1. Total and free 17-KS in normal males and females.
17 -KS
VoL. 14, No. 1, 1963
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0.60 mg. per day of free 17-KS preoperatively, with half excreting A and E in this fraction. Their mean total 17-KS (7.7 mg. per day) were normal. One patient, who had 0.47 mg. per day free 17-KS, had this increase to 1.03 mg. per day with ACTH. Medrolt then reduced this to 0.66 per day. AN DROST. TOTAL 17·KS MG./DAY 0 10 20 30 o40 50
60
70 80
90
FREE 11·KS MG./DAY 0 0.2 04 OG 08
STEIN-LEVENTHAL SYNDROME
10 12 14
AND ET10CH.
16 18 20
30F6
0.60:!: .17"'
PRE-OP 161
POST-OP Ill
ACTH 121
~O.SOIPRE-OP 1.341
NONE
==:::Jo.56
10F2
o.oo
10F2
BORDERLINE A.G.S.
1.42 + .39·
CONTROL 171
3.48
ACTH 121 I = = = : : J 4 1 . 0 ;::::::=::::::10.43
MEDROL 121
6.9
20.0 116 YR. OLDI
___.-
NONE
-- }10F2 1.5 NONE
0.0
ON THERAPY I11
HIRSUTISM UNCLASS.
po.za±.lo•
CONTROL16l
1 OF6 NONE
0.98
ACTH 121
po.M
MEDROL OR~ FF12l
CUSHING'S SYNDROMEs=====::!:~ CARCINOMA 11 l C
NONE
--:,;:
•STANDARD ERROR OF MEAN
Fig. 2. Total and free 17-KS in hirsutism patients. 0
The total17-KS changed little after ACTH and Medrol. Another patient, reexamined after ovarian wedge resection, showed a marked decrease in her total17-KS and free 17-KS. In addition, A and E disappeared from her free fraction. This would certainly suggest an ovarian contribution both to the total17-KS as well as the free 17-KS excretions. Borderline adrenogenital syndrome. 7 The clinical criteria for this group included menstrual disturbances, variable hirsutism, and polycystic ovaries in 50% of the patients. All had slight to moderate elevations of urinary 17-KS and pregnanetriol. Patients studied with the borderline adrenogenital syndrome excreted moderately elevated total17-KS ( 15.4 mg. per day). These levels rose briskly after ACTH stimulation ( 41.0 mg. per day), and then were adequately suppressed by Medrol ( 8.1 mg. per day). This is in contrast to the findings in the Stein-Leventhal syndrome. Prior to ACTH stimulation, the free fraction of these patients with adrenogenital syndrome amounted to 1.42 mg. per day, and most of these fractions contained A andjor E. ACTH then increased *Author's correction: In the final column, the fifth line should read "5 of 7"; the lOth line, ''2 of 6." tThe Upjohn Co., Kalamazoo, Mich.
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RANCE NORMAL FEMALE 1st HALF OF CYCLE 161
5.4-11.0
2nd HALF OF CYCLE 171
2.7-12.1
&
TOTAL 17-KS MG./DAY
0
5
10
1st TRIMESTER 151
6.5-16.0
2nd TRIMESTER 171
2.4-10.6
3rd TRIMESTER f91
5.9-24.2
STEIN-LEVENTHAL SYNDROME--161 5.0-13.5
20
IS
121
9.4-20.9
HIRSUTISM UNCLASSI61
6.3-19.0
RANCE
FERTILITY FREE 17-KS MC./DAY 0 1.0 2.0
3.0
& STERILITY 4.0
5.0
().0.26 trJro.lZl
16.911
0.09-0.20 0-0.70
113.011 tTO.~!I
~10.141 P=:Jro.Z2J
0-3.0
~~-'''
0.10-1.25 112.61
I
177JI
BORDERLINE A.G.S.-181 10.8-24.8 A.G.S.
25
c:::I:JTII]
NORMAL MALE-1101 6.5-21.5 NORMAL PREGNANCY
GOLDBERG
Pii' I
roJi
no.l1l
10.921
o:::::Jro.S61
0-1.30
~
0-1.34
•!0.6 )
0.46-3.06
!1.4211
l.S-6.9 0--0.47
IO J -
~10.281
Fig. 3. Consolidation of total and free 17-KS data for normal subjects and hirsutism patients.
the free fraction and Medrol suppression induced a marked fall. In the one instance where it was applicable, A and E also were no longer found after Medrol suppression. In these studies, the patients were given 20 U. of Zncortrophin"' intramuscularly daily for 5-7 days and Medrol, 8 mg. per day orally for 4 days. Adrenogenital syndrome. 8 These patients were premenarchal or had delayed puberty and characteristically demonstrated marked elevations in urinary 17-KS, pregnanetriol, and pregnanetriolone. Patients studied with the adrenogenital syndrome, showed markedly elevated excretion of free 17-KS ( 6.9 and 1.5 mg. per day), with A and E present in the older ( 16 years) patient. Steroid therapy in the younger ( 9 years old) reduced the free 17-KS from 1.5 mg. per day to zero. Unclassified hirsutism. These patients defied classification both clinically and chemically. Six patients are included whose hirsutism was unclassified etiologically. Their total17-KS ranged from normal to elevated, and the free 17-KS were essentially in the normal range. One patient had A in her free fraction; another, E. Two patients tested showed a response of the free fraction to ACTH. A male patient with Cushing's syndrome due to adrenal carcinoma demonstrated a marked elevation of the total17-KS and the free 17-KS. A female patient with Cushing's syndrome due to adrenal adenoma had comparable changes but to a lesser degree. Figure 3 shows a consolidation of data for the normal patients and those with hirsutism. Table 2 shows the results of statistical comparison between normal females and hirsute patients with the Stein-Leventhal syndrome and *Organon, Inc., Orange, N.J.
17 -KS
VoL.14, No.1,1963 TABLE 2.
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Statistical Comparison of 17-KS Values of Normals and Hirsutism Patients p
T
Normal females Stein-Leventhal syndrome
Total 17-KS Free 17-KS
.134 2.454
N.S. >.02
Normal females Borderline adrenogenital syndrome
Total 17-KS Free 17-KS
4.04 2.75
=.01 >.01
the borderline adrenogenital syndrome with regard to their total and free 17-KS excretions. The free 17-KS excretion in these hirsute patients was significantly different from that of the normal female. Renal and Hepatic Dysfunction (Table 3)
In order to evaluate effects of renal andjor hepatic dysfunction on the excretion of free 17-KS, several patients with these disorders were studied. One patient with acute hepatitis showed no real increase over normal free Zimmerman chromogen, but the patients with chronic cirrhosis excreted free Zim-:merman chromogen in mean amounts more than 3 times that found in normal individuals ( 0.83 mg. per day). However, A and E were not found in the free fractions. Two patients with chronic renal disease excreted amounts that were about twice normal ( 0.47 mg. per day) and A and E were present in one of these. DISCUSSION
Some interesting observations may be made from the data presented above on free 17-KS excretion. The normal individual, whether male or female, TABLE 3.
17-Ketosteroids in Patients with Renal and Hepatic Disorders
No.
Diagnoais
1 1 2 3 4
Acute hepatitis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) MEAN
1 2
± S.E.
OF MEAN
Chroi:llc renal dis. (male) Chronic renal dis. {male)
TotaZ17 KB (mg.jday)
Free Zimmerman chromo g. (mg.jday)
5.3 5.8 9.1 5.6 4.4
0.26 1.02 1.30 0.58 0.40
6.3 ± 2.0.
0.83 ± .31
5.0 3.2
0.50 0.44
IndivitluaZ 17-KB component• Etioch. (E) A.ndroat. (A.)
+ 0 0 0 0
0 0 0 0 0
0
0
+
+
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probably excretes little to no free 17-KS, as no 17-KS were identifiable on fractionation of the small quantity of Zimmerman chromogen found. In pregnancy however, larger amounts of Zimmerman chromogen were found, especially in the first trimester. It is less likely that this chromogenic material was nonspecific, as certain 17-KS (androsterone and etiocholanolone) were now identifiable on fractionation. In the first trimester, the total 17-KS and free 17-KS are higher than in the second trimester. In the third trimester, the total 17-KS are higher than in either of the first two and the free 17-KS are lower. This would suggest that the young placenta may play a role in conjugation mechanisms at least insofar as 17-KS are concerned. Recent placental perfusion studies23 would seem to bear this out for Porter-Silber chromogens. Patients with the Stein-Leventhal syndrome excrete more free 17-KS than normals, but less than patients with varying degrees of adrenal hyperplasia. Ovarian wedge resection in one patient apparently induced a fall in both the total and free 17-KS excretion (urine examined one week postoperatively). This would suggest that the ovary plays a role in 17-KS formation by altering adrenal activity indirectly, by actively contributing 17-KS by secreting them, or both. ACTH administered to 2 patients with the Stein-Leventhal syndrome induced minimal increases in total17-KS whereas normal individuals and patients with adrenal hyperplasia demonstrate a greater 17-KS response to ACTH stimulation. Furthermore the same 2 patients showed a minimal total 17-KS depression after Medrol administration. The latter is more difficult to interpret, however, as the control levels were relatively low initially. It would appear that the polycystic ovary characteristic of the Stein-Leventhal syndrome may actively contribute to the total17-KS and free 17-KS excretion in these patients and, furthermore (since their total17-KS are normal andrespond poorly to ACTH stimulation), this ovary may be interfering directly with normal adrenal responsiveness. The mechanism by which this might occur is obscure. Thus, in keeping with more recent studies,17 • 24 the SteinLeventhal syndrome may be basically an ovarian disease rather than a sequel of adrenal dysfunction. Patients with obvious adrenal hyperactivity, as in the borderline adrenogenital syndrome, adrenogenital syndrome, and Cushing's syndrome excrete large amounts of free 17-KS. ACTH administered to individuals with the borderline adrenogenital syndrome induced almost threefold increases in total and free 17-KS excretion. Such ACTH stimulation apparently increased the conjugate and free fractions in almost equivalent amounts as the ratio of conjugate to free changed little ( 9.8:1 to 10.8:1). Medrol suppression in the borderline adrenogenital group induced a fall in both the total and free 17KS. However, here the fall was mainly in the free steroids as the conjugate-
VoL. 14, No. 1, 1963
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81
to-free ratio jumped to 17.8:1. In a patient with the adrenogenital syndrome, Medrol also induced a striking fall in free steroids. Since the adrenal effect of Medrol is mediated via ACTH suppression, this would imply that ACTH affects free 17-KS excretion. However, the patient with Cushing's syndrome due to adrenal carcinoma, which is theoretically autonomous of ACTH, also excreted large amounts of free 17-KS. Therefore a saturation of conjugation mechanisms in the liver and kidney in all of these aforementioned situations still cannot be discarded as a major factor contributing to the increased excretion of free 17-KS. Certainly patients with chronic liver andjor kidney disease (and especially the former) demonstrate a lesser capacity to conjugate 17-KS in that there is some rise in the amount of free Zimmerman chromogen excreted, as well as a decrease in the ratio of conjugates to free fractions (6.6:1 in liver disease and 7.7:1 in kidney disease). A similar aberration has been demonstrated for 17-hydroxycorticosteroids.1 • 15 A recent report by Cohn and Hume3 demonstrated the renal conjugating capacity of 17-KS by the dog kidney. Since the free Zimmerman chromogen increased in chronic liver and renal disease, but A and E only rarely appeared in this free fraction, it would seem that this increase represents a decrease in conjugatin,g capacity rather than increased production. On the other hand, since patients with hirsutism demonstrated increases in their free fractions with contained A and E, it would seem that this increment represents an actively increased production of A and E precursors as well as a decrease in conjugating capacity. Finally, one cannot exclude an increased production of glucuronidase in these hirsute patients to account for the increased free 17-KS. Certainly this is not the case in normal controls where free Zimmerman chromogen is low initially, as borne out by our re-extraction studies (Table 1). It would thus appear that we now have more criteria available to classify the patient with hirsutism (Table 4): The patient with the Stein-Leventhal syndrome has bilateral polycystic ovaries, normal total 17-KS, slightly elevated free 17-KS, variable appearance of A and E in the free 17-KS fraction, a poor total17-KS response to ACTH stimulation, and a poor suppression response to Medrol therapy in small doses. Some excrete 11-ketopregnanetriol,4 but it is questioned whether these are truly patients with the Stein-Leventhal syndrome. Patients with the borderline adrenogenital syndrome may also have bilateral polycystic ovaries, but their total 17-KS are high-normal to slightly elevated, their free 17-KS fraction is diagnostically elevated, A and E appear frequently in the free 17-KS fraction, they respond well to ACTH stimulation and Medrol suppression, and they do not show 11-ketopregnanetriol in their urine though pregnanetriol is moderately elevated. Patients with the adrenogenital syndrome may have bilateral polycystic ovaries, but this is not
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TABLE 4.
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Criteria for Differential Diagnosis of Hirsutism SteinLeventhal syndrome
Total17-KS Free 17-KS Pregnanetriol 11-Ketopregnanetriol Total17-KS (Post-ACTH) Total17-KS (Post-Medrol) Free 17-KS (Post-ACTH) Free 17-KS (Post-Medrol) Bilat. polycystic ovaries
7.7 (Normal) 0.60 0-1 (Normal) ?Rise
Rise Fall +
Borderline A.G.S.
A.G.S.
Idiopathic hirsutism*
15.4 1.42 2-4 N Rise Fall Rise Fall +=50%
15+ 2+ 10+ Rise Rise Fall Rise Fall ±
10.1 0.28 0-1 N Var. Var. Var. Var. Var.
*Var. indicates variable.
a frequent finding. Their total17-KS are markedly elevated as are their free 17-KS and this free fraction shows A and E, at least in the adult. They show a hyperactive response to ACTH stimulation and good suppression with Medrol. In addition, they demonstrate increases in the excretion of both pregnanetrial and ll-ketopregnanetriol. 6 The unclassified hirsute group that remains in our series shows low to high total17-KS, normal to slightly elevated free 17-KS, the rare appearance of A and E in the free fraction, variable responses to ACTH stimulation and corticosteroid suppression, and finally, normal pregnanetriol and no 11-ketopregnanetriol in the urine. The fact that free 17-KS are increased in patients with hirsutism where the total17-KS excretion is not elevated or only slightly elevated and where the total17-KS fractionation may be normal12 is of potential importance both diagnostically and physiologically. It is important diagnostically in that it gives us another way of classifying hirsute patients previously termed idiopathic, particularly if the free fraction contains substances like androsterone. Furthermore, if there is an increase in free androgen (physiologically active) though these androgens are not particularly potent, such an increase possibly associated with enhanced hair follicle sensitivity, might account for previously unexplained hirsutism. Burstein and Dorfman2 have stated that in the hirsute patient there may be an increase in circulating testosterone that would be completely missed in the urine. Testosterone is a potent androgen and could certainly account for any or all the masculinization seen. In those cases where we find increases in free 17-KS and contained A and E, testosterone or androstenedione conceivably could be the precursor steroid. This is an exciting possibility. Most recently, Dorfman's group18 have demonstrated the ability of a Stein-Leventhal ovary to synthesize testosterone.
VoL. 14, No. 1, 1963
17 -KS IN HIRSUTISM
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SUMMARY
Seventy-eight patients have been studied with regard to the amount of free 17-ketosteroid they excrete in their urine. Normal females in both phases of the menstrual cycle and normal males excrete minimal amounts of free Zimmerman chromogen, and pregnant women excrete 0.92 mg. per day in the first trimester, this progressively decreasing to 0.45 mg. per day by the third trimester. Patients with the Stein-Leventhal syndrome excrete 0.60 mg. per day, those with the borderline adrenogenital syndrome excrete 1.42 mg. per day, and those with the adrenogenital syndrome, 1.5-6.9 mg. per day. Wedge resection in the Stein-Leventhal patient resulted in a fall in the total 17ketosteroid and free 17-ketosteroid excretion. Hirsute patients, including those with ovarian and adrenal disease, clearly excrete free 17-ketosteroids in an amount greater than normal. Furthermore, the ovaries may actively contribute to such excretion. The implications of this are discussed. In the hirsute group with normal to slightly elevated total17ketosteroids and normal17-ketosteroid fractionation such an increase in free 17-ketosteroids containing mild androgens might play a role in the establishment or perpetuation of the hirsutism when associated with increased hair follicle sensitivity.
Dept. of Research in Human Reproduction Michael Reese Hospital and Medical Center Ellis Ave. and 29 St. Chicago 16, Ill.
REFERENCES 1. BRoWN, H., WILLARDSON, D. G., SAMUELS, L. T., and TYLER, F. H. 17-hydroxycorticoid metabolism in liver disease. J. Clin. Invest. 83:1524, 1959. 2. BURSTEIN, s., and DoRFMAN, R. I. Biosynthesis of a16 androstene 3a ol. First International Congress of Endocrinology. Copenhagen, July, 1960. Abstract No. 346, p. 689. 3. CoHEN, G. L., and HUME, M. The in vivo glucuronide conjugation of radioactive etiocholanolone and androsterone by the dog kidney. J. Clin. Invest. 89:1584, 1960. 4. Cox, R. 1., and SHEARMAN, R. P. Abnormal excretion of pregnanetriolone and 5 pregnenetriol in the Stein-Leventhal syndrome. ]. Clin. Endocrinol. 21:586, 1961. 5. DECOURCY, C. A trichloracetic acid reagent for detection of pregnane-3a; 17a; 20 triols on paper chromatograms. J. Endocrinol. 14:164, 1956. 6. FINKELSTEIN, M., and GoLDBERG, S. A test for qualitative and quantitative estimation of pregnane 3a, 17a, 20a triol 11-one in urine and its sigriiflcance in adrenal disturbances. J. Clin. Endocrinol. 17:1063, 1957. 7. GoLD, J. J., and FRANK, R. The borderline adrenogenital syndrome. An intermediate entity. Am. J. Obst. & Gynec. 75:1034, 1958. 8. GoLD, J. J. Diagnosis of the adrenogenital syndrome. M. Clin. North America 48:1, 1959. 9. GoLD, J. J., and GoLDBERG, S. Gradient elution and paper chromatographic separation of C-21 deoxy steroids. I. Normal human studies. In preparation. 10. GoLD, J. J., and GoLDBERG, S. Gradient elution and paper chromatographic separa-
a
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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FERTILITY & STERILITY
tion of C-21 deoxy steroids. II. Studies on patients with endocrinopathy. In preparation. HoLTORFF, A. F., and KocH, F. C. The colorimetric estimation of 17-ketosteroids and their application to urine extracts. ]. Biol. Chern. 135:311, 1940. jAILER, J. W., and VANDE WEILE, R. Studies in hirsutism: urinary excretion of neutral 17-ketosteroids. Gynecologia 138:216, 1954. }AOUDE, F. A., BAULIEU, E. E., and JAYLE, N. F. Fractionnement par chromatographie fur papier des 17-cetosteroids neutres urinaire au cours de l'hyperplasie virilisante congenitale corticosurrenalienne. Acta Endocrinol. 26:30, 1957. KARL, H. J. Die 17-hydroxycorticosteroiden bei patienten mit nierenkrankungen. Klin. Wchnsch. 38:110, 1960, KLEIN, R., PAPADATOS, C., FoRTUNATO, J., BYRENS, C., and PuNTERINI, A. Serum corticoids in liver disease. ]. Clin. Endocrinol. 15:1524, 1954. KoRNEL, L. Renal clearance of endogenous 17-hydroxycorticosteroids in hypertension. ]. Clin. Endocrinol. 20:1445, 1960. LANTHIER, A., and SANDOR, T. The in vitro biosynthesis of androgenic steroids by human normal and "Stein-Leventhal type" ovarian slices. Acta Endocrinol. 39:145, 1962. LEON, M., NEVES E CAsTRo, M., and DoRFMAN, R. I. Biosynthesis of testosterone by a Stein-Leventhal ovary. Acta Endocrinol. 39:411, 1962. SAVARD, K. Paper partition chromatography of c19 and c21 ketosteroids. ]. Biol. Chern. 202:451, 1953. ScHNEIDER, J. J., and LEWBART, M. L. Fractionation and isolation of steroid conjugates. Rec. Prog. Horm. Res. 15:201, 1959. STEIN, I. F., and LEVENTHAL, M. L. Amenorrhea associated with bilateral polycystic ovaries. Am.]. Obst. & Gynec. 29:181, 1935. TELLER, W., and STEIB, W. Studies on the "differential" hydrolysis of steroid conjugates in urine. Acta Endocrinol. 32:209, 1959. TROEN, P. Perfusion studies of the human placenta. III. Production of free and conjugated Porter-Silber chromogens. ]. Clin. Endocrinol. 21:1511, 1961. WARREN, J. C., and SALHANICK, H. A. Steroid biosynthesis in the human ovary. ]. Clin. Endocrinol. 21:1218, 1961. Wonz, H. H., LEMON, H. M., MARcus, P., and SAvARD, K. The conjugated 17ketosteroids in human urine. ]. Clin. Endocrinol. 17:534, 1957.
IN
ATI'EMPTS to classify women with hirsutism into specific groups for facilitation and direction of proper therapy, numerous approaches have been utilized. It is a difficult problem to differentiate that form of hirsutism which has been clinically related to adrenal, ovarian, andjor thyroid disease from that which is familial in nature or iatrogenic in origin. Furthermore the idiopathic form, by its very name, defies classification. Members of this latter group often display completely normal urinary steroid levels, in terms of urinary 17ketosteroids ( 17-KS) and C-21 deoxysteroids. In the course of studies on normal and hirsute women, 9 • 10 the nature of our work necessitated extensive hydrolysis of urinary steroids. This required separation of urinary steriods into the free components, glucuronides and sulfates. Examination of the various fractions was then performed. It rapidly became apparent, in terms of the free 17-KS, that there were marked differences in the amount of free 17-KS excreted by normal and by hirsute women. Furthermore, there appeared to be differences in the individual17-KS contained in this free fraction. It was then logical to assume that, if certain androgenic 17-KS (even though of relatively low-potency androgenicity) in the hirsute group free fraction were not present in the normal group, these steroids might be at least partially responsible for the hirsutism. This would appear to be particularly important in that group of hirsute patients who exhibit normal to high-normal17-KS excretion with normal fractionation patterns. Few studies on free steroids have been reported specifically and the bulk of these have been related to 17-hydroxycorticosteroid excretion. 1 • 14-16 • 23 The
From Michael Reese Hospital, Chicago, Ill. This study has been supported in part by grants from the United States Public Health Service {Grant A-1670), and the Stacy H. VanValkenburgh Memorial Grant for Cancer Research from the American Cancer Society (P-185). *Present address: 19 Methodella Street, Jerusalem, Israel. We are grateful to Drs. Kenneth Thompson and Harold Upjohn for the Zn-cortrophin and Medrol used in this study and generously supplied by them, respectively. We are also grateful to Dr. Donald Holub and Dr. Fred Katz of Columbia University, College of Physicians and Surgeons for supplying us with fresh frozen urine from the patient with adrenal carcinoma.
73
74
GoLD
&
GoLDBERG
FERTILITY
&
STERILITY
fewer papers found describing studies on free and conjugated 17-KS report them only as part of another studyY· 20• 22 • 25 Two of these13 • 22 described very low to absent free 17-KS in adrenogenitals, which results are quite different from ours. In one of these studies the urine was~:¢6I1ected in the presence of a preservative. We did not take this specific precaution but collected the specimens at low temperatures. Studies to exclude the possibility of spontaneous hydrolysis prior to analysis were performed and are detailed below. METHODS AND MATERIALS
Fresh 24-hour urines were utilized for this study. The urines were extracted for free steroids as soon as feasible after the 24-hour urine collection was completed. This meant a maximal lapse of time of 4 hours between collection and analysis, unless the urines had been frozen immediately upon completion of collection. A 600-cc. aliquot of urine was extracted at neutral pH 3 times with 300 cc. of redistilled ethyl acetate. The residual urine was discarded and the ethyl acetate extracts were combined. This was then washed 3 times with IN NAOH and then twice with distilled water. The washings were discarded and the washed ethyl acetate was dried with sodium sulfate and evaporated under reduced pressure. The dried extract was dissolved in redistilled absolute methanol and 90% of the methanol extract was spotted on a wide strip ( 3 em. wide) of Whatman No.1 filter paper. The remaining 10% was spotted on a narrow strip ( 1 em. wide). The strips were run on the DeCourcy paper chromatographic system5 for 5-7 hours at room temperature. The strips were then dried at room temperature under a stream of air. The narrow strip was dipped into 70% phosphoric acid, heated at 85°C. for 10 min. and then observed for :fluorescent spots6 under a UV light source ( 3660° A). The Rf's, fluorescent color, etc. were recorded and the parallel areas on the wide strip were delineated, cut, and eluted with redistilled absolute methanol. A Zimmerman reaction was run on 10% of the methanol extract of each eluted spot. The remainder was then individually chromatographed on duplicate strips on the Savard system19 (ligroin: propylene glycol). Upon completion of the run, one strip was developed for fluorescence as noted above. Parallel areas of the duplicate strip were then eluted with methanol. Half of this eluate underwent Zimmerman quantitation and the remainder was examined for its sulfuric acid spectrum on a Cary recording spectrophotometer. The total17-KS were also measured11 on each of the 24-hr. urines studied. From this treatment it was possible to determine the total17-KS, free 17-KS, and the components of the free 17-KS fraction. In order to determine whether spontaneous hydrolysis, which would arti-
17 -KS
VoL. 14, No. 1, 1963
75
IN HIRSUTISM
ficially alter our results, had occurred during urine collection or extraction, the following studies were done. In three 24-hr. urine collections from normal women, each voiding was immediately collected and divided in half. One half was extracted for free 17-KS at collection and the other half was placed in a 24-hr. pool that was kept cold ( 4 °C.). The portion added to the 24-hr. pool was measured for free 17-KS at the end of the 24-hr. collection. The sum of the extractions of the separate voiding was then compared to the value obtained from extraction of the total24-hr. collection and no significant difference was noted. In addition three, fresh 24-hr. urines were extracted for free 17-KS and then placed in the refrigerator at 4°C. without preservative for an additional24-48 hr. They were then re-extracted for free 17-KS, and none was found. One can only conclude from these studies that spontaneous hydrolysis, under the conditions of our experiment, did not contribute to or alter in any way the results we obtained. These data are summarized in Table 1. Patients studied included females in the first and second halves of their: TABLE 1. Data from Studies to Determine Presence of Spontaneous. Hydrolysis During Urine Collection and Extraction Patient
B.J.
S.G.
F.B.
II I
Specimen
1st 8 hr. 2nd 8 hr. 3rd 8 hr.
55 0 0
1st 8 hr. 2nd 8 hr. 3rd 8 hr.
0 0 0
1st Voiding 2nd Voiding 3rd Voiding 4th Voiding 5th Voiding TOTAL
I: i I i!
ZimmeTman chTomogen (p.g.)* Delayed emtTaction:t: Immediate emtTactiont
0 33 40 34 0 107
} }
47
0
}
94
94
FRESH URINE
M.S.
FiTit emtmction
Second emtTaction
314 p.g./24 hr.
0 (24 hr. at 4°C.) 0 (48 hr. at 4°C.) 0 (48 hr. at 4°C.)
S.G.
R.B.
270 p.g./24 hr.
*Calculated on basis of dehydroepiandrosterone as a standard. t Half of each voiding. :!:Half of each voiding pooled 24 hr.
76
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& GoLDBERG
FERTILITY
& STERILITY
menstrual cycles and in the three trimesters of pregnancy; normal males; and females with hirsutism due to the Stein-Leventhal syndrome and to adrenal virilism, as well as patients with unclassified hirsutism. Finally, several patients with acute and chronic hepatic disease and chronic renal disease were also evaluated. In all, results of studies on 78 patients are included in this report. RESULTS Normals (Fig. 1 )
The normal female ( 6 patients) excreted a mean of 7.5 mg. of total17-KS per day in the first half of the menstrual cycle. Extraction for free 17-KS revealed only Zimmerman chromogen in the amount of 0.12 mg. per day, with .no identifiable 17-KS on fractionation. During the second half of the menstrual cycle the over-all pattern ( 7 patients) remained the same. Androsterone (A) andjor other mildly androgenic 17-KS were not found. Etiocholanolone (E) was found in one of the 7 patients. The normal male excreted more total 17-KS (13.0 mg. per day), as expected, and slightly more free Zimmerman chromogen ( 0.22 mg.) than the normal female. Again, fractionation revealed no identifiable 17-KS. On the other hand, the normal pregnant female excreted a mean of more than 3 times the free 17-KS (0.64 mg. per day-the mean of the three trimesters) excreted by the nonpregnant female. Furthermore, especially in the first trimester, A and E now began to be identified in this free fraction. The highest excretion of free steroid occurred at the end of the first trimester ( 0.92 mg. per day), with a progressive decrease to the third trimester ( 0.45 mg. per day). Hirsutism Patients (Fig. 2)
Stein-Leventhal syndrome.21 The clinical criteria for this group included menstrual disturbances, variable hirsutism, and bilateral polycystic ovaries. Most had normal urinary 17-KS and all had normal urinary pregnanetriol. Patients studied with the Stein-Leventhal syndrome excreted a mean of TOTAL 17-KS MC./DAY
0
NORMAL FEMALE ht HALF OF CYCLE !61 2nd HALF OF CYCLE !71
NORMALMALE-<10>
5
10
1.s + 1.sr 6.9 1.0+~
FREE 17-KS MG./DAY
15
0
1.0
05
1.5
2.0
ANDROST. ETIOCH
= .053'
NONE
NONE
po.J4 = .oza•
NONE
I OF 7
po.zz±.25'
NONE
NONE
po.12
NORMAL PREGNANCY 1st TRIMESTER !51 2nd TRIMESTER !7l 3rd TRIMESTER !91
ALL TRIMESTERS
0.92 ± .57•
10.3+3. 6.4-+- 1.43*
12.6 + 2.43*
9.8
0.56-+- .18*
~0.45±.16*
30F5
30F5
2 OF 7
lOF 7
1 OF9
30F9
0.64
!MEANS I *STANDARD ERROR OF MEAN
Fig. 1. Total and free 17-KS in normal males and females.
17 -KS
VoL. 14, No. 1, 1963
77
IN HIRSUTISM
0.60 mg. per day of free 17-KS preoperatively, with half excreting A and E in this fraction. Their mean total 17-KS (7.7 mg. per day) were normal. One patient, who had 0.47 mg. per day free 17-KS, had this increase to 1.03 mg. per day with ACTH. Medrolt then reduced this to 0.66 per day. AN DROST. TOTAL 17·KS MG./DAY 0 10 20 30 o40 50
60
70 80
90
FREE 11·KS MG./DAY 0 0.2 04 OG 08
STEIN-LEVENTHAL SYNDROME
10 12 14
AND ET10CH.
16 18 20
30F6
0.60:!: .17"'
PRE-OP 161
POST-OP Ill
ACTH 121
~O.SOIPRE-OP 1.341
NONE
==:::Jo.56
10F2
o.oo
10F2
BORDERLINE A.G.S.
1.42 + .39·
CONTROL 171
3.48
ACTH 121 I = = = : : J 4 1 . 0 ;::::::=::::::10.43
MEDROL 121
6.9
20.0 116 YR. OLDI
___.-
NONE
-- }10F2 1.5 NONE
0.0
ON THERAPY I11
HIRSUTISM UNCLASS.
po.za±.lo•
CONTROL16l
1 OF6 NONE
0.98
ACTH 121
po.M
MEDROL OR~ FF12l
CUSHING'S SYNDROMEs=====::!:~ CARCINOMA 11 l C
NONE
--:,;:
•STANDARD ERROR OF MEAN
Fig. 2. Total and free 17-KS in hirsutism patients. 0
The total17-KS changed little after ACTH and Medrol. Another patient, reexamined after ovarian wedge resection, showed a marked decrease in her total17-KS and free 17-KS. In addition, A and E disappeared from her free fraction. This would certainly suggest an ovarian contribution both to the total17-KS as well as the free 17-KS excretions. Borderline adrenogenital syndrome. 7 The clinical criteria for this group included menstrual disturbances, variable hirsutism, and polycystic ovaries in 50% of the patients. All had slight to moderate elevations of urinary 17-KS and pregnanetriol. Patients studied with the borderline adrenogenital syndrome excreted moderately elevated total17-KS ( 15.4 mg. per day). These levels rose briskly after ACTH stimulation ( 41.0 mg. per day), and then were adequately suppressed by Medrol ( 8.1 mg. per day). This is in contrast to the findings in the Stein-Leventhal syndrome. Prior to ACTH stimulation, the free fraction of these patients with adrenogenital syndrome amounted to 1.42 mg. per day, and most of these fractions contained A andjor E. ACTH then increased *Author's correction: In the final column, the fifth line should read "5 of 7"; the lOth line, ''2 of 6." tThe Upjohn Co., Kalamazoo, Mich.
78
GOLD
RANCE NORMAL FEMALE 1st HALF OF CYCLE 161
5.4-11.0
2nd HALF OF CYCLE 171
2.7-12.1
&
TOTAL 17-KS MG./DAY
0
5
10
1st TRIMESTER 151
6.5-16.0
2nd TRIMESTER 171
2.4-10.6
3rd TRIMESTER f91
5.9-24.2
STEIN-LEVENTHAL SYNDROME--161 5.0-13.5
20
IS
121
9.4-20.9
HIRSUTISM UNCLASSI61
6.3-19.0
RANCE
FERTILITY FREE 17-KS MC./DAY 0 1.0 2.0
3.0
& STERILITY 4.0
5.0
().0.26 trJro.lZl
16.911
0.09-0.20 0-0.70
113.011 tTO.~!I
~10.141 P=:Jro.Z2J
0-3.0
~~-'''
0.10-1.25 112.61
I
177JI
BORDERLINE A.G.S.-181 10.8-24.8 A.G.S.
25
c:::I:JTII]
NORMAL MALE-1101 6.5-21.5 NORMAL PREGNANCY
GOLDBERG
Pii' I
roJi
no.l1l
10.921
o:::::Jro.S61
0-1.30
~
0-1.34
•!0.6 )
0.46-3.06
!1.4211
l.S-6.9 0--0.47
IO J -
~10.281
Fig. 3. Consolidation of total and free 17-KS data for normal subjects and hirsutism patients.
the free fraction and Medrol suppression induced a marked fall. In the one instance where it was applicable, A and E also were no longer found after Medrol suppression. In these studies, the patients were given 20 U. of Zncortrophin"' intramuscularly daily for 5-7 days and Medrol, 8 mg. per day orally for 4 days. Adrenogenital syndrome. 8 These patients were premenarchal or had delayed puberty and characteristically demonstrated marked elevations in urinary 17-KS, pregnanetriol, and pregnanetriolone. Patients studied with the adrenogenital syndrome, showed markedly elevated excretion of free 17-KS ( 6.9 and 1.5 mg. per day), with A and E present in the older ( 16 years) patient. Steroid therapy in the younger ( 9 years old) reduced the free 17-KS from 1.5 mg. per day to zero. Unclassified hirsutism. These patients defied classification both clinically and chemically. Six patients are included whose hirsutism was unclassified etiologically. Their total17-KS ranged from normal to elevated, and the free 17-KS were essentially in the normal range. One patient had A in her free fraction; another, E. Two patients tested showed a response of the free fraction to ACTH. A male patient with Cushing's syndrome due to adrenal carcinoma demonstrated a marked elevation of the total17-KS and the free 17-KS. A female patient with Cushing's syndrome due to adrenal adenoma had comparable changes but to a lesser degree. Figure 3 shows a consolidation of data for the normal patients and those with hirsutism. Table 2 shows the results of statistical comparison between normal females and hirsute patients with the Stein-Leventhal syndrome and *Organon, Inc., Orange, N.J.
17 -KS
VoL.14, No.1,1963 TABLE 2.
79
IN HIRSUTISM
Statistical Comparison of 17-KS Values of Normals and Hirsutism Patients p
T
Normal females Stein-Leventhal syndrome
Total 17-KS Free 17-KS
.134 2.454
N.S. >.02
Normal females Borderline adrenogenital syndrome
Total 17-KS Free 17-KS
4.04 2.75
=.01 >.01
the borderline adrenogenital syndrome with regard to their total and free 17-KS excretions. The free 17-KS excretion in these hirsute patients was significantly different from that of the normal female. Renal and Hepatic Dysfunction (Table 3)
In order to evaluate effects of renal andjor hepatic dysfunction on the excretion of free 17-KS, several patients with these disorders were studied. One patient with acute hepatitis showed no real increase over normal free Zimmerman chromogen, but the patients with chronic cirrhosis excreted free Zim-:merman chromogen in mean amounts more than 3 times that found in normal individuals ( 0.83 mg. per day). However, A and E were not found in the free fractions. Two patients with chronic renal disease excreted amounts that were about twice normal ( 0.47 mg. per day) and A and E were present in one of these. DISCUSSION
Some interesting observations may be made from the data presented above on free 17-KS excretion. The normal individual, whether male or female, TABLE 3.
17-Ketosteroids in Patients with Renal and Hepatic Disorders
No.
Diagnoais
1 1 2 3 4
Acute hepatitis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) Chronic cirrhosis (male) MEAN
1 2
± S.E.
OF MEAN
Chroi:llc renal dis. (male) Chronic renal dis. {male)
TotaZ17 KB (mg.jday)
Free Zimmerman chromo g. (mg.jday)
5.3 5.8 9.1 5.6 4.4
0.26 1.02 1.30 0.58 0.40
6.3 ± 2.0.
0.83 ± .31
5.0 3.2
0.50 0.44
IndivitluaZ 17-KB component• Etioch. (E) A.ndroat. (A.)
+ 0 0 0 0
0 0 0 0 0
0
0
+
+
80
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& GOLDBERG
FERTILITY
&
STERILITY
probably excretes little to no free 17-KS, as no 17-KS were identifiable on fractionation of the small quantity of Zimmerman chromogen found. In pregnancy however, larger amounts of Zimmerman chromogen were found, especially in the first trimester. It is less likely that this chromogenic material was nonspecific, as certain 17-KS (androsterone and etiocholanolone) were now identifiable on fractionation. In the first trimester, the total 17-KS and free 17-KS are higher than in the second trimester. In the third trimester, the total 17-KS are higher than in either of the first two and the free 17-KS are lower. This would suggest that the young placenta may play a role in conjugation mechanisms at least insofar as 17-KS are concerned. Recent placental perfusion studies23 would seem to bear this out for Porter-Silber chromogens. Patients with the Stein-Leventhal syndrome excrete more free 17-KS than normals, but less than patients with varying degrees of adrenal hyperplasia. Ovarian wedge resection in one patient apparently induced a fall in both the total and free 17-KS excretion (urine examined one week postoperatively). This would suggest that the ovary plays a role in 17-KS formation by altering adrenal activity indirectly, by actively contributing 17-KS by secreting them, or both. ACTH administered to 2 patients with the Stein-Leventhal syndrome induced minimal increases in total17-KS whereas normal individuals and patients with adrenal hyperplasia demonstrate a greater 17-KS response to ACTH stimulation. Furthermore the same 2 patients showed a minimal total 17-KS depression after Medrol administration. The latter is more difficult to interpret, however, as the control levels were relatively low initially. It would appear that the polycystic ovary characteristic of the Stein-Leventhal syndrome may actively contribute to the total17-KS and free 17-KS excretion in these patients and, furthermore (since their total17-KS are normal andrespond poorly to ACTH stimulation), this ovary may be interfering directly with normal adrenal responsiveness. The mechanism by which this might occur is obscure. Thus, in keeping with more recent studies,17 • 24 the SteinLeventhal syndrome may be basically an ovarian disease rather than a sequel of adrenal dysfunction. Patients with obvious adrenal hyperactivity, as in the borderline adrenogenital syndrome, adrenogenital syndrome, and Cushing's syndrome excrete large amounts of free 17-KS. ACTH administered to individuals with the borderline adrenogenital syndrome induced almost threefold increases in total and free 17-KS excretion. Such ACTH stimulation apparently increased the conjugate and free fractions in almost equivalent amounts as the ratio of conjugate to free changed little ( 9.8:1 to 10.8:1). Medrol suppression in the borderline adrenogenital group induced a fall in both the total and free 17KS. However, here the fall was mainly in the free steroids as the conjugate-
VoL. 14, No. 1, 1963
17 -KS
IN HIRSUTISM
81
to-free ratio jumped to 17.8:1. In a patient with the adrenogenital syndrome, Medrol also induced a striking fall in free steroids. Since the adrenal effect of Medrol is mediated via ACTH suppression, this would imply that ACTH affects free 17-KS excretion. However, the patient with Cushing's syndrome due to adrenal carcinoma, which is theoretically autonomous of ACTH, also excreted large amounts of free 17-KS. Therefore a saturation of conjugation mechanisms in the liver and kidney in all of these aforementioned situations still cannot be discarded as a major factor contributing to the increased excretion of free 17-KS. Certainly patients with chronic liver andjor kidney disease (and especially the former) demonstrate a lesser capacity to conjugate 17-KS in that there is some rise in the amount of free Zimmerman chromogen excreted, as well as a decrease in the ratio of conjugates to free fractions (6.6:1 in liver disease and 7.7:1 in kidney disease). A similar aberration has been demonstrated for 17-hydroxycorticosteroids.1 • 15 A recent report by Cohn and Hume3 demonstrated the renal conjugating capacity of 17-KS by the dog kidney. Since the free Zimmerman chromogen increased in chronic liver and renal disease, but A and E only rarely appeared in this free fraction, it would seem that this increase represents a decrease in conjugatin,g capacity rather than increased production. On the other hand, since patients with hirsutism demonstrated increases in their free fractions with contained A and E, it would seem that this increment represents an actively increased production of A and E precursors as well as a decrease in conjugating capacity. Finally, one cannot exclude an increased production of glucuronidase in these hirsute patients to account for the increased free 17-KS. Certainly this is not the case in normal controls where free Zimmerman chromogen is low initially, as borne out by our re-extraction studies (Table 1). It would thus appear that we now have more criteria available to classify the patient with hirsutism (Table 4): The patient with the Stein-Leventhal syndrome has bilateral polycystic ovaries, normal total 17-KS, slightly elevated free 17-KS, variable appearance of A and E in the free 17-KS fraction, a poor total17-KS response to ACTH stimulation, and a poor suppression response to Medrol therapy in small doses. Some excrete 11-ketopregnanetriol,4 but it is questioned whether these are truly patients with the Stein-Leventhal syndrome. Patients with the borderline adrenogenital syndrome may also have bilateral polycystic ovaries, but their total 17-KS are high-normal to slightly elevated, their free 17-KS fraction is diagnostically elevated, A and E appear frequently in the free 17-KS fraction, they respond well to ACTH stimulation and Medrol suppression, and they do not show 11-ketopregnanetriol in their urine though pregnanetriol is moderately elevated. Patients with the adrenogenital syndrome may have bilateral polycystic ovaries, but this is not
82
GoLD
TABLE 4.
&
GoLDBERG
FERTILITY & STERILITY
Criteria for Differential Diagnosis of Hirsutism SteinLeventhal syndrome
Total17-KS Free 17-KS Pregnanetriol 11-Ketopregnanetriol Total17-KS (Post-ACTH) Total17-KS (Post-Medrol) Free 17-KS (Post-ACTH) Free 17-KS (Post-Medrol) Bilat. polycystic ovaries
7.7 (Normal) 0.60 0-1 (Normal) ?Rise
Rise Fall +
Borderline A.G.S.
A.G.S.
Idiopathic hirsutism*
15.4 1.42 2-4 N Rise Fall Rise Fall +=50%
15+ 2+ 10+ Rise Rise Fall Rise Fall ±
10.1 0.28 0-1 N Var. Var. Var. Var. Var.
*Var. indicates variable.
a frequent finding. Their total17-KS are markedly elevated as are their free 17-KS and this free fraction shows A and E, at least in the adult. They show a hyperactive response to ACTH stimulation and good suppression with Medrol. In addition, they demonstrate increases in the excretion of both pregnanetrial and ll-ketopregnanetriol. 6 The unclassified hirsute group that remains in our series shows low to high total17-KS, normal to slightly elevated free 17-KS, the rare appearance of A and E in the free fraction, variable responses to ACTH stimulation and corticosteroid suppression, and finally, normal pregnanetriol and no 11-ketopregnanetriol in the urine. The fact that free 17-KS are increased in patients with hirsutism where the total17-KS excretion is not elevated or only slightly elevated and where the total17-KS fractionation may be normal12 is of potential importance both diagnostically and physiologically. It is important diagnostically in that it gives us another way of classifying hirsute patients previously termed idiopathic, particularly if the free fraction contains substances like androsterone. Furthermore, if there is an increase in free androgen (physiologically active) though these androgens are not particularly potent, such an increase possibly associated with enhanced hair follicle sensitivity, might account for previously unexplained hirsutism. Burstein and Dorfman2 have stated that in the hirsute patient there may be an increase in circulating testosterone that would be completely missed in the urine. Testosterone is a potent androgen and could certainly account for any or all the masculinization seen. In those cases where we find increases in free 17-KS and contained A and E, testosterone or androstenedione conceivably could be the precursor steroid. This is an exciting possibility. Most recently, Dorfman's group18 have demonstrated the ability of a Stein-Leventhal ovary to synthesize testosterone.
VoL. 14, No. 1, 1963
17 -KS IN HIRSUTISM
83
SUMMARY
Seventy-eight patients have been studied with regard to the amount of free 17-ketosteroid they excrete in their urine. Normal females in both phases of the menstrual cycle and normal males excrete minimal amounts of free Zimmerman chromogen, and pregnant women excrete 0.92 mg. per day in the first trimester, this progressively decreasing to 0.45 mg. per day by the third trimester. Patients with the Stein-Leventhal syndrome excrete 0.60 mg. per day, those with the borderline adrenogenital syndrome excrete 1.42 mg. per day, and those with the adrenogenital syndrome, 1.5-6.9 mg. per day. Wedge resection in the Stein-Leventhal patient resulted in a fall in the total 17ketosteroid and free 17-ketosteroid excretion. Hirsute patients, including those with ovarian and adrenal disease, clearly excrete free 17-ketosteroids in an amount greater than normal. Furthermore, the ovaries may actively contribute to such excretion. The implications of this are discussed. In the hirsute group with normal to slightly elevated total17ketosteroids and normal17-ketosteroid fractionation such an increase in free 17-ketosteroids containing mild androgens might play a role in the establishment or perpetuation of the hirsutism when associated with increased hair follicle sensitivity.
Dept. of Research in Human Reproduction Michael Reese Hospital and Medical Center Ellis Ave. and 29 St. Chicago 16, Ill.
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