Ethinyl estradiol administration and plasma steroid concentrations in ovariectomized women DAMODAR
K.
REINHART MAJIDA A.
B.
BILLIAR
JASSANI
BRIAN
Cleveland,
MAHAJAN
LITTLE Ohio
The effect of ethinyl estradiol treatment on the plasma levels of cortisot, corticosterone, deoxycorticosterone, progesterone, testosterone, dehydroepiandrosterons sulfate, A”-androstenedione, and estrone was studied in eight women. All the subjects had undergone ovariictomy and hysterectomy at least one year prior to this study. The systemic concentration of cortisol and the binding of cortisol were significantly increased, paralleling the increased transcortin concentration due to ethinyl estradiol treatment. Corticosterone concentration was also significantly increased after three days of estrogen administration and this level continued to be higher than normal as long as patients were treated with estrogens, but there was no change in the plasma concentration of deoxycorticosterone. The plasma levels of progesterone, testosterone, dehydroepiandrosterone sulfate, A’-androstenedione, and estrone or the ratio of estrone to A4-androstenedione did not change with ethinyl estradiol treatment. These observations suggest that the administered estrogen increased the transcortin concentration and had only a limited effect on adrenocortiial steroidogenesis. (AM. J. O&WET. GYNECCIL.130: 398, 1978.)
IN THE STUDY of the influence of estrogens on the secretion rate and pattern of steroids from the adrenal cortex in women, of particular interest has been the role of estrogens during the adrenarche’r 2 and during the postmenopausal years.3-5 The effect of exogenous estrogen on plasma transcortin concentrations and the metabolic clearance rates of cortisol and progesterone in ovariectomized women have been reported.6 This latter study was extended to examine the effect of ethinyl estradiol on the plasma binding of cortisol and testosterone; the production rates of cortisol and progesterone; and the plasma concentrations of cortisol, From the Department of Reproductive Westem Reserve University. Supported HD-02378.
by United
States Public
Sponsored
by the Society for
Biolqy, Health
Gyrzecologic
Case
Service Grant Investigaation.
Re@-int requests: Dr. Damodar K. Mahaian, Defnzrtment of kep-rod&tive Biology, MacDonuEd Ho-we, Urkversity Hospitals, Case Western Reserve Universi@ Cleveland, Ohio 44106.
398
progesterone, corticosterone, deoxycorticosterone, tosterone, androstenedione, dehydroepiandrosterone (free plus sulfate), and estrone. Material
tes-
and methods
Radioactive steroids. The purity of the following radioactive steroids* was tested by paper chromatography before use: 1,2-3H-cortisol, 40.0 Ci per millimole; 1,2-3H-corticosterone, 45 Ci per millimole; 1, 2-3H-deoxycorticosterone, 47 Ci per milhmole; 1,2,6, 7-3H-progesterone, 105 Ci per millimole; 1,2-3Htestosterone, 59 Ci per millimole: 1,2-3HA4androstenedione, 60 Ci per millimole; 7-3H-dehydroepiandrosterone, 15 Ci per millimole; and 2,4,6,7-3Hestrone, 98.5 Ci per millimole. The steroids with 1 per cent or more impurities were purified by thin-layer chromatography. Radioinert steroids? for radioimmunoassays were crystallized from organic solvent. *New England Nuclear, Boston, Massachusetts. tSteraloids, Wilton, New Hampshire.
000%9378/78/04130-03981)00.50/O
0 1978 The C. V. Mosby
Co.
Volume
130
Number
t
Table
399
Effect of ethinyl estradiol on plasma steroids
I. Plasma
of ethinyl
steroid
parameters
in ovariectomized
women
before
and
during
daily
administratiotl
estradiol Oral administration of ethinyl estmdiol to ovariectomired women (naean ? standard ermri Measurement
Control
I
Cortisol binding (%) Cortisol concentration (ng./ml.) Cortisol production rate5 (mg./day) Progesterone concentration (ng./ 100 ml.) Progesterone production rate9 (mg./day) Corticosterone concentration (ng./ml.) Deoxycorticosterone concentration (ng./ 100 Testosterone binding (%I Testosterone concentration (ng./lOO ml.) Dehydroepiandrosterone concentration11 (rgi 100 ml.) Androstenedione concentration (ngJ100 ml.) Estrone concentration (ngJlOOm1.) Estroneiandrostenedione
ml.)
3 days
I
1
3 weeks
I
74.5 33.5 9.0 29.1 0.85 1.35 15.1 76.0 38.6 224.0
2 1.5 2 7.4 ” 1.2 ” 3.8 -+ 0.11 -c 0.5 2 1.2 + 1.2 f 6.9 f 28.0
84.7 81.5 13.7 31.0 1.05 2.08 12.9 78.9 27.8 272.0
t 1.2* 2 14.5* * 2.9* 2 7.4 -c 0.38 2 0.7 * 1.7 + 1.1 + 4.8 + 39.0
84.6 168.0 22.8 29.6 0.74 2.43 13.5 80.0 43.0 191.0
2 0.9* t 12*t k 3.0* t 3.4 t 0.16 + 0.2 t 2.5 + 0.7 f 12.2 + 23.0
307.0 11.3 0.048
2 72.0 * 1.7 f 0.009
286.0 14.5 0.071
-c 66.0 2 2.0 2 0.014
282.0 10.7 0.043
it 39.0 + 0.8 t 0.006
Off 3 wee4
74.0 51.7 13.4 26.8 0.95 1.38 9.4 75.4 18.8 “13.0
t 2.3t.J 2 6.5$ c 2.4* t 2.9 t 0.28 -?I 0.5 + 1.7 t 2.2 2 1.3 t 25.0 -
Blood samples were drawn from ovariectomized women before estrogen (control), on the third day of the daily oral administration of 200 mcg. of ethinyl estradiol (three days), three weeks after the daily administration of ethinyl estradiol (three weeks), and then three weeks after the withdrawal of the estrogen (off three weeks). *Values are significantly different from control values. tValues are significantly different from values of three days. SValues are significantly different from values of three weeks. §Production rate = plasma concentration X metabolic clearance rate. The metabolic clearance rate values for these studies have been published.6 /IThe dehydroepiandrosterone concentration is the total of both free and sulfated dehydroepiandrosterone. Their purity was checked by melting point determinations, which were in good agreement with the standard melting points reported in The Merck Index. Antibodies. Specific antibodies against cortisol, corticosterone, progesterone, and testosterone were prepared in rabbits as described earlier.’ The antigens injected
in rabbits
to give
antisera
against
deoxycortico-
sterone and A4-androstenedione were deoxycorticosterone-3-oxime-bovine serum. albumin and A4androstenedione-6-succinyl-bovine serum albumin, respectively. Antisera against dehydroepiandrosterone sulfate
were
obtained
from
Dr.
G.
E. Abraham*
and
antiestrone serum was supplied by Dr. C. Longcope.? The radioimmunoassays of corticosterone, deoxycorticosterone, progesterone, and testosterone were carried out as described previously.’ Dehydroepiandrosterone sulfate was measured by the method of
Buster
aration from
ing
and
Abraham8
and
radioimmunoassays
of
and estrone were carried out by charcoal sepmethod.Q The estrone fraction was separated
cortisol
estradiol-17P
the
Bush
on
B,
paper
system
chromatography
(petroleum
*UCLA School of Medicine, Torrance, t Worcester Foundation of Experimental ter, Massachusetts.
us-
ether:ben-
California. Biology,
zene: methanol: water ratio; 33: 17: 40: IO) to avoid possible cross-reaction with estradiol- 17p. Bound and f&e plasma steroids. The bound and free cortisol and testosterone were determined by equilibrium dialysis at 37” C. for 18 hours in prewashed Visking tubing (*/32 inch Arthur Thomas) according to the method of Sandberg and associates.Ln The inner phase consisted of 1 ml. of an 1: 10 dilution of plasma. The diluent was phosphate-buffered saline (PBS), pH 7.4 (O.OlM phosphate buffer in 0.14N NaCI). The outer phase consisted of 5 ml. of PBS, pH 7.4, which contained about 83,000 disintegrations per minute of either cortisol or testosterone. Subjects. The women who had ovariectomy and hysterectomy at least one year prior to this srudy ranged in age between 26 and 50 years. The metabolic clearance rate (MCR) studies of progesterone and cortisol in these subjects have been reported.” Estrogen therapy was withheld for at least three weeks and the absence of estrogen
effect
Control
blood
woman
receiving
mouth daily were repeated Worces-
tion apy.
was
and
after
Estrogen
for
confirmed
samples 200 three on the three therapy
by
were mcg.
vaginal
drawn of
ethinyl
cytology.
prior
to each
estradiol
by
weeks. The same determinations third day of estrogen administraweeks
of continuous was
then
stopped
estrogen and
therthe
de-
400
Mahajan
et al.
February 15, 1978 Am. J. Obstet. Gynecol.
Table Ii. Plasma steroid parameters in ovariectomized ethinyl estradiol administration to control values
women:
ratio of values during and after daily
Oral administration of ethyl es&did to ovariectomired women (mean 2 standard Measurement
Cortisol binding Cortisol concentration Corm01 production rate Progesterone concentration Progesterone production rate Corticosterone concentration Deoxycorticosterone concentration Testosterone binding Testosterone concentration Debydroepiandrosterone concentration Androstenedione concentration Estrone concentration Estrone/androstenedione
3 days
1.14 2 0.01* 2.96
k 0.87*
1.63 + 0.46* 0.91 & 0.08 0.88 f 0.11 1.67 f 0.31 0.86
f 0.08
1.20 +- 0.06$ k 0.08
Off 3 weeks
1.14 + 0.03* f 1.34*t
0.99 t 0.03tg 1.89 + 0.45$
i 0.59*
1.09 2 0.16 0.96 2 0.19 2.67 zt 0.67* 0.94 I!z 0.20
1.04 2 0.01 0.94
3 weeks
6.36 2.82
1.06 t 0.02 1.10 r 0.35
0.74 + 0.09
0.88 f 0.08 1.04 r 0.36
1.34 * 0.13 1.61 c 0.26
error)
1.09 -+ 0.16 1.11 -c 0.24
1.56 r 0.31* 0.85 r 0.08
0.99 zi 0.19 1.15 0.62 0.99 0.56 0.95
2 -r2 2 +-
-
0.204 0.08 0.03 0.11 0.05
*Values are significantly different from values of 1.0. tValues are significantly different from ratios of three days on ethinyl estradiol. $Values are significantly different from values of three weeks. terminations were repeated three weeks later. Vaginal cytology confirmed the effect of estrogen administration and withdrawal. All blood samples were drawn between 8:00 A.M. and 2:OO P.M. RWiUltS
Estrogen treatment resulted in a significantly increased percentage of cortisol bound to macromolecules in the plasma as measured in vitro (Tables I and II). The transcortin concentration in these subjects was increased by the estrogen treatment and the increased transcortin concentration resulted in a decreased MCR of cortisol.6 The increased cortisol binding, the decreased MCR of cortisol, and the increased plasma cortisol concentrations resulted from treatment with ethinyl estradiol (Tables I and II). Three weeks after withdrawal of estrogen the plasma cortisol was not significantly elevated in comparison with the control levels (p > 0.05). The cortisol production rate, however, was slightly higher after estrogen treatment was stopped. None of the other plasma steroid parameters was altered by the estrogen treatment when examined statistically as mean values (Table I). However, when each individual is considered as her own control and the values are expressed as ratios to control after three days, three weeks, and cessation of treatment (Table II), then the plasma corticosterone ratio after three weeks of administering ethinyl estradiol is higher than the ratios, either at three days or after stopping treatment. The dehydroepiandrosterone concentration, which is almost entirely dehydroepiandrosterone sul-
fate,‘, ’ is greater after three days of treatment than after three weeks, but there were no significant differences in the dehydroepiandrosterone concentrations between any of the other comparisons (Table II).
Comment The major effect of estrogen treatment of ovariectomized women on the plasma steroid parameters measured in this study is to increase the plasma concentrations of glucocorticoids (Tables I and II). This is associated with a lowered metabolic clearance rate of cortisol and an increased plasma concentration of transcortin6 The present study confirms the increased binding capacity of the plasma for cortisol following administration of ethinyl estradiol (Table I). The estrogen-induced increase in plasma concentration of corticosterone, which binds strongly to transcortin,‘” and the unchanged concentration of 1 l-deoxycordcosterone, which binds weakly to transcortin,rO would be expected with the change in circulating transcortin. The testosterone binding was increased from 76.0 ? 1.2 per cent to 78.9 rt 1.1 per cent after estrogen treatment for three days, and it further increased to 80.0 & 0.7 after three weeks of estrogen treatment and then returned to control level at three weeks after withdrawing the estrogen therapy. Although this increase of testosterone binding to plasma proteins at three weeks of estrogen treatment was ot statistically significant, it showed the trend of highe% -binding of testosterone than the control samples. It suggests that sex hormone-binding globulin might be increased slightly due to estrogen treatment. The testosterone
Volume Number
130 4
concentration in plasma was more variable and did not increase significantly on estrogen administration (Tables I and II). Although progesterone can also bind strongly to transcortin, estrogen administration to these women did not alter the plasma concentration or blood production rate of progesterone (Tables I and II) but estrogen administration also did not affect the blood metabolic clearance rate of progesterone.6 In a recent report by Chavarri and associates” the administration of adrenocorticotropic hormone (ACTH) was shown to increase cortisol secretion and thereby to alter aldosterone distribution between plasma protein and red cells. Since estrogen administration did not alter either the plasma concentration or the plasma MCR of progesterone, the higher plasma cortisol concentrations during ethinyl estradiol treatment do not appear to alter the plasma-red cell distribution of progesterone. This is in agreement with previous observations that progesterone is not associated with the red blood cell in women either during the menstrual cycle12 or during the third trimester of pregnancy’” when relatively high plasma cortisol and progesterone concentrations are present. It is possible, however, that the ethinyl estradiol treatment might alter the episodic adrenal secretion pattern by increasing the plasma transcortin concentrations which could theoretically alter the plasma half-time disappearance of the cortisol.‘a The production rate of cortisol was also increased by estrogen administration. This might suggest that estrogen has a direct or an indirect effect on the adrenal gland, but calculation of the production rate from plasma samples obtained at one time period must be interpreted with caution because not only is there a circadian rhythm to plasma cortisol concentrations but cortisol is secreted episodically by the adrenal gland.*5 The lack of effect of estrogen on the blood production rate of progesterone and the plasma levels of most of the other steroids suggests that estrogen is not stimulating the adrenal gland. Anderson and Yen* have also recently reported that administration of ethinyl estradiol to ovariectomized women for four to six weeks increased plasma cortisol concentrations but did not alter the plasma concentration of nine other steroids, whereas exogenous ACTH did cause alterations.
REFERENCES
1. Abraham, G. E., and Maroulis, G. B.: Effect of exogenous estrogen on serum pregnenolone, co&&, and androgens in postmenopausal women, Obstet. Gynecol. 45: 271. 1975.
Effect
of ethinyl
estradiol
on plasma
steroids
401
The report of Anderson and Yen” and the present studies are in contrast to the results of‘ ,%hraham and Maroulis’ who reported that 0. I mg. dail\ of dirthylstilbestrol or 1.25 mg. of estrogen c,onjugates caused an increase in the plasma concentration of dehydroepiandrosterone and dehydroepiandroslerone sulfate in postmenopausal women. Although three days of ethinyl estradiol administration caused d slight increase in free plus sulfated dehydroepiandrostcrone, the values were not significant in this small number of women. The difference in these results may be drle to the facts that the women were younger, the! i%‘ere ovariectomized rather than postmenopausal, and the duration, dosage, and type of estrogen treatment were ditferent. It should be noted, however. that estrogen treatment did increase the plasma concentration of cortisol in the present study and in the study by Anderson and Yen2 but it did not change the plasma cortisol concentration in the study by Abraham and Maroulis.’ The effect of exogenous estrogen on I he plasma estrone concentration was examined since exogenous estrogen is frequently administered to postmenopausal and ovariectomized women. It is now known that adrenal androstenedione secretion along with its peripheral aromatization is the major source 01’ estrone in postmenopausal and ovariecromizetl women.“’ Iti Estrogen administration may result in hepatomas,” and other workersIx. I9 have recently demonstrated that impaired hepatic function, i.e., cirrhosis, w111 result in an increase in the circulating estrogen due to an increased peripheral conversion of androgen LO estrogen. In the present study ethinyl estradiol administr;ltion for three weeks did not significantly alter the plasma concentration of androstenedione and estrone 01 rhe ratio of plasma androstenedione to estrone. It remains to be tested whether or not a more prolongetl exposure to estrogen or other estrogenic compounds will affect these parameters. We acknowledge the excellent technical assistance of Miss Barbara Brown and Mrs. Marion Wazney in carrying out steroid radioimmunoassay. Thanks to Dr. R. J. Santen for laboratory facilities to determine plasma dehydroepiandrosterone sulfate at the M. S. Hershey Medical Center.
2. Anderson, D. C., and Yen, S. S. C.: Ef’fea of estrogens on adrenal SD-hydroxy-steroid dehydrogenase in ovariectomized women. J. Clin. Endocrinol. Metab. 43: 561, 1976. 3. Vermeulen, A.: The hormonal activity of the post-
402
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9.
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11.
Mahajan
et al.
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February 15, 1978 Am. J. Obstet. Gynecol.
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