0021-972X/78/4701 -0193$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 1 Printed in U.S.A.
Increased Aldosterone Plasma Protein Binding in Women on Combined Oral Contraceptives throughout the Menstrual Cycle* W. NOWACZYNSKI, T. MURAKAMI, K. RICHARDSON, AND J. GENEST The Steroid Research Department, The Clinical Research Institute, Montreal, Quebec, Canada ABSTRACT. Plasma aldosterone concentration and the percentage of the fraction bound to a specific plasma aldosterone-binding globulin (ABG) were measured throughout the menstrual cycle in 26 healthy women aged between 19 and 38 yr who were receiving estrogencontaining oral contraceptives (OC). Midafternoon upright levels of total plasma aldosterone were similar in control and OC subjects and showed normal cyclic fluctuation in both groups. The percentage of ABG-bound aldosterone was markedly higher in OC subjects than in
I
T IS known that the administration of estrogens produces significant elevations in plasma transcortin (CBG), sex steroid, and T4binding globulins, presumably by increasing the rate of their synthesis by the liver (1-5). Estrogens also produce an elevation in the level of the aldosterone-binding protein (ABG; 6-8). This ABG, classified as an ai-glycoprotein (8-11), has been differentiated from CBG or albumin and identified (9-12) with what has been previously called a transcortin-like plasma fraction (TLPF) (8). ABG binds aldosterone reversibly and with high affinity and recent work suggests that it may be physiologically important and play a role in essential hypertension (8, 9, 11-14), but not secondary hypertension (11). Oral contraceptives (OC) have also been shown to produce significant changes in the metabolism of aldosterone. These changes are described in a recent comprehensive review by Crane (5).
controls at all stages of the cycle and showed a positive correlation with the mean blood pressure when all OC subjects studied were considered. In addition, when the plasma ABG levels were measured 6 months or more after the start of medication, they appeared to be related to the dose of estrogen in the OC, but not to the length of time of its administration. The identity of plasma ABG in OC subjects with that in control subjects was also established. (J Clin Endocrinol Metab 46: 193, 1978)
We present here a study of aldosterone binding to plasma ABG in women at various times during the menstrual cycle, some of whom were receiving estrogen- and gestagencontaining OC. Materials and Methods
The study group consisted of 26 healthy, asymptomatic women aged between 19 and 38 yr (mean, 26.7 ± 0.9 SEM) on unrestricted diet who had been taking synthetic estrogen-containing OC (Table 1) on 21 days out of every 28 for at least 6 months before the study (3.5 yr ± 0.4 (0.6-7.0)). In some women, data were also obtained during the first few weeks of the OC therapy and then again after the OC had been discontinued. Four menopausal women, aged 42-50 (44.6 ± 1.4), who were receiving conjugated equine estrogen (Premarin), were also included in the study. The control groups consisted of 59 age-matched members of our staff (26.9 ± 0.6) (19-39), who were apparently healthy and regularly menstruating with cycles of 27-31 days. All subjects were white. None of the controls had been using Received June 23, 1977. any kind of drug and members of the study group Address all correspondence and requests for reprints to: Dr. W. Nowaczynski, The Clinical Research Institute had been receiving only OC or Premarin. Blood of Montreal, 110 Pine Avenue West, Montreal H2W 1R7, samples were obtained at random throughout the Quebec, Canada. menstrual cycle from all three groups between 14:00 * This work was supported by a grant from the Medical and 16:00 h, until which time the quietly ambulatResearch Council of Canada to the Multidisciplinary Research Group on Hypertension (principle investigators: ing subjects had been carrying on their usual daily R. Boucher, J. Genest, 0. Kuchel, W. Nowaczynski, and activities. After the subjects had been seated and relaxed for at least 5 min, four blood pressure J. M. Rojo-Ortega). 193
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NOWACZYNSKI ET AL.
194 TABLE 1.
Composition (mg) of OC
No. of subjects (determinations)
% ABGbound aldosterone
a Norethindrone (1.0) Mestranol (0.08)"
8(11)
39.6 ± 1.8*-'"•''
b Norethindrone (1.0) Mestranol (0.05)
4 (6)
33.8 ± 2.3"
c Norethindrone (1.0) Ethinyl estradiol (0.05)''
2 (5)
34.8 ± 3.4''
d Ethynodiol diacetate (1.0) Ethinyl estradiol (0.05)
1 (2)
35.0 ± 5.1''
e Norgestrel (1.5) Ethinyl estradiol (0.05)
6 (7)
34.5 ± 3.4''
f Norgestrel (1.5) Ethinyl estradiol (0.03)
5 (7)
30.8 ± 2.0''
4 (5)
22.9 ± 1.1''
59 (92)
12.0 ± 0.4
Conjugated estrogen (1.25) Control subjects
"17«-Ethynyl-estra-l,3,5(10)-triene-3,17/8-diol. * P < 0.01 vs. f. c P < 0.05 vs. b + c + d + e. '' P < 0.001 vs. controls. •' 3-Methoxy-17a-ethynyl-estra-1,3,5( 10)-triene-3,17/8-diol.
readings were taken and the results were averaged. Each point in the cycle was represented by grouping some of the results into 3-5-day periods to coincide approximately with its various phases. In OC subjects, these phases are referred to by days of the cycle only. Methods Total plasma aldosterone was determined by RIA (10) and the fractional binding of aldosterone to plasma ABG or albumin was determined by the method described (8,11). An assessment of whether the increase in plasma aldosterone binding found in samples taken from subjects receiving OC was indeed a result of the increase in plasma ABG fraction was made using all previously defined criteria (8,9,11). A chromatographic ligand assay was used for plasma cortisol (15), Student's two-tailed t test was used for unpaired comparisons for statistical analysis, and the correlation between ABGbound aldosterone and blood pressure or the estrogen dose was established by the least squares method for linear regression. All results were expressed as mean ± SEM with the range shown in parentheses.
Results The cyclical variations in total plasma aldosterone were similar in both control and OC
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i 1978 Nol
subjects and no significant differences in levels were noted between the groups (Fig. 1). Throughout menstruation and the follicular phase (days 1-10 in OC subjects), total plasma aldosterone levels remained lower, at a mean of 7.3 ng/dl ± 0.6 (2.0-12.8) and 6.2 ± 0.8 (2-12) in controls and OC subjects, respectively. During the ovulatory phase (days 11-16 in OC subjects), these levels increased slightly (P < 0.05 vs. follicular), to 9.8 ±1.5 (2-20) and 10.5 ± 2.9 (2-19), and in the luteal phase (days 17-28 in OC subjects), there was a significant (P < 0.01 vs. follicular) increase to 13.5 ± 1.2 (4.8-33.0) and 10 ± 2.4 (4-27) in control and OC subjects, respectively. A striking observation in the group receiving OC was a markedly greater mean percentage of ABG-bound aldosterone at all stages of the cycle which was 35.2 ± 1.1 as compared to 12.0 ± 0.4 in the controls. This increase in the percentage of aldosterone bound to ABG was directly related to the estrogen content of the OC used (Table 1), but was independent of the duration of its administration in subjects studied 6 months or longer after the start of the medication. Consequently, in subjects on OC, the plasma concentration of free, ABGunbound aldosterone was lower, at 4.0 ± 0.5 ng/dl (1.1-7.1; days 1-10), 6.7 ± 1.3 (1.3-12.9; days 11-16), and 5.9 ± 1.5 (1.2-17.7; days 17-28) than in controls at 6.5 ± 0.6 (1.9-13.3; follicular), 10 ± 1.5 (1.7-17.9; ovulatory, and 12 ± 1.5 (4.0-36.3; luteal) (Fig. 1). This difference was statistically significant at a P < 0.05 during both the ovulatory and luteal phases of the cycle. In the four menopausal women taking conjugated estrogen, plasma aldosterone showed no significant increase at 10 ± 4.9 ng/dl (2.6-15.5), whereas the increase in the percentage of binding, although less pronounced than in the OC group, was significant (P < 0.001), averaging 22.9 ± 1.1 (21.7-25.8). There was no difference in plasma albumin binding between either of the groups (12.6% ± 0.3 and 13 ± 0.3 in controls and OC subjects, respectively). All control subjects had blood pressure readings within the normal range (86/48128/82 mm Hg, mean blood pressure was always below 97). OC subjects had significantly
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ESTROGEN AND ALDOSTERONE PROTEIN BINDING ng/IOOml
PLASMA ALDOSTERONE
15
195
PERCENTAGE ABG BOUND
FIG. 1. Mean total plasma aldosterone concentrations and the percentage of ABG-bound fraction during the normal menstrual cycle and in subjects receiving OC.
1-5
DAYS OF CYCLE
| 6-10
MENSTRUATION FOLUCULAR
higher readings ranging from 98/64-160/96 (Table 2), with only one mean blood pressure exceeding 107, but as no pretreatment readings were available, the exact increment is unknown. The percentage of ABG-bound aldosterone had a positive correlation with mean blood pressure when all OC subjects were considered (Fig. 2A). In addition, Table 3 shows the data available for four subjects at the beginning (at 2-4 weeks) or during treatment (at 24 weeks or more) and again after OC had been discontinued (for 1-12 weeks). In this group, there was a highly significant positive relationship between the percentage of ABG-bound aldosterone and the mean blood pressure (Fig. 2B). There was also a positive correlation between the estrogen content of the OC in those subjects who had been receiving treatment for 6 months or longer and the percentage of ABGbound aldosterone (Fig. 3). Moreover, the resolution of the plasma proteins effected by several different procedures clearly differentiated between the aldosterone plasma binding and that of cortisol (9). 1)
TABLE 2.
Determinations 92 Control 38 OC comprising a) 11 receiving 80 fig estrogen b) 20 receiving 50 fig estrogen c) 7 receiving 30 jug estrogen " P < 0.001 vs. controls. * P < 0.005 vs. b. c P < 0.05 vs. c.
Mean blood pressure (mm Hg) 77.2 ±0.8(58-97) 85.0" ± 1.7 (68-117)
83.0* ± 4.7 (68-117) 88.4C ± 1.7 (76-107) 78.1 ± 1.1 (73-82)
Saturation with ammonium sulfate at 0.57-1.4 M (30% saturation) plasma from subjects receiving OC precipitated all the ABG. There was no aldosterone binding in the supernatant. In repeated determinations, aldosterone concentrations measured in the precipitate corresponded very closely to the value measured as TLPF (8, 11) in the intact plasma, indicating the specificity of the TLPF method for ABG. In samples from 11 OC subjects, the total endogenous aldosterone present in the precipitate (2.58 ng/dl ± 0.5) was virtually identical to that of the total steroid (2.61 ± 0.54) bound to the intact plasma protein
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JCK&M • 1978 Vol 47 • No 1
NOWACZYNSKI ET AL.
196
TLPF. The results from these simultaneous assays gave the regression equation y = 1.056x — 0.115, and the correlation coefficient was 0.990 (P < 0.001). The percentage of aldosterone binding to ABG in these samples was 32.5 ± 2.5 and the total plasma aldosterone was 9.1 ng/dl ± 1.9. In the same samples, the saltprecipitated ABG plasma fraction, which was expected to exclude all CBG, the latter requir-
ing a much higher concentration of ammonium sulfate (beginning to precipitate at 35 with complete precipitation at 70-80% saturation), showed no cortisol or cortisol binding. In contrast, the resultant supernatants gave a value for the endogenous cortisol concentration of 21.8 jiig/dl ± 1.96, very similar to the concentration measured in the intact plasmas (22.5 ± 1.97). The linear regression equation was y = 0.977x — 0.115; with a correlation coefficient of 0.972 (P < 0.001; n = 11). 2) Other already established (9) physicochemical PERCENTAGE ABG BOUND ALDOSTERONE
45 f = 0.460 P< 0.005 4 0 I- y - 0.180x • 25.3 B r= 0.697 P< 0.005 y - 0.55* • 65.2 0C-. n - 18 45
30 PERCENTAGE ABG BOUND ALDOSTERONE
FIG. 2. Correlation between the percentage of ABGbound aldosterone and mean blood pressure in subjects taking OC for 6 months or longer (A), and in subjects at the beginning of or during OC treatment and again after medication has been discontinued (B).
0 30 50 80 DOSE OF ESTROGEN(pg/day)
FIG. 3. Correlation between the dose of estrogen in OC and the percentage of ABG-bound aldosterone fraction in subjects taking OC for 6 months or longer.
TABLE 3.
Age of subject 27
A During treatment No. of weeks after start of treatment: 2 3 4 24 or more After treatment No. of weeks after stopping of treatment: 1 2 3 4 5 8 9 12
S?
26
MBP
A
S?
19
MBP ^ 5 ?
40.6
96
26.4 41.9
81 96
19.0
67
30.1
92
18.1
71
20.0 11.5 10.4
77 79 74
19.9 12.0
79 61
21
MBP ^SP MBP
33.8 40.4
74 79
42.8
89
31.7
81
25.5
75
10.2
80
23.8
77
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ESTROGEN AND ALDOSTERONE PROTEIN BINDING . properties of this plasma ABG in samples from O the OC subjects further indicated its identity % with ABG: Sephadex G-75 chromatography and polyacrylamide gradient gel electropho* resis indicated the separation pattern without any overlapping between the aldosterone or cortisol plasma protein-bound fractions; and " electrofocusing on granular gel showed a sin„ gle homogeneous band with pi (4.75). Molecular weight determinations were 27,000 using gel filtration and trypsin inhibitor (mol wt, 28,000; Boehringer Mannheim) in addition to other standards (9). •-\,
Discussion
The results of this study indicate that the administration of OC containing only 30-80 /zg estrogen produces a very marked increase of about 280% in the plasma binding of aldosterone to ABG. This was apparent throughout the menstrual cycle in all subjects and values remained at the same high level during the tablet-free interval in OC administration, indicating that more than 7 days without them is required to noticeably lower this binding. Moreover, there seemed to be a physiological dose response between the estrogen content of the OC and the level of this binding. There was, however, no significant difference between controls and OC subjects in the concentration of total plasma aldosterone. The ABG unbound aldosterone, thus, was significantly decreased in women receiving OC. - Our original observation concerning variations in aldosterone levels during the menstrual cycle showing two peaks (16) has since been well documented (10, 12, 17, 18). In those women receiving OC in this study, there was also a normal cyclical pattern of plasma aldosterone similar to that in the control group and the percentage of the ABG-bound hormone did not change significantly throughout the cycle, - which is in agreement with our first observation (8). In a previous report, all of the four subjects receiving OC had plasma aldosterone \ peaks which were apparent by the 9th or 10th day from the onset of menstruation (19). The aldosterone measured in the ammonium sulfate precipitate (bound fraction) agreed closely with the amount of ABG-bound
197
aldosterone found in the intact plasma, and also the cortisol content of the intact plasma approximated the values found in the supernatant. Knowing that CBG is markedly increased by estrogen treatment (2, 4), the firm conclusions that may be drawn from these agreements are that the TLPF method (8) allows specific measurements of ABG and also that ABG has no binding affinity for cortisol at the physiological temperature of 37 C. Synthetic estrogens have been shown to increase from 1.5- to 2.0-fold the secretion and excretion rates of aldosterone, but not to increase the excretion rate of tetrahydroglucuronide (7) or the plasma concentration of aldosterone, as evident from this and most previous studies (5, 7). The increase in plasma aldosterone during OC administration reported by some investigators (20), therefore, could be due to different sampling times in the course of a modified circadian rhythm which, because of a marked increase in aldosterone binding to ABG, would cause delays in its elimination, especially in the morning after its early morning circadian peak, similar to the mechanism suggested in essential hypertension (EH) (9, 11, 12, 21). It would appear that OC induces definite changes (7) in the hepatic metabolism of aldosterone similar to those which accompany EH, especially in its labile form in which plasma concentration remains normal in the presence of significantly higher ABG binding, resulting in a lower ABG unbound plasma fraction and a decreased metabolic clearance rate (11, 12). Aldosterone bound to plasma ABG would be expected not to enter the hepatic extraction (22, 23), thereby lowering its rate of metabolism by the liver and, consequently, its overall metabolic clearance rate. OC has also been shown to cause some increase in blood pressure in nearly all women, and to enhance or occasionally induce overt hypertension, presumably when other predisposing factors are present (5, 7, 24-26). It has been suggested that this hypertensive action, which is apparently directly related to the length of time of administration of the OC (26), may be mediated through the renin-angiotensin II-aldosterone system (5) in which OC produces definite changes, some of which
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198
JCE & M Vol47
NOWACZYNSKI ET AL.
are very similar to those found in EH (8, 11-14). Changes in ABG binding effect the metabolism of aldosterone (8, 11-13, 21, 27) and this binding is significantly higher in EH (8, 11, 12, 21). OC consistently increases the availability of plasma renin substrate made by the liver, which is believed to sometimes increase the rate of generation of angiotensin II and aldosterone (28-33). In at least one recent study (34) in which posture and sodium intake were controlled, there was an unequivocal increase in both the plasma renin and angiotensin II levels. The fact that the increase in blood pressure may be due to higher mineralocorticoid activity is supported by their ability to cause a moderate, but significant, increase in total exchangeable sodium (35), and also by the fact that many patients who are hypertensive due to OC respond dramatically to the administration of spironolactone (8). Although the daily secretion rate of aldosterone was usually higher in women on OC, the mean total plasma concentration was usually normal and, as evident from this study, the ABG unbound aldosterone, which probably best reflects the physiologically important portion acting at the tubular level, was significantly lower. Our data were, however, obtained in midafternoon in relatively unstressed subjects and it is more than likely that markedly increased ABG binding of aldosterone would cause delays in elimination and modifications in the circadian rhythm. Therefore, this mechanism prolonging the action of aldosterone in OC subjects would result in transient elevations of its plasma concentration after circadian peaks or any other stimulation, similar to those in EH patients (8,11). As the high affinity binding to ABG is freely reversible (9), the markedly increased bound portion may also constitute a circulating reserve from which aldosterone could be dissociated whenever there is a decline in the concentration of the free fraction leading to an enhanced concentration of aldosterone in the immediate vicinity of some target tissues. The role of the bound fraction could also be to supply the hormone directly to specific target cells having receptor proteins with a higher affinity for aldosterone than that of ABG. This
1978 Nol
mechanism could be of particular importance, as already discussed (8, 22), in the outer pool of distribution (outside the extracellular fluid). The question may be raised, therefore, as to whether, in addition to causing modifications in the circadian rhythm, the ABG-bound plasma fraction of aldosterone may have some other direct and, as yet unrecognized, pressor effects leading to increased arteriolar resistance independent of its tubular sodium-retaining activity. The positive relationship between plasma ABG-bound aldosterone and the mean blood pressure in OC subjects studied and especially the highly significant correlation between these two parameters in OC subjects starting treatment or shortly after medication has been discontinued supports this possibility. Also in agreement could be the demonstration, for the first time, of specific mineralocorticoid receptors in large and small (arteriolar size) blood vessels of the rabbit (36). The use of OC seems to be associated with changes similar to those occurring during pregnancy when, as we have shown, the ABG exhibits the expected significant increase until the second trimester, thereafter maintaining a plateau (8). Estrogens as well as pregnancy appear to increase hepatic production of various other proteins, including renin substrate. It seems possible that estrogens elevate plasma ABG similar to transcortin and also other plasma carrier proteins by directly stimulating the hepatic biosynthesis (1-8). In conclusion, our studies have shown a marked increase in the percentage of aldosterone bound to a specific plasma globulin during the administration of OC drugs, but the exact relevance, if any, of the above observations to the etiology or pathogenesis of OC-induced elevation in blood pressure or hypertension requires further clarification.
Acknowledgments We wish to thank Miss P. Robinson, Mrs. J. Laberge, and Mr. R. Tremblay for their skillful technical assistance, Miss I. Morin for the drawings, and Miss. P. Day for secretarial help.
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ZALES, AND G. W. NOKES, Plasma renin concentration, activity and substrate in hypertension induced by oral contraceptives, J Clin Endocrinol Metab 34: 1067, 1972. 31. HELMER, 0. M., AND W. E. JUDSON, Influence of high renin substrate levels on renin-angiotensin system in pregnancy, Am J Obstet Gynecol 99: 9, 1967. 32. WEINBERGER, M. H., D. COLLINS, A. J. DOWDY, G. W. NOKES,
AND J. A. LUETSCHER, Hypertension induced by oral contraceptives containing estrogen and gestagen, Ann Intern Med 71: 891, 1969. 33. CAIN, M. D., W. A. WALTERS, AND K. J. CATT, Effect of oral
contraceptive therapy on the renin-angiotensin system, J Clin Endocrinol Metab 33: 671, 1971. 34. HOLLENBERG, N. K., G. H. WILLIAMS, B. BURGER, W. CHENITZ, I. HOOSMAND, AND D. F. ADAMS, An interaction between oral contraceptive agents, sodium intake, and the renin-angiotensin system in healthy young women, Circ Res 38: 35, 1976. 35. CRANE, M. G., AND J. J. HARRIS, Effect of estrogens and gestagens on exchangeable sodium, In Fregly, M. J., and M. S. Fregly (eds.), Oral Contraceptives and High Blood Pressure, Gainesville, Dolphin Press, 1974, p. 159. 36. KORNEL, L , N. KANAMARLAPUDI, R. BAUM, C. CHEN, AND T.
TRAVERS, Mineralocorticoid receptors in blood vessels, 59th Ann. Meeting of the Endocrine Society, Chicago, June 8-10, 1977 (Abstract 218).
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Vol. 47, No. 1 Printed in U.S.A.
Increased Aldosterone Plasma Protein Binding in Women on Combined Oral Contraceptives throughout the Menstrual Cycle* W. NOWACZYNSKI, T. MURAKAMI, K. RICHARDSON, AND J. GENEST The Steroid Research Department, The Clinical Research Institute, Montreal, Quebec, Canada ABSTRACT. Plasma aldosterone concentration and the percentage of the fraction bound to a specific plasma aldosterone-binding globulin (ABG) were measured throughout the menstrual cycle in 26 healthy women aged between 19 and 38 yr who were receiving estrogencontaining oral contraceptives (OC). Midafternoon upright levels of total plasma aldosterone were similar in control and OC subjects and showed normal cyclic fluctuation in both groups. The percentage of ABG-bound aldosterone was markedly higher in OC subjects than in
I
T IS known that the administration of estrogens produces significant elevations in plasma transcortin (CBG), sex steroid, and T4binding globulins, presumably by increasing the rate of their synthesis by the liver (1-5). Estrogens also produce an elevation in the level of the aldosterone-binding protein (ABG; 6-8). This ABG, classified as an ai-glycoprotein (8-11), has been differentiated from CBG or albumin and identified (9-12) with what has been previously called a transcortin-like plasma fraction (TLPF) (8). ABG binds aldosterone reversibly and with high affinity and recent work suggests that it may be physiologically important and play a role in essential hypertension (8, 9, 11-14), but not secondary hypertension (11). Oral contraceptives (OC) have also been shown to produce significant changes in the metabolism of aldosterone. These changes are described in a recent comprehensive review by Crane (5).
controls at all stages of the cycle and showed a positive correlation with the mean blood pressure when all OC subjects studied were considered. In addition, when the plasma ABG levels were measured 6 months or more after the start of medication, they appeared to be related to the dose of estrogen in the OC, but not to the length of time of its administration. The identity of plasma ABG in OC subjects with that in control subjects was also established. (J Clin Endocrinol Metab 46: 193, 1978)
We present here a study of aldosterone binding to plasma ABG in women at various times during the menstrual cycle, some of whom were receiving estrogen- and gestagencontaining OC. Materials and Methods
The study group consisted of 26 healthy, asymptomatic women aged between 19 and 38 yr (mean, 26.7 ± 0.9 SEM) on unrestricted diet who had been taking synthetic estrogen-containing OC (Table 1) on 21 days out of every 28 for at least 6 months before the study (3.5 yr ± 0.4 (0.6-7.0)). In some women, data were also obtained during the first few weeks of the OC therapy and then again after the OC had been discontinued. Four menopausal women, aged 42-50 (44.6 ± 1.4), who were receiving conjugated equine estrogen (Premarin), were also included in the study. The control groups consisted of 59 age-matched members of our staff (26.9 ± 0.6) (19-39), who were apparently healthy and regularly menstruating with cycles of 27-31 days. All subjects were white. None of the controls had been using Received June 23, 1977. any kind of drug and members of the study group Address all correspondence and requests for reprints to: Dr. W. Nowaczynski, The Clinical Research Institute had been receiving only OC or Premarin. Blood of Montreal, 110 Pine Avenue West, Montreal H2W 1R7, samples were obtained at random throughout the Quebec, Canada. menstrual cycle from all three groups between 14:00 * This work was supported by a grant from the Medical and 16:00 h, until which time the quietly ambulatResearch Council of Canada to the Multidisciplinary Research Group on Hypertension (principle investigators: ing subjects had been carrying on their usual daily R. Boucher, J. Genest, 0. Kuchel, W. Nowaczynski, and activities. After the subjects had been seated and relaxed for at least 5 min, four blood pressure J. M. Rojo-Ortega). 193
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NOWACZYNSKI ET AL.
194 TABLE 1.
Composition (mg) of OC
No. of subjects (determinations)
% ABGbound aldosterone
a Norethindrone (1.0) Mestranol (0.08)"
8(11)
39.6 ± 1.8*-'"•''
b Norethindrone (1.0) Mestranol (0.05)
4 (6)
33.8 ± 2.3"
c Norethindrone (1.0) Ethinyl estradiol (0.05)''
2 (5)
34.8 ± 3.4''
d Ethynodiol diacetate (1.0) Ethinyl estradiol (0.05)
1 (2)
35.0 ± 5.1''
e Norgestrel (1.5) Ethinyl estradiol (0.05)
6 (7)
34.5 ± 3.4''
f Norgestrel (1.5) Ethinyl estradiol (0.03)
5 (7)
30.8 ± 2.0''
4 (5)
22.9 ± 1.1''
59 (92)
12.0 ± 0.4
Conjugated estrogen (1.25) Control subjects
"17«-Ethynyl-estra-l,3,5(10)-triene-3,17/8-diol. * P < 0.01 vs. f. c P < 0.05 vs. b + c + d + e. '' P < 0.001 vs. controls. •' 3-Methoxy-17a-ethynyl-estra-1,3,5( 10)-triene-3,17/8-diol.
readings were taken and the results were averaged. Each point in the cycle was represented by grouping some of the results into 3-5-day periods to coincide approximately with its various phases. In OC subjects, these phases are referred to by days of the cycle only. Methods Total plasma aldosterone was determined by RIA (10) and the fractional binding of aldosterone to plasma ABG or albumin was determined by the method described (8,11). An assessment of whether the increase in plasma aldosterone binding found in samples taken from subjects receiving OC was indeed a result of the increase in plasma ABG fraction was made using all previously defined criteria (8,9,11). A chromatographic ligand assay was used for plasma cortisol (15), Student's two-tailed t test was used for unpaired comparisons for statistical analysis, and the correlation between ABGbound aldosterone and blood pressure or the estrogen dose was established by the least squares method for linear regression. All results were expressed as mean ± SEM with the range shown in parentheses.
Results The cyclical variations in total plasma aldosterone were similar in both control and OC
JCE & M VoU7
i 1978 Nol
subjects and no significant differences in levels were noted between the groups (Fig. 1). Throughout menstruation and the follicular phase (days 1-10 in OC subjects), total plasma aldosterone levels remained lower, at a mean of 7.3 ng/dl ± 0.6 (2.0-12.8) and 6.2 ± 0.8 (2-12) in controls and OC subjects, respectively. During the ovulatory phase (days 11-16 in OC subjects), these levels increased slightly (P < 0.05 vs. follicular), to 9.8 ±1.5 (2-20) and 10.5 ± 2.9 (2-19), and in the luteal phase (days 17-28 in OC subjects), there was a significant (P < 0.01 vs. follicular) increase to 13.5 ± 1.2 (4.8-33.0) and 10 ± 2.4 (4-27) in control and OC subjects, respectively. A striking observation in the group receiving OC was a markedly greater mean percentage of ABG-bound aldosterone at all stages of the cycle which was 35.2 ± 1.1 as compared to 12.0 ± 0.4 in the controls. This increase in the percentage of aldosterone bound to ABG was directly related to the estrogen content of the OC used (Table 1), but was independent of the duration of its administration in subjects studied 6 months or longer after the start of the medication. Consequently, in subjects on OC, the plasma concentration of free, ABGunbound aldosterone was lower, at 4.0 ± 0.5 ng/dl (1.1-7.1; days 1-10), 6.7 ± 1.3 (1.3-12.9; days 11-16), and 5.9 ± 1.5 (1.2-17.7; days 17-28) than in controls at 6.5 ± 0.6 (1.9-13.3; follicular), 10 ± 1.5 (1.7-17.9; ovulatory, and 12 ± 1.5 (4.0-36.3; luteal) (Fig. 1). This difference was statistically significant at a P < 0.05 during both the ovulatory and luteal phases of the cycle. In the four menopausal women taking conjugated estrogen, plasma aldosterone showed no significant increase at 10 ± 4.9 ng/dl (2.6-15.5), whereas the increase in the percentage of binding, although less pronounced than in the OC group, was significant (P < 0.001), averaging 22.9 ± 1.1 (21.7-25.8). There was no difference in plasma albumin binding between either of the groups (12.6% ± 0.3 and 13 ± 0.3 in controls and OC subjects, respectively). All control subjects had blood pressure readings within the normal range (86/48128/82 mm Hg, mean blood pressure was always below 97). OC subjects had significantly
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ESTROGEN AND ALDOSTERONE PROTEIN BINDING ng/IOOml
PLASMA ALDOSTERONE
15
195
PERCENTAGE ABG BOUND
FIG. 1. Mean total plasma aldosterone concentrations and the percentage of ABG-bound fraction during the normal menstrual cycle and in subjects receiving OC.
1-5
DAYS OF CYCLE
| 6-10
MENSTRUATION FOLUCULAR
higher readings ranging from 98/64-160/96 (Table 2), with only one mean blood pressure exceeding 107, but as no pretreatment readings were available, the exact increment is unknown. The percentage of ABG-bound aldosterone had a positive correlation with mean blood pressure when all OC subjects were considered (Fig. 2A). In addition, Table 3 shows the data available for four subjects at the beginning (at 2-4 weeks) or during treatment (at 24 weeks or more) and again after OC had been discontinued (for 1-12 weeks). In this group, there was a highly significant positive relationship between the percentage of ABG-bound aldosterone and the mean blood pressure (Fig. 2B). There was also a positive correlation between the estrogen content of the OC in those subjects who had been receiving treatment for 6 months or longer and the percentage of ABGbound aldosterone (Fig. 3). Moreover, the resolution of the plasma proteins effected by several different procedures clearly differentiated between the aldosterone plasma binding and that of cortisol (9). 1)
TABLE 2.
Determinations 92 Control 38 OC comprising a) 11 receiving 80 fig estrogen b) 20 receiving 50 fig estrogen c) 7 receiving 30 jug estrogen " P < 0.001 vs. controls. * P < 0.005 vs. b. c P < 0.05 vs. c.
Mean blood pressure (mm Hg) 77.2 ±0.8(58-97) 85.0" ± 1.7 (68-117)
83.0* ± 4.7 (68-117) 88.4C ± 1.7 (76-107) 78.1 ± 1.1 (73-82)
Saturation with ammonium sulfate at 0.57-1.4 M (30% saturation) plasma from subjects receiving OC precipitated all the ABG. There was no aldosterone binding in the supernatant. In repeated determinations, aldosterone concentrations measured in the precipitate corresponded very closely to the value measured as TLPF (8, 11) in the intact plasma, indicating the specificity of the TLPF method for ABG. In samples from 11 OC subjects, the total endogenous aldosterone present in the precipitate (2.58 ng/dl ± 0.5) was virtually identical to that of the total steroid (2.61 ± 0.54) bound to the intact plasma protein
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JCK&M • 1978 Vol 47 • No 1
NOWACZYNSKI ET AL.
196
TLPF. The results from these simultaneous assays gave the regression equation y = 1.056x — 0.115, and the correlation coefficient was 0.990 (P < 0.001). The percentage of aldosterone binding to ABG in these samples was 32.5 ± 2.5 and the total plasma aldosterone was 9.1 ng/dl ± 1.9. In the same samples, the saltprecipitated ABG plasma fraction, which was expected to exclude all CBG, the latter requir-
ing a much higher concentration of ammonium sulfate (beginning to precipitate at 35 with complete precipitation at 70-80% saturation), showed no cortisol or cortisol binding. In contrast, the resultant supernatants gave a value for the endogenous cortisol concentration of 21.8 jiig/dl ± 1.96, very similar to the concentration measured in the intact plasmas (22.5 ± 1.97). The linear regression equation was y = 0.977x — 0.115; with a correlation coefficient of 0.972 (P < 0.001; n = 11). 2) Other already established (9) physicochemical PERCENTAGE ABG BOUND ALDOSTERONE
45 f = 0.460 P< 0.005 4 0 I- y - 0.180x • 25.3 B r= 0.697 P< 0.005 y - 0.55* • 65.2 0C-. n - 18 45
30 PERCENTAGE ABG BOUND ALDOSTERONE
FIG. 2. Correlation between the percentage of ABGbound aldosterone and mean blood pressure in subjects taking OC for 6 months or longer (A), and in subjects at the beginning of or during OC treatment and again after medication has been discontinued (B).
0 30 50 80 DOSE OF ESTROGEN(pg/day)
FIG. 3. Correlation between the dose of estrogen in OC and the percentage of ABG-bound aldosterone fraction in subjects taking OC for 6 months or longer.
TABLE 3.
Age of subject 27
A During treatment No. of weeks after start of treatment: 2 3 4 24 or more After treatment No. of weeks after stopping of treatment: 1 2 3 4 5 8 9 12
S?
26
MBP
A
S?
19
MBP ^ 5 ?
40.6
96
26.4 41.9
81 96
19.0
67
30.1
92
18.1
71
20.0 11.5 10.4
77 79 74
19.9 12.0
79 61
21
MBP ^SP MBP
33.8 40.4
74 79
42.8
89
31.7
81
25.5
75
10.2
80
23.8
77
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ESTROGEN AND ALDOSTERONE PROTEIN BINDING . properties of this plasma ABG in samples from O the OC subjects further indicated its identity % with ABG: Sephadex G-75 chromatography and polyacrylamide gradient gel electropho* resis indicated the separation pattern without any overlapping between the aldosterone or cortisol plasma protein-bound fractions; and " electrofocusing on granular gel showed a sin„ gle homogeneous band with pi (4.75). Molecular weight determinations were 27,000 using gel filtration and trypsin inhibitor (mol wt, 28,000; Boehringer Mannheim) in addition to other standards (9). •-\,
Discussion
The results of this study indicate that the administration of OC containing only 30-80 /zg estrogen produces a very marked increase of about 280% in the plasma binding of aldosterone to ABG. This was apparent throughout the menstrual cycle in all subjects and values remained at the same high level during the tablet-free interval in OC administration, indicating that more than 7 days without them is required to noticeably lower this binding. Moreover, there seemed to be a physiological dose response between the estrogen content of the OC and the level of this binding. There was, however, no significant difference between controls and OC subjects in the concentration of total plasma aldosterone. The ABG unbound aldosterone, thus, was significantly decreased in women receiving OC. - Our original observation concerning variations in aldosterone levels during the menstrual cycle showing two peaks (16) has since been well documented (10, 12, 17, 18). In those women receiving OC in this study, there was also a normal cyclical pattern of plasma aldosterone similar to that in the control group and the percentage of the ABG-bound hormone did not change significantly throughout the cycle, - which is in agreement with our first observation (8). In a previous report, all of the four subjects receiving OC had plasma aldosterone \ peaks which were apparent by the 9th or 10th day from the onset of menstruation (19). The aldosterone measured in the ammonium sulfate precipitate (bound fraction) agreed closely with the amount of ABG-bound
197
aldosterone found in the intact plasma, and also the cortisol content of the intact plasma approximated the values found in the supernatant. Knowing that CBG is markedly increased by estrogen treatment (2, 4), the firm conclusions that may be drawn from these agreements are that the TLPF method (8) allows specific measurements of ABG and also that ABG has no binding affinity for cortisol at the physiological temperature of 37 C. Synthetic estrogens have been shown to increase from 1.5- to 2.0-fold the secretion and excretion rates of aldosterone, but not to increase the excretion rate of tetrahydroglucuronide (7) or the plasma concentration of aldosterone, as evident from this and most previous studies (5, 7). The increase in plasma aldosterone during OC administration reported by some investigators (20), therefore, could be due to different sampling times in the course of a modified circadian rhythm which, because of a marked increase in aldosterone binding to ABG, would cause delays in its elimination, especially in the morning after its early morning circadian peak, similar to the mechanism suggested in essential hypertension (EH) (9, 11, 12, 21). It would appear that OC induces definite changes (7) in the hepatic metabolism of aldosterone similar to those which accompany EH, especially in its labile form in which plasma concentration remains normal in the presence of significantly higher ABG binding, resulting in a lower ABG unbound plasma fraction and a decreased metabolic clearance rate (11, 12). Aldosterone bound to plasma ABG would be expected not to enter the hepatic extraction (22, 23), thereby lowering its rate of metabolism by the liver and, consequently, its overall metabolic clearance rate. OC has also been shown to cause some increase in blood pressure in nearly all women, and to enhance or occasionally induce overt hypertension, presumably when other predisposing factors are present (5, 7, 24-26). It has been suggested that this hypertensive action, which is apparently directly related to the length of time of administration of the OC (26), may be mediated through the renin-angiotensin II-aldosterone system (5) in which OC produces definite changes, some of which
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198
JCE & M Vol47
NOWACZYNSKI ET AL.
are very similar to those found in EH (8, 11-14). Changes in ABG binding effect the metabolism of aldosterone (8, 11-13, 21, 27) and this binding is significantly higher in EH (8, 11, 12, 21). OC consistently increases the availability of plasma renin substrate made by the liver, which is believed to sometimes increase the rate of generation of angiotensin II and aldosterone (28-33). In at least one recent study (34) in which posture and sodium intake were controlled, there was an unequivocal increase in both the plasma renin and angiotensin II levels. The fact that the increase in blood pressure may be due to higher mineralocorticoid activity is supported by their ability to cause a moderate, but significant, increase in total exchangeable sodium (35), and also by the fact that many patients who are hypertensive due to OC respond dramatically to the administration of spironolactone (8). Although the daily secretion rate of aldosterone was usually higher in women on OC, the mean total plasma concentration was usually normal and, as evident from this study, the ABG unbound aldosterone, which probably best reflects the physiologically important portion acting at the tubular level, was significantly lower. Our data were, however, obtained in midafternoon in relatively unstressed subjects and it is more than likely that markedly increased ABG binding of aldosterone would cause delays in elimination and modifications in the circadian rhythm. Therefore, this mechanism prolonging the action of aldosterone in OC subjects would result in transient elevations of its plasma concentration after circadian peaks or any other stimulation, similar to those in EH patients (8,11). As the high affinity binding to ABG is freely reversible (9), the markedly increased bound portion may also constitute a circulating reserve from which aldosterone could be dissociated whenever there is a decline in the concentration of the free fraction leading to an enhanced concentration of aldosterone in the immediate vicinity of some target tissues. The role of the bound fraction could also be to supply the hormone directly to specific target cells having receptor proteins with a higher affinity for aldosterone than that of ABG. This
1978 Nol
mechanism could be of particular importance, as already discussed (8, 22), in the outer pool of distribution (outside the extracellular fluid). The question may be raised, therefore, as to whether, in addition to causing modifications in the circadian rhythm, the ABG-bound plasma fraction of aldosterone may have some other direct and, as yet unrecognized, pressor effects leading to increased arteriolar resistance independent of its tubular sodium-retaining activity. The positive relationship between plasma ABG-bound aldosterone and the mean blood pressure in OC subjects studied and especially the highly significant correlation between these two parameters in OC subjects starting treatment or shortly after medication has been discontinued supports this possibility. Also in agreement could be the demonstration, for the first time, of specific mineralocorticoid receptors in large and small (arteriolar size) blood vessels of the rabbit (36). The use of OC seems to be associated with changes similar to those occurring during pregnancy when, as we have shown, the ABG exhibits the expected significant increase until the second trimester, thereafter maintaining a plateau (8). Estrogens as well as pregnancy appear to increase hepatic production of various other proteins, including renin substrate. It seems possible that estrogens elevate plasma ABG similar to transcortin and also other plasma carrier proteins by directly stimulating the hepatic biosynthesis (1-8). In conclusion, our studies have shown a marked increase in the percentage of aldosterone bound to a specific plasma globulin during the administration of OC drugs, but the exact relevance, if any, of the above observations to the etiology or pathogenesis of OC-induced elevation in blood pressure or hypertension requires further clarification.
Acknowledgments We wish to thank Miss P. Robinson, Mrs. J. Laberge, and Mr. R. Tremblay for their skillful technical assistance, Miss I. Morin for the drawings, and Miss. P. Day for secretarial help.
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