0021-972x/92/7404-0910$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 4 Printed in U.S.A.
JURGEN WACKER, NOUHAD E.-MISTRY, HELGA WOLFGANG STOLZ, AND GUNTHER BASTERT Departments of Gynecology and Obstetrics (J. W., N.E.-M,m University of Heidelberg, D-6900 Heidelberg, Germany
BAUER,
PAUL
To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the mineralocorticoid receptor (MR) status in mononuclear leukocytes in the 2 groups. MR count was significantly (P < 0.0005) decreased in the PIH group (148 + 9 binding sites/cell) compared with the control group (300 + 17 binding sites/cell; mean f SEM). Plasma aldosterone in women with PIH was 281 k 61 pmol/L; in normal
A
LTHOUGH pregnancy-induced hypertension (PIH) is a frequent cause of maternal and neonatal morbidity and mortality (l), its etiology is not yet clear; in particular, the role of mineralocorticoids in its pathogenesis is poorly understood. In the literature, the role of the renin-angiotensinaldosterone system (RAA) is the subject of some controversy. Some researchers describe a suppression of the RAA in PIH (2, 3), while others have found elevated aldosterone levels, especially in preeclampsia (4). Mineralocorticoid receptors (MR) have been found in tissues directly involved in sodium homeostasis, e.g. kidney, salivary gland, pituitary gland, and gut, and in other nonclassical aldosterone target tissues, e.g. brain, cultured aortic cells, pituitary tumor cells, and rat mammary glands (5-10). Armanini et al. (11) studied mineralocorticoid receptors in mononuclear leukocytes (MNL) and was able to show that in these cells the affinity of aldosterone for its receptor is similar to that reported in kidney cytosol. Received October 22,199l. Address requests for reprints to: Dr. Jurgen Wacker, Department of Gynecology and Obstetrics, University of Heidelberg, Vopstr. 9, D6900 Heidelberg, Germany. * Presented in part at the 14th Deutscher KongreP fur Perinatale Medizin, Berlin, Germany, October 1989, and the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 20-23,199O.
in
VECSEI,
WA’., G.B.) and Pharmacology
ABSTRACT.
Receptors Pregnancy-
(H.B., P. K),
pregnant women it was 697 + 172 pmol/L (P < 0.025). Plasma Shydroxycorticosterone was also significantly (P < 0.025) lower (PIH, 1071 + 149 pmol/L; controls, 1907 f 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results cannot be explained by receptor downregulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may, thus, be responsible for the hypertension and altered MR status. (J Clin Endocrinol Metub 74: 910-913,1992)
Armanini et al. (12-14) also demonstrated that MNL show a lower number of MR in patients with mineralocorticoid excess, an observation that has opened up new diagnostic possibilities. Since kidney biopsies cannot be carried out in pregnant women, we have studied MR in MNL in pregnant women with and without PIH. The present study deals with pregnant women who have already shown symptoms of PIH. In these women, we have determined the MR status as well as values of plasma aldosterone and its immediate precursor plasma l%hydroxycorticosterone (l&OH-B). Subjects and Methods Subjects We studied 25womenwith PIH with and without proteinuria and 25 healthy women with normal pregnancy. Blood samples in both groupswere taken in the third trimester. The diagnosis of PIH was based on the criterion of an elevated blood pressure (BP) read on at least three different occasions(systolic BP, >140 mm Hg; diastolic BP, >90 mm Hg). Mean arterial pressure, a valuable parameter (15) for elevated total peripheral resistancewas calculated accordingto the following formula: (systolic BP + 2 x diastolic BP)/3. Proteinuria wasfound in 15womenwith PIH, but not in normal pregnant women. Edema was present in 22 of 25 women with PIH. Normotensive women with extensive edema were excluded from the study. Details of obstetric technique and the 910
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/910/3004781 by Uppsala Universitetsbibliotek user on 10 January 2019
Mineralocorticoids and Mineralocorticoid Mononuclear Leukocytes in Patients with Induced Hypertension*
AND MR IN MNL
MINERALOCORTICOIDS
Methods The method for measuring MRs in MNL has been described by Armanini et al. (ll), and a modified method was used in our laboratory. Briefly, 50 mL peripheral blood were taken between 0800-1000 h after patients had been recumbent for at least 30 min. Blood was centrifuged at 1000 x g at 20 C for 10 min, and supernatant containing plasma was kept at 4 C for steroid determination (aldosterone and 18-OH-B). Cells were separated from blood on a Ficoll gradient, centrifuged for 40 min at 400 x g and 20 C, and washed three times with Hanks’ Balanced Salt Solution to eliminate endogenous steroids. Cell aliquots containing 3-6 millions cells/tube were incubated with increasing amounts of 3H-labeled aldosterone and a loo-fold excess of RU 28362 to exclude tracer binding from glucocorticoid receptors (16). Nonspecific binding was measured by adding a 200fold excess of nonlabeled aldosterone. After 1 h of incubation at 37 C, the reaction was stopped by adding 2 mL ice-cold saline, and then cells were washed three times. Finally, cells were transferred into scintillation vials, the radioactivity was counted, and the binding was subjected to Scatchard analysis (17). Endogenous steroids were measured by a RIA established in our laboratory. The techniques for chromatographic determination of plasma aldosterone and plasma 18-OH-corticosterone have been described previously (18, 19). The method for the determination of cortisol by ultrafiltration was adapted to TABLE 1. Technique and week of delivery of the women studied
Normal pregnancies (n = 25)
40(37-41)
1 Cesariansection 23 Spontaneouslabors
1 Forcepsdelivery PIH (n = 25)
35 (26-41)
determine aldosterone by ultrafiltration (20). Data were analyzed by Student’s t test and are specified as the mean and SEM.
Results The number of MR per cell as well as their affinity (Kd) were determined for both groups of women. MR per cell in MNL of women with normal pregnancies measured in their third trimester ranged from 200-494 sites/ cell (mean f SEM, 300 & 17). The number of MR in women with PIH ranged from 82-257 (mean + SEM, 148 f 9), a significantly lower level (P < 0.0005) than in the control group. The results are summarized in Table 2. No difference in MR affinity was seen between groups (PIH Kd, 1.0 f 0.1 nM; normal pregnancy, 1.2 f 0.1 KIM). Values of plasma aldosterone and plasma 18-OH-B are shown in Table 2 for PIH and normal pregnancies. Total aldosterone in PIH was 281 + 61 pmol/L (range, 8-1115 pmol/L), significantly lower (P < 0.025) than that in control pregnancies (697 f 172 pmol/L; range, 108-3107 pmol/L). Four of 25 aldosterone values in the latter group were higher than average. Within the PIH group and in the group of normal pregnant women there was no significant correlation between MR and plasma aldosterone concentration (r = -0.04 and r = 0.07, respectively; Fig. 1). Free aldosterone values, expressed as a percentage of the total aldosterone, did not show a significant difference. Free aldosterone, expressed as plasma concentration, differed significantly, in accordance with values of total aldosterone shown in Table 2. Like plasma aldosterone, plasma 18-OH-B was significantly (P < 0.025) lower in patients with PIH (1072 f 149 pmol/L; range, 50-2815 pmol/L) compared with that in normal pregnant women (1907 + 318 pmol/L; range, 375-3587 pmol/L). No determination of free l&OH-B was carried out.
14 Cesarian section
8 Spontaneouslabors 2 Intrauterine death 1 Vacuum extraction
Discussion It is known that plasma aldosterone levels are higher in pregnant than in nonpregnant women (21-23). It is
TABLE 2. Summary of the mean f SEM of BP, mineralocorticoids,and MR, in the two examined groups
Normal pregnancies Mean
+SEM
BP (mm Hg) Systolic 111 +2 Diastolic 71 +2 MAP (mm Hg) 85 f2 ALDO (pmol/L) 697 k171 FR ALDO absolute (pmol/L) 159 k42 FR ALDO (%) 26 18-OH-B (pmol/L) 1907 +318 MR (sites/cell) 300 f17 1.2 f0.1 Kd hM) MAP, Mean arterial pressure;BP, blood pressure;FR, fraction.
n
PIH P
25 25 24 20 20 23 25 25
Vol. 74, No. 4 Printed in U.S.A.
JURGEN WACKER, NOUHAD E.-MISTRY, HELGA WOLFGANG STOLZ, AND GUNTHER BASTERT Departments of Gynecology and Obstetrics (J. W., N.E.-M,m University of Heidelberg, D-6900 Heidelberg, Germany
BAUER,
PAUL
To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the mineralocorticoid receptor (MR) status in mononuclear leukocytes in the 2 groups. MR count was significantly (P < 0.0005) decreased in the PIH group (148 + 9 binding sites/cell) compared with the control group (300 + 17 binding sites/cell; mean f SEM). Plasma aldosterone in women with PIH was 281 k 61 pmol/L; in normal
A
LTHOUGH pregnancy-induced hypertension (PIH) is a frequent cause of maternal and neonatal morbidity and mortality (l), its etiology is not yet clear; in particular, the role of mineralocorticoids in its pathogenesis is poorly understood. In the literature, the role of the renin-angiotensinaldosterone system (RAA) is the subject of some controversy. Some researchers describe a suppression of the RAA in PIH (2, 3), while others have found elevated aldosterone levels, especially in preeclampsia (4). Mineralocorticoid receptors (MR) have been found in tissues directly involved in sodium homeostasis, e.g. kidney, salivary gland, pituitary gland, and gut, and in other nonclassical aldosterone target tissues, e.g. brain, cultured aortic cells, pituitary tumor cells, and rat mammary glands (5-10). Armanini et al. (11) studied mineralocorticoid receptors in mononuclear leukocytes (MNL) and was able to show that in these cells the affinity of aldosterone for its receptor is similar to that reported in kidney cytosol. Received October 22,199l. Address requests for reprints to: Dr. Jurgen Wacker, Department of Gynecology and Obstetrics, University of Heidelberg, Vopstr. 9, D6900 Heidelberg, Germany. * Presented in part at the 14th Deutscher KongreP fur Perinatale Medizin, Berlin, Germany, October 1989, and the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 20-23,199O.
in
VECSEI,
WA’., G.B.) and Pharmacology
ABSTRACT.
Receptors Pregnancy-
(H.B., P. K),
pregnant women it was 697 + 172 pmol/L (P < 0.025). Plasma Shydroxycorticosterone was also significantly (P < 0.025) lower (PIH, 1071 + 149 pmol/L; controls, 1907 f 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results cannot be explained by receptor downregulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may, thus, be responsible for the hypertension and altered MR status. (J Clin Endocrinol Metub 74: 910-913,1992)
Armanini et al. (12-14) also demonstrated that MNL show a lower number of MR in patients with mineralocorticoid excess, an observation that has opened up new diagnostic possibilities. Since kidney biopsies cannot be carried out in pregnant women, we have studied MR in MNL in pregnant women with and without PIH. The present study deals with pregnant women who have already shown symptoms of PIH. In these women, we have determined the MR status as well as values of plasma aldosterone and its immediate precursor plasma l%hydroxycorticosterone (l&OH-B). Subjects and Methods Subjects We studied 25womenwith PIH with and without proteinuria and 25 healthy women with normal pregnancy. Blood samples in both groupswere taken in the third trimester. The diagnosis of PIH was based on the criterion of an elevated blood pressure (BP) read on at least three different occasions(systolic BP, >140 mm Hg; diastolic BP, >90 mm Hg). Mean arterial pressure, a valuable parameter (15) for elevated total peripheral resistancewas calculated accordingto the following formula: (systolic BP + 2 x diastolic BP)/3. Proteinuria wasfound in 15womenwith PIH, but not in normal pregnant women. Edema was present in 22 of 25 women with PIH. Normotensive women with extensive edema were excluded from the study. Details of obstetric technique and the 910
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/910/3004781 by Uppsala Universitetsbibliotek user on 10 January 2019
Mineralocorticoids and Mineralocorticoid Mononuclear Leukocytes in Patients with Induced Hypertension*
AND MR IN MNL
MINERALOCORTICOIDS
Methods The method for measuring MRs in MNL has been described by Armanini et al. (ll), and a modified method was used in our laboratory. Briefly, 50 mL peripheral blood were taken between 0800-1000 h after patients had been recumbent for at least 30 min. Blood was centrifuged at 1000 x g at 20 C for 10 min, and supernatant containing plasma was kept at 4 C for steroid determination (aldosterone and 18-OH-B). Cells were separated from blood on a Ficoll gradient, centrifuged for 40 min at 400 x g and 20 C, and washed three times with Hanks’ Balanced Salt Solution to eliminate endogenous steroids. Cell aliquots containing 3-6 millions cells/tube were incubated with increasing amounts of 3H-labeled aldosterone and a loo-fold excess of RU 28362 to exclude tracer binding from glucocorticoid receptors (16). Nonspecific binding was measured by adding a 200fold excess of nonlabeled aldosterone. After 1 h of incubation at 37 C, the reaction was stopped by adding 2 mL ice-cold saline, and then cells were washed three times. Finally, cells were transferred into scintillation vials, the radioactivity was counted, and the binding was subjected to Scatchard analysis (17). Endogenous steroids were measured by a RIA established in our laboratory. The techniques for chromatographic determination of plasma aldosterone and plasma 18-OH-corticosterone have been described previously (18, 19). The method for the determination of cortisol by ultrafiltration was adapted to TABLE 1. Technique and week of delivery of the women studied
Normal pregnancies (n = 25)
40(37-41)
1 Cesariansection 23 Spontaneouslabors
1 Forcepsdelivery PIH (n = 25)
35 (26-41)
determine aldosterone by ultrafiltration (20). Data were analyzed by Student’s t test and are specified as the mean and SEM.
Results The number of MR per cell as well as their affinity (Kd) were determined for both groups of women. MR per cell in MNL of women with normal pregnancies measured in their third trimester ranged from 200-494 sites/ cell (mean f SEM, 300 & 17). The number of MR in women with PIH ranged from 82-257 (mean + SEM, 148 f 9), a significantly lower level (P < 0.0005) than in the control group. The results are summarized in Table 2. No difference in MR affinity was seen between groups (PIH Kd, 1.0 f 0.1 nM; normal pregnancy, 1.2 f 0.1 KIM). Values of plasma aldosterone and plasma 18-OH-B are shown in Table 2 for PIH and normal pregnancies. Total aldosterone in PIH was 281 + 61 pmol/L (range, 8-1115 pmol/L), significantly lower (P < 0.025) than that in control pregnancies (697 f 172 pmol/L; range, 108-3107 pmol/L). Four of 25 aldosterone values in the latter group were higher than average. Within the PIH group and in the group of normal pregnant women there was no significant correlation between MR and plasma aldosterone concentration (r = -0.04 and r = 0.07, respectively; Fig. 1). Free aldosterone values, expressed as a percentage of the total aldosterone, did not show a significant difference. Free aldosterone, expressed as plasma concentration, differed significantly, in accordance with values of total aldosterone shown in Table 2. Like plasma aldosterone, plasma 18-OH-B was significantly (P < 0.025) lower in patients with PIH (1072 f 149 pmol/L; range, 50-2815 pmol/L) compared with that in normal pregnant women (1907 + 318 pmol/L; range, 375-3587 pmol/L). No determination of free l&OH-B was carried out.
14 Cesarian section
8 Spontaneouslabors 2 Intrauterine death 1 Vacuum extraction
Discussion It is known that plasma aldosterone levels are higher in pregnant than in nonpregnant women (21-23). It is
TABLE 2. Summary of the mean f SEM of BP, mineralocorticoids,and MR, in the two examined groups
Normal pregnancies Mean
+SEM
BP (mm Hg) Systolic 111 +2 Diastolic 71 +2 MAP (mm Hg) 85 f2 ALDO (pmol/L) 697 k171 FR ALDO absolute (pmol/L) 159 k42 FR ALDO (%) 26 18-OH-B (pmol/L) 1907 +318 MR (sites/cell) 300 f17 1.2 f0.1 Kd hM) MAP, Mean arterial pressure;BP, blood pressure;FR, fraction.
n
PIH P
25 25 24 20 20 23 25 25
