Short Term Cardiovascular Effects of Aldosterone in Healthy Male

0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society

Vol. 84, No. 10 Printed in U.S.A.

Short Term Cardiovascular Effects of Aldosterone in Healthy Male Volunteers* B. M. W. SCHMIDT, A. MONTEALEGRE, C. P. JANSON, N. MARTIN, C. STEIN-KEMMESIES, A. SCHERHAG, M. FEURING, M. CHRIST, AND M. WEHLING Institute of Clinical Pharmacology (B.M.W.S., A.M., C.P.J., N.M., C.S.-K., M.F., M.C., M.W.) and First Medical Clinic (A.S.), University Hospital of Mannheim, Faculty of Clinical Medicine, University of Heidelberg, 68135 Mannheim, Germany ABSTRACT Clinical evidence of rapid, nongenomic aldosterone effects in the cardiovascular system has been provided by clinical studies; an increase in systemic vascular resistance (SVR) was shown by invasive techniques within 3 min after injection of aldosterone. Here, we study the dose dependency and the later course of the rapid aldosterone effects by noninvasive techniques. In 12 healthy male volunteers, SVR and heart rate variability were determined by impedance cardiography and digital electrocardiography, respectively, for 8 h after the injection of 0.05 or 0.5 mg aldosterone in a double blind, placebo-controlled, 3-fold cross-over study. No significant differences were observed for baseline values among the three treatments. The area under the curve of SVR during the first 45 min after injection was significantly different between the periods with the highest areas under the curve seen after the injection of 0.5 mg aldosterone

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ARDIOVASCULAR aldosterone effects have been commonly understood to be secondary to sodium and water retention due to genomic renal action of the hormone (for review, see Ref. 1). During the last 10 yr, evidence has been generated that aldosterone may also exert direct vascular effects, which are mediated by nongenomic mechanisms. Such effects are mainly characterized by their fast onset (rapid steroid effects) and their insensitivity to inhibition of DNA transcription or protein synthesis, e.g. by actinomycin D or cycloheximide (2). In vascular smooth muscle cells and porcine aortic endothelial cells, intracellular calcium concentrations are increased by physiological concentrations of aldosterone within 1 min. This nongenomic steroid effect seems to involve an initial calcium release from intracellular stores (seen to be predominant in vascular smooth muscle cells) followed by a secondary influx of extracellular calcium (predominant in porcine aortic endothelial cells) (3, 4). Even though rapid nongenomic aldosterone effects have been demonstrated consistently in several in vitro studies, few clinical studies have been performed to date to address the issue of the clinical relevance of these rapid effects. The Received December 14, 1998. Revision received April 2, 1999. Rerevision received June 16, 1999. Accepted June 25, 1999. Address all correspondence and requests for reprints to: Martin Wehling, M.D., Faculty of Clinical Medicine, University of Heidelberg, Theodor-Kutzer-Ufer, 68135 Mannheim, Germany. * This work was supported by the German Bundesministerium fu¨r Bildung, Wissenschaft, Forschung, und Technologie (FKZ 01 EC 9407/8).

(mean 6 SD, 40.4 6 12.8 vs. 36.8 6 10.3 for 0.05 mg aldosterone and 36.8 6 10.4 for placebo; P 5 0.05). Individual comparisons showed significant differences at 6 and 30 min between placebo and the 0.5 mg aldosterone period (P , 0.05), with values for the 0.05 mg aldosterone period similar to those for the placebo period. From 330 –390 min, opposite changes occurred; SVR was depressed during the 0.05 mg (P , 0.05) and 0.5 mg aldosterone periods compared with that during the placebo period. These delayed effects may reflect an increased vagal tone in the aldosterone groups, as demonstrated by higher values of the time domain parameter of heart rate variability pNN50. This study provides further evidence for clinically detectable rapid cardiovascular aldosterone effects in vivo obtained by noninvasive techniques. The data are consistent with the view of aldosterone as a rapid modulator of cardiovascular responses acting through nongenomic mechanisms. (J Clin Endocrinol Metab 84: 3528 –3533, 1999)

first clinical study dates back to 1963, when Klein and Henk demonstrated an increase in peripheral vascular resistance and blood pressure as well as a decrease in cardiac output 5 min after the application of 1 mg aldosterone (5). These findings were confirmed in a recent study by our group (6), in which an increase in peripheral vascular resistance was demonstrated within 3 min after application of 1 mg aldosterone by invasive modern methods (cardiac catheterization). In a placebo-controlled, randomized clinical trial, Zange et al. (7) showed by nuclear magnetic resonance spectroscopy a facilitated phosphocreatine recovery in calf muscles after stress (isometric exercise) within 8 min after iv administration of 0.5 mg aldosterone. To further characterize the clinical relevance of cardiovascular rapid nongenomic aldosterone effects, the present double blind, placebo-controlled, randomized 3-fold cross-over study was performed. The main aims were to evaluate the extended time course of the observed rapid aldosterone effects and the dose dependency of these effects. To assess hemodynamic parameters and heart rate variability after placebo vs. 0.05 and 0.5 mg aldosterone injection in healthy volunteers, noninvasive methods of impedance cardiography (ICG) and digital electrocardiography (ECG) were used, respectively. Subjects and Methods Study volunteers Twelve healthy male volunteers were included in the study. All volunteers were subjected to a medical examination within 2 weeks

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CARDIOVASCULAR ALDOSTERONE EFFECTS before inclusion into the study. The examination consisted of medical history, physical examination, 12-lead ECG, and determination of clinical laboratory parameters. All subjects gave their written informed consent to participate in the study.

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(HF) bands of heart rate variability was calculated by fast Fourier transformation at intervals of 256-s duration. Sympathico-vagal balance was assessed by the LF/HF ratio (10). Aldosterone levels in plasma were measured by a commercial magnetic affinity immunoassay (Biodata, Rome, Italy). The limit of detection of this assay is 6 pg/ml. Interassay variance is 4.1%, and intraassay variance is 3.5%.

Study procedures The study was designed as a randomized, double blind, placebocontrolled, 3-fold cross-over trial. It was conducted according to the guidelines for good clinical practice (ICH-GCP) and the Declaration of Helsinki. The study was approved by the ethical committee of the Faculty for Clinical Medicine Mannheim, University of Heidelberg (Heidelberg, Germany). All 12 enrolled volunteers were randomly subjected to three test periods lasting 8 h each, in which noninvasive measurements of cardiovascular parameters were performed using ICG (Cardioscreen, Medis GmbH, Ilmenau, Germany) and digital ECG (Synesis, ELA Medical, Munich, Germany) before and after application of 0.05 or 0.5 mg aldosterone or placebo, iv. Tests took place on 3 different days, with a minimum wash-out interval of 48 h. Volunteers were hospitalized at 1900 h the day before the study procedure. Urine sampling was begun at 1930 h, and the subjects received a standardized dinner. The complete dietary schedule is given in Table 1. The volunteers stayed overnight at the phase I unit of the institute. Overnight urine sampling was stopped at 0730 h by emptying the bladder completely. After a standard breakfast, study procedures were started by bringing volunteers into the supine position and inserting two indwelling catheters into forearm veins, one for drug or placebo injection and one for blood sampling. After a 30-min resting period, baseline data were obtained. Then aldosterone (0.05 and 0.5 mg) or placebo was injected into one of the catheters within 1 min, and cardiovascular parameters were measured continuously. At 3, 6, 10, 15, 20, 30, 45, and 60 min and every 30 min thereafter, blood samples for determination of the plasma aldosterone level were collected and immediately transferred on ice. During the test volunteers remained in the supine position and were only allowed to stand up three times: twice for urination at 150 and 390 min and once for taking a light standard lunch at 270 min. Urine was sampled again until 1630 h. Vital parameters were permanently monitored throughout the test. Blood pressure and heart rate were measured at the same time points (Omnicare CS24, Hewlett-Packard Co., Boblingen, Germany), as were ICG and heart rate variability (HRV). At the beginning of each test day, routine laboratory examinations (electrolytes, transaminases, urea, and creatinine) were performed using routine methods. After measuring urinary volumes, aliquots from both sampling periods were analyzed (potassium, sodium, and creatinine) using routine methods. Aldosterone for human administration was prepared according to the original recipe of the formerly registered drug Aldocorten (Ciba-Geigy, Basel, Switzerland), which became clinically unavailable only recently. The placebo was an isotonic (0.9%) NaCl solution. ICG was performed using standard methods as described previously (8). Stroke volume (SV) was computed by Bernstein’s formula (9). Cardiac output (CO) was calculated as SV 3 HR, and SVR as 80 3 (MAP 2 3)/CO (HR 5 heart rate; MAP 5 mean arterial pressure). Time domain parameters of HRV were calculated at 1-min intervals. pNN50, the percentage of interval differences of successive normal beat-to-beat intervals longer than 50 ms, was chosen as the relevant time domain parameter and used in study analyses. Power spectral density for the low frequency (LF) and high frequency

Statistical methods The statistical analysis was performed using SAS version 6.12 (SAS Institute, Inc., Cary, NC). As there was no drop out, the evaluation of data was performed on the complete set of study volunteers included. The data are given as the mean 6 sd. All variables of ICG, HRV, and urine parameters were evaluated in the following way. The effect of aldosterone compared to that of placebo was assessed at each time point by a multifactorial ANOVA, with the factors treatment, period, volunteer, and treatment-period interaction. This method takes into account the multiple measurements in the 3-fold cross-over design while also testing for eventual carryover effects (treatment-period interaction). In addition to analysis of single time points, the effect of aldosterone was evaluated by the area under the curve (AUC) method for SVR during the first 45 min after application. In the figures, ses are given that were estimated as pooled ses based on the sampling error adjusted for the above model. The level of significance was set at a 5 0.05. A descriptive evaluation was made for the safety parameters. Changes from screening to the end of the study were tested by the Wilcoxon test for paired data. Treatment differences concerning the occurrence of supraventricular and ventricular premature beats were compared by the nonparametric Friedman test. The pharmacokinetics were presented as time-concentration profile for each treatment group. The elimination half-life was estimated by fitting of a log-linear model.

Results

Important clinical characteristics, demographic features, and laboratory parameters are shown in Table 2. No significant differences between screening and the end of the study were detected. At baseline (0 min) no significant differences among the three treatment groups were found in any parameter. Moreover, no carryover effects could be detected. Pharmacodynamic data

The AUCs of SVR over the first 45 min were significantly different among the three periods (P 5 0.05). The highest value was obtained after injection of 0.5 mg aldosterone (mean 6 sd, 40.4 6 12.8) compared with those during the 0.05 mg aldosterone period (36.8 6 10.3) and the placebo period (36.8 6 10.4). The analysis at 6 and 30 min after the start of injection revealed significant differences in SVR between placebo and the 0.5 mg aldosterone treatment. Values during the 0.05 mg aldosterone treatment period were similar to those during the placebo period, but the differences compared to the 0.5 mg aldosterone period were statistically

TABLE 1. Dietary schedule Proteine (g/kg BW)

Fat (g/kg BW)

Carbohydrate (g/kg BW)

Water (mL/kg BW)

K1 (mg/kg BW)

Na1 (mg/kg BW)

Ca1 (mg/kg BW)

9.5

0.5

0.4

8.5

0.2

0.4

1.0 2.1 1.1

8.6

0.7

0.1

1.1

26.6

1.4

0.9

5.3

9.0 7.0 4.7 7 13.3 3.6 44.6

15.2 5.2 3.3 0.1 31.1 3.6 58.5

21.7 4.4 10.7 2.5 3.7 0.6 43.6

4.7 0.9 0.6 0.3 2.8 0.4 9.7

Cal/kg BW

Dinner At night Breakfast At 1st break Lunch At 2nd break Total

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significant at 30 min. Figure 1 shows the course of SVR during the entire test. At the same time points cardiac output was lower in the 0.5 mg aldosterone period than in the placebo and 0.05 mg aldosterone periods; differences between the 0.5 and 0.05 mg aldosterone periods were statistically significant at 30 min. Opposite significant differences in SVR were observed at 330, 360, and 390 min between placebo and 0.05 mg aldosterone treatment (Fig. 1). Differences in mean arterial pressure were only significant at 30 and 450 min between placebo and 0.5 mg aldosterone periods (Fig. 2). Heart rate was not different between groups at any time point. Concerning HRV parameters, pNN50 was significantly higher in the 0.5 mg aldosterone period than in the placebo period at 15, 210, 240, and 300 min. At 240 min the 0.05 mg aldosterone period was also different from the placebo period (Fig. 3). LF/HF ratios were not different between groups (not shown). During the test supraventricular and ventricular premature beats were observed. Nevertheless, these findings were sporadic, and a statistical significance for a treatment effect could not be established. Laboratory urine test results are shown in Table 3. No significant treatment differences were found for these data. Pharmacokinetics

The mean plasma aldosterone concentrations during the 0.5 and 0.05 mg aldosterone treatments were 145 6 69 and TABLE 2. Demographic data of the 12 volunteers Screening

Values are the mean 6

End of study

25.2 6 2.6 182.8 6 8.11 80.0 6 15.0 141.3 6 1.4 140.7 6 1.1 4.1 6 0.2 4.0 6 0.3 29.0 6 9.8 27.5 6 6.5 1.0 6 0.1 1.1 6 0.1

Age (yr) Ht (cm) Wt (kg) Sodium (mmol/L) Potassium (mmol/L) Urea (mg/dL) Creatinine (mg/dL) SD.

FIG. 1. Time profiles of systemic vascular resistance (mean 6 SEM) in 12 volunteers receiving placebo or 0.05 or 0.5 mg aldosterone, and results of ANOVA: P , 0.05 between placebo and 0.5 mg aldosterone (*), between the two aldosterone groups (#), or between placebo and 0.05 mg aldosterone (†).

133 6 35 pg/mL at baseline, 7430 6 2460 and 1910 6 1240 at 3 min after injection, and 1890 6 666 and 495 6 245 at 10 min after injection. The corresponding values in the placebo group were 150 6 45, 132 6 36, and 120 6 32 (Fig. 4). The elimination half-life was estimated to be 27 min (0.5 mg period) or 39 min (0.05 mg period). Discussion

This study provides further insights into the rapid nongenomic cardiovascular effects of pharmacological aldosterone doses in humans. Similar to the antecedent study of our group (6), we were able to demonstrate a higher SVR within 5 min after iv injection of 0.5 mg aldosterone. This effect persisted and was statistically significant again after 30 min. In addition, the analysis of AUC during the first 45 min after injection, a time frame that at large allows only for nongenomic effects to occur, showed a significant effect of aldosterone. After 30 min even mean arterial pressure was higher during the 0.5 mg aldosterone period than during the placebo period. These data support our previous study, in which SVR was assessed 3 and 10 min after injection of 1 mg aldosterone by invasive methods (cardiac catheterization) (6). We show here that this rapid effect lasts for about 30 min. In the later course there were no differences for SVR, which increased slightly in all periods until the lunch break. Thereafter, a postprandial vasodilatation was observed, which was more pronounced in both aldosterone periods. This later effect cannot be claimed to be nongenomic in nature, because the time course would also allow for early genomic effects to occur. Traditionally, these genomic effects would be expected to be mediated by retention of sodium and water (11). This, though, could not be observed in the urine examination, which showed no differences among the three groups during the course of the study. However, it should be pointed out that the study was not designed to rule out genomic effects by urinary examination. Urine was pooled for 9 h after the injection of aldosterone. Therefore, aldosterone action could have been effective during part of the sampling period only and thus overlooked in the whole sample. On the other hand, there are no reports in literature on vasodilatory genomic

CARDIOVASCULAR ALDOSTERONE EFFECTS

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FIG. 2. Time profiles of mean arterial pressure (mean 6 SEM) in 12 volunteers receiving placebo or 0.05 or 0.5 mg aldosterone, and results of ANOVA: P , 0.05 between placebo and 0.5 mg aldosterone (*).

FIG. 3. Time profiles of pNN50 (mean 6 SEM) in 12 volunteers receiving placebo or 0.05 or 0.5 mg aldosterone, and results of ANOVA: P , 0.05 between placebo and 0.5 mg aldosterone (*) or between placebo and 0.05 mg aldosterone (†).

TABLE 3. Urine analysis

At night before testing Vol (mL/h) Fractional sodium excretion (%) Fractional potassium excretion (%) Creatinine clearance (mL/min) Na1/K1 ratio During the test Vol (mL/h) Fractional sodium excretion (%) Fractional potassium excretion (%) Creatinine clearance (mL/min) Na1/K1 ratio Values are the mean 6

SD.

Placebo

Aldosterone (0.05 mg)

Aldosterone (0.5 mg)

94.9 6 34.3 1.1 6 0.3 14.6 6 6.8 115.6 6 21.8 3.2 6 1.7

94.7 6 35.7 0.7 6 0.2 12.8 6 5.5 129.0 6 22.5 2.4 6 1.0

100.7 6 43.6 1.0 6 0.3 14.7 6 5.4 122.4 6 35.0 2.8 6 1.0

101.3 6 40.5 1.0 6 0.6 19.6 6 8.1 117.7 6 19.7 1.8 6 0.7

114.2 6 74.5 1.1 6 0.5 23.0 6 10.6 109.8 6 28.1 1.9 6 0.7

98.8 6 44.0 1.0 6 0.3 21.8 6 6.0 121.2 6 48.5 1.7 6 0.7

No significant differences between the periods were found.

effects of aldosterone. May and Bednarik (12) showed instead that after application of aldosterone (10 mg/h) for 5 days in conscious sheep, no changes in peripheral resistance occurred, but aldosterone rather caused a mesenteric vasoconstriction. Another possible explanation is that the effects observed

in our study might be contraregulatory to the nongenomic effects observed on SVR and blood pressure. However, this interpretation cannot explain why these vasodilatory effects are more pronounced during the 0.05 mg aldosterone period. Contraregulatory effects should mirror the original effects. The differences between the two aldosterone periods were

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FIG. 4. Time profiles of aldosterone concentrations (mean 6 SEM) in 12 volunteers receiving placebo or 0.05 or 0.5 mg aldosterone.

not statistically significant, and the paradoxically greater late effect of 0.05 mg aldosterone compared with 0.5 mg aldosterone may just reflect a random order. The mechanism of these late effects remains elusive. The explanation indicating a nongenomic aldosterone action that is dependent on the autonomic nervous system, as discussed below, seems favorable, but this study warrants no conclusive interpretation of this late effect. The latter assumption would be in line with aldosterone effects on pNN50. pNN50 is an indirect measure of vagal tone (13). It represents the percentage of interval differences in successive normal beat-to-beat intervals longer than 50 ms. pNN50 has been shown to be a predictive marker for prognosis in patients suffering from chronic heart failure (14) and showed excellent reproducibility in healthy volunteers (15). pNN50 and, thus, presumably vagal tone were increased during the aldosterone periods throughout the study, with the difference being statistically significant at 15, 210, 240, and 300 min. Accordingly, the late effect of aldosterone on SVR might be the result of a nongenomically mediated (early onset within 15 min) increase in vagal tone during the aldosterone periods. This increase becomes sufficiently pronounced only during the postprandial phase, leading to the excessive postprandial vasodilatation. With vasoconstriction seen in SVR, on the one hand, and vasodilatation as seen in the postprandial situation, on the other hand, aldosterone could be termed a general amplifier of cardiovascular regulation. This idea was discussed in the very early work of Klein and Henk (5), who showed a dependency of aldosterone effects on SVR on the baseline value of CO; volunteers with normal CO tended to show a decrease in SVR, whereas in volunteers with lower CO, an increase in SVR was observed in this open uncontrolled study. The hypothesis of a general cardiovascular amplifier is supported by recent data in a supplement to the study performed by Zange et al. (7). In latter study, a rapidly facilitated phosphocreatine recovery after isometric contraction in the human calf caused by aldosterone (0.5 mg) could be dem-

onstrated. The succeeding study shows that hypoxia suppresses this effect completely (16). This effect may be interpreted as an indication that the hypoxia-induced vasodilation overruns that induced by aldosterone and thus blunts the additional vasodilatory effect of aldosterone. The concentrations of aldosterone in plasma that are reached after application of aldosterone are supraphysiological or even pharmacological. However, they are still far lower than concentrations expected to induce unspecific membrane effects ($10 mmol/L) (1). Furthermore, there is evidence for the local production of aldosterone in heart; thus, local aldosterone levels were estimated to be about 20-fold higher in heart tissue in rats than in plasma (17). In addition, there is evidence for aldosterone synthesis in rats in blood vessels (18), although no extrapolations can be made at present as to whether these results are relevant for humans. The specificity of the effects observed in this study could be tested by the use of biologically inactive analogs of aldosterone. These should also be inactive in our system if specific membrane receptors are involved in the effects observed here. The existence of a physiological in vivo membranemediated effect of aldosterone remains to be proven. In conclusion, rapid nongenomic aldosterone effects can be demonstrated in this study by noninvasive methods not only on SVR, but also on systemic blood pressure and autonomic control. On the basis of these results, it may be assumed that aldosterone induces cardiovascular responses that depend on baseline activation, e.g. by the autonomic nervous system, and thus may be diverse due to contrasting preinterventional hemodynamic states. Further studies are necessary to prove this hypothesis and to decide whether the effects of aldosterone seen in this study require pharmacological doses or may even occur to a minor extent at physiological concentrations. Acknowledgments We thank M. Krautkra¨mer and E. Kirsch for expert technical assistance.

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