Integrated Concentration of Plasma Aldosterone in Normal Subjects: Correlation with Cortisol* AVINOAM KOWARSKI, LUIZ DE LACERDA, AND CLAUDE J. MIGEON Harriet Lane Service, Children's Medical and Surgical Center, The Johns Hopkins Hospital and University, Baltimore, Maryland 21205 were studied a second time while staying in bed most of the day was lower than the I.C. observed during normal activity. Furthermore, there was a significant correlation between 24-h I.C. and percentage of time spent in supine position, c) A weak, but significant correlation was found between the 30-min I.C. of aldosterone and cortisol in 4 out of the 7 subjects tested. The overall correlation for all experiments was also significant (R = 0.3, P < 0.001). d) The 4-h I.C. of aldosterone and cortisol showed that the lowest mean values were between 4 PM and 4 AM and the highest values between 4 AM and 4 PM for both steroids. (J Clin Endocrinol Metab 40: 205, 1975)
ABSTRACT. The integrated concentration (I.C.) of plasma aldosterone ' and cortisol was determined every 30 min during a 24-h period, using a blood collection system composed of a nonthrombogenic catheter and a small, portable withdrawal pump. The experiments were carried out in 8 normal adult men during daily routine life, and repeated in 2 of the subjects while recumbent in bed most of the day. The following conclusions were made: a) The 30-min I.C. of aldosterone fluctuated widely throughout the day. b) Although peaks of increased concentration occurred after a change in posture from supine to erect, there were many peaks of concentration that occurred during the supine posture. The 24-h I.C. of aldosterone in 2 subjects that
C
ORTISOL is secreted in man in a series of pulses of variable amplitude and duration (1-4). Aldosterone, another major steroid hormone of the human adrenal cortex, has also been reported by Katz et al. (5) to be secreted in similar fashion in two normal subjects who were studied every 20 min while staying in bed for 24 h. Although the number of subjects studied by Katz et al. (5) was too small for definite conclusions, it was suggested that the level of plasma cortisol, in the same blood samReceived July 30, 1974. Supported by Research Grants RO1-HD 06284-03, AM-00180-21, Traineeship Grant Tl-AM-5219, and Research Career Award 5K06-AM-21, 855 (CJM) of the National Institutes of Health, United States Public Health Service. The patients were studied at the Clinical Research Center of Pediatrics, supported by Grant 5MO1-RR-0052 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Dr. de Lacerda was supported in part by a Grant of Coordenacao Do Aperfeicoamento de Pessoal de Nivel Superior (Capes), M.E.C., Brasil. * This work was presented in part at the 12th meeting of the European Society for Paediatric Endocrinology 1973.
pies, was synchronous with the fluctuations of plasma aldosterone. Vagnucci et al. (6) published a somewhat similar study in 4 normal supine subjects during sodium restriction. These authors reported that aldosterone production rose with onset of sleep, while the frequency, timing and magnitude of its secretory episodes were variable during the awake hours. Furthermore, they observed that aldosterone levels were closely related to those of renin activity but were poorly correlated with those of cortisol and corticosterone (6). The purpose of this work was to study the fluctuations in the concentration of aldosterone in plasma during normal daily activity under normal sodium diet, to estimate the role of posture and to correlate these fluctuations with those of cortisol. Materials and Methods Subjects and blood collections Eight healthy male subjects ranging in age from 21 to 26 yr were studied. Three of them were laboratory technicians and the others were medical students or physicians.
205
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
206
KOWARSKI, DE LACERDA AND MIGEON
All subjects continued normal routine activity with sleep starting between midnight and 1 AM and ending between 7 and 9 AM. During the waking hours, the subjects were not confined to bed; instead they carried on their usual activities including their regular work. Two subjects (#7 and 8) were studied twice. During the repeat study they were asked to remain in bed supine or sitting for most of the time. Blood was collected through an intravenous, nonthrombogenic catheter inserted in an antecubital vein. The catheter was connected to a small, portable, withdrawal pump, by means of a 70 cm long tube, which permitted free movement of the subjects.* The pump was adjusted to withdraw blood at a constant rate of 3 ml/h. The first hour of collection was discarded. Subsequently blood was collected every 30 min for a 24-h period. During the waking period the pump was strapped to the chest of the subjects. During the sleeping period, the pump was set on the side of the bed, one of the investigators collecting the 30-min blood samples. Once obtained, the samples were stored temporarily at 4 C until six of them were available for centrifugation. The plasma was then stored at - 2 0 C until tested. The body posture of the subjects was recorded for every 30-min period. The times at which the subjects fell asleep and woke up were also noted. Determination of plasma aldosterone and cortisol concentrations The concentrations of plasma aldosterone and cortisol in the 48 samples collected on each subject were determined by a previously described radioimmunoassay (7,8) and a previously described competitive protein binding assay (9), respectively. Since aldosterone and cortisol concentrations were measured in samples collected continuously every 30 min, the values obtained represent the integrated concentration (I.C.) during that period. In addition, the 30-min I.C. were combined in order to obtain 4-h I.C. for the following periods: 1/ Midnight to 4 AM, 11/ 4 AM to 8 AM, III/ 8 AM to noon, IV/ noon to 4 PM, VI 4 PM to 8 PM and VI/ 8 PM to midnight. Finally, the mean of all * This system was purchased from Sigmamotor, Inc., 14 Elizabeth Street, Middleport, New York 14105.
JCE & M • 1975 Vol 40 • No 2
samples was calculated in order to obtain the value of the 24-h I.C. Stability of cortisol and aldosterone within the pump system The stability of cortisol within the pump system has been studied and reported previously (4). In order to determine if the system employed for collection of blood would change the actual concentration of plasma aldosterone in the 30-min samples, the following experiment was undertaken: 2 x 106 cpm of 3H-aldosterone was added to 40 ml of heparinized blood contained in a glass beaker which was placed on a horizontal shaker at room temperature. The tip of an intravenous catheter was immersed in the blood and the other end was connected to the pump adjusted to withdraw 3 ml/h. Six 1-h samples were obtained in this fashion. Simultaneously, at the end of collection of each sample through the pump, one corresponding sample was directly pipetted out of the beaker. The blood was centrifuged and the plasma stored at —20 C until processing. To 1 ml of plasma, a tracer amount of 14 C-aldosterone was added before extraction, for correction of losses during extraction and chromatography. The plasma was extracted twice with 10 ml of dichloromethane. The solvent was washed twice with 2 ml of O.lN NaOH and once with 2 ml of O.lN HC1 and then evaporated. The residue was dissolved in methanol and 10 /xg of nonradioactive aldosterone was added to it. The solution was chromatographed in the Bush System Benzene: Methanol: Water (2:1:1) for a period of 5 h, at 37 C. The aldosterone was visualized under UV light and the area cut off. The paper strip was eluted with methanol in a counting vial, the eluate was dried with a current of air at 37 C, and 10 ml of a solution of PPO-POPOP was added. The amount of 3H and 14C-aldosterone was counted as previously described (10), in a Nuclear Chicago liquid scintillation counter, Mark II. The concentration of 3H-aldosterone (cpm/ml plasma) in the six samples collected after passage through the tubing and pump system was constant (linear regression not statistically different from zero) and similar to that observed in samples collected directly from the beaker.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
INTEGRATED CONCENTRATION OF PLASMA ALDOSTERONE
207
FIG. 1. Fluctuations of I.C. of plasma aldosterone in three normal subjects. Black areas represent period of sleep.
MN
Results and Discussion Figure 1 shows the variations of the I.C. of three normal men chosen as representative examples of our findings. The levels of aldosterone fluctuated with no apparent
6AM
I2N
6PM
MN
pattern, several variable peaks being observed. Often, the levels of aldosterone rose during sleep, a finding which is contrary to what would be expected during supine position (7). This feature can also be seen in Figs. 2 and 3.
10-
FIG. 2. Fluctuations of I.C. of plasma aldosterone in subject #6. The study was carried out on two occasions, once during normal activity, then when in bed during most of the day. The body posture during each 30-min period is indicated by the shading and explained in the figure. The black areas represent periods of sleep; the heavy shaded bars are periods when the subject was awake but supine; the light shaded bars are periods when the subject was sitting and the white bars when he was standing and walking.
6AM
12 N
6PM
MN
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1975 Vol 40 • No 2
KOWARSKI, DE LACERDA AND MIGEON
208
FIG. 3. Fluctuations of the I.C. of plasma aldosterone and cortisol in subject # 3 who showed significant correlation between the I.C. of the two steroids. However, a number of subjects did not show any correlation.
MN
6AM
6PM
I2N
An example of the relationship between the level of plasma aldosterone and the body posture assumed by the experimental subject is given in Fig. 2. This figure represents the result of two separate studies in the same individual. The first study was carried out during normal daily activity of the subject who is a laboratory technician. The second study was carried out with the subject spending most of the day in bed, mainly in a recumbent position. Changes in posture were allowed only after completion of a 30-min blood collection, and once assumed the posture was maintained throughout the next period. The four
MN
basic body postures are shown in Fig. 2. Of the several peaks of aldosterone observed in this individual, only one could be related to the assumption of the erect posture. Several peaks occurred while in bed, including during sleep. This is in agreement with the finding of Katz et al. (5) and Vagnucci et al. (6) who reported an elevation of the plasma aldosterone concentrations during sleep in the subjects studied. Although no consistent immediate effect of posture was detected in the ten experiments that we have conducted, the 24-h I.C. of aldosterone of the two subjects that were studied twice (#6 and #7) was
TABLE 1. Correlation between the 30-min I.C. of aldosterone and cortisol in plasma and 24-hr I.C. of aldosterone and cortisol Correlation 30-min I.C. al do.-cortisol
24-hr I.C.
Subject no.
Percent supine
R
P two-tailed
Aldosterone ng/100 ml
Cortisol /xg/100 ml
1
6A Active 6B Supine 7A Active 7B Supine
53% 42% 60% 62% 43% 35% 69% 30% 81%
0.02 0.35 0.62 0.35 0.16 0.44 0.67 0.16 0.16
NS
ABSTRACT. The integrated concentration (I.C.) of plasma aldosterone ' and cortisol was determined every 30 min during a 24-h period, using a blood collection system composed of a nonthrombogenic catheter and a small, portable withdrawal pump. The experiments were carried out in 8 normal adult men during daily routine life, and repeated in 2 of the subjects while recumbent in bed most of the day. The following conclusions were made: a) The 30-min I.C. of aldosterone fluctuated widely throughout the day. b) Although peaks of increased concentration occurred after a change in posture from supine to erect, there were many peaks of concentration that occurred during the supine posture. The 24-h I.C. of aldosterone in 2 subjects that
C
ORTISOL is secreted in man in a series of pulses of variable amplitude and duration (1-4). Aldosterone, another major steroid hormone of the human adrenal cortex, has also been reported by Katz et al. (5) to be secreted in similar fashion in two normal subjects who were studied every 20 min while staying in bed for 24 h. Although the number of subjects studied by Katz et al. (5) was too small for definite conclusions, it was suggested that the level of plasma cortisol, in the same blood samReceived July 30, 1974. Supported by Research Grants RO1-HD 06284-03, AM-00180-21, Traineeship Grant Tl-AM-5219, and Research Career Award 5K06-AM-21, 855 (CJM) of the National Institutes of Health, United States Public Health Service. The patients were studied at the Clinical Research Center of Pediatrics, supported by Grant 5MO1-RR-0052 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Dr. de Lacerda was supported in part by a Grant of Coordenacao Do Aperfeicoamento de Pessoal de Nivel Superior (Capes), M.E.C., Brasil. * This work was presented in part at the 12th meeting of the European Society for Paediatric Endocrinology 1973.
pies, was synchronous with the fluctuations of plasma aldosterone. Vagnucci et al. (6) published a somewhat similar study in 4 normal supine subjects during sodium restriction. These authors reported that aldosterone production rose with onset of sleep, while the frequency, timing and magnitude of its secretory episodes were variable during the awake hours. Furthermore, they observed that aldosterone levels were closely related to those of renin activity but were poorly correlated with those of cortisol and corticosterone (6). The purpose of this work was to study the fluctuations in the concentration of aldosterone in plasma during normal daily activity under normal sodium diet, to estimate the role of posture and to correlate these fluctuations with those of cortisol. Materials and Methods Subjects and blood collections Eight healthy male subjects ranging in age from 21 to 26 yr were studied. Three of them were laboratory technicians and the others were medical students or physicians.
205
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
206
KOWARSKI, DE LACERDA AND MIGEON
All subjects continued normal routine activity with sleep starting between midnight and 1 AM and ending between 7 and 9 AM. During the waking hours, the subjects were not confined to bed; instead they carried on their usual activities including their regular work. Two subjects (#7 and 8) were studied twice. During the repeat study they were asked to remain in bed supine or sitting for most of the time. Blood was collected through an intravenous, nonthrombogenic catheter inserted in an antecubital vein. The catheter was connected to a small, portable, withdrawal pump, by means of a 70 cm long tube, which permitted free movement of the subjects.* The pump was adjusted to withdraw blood at a constant rate of 3 ml/h. The first hour of collection was discarded. Subsequently blood was collected every 30 min for a 24-h period. During the waking period the pump was strapped to the chest of the subjects. During the sleeping period, the pump was set on the side of the bed, one of the investigators collecting the 30-min blood samples. Once obtained, the samples were stored temporarily at 4 C until six of them were available for centrifugation. The plasma was then stored at - 2 0 C until tested. The body posture of the subjects was recorded for every 30-min period. The times at which the subjects fell asleep and woke up were also noted. Determination of plasma aldosterone and cortisol concentrations The concentrations of plasma aldosterone and cortisol in the 48 samples collected on each subject were determined by a previously described radioimmunoassay (7,8) and a previously described competitive protein binding assay (9), respectively. Since aldosterone and cortisol concentrations were measured in samples collected continuously every 30 min, the values obtained represent the integrated concentration (I.C.) during that period. In addition, the 30-min I.C. were combined in order to obtain 4-h I.C. for the following periods: 1/ Midnight to 4 AM, 11/ 4 AM to 8 AM, III/ 8 AM to noon, IV/ noon to 4 PM, VI 4 PM to 8 PM and VI/ 8 PM to midnight. Finally, the mean of all * This system was purchased from Sigmamotor, Inc., 14 Elizabeth Street, Middleport, New York 14105.
JCE & M • 1975 Vol 40 • No 2
samples was calculated in order to obtain the value of the 24-h I.C. Stability of cortisol and aldosterone within the pump system The stability of cortisol within the pump system has been studied and reported previously (4). In order to determine if the system employed for collection of blood would change the actual concentration of plasma aldosterone in the 30-min samples, the following experiment was undertaken: 2 x 106 cpm of 3H-aldosterone was added to 40 ml of heparinized blood contained in a glass beaker which was placed on a horizontal shaker at room temperature. The tip of an intravenous catheter was immersed in the blood and the other end was connected to the pump adjusted to withdraw 3 ml/h. Six 1-h samples were obtained in this fashion. Simultaneously, at the end of collection of each sample through the pump, one corresponding sample was directly pipetted out of the beaker. The blood was centrifuged and the plasma stored at —20 C until processing. To 1 ml of plasma, a tracer amount of 14 C-aldosterone was added before extraction, for correction of losses during extraction and chromatography. The plasma was extracted twice with 10 ml of dichloromethane. The solvent was washed twice with 2 ml of O.lN NaOH and once with 2 ml of O.lN HC1 and then evaporated. The residue was dissolved in methanol and 10 /xg of nonradioactive aldosterone was added to it. The solution was chromatographed in the Bush System Benzene: Methanol: Water (2:1:1) for a period of 5 h, at 37 C. The aldosterone was visualized under UV light and the area cut off. The paper strip was eluted with methanol in a counting vial, the eluate was dried with a current of air at 37 C, and 10 ml of a solution of PPO-POPOP was added. The amount of 3H and 14C-aldosterone was counted as previously described (10), in a Nuclear Chicago liquid scintillation counter, Mark II. The concentration of 3H-aldosterone (cpm/ml plasma) in the six samples collected after passage through the tubing and pump system was constant (linear regression not statistically different from zero) and similar to that observed in samples collected directly from the beaker.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
INTEGRATED CONCENTRATION OF PLASMA ALDOSTERONE
207
FIG. 1. Fluctuations of I.C. of plasma aldosterone in three normal subjects. Black areas represent period of sleep.
MN
Results and Discussion Figure 1 shows the variations of the I.C. of three normal men chosen as representative examples of our findings. The levels of aldosterone fluctuated with no apparent
6AM
I2N
6PM
MN
pattern, several variable peaks being observed. Often, the levels of aldosterone rose during sleep, a finding which is contrary to what would be expected during supine position (7). This feature can also be seen in Figs. 2 and 3.
10-
FIG. 2. Fluctuations of I.C. of plasma aldosterone in subject #6. The study was carried out on two occasions, once during normal activity, then when in bed during most of the day. The body posture during each 30-min period is indicated by the shading and explained in the figure. The black areas represent periods of sleep; the heavy shaded bars are periods when the subject was awake but supine; the light shaded bars are periods when the subject was sitting and the white bars when he was standing and walking.
6AM
12 N
6PM
MN
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 02:53 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1975 Vol 40 • No 2
KOWARSKI, DE LACERDA AND MIGEON
208
FIG. 3. Fluctuations of the I.C. of plasma aldosterone and cortisol in subject # 3 who showed significant correlation between the I.C. of the two steroids. However, a number of subjects did not show any correlation.
MN
6AM
6PM
I2N
An example of the relationship between the level of plasma aldosterone and the body posture assumed by the experimental subject is given in Fig. 2. This figure represents the result of two separate studies in the same individual. The first study was carried out during normal daily activity of the subject who is a laboratory technician. The second study was carried out with the subject spending most of the day in bed, mainly in a recumbent position. Changes in posture were allowed only after completion of a 30-min blood collection, and once assumed the posture was maintained throughout the next period. The four
MN
basic body postures are shown in Fig. 2. Of the several peaks of aldosterone observed in this individual, only one could be related to the assumption of the erect posture. Several peaks occurred while in bed, including during sleep. This is in agreement with the finding of Katz et al. (5) and Vagnucci et al. (6) who reported an elevation of the plasma aldosterone concentrations during sleep in the subjects studied. Although no consistent immediate effect of posture was detected in the ten experiments that we have conducted, the 24-h I.C. of aldosterone of the two subjects that were studied twice (#6 and #7) was
TABLE 1. Correlation between the 30-min I.C. of aldosterone and cortisol in plasma and 24-hr I.C. of aldosterone and cortisol Correlation 30-min I.C. al do.-cortisol
24-hr I.C.
Subject no.
Percent supine
R
P two-tailed
Aldosterone ng/100 ml
Cortisol /xg/100 ml
1
6A Active 6B Supine 7A Active 7B Supine
53% 42% 60% 62% 43% 35% 69% 30% 81%
0.02 0.35 0.62 0.35 0.16 0.44 0.67 0.16 0.16
NS
