Europ.J.clin.Invest. 6, 51-57 (1976)
Effect on Plasma Aldosterone, Renin Activity and Cortisol of Acute Volume Depletion Induced by Ethacrynic Acid under Constant Infusion of Angiotensin I1 and Dexamethasone in Man R.C. Gaillard, U. Merkelbach, A.M. Riondel, M.B. Vallotton, and A . F . Muller Division of Endocrinology, Department of Medicine, H6pital Cantonal, Geneva, Switzerland Received: February 6 , 1975, and in revised form: June 13, 1975
Abstract. The influence on plasma aldosterone of acute volume depletion induced by ethacrynic acid was studied in man. The experiments were performed during the morning in supine healthy males receiving a control infusion of 5 Z glucose or an infusion of angiotensin I1 (AII) to suppress endogenous renin production or an infusion of dexamethasone to suppress endogenous ACTH. Ethacrynic acid induced in all circumstances a similar diuresis and volume depletion. The rise of plasma renin activity (PRA) was effectively suppressed by A11 and the rise of plasma cortisol by dexamethasone. Plasma aldosterone (PA) rose markedly even when the elevation of PRA or cortisol were suppressed. Yet when both endogenous renin and ACTH secretion were blocked, PA rose much less after ethacrynic acid. This residual increase could be attributed mainly to a decrease of the metabolic clearance rate (MCR) of aldosterone which had been measured before and after ethacrynic acid administration. The data presented indicate that multiple factors influencing PA after acute volume depletion could be dissected out and that renin, ACTH and a decrease of the MCR each contribute to the.elevation of PA. Key words: Renin-angiotensin system, ACTH, metabolic clearance rate, radioimmunoassays, potassium, dexamethasone.
Introduction The importance of the extracellular fluid volume or of one of its compartments in the regulation of aldosterone secretion has been widely recognized. In man the renin-angiotensin system is generally considered to be the dominant factor regulating aldosterone when extracellular volume is altered by changes in sodium balance. Yet it has been pointed out that under certain circumstances of extracellular volume change the values of renin and aldosterone are opposite (1, 2, 3 ) . Therefore, the possibility was considered that factor(s) other than the reninangiotensin system could mediate the effect of volume variations upon aldosterone secretion ( 4 , 5, 6 ) . Outside the known factors renin, ACTH, potassium and MCR a fifth factor of unknown origin has been evoked (7, 8 ) . The purpose of the present study is to investigate the effect of PA of an acute volume depletion induced by ethacrynic acid and to deternitie-therelative contributions of the known factors.
plasma aldosterone, ABPR: aldosterone blood production rate, MCR: metabolic clearance rate.
Experimental Design and Methods
Abbreviations: PRA: plasma renin activity, AII: yenous immunoreactive angiotensin 11, PA:
Experimental Design. The experiments were performed in normal young male volunteers previously on an ad libitwn diet who were fully informed of the purpose of the study and gave their consent. The subjects remained fasting and recumbent in the metabolic ward from 11 p.m. until the end of the experiment the next morning. The experiments started between 7 and 8 a.m. when a catheter was placed in a vein of the forearm into which infusiop was delivered using a constant infusion pump ([email protected]
) ; another small catheter specially designed for drawing blood into evacuated tubes ([email protected]
and [email protected]
Becton and Dickinson) was placed in the other forearm. The effects of an acute volume depletion produced by 100 mg of ethacrynic acid were studied in the following situations: - during a glucose infusion (A) - during an A11 infusion (B) - during a dexamethasone infusion (C) - during an infusion of A11 and dexamethasone (D)
Supported by grants number 4485 and 3.2300.74 from the Swiss National Foundation for Scientific Research, by the Sandoz Foundation and the Birkigt-Bickel Foundation,
Each experiment was divided into two periods of 2-hours: (I) A first 2 hour equilibration period. (2) A second period starting at time 120 min.
Acute Volwne Dep,letion by Etkacrynic Acid.
R.C. Gaillard et aZ.: Effect on Plasma Aldosterone
when 100 mg ethacrynic acid was infused over 10 min. and its effect observed for the next 2 hours. During the four hours the subjects received a continuous infusion of 5 % glucose at a rate of 0.14 ml/kg/min. Blood samples (30 ml) were drawn at time 0, 60, 90, 120, 135, 150, 180, 210, 225, 240 min. for PA, PRAY AII, serum electrolytes, haematocrit (Hct) , haemoglobin (Hb) and proteins. AII I n f u s i o n . A constant intravenous infusion of angiotensin I1 ([email protected]
Ciba) in a 5 % glucose solution was given at a rate of 7 ng/kg/ min. (43 - 75 ml/h) during the 4 hours of the study. At this rate diastolic arterial pressure increased by 10 to 25 mm Hg. Dexmethasone Administration. 2 mg dexamethasone ([email protected]
) was given per 0s at midnight (except for subject KM) and I mg dexamethasone I.V. per hour during the 4 hours of the experimental period. Dexamethasone was added to the 5 % glucose solution. Methods Determination of AZdosterone MCR. The KCR of aldosterone was measured before and after volume depletion in subjects whose endogenous ACTH was suppressed by dexamethasone in two situations: without (2 subjects) and with ( 2 subjects) A11 infusion. The basic protocol therefore corresponds to protocols C and D respectively In addition the subjects received at time 60 min. a priming dose of 10 pCi of [H31 aldosterone and 30 min. later a constant infusion of H3 aldosterone (10 $if30 ml 5 X glucose) at a rate of 10 pCi/h (9). Immediately after withdrawing the last blood sample at time 230 min. the infusion was stopped and its exact flow rate was checked. The blood samples were drawn at time 100, 110, 120, 150, 190, 230 min. The aldosterone MCR was determined by measuring the radioactivity concentration in the plasma assessed specifically as H3-aldosterone after purification through 5 chromatography systems‘and two derivative formations, using the following equation: cpm H3 aldosterone infused/24h MCR = cpm Hj aldosterone/L plasma = Lf24 h Blood samples for the hormone assays were drawn quickly into chilled vacutainer tubes containing 12 mg Na EDTA solubilised in 0.05 ml water. They were centrifuged at 4OC and the plasma separated and kept frozen until assay. The samples for plasma electolytes (Na, K, Cl) proteins, osmolality, Hct and Kb were collected into tubes containing heparin. Urine was collected each time the subject could void spontaneously. Urinary flow and urinary sodium and potassium excretion were measured. PRA measured by generation of A1 and venous A11 were determined by radioinnnunoassay ( 1 0) Plasma cortisol was measured by a protein binding method ( 1 1 ) . PA was measured by the double isotope dilution method of Bojesen and Thuneberg (12) or by the radioimmunoassay method reported by Underwood et aZ. (13). The characteristics of the double isotope dilution method applied to human plasma have been described in detail (14).
The validity in our hands of the radioimmunoassay method using antiserum Nr 088 from “LAND was checked and the following results were obtained : recovery of H3 aldosterone after extraction and chromatography was 64.6 k 5 % (n = 60) ; water and charcoal-treated plasma blanks measured on 4 ml were less than the sensitivity of the standard curve; accuracy determined by adding known amount of steroid to water o r to plasma gave highly significant coefficients of correlation, r = 0.989 and r = 0.992 respectively; the intraassay precision was determined on three pools of plasma, values found were 26.3 ? 1.14 ng/100 ml (n = 8), variation coefficient t 4.3 %, 18.1 2 0.96 ng/100 ml (n = 8), variation coefficient 2 5 . 3 % and 3.3 2 0.66 ng/100 ml (n = 8), variation coefficient 2 20 %; comparison of 33 plasma concentrations measured by both methods is shown in Fig. 1 . In 84 healthy subjects on an unrestricted salt diet and in supine position overnight and fasting the mean value of plasma aldosterone between 7:30 and 8:OO a.m. was 7.5 t 0.38 ng/100 ml. Standard methods were used for determination of the other plasma and urine parameters. The blood pressure was measured repeatedly throughout the tests by an auscultatory method. Side Effgct. In one patient, the marked diuresis occasioned abdominal cramps requiring the injection of a spasmolytic drug. In the first subject studied, we observed after completion of the experiment a marked hypotension in the supine position when the infusion of A11 was abruptly stopped. We therefore slowed down progressively the infusion of A11 f o r the other subjects after completion of the experiment ( 1 5 ) . Statistics. The Student t test was applied to assess the statistical significance of the results which are expressed as mean 2 SD. so
RIA ALDOSTERONE ng/CUml
30 40 50 010 ALOOSTERONE q / 100 mi
Fig. 1 . Comparison of the results obtained in the same plasma sample with radioimmunoassay (RIA) and the double isotope derivative method (DID) for aldosterone. The regression line calculated by the least square fit method and its confidence limits have been computed by the REGFIT program (Centre d’Informatique de 1’ Universiti? de GenGve)
R.C. Gaillard e t aZ.: Effect on Plasma Aldosterone
Results Effect o f Administration of Ethacrynic Acid (A, B, C, 0 ) . Following the injection of ethacrynic acid a prompt and marked diuresis occurred within minutes. The urinary volume, the urinary sodium and potassium excretion remained elevated till the end of the experimental period and then fell to lower values. The mean total urinary volume excreted during the two hours after ethacrynic acid infusion was 1745 f 295 ml (n = 1 8 ) . The urinary flow increased from a mean of 0.70 f 0.27 ml/min. before to 13.3.6 -C 3.43 ml/min. with a mean maximum of 24.21 f 8.85 ml/min. after administration of the diuretic. Concomitantly with the rise in the diuresis, there was an increase in the excretion of sodium from a mean of 0 . 9 3 f 0.04 mEq/min. to 1.59 f 0.45 mEq/min. and an increase in the excretion of potassium from a mean of 0.04 f 0.03 mEq/min. before to 0.25 f 0.11 mEq/min. after the administration of the diuretic. Plasma values for sodium revealed no significant changes, while potassium was slightly but significantly lower after the volume depletion. The values of plasma potassium decreased from a mean of 4.26 f 0.27 mEq/1 to 3.96 f 0.16 mEq/l (mean A 0.32 mEq/l It: 0 . 2 9 ) . The plasma potassium decline after the administration of ethacrynic acid appears in each group. It reaches a statistical significance in the
studies B and C (P < 0.02) but not in the studies A and D (0.05 < P < 0.1). The plasma volume depletion was indicated by the rise in Hct, Hb and plasma proteins from the stable values observed between 6 0 min. and 120 min.: the Hct increased from 46.08 f 2.11 % t o 49.07 f 2.46 % (mean A 2.86 2 1.06 % ) , the Hb from 15.07 f 0.96 g/100 ml to 16.08 k 1.4 g/100 ml (mean A 0 . 9 8 f 0 . 3 5 ) , the plasma proteins from 68.24 f 2.79 g/lOO ml to 75.0 ? 2.37 g/100 ml (mean A 6.76 f 1.86.g/100 ml). A l l these increases are highly significant as assessed by the paired Student "t" test. Acute Volume Depletion without A I I Infusion (A, C) Fig. 2 and 3 . The acute volume depletion observed with protocols A and C produced a significant increase in PA and PRA occurring within 15 - 30 minutes. In the absence of dexamethasone (A) the mean maximum for PRA was 2.55 k 0.75 ng/ ml/h and for PA 21.60 2 8.0 ng/100 ml. A rise of plasma cortisol was observed suggesting stimulation of ACTH secretion. The latter could be blocked by administration of dexamethasone. Under dexamethasone (C) PA increases to a mean maximum of 25.22 f 3.99 ng/100 ml and PRA to a mean maximum of 3.10 f 0.77 ng/ml/h. There was no statistical difference for PA and PRA between the 2 protocols. All the values of PRA have therefore been grouped together in Fig. 3 (right part).
EFFECT OF ETHACRYNIC ACID WITH AND WITHOUT DECADRON. MINUTES 0
5 Y. GLUCOSE
ETHACRYNIC AClO I
' i i
nglml l h
ng I 106 ml
Fig. 2 . Effect of the administration of ethacrynic acid (100 mg intravenously in 10 min.) in healthy recumbent males under continuous infusion of 5 X glucose (left: protocol A) or dexamethasone I mg/h in glucose (right: protocol C ) . One subject in protocol C (m) had high
starting values of PRA but displayed a similar response to ethacrynic acid in every other respect; the PRA values, not reported on the figure, were 2.4, 3.0, 3.0, 3.0, 7 . 3 , 7.8, 9,5 ng/ml/h at time'0, 100, 110, 120, 150, I90 and 230 min
R.C. Gaillard e t el : Effect on Plasma Aldosterone
EFFECT OF VOLUME DEPLETION ON PLASMA RENIN ACTIVITY. MINUTES
PRA ng /ml / h
5 % GLUCOSE
I ’ ETHACRWC ACID
ETHACRYNIC AClDm i t 100 mg I
Fig. 3. Effect of the administration of ethacrynic acid on plasma renin activity in healthy recumbent males receiving (left: protocol B and D) or not receiving (right: protocol A and c) a continuous infusion O f angiotensin 11. The shaded area indicates the range of values for normal recumbent suhjects On a regular diet
Acute V o b e DepLatisn under A I I Infksion !E, 0 ) ( F i g . 3 and 4 ) . The effects of the i n fusion of A11 during the equilibration period o n PRA, A I L and PA have been described (6). The mean steady state value for aldosterone was 20.06 i: 5 . 3 7 ng/100 ml (n = 106) between 60 and 120 min., while PRA decreased at time 60, 90 and 120 min. to 0.38 2 0.30 ng/ml/h, 0.32 ? 0.27 ng/rnl/h and t o 0 . 2 3 It 0.19 ng/ml/h respectively. In protocol B PA increased markedly in all 4
subjects reaching a mean maximum of 4 8 . 9 ? 15.0 ng/100 ml. PRA escaped slightly from the inhibition by the infused AII. Yet all these PRh values remained within the normal range For recumbent men. Furthermore the higher PRA values were often concomitant with the lower values o f aldosterone. In 3 out of 4 subjects the administration of the diuretic was followed by an increase of plasma cortisol above the low levels normally observed at that time of the day in
R.C. Gaillard e t a1 : Effect on Plasma Aldosterone
Table 1. Plasma aldosterone (PA), aldosterone metabolic clearance rate (MCR) and calculated aldosterone blood production rate (ABPR) during acute volume depletion
PA ng/l00 ml
ABPR id24 h
Volume depletion 7 0 min. 110 min. after ethacrynic acid
8.2 16.0 22.0 21.6
22.9 23.2 29.5 23.0
27.1 23.0 27.3 27.8
30.5 17.4 31.5 36.2
I 289 I47 03 I 828
1360 I195 a95 712
549 50 I 06 1
C C D D
I55 248 330 229
295 266 304 190
368 275 244 198
413 200 277 24 1
J.F.H. B.R.M. D.M.
Control (mean of 3 values)
fC: ethacrynic acid + dexamethasone
I155 880 667
D: ethacrynic acid + dexamethasone and angiotensin 11
view of the circadian rhythm. This increase of plasma cortisol (mean maximum 13.07 C 5.26 pg/ 100 ml) was similar to the one observed in the 4 subjects studied in experiment A (mean maximum 10.00 -+ 4 . 7 8 pg/IOO ml). In protocol D, that is under dexamethasone perfusion, plasma cortisol values were suppressed, particularly in the 3 subjects who had received 2 mg dexamethasone at midnight. PA rose significantly from a mean of 21.6 ? 1 . 7 5 ng/100 ml before ethacrynic acid to 28.6 2 2.95 ng/100 ml (p < 0.001) but the values reached were not as high as in protocol B (Fig. 4 ) . The mean maximum of 31.9 f 2.98 ng/100 ml was also statistically different from the mean maximum in protocol B. Dexamethasone had no effect on PRA values, therefore PRA values of protocol B and D have been grouped in Fig. 3 (left part). Measurement of Atdosterone MCR before and
a f t e r Acute Volme Depletion with and withact 4 1 1 Infusion and with Blockade of Endogenous 4CTH. Results for aldosterone MCR and calculated aldosterone blood production rate (ABPR) are summarized in Table 1 . As in experiments C and I all 4 subjects showed an increase of PA. A iecrease in the KCR of 25 to 41 was observed. In the 2 subjects with an intact renin-angiorensin system the calculated ABPR increased in m e but did not change in the other subject 3fter volume depletion. Under A11 infusion the :alculated ABPR did not change after volume de?letion.
)iscussion The importance of the known factors influencing )lasma.aldosterone has been evaluated during acute olume depletion. In control experiments (A) the
injection of ethacrynic acid alone induced the expected acute plasma volume depletion and an intense activation of the renin-angiotensin system as well as a marked increase of PA. The time course and the magnitude of the PRA and PA response of the administration of this potent diuretic is similar to that described by other authors (16 - 1 9 ) . The concomitant rise of plasma cortisol values, which is even more striking when they are compared to the normal decline of plasma cortisol during the morning hours ( 2 0 ) , cannot be attributed to any clinical manifestation of stress or anxiety. In the study of Fraser e t at., plasma cortisol is uninfluenced after frusemide ( 2 1 ) , while in the study of Williams e t a t . ( 1 9 > , plasma cortisol appears higher than the corresponding control values. In the ex, periments with ethacrynic acid and dexamethasone (C) the increases of plasma renin activity and of aldosterone were similar to those observed in control experiments, yet the complete suppression of cortisol confirmed the inhibition of endogenous ACTH. This indicates that an increased ACTH release was not the main cause f o r the rise of PA, although it may have been contributing. In the experiments performed under constant infusion of angiotensin I1 (B) , the acute volume depletion still produced an increase in PA well above the "plateau" reached under angiotensin I1 alone. PRA escaped slightly from the A11 inhibition during the hours following the administration of ethacrynic acid; yet all these values remained within the normal range for recumbent men and the renin-angiotensin system can be considered as effectively blocked and therefore discounred as a major contributing factor for the elevation of PA. In the experiments where both endogenous renin and ACTH were suppressed ( D ) , PA still increased significantly
R.C. Gaillard et a2 : Effect on Plasma Aldosterone
erone still rises though less. An increase in the sensitivity of the zona glomerulosa secondary to sodium depletion as demonstrated in chronic studies ( 2 4 , 25) is not excluded in the y = 1 . 3 7 ~-90.2 present acute experiments. However, a decrease r = 0.643 of the MCR was found then to play a major role n = 99 for this residual rise. The role of decreased MCR and of haemoconcentration is demonstrated /on Fig. 5, which shows the correlation between / the degree of volume depletion as assessed by the elevation of plasma proteins and the rise of aldosterone. The regression lines for the values taken from protocol A , B and C have identical slopes whereas when angiotensin XI and dexamethasone (protocol D) were administered, the slopes of the regression lines were significantly changed, yet the correlation remained significant. From this study we can conclude PROTEINS gll that in men and in those conditions there is no Fig. 5 . Regression lines of the correlation between need to call for a hypothetical additional facplasma proteins and plasma aldosterone during study tor as a stimulus for aldosterone (7, 8). of volume depletion. Continuous regression line: Acknowledgments. The authors are indebted protocol D. Dashed regression line: protocol A, B to U. Hufschmid, C. Ryser, M. Stempfel, R. and C Sultan, F. Terrapon-Vogt for their skilful technical assistance, and the Clinical Chemistry following plasma volume depletion, but less than Laboratory (Dr. M. Roth) for the facilities in in the protocols with dexamethasone or with the determinations of the non-hormonal paraangiotensin I1 alone. The role of the potassium meters. The antiserum Nr 088 used for the radioion in the regulation of aldosterone secretion immunoassay of aldosterone was generously suphas been previously stressed (6). During the plied by the NIAMD (Bethesda, USA), period following the administration of ethacrynic acid, there was a decline of plasma potassium, which could be due to the kaliuresis actually References observed and/or to a passage of potassium into the cells. In the first case the external potI. Boyd, G.W., Adamson, A.R., Arnold, M., assium l o s s would rather result in a decrease James, V.H.T., Peart, W.S.: The role of of aldosterone secretion. In the second case angiotensin I1 in the control of aldosterone the entry of potassium into the glomerulosa in man. Clin. Sci. 4 2 , 91 (1972) cells could participate in the stimulation of 2 . Chinn, R . H . , Brown, J.J., Fraser, R., Heron, aldosterone secretion. Yet this latter possiS.M., Lever, A.F., Murchison, L., Robertson, bility remains to be proven (4, 5). J.I.S.: The natriuresis of fasting: relationPlasma concentration of aldosterone reship to changes in plasma renin and plasma presents the dynamic state between adrenal aldosterone concentrations. Clin. Sci. 39, 437 (1970) secretion and metabolic clearance rate (MCR) of aldosterone (22, 2 3 ) . MCR was measured before 3 . Epstein, M., Satura, T.J.: Effect of water and after administration of ethacrynic acid, immersion on renin aldosterone and renal while blocking endogenous renin and ACTH. The handling in normal man. J. appl. Physiol. 31, MCR effectively decreased, most likely because 368 (1971) of a diminished hepatic blood flow. In conse4 . Vallotton, M.B.: R'ecents concepts de la rgquence, the acute change in the half-life of gulation hormonale de l'gquilibre hydroaldosterone can account for the elevation of min'eral. In: L'gtude des hormones et r'eplasma aldosterone, since the calculated ABPR gulations mgtaboliques. Rapports de la XIIe did not increase during the plasma volume dergunion des endocrinologistes de langue pletion. No change of cortisol was expected, francaise. Paris: Masson and Cie (Eds.) 1974 since in contrast to aldosterone, it is incom5. Muller, A.F., Vallotton, M.B.: Mode d'action et rkgulation de l'aldostgrone. Schweiz. pletely extracted from blood in the liver, the metabolic clearance rate being therefore indemed. Wschr. 104, 905 (1974) pendent of liver blood flow rate ( 2 2 ) . 6 . Birkhguser, M., Gaillard, R., Riondel, A.M., Summarizing, it can be stated that acute Scholer, D., Vallotton, M.B., Muller, A.F. : volume depletion induced by the administration Effect of volume expansion by hyperosmolar of ethacrynic acid resulted in an elevation of and hyperoncotic SoluKiOnS under constant PA which cannot be attributed t o a single facinfusion of angiotensin 11 on plasma aldosttor. Bath the renin-angiotensin system and to erone in man and its counterbalance by potasa lesser extent ACTH are contributing factors, sium administration. Europ. J. clin. Invest. but, when both are effectively blocked, aldost3 , 307 (1973) ALDOSTERONE
R.C. Gaillard e t a2 : Effect on Plasma Aldosterone 7. Blair-West, J.R., Coghlan, J.P., Cran, E., Denton, D.A., Funder, J.W., Scoggins, B.A.: Increased aldosterone secretion during sodium depletion with inhibition of renin release. h e r . J. Physiol. 224, 1409 (1973) 8. McCaa, R.E., Young, D.B., Guyton, A.C., McCaa, C.S.: Evidence for a role of an unidentified pituitary factor in regulating aldosterone secretion during altered sodium balance. Circulat. Res. Suppl. 1, 34-35, 15 ( I 974) 9. Tait, J.F., Tait, S.A.S., Little, B., Laumas, K.R. : The disappearance of 7-3H-d-Aldosterone in the plasma of normal subjects. J. clin. Invest. 40, 72 (1961) 10. Vallotton, M.B.: Parallel radioimmunoassays of angiotensin I and of angiotensin I1 for measurement of renin activity and of circulating active hormone in human plasma. In: Immunological Methods in Endocrinology. Symposium in Ulm 1970. K. Federlin, C.N. Hales and J. Kracht (Eds.). Hormone and Metabolic Research 3 (Suppl.), p. 94. Stuttgart, New York, London: Georg Thieme/ Academic Press 1971 1 1 . Leclercq, R., Copinschi, G., Franckson, J. R.M.: Le dosage par competition du cortisol plasmatique. Modification de la mhthode de Murphy. Rev. franc. Etud. clin. biol. 14, 815 (1969) 12. Bojesen, E., Thuneberg, L.: Isotope derivative methods for the determination of steroid hormones with 35s-sulfonylating reagents. In: Steroid hormone analysis. H. Carstensen (Ed.), Vol. 1, p. 39. New York: Marcel Dekker 1967 13. Underwood, R.H., Williams, G.H.: The simultaneous measurement of aldosterone, cortisol and corticosterone in human peripheral plasma by displacement analysis. J. Lab. clin. Med. 79, 848 (1972) -14. Scholer, D., Riondel, A.M., Manning, E.L.: Plasma aldosterone determination with 35sp-toluene-sulfonic anhydride. Acta endocr. (Kbh) 7 0 , 552 (1972) 15. Gaiilard, R., Vallotton, M.B., Muller, A.F.: Hypotension after angiotensin-I1 infusion and hypovolemia induced by diuretic. Lancet 1, 1349 (1974) 16. Espiner, E.A., Tucci, J.R., Jagger, P.I., Pauk, G.L., Lauler, D.P.: The effect of acute diuretic-induced extracellular volume
57 depletion on aldosterone secretion in normal man. Clin. Sci. 33, 125 (1967) 17. Rosenthal, J., Boucher, R., Nowaczynski, W., Genest, J.: Acute changes in plasma volume, renin activity, and free aldosterone levels in healthy subjects following fursemide administration. Can. J. Physiol. Pharmacol. 46. 85 (1968) 18. RadS, J . P . , Szende, L., Borbgly, L.: Effects of ethacrynic acid on specific renal functions without and during angiotensin infusion in man. Arch. int. Pharmacodyn. 186, 142 (1970) 19. Williams, G.H., Cain, J.P., Dluhy, R.G., Underwood, R.H.: Studies of the control of plasma aldosterone concentration in normal man. 1. Response to posture, acute and chronic volume depletion and sodium loading. J. clin. Invest. 51, 1731 (1972) 20. Scholer, D., Birkhauser, M., Peytremann, A . , Riondel, A.M., Vallotton, M.B., Muller, A. F.: Response of plasma aldosterone to angiotensin 11, ACTH and potassium in man. Acta endocr. (Kbh) 72, 293 (1973) 21. Fraser, R., James, V.H.T., Brown, J.J., Isaac, P., Lever, A.F., Robertson, J.I.S.: Effect of angiotensin and of frusemide on plasma aldosterone, corticosterone, cortisol, and renin in man. Lancet 13, 989 (1965) 22. Yates, F.E.: Contribution of the liver to steady-state performance and transient responses of adrenal cortical system. Fed. Proc. 24, 723 (1965) 23. Balikian, H.M.: Metabolism of aldosterone by the splanchnic organs of the dog. Endocrinology 89, 1309 (1971) 24. Hollenberg, N.K., Chenitz, W.R., Adams, D.F., Williams, G.H.: Reciprocal influence of salt intake on adrenal glomerulosa and renal vascular responses to Angiotensin 11 in normal man. J. clin. Invest. 5 4 , 34 (1974) 25. Celkers, W., Brawn, J.J., Fraser, R., Lever, F.F., Morton, J.J., Robertson, J.I.S.: Sensitization of the adrenal cortex to angiotension I1 in sodium-deplete man. Circulat. Res. 34, 69 (1974) Dr. M.B. Vallotton Laboratoire d'investigation clinique Clinique mhdicale H8pital cantonale CH-1211 Geneve 4 Switzerland