The Effect of ACTH and Cortisol on Aldosterone and Cortisol Clearance and Distribution in Plasma and Whole Blood ROBERT D. ZIPSER, PAUL F. SPECKART, PRISCILLA K. ZIA, W. ALLAN EDMISTON, FRANCIS Y. K. LAU, AND RICHARD HORTON Sections of Endocrinology and Cardiology, Department of Medicine, Los Angeles C ounty-University of Southern California Medical Center, Los Angeles, California 90033 ABSTRACT. The mechanisms of increased aldosterone and cortisol metabolic clearance rates (MCR) following ACTH or cortisol administration were studied in 13 subjects undergoing cardiac catheterization and in 9 healthy controls. In control subjects, the MCR (plasma) of both steroids increased by 29% (aldosterone: from 936 ± 57 to 1204 ± 55 1/day/m2, cortisol: from 205 ± 12 to 264 ± 17 1/day/m2 ± SE) after ACTH (12 units/h) for 1 to 4 h, and by 20 and 32%, respectively, after cortisol (12 mg/h) for 1 to 2 h. In contrast, aldosterone MCR (whole blood) did not change with ACTH or cortisol administration (from 1276 ± 57 to 1330 ± 59 1/day/m2), indicating that the plasma MCR increase results from a redistribution of aldosterone from plasma to red cells. Aldosterone splanchnic extraction was 92 ± 1% (n = 12) with normal morning cortisol levels, and extraction was unchanged after ACTH administration. For cortisol, however, the splanchnic extraction

R

ECENT REPORTS indicate that aldosterone metabolic clearance rate (MCR) is increased after ACTH administration (1-3), but the mechanism of this effect is not established. Previous in vitro studies suggested that a fraction of aldosterone is bound to corticosteroid binding globulin (CBG) and that cortisol displaces aldosterone from this binding (3-7). In vivo displacement of aldosterone from CBG with increased availability for hepatic metabolism could explain the ACTH effect. Other possible mechanisms are increased extrasplanchnic metabolism or increased liver blood flow. Reports in man and in sheep support the possibility of increased liver blood flow after ACTH (1,8). A redistribution of steroid from plasma to other body spaces is also possible (9,10). The purpose of this study was to investigate the mechanism of increased plasma aldosterone and cortisol MCR following ACTH. Received April 6, 1976.

increased from 8 ± 0.8% to 17.8 ± 5.0%, and the MCR (whole blood) likewise increased by 15 to 31% (from 295 ± 23 to 357 ± 30 1/day/m2), after ACTH or cortisol administration. In vivo and in vitro measurements (at 37 C) of tracer aldosterone concentration in plasma and in red cells showed an increase in distribution to red cells with increasing cortisol concentrations. The results suggest that a fraction of aldosterone is bound in plasma and displaced by cortisol into red cells. There is an increased aldosterone plasma MCR, but unaltered whole blood MCR, since the liver extracts aldosterone almost completely from both plasma and red cells. The increase in cortisol MCR (plasma) results from both an increased splanchnic extraction as plasma binding sites approach saturation and a redistribution into red cells (J Clin Endocrinol Metab 43: 1101, 1976)

Materials and Methods Material Methanol and dichloromethane were purchased from Matheson Coleman & Bell and were of spectro quality. l,23H-labeled aldosterone (sp act 160 ixCi/(jig) and cortisol (sp act 110 /u,Ci//u,g) obtained from the New England Nuclear Co., Boston, were separately purified prior to use by LH-20 Sephadex chromatography (methylene chloride: methanol, 98:2). Subjects Metabolic clearance rate studies were performed on 9 healthy men who gave informed consent. They constituted the control group. Metabolic clearance and hepatic extraction studies were also performed on 13 men who were scheduled for elective cardiac catheterization. Informed consent was obtained after the subjects were thoroughly acquainted with the procedure. They constituted the catheterization group. At the time of study, there was no evidence of hypertension or congestive heart failure. Liver and renal function tests were normal (Table 1).

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JCE & M • 1976 Vol 43 • No 5

ZIPSER ET AL. TABLE 1. Aldosterone MCR and splanchnic extraction in catheterizec1 subjects

Splanchnic extraction (%) P 1 Q CTM Cl

ITlclMIlcl t u u i a u i

(ug/dl) Subject 1 2 3 4

Age

6 7 8 9 10 11

22 40 50 61 48 45 51 46 59 20 43

12

48

13

43

5

Cardiac diagnosis

index (1/min/m2)

Before ACTH

Aortic regurg. Pain syndrome* Angina Angina Pain syndrome* Angina Angina Pain syndrome* Hypertension Aortic regurg. Pain syndrome* Pain syndrome* Angina

2.6 2.6 3.2 2.2 3.2 2.6 2.2 3.7

15.3 25.0 11.9

2.8

9.8

2.9

12.9

2.6

7.1 9.4 9.2

Mean SE

2.9 3.3 2.8 0.1

After ACTH

Plasma After ACTH

Before ACTH

After ACTH

730 719

85 88 95 87 91

95 92 90

76 93

80

1399

92

92

87

93 92

907

95 93 88

79

95

78

90 82

93 87

94 88

92 1

89 2

89 3

87 3

MCR

Before ACTH

17.1

983

6.6

1007 1011

96 96

(1/day/m2)

14.4 7.4 9.3

12.0

Whole blood

Plasma

818

25.9 31.8 17.4 15.9 21.6 18.9 22.8 21.4 22.0

1293 1373

1006 704 996

71

Chest pain without angiographic evidence of cardiac disease.

Metabolic clearance studies

at 60 and 120 min. At the conclusion of each study, infusion fluid was obtained from the All studies were begun between 0700 and patient's iv tubing for determination of the 0800 h. The subjects fasted overnight and main- infusion rate of labeled steroid. tained a recumbent position for several hours In catheterized subjects, the infusions were before and during the procedure. Determination begun as soon as the patient was positioned of MCR was by the constant infusion method on the radiography table. The cardiology staff (11). Infusion solutions for control patients were then performed the indicated catheterization prepared by dissolving 30 /iCi of 3H-aldosterone procedures. In 5 subjects, peripheral samples and 5 /xCi of 3H-cortisol in 4 ml of absolute were obtained at approximately 75, 90, and 105 ethyl alcohol and then diluting this in 50 ml min. Two samples from the hepatic vein, of normal saline. For catheterized patients, separated by 15 min, were obtained by catheter 50 jiiCi and 10 ^iCi, respectively, were used during this period. In the other 8 subjects, to assure an adequate number of counts in 2 peripheral and 2 hepatic vein samples were hepatic venous blood. After a 4 ml priming obtained between 70 and 100 min, ACTH dose, infusion into an antecubital vein was infusion at 12 units per h was added, and maintained at either 0.382 or 0.191 ml/min samplings repeated 30 and 40 min later. To (infusion pump, Harvard Apparatus Co.). Blood provide further evidence that these studies was drawn during the procedure from an in- were under steady state conditions, the last dwelling needle in an antecubital vein of the subject was infused for 210 min. opposite arm. In control patients, blood was drawn 105 Sample handling and 120 min after beginning the infusion. A constant infusion of synthetic 1-24 ACTH From each sample, 5 ml of heparinized whole (Cortrosyn, Organon) diluted in normal saline blood was removed, lysed with 5 ml of deionized was then added at 12 U per h and blood drawn water and analyzed separately. The remainder 60, 120, 180, and 240 min later. On a different was centrifuged at 37 C at 2500 rpm for 15 day, the procedure was repeated with the sub- min and the plasma separated. 14C-aldosterone stitution of hydrocortisone phosphate (Hydro- and 14C-cortisol were added to each sample. cortone®, Merck, Sharp & Dohme) for ACTH, The mixtures were extracted twice with 5% infused at 12 mg perh and with blood drawn methanol in dichloromethane and the organic

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ALDOSTERONE AND CORTISOL CLEARANCE AND BINDING solvent separated by filtration through glass wool and anhydrous sodium sulfate. After vacuum evaporation, the dried extract was chromatographed on a Sephadex LH-20 system (12), dried under nitrogen and counted in a Mark I dual channel scintillation counter (Nuclear Chicago Corp.) (3H efficiency 36%, HC efficiency 54%, '"C to 3H feedthrough 20%, and 3H to I4 C feedthrough less than 0.1%). Samples were counted for 120 min (40 x 3). The recoveries for aldosterone and cortisol averaged 73% and 60%, respectively. Calculations Plasma and whole blood MCR was determined by the formula: MCR= — i where R is the infusion rate (counts/day) and i is the plasma or whole blood concentration of 3H steroid per liter after purification and correction of 14C losses. The concentration of tracer steroid in the hepatic vein blood was used to calculate the splanchnic extraction: PV-HV x 100 PV where PV is the 3H steroid concentration in peripheral venous blood and HV is the concentration in hepatic vein blood. The peripheral concentration is assumed to be the same as portal vein concentration. The distribution of steroids between plasma and red cells was calculated from die simultaneously obtained measurement on whole blood and plasma samples and expressed by the following formula:

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In vitro studies To determine the distribution of aldosterone in vitro, 5 ml samples of heparinized whole blood were incubated for 30 min at 37 C with 0.001 /xCi of 3H-aldosterone. One ml of whole blood was separated and the rest centrifuged for plasma at 37 C. 14C-aldosterone was added to monitor recovery, and the whole blood and plasma samples were extracted and counted as above. The red cell distribution of aldosterone was then calculated. In a different study, 0.1 to 10 /xg of unlabeled cortisol were added to separate 5 ml aliquots of whole blood obtained from a control subject. Each sample was incubated with 3Haldosterone for 30 min. Samples were separated, extracted, and counted as above. The study was repeated in duplicate on two other days with blood obtained in the evening. Results were pooled and a dose response curve was calculated for aldosterone distribution coefficient with the different plasma cortisol concentrations. Plasma assays Plasma cortisol was measured by a specific radioimmunoassay procedure (13).

Splanchnic Extraction =

X

Dist. Coeff. =

WB

- (1 - Hct)

(Red Cells)_ (Plasma)

Hct

(Red Cells)

% Red Cell Uptake =

(Whole Blood) X

WB - XP1 (1 - Hct)

x 100

where XWB is counts per ml in whole blood X P1 is the counts per ml in plasma Hct is the hematocrit.

Results MCR The plasma MCR of aldosterone in 9 control patients averaged 963 ± 39 1/day/m2 (SE) or 1692 ± 61 I/day. Attainment of a steady state was suggested by less than 1% difference between the averages of the 105 and the 120 min MCR calculations. In 12 catheterized patients, the MCR was not significantly different at 996 ± 7 1 I/day/ m2. The plasma cortisol MCR in control patients was 220 ± 14 1/day/m2 or 384 ± 2 1 I/day and the difference between the average 105 and 120 min samples was less than 3%. In 6 catheterized patients, the MCR was not significantly different at 210 ± 29 1/day/m2. During ACTH infusion in control patients, the plasma aldosterone MCR increased by an average of 29% (P