ALDOSTERONISM - dm5migu4zj3pb.cloudfront.net€¦ · ALDOSTERONISM* By NICHOLAS A. YANKOPOULOS,...

EVIDENCE THAT A HUMORALAGENTSTIMULATES THEADRENALCORTEXTO SECRETEALDOSTERONEIN

EXPERIMENTALSECONDARYHYPER-ALDOSTERONISM*

By NICHOLAS A. YANKOPOULOS,JAMES 0. DAVIS, BERNARDKLIMAN ANDRALPHE. PETERSONWITH THE SURGICAL ASSISTANCE OF ALFREDCASPER

(From the Section on Experimental Cardiovascular Disease, Laboratory of Kidney and Electro-lyte Metabolism, National Heart Institute and the National Institutte of

Arthritis and Metabolic Diseases, Bethesda, Md.)

(Submitted for publication December 16, 1958; accepted April 2, 1959)

The mechanisms which effect alterations in thesecretion of aldosterone by the adrenal cortex maybe nervous, humoral or both. The present studywas undertaken to examine the possibility that ahumoral agent stimulates the adrenal cortex tosecrete aldosterone in dogs with experimental sec-ondary hyperaldosteronism produced by constric-tion of the thoracic inferior vena cava. These ani-mals secrete large amounts of aldosterone (1) andshow almost complete retention of Na (2). Totest the hypothesis of a humoral mechanism, theeffect on aldosterone secretion was studied in nor-mal recipient animals or in the isolated adrenals ofnormal dogs during cross-circulation of blood fromdogs with constriction of the thoracic inferior venacava. Corticosterone secretion was also measuredto evaluate the possible role of adrenocortico-tropic hormone (ACTH) in the aldosterone regu-latory system.

METHODS

The basic plan of the experiments consisted of cross-circulation of blood from a donor into a recipient animalor into the isolated adrenal glands of a recipient animal.Secondary hyperaldosteronism was produced in the donoranimals by either acute or chronic constriction of thethoracic inferior vena cava. Acute caval constriction wasperformed on normal dogs. In the chronic donor dogs,ascites had been present for one to three weeks, and Naexcretion on the day before the experiment was less than4 mEq. per day; Na and K intakes were 60 and 18 mEq.per day, respectively. Also, aldosterone secretion fromthe right adrenal gland was measured in a group ofeight dogs with chronic thoracic inferior vena cava con-striction to determine the magnitude and consistency ofhypersecretion. All animals were mongrel dogs whichweighed 15 to 20 Kg. Nembutal® anesthesia was used.Each dog received 15,000 I.U. of heparin before collec-tion of adrenal vein blood and before cross-circulation.

* A preliminary report was given at the LaurentianHormone Conference, September, 1958.

Two types of experiments were performed. In thefirst group of experiments (three pairs of dogs), cross-circulation was achieved through the femoral vessels bypolyethylene tubing (i.d., 3.5 mm.). Exchange of bloodwas regulated by screw clamps so that arterial andvenous pressures remained similar to the values recordedbefore cross-circulation. Adrenal vein blood was col-lected from both animals from the right adrenal glandby cannulation of the right adrenolumbar vein as de-scribed previously (1). In the first pair of dogs, cross-circulation was performed between two normal animals.After control determinations in both animals, the thoracicinferior vena cava was constricted in one of the dogswhich became the donor. In the two other experimentsof this group, dogs with chronic thoracic caval con-striction were the donor animals. Under these circum-stances, control observations were made in both thechronic donor and the normal recipient before cross-circulation was established.

In the second group of experiments (15 pairs of dogs),blood was circulated from a donor animal with chronicthoracic caval constriction and ascites or from a normaldog into the isolated adrenals of a normal recipient ani-mal (Figure 1). Cross-circulation of blood from normaldogs provided a control on the effect of cross-circulationper se. The recipient's adrenals were isolated in situwith segments of aorta and inferior vena cava by theprocedure of Hilton and co-workers (3). Care was takento include in the arterial circulation to the lower poleof the right adrenal gland a small artery arising from theaorta. Polyethylene tubing was used for all connections.During the control and recovery periods, blood was circu-lated from the recipient's carotid artery (autoperfusion)into the isolated aorta, through the adrenal glands, intothe inferior vena cava and, subsequently, into the systemiccirculation of the recipient by way of the external jugu-lar vein. During the period of cross-circulation, bloodperfused the isolated adrenals of the recipient from thefemoral artery of the donor and returned to the donor'sfemoral vein. An infusion of norepinephrine (100 ,ug.per ml.) was given in some instances (Tables I and II)during the latter part of the experiment to support arterialpressure and adrenal blood flow.

Collections of 10 ml. of adrenal vein blood and 10 ml.of peripheral blood were made at approximately one-half

1278

EVIDENCE FOR AN ALDOSTERONESTIMULATING HORMONE

I 0

FIG. 1. CROSS-CIRCULATION OF BLOODFROMA DOGWITH CHRONICTHOR-ACIC INFERIOR VENA CAVA (IVC) CONSTRICTION INTO THE ISOLATEDADRENALSOF A NORMALRECIPIENT ANIMAL

hour intervals; blood was replaced immediately. Adrenalblood flow was measured by collection of blood into agraduated tube; the hematocrit was determined andadrenal plasma flow was calculated. The concentra-tions of aldosterone and corticosterone in adrenal veinplasma were determined and the rates of secretion calcu-lated. The concentrations of plasma Na and plasma Kwere determined during cross-circulation of blood fromdogs with hyperaldosteronism. Arterial and venous pres-sures were measured continuously with Statham straingages and a Sanborn recording system. During cross-circulation via the femoral vessels, aortic and inferiorvena caval pressures were determined in both donor andrecipient animals. During perfusion of the isolated adre-nals, pressures were recorded from the isolated segmentsof the aorta and inferior vena cava associated with theadrenals. The height of the animal table of the recipientdog was adjusted to maintain a constant venous pressurein the inferior vena cava or in the isolated segment of theintferior vena cava.

Procedure for the double isotope derivative assay foraldosterone and corticosterone. Aldosterone and corti-costerone were measured in 3 ml. of adrenal vein plasmaby the double isotope derivative procedure of Kliman,Peterson, Davis and Yankopoulos (4, 5). Each 3 ml.sample of plasma was extracted with 22 ml. of purifieddichloromethane. The dichloromethane extract waswashed successively with 2 ml. of 0.05 N NaOHand with2 ml. of 0.1 N acetic acid. The dichloromethane extractwas dried at 370 C. with a stream of nitrogen. The dried

extract was transferred to a 6.5 ml. conical glass-stopperedtube with 2 to 3 ml. of absolute ethanol and two rinses of2 ml. each of dichloromethane; the sample was driedwith a stream of nitrogen. Each sample was acetylatedwith 0.03 ml. of tritium-labeled acetic anhydride (15 percent acetic anhydride in dry benzene) in the presence of0.025 ml. of anhydrous pyridine. The specific activity ofthe acetic anhydride was approximately 100 mc. permMole. Each sample was incubated for 60 to 70 hours at370 C.; the acetylation tube was rotated on one occasionduring incubation to insure complete acetylation. Al-dosterone diacetate-H3, corticosterone monoacetate-H3and the labeled acetates of other adrenal steroids wereformed. About 2,000 cpm of aldosterone diacetate-C1'in 0.1 ml. of ethanol and 5,000 cpm of corticosteronemonoacetate-C1' in 0.1 ml. of ethanol were added to eachsample to correct for losses of aldosterone and cortico-sterone during all subsequent chromatographies. The ex-act number of cpm for each steroid in 0.1 ml. of ethanolwas determined when the labeled steroids from the plasmasamples were counted. Distilled water (0.8 ml.) wasadded to each tube, extraction was carried out with about5 ml. of dichloromethane and the dichloromethane extractwas washed once with 1 ml. of distilled water. The di-chloromethane was dried under a stream of air at 37°.Adrenosterone (0.02 ml.) and corticosterone monoacetate(0.02 ml.) in concentrations of 1 mg. per ml. in absoluteethanol were added to each sample to provide a visiblemarker in ultraviolet light for aldosterone and enoughcorticosterone monoacetate for it to be visible.

1279

YANKOPOULOS,DAVIS, KLIMAN AND PETERSON

TABLE I

Effects of cross-circulation of blood from dogs with chronic thoracic inferior vena cava constrictioninto the isolated adrenals of normal dogs

Control Cross-circulation RecoveryDogno. 1 2 3 1 2 3 4 1 2

Aldosterone secretion (,ug./min.) *

.017 .025 .037 .019

.010 .051 .038 .014

.026 .064 .046 .021

.011 .034 .042 .039

.005 .037

.049 .100 .141 .146.039 .047 .058

.020 .050 .059 .050

Corticosterone secretion (jAg./min.)*1.9 5.5 3.0 1.72.3 3.1 3.4 2.6

3.03.2 4.6 6.0 5.6

2.5

5.5010.17

7.707.067.106.135.81

7.07

2.0 2.3 2.1

3.6 3.7 3.0

Adrenal blood flow (ml./min.) *

6.80 3.22 1.8416.24 7.50 4.03

8.11 4.80 3.3310.00 8.11 5.36

9.5012.00 11.76 13.60

2.93 2.89 3.01

9.37 6.38 5.19

Mean arterial pressure (mm. Hg) *

120 70 70125 95 9095 lOOt loot95 95 80

15st100 96 87

92 86 96

112 90 87

Venous pressure (mm. water) *

40 30 2530 45 5055 50 705045 30 3035 20 15

43 35 38

Plasma Na (mEq./L.)145 149143 145149 154140 149140 141136 136

149144151149146139

.045

.035

.040

3.32.4

2.9

9.60

8.83

9.21

130t

140t

135

20

80

50

165146

146

142 146 146 152

.019

.018

.011

.063

.043

.031

1.52.0

2.21.9

1.9

10.528.117.06

4.593.54

6.76

.011

.016

.022

.053

.024

.025

1.02.6

2.01.1

1.7

7.698.008.00

3.572.62

5.98

200 170120 130160 135

98 90114 112

138 127

145 12070 5580 60

45 3840 45

76 63

149 152152 151146 150146 143144 147

147 149

* Steroid secretion, adrenal blood flow and arterial and venous pressures from isolated adrenals and associated vessels.t Norepinephrine infusion given to maintain arterial pressure and adrenal blood flow.

.006

.019

.029

.022

.011

.054

.027

.024

.015

.015

.047

.012

.005

.048

.018

.023

2.22.2 2.22.7 3.13.4 3.22.0 2.0

2.6 2.5

6.6711.33

8.447.418.906.176.54

7.92

456789

10

NMean

5789

10

Mean

456789

10

Mean

4S6789

10

Mean

56789

10

Mean

45679

10

Mean

125140135145120103130

128

12514013013510092

127

121

7.4012.5011.3013.3310.75

7.507.50

10.04

125140120135115114122

124

553565904565

59

152142149150136145

146

60 6030 3565 5575 7050 5555 50

56 54

149 152144 144147 147148 145145 146150 146

147 147

1280


TABLE I-Continued

Control Cross-circulation RecoveryDogno. 1 2 3 1 2 3 4 1 2

Plasma K (mEq./L.)4 3.4 3.8 3.6 3.4 4.0 3.7 3.75 4.1 3.6 3.9 4.5 3.7 4.2 3.4 2.86 3.9 3.2 3.0 3.9 4.0 4.7 2.57 3.7 3.3 3.3 3.4 4.1 5.0 4.5 5.5 5.09 3.2 3.0 2.8 2.9 2.6 4.1 3.7 3.9

10 4.3 4.0 3.8 4.3 4.5 4.7 4.0 4.2

Mean 3.8 3.5 3.4 3.7 3.8 4.4 3.9 3.7 4.4

Each sample was transferred to Whatman No. 1 filterpaper (7.5 X 22.5 inches) with four drops of absoluteethanol and eight to 10 drops of dichloromethane andchromatographed in a cyclohexane (100 parts), benzene(40 parts), methanol (100 parts) and water (20 parts)system by descending chromatography. After a mini-mal period of 45 minutes for equilibration, the chromato-gram was developed at room temperature (750 F.) for16 to 18 hours by adding 90 ml. of the mobile phase. Thepaper was scanned with ultraviolet light, each spot ofcorticosterone monoacetate and adrenosterone was markedand areas of paper containing corticosterone monoacetateand aldosterone diacetate were cut out. The relationof adrenosterone to aldosterone diacetate had been previ-ously ascertained by locating the position of aldosteronediacetate-C'4 with a strip counter and the visible spot ofadrenosterone by ultraviolet light. This relationship waschecked periodically. The samples of corticosterone mon-oacetate and aldosterone diacetate were eluted into sepa-rate tubes with 10 ml. of absolute methanol. Both sampleswere dried.

For aldosterone, the second chromatography was car-ried out with a cyclohexane (100 parts), dioxane (100parts), methanol (50 parts) and water (25 parts) system.The reference marker for aldosterone diacetate in thischromatographic system was 11-keto, 17ahydroxypro-gesterone (0.02 ml. of a 1 mg. per ml. solution); the ex-act relation of the mobility of this steroid and aldosteronehad been determined previously. The chromatogram wasdeveloped for 14 to 16 hours, the aldosterone diacetatewas eluted with methanol and the sample was dried underair. To alter the mobility of the steroid and to insurebetter purification, each sample was treated with 0.1 ml.of 0.5 per cent chromic trioxide in glacial acetic acid.The tube was rotated to allow the chromate solution towet the residue and allowed to stand for 10 minutes.The reaction was stopped with 2 ml. of 20 per cent ab-solute ethanol in water. Approximately 10 ml. of di-chloromethane was added and each tube was shakenvigorously; the aqueous layer was aspirated and discarded.Each sample was washed twice with 1 ml. of distilledwater and the dichloromethane was dried under an airstream.

The third chromatography for the oxidation productof aldosterone diacetate was performed in a cyclohexane

(100 parts), benzene (50 parts), methanol (100 parts)and water (25 parts) system. The reference marker was0.02 ml. of a 1 mg. per ml. solution of llcahydroxyproges-terone. The chromatogram was developed for 24 to 26hours. Each sample of the oxidation product of aldo-sterone diacetate was eluted into a counting vial and driedunder an air stream. To each vial, 5 ml. of a phosphorsolution (0.4 per cent solution of 2,5-diphenyloxazole and0.004 per cent solution of 1,4, di-2-5 phenyloxazole in rea-gent grade toluene) was added.

A second chromatography for corticosterone mono-acetate was performed with a cyclohexane (100 parts),dioxane (100 parts), methanol (50 parts) and water (25parts) system. The chromatogram was developed for 12to 15 hours. The corticosterone was eluted into countingvials, dried and the phosphor solution added.

The samples of aldosterone and of corticosterone werecounted to determine the tritium and carbon-14 radio-activity in a liquid scintillation spectrometer (PackardTri-Carb Scintillation Spectrometer). Each sample wascounted at a voltage of 890 (discriminator setting of 10to 50) to determine the radioactivity of carbon-14 andat 1,270 volts (discriminator setting of 10-00) to obtainthe combined activity of carbon-14 and tritium. Thenumber of tritium counts at 1,270 volts was obtained bysubtracting the contribution of carbon-14 at this voltagefrom the total counts; the counts of carbon-14 at the highvoltage were calculated from the ratio of pure carbon-14counts at 1,270 volts to carbon-14 counts at 890 volts.The quantity of aldosterone or corticosterone in a samplewas calculated from the yield of tritium radioactivity, theamount of carbon-14 lost during chromatography and thespecific activity of the tritium-labeled acetic anhydride.

The specific activity of the tritium-labeled acetic an-hydride was determined by acetylating hydrocortisone anddetermining the specific activity of the acetylated steroid.Dry hydrocortisone (0.5 mg.) was acetylated for 20hours at room temperature with 0.02 ml. of tritium-la-beled acetic anhydride in the presence of 0.02 ml. of an-hydrous pyridine. After acetylation, 1 ml. of 20 per centethanol in water was added to the reaction mixture. Thehydrocortisone acetate was extracted from the ethanolsolution with five volumes of dichloromethane. Thedichloromethane was washed with 1 ml. of distilled waterand dried under an air stream. The sample of hydrocor-

1281


TABLE II

Effects of cross-circulation of blood from normal dogs into the isolated adrenals of normal animals

Control Cross-circulation RecoveryDog -no. 1 2 3 1 2 3 1 2

Aldosterone secretion (ug./min.) *

.032 .034 .036 .047 .024

.062 .062 .056 .034 .023

.037 .045 .051 .022 .017

.043 .010 .019 .016

.038 .034 .032 .047 .047

.073 .060 .053 .053 .064

.044 .014 .012 .009 .011

.012 .005 .005 .019 .022

.043 .033 .033 .031 .030

Corticosterone secretion (Ag./min.)*3.5 4.2 3.6 3.1 2.93.8 3.8 3.3 4.7 4.53.9 3.4 3.6 4.3 3.33.3 2.4 2.5 4.54.4 4.0 4.2 4.4 3.34.1 3.4 3.6 4.0 4.12.6 1.7 1.6 1.9 2.51.4 1.0 0.8 3.4 2.5

3.4 3.0 2.9 3.8 3.3

4.775.333.484.803.757.804.927.14

5.24

Adrenal blood flow (ml./min.)*4.59 3.31 3.484.93 8.33 6.373.00 5.35 3.644.30 15.003.02 5.82 3.179.10 11.10 7.804.00 5.22 3.835.72 7.57 6.26

4.83 7.71 4.93

Mean arterial pressure (mm. Hg) *

113 110 100 80 92t135 132 122 118 110122 112 100 104 80110 105 100 11080 96 101 125 115

125 125 116 116 94110 127 125 112 81110 123 116 110 92

113 116 110 109 95

S2020

-2035352065

23

202020

-2530252555

21

Venous pressure (mm. water) *-10 -10

15 2020 202540 4025 20

0 -1520 30

17 15

.033t

.025

.053

.019

.032

.032

4.42.6

3.6

1.72.8

3.0

10.303.33

2.92

4.426.89

5.57

10880

103

95102

98

100

40

-1030

14

1112131415161718

Mean

1112131415161718

Mean

.043

.065.043

.032

.078

.024

.010

.042

.024

.063

.070

.047

.118

.051

.009

.055

3.7 1.93.2 3.22.4 3.2

1.6 1.93.3 4.31.1 1.80.9 0.9

2.3 2.5

1112131415161718

Mean

6.109.505.358.603.92

11.3012.8010.20

8.47

3.164.353.17

1.1010.00

2.223.94

3.99

1112131415161718

Mean

2.323.854.05

2.409.803.333.45

4.17

140140105t

77134106t118

117

1112131415161718

Mean

122$1351324

140t134114t116

128

03030

-1530452080

-28

0 5025 20

0 10

0 11045 4090 10070 75

33 58

* Steroid secretion, adrenal blood flow and arterial and venous pressures from isolated adrenals and associated vessels.t The thoracic inferior vena cava was constricted for one hour and studies conducted between the experimental and

recovery periods in this experiment (see Figure 4).t Norepinephrine infusion given to maintain arterial pressure and adrenal blood flow.

i1282


tisone acetate was chromatographed on Whatman No. 1

paper for 15 to 18 hours in a carbon tetrachloride (100parts), methanol (100 parts) and water (25 parts) sys-tem. The hydrocortisone acetate was eluted with ethanoland rechromatographed for 20 to 25 hours on alcoholwashed paper in the second solvent system used for aldo-sterone. The hydrocortisone acetate was eluted with re-distilled absolute ethanol and diluted to 25 ml. The con-centration of hydrocortisone acetate in this eluate was

measured by three methods: 1) absorption in the ultra-violet in a Beckman DU spectrophotometer at 242 m,u(e = 15,850); 2) phenylhydrazine reaction in sulfuricacid-ethanol (Porter-Silber) (6) ; and 3) fluorometricassay with sulfuric acid-ethanol. The specific activitywas determined by counting an aliquot of the eluate in theliquid scintillation spectrometer. The specific activity was

expressed in terms of cpm per ,ug. at the 1,270 voltagesetting.

The aldosterone diacetate-C1" was prepared from 1.0mg. of aldosterone. The aldosterone was acetylated for44 hours at 370 C. with acetic anhydride-1-C"' (5 mc. permMole) in dry benzene (15 per cent) in the presence ofanhydrous pyridine. The aldosterone diacetic-C' was

partitioned between aqueous alcohol and dichloromethaneas described previously and chromatographed on What-man No. 1 paper in the first and second systems describedpreviously for aldosterone. The steroid was eluted withredistilled ethanol and diluted to a volume such that each0.1 ml. yielded approximately 2,000 cpm. The solutionwas stored at -200 C. where it remained stable formonths.

The corticosterone monoacetate-C"' was prepared from2 mg. of corticosterone. Corticosterone was acetylatedfor 44 hours at 370 C. with 0.02 ml. of acetic anhydride-1-C14 (5 mc. per mMole) in benzene (15 per cent) in the

presence of anhydrous pyridine. The corticosteronemonoacetate-C1' was partitioned between aqueous alcoholand dichloromethane and chromatographed on WhatmanNo. 1 paper for 18 hours in the solvent system used forthe first chromatography of aldosterone and corticosterone.The steroid was eluted, dried with a stream of nitrogenand chromatographed for 15 hours on the second sol-vent system used for aldosterone. The corticosteronewas eluted with redistilled absolute ethanol and dilutedto yield approximately 5,000 cpm per 0.1 ml.

Specificity, reproducibility and recovery data for thedouble isotope derivative assay for aldosterone. Evi-dence for the specificity of this procedure for aldosteronewas obtained by demonstrating the constancy of H2/C1"ratios for each sample with repeated chromatography.Aldosterone was added to each of the eight samples ofpooled adrenal vein plasma in amounts to produce fourlevels of concentration (Table III) ; assays were carriedout at each level and on the same pool of adrenal veinplasma with no added aldosterone. The H'/C1" ratio was

determined after each of the three chromatographies bydetermining tritium and carbon counts from an aliquotof the eluate at the 1,270 voltage setting. The solventsystems and time for development of the chromatogramswere the same as those described previously for al-dosterone. The ratio of H'/C1' was essentially the same

after the second and third chromatographies (Table III).This finding indicates that no tritium-labeled impuritieswere present after the second chromatography. Never-theless, a third chromatography has been used in all rou-

tine studies. Recovery data on the same plasma samplesare presented in Table III.

A similar study of the specificity of this technique forcorticosterone was made. The first two chromatographieswere the same as those used for corticosterone in routine

TABLE III

Recovery, reproducibility and specificity data for aldosterone with the double isotope derivative assay procedure

Aldosterone recoveries Purification during chromatography

Recovery of H3/C4 after each chromatographytAldosterone Determined aldosterone %

Sample added* aldosterone added Recoveryt 1st 2nd 3rd

pg./ml. of og./ml. pg./mi.plasma

N* .025 21.1 2.14 2.321 A .012-.015 .039 .014 104 34.7 4.18 4.40

B .012-.015 .041 .016 119 15.0 1.88 2.212 A .024-.030 .047 .022 82 36.4 5.58 5.28

B .024-.030 .052 .027 100 17.2 2.67 2.903 A§ .040-.050 .075 .050 111 18.0 3.75 4.03

B .040-.050 .065 .040 89 16.8 3.36 3.474 A§ .080-.100 .105 .080 89 16.6 5.77 5.62

B .080-.100 .111 .086 96 15.3 6.13 5.96s

* Three ml. of the same pool of adrenal vein plasma from the isolated adrenals of a normal dog were used for eachsample. No aldosterone was added to Sample N. The concentration of the aldosterone added was determined bymeasuring a stock solution in the ultraviolet at 242 m,u (e = 15,850). The higher values given represent addedaldosterone calculated on this basis. The lower values were calculated from data obtained by double isotope derivativeanalysis of the same stock solution of aldosterone after addition to plasma (see Table IV).

t Per cent recovery is calculated by use of the average of the two values for aldosterone from Column 2.t H3/C14 is the ratio of the cpm of H3 and C14 at the 1,270 voltage setting; see text.§ Five thousand cpm of corticosterone monoacetate-C14 was added initially to provide an artificial contaminant

of the sample.

1283


TABLE IV

Reproducibility of aldosterone determinations inplasma

Lots of Average valuetritium- for aldosteronelabeled for each lotacetic Aldosterone in each of acetic

anhydride sample anhydride

psg./mi. pg./ml.1 .063, .071,.072,.062 .0672 .076, .069 .0733 .079,.081,.077 .0794 .077, .082, .075 .0785 .103,.082,.092,.080, .100 .0916 .080 .080

Mean = 0.079 4 .011 S. D. Mean = .078 i .008 S. D.

analyses. The samples were then oxidized with chromictrioxide as for aldosterone except for onily five minutes;corticosterone monoacetate was converted to 11-dehy-drocorticosterone acetate. The first solvent system was

used for the third chromatography and the chromatogramwas developed 14 to 16 hours. The ratio of H3/C14 was

unchanged after the second chromatography. The dataindicate that only two chromatographies were needed forpurification at the concentrations of corticosterone ob-served in the present study.

Data on reproducibility are presented for duplicatesamples of plasma aldosterone analyzed concurrently(Table III); the agreement is satisfactory. Similarresults were obtained for corticosterone. Additional dataon reproducibility have been obtained by analyzing one

sample of plasma for aldosterone with each weekly groupof 10 to 15 samples of adrenal vein plasma from an ex-

periment. A pool of 150 ml. of peripheral plasma towhich aldosterone had been added was the source foreach control sample which was analyzed weekly. Sixdifferent lots of tritium-labeled acetic anhydride were usedand analyses were performed weekly over a period of fivemonths. The results are presented in Table IV. Themean value for the 18-aldosterone determinations with sixlots of acetic anhydride was 0.079 0.011 ,ug. per ml.

RESULTS

Cross-circulation of blood from dogs zwith thoraciccaval constriction into normal recipients via thefemoral vessels

Aldosterone secretion increased in two of thethree recipient dogs following cross-circulation ofblood from a donor dog with thoracic caval con-

striction (Table V). During study of the firstpair of animals, acute constriction of a normal dogwhich became the donor resulted in a 175 per centincrease (comparison of average control withaverage experimental value) in aldosterone secre-

tion while a 251 per cent elevation in aldosteroneproduction occurred in the recipient animal. Inthe second pair of dogs, aldosterone secretion inthe chronic donor animal with thoracic caval con-

striction was markedly elevated above the average

rate of aldosterone secretion for normal dogs(Table V, Figure 2); during cross-circulation, al-dosterone secretion was not altered appreciably inthe donor. In the normal recipient dog of thispair, a 146 per cent increase in aldosterone se-

cretion occurred during cross-circulation. Failureof a response to occur in the third recipient may bea reflection of the abnormally high control valuesof 0.077 to 0.142 pg. per minute in the recipient;the initial hematocrit in this animal was 39 per

cent which was considerably lower than the he-matocrit for the other dogs. It seems likely thatthe initial high values for aldosterone resultedfrom intra-abdominal bleeding.

The alterations in corticosterone secretion were

slight and variable. In the first pair of dogs, thevalues of corticosterone secretion in the recipientwere 33 per cent higher during cross-circulationthan during the control period. Also, in the donora similar slight increase in corticosterone secretionoccurred. In the other four animals, cortico-sterone secretion was either unchanged or fellslightly in both donor and recipient.

The changes in adrenal blood flow after acute

.30

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0 8- - "L__q

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DOGNO.-I 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8

NORMAL THORACIC IVC

CONSTRICTION

FIG. 2. THE RATES OF ALDOSTERONESECRETION IN

ADRENAL VEIN PLASMA FROM THE RIGHT ADRENALGLANDOF NORMALDOGSAND FROMDoGS WITH CHRONICTHORACIC INFERIOR VENA CAVA (IVC) CONSTRICTION

The three determinations for each animal were made at30 minute intervals.

1284

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ALOOSTERONESECRETION 03

({Lg/min. )

ALDOSTERONEINADRENALVEIN PLASMA I.

0.CORTICOSTERONE

SECRETION(;ig/min.)

ALDOSTERONECORTICOSTERONE

SECRETION

ADRENALBLOODFLOW(cc/min)

PLASMANo 0

PLASMAK ° 16(mEq./LJ :40 15

ARTERIAL PRESSURE 150'oOF ISOLATED ADRENALS125

(mm/Hg) 0075

VENOUSPRESSURE 80IOF ISOLATED ADRENALS60

(mm.Woter) NOREPINEPHRINE

_ aur71#CROSS

ONPRFUSION ClCULATIO PERFUSION

0 60 120 180 240MINUTES

FIG. 3. THE EFFECTS OF CIRCULATION OF BLOODFROM

A DoG WITH CHRONIC THORACIC CAVAL CONSTRICTIONINTO THE ISOLATED ADRENALSOF A NORMALRECIPIENTANIMAL (DoG No. 7 IN TABLE I)

The actual aldosterone ratio is multiplied by 10'.corticosterone

caval constriction or after establishment of cross-

circulation were slight and apparently were with-out effect on hormone secretion (Table V). In-ferior vena caval pressure increased in the donordog only in the first pair of animals, and the highvenous pressure present in the other donor dogswith chronic thoracic caval constriction increasedfurther during cross-circulation while venous pres-

sure in the recipient animals was maintained at thecontrol level. Only slight fluctuations in arterialpressure occurred. No consistent alterations inplasma Na or K were detected.

Circulation of blood from dogs with chronic con-

striction of the thoracic inferior vena cava

through the isolated adrenals of normal recipientanimals

Observations were first made on a group ofeight dogs with chronic thoracic caval constriction,ascites and marked Na retention to determine bythe present radioisotope technique the magnitudeand consistency of hypersecretion of aldosterone.

The data that were obtained are compared (Fig-ure 2) with those reported previously (5) fornormal dogs. Aldosterone secretion was elevatedin every animal with caval constriction and themean rate of aldosterone secretion of 0.146 ,Lg.per minute for dogs with caval constriction wassixfold greater than the average rate of 0.024 ug.per minute for normal dogs (t = 5.8; p < 0.001 ) .It was concluded that the consistency and magni-tude of the hyperaldosteronism was sufficient tojustify performance of cross-circulation withoutmeasurements of aldosterone secretion in thechronic donor dogs.

Seven cross-circulation experiments were con-ducted using dogs with chronic thoracic cavalconstriction as donors (Table I). After threecontrol determinations during autoperfusion of therecipient's isolated adrenals, cross-circulation wasestablished with a dog with chronic thoracic cavalconstriction. In five of the seven experiments, arecovery period with autoperfusion was obtained.

Aldosterone secretion increased in every in-stance (average increase was 129 per cent) andreturned to the control level during the recoveryperiods (Table I, Figure 3). A statistical analy-sis of variance showed that the increase was sig-nificant at the 1 per cent level (F = 14.50, t =

3.80). The increase in aldosterone secretion wasassociated with a greater increase percentage-wise(196 per cent) in the concentration of aldosteronein adrenal vein plasma. Since blood from thedonor dogs perfused isolated adrenal glands, thedata show a direct effect upon the recipient'sadrenals of a humoral agent in donor blood. Therate of corticosterone secretion was slightly higherduring cross-circulation in three of the five ex-periments and fell during the recovery period.Variations in adrenal blood flow occurred and thelow values for aldosterone secretion for the thirddetermination during the cross-circulation periodin Dogs 4, 5 and 6 were associated with lowadrenal blood flows (Table I). Arterial pressurewas slightly lower during cross-circulation thanduring control and recovery periods unless thepressure was maintained by a norepinephrine in-fusion. Inferior vena caval pressure was un-changed or slightly reduced during cross-circula-tion and slightly elevated during recovery. Noconsistent changes in plasma Na were detectedduring cross-circulation. Plasma K was un-

l il

4-

2~~ ~ ~~ie--n-t-----I--s-

,0 s1 -nnn5:, ~ 1 N 1

I

1286


changed or increased slightly during cross-circu-lation; in one instance (Figure 3) the slight in-crease continued during recovery.

Circulation of blood from normal dogs into theisolated adrenals of normal recipient animals

To determine the effects of cross-circulation perse on adrenocortical function, eight studies wereperformed by circulation of blood from a normalanimal into the isolated adrenals of a normalrecipient. Aldosterone secretion remained un-changed or decreased in all but two dogs andthe mean values for aldosterone secretion wereunchanged (Table II). During recovery, aldo-sterone production was slightly elevated in someinstances. The variable response is probably at-tributable to factors producing variation in aldo-sterone secretion in the normal donors. Cortico-sterone output was increased slightly (four dogs)or unchanged (four dogs) during cross circula-tion (Table II, Figure 4) and decreased duringrecovery. Adrenal blood flow was not appreciablydifferent during the control and cross-circulationperiods but was slightly reduced during recovery.The slight decline in arterial pressure was simi-lar to that observed during cross-circulation ofblood from dogs with secondary hyperaldosteron-ism into normal isolated adrenals. Inferior venacaval pressure was unchanged during cross-circu-lation but was frequently slightly elevated duringrecovery. In one experiment (Figure 4), thethoracic inferior vena cava was constricted in thenormal donor after three determinations hadbeen made during cross-circulation; a 100 percent increase in aldosterone secretion resulted.The data for arterial and venous pressures andadrenal blood flow on this animal are presentedin Table II (see Dog No. 12).

DISCUSSION

The nmechanisms regulating aldosterone secre-tion by the adrenal cortex may be neural, humoralor both. That neural connections to the adrenalgland are unnecessary for aldosterone secretionand maintenance of electrolyte metabolism wassuggested more than two decades ago by Levyand Blalock (7). They transplanted one adrenalgland to the neck and removed the other adrenal;

ALDOSTERONESECRETION(gJmin.)

CORTICOSTERONESECRETION

(pLg/min.)

.06

03

0

4

0 60 120 180 240 300

MINUTES

FIG. 4. ALDOSTERONEAND CORTICOSTERONESECRETIONFROM NORMAL ISOLATED ADRENALS BEFORE (AUTOPER-FUSION) AND DURINGCROSS-CIRCULATIONOF BLOODFROM

A NORMALDoG

Subsequently, the thoracic inferior vena cava in thenormal animal was constricted. A recovery period was

obtained with the recipient's blood (autoperfusion).

several animals lived for months in a normalhealthy condition. More recently, Fleming andFarrell (8) showed that acutely transplantedadrenals secrete normal or above normal amounts

of aldosterone. Additional data were provided byMcDonald, Goding and Wright (9) from chronicstudies in unanesthetized sheep. Unilaterallyadrenalectomized sheep with the remaining adrenaltransplanted to the neck showed maintenance ofnormal plasma electrolyte concentrations and a

normal response to Na depletion as indicated bya marked drop in the salivary Na: K ratio. If a

humoral mechanism is involved, it might be an

alteration in the concentrations of plasma electro-lytes, or it might be hormonal. Slight fluctuationsin plasma Na and K occurred in the present studybut no consistent changes were detected. Meas-urements of total plasma osmolality in previousstudies of dogs with acute thoracic inferior vena

cava constriction and hyperaldosteronism (5)showed no change.

The present experiments demonstrate thatblood from dogs with secondary hyperaldosteron-ism stimulated aldosterone secretion in normalrecipient animals and in the isolated adrenals ofnormal dogs. The increase was associated withan elevation in the concentration of aldosterone inadrenal vein plasma. During the preliminarystudy of the effects of cross-circulation via thefemoral vessels, the volume of blood in both donor

DURINGTHORACICIVCCONSTRICTION

IN DONOR

AU--H CROSSCIRCULATION -] AUTO- -PERFUSION PERFUSION

1287

2


and recipient animals was regulated by means ofscrew clamps on the interconnecting tubing in anattempt to maintain a constant arterial pressure.Since changes in blood volume could not be ex-cluded, the definitive study was performed bycross-circulation of blood from dogs with thoraciccaval constriction into the isolated adrenals ofnormal dogs. As a control on the effects ofcross-circulation per se or for any event associ-ated with this experimental design, blood fromnormal dogs was circulated through normalisolated adrenals. Arterial pressure fell slightlyduring cross-circulation in most experiments butthe decline in pressure was essentially the samefor both experimental (Table I) and control(Table II) groups. The arterial pressure in thedonor animals was the same as the recorded pres-sure of blood which perfused the isolated adrenalssince the blood pressure in the isolated aorta wasderived entirely from blood from the donor.Aldosterone secretion increased in every instanceand the change was highly significant duringcross-circulation of blood from hyperaldosterone-mic donors into isolated adrenals, whereas cross-circulation of blood from normal animals producedno consistent alteration in aldosterone secretionand the mean control and mean experimental val-ues were the same. The present study demon-strates, therefore, the existence of an aldosteronestimulating agent in blood of dogs with experi-mental secondary hyperaldosteronism and it issuggested that this humoral agent is a hormone.The data agree with the suggestion of Orti, Ralli,Laken and Dumm(10) that a humoral substanceprovides the stimulus to aldosterone production.Orti and associates reported evidence from stud-ies of rats on a low Na diet that a substance inurine increased aldosterone excretion in feces.

In most of the present experiments, cortico-sterone secretion was measured to examine thepossibility that the circulating hormone is ACTH.Corticosterone rather than hydrocortisone wasstudied because of the similar polarity of cortico-sterone monoacetate and aldosterone diacetate dur-ing descending chromatography; these two ster-oids were separated with the same solvent systemduring the first chromatography. In the dog, theeffects of ACTHon corticosterone and hydrocorti-sone release are very similar. Farrell, Rausch-kolb and Royce ( 11 ) showed similar marked drops

in corticosterone and hydrocortisone after hypo-physectomy and an increase in both steroids afterACTHadministration. This marked drop in cor-ticosterone after hypophysectomy in the dog andthe response to ACTHhave been confirmed (Ref-erence 12, and unpublished observations). Also,in an earlier report (5), concurrent measurementsof Porter-Silber reacting steroids and corticoster-one in adrenal vein plasma showed similar direc-tional changes in every instance. Available evi-dence indicates, therefore, that alterations in eithercorticosterone or hydrocortisone secretion may re-flect changes in circulating ACTHin the dog.

Increased circulating ACTH alone does notappear to explain the observed increase in aldo-sterone output, Aldosterone secretion increasedmarkedly in every instance during cross-circula-tion of blood from dogs with secondary hyperal-dosteronism into isolated adrenal whereas cortico-sterone secretion increased slightly in 60 per centof the experiments. During the present recoveryobservations, aldosterone secretion was at the con-trol level or slightly elevated whereas cortico-sterone output declined further and in many in-stances this decline was associated with a decreasein adrenal blood flow. In one experiment (Figure4) in which blood from a normal dog was circu-lated through normal isolated adrenals, aldosteronesecretion fell while corticosterone output increased;subsequent constriction of the thoracic inferiorvena cava of the normal donor resulted in a 100per cent increase in aldosterone production butcorticosterone secretion was unchanged. Also,the alterations in corticosterone secretion were sim-ilar during cross-circulation of blood from nor-mal dogs into isolated adrenals to the changesfound during cross-circulation of blood from dogswith chronic caval constriction. During previousobservations (5), acute constriction of the thoracicinferior vena cava produced an increase in aldo-sterone secretion while corticosterone output fell.Although these findings suggest different regula-tory mechanisms for aldosterone and corticoster-one secretion, the data do not exclude an influenceof ACTH on aldosterone production. Both al-dosterone and corticosterone output may have beenincreased during the control observations as a re-sult of surgery. Hilton and associates (3) havereported near maximal output of hydrocortisone inthe isolated adrenal preparation. Additional stud-

1288


ies are needed to identify this aldosterone stimulat-ing substance and to determine its origin.

SUMMARYAND CONCLUSIONS

To test the possibility of a humoral mechanismin the regulation of aldosterone secretion, cross-circulation experiments were performed betweendogs with thoracic inferior vena cava constrictionand secondary hyperaldosteronism and normal re-cipient animals. Cross-circulation was establishedthrough the femoral vessels, or the isolated adrenalsof normal animals were perfused with blood from ahyperaldosteronemic donor. Control and recov-ery periods were obtained with the recipient's bloodor by cross-circulation of blood from a normal dog.Repeated determinations of aldosterone and corti-costerone were made in adrenal vein plasma by thedouble isotope derivative method. Aldosteronesecretion in the recipient or in the recipient's iso-lated adrenals increased markedly in nine of 10experiments, and returned to the control level inthe isolated adrenals of five dogs with recoveryobservations. Corticosterone secretion increasedonly slightly in four of eight experiments. Noconsistent alterations in the concentrations ofplasma Na and K were detected. During cross-circulation of blood from eight normal dogs intothe isolated adrenals of normal animals, aldosteronesecretion was unchanged or decreased in all buttwo dogs; a slight increase in corticosterone secre-tion occurred in one-half of the animals. It is con-cluded that a humoral agent, possibly a hormone,stimulates the adrenals to secrete aldosterone indogs with secondary hyperaldosteronism.

ACKNOWLEDGMENTS

Weare indebted to Dr. J. E. Lieberman for the statisti-cal analysis of variance.

REFERENCES

1. Davis, J. O., Pechet, M. M., Ball, W. C., Jr., andGoodkind, M. J. Increased aldosterone secretionin dogs with right-sided congestive heart failureand in dogs with thoracic inferior vena cava con-striction. J. clin. Invest. 1957, 36, 689.

2. Davis, J. O., and Howell, D. S. Mechanisms of fluidand electrolyte retention in experimental prepara-tions in dogs. II. With thoracic inferior venacava constriction. Circulat. Res. 1953, 1, 171.

3. Hilton, J. G., Weaver, D. C., Muelheims, G., Glavi-ana, V. V., and Wegria, R. Perfusion of the iso-lated adrenals in situ. Amer. J. Physiol. 1958, 192,525.

4. Kliman, B., and Peterson, R. E. Isotope derivativeassay of aldosterone in biological extracts. Fed.Proc. 1958, 17, 255.

5. Davis, J. O., Kliman, B., Yankopoulos, N. A., andPeterson, R. E. Increased aldosterone secretionfollowing acute constriction of the inferior venacava. J. clin. Invest. 1958, 37, 1783.

6. Peterson, R. E., Karrer, A., and Guerra, S. L.Evaluation of Silber-Porter procedure for deter-mination of plasma hydrocortisone. Analyt. Chem.1957, 29, 144.

7. Levy, S. E., and Blalock, A. A method for trans-planting the adrenal gland of the dog with re-establishment of its blood supply. Ann. Surg.1939, 109, 84.

8. Fleming, R., and Farrell, G. Aldosterone and hydro-cortisone secretion by the denervated adrenal. En-docrinology 1956, 59, 360.

9. McDonald, I. R., Goding, J. R., and Wright, R. D.Transplantation of the adrenal gland of the sheepto provide access to its blood supply. Aust. J.exp. Biol. med. Sci. 1958, 36, 83.

10. Orti, E., Ralli, E. P., Laken, B., and Dumm, M. E.Presence of an aldosterone stimulating substancein the urine of rats deprived of salt. Amer. J.Physiol. 1957, 191, 323.

11. Farrell, G. L., Rauschkolb, E. W., and Royce, P. C.Secretion of aldosterone by the adrenal of the dog.Amer. J. Physiol. 1955, 182, 269.

12. Davis, J. O., Bahn, R. C., Yankopoulos, N. A., Kli-man, B., Peterson, R. E., and Ball, W. C., Jr.Effects of acute and chronic hypothalamic lesionson aldosterone secretion in dogs with chronic ex-perimental ascites. The Physiologist 1958, 1, 15.

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