Journal of Clinical InvestigationVol. 46, No. 5, 1967

The Steroid Content of Adrenal Adenomas and Measure-ments of Aldosterone Production in Patients with

Essential Hypertension and PrimaryAldosteronism *

NORMANM. KAPLANt WITH THETECHNICALASSISTANCEOF ROBERTCOOKANDSTEVONNEGULLEY

(From the Department of Internal Medicine, The University of Texas Southwestern MedicalSchool, Dallas, Texas)

Summary. The content of aldosterone, corticosterone, and cortisol has beenassayed in normal adrenal tissue obtained at autopsy and in adrenal adenomasfrom both normotensive and hypertensive patients obtained at autopsy andfrom patients with primary aldosteronism obtained at surgery. The contentof aldosterone and corticosterone in the adenomas of patients with essentialhypertension was similar to that of normal adrenal tissue and much less thanthat of adenomas from patients with primary aldosteronism.

Since these results do not appear to reflect various extraneous factors suchas assay technique, degradation of steroids, and patient selection, they sug-gest that most adrenal adenomas found in patients with essential hyperten-sion are not associated with increased aldosterone production. The presenceof such adenomas should not, in itself, be considered evidence for primaryaldosteronism.

The excretion or secretion of aldosterone in 43 hypertensive patients wasfound to be similar to that of 39 normotensive patients. These results sug-gest that primary aldosteronism is rarely the cause of "essential" hypertension.

Introduction

Benign adrenocortical adenomas have long beenrecognized as occurring more frequently in pa-tients with hypertension than in normotensivesubjects (1). These adenomas have generally

* Submitted for publication August 5, 1966; acceptedJanuary 4, 1967.

Presented in part at the 38th Annual Meeting of theCentral Society for Clinical Research, November 5, 1965,and the endocrinology sectional meeting of the Ameri-can Society for Clinical Investigation and the AmericanFederation for Clinical Research, May 1, 1966.

Supported by grants from the Dallas Heart Associa-tion and the U. S. Public Health Service, National In-,stitute of Arthritis and Metabolic Diseases (AM 06938-04) and General Medical Sciences (NIH STO1 GM142101).

t Address requests for reprints to Dr. Norman M.Kaplan, Dept. of Internal Medicine, The University ofTexas Southwestern Medical School, 5323 Harry HinesBlvd., Dallas, Texas 75235.

been considered to be "incidental" and of no func-tional significance (2), since none of the expectedclinical features of adrenal hormone excess wereusually observed in patients found to have them atautopsy.

However, the possibility that such adenomasmay be causally related to the hypertensive processhas been proposed (3). The reports by Conn andhis associates have strongly suggested that suchadenomas producing excess amounts of aldosteronemay be the cause of as much as 20%o of all essentialhypertension (4).

In view of this suggestion, we have attemptedto determine the functional significance of adrenaladenomas in patients with essential hypertensionby comparing the steroid content of such adenomaswith that of normal adrenal tissue and adenomasremoved from patients with primary aldosteronism.The results suggest that most adrenal adenomasfound in patients with essential hypertension prob-

728

STEROID CONTENTOF ADRENALADENOMAS

ably are not producing excess amounts of aldos-terone.

We sought additional evidence concerning theincidence of primary aldosteronism by measuringeither urinary aldosterone excretion or the aldos-terone secretory rate in patients with essential hy-pertension. No significant difference in aldos-terone production between such patients and nor-

motensive subjects was found.

Methods

Source of adrenal tissue. Adrenocortical adenomas are

here defined as solitary nodules greater than 5 mmindiameter composed of cords of lipoid-engorged cellswithout identifiable zonation, which are demarcated by a

partial capsule or compression of adjacent adrenal tissue.Adenomas were removed surgically from seven patients

with the classical clinical and laboratory features of pri-mary aldosteronism, including hypokalemia and elevatedurinary aldosterone excretion. These features disappearedafter unilateral adrenalectomy.

All other adrenal glands were obtained at autopsy.The "normal" glands were obtained from seven patientswho died of the following causes: acute myocardial in-farction (two), sudden trauma (two), chronic renalinsufficiency (one), pulmonary embolus (one), and car-

cinoma of the stomach (one). Three of these patientshad been hypertensive. Two glands with solitary adeno-mas were found in patients who had been normotensive,and 11 were found in patients whose blood pressures hadbeen above 140/90 mmHg and whose heart weights were

above 500 g for men or 450 g for women. These pa-

tients had had normal serum potassium concentrationswhen measured at least twice during the year beforedeath, and did not have hemorrhages, exudates, or papil-ledema in the optic fundi; there were no specific causes

for hypertension found post-mortem. The living patientsstudied met similar criteria, except that specific causes

for hypertension were excluded by the usual clinical tech-niques, including intravenous pyelography.

Patient selection. Assays of aldosterone excretion or

secretion were performed on 39 normotensive and 43 hy-pertensive patients. They had been hospitalized for vari-ous medical problems, but all were ambulatory, eating a

regular diet, off medication, and free of overt manifes-tations of disease when studied. All such patients avail-able during a 6-month interval were studied. Duringthis interval, none of the seven patients with primaryaldosteronism were diagnosed.

We considered the hypertensive patients to have es-

sential hypertension by excluding specific causes of hy-pertension by physical examination, intravenous pyelog-raphy, urinary 17-hydroxycorticoid and catecholamine as-

says, and, in 16 patients, renal arteriography. Patientswere excluded who had hemorrhages, exudates or papil-ledema in the optic fundi, renal disease with azotemia,or heart failure.

Serum potassium had been below 3.5 mEq per L in fiveof the normotensive and seven of the hypertensive pa-tients when they were admitted to the hospital. An ex-trarenal cause of potassium loss was known for all twelvepatients and was demonstrated by the finding of less than30 mEq of potassium in a 24-hour urine collection.Supplemental potassium had been given to ten of thesepatients but had been discontinued for at least 1 weekbefore study of aldosterone production. At that time,the serum potassium was between 3.8 and 5.0 mEq per .Lin all patients, and all were on regular hospital diets withunrestricted access to salt. The adequacy of sodium in-take was determined by the presence of more than 100mEq of sodium in the 24-hour urine collection.

Steroid measurements. Urinary aldosterone excretionwas measured by the technique of Kliman and Peterson(5). Aldosterone secretion was measured by an isotopicdilution technique, in which 1 Ac of tritium-labeled al-dosterone diluted in 20 ml of 5% ethanol was injected in-travenously at the start of the urine collection. The al-dosterone released by incubation at pH 1 for 24 hours wasidentified by chromatography in the Bush C system,eluted, acetylated with acetic anhydride-"C of knownspecific activity, and thereafter purified in the same man-ner as in the urinary excretion measurements. Usingstandard isotopic dilution equations, we determined the24-hour secretion rate from the 'H to "4C ratio of thepurified sample.

Assay of adrenal tissue. The adrenal tissue was keptfrozen until assayed by a modification of the double iso-topic derivative assay of Kliman and Peterson (5).Only cortical tissue from the normal glands was used.From 200 to 800 mg of tissue was placed in 5 ml water,and 2,000 cpm of "C-labeled aldosterone, corticosterone,and cortisol was added. The tissue was homogenized,and the steroids were extracted with methylene dichloride.The extract was washed with 0.05 N NaOH, 0.1 N aceticacid, and petroleum ether. The methylene dichloride wasevaporated and the steroid residue kept in a dessicatorfor 24 hours.

The extract was streaked onto chromatography paperwith appropriate nonradioactive steroid markers on eitherside and run for 4 hours in the Bush C system (toluene450, methanol 250, water 250, ethyl acetate 50 vol/vol).The appropriate areas were eluted together in ethanol,and the extract was dried and acetylated with aceticanhydride-3H, 25 mc per mmole. After the addition ofthe three nonradioactive steroid acetates as markers, thesamples were chromatographed in a cyclohexane: dioxane:methanol: water (100: 75: 50: 25 vol/vol) system for15 hours, which provided separation of aldosterone diace-tate, corticosterone acetate, and cortisol acetate. Theseparate areas of each steroid were then individuallychromatographed, first in the cyclohexane: benzene:methanol: water (100: 33: 100: 25 vol/vol) system andthen after oxidation with chromium trioxide, in thecyclohexane: benzene: methanol: water (100: 75:100: 25vol/vol) system. The appropriate areas were eluted andcounted for 'H and "C in a liquid scintillation spectrom-

729

NORMANM. KAPLAN

eter. All values were expressed as micrograms steroidper gram tissue.

Stability of steroids in adrenal tissue. Since compari-sons were to be made from adrenal tissue frozen withinan hour after surgical removal from living patients withprimary aldosteronism and after removal at autopsy fromcadavers kept at 20 C for 6 to 18 hours, the effects oftime and temperature on steroid content were firststudied.

Adrenal glands from four cattle were obtained within15 minutes after death, and portions were frozen immedi-ately or kept at 25 or 370 C for 6 to 24 hours and thenfrozen. A similar procedure was followed for one adrenaladenoma surgically removed from a patient with primaryaldosteronism. Steroid content was assayed in the man-ner previously described. As seen in Table I, only inthose tissues kept at 370 for 24 hours was a marked de-crease in aldosterone and corticosterone content noted.In the adenoma kept at 370 for 24 hours, the level ofaldosterone fell by 22%, the level of corticosterone 45%,of that in the tissue frozen immediately. The cortisolcontent fell sharply within 6 hours at 370 and 24 hours at250 C in the cattle glands, but only after 24 hours at bothtemperatures in the adenoma.

The rate of cooling in the inner core of the humancadaver in refrigerated vaults is variable, depending onthe cause of death, amount of body fat, and so forth.However, it can be assumed that the adrenal glands werenear 370 C for only a few hours and probably below250 C for most of the time between death of the patientand removal of the tissue. Therefore, any observed dif-ferences between the "fresh" and postmortem tissues areprobably not caused simply by loss of steroid from theglands obtained at autopsy.

ResultsSteroid content. The results of the assays on

the nonadenomatous adrenal tissue from the sevennormal patients and the solitary adenomas removedat surgery from seven patients with primary aldos-

TABLE I

Effects of time and temperature on steroidcontent of adrenal tissue

TimeSource of after Tem- Aldos- Corti-

tissue death perature terone costerone Cortisol

hours 0 C gA/1tissue

Cattle 0 0.64 5.63 10.336 25 0.53 5.57 8.28

24 25 0.43 5.01 2.766 37 0.46 3.08 2.71

24 37 0.13 2.15 1.53

Adenomafrom patient 0 5.03 15.41 24.65

6 25 5.21 17.50 22.9624 25 4.65 11.67 14.43

6 37 4.88 12.33 20.0224 37 3.96 8.40 4.53

20p

Tug/gTtssueYO

C

ALDOSTERONE

* (30.4)

00/

I'd4

CORTICOSTERONE

* (55.0)* (28.5)* (26.9)

ol-

* i*

I

Normal Esse.tiol Primary Noroml Esseltiol PmoryH.T. Aldo I H.T. Aldo

FIG. 1. CONTENTOF ALDOSTERONEAND CORTICOSTERONEIN NORMALADRENAL TISSUE, CORTICAL ADENOMASFROM

PATIENTS WITH ESSENTIAL HYPERTENSION, AND ADENOMASFROMPATIENTS WITH PRIMARY ALDOSTERONISM.

teronism and at autopsy from two previously nor-motensive and eleven previously hypertensive pa-tients are shown in Table II. The individual valuesfor aldosterone and corticosterone are shown inFigure 1.

Compared with the normal glands, the contentof aldosterone and corticosterone was markedlyincreased in the adenomas of patients with pri-mary aldosteronism. However, the adenomasfound in the normotensive patients and those withessential hypertension contained amounts of aldos-terone and corticosterone similar to those in nor-mal tissue. Adrenal tissue adjacent to the ade-nomas of three of the patients with essentialhypertension was also analyzed. The content ofaldosterone and corticosterone was slightly lowerin the nonadenomatous tissues than in the ade-nomas, with mean values of 0.29 and 3.86 ,ug perg tissue, respectively. The content of cortisol wassimilar in all four types of adrenal tissue. Sincethe level of cortisol was most markedly altered inadrenal tissue left for 6 to 24 hours at 25 or 37°,the finding of similar cortisol contents in the freshand postmortem tissues should be strong evidencethat the observed changes in aldosterone contentwere not simply the result of degradation in thepostmortem tissue.

These levels of steroid content of both normaladrenal tissue and adenomas of patients with pri-mary aldosteronism are higher than most of thosepreviously reported (6-8). However, these re-sults were obtained with a more sensitive tech-nique than that used in the first two studies (6,

730

STEROID CONTENTOF ADRENALADENOMAS

TABLE II

Steroid content of adrenal tissue

Blood Serum TissuePatient Age Sex pressure Na/K weight Aldosterone Corticosterone Cortisol

mmHg mEq1L g Pg/g tissue

M 130/ 80M 180/110M 160/ 95F 175/100F 120/ 75F 120/ 80F 130/ 85

142/4.6138/4.0140/3.8136/3.5141/4.3140/4.5138/4.2

3.5 0.264.1 0.333.2 0.253.0 0.184.8 0.623.8 0.114.1 0.15

0.27

4.8 9.440.7 30.402.1 4.081.6 11.711.5 4.531.2 9.520.5 2.70

10.34

6.523.032.084.650.822.002.88

3.14

2.3011.2555.0226.8814.4128.4715.81

22.02

4.443.69

4.06

7.364.083.442.234.346.086.021.684.413.026.21

4.44

9.925.46

12.2817.38

5.756.66

13.60

10.15

2.3911.52

8.1115.0324.6510.434.48

10.94

6.235.13

5.68

8.7810.06

9.038.157.77

11.403.56

12.226.038.476.51

8.36

"Normal" adrenal glandsL.V. 71W.B. 27R.T. 34C.W. 85R.W. 79A.C. 45P.L. 50

Mean

Adenomas from patients with primary aldosteronismW.E. 42 M 210/140 145/2.6L.W. 48 M 180/120 142/3.0M.J. 34 F 180/ 95 137/2.2D.D. 55 M 180/110 148/2.5M.B. 48 F 200/110 145/3.2J.T. 52 M 180/115 144/2.9M.S. 43 M 170/100 142/2.6

Mean

Adenomas from normotensive patientsW.F. 63 M 12M.Y. 56 F 1

Mean

20/ 80 140/3.6 1.6 0.3130/ 85 139/4.3 1.2 0.18

0.24

Adenomas from patients with essential hypertensionB.M. 79 F 200/120 136/4.1A.S. 48 M 190/120 140/4.7E.R. 66 F 180/100 135/3.6B.Y. 56 F 145/ 95 138/4.0I.H. 50 M 160/ 95 136/3.6L.J. 55 F 170/105 140/4.1M.O. 86 M 210/130 142/4.0L.S. 76 F 160/100 136/4.8C.C. 33 F 175/105 140/4.5A.S. 56 F 150/100 138/4.1P.Q. 65 M 190/110 140/3.7

Mean

2.3 0.360.8 0.300.9 0.521.3 0.231.0 0.111.1 0.750.6 0.230.5 0.292.8 0.681.8 0.241.1 0.35

0.37

7). No normal tissue was measured by Biglieri,Hane, Slaton, and Forsham, so comparisons can-not be made (8).

Measurements of aldosterone production. The24-hour urinary aldosterone excretion or aldos-terone secretion rates of 39 normotensive and 43hypertensive patients are shown in Figure 2.Although the mean values for the patients withessential hypertension are slightly higher (10.9and 112 compared to 10.1 and 90), the differ-ences are not statistically significant (p > 0.2).Moreover, the distributions of the values withinthe two groups appear similar.

Aldosterone secretion was above 150 ug per

day in one normotensive and two hypertensivepatients, and aldosterone excretion was above 15ytg per day in four normotensive and six hyper-tensive patients. In an attempt to demonstratethat these higher values were normal, plasmarenin activity was measured in six of these thir-teen patients, one normotensive and five hyper-tensive. The blood was obtained first while theywere on a regular diet and supine, and then after3 days on a 500-mg low salt diet and 4 hoursupright. The assays were performed by a modi-fication of the Helmer technique.' All six pa-

1 Performed in the laboratory of Dr. J. Caulie Gunnelsat Duke Medical Center.

731

NORMANM. KAPLAN

200

d

c ai*40 l

U)(I)

150

100

50

0

ALDOSTERONE

I - 2C0 0o

1

I) _ ° - 150 A

o I>~~~~0 ..... ..........

50

.c

FIG. 2. ALDOSTERONESECRETION (OPEN DOTS) OR EX-

CRETION (CLOSED DOTS) IN 39 NORMOTENSIVEPATIENTS AND

43 WITH "ESSENTIAL" HYPERTENSION.

tients had a greater than threefold rise in plasmarenin activity to a value above 300 ng per 100 mlplasma after a 1-hour incubation. These valuesdemonstrate a normal responsiveness of plasmarenin activity and are further evidence that thesepatients with relatively high aldosterone assays

are, in fact, normal.

Discussion

Autonomous hypersecretion of aldosterone froma benign adrenocortical adenoma was first re-

ported in 1955 (9, 10). Since then, over 200cases of primary aldosteronism have been re-

ported, and all have been associated with hyper-tension (11). The hypertension seen with pri-mary aldosteronism has been, with rare excep-

tions, of a benign type, indistinguishable fromessential hypertension. The presence of hypo-kalemia has been the usual major diagnostic cri-terion for the recognition of this syndrome among

patients with hypertension. However, Conn,Cohen, Rovner, and Nesbit have recently dis-covered aldosterone-secreting adenomas in nor-

mokalemic patients (12). They suggest that pri-mary aldosteronism may be the cause of as muchas 20%o of all essential hypertension.

One of the reasons given for this view on thelarge incidence of primary aldosteronism is thefinding of adrenocortical adenomas greater than1.0 cm in diameter in as many as 20% of hyper-tensive subjects but in only 1.8%o of normotensivesubjects at autopsy (3). However, if smalleradenomas that measure between 0.5 and 1.0 cm

are included, their incidence in this study in thehypertensive patients was 88% and in normo-

tensive patients, 44% (3). These adenomas are

indistinguishable grossly and microscopically fromthose seen in overt primary aldosteronism. Otherauthors have found only a twofold increased in-cidence of adenomas among hypertensive patients(13-15). In the two large series, the incidencesof adenomas among hypertensive patients wereonly 5 and 7.4% (15, 16). Nonetheless, all agreethat such adenomas are found in many patientswith hypertension, many more than have beenthought to have primary aldosteronism. How-ever, it is not yet known whether these adenomasare the cause of the hypertension or simply inci-dental findings of no functional significance.

The steroid analyses performed on adenomasfrom patients with essential hypertension suggestthat they do not produce increased amounts ofaldosterone and therefore are not the cause ofprimary aldosteronism. These adenomas con-tained no more aldosterone than normal adrenaltissue, and only about one-twentieth that foundin adenomas removed from patients with primaryaldosteronism.

Certain limitations on the use of these data asan indication of the role of adrenal adenomas inhypertension are obvious. First, the assay ofsteroid content is a static measurement, whichmay have little relationship to the secretory func-tion of these adenomas during life. However,as also noted in this study, aldosterone excretionand secretion from patients with essential hyper-tension have been normal in all series that hadcareful selection of patients and reliable assayprocedures (17-21). Secondly, the adenomasfrom patients with essential hypertension were ob-tained after death, when considerable degradationof steroids may have occurred. However, thepossibility that such postmortem degradationcould explain these differences appears unlikely,since such degradation was not found in adrenaltissue subjected to similar conditions. Moreover,any differences between the adenomas from pa-tients with essential hypertension and normaltissue should have persisted even though the totalcontent decreased, since they were obtained andhandled in the same manner. Thirdly, the pa-tients with essential hypertension and adrenaladenomas may have had aldosterone excess dur-ing life, since neither urinary aldosterone norplasma renin activity was measured. Conn sug-gests that the serum potassium in primary aldos-

732

STEROID CONTENTOF ADRENALADENOMAS

teronism may remain normal for many years, and,so, the essential hypertensive patients in this studymay have been in the normokalemic phase of thedisease. This appears unlikely since, despite thepresence of significant hypertension for from 6 to

24 years, none of the patients had abnormal serum

sodium or potassium levels.The adrenocortical adenomas found at autopsy,

more commonly in hypertensive but also in nor-

motensive patients, do not then appear to beassociated with aldosterone excess. They couldbe the cause of hypertension by producing othersteroids not measured in this study, such as de-oxycorticosterone.

The pathogenesis of these adenomas is un-

known. Adenomas in other endocrine tissues are

frequently found at autopsy without evidence ofhyperfunction during life. The possibility thatthese adenomas are secondary to the hypertensiveprocess should be considered, particularly sinceautonomous adenomas of the thyroid and para-

thyroid glands have been noted after prolonged"secondary" stimulation. A form of secondaryaldosteronism does appear with malignant hyper-tension, presumably from stimulation of theadrenal cortex by increased amounts of renin re-

leased from the juxtaglomerular apparatus in re-

sponse to the ischemia produced by intrarenalvascular disease (17, 22). However, this sec-

ondary stimulation produces a diffuse nodularhyperplasia, not a solitary adenoma, and the hy-pertensive process is of a malignant nature, notthe long-standing benign hypertension almost al-ways seen with primary aldosteronism. Conn,Cohen, and Rovner could find no evidence tosupport the view that autonomous adrenal ade-nomas arise from prolonged secondary stimula-tion (23). The issue is confused by the pro-

duction of typical primary aldosteronism withbenign hypertension by diffuse adrenocortical hy-perplasia (24), but this has been a relatively rare

finding (11) and there is no reason to believethat such diffuse hyperplasia would later giverise to a solitary adenoma. It appears, then, thatsuch adrenal adenomas in hypertensive patientsare not secondary to the hypertensive process it-self, particularly since adenomas of similar ap-

pearance and steroid content occur frequently innormotensive subjects.

It may be that these incidental adenomas com-monly appear and, in most patients, simply sup-press the function of the remainder of the glandbut leave the patient in a normal state. Such amechanism has been suggested in a report of anormotensive normokalemic subject found to havea solitary adrenocortical adenoma and atrophy ofthe zona glomerulosa (25). Such marked at-rophy of the rest of the adrenal tissue was notobserved in the glands we studied.

Another piece of evidence used in support ofthe 20 to 25%o incidence of primary aldosteronismin essential hypertension (12) is the report thataldosterone excretion was elevated in 25%o of 38hypertensive patients (26). In that study aswell as three other reports of aldosterone excessin hypertension (27-29), the aldosterone mea-surements were performed by a less specific andsensitive physicochemical technique, and the selec-tion of patients was not delineated to ensure theexclusion of advanced or malignant hypertension,where aldosterone excess is expected. On theother hand, when isotopic techniques are used andpatient selection is careful, the incidence of al-dosterone excess in essential hypertension hasbeen found to be one in 114 patients (17-21).Our data show no increase in the excretion orsecretion of aldosterone in 43 essential hyper-tensive patients and no difference in their meanvalues when compared with 39 normotensive pa-tients. There are other findings which suggestthat aldosterone excess is not a frequent cause ofessential hypertension. These include the fol-lowing: normal plasma volume, serum sodium,and total body exchangeable sodium (30), allusually elevated in primary aldosteronism (11,31); and normal plasma renin activity (21), sup-pressed in primary aldosteronism (4). Further-more, whereas primary aldosteronism has beennoted to be almost as common below the age of40 as beyond that age (11), the occurrence ofincidental adrenal adenomas is rare below 40 butincreases progressively thereafter (15, 16). Forthese reasons it appears that primary aldosteron-ism is only rarely the cause of essential hyper-tension, and the finding of an adrenal adenomashould not be used as evidence for the presenceof primary aldosteronism in the absence of sup-porting clinical or laboratory data.

733

NORMANM. KAPLAN

Acknowledgment

The assistance of Dr. J. Caulie Gunnels in the per-

formance of the assays of plasma renin activity is grate-

fully acknowledged.

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