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Journal of Clinical InvestigationVol. 42, No. 5, 1963

PLASMAANGIOTENSINASEACTIVITY IN PATIENTS WITHHYPERTENSIONANDEDEMA*

By ROGERB. HICKLER,t DAVID P. LAULER, AND GEORGEW. THORN

(Fromn the Hypertension Laboratory, Peter Bent Brigham Hospital, and the Departmentlt ofMedicine, Harvard Medical School, Boston, Mass.)

(Submitted for publication October 17, 1962; accepted January 10, 1963)

An abnormal metabolism of angiotensin II inessential hypertension has been suggested by sev-eral studies. Wood (1) reported that the venousblood of hypertensive patients showed a decreasedcapacity to inactivate an in vitro admixture withsynthetic angiotensin' as judged by the pressorresponse on reinfusion into the hypertensive do-nors. Wolf and associates reported a prolongedradiochemical half-life of I131-labeled angiotensinin hypertensive patients after intravenous adminis-tration (2), but found a shortened radiochemicalhalf-life in a purely in vitro system (3). Klaus(4) described a system in which the measurementof the amount of valine freed from synthetic valyl-5-angiotensin II after an in vitro admixture withplasma was equated to angiotensinase activity andreported no significant difference between hyper-tensive and normotensive plasma.

Thus, in the in vitro system employed, there hasbeen suggested an increase, a decrease, and nochange in the angiotensinase activity of the hyper-tensive plasma.2 Using the pressure response ofthe subject on reinfusion of an in vitro admixtureleaves the difficult problem of controlling differ-ences in vascular tone and reactivity between hy-pertensive and normotensive individuals. The

* Presented in part at the Fifty-fourth Annual Meetingof the American Society for Clinical Investigation, April29, 1962, and published as an abstract (J. clin. Invest.1962, 41, 1365). This work was supported by the JohnA. Hartford Foundation and by U. S. Public HealthService grant HSG 14,903. The clinical studies werecarried out in the Clinical Research Center of the Har-vard Medical School and Peter Bent Brigham Hospital,supported by grant OG-6 of the National Institutes ofHealth.

t Career Development Award Program, U. S. PublicHealth Service grant HE-K-14,903-C1.

1 Ciba Pharmaceutical Co., Summit, N. J.2 Plasma angiotensinase activity is used in this paper

for convenience to designate the capacity of plasma to in-activate synthetic angiotensin, although a specific plasmaenzyme, as such, has not been isolated to date.

in vitro chemical methods leave the difficult prob-lem of equating the chemical end-point to residualbiological activity. It seemed that an in vitrosystem employing the loss of pressure response ina standard bioassay rat preparation to the serialinjection of an admixture of plasma and angioten-sin might obviate some of these difficulties andcontribute to an understanding of this intricateproblem.

MATERIALS AND METHODS

A. The assay. An in vitro admixture of syntheticangiotensin II 1 and plasma was studied for the progres-sive loss of pressor activity by serial inj ections into a250-g vagotomized rat prepared with 'Nembutal and pen-tolinium. The left jugular vein was cannulated withpolyethylene tubing and injected with a microsyringecontaining a standard (1 /Ag per ml) dilution of angio-tensin II (preserved as a 20%o ethanol solution). Theright jugular vein was similarly connected to a micro-syringe containing a mixture of 1 ml of the standardangiotensin solution and 1 ml of plasma. A stop watchwas started at the time of the admixture, and room tem-perature was constant at 25 + 10 C. Alternate equaldoses of standard angiotensin solution and standard solu-tion mixed with plasma were administered every 3 to 4minutes. The right carotid artery was cannulated withpolyethylene tubing and the arterial pressure responserecorded on a direct-writing pen-float mercury manometer.At the top of Figure 1 is shown the rat's pressure re-cording in a study on the plasma from a normotensivesubject. The hatched spikes represent the serial pressureresponse of the plasma-angiotensin mixture and show theprogressive reduction in the response; the nonhatchedspikes represent the serial pressure response of the stand-ard angiotensin solution alone and show full sensitivity ofthe rat to the serial injection of 10 m/Ag throughout thestudy. Variations, particularly a decrease of the stand-ard response by more than 2 mm, are unsatisfactory andindicate that the study should be repeated. A minimalrat sensitivity of 1 mmHg pressure rise per m/Ag of in-jected angiotensin is required. In a given study the serialdose was either 5, 10, or 15 m/Ag, whichever was neces-sary to ensure a pressure response in the range of 10 to20 mmHg, the same dose being held throughout thestudy for both the standard and the standard plus plasma

635

ROGERB. HICKLER, DAVID P. LAULER, AND GEORGEW. THORN

P.C. NORMOTENSIVEV 24 yr.

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FIG. 1. ARTERIAL PRESSURETRACING IN THE BIOASSAY RAT PREPARATION,

SHOWINGPROGRESSIVELOSS OF RESPONSE(HATCHEDAREAS) TO THE PLASMA-

ANGIOTENSIN MIXTURE COMPAREDWITH THE FULL RESPONSE (NONHATCHEDAREAS) TO THE ANGIOTENSIN STANDARD. Shown below is the plot of the dataand the determination of the biological half-life of angiotensin in humanplasma.

mixture. At this low dose range, the steep or linear partof the logarithmic relationship between dose and re-

sponse pertains. Figure 2 demonstrates this linearity in16 different test rats, representing the rise in pressure to

DOSE RESPONSECURVES FOR ANGIOTENSININ THE RAT

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TERED (0 TO 15 M/.AG) IN 16 DIFFERENT BIOASSAY RAT

PREPARATIONS.

increments in the angiotensin dose from 0 to 15 mpg.

Thus, a given reduction in pressure response during theassay for angiotensinase activity may be equated to a

proportionately equal reduction in the amount of angio-tensin remaining in the plasma-angiotensin mixture. Theregression curve for this progressively smaller pressure

response is shown on the lower left of Figure 1, indicatingfirst-order' kinetics, which has been the characteristic re-

sponse in all plAsma samples tested. This was correctedto the starting point of the standard response beforeplotting to compensate for any slight variation in thestandard curve, and plots as a straight line against a

logarithmic ordinate, shown on the lower right of Fig-ure 1. From this, the in vitro biological half-life of syn-thetic angiotensin II in plasma may be read as the min-utes required for a 50% reduction in pressure response;'the shorter the biological half-life, the greater the plasmaangiotensinase activity presumed to be present.

Since there was no observed pressode response to ad-ministration of plasma alone in the volumes involved inthe assay (.003 to .075 ml), it is assumed that therewas no error introduced by the possible presence -f en-

dogenous renin or angiotensin 'in the plasma beingassayed.

Evidence suggesting that the reaction may be enzy-matic in nature is cited as follows. Chilling the angio-tensin-plasma mixture in ice water almost completely in-hibited the"' degradation of the angiotensin, and pre-heating the plasma at 570 for 30 minutes before'adding

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FIG. 3. DEMONSTRATIONOF THE PHENOMENONOF SUBSTRATESATURATION, EVIDENCED BY THE

PLATEAU IN THE VELOCITY OF THE ENZYMATIC REACTION (MOLES ANGIOTENSIN DESTROYEDPER

LITER PLASMA PER MINUTE) WHENTHE SUBSTRATE CONCENTRATIONREACHES THE RANGE OF

X 10 MANGIOTENSIN.

angiotensin markedly decreased its capacity to inactivatethe angiotensin. The velocity of the reaction was stud-ied with increasing concentrations of added angiotensinsolutions (1 ml of solution added to 1 ml of plasma) inone normal subject. An increasing rate of angiotensininactivation was found up to a concentration of 10 mug

per ml added to the plasma, above which concentration therate leveled off sharply in a manner characteristic of thephenomenon of enzyme saturation. This relationship isplotted in the conventional fashion in Figure 3, showingsubstrate concentration as moles per L angiotensin in thefinal solution and enzyme velocity as moles of angio-tensin destroyed per L plasma per minute. Finally, theeffect of varying enzyme concentration on the reactionvelocity was studied by varying the plasma concentrationin the final mixture with angiotensin standard solutionbetween 25 and 60%. In the two plasmas so studied, a

linear relationship was found between the plasma concen-

tration and the percentage of angiotensin destroyed (Fig-ure 4). This is characteristic of an enzyme reaction, andthe linearity of the response is evidence against the pres-

ence of an enzyme inhibitor or activator.A simplified calculation of rate of angiotensin de-

struction as a measure of angiotensinase activity has beenadopted, which obviates the necessity of correcting theregression curve back to the starting point to compensatefor slight variations in the standard response and thenplotting on semilog paper. At 20 minutes of incubationat room temperature the slope of the regression curve

is still relatively steep (Figure 1), and a measure of theangiotensinase activity is given by determining the per-

centage of difference between this curve and the stand-ard curve at precisely 20 minutes. Thus, for the clini-

cal material to follow, the percentage of destruction ofangiotensin in 20 minutes is calculated and used as theindex of plasma angiotensinase activity. By comparingthe mean value for a group of normal subjects with thatof the groups of patients studied, an index is then ob-tained of the percentage of increase (or decrease) inplasma angiotensinase activity above (or below) the nor-

mal mean. The reaction may be stopped at the 20-minutepoint by -placing the plasma-angiotensin mixture in boil-ing water prior to assaying.

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LOSS OF ENZYMEACTIVITY (IN TERMS OF PERCENTAGEOF

ANGIOTENSIN DESTROYED) IN THE PLASMA FROM TWO

SUBJECTS.

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Duplicate determinations have been performed on 20patients. The same plasma sample was used for the re-

peat determination in 11 instances, and a new plasmasample drawn for the repeat determination in 9 instances(frequently on a later day). The same range of variationbetween the 2 duplicate determinations was found inboth groups (0 to 11%o, average 3%) in terms of thepercentage of angiotensin destroyed in 20 minutes. Repli-cate determinations were performed 15 times on the same

plasma sample, giving a mean of 38.4% with SD of+ 4.0%. This gives a relative SD (coefficient of varia-tion) of 10.4%o. However, angiotensinase activity of redblood cells is higher than that of plasma (5), and imme-diate and complete separation of red cells from the hepari-nized venous blood sample without hemolysis is essential;a centrifuged but unseparated blood sample standing atroom temperature for 4 hours showed a 20-minute angio-tensin destruction of 44% on the initial assay and 60%after standing. Plasma samples may be stored at 40 Cwithout affecting the angiotensinase activity, but storingplasma at - 180 C has consistently resulted in a depres-sion of activity and should be avoided. Heparin per se

has no effect on the pressor activity of standard angio-tensin.

B. Clinical material. Several groups of subjects havebeen studied for plasma angiotensinase activity: 18 nor-

mal control subjects, 33 patients with essential hyper-tension, 26 patients with renal hypertension, and 4 pa-

tients with adrenal hypertension (2 with Cushing's syn-drome and 2 with pheochromocytoma). Two groups ofpatients with conditions commonly associated with sec-

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ondary hyperaldosteronism have been studied as well: 5

with refractory edema (6-8) (nephrosis, 3; cirrhosis withascites, 1; congestive heart failure with anasarca, 1), and5 with pregnancy (9) (normal third trimester, 2; toxe-mia, 2; hypertension and pregnancy, 1). One Addisonianpatient who was deficient in steroid replacement therapyat the time of study is included.

The normal control subjects were all healthy individu-als (6 females, 12 males) ranging in age from 24 to 38years. The clinical data for the patients with essentialhypertension are shown in Table I. Each patient was

subj ected to an extensive hospital study to rule out an

identifiable cause before being classified as essential.The group is divided into labile hypertension (Class A),hypertensive cardiovascular disease of moderate severity(Class B), and severe hypertensive cardiovascular disease(Class C). The labile group had blood pressure read-ings fluctuating between normotensive and hypertensivelevels and was completely lacking in evidence of cardiac,cerebral, or renal complications. Twelve of the patientswith essential hypertension fulfilled these criteria. Thegroup with hypertensive cardiovascular disease of mod-erate severity all had evidence of cardiac hypertrophy byelectrocardiogram or cardiac enlargement on chest film,but no evidence of hemorrhages, exudates, or papilledemaon funduscopic examination and no nitrogen retention.Thirteen of the patients with essential hypertension ful-filled these criteria. The group with severe hypertensivecardiovascular disease all had evidence of cardiomegalyand funduscopic changes, grade 3 or 4 (hemorrhage andexudates with or without papilledema). Eight of the

HYPERT. CARDIOVASC. DIS.SEVERE

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upper limit norm.

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FIG. 5. PLASMA ANGIOTENSINASE ACTIVITY IN TERMS OF PERCENTAGEOF

CHANGEFROMTHE NORMALMEANLEVEL FORTHE INDIVIDUAL SUBJECTSIN THE

NORMALGROUPAND THE GROUPS WITH ESSENTIAL HYPERTENSION OF IN-

CREASING SEVERITY.

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CHANGEFROMTHE NORMALMEANLEVEL FOR THE INDIVIDUAL PATIENTS WITH

THE DIFFERENT FORMSOF RENALHYPERTENSION. The normal range and mean

are shown to the left.

patients with essential hypertension fulfilled these cri-teria; none was in obvious congestive heart failure and6 had nitrogen retention' by blood urea nitrogen deter-mination.

Table II outlines the clinical data for the patients withrenal hypertension. After thorough hospital evaluationthese patients were grouped as follows: 9 with chronicpyelonephritis, 4 with unilateral pyelonephritis, 7 withchronic glomerulonephritis, and 5 with renal artery steno-sis. Surgical exploration and pathologic examinationconfirmed the diagnosis in 3 of the 4 patients with uni-lateral pyelonephritis and 4 of the 5 patients with renalartery stenosis. Retrograde pyelography and aortographywere diagnostic in the remaining two patients with uni-lateral renal disease. For purposes of comparing resultsbetween the group with renal hypertension and the groupwith essential hypertension, the renal hypertensive pa-tients are also designated as either Class A, B, or C inseverity according to the same criteria outlined for thepatients with essential hypertension.

RESULTS

A. Essential hypertension. The mean per-

centage of destruction of angiotensin in 20 min-utes for the normal control group of 18 subjectswas 27%o, with a range of 7 to 40%. Thus, 40%destruction defines the upper limit of normal. Theindividual values were then calculated in terms of

percentage of increase or decrease from this nor-

mal mean (Figure 5), which gave a range of - 74to + 48%,o which constitutes a measure of the in-

crease or decrease from the average value in plasmaangiotensinase activity for each individual in a

normal group. An increase in plasma angioten-sinase activity of over 48% from the normal mean,

then, may be considered abnormal. The group of12 patients with labile hypertension had a mean

activity that was only 7%o above the normal mean,

with a range of - 70 to + 82%. Only 2 valueswere above the upper limit of normal, and therewas no significant difference between the mean

percentage of destruction in 20 minutes for thenormotensive group and the group with labile hy-pertension (p >.5). The group of 13 patientswith hypertensive cardiovascular disease of mod-erate severity had a mean activity that was 48%above the normal mean, with a range of from - 56to + 175%; 6, or nearly 50%o, of the values were

above the upper limit of normal, and the mean forthe 20-minute percentage of destruction was sig-nificantly elevated above the normal (p <.05 >.025). The group of 8 patients with severe hy-pertensive cardiovascular disease had a mean ac-

tivity that was 100%o above the normal mean, witha range of from + 15 to + 185%o. All but 1 ofthe values were above the upper limit of normal, andthe difference in the mean from the normal mean

for the 20-minute percentage of destruction was

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641


highly significant (p <.001). The elevation inplasma angiotensinase activity in essential hyper-tension appears to be a graded characteristic, be-coming progressively elevated as the disease pro-gresses (Figure 5).

B. Renal hypertension. Figure 6 shows the re-

sults for the 4 groups with renal hypertension incomparison with the normal. The group of 9patients with chronic pyelonephritis had an ele-vation of mean activity that was only 7% abovethe normal mean, with a range of - 59 to + 44%o,which is entirely within the normal range. Thedifference in the mean from the normal mean forthe 20-minute percentage of destruction was in-significant (p >.5). This could not be attributedto a lack of severity of the hypertensive disease;5 of the cases were in Class B (comparable to thegroup with essential hypertension with moderatelysevere hypertensive cardiovascular disease) and 3in Class C (comparable to the group with essentialhypertension with severe hypertensive cardiovas-cular disease). The group of 4 patients with uni-lateral pyelonephritis had a mean activity that was

30% above the normal mean, with a range of from- 56 to + 70%. There was no significant dif-ference between the 20-minute mean percentage of

destruction for this group and the normotensivegroup (p > 0.2). However, 2 of the 4 valueswere in the range above the upper limit of normal,and the implications in terms of a possible renalpressor mechanism in certain instances of pyelo-nephritis will be considered below. The group of7 patients with chronic glomerulonephritis had an

elevation of mean activity that was 63% abovethe normal mean, with a range of + 22 to + 100%.The mean for the 20-minute percentage of destruc-tion was significantly elevated above the normal(p <.01 >.005). This also represented a sig-nificant difference from the group with chronicpyelonephritis (p <.025 >.01). Of the 4 patientswith chronic glomerulonephritis with severe hy-pertensive disease (Class C), all fell above theupper limit of the normal range. As seen with thegroup with essential hypertension, the elevation ofserum angiotensinase activity in chronic glomeru-lonephritis would seem to be a graded character-istic, increasing with the progressive severity ofthe disease. The group of 5 patients with hyper-tension related to renal artery stenosis had a mean

activity that was 93% above the normal mean,

with a range of from + 74 to + 122%. All ofthe values were above the upper limit of normal,

TABLE III

Adrenal disease and clinical states associated with hyperaldosteronism

ChangeDestruc- in angio-tion of tensinaseangio- activity from

Patient Age Sex Diagnosis tensin normal mean

%in %20 min

Secondary hyperaldosteronism, refractory edemaH.R. 64 M Nephrotic syndrome 90 +233E.D. 60 F Nephrotic syndrome 71 +160N.H. 64 F Nephrotic syndrome 52 + 93J.S. 70 F Cirrhosis with ascites 44 + 65E.Z. 80 F Congestive heart failure with refractory anasarca 50 + 85

Secondary hyperaldosteronism, pregnancyA.M. 25 F Normal pregnancy, trimester3 67 +148B.P. 25 F Normal pregnancy, trimester3 78 +188A.E. 16 F Hypertension in pregnancy 58 +115C.L. 20 F Toxemia of pregnancy 83 +207O.L. 23 F Toxemia of pregnancy 48 + 78A.L. 63 F Cushing's syndrome 23 - 15C.S. 60 M Cushing's syndrome 27 0M.N. 52 F Addison's disease 47 + 74L.M. 22 M Pheochromocytoma preop. 65 +140

postop. 88 +224L.E. 65 F Pheochromocytoma preop. 48 + 78

postop. 52 + 92

642

HUMANIN PLASMAANGIOTENSINASEACTIVITY IN HYPERTENSIONAND EDEMA

SECONDARYHYPERALDOSTERONISMAND OTHERADRENALFACTORS

ADRENO-CORTICAL REFRACT.

NORMAL DYSFUNCT. PHEO. EDEMA

0

upper limit norm.

* post-op.

I*Opre -op.

.....................................................................................................................

40 -

0 * Cushing's* J Syndrome

40S


CHANGEFROMTHE NORMALMEANLEVEL FOR THE INDIVIDUAL PATIENTS WITHADRENAL DISORDERS AND STATES OF SECONDARYHYPERALDOSTERONISM(RE-FRACTORYEDEMAAND PREGNANCY). The normal range and mean are shown

to the left.

and the difference in the mean 20-minute per-

centage of destruction from the normal was highlysignificant (p <.001).

C. Adrenal diseases and clinical states associ-ated zw4th hyperaldosteronism. Table III showsthe clinical data and assay results in the group ofpatients with disorders of the adrenal gland andclinical states commonly associated with secondaryhyperaldosteronism. The angiotensinase activityfor the individual patients is plotted in; Figure 7for the various subgroups studied and contrastedwith the normal control group. The 2 patientswith Cushing's syndrome had an angiotensinaseactivity in the normal range, whereas the 1 Ad-disonian patient who was undertreated at the timeof study showed an activity above the upper limitof normal. The 2 patients with pheochromocy-toma had an elevation of activity above the upper

limit of normal, which was shown to persist in

the one instance at 10 days and in the other at 2months after surgical cure. The 2 groups of pa-

tients with clinical states that have been shown tobe associated with an increase in aldosterone se-

cretory rate are divided into the group with re-

fractory edema and the group with normal and ab-normal pregnancy. The group with refractoryedema consisted of 3 patients with nephrosis, 1with cirrhosis and ascites, and 1 with congestiveheart failure and refractory anasarca. All 5 valuesfor plasma angiotensinase activity were consider-ably above the normal range& (+ 56 to +-I 233%o)with a mean activity that was 126% above thenormal mean. The difference in the mean fromthe normal mean for the 20-minute percentage ofdestruction was highly significant (p <.001). TIegroup of pregnant patients, all in their third tri-mester, consisted of 2 normal pregnant females,2 with toxemia, and 1 with hypertension starting

+ 240

+200

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NORMALandABNORMALPREGNANCY

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643


TABLE IV

Summary of plasma angiotensinase activity in hypertension

Mean Mean increasedestruction in angiotensinase

of angio- activity abovePatients Diagnosis tensin SD SEM* p factort normal mean

no. %in %20 min

Essential hypertension18 Normal 27 11 2.612 Labile hypertension 29 12 3.5 >.5 713 Hyperten. cardiovasc. 40 19 5.5 <.05 >.025 48

Disease-moderate8 Hyperten. cardiovasc. 54 15 5.3 <.001 100

Disease-severe

Renal hypertension9 Chronic pyelonephritis 29 8 2.7 >.5 74 Unilateral pyelonephritis 35 14 7.0 >.2 <.4 307 Chronic glomerulonephritis 44 8 3.0 <.01 >005 635 Renal artery stenosis 52 5 2.0 <.001 93

Secondary hyperaldosteronism5 Refractory edema 61 19 8.5 <.001 1265 Pregnancy, normal and 64 11 5.5 <.001 148

abnormal

* Standard error of the mean.t Significance of the difference between the patient groups and the normal group.

during the first trimester. All 5 values forplasma angiotensinase activity were considerablyabove the normal range (+ 78 to + 148%), witha mean activity that was 148% above the normalmean. The difference in the mean from the nor-mal mean for the 20-minute percentage of de-struction was highly significant (p <.001).

The angiotensinase activity data for all groupsstudied are summarized in Table IV.

DISCUSSION

Our working hypothesis has been that stateswhich may have in common an underlying in-creased rate of angiotensin elaboration, with orwithout hypertension, may develop an increasedplasma capacity to inactivate angiotensin througha process of substrate induction, that is, througha specific enzymatic adaptation at the cellularlevel. Knox, Auerbach, and Lin (10) have re-viewed the subject of enzymatic adaptation, citinganimal work in which amino acid oxidase activityincreased in response to the administration of pro-tein and specific amino acids. Whether the angio-tensinase activity in plasma is represented by asingle specific protease or several has not been es-tablished as yet. Since a variety of highly purifiedproteolytic enzymes have the capacity of inactivat-

ing angiotensin (5), the degree of specificity ofplasma angiotensinase activity will have to awaitthe isolation of such activity from the plasma pro-teins. Other possibilities exist, such as the elab-oration of specific angiotensinase inhibitors or ac-tivators to account for-the observed alterations inplasma angiotensinase activity.

The 4 groups of patients with hypertension thatdemonstrated a significant elevation in plasmaangiotensinase activity were 1) those with essentialhypertension that had progressed to a stage ofhypertensive cardiovascular disease of- a moderateto severe degree, 2) those with hypertension re-lated to chronic glomerulonephritis, particularlyin its advanced stages, 3) those with hypertensionrelated to renal artery stenosis, and 4) the 2 pa-tients with pheochromocytoma. No significantchange was found in the group with hypertensionassociated with chronic pyelonephritis and uni-lateral pyelonephritis, irrespective of the severityof the hypertensive disease. Thus, the group withchronic bilateral pyelonephritis serves as a sickcontrol group, where no elevation of angiotensi-nase activity was found, despite the presence ofuremia in 7 of the 9 patients, 3 of whom had thechanges of malignant hypertension and were stud-ied shortly before their death. One uremic pa-

644

HUMANPLASMAANGIOTENSINASEACTIVITY IN HYPERTENSIONANTD EDEMA

tient with an elevated angiotensinase activity as-

sociated with chronic glomerulonephritis and hy-pertension had no change in his angiotensinase ac-

tivity after hemodialysis, despite a marked reduc-tion in his blood urea nitrogen concentration.

There is a striking correlation between thesefindings and the degree of juxtaglomerular hyper-trophy as determined by juxtaglomerular cellcounts, reported by Turgeon and Sommers (11).These workers reported juxtaglomerular hyper-plasia in the same 4 hypertensive groups in whicha significant increase in plasma angiotensinase ac-

tivity is reported here (essential hypertension,chronic glomerulonephritis, renal artery stenosis,and pheochromocytoma). Further, they failed to

find hyperplasia in the pyelonephritic group withhypertension. corresponding with the lack of a

significant increase in plasma angiotensinase ac-

tivity reported here. The evidence that the reninsecretory mechanism is situated in the juxtaglo-merular apparatus has been reviewed by Tobian(12) and is substantial; it is assumed that a highrate of renin secretion may be associated with a

high rate of angiotensin elaboration (13).Although a modest elevation of angiotensin

blood levels in humans with severe essential hy-pertension has been reported in the past (14),Genest and co-workers, employing a more specificmethod (15), found an elevation in only 30% oftheir determinations (16). There is more uni-form agreement on a consistent elevation in therenal vein blood in hypertension due to renal ar-

tery stenosis (17. 18). Angiotensin data in hy-pertension associated with chronic nephritis andpheochromocytoma are not as yet available. Thefailure to show consistently elevated blood angio-tensin levels in essential hypertension in no way

precludes the possibility of an elevated rate of elab-oration, particularly in the more advanced stagesof the disease, in the light of the finding of a pro-

gressive elevation in plasma angiotensinase activityas the disease advances, which would tend to masksuch a phenomenon. It does suggest, however,that angiotensinemia is not the underlying etiologi-cal factor in essential hypertension, but more likelya secondary manifestation of the progressive in-trarenal vascular disease attending its more ad-vanced stages. A stronger argument can be madefor angiotensinemia as the etiological factor in hy-pertension relating to renal artery stenosis, where

the angiotensin-angiotensinase levels may be ele-vated in even the early stages of the disease.

It is possible that in the very late stages of hy-pertensive disease associated with progressive re-nal failure the renin mechanism is finally obliter-ated, as suggested by the experimental work inrats of Omae, Masson, and Page (19), where"'morphological examination showed a relationshipbetween the presence of nephrosclerotic lesionsand absence of, or decrease of, renin-like pressorsubstance released." This may account for theonly instance of a normal angiotensinase level inthe group with severe hypertensive cardiovasculardisease, Case J.R., who was admitted with grade 4eyegrounds, hypertensive encephalopathy. and pro-gressive azotemia.

This leads to the question of the mechanism ofthe hypertension in the group with chronic pyelo-nephritis, where juxtaglomerular hypertrophy andelevated plasma angiotensinase activity are strik-ingly absent. Assuming a predominantly renalmedullary destruction in this disease, one mayspeculate that this represents a human manifesta-tion of renoprival hypertension as suggested byseveral studies (20, 21), where a protecting, anti-hypertensive medullary factor, as identified byMuirhead, Jones, and Stirman (22), may havebeen eliminated by the disease. Although a figureis hard to arrive at, a significant reduction of pres-sure following extirpation of a unilaterally pyelo-nephritic kidney may be expected in "less thanhalf of such cases" (23), whereas a significantimprovement in the range of 75% has been re-ported (24) following surgery for occlusive re-nal arterial disease. However, one must accountfor those isolated reports of definite improvementfollowing excision of a unilaterally pyelonephritickidney (25). In this regard it is of interest that,although the mean angiotensinase level was not

significantly elevated above the normal mean, 2 ofthe 4 cases of unilateral pyelonephritis had a levelabove the normal range. A renal pressor mecha-nism may be contributing to the hypertension incertain instances of pyelonephritis through a proc-ess of proliferative endarteritis, as emphasized bySmythe (26). Saphir (27) proposes the existenceof 2 types of pyelonephritis, one being clinicallyevident with the predominant feature of medullarvdestruction, and the other, "pyelonephritis lenta,"being clinically silent with a prominent feature of

645


vascular intimal fibrosis and hyalinization leadingto hypertension (and often misdiagnosed as es-sential hypertension). Sommers (21) on the ba-sis of juxtaglomerular cell counts, states that"pyelonephritis that causes an intrarenal Gold-blatt-type phenomenon does occur but it is rela-tively rare in the material analyzed so far." If theplasma angiotensinase level proves to be differ-entiating, a greater degree of sophistication maybe obtained in the surgical selection of those pa-tients with hypertension associated with unilateralrenal disease. The degree of surgical success maybe proportional to the degree to which the renalpressor mechanism is responsible for the hyper-tension.

The common denominator for those instances ofan elevated plasma angiotensinase activity in thevarious hypertensive groups may prove to be arenal pressor mechanism resulting from extrinsicor intrinsic renovascular disease. This may beeither a cause or a consequence of the elevatedarterial pressure. In pheochromocytoma, persis-tent renal vasoconstriction may be sufficient to ac-count for the mechanism, even in the absence ofnephrosclerosis.

In a recent review, Davis, Carpenter, and Ayers(28) have summoned-up convincing evidence forthe role of the renin-angiotensin system in the con-trol of aldosterone secretion. It is of interest thatLaragh (29) found elevated aldosterone secretoryrates in the advanced and particularly the malig-nant phase of essential hypertension, but not inthe milder stages. This report is quite parallel tothe plasma angiotensinase data presented above.If an angiotensinemia is assumed to apply in gen-eral to clinical states associated with secondaryhyperaldosteronism, it seemed reasonable to lookfor an elevated angiotensinase activity in patientswith refractory edema and pregnancy. As citedabove, the highest mean levels observed for plasmaangiotensinase activity were found in these con-ditions. The absence of hypertension in thesegroups (excepting the toxemic patients), doesnot preclude the possibility of an increased rateof angiotensin elaboration in the light of Laragh'sfinding (30) of a refractoriness to the pressor ef-fects of infused angiotensin in cirrhotic patientswith ascites. The finding of hypergranulation ofthe juxtaglomerular cells from kidneys of sodiumdeficient rabbit (31), of dogs with hyperaldoster-

onism secondary to thoracic caval construction(32), and of rats with experimental nephrosis(33) is supportive evidence.

If Tobian's view (12) that the juxtaglomerularcells act as stretch receptors is sustained, then thecommon denominator between hypertension asso-ciated with renal vascular disease on the one handand edematous states on the other for the activationof the renin-angiotensin-aldosterone system wouldbe a decreased renal perfusion pressure, whetherthrough a vascular obstruction at the renal levelor through the sequestration of effective circulatingvolume as edema fluid. Such a contraction ofplasma volume has been determined in the ne-phrotic syndrome and cirrhosis with ascites; insevere congestive failure the redistribution of bloodon the venous side of the circulation has the sameeffect (34). In pregnancy, however, cardiac out-put and renal blood flow are increased until week32 except in severe pre-eclampsia (35), and in thissituation this line of reasoning cannot be applied.The possibility exists that an unidentified placentalfactor may be operative in activating the reninmechanism to meet the need for a positive sodiumbalance during pregnancy. An increase in plasmaoxytocinase activity at term is another possibility.

The finding of an elevated angiotensinase ac-tivity in an undertreated patient with Addison'sdisease is probably indicative of an activation of therenal pressor system due to volume depletion,comparable to the observations in hemorrhagicshock (36). Experimental support is offered bythe observation of increased juxtaglomerular gran-ularity in the adrenalectomized cat (37). Thefinding of a normal level in 2 patients with hyper-tension secondary to Cushing's syndrome sug-gests that the assay may be helpful in identifyingforms of secondary hypertension where the renalpressor system is not involved.

It is hypothesized than angiotensinemia and hy-peraldosteronism may only develop in those con-ditions where an increased rate of angiotensinelaboration exceeds an adaptive increase in the rateof angiotensin destruction.

The authors are aware that there may be otherdisease states not associated with hypertension oredema which may also prove to have alterationsin the plasma angiotensinase activity. Any pro-found disturbance in the plasma proteins and al-teration in circulating proteases such as may at-

646

HUMANPLASMAANGIOTENSINASEACTIVITY IN HYPERTENSIONAND EDEMA

tend severe pancreatic or hepatobilary disease car-ries with it this potential. A systematic appraisalwill be a subject for further investigation.

SUMMARY

1) An in vitro biological method for the deter-mination of plasma angiotensinase activity is de-scribed. Plasma angiotensinase activity is usedfor convenience to designate the capacity of plasmato inactivate synthetic angiotensin, although aspecific plasma enzyme, as such, has not been iso-lated to date.

2) A significant elevation was found in the fol-lowing hypertensive disorders: essential hyper-tension complicated by moderately severe to se-vere hypertensive cardiovascular disease, chronicglomerulonephritis, occlusive renal arterial disease,and pheochromocytoma.

3) Evidence is cited from the literature for theincreased activity of the renin secretory mecha-nism in each of these diseases, which may have incommon an ischemic or obstructive renal vasculardisorder.

4) In the two groups with conditions com-monly associated with secondary hyperaldosteron-ism, refractory edema and pregnancy (normal andabnormal), marked elevations of plasma angio-tensinase activity were also found.

5) It is suggested that the increased plasmaangiotensinase activity in these states may ensuefrom an increased rate of angiotensin elabora-tion through a process of enzymatic adaptation.

6) In the light of these findings, reports of thefailure to find a consistently elevated blood angio-tensin level in even the more severe stages of es-sential hypertension and in malignant hypertensiondo not preclude the possibility of an increasedrate of angiotensin elaboration.

ACKNOWLEDGMENTS

The authors express their appreciation to Dr. JacquesGenest and to his associates, Dr. Pierre Biron and Dr.Roger Boucher, for their helpful instruction in the prepa-ration of the bioassay rat, and to Miss Barbara Jacobsfor her technical assistance. They also gratefully ac-knowledge the generous supply of synthetic angiotensin IIfrom Mr. Neal J. Sullivan of the Ciba PharmaceuticalCompany, Summit, N. J.

REFERENCES

1. Wood, J. E. Genetic control of neutralization ofangiotensin and its relationship to essential hyper-tension. Circulation 1962, 25, 225.

2. Wolf, R. L., M. Mendlowitz, J. Pick, S. E. Gitlow,and N. Naftchi. Metabolism and distribution ofI,3,-labeled angiotension II. J. Lab. clin. Med.1962, 60, 150.

3. WVolf, R. L., M. Mendlowitz, S. Gitlow, and E. Naft-chi. In vitro degradation of angiotensin II-L3` innormotensive and hypertensive human sera (ab-stract). Circulation 1961, 24, 1074.

4. Klaus, D. Untersuchungen uber die Angiotensinaseim Serum gesunder and kranker. Klin. Wschr.1962, 40, 701.

5. Page, I. L., and F. Merlin Bumpus. Angiotensin.Physiol. Rev. 1961, 41, 331.

6. McCall, M. F., and B. Singer. Studies in nephrosis:chemical corticoids, salt-retaining factor, and ef-fect of ACTH. J. clin. Endocr. 1953, 13, 1157.

7. Dyrenfurth, I., C. H. Stacey, and E. H. Venning. Al-dosterone content of urine and abdominal fluidin patients with cirrhosis of the liver. Fed. Proc.1956, 15, 245.

8. Wolff, H. P., Kh. R. Koczorek, and E. Buchborn.Hyperaldosteronism in heart disease. Lancet 1957,2, 63.

9. Watanabe, M., 0. V. Dominguez, C. I. Meeker, E. A.H. Sims, M. J. Gray, and S. Solomon. Aldosteroneand progesterone secretion in pregnancy (abstract).J. clin. Invest. 1962, 41, 1408.

10. Knox, W. E., V. H. Auerbach, and E. C. C. Lin.Enzymatic and metabolic adaptations in animals.Physiol. Rev. 1956, 36, 164.

11. Turgeon, C., and S. C. Sommers. Juxtaglomerularcell counts and human hypertension. Amer. J.Path. 1961, 38, 227.

12. Tobian, L. Relationship of juxtaglomerular appara-tus to renin and angiotensin. Circulation 1962, 25,189.

13. Page, I. H., and 0. M. Helmer. A crystalline pres-sor substance (angiotonin) resulting from the re-action between renin and renin-activator. J. exp.Med. 1940, 71, 29.

14. Kahn, J. R., L. T. Skeggs, N. P. Shumway, andP. E. Wissenbaugh. The assay of hypertensinfrom the arterial blood of normotensive and hyper-tensive human beings. J. exp. Med. 1952, 95, 523.

15. Boucher, R., P. Biron, and J. Genest. Procedurefor isolation and determination of human bloodangiotensin. Canad. J. Biochem. 1961, 39, 581.

16. Genest, J., P. Biron, M. Chretien, R. Boucher, andE. Koiw. Blood angiotensin levels in normal sub-jects and hypertensive patients (abstract). J. clin.Invest. 1962, 41, 1360.

17. Langford, H. G., L. H. Day, I. Conner, and J. E.Howard. A pressor substance, probably angio-tensin found in renal venous blood. Clin. Res.1961, 9, 57.

647


18. Morris, R. E., Jr., P. A. Ransom, and J. E. Howard.Studies on the relationship of angiotensin to hy-pertension of renal origin (abstract). J. clin. In-vest. 1962, 41, 1386.

19. Omae, T., G. M. C. Masson, and I. H. Page. Re-lease of pressor substances from renal grafts origi-nating from rats with renal hypertension. Circulat.Res. 1961, 9, 441.

20. Grieble, H. G., and G. G. Jackson. Bacteriuria, pye-lonephritis, and hypertension: a clinical and patho-logical study in Biology of Pyelonephritis., E. L.Quinn and E. H. Koss, Eds. Boston, Little, Brown,1960, 485.

21. Sommers, S. C. Some pathologic conditions associ-ated with renal and adrenal hypertension. J.Amer. med. Ass. 1961, 178, 715.

22. Muirhead, E. E., F. Jones, and J. A. Stirman. Anti-hypertensive property in renoprival hypertensionof extract from renal medulla. J. Lab. clin. Med.1960, 56, 167.

23. Pickering, G., W. I. Cranston, and M. A. Pears.The nature of hypertension in The Treatment ofHypertension, I. M. Kugelmass, Ed. Springfield,Ill., Charles C Thomas, 1961, p. 15.

24. Dustan, H. P. Some aspects of occlusive renal ar-terial disease in man. Circulat. Res. 1962, 11, 221.

25. Butler, A. M. Chronic pyelonephritis and arterialhypertension. J. clin. Invest. 1937, 16, 889.

26. Smythe, C. McC. Pyelonephritis as a cause of hyper-tension. Amer. J. Cardiol. 1962, 9, 692.

27. Saphir, 0. Pyelonephritis lenta and its relationshipto malignant hypertension. Sth. med. J. (Bgham,Ala.) 1961, 54, 834.

28. Davis, J. O., C. C. J. Carpenter, and C. R. Ayers.Relation of renin and angiotensin II to the con-

trol of aldosterone secretion. Circulat. Res. 1962,11, 171.

29. Laragh, J. H. Relation of aldosterone secretion tohypertensive vascular disease. Circulat. Res. 1961,9, 792.

30. Laragh, J. H. Interrelationships between angioten-sin, norepinephrine, epinephrine, aldosterone se-cretion, and electrolyte metabolism in man. Cir-culation 1962, 25, 203.

31. Hartroft, P. M., and R. Edelman. Renal juxtaglo-merular cells in sodium deficiency: effects of hy-pophysectomy: localization of renin by fluorescentantibody techniques in Edema Mechanisms andManagement, J. H. Moyer and M. Fuchs, Eds.Philadelphia, W. B. Saunders, 1960, p. 65.

32. Davis, J. O., P. M. Hartroft, E. 0. Titus, C. C. J.Carpenter, C. R. Ayers, and H. E. Spiegel. Therole of the renin-angiotensin system in the controlof aldosterone secretion. J. clin. Invest. 1962, 41,378.

33. Tobian, L., S. Perry, and J. Mork. The relationshipof the juxtaglomerular apparatus to sodium re-tention in experimental nephrosis. Ann. intern.Med. 1962, 57, 382.

34. Welt, L. G. Pathogenesis and management of edemain Clinical Disorders of Hydration and Acid-BaseEquilibrium. Boston, Little, Brown, 1955, p. 176.

35. Friedberg, V. Renal function in normal pregnancyand in toxemia. Geburtsh. u. Frauenheilk. 1961,21, 333.

36. Collins, D. A., and A. S. Hamilton. Changes in therenin-angiotensin system in hemorrhagic shock.Amer. J. Physiol. 1944, 140, 499.

37. Dunihue, F. W. The effect of adrenal insufficiencyand of desoxycorticosterone acetate on the juxta-glomerular apparatus. Anat. Rec. 1949, 103, 442.

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