Methodologic Problems in Plasma Renin Activity Measurements
ANNE DELORME, M.D. PHAN TAM GUYENE, M.D. PIERRE CORVOL,
M.D.
JOEL MENARD, M.D. Paris, France
From the HBpital Louis Mourier and the Unit& de Recherches INSERM U3s. Paris, France. This work was supported by grants from DGRST and INSERM. Requests for reprints should be addressed to Dr. Joel Menard, Unit6 de Recherches INSERM Uss. 17 rue du Fer&Moulin, 75005 Paris, France.
The influence of pH and angiotensinase inhibitors on the in vitro generation of angiotenstn I during PRA measurements has been investigated. PRA values obtained at pH 5.7 are higher than those obtained at pH 7.4. At pH 5.7, values obtained using diisopropylfluorophosphate (DFP 9 mM) as an angiotensinase inhibitor are higher than values obtained with a mixture of dimercaprol (BAL, 1.6 mM) and hydroxyquinoline (6-OHQ, 3 to 4 mM). Since the two methods for inhibiting angiotensinase are completely and equally efficient, it is suggested that these inhibitors might interfere with the renin angiotensinogen reaction. Significant correlations are observed between the PRA values obtained by the different methods which have been studied. Using an incubation pH of 5.7, and BAL and 6-OH quinoline as angiotensinase inhibitors, the distribution of PRA values in a population of 124 hospitalized hypertensive patients ingesting a normal sodium diet has been studied, and it has been demonstrated that the sensitivity of this method of measurement can detect small changes in PRA in patients with low renin activity. A great dispersion is being observed in the values of plasma renin activity (PRA) obtained by different methods of measurement [I]. Many factors can explain this dispersion, some are physiologic, others biochemical. Because it was proposed that PRA could have a prognostic meaning in hypertension [2] and that it could direct the choice of treatment with diuretic [3] or beta-blocking [4] agents, it is becoming important to assert that it is actually the same parameter that is being measured by the different methods. Otherwise, normal values in different laboratories are determined under various physiologic circumstances: upright or supine position, during normal sodium intake or during sodium restriction, intravenous or oral diuretic treatment 151. This variability becomes an obstacle when the classifications of patients must be compared and may make this comparison illusive. Apart from the sampling circumstances, the in vitro-incubation conditions of plasma for the determination of PRA must be rigorously controlled. PRA means that no homologous or heterologous substrate has been added, and no inhibitor or activator of the angiotensinogen-renin reaction has been eliminated by a previous extraction of the renin. Contradictory results dealing with the influence of various factors in this method of measurement have already been related [6,7]. The effects of pi-l and of two inhibitors of angiotensinases and converting enzyme on the in vitro generation of angiotensin I are now examined.
November 1976
The American Journal of Medicine
Volume 61
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PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL
201 I
/,
10 ANGIOTENSIN
20
1’0
1’00
2tlo
I P”=og’-ams
Figure 1. Binding of antiangiotensin I antiserum with angiotensin I. The standard curve was determined by adding increasing amounts of angiotensin (0). Three binding curves were determined after the a&Won to the stano!ard curves of 25 plaliquots of the nonincubatedmixture which contained all reagents for PRA measurements: (0) mixture without inhibitors; ( H ) mixture with BAL and 8-Ol-fQ (A) mixture with DFP.
MATERIAL AND METHODS Determination of Plasma Renin Activity. Endogenous angiotensin I was removed from plasma samples by passage through a column of moist resin Dowex WXp (ammonium ion). When PRA was measured at pH 5.7, 250 ~1 of stripped plasma were added to 200 1.11of 0.2 M citrate-phosphate buffer, pH 5.7, containing 1.5 per cent EDTA (ammonium ion) (2. IO-* M final concentration), 25 PI of 0.6 N hydrochloride, and 25 PI of angiotensinase inhibitor. When PRA was measured at pH 7.4, the incubation mixture contained 200 ~1 of 0.2 M citrate-phosphate buffer, pH 7.4 with 1.5 per cent EDTA (ammonium ion), 25 ~1 of 0.2 M citrate-phosphate buffer, pH 7.4 and 25 ~1 of angiotensinase inhibitor. The incubation lasted 3 hours at 37’C. The reaction was stopped by cold. Ten and 25 &I of the incubation mixture were put into tubes for immediate angiotensin I radioimmunoassay. The results were expressed as nanograms of angiotensin I produced per milliliter of plasma per hour. Two inhibitorsof angiotensinases and converting enzyme were used. One was the mixture of dimercaprol (BAL) (1.6 mmol final concentration) and 8hydroxyquinoline (8-OHQ) (3.4 mmol final concentration) in water-ethanol; the other was diisopropyl fluorophosphate (DFP) (9 mmol final concentration) in isopropyl alcohol. Determination of Plasma Renln Substrata. Ten microliters of I:50 plasma renin samples were incubated at pH 5.7 in the same mixture as the one used for the measurement of PRA
726
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in the presence of an excess of human renin (0.05 Goldblatt Units (GU) of a renin purified until step 5 of the Haas et al. method [8]). The incubation lasted 2 hours. The angiotensin concentrations were expressed as nanograms of angiotensin I which could be liberated per milliliter of plasma by an excess of renin. Radioimmunoassay of Angiotensin I. A 1251-monoiodinated angiotensin I, purified by polyacrylamide gel electrophoresis [9], was used. Its specific activity was 1,750 &i/pg. The angiotensin I antiserum was obtained by rabbit immunization against angiotensin I coupled to bovine gamma globulin by carbodiimide condensation; it does not recognize angiotensin II [IO]. When utilized for the purification of hog renin, this antiserum permitted the realization of successive steps resulting in a highly purified and stable preparation which contained 240 GU/mg protein [ 11,121. Assay of this purified renin in the laboratory of Haas and Goldblatt verified that the results obtained with this bioassay in dogs were the same as those obtained with radioimmunoassay of angiotensin I produced in vitro [ 121. Radioimmunoassays were performed with standards of synthetic ileud angiotensin I (Schwartz-Mann). One milliliter aliquots of diluted antiangiotensin I serum (diluted: 1:l10,000 in 0.1 M Tris hydrochloride buffer pH 8.5 containing bovine albumin 0.5 mg/ml) was added to tubes containing angiotensin I standards (5 to 150 pg) or samples to be assayed. After mixing, 1 ml aliquots of tracer solution containing 20,000 dpm of 1251-angiotensin I were added, followed by incubation at 4OC for 48 hours. Bound and free tracer were then separated by the addition of 0.3 ml of a suspension containing 2.5 per cent Norit A (Fisher) and 0.5 per cent Dextran 70 (Pharmacia) in 0.1 M Tris hydrochloride buffer pH 8.5. After 10 minutes the tubes were centrifuged at 1,200 g for 15 minutes at 4’C; the supernatant solutions were aspirated, and the charcoal counted in a Packard automaticgamma spectrometer with 1251counting efficiency of 55 per cent. The complete absence of nonspecific interference in the radioimmunoassay was a characteristic of the standard curve using this tracer and this antiserum. The addition of a 25 @I aliquot of the nonincubated mixture which was used for PRA measurement did not change the standard curve (Figure 1). Destruction of Endogenous Angiotensinogen. Two plasma samples were treated according to Skinner’s method of substrate denaturation [ 131. In these conditions, more than 97 per cent of the endogenous substrate was destroyed, and the angiotensinases were inactivated. RESULTS Influence of the incubation pH on the Angiotensin I Generation. Whatever the angiotensinase inhibitor used is, the amount of angiotensin I generated is greater at pH 5.7 than at pH 7.4 (Figure 2). The correlation coefficient between incubations performed at pH 5.7 and pH 7.4 is r = 0.97 in the presence of DFP and r = 0.93 in the presence of BAL and 8-OHQ. Influence of the Angiotensinase Inhibitors on Anglotensin I Generation. At pH 5.7, the amount of angio-
Volume 61
PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL.
/
2y4 PRA Figure 2. angiotensin conditions. &OH0 (0) tween BAL right side.
6
B
l‘.
,,‘k
6
PRA’EIAL
PH 7.L
,
l‘.
1’2
8 0H:
Influence of the incubation pH and the angiotensinase inhibitors on I generation. Sixteen plasma samples were incubated in standard Correlations between pH 5.7 and pH 7.4 in the presence of BAL and or of DFf (0) are shown at the left side of the graph. Correlations beand 8-OHQ, and DFP at RH 5.7 (A) or at pH 7.4 (A) are shown at the
tensin I generated is greater in the presence of DFP than of BAL and 8-OHQ. On the contrary, at pH 7.4, the amount of angiotensin I produced does not depend on the inhibitor (Figure 2). This correlation coefficient between BAL and &OHQ, and DPF is r = 0.99 at pH 5.7 and r = 0.98 at pH 7.4. The correlation is highly significant among 70 plasma samples from hypertensive patients with low, normal or high renin activity levels: r = 0.99, Yorp = 1.55 XBAL-8 OHQ.
Influence of the Incubation lime on Angiotenstn I Generation. At pH 5.7, in the presence of BAL and 8-OHQ as well as DFP, the amount of angiotensin I increases with the incubation time in a linear manner. More angiotensin I is produced in the presence of DFP (Table I). Influence of the Angiotensinase Inhibitors on the Recovery of Angiotenetn I at Various pH. The production of angiotensin I at pH 5.7, which differs according to the inhibitor used, is not due to an unequal inhibition of angiotensinases and converting enzyme. Two aliquots of a renin-free plasma, obtained from a binephrectomized male patient were incubated at pH 5.7 after having added 0.25 ng and 5 ng of angiotensin I, respectively. A pool of plasma was incubated under standard conditions, with or without 2.5 ng of angiotensin I. In the two experiments, the amounts of angiotensin I added were completely recovered after 3 hours of incubation, at pH 5.7 and pH 7.4, in the presence of BAL and 8-OHQ as well as DFP (Table II). Influence of the Incubation pH and Angiotensinase Inhibitors on the Reaction of Acidified Plasma Renin with Human Angiotensinogen. PRA and plasma renin
substrate (PPS) of two plasma samples were measured at pH 5.7, in the presence of BAL and 8-OHQ, and of DFP. Afterwards, the substrate of these plasmas was denatured according to Skinner’s method [ 131. In a 500 ~1 volume, 50 PI of these acidified samples were added to 100 ~1 of binephrectomized human plasma which contained 2,800 ng of angiotensin I liberable/ml. The angiotensin I production was measured after 2 hours of incubation. The angiotensin I produced by the substrate-free plasma incubated with similar amounts of binephrectomized plasma substrate, is two to four times greater than that produced by the incubation of the plasma by itself, before any acidification. However, the effects of pH and inhibitors remain the same: at pH 5.7, but not at pH 7.4, more angiotensin I is generated in the presence of DFP than of BAL and 8-OHQ (Table Ill).
TABLE I
Kinetics of Angiotensin Generation (ng angiotensin I/ml) of Three Different Plasmasat pH 5.7 incubation Time
Plasma
Inhibitor
0 hr
2 hr
4 hr
15.3 26.0
28.9 49.3
54.5 104
0
2.4
0
3.8
4.1 7.5
8.6 13.8
0 0
0.5
. .
0.9 1.3
1
BAL + 8-QHQ DFP
0 0
2
BAL + 8-QHQ DFP BAL+8-QHQ DFP
3
1 hr
0.7
NOTE: BAL = British antilewisite; 8-OHQ = 8-hydroxyquinoline; DFP = diisopropyl (dimercaprol)fluorophosphate.
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influence of Angiotensinase Inhibitors on the Recovery of Angiotensin I (ng angiotensin l/500 PI incubation) at Two Different pH’s
TABLE II
Incubation
Time 0 hr
Plasma Plasma*
Plasma + 2.5 ng angiotensin
I
Renin-free
plasma + 0.25
Renin-free
plasma + 5 ng angiotensin
NOTE:
Abbreviations
*Five determinations the renin-free plasma.
ng anglotensin
as in Table
I
I
BAL + 8-OHQ
5.7
0
0
7.4
0
0
5.7
2.51 k 0.13
2.60 + 0.08
7.4
2.40
2.40 + 0.18
f 0.18
+ 0.19 + 0.24
6.44 + 0.36 4.35 r 0.34
4.96 4.37
0.22
0.21
0.29
0.24
5.7
5.0
5.0
4.75
4.79
deviation)
were made on each plasma containing
Renin-Free
Inhibitors
on the Reaction
Between
Two Substrate-
Renin-Substrate
After Acidification PRA
PRA
Renin
pH 5.7
Substrate
Added Renin Substrate
--~_____
pH 5.7
pH 1.4
1
BAL + 8-OHQ DFP
3.0 5.1
1,120 1.120
1,120 1,120
14.6 19.0
9.6 10.1
2
BAL + 8-OHQ DFP
7.7 11.3
1,380 1,380
1,120 1,120
18.6 22.0
14.2 13.2
Abbreviations
as in Table
November 1978
on
The investigation of the renin-angiotensin system in hypertensive diseases supplies three types of information. It is very useful in making the diagnosis of surgically curable hypertension due to renal artery disease or to overproduction of mineralocorticoids [ 11. It helps in the choice of treatment with diuretics or beta-blocking agents [3,4]. It is a part of the cardiovascular prognosis of the disease [ 21. The PRA measurement is now very often carried out using the easy assay of angiotensin I by radioimmunoassay. Although it is a very incomplete method by which to explore the renin-angiotensin system, it is a sufficient routine index to appreciate the basal or stimulated state of this system. The comparison of the PRA results obtained in several laboratories shows a big dispersion [ 11. A critical study of the different parameters in assay methods is thus necessary, especially as opposite conclusions concerning PRA
BeforeAcidification
Inhibitors
renin and one determination
COMMENTS
Influence of the Incubation pH and Angiotensinase Free Plasmas with
endogenous
of 107 f 27 meq/day. During the same period of time, among 124 essential hypertensive patients aged 25 to 55 years and investigated under the same conditions, PRA was lower in 35 and higher in 25 than in control normotensive subjects.
(at 37’C 2 or 4 hours before the addition of renal renin) or not preincubated. Kinetics of plasma angiotensinogen exhaustion do not depend on the inhibitors (Figure 3). Searching out Patients with Low Renin Activity. From 1972 to 1974, among 180 patients who were investigated, a solitary adenoma of the adrenal glands overproducing aldosterone was found in 16 patients. PRA was measured at pH 5.7, with BAL and 8-OHQ as inhibitors, after a 3 hour incubation time. It has been observed that PRA was always detectable in patients adhering to a diet containing under 110 meq of sodium, and that the PRA could be increased by having them assume the upright posture. The same observation was made in four cases of licorice-induced hypertension (Table IV). In these two syndromes, PRA is sharply decreased by comparison with a mean PRA (measured with the patient upright) of 1.59 f 0.7 ng of angiotensin I/ml of plasma/hour in 20 normotensive subjects, aged 25 to 55 years, with a mean urinary sodium excretion
728
3.77 Ik 0.10 2.28 + 0.15
5.7
exhaustion of plasma by an excess of renal renin is not altered by the presence of the angiotensinase inhibitors, whether the plasma is preincubated in their presence
NOTE: ml.
2.77 i- 0.14 2.47 + 0.18
I.
(mean * standard
Plasma
DFP
.._..
._____
Influence of the Angiotensinase Inhibitors on the Renin Substrate. The amount of angiotensin I liberated by
TABLE III
OFP
BAL + 8-OHQ
PH
-.-
3 hr
.____
I. PRA values are given in ng/angiotensin
The American Journal of Medlclne
Volume 81
I/ml/hr.
Renin substrate values are given in ng/angiotensin
I/
PLASMA
meanings for the choice of treatments [ 14,151 as well as for the prognosis [2,16-181 were reported. As for any radioimmunoassay, a great sensitivity and a high specificity of the antibody for its hapten are absolutely required. Another difficulty appears as some antibodies cross react with nonspecific substances in plasma. This “blank” leads, if it is not subtracted, to falsely high values, or, if it is substracted from low renin plasma, to negative values [ 71. In the present assay, the minimum detectable dose defined as a dose 2 standard deviations away from mean for zero-dose tubes is 5 pg, and the presence of 25 PI of incubated stripped plasma does not interfere. Consequently, the sensitivity of this PRA measurement is 0.08 ng of angiotensin I/ml of plasma/hour, and it makes it easy to detect patients with low renin activity with 3 hours’ incubation time. In order to measure PRA, it is important to protect the angiotensin I produced in vitro from the action of converting enzyme and angiotensinases. BAL and 8-OHQ mixture and DFP are the generally used inhibitors [20,21]. Oparil et al. [6] recently reported that the efficiency of these inhibitors was incomplete and that it varied with pH at incubation. At pH 7.4, the BAL and 8-OHQ mixture would permit a 1251-angiotensinrecovery of 90 per cent, DFP of only 10 per cent. At pH 5.5, the BAL and 8-OHQ mixture is less efficient, and DFP would result in a 90 per cent recovery. Using nonradioactive angiotensin I amounts which are detectable by radioimmunoassay, the present study shows that the two inhibitors are completely and equally efficient, at pH 5.7 as well as pH 7.4. An incomplete inhibition of converting enzyme and angiotensinases does not explain the different PRA values observed in the presence of these two different inhibitors at two different pH’s. More angiotensin I is always detected at pH 5.7 than at pH 7.4, regardless of the angiotensinase inhibitors used. At pH 5.7, angiotensin I generation is always greater in the presence of DFP than of BAL and 8-OHQ, during incubation times from 1 to 4 hours. This difference persists when plasma, free of endogenous substrate and angiotensinases through acidification [ 131, is then incubated in the presence of the same amount of a renin-free substrate obtained in plasma from a binephrectomized male patient. In these conditions, the angiotensin I production becomes more important. This may be due to the use of binephrectomized plasma as substrate, or to the activation of the plasma renin by the acidification at pH 3.3 [22]. Whatever the cause of this increased angiotensin I production, the incubation in the presence of DFP at pH 5.7 leads once more to greater values than in the presence of BAL and 8-OHQ. At pH 7.4, production of the angiotensin I is smaller than at pH 5.7 and similar in the presence of one or another of the inhibitors. It was proved that the use of these inhibitors does
RENIN ACTIVITY
MEASUREMENTS-DELORME
ET AL.
2000
3’0 INCUBATION
6-0 TIME(minutes)
120
inhibitors on plasma Figure 3. Influence of angiotensinase renin substrate measurements. No preincuba tion (0); 3 hours preincubations {A); 4 hours preincubations (0). /n the presence of BAL and 8-QHQ, white; of DFP, black.
not change endogenous substrate, thus it is concluded that the addition of DFP to plasma at pH 5.7 might modify,, directly or not, the renin-substrate reaction and increase the rate of the enzymatic reaction. The correlations between the PRA values obtained at two pH’s, and with two inhibitors are highly significant. Within the same laboratory, the high or low PRA values determined in hypertensive patients are compared to the values obtained in normal subjects. The correlations
TABLE
IV
Influence
of Upright
Posture on PRA
16 Cases of Surgically Adenomas
(p 0.01)
Licorice-Induced
Condition Primary
aldosteronism
Licorice-induced hypertension
No. of Cases
Confirmed
in
Adrenal
and in Four Cases of
Hypertension
Recumbent
Upright
16
0.15
+ 0.09
0.20
+ 0.21
4
0.23
f 0.09
0.37
f 0.09
*p < 0.01,
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PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL.
just described might suggest that different methods should lead to similar clinical conclusions. Thus in our laboratory, when PRA is measured at pH 5.7 in the presence of BAL and 8-OHQ, in 124 subjects with essential hypertension receiving 110 meq of sodium a day, PRA was lower in 28 per cent and higher in 23 per cent than the PRA of control normotensive subjects of the same age; this distribution approaches the one reported by Buhler et al. [4] using a different method. Moreover, among 20 low renin hypertensive cases secondary to primary aldosteronism or licorice intoxication, it is possible to demonstrate that even in this low range, renin secretion in such patients is regulated in the same way as in normal subjects. Using a sensitive method of measurement for plasma renin concentration, Stockigt et al. [ 221 have already reported postural responses in patients with aldosterone-producing adenomas and hyperplastic aldosteronism. In conclusion, the results show that absolute PRA
values can be modified either by changing the incubation pH or by utilizing various angiotensinase inhibitors. However, the fact that PRA values correlate significantly within different methods suggest that differences in methodology cannot explain differences in clinical conclusions. Indeed, using its own method, in reference to its own normal range, each laboratory should obtain a similar classification of patients. This is illustrated by the identity of PRA distribution as measured by two different methods in two different hospitalized groups of hypertensive patients. Moreover, when the method is sufficiently sensitive, PRA can be equally valuable as plasma renin concentration changes for detecting small variations in renin secretion of patients with low renin activity. ACKNOWLEDGMENT We wish to thank Mrs. M. F. Gonzales for her skillful technical help.
REFERENCES
8.
9.
10.
11.
12.
730
Oparil S, Haber MD: The renin angiotensin system. N Engl J Med 292: 389, 1974. Brunner HR. Laragh JH, Baer L, et al.: Essential hypertension: renin and akiosterone, heart attack and stroke. N Engl J Med 286: 441, 1972. Dunn MJ, Tanner RL: Low renin hypertension. Kidney Int 5: 317,1974. Buhler FR, Laragh JH, Baer L, et al.: Propranolol inhibition of renin secretion. A specific approach to diagnosis and treatment of reni Mt hypertensive diseases. N Engl J Med 287: 1209, 1972. Drayer J, Kloppenborg PWC, Benraad TJ: Detection of lowrenin hypertension. Evaluation of out-patient renin-stimulation methods. Clin Sci Mot Med 48: 91, 1975. Oparil S, Koerner JT, Haber E: Effects of pH and enzymes inhibitors on apparent generation of angiotensin I in human plasma. J Clin Endocrinol Metab 39: 965, 1974. Sealey JE, Laragh JH: Searching out low renin patients: limitations of some commonly used methods. Hypertension Manual (Laragh JH, ed), New York, Yorke Medical Books, 1974. p 87. Haas E, Goldblatl H, Gipson EC, et al.: Extraction, purification, and assay of human renin free of angiotensinase. Circ Res 19: 739, 1966. Corvol P, Rodbard D, Drouet J, et al.: Monoiodinated angiotensins: preparation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing. Biochim Biophys Acta 322: 392. 1973. Menard J, Catt KJ: Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I. Endocrinology 90: 422, 1972. Corvol P, Devaux C, Menard J: Pep&tin, an inhibitor for renin purification by affinity chromatography. FEBS Lett 34: 189, 1973. Devaux C, Menard J, Sicard A, et al.: Partial characterization
November 1976
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13.
14. 15.
16.
17. 18.
19.
20.
21.
22.
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of hog-renin purified by affinity chromatography. Eur J Biochem 64: 621, 1976. Skinner SL: Improved assay methods for renin concentration and activity in human plasma: methods using selective denaturation of renin substrate. Circ Res 20: 391, 1967. Stokes GS, Weber MA, Thomel IR: Beta-blockers and plasma renin activity in hypertension. Br Med J 1: 60, 1974. Hansson L: Beta-adrenergic blockade in essential hypertension. Effects of propranolol on hemodynamic parameters and plasma renin activity. Acta Msd Stand 194 (suppl): 550. 1973. Doyle AE, Jerums G. Johnston Cl: Plasma renin levels and vascular complications in hypertension. Br Med J 2: 206, 1973. Genest J, Boucher R, Kuchel 0:Renin in hypertension. How important as a risk factor? Br Med J 2: 206, 1973. Mroczek WJ, Finnerty FA, Catt KJ: Lack of association between plasma-renin and history of heart-attack or stroke in patients with essential hypertension. Lancet 2: 464, 1973. Stockigt JR, Collins RD, Biglieri EG: Determination of plasma renin concentration by angiotensin I immunoassay; diagnostic importance of precise measurements of subnormal renin hyperaldosteronism. Circ Res 28 (suppl II): 175, 1970. Haber E, Koerner T, Page LB: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J Clin Endocrinol Metab 29: 1349, 1969. Sealey JE, Gerten-Banes J, Laragh JH: The renin-system: variations in man measured by radioimmunoassay or bioassay. Kidney Int 1: 240, 1972. Day RP, Luetscher JA: Biochemical properties of big renin extracted from human plasma. J Clin Endocrinol Metab 40: 1085, 1975.
ANNE DELORME, M.D. PHAN TAM GUYENE, M.D. PIERRE CORVOL,
M.D.
JOEL MENARD, M.D. Paris, France
From the HBpital Louis Mourier and the Unit& de Recherches INSERM U3s. Paris, France. This work was supported by grants from DGRST and INSERM. Requests for reprints should be addressed to Dr. Joel Menard, Unit6 de Recherches INSERM Uss. 17 rue du Fer&Moulin, 75005 Paris, France.
The influence of pH and angiotensinase inhibitors on the in vitro generation of angiotenstn I during PRA measurements has been investigated. PRA values obtained at pH 5.7 are higher than those obtained at pH 7.4. At pH 5.7, values obtained using diisopropylfluorophosphate (DFP 9 mM) as an angiotensinase inhibitor are higher than values obtained with a mixture of dimercaprol (BAL, 1.6 mM) and hydroxyquinoline (6-OHQ, 3 to 4 mM). Since the two methods for inhibiting angiotensinase are completely and equally efficient, it is suggested that these inhibitors might interfere with the renin angiotensinogen reaction. Significant correlations are observed between the PRA values obtained by the different methods which have been studied. Using an incubation pH of 5.7, and BAL and 6-OH quinoline as angiotensinase inhibitors, the distribution of PRA values in a population of 124 hospitalized hypertensive patients ingesting a normal sodium diet has been studied, and it has been demonstrated that the sensitivity of this method of measurement can detect small changes in PRA in patients with low renin activity. A great dispersion is being observed in the values of plasma renin activity (PRA) obtained by different methods of measurement [I]. Many factors can explain this dispersion, some are physiologic, others biochemical. Because it was proposed that PRA could have a prognostic meaning in hypertension [2] and that it could direct the choice of treatment with diuretic [3] or beta-blocking [4] agents, it is becoming important to assert that it is actually the same parameter that is being measured by the different methods. Otherwise, normal values in different laboratories are determined under various physiologic circumstances: upright or supine position, during normal sodium intake or during sodium restriction, intravenous or oral diuretic treatment 151. This variability becomes an obstacle when the classifications of patients must be compared and may make this comparison illusive. Apart from the sampling circumstances, the in vitro-incubation conditions of plasma for the determination of PRA must be rigorously controlled. PRA means that no homologous or heterologous substrate has been added, and no inhibitor or activator of the angiotensinogen-renin reaction has been eliminated by a previous extraction of the renin. Contradictory results dealing with the influence of various factors in this method of measurement have already been related [6,7]. The effects of pi-l and of two inhibitors of angiotensinases and converting enzyme on the in vitro generation of angiotensin I are now examined.
November 1976
The American Journal of Medicine
Volume 61
725
PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL
201 I
/,
10 ANGIOTENSIN
20
1’0
1’00
2tlo
I P”=og’-ams
Figure 1. Binding of antiangiotensin I antiserum with angiotensin I. The standard curve was determined by adding increasing amounts of angiotensin (0). Three binding curves were determined after the a&Won to the stano!ard curves of 25 plaliquots of the nonincubatedmixture which contained all reagents for PRA measurements: (0) mixture without inhibitors; ( H ) mixture with BAL and 8-Ol-fQ (A) mixture with DFP.
MATERIAL AND METHODS Determination of Plasma Renin Activity. Endogenous angiotensin I was removed from plasma samples by passage through a column of moist resin Dowex WXp (ammonium ion). When PRA was measured at pH 5.7, 250 ~1 of stripped plasma were added to 200 1.11of 0.2 M citrate-phosphate buffer, pH 5.7, containing 1.5 per cent EDTA (ammonium ion) (2. IO-* M final concentration), 25 PI of 0.6 N hydrochloride, and 25 PI of angiotensinase inhibitor. When PRA was measured at pH 7.4, the incubation mixture contained 200 ~1 of 0.2 M citrate-phosphate buffer, pH 7.4 with 1.5 per cent EDTA (ammonium ion), 25 ~1 of 0.2 M citrate-phosphate buffer, pH 7.4 and 25 ~1 of angiotensinase inhibitor. The incubation lasted 3 hours at 37’C. The reaction was stopped by cold. Ten and 25 &I of the incubation mixture were put into tubes for immediate angiotensin I radioimmunoassay. The results were expressed as nanograms of angiotensin I produced per milliliter of plasma per hour. Two inhibitorsof angiotensinases and converting enzyme were used. One was the mixture of dimercaprol (BAL) (1.6 mmol final concentration) and 8hydroxyquinoline (8-OHQ) (3.4 mmol final concentration) in water-ethanol; the other was diisopropyl fluorophosphate (DFP) (9 mmol final concentration) in isopropyl alcohol. Determination of Plasma Renln Substrata. Ten microliters of I:50 plasma renin samples were incubated at pH 5.7 in the same mixture as the one used for the measurement of PRA
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1976
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American Journal of
Medicine
in the presence of an excess of human renin (0.05 Goldblatt Units (GU) of a renin purified until step 5 of the Haas et al. method [8]). The incubation lasted 2 hours. The angiotensin concentrations were expressed as nanograms of angiotensin I which could be liberated per milliliter of plasma by an excess of renin. Radioimmunoassay of Angiotensin I. A 1251-monoiodinated angiotensin I, purified by polyacrylamide gel electrophoresis [9], was used. Its specific activity was 1,750 &i/pg. The angiotensin I antiserum was obtained by rabbit immunization against angiotensin I coupled to bovine gamma globulin by carbodiimide condensation; it does not recognize angiotensin II [IO]. When utilized for the purification of hog renin, this antiserum permitted the realization of successive steps resulting in a highly purified and stable preparation which contained 240 GU/mg protein [ 11,121. Assay of this purified renin in the laboratory of Haas and Goldblatt verified that the results obtained with this bioassay in dogs were the same as those obtained with radioimmunoassay of angiotensin I produced in vitro [ 121. Radioimmunoassays were performed with standards of synthetic ileud angiotensin I (Schwartz-Mann). One milliliter aliquots of diluted antiangiotensin I serum (diluted: 1:l10,000 in 0.1 M Tris hydrochloride buffer pH 8.5 containing bovine albumin 0.5 mg/ml) was added to tubes containing angiotensin I standards (5 to 150 pg) or samples to be assayed. After mixing, 1 ml aliquots of tracer solution containing 20,000 dpm of 1251-angiotensin I were added, followed by incubation at 4OC for 48 hours. Bound and free tracer were then separated by the addition of 0.3 ml of a suspension containing 2.5 per cent Norit A (Fisher) and 0.5 per cent Dextran 70 (Pharmacia) in 0.1 M Tris hydrochloride buffer pH 8.5. After 10 minutes the tubes were centrifuged at 1,200 g for 15 minutes at 4’C; the supernatant solutions were aspirated, and the charcoal counted in a Packard automaticgamma spectrometer with 1251counting efficiency of 55 per cent. The complete absence of nonspecific interference in the radioimmunoassay was a characteristic of the standard curve using this tracer and this antiserum. The addition of a 25 @I aliquot of the nonincubated mixture which was used for PRA measurement did not change the standard curve (Figure 1). Destruction of Endogenous Angiotensinogen. Two plasma samples were treated according to Skinner’s method of substrate denaturation [ 131. In these conditions, more than 97 per cent of the endogenous substrate was destroyed, and the angiotensinases were inactivated. RESULTS Influence of the incubation pH on the Angiotensin I Generation. Whatever the angiotensinase inhibitor used is, the amount of angiotensin I generated is greater at pH 5.7 than at pH 7.4 (Figure 2). The correlation coefficient between incubations performed at pH 5.7 and pH 7.4 is r = 0.97 in the presence of DFP and r = 0.93 in the presence of BAL and 8-OHQ. Influence of the Angiotensinase Inhibitors on Anglotensin I Generation. At pH 5.7, the amount of angio-
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PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL.
/
2y4 PRA Figure 2. angiotensin conditions. &OH0 (0) tween BAL right side.
6
B
l‘.
,,‘k
6
PRA’EIAL
PH 7.L
,
l‘.
1’2
8 0H:
Influence of the incubation pH and the angiotensinase inhibitors on I generation. Sixteen plasma samples were incubated in standard Correlations between pH 5.7 and pH 7.4 in the presence of BAL and or of DFf (0) are shown at the left side of the graph. Correlations beand 8-OHQ, and DFP at RH 5.7 (A) or at pH 7.4 (A) are shown at the
tensin I generated is greater in the presence of DFP than of BAL and 8-OHQ. On the contrary, at pH 7.4, the amount of angiotensin I produced does not depend on the inhibitor (Figure 2). This correlation coefficient between BAL and &OHQ, and DPF is r = 0.99 at pH 5.7 and r = 0.98 at pH 7.4. The correlation is highly significant among 70 plasma samples from hypertensive patients with low, normal or high renin activity levels: r = 0.99, Yorp = 1.55 XBAL-8 OHQ.
Influence of the Incubation lime on Angiotenstn I Generation. At pH 5.7, in the presence of BAL and 8-OHQ as well as DFP, the amount of angiotensin I increases with the incubation time in a linear manner. More angiotensin I is produced in the presence of DFP (Table I). Influence of the Angiotensinase Inhibitors on the Recovery of Angiotenetn I at Various pH. The production of angiotensin I at pH 5.7, which differs according to the inhibitor used, is not due to an unequal inhibition of angiotensinases and converting enzyme. Two aliquots of a renin-free plasma, obtained from a binephrectomized male patient were incubated at pH 5.7 after having added 0.25 ng and 5 ng of angiotensin I, respectively. A pool of plasma was incubated under standard conditions, with or without 2.5 ng of angiotensin I. In the two experiments, the amounts of angiotensin I added were completely recovered after 3 hours of incubation, at pH 5.7 and pH 7.4, in the presence of BAL and 8-OHQ as well as DFP (Table II). Influence of the Incubation pH and Angiotensinase Inhibitors on the Reaction of Acidified Plasma Renin with Human Angiotensinogen. PRA and plasma renin
substrate (PPS) of two plasma samples were measured at pH 5.7, in the presence of BAL and 8-OHQ, and of DFP. Afterwards, the substrate of these plasmas was denatured according to Skinner’s method [ 131. In a 500 ~1 volume, 50 PI of these acidified samples were added to 100 ~1 of binephrectomized human plasma which contained 2,800 ng of angiotensin I liberable/ml. The angiotensin I production was measured after 2 hours of incubation. The angiotensin I produced by the substrate-free plasma incubated with similar amounts of binephrectomized plasma substrate, is two to four times greater than that produced by the incubation of the plasma by itself, before any acidification. However, the effects of pH and inhibitors remain the same: at pH 5.7, but not at pH 7.4, more angiotensin I is generated in the presence of DFP than of BAL and 8-OHQ (Table Ill).
TABLE I
Kinetics of Angiotensin Generation (ng angiotensin I/ml) of Three Different Plasmasat pH 5.7 incubation Time
Plasma
Inhibitor
0 hr
2 hr
4 hr
15.3 26.0
28.9 49.3
54.5 104
0
2.4
0
3.8
4.1 7.5
8.6 13.8
0 0
0.5
. .
0.9 1.3
1
BAL + 8-QHQ DFP
0 0
2
BAL + 8-QHQ DFP BAL+8-QHQ DFP
3
1 hr
0.7
NOTE: BAL = British antilewisite; 8-OHQ = 8-hydroxyquinoline; DFP = diisopropyl (dimercaprol)fluorophosphate.
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PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
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influence of Angiotensinase Inhibitors on the Recovery of Angiotensin I (ng angiotensin l/500 PI incubation) at Two Different pH’s
TABLE II
Incubation
Time 0 hr
Plasma Plasma*
Plasma + 2.5 ng angiotensin
I
Renin-free
plasma + 0.25
Renin-free
plasma + 5 ng angiotensin
NOTE:
Abbreviations
*Five determinations the renin-free plasma.
ng anglotensin
as in Table
I
I
BAL + 8-OHQ
5.7
0
0
7.4
0
0
5.7
2.51 k 0.13
2.60 + 0.08
7.4
2.40
2.40 + 0.18
f 0.18
+ 0.19 + 0.24
6.44 + 0.36 4.35 r 0.34
4.96 4.37
0.22
0.21
0.29
0.24
5.7
5.0
5.0
4.75
4.79
deviation)
were made on each plasma containing
Renin-Free
Inhibitors
on the Reaction
Between
Two Substrate-
Renin-Substrate
After Acidification PRA
PRA
Renin
pH 5.7
Substrate
Added Renin Substrate
--~_____
pH 5.7
pH 1.4
1
BAL + 8-OHQ DFP
3.0 5.1
1,120 1.120
1,120 1,120
14.6 19.0
9.6 10.1
2
BAL + 8-OHQ DFP
7.7 11.3
1,380 1,380
1,120 1,120
18.6 22.0
14.2 13.2
Abbreviations
as in Table
November 1978
on
The investigation of the renin-angiotensin system in hypertensive diseases supplies three types of information. It is very useful in making the diagnosis of surgically curable hypertension due to renal artery disease or to overproduction of mineralocorticoids [ 11. It helps in the choice of treatment with diuretics or beta-blocking agents [3,4]. It is a part of the cardiovascular prognosis of the disease [ 21. The PRA measurement is now very often carried out using the easy assay of angiotensin I by radioimmunoassay. Although it is a very incomplete method by which to explore the renin-angiotensin system, it is a sufficient routine index to appreciate the basal or stimulated state of this system. The comparison of the PRA results obtained in several laboratories shows a big dispersion [ 11. A critical study of the different parameters in assay methods is thus necessary, especially as opposite conclusions concerning PRA
BeforeAcidification
Inhibitors
renin and one determination
COMMENTS
Influence of the Incubation pH and Angiotensinase Free Plasmas with
endogenous
of 107 f 27 meq/day. During the same period of time, among 124 essential hypertensive patients aged 25 to 55 years and investigated under the same conditions, PRA was lower in 35 and higher in 25 than in control normotensive subjects.
(at 37’C 2 or 4 hours before the addition of renal renin) or not preincubated. Kinetics of plasma angiotensinogen exhaustion do not depend on the inhibitors (Figure 3). Searching out Patients with Low Renin Activity. From 1972 to 1974, among 180 patients who were investigated, a solitary adenoma of the adrenal glands overproducing aldosterone was found in 16 patients. PRA was measured at pH 5.7, with BAL and 8-OHQ as inhibitors, after a 3 hour incubation time. It has been observed that PRA was always detectable in patients adhering to a diet containing under 110 meq of sodium, and that the PRA could be increased by having them assume the upright posture. The same observation was made in four cases of licorice-induced hypertension (Table IV). In these two syndromes, PRA is sharply decreased by comparison with a mean PRA (measured with the patient upright) of 1.59 f 0.7 ng of angiotensin I/ml of plasma/hour in 20 normotensive subjects, aged 25 to 55 years, with a mean urinary sodium excretion
728
3.77 Ik 0.10 2.28 + 0.15
5.7
exhaustion of plasma by an excess of renal renin is not altered by the presence of the angiotensinase inhibitors, whether the plasma is preincubated in their presence
NOTE: ml.
2.77 i- 0.14 2.47 + 0.18
I.
(mean * standard
Plasma
DFP
.._..
._____
Influence of the Angiotensinase Inhibitors on the Renin Substrate. The amount of angiotensin I liberated by
TABLE III
OFP
BAL + 8-OHQ
PH
-.-
3 hr
.____
I. PRA values are given in ng/angiotensin
The American Journal of Medlclne
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I/ml/hr.
Renin substrate values are given in ng/angiotensin
I/
PLASMA
meanings for the choice of treatments [ 14,151 as well as for the prognosis [2,16-181 were reported. As for any radioimmunoassay, a great sensitivity and a high specificity of the antibody for its hapten are absolutely required. Another difficulty appears as some antibodies cross react with nonspecific substances in plasma. This “blank” leads, if it is not subtracted, to falsely high values, or, if it is substracted from low renin plasma, to negative values [ 71. In the present assay, the minimum detectable dose defined as a dose 2 standard deviations away from mean for zero-dose tubes is 5 pg, and the presence of 25 PI of incubated stripped plasma does not interfere. Consequently, the sensitivity of this PRA measurement is 0.08 ng of angiotensin I/ml of plasma/hour, and it makes it easy to detect patients with low renin activity with 3 hours’ incubation time. In order to measure PRA, it is important to protect the angiotensin I produced in vitro from the action of converting enzyme and angiotensinases. BAL and 8-OHQ mixture and DFP are the generally used inhibitors [20,21]. Oparil et al. [6] recently reported that the efficiency of these inhibitors was incomplete and that it varied with pH at incubation. At pH 7.4, the BAL and 8-OHQ mixture would permit a 1251-angiotensinrecovery of 90 per cent, DFP of only 10 per cent. At pH 5.5, the BAL and 8-OHQ mixture is less efficient, and DFP would result in a 90 per cent recovery. Using nonradioactive angiotensin I amounts which are detectable by radioimmunoassay, the present study shows that the two inhibitors are completely and equally efficient, at pH 5.7 as well as pH 7.4. An incomplete inhibition of converting enzyme and angiotensinases does not explain the different PRA values observed in the presence of these two different inhibitors at two different pH’s. More angiotensin I is always detected at pH 5.7 than at pH 7.4, regardless of the angiotensinase inhibitors used. At pH 5.7, angiotensin I generation is always greater in the presence of DFP than of BAL and 8-OHQ, during incubation times from 1 to 4 hours. This difference persists when plasma, free of endogenous substrate and angiotensinases through acidification [ 131, is then incubated in the presence of the same amount of a renin-free substrate obtained in plasma from a binephrectomized male patient. In these conditions, the angiotensin I production becomes more important. This may be due to the use of binephrectomized plasma as substrate, or to the activation of the plasma renin by the acidification at pH 3.3 [22]. Whatever the cause of this increased angiotensin I production, the incubation in the presence of DFP at pH 5.7 leads once more to greater values than in the presence of BAL and 8-OHQ. At pH 7.4, production of the angiotensin I is smaller than at pH 5.7 and similar in the presence of one or another of the inhibitors. It was proved that the use of these inhibitors does
RENIN ACTIVITY
MEASUREMENTS-DELORME
ET AL.
2000
3’0 INCUBATION
6-0 TIME(minutes)
120
inhibitors on plasma Figure 3. Influence of angiotensinase renin substrate measurements. No preincuba tion (0); 3 hours preincubations {A); 4 hours preincubations (0). /n the presence of BAL and 8-QHQ, white; of DFP, black.
not change endogenous substrate, thus it is concluded that the addition of DFP to plasma at pH 5.7 might modify,, directly or not, the renin-substrate reaction and increase the rate of the enzymatic reaction. The correlations between the PRA values obtained at two pH’s, and with two inhibitors are highly significant. Within the same laboratory, the high or low PRA values determined in hypertensive patients are compared to the values obtained in normal subjects. The correlations
TABLE
IV
Influence
of Upright
Posture on PRA
16 Cases of Surgically Adenomas
(p 0.01)
Licorice-Induced
Condition Primary
aldosteronism
Licorice-induced hypertension
No. of Cases
Confirmed
in
Adrenal
and in Four Cases of
Hypertension
Recumbent
Upright
16
0.15
+ 0.09
0.20
+ 0.21
4
0.23
f 0.09
0.37
f 0.09
*p < 0.01,
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PLASMA RENIN ACTIVITY MEASUREMENTS-DELORME
ET AL.
just described might suggest that different methods should lead to similar clinical conclusions. Thus in our laboratory, when PRA is measured at pH 5.7 in the presence of BAL and 8-OHQ, in 124 subjects with essential hypertension receiving 110 meq of sodium a day, PRA was lower in 28 per cent and higher in 23 per cent than the PRA of control normotensive subjects of the same age; this distribution approaches the one reported by Buhler et al. [4] using a different method. Moreover, among 20 low renin hypertensive cases secondary to primary aldosteronism or licorice intoxication, it is possible to demonstrate that even in this low range, renin secretion in such patients is regulated in the same way as in normal subjects. Using a sensitive method of measurement for plasma renin concentration, Stockigt et al. [ 221 have already reported postural responses in patients with aldosterone-producing adenomas and hyperplastic aldosteronism. In conclusion, the results show that absolute PRA
values can be modified either by changing the incubation pH or by utilizing various angiotensinase inhibitors. However, the fact that PRA values correlate significantly within different methods suggest that differences in methodology cannot explain differences in clinical conclusions. Indeed, using its own method, in reference to its own normal range, each laboratory should obtain a similar classification of patients. This is illustrated by the identity of PRA distribution as measured by two different methods in two different hospitalized groups of hypertensive patients. Moreover, when the method is sufficiently sensitive, PRA can be equally valuable as plasma renin concentration changes for detecting small variations in renin secretion of patients with low renin activity. ACKNOWLEDGMENT We wish to thank Mrs. M. F. Gonzales for her skillful technical help.
REFERENCES
8.
9.
10.
11.
12.
730
Oparil S, Haber MD: The renin angiotensin system. N Engl J Med 292: 389, 1974. Brunner HR. Laragh JH, Baer L, et al.: Essential hypertension: renin and akiosterone, heart attack and stroke. N Engl J Med 286: 441, 1972. Dunn MJ, Tanner RL: Low renin hypertension. Kidney Int 5: 317,1974. Buhler FR, Laragh JH, Baer L, et al.: Propranolol inhibition of renin secretion. A specific approach to diagnosis and treatment of reni Mt hypertensive diseases. N Engl J Med 287: 1209, 1972. Drayer J, Kloppenborg PWC, Benraad TJ: Detection of lowrenin hypertension. Evaluation of out-patient renin-stimulation methods. Clin Sci Mot Med 48: 91, 1975. Oparil S, Koerner JT, Haber E: Effects of pH and enzymes inhibitors on apparent generation of angiotensin I in human plasma. J Clin Endocrinol Metab 39: 965, 1974. Sealey JE, Laragh JH: Searching out low renin patients: limitations of some commonly used methods. Hypertension Manual (Laragh JH, ed), New York, Yorke Medical Books, 1974. p 87. Haas E, Goldblatl H, Gipson EC, et al.: Extraction, purification, and assay of human renin free of angiotensinase. Circ Res 19: 739, 1966. Corvol P, Rodbard D, Drouet J, et al.: Monoiodinated angiotensins: preparation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing. Biochim Biophys Acta 322: 392. 1973. Menard J, Catt KJ: Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I. Endocrinology 90: 422, 1972. Corvol P, Devaux C, Menard J: Pep&tin, an inhibitor for renin purification by affinity chromatography. FEBS Lett 34: 189, 1973. Devaux C, Menard J, Sicard A, et al.: Partial characterization
November 1976
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13.
14. 15.
16.
17. 18.
19.
20.
21.
22.
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of hog-renin purified by affinity chromatography. Eur J Biochem 64: 621, 1976. Skinner SL: Improved assay methods for renin concentration and activity in human plasma: methods using selective denaturation of renin substrate. Circ Res 20: 391, 1967. Stokes GS, Weber MA, Thomel IR: Beta-blockers and plasma renin activity in hypertension. Br Med J 1: 60, 1974. Hansson L: Beta-adrenergic blockade in essential hypertension. Effects of propranolol on hemodynamic parameters and plasma renin activity. Acta Msd Stand 194 (suppl): 550. 1973. Doyle AE, Jerums G. Johnston Cl: Plasma renin levels and vascular complications in hypertension. Br Med J 2: 206, 1973. Genest J, Boucher R, Kuchel 0:Renin in hypertension. How important as a risk factor? Br Med J 2: 206, 1973. Mroczek WJ, Finnerty FA, Catt KJ: Lack of association between plasma-renin and history of heart-attack or stroke in patients with essential hypertension. Lancet 2: 464, 1973. Stockigt JR, Collins RD, Biglieri EG: Determination of plasma renin concentration by angiotensin I immunoassay; diagnostic importance of precise measurements of subnormal renin hyperaldosteronism. Circ Res 28 (suppl II): 175, 1970. Haber E, Koerner T, Page LB: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J Clin Endocrinol Metab 29: 1349, 1969. Sealey JE, Gerten-Banes J, Laragh JH: The renin-system: variations in man measured by radioimmunoassay or bioassay. Kidney Int 1: 240, 1972. Day RP, Luetscher JA: Biochemical properties of big renin extracted from human plasma. J Clin Endocrinol Metab 40: 1085, 1975.
