Plasma Renin Activity Assay Research Articles

Effects of Angiotensinase Inhibitors on Plasma Protein Binding and IC50 Determinations of Renin Inhibitors

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.

Plasma renin activity and plasma prorenin assays

Sensitivity and accuracy are essential features of an assay of plasma renin activity (PRA) because the normal concentration of PRA is only 1 pmol/L, and subnormal concentrations have diagnostic relevance. Conditions for blood collection need to be standardized but the conditions are not difficult for outpatients. For routine diagnostic purposes blood should be collected from ambulatory (ideally, untreated) patients on moderate sodium intake. To avoid irreversible cryoactivation of plasma prorenin (which is present in 10-fold greater concentrations than renin), samples should be processed at room temperature and stored completely frozen. Cryoactivation occurs when plasma is liquid at temperatures less than 6 degrees C. PRA is commonly measured with an enzyme kinetic assay in which angiotensin I (Ang I) is formed by the reaction of plasma renin with endogenous renin substrate (angiotensinogen). The Ang I so formed is measured by RIA; results are expressed as an hourly rate (micrograms/L formed per hour). This method, which is provided by most commercial kits, has the potential for unlimited sensitivity because the step for Ang I generation can be prolonged as long as necessary, so that enough Ang I forms to be measured accurately. Unfortunately, that sensitivity is not always exploited. Dilution of plasma during pH adjustment should be kept to a minimum. The Ang I generation step should last at least 3 h. The step should last 18 h for samples with PRA less than 1.0 micrograms/L per hour, to eliminate the errors inherent in the measurement and subtraction of immunoreactive Ang I in the untreated plasma (blank subtraction). These changes actually simplify PRA measurements because they eliminate the need for ice in the clinic and reduce by almost half the number of samples to be assayed by RIA. I also describe the method for measurement of plasma prorenin, which may be an important marker for patients with diabetes mellitus who subsequently develop vascular complications.

Nonparallel Effects of Renin Inhibitor Treatment on Plasma Renin Activity and Angiotensins I and II in Hypertensive Subjects

Nonparallel effects of renin inhibitor treatment on plasma renin activity (PRA) and the plasma levels of angiotensins (ANG), as well as on blood pressure, have been observed in subjects with hypertension. This study addresses the possibility that renin inhibitors may show a high degree of plasma protein binding in vivo and that displacement of protein-bound inhibitor during the assay of PRA in vitro may lead to overestimation of renin inhibition. Indeed, with the ultrafiltration technique it was found that 96% of the novel renin inhibitor Ro 42-5892, when added to EDTA plasma, was bound to protein. The angiotensinase inhibitors phenylmethylsulfonyl fluoride (PMSF) and 8-hydroxy-quinoline sulfate (8-OHQ), which are currently used in PRA assays, caused a displacement of protein-bound inhibitor, thereby increasing its free concentration. This displacement was sufficient to explain the reduction in IC 50 of Ro 42-5892, which was seen in the PRA assay when PMSF and 8-OHQ were added to plasma. Such reductions in IC 50 were also seen with the renin inhibitors CGP 29-287, CGP 38-560A, and SR 43-845. When Ro 42-5892 was given, 1 mg/kg intravenously in 10 min, to subjects with hypertension, it appeared that plasma ANG I and II returned to baseline after 6-8 h, whereas PRA measured in the presence of PMSF and 8-OHQ was still suppressed. However, when PRA was measured without these angiotensinase inhibitors, the inhibition of PRA was parallel to the suppression of ANG I and II.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurements of enzyme activity and immunoreactivity of plasma renin. Limitations of the conventional method for assay of plasma renin catalytic activity.

The values for "plasma renin activity", "plasma renin concentration" (both determined by the rate of angiotensin I generation) and the plasma active renin protein mass concentration (active enzyme protein measured by a direct radioimmunoassay) were compared. The values for "plasma renin activity" (angiotensin I generation from endogeneous renin substrate) and "plasma renin concentration" (angiotensin I generation from exogeneous excess of renin substrate) showed a significant correlation. However, the correlation was lost in samples with high (plasma renin activity greater than or equal to 10 micrograms/l.h) and low (plasma renin activity less than or equal to 1 microgram/l.h) renin contents. In another study, values for "plasma renin activity" were correlated with those for plasma active renin protein mass concentration, and values for "plasma renin concentration" were also correlated to those for plasma active renin protein mass concentration. However, in samples with low renin contents (plasma active renin protein mass concentration less than or equal to 20 ng/l), there was no correlation between values for "plasma renin activity" and plasma active renin protein mass concentration, although those for "plasma renin concentration" and plasma active renin protein mass concentration showed a significant correlation. These results suggest that the current method for the assay of plasma renin activity gives erroneous values for low and high plasma renin concentrations. The new radiometric method for the determination of the plasma active renin protein mass concentration seems to be applicable to plasma samples with a wide range of renin concentrations.

Blood pressure and plasma renin activity responses to chronic stress in the borderline hypertensive rat

The purpose of this study was to examine the systolic blood pressure and plasma renin activity (PRA) responses to chronic stress in normotensive rats and in rats with one hypertensive parent. Twenty-four male Wistar-Kyoto (WKR) and 24 male F 1 offspring of spontaneously hypertensive and WKY rats (BHR) were randomly assigned to 3 groups of 8 each. Experimental (E) animals were subjected to 2 hr daily of shock-shock conflict. Each response produced a 0.2 sec, 0.2–0.4 mA cutaneous electric shock. Failure to respond in 10 sec resulted in a train of 5 shocks (0.2 sec each sec). Yoked animals (Y) received the same shocks as E but had no control over their presentation. Finally, a control group (C) for maturation received no shocks. The E and Y animals were subjected to 14 weeks of conflict and were then monitored an additional 14 weeks in the absence of shock. All animals had their tail cuff blood pressures taken weekly except for 3 times when bloods were obtained for PRA assays. Analysis of blood pressure data revealed that: (1) BHR animals showed more of a blood pressure response to shock than WKY animals; (2) Y animals showed more of a response to conflict than E, especially for the BHR group; and (3) BHR shocked animals remained permanently elevated compared to BHR control animals even in the 14 week post-conflict period during which no shocks were given. Although PRAs for BHR animals were significantly higher than for WKY at the beginning of study, the stress-induced hypertension was associated with either normal or suppressed PRA values, suggesting that the hypertension in these animals is not a high renin hypertension.

Antihypertensive effects of 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on on plasma renin activity and catecholamine responses in spontaneously hypertensive rats

Fourteen 23 week old male spontaneously hypertensive rats (SHR) were randomly divided into saline control or phospholipid (1-hexadecyl-2-acetyl- sn -glycero-3-phosphocholine) treatment groups. Four weeks of baseline systolic blood pressure (SBP) and heart rate (HR) measurements were determined via tail plethysmography. On week 25 of the baseline period a 1.5 ml blood sample was taken by tail clip for analysis of norepinephrine (NE), epinephrine (E), and plasma renin activity (PRA). On the following week, a single injection of phospholipid (11 ug/kg, s.c.) was given to the experimental animals following baseline SBP and HR determinations. A similar procedure was employed for control subjects, except they received an injection of normal saline (0.5 ml, s.c). Systolic BP and HR responses were monitored for 24 minutes following the injection. A 1.5 ml blood sample was taken at the end of the 4th minute for NE, E, and PRA assays. A significant drop in SBP (202 ± 5 mmHg to 124 ± 6 mmHg) and an increase in HR (431 ± 17 bpm to 519 ± 21 bpm) were observed for experimental animals, but not for control subjects. Plasma NE increased significantly (446 ± 42 pg/ml to 1099 ± 77 pg/ml), but E remained unchanged following treatment with the phospholipid. Plasma renin activity increased for both groups, but this change was only significant for the experimental group (18.1 ± 5.7 ng Al/ml/hr to 34.3 ± 3.6 ng Al/ml/hr). Thus, it appears that 1-hexadecyl-2-acetyl- sn -glycero-3-phosphocholine i is a potent antihypertensive vasodilating agent which stimulates baroreceptor mediated sympathetic discharge to the heart and kidneys of the SHR.

Spontaneous variations in renal vein renin activity in man

The spontaneous variations in renal vein renin activity (RVRA) and in peripheral vein renin activity (PVRA) were studied in one normotensive and nine hypertensive patients. Eight of the hypertensive patients had renal artery stenosis on one or both sides. Blood samples were drawn simultaneously from the two renal veins and from a peripheral vein every fifth or tenth minute for one hour. Plasma renin activity (PRA) was measured by radioimmunoassay. The precision of the PRA assay, expressed as coefficient of variation, was related to the PRA level. A large intra-individual variation was found in RVRA, the RVRA ratio and PVRA even in patients with unilateral renovascular hypertension. The intra-individual variation could not be explained by specimen collection error or by error of the assay procedure. The variation seems to reflect a biological fluctuation. The clinical implication of these findings is that repeated, simultaneous collection from the two renal veins, avoidance of factors known to decrease renin secretion, and consideration of the relation between the RVRA ratio and RVRA level are of importance in the preoperative evaluation of patients with renal artery stenosis.

Effects of di-isopropylfluorophosphate and isopropanol on the measurement of plasma renin activity

Since the protease inhibitor, di-isopropylfluorophosphate (DFP), inhibits the cryoactivation of inactive renin, its addition to stored plasma should prevent unintended renin activation and thereby minimize this source of inaccuracy in the assay of plasma renin activity. However, we have found that 6 mmol/1 DFP in stored frozen plasma actually reduced plasma renin activity from 4.89 ± 0.13 (S.D.) to 2.48 ± 0.17 ng · ml −1 · h −1 ( p < 0.001). This effect did not occur if DFP was added immediately before assay, suggesting that the decreased renin activity reflected an action of DFP on components of the renin system during storage rather than any interference with the assay method itself. The organic solvent isopropanol, which is required to dilute the DFP, appeared responsible for this phenomenon, for when the isopropanol alone, in concentrations of 20 and 40 μl/ml, was added to stored frozen plasma it decreased plasma renin activity from 3.6 ± 0.3 to 2.2 ± 0.1 and 0.6 ± 0.1 ng · ml −1 · h −1, respectively ( p < 0.001 in each case). Correspondingly, plasma renin substrate concentrations were decreased to 71% and 6% of control, indicating that the renin activity reductions produced by isopropanol were due to its denaturation of substrate. Moreover, addition of exogenous sheep renin substrate (1400 ng/ml) immediately before assay restored plasma renin activity to control. Thus, although DFP effectively prevents inadvertent renin activation in stored frozen plasma, it would seem important that subsequent assays for plasma renin activity be performed in the presence of added exogenous substrate.

Should blood samples for assay of plasma renin activity be chilled?

Collecting blood on ice for renin determination reportedly may produce falsely high results. To assess the probability of this occurring under actual collection conditions, we measured renin activity in duplicate aliquots of plasma from blood samples from 25 hypertensive patients, both supine and upright, and in 10 supine normotensive controls. One aliquot of the blood was collected on ice and processed at 4 degrees C, the other at room temperature. The two aliquots showed no significant differences in renin activity. If anything, values for samples collected at room temperature were higher. Repeat determination on the same specimens stored at--20 degrees C for nine and 12 months revealed no significant changes in results for any samples, although the amount of angiotensin I found in the sample before incubation at 37 degrees C significantly increased. We conclude that it makes little difference at what temperature one collects blood for renin determination, but because of the wide fluctuations in "room" temperature we recommend that blood samples be collected on ice.

Faster radioimmunoassay of angiotensin I at 37 degrees C.

We applied a 1-h radioimmunoassay incubation at 37 degrees C in determining generated angiotensin I in an assay for plasma renin activity. Under these nonequilibrium conditions, 26% of the 125I-labeled angiotensin I was bound at zero dose of unlabeled angiotensin, as compared to 57% at equilibrium after 18 h at 4 degrees C. Sensitivity and useful range for the standard curve remained unchanged. Blanks were not altered. There was a good (r = .971) correlation between renin values in 120 plasma samples from hypertensive patients as measured with both procedures. With isoelectric focusing, we detected no damage to the labeled angiotensin I during incubation for 1 h at 37 degrees C in the presence of diluted plasma, disodium ethylenediaminetetraacetate, hydroxyquinoline, and neomycin. Analytical recovery of unlabeled angiotensin I added to the assay mixture was 98 +/- 2.3% (mean +/- SD). We conclude that our incubation conditions allow rapid and accurate assay of plasma renin activity to be completed in one working day.

The effect of proteolytic activity on plasma renin activity assay

Carrier proteins are used in the radioimmunoassay of angiotensin I. It has been shown that the proteolytic activity found in commercial lysozyme will falsely elevate the observed plasma renin activity. This effect is probably due to the fact that human plasma contains proteinase inhibitors which inhibit this activity during radioimmunoassay of angiotensin I. This proteolytic activity can be inhibited by diisopropyl fluorophosphate or by heating lysozyme.

Plasma renin concentration: comparison of angiotensinase inhibitors and correlation with plasma renin activity and aldosterone.

Abstract There are wide variations between laboratories in the results obtained for the estimation of the renin content of plasma. Much of this is due to the many different methods employed for inhibiting angiotensinases. In comparing those different methods in the context of a plasma renin concentration assay, phenylmethylsulfonylfluoride was found to be superior to dimercaprol, 8-hydroxyquinoline, and acidification in preserving angiotensin I. In the presence of adequate angiotensinase inhibition there was an excellent correlation between plasma renin activity and plasma renin concentration determined in plasma from 30 male hypertensive patients with low or normal levels of renin. The plasma renin concentration assay method can also be used for samples with high levels of renin. Plasma renin concentration and plasma renin activity correlated equally well with plasma aldosterone. This suggests that in routine clinical screening the plasma renin activity assay gives adequate information and the more complex plasma renin concentration assay need not be done unless aberrations in renin substrate, which could be responsible for dissociation of renin concentration from renin activity, are suspected.

Assay of plasma renin activity using commercially available reagents

1. The rate of generation of Angiotensin 1 by plasma samples at 37° is a measure of their plasma renin activity. The generated Angiotensin 1 has been measured by radio-immunoassay using commercially available 125 I-Angiotensin 1 and anti-Angiotensin 1 antiserum. 2. Both the generation and radio-immunoassay phases of the procedure have been studied and alterations to the manufacturer's recommended protocol have been found necessary. 3. Failure to control the pH of the generation phase of the assay results in a deviation from zero order kinetics. 4. Use of damaged labelled peptide in the radio-immunoassay system results in a loss of sensitivity of the assay. 5. Results are presented from normal subjects and patients with a variety of clinical conditions.