A rapid simple method for the assay of renin in rabbit plasma

1. EDTA (10mm), 2,3-dimercaptopropan-1-ol (10mm) and chlorhexidine gluconate (0.005%, w/v) cause complete inactivation of plasma enzymes that degrade angiotensin I, but have no effect on the reaction of renin with its substrate. The reagents were termed the selective inhibitors. 2. Thus it is possible to measure renin in plasma by its ability to catalyse the release of angiotensin I. 3. Sterile plasma, treated with the selective inhibitors, is incubated with renin substrate (500-1000ng. of angiotensin content/ml.) at pH6 at 42 degrees for 6hr. 4. Under these conditions the reaction obeys first-order kinetics. Renin activity is calculated in terms of the percentage release of the angiotensin content/hr. 5. As described, the assay is sufficiently sensitive to measure renin in the plasma of all normal rabbits. By extending the length of the incubation, much lower activities can be measured.     

Differences in the kinetic rate constants of normal and high molecular weight renin substrate from term pregnancy human plasma

We have previously reported on the differences in physical and chemical characteristics between the high-molecular weight renin substrate (HMS greater than 150,000 daltons) and the normal substrate (NMS = 60,000). In this study, the kinetic constants were determined in both HMS and NMS which were prepared by gel exclusion chromatography from the plasma of pregnant women at term. Renin substrate (angiotensinogen) levels were expressed by radioimmunoassay of angiotensin I after incubation of samples with added semi-purified human kidney renin in the presence of angiotensinase inhibitors. The kinetic constants (Km and Vmax) were determined by the method of Line-weaver-Burk plots and also the method of Wilkinson. The Km for the HMS was 1.79 micrograms angiotensin 1 equivalents ( AIeq )/ml and the Vmax = 41.2 ngAIeq /ml/h, and the Km for the NMS was 3.52 micrograms AIeq /ml and the Vmax = 138 ng/ml/h. When adding small amounts of the HMS to the NMS, the production of angiotensin I was found to increase more than that in the NMS alone. It was also observed that the renin substrate reactivities of the plasma of pregnant women, which contained small amounts of the HMS, were higher than that found in the plasma of normotensive women not taking oral contraceptives. It is suggested that the existence of small amounts of the HMS may therefore contribute to the elevation in blood pressure under the influence of estrogens.     

Kinetics of renin reaction in rat plasma (author's transl)]

Important kinetic aspects of renin reaction were studied in order to evaluate the parameters that regulate the formation rate of angiotensin I. This rate decreased throughout the incubation period of normal rat plasma and it showed a linear increase when plasma was incubated with renin-substrate. When renin was added to normal rat plasma a plateau in the angiotensin I formation rate occurred after 4-6 hours. When plasma samples containing increasing amounts of renin-substrate were incubated, the velocity of their reaction increased in proportion to the renin-substrate concentration. Under these incubation conditions, the reaction between endogenous renin and renin-substrate in normal rat plasma, proved to be a first kinetic order with respect to the substrate.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

Changes of Blood Pressure,Renin, and Angiotensin after Bilateral Nephrectomy in Patients with Chronic Renal Failure

Circulating levels of renin, angiotensin I, and angiotensin II were increased in six patients with chronic renal failure and hypertension uncontrolled by dialysis and hypotensive drugs. Lower and often normal levels were found in 10 patients whose blood pressure was controlled by dialysis treatment. For a variety of reasons all patients were subjected to bilateral nephrectomy. The logarithm of the decrease in plasma concentrations of renin and angiotensin II was significantly related to the fall of blood pressure after operation. Plasma renin concentration correlated significantly with blood angiotensin I concentration and with plasma angiotensin II in samples taken before and after nephrectomy. Renin, angiotensin I, and angiotensin II were measurable in samples of blood taken 48 hours or more after the operation.     

An increase in high-molecular weight renin substrate associated with estrogenic hypertension

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form (Km = 1800 +/- 290 versus 3520 +/- 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Recurrent Hyperkalaemia due to Selective Aldosterone Deficiency: Correction by Angiotensin Infusion

A patient with recurrent weakness and blurring of consciousness associated with hyperkalaemia due to aldosterone deficiency is reported. The plasma concentrations of renin, angiotensin II, and aldosterone were low and did not increase during sodium deprivation. Blood angiotensin I was also low while renin-substrate concentration was normal. Infusion of angiotensin produced a distinct rise in plasma aldosterone. The patient was treated successfully with fludrocortisol.The results support the concept that the renin-angiotensin system is an important regulator of aldosterone secretion and that in the syndrome of acquired selective hypoaldosteronism the primary abnormality may be a deficiency of renin. It is suggested that a selective lack of aldosterone should be considered in all cases of otherwise unexplained hyperkalaemia.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.     

Peptide inhibitors of converting enzyme.

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp¹ angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp¹-Arg² angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.     

Resolution of rat renin substrates by isoelectric focusing.

Pooled plasmas from normal or binephrectomized rats and perfusates of isolated livers were used as sources of renin substrate for isoelectric focusing. After desalting, preliminary fractionation (plasma only), and concentration, the preparations were focused in a pH 3--10 gradient on 20-cm glass plates layered with Sephadex slurry. The pH 4--6 region, containing all the substrate, was scraped from this plate and refocused in a pH 4--6 gradient. Substrate content of 1-cm strips of slurry from half of the plate was determined by both radioimmunoassay and bioassay of angiotensin resulting from incubation with added renin. Corresponding strips from the other half of the plate were incubated without renin as a control for any preformed angiotensin. The asymmetry and broad distribution (pH 4--5) of substrate from different sources suggested the existence of more than one form. Higher resolution achieved by using the high substrate concentration of postnephrectomy plasma and 0.5-cm strips of slurry on 20-cm or 40-cm plates revealed peaks and shoulders of substrate activity. Our data suggest that multiple forms of substrate are synthesized by the liver and circulate in plasma. Postnephrectomy rat plasma appears to contain relatively more substrate(s) with higher isoelectric points than in normal plasma, possibly an accumulation of forms ordinarily degraded by endogenous renal renin.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Plasma renin activity, active and inactive renin concentrations, and their responses to beta 1-adrenoceptor blockade with metoprolol in hyperthyroidism

Plasma renin activity (PRA), plasma renin concentration (PRC), inactive renin concentration (IRC) and total renin concentration (TRC) were measured in 31 normal controls and in 8 patients with hyperthyroidism. TRC was determined as angiotensin I generated with sheep renin substrate after an acid activation of plasma. The angiotensin I of non-acidified plasma was expressed as PRC. IRC was calculated as TRC minus PRC. The mean values for PRA, PRC, IRC and TRC were significantly (P less than 0.05 to P less than 0.01) higher in the hyperthyroid patients than in the normal or euthyroid controls. The administration of a beta 1-adrenergic blocker, metoprolol (120 mg/day for 14 days), produced a significant (P less than 0.05 to P less than 0.01) fall in levels of T4, PRA and TRC, and reduced the active renin ratio calculated from PRC/TRC significantly (P less than 0.025), as compared to the pretreatment values. Our observations support the idea that the higher PRA in hyperthyroidism is due to an increased secretion of renin. Furthermore, the results may indicate that the conversion of inactive to active renin is accelerated in hyperthyroidism, possibly by an increased sympathetic activity.     

Could angiotensin I be produced from a renin substrate by the HIV-1 protease?

The standard angiotensin I (Ang I) radioimmunoassay for renin activity determination is a useful clinical tool for the diagnosis of high renin levels in certain cases of hypertension. It depends upon the liberation of Ang I from human plasma angiotensinogen. We considered whether a commercially available synthetic tetradecapeptide (TDP), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, would produce authentic Ang I upon incubation with protease from human immunodeficiency virus type 1 (HIV-1). This peptide is also known to be cleaved by renin at the Leu-Leu bond to yield the decapeptide Ang I. When the TDP is incubated with the HIV-1 protease, the peptide is readily hydrolyzed. Product formation is linear with respect to time and enzyme concentration. HPLC analysis of reaction products showed two new peaks, as one would expect from the cleavage of a TDP into a decapeptide and a tetrapeptide. Amino acid analysis of HPLC-purified peaks confirmed that the HIV-1 protease cleaves TDP at the Leu10-Leu11 site to produce the desired decapeptide, Ang I. Production of Ang I by the HIV-1 protease, like human renin, is inhibited in the presence of a protease inhibitor. Implications of the discovery of an HIV-1 protease substrate that produces authentic Ang I are discussed in light of a screening assay for soluble HIV-1 protease inhibitors.     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.     

Measurement of renin and prorenin in cattle, hog and horse.

1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-exchange resin, allowing an assay of the plasma prorenin concentration to be constructed in these species. 5. Another strategy has to be followed in order to measure prorenin and renin concentrations in horse plasma.     

Indomethacin potentiates the vasoconstrictor actions of angiotensin II in normal man

The blood pressure response to graded infusions of angiotensin II was assessed under control conditions and following short term (16 hour) indomethacin treatment utilizing normal men equilibrated on a constant diet of normal sodium and potassium content. Although basal mean blood pressure was unchanged, the increase in blood pressure with all rates of angiotensin II infusion ranging from 200 to 1000 ng/min was significantly greater with indomethacin treatment. Pre-infusion body weight and plasma renin activity were similar under the two conditions. These results suggest that prostaglandins modulate the systemic vasoconstrictor effects of angiotensin II.     

Enalapril in the treatment of hypertension with renal artery stenosis.

The converting enzyme inhibitor enalapril, in single daily doses of 10-40 mg, was given to 20 hypertensive patients with renal artery stenosis. The blood pressure fall six hours after the first dose of enalapril was significantly related to the pretreatment plasma concentrations of active renin and angiotensin II and to the concurrent fall in angiotensin II. Blood pressure fell further with continued treatment; the long term fall was not significantly related to pretreatment plasma renin or angiotensin II concentrations. At three months, 24 hours after the last dose of enalapril, blood pressure, plasma angiotensin II, and converting enzyme activity remained low and active renin and angiotensin I high; six hours after dosing, angiotensin II had, however, fallen further. The rise in active renin during long term treatment was proportionally greater than the rise in angiotensin I; this probably reflects the fall in renin substrate that occurs with converting enzyme inhibition. Enalapril alone caused reduction in exchangeable sodium, with distinct increases in serum potassium, creatinine, and urea. Enalapril was well tolerated and controlled hypertension effectively long term; only two of the 20 patients required concomitant diuretic treatment.     

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 μl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC₅₀ values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC₅₀ values obtained correlated well with those obtained using EIA methodology (r² = 0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.