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Technical Briefs |
1 Blood Pressure Unit, Department of Cardiac and Vascular Medicine, St. George’s Hospital Medical School, London, UK;2 Nichols Institute Diagnostics Ltd, Unit B1, Heston, Middlesex, UK;
aaddress correspondence to this author at: Blood Pressure Unit, Department of Cardiac and Vascular Medicine, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK; fax 44-208-725-2959, e-mail dhartman{at}sghms.ac.uk
The enzyme renin (Mr 
40 000) is released in an active form from the renal juxtaglomerular cells in response to physiologic factors, including sodium depletion, decreased blood volume and blood pressure, and ß-adrenergic stimulation (1). Although several local angiotensin II-generating systems exist within various tissues (including the heart, brain, and adrenal glands), the concentration of active renin in plasma depends on the rate of renin secretion from the kidneys (2). Renin catalyzes the formation of angiotensin I (AngI) by cleavage of the renin substrate called angiotensinogen. Plasma renin is therefore the initiator of the renin-angiotensin-aldosterone system, which has an important role in the homeostasis of water and electrolyte balance and in the regulation of arterial pressure.
In most studies, circulating renin has been estimated by assays of plasma renin activity (PRA). PRA is measured by generating AngI from endogenous angiotensinogen, followed by measurement by RIA of the generated AngI. Although PRA measurement is convenient for estimating the biological activity of the renin system, it may not necessarily reflect the real concentration of active renin. The concentration of substrate rarely affects the PRA result, but exceptions do occur (3). More importantly, PRA depends not only on renin, but also on factors that influence the renin–renin substrate interaction.
An additional difficulty occurs in measuring low concentrations of renin. In the PRA method, prolonged incubation is needed to generate measurable AngI. This is specifically important when measuring PRA in black people because their values are often below the limit of detection of the routine PRA method. This complicates and prolongs the method and makes it impractical for large throughput for population-based studies.
Immunoassays are available to quantify renin directly with use of monoclonal antibodies. In addition, interlaboratory CVs are lower with immunoassays than with PRA assays (4).
A new method for measuring direct renin (DR) by an automated immunochemiluminometric assay may provide an alternative, but few studies have made a clear-cut comparison of the two methods, specifically for PRA <0.65 ng · mL–1 · h–1. The purpose of the current study was to determine the relationship between DR and PRA in hypertension, specifically in patients with very low PRA values, with the aim of finding assays for rapid screening.
Plasma was obtained from 111 individuals of mixed ethnic origin of whom 34 were on treatment for hypertension and 77 were not. At least three internal quality-control samples were analyzed in duplicate within each assay run. Blood samples were collected in potassium EDTA tubes and centrifuged immediately for 10 min at 1000g; the plasma was then removed and stored at –20 °C. Sample preparation before the assay run involved rapid thawing of the samples in a 26 °C water bath to reach room temperature. Before analysis for DR, the thawed samples were centrifuged briefly at 1000g for 60 s.
The Diasorin RENCTK (AngI) RIA was used to measure PRA in EDTA plasma, expressed in units of ng · mL–1 · h–1. During the incubation step, 500 µL of plasma sample was added in duplicate to tubes containing 500 µL of citrate buffer (100 mmol/L, pH 6.0) and 50 µL of a saturated solution of the enzymatic inhibitor phenylmethylsulfonyl fluoride. The sample contents of each of the duplicate samples were mixed. One sample set was immediately frozen at –20 °C (sample blank), and the second sample set was incubated for 3 h at 37 °C and subsequently frozen at –20 °C. During the RIA step, 50 µL of each of the thawed samples was placed into antibody-coated tubes, and 500 µL of 125I-labeled AngI was added. The tube contents were mixed and incubated at room temperature for 18 h before being counted for radioactivity in a gamma counter for 60 s. An eight-point calibration curve was constructed from the reconstituted AngI calibrators provided with the assay reagents (0–50 µg/L), which were treated similarly to the incubated samples. Samples that gave low PRA values, between 0.10 and 0.65 ng · mL–1 · h–1, were remeasured after a longer (6 h) incubation for the enzymatic reaction.
DR was measured in 200 µL of EDTA plasma by the Nichols Advantage® fully automated immunochemiluminometric assay (Nichols Institute Diagnostics); the results were expressed in units of mU/L, with 1 U generally considered to equal 0.6 µg of renin.
The DR assay uses two specific monoclonal antibodies. The first monoclonal antibody is biotin-labeled and is used to capture renin by recognizing both active renin and prorenin. The second monoclonal antibody is labeled with acridinium ester, and this labeled antibody detects active renin and activated prorenin. The acridinium ester emits light on treatment with hydrogen peroxide and an alkaline solution. Into each well of the cuvette strip, we added 200 µL of sample, 30 µL of biotinylated antibody, 30 µL of acridinium ester-labeled antibody, and 40 µL of assay buffer (containing normal saline and sheep serum with 1 g/L of sodium azide and 0.5 g/L ProClin-300® as preservative). The cuvette strip was then incubated for 20 min at 37 °C, after which streptavidin-coated magnetic particles and buffer (phosphate-buffered saline containing the same preservatives as the buffer mentioned above) were added and the reaction mixture was incubated for an additional 10 min. The magnetic particles are superparamagnetic polystyrene beads with streptavidin covalently attached to the hydrophilic bead surface.
The captured complex bound to the magnetic particles was then washed by the instrument to remove unbound plasma components and acridinium-ester-labeled antibody. The chemiluminescence reaction was initiated by the addition of alkaline peroxide. The amount of bound labeled antibody, as determined by relative light units, was directly proportional to the concentration of active renin in the sample. The results were interpolated by use of a manufacturer-generated 10-point master curve, which was instrument-specifically adjusted by running 2-point calibrators. The manufacturer estimates that the dynamic range for this assay is up to 500 mU/L and the limit of detection is 0.8 mU/L.
The imprecisions of the PRA and DR assays were determined by measurement of 10 samples with PRA between 0.10 and 1.09 ng · mL–1 · h–1. The mean (SD) interassay CV for the PRA assay was 6.2 (3.0)%. The mean (SD) intra- and interassay CVs for the DR assay were 6.8 (13)% and 6.6 (5.6)%, respectively, at 1.6–24 mU/L. Intraassay CVs were 7.2% for a mean DR value of 7.8 mU/L and 4.0% for a mean DR value of 16.3 mU/L, similar to the manufacturer’s stated interassay CVs of 10.0% at a mean DR value of 7.6 mU/L and 6.2% at a mean DR value of 17.9 mU/L.
For 110 samples, the mean (SD) renin concentrations measured by these two methods were 1.04 (2.22) ng · mL–1 · h–1 and 26.5 (53.8) mU/L for the PRA and DR assays, respectively.
We obtained an overall correlation coefficient of 0.98 (n = 110) for all results measured over the wide range of PRA samples (Fig. 1
, A and B). For the linear regression line in Fig. 1A
, the SD of the residuals was 11.8 mU/L. A single outlier (out by more than 5 SD of the residuals) was excluded from all calculations. The clustering of points close to the linear regression line for PRA values <2.0 ng · mL–1 · h–1 (n = 99) is shown in Fig. 1B
.
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For samples with low PRA <0.65 ng · mL–1 · h–1 (which were incubated for 6 h rather than 3 h), the correlation coefficient (r) was 0.77 (n = 25) for PRA and corresponding DR values (Fig. 1C
). PRA results from the 6 h-incubation assay correlated strongly (r = 0.98) with corresponding 3-h incubation results.
The DR assay can measure low concentrations of renin in samples with PRA <0.65 ng · mL–1 · h–1 with a good correlation and low CV. This suggests that the DR method is an adequate method for measuring samples with low renin concentrations and is possibly a suitable alternative for measuring renin in black individuals. The advantage of higher throughput with the DR assay could facilitate the measurement of renin in population-based studies and aid in the screening for secondary forms of hypertension, including primary aldosteronism and Liddle syndrome.
Acknowledgments
We wish to thank Nichols Institute Diagnostics for providing us with the reagents and the Department of Chemical Pathology (SGHMS) for help within their laboratory and use of their facilities.
References
The following articles in journals at HighWire Press have cited this article:
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  E. Singer, S. Strohm, U. Gobel, M. Bieringer, D. Schmidt, W. Schneider, R. Kettritz, and F. C. Luft Cushing's Disease, Hypertension, and Other Sequels Hypertension, December 1, 2008; 52(6): 1001 - 1005. [Full Text] [PDF]
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I. Quack, O. Vonend, L. Sellin, J. Stegbauer, G. Dekomien, and L. C. Rump A Tale of Two Patients With Mendelian Hypertension Hypertension, March 1, 2008; 51(3): 609 - 614. [Full Text] [PDF]
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M. J Brown Direct renin inhibition -- a new way of targeting the renin system Journal of Renin-Angiotensin-Aldosterone System, June 1, 2006; 7(2_suppl): S7 - S11. [Abstract] [PDF]
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