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Endocrinology and Metabolism |
1 Clinical Chemistry and Pathobiochemistry and 2
Endocrinology, Diabetes, and Nutritional Medicine, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
3 Nichols Institute Diagnostics, Bad Vilbel, Germany.
4 Department of Internal Medicine II, University of Freiburg, Freiburg, Germany.
5 Department of Medicine, University Medical Center Nijmegen, Nijmegen, The Netherlands.
6 Department of Pharmacology, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany.
aAddress correspondence to this author at: Institut für Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Fax 49-30-8445-4152; e-mail frank.perschel{at}charite.de.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We studied 76 healthy normotensive volunteers and 28 patients with confirmed PHA. PAC and PRC were measured immunochemically in EDTA plasma on the Nichols Advantage® chemiluminescence analyzer, and PRA was determined by an activity assay.
Results: In volunteers, PAC varied from 33.3 to 1930 pmol/L, PRA from 1.13 to 19.7 ng · mL–1 · h–1 (0.215 ng · mL–1 · h–1 = 1 pmol · L–1 · s–1), and PRC from 5.70 to 116 mU/L. PAC/PRA ratios ranged from 4.35 to 494 (pmol/L)/(ng · mL–1 · h–1) and PAC/PRC ratios from 0.69 to 71.0 pmol/mU. In PHA patients, PAC ranged from 158 to 5012 pmol/L, PRA from 0.40 to 1.70 ng · mL–1 · h–1, and PRC from 0.80 to 11.7 mU/L. PAC/PRA ratios were between 298 and 6756 (pmol/L)/(ng · mL–1 · h–1) and PAC/PRC ratios between 105 and 2328 pmol/mU. Whereas PAC or PRC showed broad overlap between PHA patients and volunteers, the PAC/PRC ratio indicated distinct discrimination of these two groups at a cutoff of 71 pmol/mU.
Conclusion: The PAC/PRC ratio offers several practical advantages compared with the PAC/PRA screening method. The present study offers preliminary evidence that it may be a useful screening test for PHA. Further studies are required to validate these results, especially in hypertensive cohorts.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most common screening test for PHA is the ratio of plasma aldosterone concentration to plasma renin activity (PAC/PRA)(7)(9). Because the measurement of PRA requires special preanalytical prerequisites, is time-consuming, and shows poor interlaboratory reproducibility(10)(11)(12), this strategy is not well recommended for screening of hypertensive patients in primary care centers. The lower limit of detection varies among the different PRA assays; therefore, the effect on the PAC/PRA ratio and the resulting cutoff values for PHA can be dramatic(12). The validity and usefulness of the PAC/PRA ratio for screening for PHA has therefore been challenged(13)(14)(15).
Recently developed immunoassays for measurement of circulating renin in plasma (PRC) may overcome this limitation. However, studies evaluating these assays with regard to the diagnosis of PHA are rare(16)(17). An additional problem is that the proposed cutoff values published to date are derived from measurements of aldosterone in either plasma(2)(3)(6)(11)(13)(15)(17) or serum(1)(4)(16) and are based on different assays. Because both PRA and PRC are measured in plasma and because a simple screening test should be done from only a single specimen, aldosterone should also be measured in plasma.
We therefore compared the "new" ratio between plasma aldosterone concentration and plasma renin concentration (PAC/PRC) with the established PAC/PRA screening test in patients with PHA and in healthy volunteers to evaluate its clinical and diagnostic significance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PRC was measured with the Nichols Advantage Direct ReninTM assay. This two-site immunometric assay uses an acridinium-ester-labeled monoclonal antibody, a second biotinylated monoclonal antibody, and streptavidin-coated magnetic particles. To avoid prorenin activation during the assay, the incubation time is limited to 30 min at 37 °C. This assay is calibrated to the WHO reference material (National Institute for Biological Standards and Control code 68/356).
In an evaluation study(18) this PRC assay showed the following performance:
Precision.
The intraassay variation for three samples with various concentrations of renin was 1.7–5.3%. Interassay variation in seven samples with renin concentrations from 10 to 466.5 mU/L was between 2.7–8.2%.
Accuracy.
Dilution of four plasma samples with the sample diluent gave parallel lines whose slopes did not differ. Renin measurements in two series of mixtures prepared from two plasma samples with low and high renin concentrations, respectively, yielded renin values between 86% and 111%.
Limits of detection and functional sensitivity.
The limits of detection, defined as the means plus 3 SD in two series of 20 runs of the sample diluent, were 0.013 and 0.094 mU/L, respectively. The functional sensitivity, defined as the sample renin concentration at which the CV for four series of five assay runs is <20%, was 2.65 mU/L; 1 mU/L is equivalent to 0.6 ng/L(18)(19).
The aldosterone assay on Nichols Advantage is a competitive one-site immunometric assay that uses a biotinylated monoclonal antibody bound to streptavidin-coated magnetic particles. Acridinium-ester-labeled aldosterone competes with sample aldosterone for the limited amount of biotinylated antibody. The cross-reactivity to any steroid hormones is negligible. According to the manufacturer’s package insert, the assay has an analytical sensitivity of 33.3 pmol/L, and the dynamic range is 0–3330 pmol/L. The within-run imprecision (CV) at 119, 228, 547, 835, and 1990 pmol/L is 14.0%, 5.4%, 4.1%, 4.4%, and 2.9%, respectively. The total CV at 119, 228, 547, 835, and 1990 pmol/L is 18.6%, 8.5%, 5.2%, 6.3%, and 4.9%, respectively. Addition and dilution study results are between 88% and 110%. Parallelism results are between 91% and 116%. A method comparison with a commercially available RIA gave a correlation coefficient of 0.96 and a slope of 1.04; 1 pmol/L is equivalent to 0.36 ng/L.
PRA was measured as described previously(10)(11). The intraassay variation of this assay was 3.9% at 11.7 ng · mL–1 · h–1; the interassay variation was 3.6% at 11.0 ng · mL–1 · h–1 and 6.2% at 3.29 ng · mL–1 · h–1, respectively. The detection limit was 0.40 ng · mL–1 · h–1 (0.215 ng · mL–1 · h–1 = 1 pmol · L–1 · s–1).
All laboratory testing was done by qualified staff of the Charité. The study was not performed as a blind study. Data analysis and estimation of cutoff values by ROC curve analysis were done with the Analyze-It software package (Analyze-It Software Ltd.).
volunteers and patients
We tested 76 healthy, normotensive volunteers [employees and students of the Charité; 23 males (age range, 22–75 years) and 53 females (age range, 16–69 years)], who were not taking any relevant medications. Smoking and use of oral contraceptives were not exclusion criteria. Blood samples were drawn in an upright sitting posture.
The patients studied included 28 patients with confirmed PHA [16 males and 12 females; mean (SD) age, 51 (13.1) years; age range, 26–72 years]: 9 patients had aldosterone-producing adenomas (APAs), 18 had idiopathic hyperaldosteronism (IHA), and 1 patient had glucocorticoid-suppressible hyperaldosteronism. The characteristics of these PHA patients are summarized in Table 1
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The screening of our patients was done according to a generally accepted protocol(7)(8)(9), in which plasma aldosterone was measured with a commercially available RIA (not with the new immunometric assay) and had to be >416 pmol/L (150 ng/L). The ratio between PAC and PRA had to be >555 (pmol/L)/(ng · mL–1 · h–1) [>(200 ng/L)/(ng · mL–1 · h–1)].
Diagnosis was confirmed by successful surgery (all APAs), pathologic saline infusion test(7)(8)(9), and increased 24-h urinary aldosterone excretion on a high- sodium diet(12) (for details see Table 1
). Seven of the 28 patients were normokalemic [mean (SD) serum potassium, 3.4 (0.5) mmol/L; range, 2.0–4.3 mmol/L]. Systolic blood pressure was between 140 and 220 mmHg [mean (SD), 179 (21) mmHg]; diastolic blood pressure was between 80 and 140 mmHg [mean (SD), 104 (12.5) mmHg].
The described characterization of the PHA patients was done routinely in the outpatient clinics of the departments of endocrinology at the Universities of Freiburg and Berlin.
For this evaluation study, separate blood samples were drawn from all PHA patients in an upright sitting position at 0800. The results presented in Tables 2–4
and Fig. 1
are from this standardized blood sampling. Although several authors have recommended PAC/PRA screening without discontinuing hypertensive medications(20)(21)(22), our study protocol for the evaluation of PAC/PRC was the following: Most of the patients were on antihypertensive therapy, but ß-blockers, angiotensin-converting enzyme inhibitors, angiotensin antagonists, and diuretics were stopped for at least 3 days and spironolactone for at least 4 weeks before blood sampling.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The linearity on dilution was evaluated in two samples with concentrations of 525 and 1141 pmol/L. The measured concentrations were 76–100% of the expected. Parallelism in two samples of different concentrations was 89.5–118.3%.
Calibration stability was evaluated with eight different calibrations. The CV was 3.0% for calibrator A (mean of 60 127 RLU) and 3.2% for calibrator B (mean of 23 062 RLU). The analytical sensitivity was 25 pmol/L.
clinical results
All methods tested (Fig. 1
and Table 2
) differed significantly between PHA patients and healthy volunteers. Whereas single measurements of PAC, PRA, or PRC concentrations showed broad overlap between both groups, the ratios PAC/PRA and PAC/PRC provided distinct discrimination between these two groups.
PRC and PRA showed good correlation [Pearson regression coefficient (r) = 0.72]. Because the same PAC values served for calculation of the ratios, PAC/PRC and PAC/PRA correlated similarly (r = 0.73). As expected, differences occurred more frequently at the ends of lower renin concentration and activity ranges near the detection limits of both methods.
ROC analysis (Table 3
) showed the superiority of the ratios compared with PAC, PRA, and PRC alone. The PAC/PRC ratio performed at least as well as the PAC/PRA ratio for differentiating PHA patients from healthy volunteers. With respect to the requirements of a screening test, we evaluated the cutoff values at a sensitivity of 100% (Tables 3
and 4
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At present, the most common screening test is the ratio between serum or plasma aldosterone (PAC) and PRA. Because several authors have determined PAC/PRA cutoffs to screen for PHA(1)(2)(3)(4)(5)(6)(7)(8)(9), this procedure is widely accepted as the best validated screening protocol. However, simultaneous measurement of PAC and PRA has some disadvantages: The method for measuring PRA requires cooling of the specimen during transport and storage, is time-consuming, and shows weak interlaboratory reproducibility(10)(11)(12). Because aldosterone is usually measured in serum, determination of the PAC/PRA ratio requires an additional, simultaneously drawn, blood tube. In addition, PAC, PRA, and the PAC/PRA ratio show large intra- and interpatient variations in patients with PHA(15); therefore, several authors have cast doubt on the validity and sensitivity of the PAC/PRA ratio(13)(14)(15).
Alternative screening procedures that overcome these disadvantages seem to be necessary. Measurement of PRC instead of PRA may reduce some preanalytical and analytical problems and, therefore, improve intra- and interlaboratory reproducibility(23)(24). Major advantages of the new PAC and PRC assays evaluated in our study include the ease and performance of the test (no cooling, a single plasma sample, and good interlaboratory reproducibility because the assays are automated) and the rapid availability of results (automated immunochemiluminometric assays), which is a prerequisite for extensive screening of hypertensive patients for PHA.
The performance of the new PAC assay was evaluated with a preliminary reagent lot. However, our results confirmed the assay specifications given by the manufacturer.
Because this is the first study with a fully automated system and only two studies with manual PRC assays for screening for PHA have been published to date(16)(17), the comparison of our data with data from the literature is limited. Trenkel et al.(16) suggested a cutoff value of 50 (aldosterone measured in serum by RIA, expressed in ng/L; PRC measured by IRMA, expressed in ng/L), which corresponds to 83 when using the units in Table 2
(PAC expressed in pmol/L, PRC in mU/L). Ferrari et al.(17) recommended a cutoff of 150 (aldosterone measured in plasma by RIA, expressed in ng/L; PRC measured by IRMA, expressed in ng/L) corresponding to 90 when using the units in Table 2
. Our data analysis (Tables 2
and 3
; Fig. 1
) produced a tentatively proposed cutoff value of 71. Subsequent studies including essential hypertensive cohorts may necessitate readjustment, although there is good concordance with the recommendations made by Trenkel et al.(16) and Ferrari et al.(17).
Although the simultaneous measurement of PAC/PRC in plasma samples from 28 patients with known PHA showed no overlap with healthy volunteers (Fig. 1
), there are certain limitations to our study: The PAC/PRC ratio seems to be superior to the PAC/PRA ratio. Although the PAC/PRA ratio of our PHA patients in the initial outpatient characterization was clearly above the cutoff value of 555 (pmol/L)/(ng · mL–1 · h–1) [200 (ng/L)/(ng · mL–1 · h–1)], the standardized reexamination of these patients by use of a second blood sample showed that some patients would have failed this diagnostic criterion. Our proposed cutoff value (Table 3
), which was chosen to achieve a 100% sensitivity with the new automated assay, was therefore somewhat lower than the values recommended in the literature. In addition, some patients with confirmed PHA had PACs in the standardized reexamination that were <414 pmol/L (150 ng/L) and therefore would also fail the second diagnostic criterion for positive screening of PHA.
Because there is no doubt in the correct characterization of our patients as having PHA, we have two explanations for these findings: (a) a recent study by Tanabe et al.(15) has demonstrated high intraindividual variability of PAC, PRA, and the PAC/PRA ratio in patients with PHA; and (b) we measured plasma aldosterone with the new immunochemiluminometric assay, these data show that each PAC and PRC assay requires separate validation of cutoff values.
Evaluating diagnostic tests in a group of patients already known to have the disease and in a group of healthy volunteers can lead to overestimation of diagnostic accuracy(25). Thus, our data necessarily need to be complemented by further studies in hypertensive cohorts, e.g., in groups of patients with essential hypertension and groups with renovascular disease.
The clinical conditions necessary for testing of the PAC/PRC or PAC/PRA ratios, such as discontinuing drug therapy, recording of dietary sodium intake, or time of day for sampling, are not yet sufficiently standardized, which contributes to the different published cutoff values and complicates the use of the screening test in clinical routine. An adequate standardization that meets general acceptance is required.
In summary, our results suggest that the simultaneous measurement of PAC and PRC with automated immunochemiluminometric assays may be useful in screening for PHA. Nevertheless, subsequent studies are required for further comparison of the PAC/PRC ratio vs the PAC/PRA ratio used for screening at present. These studies will have to deal with the following questions: (a) Because we tested only for the differentiation of patients from normotensive volunteers, has the PAC/PRC ratio similar sensitivities for screening of large hypertensive populations? Prospective studies are needed. (b) Is the newly developed assay for PAC really a method that offers reliable results if performed by different laboratories in clinical routine? (c) How is the PAC/PRC ratio influenced by clinical conditions, especially drug effects? Regardless of the answers to these questions, we expect that the new ratio method will evolve as a rapid and easily performed screening test for PHA.
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Acknowledgments |
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Footnotes |
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, healthy volunteers; •, patients with PHA.