Endocrine Care 663
Authors
J. Ceral1, E. Malirova2, M. Ballon1, M. Solar1
Affiliations
1 st
1 Department of Internal Medicine – Cardioangiology, Charles University, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic 2 Department of Nuclear Medicine, Charles University, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
Key words ▶ primary aldosteronism ● ▶ urinary aldosterone ● ▶ secondary arterial hyperten● sion
Abstract
received 29.11.2013 accepted after second revision 02.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1374638 Published online: May 8, 2014 Horm Metab Res 2014; 46: 663–667 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence M. Solar MD 1st Department of Internal Medicine – Cardioangiology University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Kralove Czech Republic Tel.: + 420/495/833 3249 Fax: + 420/495/832 006 [email protected]
▼
When diagnosing primary aldosteronism, the measurement of urinary aldosterone after oral sodium loading is one of the currently recommended confirmatory tests. The aim of the study was to assess the repeatability and interpretation of urinary aldosterone in patients examined for suspected primary aldosteronism. Sixty-four hypertensive patients with suspected primary aldosteronism were prospectively enrolled and examined according to the study protocol. After antihypertensive medications interfering with renin-angiotensin-aldosterone system were withdrawn for at least 2 weeks, the confirmatory testing was performed: oral sodium loading preceded the collection of 24-h urine sample and subsequent saline infusion test. The identical procedures were repeated after 2 weeks. The concordant results of both saline infusion tests served for confirmation/exclusion of pri-
mary aldosteronism. Forty-nine patients were included in data analysis. Primary aldosteronism was excluded in 16, and confirmed in 33 individuals. The repeatability of urinary aldosterone was evaluated in 44 patients: the difference of urinary aldosterone levels ranged between 1 and 88 % (median 31 %). Ninety-three urine samples from 49 patients were used to validate the interpretation of urinary aldosterone in respect to the diagnosis of primary aldosteronism made by saline infusion testing; 96 % sensitivity was characterized by urinary aldosterone ≥ 19 nmol/day, and 96 % specificity was associated with urinary aldosterone ≥ 92 nmol/day. In 22 (45 %) patients, urinary aldosterone remained in the “gray” zone between 19 and 92 nmol/day in all provided samples. The estimation of urinary aldosterone excretion after oral sodium loading is associated with marked intraindividual variability, and significant number of inconclusive results.
Introduction
Subjects and Methods
Primary aldosteronism (PA) is not an infrequent form of secondary arterial hypertension [1]. The cardiovascular consequences of this disorder go beyond the blood pressure elevation [2]. The identification of PA cases is important as the affected individuals can profit from the specific medical or surgical treatment. The estimation of urinary aldosterone excretion is an easy to perform test that is currently recommended for the confirmation of PA diagnosis [3]. However, the data describing the clinical use of urinary aldosterone are limited, and the published reports show discrepant results [4–8]. These facts inspired us to perform a clinical study to contribute to the clinical evaluation of this diagnostic test.
The study enrolled both the healthy volunteers (Group A) and the patients with suspected PA who were referred for confirmatory testing (Group B). The study was approved by the local Ethics committee and all participants gave written informed consent with the study procedures.
▼
▼
Group A The healthy volunteers served as a control group to assess the physiological aldosterone secretion following oral sodium loading. Repeated blood pressure measurement confirmed normal, that is not elevated, blood pressure. The enrolled individuals had to have no history of any chronic disease potentially influencing the renin-angiotensin-aldosterone system. The only allowed
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
The Role of Urinary Aldosterone for the Diagnosis of Primary Aldosteronism
664 Endocrine Care
Group B Group B prospectively enrolled the patients with suspected PA who were, based on the screening tests, referred for the confirmatory testing. The referral for the confirmatory testing resulted mainly from repeatedly elevated aldosterone-to-renin ratio but the tests were also indicated in selected hypertensive individuals who presented with unexplained hypokalemia or an adrenal adenoma. Two identical confirmatory tests were performed in all group B patients. Both procedures were performed under the identical conditions: antihypertensive drugs interfering with renin-angiotensin-aldosterone system had to be stopped for at least 2 weeks before the first test with the exception of spironolactone that had to be stopped for at least 8 weeks before the testing. When the risks of the uncontrolled blood pressure had been considered high, the patients were given doxazosin and/or verapamil. No changes in the antihypertensive medications were allowed in the period between both tests. The suggested period between the 2 tests was 2 weeks. All confirmatory tests were performed according to the protocol that is routinely used in our centre: oral sodium loading preceded the intravenous administration of normal saline. The patients were advised to enhance their oral salt intake after adjusting their medication before testing. Three days before the saline infusion was administered, oral salt intake was further increased by at least 6 g/day. The day before the saline infusion, 24-h urine sample was collected in order to estimate sodium and aldosterone excretion. At the end of urine collection plasma renin and serum aldosterone were taken when the patients had to be in the upright position for at least 30 min. Saline infusion was administered in the morning hours. After 2 h of bed rest, 2 liters of normal saline were infused during the period of 4 h. The patients remained in supine position till the end of infusion when the blood samples were taken for the estimations of plasma renin and serum aldosterone. The test was appropriate only if the adequate suppression of plasma renin ( < 7 ng/l) was achieved. Serum aldosterone was considered nonsuppressible when > 100 pmol/l at the end of saline infusion. The respective cutoff values are based on our previous observation in healthy volunteers [10]. The clinical decision-making was based solely upon the results of serum aldosterone and plasma renin at the end of saline infusion. The results of urinary aldosterone measurements played no role in this process.
Adrenal venous sampling was offered to all patients who had a nonsuppressible serum aldosterone at the end of any suppression test. The protocol of adrenal venous sampling used in our center was described in detail previously [11].
Laboratory measurement Urinary aldosterone concentrations were measured using commercially available kits (Coat-A-Count® Aldosterone, Siemens/ DPC, Los Angeles, CA, USA). The samples were processed as suggested by the manufacturer (www.siemens.com/diagnostics). In brief: after acid hydrolysis of aldosterone-glucuronide, the aldosterone was extracted using ethyl acetate. The hormonal concentrations were subsequently estimated by radioimmunoassay. The range of the assay was 3–300 nmol/l using the recommended dilution of the samples. The observed interassay coefficients of variation for urinary aldosterone concentrations of 7.8 and 16.6 nmol/l were 11.4 % and 9.7 %. Plasma renin measurements were performed using the Renin III generation radioimmunometric assay (CIS Biointernational, Gif sur Yvette Cedex, France). The normal range of the assay was 0–60 ng/l. When the estimated renin concentration was below the assay’s lower limit of detection, the renin concentration was considered the lowest detectable value; that is 2.7 ng/l. Serum aldosterone concentrations were measured using a commercially available radioimmunoassay (Coat-A-Count® aldosterone Siemens/DPC, Los Angeles, CA, USA). The normal range of the assay was 0–450 pmol/l. When the estimated aldosterone concentrations were below the lower detection limit, the samples were considered to have the lowest detectable serum aldosterone concentration; that is 30 pmol/l. The details of the estimations of plasma renin and serum aldosterone were described in more detail previously [12].
Data analysis Group A The upper reference value was calculated according to the International Federation of Clinical Chemistry and Laboratory Medicine/ The Clinical and Laboratory Standards Institute recommendations using Robust method for smaller sample sizes [13].
Group B In order to eliminate any confounding factors that might influence the interpretation of the acquired data, the final analysis included only the patients who: a) did not require the adjustment of antihypertensive medication before the second confirmatory test; b) achieved adequate renin suppression at the end of both saline infusion tests ( < 7 ng/l); c) exhibited concordant results of both saline infusion tests; d) had urinary sodium > 200 mmol/day [8] in at least one provided urine sample. The data analysis was aimed at: a) the assessment of the repeatability of urinary aldosterone secretion. Bland-Altman plot was used to describe the intra-individual differences that were expressed as the percentage of the absolute difference related to the mean of 2 compared values, b) the interpretation of urinary aldosterone excretion in respect to the results of saline infusion testing. All the statistical calculations were done using the MedCalc® software, version 11.6 (Mariakerke, Belgium).
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
chronic medications were antihistamines and hormonal contraception. We were aware of possible interactions between exogenous estrogens and progestins and the renin-angiotensinaldosterone system [9]. However, our primary aim was to obtain data applicable to real life clinical conditions. In our center, we prefer not to withdraw the contraceptives in females undergoing examination for suspected PA, as an unplanned pregnancy in PA could present a clinically difficult situation. Each individual provided a sample of urine that was collected during the period of 24 h in order to estimate sodium and aldosterone excretion. Two days before and during the day of urine collection, the participants were recommended to increase oral salt intake. In addition (to high salt diet), they were given 6 g daily of sodium chloride in capsules. The analysis included only those individuals who had urinary sodium secretion ≥ 200 mmol/24 h.
Results
▼
Group A Forty healthy volunteers that were recruited mainly from the medical students of our university were enrolled in Group A. All of them provided urine samples as suggested by the protocol. Ten individuals were excluded from the study because of low urinary sodium content ( < 200 mmol/day). The characteristics and the principal results of Group A individuals are summarized ▶ Table 1. Using the above mentioned statistical method, the in ● upper reference limit for urinary aldosterone secretion, reflecting 97.5 percentile of acquired values, was 36 nmol/day.
Group B Group B prospectively enrolled 64 consecutive patients; all of them underwent 2 confirmatory tests as suggested by the protocol. Fifteen individuals were excluded from the final analysis. In 2, the plasma renin remained unsuppressed at the end of either test. In 4, the antihypertensive medication was modified after the first test. In 8, the results of saline infusion testing were disTable 1 The principal characteristics of healthy volunteers (Group A). Number of subjects (women) Age (years) Body mass index (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Diuresis (ml/24 h) Urinary sodium (mmol/24 h) Urinary aldosterone (nmol/24 h)
30 (8) 23 (20–29) 23.7 (18.8–26.4) 120 (100–135) 80 (58–86) 2 100 (1 100–4 100) 295 (200–500) 9.3 (1.9–63.8)
The data are expressed as numbers or medians (ranges)
cordant, and in one patient, the daily urinary sodium excretion was < 200 mmol in both collected urine samples. The data of the remaining 49 patients were subjected to the analysis. The principal results of this group are shown in ▶ Table 2. Based on the results of saline infusion testing, the ● primary aldosteronism was suppressible in 16 (33 %) and nonsuppressible in 33 (67 %). Thirty-two patients underwent successful adrenal venous sampling and the lateralization of aldosterone secretion was diagnosed in 23. Twenty patients underwent adrenalectomy, adrenal adenoma was diagnosed in 17, and nodular hyperplasia in 2, and normal histological finding was reported in 1. Improvement of blood pressure control was noted in all but one, that is in 95 %, of operated patients. The patient who did not improve after adrenalectomy had borderline results of adrenal venous sampling and an adenoma was found at histological examination. The repeatability of the urinary aldosterone excretion was evaluated in 44 patients who fulfilled the above mentioned eligibility criteria. The observed intraindividual differences are ▶ Fig. 1. The significant described by the Bland-Altman plot in ● variations in urinary aldosterone secretion were observed not only in patients with excluded PA, but also in individuals with proven lateralization of aldosterone secretion who eventually ▶ Table 3). The profited from unilateral adrenalectomy as well (● significant differences persisted when the urinary aldosterone was corrected by the urinary sodium and/or creatinine concentrations. No difference between genders was identified. Marked differences in urinary aldosterone were also noted in individuals who had minimal differences in urinary volumes, sodium and creatinine excretions. Similarly, significant variations in urinary aldosterone were noted in patients who had very high urinary sodium excretion ( > 400 mmol/day) in both collected samples.
Table 2 Data of group B patients who fulfilled the inclusion criteria and were enrolled in the analysis. Excluded PA
Confirmed PA
Confirmed PA – successful
16 (11) 47 (21–62)
33 (8) 50 (32–68)
19 (4) 46 (32–68)
144 (126–160) 86 (68–116) 2 (0–4) 93 (61–118) 42 (19–71)
145 (100–196) 90 (64–104) 4 (0–7) 96 (67–151) 74 (24–205)
151 (100–170) 89 (64–104) 4 (0–5) 96 (68–151) 84 (24–205)
2 9 (6.9 mg) 13 (295 mg) 4.0 (3.5–4.6) 2 350 (750–5 300) 356 (278–738) 24.7 (4.9–101.9)
5 21 (7.3 mg) 23 (264 mg) 3.4 (2.7–4.1) 2 734 (1 500–5 000) 358 (202–688) 81.9 (14.8–430)
3 15 (7.4 mg) 15 (235 mg) 3.4 (2.7–4.1) 2 800 (1 700–5 000) 382 (202–688) 84.1 (25.2–430)
285 (30–1 070) 4.3 (2.7–10.8)
675 (160–2 080) 2.7 (2.7–35.0)
480 (180–980) 2.7 (2.7–5.9)
35 (30–100) 2.7 (2.7–6.9)
280 (110–2 360) 2.7 (2.7–6.1)
385 (110–910) 2.7 (2.7–4.7)
surgical treatment Number of patients (women) Age (years) Screening Systolic blood pressure (mm Hg)1 Diastolic blood pressure (mm Hg)1 Number of antihypertensive drugs1 Creatinine clearance (ml/min) Aldosterone-to-renin ratio1,2 Confirmatory testing No. of patients on no antihypertensive therapy No. of patients on doxazosin (mean dose) No. of patients on verapamil (mean dose) Serum potassium (mmol/l) 3 Amount of urine (ml/24 h) 4 Urinary sodium (mmol/24 h) 4 Urinary aldosterone (nmol/24 h) 4 End of urine collection Serum aldosterone (pmol/l) Plasma renin (ng/l) End of saline infusion Serum aldosterone (pmol/l) Plasma renin (ng/l)
Unless otherwise stated, the data are expressed as medians (range) 1
Denotes the data obtained during the screening for primary aldosteronism
2
To calculate the aldosterone-to-renin ratio, both aldosterone and renin were expressed in ng/l
3
Serum potassium was taken at the end of urine collection, before saline infusion was given
4
Denotes only urinary samples enrolled in the analysis, that is containing ≥ 200 mmol/day of sodium
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
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Endocrine Care 665
Urinary aldosterone was analyzed in 93 samples of 49 patients. The receiver operator analysis curve (ROC) of urinary aldosterone in respect to PA diagnosis made by saline infusion testing is ▶ Fig. 2. depicted in ● Upper reference limit defined by the data of healthy volunteers (36 nmol/day) was characterized by 86 % sensitivity and 50 % specificity for the PA diagnosis; the concordant assessment was observed in 70 samples of 40 patients. Urinary aldosterone ≥ 19 nmol/day was associated with 96 % sensitivity and urinary aldosterone ≥ 92 nmol/day was characterized by a 96 % specificity in respect to the PA diagnosed by saline infusion testing. In 22 (45 %) patients, urinary aldosterone remained in the “gray” zone between 19 and 92 nmol/l. Among them, there were 9 cases that had positive AVS results and eventually profited from unilateral adrenalectomy. At the end of oral sodium loading and urinary collection, the plasma renin in the upright position was generally ≤ 11 ng/l, which reflects 97.5 percentile of the observed values.
Discussion
▼
Several approaches are currently recommended for the confirmatory testing in patients with suspected primary aldosteronism, none of them is generally accepted as superior to others [14]. While the fludrocortisone testing [15] is probably highly effective in suppressing an autonomous aldosterone secretion it may be considered difficult or inconvenient for many patients. The short-lasting saline infusion may not be sufficient for adequate suppression of nonautonomous aldosterone secretion. The limited repeatability of this test was observed even when the saline infusion was preceded by oral sodium loading [12].
Fig. 1 The Bland-Altman plot describes the differences in urinary aldosterone between 2 collected samples (UA1 and UA2).
The aim of our study was to elucidate the value of urinary aldosterone secretion in this diagnostic process. The study protocol addressed both the evaluation of repeatability and the interpretation of the results. Concerning the repeatability testing, our results show that there is a high variation in urinary aldosterone secretion in a large number of examined patients. We cannot offer any definite explanation for this finding. The observed intraindividual differences are well beyond the imprecision of laboratory measurements. Although the imperfections of urinary collection might be responsible for some of the observed discrepancies, urinary aldosterone markedly varied also in patients who had little differences in the urine volumes and creatinine excretion. Concerning the interpretation of the results, we were not able to identify a single cutoff value applicable both for excluding and confirming PA. The ROC analysis of our data showed a large “gray zone” between acceptable sensitivity and specificity. The high sensitivity value (19 nmol/day) approximately corresponds to the respective cutoff mentioned in the current guidelines (28 nmol/day) [3]. Similarly, the high specificity cut off (94 nmol/ day) is not much different from the reported urinary aldosterone in patients with aldosterone producing adenomas ( > 83 nmol/ day) [16]. In our study, the assessment of urinary aldosterone would confirm or exclude PA in 27 (55 %) examined patients. In the remain-
Fig. 2 The graph describes the receiver operator analysis of the urinary aldosterone in respect to the PA diagnosis based on 2 concordant results of saline infusion tests. The shape of the curve does not enable to identify a single cutoff characterized by acceptable sensitivity and specificity.
Excluded PA
Confirmed PA
Confirmed PA – success-
14 33 % (1–87 %) 25 % (8–66 %)
30 30 % (1–88 %) 17 % (0–55 %)
16 28 % (1–81 %) 8 % (0–55 %)
ful surgical treatment Number of patients Intra-individual variations in urinary aldosterone Intra-individual variations in urinary sodium
The percentage values are calculated as follows: 100 × (the absolute difference divided by the mean of 2 obtained values). The data are expressed as medians (ranges)
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
Table 3 The intra-individual variation in urinary aldosterone and sodium.
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666 Endocrine Care
Endocrine Care 667
Study Limitations
▼
First, our data are closely related to the above-mentioned diagnostic protocol and the laboratory assays used for the estimations of the hormonal concentrations [17]. The accuracy of the urinary collection might be questioned as the study individuals were examined on an outpatient basis. The project was conceived as reflecting a real-life practice. Even if the improper urine collection was partly responsible for observed differences, this fact has to be taken into consideration. Second, one quarter of enrolled patients was eventually excluded from the study based on the stringent exclusion criteria, which may limit the general applicability of the study results. The primary aim of our study was to eliminate any potentially confounding factors in order to increase the quality of acquired data. Third, the lower number of individuals with excluded PA might have led to less accurate estimation of test specificity. On the other hand, a high number of patients with confirmed PA provide more precise information on test sensitivity. Fourth, the number of healthy volunteers was not high. However, the calculated upper reference value is not markedly different from the published data [8, 18]. Moreover, we do not assume that the augmentation of the number of healthy individuals would significantly change the study results.
Conclusions
▼
The confirmatory testing for suspected primary aldosteronism performed by the estimation of urinary aldosterone after oral sodium loading exhibits a marked intraindividual variability and is associated with inconclusive results in nearly half of examined patients. Based on our results, we cannot recommend urinary aldosterone as a confirmatory test in patients with suspected primary aldosteronism.
Acknowledgements
▼
This work was supported by the research project PRVOUK P037/03.
Conflict of Interest
▼
References 1 Hannemann A, Bidlingmaier M, Friedrich N, Manolopoulou J, Spyroglou A, Volzke H, Beuschlein F, Seissler J, Rettig R, Felix SB, Biffar R, Doring A, Meisinger C, Peters A, Wichmann HE, Nauck M, Wallaschofski H, Reincke M. Screening for primary aldosteronism in hypertensive subjects: results from two German epidemiological studies. Eur J Endocrinol 2012; 167: 7–15 2 Milliez P, Girerd X, Plouin PF, Blacher J, Safar ME, Mourad JJ. Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism. J Am Coll Cardiol 2005; 45: 1243–1248 3 Funder JW, Carey RM, Fardella C, Gomez-Sanchez CE, Mantero F, Stowasser M, Young WF Jr, Montori VM. Case detection, diagnosis, and treatment of patients with primary aldosteronism: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2008; 93: 3266–3281 4 Abdelhamid S, Blomer R, Hommel G, Haack D, Lewicka S, Fiegel P, Krumme B. Urinary tetrahydroaldosterone as a screening method for primary aldosteronism: a comparative study. Am J Hypertens 2003; 16: 522–530 5 Collins RD, Weinberger MH, Dowdy AJ, Nokes GW, Gonzales CM, Luetscher JA. Abnormally sustained aldosterone secretion during salt loading in patients with various forms of benign hypertension; relation to plasma renin activity. J Clin Invest 1970; 49: 1415–1426 6 Bravo EL, Tarazi RC, Dustan HP, Fouad FM, Textor SC, Gifford RW, Vidt DG. The changing clinical spectrum of primary aldosteronism. Am J Med 1983; 74: 641–651 7 Stowasser M, Taylor PJ, Pimenta E, Ahmed AH, Gordon RD. Laboratory investigation of primary aldosteronism. Clin Biochem Rev 2010; 31: 39–56 8 Young WF Jr, Hogan MJ, Klee GG, Grant CS, van Heerden JA. Primary aldosteronism: diagnosis and treatment. Mayo Clin Proc 1990; 65: 96–110 9 Ahmed AH, Gordon RD, Taylor PJ, Ward G, Pimenta E, Stowasser M. Effect of Contraceptives on Aldosterone/Renin Ratio May Vary According to the Components of Contraceptive, Renin Assay Method, and Possibly Route of Administration. J Clin Endocrinol Metab 2011; 96: 1797–1804 10 Ceral J, Malirova E, Kopecka P, Pelouch R, Solar M. The Effect of Oral Sodium Loading and Saline Infusion on Direct Active Renin in Healthy Volunteers. Acta Endocrinologica (Buc) 2010; 7: 33–38 11 Ceral J, Solar M, Krajina A, Ballon M, Suba P, Cap J. Adrenal venous sampling in primary aldosteronism: a low dilution of adrenal venous blood is crucial for a correct interpretation of the results. Eur J Endocrinol 2010; 162: 101–107 12 Solar M, Malirova E, Ballon M, Pelouch R, Ceral J. Confirmatory testing in primary aldosteronism: extensive medication switching is not needed in all patients. Eur J Endocrinol 2012; 166: 679–686 13 Henny J. The IFCC recommendations for determining reference intervals: strengths and limitations. J Lab Med 2009; 33: 45–51 14 Mulatero P, Stowasser M, Loh KC, Fardella CE, Gordon RD, Mosso L, Gomez-Sanchez CE, Veglio F, Young WF. Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab 2004; 89: 1045–1050 15 Stowasser M, Gordon RD. Primary aldosteronism – careful investigation is essential and rewarding. Mol Cell Endocrinol 2004; 217: 33–39 16 Young WF Jr. Minireview: primary aldosteronism – changing concepts in diagnosis and treatment. Endocrinology 2003; 144: 2208–2213 17 Schirpenbach C, Seiler L, Maser-Gluth C, Beuschlein F, Reincke M, Bidlingmaier M. Automated chemiluminescence-immunoassay for aldosterone during dynamic testing: comparison to radioimmunoassays with and without extraction steps. Clin Chem 2006; 52: 1749–1755 18 Baas SJ, Endert E, Fliers E, Prummel MF, Wiersinga WM. Establishment of reference values for endocrine tests. III: Primary aldosteronism. Neth J Med 2003; 61: 37–43
The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
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ing individuals, the urinary aldosterone remained in the gray zone of inconclusive results. Among them there was a significant number of those who eventually profited from adrenalectomy (9 out of 22).
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Authors
J. Ceral1, E. Malirova2, M. Ballon1, M. Solar1
Affiliations
1 st
1 Department of Internal Medicine – Cardioangiology, Charles University, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic 2 Department of Nuclear Medicine, Charles University, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
Key words ▶ primary aldosteronism ● ▶ urinary aldosterone ● ▶ secondary arterial hyperten● sion
Abstract
received 29.11.2013 accepted after second revision 02.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1374638 Published online: May 8, 2014 Horm Metab Res 2014; 46: 663–667 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence M. Solar MD 1st Department of Internal Medicine – Cardioangiology University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Kralove Czech Republic Tel.: + 420/495/833 3249 Fax: + 420/495/832 006 [email protected]
▼
When diagnosing primary aldosteronism, the measurement of urinary aldosterone after oral sodium loading is one of the currently recommended confirmatory tests. The aim of the study was to assess the repeatability and interpretation of urinary aldosterone in patients examined for suspected primary aldosteronism. Sixty-four hypertensive patients with suspected primary aldosteronism were prospectively enrolled and examined according to the study protocol. After antihypertensive medications interfering with renin-angiotensin-aldosterone system were withdrawn for at least 2 weeks, the confirmatory testing was performed: oral sodium loading preceded the collection of 24-h urine sample and subsequent saline infusion test. The identical procedures were repeated after 2 weeks. The concordant results of both saline infusion tests served for confirmation/exclusion of pri-
mary aldosteronism. Forty-nine patients were included in data analysis. Primary aldosteronism was excluded in 16, and confirmed in 33 individuals. The repeatability of urinary aldosterone was evaluated in 44 patients: the difference of urinary aldosterone levels ranged between 1 and 88 % (median 31 %). Ninety-three urine samples from 49 patients were used to validate the interpretation of urinary aldosterone in respect to the diagnosis of primary aldosteronism made by saline infusion testing; 96 % sensitivity was characterized by urinary aldosterone ≥ 19 nmol/day, and 96 % specificity was associated with urinary aldosterone ≥ 92 nmol/day. In 22 (45 %) patients, urinary aldosterone remained in the “gray” zone between 19 and 92 nmol/day in all provided samples. The estimation of urinary aldosterone excretion after oral sodium loading is associated with marked intraindividual variability, and significant number of inconclusive results.
Introduction
Subjects and Methods
Primary aldosteronism (PA) is not an infrequent form of secondary arterial hypertension [1]. The cardiovascular consequences of this disorder go beyond the blood pressure elevation [2]. The identification of PA cases is important as the affected individuals can profit from the specific medical or surgical treatment. The estimation of urinary aldosterone excretion is an easy to perform test that is currently recommended for the confirmation of PA diagnosis [3]. However, the data describing the clinical use of urinary aldosterone are limited, and the published reports show discrepant results [4–8]. These facts inspired us to perform a clinical study to contribute to the clinical evaluation of this diagnostic test.
The study enrolled both the healthy volunteers (Group A) and the patients with suspected PA who were referred for confirmatory testing (Group B). The study was approved by the local Ethics committee and all participants gave written informed consent with the study procedures.
▼
▼
Group A The healthy volunteers served as a control group to assess the physiological aldosterone secretion following oral sodium loading. Repeated blood pressure measurement confirmed normal, that is not elevated, blood pressure. The enrolled individuals had to have no history of any chronic disease potentially influencing the renin-angiotensin-aldosterone system. The only allowed
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
The Role of Urinary Aldosterone for the Diagnosis of Primary Aldosteronism
664 Endocrine Care
Group B Group B prospectively enrolled the patients with suspected PA who were, based on the screening tests, referred for the confirmatory testing. The referral for the confirmatory testing resulted mainly from repeatedly elevated aldosterone-to-renin ratio but the tests were also indicated in selected hypertensive individuals who presented with unexplained hypokalemia or an adrenal adenoma. Two identical confirmatory tests were performed in all group B patients. Both procedures were performed under the identical conditions: antihypertensive drugs interfering with renin-angiotensin-aldosterone system had to be stopped for at least 2 weeks before the first test with the exception of spironolactone that had to be stopped for at least 8 weeks before the testing. When the risks of the uncontrolled blood pressure had been considered high, the patients were given doxazosin and/or verapamil. No changes in the antihypertensive medications were allowed in the period between both tests. The suggested period between the 2 tests was 2 weeks. All confirmatory tests were performed according to the protocol that is routinely used in our centre: oral sodium loading preceded the intravenous administration of normal saline. The patients were advised to enhance their oral salt intake after adjusting their medication before testing. Three days before the saline infusion was administered, oral salt intake was further increased by at least 6 g/day. The day before the saline infusion, 24-h urine sample was collected in order to estimate sodium and aldosterone excretion. At the end of urine collection plasma renin and serum aldosterone were taken when the patients had to be in the upright position for at least 30 min. Saline infusion was administered in the morning hours. After 2 h of bed rest, 2 liters of normal saline were infused during the period of 4 h. The patients remained in supine position till the end of infusion when the blood samples were taken for the estimations of plasma renin and serum aldosterone. The test was appropriate only if the adequate suppression of plasma renin ( < 7 ng/l) was achieved. Serum aldosterone was considered nonsuppressible when > 100 pmol/l at the end of saline infusion. The respective cutoff values are based on our previous observation in healthy volunteers [10]. The clinical decision-making was based solely upon the results of serum aldosterone and plasma renin at the end of saline infusion. The results of urinary aldosterone measurements played no role in this process.
Adrenal venous sampling was offered to all patients who had a nonsuppressible serum aldosterone at the end of any suppression test. The protocol of adrenal venous sampling used in our center was described in detail previously [11].
Laboratory measurement Urinary aldosterone concentrations were measured using commercially available kits (Coat-A-Count® Aldosterone, Siemens/ DPC, Los Angeles, CA, USA). The samples were processed as suggested by the manufacturer (www.siemens.com/diagnostics). In brief: after acid hydrolysis of aldosterone-glucuronide, the aldosterone was extracted using ethyl acetate. The hormonal concentrations were subsequently estimated by radioimmunoassay. The range of the assay was 3–300 nmol/l using the recommended dilution of the samples. The observed interassay coefficients of variation for urinary aldosterone concentrations of 7.8 and 16.6 nmol/l were 11.4 % and 9.7 %. Plasma renin measurements were performed using the Renin III generation radioimmunometric assay (CIS Biointernational, Gif sur Yvette Cedex, France). The normal range of the assay was 0–60 ng/l. When the estimated renin concentration was below the assay’s lower limit of detection, the renin concentration was considered the lowest detectable value; that is 2.7 ng/l. Serum aldosterone concentrations were measured using a commercially available radioimmunoassay (Coat-A-Count® aldosterone Siemens/DPC, Los Angeles, CA, USA). The normal range of the assay was 0–450 pmol/l. When the estimated aldosterone concentrations were below the lower detection limit, the samples were considered to have the lowest detectable serum aldosterone concentration; that is 30 pmol/l. The details of the estimations of plasma renin and serum aldosterone were described in more detail previously [12].
Data analysis Group A The upper reference value was calculated according to the International Federation of Clinical Chemistry and Laboratory Medicine/ The Clinical and Laboratory Standards Institute recommendations using Robust method for smaller sample sizes [13].
Group B In order to eliminate any confounding factors that might influence the interpretation of the acquired data, the final analysis included only the patients who: a) did not require the adjustment of antihypertensive medication before the second confirmatory test; b) achieved adequate renin suppression at the end of both saline infusion tests ( < 7 ng/l); c) exhibited concordant results of both saline infusion tests; d) had urinary sodium > 200 mmol/day [8] in at least one provided urine sample. The data analysis was aimed at: a) the assessment of the repeatability of urinary aldosterone secretion. Bland-Altman plot was used to describe the intra-individual differences that were expressed as the percentage of the absolute difference related to the mean of 2 compared values, b) the interpretation of urinary aldosterone excretion in respect to the results of saline infusion testing. All the statistical calculations were done using the MedCalc® software, version 11.6 (Mariakerke, Belgium).
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
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chronic medications were antihistamines and hormonal contraception. We were aware of possible interactions between exogenous estrogens and progestins and the renin-angiotensinaldosterone system [9]. However, our primary aim was to obtain data applicable to real life clinical conditions. In our center, we prefer not to withdraw the contraceptives in females undergoing examination for suspected PA, as an unplanned pregnancy in PA could present a clinically difficult situation. Each individual provided a sample of urine that was collected during the period of 24 h in order to estimate sodium and aldosterone excretion. Two days before and during the day of urine collection, the participants were recommended to increase oral salt intake. In addition (to high salt diet), they were given 6 g daily of sodium chloride in capsules. The analysis included only those individuals who had urinary sodium secretion ≥ 200 mmol/24 h.
Results
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Group A Forty healthy volunteers that were recruited mainly from the medical students of our university were enrolled in Group A. All of them provided urine samples as suggested by the protocol. Ten individuals were excluded from the study because of low urinary sodium content ( < 200 mmol/day). The characteristics and the principal results of Group A individuals are summarized ▶ Table 1. Using the above mentioned statistical method, the in ● upper reference limit for urinary aldosterone secretion, reflecting 97.5 percentile of acquired values, was 36 nmol/day.
Group B Group B prospectively enrolled 64 consecutive patients; all of them underwent 2 confirmatory tests as suggested by the protocol. Fifteen individuals were excluded from the final analysis. In 2, the plasma renin remained unsuppressed at the end of either test. In 4, the antihypertensive medication was modified after the first test. In 8, the results of saline infusion testing were disTable 1 The principal characteristics of healthy volunteers (Group A). Number of subjects (women) Age (years) Body mass index (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Diuresis (ml/24 h) Urinary sodium (mmol/24 h) Urinary aldosterone (nmol/24 h)
30 (8) 23 (20–29) 23.7 (18.8–26.4) 120 (100–135) 80 (58–86) 2 100 (1 100–4 100) 295 (200–500) 9.3 (1.9–63.8)
The data are expressed as numbers or medians (ranges)
cordant, and in one patient, the daily urinary sodium excretion was < 200 mmol in both collected urine samples. The data of the remaining 49 patients were subjected to the analysis. The principal results of this group are shown in ▶ Table 2. Based on the results of saline infusion testing, the ● primary aldosteronism was suppressible in 16 (33 %) and nonsuppressible in 33 (67 %). Thirty-two patients underwent successful adrenal venous sampling and the lateralization of aldosterone secretion was diagnosed in 23. Twenty patients underwent adrenalectomy, adrenal adenoma was diagnosed in 17, and nodular hyperplasia in 2, and normal histological finding was reported in 1. Improvement of blood pressure control was noted in all but one, that is in 95 %, of operated patients. The patient who did not improve after adrenalectomy had borderline results of adrenal venous sampling and an adenoma was found at histological examination. The repeatability of the urinary aldosterone excretion was evaluated in 44 patients who fulfilled the above mentioned eligibility criteria. The observed intraindividual differences are ▶ Fig. 1. The significant described by the Bland-Altman plot in ● variations in urinary aldosterone secretion were observed not only in patients with excluded PA, but also in individuals with proven lateralization of aldosterone secretion who eventually ▶ Table 3). The profited from unilateral adrenalectomy as well (● significant differences persisted when the urinary aldosterone was corrected by the urinary sodium and/or creatinine concentrations. No difference between genders was identified. Marked differences in urinary aldosterone were also noted in individuals who had minimal differences in urinary volumes, sodium and creatinine excretions. Similarly, significant variations in urinary aldosterone were noted in patients who had very high urinary sodium excretion ( > 400 mmol/day) in both collected samples.
Table 2 Data of group B patients who fulfilled the inclusion criteria and were enrolled in the analysis. Excluded PA
Confirmed PA
Confirmed PA – successful
16 (11) 47 (21–62)
33 (8) 50 (32–68)
19 (4) 46 (32–68)
144 (126–160) 86 (68–116) 2 (0–4) 93 (61–118) 42 (19–71)
145 (100–196) 90 (64–104) 4 (0–7) 96 (67–151) 74 (24–205)
151 (100–170) 89 (64–104) 4 (0–5) 96 (68–151) 84 (24–205)
2 9 (6.9 mg) 13 (295 mg) 4.0 (3.5–4.6) 2 350 (750–5 300) 356 (278–738) 24.7 (4.9–101.9)
5 21 (7.3 mg) 23 (264 mg) 3.4 (2.7–4.1) 2 734 (1 500–5 000) 358 (202–688) 81.9 (14.8–430)
3 15 (7.4 mg) 15 (235 mg) 3.4 (2.7–4.1) 2 800 (1 700–5 000) 382 (202–688) 84.1 (25.2–430)
285 (30–1 070) 4.3 (2.7–10.8)
675 (160–2 080) 2.7 (2.7–35.0)
480 (180–980) 2.7 (2.7–5.9)
35 (30–100) 2.7 (2.7–6.9)
280 (110–2 360) 2.7 (2.7–6.1)
385 (110–910) 2.7 (2.7–4.7)
surgical treatment Number of patients (women) Age (years) Screening Systolic blood pressure (mm Hg)1 Diastolic blood pressure (mm Hg)1 Number of antihypertensive drugs1 Creatinine clearance (ml/min) Aldosterone-to-renin ratio1,2 Confirmatory testing No. of patients on no antihypertensive therapy No. of patients on doxazosin (mean dose) No. of patients on verapamil (mean dose) Serum potassium (mmol/l) 3 Amount of urine (ml/24 h) 4 Urinary sodium (mmol/24 h) 4 Urinary aldosterone (nmol/24 h) 4 End of urine collection Serum aldosterone (pmol/l) Plasma renin (ng/l) End of saline infusion Serum aldosterone (pmol/l) Plasma renin (ng/l)
Unless otherwise stated, the data are expressed as medians (range) 1
Denotes the data obtained during the screening for primary aldosteronism
2
To calculate the aldosterone-to-renin ratio, both aldosterone and renin were expressed in ng/l
3
Serum potassium was taken at the end of urine collection, before saline infusion was given
4
Denotes only urinary samples enrolled in the analysis, that is containing ≥ 200 mmol/day of sodium
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
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Endocrine Care 665
Urinary aldosterone was analyzed in 93 samples of 49 patients. The receiver operator analysis curve (ROC) of urinary aldosterone in respect to PA diagnosis made by saline infusion testing is ▶ Fig. 2. depicted in ● Upper reference limit defined by the data of healthy volunteers (36 nmol/day) was characterized by 86 % sensitivity and 50 % specificity for the PA diagnosis; the concordant assessment was observed in 70 samples of 40 patients. Urinary aldosterone ≥ 19 nmol/day was associated with 96 % sensitivity and urinary aldosterone ≥ 92 nmol/day was characterized by a 96 % specificity in respect to the PA diagnosed by saline infusion testing. In 22 (45 %) patients, urinary aldosterone remained in the “gray” zone between 19 and 92 nmol/l. Among them, there were 9 cases that had positive AVS results and eventually profited from unilateral adrenalectomy. At the end of oral sodium loading and urinary collection, the plasma renin in the upright position was generally ≤ 11 ng/l, which reflects 97.5 percentile of the observed values.
Discussion
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Several approaches are currently recommended for the confirmatory testing in patients with suspected primary aldosteronism, none of them is generally accepted as superior to others [14]. While the fludrocortisone testing [15] is probably highly effective in suppressing an autonomous aldosterone secretion it may be considered difficult or inconvenient for many patients. The short-lasting saline infusion may not be sufficient for adequate suppression of nonautonomous aldosterone secretion. The limited repeatability of this test was observed even when the saline infusion was preceded by oral sodium loading [12].
Fig. 1 The Bland-Altman plot describes the differences in urinary aldosterone between 2 collected samples (UA1 and UA2).
The aim of our study was to elucidate the value of urinary aldosterone secretion in this diagnostic process. The study protocol addressed both the evaluation of repeatability and the interpretation of the results. Concerning the repeatability testing, our results show that there is a high variation in urinary aldosterone secretion in a large number of examined patients. We cannot offer any definite explanation for this finding. The observed intraindividual differences are well beyond the imprecision of laboratory measurements. Although the imperfections of urinary collection might be responsible for some of the observed discrepancies, urinary aldosterone markedly varied also in patients who had little differences in the urine volumes and creatinine excretion. Concerning the interpretation of the results, we were not able to identify a single cutoff value applicable both for excluding and confirming PA. The ROC analysis of our data showed a large “gray zone” between acceptable sensitivity and specificity. The high sensitivity value (19 nmol/day) approximately corresponds to the respective cutoff mentioned in the current guidelines (28 nmol/day) [3]. Similarly, the high specificity cut off (94 nmol/ day) is not much different from the reported urinary aldosterone in patients with aldosterone producing adenomas ( > 83 nmol/ day) [16]. In our study, the assessment of urinary aldosterone would confirm or exclude PA in 27 (55 %) examined patients. In the remain-
Fig. 2 The graph describes the receiver operator analysis of the urinary aldosterone in respect to the PA diagnosis based on 2 concordant results of saline infusion tests. The shape of the curve does not enable to identify a single cutoff characterized by acceptable sensitivity and specificity.
Excluded PA
Confirmed PA
Confirmed PA – success-
14 33 % (1–87 %) 25 % (8–66 %)
30 30 % (1–88 %) 17 % (0–55 %)
16 28 % (1–81 %) 8 % (0–55 %)
ful surgical treatment Number of patients Intra-individual variations in urinary aldosterone Intra-individual variations in urinary sodium
The percentage values are calculated as follows: 100 × (the absolute difference divided by the mean of 2 obtained values). The data are expressed as medians (ranges)
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
Table 3 The intra-individual variation in urinary aldosterone and sodium.
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666 Endocrine Care
Endocrine Care 667
Study Limitations
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First, our data are closely related to the above-mentioned diagnostic protocol and the laboratory assays used for the estimations of the hormonal concentrations [17]. The accuracy of the urinary collection might be questioned as the study individuals were examined on an outpatient basis. The project was conceived as reflecting a real-life practice. Even if the improper urine collection was partly responsible for observed differences, this fact has to be taken into consideration. Second, one quarter of enrolled patients was eventually excluded from the study based on the stringent exclusion criteria, which may limit the general applicability of the study results. The primary aim of our study was to eliminate any potentially confounding factors in order to increase the quality of acquired data. Third, the lower number of individuals with excluded PA might have led to less accurate estimation of test specificity. On the other hand, a high number of patients with confirmed PA provide more precise information on test sensitivity. Fourth, the number of healthy volunteers was not high. However, the calculated upper reference value is not markedly different from the published data [8, 18]. Moreover, we do not assume that the augmentation of the number of healthy individuals would significantly change the study results.
Conclusions
▼
The confirmatory testing for suspected primary aldosteronism performed by the estimation of urinary aldosterone after oral sodium loading exhibits a marked intraindividual variability and is associated with inconclusive results in nearly half of examined patients. Based on our results, we cannot recommend urinary aldosterone as a confirmatory test in patients with suspected primary aldosteronism.
Acknowledgements
▼
This work was supported by the research project PRVOUK P037/03.
Conflict of Interest
▼
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
Ceral J et al. Urinary Aldosterone in Aldosteronism … Horm Metab Res 2014; 46: 663–667
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ing individuals, the urinary aldosterone remained in the gray zone of inconclusive results. Among them there was a significant number of those who eventually profited from adrenalectomy (9 out of 22).
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