Sodium interference in the determination of urinary aldosterone

Clinical Biochemistry 49 (2016) 295–297

Contents lists available at ScienceDirect

Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem

Short Communication

Sodium interference in the determination of urinary aldosterone Marta Lucía Aldea ⁎, Jaume Barallat, María Amparo Martín, Irene Rosas, María Cruz Pastor, María Luisa Granada Department of Clinical Biochemistry, Hospital Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain

a r t i c l e

i n f o

Article history: Received 25 September 2015 Received in revised form 30 October 2015 Accepted 4 November 2015 Available online 10 November 2015 Keywords: Aldosterone Immunoassay Liaison® analyzer Sodium interference Primary hyperaldosteronism

a b s t r a c t Objectives: Primary hyperaldosteronism (PHA) is one of the most common endocrine forms of secondary hypertension. Among the most used confirmatory tests for PHA is urinary aldosterone determination after oral sodium loading test. The primary aim of our study was to investigate if sodium concentrations interfere with urinary aldosterone in an automated competitive immunoassay (Liaison®) as well as to verify the manufacturer's specifications. Design and methods: 24-hr urine samples were collected and stored frozen until assayed. Two pools at low and high aldosterone concentrations were prepared. Verification of performance for precision was tested according to Clinical and Laboratory Standards Institute (CLSI) document EP15-A2 and interference with increasing concentrations of NaCl according to CLSI EP7-A2. Results: The assay met the quality specifications according to optimal biological variation. Our results show that sodium concentrations up to 200 mmol/L do not interfere on urinary aldosterone quantification, but sodium concentrations above 486 mmol/L negatively interfere with the test. Conclusions: The Liaison® automated method is useful for aldosterone determination in the PHA confirmatory test, but interferences with NaCl may occur. It is therefore recommended to determine urinary NaCl before measuring urinary aldosterone to avoid falsely low results. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

1. Introduction The steroid hormone aldosterone, secreted by the adrenal cortex, is the major bioactive mineralocorticoid in humans. It decreases the excretion of sodium and increases the excretion of potassium in the kidney and the sweat and salivary glands. At these sites, aldosterone acts by binding to mineralocorticoid receptors, particularly to the cortical collecting ducts in the kidney. In the liver and kidney, aldosterone is transformed into watersoluble and less active products. It is conjugated to glucuronic acid, producing aldosterone 18-oxo-glucuronide, which is then excreted into the urine. A small amount of unmetabolized (‘free’) aldosterone also remains. Primary hyperaldosteronism (PHA) is the most common endocrine cause of secondary hypertension with a prevalence rate of 5% to 15% among the hypertensive population. Sequential steps are involved in the diagnosis of PHA: screening tests followed by confirmatory tests. The ratio of plasma aldosterone concentration to plasma renin activity (PAC/PRA) is considered the screening test of choice for PHA [1]. Among the confirmatory tests, one of the most used is the oral salt loading test [2,3], in which the patient increases sodium intake to N200 mmol/d (6 g/d) for 3 days, verified through 24-h urine sodium ⁎ Corresponding author at: Department of Clinical Biochemistry, Germans Trias i Pujol Hospital, Autonomous University of Barcelona, Crta Canyet s/n, Badalona 08916, Spain. E-mail address: [email protected] (M.L. Aldea).

content. Urinary aldosterone is measured from the 24-h urine samples collected between the morning of Day 3 and the morning of Day 4. PHA is considered when high urinary aldosterone excretion (N 12 μg/24 h) is determined [1]. The gold standard for aldosterone determination is the combination of gas chromatography or HPLC with mass spectrometry. Before, aldosterone measurement assays were mainly carried out by radioimmunoassay (RIA), for which a previous extraction step and a hydrolysis step were necessary before sample processing. Recently, an automated competitive immunoassay, Liaison® (DiaSorin, Saluggia, Italy), has been developed that uses a chemiluminescence procedure (CLIA) that requires only the hydrolysis step. The aims of the study are 1) to investigate whether at certain concentrations, sodium interferes with the detection of urinary aldosterone, thus affecting the clinical diagnosis, and 2) to assess the precision of the assay and verify that the assay's analytical variation meets the quality requirements desirable for biological variation. 2. Materials and methods 2.1. Sample preparation Complete 24-h urine collections were carried out with 10 g of boric acid to maintain urine's pH below 7.5. Urine was kept at 4 °C during the collection period. After mixing thoroughly the 24-hr pool, an aliquot was taken and stored at −20 °C until assayed.

http://dx.doi.org/10.1016/j.clinbiochem.2015.11.004 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

296

M.L. Aldea et al. / Clinical Biochemistry 49 (2016) 295–297

Two urine pools containing either high or low levels of aldosterone (~2 μg/L and 15 μg/, respectively) were prepared. The final concentrations of aldosterone in each of the pools, as measured by the Liaison®, were 1.37 μg/L and 17.01 μg/L, respectively. The initial sodium concentrations were 89 mmol/L for the low pool and 73 mmol/L for the high pool, respectively. Urinary aldosterone measurements were done using the competitive aldosterone CLIA assay (Liaison® Aldosterone Ref: 310,450, DiaSorin, Saluggia, Italy) following the manufacturer's recommendations. Hydrolysis of the urine samples was performed with 0.2 N HCl and subsequent overnight incubation at 30 °C. After the hydrolysis, the samples were neutralized with the Liaison® Aldo Neutralization Buffer (Ref: 310452) and processed immediately. Urinary samples were obtained using leftover human specimens that are not individually identifiable, as defined by the FDA [4]. The study was approved by the Hospital's Ethics Committee for Clinical Research. 2.2. Assay precision and quality specifications Verification of performance for precision was tested following the Clinical and Laboratory Standards Institute (CLSI) Evaluation Protocol 15 (EP15-A2) specifications to establish that the laboratory's performance was consistent with the manufacturer's claims. For five consecutive days, a thawed aliquot from each pool was assayed three times [5]. Furthermore, optimal coefficient of variation (CVo) and desirable coefficient of variation (CVd) for aldosterone assays were calculated as quality specifications based on the biological variability (CVbi = 39.4%, within-subject biological variation) [6] as CVo = 0.25*CVbi = 9.85% and CVd = 0.5 ∗ CVbi = 19.7%, respectively, in order to assess if the interassay analytical CV (CVa1) is appropriate [7]. 2.3. Interference assay Interference test laboratory procedures were conducted strictly according to the CLSI document EP7-A2, the most recent guideline on interference testing, approved in 2005 by the U.S. Committee for Clinical Standards [8]. A 5-point interference curve for each pool was prepared. Solutions with increasing concentrations of sodium chloride (NaCl) (0, 200, 400, 600, and 800 mmol/L) from a 20%v saline solution were prepared. The high NaCl concentration sample (800 mmol/L) of the interference curve was prepared by adding 94 μL of NaCl saline solution (20%v) to 306 μL of urine. For the low NaCl concentration sample (0 mmol/L), 94 μL of deionized water was added to 306 μL of urine. For the 400, 200, and 600 mmol/L concentrations the following dilutions were made: equal volumes of i) 800 and 0 mmol/L, ii) 400 mmol/L and 0 mmol/L, and iii) 800 and 400 mmol/L samples,

respectively. This was performed for both the high and the low aldosterone urine pools. Aldosterone concentrations were measured in triplicate. For each interfering concentration, the percentage of recovery and the percentage of interference were calculated as % Recovery = (measured value / true value) ∗ 100 and % Interference = ((measured value − true value) / true value) ∗ 100 respectively. 3. Results 3.1. Assay precision The within-run CVs also were lower than those reported by the manufacturer (2.22% vs. 2.5% for the high pool and 3.52% vs. 3.6% for the low pool). Within-laboratory CVs also complied with the provided specifications (1.83% vs. 8.6% for the high pool and 8.84% vs. 9.8% for the low pool). It must be noted that aldosterone urine concentrations tested by the manufacturer were not exactly the same as those used in our experiment. High pool CVa1 was 1.72% and low pool 8.84%. All assays showed a lower CVa1 than optimal or desirable CV, meeting the quality specifications. 3.2. Interference assay Table 1 shows aldosterone concentration, recovery, and percentage of interference for each point of the interference curve. A significant decrease of aldosterone concentration is seen for both pools from the third point of the curve ([NaCl] = 400 mmol/L) (Fig. 1). Sodium concentrations above 400 mmol/L negatively interfere on urinary aldosterone determination. 4. Discussion This study aimed to verify the precision of the Liaison® CLIA automated immunoassay method for urinary aldosterone determination and analyze NaCl interference. The Liaison® system has already been validated [9,10], both for sensitivity and for linearity, and has proven its usefulness in determining urinary aldosterone. In this study, we showed that the imprecision of the method was lower than that indicated by the manufacturer, based on the CLSI document EP7-A2. Biological variation was used to define the aims of the analytical quality specifications. The analytical variation meets the optimal quality specifications; thus, the analytical results are adequate for patient screening, diagnosis, and monitoring.

Table 1 Aldosterone concentrations, recovery, and interference for both pools. High concentration pool 0 mmol/L Expected urinary sodium concentration(mmol/L) Aldosterone (μg/L) run 1 Aldosterone (μg/L) run 2 Aldosterone (μg/L) run 3 Mean aldosterone concentration of (μg/L) % of recovery % of interference

73 17.34 16.77 16.92 17.01 – –

Low concentration pool

200 mmol/L

400 mmol/L

600 mmol/L

800 mmol/L

0 mmol/L

264 16.95 16.03 16.18 16.39 96.33 −3.67

479 12.49 12.75 12.51 12.59 73.99 −26.01

681 13.39 12.73 12.60 12.91 75.88 −24.12

876 11.94 11.72 11.61 11.76 69.12 −30.88

89 1.40 1.38 1.32 1.37 – –

200 mmol/L

400 mmol/L

600 mmol/L

800 mmol/L

295 1.34 1.34 1.37 1.35 98.43 −1.57

486 1.00 1.10 1.02 1.04 76.03 −23.97

682 1.04 1.04 1.02 1.03 75.49 −24.51

895 0.92 0.90 0.91 0.91 66.47 −33.53

The table shows mean aldosterone concentrations (μg/L) for each point of the interference curve. % of recovery: recovery percentage obtained as: (mean measured aldosterone at each interfering point / mean aldosterone at 0 mmol/L) ∗ 100. % of interference: interference percentage obtained as: (mean measured aldosterone at each interfering − mean aldosterone at 0 mmol/L / mean aldosterone at interfering NaCl concentration of 0 mmol/L) ∗ 100. The “measured value” corresponds to the mean concentration of aldosterone in each point of the curve. The “real value” is the mean concentration of aldosterone in the first point of the curve, with an interfering concentration of 0 mmol/L.

M.L. Aldea et al. / Clinical Biochemistry 49 (2016) 295–297

297

Authors' conflict of interest disclosure The authors declare that there are no conflicts of interest.

Research funding None declared.

Employment or leadership None declared.

Fig. 1. Aldosterone recovery % related to matrix NaCl concentration. The figure shows the representation of the interfering effect of NaCl on aldosterone concentrations. With increasing concentrations of NaCl, the aldosterone recovery is less and therefore greater interfering effect. Aldosterone concentrations for each point of the interference curve are shown in Table 1.

Honorarium None declared.

References Urine is a highly variable matrix and NaCl concentrations fluctuate widely even in a healthy population [11]. To our knowledge, NaCl interference in urinary aldosterone has not been evaluated and no information is provided by the manufacturer. Here, we evaluated the interference of NaCl and found that the method is useful around the cutoff conditions required by the confirmatory test (NaCl = 200 mmol/L). However, it should be considered that NaCl urinary concentrations above 476 mmol/L can decrease the values of urinary aldosterone up to 30%. This could lead to a false negative in the confirmatory test of primary hyperaldosteronism. It is widely known that ionic concentrations may modify antigen–antibody affinity [12]. This could explain the interference reported in this study. Further experiments would be required in order to elucidate the nature of this interaction. According to the coefficient of variation, when determining urinary sodium in our laboratory maximum allowable concentrations of urinary NaCl for aldosterone measurements on urinary samples should be 295 ± 4.9 mmol/L. We have shown that urine NaCl concentrations higher than 486 mmol/L negatively interfere with urinary aldosterone determination. Unfortunately, we have not studied what happens at intermediate points of the curve, between 300 mmol/L and 486 mmol/L. In conclusion, the Liaison® automated method is useful for aldosterone determination during PHA confirmatory tests. However, sodium concentrations above 400 mmol/L negatively interfere with diagnostic performance. It is therefore recommended to determine urinary NaCl before measuring urinary aldosterone to avoid falsely low results.

[1] J.W. Funder, R.M. Carey, C. Fardella, C.E. Gomez-Sanchez, F. Mantero, M. Stowasser, et al., Endocrine Society. Case detection, diagnosis, and treatment of patients with primary aldosteronism: an endocrine society clinical practice guideline, J. Clin. Endocrinol. Metab. 93 (9) (Sep 2008) 3266–3281. [2] G. Giacchetti, P. Mulatero, F. Mantero, F. Veglio, M. Boscaro, F. Fallo, Primary aldosteronism, a major form of low renin hypertension: from screening to diagnosis, Trends Endocrinol. Metab. 19 (3) (Apr 2008) 104–108. [3] P. Mulatero, S. Monticone, C. Bertello, G. Mengozzi, D. Tizziani, et al., Confirmatory tests in the diagnosis of primary aldosteronism, Horm. Metab. Res. 42 (6) (Jun 2010) 406–410. [4] Guidance on informed consent for in vitro diagnostic device studies leftover human specimens that are not individually identifiable. Available at: http:// www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/ GuidanceDocuments/ucm078384.htm. [5] Clinical and Laboratory Standards Institute, User Verification of Performance for Precision and Trueness; Approved Guideline, 2nd ed. Clinical and Laboratory Standards Institute, Wayne, PA, 2005 (EP15-A2). [6] C.G. Fraser (Ed.), Biological Variation: From Principles to Practice, American Association for Clinical. Chemistry Press, Washington DC, 2001. [7] C. Ricos, V. Alvarez, F. Cava, Biological variation and desirable specifications for quality controlAvailable at: http://www.westgard.com/guest17.htm (Accessed: ) 25 Sept 2013. [8] Clinical and Laboratory Standards Institute, Interference Testing in Clinical Chemistry; Approved Guideline, 2nd ed. Clinical and Laboratory Standards Institute, Wayne, PA, 2005 (EP07-A2). [9] N. Belaidi, A. Georges, J. Brossaud, J.B. Corcuff, Aldosterone determination: comparison of a RIA assay and a CLIA assay, Clin. Biochem. 48 (1–2) (Jan 2015) 89–92. [10] F. Derlet, T. Lepoutre, D. Gruson, Aldosterone testing: evaluation of a novel automated immunoassay, Biomarkers 19 (1) (Feb 2014) 86–91. [11] R. Sam, I. Feizi, Understanding hypernatremia, Am. J. Nephrol. 36 (1) (2012) 97–104. [12] W.E. Paul, Fundamental Immunology, 7th Ed. Lippincott Williams and Wilkins, Philadelphia, 2013.