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Technical Briefs |
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Aldosterone, a steroid hormone secreted by the adrenal cortex, is important in the overall control of sodium and potassium balance. Abnormalities in aldosterone concentrations are seen in a variety of clinical conditions. Overproduction (hyperaldosteronism) may result both from a primary cause such as adrenal adenoma (1) or a secondary cause, e.g., stimulation by increased renin release (2). Underproduction of the hormone is seen in such diseases as Addison disease (3).
Many methods for measurement of plasma aldosterone have been based on the extraction method of James and Wilson (4). However, technical progress has enabled the production of direct kit methods for measuring plasma aldosterone. Previous studies (5)(6) have demonstrated that direct methods gave anomalous results in certain circumstances, possibly because of the presence of polar metabolites. The purpose of the current study was to compare a direct method with an existing extraction method for measuring aldosterone—first, in a large number of samples from clinical sources, and second, after physiological alterations of sodium intake.
Plasma was obtained in the morning from 89 individuals after they had sat for 5–10 min: 40 patients with essential hypertension, average age 52 years (range 25–80 years; 21 men, 19 women); 28 presumed healthy volunteers, average age 45 years (range 24–77 years; 19 men, 9 women); 17 patients with chronic renal failure (serum creatinine 349 ± 32.2; range 189–535 µmol/L), average age 59 years (range 29–83 years; 9 men, 8 women); a 42-year-old woman with a pheochromocytoma; 2 patients with Conn syndrome (a 37-year-old woman and a 35-year-old man); and a 75-year-old woman with persistent hypertension and hypokalemia. Except for the chronic renal failure patients, none of the subjects had any sign of renal failure as determined by serum creatinine values (all <110 µmol/L). In addition, two or three quality-control pools were analyzed in duplicate in each assay run.
We also determined the effect of sodium alteration on the measurement of plasma aldosterone concentrations in 8 hypertensive individuals (4 women, 4 men). Measurements were made after 1 month on a low-sodium diet and 1 month on a high-sodium diet in a double-blind, crossed-over, randomized designed study.
Blood samples (10 mL) for the plasma aldosterone determinations were obtained from a subcutaneous vein in the forearm; collected into lithium-heparin tubes, the samples were immediately centrifuged at 1200g for 15 min at 4 °C. The plasma was removed and stored at -20 °C until assay. Blood was also taken for routine biochemical determinations. Urine samples (24 h) were collected for the measurement of sodium and creatinine. Blood pressure recordings were made with an ultrasound sphygmomanometer (Arteriosonde; Roche). Ethical approval was obtained from our institution.
The extraction-based method for RIA of plasma aldosterone was based on that of James and Wilson (4). In brief, plasma samples (0.5 mL) were extracted with 10 mL of dichloromethane (BDH/Merck; AnalaR) to which had been added [3H]aldosterone (Radiochemical Centre, Amersham), 1000 counts/min, to assess the recovery of the process for each sample. The aqueous phase was dried under air and the extracts were reconstituted in 500 µL of phosphate-buffered saline (PBS; NaH2PO4 · 2H2O 7.5 mmol/L, anhydrous Na2HPO4 32.4 mmol/L, NaCl 103 mmol/L, sodium azide 15.4 mmol/L, bovine serum albumin 2 g/L). The reconstituted samples were then assayed by RIA, 100-µL samples being placed, in duplicate, into assay tubes. Additionally, 100 µL of sample was placed into scintillation vials to which was added 4.5 mL of scintillation fluid (Ultima-gold; Packard) to assess recovery. To the assay tubes was added 100 µL of diluted antiserum (obtained from St. Mary's Hospital, Paddington, UK) and 100 µL of 125I-labeled aldosterone (Amersham) that had been diluted in PBS to 2000 counts/min. The maximum binding was 76.0% ± 0.9% (n = 85). A 9-point calibration curve covering the range 0–160 pg/tube (0–4440 pmol/L) was prepared from dilutions of aldosterone (Sigma) in PBS. The effective limit of detection of the assay was given as equivalent to the lowest-concentration calibrator [1.25 pg/tube (35 pmol/L)]. The bound and unbound fractions were separated by adding 1 mL of cold dextran-coated charcoal [250 mg of activated charcoal (Sigma) and 25 mg of dextran T-70 (Pharmacia) in 100 mL of PBS] to the tubes. After 5 min the samples were centrifuged at 1200g for 15 min at 4 °C, and the radioactivity in the bound fractions was counted in a gamma counter (1261 Multigamma; Wallac) for 5 min. The extracted values were all corrected for recovery.
To measure aldosterone by a direct method, we used a Coat-A-Count kit (DPL Division, Euro/DPC), according to the manufacturer's instruction. Plasma (200 µL) was placed into antibody-coated tubes, to which was then added 1 mL of 125I-labeled aldosterone. The samples were mixed and incubated at 37 °C for 3 h before being decanted and counted for radioactivity in the gamma counter for 1 min. A 7-point calibration curve was constructed from the reconstituted calibrators provided with the kit (0 to 3300 pmol/L) and treated similarly. The minimal detectable limit for this assay as given by the manufacturer is 44 pmol/L.
Group comparisons were performed by using Student's paired t-tests. Group results are expressed as means ± SE. Method comparisons were carried out by linear least squares. P <0.05 was taken to be significant.
Plasma aldosterone determined by the direct method in 40 essential
hypertensive individuals—blood pressures of 166.8 ±
3.4/99.0 ± 1.9 mmHg (reclining systolic/reclining diastolic)—was
not significantly different from that determined by the extraction
method: 610.3 ± 48.8 vs 607.8 ± 54.2 pmol/L, respectively.
Likewise, results for the 28 volunteers (blood pressure 121.3 ±
2.5/75.0 ± 1.4 mmHg) showed no significant difference between the
direct method (462.5 ± 41.4 pmol/L) and the extraction method
(432.6 ± 39.5 pmol/L). A plot of all the measured values is shown
in Fig. 1
. Samples from two subjects gave values below the assay
detection limits and were therefore excluded from the analysis. Linear
regression analysis revealed a combined correlation coefficient for all
the subjects of 0.91 (n = 83) and a best-fit line equation of
y = (1.14 ± 0.06)x + (-9.22 ±
42.52). However, further analysis of the 17 patients with chronic renal
failure (creatinine clearance 27.6 ± 4.7 mL/min) showed that,
although the correlation between the two methods was good
(r = 0.96) with a regression line of y
= (1.26 ± 0.10)x +(91.96 ± 108.3), the mean
value obtained with the direct method (1154 ± 221 pmol/L) was
significantly higher than that obtained with the extraction assay
(842.0 ± 168 pmol/L; P = 0.001; Fig. 1
). On
average, concentrations measured in the patients with chronic renal
failure were 36.7% ± 7.0% higher by the direct method. However,
there was no association between the difference in values, expressed as
a percentage of the extracted value, and the degree of renal failure,
as measured by creatinine clearance (r = 0.06; ns).
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Additional analysis of the comparisons between the two assays for the 12 individuals whose aldosterone was <300 pmol/L revealed no significant difference (P = 0.15) between the mean reported by the direct method (232.8 ± 14.6 pmol/L) and that by the extraction assay (196.6 ± 19.6 pmol/L).
The patient with pheochromocytoma had a plasma aldosterone of 1267 pmol/L as determined by the direct method and 1492 pmol/L by the extraction method. The two patients with Conn syndrome had similar results on both assays: 2662 vs 2812 pmol/L and 1539 vs 1716 pmol/L. The aldosterone concentration in the patient with persistent hypertension and hypokalemia was extremely high: The undiluted sample value obtained with the direct method was 8834 pmol/L and that determined by the extraction method was 7025 pmol/L.
Although urinary sodium during the low-salt diet (70.3 ± 13 mmol/24 h) was significantly different from that during the high-salt diet (159.9 ± 22.8 mmol/24 h; P <0.001), the plasma aldosterone concentration as determined by the extraction method (636.1 ± 96.6 pmol/L) on the low-salt diet was not significantly different from that determined by the direct method (618.9.3 ± 12.9 pmol/L). The values determined on the high-salt diet by both methods were also comparable (476.3 ± 58.7 vs 458.3 ± 77.1 pmol/L).
Regarding analyses of the quality-control pools, the mean value obtained for the high-concentration pool by the direct method (2724.3 ± 44.8 pmol/L; n = 50; CV 11.6%) was not significantly different from that measured by the extraction method (2721.7 ± 57.9 pmol/L; n = 50; CV 15%). Similarly, there was no significant difference between the normal pool value obtained by the direct method (697.2 ± 12.1 pmol/L; n = 50; CV 12.3%) or by the extracted method (709.9 ± 14.0 pmol/L; n = 50; CV 13.9%). The same was true for the low-concentration pool mean: direct assay 143.2 ± 6.1 pmol/L (n = 16; CV 17.0%), extraction method 142.8 ± 5.3 pmol/L (n = 50; CV 26.1%).
Compared with the extraction method, the Coat-A-Count RIA kit measures
plasma aldosterone directly and is technically easier to perform. The
kit allows for a greater number of samples to be processed in an assay,
is far less time-consuming, and is cost effective. The CVs for the
pooled samples were similar in both methods. Results from these two
methods in normotensive and hypertensive individuals were close to the
line of identity (see Fig. 1
). The two methods also yielded comparable
results during physiological alterations in circulating aldosterone as
induced by dietary sodium alteration. However, in chronic renal failure
patients, although the correlation between the two methods was good
(r = 0.96; n = 17), the results obtained by the
direct method were significantly higher, on average 36.7% ± 7.0%,
than those obtained with the extraction method—possibly resulting from
the accumulation of polar metabolites in patients with renal failure
(5). Our study, in conjunction with previous work
(5)(6), highlights the problem of using direct
kits to assay plasma from patients with chronic renal failure, which
may overestimate the plasma aldosterone. In these patients, therefore,
a suitable extraction of plasma aldosterone before RIA should be
considered.
Footnotes
Blood Pressure Unit, Dept. of Med., St. George's Hosp. Med. Sch., Tooting, London SW17 ORE, UK
References
The following articles in journals at HighWire Press have cited this article:
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  J Manolopoulou, M Bielohuby, S J Caton, C E Gomez-Sanchez, I Renner-Mueller, E Wolf, U D Lichtenauer, F Beuschlein, A Hoeflich, and M Bidlingmaier A highly sensitive immunofluorometric assay for the measurement of aldosterone in small sample volumes: validation in mouse serum J. Endocrinol., February 1, 2008; 196(2): 215 - 224. [Abstract] [Full Text] [PDF]
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 D. Hartman, T. W. Doulton, C. A. Chesters, G. A. Sagnella, and G. A. MacGregor Plasma Aldosterone: Comparison of a New Automated Assay with a Standard Extraction Method. Clin. Chem., November 1, 2006; 52(11): 2118 - 2119. [Full Text] [PDF]
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C. Schirpenbach, L. Seiler, C. Maser-Gluth, F. Beuschlein, M. Reincke, and M. Bidlingmaier Automated Chemiluminescence-Immunoassay for Aldosterone during Dynamic Testing: Comparison to Radioimmunoassays with and without Extraction Steps Clin. Chem., September 1, 2006; 52(9): 1749 - 1755. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and 17 patients with chronic renal
failure (
).