STUDIES ON A DIRECT RADIOIMMUNOASSAY OF PLASMA ALDOSTERONE

Abstract

The plasma level of aldosterone has been measured by radioimmunoassay (RIA) with Sephadex LH-20 column chromatography in our laboratories. However, the techniques of chromatography are complicated. Recently, a direct RIA of plasma aldosterone using a highly specific antiserum has been reported from a few laboratories. The direct assay is thought to been useful to treat a large number of samples.
In this report, a direct RIA of plasma aldosterone was achieved by using anti-aldosterone-18, 21-diacetate-3-oxime bovine serum albumin provided from CIS as an antiserum. Procedure of extraction in a direct method is as usual. Plasma aldosterone was extracted with dichloromethane. The extract was washed with 0.1N acetic acid and distilled water. The solvent was evaporated, and the dried residues were dissolved in buffer solution for RIA. Aldosterone values obtained from the direct method were compared to those obtained by the chromatographic method. The most suitable conditions of the direct method are as follows:
1. Preparation of standard curve must be treated in exactly the same way as the extraction procedure of plasma sample. The most suitable standard curve was obtained from the case in which the standard aldosterone was added to 5% bovine serum albumin.
2. Incubation for 30 minutes at 37°C followed by 2 hours at 4°C was confirmed as the most suitable condition in this assay procedure.
3. A large quantity of cortisol added to plasma in vitro did not interfere with aldosterone values by this method. Dilution curve of the high concentration of plasma aldosterone was paralleled with standard curve. High specificity of this antiserum was clear from these observations.
4. Recovery of added aldosterone to pool plasma was over 93%.
5. Reproducibility of this assay was satisfactory: The intra assay and inter assay variations were 5% to 14% and 10% to 19% respectively.
6. Comparing between the direct assay and Sephadex LH-20 column chromatography was established to measure in 123 samplex which contained aldosterone valued from 0 to 160ng/dl. Correlationship between these two methods was excellent: r=0.96; y=1.08x-2.55.
7. By the direct method, the mean level of plasma aldosterone in normal subjects was 6±3ng/dl at lying, and was 8±4ng/dl at standing. The mean concentration from 19 patients of primary aldosteronism was 71ng/dl. After operation in 4 patients of primary aldosteronism, plasma aldosterone was under 6ng/dl.
These results demonstrated that this direct method is a rapid, simple, efficient and accurate one for measuring plasma aldosterone as a routine test.