A Method for the Measurement of Aldosterone in Peripheral Plasma Using3H-acetic Anhydride

Abstract

A double isotope derivative method suitable for the estimation of aldosterone in human peripheral plasma has been developed. ³H-acetic anhydride was used as labeled reagent (1050 mc/mmole) and 4-¹⁴C-aldosterone (46 mc/mmole; 0.8 mμg added to the sample) as indicator. Only 0.25 μl (2.5 mc) acetic anhydride in 5μl benzene is required per reaction as aldosterone 21-monoacetate is the initial derivative formed. Purification is achieved by 2-dimensional silica gel thin layer chromatography of aldosterone 21-monoacetate and of the 18,21-diacetate then formed using nonisotopic acetic anhydride, followed by 2 paper chromatograms of the diacetate. The over-all recovery of the method with these 4 chromatographic steps is 31 ±9 (sd)%. Adequate specificity of the method was shown by no significant change in the values when aliquots of all samples estimated were further purified by acid hydrolysis of the diacetate to the 21-monoacetate followed by paper chromatography, and then after oxidation of the monoacetate to the aldosterone lactone 21-monoacetate followed by a 2-dimensional thin layer chromatogram. The nonspecific blank of the method was 0.09±0.06 (sd) mμg/20 ml plasma from 7 adrenalectomized subjects. The mean values for replicate estimates on 20 ml aliquots of plasma pools from 4 groups of normal supine male subjects were 0.71±0.07 (sd) (5 assays); 0.66±0.07 (sd) (6 assays); 0.93±0.06 (sd) (6 assays); and 0.77±0.07 (sd) mμg/20 ml (8 assays) (uncorrected for the blank of 0.09 mμg). As the values from plasma from adrenalectomized patients gave the same standard deviation, the standard error of the estimate in the lower range of values is about 0.06 mμg/20 ml plasma. The analysis of variance of estimates of plasma (low normal) with known amounts of aldosterone added gave y =0.85±0.22+x (0.99±0.02), where y=estimated amount and x = known amount added; this shows there is no systematic error in the method. The mean value for individual plasma samples from 9 normal male subjects in the sitting position and receiving ad lib. salt intakes was 6.96±4.05 (sd) ±1.35 (se) mμg/100 ml. The mean value for 4 of these normal subjects before receiving 20 g sodium chloride in addition to the ad lib. diet was 6.01±4.15 (sd) ±2.05 (se) mμg/100 ml, and after salt loading was 0.65±0.75 (sd) ±0.40 (se) mμg/100 ml. All these values are corrected for blank.