Abstract
The urinary free aldosterone determination is one of the tests used for assessing adrenocortical function in clinical medicine.
Several workers have obtained the measurement but these methods are too elaborate and time-consuming. To obtaina rapid and simple method I compared the fluorometric method with the colorimetric determination with B.T.
The fluorescence of aldosterone in strong acid medium is not yet definitely known. Its absorption peak in sulphric acid was obtained at 288 mμ, and after heating the same solution to 100°C for 5 minutes its peak was detected at 340 mμ (Fig. 2, 3).
Because of these results it was thought that the fluorescence of aldosterone in acid medium could be produced.
The dry samples of D-aldosterone at three concentrations, 2, 1 and 0.5 μg per ml, were used.
75% sulpuric acid ethanol (v/v) was used as acid medium.
The measurements were carried out with spectrophoto-fluorometer at an exitation wave length of 470 mp and emission wave length of 520 mμ.
As a result of the study the most suitable temperature and time were obtained by heating aldosterone in sulphuric acid medium at 75°C for 5 minutes (Fig. 4, 5). The fluorescence of the cooled aldosterone solutions proved to be stable for at least 5~25 minutes.
Concentration-fluorescence curve was obtained as a straight line in the range of 0.25~3 μg per ml (Fig. 8). The samples, at least 3 ml of acid medium are required, were examined with the Aminco Bowman fluorophotometer.
The purification of the urinary aldosterone was carried out by the modified method of Oda & Suzuki which uses florisil column, three T.L.C. systems and acetylation (Table 1).
The results of determination on the urines of four normal subjects were higher (average : 25 μg per 24 hrs.) than the normal range (average : 10±3 μg per 24 hrs.) by the colorimetric determination with B.T. (Table 3).
At the same time, a modification of the B.T. method which is based on Oda & Suzuki's method was tried to obtain a rapid and simple technique.
Aldosterone concentration curve by the colorimetric determination with B.T. was obtained as a straight line in the range of 2.5~30 μg (Fig. 11).
500 ml of 24 hrs pooled urines were used, and they were extracted three times with 100 ml chloroform (Table 4).
The excellent recovery rate was average : 50~55%
The excretion of urinary aldosterone was measured by this method, in one patient with hypertension with nonsuppressive plasma renin activity, one case of Conn's syndrome and three healthy adults. Aldosterone values of three normal cases were from 13 to 14 pg per 24 hrs., hypertensive case was 33 μg per 24 hrs. and Conn's syndrome was 17 μg per 24 hrs. (Table 5).
The results of the fluorimetric method have been compared with those of the B.T. method on several urinary samples. In spite of the purification of the urinary aldosterone at several stages, the relatively high and unstable data were obtained in four normal human urines by the fluorimetric method (Table 3). It was thought that the fluorescence of several unknown materials interacted for the determination of urinary aldosterone. The result of the B.T. method was better than that of the fluorimetric method. For instance, the B.T. method was relatively quickly performed and the measurement data of urinary excretion of aldosterone were exact in a variety of clinical circumstances.
