A Procedure for the Measurement of Urinary 17-Hydroxycorticosteroids, Aldosterone Acid-labile Conjugate, and Free Plasma Cortisol in Patients with Chronic Illness

Abstract

The usual steps for steroid isolation are: (1) hydrolysis, acid for the urinary acid-labile aldosterone conjugate, and enzymatic for the urinary 17-hydroxycorticosteroids (17-OHCS); (2) organic solvent extraction of the corticosteroids; and (3) alkaline, acid, and aqueous washing of the organic extracts of steroids.

In the method described the crude extracts are purified by 3 or 4 sequential partitions using solvent systems of increasing polarity. After three chromatographic separations the 17-OHCS are still near the origin, and the fourth solvent system will separate these from each other. For the measurement of 17-OHCS, the mixed steroids are eluted after the third separation. Half of each eluate is analyzed by the Porter-Silber reaction; the other half is used as a blank to obtain the absorbance of the phenylhydrazones by spectrophotometry. For the measurement of aldosterone or cortisol, aldosterone (mixed with cortisone) or cortisol (mixed with tetrahydrocortisone) is eluted after the fourth paper chromatographic separation and acetylated with 100 µg (2 µc) of ¹⁴C-acetic anhydride (specific activity, 2 mc/mmol). The labeled acetates are extracted by liquid partition and isolated by paper chromatography after addition of stable carriers. The beta emissions of the isotopic derivatives are counted by liquid scintillation. Concentrations are calculated from data obtained by acetylation and chromatographic separation of pure steroid standards and suitable blanks.