The Quantitative Determination of 3-7-Hydroxycorticosterone

Abstract

Various methods have been devised in an attempt to estimate the level of adrenal cortical function by measurement of urinary steroids (1-5). Difficulties with interfering non-steroidal contaminants enter into the different methods of steroid analysis. The applications of color reactions to urine extracts purified by solvent partition method leads to high and irregular values, even in Addison's disease, because of non-steroidal contaminants(2). The Porter-Stilber reaction using; phenyl hydrazine and sulfuric acid(6) is one cf the most widely used and specific reactions for estimations of 17 — hyciroxycorticoids. However, interfering chromagenic material is also a problem using this reaction(2). | 17-hvdroxycorticosterone (Compound F or hydrocortisone) reacts with 85% phosphoric acid with or without glaciai acetic acid to form a bright yellow color. This reaction is not given by any other member of the C21 corticosteroids: cortisone acetate (Comp. 1 ); 11-desoxy-17-hydroxy-corticosterone acetate; 11-dehydrocorticosterone acetate; desoxycortisone; 21-desoxycortisone; allopregnanediol- 3 (alpha), 20(alpha) ; allopregnanedione-3,20 (7). It was the purpose in this project, first of all, to determine the sensitivity of the phosphoric acid---Comp. F reaction and whether this reaction is quantitative. Secondly, this reaction was applied to urine extracts to determine if it could be used to measure the level of Comp. F excretion. Since phosphoric acid is much less active than the phenyl hydrazine---sulfuric acid reagent, it was thought that the interference from non-steroidal chromagens could be avoided by using this reaction in determining the level of Comp. F in urine.