DETERMINATION OF Δ4-3-KETO-CORTICOSTEROIDS IN HUMAN PERIPHERAL PLASMA BY THE ISONICOTINIC ACID HYDRAZIDE (INH) REACTION*

Abstract

THE purpose of this communication is to extend a previous report by the authors (1) on quantitative studies of various adrenocortical steroids in small samples of peripheral human blood plasma, by presenting the findings based on a new reaction (INH) which involves the formation of the isonicotinyl hydrazones of Δ⁴-3-keto-corticosteroids. In conjunction with the Porter-Silber reaction (2) it provides an improved quantitative guide to the estimation of plasma corticosteroids. Actually, our application of this procedure has been part of an attempt to isolate and identify the various corticosteroids found in blood plasma. Previous work. A new color reaction for the determination of Δ⁴-3-ketocorticosteroids has recently been described by Umberger (3), who utilized the earlier findings by Ercoli, DeGiuseppe and De Ruggieri (4). When the isonicotinyl hydrazones of ketosteroids having a double bond conjugated with a carbonyl group are dissolved in acidified absolute ethyl alcohol, a yellow color (absorption maximum at 380 mμ) is produced. Under the conditions described by Umberger, this color is not produced by the hydrazones of the saturated ketosteroids or by those having a double bond in positions other than the alpha-beta position. The presence of the Δ⁴-3-keto grouping is generally accepted as essential in all biologically active steroids. We have confirmed the claims of Umberger (3) for the specificity of this reaction and have further expanded these claims by demonstrating that there is no INH color reaction with allo-dihydro-hydrocortisone, a compound which contains the 3-keto group but not the double bond in the alpha-beta position.