Chromogenic and fluorogenic reactions of adrenocortical and other steroids in concentrated acids

Abstract

Spectrophotometric and spectrofluorometric studies were made of the reaction mixtures obtained by dissolving various steroids in concentrated sulfuric and concentrated phosphoric acids. The effects of time, heat and subsequent dilution of the reaction mixtures were also studied, with respect to the absorption and fluorescence properties. An attempt was made to isolate the reaction products of cortisone and of hydrocortisone in concentrated sulfuric acid, and to correlate their light-modifying properties with those of the parent steroids. The mechanism of reaction of a given steroid is essentially the same in either acid, differing in rate rather than in nature. Appropriate variations of the time and temperature of reaction, and dilution of the reaction mixture with water or with ethanol, tend to diminish or remove the differences observed in the behavior of any steroid in these 2 acids. The absorption and fluorescence spectra of each reaction mixture are complex spectra resulting from the contributions of 2 or more reaction products in each case. With hydrocortisone, the most characteristic fluorescence band is associated with a stable product which resists change on dilution and can be isolated chromatographically. The characteristic blue fluorescence of the cortisone reaction mixture is destroyed by dilution. The observed changes are tentatively explained on the basis of a three-stage-reaction pattern. Reversible protonation of the [DELTA]4 -C-3 ketone of corticosteroids or testosterone, or of the phenolic C-3 hydroxyl of estriol, is the first step. The protonated steroid is assumed to form intermolecular association complexes at reactive substituent sites of other steroid molecules. These postulated complexes are thought to break down irreversibly to yield a series of products, via reactions which are markedly affected by dilution.