Formation of hydrogen sulphates as a means of separating alcoholic from non-alcoholic steroids

Abstract

Sobel, Drekter & Natelson (1936) determined plasma cholesterol by a method that involved the formation of cholesteryl hydrogen sulphate as a means of separating the sterol from its esters and from other non-alcoholic substances. It is now shown that under appropriate conditions the analytical approach of Sobel et al. (1936) is capable of wider application. Each compound tested (see Table 1) was dissolved in dry pyridine (0.2ml), and the solution was chilled in ice-water and mixed with a saturated solution of chlorosulphonic acid in carbon tetrachloride (0.1 ml; a fresh solution was prepared weekly). After 30min at room temperature, 0.4M-NaHCO3 (1.0ml) was added and the non-alcoholic fraction extracted with benzene (2 x 2.5ml) or (in the cases of cortisol and aldosterone) with dichloromethane (2 x 2.5ml). The extract was washed with water (0.5ml) and evaporated to dryness. For solvolysis the aqueous phases were combined and diluted with 2M-pyridinium sulphate (1.0ml) and extracted with dichloromethane (2 x 2.5ml), and the extract was evaporated to dryness; the residue was kept in dioxan (1 ml) for 4h at 370C, and the solution was diluted with dichloromethane (4ml), washed with 0.4M-NaHCO3 (0.5ml) and evaporated to dryness (McKenna & Norymberski, 1960), giving the alcoholic fraction. In the case of pregnanediol the aqueous phase was adjusted to pHl, mixed with ammonium sulphate (0.75g) and extracted with ether-ethanol (3: 1, v/v; 3 x 2ml) (Edwards, Kellie & Wade, 1953); solvolysis was in dioxan containing 2% (v/v) ofethanol at 470C. For enzymic hydrolysis the aqueous phase was adjusted to pH4.9 with acetic acid; 2M-sodium acetate buffer, pH4.9 (0.2ml), was added and then the gastric juice of Helix pomatia (Henry & Thevenet, 1952; supplied by l'Industrie Biologique Fran9aise, Gennevilliers, France) containing 8000 sulphatase units (Roy, 1956). After 48h at 370C the mixture was extracted with dichloromethane (3 x 2ml), and the extract was washed with 0.4M-NaHCO3 (0.5ml) and evaporated to dryness. The minor fraction and part of the major fraction (see Table 1