Occurrence of a 5,7-Diene Sterol in Leishmania donovani

Abstract

Ergosterol, a 5,7-diene sterol, has been isolated from a number of the Trypanosomidae. It is the major sterol in Crithidia fasiculuta (Kusel & Weber, 1965) Williamson & Ginger, 1965) and concluded that cholesterol is the major sterol in the organism. However, Ghosh (1963) working with membranes from L. donovani, found a sterol which was 'fastreacting' with the Lieberman-Buchard reagent indicating the presence of a A' or As structure (Cook & Rattray, 1958), but did not obtain an ultraviolet spectrum showing the presence of a 5,7-diene and concluded that 5,7-dienes and cholesterol were absent.Our studies on the lipids of Leishmania donovani have produced results contrary to those reported by Williamson (1963) and we have obtained an ultraviolet spectrum confirming the presence of 5,7-dienes in the sterol fraction from L. donovani. METHODSLeishmania donovani (National Institutes of Health, U.S.A., strain, obtained from Dr E. J. Tobie) was cultured in a diphasic medium consisting of an agar supplemented brain-heart infusion agar (Difco) containing 2 % (w/v) glucose and 10 % (v/v) defibrinated rabbit blood as the solid base. Locke's solution was used as an overlay. The cultures were grown at room temperature for 8 to 10 days before harvesting by centrifugation at 3500 g for 10 min. at room temperature.The harvested organisms were suspended in 0-1 culture volumes of IOO mM-KCl at 5" and recentrifuged.The washed organisms were resuspended in 0-01 culture volurneof cold IOO mM KCL and extracted by the method of Bligh & Dyer (1959).The dry, total lipid extract from I O~O organisms was added to a 15 g. column of silicic acid (CC-4; Mallinkrodt Chemical) and the neutral lipids were eluted with chloroform. Chloroform was removed with a rotary evaporator and the neutral lipids were dissolved in hexane. The sample was then chromatographed on a 10 g. column of Florisil (Floridin Co., Tallahassee, Florida) using the elution scheme given by Carroll (1961). The sterols were eluted with 15 % (v/v) diethyl ether in hexane.Thin-layer chromatography was performed on silica gel G (Merck, Darmstat, Germany).