REPLACEMENT OF CHLOROFORM THROUGHOUT GLYCOSPHINGOLIPID ISOLATION

Chloroform, the predominant constituent of solvents used for lipid ext raction and chromatography, is believed to give rise to birth defects and serious damage to health, and may also be carcinogenic. Therefore, simple and successful methods have been developed to replace chlorofo rm throughout the isolation of glycosphingolipids (GSLs) by less harmf ul solvents, Gangliosides of sheep brain (ganglio-series gangliosides G(M1), G(D1a), G(D1b) and G(T1b)) and of lymphocyte-derived mouse hybr idoma cells (namely G(M3)) were extracted with six different solvent m ixtures. Chloroform:methanol:water (40:80:30, v/v/v) was employed as r eference (solvent I). Combinations without chloroform were: n-propanol :water (40:10, v/v) (II), methylisobutylketone:methanol:water (40:80:3 0, v/v/v) (III), ethylacetate:methanol:water (40:72:28, v/v/v) (IV), m ethylacetate:methanol:water (40:72:28, v/v/v) (V) and petroleum ether: isopropanol:water (40:112:38, v/v/v) (VI). After extraction and dialys is, the weight of lipid extract as well as the content of sialic acid, gangliosides, sulphatides and phospholipids were determined. Quantita tion of GSL yields in crude extracts obtained by the alternative solve nt mixtures II to VI showed recoveries of brain gangliosides from near ly 67% up to 104% compared with the reference solvent I. Extraction of hybridoma cells by means of the alternative combinations without chlo roform revealed at least the same and mostly better ganglioside yields in the range from 98% to 116% with regard to the reference solvent I. n-Propanol:water (II) and methylisobutylketone:methanol:water (III) w ere the recommended extractants for both tissues. Therefore, the metho ds described offer simple, less hazardous and successful strategies fo r GSL extraction in excellent yield without the need for using chlorof orm.