A new method for the isolation of the simple and highly complex glycosphingolipids from animal tissue

The most widely used methods for the extraction of glycosphingolipids from animal tissues are based on the use of chloroform/methanol mixtures. These methods, although suitable for a great majority of lipids, fail to remove highly complex glycosphingolipids. Reported here is a method for the isolation of the entire population of glycosphingolipids by means of a gradual degradation of tissue components and enrichment in carbohydrate conjugates resistant to alkali and proteases. Fresh gastric mucosa was homogenized and treated with alkali (beta-elimination) and RNAase and DNAase to decrease the viscosity of the homogenate, followed by pronase digestion. Each treatment was completed by exhaustive dialysis against distilled water. The resultant tissue digest was partitioned with chloroform/methanol (2:1) to remove simple glycosphingolipids. The aqueous portion of the system was adjusted to 1.0% with Zwittergent TM- 314 and solubilized for 24 h by mixing. Thus, prepared sample subjected to Bio-Gel P60 column chromatography afforded five fractions. Of these, three were free of protein and contained carbohydrates, fatty acids and sphingosine. Further fractionation on Bio-Gel P 10 and P6 columns followed by thin-layer chromatography afforded homogeneous components with all the characteristics of highly complex glycosphingolipids.