Successive Isolation and Separation of the Major Lipid Fractions Including Gangliosides from Single Biological Samples

Abstract

Currently available techniques concerning extraction and characterization of the different lipids from biological specimens are designed for particular families and do not address consecutive isolation of lipid constituents in their globality. We describe here a simple, nondestructive chromatographic procedure that allows efficient elution and further analysis of the major lipid classes (neutral lipids, phospholipids, nonsialylated sphingolipids, and gangliosides) in their natural states from the same starting material. The procedure describes the use of solvent mixtures adapted to silicic acid column chromatography and permits 90–97% recovery of each of the above lipid groups. We have particularly concentrated on optimizing the efficient recovery of the diverse minor forms of gangliosides, free of other contaminants, from relatively small amounts of neural tissue. As model systems we have usedin vivoandin vitropreparations of mammalian retina for which only fragmentary data are available on lipid composition. We show that relative to brain, retina contains, for example, twofold more sphingomyelin and sixfold more GD3 ganglioside. In turn, cultured retinal glial cells contain twofold higher levels of globoside and eightfold higher amounts of GM3 ganglioside with respect to intact retina. Compared to previously published techniques, we obtain improved total ganglioside recovery, with enrichment of poly-sialogangliosides. The technique presented here should be widely applicable to analyze global lipid composition of diverse biological samples.