Abstract
A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes. Total lipids extracted from 50 ml of plasma or packed erythrocytes were separated by silicic acid chromatography into neutral lipids, a fraction of mixed glycolipids that was eluted with acetone-methanol 9:1, and phospholipids. After mild alkali-catalyzed methanolysis to remove contaminants from the crude fraction of glycolipids, individual glycosyl ceramides were isolated by preparative thin-layer chromatography. The oligosaccharide portions of these lipids were characterized by cleavage with methanolic hydrogen chloride and gas chromatography of the O-trimethylsilyl methyl glycosides. It was possible to study the composition of the carbohydrate and sphingolipid base fractions in the same gas chromatographic analysis. With mannitol as an internal standard for gas chromatographic estimation of glucose, concentrations of each of the glycosyl ceramides were determined with a precision of about 10%. Recoveries of the lipids from plasma varied with the complexity of the oligosaccharide moiety and ranged from 94% with glucosyl ceramide to 71% with globoside. Concentrations of the four glycosyl ceramides in plasma and in erythrocytes were determined for samples from young, healthy males. Amounts of glycolipid as low as 0.1 micromole can be determined conveniently by this procedure.
Highlights
A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes
Extraction of the erythrocytes a t room temperature with chloroformmethanol 2 : l was used routinely in these studies, but one extraction did not suffice to remove all of the glycosyl ceramides
A second extraction obtained at reflux temperature contained measurable quantities of all four neutral glycosyl ceramides ; further extractions of the residue failed to yield glycolipid
Summary
A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes. Yamakawa and Suzuki found the same substance, which they called globoside, in washed stroma from fresh human erythrocytes [2] and described the isolation of a related glycolipid (hematoside), containing neuraminic acid instead of galactosamine, from horse erythrocytes [3]. Chromatographic procedures for the isolation of individual glycosyl ceramides were subsequently described [8] and used for a detailed study of the glycolipids from 17 liters of human serum [9]. Glucosyl ceramide and a dihexosyl ceramide with equimolar amounts of glucose and galactose were present in approximately equal quantities in the mixture, along with much lesser proportions of a trihexosyl ceramide and globoside or a related aminoglycolipid with the same molar ratio of glucose, galactose, and galactosamine (1:2:1)
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