Quantitative Determination of Glycosphingolipids Illustrated by Using Erythrocyte Membranes of Various Mammalian Species

Abstract

A procedure for the quantitative determination of glycosphingolipids is described, involving extraction of total lipids, fractionation on a column, acid cleavage and photometry of a complex formed between sphingosine and methyl orange. The level of glycosphingolipid sphigosine of erythrocyte membranes of a variety of mammals ranges from 59.1 (rat) to 410.1 (pig) nmol/ml packed cells.There is no simple method for the quantitative determination of glycosphingolipids as a whole. Since lipid sugar of animal tissues is predominantly a moiety of glycosphingolipids, one should expect that a determination of sugar in a lipid extracted from animal tissues might serve as a method for the determination of glycosphingolipids. However, glycosphingolipids contain various kinds of hexoses and N-acetylhexosamines, and there is no reagent known that indicates various hexoses and N-acetylhexosamines to the same extent. Thus, a reagent, such as anthrone, that reacts more readily with galactose than with glucose, would indicate a higher amount of a galactose-rich glycosphingolipid than of a glucose-rich glycosphingolipid. If the anthrone reaction is based on a mixed galactose glucose standard, then this would be a poor method, because there is rarely an equal amount of galactose and glucose in natural samples. Moreover, the anthrone reagent gives almost no color reaction with N-acetylhexosamines.Another possibility is given by one of the various methods of sphingosine determination. The formation of a complex between sphingosine and methyl orange appears to be the basis of a convenient procedure [l]. However, application of a sphingosine determination for the determination of glycosphingolipids involves the quantitative separation of sphingomyelins from glycosphingolipids.This paper reports a procedure that was adopted from the column chromatographic fractionation method of Saito and Hakomori [2] and the spectrophotometric method of sphingosine determination of Lauter and Trams [I]. This procedure was used in the determination of glycosphingolipids of erythrocyte membranes of various mammalian species. Both methods were modified in various ways. MATERIALS AND METHODS Blood Samples and ChemicalsBlood from man, cattle, pig, and llama was drawn by venepuncture. Blood from other animals (cat, sheep, rat) was obtained on occasion when the animals were sacrificed for other reasons. Acid &rate/ dextrose solution was used as an anticoagulant. Erythrocytes were isolated as described earlier [3].Cerebrosides and ceramide oligohexosides, isolated from human brain and bovine spleen respectively, were used as standard samples. Sphingosine sulfate and sphingomyelin were purchased from Koch Light (Colnbrook, England). Procedure of Determination20ml of a suspension of washed erythrocytes (hematocrit 40-60) was extracted three times with chloroform and methanol, as described elsewhere [3]; the total lipids obtained were purified by passing them through Sephadex G-25 fine [4], thus saving the gangliosides, and then transferred to a 25-ml volumetric ...

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