Gangliosides separation

Analysis of 3- and 6-linked sialic acids in mixtures of gangliosides separation of 62... 

The individual gangliosides separated differ mainly in the structure of their carbohydrate moieties, although the sphingosine part may also differ in length and size of the saturated or unsaturated chain173-176 and is dependent on the age of the organism.177... 

The isolation of gangliosides from brain has been reviewed by Kanfer (1969) and by Bergelson (1980). Other specific references are Suzuki (1965), Sven-nerholm et al. (1972) and Laine et al (1974), and general comments on ganglioside separations will be found in Kates (1972) and Christie (1982). 

A high-accuracy analysis represents an approach in which polysialylated gangliosides separated by HP-TLC were directly analyzed using a MALDI-Fowrier transform-ion cyclotron resonance (FTICR)-MS analyzer with UV lasers and soHd MALDI matrices [88-90],... 

Figure 8 Two-dimensional separation of a mixture of rat brain gangliosides plus authentic standards on an HPTLC plate (a) photograph of the chromatogram (b) tracing. See text for chromatographic conditions and Ref. 63 for a description of the symbols used to designate the major and minor gangliosides separated by TLC. Reproduced from Ref. 63 with the permission of Elsevier. Inc.

Gangliosides are associated with varions oligosaccharides, and nsnally their complete separation is not satisfactory nsing one-dimensional developments on TLC. 

The polysaccharide component of a lipopolysaccharide can be separated from the lipid component by selective hydrolysis of the glyco-sidic linkages of the 3-deoxy-D-manno-octulosonic acid residues connecting these two components. The conditions for the hydrolysis are mild, namely, 0.1 M acetic acid for 1.5 h at 100° (Ref. 18). Similar conditions, namely, M formic acid for 1 h at 100° or 0.05 M hydrogen chloride in methanol for 1 h at 85°, were used to split off the sialic acid residues from gangliosides.19,20... 

Svennerholm, L. 1963. Chromatographic separation of human brain gangliosides. J. Neu-rochem. 10, 613-623. 

The situation for gangliosides is also complex, with a number of separate species. However, two of these are quite dominating, and are hematoside with N-acetyl and N-glycoloyl substitution, respectively. Fig. 12 shows that the N-acetyl type exists in non-epithelial while the N-glycoloyl type is mostly present in epithelial cells. 

Comparison of glycosphingolipids from human and chicken skeletal muscle. The elution of gangliosides from DEAE-Sephadex A 50 column with these 0.01, 0.02 and 0.2 M sodium acetate concentrations separated the gangliosides into mono-, di- and poly-sialo- fractions. The gangliosides of human muscle are shown in Fig. 1A. The monosialogangliosides GM3, GM2 and GM1 were eluted with 0.01 M sodium acetate in methanol (lane 2), GD3 and GDla with the 0.02 M solvent (lane 4) and others with 0.2 M acetate... 

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Thin Layer Chromotography. Unless otherwise stated, all thin layer chromotography was on plates coated with silica Gel G (E. Merck, Dramstadt). All the solvents were mixed on a volume basis. Neutral glycolipid fractions were developed in chloroform methanol water (60 35 6.5). Labelled fucolipid fractions were developed in chloroform methanol water (40 40 10). For separation of gangliosides, chloroform methanol 2.5N aqueous NH4OH (60 40 9) was used. 

Figure 11. Effect of incubation time (at 37°C) and of ganglioside concentration on the incorporation of gangliosides (G.y, G m, GTib) into phosphatidylcholine mono-lamellar vesicles. Phosphatidylcholine (as vesicles) 9 nmol. Ganglioside from 0.5 to 2 nmol. After incubation the mixtures were passed through a 1 X 20 cm Sepharose 4B column to separate vesicles from ganglioside micelles.

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

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