Glycosphingolipids separation

Liquid chromatography (l.c.) under elevated pressure has now been introduced into glycosphingolipid separation. Glycolipids are analyzed as perbenzoylated,112-116 N-acetylated O-benzoylated,"7 "8 and N-(p-nitro-benzoyl)ated O-acetylated119-122 derivatives, as well as in their native... 

FIGURE 9.4 TLC-blotting/SIMS of various glycosphingolipids. (A) TLC profile of the separated glycosphingolipids, (B) MS profiles obtained by TLC-blotting/SIMS (a) spectrum of sulfatide peaks at m/z 862, 876, 890, 906, and 918 are molecular ions with different fatty acid species indicated in the figure (asterisks are peaks of triethanolamine as matrix) (b) spectrum of Gb Cer peaks at m/z 1309 and 1337 are molecular ions with different fatty acid species as indicated (c) spectrum of G peaks at m/z 1151, 1235, and 1263 are molecular ions with different fatty acid species as shown. Abbreviations LCB — long chain base, FA — fatty acid, hFA — hydroxyfatty acid. Hex — hexose, HexNAc — /V-acetylhexosamine. (From Taki, T. et al.. Anal. Biochem., 225, 24—27, 1995. With permission.) Continued. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

Glycosphingolipids of cestode species (tapeworms) analysed so far can be structurally divided into two different groups and are, therefore, separately discussed. As far as available, structural data on glycoprotein-glycans are presented independently. 

Isolation of the neutra-L glycosphingolipids. In a typical extraction procedure, 10purified human neutrophils yielded 100-150 mg of total glycosphingolipids. As shown in Table I, glycosphingolipids account for approximately 10% of the total cellular dry weight of the neutrophil. Separation of the total neutrophil lipids by DEAE-sephadex and silicic acid column chromatography yielded 70-100 mg of neutral glycosphingolipids from lO cells. 

Figure 1. Thin-layer chromatography of fractions 1-lV isolated from human neutrophils. The separation is on a plate of silica gel 60 (HPTLC) in solvent system A. S erythrocyte glycosphingolipid standards 1-4 human neutrophilglycosphingo-...

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... 

Application of buffered tetrahydrofuran extraction to gastric mucosa, followed by careful examination of the aqueous phase for various glycosphingolipids, indicated that this phase contained sialoglycosphingolipids and considerable quantities of neutral glycosphingolipids (22). These were separated from the acidic glycosphingolipids by DEAE-Sephadex column chromatography (23). 

The development of methods for separating lipid substances from natural sources has opened up new aspects in this field, (a) The use of chromatography has led to rapid and exact separation of compounds on a preparative scale, as well as in micro quantities, (b) It was found that, in tissues other than the central-nervous material (of mammals), there exists a group of glycosphingolipids having a sulfate group. 

In the case of acidic glycolipids the relative proton affinity of chemicals can shift the balance for negative ionisation in favor of co-eluted compounds. Pre-analytical separation under acidic conditions serves also to reduce as much as possible the dispersion in the MS spectrum of the metabolite into multiple m/ z representing the various adducts of counterions Na, K, NH4, organic amines, ... which improves sensitivity of the test. Sulfatides are lost during the partition between the hexane and the methanol/water phase. The analysis of sulfatides involves the isolation of the glycosphingolipid fraction and the subsequent separation of sulfatides from neutral lipids by chromatography on DEAE-sephadex or DEAE-cellulose column (the variety of methods are referenced in the website CyberLipid (http //www.cyberlipid.org/). 

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