Glycosphingolipids determination

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]. 

For the determination of glycosphingolipids in urine, a 24-h or a first-morning voiding urine is collected. 

Glycolipids (5,14) are primarily glycosphingolipids, molecules that have oligosaccharide groups attached to ceramide [104404-17-3]. They are present, at least in small amounts, in the membranes of most, if not all, tissues. They too, like cell-membrane glycoproteins, are recognition determinants. 

Structural studies of glycosphingolipids involves determination of the structure of the oligosaccharide chain and of the lipid moiety. For the oligosaccharide chain, it is necessary to determine the composition, molar ratio, and sequence of the monosaccharides, their pyranose or furanose nature, and the position of glycosidic bonds and their configuration for the lipid moiety, the composition of the fatty acids and sphingosine bases must be determined. Used for these purposes are the classical, chemical methods, conventionally accepted in the chemistry of carbohydrates and lipids and based on the degradation of compounds, enzymic, and physicochemical methods, primarily mass spectrometry and n.m.r. spectroscopy. 

Sphingosine bases are usually isolated from glycosphingolipids by treatment with methanolic HC1 containing some water,148 and characterized, in the form of the free bases or their 2,4-dinitrophenyl (DNP) derivatives,149 151 by t.l.c. For quantitative determination of bases, a colorimetric procedure is used, based on color formation of the base with Methyl Orange.152 The composition of the sphingosine bases, as their Me3Si derivatives, is determined by g.l.c. and g.l.c.-m.s.,148,153,154 as well as by using for this purpose... 

To determine the position of glycosidic bonds in glycosphingolipids, periodate oxidation and methylation are widely used. A characteristic feature of the periodate oxidation of glycosphingolipids is the stability, to oxidation under mild conditions,179 of the D-glucose residue that is located at the beginning of the carbohydrate chain and substituted at 0-4 this seems to be associated with steric hindrance caused by the close location of the lipid moiety. 

The studies discussed above have all dealt with glycosphingolipids isolated from neutrophils that were derived from normal whole blood. Wherrett (16) has presented detailed structural analysis of a tetraglycosylceramide isolated from polymorphonuclear leukocytes obtained from the urine of a patient with a urinary tract infection. This glycosphingolipid was determined to be lactoneotetraosylceramide. 

This information was obtained from samples of less than 5 pg by electron impact/desorption direct probe mass spectrometry. On the basis of complete structural analyles, to be presented elsewhere, we have been able to determine that the four fractions isolated thus far actually contain six different glycosphingolipids with the following structures ... 

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