Sphingolipids separation

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

In brief [43, 44], primary neurons were prepared from the cerebellum of 6-day-old mice and the cells were treated with different concentrations of the target compounds for 24 h. Radiolabeled 3-[ 14C] serine or [14C]galactose was added to the culture medium and incorporation into newly synthesized sphingolipids was analyzed after labeling for 24 h. Lipids were extracted, separated by thin-layer chromatography, and visualized with a phosphoimager. Radioactivity found in the selected lipids is expressed in relation to untreated cells. 

Lipids are made up of many classes of very different molecules that all show solubility properties in organic solvents. Mass spectrometry plays a key role in the biochemistry of lipids. Indeed, mass spectrometry allows not only the detection and determination of the structure of these molecules but also their quantification. For practical reasons, only the fatty acids, acylglycerols and bile acids are discussed here, although other types of lipids such as phospholipids, [253-256] steroids, [257-259] prostaglandins, [260] ceramides, [261,262] sphingolipids [263,264] and leukotrienes [265,266] have been analysed successfully by mass spectrometry. Moreover, the described methods will be limited to those that are based only on mass spectrometry, even if the majority of these methods generally are coupled directly or indirectly with separation techniques such as GC or HPLC. A book on the mass spectrometry of lipids was published in 1993. [267]... 

Lipids are bioorganic substances related to fatty acid esters and include a variety of compounds such as glycerol esters, waxes, phosphoglycerides, sphingolipids, natural hydrocarbons, some vitamins, etc. This diversity of compounds is explained by the fact that initially the term lipids was used to describe natural bioorganic substances soluble in hydrocarbons and insoluble in water. Lipids include both small molecules and polymeric materials. Because some simple lipids are not polymeric, their pyrolysis will be discussed only to the extent of being associated with the pyrolysis of complex lipids. However, non-polymeric lipids are commonly associated with polymeric ones, and pyrolytic techniques were frequently applied on the whole lipid without separation for purposes such as classification or identification of microorganisms based on the pyrolysis pattern of their lipids [1]. 

Lipids are a diverse group of biomolecules that dissolve in nonpolar solvents. They can be separated into the following classes fatty acids and their derivatives, triacylglycerols, wax esters, phospholipids, lipoproteins, sphingolipids, and the isoprenoids. 

Structure of sphingolipids in foods, especially foods of plant origin, is sparse. Recently, a developed LC/MS method with APCI positive ion mode has been nsed to separate, identify, and qnantify cer-amide and ceiebroside species simnltaneonsly in nnt samples inclnding almond, cashew, hazelnut, peanut, and walnnt. The ceiebroside consisting of 4,8-sphingadienine, a-hydroxypalmitic acid, and glucose as the head group was found to be the most abundant cerebroside in nuts. 

Once separation of a phospholipid mixture or individual phosphatide has been obtained then the compounds(s) need to be identified and also to be quantified. Provisional identification is made by comparing Rf values with known standards and by differential colour spray reagents (cf. Kates, 1972 Christie, 1982). Useful stains/sprays are those for phosphate, primary amino groups (ninhydrin), choline (Dragendorff) and sphingolipid. 

Most of the early tests were based on the hydrolysis of the natural sphingolipid which had been labelled in the hydrophilic portion. Thus, the substrate and products could be easily separated by two-phase solvent partition or by precipitation of the product. In many cases artificial chromogenic or fluorogenic glycosides could be used instead of radiolabelled substrates. However, in these cases it is very important to demonstrate that the conditions and specificities of the enzynie reactions are such that the results can be extrapolated to the natural substrate (e.g. Peters et al., 1975). Thus, in the case of Niemann-Pick or Krabbe s diseases the unsubstituted... 

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