Sphingolipid, detection

While the fluid mosaic model of membrane stmcture has stood up well to detailed scrutiny, additional features of membrane structure and function are constantly emerging. Two structures of particular current interest, located in surface membranes, are tipid rafts and caveolae. The former are dynamic areas of the exo-plasmic leaflet of the lipid bilayer enriched in cholesterol and sphingolipids they are involved in signal transduction and possibly other processes. Caveolae may derive from lipid rafts. Many if not all of them contain the protein caveolin-1, which may be involved in their formation from rafts. Caveolae are observable by electron microscopy as flask-shaped indentations of the cell membrane. Proteins detected in caveolae include various components of the signal-transduction system (eg, the insutin receptor and some G proteins), the folate receptor, and endothetial nitric oxide synthase (eNOS). Caveolae and lipid rafts are active areas of research, and ideas concerning them and their possible roles in various diseases are rapidly evolving. 

There are also differences in the composition of the phospholipids in human platelets. These cells contain a little over twice as much phosphatidylcholine as sphingomyelin. Another dramatic difference is noted in the ether phospholipid content in the human platelets, where there is approximately 37% vinyl ether-containing components in the ethanolamine fraction whereas there only are 10% or less in the other phosphoglyceride species. The vinyl ether linkage has not been detected in the sphingolipids of these 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]... 

The nanosized detection area Ar or volume created by STED also extends the power of fluorescence correlation spectroscopy (FCS) and the detection of molecular diffusion [74,95]. For example, STED microscopy has probed the diffusion and interaction of single lipid molecules on the nanoscale in the membrane of a living cell (Fig. 19.6). The up to 70 times smaller detection areas created by STED (as compared to confocal microscopy) revealed marked differences between the diffusion of sphingo- and phospholipids [74]. While phospholipids exhibited a comparatively free diffusion, sphingolipids showed a transient ( 10 ms) cholesterol-mediated trapping taking place in a < 20-nm diameter area, which disappeared after cholesterol depletion. Hence, in an unperturbed cell putative cholesterol-mediated lipid membrane rafts should be similarly short-lived and smaller. 

Colsch et al., 2007). Because the diagnosis may be complicated in cases of arylsulfatase A pseudodeficiency and sphingolipid activator protein deficiency, this measurement of sulfatide in the urinary sediment of affected individuals by a rapid, sensitive, and specific mass spectrometric method has been long wanted (Whitfield et al., 2001). Urinary sulfatides are now commonly detected using electrospray ionization-tandem mass spectrometry by means of the precursor ion scan 97. Levels are considerably increased to X20-30 folds as compared to controls which allows the rapid screening of a large number of samples. 

FIGURE 11-2 Lipid composition of the plasma membrane and organelle membranes of a rat hepatocyte. The functional specialization of each membrane type is reflected in its unique lipid composition. Cholesterol is prominent in plasma membranes but barely detectable in mitochondrial membranes. Cardiolipin is a major component of the inner mitochondrial membrane but not of the plasma membrane. Phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol are relatively minor components (yellow) of most membranes but serve critical functions phosphatidylinositol and its derivatives, for example, are important in signal transductions triggered by hormones. Sphingolipids, phosphatidylcholine, and phosphatidylethanolamine are present in most membranes, but in varying proportions. Clycolipids, which are major components of the chloroplast membranes of plants, are virtually absent from animal cells. 

A similar structure was seen in Aspergillus oryzae, the Japanese yellow mold (75). Trans-unsaturated hydroxy fatty acids ( 2-hydroxyoctadec-3-enoic acid) found in F. amygdali and A. oryzae cerebrosides have not been detected in sphingolipids of animals and plants. 

Pan and Charych, after their pioneering work for virus detection by PDA platform, extended its application for detection of cholera toxins. Initially, they employed sphingolipid gangliosides (Figure 10.7) along with 5,7-dicosadiynoic acid for him... 

Figure 10.7. The structure of the sphingolipid ganglioside GMl used for detecting cholera toxin is shown.

In recent years, HPLC coupled with ESI-MS has become a well-established method for the identification and detection of chemical stmctures of lipids, including sphingolipids. Recently, Sugawara and coworkers [44] identified the chemical structures of glucosylceramides from maize, rice, mushroom, and sea cucumber by liquid chromatography-ion trap mass spectrometry with an ESI interface. In the positive full-scan mode, [M-t-HJ" ", [M-l-H—H2O ] , or [M-l-H—162]" (loss of glucose) was used for MS/MS analysis to obtain the product ions, which were used for the identification of the glucocerebrosides. 

Fig. 140. Thin-layer chromatogram of sphingolipids [210]. Adsorbent silica gel G solvent chloroform-methanol-water (60 + 35 + 8) time of run 2 h spray reagents diphenylamine solution and Dragendorff reagent for detection of sphingomyelin amounts 50—100 jxg of each 2 sphingolipid mixture from beef brain 2 non-esterified cerebrosides 3 cerebroside sulphates 4 sphingomyelin 5 gang-...

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