Sphingomyelin determination

Karlsson AA, Michelsen P, Odham G. 1998. Molecular species of sphingomyelin determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization. J Mass Spectrom 33 1192. 

It can be seen from Figure 1 that the choline-containing phospholipids, phosphatidylcholine and sphingomyelin are localized predominantly in the outer monolayer of the plasma membrane. The aminophospholipids, conprising phosphatidylethanolamine and phosphatidylserine, by contrast, are enriched in the cytoplasmic leaflet of the membrane (Bretcher, 1972b Rothman and Lenard, 1977 Op den Kamp, 1979). The transmembrane distribution of the minor membrane lipid components has been determined by reaction with lipid-specific antibodies (Gascard et al, 1991) and lipid hydrolases (Biitikofer et al, 1990). Such studies have shown that phosphatidic acid, phosphatidylinositol and phosphatidylinositol-4,5-fc -phosphate all resemble phosphatidylethanolamine in that about 80% of the phospholipids are localized in the cytoplasmic leaflet of the membrane. 

Caveolae-dependent endocytosis occurs in cholesterol- and sphingomyelin-rich flask-shaped invaginations of the cell membrane known as caveolae (67). The shape is determined by a framework constructed by the protein caveolin. The protein binds plasma cholesterol, inserting into... 

Lipids also show asymmetrical distributions between the inner and outer leaflets of the bilayer. In the erythrocyte plasma membrane, most of the phosphatidylethanolamine and phosphatidylserine are in the inner leaflet, whereas the phosphatidylcholine and sphingomyelin are located mainly in the outer leaflet. A similar asymmetry is seen even in artificial liposomes prepared from mixtures of phospholipids. In liposomes containing a mixture of phosphatidylethanolamine and phosphatidylcholine, phosphatidylethanolamine localizes preferentially in the inner leaflet, and phosphatidylcholine in the outer. For the most part, the asymmetrical distributions of lipids probably reflect packing forces determined by the different curvatures of the inner and outer surfaces of the bilayer. By contrast, the disposition of membrane proteins reflects the mechanism of protein synthesis and insertion into the membrane. We return to this topic in chapter 29. 

The spectrum of sphingomyelin (Figure 11) taken at higher emitter temperatures (25-28 ma) shows a transition from the pattern described in Scheme A to one dominated by ions at m/is 834 and 916 and 918. These correspond to addition of cholTne (mass 104) to the major molecular species CigiO (MW 730), C24 1 (MW 812) and C24 0 (MW 814). This assignment of (M + choline)+ has been confirmed by determination of the exact mass of the m/e 832 ion as m/e 834.7106 (calculated for C46Hg707N3P 834.7060J. 

A series of studies show that sphingolipids derived from milk protect against development of colon tumors in mice. In the initial study (Dillehay et al., 1994), mice fed diets supplemented with 0.025, 0.050 or 0.1% sphingomyelin had less than half the incidence of 1,2-dimethylhydrazine (DMH)-induced colon tumors than mice fed non-supplemented diets. In addition, a diet supplemented with 0.025% of the ganglioside GMj produced significantly fewer DMH-induced aberrant crypt foci (ACF) than control animals. ACF are microscopically determined pre-neoplastic lesions that can develop into colon tumors. 

The skin is cleaned and anesthetized as for other similar procedures, and lOmL of fluid is aspirated into a syringe connected to the spinal needle that is typically used. Sterile containers such as polypropylene test tubes or urine cups are used to transport the fluid to the laboratory. If a specimen is for the determination of the lecithin-sphingomyelin (L/S) ratio, the container is immediately placed in ice if it is for spectrophotometric analysis, the specimen should be transferred to a brown tube or bottle to prevent photodegradation of bilirubin. Alternatively the specimen container may be wrapped in aluminum foil. 

Determination of Lecithin/Sphingomyelin Ratio and Saturated Phosphatidylcholine ... 

SpiUman T, Cotton DB, Lynn SC, Bretaudiere JP. Influence of phospholipid saturation on classical thin-layer chromatographic detection methods and its effect on amniotic fluid lecithin/sphingomyelin ratio determinations. Clin Chem 1983 29 250-5. 

Antonis (A3) has estimated phospholipids by a procedure for determining fatty acids. This technique requires a total serum extract and a phospholipid-free extract for the measurement of both total and free fatty acids, the difference between them being a measure of the phospholipid content. Free fatty acids (A4) are determined on a phospholipid-free extract by a procedure based on partitioning the fatty acids as copper soaps into chloroform, and subsequent photometric determination of the copper with diethyldithiocarbamate. Phospholipids, as well as the free fatty acids present in the total lipid-extract, are measured by the same method, since they also form a complex with copper that is soluble in chloroform. A criticism of this technique is that equal response is not given by dipalmitoyl lecithin, dipalmitoyl cephalin, or beef brain sphingomyelin. 

CETP activity can be measured in vitro by a variety of methods, conditions, and substrates, but a common method is to monitor the transfer of [ C]CE between HDL and LDL. The donor and receptor lipoproteins are separated by heparin-MnCl2 precipitation and aliquots are counted [66,72]. CETP activity is determined by the difference between CE transfer with and without CETP. Fielding and coworkers [74] have devised an equally useful method, measuring CE transfer from cholesterol-lecithin liposomes to sphingomyelin-cholesterol liposomes. 

Other interfacial chemistries are found in juxtamem-brane domains of certain proteins that show a preferential interaction with specific lipids in their head group region. Important lipids involved in these interactions include the polyphosphate phosphatidylinositol lipids, cholesterol, gangliosides, and sphingomyelin. Interfacial chemistries play a role in the formation of microdomains and can determine the effective concentration of certain amphiphilic drugs in different membranes or even in different leaflets of the same membrane. 

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