Sphingomyelin isolation

Several analytical techniques can be employed to characterize and prove the structure of sphingomyelin isolated from cells. 

Sphingatrienes also occur naturally but in minor amounts. Sphingomyelin isolated from squid nerve and from starfish contains a branched, triunsaturated sphingoid base, 2-amino-9-methyl-4 , 8 , 10 -octadecatriene-l,3-diol (10), and glycosphingolipids from starfish contain the same long-chain base without the methyl branch (9). 

The actions of proteins isolated from sea anemones, or other coelenterates, involve mechanisms different from those described for saponins. Thus, hemolysins from sea anemone R macrodactylus are capable of forming ion channels directly in membranes (98). The basic protein from S. helianthus also forms channels in black-lipid membranes. These channels are permeable to cations and show rectification (99). This ability of S. helianthus toxin III to form channels depends upon the nature of the host lipid membrane (100). Cytolysin S. helianthus binds to sphingomyelin and this substance may well serve as the binding site in cell membranes (101-106). 

FIGURE 10.12 The mole ratio of carotenoid/phospholipid and carotenoid/total lipid (phospholipid + cholesterol) in raft domain (detergent-resistant membrane, DRM) and bulk domain (detergent-soluble membrane, DSM) isolated from membranes made of raft-forming mixture (equimolar ternary mixture of dioleoyl-PC (DOPC)/sphingomyelin/cholesterol) with 1 mol% lutein (LUT), zeaxanthin (ZEA), P-cryptoxanthin (P-CXT), or P-carotene (P-CAR). 

Proulx [30] summarized the published lipid compositions of BBM isolated from epithelial cells from pig, rabbit, mouse and rat small intestines. Table 3.1 shows the lipid make-up for the rat, averaged from five reported studies [30], On a molar basis, cholesterol accounts for about 50% of the total lipid content (37% on a weight basis). Thus, the cholesterol content in BBM is higher than that found in kidney epithelial (MDCK) and brain endothelial cells (Table 3.1). Slightly different BBM lipid distribution was reported by Alcorn et al. [31] here, the outer (luminal) leaflet of the BBM was seen to be rich in sphingomyelin content, while the inner leaflet (cytosol) was rich in PE and PC. Apical (brush border) and basolateral lipids are different in epithelia. The basolateral membrane content (not reported by... 

The data in Table 4-1 indicate that myelin accounts for much of the total lipid of white matter, and that the lipid composition of gray matter is quite different from that of myelin. The composition of brain myelin from all mammalian species studied is very much the same. There are, however, some species differences for example, myelin of rat has less sphingomyelin than does that of bovine or human (Table 4-1). Although not shown in the table, there are also regional variations for example, myelin isolated from the spinal cord has a higher lipid-to-protein ratio than brain myelin from the same species. 

Sphingomyelins were first isolated from brain and nervous tissue, where they are abundant, but they also occur in many other animal tissues. 

This zinc-dependent enzyme [EC 3.1.4.3] (also known as lipophosphodiesterase I, lecithinase C, Clostridium welchii ce-toxin, and Clostridium oedematiens 13- and y-toxins) catalyzes the hydrolysis of a phosphatidylcholine to produce 1,2-diacylglycerol and choline phosphate. The enzyme isolated from bacterial sources also acts on sphingomyelin and phosphatidylinositol however, the enzyme isolated from seminal plasma does not act on phosphatidylinositol. See Micelle... 

Droplets of different density and lipid protein ratios ranging from about 1.5 1 to 40 1 have been isolated from bovine mammary gland. Triglycerides are the major lipid class in droplets of all sizes and represent increasingly greater proportions of total droplet mass in increasingly less dense droplet preparations. Surface coat material of droplets contains cholesterol and the major phospholipid classes found in milk, i.e. sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidyl-serine. 

Morrison (1970) presented earlier data on the fatty acid composition of these lipids. Morrison and Hay (1970) described the isolation and analyses of milk sphingomyelin, glucosylceramide, and lactosylcera-... 

A considerable amount of data has been reported on the substrate preference of the phospholipase C present in the organism Bacillus cereus. Interestingly, three phospholipases C have been isolated and purified, the first of which has high specificity for phosphatidylcholine, the second for phospha-tidylinositol, and the third for sphingomyelin (often termed sphingomyelinase). Similar substrate requirements have been noted in the phospholipase C isolated from other bacteria. 

In chapter 3 the experimental route to isolation of individual classes of phospholipids from cellular preparations was described in some detail. Either a column-chromatographic or a thin-layer chromatographic (TLC) procedure can be used here. If preparative TLC plates (normally silica gel G) are available, the separation of sphingomyelin from its most likely contaminant, phosphatidylcholine, is easily accomplished. The only other probable contaminant would be monoacylglycerophosphocholine (lysolecithin), but it is usually present in very, very low concentrations. If only a relatively few cells are available for lipid extraction, the TLC route is the procedure of choice. If milligram quantities of sphingomyelin are desired, then the cell of choice is the bovine erythrocyte and the isolation can be accomplished as described by Hanahan (1961). 

Improvement of membrane separation technology has resulted in the isolation of MFGM-enriched material from commercially available products. A phospholipid-rich fraction can be extracted from whey (Boyd et al., 1999) and buttermilk (Sachedva and Buchheim, 1997) with a reported yield of 0.25 g of phospholipids/g of protein in buttermilk (Sachdeva and Buchheim, 1997). Microfiltration of whey derived from the Cheddar cheese process, using 0.2 pm ceramic filters results in a fraction containing two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, and lesser amounts of phosphatidylinositol, phosphatidylserine, sphingomyelin and cerebrosides (Boyd et al., 1999). The phospholipid fraction separated from the total lipids contains a larger proportion of mono- and polyunsaturated fatty acids (mainly oleic, Cig i and linoleic, C ) compared to the total lipid and the neutral lipid fraction (Boyd et al., 1999). 

Schmelz et al. (2000) then isolated the complex sphingolipids, gluco-sylceramide, lactosylceramide and the ganglioside GD3, from milk. These sphingolipids were added individually to the diet of mice at a level of 0.025 or 0.1%. All three sphingolipids reduced the number of DMH-induced ACF by about 40%, a reduction that was comparable to that obtained by sphingomyelin in earlier experiments. 

Differences in nutritional effects between PLs and TAGs can be caused by several factors not related to their fatty acid composition, such as the presence of a phosphate group and a nitrogen base (mainly chohne) that may interact in several metabolic pathways (82). Moreover, several glycerophospholipid preparations studied can contain other components such as cholesterol, cerebrosides, sphingomyelins also depending on their source, method of isolation, and purification. These components may also affect the nutritional properties. In this chapter, the metabolic fate of constituent fatty acids of PLs and TAGs will be compared. 

Christomanou, H., and Kleinschmidt, T., Isolation of two forms of an activation protein for the enzymic sphingomyelin degradation from human Gaucher spleen. Biol. Chem. Hoppe-Seyler 366, 245-256 (1985). 

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