Myelin cerebrosides

In this manuscript, we will first describe the newly developed high performance liquid chromatography of cerebroside, sulfatide, and other minor galactolipids. This method allows complete analysis of a very small amount of these glycolipids in cell or membrane preparations. This will be followed by a description of our new method of determining surface galactolipids and its application to myelin cerebrosides. 

Using the new procedure, attempts were made to quantitate the cerebrosides located on the surface of myelin. Myelin is composed of multilamellar bilayers of membrane of approximately 70% lipid and 30% protein (16). About 20% of the total lipid consists of cerebroside and sulfatide. Because of the lipophilic nature of the ceramide moiety and the hydrophilic nature of galactose, it has been postulated that the galactose moiety of myelin cerebrosides is facing the surface while the ceramide moiety is buried within the bilayer. Even considering the multilamellar structure of myelin, at least several percent of the cerebrosides should be present on the myelin surface. The method described in this manuscript should allow us to determine surface cerebrosides to as little as 0.5% of the total cerebrosides. 

Cerebrosides are found in the brain and in the myelin sheath of nerve tissue The structure of the cerebroside phrenosine is... 

Component of the myelin sheath surrounding the axons of nerve cells. Additional compounds of the myelin sheath are phospholipids, cholesterol, cerebrosides, and specific keratins. The myelin sheath constitutes an isolating barrier during electrophysiological axonal signaling. 

Central nervous system myelin is enriched in certain lipids. Table 4-1 lists the composition of bovine, rat, and human myelin compared to bovine and human white matter, human gray matter, and rat whole brain [1] (see Ch. 3). While there are no absolutely myelin-specific lipids, cerebroside (galactosyl ceramide) is the most typical of myelin. With the exception of early development,... 

Peripheral and central nervous system myelin lipids are qualitatively similar. However, there are quantitative differences. PNS myelin has less cerebroside and sulfatide and considerably more sphingomyelin than CNS myelin. Of interest is the presence of the LM1 ganglioside, sialosyl-lactoneotetraosylceramide, as a characteristic component of myelin in the PNS of some species. These differences in lipid composition between CNS and PNS myelin are not, however, as dramatic as the differences in protein composition discussed below. 

Imbalances of brain amino acids may hinder the synthesis of brain lipids, leading to a diminution in the rate of myelin formation. Decreases of lipids, proteolipids and cerebrosides (Ch. 3) have been noted in several of these syndromes, e.g. maple syrup urine disease, when intra-myelinic edema is a prominent finding, particularly during the acute phase of metabolic decompensation [9]. Pathological changes in brain myelin are common, especially in infants who die early in life. The fundamental... 

Glycolipids are important constituents of the plasma membranes, of the endoplasmic reticulum, and of chloroplasts. The cerebrosides and their sulfate esters, the sulfatides, are especially abundant in myelin. In plant membranes, the predominant lipids are the galactosyl diglycerides.29 74 The previously described ether phospholipids (archaebacteria), ceramide arnino-ethylphosphonate (invertebrates), and sulfolipid (chloroplasts) are also important membrane components. 

The diseases considered here affect only a small fraction of the problems in the catabolism of body constituents. On the other hand, fewer cases are on record of deficiencies in biosynthetic pathways. These are more often absolutely lethal and lead to early spontaneous abortion. However, blockages in the biosynthesis of cerebrosides are known in the special strains of mice known as Jimpy, Quaking, and msd (myelin synthesis deficient).377 378 The transferases (points 11 and 12 of Fig. 20-11) are not absent but are of low activity. The mice have distinct neurological defects and poor myelination of nerves in the brain. 

Treatment with galactose oxidase. Oxidation of myelin with galactose oxidase was performed as described previously for similar oxidation of rat spinal cord preparations (4). Typically, myelin containing 0.2-1.1 mg protein is incubated with 100-500 units of galactose oxidase in 1-3 ml of phosphate buffer (10-100 mM, pH 7.2-7.4) with or without catalase. After the incubation at room temperature to 30°C for the duration of 30 min to overnight, myelin is recovered by centrifugation, washed, and lyophilized. Total lipids were extracted from the dried residue and the oxidized cerebroside as well as unaltered cerebrosides were analyzed as described above. Alternatively, the incubation was stopped by the addition of 5 volumes of chloroform/methanol (2/1, v/v) and mixed. The lower layer after centrifugation of the mixture is washed and then evaporated to dryness, and the total lipids obtained were analyzed as described above. 

Oxidation of myelin surface cerebrosides by galactose oxidase. Fig. 4 shows silica HPLC of a mixture containing benzoylated-non-hydroxy and hydroxycerebroside and benzoylated derivatives of 2,4-dinitrophenylhydrazone of oxidation products from nonhydroxy- and hydroxycerebroside. Standard curves of two 6-dehydro-derivatives were shown in Fig. 5. These standard curves demonstrate that the response of the benzoylated dinitrophenylhydrazones are linear between 0.025 nmol and 0.6 nmol. Since cerebrosides containing 5 nmol can be determined without tailing to these peaks, this method should allow the determination of as little as 0.5% of the oxidation product. The fact that each curve intersects 0 point in both the abscissa and ordinate indicates that even smaller amounts of these compounds can be detected by this technique. 

We obtained unexpected findings using this method to study myelin the oxidation by galactose oxidase of myelin-bound cerebrosides could not be detected. The oxidation did not occur either with the intact spinal cord preparation, with isolated myelin, or even with lyophilized myelin. In one experiment, lyophilized myelin containing 5 mg each of dry weight was incubated with 100, 200, and 500 units of galactose oxidase for 60 min at room temperature, and no cerebroside oxidation occurred. 

To further examine the inability of galactose oxidase in oxidizing myelin-bound cerebrosides, one mg each of lyophilized... 

Figure 7. Silica HPLC of product from galactose oxidase-treated myelin which were "coated by cerebrosides. See caption to Figure 4 for peak identification.

The second cause may be due to steric hinderance of neighboring components within the myelin sheath. However, we found that digestion of trypsin or phospholipase C cannot alleviate the problem. In addition, hypotonic treatment of myelin which affects the integrity of the bilayer and also causes the splitting of the lamellae at the external apposition (19, 20), did not result in the oxidation of the cerebrosides. Even cerebrosides in liposomes made from pure phosphatidyl choline could not be oxidized. Incidentally, this finding also contradicts Linington and Rumsby who reported significant oxidation of cerebrosides in liposomes made from myelin lipids. 

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