Cerebrosides determination

From the sea anemone M. senile was isolated j3-glucopyranosyl-( 1 - l)-ceramide, the structure of which was determined by mass spectrometry, H-n.m.r. spectroscopy, and chemical splitting. The major sphingosine in the cerebroside is a new base containing two double bonds and branching at one double bond, namely, 2-amino-D-eryf/jro-l,3-dihydroxy-9-methyl-( , E)-octadeca-4,8-diene. The major fatty acids are Ci6 0 and C20 o a-hydroxy acids.133... 

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. 

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. 

Our new method of sphingolipid analysis using high performance liquid chromatography allows us to determine not only their quantities but also their homolog compositions in a small amount of tissue. We now feel that less than 1 mg of fresh brain or nerve tissue is sufficient for complete analysis. The application of this new method for analyzing cerebroside and sulfatide-in plaques of brain from a patient with multiple sclerosis has been recently described (15). 

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. 

Porter, M. T., Fluharty, A. L., Flor Dela, S. D., and Kihara, H., Cerebroside sulfate determination in human fibroblast. Biochim. Biophys. Acta 258, 769-778 (1972). 

The diastereoisomeric configuration of the sugar-free cerebrosides (ceramides) was determined by Carter and coworkers,80 and found to be erythro, in accordance with the structure of sphingosine, which is isolated after drastic hydrolysis of sphingolipid mixtures or purified cerebrosides with acid. 

Menkes, Philippart, and Concone110 determined the relationship between the concentration of the sulfatides (and cerebrosides) of the human brain and the age of the human, and found a progressive increase from birth to maturity.111 Furthermore, the proportion of the a-hydroxy fatty acids increases, together with that of the C24-fatty acids. 

Moschidis et al. (1984) found small amounts of phosphonohpids in the egg yolk. The comparison of the FA composition of minor components shows that in sphingomyelins the amount of palmitic acid is twice that in the egg yolk lipids, whereas there is almost 40% less oleic acid. Linoleic acid at 30% is found at concentrations about three times higher in lysophosphatidylcholines than in egg yolk hpids, and twice as much as in the PC fraction. At 28%, docosanoic acid (C20 o = behenic acid) is present in considerable amounts in gangliosides, as opposed to lower concentrations of oleic and linoleic acids in comparison with the FA spectrum of egg yolk lipids. Momma et al. (1972) also found a high proportion of approximately 67% hydroxy FA in the cerebrosides. Many of the hydroxy FA have a chiral center. In any case. Momma et al. (1972) determined a relatively high concentration (15.7%) of tetracosanoic acid (C24 o = lignoceric acid), while smaller concentrations of the main FA of the egg yolk lipids were found in the cerebrosides. Table 14.5 shows an overview of the FA spectrum of the minor components. 

Based on the evidence of positive and negative ion mode FAB MS, the structure of cerebroside (1) was determined to be (4E,8E)-N-2-hydroxyhexadecanoyl-l-0-P-gluco-pyranosyl-9-methyl-Ci8-sphinga-4,8-dienine (Fig. 1), which had been isolated from Schizophyllum commune by Kawai and Ikeda [8] as a fungal fruiting body inducer. 

These data might contribute to the determination of structures of cerebrosides having the long-chain base of 9-methyl-Cu-sphinga-4,8-dienine, which are widely distributed among fiingi. 

Applications of h.p.l.c. to benzoyl derivatives of alditols, glycosides, amino-sugars, aldoses, and alditols has been examined the presence of benzoyl groups has the advantage that u.v. detection methods can be used, and indeed a method for the simultaneous determination of picomole levels of gluco- and galacto-cerebrosides involves benzoylation followed by h.p.l.c.Similarly, tritiated mannitol and fucitol were easily separated after O-benzoylation. ... 

The absolute stereochemistry of oceanapiside, the antifungal compound 83 isolated from a marine sponge, has been determined.Four new cerebrosides which induce neuronal differentiation in rat PC 12 cells have been isolated from an edible Chinese mushroom and found to have structures 84-87. ... 

The presence of 2-hydroxylated fatty acids can be easily determined by C NMR spectroscopy in cerebrosides containing sphingosine-type bases due to the chemical shift of C-2 (72.5 ppm). In cerebrosides with phytosphingosine-type bases, C-4 in the long-chain base and C-2 hydroxy-lated in the fatty acid moiety show similar chemical shifts [6]. 

ParkT, Park YS, Rho JR, Kim YH. Structural determination of cerebrosides isolated fi om Asterias amuremis starfish eggs using high-energy collision-induced dissociation of sodium-adducted molecules. Rapid Commun Mass Spectrom. 2011 25 572-8. 

---