Composition of long chain bases in ceramide of the guinea pig Harderian gland.

Ceramide of the guinea pig Harderian gland was isolated and characterized. The purified ceramide gave two spots on thin-layer chromatography. Ceramide with the higher Rf value (NHCer) contained non-hydroxy fatty acids and that with the lower Rf value (HCer) contained 2-hydroxy fatty acids. The ratio of NHCer to HCer was 6:1. The non-hydroxy fatty acids of NHCer were composed of straight-chain acids (94.9%) and branched-chain acids (5.1%). The 2-hydroxy fatty acids were also composed of straight-chain acids (94.2%) and branched-chain acids (5.8%). The ratio of straight-chain acids to branched-chain acids was similar in NHCer and HCer. The long chain bases of NHCer and HCer consisted of straight chain sphinganines and sphingenines, and methyl-branched long chain bases. In NHCer, 59.9% of the total bases were methyl branched, and in HCer, 48.3%. The characteristics of ceramide, that is, the large amount of methyl-branched long chain bases and relatively small amount of methyl-branched fatty acids, are similar to those of cerebroside and sphingomyelin isolated from the same organ, although the ratios of constituents are different among these sphingolipids.     

Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.     

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

Fatty acid composition of cardiolipin from guinea pig Harderian gland

The fatty acid composition of cardiolipin from the Harderian gland of guinea pig was examined by gas chromatography and gas chromatography-mass spectrometry. At least 33 kinds of fatty acids were detected. Oleic acid was the most prominent component, accounting for 18.2 mol% of the total fatty acids. About 70.2 mol% of fatty acids had methyl branches. Ethyl branches were also detected (1.3 mol%). Straight chain saturated acids comprised only 10.3 mol%. On the other hand, linoleic, linolenic, and arachidonic acids were not found in this lipid. The 2-(2'-) acyl moieties contained larger amounts of oleic acid and smaller amounts of branched chain acids than the 1-(1'-)acyl moieties, but the saturated straight chain acids showed even distribution between the 1-(1'-) and 2-(2'-)positions. The fatty acids of cardiolipin from the liver of the same animal were also examined. Linoleic acid was the most abundant component (66.9 mol%), and saturated straight chain acids occupied 21.9 mol%. Branched chain acids were detected but comprised only 11.2 mol%.     

The identification of aminoalkylphosphonyl cerebrosides in the marine gastropod, Monodonta labio.

From Monodonta labio a mixture of aminoalkylphosphonyl cerebrosides was isolated in a yield of 1.0% of the total complex lipids. The aminoalkylphosphonyl cerebrosides were found to be composed of a major portion of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactopyranosyl]ceramide and a minor portion of 1-O-[6'-O-(aminoethylphosphonyl)galactopyranosyl]ceramide. Palmitic (49.7%) and 2-hydroxy palmitic (12.2%) acids were the main fatty acid constituents and a small amount of oleic acid was also detected. The long chain bases had a characteristic pattern with 16-22 carbon chain lengths, and were classified into four groups: normal dihydroxy monounsaturated bases (40.6%), branched dihydroxy monounsaturated bases (34.0%), dihydroxy diunsaturated bases (22.0%), and trihydroxy bases (2.2%). Among them dihydroxy 18 : 1 and 18 : 2 and branched 19 : 1 bases were predominant.     

Assay of fatty acid synthetase by mass fragmentography using [13C]malonyl-CoA

A new assay method for fatty acid synthetase using mass fragmentography was described. [2-13C]Malonyl-CoA was chemically synthesized from [2-13C]malonic acid and used as a substrate. The newly synthesized fatty acids were quantitated with a GC-MS instrument after methyl esterification. Monitoring of molecular ions of the newly synthesized fatty acids enabled us to determine the absolute amounts with heptadecanoic acid as an internal standard. Multiple products (14 : 0, 16 : 0, and 18 : 0) were measured individually. Using this technique, we obtained information about production profiles such as that of chain length against incubation temperature and about malonyl-CoA decarboxylation activity in enzyme preparations, and we also confirmed the presence of malonyl-CoA decarboxylation activity even in purified fatty acid synthetase from guinea pig Harderian gland. Compared with the conventional assay methods (spectrophotometric and radioisotopic), this method was more reliable and useful.     

Lipids from the guinea pig Harderian gland: use of picolinyl and other pyridine-containing derivatives to investigate the structures of novel branched-chain fatty acids and glycerol ethers

The lipid extracted from guinea pig Harderian glands was hydrolysed and the constituents were examined as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS, acetonide/TMS, nicotinate/TMS, picolinyl/TMS and nicotinylidene/TMS derivatives by capillary gas-liquid chromatography and gas chromatography/mass spectrometry. Over 70 compounds amounting to over 93% of the extract were identified. These consisted of 1-O-alkyl glycerols (glycerol ethers) with alkyl chains containing from 17 to 21 carbon atoms and fatty acids ranging from 14 to 26 carbon atoms. The alkyl chains in the glycerol ethers were straight, mono- and dimethyl-branched with the major site of branching being at C-14. All straight-chain acids from C14 to C26 were present, with the most abundant being n-24:0. Again mono- and dimethyl branched structures comprised the bulk of the remaining acids. Methyl groups tended to be towards the middle of the chain rather than in the more usual omega-1 (iso) and omega-2 (anteiso) positions, with C-14 again being a major site. The shorter-chain acids tended to have methyl groups closer to the acid group, with several of the short-chain compounds being substituted at C-2. Structural information on the acids was provided by the picolinyl derivatives and the sample provided an opportunity to evaluate these derivatives with branched acids other than the iso and anteiso compounds studied previously. They were found to be satisfactory for analysis of both mono- and dimethyl branched acids with the possible exception of compounds containing a methyl branch at C-4. However, in this case, structural information was provided by the methyl ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

FATTY ACIDS AND LONG CHAIN BASES OF VERTEBRATE BRAIN GANGLIOSIDES

Abstract— Fatty acid and long-chain base composition of gangliosides from brains of animals belonging to various vertebrate classes (fishes, amphibia, reptiles, birds and mammals) was studied by gas-liquid chromatography. Stearic acid was found to be the main fatty acid everywhere. Brain gangliosides of cold-blooded animals contain lesser amounts of stearic and higher amounts of palmitic and monoenoic acids as compared to those of mammals. Long-chain bases were separated as trimethylsilyl derivatives. Large amounts (23-48 per cent) of long chain bases with 20 carbon atoms were found in brain gangliosides of mammals while in those of cold-blooded animals they constitute 3-12 per cent of the total. The comparison of the composition of gangliosides with that of cerebrosides and sulphatides indicates, that the fatty acid and long chain base composition of gangliosides from mammalian brain differs from that of other glycosphingolipids, whereas in brains of cold-blooded animals they are much more similar.     

THE STRUCTURE OF BRAIN DIHEXOSYLCERAMIDE IN GLOBOID CELL LEUKODYSTROPHY1

Abstract— Dihexosylceramide from the brain of a case of globoid cell leukodystrophy was isolated and characterized. Studies utilizing partial acid hydrolysis and methylation indicated the carbohydrate sequence to be galactosylglucosylceramide. The carbohydrate moiety was liberated by ozonolysis and identified to be lactose by GLC. 4-Sphingenine and sphinganine were the only long chain bases detected. The chain length of the fatty acids present ranged from 14 to 25 carbons; C₁₈, and C₂₁ fatty acids composed one-half of the mixture. All analyses were performed by GLC and TLC utilizing micro-techniques.     

6-acyl galactosyl ceramides of pig brain: structure and fatty acid composition

Two glycolipids were isolated from pig brain and were shown to be the fatty acid esters of kerasin and cerebron in which the second fatty acid moiety is attached to the 6-position of the galactose. The point of attachment was shown in two ways: by permethylation and by cleavage with periodate. Methanolysis of the permethylated cerebroside esters yielded O-methyl sphingosines, methyl esters of nonhydroxy or 2-methoxy acids, and methyl 2,3,4-trimethyl galactoside. Cleavage of the cerebron ester with periodate, followed by treatment with sodium borohydride and dilute HCl, yielded ceramide plus 1-monoglyceride. The ester-linked fatty acids were primarily 16:0, 18:0, and 18:1, while the amide-linked fatty acids showed the wide assortment of chain lengths typical of brain cerebrosides. The methylation step, with silver oxide and methyl iodide, yielded two derivatives with the cerebroside esters, but the structural explanation for the difference was not elucidated. The galactose in the cerebron ester was shown to exist in the beta-pyranoside form.     

The existence of galactosylceramide I3-sulfate in serums of various mammals and its anticoagulant activity.

Human, porcine, goat, sheep, bovine, horse, canine, rat, mouse, guinea pig, and chicken serums were investigated for the existence of sulfatide. Among the ten mammal serums, seven were found to be sulfatide positive, and the amounts of sulfatide were determined to be: 16.29 nmol/ml serum (porcine), 9.39 (bovine), 12.71 (goat), 7.75 (horse), 1.21 (sheep), 0.64 (human), and 0.16 (dog). The existence of sulfatide in the serums of human, goat, sheep, cow, horse, and dog is here reported for the first time. It is suggested that sulfatide is widely distributed in the serums of various mammals except for rodents and that it takes part in the anticoagulant systems. The fatty acids of those sulfatides comprised mainly non-hydroxy fatty acids and a significant amount (18-53% of the total fatty acid) of hydroxy fatty acids with chain lengths of C16, C22, C23, and C24. The long chain bases comprised sphingenine, sphinganine, and 4-D-hydroxysphinganine. Experiments to elucidate the mechanism of the anticoagulant activity of sulfatide revealed that it was specific to sulfatide and that the galactose-bound sulfate group is essential for this activity. The activity of clusters of sulfatide molecules was much more pronounced than that of single molecules.     

Long-chain and very long-chain fatty acyl-coa synthetase activity in microsomes of jimpy mouse brain

The objective of this study was to determine whether the conversion of free, very long chain fatty acids (C₂₂–C₂₆) to their CoA-esters are involved in cerebroside synthesis, since cerebrosides are uniquely rich in very long chain fatty acids including lignoceric acid (C_24:0). We have studied lignoceroyl-CoA synthetase activity in the microsomes isolated from normal and jimpy mouse brain. The jimpy mouse lacks the ability to make myelin and is deficient in enzyme activities involved in the synthesis of myelin components, including cerebrosides. Unexpectedly, the lignoceroyl-CoA synthetase activity in jimpy brain microsomes was slightly higher than that in control microsomes. The palmitoyl (C_16:0)-CoA synthetase activity in jimpy brain was not different from the control. The level of cerebrosides in microsomes was grossly lower in jimpy brain. The implication of these findings and the involvement of lignoceric acid activation in cerebroside synthesis is discussed.     

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.     

Reptile brain cerebrosides and cerebroside sulfates

Studies have been made on the content of cerebrosides and cerebroside sulfates, as well as on their fatty acid composition in the brain of reptiles, subclass Anapsida (tortoises Emys orbicularis and Testudo horsfieldi) and subclass Lepidosauria (lizards Agama caucasica, A. sanguinolenta, Phrynocephalus mystaceus and snake Natrix tesselata). Total content of cerebrosides and cerebroside sulfates is higher in the brain of Lepidosaurians than in that of Anapsids. In the brain of tortoises, the content of cerebroside fraction with hydroxy fatty acids is significantly higher than of the fraction with normal fatty acids, which is also typical of the brain of homoiothermic mammals and birds. In the brain of Lepidosaurians, concentration of hydroxycerebrosides is considerably lower than of cerebrosides with normal fatty acids, which is similar to lower vertebrates -- amphibians and fishes. Low content of hydroxycerebrosides was found in all the Lepidosaurians investigated, irrespectively of their ecological conditions, being therefore dependent on their phylogenetic position. The composition of fatty acids, both normal and hydroxyderivates, as well as that of glycolipids from the brain of Anapsids and Lepidosaurians is essentially similar. However, some interspecific differences were noted in the pattern of fatty acids of cerebrosides and cerebroside sulfates of the brain, which concern the content of saturated and long chain fatty acids.     

PELIZAEUS-MERZBACHER DISEASE: BRAIN LIPID AND FATTY ACID COMPOSITION

Abstract— Biochemical analysis of the leukodystrophy brain from a case of Pelizaeus-Merzbacher disease, classical type, was performed. A decrease in the amount of solid material present was found. The lyophilized brain weight was reduced to 76% of normal with a slightly greater decrease in the amount of extractable lipid. Total myelin was diminished to 7% of normal. Among specific lipids plasmalogens were present in slightly lowered amounts. Cerebrosides and sulphatides were drastically reduced to 8% of normal, whereas sphingomyelin was less severely affected. Fatty acids from phospholipids were close to normal, only enols being slightly diminished. Analysis of pure cerebrosides and sulphatides revealed that the a-hydroxylated compounds as well as very long chain fatty acids (over C₁₈, especially C₂₃ to C₂₆) were greatly reduced. For chain lengths over C₁₈, the ratio of leukodystrophy fatty acid to normal fatty acid was close to 10%. The defect in very long chain fatty acids is estimated at 99.2% in total brain.
Thus, we have found a marked decrease in the amount of very long chain fatty acids and a less marked decrease in sphingolipids. The reduced amount of these acids appears to be partially offset by an increase in the amount of medium-chain fatty acids in sphingolipids. We conclude that one aspect of Pelizaeus-Merzbacher disease may be a defect in the synthesis of myelin very long chain fatty acids (as these acids are far much reduced than any other myelin molecule).     

FATTY ACID COMPOSITION OF CEREBROSIDES IN MICROSOMES AND MYELIN OF MOUSE BRAIN

Abstract— The fatty acid composition of cerebrosides isolated from myelin and from light and heavy microsomes of adult mouse brain was determined. 2-Hydroxy fatty acids represented 80 per cent of the fatty acids in myelin cerebrosides and approximately 55 per cent of the fatty acids in both light and heavy microsomes. In myelin, the majority of the fatty acids, both normal and hydroxy, were of chain length > C-20; in microsomes, shorter chain acids (C-16 to C-20) predominated.     

Cerebrosides of<Emphasis Type="Italic"> Candida lipolytica</Emphasis> yeast

Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.     

Synthesis of methyl-branched fatty acids from methylmalonyl-CoA by fatty acid synthase from both the liver and the harderian gland of the guinea pig

Partially purified fatty acid synthase preparations from both the liver and the harderian gland of guinea pig showed the same relative rates of utilization of methylmalonyl-CoA when compared to malonyl-CoA. Radio gas-liquid chromatographic analysis of the products generated from [methyl-14C]methylmalonyl-CoA and from [2-14C]malonyl-CoA in the presence of unlabeled methylmalonyl-CoA showed that the enzyme from both tissues generated identical mixtures of branched fatty acids. Therefore, it is concluded that the production of methyl-branched acids only by the harderian gland is not due to any unique specificity of the fatty acid synthase of this gland, in contrast to the conclusion reached from results obtained from mass spectrometry of the products generated by crude extracts (Y. Seyama, H. Otsuka, A. Kawaguchi, and T. Yamakawa J. Biochem. 90, 789-797, 1981).     

A new procedure for synthesis of 3-keto derivatives of sphingolipids and its application for study of fatty acid composition of brain ceramides and cerebrosides containing dihydrosphingosine of sphingosine

3-Keto derivatives were prepared in good yield by the oxidative procedure with 2,3-dichloro-5,6-dicyanobenzoquinone from N-acetyl sphingosine, N-palmitoyl sphingosine, N-lignoceroyl sphingosine, and N-lignoceroyl psychosine. None of these 3-keto derivatives, except the one from N-acetyl sphingosine, have been previously reported. Ceramides were isolated from a calf brain and reacted with 2,3-dichloro-5, 6-dicyanobenzoquinone. Ceramides containing sphingosine (4-sphingenine) were converted to 3-keto derivative, while those containing dihydrosphingosine (sphinganine) remained intact under these conditions. The 3-keto ceramides were then separated from the ceramides containing dihydrosphingosine by preparative thin layer chromatography. Similarly cerebrosides from the same calf brain were oxidized and fractionated to 3-ketocerebrosides (from cerebrosides containing sphingosine) and unreacted cerebrosides (cerebrosides containing dihydrosphingosine). The fatty acid composition of these four sphingolipids were determined. Both the ceramides and the cerebrosides containing sphingosine had more unsaturated fatty acids than the corresponding dihydrosphingosine-containing compounds. The ratio of C₁₆-C₂₀ fatty acids to C₂₂-C₂₆ acids was higher in the ceramides containing sphingosine than in ceramides containing dihydrosphingosine, while the ratio was reversed in cerebrosides. The possible precursor-product relationship among these lipids is discussed.