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.     

Glycosphingolipids from rabbit aorta, plasma, and red blood cells: effects of high cholesterol-high fat diets on fatty acid distribution and quantity of glycosphingolipids

Four glycosphingolipids were isolated from rabbit aorta, plasma, and red blood cells. They were identified, by thin-layer chromatography and by quantitative analysis of hexose and fatty acid, as cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The rabbits had been maintained on a normal diet or on one of three high cholesterol diets for 180 days. The quantities of the glycosphingolipids and their fatty acid distributions were determined, and comparisons were made between the control and experimental animals. Aorta and plasma glycosphingolipids were more affected by the high cholesterol diets than were those from red blood cells. The effects on aorta and plasma glycosphingolipids were similar. The amount of cerebroside was increased in aorta and plasma in all animals in the experimental groups. The amount was also increased in red blood cells in rabbits from two of the experimental groups. The average fatty acid chain length was greater in the lipids from the experimental animals than in those from the control animals for all measured glycosphingolipids from aorta. The average chain length was also greater in cerebrosides from the experimental animals from all three tissues. Probably the most notable differences in the experimental animals were the increased 24:1/24:0 ratios and the increased concentrations of 24:2. These increases occurred in nearly all samples from plasma and aorta, but not in red blood cells. There was also an increase of total unsaturated fatty acids in aorta cerebrosides from the experimental animals. Except for the increase in 24:2, lard generally caused more deviation from normal than did cottonseed oil when the level of cholesterol in the diet was 1%.     

Brain sphingoglycolipids in Krabbe's globoid cell leucodystrophy

Abstract— Seven sphingoglycolipids were isolated from the white matter of a patient with globoid cell leucodystrophy (Krabbe's disease). After purification by saponification and column and preparative thin-layer chromatography, these compounds were analysed for the carbohydrate composition and sequence and for fatty acid composition by paper and gas-liquid chromatography. The compounds were identified as gluco- and galactocerebrosides, lactosyl-ceramide, digalactosy I-glucosyl-ceramide, two types of tetrahexosyl-ceramides (asialo-ganglioside and globoside), and sulphatide. Glucocerebrosideconstituted 13 percent of total cerebroside in white matter, but sulphatide contained only galactose. Galactocere-broside and sulphatide exhibited compositions of fatty acids similar to those in normal white matter, with only minor abnormalities. Other sphingoglycolipids showed fatty acid patterns with relatively high proportions of longer-chain fatty acids, rather than the predominant C_18:0 acid usually found in ceramide hexosides of the brain. Hematoside, also found in the white matter in a significant amount, similarly contained a large proportion of longer-chain fatty acids, whereas other gangliosides contained predominantly C_18:0 acid. The abnormal ceramide hexoside pattern was restricted mostly to white matter except for glucocerebroside, which constituted 32 per cent of grey matter cerebroside. We postulate that the visceral type of sphingoglycolipids may be constituents of globoid cells, abundantly present in white matter and considered to be cells of mesenchymal origin.     

Isolation of Cerebroside from Pea Seeds

Cerebroside was isolated from pea (Pisum sativum L.) seeds by solvent extraction, mild alkaline hydrolysis and silicic acid column chromatography. The purified material was identified as cerebroside by thin-layer chromatography and infrared spectrometry. Hydrolysates of the cerebroside were divided into fatty acid, sphingosine base and sugar fractions, and analysed, mainly by gas-liquid chromatography. The major fatty acid components were hydroxytricosanoic, hydroxydocosanoic and hydroxytetracosanoic acids. Dihydrosphingosine was the predominant sphingosine base. Only glucose was detected in the sugar fraction. Based on these results, one of the major species of pea cerebroside is suggested to be N-hydroxytricosanoyl-glucopyranosyl-dihydrosphingosine.     

Content and chemical compositions of cerebrosides in lactose-assimilating yeasts

Cerebrosides were found in ten lactose-assimilating yeasts. Representative component ceramide moieties of cerebrosides from nine of these yeasts contained 9-methyl-4-trans, 8-trans-sphingadienine, and 2-hydroxy fatty acid with carbon chain lengths of 16 or 18. The major ceramide moieties in Brettanomyces anomalus, however, differed from those in other yeasts, and were predominately moieties containing 2-hydroxymyristic acid. Thus we found that various cerebroside molecular species are present in yeasts.     

The content of lipoperoxidation products in normal and atherosclerotic human aorta.

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 +/- 18 and 92 +/- 18 vs. 70 +/- 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 ± 18 and 92 ± 18 vs 70 ± 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

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.     

Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune

Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum commune were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 μg of the cerebroside fractions showed a discriminative stimulating activity on S. commune when tested by the method these authors adopted. The active substance in the fraction II was N-2′-hydroxypalmitoyl-1-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccum amygdali showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.     

Lipids of higher fungi. II. The nature of cerebrins and cerebrosides from the mushroom Clitocybe tabescens Scop

Four cerebrins (ceramides) and two cerebrosides were isolated from the mushroom Clitocybe tabescens by combining column chromatography and preparative thin-layer chromatography. Quantification of the amide-linked nonhydroxy and hydroxy fatty acids (NFA and HFA) was obtained by gas—liquid chromatography. The nature of the long-chain bases was determined by thin-layer chromatography. The major acids in cerebrins were: NFA 16:0, 17:0, 18:X, 22:0 and HFA h16:0, h18:0, h22:0, h24:0. In cerebrosides predominated 17:0, 22:0 and h16:0. The component long-chain bases in all samples were sphingosines, dihydrosphingosines, phytosphingosines and unidentified more polar bases. Glucose was detected as the sole sugar constituent in cerebrosides.     

LIPID COMPOSITION OF THE CENTRAL NERVOUS SYSTEM OF MARINE VERTEBRATES

—The lipid composition of the central nervous system of some marine vertebrates and two mammalian species (rat and man) was analysed by one- and two-dimensional quantitative thin-layer chromatography, and the cerebroside fatty acids were analysed by gas chromatography. The concentrations of sphingomyelin and cerebrosides are higher in mammals than in fishes, while no significant differences are observed for other lipid classes. Furthermore, in mammals the ratio between hydroxy and normal fatty acids in the cerebrosides is much higher than in fishes. The cerebrosides of mammals contain more very long chain fatty acids than those of marine vertebrates.     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.     

Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells.

Free ceramide, glucosylceramide, and sphingomyelin were isolated from mature cells of adult rat small intestine. Free ceramide and ceramide cleaved from sphingomyelin by enzymatic hydrolysis were fractionated by thin-layer chromatography on borate-impregnated silica gel plates. Sphingoid bases were characterized by gas-liquid chromatography of aldehydes formed upon periodate oxidation. Fatty acids were quantified as methyl esters. Ceramide structures were confirmed by direct-inlet mass spectrometry. Free ceramide was found to contain two major long-chain bases in nearly equal quantity: sphingosine, mainly linked to palmitic acid, and 4D-hydroxysphinganine associated with C20 to C24 fatty acids, 22% being hydroxylated. Sphinganine occurred as a minor component linked to nonhydroxy fatty acids. Sphingomyelin contained the three long-chain bases and 63% of its ceramide was N-palmitoyl-sphingosine. Mass spectrometry of glucosylceramide confirmed 4D-hydroxyshingamine as the major sphingoid base associated preferentially with longer chain hydroxy fatty acids.     

Splenic lipids in Gaucher's disease

Column chromatography (on cellulose, silicic acid, and Florisil) and thin-layer chromatography were employed for the separation and purification of lipid fractions from normal and Gaucher spleens. A new hydrolysis procedure, followed by paper chromatography, was used for identification of sugar moieties. A nonhydrolytic combined colorimetric procedure, with anthrone and orcinol, was used for the estimation of glucose and galactose separately in glycolipids. The limitations of this method were examined. Spleens from two control subjects and three patients with Gaucher's disease have been examined in detail. In all Gaucher spleens, the predominant feature was the massive acccumulation of glucocerebroside; neutral ceramide oligohexoside levels were probably within the normal range, as were other neutral lipids and phospholipids. In one case examined for gangliosides, these were increased twentyfold. One Gaucher spleen, in which others had reported that the stored "cerebroside" contained predominantly lactose as the saccharide moiety, has been examined in detail and it has been established that the stored material was, in fact, glucocerebroside, ceramide lactoside levels not being significantly elevated. In a further nine cases glucose was the major sugar detected in the splenic lipids.     

Spontaneous Aortic Lesions in Roe Deer (Capreolus Capreolus L)

In 34 out of 60 aortas from roe deer, aged from 6 months to more than 8 years, aortic lesions were found. The frequency of affected regions involved were, in the posterior abdominal portion 53.3 %, in the aortic arch 18.3 %, in the anterior abdominal portion 16.7 %, in the posterior thoracic portion 8.33 % and in the anterior thoracic portion 3.3 %. Of the observed lesions, fatty streaks were seen in 3, fatty streaks and fibrous plaques in 3, fatty streaks with complicated lesions (calcification and acid mucopolysaccharides) in 14 and fibrous plaques with complicated lesions in 14 of the aortas. Elastic tissue degeneration of the inner two thirds of the tunica media was principally found in the aorta of the animals beyond 4 years of age. The lesions significantly (P < 0.001) increased in number and severity with age and appeared to be more progressive in pregnant animals. There was, however, no significant difference between affected male and female animals in the different age groups.     

The isolation and partial characterization of glycolipids of normal human leucocytes

1. The lipids of purified human leucocytes were extracted with chloroform-methanol and the extract was washed with water. Glycolipids, isolated by Florisil chromatography, were subjected to mild alkaline hydrolysis and the alkali-resistant fraction was fractionated on a silicic acid column. 2. Three classes of glycolipid were separated. The less polar, containing 3.6% of the total glycolipid hexose as galactose, was tentatively identified as ceramide monohexoside. The major glycolipid fraction was characterized as ceramide dihexosides. The more polar glycolipids comprised 1.6% of the total glycolipid hexose as galactose and glucose (in the molar ratio 2:1) and were non-acidic. This class was separated as a mixture containing ninhydrin-positive glycolipids. 3. The ceramide dihexosides taken from two leucocyte preparations accounted for 15.2% and 16.4% by weight of the total lipids. 4. The carbohydrate moiety of the ceramide dihexosides contained galactose and glucose in the molar ratio 2:1. Partial acid hydrolysis and paper chromatography indicated that the hexoses are present as disaccharides, lactose being identified as one of them. 5. Palmitic acid (C(16:0)) and nervonic acid (C(24:1)) were the major fatty acids of this glycolipid. Hydroxy fatty acids were not detected.     

Kinetic Changes in Free Ceramide and Cerebroside during Germination of Black Gram

Free ceramide and cerebroside were isolated from black gram sprouts of all germinating stages. Free ceramide and cerebroside were found to increase during germination.

The major sphingosine bases of free ceramide were 4-hydroxysphingenine and 4-hydroxy-sphinganine (trihydroxy type) while that of cerebroside was sphinga-4,8-dienine (dihydroxy type). A change in the component sphingosine base was that 4-hydroxysphingenine in free ceramide and cerebroside increased slightly after germination.

The major fatty acid of free ceramide was α-hydroxylignoceric acid while that of cerebroside was α-hydroxypalmitic acid. Changes in component fatty acid were that α-hydroxylignoceric acid in both sphingolipids increased after germination.     

Characterization of Alkylgalactolipids from Calf Brain by High Performance Liquid Chromatography

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11 :9. After perbenzoylation. they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor components were oleyl. myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

Heterogeneity of ciguatoxins extracted from fish caught in the French Antilles

Ciguatoxin-like substances were extracted from the viscera or the flesh of eight Caribbean fish species, including small invertebrate feeders and large carnivores. The had similar properties, i.e. pharmacological action, solubility, chromatographic behaviour on silicic acid or Sephadex LH 20 column, stability in a weak acid solution and instability in alkaline medium. However, Florisil column and thin-layer chromatography showed different ciguatoxins whose number depended on tissue or species but not on fish trophic level. Less polar ciguatoxins appeared in salted and dried flesh. Thus, fish ciguatoxins are believed to be closely related substances, possibly changing in structure according to particular experimental conditions.