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.     

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.     

Isolation and Anti-Fatty Liver Activity of a Novel Cerebroside from the Sea Cucumber Acaudina molpadioides

Cerebrosides are a kind of important bioactive substance in sea cucumber. A novel cerebroside, AMC-2, was purified from the less-polar lipid fraction of the sea cucumber Acaudina molpadioides by repeated column chromatography. The major structure of AMC-2 was analyzed by gas chromatography-mass spectra. The amide-linked fatty acid unit was confirmed to be four saturated and monounsaturated α-hydroxy fatty acids, the long-chain base was dihydroxy sphingoid base with one double bond, and the glycosyl group was glucose. We also investigated the anti-fatty liver activity of AMC-2 in rats with fatty liver induced by orotic acid. AMC-2 significantly reduced hepatic triglyceride (TG) and total cholesterol (TC) levels at a diet supplement of 0.03% and 0.006%. The indexes of stearoyl-CoA desaturase (SCD) activity and mRNA expression were significantly decreased by AMC-2. This indicates that AMC-2 ameliorated nonalcoholic fatty liver disease (NAFLD) through suppression of SCD activity and impaired the biosynthesis of monounsaturated fatty acids in the livers of the rats.     

Sphingolipids in bean leaves (Phaseolus vulgaris)

Phytoglycolipid has been isolated for the first time from plant leaves (Phaseolus vulgaris). The purified product (almost identical with the phytoglycolipid isolated from flax seed) was a ceramide attached through phosphate diester linkage to an oligosaccharide, which consisted of the usual trisaccharide unit (inositol, hexuronic acid, hexosamine) to which were attached mannose, galactose, and arabinose. The major fatty acids were the saturated 2-hydroxy C(22), C(24), and C(26) acids; the major long-chain bases were dehydrophytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-8-trans-octadecene) (53%) and phytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-octadecane) (32%). A ceramide and a cerebroside were also isolated. In the ceramide the major fatty acids and the major long-chain bases were the same as in the phytoglycolipid. In the cerebroside, the fatty acid composition was similar to that in the ceramide and phytoglycolipid, but the long-chain bases consisted of dehydrophytosphingosine and phytosphingosine (7:1) with a substantial amount of unidentified long-chain base. The sugar component was glucose.     

FATTY ACID AND FATTY ALDEHYDE COMPOSITION OF GLIAL CELL LIPIDS ISOLATED FROM BOVINE WHITE MATTER

Abstract— Glial cells were isolated from bovine white matter by differential centrifugation. The fatty aldehyde and fatty acid compositions of ethanolamine glycerophosphatides (EGP), serine glycerophosphatides (SGP) and choline glycerophosphatides (CGP) were determined by gas-liquid chromatography. The fatty acid compositions of the sphingo-lipids including sphingomyelin, cerebroside and cerebroside sulphate, and of minor lipid components including cholesterol esters and triglycerides, were also determined by gas-liquid chromatography. The relative proportions correlated closely with the results obtained by O'B rien and S ampson (1965 b ) for adult human brain. The fatty aldehyde compositions of the glycerophosphatides were more closely related to the corresponding fatty acid compositions of the plasma membrane than of the mitochondria. Long-chain fatty acids (19–26 carbon atoms) were detected in sphingomyelin, cerebroside and cerebroside sulphate; this indicates that chain-elongation beyond C₁₈ occurs in the glial cells.     

Cerebroside of the dimorphic human pathogen, Candida albicans

Structural studies on the cerebroside isolated from the yeast form of a dimorphic pathogen, Candida albicans were carried out using fast atom bombardment mass spectrometry (FAB/MS), proton magnetic resonance spectrometry, gas chromatography-mass spectrometry and usual chemical methods. The component sugar was only glucose attached to ceramide in a beta-configuration. The major fatty acid was 2-hydroxystearic acid (62%). The predominant long chain base was identified as 9-methyl-C18-sphinga-4,8-dienine which is widely distributed in fungi and reported to be essential to the fruit-inducing activity of fungi. Therefore, the structure of the main molecular species of the cerebroside was determined to be N-2-hydroxystearoyl-1-O-beta-glucosyl-9-methyl-C18-sphinga-4 ,8-dienine. Cerebroside prepared from the mycelial form of C. albicans has the same structure.     

Cerebrosides of human aorta: isolation, identification of the hexose, and fatty acid distribution

Cerebrosides have been isolated from adult human aortic tissue. Each aorta was divided into portions classified as normal, fatty streaks, fibrous plaques, or complicated lesions. The cerebrosides were isolated by Florisil column chromatography, mild alkaline methanolysis, a second Florisil column, and preparative thin-layer chromatography. The concentration of cerebrosides was higher in fatty streaks than in the more advanced plaques; apparently normal tissue gave the same cerebroside content as plaques found in the same aorta. The quantities of cerebrosides ranged from 0.01 to 0.73% of the total lipid. Of the 16 cerebroside samples isolated, 10 contained glucosyl ceramide, 1 contained galactosyl ceramide, and 5 were not analyzed for specific hexose. The fatty acid distribution was determined for 11 of the samples; it was similar to that of spleen cerebrosides. We suggest that aortic cerebrosides originate in the plasma. "Normal tissue" cerebrosides contained less unsaturated fatty acid than cerebrosides from a diseased area of the same aorta. Preparative thin-layer chromatography, the last step of cerebroside isolation, always separated at least two unidentified substances. One of these substances yielded both glucose and galactose on acid hydrolysis. Their removal from the cerebrosides accounts for the lower values for cerebroside compared to other authors' determinations.     

Isolation and anti-fatty liver activity of a novel cerebroside from the sea cucumber Acaudina molpadioides

Cerebrosides are a kind of important bioactive substance in sea cucumber. A novel cerebroside, AMC-2, was purified from the less-polar lipid fraction of the sea cucumber Acaudina molpadioides by repeated column chromatography. The major structure of AMC-2 was analyzed by gas chromatography-mass spectra. The amide-linked fatty acid unit was confirmed to be four saturated and monounsaturated α-hydroxy fatty acids, the long-chain base was dihydroxy sphingoid base with one double bond, and the glycosyl group was glucose. We also investigated the anti-fatty liver activity of AMC-2 in rats with fatty liver induced by orotic acid. AMC-2 significantly reduced hepatic triglyceride (TG) and total cholesterol (TC) levels at a diet supplement of 0.03% and 0.006%. The indexes of stearoyl-CoA desaturase (SCD) activity and mRNA expression were significantly decreased by AMC-2. This indicates that AMC-2 ameliorated nonalcoholic fatty liver disease (NAFLD) through suppression of SCD activity and impaired the biosynthesis of monounsaturated fatty acids in the livers of the rats.     

Characteristic Constituents of Glycolipids from Frog Brain and Sciatic Nerve

Cerebroside, sulfatide, monoglycosyl glyceride, and ester cerebroside were isolated from frog brain and sciatic nerve, and their distribution and chemical constituents were determined. The long-chain base compositions of cerebroside, sulfatide, and ester cerebroside were unique in the presence of branched-base components (5-15% of the total bases) and in the abundance of saturated dihydroxy base components (15-45% of the total). The amount of branched long-chain bases was greater in sciatic nerve than in brain. The hexose composition of the glycolipids consisted entirely of galactose except for brain cerebroside, in which a small amount of glucose was detected. Monogalactosyl glyceride consisted of the diacyl and alkylacyl forms, in a molar ratio of 81:19 for brain and 62:38 for sciatic nerve. The fatty acid composition of glycosphingolipids was characterized by the predominance of hydroxy and nonhydroxy 24:1 acids, and the concentration of 24:0 was extremely low. The proportion of unsaturated fatty acids accounted for 80% of the total. Major fatty acids of monogalactosyl glyceride were palmitic, oleic, stearic, and palmitoleic acids; the highest concentration was that of palmitic acid. Ester cerebroside was separated into three subfractions mainly on the basis of the proportion of hydroxy and nonhydroxy components in the amide-linked fatty acids.     

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.     

Linoleic acid biosynthesis in the pea aphid,Acyrthosiphon pisum (Harris)

The pea aphid Acyrthosiphon pisum (Harris) incorporated [1-¹⁴C]acetate into a phospholipid dienoic fatty acid in a time-dependent manner. In 2-h incubations, the incorporation of radioactivity into the 18:2 fraction was minimal, whereas after 45 h 18:2 was the major fatty acid labeled. Ozonolysis of the isolated dienoic fatty acid methyl ester followed by radio-gas-liquid chromatography showed that radioactivity was associated with fragments containing carbons 1–9 and 13–18. These data established the location of the double bonds in the 9,12 positions and indicated that the entire molecule was labeled from [1-¹⁴C]acetate. Tetracycline-treated aphids synthesized linoleic acid in the same proportions as untreated controls. Scanning electron microscopy showed that over 50% of the treated insects had greatly reduced numbers of intracellular symbiotes or lacked them or most of the existing symbiotes had an abnormal appearance. Therefore, we conclude that intracellular symbiotes are not involved in the biosynthesis of linoleic acid in the pea aphid.     

Identification of a xylose-containing cerebroside in the salt gland of the herring gull

A pentose-containing cerebroside has been identified in the salt gland of the herring gull, using mass spectrometry of acetyl and trimethylsilyl derivatives. A detailed interpretation of the spectra allowed a conclusion concerning the major long-chain base (the C(20) homolog of sphingosine) and the major fatty acids (C(22)-C(25) 2-hydroxy fatty acids), using reference spectra of synthetic galactosylceramides. A six-membered glycose ring (aldopyranose) was demonstrated by mass spectrometry of the acetyl derivative of periodic acid-oxidized and sodium borodeuteride-reduced pentosylceramide. By gas-liquid chromatography and mass spectrometry of methanolysis products, the pentose was shown to be identical with xylose. The procedures were applied to 25-50 micro g of glycolipid.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

IN VIVO STUDIES ON THE METABOLISM OF [15,16-3H]TETRACOSANOIC ACID (LIGNOCERIC ACID) IN ADULT RAT BRAIN

—Adult rats were killed 16 h, 48 h, 6 days and 21 days after intracerebral application of n-[15,16-³H]tetracosanoic acid (lignoceric acid). After incorporation into complex lipids with a strong preference for the ester-bound fatty acids of glycerophospholipids, radioactivity decreased with time. The incorporated activity into the amide-bound fatty acids of sphingolipids was also shown to decrease, with exception of the cerebroside of the hydroxy fatty acid type (cerebron fraction). Only negligible amounts of labelled triglyceride and cholesterol ester could be detected. The fatty acids derived from the complex lipids were analysed by radio gas chromatography. It was revealed that some of the applied labelled lignoceric acid was hydroxylated and incorporated into the cerebron fraction while the rest had their chains shortened. In the latter case all even and odd numbered chain lengths down to C₁₈ and C₁₆ (stearic and palmitic acid) were detected. At this stage, the pool of the degradation products of lignoceric acid is stabilized by the preferred incorporation of fatty acids of these chain lengths into glycerophospholipids. A time-dependent desaturation to oleic acid from stearic acid was observed.     

Composition of human cerebrospinal fluid cerebroside

A technique was developed to isolate sufficient material for compositional analysis of cerebroside from pooled human cerebrospinal fluid. The carbohydrate moiety was principally galactose. The sphingosine base and fatty acid compositions were found to be similar to that of brain cerebroside. The presence of a contaminant in commercial silica gel which chromatographed like the trimethylsilyl derivative of glucose is described.     

Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.     

Lipid composition and sphingolipid fatty acids of myelin in fruit bat brain

Myelin was isolated from the brain of adult fruit bats (Rousettus aegyptiacus) in a discontinuous sucrose gradient. Cholesterol comprised 189.0 mol/100 mol lipid phosphorus, galactolipids 60.3 mol/100 mol phosphorus and plasmalogens 32.5 mol/100 mol phosphorus. Choline and ethanolamine glycerophosphatide were present in nearly equal amounts followed by serine glycerophosphatide, sphingomyelin and inositol glycerophosphatides.The fatty acid composition of sphingomyelin and non-hydroxy cerebroside was determined by gas-liquid chromatography. Fatty acids were mainly saturated or mono-unsaturated with a small percentage of polyunsaturated fatty acids present.The lipid composition and sphingolipid fatty acid distribution in bat myelin was fairly similar to that of other species.     

Characteristic distribution of glycolipids in gadoid fish nerve tissues and its bearing on phylogeny.

Glycolipids were isolated from nerve tissues of gadoid fishes including Alaskan pollack and Pacific cod. Their chemical structures were determined by gas-liquid chromatography and gas chromatography-mass spectrometry, and their constituents were analyzed in detail and compared with those of glycolipids from other fish groups. The results revealed that gadoid fish nerve membranes contain peculiar glycolipid molecular species that are distinctly different from those in other teleostean fishes and higher vertebrates. The mole percentage ratio of the four major glycolipids (cerebroside-sulfatide-galactosylglyceride-sulfogalactosylglyce ride) was 48:12:25:15, indicating profound accumulation of glycoglycerolipids. Galactosylglyceride and sulfogalactosylglyceride were primarily of the diacyl type (greater than 90%), the major fatty acids being 16:0 and 18:1. An abundance of glucocerebroside (25 to 55% of cerebroside) and its fatty acid ester (37 to 47% of ester cerebroside) was noted. Cerebroside and sulfatide were characterized by the absence of hydroxy and odd numbered fatty acids, and 24:1 acid was a predominant component of both glucocerebroside and galactocerebroside. Subcellular fractionation revealed that myelin membranes comprised such unusual glycolipid constituents as those seen in whole nerve tissues. A vertebrate whose nerve membranes consist of such peculiar glycolipid molecules has not previously been reported. The characteristics of the glycolipid composition in gadoid fishes are discussed in relation to myelin functions, physicochemical properties of nerve membranes, and the phylogenic significance of this fish group.     

Isolation ofN-2′-Hydroxyhexadecanoyl-1-O-β-d-glucopyranosyl-9-methyl-4,8-d-erythro-sphingadienine from fruiting bodies of twoBasidiomycetes fungi

Fruiting bodies ofClitocybe geotropa andClitocybe nebularis were extracted with chloroform-methanol and the extracts were fractionated by chromatography. The title compound was isolated in pure form from both extracts. The structure was verified by NMR spectroscopy, sugar and fatty acid analysis, and FAB-MS.Abbreviations TMS trimethylsilane - DMSO dimethylsulfoxide     

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.