Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract.

Sphingomyelins were isolated from mucosal layers of bovine rennet stomach, duodenum, jejunoileum, and colon ascendens. The ceramides obtained after phospholipase degradation were characterized by thin-layer chromatography, mass spectrometry, and gas-liquid chromatography. The main ceramide group from all regions consisted of dihydroxy long-chain bases and normal fatty acids. Sphingosine was the predominant base in all these fractions, and only in rennet stomach were smaller amounts of the C17 and C20 homologs present. Normal saturated C16, C18, C22, and C24 fatty acids were most abundant. In rennet stomach there was in addition a ceramide group having dihydroxy long-chain bases in combination with hydroxy fatty acids. Sphingosine was the predominant long-chain base and the fatty acids were 2-hydroxy C16, C22, C23, and C24. From jejunoileum three minor ceramide fractions were isolated; these consisted of phytosphingosine and normal fatty acids C22-C24), sphingosine and 2-hydroxy fatty acids (C16-C24), and phytosphingosine and 2-hydroxy fatty acids (C22-C24), respectively. No branched paraffin chains were found in significant amounts. Sphingomyelins with trihydroxy long-chain bases and 2-hydroxy fatty acids found in jejunoileum were also detected in bovine kidney and have not been demonstrated before. These sphingomyelins from both kidney and jejunoileum showed a preferential combination of trihydroxy bases and fatty acids with very long chains (C22-C24).     

Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori.

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of 相似文献   

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.     

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.     

Sphingosine Forms Channels in Membranes That Differ Greatly from Those Formed by Ceramide

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.     

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.     

Effects of Fat-Enriched Diet on the Content of Sphingolipids in the Brain and on Cognitive Functions in Old Rats

It was demonstrated that levels of free fatty acids and radioactively labeled ([¹⁴C]) ceramide in the liver, muscles, and brain of rats fed a saturated fat-enriched diet for 36 weeks (from 15 to 24 months) increased, as compared with the corresponding levels in control animals of the same age (24 months old). A high-saturated fat diet resulted in a rise in the content of sphingolipids synthesized de novo in the neocortex and hippocampus of 24-month-old rats. High levels of ceramide and sphingosine in the neocortex and hippocampus induced in these rats by the above diet correlated with considerable worsening of the conditioned reflex activity of animals (reflex of active avoidance in the shuttle chamber).     

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.     

Developmental changes of the lipidic part of the neutral glycosphingolipids of the rat stomach

Neutral glycolipids were purified from the glandular part of the stomach of rats of different ages from 20 days of gestation to 60 days after birth. The two major glycolipids were identified as glucosylceramide and isogloboside. Free ceramide was also detected. The concentrations of these sphingolipids remained almost stable with development. Monohexosylceramide contained 55 and 68% of 2-hydroxylated fatty acids at 20 and 22 days of gestation, respectively, and 82% in the adult. Its three major bases, C18-sphingenine, C18- and C20-4D-hydroxysphinganine were characterized by gas-liquid chromatography and mass spectrometry of their N-acetyl-O-trimethylsilyl derivatives. The occurrence of the bases changed with development. C18-sphingenine contributed for 26% of the bases at birth and 65% in the adult. Conversely, C18-4D-hydroxysphinganine contributed for 35% of the bases at birth and 9% in the adult. The ceramide part of isogloboside consisted of nonhydroxylated fatty acids and mainly C18-sphingenine throughout development. The percentage of long-chain fatty acids was higher in older animals. These results stressed the specificity of the lipidic part of the rat gastric glycolipids and their specific evolution during the development.     

Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta

NMR and electrospray ionization tandem mass spectrometry were used to show for the first time the presence of sphingomyelins in extracts of the tobacco hornworm Manduca sexta (Lepidoptera). The sphingosine in the ceramide was identified as tetradecasphing-4-enine, and the fatty acids were C18:0, C20:0, C22:0, and C24:0 (compound 1). Heterogeneity in the ceramide was observed in sphingomyelins from M. sexta. All of the sphingomyelins were associated with their doubly unsaturated sphingosine, tetradecasphing-4,6-dienine (compound 2), which contained the same set of fatty acids as compound 1 and represents a novel set of sphingomyelins not previously reported in Lepidoptera. Lipid rafts were isolated from brains of M. sexta, and the association of these novel sphingomyelins with rafts was confirmed. The existence of the additional double bond was also observed in ceramide and ceramide phosphoethanolamine isolated from M. sexta. The levels of the doubly unsaturated ceramide showed modest changes during metamorphosis of M. sexta. These results suggest that Manduca sphingomyelins may participate in the formation of lipid rafts, in keeping with their function in vertebrates.     

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.     

The chemistry and control of hereditary lipid diseases

Direct inlet mass spectrometry has been performed on different derivatives of a hematoside (a triglycosylceramide of a tumour) and the major monosialoganglioside of brain (a pentaglycosyl-ceramide). As a confirmation of earlier results it was shown that trimethylsilyl derivatives gave information on ceramide structure (fatty acids and long-chain bases) but no specific information on carbohydrate structure. Fully methylated derivatives on the other hand, not analyzed before, gave in addition to ceramide fragments, specific ions for the sialic acid as well as carbohydrate sequence and branching. Using these derivatives molecular ions were not obtained for the brain ganglioside. However, by reduction of the methylated derivatives with LiA1H₄ (amide groups of ceramide and amino sugars were reduced to the corresponding amines) and trimethylsilylation of the converted sialic acid ester group, molecular weight ions were obtained for both gangliosides. In addition very strong peaks were found for the complete carbohydrate plus the fatty acid, of importance for the determination of the type and exact ratio of sugars, and also the fatty acid composition of the molecules. Ions were also obtained for a conclusive information on carbohydrate sequence and branching. It is concluded that a combined mass spectrometric use of methylated and methylated plus reduced ganglioside derivatives affords structural information on the complete molecules, which will be of considerable help in the characterization of gangliosides on a microscale.     

Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS

By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.     

Ceramide Synthesis from Free Fatty Acids in Rat Brain: Function of NADPH and Substrate Specificity

At the subcellular level, the synthesis of ceramide from free lignoceric acid and sphingosine in brain required reconstituted enzyme system (particulate fraction, heat-stable and heat-labile factors) and pyridine nucleotide (NADPH). The mitochondrial electron transfer inhibitors (KCN and antimycin A), energy uncouplers (oligomycin and 2,4-dinitrophenol), and carboxyatractyloside, which prevents the transport of ATP and ADP through the mitochondrial wall, inhibit the synthesis of ceramide in the presence of NADPH but have very little effect in the presence of ATP. Similar to the synthesis of ceramide, the synthesis of ATP from NADPH and NADH by the particulate fraction also required cytoplasmic factors (heat-stable and heat-labile factors). Moreover, ATP, but not its analog (AMP-CH2-P-O-P), can replace NADPH, thus suggesting that the function of the pyridine nucleotide is to provide ATP for the synthesis of ceramide. The cytoplasmic factors were not required for the synthesis of ceramide in the presence of ATP. The maximum velocity for synthesis of ceramide from free fatty acids of different chain lengths (C16-C26) was bimodal, with maxima around stearic acid (C18) and behenic acid (C22). The relative rate of synthesis of ceramide parallels the relative distribution of these fatty acids in brain cerebrosides and sulfatides.     

Ceramide profiles in the brain of rats with diabetes induced by streptozotocin

Diabetes is associated with disturbances of brain activity and cognitive impairment. We hypothesize that ceramides may constitute an important contribution to diabetes-linked neuro-dysfunction. In our study we used rats injected with streptozotocin (STZ) as a model of severe hyperglycemia. Using the gas-liquid chromatography technique we found a significant increase of ceramide content in brains and a decrease in plasma of diabetic rats. The inhibitor of serine palmitoyltransferase, myriocin, reduced ceramide generation in hyperglycemic brains, although injected alone it exerted a paradoxical effect of ceramide upregulation. Myriocin had no impact on ceramide concentration in the plasma of either control or diabetic rats. The level of ceramide saturated fatty acids was elevated whereas the level of ceramide poly-unsaturated fatty acids was downregulated in brains of all experimental groups. The concentration of ceramide mono-unsaturated fatty acids remained unchanged. The pattern of individual ceramide species was altered depending on treatment. We noted an STZ-evoked increase of brain ceramide C16:0, C18:0 and C20:0 and a strong decline in ceramide C18:2 fatty acid levels. Some changes of brain ceramide pattern were modified by myriocin. We found a decreased amount of total ceramide-ω-6 fatty acids in STZ-treated rat brains and no changes in ceramide-ω-3 concentration. We conclude that ceramides may be important mediators of diabetes-accompanied brain dysfunction.     

Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.     

Identification of ceramide phosphorylethanolamine and ceramide phosphorylglycerol in the lipids of an anaerobic bacterium

Nearly half the phospholipids isolated from the anerobic bacterium Bacteroides melaninogenicus are phosphosphingolipids. The two major phosphosphingolipids have been characterized as ceramide phosphorylethanolamine and ceramide phosphorylglycerol. The long-chain bases of these phosphosphingolipids appear to have branched and normal saturated carbon chains of 17, 18, and 19 atoms; the phosphate is at the 1-position of the long-chain base. The composition of the amide-linked fatty acids of the phosphosphingolipids differs from that of the ester-linked fatty acids of the diacylphosphoglycerides in having a higher percentage of 14:0, 17:0, and 18:0 acids as well as containing nearly all the monoenoic fatty acids found in the bacterial lipids. The finding of phosphosphingolipids in bacteria is exceedingly rare and to our knowledge ceramide phosphorylglycerol has not been previously found in nature.     

In vivo metabolism of 3-ketoceramide in rat brain

Eight hours after intracerebral injection of a double-labeled 3-ketoceramide⁴, [1-¹⁴C]lignoceroyl 3-keto [1-³H]sphingosine, various brain sphingolipids were isolated. Free ceramide and the ceramide portions of nonhydroxy cerebroside and sphingomyelin were further fractionated into subgroups containing longer-chain or shorter-chain fatty acids. Nonhydroxy ceramide, nonhydroxy cerebroside and sphingomyelin containing longer-chain fatty acids had significant quantities of radioactivity with ³H/¹⁴C ratios similar to each other but lower than that of the injected material. The sphingolipids containing shorter-chain fatty acids were also significantly labeled; however, the ³H/¹⁴C ratios were much higher than that of the injected material. Hydroxy-ceramide and sulfatides contained very little radioactivity. However, hydroxy-cerebroside contained an amount of radioactivity comparable to that of the longer-chain nonhydroxy cerebroside with a similar ³H/¹⁴C ratio. It is proposed that the injected 3-ketoceramide was converted into ceramide, cerebroside, and sphingomyelin and that the fatty acids of these lipids were partly replaced by other fatty acids during the metabolic conversions.     

Glycosphingolipids in cestodes. Chemical structures of ceramide monosaccharide, disaccharide, trisaccharide and tetrasaccharide from metacestodes of the fox tapeworm, Taenia crassiceps (Cestoda: Cyclophyllidea).

The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal-phase TLC pattern of the neutral-fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Gal beta 1Cer, Gal beta 6Gal beta 1Cer and Gal beta 6Gal beta 6Gal beta 1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Gal beta 6Gal beta 6Gal beta 6Gal beta 1Cer and the component Gal alpha 4Gal beta 6Gal beta 6Gal beta 1Cer (both occurring in approximately equimolar proportions). The long-chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse-phase TCL, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.