Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae

Immune responses induced by glycans upon infection with Schistosoma mansoni may be mediated by either schistosomal glycoproteins or glycosphingolipids. In this study, we have elucidated the structural features of both carbohydrate moieties and respective ceramide units of complex glycosphingolipids from adult S. mansoni. Obtained data revealed a vast structural heterogeneity due to manifold combinations of different oligosaccharides and ceramide entities. Observed carbohydrate moieties included Lewis(X) (Le(X); Gal(β1-4)[Fuc(α1-3)]GlcNAc) as well as, in part, multiply fucosylated LacdiNAc (LDN; GalNAc(β1-4)GlcNAc) carbohydrate epitopes. Corresponding lipid portions comprised predominantly C18-sphingosine as well as C18- and C20-phytosphingosine derivatives. Intriguingly, glycosphingolipids carrying an Le(X) epitope contained predominantly C18-sphingosine, whereas LDN-based species exhibited mostly phytosphingosine derivatives, in addition to C18-sphingosine, indicating that the two classes of glycosphingolipids might be synthesized via different biosynthetic routes. Compared with literature data, adult worm glycosphingolipids with Le(X) epitopes revealed clear structural differences in comparison to corresponding cercarial species which have been shown to exhibit mainly sphinganine bases with 18-21 carbon atoms. Therefore, it may be hypothesized that the divergent structural features of the respective ceramide moieties are responsible for the published observation that only adult worm, but not cercarial glycosphingolipids are able to induce dendritic cell activation skewing the T-cell response toward a Th1 profile.     

Glycosphingolipids from Magnaporthe grisea cells: expression of a ceramide dihexoside presenting phytosphingosine as the long-chain base

Magnaporthe grisea is a fungal pathogen that infects rice leaves and causes rice blast, a devastating crop disease. M. grisea produces active elicitors of the hypersensitive response in rice that were previously identified as ceramide monohexosides (CMHs). Using several chromatographic approaches, mass spectrometry, and nuclear magnetic resonance, we identified ceramide mono- and dihexosides (CDH) in purified lipid extracts from M. grisea cells. As described by other authors, CMH consists of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecenoic or 2-hydroxyhexadecenoic acids and a carbohydrate segment consisting of one residue of glucose. CDHs, however, contain beta-galactose (1-->4)-linked to beta-glucose as sugar units and phytosphingosine as the long-chain base, bound to a C24 alpha-hydroxylated fatty acid. To our knowledge, this is the first report on the occurrence of CDH in a fungal species and illustrates the existence of an alternative path of ceramide glycosylation in fungal cells.     

The Chemical Structure of Glycolipids of Bovine Milk

Experiments were executed to elucidate the chemical structure of ceramide monohexoside (CMH) and ceramide dihexoside (CDH) isolated from cow’s milk, especially with regard to the nature of the sugar moiety of the molecules. The results have shown that the structure of CMH and CDH in bovine milk is β-glucosyl-(l→l)-N-acyl-sphingosine, namely ceramide glucoside, and β-galactosyl-(1→4)-β-glucosyl-(1→1)-N-acyl-sphingosine, namely ceramide lactoside, respectively.     

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.     

Divergent fate of unsaturated and saturated ceramides and sphingomyelins in rat liver and cells in culture

The metabolism of sphingomyelins and ceramides with defined labeled fatty acids was compared after injection in vivo or incubation with cultured cells. The liver was the major site of uptake of sphingomyelins and ceramides with 18:2 or 16:0 fatty acids, but with both sphingolipids a higher recovery of radioactivity was found with 16:0 species. The distribution of radioactivity among liver lipids showed that 1.5 h after injection of 18:2 sphingomyelin, only 21% of the label was found as sphingomyelin, and this value was 37% in the case of 16:0 sphingomyelin. There was a very marked difference in the metabolism of 18:2 and 16:0 ceramides. After injection of 18:2 ceramide only 14% of the radioactivity was recovered as sphingomyelin, and this value was more than 50% with 16:0 ceramide. [14C]18:2 ceramide was converted also to glucoceramide and hydrolyzed more extensively than 16:0 ceramide. These observations were extended to sphingomyelins and ceramides with other fatty acids, using Hep-G2 cells in culture. Significantly more radioactivity was recovered as labeled sphingomyelin after incubation with 16:0, 18:0, 20:0 and 24:0 sphingomyelins than with 18:1 and 18:2 sphingomyelins, while more labeled phosphatidylcholine and phosphatidylethanolamine were found with the unsaturated sphingomyelins. In analogy to the findings in vivo, in the Hep-G2 cells more 16:0, 18:0 and 24:0 ceramides were converted to sphingomyelin than 18:1 or 18:2 ceramides. These differences were also seen with cultured macrophages, in which a more marked reutilization for sphingomyelin formation was found with the saturated ceramide series. The sphingomyelin liposomes were tested also for their capacity to mobilize cholesterol, and a rise in plasma unesterified cholesterol occurred after injection of 18:2 sphingomyelin. Marked enhancement of cholesterol efflux from cholesterol ester-loaded macrophages was also seen with 18:1 and 18:2, 20:0 sphingomyelin in the presence of delipidated high-density lipoprotein. The present results demonstrate that the metabolic fate of sphingolipids is related to their fatty acid composition. While ceramides with saturated fatty acids are predominantly reutilized for sphingomyelin formation, those with unsaturated fatty acids undergo probably more rapid hydrolysis with liberation of fatty acids and channeling into glycerolipids.     

Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus)

Of the lipid extracts of the defense secretion from the Formosan subterranean termite, Coptotermes formosanus Shiraki, on high-performance thin-layer chromatography analysis, no glycolipids or phospholipids were detected, but free fatty acids and three novel ceramides were found (termed TL-1, TL-2, and TL-3). Free fatty acids were confirmed to be lignoceric acid (C24:0) and hexacosanoic acid (C26:0), as described previously [Chen, J., G. Henderson, and R. A. Laine. 1999. Lignoceric acid and hexacosanoic acid: major components of soldier frontal gland secretions of the Formosan subterranean termite (Coptotermes formosanus). J. Chem. Ecol. 25: 817-824]. TL-1, TL-2, and TL-3 were characterized as ceramides differing in hydrophobicity based on results of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, mild alkaline treatment, GC-MS analysis of fatty acid methylesters, and GC-MS analysis of sphingoid long-chain bases (LCBs) as trimethylsilyl derivatives. Fatty acids in TL-1 and TL-2 were C18:0, C20:0, and C22:0, and those in TL-3 were 2-hydroxy C18:0, C20:0, and C22:0. The most predominant LCB in TL-2 was a novel trihydroxy C(14)-sphingosine, 1,3,9-trihydroxy-2-amino-6-tetradecene. TL-3 contained C(18)-sphinganine and two kinds of novel sphingadienines, 1,3-dihydroxy-2-amino-7,10-hexadecadiene and 1,3-dihydroxy-2-amino-11,14-eicosadiene. Although examination of the biological activities of these novel ceramides was beyond the scope of these studies, because of the minuscule quantities available from termite secretions, it will be interesting in the future to synthesize these molecules for biological testing.     

The liver flukes Fasciola gigantica and Fasciola hepatica express the leucocyte cluster of differentiation marker CD77 (globotriaosylceramide) in their tegument

Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase treatment, on-target exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen. To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.     

Glycosphingolipids in murine lymphocytes from thymus and spontaneous and X-ray induced thymomas

Lymphocytes from spontaneous thymoma in AKR mice and from X-ray induced thymoma in C57B1/6 mice showed elevated levels (by 50% and 100%, respectively) of lipid-bound sialic acid as compared with lymphocytes from normal thymuses used as controls. Some ganglioside fractions in thymomas were elevated 4-6-fold over those in normal thymuses while other fractions decreased or disappeared. Neutral glycosphingolipid (NGSL) content in lymphocytes from thymomas was also changed. Thin-layer chromatography of NGSLs showed that the fractions migrating as ceramide monohexoside (CMH), dihexoside (CDH) and below globoside standards were increased, respectively, 2-3-fold, 3-6-fold and 2-fold in both types of thymomas. Methylation and gas-liquid chromatography analysis confirmed the presence of CMH, CDH and globoside in NGSLs isolated from X-ray induced thymoma.     

Rabbit muscle gangliosides

Four ganglioside fractions were isolated from rabbit muscle: one hematoside and three hexosamine-containing species. They were analyzed for hexoses, hexosamine, sialic acid, fatty acids, and long-chain base content. The molar ratios of sphingosine-hexose-hexosamine-sialic acid were: for hematoside, 1:2:0:1; for the disialogangliosides, 1:3:1:2; and for trisialoganglioside, 1:3:1:3. The carbohydrates were studied by thin-layer and paper chromatography. The hexoses were glucose and galactose; the hexosamine was N-acetylgalactosamine and the sialic acid was N-acetylneuraminic acid. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography. The fatty acid composition was similar in all of the four gangliosides. The most abundant fatty acids were 16:0 and 18:0, but significant amounts of 16:1, 18:1, 20:0, and 22:0 were also found. Hydroxy fatty acids were not detected. In all of the muscle gangliosides the main long-chain bases were C(18)-sphingenine and C(20)-sphingenine. In hematoside there were also measurable amounts of C(18)-sphinganine and C(20)-sphinganine, whereas in the major gangliosides only traces of C(18)-sphinganine were detected.     

Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes

The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.     

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.     

New ceramides from sea sponge Oceanapia sp

Total ceramides containing nonbranched and iso-branched C18- and C19-phytosphingosines acylated with non-hydroxylated fatty acids were isolated from a marine sponge Oceanapia sp. The structures of these compounds were determined by HPLC, NMR, MALDI-TOF MS, and GLC-MS of the Me3Si derivatives and by chemical transformations. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B(1), an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-(3)H]sphinganine or L-3-[(3)H]serine demonstrated that uog1 conferred fumonisin B(1) resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB(1), and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B(1) resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.     

Ceramide as a Mediator of Non-Alcoholic Fatty Liver Disease and Associated Atherosclerosis

Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Since plasma ceramides are increased in NAFLD and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, the role of ceramides in dyslipidemia was assessed using LDLR-/- mice, a diet-induced model of NAFLD and atherosclerosis. Mice were fed a standard or Western diet (WD), with or without myriocin, an inhibitor of ceramide synthesis. Hepatic and plasma ceramides were profiled and lipid and lipoprotein kinetics were quantified. Hepatic and intestinal expression of genes and proteins involved in insulin, lipid and lipoprotein metabolism were also determined. WD caused hepatic oxidative stress, inflammation, apoptosis, increased hepatic long-chain ceramides associated with apoptosis (C16 and C18) and decreased very-long-chain ceramide C24 involved in insulin signaling. The plasma ratio of ApoB/ApoA1 (proteins of VLDL/LDL and HDL) was increased 2-fold due to increased ApoB production. Myriocin reduced hepatic and plasma ceramides and sphingomyelin, and decreased atherosclerosis, hepatic steatosis, fibrosis, and apoptosis without any effect on oxidative stress. These changes were associated with decreased lipogenesis, ApoB production and increased HDL turnover. Thus, modulation of ceramide synthesis may lead to the development of novel strategies for the treatment of both NAFLD and its associated atherosclerosis.     

A novel GlcNAcalpha1-HPO3-6Gal(1-1)ceramide antigen and alkylated inositol-phosphoglycerolipids expressed by the liver fluke Fasciola hepatica

The acidic (glyco)lipids of the parasitic liver fluke Fasciola hepatica exhibited two different phosphate-containing species, designated AL-I and AL-II, which were analyzed by MALDI-TOF MS, ESI MS, NMR, methylation analysis, and combined GC-MS in conjunction with HF treatment. AL-I was structurally determined as 1-O-hexadecyl-sn-glycerol-3-phosphoinositol, an ether bond variant of lysophosphatidylinositol. The structure of AL-II was shown to be GlcNAcalpha1-HPO3-6Gal(1-1)ceramide. Ceramide analysis revealed as major components 2-hydroxyoctadecanoic acid [18:0(2-OH)] together with C18- and C20-phytosphingosines. AL-II was apparently highly antigenic and strongly recognized by both animal- and human-F. hepatica infection sera. Furthermore, inhibition ELISAs revealed that the unusual antigenic determinant GlcNAcalpha1-HPO3- phosphate might have a potential in the serodiagnosis of F. hepatica infections.     

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida)

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(β1-1)ceramide, Man(β1-4)Glc(β1-1)ceramide, GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide and Gal(α1-3)GalNAc(β1-4)GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine. Abbreviations: CDH, ceramide dihexoside; Cer, ceramide; CMH, ceramide monohexoside; CPH, ceramide pentahexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; Hex, hexose; HexNAc, N-acetylhexosamine; HPTLC, high-performance thin-layer chromatography; LSIMS, liquid secondary-ion mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; N-, Nz- and A-glyco(sphingo)lipids, neutral, neutralzwitterionic and acidic glyco(sphingo)lipids, respectively This revised version was published online in November 2006 with corrections to the Cover Date.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.     

Changes of Free Long-Chain Bases in Neuronal Cells During Differentiation and Aging in Culture

Abstract: Changes in the free long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o -phthalaldehyde and fractionated by reversed-phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: C18-sphingosine, C18-sphinganine, C20-sphingosine, and C20-sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18-sphingosine> C20-sphinganine and varying from 0.02 ± 0.015 pmol/mg of cell protein for C20-sphinganine at day 0 to 223 ± 22 pmol/mg of cell protein for C18-sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20-sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20-sphinganine progressively increased from day 0 to 22 of culture; that of C18-sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18- and C20-sphingosine levels during cell culture were qualitatively similar to those of C18- and C20-sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.     

Gas--liquid chromatography-mass spectrometry of synthetic ceramides

Two series of ceramides with either sphingosine (sphing-4-enine) or sphinganine as base and with one of the saturated fatty acids C(16), C(18), C(20), C(22), C(24), C(26), or oleic acid were analyzed as the 1,3-di-O-trimethylsilyl ether derivatives by gas chromatography-mass spectrometry. The fragments formed on electron impact can be divided into three main groups, namely "molecular weight fragments," "long-chain base fragments," and "fatty acid fragments." The m/e values of these fragments can be used to determine unequivocally the structures of the long-chain base and fatty acid of a ceramide derived from a sphingolipid.