Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.     

Fluorine-, pyrene-, and nitroxide-labeled sphingomyelin: semi-synthesis and thermotropic properties

A rapid, high-yield method has been developed for the N-acylation of sphingosine-1-phosphocholine (SPC) to obtain a series of sphingomyelin (SM) derivatives bearing different reporter groups in the N-acyl chain. The procedure utilizes a fatty acid activated as the N-hydroxysuccinimide ester. A 1:1 molar mixture of the activated fatty acid and SPC is refluxed in 5% aqueous NaHCO3-ethanol 9:1 (v/v) for 2-3 hr. After acidification, the precipitated SM is purified by column chromatography over silica gel. This procedure offers significant advantages over those reported for the synthesis of well-defined SM: i) only the amino (not the hydroxyl) group is acylated; ii) only one equivalent of fatty acid is required; and iii) the time necessary for the reaction to go to completion is short. The transition temperature and enthalpy of each SM derivative has been measured by differential scanning calorimetry and compared to its unlabeled analog.     

Preparation and characterization of well defined d-erythro sphingomyelins

A simple semisynthetic procedure for the preparation of various d-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The d-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of d-erythro well defined simple sphingolipids.     

X-ray scattering of vesicles of N-acyl sphingomyelins. Determination of bilayer thickness.

A series of N-acyl sphingomyelins (C16:0, C18:0, C20:0, C22:0, and C24:0) have been synthesized and single bilayer vesicles formed by sonication and ultracentrifugation. X-ray scattering data have been collected from the sphingomyelin vesicles at 50 degrees C in the melted-chain state. The x-ray scattering data have been transformed to the corresponding Patterson functions and Fourier electron density profiles; analysis of these functions has provided the intrabilayer phosphate-phosphate separation dp-p, a measure of the lipid bilayer thickness. The bilayer thickness increases linearly with increasing chain length (increment 1.3-1.4 A) and the intercept, 14.3-15.0 A, suggests a contribution of 7.0-7.5 A for each phosphorylcholine group to the bilayer thickness. The electron-density profiles have features suggestive of chain interdigitation when the length of the N-acyl chain (C20:0, C22:0, and C24:0) exceeds significantly the length of the invariant sphingosine chain.     

METABOLISM OF BRAIN SPHINGOMYELINS: HALF-LIVES OF SPHINGOSINE, FATTY ACIDS AND PHOSPHATE FROM TWO TYPES OF RAT BRAIN SPHINGOMYELIN

Abstract— The metabolism of rat brain sphingomyelins containing short-chain (C₁₆-C₁₈) and long-chain (C₂₀- C₂₄) fatty acids has been studied by determination of the content of radioactivity in the sphingo-sinc. fatty acids and phosphate of the sphingomyelins over a period of 60 days following the intracisternal injection of [¹⁴C]acetate and [³²P]phosphate. From the rate of decrease of the specific radioactivities of the different constituents of short-chain fatty acid sphingomyelins, we have calculated a half-life of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. For the long-chain fatty acid sphingomyelins the half-life of sphingosine was approximately 465 days. The fatty acids and phosphate from these sphingomyelins had fast and slow turnover pools. The half-life for the fast pool was 7 days for the two constituents and the estimated half-lives for the slow pool were 220 days for fatty acids and 480 days for phosphate. These results suggest that one can distinguish at least three metabolic pools of brain sphingomyelins: (a) sphingomyelins with long-chain fatty acids situated in myelin whose half-lives are 465 days for sphingosine, 220 days for fatty acids and 480 days for phosphate; (b) sphingomyelins with long-chain fatty acids located mainly in non-myelin structures having half-lives of 465 days for sphingosine. 7 days for fatty acids and 7 days for phosphate; (c) sphingomyeiins with short-chain fatty acids with half-lives of 65 days for sphingosine. 41 days for fatty acids and 62 days for phosphate. The differences between the half-lives of the three metabolic pools of sphingomyelin, together with the subcellular localizations of the two molecular species of these compounds, suggest that the metabolism of the different molecular species of sphingomyelin are independent and that in various subcellular fractions the long-chain fatty acid and short-chain fatty acid sphingomyelins have different turnover rates.     

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.     

Fatty acid composition and thermal behavior of natural sphingomyelins

We found significant differences in the fatty acid composition of several bovine brain, egg yolk and sheep erythrocyte sphingomyelins. These differences in fatty acid composition influence the thermal behavior of hydrated sphingomyelin as recorded by differentail scanning calorimetry. Significant differences were also found in the temperature and complexity of the order-disorder phase transitions of bovine brain sphingomyelin obtained from different sources which, in general, correlate with the relative content of the saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0) acids) and the long unsaturated nervonic acid (C24:1).     

Heterogeneous N-terminal acylation of retinal proteins results from the retina's unusual lipid metabolism

Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1, and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca(2+)-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates--C14:2- and C14:1-coenyzme A to C14:0-coenzyme A--were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.     

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

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

Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.     

Membrane bilayer properties of sphingomyelins with amide-linked 2- or 3-hydroxylated fatty acids

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(D)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5-10 °C) gel-liquid transition temperatures (T(m)) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(D)-palmitoyl SM, however, had lower T(m) (5 °C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner.     

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).     

Changing fatty acid composition during somatic embryogenesis in cultures of Daucus carota

The fatty acid composition of five embryo stages following the induction of embryogenesis in cultures of Daucus carota have been studied. Quantitative rather than qualitative changes in fatty acid content were observed, although globular embryos do possess some fatty acids of long (C 20 and above) chain length not observed in earlier or later stages in development. The major fatty acids present were C 16:0, 18:0, 18:1, 18:2 and 18:3.Abbreviations SM small meristematic - LV large vacuolated - GLC gas-liquid chromatography - TLC thin lager chromatography     

Exploring the molecular mechanisms of OSU-03012 on vascular smooth muscle cell proliferation

Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein (UCV). We evaluate the sphingolipid composition of UCV and its alteration in preeclampsia. The veins were taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Thin layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed for sphingolipid analyses. The UCV walls of newborns delivered by healthy mothers are abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases are rather low. Preeclampsia is associated with a significant decrease in sphingomyelins and ceramides, whereas the sphingoid bases changed in uncharacteristic manner. The increase in sphinganine and sphingosine 1-phosphate was accompanied with a decrease in sphingosine, hydroxysphinganine and sphinganine 1-phosphate. Stearate is the dominating fatty acid in sphingomyelins and ceramides of both control and preeclamptic veins. Sphingolipids and some sphingoid bases are bioactive molecules which contribute to regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of UCV wall resulting in remodelling of its composition.     

Sphingomyelins of Rat Liver: Biliary Enrichment with Molecular Species Containing 16:0 Fatty Acids as Compared to Canalicular-Enriched Plasma Membranes

We harvested canalicular-enriched plasma membranes of hepatocytes and collected fistula bile from male rats and isolated the sphingomyelins. Following sphingomyelinase hydrolysis, we identified the sphingomyelin molecular species on the basis of their benzoylated ceramide derivatives employing high performance liquid chromatography. Sphingomyelin constitutes ≤3% of total biliary phospholipids (which are mostly sn-1 16:0 long-chain phosphatidylcholines) and approximately 30% of canalicular-enriched membranes. In both cases, the principal molecular species were composed of 16:0, 18:0, 20:0, 22:0, 23:0, 24:0, 24:1 and 24:2 fatty acid classes. However, the 16:0 fatty acid species was enriched in biliary sphingomyelin to a significantly greater degree than in sphingomyelins of canalicular-enriched plasma membranes (46% vs. 25% of total). We argue a physical-chemical case for laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane. We bolster our hypothesis by the likelihood that the least hydrophobic, e.g., 16:0 sphingomyelin molecular species, are miscible with biliary phosphatidylcholines, and are secreted into bile. Laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane could provide a means of sequestering cholesterol molecules prior to secretion into bile. Received: 19 March 1998/Revised: 8 October 1998     

Isolation of a novel sphingoglycolipid containing glucuronic acid and 2-hydroxy fatty acid from Flavobacterium devorans ATCC 10829

A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).     

Differential scanning calorimetry of chain-melting phase transitions of N-acylphosphatidylethanolamines.

Phosphatidylethanolamines in which the polar headgroup is N-acylated by a long-chain fatty acid (N-acyl PEs) are present in many plasma membranes under normal conditions, and their content increases dramatically in response to membrane stress in a variety of organisms. The thermotropic phase behavior of a homologous series of saturated N-acyl PEs, in which the length of the N-acyl chain is equal to that of the O-acyl chains attached at the glycerol backbone, has been investigated by differential scanning calorimetry (DSC). All fully hydrated N-acyl PEs with even chain lengths from C-12 to C-18 exhibit sharp endothermic chain-melting phase transitions in the absence of salt and in 1 M NaCl. Cooperative chain-melting is demonstrated directly by the temperature dependence of the electron spin resonance spectra from probe phospholipids bearing a spin label group in the acyl chain. The calorimetric transition enthalpy and the transition entropy obtained from DSC depend approximately linearly on the chain length with incremental values per CH2 group that exceed those of normal diacyl phosphatidylethanolamines, but to an extent that underrepresents the additional N-acyl chain. A thermodynamic model is constructed for the chain-length dependences and end effects of the calorimetric quantities, which includes a deficit proportional to the difference in O-acyl and N-acyl chain lengths for nonmatched chains, as is found and justified structurally for mixed-chain diacyl phospholipids. From data on the chain-length dependence of N-acyl diC16PEs, it is then deduced that the N-acyl chains are less well packed than the O-acyl chains and, from the data on the matched-chain N-acyl PEs, that the O-acyl chain packing is similar to that in normal diacyl PEs. The gel-to-fluid phase transition temperatures of the N-acyl PEs in the absence of salt are practically the same as those of the normal diacyl PEs of the corresponding chain lengths, although the transition enthalpies and entropies are appreciably greater, indicating entropy-enthalpy compensation. In 1 M NaCl, the transition temperatures are 3-4.5 degrees higher than in the absence of salt, representing the contribution of the electrostatic surface potential of the N-acyl PEs.     

Isolation and characterization of a ganglioside containing fucose from boar testis.

A ganglioside containing fucose (fucoganglioside) was obtained from boar testis and purified by silicic acid and DEAE-cellulose column chromatographies and preparative thin layer chromatography. The structure of this ganglioside, determined by chemical and enzymatic methods was: (see article). Its fatty acids were mainly long chain saturated ones (20 : 0, 22 : 0, 24 : 0). Its long chain bases consisted of 27% C(16:1) sphingosine and 68% C(18:1) sphingosine.     

Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34 % of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80 % of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.