Characterization of a new phosphonocerebroside, N-methyl-2-aminoethylphosphonylglucosylceramide, from the antarctic krill, Euphausia superba

A novel phosphonglycosphingolipid was purified from the whole tissue of the antarctic krill, Euphausia superba by successive column chromatography on DEAE- and QAE-Sephadex and silicic acid (Iatrobeads). The structure was elucidated by means of IR, FAB-MS, 1H-NMR, GC and GC-MS analyses of the water-soluble products after complete and partial acid hydrolysis, and methylation analysis of a product of hydrogen fluoride degradation; it was identified to be a phosphonocerebroside, 6'-O-(N-methyl-2-aminoethylphosphonyl)Glcp beta 1----1ceramide. The ceramide moiety was composed of tetradecasphingenine and octadecasphingatriene as the main sphingoids, and monounsaturated C22- and C24-acids and their 2-hydroxy homologues as the major fatty acids.     

Sphingolipid profile in the CNS of the twitcher (globoid cell leukodystrophy) mouse: a lipidomics approach.

Globoid cell leukodystrophy (Krabbe disease) is caused by mutations in galactosylceramidase, a lysosomal enzyme that acts to digest galactosylceramide, a glycolipid concentrated in myelin, and psychosine (galactosylsphingosine). Globoid cell leukodystrophy has been identified in many species including humans and twitcher mice. Several studies on human tissue have examined the lipid profile in this disease by gas, liquid or thin layer chromatography. Electrospray ionization tandem mass spectrometry combined with reverse phase HPLC has become a powerful alternative strategy, used here to compare the sphingolipid profile of pons/medulla tissue from twitcher mice with control tissue. In this lipidomics LC-MS approach, we scanned for precursors of m/z 264 to obtain a semi-quantitative profile of ceramides and galactosylceramides. Sphingosine-1-phosphate, C18:0 ceramide, C22:0 ceramide and C24:0 ceramide levels were reduced in the pons/medulla of twitcher mice compared to levels in control mice at 31 and 35-37 days of age. The levels of C22:0 and C24:0 galactosylceramide were similar between twitcher and control specimens and there was a trend toward reduced levels of C24:1 galactosylceramide and C24:1 hydroxy-galactosylceramide in twitcher specimens. Psychosine, C 16:0 ceramide and C 18:0 galactosylceramide levels were increased in the CNS of twitcher mice compared to levels in control mice. These data indicate that there is a trend toward decreased levels of long chain fatty acids and increased levels of shorter chain fatty acids in galactosylceramides and ceramides from twitcher mice compared with control mice, and such changes may be due to demyelination characteristic of acute pathology.     

Biomass measurement of methane forming bacteria in environmental samples

Methane-forming bacteria contain unusual phytanylglycerol ether phospholipids which can be extracted from the bacteria in sediments and assayed quantitatively by high performance liquid chromatography (HPLC). In this procedure the lipids were extracted, the phospholipids recovered, hydrolyzed, purified by thin layer chromatography, derivatized and assayed by HPLC. Ether lipids were recovered quantitatively from Methanobacterium thermoautotrophicum and sediments at levels as low as 8 × 10^?14 moles. In freshwater and marine sediments the flux of methane to the atmosphere and the methane levels in the pore water reflects the recovery of the phytanyl glycerol ether lipid ‘signature’. The proportion of the ether phospholipid to the total recoverable phospholipid was highest in anaerobic digester sewage sludge and deeper subsurface freshwater sediment horizons.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

High-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography of glycosphingolipids

A method combining high-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography is introduced for determination of glycosphingolipids. Glycolipids in crude extract from rat liver were separated quantitatively from neutral lipids and phospholipids with a phenylboronic acid-derivatized silica gel column. Glycolipids were eluted quantitatively with approximately 98% of crude extract recovered. This column is useful for selective cleanup of glycosphingolipids in crude extract from tissue. Simultaneously, a fluorometric determination of glycosphingolipids with 7-amino-4-methylcoumarin after NaIO4 oxidation on a TLC plate was introduced and its condition was optimized. Glycolipids in amounts ranging from 1 to 100 pmol are easily detectable and give linear responses over the respective ranges. The method is fast and useful for the determination of glycolipids from small amounts of biological samples and requires a minimum amount of about 1 mg of biological specimen for determination of glycolipids.     

Glycosphingolipids of Human Peripheral Nervous System Myelins Isolated from Cauda Equina

Abstract: Compositions of neutral and sulfated glucuronyl glycosphingolipids purified from human motor and sensory nerves and myelins were studied. Higher neutral glycosphingolipids (fraction B), which were separated from GazlCer (fraction A), were analyzed by TLC and TLC-immunostaining. Both nerve myelins contained paragloboside (nLc₄Cer) and nLc₆Cer dominantly as major higher glycosphingolipids and very little globoside (Gb₄Cer), whereas both nerves contained Gb₄Cer and nLc₄Cer. Besides these major glycosphingolipids, a neutral glycolipid containing asialoGMI (Gg₄Cer) epitope and other minor components such as ceramide trihexoside and ceramide dihexoside were detected in both nerves and their myelins. Furthermore, sulfated glucuronyl nLc₄Cer and n Lc₆Cer, which were monoclonal antibody HNK-1 reactive glycolipids, were detected in both nerves and myelins.     

Self-recognition of N-linked glycans with multivalent GlcNAc, determined as ceramide mimetic conjugate

Aminoceramide mimetic was synthesized and conjugated to N-linked oligosaccharides having multivalent GlcNAc by reductive amination. Ceramide mimetic conjugates with "complex-type" glycan having five or six GlcNAc termini (termed Os Fr. B-Cer) were purified, analyzed by thin-layer chromatography (TLC), and finally characterized by MS/MS analysis through liquid chromatography/mass spectrometry. Binding of Os Fr. B-Cer placed on solid phase polystyrene surface with [3H]cholesterol-labeled liposomes containing Os Fr. B-Cer, or containing various glycosphingolipids (GSLs) was determined. The binding of Os Fr. B-Cer liposomes to Os Fr. B-Cer coated plate was significantly higher than binding of GM3 liposomes. Other GSL liposomes showed no binding. Thus, self-recognition of Os Fr. B-Cer was clearly demonstrated using ceramide mimetic conjugates.     

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.     

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.     

Versatile synthesis of phenoxydiazirine-based fatty acid analogues and photoreactive galactosylceramide.

A versatile synthesis of diazirine-based photoreactive fatty acid analogues is reported. The key step is phenoxy alkylation of diazirine with halo alkyl acid esters. The conditions described will be acceptable for the synthesis of various alkyl-length derivatives. The fatty acid derivatives are acceptors for reverse reactions of sphingolipid ceramide N-deacylase (SCDase), which catalyzes the condensation of psychosine and fatty acids to form photoreactive galactosylceramide. The photoreactive galactosylceramide can also be prepared with chemical synthesis, condensation of psychosine and fatty acid succinimidyl ester, and is recognized with anti-GarCer antibody both before and after irradiation.     

The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection

Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.     

Analysis of underivatized glycosphingolipids by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Analytical conditions for underivatized glycosphingolipids by using high-performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC/API-MS) were investigated. The analysis was performed by using an ordinary reversed-phase column (4.6 X 150 or 4.6 X 250 mm) at a flow rate of 1 ml/min. The glycosphingolipids could be characterized from the HPLC/API-MS in terms of molecular weight, ceramide composition, and partial oligosaccharide sequence. In order to obtain an adequate spectrum the amount of material needed is in the range of a few micrograms of lipid. By selected ion monitoring the sensitivity of the method allowed characterization of only 60 ng of glycosphingolipid. The method will be very useful in the characterization of small quantities of glycosphingolipids from biological samples.     

Purification and spectroscopic properties of pyrene fatty acids

Pyrene fatty acids are routinely purified by silica based column chromatography and analyzed on thin-layer silica plates (H.-J. Galla et al., Chem. Phys. Lipids, 23 (1979) 239-251). Although pyrene decanoic acid runs as a single spot on thin-layer chromatography (TLC), gas-liquid chromatography (GC) of the methyl ester derivatives of a representative sample revealed four separate peaks with the major component only 92% of the total. High performance reverse phase liquid chromatography (HPLC) was used to purify pyrene decanoic acid and separate the contaminants. After two passes on a C18 reverse phase HPLC column, pyrene decanoic acid is 99.98% pure by GC analysis. Absorption, fluorescence, and NMR spectra were recorded for pyrene decanoic acid and the major impurities. The results indicate that one impurity is a C10 fatty acid with an altered aromatic moiety. Two other impurities are pyrene derivatives but their acyl chains probably are not decanoic acid.     

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.     

银耳多糖单糖组成分析的三种色谱方法比较

为选择一种准确快捷的方法测定银耳多糖的单糖组成,对薄层色谱法(TLC)、气相色谱法(GC)、高效液相色谱法(HPLC)三种色谱方法进行比较。结果表明,前两种方法的测定结果均不理想,而HPLC法,操作简便,灵敏度高,分离效果好,信息完整。测定结果为由葡萄糖、甘露糖、葡萄糖醛酸、木糖、岩藻糖组成,其摩尔比为0.24∶1.00∶0.06∶0.29∶0.25。HPLC法对酸性杂多糖组成糖分析是一种比较理想的选择。     

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.     

Integration of glycosphingolipid metabolism and cell-fate decisions in cancer and stem cells: Review and Hypothesis

The metabolism of glycosphingolipids is strictly regulated during the mitotic cell cycle. Before the G1-to-S transition, the ceramide and glucosylceramide concentration is elevated. Ceramide induces apoptosis synergistically with the pro-apoptotic protein prostate apoptosis response 4 (PAR-4) that may be asymmetrically inherited during cell division. Only one daughter cell dies shortly after mitosis, a mechanism we suggested to regulate the number of neural stem cells during embryonic development. The progeny cells, however, may protect themselves by converting ceramide to glucosylceramide and other glycosphingolipids. In particular, complex gangliosides have been found to sustain cell survival and differentiation. The cell cycle may thus be a turning point for (glyco)sphingolipid metabolism and explain rapid changes of the sphingolipid composition in cells that undergo mitotic cell-fate decisions. In the proposed model termed "Shiva cycle", progression through the cell cycle, differentiation, or apoptosis may rely on a delicate balance of (glyco)sphingolipid second messengers that modulate the retinoblastoma-dependent G1-to-S transition or caspase-dependent G1-to-apoptosis program. Ceramide-induced cell cycle delay at G0/G1 is either followed by ceramide-induced apoptosis or by conversion of ceramide to glucosylceramide, a proposed key regulatory rheostat that rescues cells from re-entry into a life/death decision at G1-to-S. We propose a mechanistic model for sphingolpid-induced protein scaffolds ("slip") that regulate cell-fate decisions and will discuss the biological consequences and pharmacological potential of manipulating the (glyco)sphingolipid-dependent cell fate program in cancer and stem cells.     

Glycosphingolipids of cultured human colon carcinoma cells and their drug-resistant sublines

Human colon carcinoma cells were analyzed for lipid phosphorus, cholesterol and glycosphingolipids. Ceramide mono-, di- and trihexosides and sulfatides were isolated by column and thin-layer chromatography and determined quantitatively on the basis of their hexose content. The complex lipid fractions so isolated were only partially resolved with the material available. Gangliosides GM2 and GM3 and globoside were major components of the fraction and were determined on the basis of their hexose, hexosamine and neuraminic acid content. The HCT 116, 116a and 116b cells contained no fucolipids. Cell lines resistant to mitomycin C, teniposide and etoposide were developed and analyzed. Over the 5 year period of the study sulfatides declined to about one-fourth of their original amounts in both parent and drug-adapted cells. HCT 116 cells adapted to mitomycin C and teniposide had 30% less ceramide monohexoside and a 45% greater cholesterol to lipid phosphorus ratio than the parent cells. Reductions in ceramide dihexoside in the drug-adapted cells were greater than those of the ceramide monohexoside. Galabiosyl ceramide was the major ceramide dihexoside in all the cells and accumulated in HCT 116a to levels 4-6-fold greater than that of the other lines as the only dihexoside.     

Yeast sphingolipids do not need to contain very long chain fatty acids

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.     

Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC). A streamlined and more sensitive method for the measurement of esterified plasma F2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 microL) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF2alpha being identified as a major isomer. With this procedure, esterified plasma F2-isoprostanes were found to be 8.3-fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF2alpha was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay.