Stimulatory effect of certain plant sphingolipids on fruiting of Schizophyllum commune

We reported previously that certain cerebrosides and ceramides from fungi were active upon fruiting of Schizophyllum commune (Kawai, G., and Ikeda, Y. (1982) Biochim. Biophys. Acta 719, 612-618; Kawai, G., and Ikeda, Y. (1983) (Biochim. Biophys. Acta 754, 243-248). This work was undertaken to extend our study to sphingolipids in wheat grain. The cerebrosides from wheat grain were fractionated by high-performance liquid chromatography into at least 40 components with and without the fruiting-inducing activity. Four major active fractions were characterized by thin-layer chromatography, infrared spectroscopy, gas-liquid chromatography, gas-liquid chromatography-mass spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The active cerebrosides consist of glucose, 2-hydroxyhexadecanoic acid or 2-hydroxyoctadecanoic acid acid, and (4E,8Z)-sphingadienine or (8Z)-sphingenine. The cerebroside with (8Z)-sphingenine became inactive when the double bond was hydrogenated. Diglycosylceramides were as active as the monoglycosylceramides, but triglycosylceramides were only about 10% as active. The relationship between the structure and the activity is discussed.     

A method for the quantitative determination of neutral glycosphingolipids in urine sediment

A method is described for the isolation and quantitation of six neutral glycosyl ceramides from human urinary sediment. Total lipids were extracted from sediments of 24-hr urine collections, and the glycosyl ceramides were isolated by silicic acid column chromatography followed by thin-layer chromatography. Methanolysis of the individual glycosyl ceramides yielded methyl glycosides which were quantitated as the trimethylsilyl ethers by gas-liquid chromatography. By this technique, the submicromolar concentrations of six glycosyl ceramides in normal subjects and in individuals with Fabry's disease, an hereditary glycosphingolipid storage disease, were determined. Trihexosyl ceramide (galactosyl-galactosylglucosyl ceramide) and a digalactosyl ceramide accumulated in the urinary sediment of patients with Fabry's disease.     

Identification and quantitation of free ceramides in human platelets

Free ceramides were isolated from human platelets. Their structures were unequivocally determined by gas-liquid chromatography-mass spectrometry of the trimethylsilyl ether derivatives. The major components were N-(palmitoyl) sphingosine, N-(stearoyl) sphingosine, N-(eicosanoyl) sphingosine, N-(docosanoyl) sphingosine, N-(tetracosanoyl) sphingosine, and N-(tetracosenoyl) sphingosine. Sphinganine-and sphingadienine-containing ceramides as well as ceramides containing other unsaturated acids were also present. The amount of ceramides was determined by quantitative gas-liquid chromatography, using radioactive ceramide as internal standard and synthetic crystalline ceramides for comparison of peak areas. The concentration of ceramides was found to be 1.31 micro g/10(9) platelets or 0.47 micro g/mg of platelet protein.     

Effect of growth phase on the content and composition of ceramides of the hydrocarbon-assimilating yeast Candida lipolytica.

Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography. After acid methanolysis their composition was analyzed by gas-liquid chromatography. The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells. The composition of long-chain base moiety of ceramides did not change significantly during the growth. In both growth phases 19-phytosphingosine was the major long-chain base. However, the fatty acid composition of ceramides changed greatly during the growth. In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one. In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids. However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase. The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase.     

Ceramides, sphinganine, sphingosine and acid sphingomyelinases in the human umbilical cord blood.

Ceramides, sphingosine, sphinganine, as well as Zn (++)-dependent and Zn (++)-independent acid sphingomyelinase are present in the plasma of adults. The aim of the present study was to examine the concentrations of these compounds and activities of both enzymes in the umbilical cord blood in humans. Twenty-two women with uncomplicated term pregnancy volunteered for the study. Blood was taken from the umbilical cord artery and from the antecubital vein of the mother immediately after delivery. Free ceramides were isolated by thin layer chromatography, and their fatty acids were identified and quantified by gas-liquid chromatography. Free sphingosine and sphinganine concentrations were determined using high-performance liquid chromatography. Acid Zn (++)-dependent and Zn (++)-independent sphingomyelinase activity was measured using sphingomyelin [choline-methyl-14C] as a substrate. We found that the compounds examined are present in the umbilical cord blood. The total fatty acid-containing ceramide concentrations in fetal blood were lower than in mother's blood. The mean sphingosine and sphinganine concentrations in the fetal and maternal serum were similar. The examined enzymes were present in the fetal serum, and their mean activity did not differ from that in the mother. In conclusion, we have shown the presence of ceramides, sphingosine and sphinganine and both isoforms of acid sphingomyelinase in the human fetal cord blood. They are most likely the product of the fetus itself.     

Human urinary sulfatides in patients with sulfatidosis (metachromatic leukodystrophy)

The excretion of sulfatides in human urine was studied. 24-hr urine collections were filtered. Urinary glycolipids were extracted from the filter paper and fractionated on diethylaminoethyl cellulose and silicic acid columns, and by thin-layer chromatography. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography of the corresponding esters and aldehydes. Glycosyl ceramide concentration was determined by gas-liquid chromatography of the trimethylsilyl ethers of the methyl glycosides. Normal females were found to excrete larger amounts of dihexosyl ceramides than males. Sulfatides were detected in all urine specimens. In sulfatidosis, a hereditary sulfatide storage disorder known as metachromatic leukcdystrophy, a large increase in sulfatide was readily apparent on a thin-layer chromatogram of the crude lipid extract. On comparing samples from normal individuals and patients with sulfatidosis, urinary sulfatide composition was remarkably similar to that previously reported in the kidney, including differences in fatty acid pattern. The determination of urinary sulfatides was a valuable confirmation of the deficiency in arylsulfatase A activity characteristic of sulfatidosis.     

A convenient procedure for the synthesis of ceramides

A procedure for the preparation of ceramides by direct coupling of long-chain bases and fatty acids in the presence of a mixed carbodiimide is described. This method has been used to prepare ceramides containing sphing-4-enine or sphinganine and various saturated and unsaturated fatty acids as well as saturated 2-hydroxy acids. Ceramides containing 4-hydroxy sphinganine and saturated nonhydroxy acids have also been prepared. The yields were 60-75%. The characterization of these compounds by gas-liquid chromatography-mass spectrometry as trimethylsilyl derivatives has been previously reported. Some of the ceramides are further characterized in this report by infrared spectroscopy and one compound, in addition, by elementary analysis. Use of racemic constituents for 2-hydroxy acid ceramide syntheses leads to the formation of diastereoisomers which separate by thin-layer chromatography. These were characterized by gas-liquid chromatography-mass spectrometry as the trimethylsilyl derivatives and by infrared spectroscopy. Their configurations were established by syntheses with optically active constituents.     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

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.     

Separation and identification of ceramides derived from human plasma sphingomyelins

Sphingomyelins from human blood plasma have been converted into ceramides by enzymatic hydrolysis with phospholipase C. After acetylation the ceramides were fractionated by thin-layer chromatography on silica gel containing silver nitrate. Four main fractions obtained by this method were subsequently converted to di-O-trimethylsilyl ether derivatives and separated by gas-liquid chromatography on 1% OV-1. 2-11 components could be distinguished in each of the four fractions. The major fractions emerging from the gas chromatograph were analyzed by mass spectrometry and their main molecular species were identified. Two of the gas chromatographic fractions contained essentially pure molecular species, namely N-tetracosenoyl sphingosine and N-tetracosenoylsphinga-4, 14-dienine.     

Ceramides of human normal and cataractous lens.

Ceramides were quantitatively isolated from human normal and cataractous lens by solvent extraction, silicic acid chromatography, thin-layer chromatography, and gas-liquid chromatography. Only two species of ceramides with normal fatty acids were detected. In the mature cataracts, there was an increase in palmitate and nervonate at the expense of the other fatty acids. Due to the increase of 24 : 1, the ratio of 24 : 1/24 : 0 increased significantly from normals to cataracts. Sphinganine was the major long-chain base, but 4-sphingenine was also present. The total amount of ceramides in the immature and mature cataracts was 1.8 and 3.0 times higher than the normals of the same age group. Such an increase does not seem to be the result of an age-dependent process.     

Molecular Species of Free Ceramides in Aspergillus oryzae

Free ceramides isolated from A. oryzae were fractionationed into three groups by thin-layer chromatography on silica gel G according to degree of hydroxylation of the molecules. Each group was converted to trimethylsilyl ether derivatives, which were analyzed by gas-liquid chromatography-mass spectrometry for the structure of the molecular species. As a result, the representative molecular species of ceramides were characterized as N-lignoceroyl-phytosphingosine (1%), N-2-hydroxylignoceroyl-phytosphingosine (31%) and N-2,3-dihydroxylignoceroyl-phytosphingosine (31%).     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C₁₉ phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Received: 20 April 1998 / Received revision: 10 July 1998 / Accepted: 29 July 1998     

Immunochemical characteristics of a lipopolysaccharide from Pseudomonas aurantiaca

A lipopolysaccharide was isolated from Pseudomonas aurantiaca IMB 31 by extraction with aqueous phenol and purified by ultracentrifugation. The lipopolysaccharide was confined to the phenol phase. Fucosamine (2-amino-2,6-dideoxygalactose) (36%) and bacillosamine (2,4-diamino-3,4,6-trideoxyglucose) (23%) were identified as hypothetic components of the O-chain in the carbohydrate moiety of the macromolecule using the techniques of paper chromatography, gas-liquid chromatography and ion-exchange chromatography on an amino acid analyser. Rhamnose, glucose, galactose, glucosamine and galactosamine were detected as hypothetical components of the core in the lipopolysaccharide composition, as well as 2-keto-3-deoxyoctonic acid, heptose, alpha-alanine and phosphorus, usual components of the core in Pseudomonas. The following predominant fatty acids were identified in the composition of lipid A using the techniques of gas-liquid chromatography with standard compounds and gas-liquid mass spectrometry: 3-OH C10:0 (14.4%), C12:0 (30.5%), 2-OH C12:0 (14.9%), 3-OH C12:0 (17.4%), C16:0 (9.9%). The serological relationship between P. aurantiaca strains was studied, and their phylogenetic relationship with P. fluorescens is discussed.     

Fruiting of Schizophyllum commune Induced by Certain Ceramides and Cerebrosides from Penicillium funiculosum

Fruiting bodies were formed around a Penicillium colony which appeared as a contaminant in a culture of Schizophyllum commune, and this phenomenon was reproduced with a synthesized system consisting of S. commune IAM 9006 and P. funiculosum A-l. The active substances were recovered in an acetone extract of the mycelia of P. funiculosum, purified by silica gel column chromatography and reverse-phase high-performance liquid chromatography, and characterized by infrared spectroscopy, gas-liquid chromatography, gas-liquid chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy. They were ceramides and cerebrosides having nonadecasphingadienine and 2-hydroxy fatty acid moieties in common. The major component was identified as (4E,8E)-N-2^′-hydroxy-(E)-3^′-octadecenoyl-1-O-β-glucopyranosyl-9-methyl-4,8-sphingadienine.     

New technique for enzymic hydrolysis of glycosphingolipids

A method is described for the study of glycosyl ceramide glycosyl hydrolases. Problems arising from the limited solubility of glycosyl ceramides in aqueous media were overcome by coating the substrate on a filter paper disc that had been treated with phosphatidyl choline. A comparison between the disc method and conventional dispersion of the substrate by detergents was made with two enzymes, galactosylgalactosyl-glucosyl ceramide galactosyl hydrolase (trihexosyl ceramide galactosyl hydrolase) from lysosomes of human and rat small intestine and human spleen, and d-galactose oxidase. In both cases enzymatic activity was greater with the paper disc method than it was with substrates dispersed by detergents. The galactose liberated by the glycosyl hydrolase was determined as the trimethylsilyl derivative of the free sugar by gas-liquid chromatography.     

α-Hydroxylation of Fatty Acids in Brain: Characterization of Heat-Labile Factor

Abstract: The particulate fraction, heat-labile factor, heat-stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (α-hydroxylignoceric acid). The heat-labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD-Agarose affinity chromatography. The specific activity of the heat-labile factor was increased 105-fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS-polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat-labile factor was incubated with 1 mM- N -ethylmaleimide. This inhibition was prevented by preincubating the heat-labile factor with 1 mM-NADH. These observations indicate that the heat-labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.     

Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl-D-glucosyl phosphate to ceramides by BHK-21 cell microsomes

Enzymatic glucosylation of dolichol monophosphate (dolichol-P) from UDP-D-[3H]glucose was studied using the microsomal fraction of BHK-21 cells. The reaction product was separated by preparative thin-layer chromatography, further purified by DEAE-cellulose acetate column chromatography, and characterized as dolichyl-beta-D-glucosyl phosphate (Dol-P-Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP-[3H]glucose into ceramides (endogenous and exogenous) and Dol-P; both reactions required Mn2+. Maximal glucosylation of Dol-P was achieved at pH 5.6-5.8 in the presence of a non-ionic detergent, Zonyl A. Glucosylation of exogenous Dol-P, from UDP-Glc, was non-competitively inhibited by exogenous ceramides. Incubation of Dol-P-[3H]Glc or Dol-P-[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK-21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc-Cer) on silica gel thin-layer chromatography. Transfer of [14C]glucose from Dol-P-[14C]Glc to exogenous ceramides was confirmed by double-labeling techniques. The pH dependence for transfer of radio-labeled glucose from Dol-P-[3H]Glc to ceramides was multi-phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol-P-Glc may also participate in the synthesis of ceramide hexosides.     

Synthesis of alpha,beta-unsaturated C24 bile acids

Synthesis of the alpha,beta-unsaturated analogues of cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid is described. Each common bile acid was converted to the corresponding C22 aldehyde which was then converted to the delta 22 bile acid by Wittig reaction with methyl (triphenylphosphoranylidene)acetate. The synthetic unsaturated bile acids were characterized by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.