Ceramide Acid Research Articles

Anti-tumor activity of ceramides and glycosphingolipids in a murine tumor system.

The anti-tumor activity of 7 sphingolipids, 2 ceramides and 5 glycosphingolipids against the syngeneic murine ascitic tumors MH134 and MM102 in C3H mice was examined. Five of these compounds showed anti-tumor activity against the tumors, ceramide type-IV (Cer-IV) having the highest activity without cytotoxic or cytostatic activity. These results indicate that the fatty acid in ceramide and sugar chains binding to it affect the anti-tumor activity in vivo. The anti-tumor activity of Cer-IV depended on the time of treatment. Mice treated with Cer-IV one day after tumor implantation showed the highest rate of survival. The cured mice were resistant to rechallenge with the same tumor (MH134-->MH134, MM102-->MM102) but not with a heterologous tumor (MH134-->X5563, MM102-->X5563), indicating that the effect of Cer-IV may be due to in vivo induction of specific immunity. Studies with various antibodies demonstrated that the anti-tumor effect of Cer-IV was inhibited by all the antibodies tested (L3T4, Lyt-2, and Thy-1,2 T cells, macrophages, and TNF alpha) in the induction phase (before Cer-IV administration) and by the antibodies of L3T4 and TNF alpha in the effector phase (after Cer-IV administration). Therefore, the anti-tumor effect of Cer-IV in this system depended on the host immune response rather than on its direct cytotoxic and/or cytostatic action.

Hypoxic injury to oligodendrocytes: reversible inhibition of ATP-dependent transport of ceramide from the endoplasmic reticulum to the Golgi.

Previous studies have shown that gradual progressive hypoxia specifically inhibits the synthesis of the major myelin lipid galactosylceramide (GalCer) in cultured neonatal rat oligodendrocytes (OLG) (Kendler and Dawson, J Biol Chem 265:12259-12266, 1990). The inhibition of de novo synthesized GalCer (measured by [3H]palmitate incorporation) was accompanied by an increase in the [3H]labeled pool of nonhydroxy fatty acid ceramide, the precursor of GalCer. The decreased galactosylation of NFACer was not due to an inhibition of UDP-Gal:ceramide:galactosyltransferase activity or to a depletion in available UDP-Gal. Analysis of subcellular fractionations of OLG membranes on Percoll gradients indicated that NFA ceramide was accumulating in the endoplasmic reticulum (ER) during hypoxia, suggesting that the transport of NFACer from its site of synthesis (ER) to its site of galactosylation, presumably the Golgi, was blocked by hypoxia. This accumulation of ceramide was replicated by lowering ATP levels to 80-90% of control by treating OLG with 12 nM oligomycin, and was reversed by reoxygenation of the cells. Conversion of [3H]palmitate-labeled NFACer to GalCer in semi-intact OLG required both exogenous UDP-Gal and ATP, further suggesting that the transport of NFACer from the ER to its site of synthesis (cis-Golgi) is an energy-dependent step that is highly susceptible to relatively minor ATP depletion associated with early hypoxic injury. Our results further suggest that ceramide appears to be a good marker for ER and GalCer is a good marker for the cis-Golgi.

51] Enzymes of ceramide biosynthesis

Ceramides are the lipid backbones of sphingomyelin, ceramide phosphoinositides, and other glycosphingolipids. They are composed of a long-chain (sphingoid) base with an amide-linked fatty acid. Sphingosine (trans-4-sphingenine) is the major long-chain base of most sphingolipids, but lesser amounts of other chain length homologs, dihydrosphingosines (sphinganines), and 4-hydroxysphinganines (phytosphinganines) are also found. The amide-linked fatty acids of ceramides typically have alkyl chain lengths from 16 to 24 carbon atoms (unbranched); small percentages are branched or monounsaturated, but α-hydroxy fatty acids are common in some sphingolipid subclasses. An overall scheme for ceramide biosynthesis is discussed and assays for each of the three enzymes of this pathway are described in this chapter. Ceramide synthesis occurs very fast in vivo because very low amounts of free long-chain bases occur as intermediates in sphingolipid biosynthesis. This may be advantageous as sphingosine and other long-chain bases are very bioactive compounds that inhibit protein kinase C, activate the epithelial growth factor (EGF) receptor kinase, and affect diverse other systems in cells.

Characterization of sphingolipids in spinach leaves

Ceramide, mono- and diglycosylceramide were isolated from spinach leaves, and their components and principal molecular species were studied. The major fatty acid components were 2-hydroxylignoceric and 2-hydroxybehenic acids in ceramide and diglycosylceramide, and 2-hydroxypalmitic and 2-hydroxylignoceric acids in monoglycosylceramide. As component sphingoids at least eight species of dihydroxy and trihydroxy bases were commonly found. The principal sphingoid components of ceramide were 4-hydroxy-8-sphingenine and 4-hydroxysphinganine, whereas those of glycosylceramides were 4,8-sphingadienine and 4-hydroxy-8-sphingenine. The sugar component of monoglycosylceramide was only glucose, but in diglycosylceramide glucose and mannose were found in the ratio of 85:15. The main ceramide species was N-2′-hydroxylignoceroy1-4-hydroxy-8-sphingenine. Generally, monoglycosylceramide consisted of two types of component ceramide species, viz., hydroxypalmitoyl-dihydroxysphingoid and hydroxylignoceroyl-trihydroxysphingoid. The principal species of monoglycosylceramide were found to be 1- O-β-glucosyl- N-2′-hydroxypalmitoyl-4, 8-sphingadienine and 1- O-β-glucosyl- N-2′-hydroxylignoceroyl-4-hydroxy-8-sphingenine. The major species of diglycosylceramide was postulated as 1- O-β-cellobiosyl- N-2′-hydroxylignoceroyl-4-hydroxy-8-sphingenine.

The use of liposomes as acceptors for the assay of lipid glycosyltransferases from rat brain

Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first tune as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose:ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1: 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucosyltransferase. For the assay of UDPgalactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing α-hydroxy fatty acids, in a molar ratio of 6: 0.57: 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200–500 Å. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesul fonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography.

Isolation and characterization of inositol-containing glycosphingolipids from Aspergillus niger.

The glycophosphosphingolipids are a group of sphingolipids characterized by the presence of equimolar amounts of long-chain base and fatty acid (ceramide) and containing either one or two mol of inositol phosphate per mol of lipid. From one to several mol of sugar are also present. Glycophosphosphingolipids have been isolated from both plant and fungal sources. Work in this area had for years been the province of Herbert Carter and colleagues. They showed that such glycolipids occurred in seeds from corn, flax, soya beans and bean leaves (Carter et al., 1958; Carter & Koob, 1969). The major glycolipid from corn seed had the structure (Carter et al., 1969):

Rat intestinal glycolipids: II. Distribution and biosynthesis of glycolipids and ceramide in villus and crypt cells

Intestinal epithelial cells were isolated from rat intestine and grouped into villus and crypt cell fractions. Glycolipids were purified from each cell fraction and quantitated by fluorimetric determination of glycolipid sphingosine. Significant quantities of ceramide were found in all cell fractions and accounted for approximately 15% of total glycolipid sphingosine. While villus and crypt cell fractions quantitatively contained differing amounts of sphingosine, all cell fractions contained proportionally similar quantities of sphingosine when compared to cellular cholesterol or phospholipid. Individual glycolipids, however, showed significant differences in distribution between villus and crypt cells. Hematoside and glucosylceramide were proportionally increased in villus cells, while crypt cells showed an increase in trihexosylceramide and ceramide content. The rate of UDPglucose: hydroxy fatty acid ceramide glucosyltransferase was higher in villus cells while the rate of UDPgalactose:lactosylceramide galactosyltransferase was 3–4 times increased in crypt cells. These studies demonstrate that significant differences in both the distribution and biosynthesis of individual glycolipids occur in crypt and villus cells of rat intestine and are of possible importance in the process of intestinal cell differentiation.

Stimulation of erythroblast maturation in vitro by sphingolipids

A lipid factor previously isolated from leukocytes and found to stimulate basophilic erythroblast formation in an in vitro system of incubated rabbit bone marrow cells has been analyzed by thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry. The biologically active components are sphingosine ceramides of tetracosanoic and dehydrotetracosanoic acids. Tests of a series of related ceramides show a high degree of structural specificity for the C(24)-V-acyl compounds with significant but markedly lower activity of the C(22) analog. Commercially available sphingomyelin shows activity comparable to that of the tetracosanoic acid ceramide. Sphingosine and tetracosanic acid supplied in equimolar amounts have negligible activity. The results, in the context of other findings, suggest a possible supportive role of plasma ceramides and sphingomyelins in red cell maturation.

Glycosphingolipids in Secondary Lysosomes Prepared from Rat Liver

Glycosphingolipid classes were isolated from unfractionated liver and from the buoyant fraction of secondary lysosomes following pretreatment of rats with Triton WR-1339. They were identified by thin-layer chromatography and by sugar analysis using gas chromatography and were quantified by estimating long chain base. Hematoside containing N-acetyl neuraminic acid, glucosyl, lactosyl, trihexosyl, and galactosamine-containing tetrahexosyl ceramides were identified in unfractionated liver in the following concentrations, respectively: 0.89, 0.59, 0.19, 0.06, and 0.1 nmol/mg protein. The same glycosphingolipid classes were identified in buoyant Triton-filled lysosomes where their respective concentrations in terms of protein were 8.6, 21, 28, 34, and 28 times as great as in unfractionated liver. The lysosome fraction was enriched 28-fold in acid phosphatase. These results demonstrate that some glycosphingolipid classes are concentrated in secondary lysosomes to a degree expected of markers for primary lysosomes and plasma membrane. Secondary lysosomes are thought to be derived from these latter two structures.

Changes in cerebroside synthetic activities of the brain with development of the rhesus monkey (macaca mulatta)

Cerebroside synthetic activities vary according to age in homogenates of the pons and cortical gray matter from brains of rhesus monkeys. The rate of utilization of uridine diphosphate (UDP) galactose and hydroxy fatty acid (HFA) ceramide for cerebroside synthesis by the pons was very low until 100 days after conception and reached a maximum at about 150 days (15 days before birth). This activity was very low in gray matter.There were marked effects of the type of ceramide and UDP sugar added, as well as of the origin of the brain homogenates from gray matter or pons on the quantity and type of cerebroside synthetized.

Cholesterol-cerebroside interaction: the role of -hydroxy fatty acids.

AbstractThe esterification of cholesterol by the plasma phosphatidyl choline‐cholesterol acyltransferase reaction was studied by two methods, radioisotopic and colorimetric, in the presence of cerebroside, ceramide, or methyl esters of lignoceric or α‐hydroxy lignoceric acid. The radioisotopic method measures esterification of exogenous labeled cholesterol which must be taken up into the lipoprotein‐bound pool prior to its utilization as a substrate. The colorimetric method measures esterification of endogenous lipoprotein‐bound free cholesterol since the exogenous labeled cholesterol is negligible in concentration. Cerebroside and ceramide containing α‐hydroxy fatty acids reduced the utilization of exogenous labeled cholesterol as substrate, but had no effect on lipoprotein‐bound exogenous cholesterol esterification. Cerebroside and ceramide containing no α‐hydroxy fatty acid had no effect on exogenous labeled cholesterol esterification. The methyl esters of lignoceric acid and α‐hydroxy lignoceric acid had no effect on the esterification of exogenous cholesterol in plasma. There is a decrease in esterification of exogenous labeled cholesterol with increasing concentration of α‐hydroxy fatty acid ceramide. Increasing the concentration of exogenous cholesterol tends to counteract the effect of the ceramide on cholesterol esterification. There was little effect on exogenous cholesterol esterification when the α‐hydroxy fatty acid ceramide was exposed to plasma before adding the labeled cholesterol. The findings demonstrate an interaction between free cholesterol and cerebroside or ceramide containing α‐hydroxy fatty acids, but the nature of the interaction is not elucidated.

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.

Effects of cycloheximide and puromycin on glycosyltransferases of microsomal fractions from rat brain

Injection of cycloheximide or puromycin into immature rats reduced the capacity of cerebral microsomal system to transfer glucose from UDP-glucose to either non-hydroxy fatty acid or hydroxy fatty acid ceramide. Cycloheximide was effective whether given intramuscularly or intracerebrally, and puromycin was effective if given intracerebrally. Injection of the antibiotics also reduced the rate of amino acid incorporation into proteins by the cerebral microsomal systems. The antibiotics had no effect on the transfer of galactose from UDP-galactose to hydroxy fatty acid ceramide. The results support the view that the glucose transfer enzyme in brains of immature rats has a short turnover rate, and becomes depleted under conditions in which cerebral protein synthesis is impaired.

Glycosyl transferases of microsomal fractions from brain: synthesis of glucosyl ceramide and galactosyl ceramide during development and the distribution of glucose and galactose transferase in white and grey matter.

Abstract— The substrate specificity for glycosyl transferases of microsomal fractions from brain was investigated. Ceramides were found to be better acceptors than sphingosine for both glucose and galactose when a Celite dispersion of lipid substrate was used. For galactose transfer only hydroxy fatty acid ceramide served as an acceptor. For transfer of glucose both non‐hydroxy and hydroxy fatty acid ceramide served as acceptors, but the hydroxy fatty acid ceramide was the more effective of the two. Glucose transferase activity was found to be highest between birth and 15 days of age and declined slowly with later development. Galactose transferase activity did not appear until the 10th day of postnatal age and reached a peak at about the 30th day. Galactose transferase activity was present principally in white matter microsomes, but glucose transferase activity was present in the microsomal fractions of both white and grey matter. The developmental alteration in the activities of galactosyl and glucosyl transferases and their distribution in white and grey matter correlated with development and distribution of cerebroside and ganglioside, respectively.

On the biosynthesis of cerebrosides from 2-hydroxy acid ceramides: Use of deuterium labeled substrate and multiple ion detector

N (7′,8′,10′,11′,13′ ,14′,16′,17′-octadeutero 2′D-hydroxy docosanoyl) 4,5-dideutero D-erythro- trans sphingosine and N (2′D-hydroxy docosanoyl) 3-tritio D-erythro- trans sphingosine were prepared. A mixture of these ceramides was enzymically converted to cerebroside with mouse brain microsomes. The product was degraded to ceramides which were separated into molecular species by gas-liquid chromatography and analyzed by mass spectrometry. This analysis conclusively showed that the substrate was transformed into cerebroside and that there was no hydrolysis of the amide bond prior to galactosylation.