Lipid components of sialosylgalactosylceramide of human brain

A ganglioside, previously designated HG-B in our laboratory, was isolated from mixed human brain ganglioside preparations and shown to contain equimolar quantities of sialic acid, galactose, and sphingosine. Treatment of this material with neuraminidase yielded a galactosylceramide. The ganglioside, now referred to as sialosylgalactosylceramide, thus appears to be identical with G(gal) reported by Kuhn and Wiegandt. The fatty acids and long-chain bases of this material were analyzed by gas-liquid chromatography. Approximately equal amounts of normal and hydroxy acids were found. Oleic, palmitic, and stearic acids were the only normal fatty acids present. In the hydroxy series, the C(24) and C(23) saturated acids were the major components. The ratio of C(20) to C(18) long-chain base was approximately 5:3. These data suggest that sialosylgalactosylceramide has no direct metabolic relationship with either the major brain gangliosides or adult brain cerebroside.     

Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues

Serine palmitoyltransferase [EC 2.3.1.50] catalyzes the first unique reaction of sphingolipid biosynthesis. To determine whether or not different rat tissues are capable of initiating this pathway, its activity was determined for microsomes from rat liver, lung, brain, kidney, intestine, spleen, muscle, heart, pancreas, testes, ovary, and stomach. Serine palmitoyltransferase was found in every tissue, and, when compared to the microsomal glycerol 3-phosphate acyltransferase, the activities correlated directly with their sphingomyelin levels as a percentage of total phospholipids. This suggests that the activities were comparable to expected cellular needs for long-chain bases, if the initial enzymes of glycerolipid and sphingolipid biosynthesis influence the phospholipid composition of cells by determining the relative partitioning of fatty acyl-CoA's toward these two lipid classes. Serine palmitoyltransferase activities were also determined using different fatty acyl-CoA's and were consistently greatest with CoA thioesters of saturated fatty acids with 16 +/- 1 carbon atoms. This suggests that the predominance of 18-carbon long-chain bases in vivo is due to the higher activity of this enzyme with palmitoyl-CoA. Together, these findings indicate a role for serine palmitoyltransferase in regulating both the type and amount of long-chain bases found in tissues.     

Lack of the Ganglioside Molecular Species Containing the C20-Long-Chain Bases in Human, Rat, Mouse, Rabbit, Cat, Dog, and Chicken Brains During Prenatal Life

The long-chain base composition of the total gangliosides from fetal brain of human, rat, mouse, rabbit, cat, dog, and chicken has been investigated during prenatal life. All the analyzed ganglioside mixtures contained the C18:1- and C18:0-long-chain bases, the latter covering 2-5% of the total long-chain base content. The C20-long-chain bases, which are components of the total ganglioside mixture from adult animals, were absent.     

FATTY ACIDS AND LONG CHAIN BASES OF VERTEBRATE BRAIN GANGLIOSIDES

Abstract— Fatty acid and long-chain base composition of gangliosides from brains of animals belonging to various vertebrate classes (fishes, amphibia, reptiles, birds and mammals) was studied by gas-liquid chromatography. Stearic acid was found to be the main fatty acid everywhere. Brain gangliosides of cold-blooded animals contain lesser amounts of stearic and higher amounts of palmitic and monoenoic acids as compared to those of mammals. Long-chain bases were separated as trimethylsilyl derivatives. Large amounts (23-48 per cent) of long chain bases with 20 carbon atoms were found in brain gangliosides of mammals while in those of cold-blooded animals they constitute 3-12 per cent of the total. The comparison of the composition of gangliosides with that of cerebrosides and sulphatides indicates, that the fatty acid and long chain base composition of gangliosides from mammalian brain differs from that of other glycosphingolipids, whereas in brains of cold-blooded animals they are much more similar.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

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).     

Branched long-chain bases from the bivalve Corbicula sandai.

Long-chain bases were liberated from a crude mixture of sphingolipids from whole tissue of the fresh-water bivalve C. sandai, and conversion of the bases into N-acetyl-0-trimethylsily derivatives was accomplished. The derivatized bases were analyzed by combined gas-liquid chromatography and mass spectrometry. A portion of the sphingolipids was subjected to catalytic hydrogenation from whch saturated long-chain bases (sphinganines) were obtained. The saturated bases were oxidized with lead tetra-acetate and the aldehydes produced were analyzed by gas-liquid chromatography. The aldehydes were further oxidized to acids with silver oxide, the resulting fatty acids methylated and also analyzed by gas-liquid chromatography. By these analyses, altogether five long-chain bases were identified, consisting of hexadeca-4-sphingenine (15%), heptadeca-4-sphingenine (2%), iso-octadeca-4-sphingenine (13%), octadeca-4-sphingenine (39%) and anteiso-noadeca-4-sphingenine (31%). So far no branches have been found in shellfish spingolipid long-chain bases.     

Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells.

Free ceramide, glucosylceramide, and sphingomyelin were isolated from mature cells of adult rat small intestine. Free ceramide and ceramide cleaved from sphingomyelin by enzymatic hydrolysis were fractionated by thin-layer chromatography on borate-impregnated silica gel plates. Sphingoid bases were characterized by gas-liquid chromatography of aldehydes formed upon periodate oxidation. Fatty acids were quantified as methyl esters. Ceramide structures were confirmed by direct-inlet mass spectrometry. Free ceramide was found to contain two major long-chain bases in nearly equal quantity: sphingosine, mainly linked to palmitic acid, and 4D-hydroxysphinganine associated with C20 to C24 fatty acids, 22% being hydroxylated. Sphinganine occurred as a minor component linked to nonhydroxy fatty acids. Sphingomyelin contained the three long-chain bases and 63% of its ceramide was N-palmitoyl-sphingosine. Mass spectrometry of glucosylceramide confirmed 4D-hydroxyshingamine as the major sphingoid base associated preferentially with longer chain hydroxy fatty acids.     

Fatty acids and long-chain bases of gangliosides of human gastrointestinal mucosa

The fatty acid and long-chain base composition of five major gangliosides from human stomach and small and large intestine mucosa were analyzed with gas chromatography. All the gangliosides greatly resembled each other in the fatty acid pattern. The main fatty acids were C_16:0, C_18:0 and C_24:0. No hydroxy fatty acids could be detected. In all the gangliosides 4-sphingenine was the predominant long-chain base (70–75%). About 15% of the long-chain bases had 20 carbon atoms in their chain. No trihydroxy long-chain bases could be detected.     

Sphingolipids in bean leaves (Phaseolus vulgaris)

Phytoglycolipid has been isolated for the first time from plant leaves (Phaseolus vulgaris). The purified product (almost identical with the phytoglycolipid isolated from flax seed) was a ceramide attached through phosphate diester linkage to an oligosaccharide, which consisted of the usual trisaccharide unit (inositol, hexuronic acid, hexosamine) to which were attached mannose, galactose, and arabinose. The major fatty acids were the saturated 2-hydroxy C(22), C(24), and C(26) acids; the major long-chain bases were dehydrophytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-8-trans-octadecene) (53%) and phytosphingosine (d-ribo-1,3,4-trihydroxy-2-amino-octadecane) (32%). A ceramide and a cerebroside were also isolated. In the ceramide the major fatty acids and the major long-chain bases were the same as in the phytoglycolipid. In the cerebroside, the fatty acid composition was similar to that in the ceramide and phytoglycolipid, but the long-chain bases consisted of dehydrophytosphingosine and phytosphingosine (7:1) with a substantial amount of unidentified long-chain base. The sugar component was glucose.     

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.     

Structure and composition of sulfatides isolated from livers of patients with metachromatic leukodystrophy: galactosyl sulfatide and lactosyl sulfatide

The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.     

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     

Gas-chromatography/mass-spectrometry analysis of human skin constituents as heptafluorobutyrate derivatives with special reference to long-chain bases

The composition of the constituents (monosaccharides, long-chain bases, and fatty acids) found in an ethanol extract of the human skin could be determined, without time-consuming steps of purification, after acid-catalyzed anhydrous methanolysis, followed by the formation of volatile derivatives with heptafluorobutyric anhydride and gas-chromatography/mass-spectrometry analysis. Despite the extreme heterogeneity of such extracts, the electron impact analysis of the constituents allowed qualitative and quantitative determinations of monosaccharides, long-chain bases, fatty acids, and alkyl-glycerols. Throughout the different long-chain bases, sphingenines (Sphes), sphinganines, phytosphingosines, and 6-hydroxy-Sphes (6oh-Sphes) can be identified and quantified. Long-chain bases with a chain-length up to 28 carbon atoms can be identified through specific fragmentation patterns in the electron impact mode. Particular attention was drawn to the behavior of compounds of the family of 6oh-Sphes upon acid-catalyzed methanolysis.     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.     

Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.     

Lipids of a Sterol-Nonrequiring Mycoplasma

The lipids of the sterol nonrequiring Mycoplasma strain S743 were found to include both ester glycerophosphatides (phosphatidylglycerol, acylphosphatidylglycerol, and diphosphatidylglycerol) and ceramide glycerophosphate compounds containing N-hydroxyacyl groups. The major phosphosphingolipid was tentatively identified as a hydroxyceramidephosphorylglycerol containing an O-acyl group. These compounds became labeled during growth in the presence of (32)P-orthophosphate, (14)C-glycerol, or (14)C-palmitate. The lipid fraction also contained free long-chain base. (14)C-palmitate was converted to labeled sphinganine. The long-chain base composition of the lipids was modified by growing the organisms in media containing different fatty acids, which were converted to bases containing two more C atoms per molecule. Ninety per cent of the long-chain base from cells grown in medium supplemented with elaidate consisted of monounsaturated C(20) base.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.