Distribution of glycosphingolipids and ceramide of rat small intestinal mucosa

Previous studies have suggested that glycosphingolipids may be involved in a number of physiological functions of the small intestinal mucosa. Regional variations in many of these processes exist along the length of this organ. In the present studies, the glycosphingolipid and ceramide composition of the proximal, middle and distal thirds of the rat small intestine were characterized and compared. Mono- and trihexosylceramide were the major neutral glycolipids and hematoside (GM₃), the principal ganglioside of this organ. Monohexosylceramide was the major glycolipid of the proximal segment, whereas trihexosylceramide predominated in the distal segments. The total content of neutral glycolipids, ceramide and gangliosides as well as the content of the individual glycosphingolipids and ceramide were highest in the distal segment, intermediate in the middle and lowest in the proximal segment. Additionally, regional variations were noted in the fatty acid composition of the major glycosphingolipids. These differences in the composition of glycolipids and ceramide along the length of the intestine may be responsible, at least partially, for the regional functional specialization seen in this organ.     

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x₂ glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Le^b determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.     

Distribution of glycosphingolipids of monkey small and large intestinal mucosa

The ganglioside and neutral glycosphingolipid composition of adult monkey small and large intestinal mucosa were characterized and compared. GM₃, GM₂ and GD_1A were found to be the principal gangliosides in each of these tissues. Dihexosylceramide was the major neutral glycosphingolipid of both organs. The total content of gangliosides and neutral glycolipids/ceramide, however, was ca. four-fold and two-fold higher, respectively, in small intestinal than colonic mucosa. While all glycosphingolipids examined contained hydroxy and nonhydroxy fatty acids, the former fatty acids accounted for 60–90% of the total fatty acids in both organs. Sphingosine was the predominant long chain base of ceramide, mono-, di-, tri- and tetrahexosylceramide, whereas phytosphingosine was the major base of GM₃ in both tissues. The results of these studies demonstrate that while many similarities of monkey small and large intestinal glycosphingolipids exist, qualitative and quantitative differences are present along the length of the monkey gut. These differences may be at least partially responsible for certain of the well-recognized variations in normal physiological and pathological processes that occur in these organs.     

Occurrence of bioactive sphingolipids in meat and fish products

Recent research has revealed that the degradation products of dietary sphingolipids are biologically highly active and have the capacity to inhibit the development of colon cancer in mice. Nevertheless, the content of sphingolipids in common foodstuff has never been systematically analyzed. Therefore we investigated the contents of sphingomyelin and neutral glycosphingolipids in commonly consumed meat and fish products. Sphingomyelin and glycosphingolipids were found in all foodstuffs studied. The total amount varied between 118 ± 17 nmol/g (cod) to 589 ± 39 nmol/g (chicken leg). Generally, lower amounts of sphingolipids were determined in fish meat than in red meat and poultry, while poultry was the richest source of this class of lipids. However, fish meat contained a relatively high content of neutral glycolipids compared with other types of meat. Thus, in fish the ratio sphingomyelin/neutral glycolipids varied from 1 to 2.9, while in poultry this ratio varied between 5.2 to 19.2 and in red meat it varied from 1.6 to 8.3. The fatty acid composition of sphingomyelin in fish was dominated by C24:1 (Δ⁹) or C22:1 (Δ⁹), while C16:0 and C18:0 were the dominating sphingomyelin species in poultry and C18:0 in the meat of mammal origin.     

Characterization of porcine omental lipids

Porcine omental lipid extracts were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1, and 18∶2 fatty acids. Small quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry, was found to consist primarily of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by HPTLC and HPLC, were found to consist primarily of di-, tri-and tetraosylceramides. The complex glycolipid fraction, obtained from Folch upper phase solvent partition and characterized by HPTLC and immunoblotting, was found to consist primarily of ganglio-, globo-, and neolacto- neutral glycolipids and ganglio-, globo-, neolacto- and fucosylated gangliosides.     

Isolation and partial characterization of the neutral glycosphingolipids and gangliosides of the human heart

The glycosphingolipids (GLS) of the human heart muscle have been isolated from total lipids by column and thin layer chromatography and their sugars and fatty acids analyzed by gas liquid chromatography. Hearts from traffic victims were obtained at autopsy between 12 and 16 hr after death and dissected into parts (left and right ventricular walls, intraventricular septum and papillary muscle). The neutral GSL content for those parts of the hearts of two males aged 22 and one female aged 14 ranged from about 90 to 160 nmoles/g wet weight. Trihexosyl ceramide and globoside were the most abundant neutral GSL. Total ganglioside content was about 50 nmoles/g wet weight, and the most abundant gangliosides were partially characterized as GM₃ and GM₁; other mono-, di- and trisialogangliosides were also present. Differences in the content and composition of neutral GSL and gangliosides between the heart and other human tissues are discussed. It is concluded that the patterns of these two GSL fractions of the heart are more complex than those of most of the extraneural human tissues.     

Dexamethasone-induced alterations in the glycosphingolipids of rat kidney

To determine whether glucocorticoids would influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 μg/100 g body wt/day) or diluent for four days. The compositions of ceramide and of acidic and neutral glycosphingolipids of the kidneys of these animals were then examined and compared. The results demonstrated that dexamethasone administration: 1) increased the content of ceramide and of acidic and neutral glycosphingolipids in kidney; 2) increased the relative percentage of globotriaosyl- and globotetraosylceramide, but decreased the relative percentages of glucosylceramide; 3) decreased the relative percentages of G_M3 and increased other gangliosides; 4) increased the relative percentages ofN-glycolylneuraminic acid in G_M3; 5) did not appear to influence significantly the long-chain bases of the major glycosphingolipids; and 6) altered the relative percentages and chain length of the hydroxy and nonhydroxy fatty acids of the major acidic and neutral glycosphingolipids in this tissue. The data show that dexamethasone administration induces quantitative and qualitative changes in the glycosphingolipids of the rat kidney.     

Lipid composition of murraya koenigii seed

Total seed lipids extracted fromMurraya koenigii (Linn), Rutaceae amounted to 4.4% of the dry seed. The total lipids consisted of 85.4% neutral lipids, 5.1% glycolipids and 9.5% phospholipids. Neutral lipids consisted of 73.9% triacylglycerols, 10.2% free fatty acids and small amounts of diacylglycerols, monoacylglycerols and sterols. At least five glycolipids and seven phospholipids were identified. Sterylglucoside and acylated sterylglucoside were major glycolipids, while digalactosyldiacylglycerol, monogalac-tosyldiacylglycerol and monogalactosylmonoacylglycerol were present in small quantities. The phospholipids consisted of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylcholine as major phospholipids and minor quantities of phosphatidylinositol, phosphatidylglycerol and phosphatidic acid. The fatty acid composition of these different neutral lipids, glycolipids and phospholipids were determined.     

Monohexosylceramides of larval and adult forms of the tapeworm,spirometra erinacei

The occurrence of glycosphingolipids with unique carbohydrate structures in different species of cestode, Platyhelminth, which had been shown, previously, prompted us to study the molecular species of the monohexosylceramides (cerebrosides) in the pseudophyllidean cestode,Spirometra erinacei. The purpose of the study was to obtain a basis for future investigations of the physiological role of glycolipids in parasitism. Cerebrosides were isolated froms. erinacei at two growth stages, i.e., from the larval form (plerocercoid) and from the adult tapeworms (intestinal form). The cerebrosides were separated into four subfractions by silica gel column chromatography, and their constituents were analyzed by gasliquid chromatography, gas chromatography/mass spectrometry, and high-performance liquid chromatography/mass spectrometry. The hexoses of the cerebrosides consisted primarily of galactose in both growth stages, while only a small amount of glucose was detected. The ceramides were composed of sphinganine (d18∶0) and phytosphingosine (t18∶0) as sphingoid bases, and of nonhydroxy fatty acids ranging from C₁₆ to C₃₀ and hydroxy stearic acid (18h∶0). The cerebrosides of adult tapeworms contained more 18h∶0 than those of plerocercoids. The combination of hexoses and ceramides in the cerebroside molecules was slightly different in the two growth stages: the glucocerebrosides of plerocercoids contained only d18∶0-nonhydroxy fatty acids in their ceramide moieties, whereas those of adult tapeworms contained varying ceramide moieties. Our data indicate that the molecular species of glycolipids present were essentially homeostatic throughout growth in spite of the entirely different environmental conditions, although there were slight differences in the hexose distribution in the two growth stages.     

Composition of lipids of bovine optic nerve

Lipids from bovine optic nerve were analyzed. The total content of 16.5% by weight included 27.2% nonpolar lipids, 26.1% glycolipids, and 46.7% phospholipids by weight. Free cholesterol was the major component of the nonpolar lipid fraction. The cerebrosides, 73.5% of total glycolipids, were separated by thin layer chromatography (TLC) into two bands (upper and lower) that were present in equal proportion. Cerebroside sulfates comprised about 27.5% of total glycolipids. Gangliosides were also detected in the glycolipid fraction. In order of predominance, choline glycerophospholipids, ethanolamine glycerophospholipids, ethanolamine plasmalogens, serine glycerophospholipids, sphingomyelins, and inositol glycerophospholipids were the major phospholipids. Palmitoyl (16∶0), stearoyl (18∶0), and oleoyl (18∶1) groups were the major acyl groups in all neutral and phospholipid classes. However, ethanolamine glycerophospholipids, serine glycerophospholipids, and inositol glycerophospholipids contained a large percentage of 22∶6 (docosahexaenoyl) group. The major alk-1-enyl groups of the plasmalogens were 16∶0, 18∶0, and 18∶1. Steroyl (18∶0), lignoceroyl (24∶0), and nervonoyl (24∶1) were the major acyl groups in all sphingolipids. Lower cerebroside band and cerebroside sulfates contained large amount of hydroxylignoceroyl (cerebronoyl) and hydroxynervonoyl groups. This investigation was supported by Grants DE-03191 and 2S06-RR-08037 from the National Institutes of Health, Bethesda, MD.     

Isolation of cerebroside containing glucose (Glucosyl ceramide) and its possible significance in ganglioside synthesis

A small amount of cerebroside containing glucose (glucosyl ceramide) was isolated from bovine brain by Florisil column chromatography and thin-layer chromatography. The fatty acids of the glucosyl ceramide were palmitic and stearic acids; small amounts of oleic and linoleic acids were present. Rat brain tissue slices, incubated with U-¹⁴C-glucose, incorporated more radioacivity into glucosyl ceramide than into galactosyl ceramide. From these results the possible metabolic significance of the brain glucosyl ceramide in ganglioside metabolism is discussed.     

Composition and biosynthesis of the major hexose containing sphingolipids of pig leucocytes

The oligohexosyl ceramides of two preparations of blood leucocytes taken from large white pigs, weighing 80 kg were analyzed and found to comprise as major constituents trihexosyl ceramide, with glucose-galactose in a molar ratio of 1∶2, and glucosyl ceramide. The mean value of the two preparations was 4.9% and 1.35% of the total lipids, respectively, for the two glycolipids. Lactosyl ceramide is the major component of human leucocytes, in contrast to the pig leucocytes where it was detected in minor quantities. U-¹⁴C-glucose, incubated with bone marrow cells of young pigs (20 kg living weight), was mainly incorporated into the carbohydrate moiety of glucosyl ceramide and trihexosyl ceramide. Further analysis of this compound indicated that all three hexoses were labeled with¹⁴C and that the glucose attached to the ceramide had the highest specific activity. Time studies showed a decline in the incorporation of¹⁴C into glucosyl ceramide as compared to its incorporation into trihexosyl ceramide.     

Neutral glycolipids of human and bovine milk

The neutral glycolipids of milk, a small fraction of the total lipids, are of potential biological importance. The simultaneous quantitation of the simple (less than five sugars) glycosphingolipids of human milk samples was achieved by high-pressure liquid chromatography. The samples, representing various stages of lactation, parity of the nursing child, and age of the mother, contained similar glycolipid patterns, but with varying individual glycolipid concentrations. The cerebrosides are major glycosphingolipids of human milk: the non-hydroxylated fatty acid (NFA)-containing species are present at 1.8 μM, and the hydroxylated and/or short-chain fatty acid-containing species (HFA) are present at 1.7 μM; NFA lactosylceramide is present at 931 nM. The cerebrosides appear to be primarily galactosylceramides (galactocerebrosides); glucosylceramides (glucocerebrosides) are a minor component. Globotriaosylceramide (Gb₃) is found at 50 nM and 73 nM for the NFA and HFA species, respectively, while globoside (Gb₄) is found at 45 nM and 46 nM for the NFA and HFA species. Bovine milk glycosphingolipids differ from those of human milk, with bovine milk containing mainly NFA glucosylceramide (8 μM) and NFA lactosylceramide (17 μM); bovine milk contains little Gb₃ or Gb₄. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

The distribution and excretion of tritium labeled cerebroside (galactosyl ceramide) radioactivity by the monkey

Tritium labeled galactosyl ceramide was administered intravenously to three male monkeys (Macaca mulatta). Blood, urine, feces and organs obtained at autopsy were examined for radioactivity after 1 hr, 8 hr and 37 days. The radioactive galactosyl ceramide was taken up by the reticuloendothelial system. Very little radioactivity appeared in brain tissue. The radioactivity was excreted primarily in urine. Presented at the 137th Meeting of the American Chemical Society.     

Lipid composition of millet (Pennisetum americanum) seeds

The composition of lipids extracted from a sample of millet seeds by each of 8 solvent systems is reported. Lipid components were separated by silicic acid column and thin layer chromatography (TLC) and quantitated by analysis of fatty acid methyl esters by gas liquid chromatography (GLC), with heptadecanoic acid as internal standard. Best results were obtained by extraction with hot water-saturated butanol. Lipids extracted amounted to 7.2% of the seed dry weight and consisted of 85% neutral lipids, 12% phospholipids and 3% glycolipids. Neutral lipids contained mostly (85%) triacylglycerols and small amounts of mono- and diacylglycerols, sterols and free fatty acids. Sterols consisted of campesterol, stigmasterol and 2 unidentified sterols, occurring in the same proportions in free and esterified forms. Ten glycolipid and 10 phospholipid components were separated and characterized. Contrary to previously published observations, lysophosphatidylcholine was the major phospholipid (42%) in millet seeds; smaller amounts of phosphatidylcholine (24%), lysophosphatidylethanolamine (21%) and trace amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine and phosphatidylinositol also were present. The major glycolipids were esterified sterol glycoside, sterol glycoside, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and cerebrosides (ceramide monohexosides).     

Sphingolipid metabolism in leucocytes. I. Incorporation of14C-glucose and14C-galactose into glycosphingolipids by intact human leucocytes

Glucosyl ceramide and lactosyl ceramide have been isolated from intact human leucocytes. Incubation of intact white blood cells with either¹⁴C-glucose or¹⁴C-galactose resulted in the incorporation of these tracers into the glycosphingolipids. The products were extracted by conventional procedures and purified by combined silicic acid column and thin-layer chromatography. The bulk of the radioactivity was found in the monohexoside and dihexoside ceramide fractions. Acid hydrolysis yielded glucose as the principal carbohydrate of the monohexoside ceramide, regardless of the sugar precursor employed. In the dihexoside ceramide fraction, galactose was liberated as the major sugar component. The specific activities of the lactosyl ceramide was found to be greater than that of the corresponding glucosyl ceramide. A preliminary report was presented at the 51st Meeting, Federation of American Societies of Experimental Biology, Chicago, 1967     

Spectrophotometric determination of molar amounts of glycosphingolipids and ceramide by hydrolysis and reaction with trinitrobenzenesulfonic acid

Three spectrophotometric procedures for determination of chromatographically separated glycosphingolipids (cerebroside, sulfatide, ganglioside) are described and compared. Hydrolysis to release long chain base followed by reaction of the amino group with trinitrobenezene sulfonic acid (TNBS) and measurement at 340 mμ was shown to give values in good agreement with those obtained by reaction of the carbohydrate moiety with α-naphthol or anthrone and measurement at 550 and 625 mμ respectively. The TNBS method is also applicable to ceramide.     

The isolation and partial characterization of gangliosides and ceramide polyhexosides from the lens of the human eye

The first isolation of glycolipids from the lens of the human eye is described. Neutral (ceramide polyhexosides) and acidic (gangliosides) glycolipids were separated by column chromatography and further resolved by thin-layer chromatography. The components were methanolyzed, converted to trimethyl silyl ethers and the ratios of the components determined. Two types of monosialogangliosides were found. The most abundant ganglioside contained long chain base/fatty acid/glucose/galactose/neuraminic acid in the ratio 1/1/1/2/1. The ratio of components of the minor ganglioside fraction was 1/1/1/1/1. Dihydrosphingosine was the major base and the major fatty acids were palmitate and nervonate. The ceramide polyhexosides all had a glucose/galactose molor ratio of 1/1 and the mixture of ceramide polyhexosides had a dihydrosphingosine/sphingosine molar ratio of 7.85. The fatty acids ranged from C₁₀ to C₂₅ with both odd and even carbon chains and were saturated or monounsaturated with palmitate, oleate, and nervonate predominating.     

Sterol lipids in finger millet(Eleusine coracana)

Neutral lipids, glycolipids, and phospholipids (1.3%, 0.25%, and 0.10% of seed weight) were isolated from the total lipids (chloroform-methanol) of finger millet seeds(Eleusine coracana), and four sterol-containing lipids further isolated from neutral and glycolipids by preparative column and thin layer chromatography. On seed weight, these comprised: free sterols (S) 0.091%, sterol esters (SE) 0.013%, sterol glycosides (SG) 0.025%, acyl sterol glycosides (ASG) 0.020%, and total 0.149%. The major fatty acids, totaling 85-90%, were the same in both esterified sterols, but the proportions varied: 16:0, 18:1, and 18:2 comprising 24, 49, and 17% in SE (calculated iodine value 75) and 43, 36, and 7% in ASG (calculated iodine value 46). All four sterol lipids contained 80-84% of β-sitosterol, the remainder being stigmasterol. The only sugar in SG and ASG was D-glucose. It is deduced that the major representative species are: SE, β-sitosterol oleate/palmitate; SG, β-D-glucopyranosyl-(l → 3)-β-sitosterol; and ASG, 6-0-palmitoyl-β-D-glucopyranosyl-(l → 3)-β-sitosterol.     

Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer-chain fatty acids in the gastric and duodenal mucosa

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb₃Cer, Gb₄Cer and more polar ones with more than four sugars, whereas neither Gg₃Cer nor Gg₄Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as G_M3, G_M1, G_D3 and G_D1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I³SO₃-GalCer with hydroxylated longer-chain fatty acids based on the analyses by gas-liquid chromatography and negative ion fast-atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection. The nomenclature used for gangliosides and other glycosphingolipids follows the system of Svennerholm (Ref. 1) and the recommendation of the IUPAC-IUB Commission (Ref. 2), respectively.