Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Metabolism of exogenous gangliosides GM1 and chemically modified GM1 in mice

Ganglioside GM1(NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [3H]GM1(NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside GM1(NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered GM1(NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorporated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of GM1(NeuAc) administered to the three strains of mice were different from each other. In other organs, GM1(NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside GM1(GM1(NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [3H]GM1(NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3(NeuAc)-NMe, via GM2(NeuAc)-NMe, within 24 h. GM3(NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradually. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetylneuraminic acid by monooxygenase.     

Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides.

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGc alpha 2----3Gal- terminal structures, such as GM3(NeuGc), IV3NeuGc alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-Gb5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGc alpha2----3Gal- sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAc alpha 2----3Gal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGc alpha 2----3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GD3(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal sequences, such as GD3(NeuGc-NeuGc-), IV3NeuGc alpha 2-Gg4Cer, IV3NeuGc alpha 2-nLc4Cer, and V3NeuGc alpha 2-Gb5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR and computational studies of interactions between remote residues in gangliosides

Conformational preferences of the gangliosides GM1, GM1b, and GD1a have been investigated by using a systematic combination of NMR distance constraints and molecular mechanics calculations. These gangliosides share a common four-sugar core but differ in the number or placement of sialic acid residues attached to the core. Placement of the sialic acid residues is shown to influence the preferred core conformation. The origin of these effects is postulated to be intramolecular interactions of the sialic acid residues with other remote residues. In the case of GM1, hydrogen bonds between the internal sialic acid and an N-acetyl group on GalNAc are suggested. In the case of GD1a, a hydrogen-bonding network between the terminal and internal sialic acids is suggested to play a role.     

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GD1a and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo-GM2 and asialo-GM1, respectively, by thin-layer chromatography.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

Characterization of two molecular species GD3 ganglioside from bovine buttermilk

Two gangliosides, representing 85% of total lipid-bound sialic acid, have been isolated from bovine buttermilk and characterized. Both contained long-chain base, glucose, galactose and sialic acid in the molar ratio 1:1:1:2, and gave, upon sialidase treatment, a neutral glycolipid, characterized as lactosylceramide. Partial acid hydrolysis, permethylation analysis and chromium trioxide oxidation indicated their basic oligosaccharide portion to be NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc. The difference between the two forms was exclusively in the ceramide moiety of the molecule, one containing mainly long-chain (C22-C25) fatty acids and an equimolar proportion of C16 and C18 long-chain bases, and the other mainly palmitic acid and C18 long-chain base.     

Gangliosides in bovine milk. Changes in content and distribution of individual ganglioside levels during lactation.

Bovine milk undergoes changes in its ganglioside contents during the different stages of lactation. These contents are higher in colostrum (7.5 mg of lipid-bound NeuAc/kg) than in transitional (2.3 mg) or mature (1.4 mg) milk. The sialic acid content of milk follows a similar profile to that of gangliosides with the highest content during the first few days post partum followed by a gradual decrease towards the end of the period studied. When the individual distribution of gangliosides was examined throughout the course of lactation, several changes were also found. GD3 is the major ganglioside (about 60-70%) found; its content decreases from the first to the fifth day, increasing towards the end of the period considered. GM3, GD3 and GT3, sialyllactosylceramide-containing gangliosides account for 80-90% of the total lipid-bound NeuAc content. The most striking change in the ganglioside pattern was the gradual increase in G3.     

Biochemical properties of N-methylamides of sialic acids in gangliosides

A simple and rapid method for the preparation of N-methylamides ( - CONHCH3) of sialic acids in gangliosides and biochemical properties of the modified gangliosides are described. The sialic acid carboxyl groups of gangliosides were esterified with CH3I-dimethylsulfoxide, followed by heating with monomethylamine. The modified gangliosides were chemically identified by TLC, IR spectroscopy, GLC-mass spectrometry and NMR spectroscopy. The N-methylamide derivative of GM1 produced a high titer IgG antibody. The antibody weakly cross-reacted with the methylester of GM1 and its reductive derivative but did not react with the intact GM1. A monoclonal antibody (M2590) specific for GM3 did not react with carboxyl-modified GM3 (methylester, N-methylamide, and reduced GM3), but it reacted with modified GM3 which contains the C7-analog of the sialic acid. Clostridium perfringens and Arthrobacter ureafaciens sialidases did not hydrolyze the N-methylamide derivatives, methylesters or reductive derivatives of the gangliosides and, furthermore, these derivatives did not inhibit the actions of these sialidases.     

Differences in liver ganglioside patterns in various inbred strains of mice

The ganglioside patterns in the liver of different inbred and hybrid strains of mice were investigated. The inbred strains were Balb/cAnNCr1BR, C57BL/6NCr1BR, DBA/2NCr1BR. C3H/HeNCr1BR; the hybrid strain was the Swiss albino. The following major gangliosides were found to be present in mouse liver: GM3-NeuAc; GM3-NeuGl, GM2 [a mixture of one species carrying N-acetylneuraminic acid (NeuAc) and one carrying N-glycollylneuraminic acid (NeuGl)], GM1 and GD1a-(NeuAc,NeuGl). The qualitative and quantitative patterns of liver gangliosides were markedly different in the various inbred strains of mice; in Balb/cAnNCr1BR strain, ganglioside GM2 was preponderant (99.2% of total ganglioside content); in C57BL/6NCr1BR, the major ganglioside was GM2 (90.4%), followed by GM3-NeuAc (5.6%) and GM3-NeuGl (4.0%); in DBA/2NCr1BR, GM2 accounted for 77.1%, GD1a-(NeuAc,NeuGl) 18.9% and GM1 3.1% of gangliosides; in C3H/HeNCr1BR, GM2 constituted 50.6%, GM1 22.8% and GD1a-(NeuAc,NeuGl) 22.1%. In the hybrid Swiss albino mice, liver ganglioside composition markedly varied from one animal to another, GM3-NeuGl, GM2 and GD1a-(NeuAc,NeuGl) being the predominant gangliosides in the various cases.     

O-acetylated sialic acids in gangliosides from pig spleen lymphocytes

The sialic acid content of gangliosides from pig spleen lymphocytes was studied by thin-layer chromatography. N-glycolylneuraminic acid and N-acetylneuraminic acid were detected for the first time in this material as the major sialic acids. In addition, two other sialic acids, tentatively designated O-acetylated sialic acids, according to their RF values on cellulose plates, were also found. We have detected several gangliosides showing a retarded migration pattern in two dimensional thin-layer chromatography with an intermediate ammonia treatment. One of these gangliosides could be an O-acetylated derivative of the disialoganglioside GD3, since after de-O-acetyation it co-migrates with GD3. Another ganglioside co-migrated with GM2 before the alkaline treatment; however, after the treatment it was also retarded and co-migrates with GD3.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Occurrence of glycosylation and deglycosylation of exogenously administered ganglioside GM1 in mouse liver.

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc,NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc,NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc,NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc,NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.     

Gangliosides in rat kidney: composition, distribution, and developmental changes

Gangliosides in rat kidney were analyzed for their composition, regional distribution, and developmental changes. Renal tissue from 7-week-old rats showed a GM3-dominant pattern with GD3 and several minor ganglioside components including GM4, GM2, GD1a, and an unknown ganglioside (ganglioside X). The tissue also contained c-series gangliosides that included GT3 as the main component with GT2 in a lesser amount. Ganglioside analysis of cortical and medullary regions of renal tissue suggested the restricted localization of some gangliosides. While GM4 and GD3 were enriched in the cortical region, GM2 was distributed mainly in the medullary area. Renal gangliosides showed unique developmental profiles during a period from Embryonic Day 20 (E20) to 7 weeks postnatal. The content of renal gangliosides increased from E20, reached the highest around Postnatal Day 1, and thereafter, decreased rapidly to the adult level. The ratio of N-glycolylneuraminic acid to total sialic acids in gangliosides tended to change in inverse proportion to the amount of total sialic acids. The composition of major gangliosides in renal tissues shifted from GD3-dominant to GM3-dominant patterns with advancing ages. While GM1 was expressed only at early stages of the development, GM4, GM2, and ganglioside X appeared after Postnatal Day 3. The expression of c-series gangliosides was less affected through the period examined. These results suggest that gangliosides may be implicated with development and function of rat kidney.     

Major gangliosides in normal and pathological human thyroids

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.