Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).     

Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma.

Acidic and neutral glycosphingolipids were isolated from a human gastric adenocarcinoma, and binding of Helicobacter pylori to the isolated glycosphingolipids was assessed using the chromatogram binding assay. The isolated glycosphingolipids were characterized using fast atom bombardment mass spectrometry and by binding of antibodies and lectins. The predominating neutral glycosphingolipids were found to migrate in the di- to tetraglycosylceramide regions as revealed by anisaldehyde staining and detection with lectins. No binding of H. pylori to these compounds was obtained. The most abundant acidic glycosphingolipids, migrating as the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori selectively interacted with slow-migrating, low abundant gangliosides not detected by anisaldehyde staining. Binding-active gangliosides were isolated and characterized by mass spectrometry, proton nuclear magnetic resonance, and lectin binding as sialyl-neolactohexaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and sialyl-neolactooctaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer).     

Influence of sialic acid groups on the retention of glycosphingolipids in blood plasma.

The removal of several glycosphingolipids from the circulation and their disposal in different tissue and fluid compartments was studied in adult rats. 3H-labeled dihydro analogs of several glycosphingolipids were injected intravenously and radioactivity was measured in arterial blood samples at subsequent time intervals, to obtain half life values for the labeled compound in the plasma. Half life values of less than 1 min were obtained for neutral glycosphingolipids whereas the half lives of labeled gangliosides were much longer and ranged from 3.8 to 21 h. The prompt removal of labeled neutral glycosphingolipids but not of the gangliosides indicates that sialic acid groups play a significant role in the retention of glycosphingolipids in the circulation. The results suggest that neutral glycosphingolipids are rapidly exchanged with their counterparts in a large extraplasma pool and that a major portion of this exchange could occur between plasma and liver. The detection of only a minute fraction of the injected glycosphingolipids in the cerebrospinal fluid indicates that a blood-cerebrospinal fluid barrier exists for these compounds in the rat.     

Recent advances in identifying the functions of gangliosides

The recent development of several new approaches has proven extremely useful in identifying functions for gangliosides, the sialic-acid containing glycosphingolipids. The first is the incorporation of exogenous gangliosides into the plasma membrane of ganglioside-deficient cells. Using this approach, specific gangliosides have been identified as the receptors for certain bacterial toxins and viruses and as important factors in the organization of fibronectin into an extracellular matrix. The second approach has been a ligand blotting technique which allows detection of ganglioside-binding proteins such as toxins and antibodies. Gangliosides are separated by thin-layer chromatography and overlain with the protein of interest. Specific binding of the ligand to gangliosides can then be detected by either direct or indirect methods. The third approach is the use of the B or binding subunit of cholera toxin as a specific probe for endogenous plasma membrane ganglioside function. The ability of the B subunit to alter the growth of cells directly demonstrates a role for gangliosides as biotransducers of signals for the regulation of cell growth.     

Sialyl-lactotetra,a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10⁹ cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.     

Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits

Neutral glycosphingolipids and gangliosides in rabbit thymus, spleen, bone marrow, and erythrocyte ghosts were analyzed by conventional chemical and enzymatic procedures and negative ion fast atom bombardment mass spectrometry (FABMS). Thymus gangliosides showed a characteristic composition. Major gangliosides comprising 75% of the total thymus gangliosides were sialosyl lacto-N-neo-tetraosyl- and sialosyl lacto-N-nor-hexaosylceramides containing NeuGc and palmitic acid. These major thymus gangliosides were not detected in spleen, bone marrow, or erythrocytes, whereas GD1a, which was not present in the thymus even in a trace amount, was present in spleen and bone marrow. In addition, the major gangliosides in rabbit thymus were preferentially reduced when an animal was given an intraperitoneal injection of cortisone acetate, as found on analysis 48 h later. The decrease was accompanied by a concomitant increase in NeuAc-containing GM3 with longer chain fatty acids.     

Glycosphingolipid composition of human semen

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.     

Differential expression patterns of gangliosides in the ischemic cerebral cortex produced by middle cerebral artery occlusion

Neuronal damage subsequent to transient cerebral ischemia is a multifactorial process involving several overlapping mechanisms. Gangliosides, sialic acid-conjugated glycosphingolipids, reduce the severity of acute brain damage in vitro. However their in vivo effects on the cerebral cortex damaged by ischemic infarct are unknown. To assess the possible protective role of gangliosides we examined their expression in the cerebral cortex damaged by ischemic infarct in the rat. Ischemia was induced by middle cerebral artery (MCA) occlusion, and the resulting damage was observed by staining with 2, 3, 5-triphenylterazolium chloride (TTC). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3 and GM1 increased in the damaged cerebral cortex, and immunofluorescence microscopy also revealed a significant change in expression of GM1. In addition, in situ hybridization demonstrated an increase in the mRNA for ganglioside GM3 synthase. These results suggest that gangliosides GM1 and GM3 may be synthesized in vivo to protect the cerebral cortex from ischemic damage.     

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Chemokines bind to sulfatides as revealed by surface plasmon resonance

Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1alpha/CXCL12, MIP-1alpha/CCL3 and MIP-1beta/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain 'rafts', i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.     

Antibodies against ganglioside GT3 in the sera of patients with type I diabetes mellitus

Clinical and experimental data support the concept that type I diabetes mellitus results from autoimmune destruction of pancreatic beta cells. Although both proteins and glycolipids are targets of anti-islet cell antibodies, the Ag have not been purified or characterized. Previously, we observed that rat insulinoma (RIN) cell lines varied in their reactivity with both human antibodies and murine mAb A2B5, which binds to polysialo gangliosides. To determine the chemical basis of the varied immunoreactivity, we analyzed the glycosphingolipids of 5 RIN lines. Glycolipids bound by two mAb and by antibodies in the sera of type I diabetics were identified. The more immunoreactive RIN lines contained a much higher content of gangliosides and a higher proportion of complex gangliosides. The major gangliosides were GM3, GD3, and GT3. By high performance TLC immunostaining, we demonstrated that A2B5 and R2D6, an anti-beta cell murine mAb, bound most strongly to ganglioside GT3. The binding of human sera to gangliosides was analyzed by an ELISA assay. Although both normal and diabetic sera contained antibodies to various glycolipids, binding to GT3 was significantly elevated in 31 new-onset type I diabetics (p less than 0.001). The presence of the GT3 trisialosyl epitope on human islet cells was shown by immunofluorescent staining by both R2D6 and A2B5. These findings support previous suggestions that gangliosides play an important role in the immunopathology of type I diabetes, and identify for the first time a specific ganglioside Ag that is the target for autoantibodies in a subset of diabetic patients.     

Separation of derivatized glycosphingolipids into individual molecular species by high performance liquid chromatography

The high performance liquid chromatography separation of the perbenzoyl derivatives of the neutral glycosphingolipids (GlcCer, LacCer, GbOse3Cer, GbOse4Cer, and GgOse3Cer) and the p-bromophenacyl and 2,4-dinitrophenyl hydrazide derivatives of the gangliosides (GM4, GM3, GM2, GM1, GD1a) into individual molecular species on a C18 reversed-phase column is described. Peaks were identified by comparing their relative retention times to the relative retention time of the corresponding glycosphingolipid of known molecular species composition. As little as 5 to 10 pmol of each molecular species of neutral glycosphingolipids and 3 to 5 pmol of the gangliosides can be detected. The effects of changes in the proportion of acetonitrile, methanol, and water in the mobile phase and of column temperature on the molecular species separation are described. A procedure for the tentative identification of glycosphingolipid molecular species based on their relative retention times is presented.     

Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.