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Small intestine of cat was homogenized under denaturating condition (6.0 M guanidinium chloride) in the presence of protease inhibitors and molecules in the 500 to 10 000 Da mass range were obtained by sequential ultrafiltrations. This material was separated by gel chromatography (Sephadex G-25), where fractions eluted in the mass range of 500–1000 Da inhibited 86Rb uptake into kidney slices in vitro. Furthermore, the same fractions exhibited a natriuretic effect when given intravenously to anesthetized rats. The gel chromatography fractions were further purified by cation exchange chromatography (CM-Sephadex C-25). Fractions eluted with a NaCl solution of around 250 mM inhibited 86Rb uptake into renal cortical tissue in vitro and showed natriuretic activity when tested in vivo in rats. It is proposed that intestine contains a water soluble natriuretic factor with an apparent molecular mass of 500-1000 Da. This material evokes a natriuresis in vivo and inhibits 86Rb uptake in vitro.

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.

A simple one-step method for the analysis of monosaccharides including galactosaminitol after acidic hydrolysis is described. The hydrolyzate was re-N-acetylated and analyzed by high-performance anion-exchange chromatography and the sugars were detected by a pulsed amperometric detector. The method was applied on a mixture of neutral oligosaccharides released from mucin glycopeptides of rat small intestine by alkaline borohydride. Sugars were detected down to the nanomole range and the results were compared with monosaccharide compositional analysis performed by gas chromatography of acetylated alditols.

Sialyl-Lewis x and a are known as ligands for E-selectin (ELAM-I) involved in leukocyte-endothelial adhesion. L-CanAg (light cancer antigen), secreted by a colon carcinoma cell line COLO 205, is a soluble mucin-type glycoprotein expressing sialyl-Lewis a antigens. L-CanAg was purified from spent culture medium by trichloracetic acid precipitation and Superose 6 gel filtration. With a monoclonal antibody against E-selectin (BBAI) as a positive control, the purified L-CanAg was shown to bind to E-selectin-Fc coated into plastic microtiter wells and to the surface of transiently E-selectin-transfected COS-I cells in a Ca(2+)-dependent way. Immunofluorescent double labelling showed that both BBAI and L-CanAg stained the same cells and morphological co-localization on E-selectin-transfected COS-I cells. Like BBAI, L-CanAg can inhibit leukocyte HL-60 cell adhesion to E-selectin-transfected COS-I cells, and this inhibition can be blocked by a F(ab')2 fragment directed against the sialyl-Lewis a epitope.

Leukotriene B4 (LTB4) and leukotriene C4 (LTC4) are considered to be important mediators in the pathophysiology of asthma. Theophylline, terbutaline, and hydrocortisone are drugs commonly used in the treatment of asthma. In the present study we have investigated the in vitro inhibitory effects of theophylline, terbutaline, and hydrocortisone on LTB4 and LTC4 generation from human leukocytes. After preincubation in the presence of these drugs, the cells were stimulated with the calcium ionophore A 23187 and the supernatants were analyzed for their LTB4 and LTC4 content using reverse-phase high-performance liquid chromatography (HPLC). Total leukotriene (LT) production (the combined amounts of LTB4 and LTC4) was dose-dependently inhibited by pretreatment with theophylline, terbutaline or hydrocortisone. Therapeutic levels of hydrocortisone (5 x 10(-6) M) plus theophylline (5 x 10(-5) M) inhibited LTB4 and LTC4 production in an additive way, as did the combination of hydrocortisone plus terbutaline (5 x 10(-8) M). A statistically significant effect of diminished LTB4 generation was obtained after preincubation with therapeutic levels of theophylline plus terbutaline, but no such effect was seen for LTC4 levels. The in vitro inhibitory effects on LTB4 and LTC4 generation from human leukocytes by theophylline, terbutaline, and hydrocortisone, as well as the additive effect of hydrocortisone plus theophylline or terbutaline, add to our understanding of the therapeutic effects of these drugs in the treatment of bronchopulmonary obstruction.

In 1975, reports were published that suggested that the gastrointestinal tract can "taste" the intake of sodium and in some unknown way influence the kidneys to increase sodium excretion. To test whether the intestine contained a natriuretic factor, intestinal tissue from cats was homogenized and fractionated by ultrafiltration to a molecular range of approximately 500-10,000 Da and separated by gel chromatography (Sephadex G25). The fractions were pooled into four large fractions that were assayed for "natriuretic" activity on anesthetized rats. The fraction containing the material with an apparent molecular mass of 500-1,000 Da augmented renal excretion of sodium and water, whereas the other pooled fractions did not exhibit any consistent natriuretic effect. The "natriuretic" fractions from gel filtration were further purified by ion exchange chromatography using a cation exchanger. The natriuretic activity was eluted from the ion exchange chromatography column at a NaCl concentration of 250 mM. Preliminary experiments on Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) suggest that the intestinal influence on renal sodium excretion is more pronounced in SHR than in WKY rats.

Background Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described. Principal Findings When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells. Conclusion DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.

The N-linked type of glycans were prepared as their glycopeptides after pronase digestion of the epithelial cells from the small and large intestine of two inbred strains of rat. These glycopeptides were analysed for sugar composition, for blood-group activity, by 1H-NMR spectroscopy, and after permethylation by electron-impact mass spectrometry. The glycopeptides were of the triantennary and tetraantennary types with intersected GlcNAc. The terminal parts were, in contrast to most N-linked glycans, devoid of neuraminic acid residues. Instead they contained blood-group determinants. Blood-group-H types 1 (Fuc alpha 1-2Gal beta 1-3GlcNAc) and 2(Fuc alpha 1-2Gal beta 1-4GlcNAc) were found in the small and large intestines of both strains, although type-1 predominated. One rat strain (GOT-W) did not express blood-group-A glycopeptides in the small intestine, but the large intestine from the same strain did. The other strain (GOT-BW) expressed blood-group-A determinants in the small intestine. The lack of neuraminic acid residues in the small and large intestine and of blood-group-B activity in the large intestine differed from that found in glycosphingolipids obtained from the same organs.

Acetylation of sialic acid-containing oligosaccharides lactonises the sialic acid residue quantitatively for all oligosaccharides studied except for 6′-sialyl-lactose. The modified, unsulphated, sialylated and sulphated oligosaccharides can then be fractionated by anion-exchange chromatography. Ammonolysis of the lactones followed by methylation yielded the dimethylamides, which are amenable to g.l.c.-m.s. and give intense and informative mass spectra. This approach has been used to characterise the sialic acid-containing O-linked oligosaccharides obtained from the mucin glycopeptides of the small intestine of the pig. At least 28 structures were found, having NeuAc or NeuGc 6-linked to the HexNAc attached to the peptide core or to a Hex 3-linked to HexNAc. Four different disialylated oligosaccharides were found having NeuAc or NeuGc on the Hex residue 3-linked to HexNAc and 6-linked to HexNAc.

Mucus is a highly hydrated gel which covers the mucosal surfaces of the body and provides a protective barrier against the external environment. The major part (>95%) of mucus is water and the gel matrix is formed by large and complex glycoproteins referred to as the mucus glycoproteins or the mucins, which account for about 0.5–5% of the secretion. In addition, proteins such as secretory IgA, lysozyme, lactoferrin and proteinase inhibitors are often present in the gel.

Publisher Summary This chapter discusses high-mass gas chromatography-mass spectrometry of permethylated oligosaccharides. Capillary gas chromatography-mass spectrometry (GC/MS) is a fast, sensitive, and high-resolving method for the characterization of biomolecules. It has been used in glycoconjugate research for analyzing the partially methylated alditol acetates after degradation of the glycoconjugates and, in a few cases, for the analysis of oligosaccharides after permethylation or trifluoroacetolysis. The advent of high-temperature capillary GC using thin-film thermostable bonded stationary phases now allows the analysis of large permethylated oligosaccharides, an adaptation that should also be useful in other fields of biomedical research. Oligosaccharides released from neutral glycosphingolipids and gangliosides have been successfully analyzed, as well as similar oligosaccharides found in free form in, for example, milk. Oligosaccharides released from mucins and other glycoproteins by alkaline-NaBH4 treatment can be analyzed and the high resolution achieved is necessary for separating the complex mixtures found in nature. Permethylation using solid NaOH gives good yields for neutral oligosaccharides with a GalNAc-alditol, and neutral and sialic acid-containing oligosaccharides with a Glc-aldose.

COMPARISON STUDY ON EXPRESSION OF RMUC2 AND RMUC3 IN RAT WITH ULCERATIVE COLITIS. Toshihiko Kubota. Susumu Ohara, Yuuji Hinoda, Gunnar C. Hansson, Hideaki Tamaki, Tomoe Katumata, Katunori Saigenji, Dept of Int Med, Fuchinobe Gen Hosp, Sagamihara, Japan; Dept of Biochem, Univ of Kitasato, Sagamihara, Japan; Oept of Med Technol, Univ of Yamaguchi, Yamaguchi, Japan; Dept of Med Biochem, Univ of Goteborg, Gothenburg, Sweden; Dept of Anat, Univ of Kitasato, Sagamihara, Japan; Dept of Int Med, Univ of Kitasato, Sagamihara, Japan.

Acid and neutral glycosphingolipids (0.01–1 μmol) were completely methylated in high yields and with little formation of by-products in 10 min at room temperature, using methyl sulphoxide, methyl iodide, and powdered NaOH. Re-methylation of methylated and LiAlH 4 -reduced gangliosides gave a new derivative that was useful for the analysis of gangliosides by mass spectrometry.

The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by thin-layer chromatography, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subfraction. As for the small intestine, the two separated compartments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Gal alpha being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major glycolipid with the structure Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4Glc beta 1----1Cer. as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectroscopy to have the probable structure Gal alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass spectrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The four- and six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-based blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Epithelial cells of rat small intestine have been separated into three intervals of different maturity correlated to cell migration from the crypt to the villus tip. The total acid and non-acid glycosphingolipids were isolated and analysed by thin-layer chromatography. The amount of glucosylceramide and N-glycoloylneuraminosyllactosylceramide was higher, while the amount of globotriaosylceramide and tetrahexosylceramide was lower in villus tip cells (more differentiated) compared to crypt cells (less differentiated). In addition to these alterations the lipophilic composition changed, as shown by a comparison by mass spectrometry of pennethylated and LiA1H4-reduced, permethylated derivatives of two of the non-acid glycolipid mixtures (crypt cells and villus tip cells). The components of ceramide were mainly trihydroxy 18:0 long-chain base (phytosphingosine) and hydroxy and non-hydroxy fatty acids. The only significant change concerned the fatty acids. In the crypt cell glycolipids the most abundant fatty acid was 20:0 non-hydroxy fatty acid. In the villus tip cells there was a relative increase of hydroxy fatty acids, with the 24:0 species in dominance. This change occurred for most glycolipids, but the fatty acids of glucosylceramide were villus tip-like already in the crypt cells. The blood group A-active tetraglycosylceramide, and probably the hematoside, did not show any alteration in the lipophilic part. The results indicate that the turnover of some glycolipids (or only their lipophilic part) is more rapid than the epithelial cell turnover.

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.

Electron ionization after direct inlet of permethylated and LiAlH4-reduced permethylated glycolipids allows a detailed structural interpretation of up to 12-sugar species, including size, sugar sequence and lipophilic components. A 25-sugar glycolipid gave partial information on sequence, with 13 sequences from the non-reducing end and 9 sequences from the ceramide. The specific sequence ions allow an analysis of mixtures, improved by a fractionation into separate species with programmable probe heating.

The hematosides (sialyl-lactosylceramides) of rat small intestine were separated as their acetylated derivatives. The isolated fractions were characterized by mass spectrometry and degradative methods, and the two major fractions also by NMR spectroscopy. From these results hematosides with different sialic acid and ceramide type could be assigned to thin-layer chromatographic bands. This allowed a structural interpretation of the chromatographic patterns observed for different parts of the small intestine. Thus, epithelial cells of ileum contained only hematoside with N-glycoloylneuraminic acid. Duodenum lacked this compound and instead the epithelial cells contained hematoside with N-acetylneuraminic acid. In non-epithelial tissue or both duodenum and jejunum-ileum the major hematoside had N-acetyl-neuraminic acid. The hematosides of epithelial cells had ceramide containing 18 : 0 trihydroxy base combined with 16, 20, 22, 24 : 0, and 24 : 1 hydroxy fatty acids (major part) or non-hydroxy fatty acids. In the non-epithelial hematosides the ceramide consisted of 18 : 1 dihydroxy base combined with 16, 18, 20, 22, 24 : 0, and 24 : 1 non-hydroxy fatty acids.

A novel series of glycosphingolipids has been isolated from the nonepithelial part of rat small intestine. A mixed fraction containing 3 major components corresponding to glycolipids with 5, 6, and 7 sugars and 2 minor components with 8 and 9 sugars was characterized. The structure of the major components was deduced by mass spectrometry and proton NMR spectroscopy of nondegraded permethylated and permethylated-reduced (LiAlH4) derivatives and gas-liquid chromatography of degradation products of native, permethylated, and permethylated-reduced glycolipids. The structures of the penta-, hexa-, and hepta-glycosylceramides were found to be GalNAcp beta 1 leads to (3Galp alpha 1 leads to)2-44Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. By analogy reasoning, supported by mass spectrometry, the octa- and nonaglycosylceramides were concluded to have 1 and 2 additional internal leads to 3Galp alpha 1 leads to 3 structures, respectively. A pentaglycosylceramide fraction from another rat strain was also isolated. The NMR spectra were in agreement with 2 isomeric structures of which 1 had the internal alpha 1 leads to 4 linkage replaced by an alpha 1 leads to 3 linkage. The fatty acids of all components were nonhydroxy 16:0 to 24:0 acids with the 18:0 homologue as dominating species. The major base was sphingosine and possibly monohydroxy 18:1 base in the larger glycolipids. This is a novel series of structures with a terminal saccharide identical with isoglobotetraoxylceramide (cytolipin R). The glycosyltransferase for the terminal GalNAc beta 1 leads to 3 of cytolipin R may possibly be identical with the enzyme adding the terminal sugar of this novel series. This is supported by the presence in the same tissue of probable precursor glycolipids with 4 to 8 hexoses.

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.

1. Introduction The finding of a relatively high glycolipid content in human small intestine [ I] and the fact that epithelial cells of intestine offer a system for study of the differ- entiation of eukaryotic cells [2], opens a unique possibility to test the postulated role of cell-surface carbohydrates in differentiation and regulation [3]. We are therefore developing specific preparative and analytical techniques for microscale characterization of complex glycosphingolipids. The present work is an application of a novel technique of mass frag- mentography on a glycolipid mixture from an individ- ual cat small intestine. 2. Experimental One lyophilized small intestine was extracted with chloroform-methanol in a Soxhlet apparatus. The total non-acid glycolipid fraction was prepared by conventional steps like mild alkaline degradation, sili- tic acid and DEAE-cellulose chromatography [4], and a combinatory use of native and acetylated [5] sub- stances. Methylation and reduction was done as in [6]. The mass spectrometer (MS 902, AEI Ltd, England) was connected on line to a data system (Instem Ltd, England). The sample probe temperature was raised linearly l”C/min or S”C/min and scans were taken every 2 min. A detailed description of mass scale cali

Small intestinal epithelial cells (enterocytes) were isolated from specimens obtained at operation from four human individuals with different blood group ABO, Lewis, and secretor phenotypes. The non-acid glycolipids were isolated and characterized by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy and for reactivity with monoclonal antibodies on thin-layer chromatograms. Monohexosylceramides and blood group ABH (type 1 chain) and Lewis glycolipids with 5-7 sugar residues were the major compounds present in all cases, and the expression of the major blood group glycolipids was in agreement with the ABO, Lewis, and secretor phenotype of the individual donors. Small amounts of more complex glycolipids with up to 10 sugar residues were identified by mass spectrometry in all cases. In addition, small amounts of lactotetraosylceramide, a blood group H-active triglycosylceramide with the structure of Fuc alpha 1-2Gal-Hex-Cer (where Fuc is fucose, Hex is hexose, and Cer is ceramide), and dihexosylceramides were identified in some cases. Globotriaosyl- and globotetraosylceramides were absent from the epithelial cells. Small amounts of Leb-active glycolipids in blood group OLe(a+b-), non-secretor and OLe(a-b-), secretor individuals as well as trace amounts of type 2 carbohydrate chain compounds in all individuals were detected by specific monoclonal antibodies.

The total non-acid glycosphingolipids were isolated from the small intestine of cat, cod-fish, guinea-pig, hen, mouse, rabbit, and two strains of rat. The samples were analyzed by thin-layer chromatography and mass spectrometry and for immunological activity. Mass spectrometry of permethylated and LiAlH4-reduced permethylated derivatives allowed the interpretation of the structures (carbohydrate sequence and ceramide composition) of up to 9 glycolipid species in one mixture. The interpretation was facilitated by a temperature programming of the direct inlet probe, leading to a successive evaporation of glycolipid species mainly according to the number of sugars. The structures concluded could in most cases be assigned to the separate bands revealed by thin-layer chromatography. Antigenic determinants proposed by the spectra were settled by immunological analysis. Thus, Forssman glycolipid was identified in cat, guinea-pig, hen and mouse, blood group A glycolipids in cat, rabbit, and rat and blood group B glycolipids in rabbit and rat. No Lewis activity was found. Certain ceramide types were demonstrated to exist preferentially in some glycolipids. Globoside and Forssman glycolipids (globo series) had a less hydroxylated ceramide (one free hydroxyl) compared to most fucolipids and other glycolipids (two or three hydroxyls). In conclusion, glycolipid patterns of intestine vary between species, and individuals of the same species.

Blood group A and H active glycosphingolipids have been isolated from rat small intestine. By mass spectrometry of the permethylated and LiAlH4-reduced permethylated glycolipid derivatives, the A glycolipids were shown to contain four (A-4), six (A-6), and 12 (A-12) sugar residues, respectively. The anomeric structure of the A-4 and A-6 glycolipids was established by proton NMR spectroscopy of the permethylated-reduced derivatives. Acid degradation and gas chromatography were used for analysis of binding positions. The structures of the A-4 and A-6 glycolipids were GalNAcp alpha 1 leads to 3Galp(2 comes from 1Fucp alpha) beta 1 leads to Glcp beta 1 leads to 1Cer and GalNAcp alpha 1 leads to 3Galp(2 comes from 1Fucp alpha) beta 1 leads to 3GlcNAcp beta 1 leads to 4Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The third glycolipid (A-12) was a branched dodecaglycosylceramide with two blood group A determinants. The complete structure of this glycolipid has not yet been solved. The blood group A activity was the same for the A-6 and A-12 glycolipids based on an equal number of blood group A determinants, but the activity of the A-4 compound was only about half of the others. The A-6 glycolipid was based on a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, thus differing from the already known isomer based on a type 2 chain (Gal beta 1 leads to 4GlcNAc) present in human erythrocyte. The blood group A activity of these two glycolipids was found to be identical. The three rat intestinal blood group A active glycolipids were exclusively located to the mucosa epithelial cells. The blood group H active tri- and pentaglycosylceramides (H-3 and H-5), presumed to be the precursors of the A-4 and A-6 glycolipids, were also identified. A 10-sugar glycolipid (H-10), a possible precursor of A-12, was not detected.

A total neutral (non-acid) glycolipid fraction has been isolated from rat small intestine. By silicic acid column chromatography of the acetylated glycolipid derivative, 7 different partly purified fractions were obtained. Thin-layer chromatography of both the acetylated and native glycolipid fractions revealed a highly complex pattern with at least 30 different glycolipid bands having a thin-layer mobility as for mono- to dodecaglycosylceramides. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) derivatives showed the presence of several glycolipid species not known before, including olighexosylceramides with 4, 5, 6 and 7 sugar residues and a tetraglycosylceramide with a blood group A determinant. This is the first report on such a complex glycolipid composition of a single organ.

A novel blood group H-active triglycosylceramide has been isolated from rat small intestine. It was present exclusively in the epithelial cells. The structure was established by mass spectrometry, NMR spectroscopy and degradative methods to be Fucpα1 → 2Galpβl → 4Glcpβ1 → 1Cer. The lipophilic part was made up of mainly trihydroxy base (phytosphingosine) and 16 : 0–24 : 0 fatty acids.

A strain of Escherichia coli originally isolated from urine of a patient with acute pyelonephritis was studied in detail for binding to glycosphingolipids. Bacteria labeled metabolically with [14C]glucose were layered over a glycolipid chromatogram and bound bacteria were detected by autoradiography. The detection was down to a few ng of glycolipid (pmol level) under these assay conditions. At a test level of 500 ng all glycolipids (more than a dozen molecular species analyzed) with Gal alpha 1----4Gal as an internal or terminal part bound the bacteria strongly while glycolipids known to lack this sequence were negative. Conformational analysis using hard sphere calculations including the exo-anomeric effect showed a bend in the saccharide chain at this disaccharide with a largely hydrophobic surface of the convex side, probably being part of the binding epitope. Mixtures of glycolipids isolated from a human ureter scraping and from urinary sediments bound bacteria in the 2- to 7-sugar interval. Thus, this infectious strain of E. coli recognizes glycolipids being present in epithelial cells lining the urinary tract.

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH4) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.

Utilisation de la chromatographie en phase gazeuse a haute temperature, couplee a la detection par spectrometrie de masse pour mettre en evidence des oligosides provenant des glycosphingolipides et mucines de Rat ou de Porc

A glucose-containing tetrasaccharide (13 mg/24 h) was isolated from the urine of a ten year old boy with the “childhood form” of glycogen storage disease type II (Pompe's disease). The structure, established by sugar analysis, methylation analysis, optical rotation and enzymatic degradation was found to be α-D-Glc p (lr6)-α-D-Glc p (l r 4).α-D-Glc p (1r4)-D-Glc. The same compound was also isolated in small amounts (1 mg/24 h) from normal urine. Larger glucose-containing oligosaccharides, not detected in normal urine were also present in the urine of the Pompe case.

Changes in lipid-bound cell surface carbohydrates during cell differentiation were studied in epithelial cells of rat small intestine (crypt and villus tip cells). The identity of the cells was established by morphological and enzymatic criteria. Acid and non-acid glycosphingolipids were analysed by thin-layer chromatography, with a separate study by mass spectrometry for identification purposes. The amount of glucosylceramide in the epithelial cell increased 2–3-fold and N -glycoloylneuraminosyllactosylceramide (hematoside) 6–7-fold from crypt to villus tip, but globotriaosyl- and tetrahexosylceramide decreased. Fucolipids with blood group A activity (black-white strain) and H activity (white and black-white strain) showed no difference in this respect. No lengthening of the carbohydrate chains was noted, in contradiction to earlier suggestions concerning cell surface carbohydrate and differentiation. The increased amount of sialic acid of the villus tip cells may facilitate the cell-cell dissociation needed for cell extrusion. There was an increase in 2-hydroxy fatty acids vs non-hydroxy fatty acids of major glycolipids from crypt to villus tip.

Two novel blood group H-type decaglycosylceramides with a branched core saccharide have been identified in mixture in a fraction isolated from rat small intestine. They were present exclusively in the epithelial cells. The number and sequence of sugars were established by direct inlet mass spectrometry of the permethylated and LiAlH4-reduced permethylated derivatives. Gas-liquid chromatography of the products after degradation of the native and permethylated glycolipids gave the type of sugars and the binding positions. A di- and a trisaccharide were identified by mass spectrometry after degradation of the permethylated-reduced derivative. One trisaccharide had the structure (formula see text) and was therefore additional evidence for a branched structure. Treatment of the decaglycosylceramide fraction with alpha-L-fucosidase gave free fucose and an octaglycosylceramide identified by mass spectrometry. Proton NMR spectra of the permethylated and permethylated-reduced octa- and decaglycosylceramides provided evidence for the binding configurations and the localization of type 1 and type 2 sequences in the two branches. The 3-linked branch was homogeneous with a type 1 saccharide (Gal beta 1 leads to 3GlcNAc) but the 6-linked branch had both type 1 and type 2 (Gal beta 1 leads to 4GlcNAc) sequences. Two glycolipids with the following probable structures were therefore present, making up 60 and 40% of the mixture, respectively: (formula see text) The lipophilic part contained mainly trihydroxy 18:0 long chain base (phytosphingosine) and 16:0 to 24:0 nonhydroxy fatty acids.

Rat stomach gangliosides were purified and their distribution in the different tissue compartments was established. Three major monosialogangliosides were found: GM3, GM1, and a ganglioheptaosylceramide carrying a blood group B determinant. This latter structure was characterized by exoglycosidase degradation, immunostaining with a monoclonal anti-blood group B antibody on thin layer chromatogram, permethylation analysis, electron-impact mass spectrometry of the permethylated-reduced and trimethylsilylated molecule, and 1H NMR spectroscopy of the native ganglioside. It was found to be (Formula: see text) i.e. a GM1 structure substituted with the blood group B determinant and was called B-GM1. A similar structure has been previously identified in precancerous rat liver and chemically induced rat hepatoma (Holmes, E. H., and Hakomori, S. (1982) J. Biol. Chem. 257, 7698-7703). Fucosyl-GM1 was also detected as a minor ganglioside in rat gastric mucosa. The ganglioside profile was modified during the postnatal development. The contribution of GM3 and GD3, which accounted for 95% of the ganglioside sialic acid at birth, decreased during the first 3 weeks of life. GM1, fucosyl-GM1, and B-GM1 were not detected at birth. The concentration of the fucogangliosides increased during the 2nd and 3rd weeks after birth, was stable during the 4th week and then decreased, whereas that of GM1 increased steadily between 6 days and 2 months of age. B-GM1, which has been defined as a tumor-associated ganglioside in the rat liver, was found to be a developmentally regulated antigen of the normal rat stomach.

The epithelial cells of rat small intestine (jejunum-ileum) were separated from their supporting stroma (residue). Total nonacid and acid glycosphingolipids were prepared from the two compartments. The acetylated nonacid glycolipids were separated into 10-12 fractions by column chromatography. These were analyzed by chromatographic methods, mass spectrometry, and proton NMR spectroscopy and compared with glycolipids isolated from whole rat small intestine. The sialic acid-containing glycosphingolipids were compared in the same way without subfractionation. At least 37 different glycosphingolipids (different carbohydrate moieties) were found, 23 in the nonepithelial residue and 17 in the epithelial cells of one rat strain. In a second rat strain, another 4 structures were detected. The glycosphingolipids of epithelial cells and nonepithelial residue were distinctly different. Glucosylceramide, lactosylceramide, and globotriaosylceramide were found in both compartments, while isoglobotriaosylceramide was restricted to the nonepithelial residue. A tetrahexosylceramide with a terminal Gal alpha 1 leads to 3 on a globotriaosylceramide core was found in both compartments as were homologues with 1 or 2 additional internal leads to 3Gal alpha 1 leads to units, but homologues with 3 or 4 additional internal Gal were only nonepithelial. Glycosphingolipids with terminal beta-GalNAc were restricted to the nonepithelial residue comprising globotetraosylceramide, isoglobotetraosylceramide, and a series of glycolipids with 5 to 9 sugars having the above-mentioned oligohexosylceramides as core structures. Fucolipids (blood group H) having 3, 5, 6, and 7 sugars and lacking amino sugars, and fucolipids with 5 and 10 sugars containing N-acetylglucosamine were restricted to the epithelial cells. Fucolipids (blood groups H and B) with 5 and 6 sugars containing N-acetylgalactosamine were restricted to the nonepithelial residue. In a 4, 6, and 12 sugars were found in the epithelial cells. N-Glycoloylneuraminosyllactosylceramide was the only ganglioside found in the epithelial cells while N-acetylneuraminosyllactosylceramide was nonepithelial together with gangliosides based on gangliotetraosylceramide and isoglobotetraosylceramide.