Heinz Egge

Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula.

At present, not much is known about the absorption and metabolism of human milk (HM) oligosaccharides in term and preterm infants. We investigated the renal excretion of lactose and complex oligosaccharides in preterm infants fed HM (n= 9, mean actual body weight 2290 g) or a cows milk‐based infant formula (n= 9, mean actual body weight 2470 g). We found that the renal excretion of lactose in HM‐fed infants was slightly lower than in formula‐fed infants (14.0 ± 7.4 versus 20.4 ± 8.7 mg kg‐1 day‐1, mean ± SD). The excretion of neutral sugars deriving from oligosaccharides was similar in HM‐fed and formula‐fed infants (3.8 ± 2.1 versus 2.9 ± 0.9mgkg‐1 day1‐); the difference between means was not statistically significant. The separation and characterization of oligosaccharides by high‐pH anion exchange chromatography with pulsed amperometric detection (HPAE‐PAD) and subsequent analysis by fast atom bombardment‐mass spectrometry (FAB‐MS) revealed a more complex pattern in HM‐fed infants compared to the formula‐fed group. Lactose‐derived oligosaccharides characteristic for HM (e.g lacto‐N‐tetraose, and lacto‐N‐fucopentaoses I and II) were excreted in HM‐fed but not in formula‐fed infants. These results indicate that nutrition has a significant impact on the oligosaccharide composition in urine of preterm infants.

Fast-atom-bombardment mass-spectrometry for carbohydrate-structure determination

Abstract The potential of fast-atom-bombardment (f.a.b.) mass-spectrometry in the carbohydrate field was assessed with the aid of unmodified, permethylated, and peracetylated oligosaccharides and glycosphingolipids. F.a.b. spectra are presented for ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 → d -GlcNAc, permethylated ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 - d -[ 2 H]GlcNAcol, permethylated gangliotetraosylceramide, and reduced and peracetylated tetra- to hepta-saccharides from human milk. From unmodified oligosaccharides, pseudomolecular ions (M + Na + ) were obtained as major ions in the high-mass range. Permethylated oligosaccharides and glycosphingolipids gave pseudomolecular ions (M + H + ) of high intensity, together with fragment ions of high diagnostic importance. At ambient temperature, f.a.b. spectra could only be obtained for the lower homologs of per- O -acetylated oligosaccharides. Reduced and peracetylated penta- to hepta-saccharides from human milk gave f.a.b. spectra only after heating of the target.

Neuroaxonal Dystrophy Due to Lysosomal α-N-Acetylgalactosaminidase Deficiency

AMONG the inherited neurologic diseases, the neuroaxonal dystrophies constitute a group of neurodegenerative disorders characterized by a common axonal lesion.1 These disorders include the infantil...

Fucose containing oligosaccharides from human milk: I. Separation and identification of new constituents

Neutral oligosaccharides isolated from pooled human milk were subjected to fractionation on high-performance thin-layer chromatography (HPTLC) plates, Iatrobeads, and reverse-phase chromatography after borohydride reduction and peracetylation. By the combined HPLC and HPTLC separation a mixture of pooled human milk oligosaccharides was separated into 101 fractions. These fractions were characterized by field desorption or fast atom bombardment (FAB)-mass spectrometry. Each of the carbohydrate constituents, the peracetylated glucitol, the galactose, the glucosamine, and the fucose contribute specific mass increments to the molecular weight of the oligosaccharide. Therefore, the exact carbohydrate composition can be calculated from the molecular weight determined by mass spectrometry. Among the fractions obtained one trifucosyl-lacto-N-tetraose, five monofucosyl-, eleven difucosyl-, and nine trifucosyl-lacto-N-hexaoses, one monofucosyl-, eight difucosyl-, seven trifucosyl-, four tetrafucosyl-, and two pentafucosyl-lacto-N-octaoses, one trifucosyl-, and two difucosyl-lacto-N-decaoses could be identified. FAB spectra furnished additional data on structural features of the isolated oligosaccharides.

Heterogeneity of bovine lactotransferrin glycans. Characterization of α-D-Galp-(1→3)-β-D-Gal- and α-NeuAc-(2→6)-β-D-GalpNAc-(1→4)-β-D-GlcNAc-substituted N-linked glycans

Abstract Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1 → 6)-α- l -fucosylated structures in Fractions A and B. Sequences containing α- d -Galp-(1 → 3)-β- d -Gal on the α- d -Man-(1 → 6) antenna, and β- d -GalpNAc-(1 → 4)-β- d -GlcNAc and α-NeuAc-(2 → 6)-β- d -GalpNAc-(1 → 4)-β- d -GlcNAc on the α- d -Man-(1 → 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4 − 9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-β- d -glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le(a) and A, Le(b). Neutral and acidic components were separated from each other by anion-exchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore the moral response factors after HPAEC-PAD have been determined for 28 components.

Fucose-containing oligosaccharides from human milk from a donor of blood group 0 Le(a) nonsecretor.

The neutral oligosaccharides from the milk of a single donor with blood group 0, Lewis(a+b-) nonsecretor were separated into 18 fractions essentially according to the number of carbohydrate constituents using gel permeation chromatography on Biogel P-4 and Fractogel TSK HW-40. Further separation was achieved by HPLC and by HPTLC after reduction and peracetylation. The fractions obtained were analysed by FAB mass spectrometry with and without derivatization and by one- and two-dimensional proton NMR. Besides the already described 3-fucosidolactose, fucopentaose II (2), fucopentaose III (6) and difucohexaose II (4) the following fucosylated oligosaccharides could be identified. Among the higher oligosaccharides a branched lacto-N-decaose (12) was obtained in pure form after removal of the fucose residues by mild acid treatment.

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances☆☆☆

The Lea-, Leb-, and H-type 1 (LedH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by 1H nuclear magnetic resonance at 360 MHz in dimethyl-d6 sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.

[4] Analysis of glycosphingolipids by high-resolution proton nuclear magnetic resonance spectroscopy

Publisher Summary Nuclear magnetic resonance (NMR) is a nondestructive method capable of furnishing many-sided structural information; it is therefore highly desirable fully to exploit its potential, aiming at complete structure determinations without resorting to data obtained by other methods. This chapter presents examples of such structure elucidation and discusses the prospects of proton NMR becoming a self-sufficient method in the field of structural research of glycosphingolipids. In this study spin decoupling difference spectroscopy is applied because it is reasonable balance between the measuring time invested and the spectral information obtained. In this way one can identify most of the signals of the sugar ring protons for several glycosphingolipids containing up to 10 sugar residues. Another successful approach is the use of the nuclear Overhauser effect. The second, decisive, part of the problem is to extract structural information from those data. This task is greatly facilitated if series of structurally related substances are available. The work on Forssman glycolipid and the pentasaccharide ceramide from rabbit erythrocytes with B-like blood group activity offers a good opportunity to illustrate the approach. The results obtained revealed regular changes of chemical shifts related to the structural features a-e listed in this chapter. These regularities are shown to be applicable to structure determination of a bifurcate ceramide decasaccharide and of a series of highly crowded fucose-containing glycosphingolipids of the Lewis blood-group system.

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.