Neutral Human Milk Oligosaccharides Research Articles

Mass spectrometric detection of multiple extended series of neutral highly fucosylated N-acetyllactosamine oligosaccharides in human milk

Complex mixtures of high-molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)–Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly- N-acetyllactosamine cores are substituted with up to 10 fucose residues on an oligosaccharide that has 7- N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.

Selected oligosaccharides from human milk induce growth inhibition, differentiation and apoptosis in intestinal cells

Human milk oligosaccharides (HMO) have recently been discussed with regard to prebiotic effects and their immune modulating potential for breast-fed infants. However, little is known about their direct effects on the infant’s intestine. Therefore, we investigated the growth-promoting activity of HMO as well as their impact on cell differentiation and apoptosis by using human transformed and non-transformed intestinal cells (HT-29, Caco-2 and HIEC). Cells were exposed to neutral and acidic oligosaccharides in concentrations of 0–15mg/mL for 24–72 h. The highest concentrations of neutral HMO decreased intestinal cell proliferation by 41.7 ± 8.2 % in HT-29, 62.5 ± 3.5 % in Caco-2 and 77.3 ± 4.0 % in HIEC cells. Inhibition of proliferation by acidic HMOs was also distinctive: 31.6 ± 4.6 % in HT-29, 53.8 ± 8.4 % in Caco-2 and 65.7 ± 4.0 % in HIEC cells. This inhibition by neutral HMO was associated with an induction of differentiation and apoptosis, whereas acidic HMO only induced differentiation in HT-29 and HIEC cells. In addition, we investigated 18 of the most abundant HMOs in order to identify possible key compounds. All substances tested displayed significant growth reduction but few were able to induce differentiation and/or apoptosis. In conclusion, selected HMOs were shown to induce growth inhibition in different epithelial intestinal cells by two different mechanisms, i.e. by suppressing cell cycle progression through induction of differentiation and/or apoptosis.

Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression.

Human milk is thought by many authorities to be preferable to formula as a source of nutrients for infants. Some of the benefits may stem from its high concentration of unbound oligosaccharides (5-10 g/L). These sugars have structural similarities to selectin ligands, known to mediate important cell-cell interactions in the immune system. Platelet-neutrophil complexes (PNC) exist in healthy individuals but have been implicated in disease states. Formation of these complexes requires selectins and as such, could be influenced by human milk oligosaccharides (HMO). Here, we investigate this possibility by examining the effect of HMO on the formation of PNC and activation of associated neutrophils. We collected blood from 10 healthy volunteers, activated platelets with adenosine 5'-diphosphate, and added HMO, oligosaccharide standards, or phosphate-buffered saline as a control. We determined the influence of HMO on PNC formation and adjacent neutrophil activation with fluorescein-activated cell sorter analysis after labeling with antibodies for the platelet marker CD42a and the neutrophil activation marker CD11b. Within physiologically achievable concentrations (6.25-125 microg/mL), an acidic HMO fraction reduced PNC formation up to 20%, which was similar to the effect seen with high concentrations of sialyl-Lewis x. Associated neutrophils showed a dose-dependent decrease in beta 2 integrin expression, up to 30%, at high but physiological concentrations. The neutral HMO fraction had no effect. These results support the hypothesis that acidic HMO serve as anti-inflammatory components of human milk and thus, contribute to the lower incidence of inflammatory diseases such as necrotizing enterocolitis in breast-fed versus formula-fed infants.

Prebiotics and immune responses.

Feeding breast milk of healthy mothers is associated with a lower incidence of infectious and allergic diseases. Although this effect is of multifactorial origin, it is widely accepted that the entire intestinal flora of breastfed infants provides anti-infective properties and is an important stimulating factor for the postnatal development of the immune system. Clearly, the effect of human milk on the postnatal development of the intestinal flora cannot be attributed to a single ingredient. It is generally accepted, however, that human milk oligosaccharides play a key role in this matter (1). The composition of human milk oligosaccharides is very complex. As they are resistant against digestion, they can be detected in the faeces as well as in the urine of breast-fed infants (2). This low or absent digestibility is a prerequisite for prebiotic activities of dietary compounds (3). Apart from their prebiotic effects, there is also evidence that human milk oligosaccharides act as receptor analogues to inhibit the adhesion of pathogens on the epithelial surface (1) and interact directly with immune cells (Eiwegger Th, et al., unpublished data). Due to their complexity, oligosaccharides with structures indentical with human milk oligosaccharides are not available as dietary ingredients. Searching for alternatives, a mixture of neutral galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) have been identified as effective prebiotic ingredients during infancy (5). More recently, also acidic oligosaccharides such as pectin hydrolysate are under investigation (6). In several clinical trials it could be demonstrated that feeding a formula supplemented with a mixture of GOS and long-chain FOS results in an entire intestinal flora similar to that found in breast fed infants (7). Comparable to human milk oligosaccharides, GOS and FOS stay present during the whole gastrointestinal passage and are therefore detectable in the faeces of infants fed a GOS/FOS supplemented formula. With regard to stool characteristics, there was a significant influence of GOS/FOS in preterm as well as term infants: feeding the oligosaccharides-supplemented formula increased the stool frequency and softened the stool consistency in comparison to a standard formula so that stool characteristics could be achieved which were very similar to those of human milk fed infants (5). In a study in term infants the effect of different concentrations of the GOS/FOS mixture was tested in parallel by a traditional plating technique and a modern molecular biologic technique (7). Whereas with the plating technique, only living bacteria can be detected, all bacteria are counted with the molecular biologic technique. The results obtained by the two methods are shown in Figure 1. They clearly indicate that with the use of the GOS/FOS mixture, the number of bifidobacteria increases and the effect is much more pronounced for the living bacteria than for the total number of bacteria. Short-chain fatty acids (SCFA) are an important marker of the bacterial metabolism in the intestine. Invitro experiments focusing on the short SCFA synthesis by the faecal flora could demonstrate that the SCFA synthesis from GOS/FOS mixture was comparable to the synthesis from a quantitatively equivalent neutral human milk oligosaccharides fraction (7). Similar results were obtained in-vivo by measuring the fatty acid pattern in faeces from infants fed a formula supplemented with GOS/FOS: The faecal SCFA pattern was similar to that observed in breast fed infants (8). The results are in line with the observation that during a 28-day feeding period the faecal pH decreased in a group of term infants fed a GOS/FOS supplemented formula similar to breast fed infants, whereas the faecal pH in infants fed a nonsupplemented standard formula increased (5). Increased numbers of bifidobacteria and a decreased faecal pH are contributing to a reduction of clinically relevant pathogens, either expressed in absolute numbers or as percentage of total bacteria. In an animal study using the mouse vaccination model as recommended by international governmental guidelines (6), feeding GOS/FOS significantly stimulated the cellular (i.e., Th1/Th2) immune balance. In an animal model for allergic hypersensitivity using ovalbumin as allergen, dietary GOS/FOS inhibited the allergic inflammation (7). In summary, the data demonstrate that an infant formula supplemented with a mixture of GOS/FOS stimulates the entire intestinal flora and results in the production of physiologically important bacterial metabolites as well as a faecal pH similar to influences of human milk feeding. These effects are accompanied by stool characteristics close to those of breast-fed infants and by a Address correspondence and reprint requests to Prof. Gunther Boehm, Director Infant Nutrition Research Germany, Milupa GmbH & Co, KG Bahnstrasse 14-30, D-61381 Friedrichsdorf; Fax: 0049-6172991862 (e-mail: guenther.bohem@milupa.de). Journal of Pediatric Gastroenterology and Nutrition 39:S772–S773 © June 2004 Lippincott Williams & Wilkins, Philadelphia

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS n (part 2: application to isomeric mixtures)

A complex mixture of isomeric neutral oligosaccharides from pooled human milk was analyzed by nano-electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer (QIT-MS) in the negative ion mode. Since deprotonated molecules of neutral oligosaccharides follow distinct fragmentation rules, which have been elucidated by using model compounds (see [1]), spectra obtained from consecutive CID experiments allowed the differentiation of isomers out of this highly complex mixture. With this method new human milk oligosaccharides of previously unknown isomeric structures have been identified, e.g., the occurence of three isomeric fucosylated lacto-N-hexaoses could be determined precisely, which have not been described before: (1) Fuc (α1 → 2) Gal (β1 → 3) GlcNac ( β1 → 3) Gal ( β1 → 4) GlcNac ( β1 → 3) Gal ( β1 → 4) Glc, (2) Gal ( β1 → 4) GlcNAc [( α1 → 3) Fuc] ( β1 → 3) Gal ( β1 → 4) GlcNac ( β1 → 3) Gal ( β1 → 4) Glc, (3) Gal ( β1 → 4) GlcNAc ( β1 → 3) Gal ( β1 → 4) GlcNac [( α1 → 3) Fuc] ( β1 → 3) Gal ( β1 → 4) Glc.

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS n (Part 1: methodology)

Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M − H] − with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a “new” reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS n capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal β1→3 GlcNAc or Gal β1→4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.

Glycoconjugates in human and transgenic animal milk.

Human milk samples contain a particularly rich collection of oligosaccharides compared with other milk samples. The synthesis of these molecules should depend on the expression of glycosyltransferases and the presence of sugar nucleotides in lactating mammary glands. We set out to produce transgenic animals expressing glycosyltransferases during lactation with the purpose of exploring the following issues: a) Is it possible to synthesize human milk oligosaccharides in lactating mammary glands of nonhuman animals?, b) Is it possible to express during lactation homologous, tissue specific glycosyltransferases that are not normally expressed in lactating mammary tissue?, and c) What is the effect of expressing a human glycosyltransferase in different animal species? Simultaneously, we embarked on a research program to study short-chain neutral human milk oligosaccharides--no larger than hexasaccharides--to understand the natural variation of milk sugars and glycoproteins. The reagents and methods developed to study human milk oligosaccharides and glycoproteins were also applied to the study of milk from transgenic animals. Our results indicate that mice predictably express transgene-encoded glycosyltransferases and their secondary gene products, oligosaccharides and remodeled glycoproteins. This was true even when the transgene encoded a homologous galactosyltransferase. Also, it was possible to synthesize fucosylated glycoconjugates in mouse milk using two different fucosyltransferases, thus demonstrating that is feasible to emulate the synthesis occurring in the human lactating mammary gland. Experiments with transgenic rabbits yielded different phenotypes, some of them unexpected. Taken together, our results answer the questions stated above but open even more intriguing areas of inquiry.