Clemens Kunz

Biological functions of oligosaccharides in human milk

Breast-fed infants appear to have fewer or less severe gastrointestinal and respiratory infections during the first year of life than formula-fed infants, an effect which has been shown to be most pronounced in developing countries (e.g. 1-3). Recently, the protective effect of breast feeding has been suggested for infections of the urinary tract also (4, 5) . However, only few studies have actually proven that the intake of specific antibodies in breast milk protects infants against colonization by pathogenic bacteria (6-8). Besides immunological components, there are many non-immunological factors in human milk which are thought to be anti-infective agents for the newborn (9). Compared with milk from other species, human milk is unique with regard to its content of complex oligosaccharides (for review see 10-12). For many years these components have been thought to play a role only in the development of a specific intestinal flora in breast-fed infants. Today, there is striking evidence that free oligosaccharides, as well as glycoproteins, are potent inhibitors of bacterial adhesion to epithelial surfaces, an initial stage of the infective process. Therefore, these oligosaccharides are considered to be soluble receptor analogues of epithelial cell-surface carbohydrates. This review summarizes recent data on biological functions of oligosaccharides. As these functions are closely related to the conformation of the oligosaccharide, a brief description of the structural diversity is given also. The following topics will be discussed: qualitative and quantitative aspects of free oligosaccharides in term and preterm milk; influence of infant feeding on the development of a specific flora in newborns; oligosaccharides as soluble receptors for pathogenic bacteria and viruses; renal excretion of oligosaccharides in preterm infants; role of oligosaccharides as ligands for cell adhesion molecules.

Nutritional and biochemical properties of human milk: II. Lipids, micronutrients, and bioactive factors.

Human milk lipids contain preformed LCPUFA in considerable amounts, which serve as precursors for the formation of prostaglandins, prostacyclins, and other lipid mediators, as well as essential components in membrane-rich tissues (such as the brain and the retina), thus affecting functional outcomes. Besides a balanced nutrient composition and a number of conditionally essential nutrients, human milk provides different types and classes of bioactive factors, such as enzymes, hormones, and growth factors, many of which appear to have a role in supporting infantile growth and development. The bioactive agents include antimicrobial factors (e.g., secretory IgA, oligosaccharides, FA); anti-inflammatory agents; transporters (e.g., lactoferrin); and digestive enzymes (e.g., BSSL). Several nonpeptide hormones (thyroid hormones, cortisol, progesterone, pregnanediol, estrogens, and artificial contraceptive) and peptide hormones and growth factors (erythropoietin, hHG, gonadotropin-releasing hormone, epidermal growth factor insulin, insulin-like growth factor-I, nerve growth factor, transforming growth factor-alpha, gastrointestinal regulatory peptides and thyroid-parathyroid hormones) have been isolated and quantitated in human milk. Some of these components are also involved in the maturation of the gastrointestinal tract of the infant. In addition to the passive benefits provided by human milk, several data support the hypothesis that breastfeeding promotes the development of the infants own immune system, which might confer long-term benefits for the newborn infant. The risk of IDDM, Crohns disease, and atopic disease is lower in individuals who had been breastfed during infancy. Areas of major interest in human milk research include the study of human milk synthesis and the contributions of dietary composition and maternal metabolism to human milk composition, infantile utilization of human milk components, and the study of bioactive components, such as oligosaccharides, proteins and peptides, and lipids and their in vivo fate and biologic effects in the recipient infant.

Human milk oligosaccharides reduce platelet‐neutrophil complex formation leading to a decrease in neutrophil β 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 μg/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 β 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.

Re‐evaluation of the whey protein/casein ratio of human milk

Total casein subunits as well as whey proteins were quantitated in human milk samples during lactation. Two independent methods were used: precipitation at pH 4.3 in the presence of Ca2+ followed by Kjeldahl analysis and polyacrylamide gradient gel electrophoresis (PAGGE) followed by densitometric scanning. Both methods yielded similar results: casein synthesis is low or absent in early lactation, then increases rapidly and subsequently decreases. The concentration of whey proteins decreases from early lactation and continues to fall. These changes result in a whey protein/casein ratio of about 90: 10 in early lactation, 60: 40 in mature milk and 50: 50 in late lactation. These observations indicate that the synthesis and/or secretion of caseins and whey proteins is regulated by different mechanisms. In addition, the relative proportion of the different β‐ and k‐casein subunits was found to vary throughout lactation.

Oligosaccharides from human milk influence growth-related characteristics of intestinally transformed and non-transformed intestinal cells.

Human milk oligosaccharides (HMO) are considered to influence the composition of the gut microflora in breastfed infants. We investigated direct effects of milk HMO fractions or individual oligosaccharides on proliferation, differentiation and apoptosis in transformed human intestinal cells (HT-29 and Caco-2) and non-transformed small intestinal epithelial crypt cells of fetal origin (human intestinal epithelial cells; HIEC). We observed growth inhibition induced by neutral and acidic HMO fractions in HT-29, Caco-2 and HIEC cells in a dose dependent manner. However, the effects varied between cell lines, i.e. HT-29 and Caco-2 cells were more sensitive than HIEC cells. In HT-29, all 16 individual neutral and acidic oligosaccharides except from the two fucosyllactoses had an inhibitory effect on cell growth. Regarding the induction of differentiation in HT-29 and HIEC cells a threshold concentration was observed at 7.5 mg/ml for neutral and acidic HMO fractions. Among individual oligosaccharides, only sialyllactoses induced differentiation in HT-29 and HIEC cells; no effect neither of fractions nor of individual oligosaccharides was found in Caco-2 cells. A strong induction of apoptosis was only detected in HT-29 and HIEC cells for neutral oligosaccharide but not for acidic fractions. HMO were shown to induce growth inhibition in intestinal cells through two different mechanisms, by suppressing cell cycle progression through induction of differentiation and/or by influencing apoptosis. As the development and maturation of digestive and absorptive processes depend on differentiation our experiments show that oligosaccharides are effective at influencing various stages in gastrointestinal development in vitro.

Nutritional and Biochemical Properties of Human Milk, Part I

Human milk provided by healthy and well-nourished mothers is believed to cover the infants nutrient requirements during the first half year of life. It is composed of a mixture of nutritive components as well as other bioactive factors with relevant physiologic effects in the neonate infant. Human milk composition has a dynamic nature and varies with time postpartum, during a nursing, and with the mothers diet and certain diseases. The changes of human milk composition with time of lactation seem to match the changing needs of the growing infant over time. Human milk proteins are a source of peptides, amino acids, and nitrogen for the infant, but also in the protein fraction reside other properties of human milk that may benefit the breastfeeding infant. Specific whey proteins are involved in the development of the immune response (immunoglobulins), whereas others participate in the nonimmunologic defense (lactoferrin). In addition, human milk contains a complex mixture of oligosaccharides that are present only in minute amounts in other mammals milk. They may act as inhibitors of bacterial adhesion to epithelial surfaces, and thus play an important role in preventing infectious diseases in the newborn infant. Oligosaccharides may also promote the development of a so-called bifidus flora. In the next years, future research will lead to improved characterization of human milk components and elucidation of their individual mechanisms of action, which will increase our knowledge about the properties of human milk and the benefits of breastfeeding for the infant.

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.

Potential Anti-Inflammatory and Anti-Infectious Effects of Human Milk Oligosaccharides

There is increasing evidence of the local effects within the gastro intestinal tract and the systemic functions of human milk oligosaccharides (HMO). In addition to the vast majority of in vitro data, animal studies underline the high potential of HMO to influence very different processes. HMO probably influence the composition of the gut microflora through effects on the growth of bifidus bacteria. Whether the concomitant low number of pathogenic microorganisms in breastfed infants is also caused by HMO is an intriguing question that still has yet to be proven. Due to the similarity of HMO to epithelial cell surface carbohydrates, an inhibitory effect on the adhesion of pathogens to the cell surface is most likely. If this could be shown in humans, HMO would provide a new way to prevent or treat certain infections. It would also indicate supplementing infant formula based on cows milk with HMO, as those oligosaccharides are either not detectable or present only in low numbers in bovine milk. As some HMO can be absorbed and circulate in blood, systemic effects may also be influenced. Due to their similarities to selectin ligands, HMO have been tested in in vitro studies demonstrating their anti-inflammatory abilities. For example, it has been shown that sialic acid-containing oligosaccharides reduce the adhesion of leukocytes to endothelial cells, an indication for an immune regulatory effect of certain HMO. We cover these topics after a short introduction on the structures of HMO, with a particular emphasis on their blood group and secretor specificity.

Oligosaccharides from human milk induce growth arrest via G2/M by influencing growth-related cell cycle genes in intestinal epithelial cells

Oligosaccharides are present in human milk in large amounts and in a high variety. We have previously shown that these oligosaccharides are strong inhibitors of proliferation and inducers of differentiation in intestinal cell lines. To elucidate the molecular mechanism, we investigated the influence on cell cycle events via flow cytometry and expression levels by using quantitative real-time RT-PCR. Human intestinal cells, i.e. HT-29, HIEC and Caco-2 cells, were exposed to neutral or acidic human milk oligosaccharides. Both fractions induced a concentration-dependent G2/M arrest. Cell cycle analysis for HT-29 revealed 37 % of cells in G1 and 35 % in G2/M (neutral oligosaccharides) and incubation with acidic oligosaccharides led to 42 % cells in G1 and 40 % in G2/M. In control experiments without oligosaccharides we found 71 % of cells to be in G1 and 17 % in G2/M. This G2/M arrest was associated with changes in mRNA expression of cyclin A and B. A G2/M arrest with concomitant alterations of cell cycle gene expression could also be shown for HIEC and Caco-2 cells. Analysing the expression of cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1) and the tumour suppressor p53 we observed that the expression of p21(cip1) was p53-independent and necessary for arresting cells in the G2/M phase, while p27(kip1) was associated with differentiation effects. Both neutral and acidic human milk oligosaccharides were able to induce epidermal growth factor receptor, extracellular signal-regulated kinase 1/2 and p38 phosphorylation. These results suggest that oligosaccharides from human milk inhibited intestinal cell proliferation and altered cell cycle dynamics by affecting corresponding regulator genes and mitogen-activated protein kinase signalling.

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.