The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats

Background Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. Objective To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. Methods Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. Results Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98±1.11 to 0.44±0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. Conclusion HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mother's milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

[1]  R. Schanler,et al.  Randomized Trial of Donor Human Milk Versus Preterm Formula as Substitutes for Mothers' Own Milk in the Feeding of Extremely Premature Infants , 2005, Pediatrics.

[2]  C. Lebrilla,et al.  Development of an annotated library of neutral human milk oligosaccharides. , 2010, Journal of proteome research.

[3]  G. Ruiz-Palacios,et al.  Human milk glycans protect infants against enteric pathogens. , 2005, Annual review of nutrition.

[4]  L. Bode.  Recent advances on structure, metabolism, and function of human milk oligosaccharides. , 2006, The Journal of nutrition.

[5]  C. Lebrilla,et al.  Annotation and structural analysis of sialylated human milk oligosaccharides. , 2011, Journal of proteome research.

[6]  S. Thurl,et al.  Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides , 1997, Glycoconjugate Journal.

[7]  N Klein,et al.  Oligosaccharides in human milk: structural, functional, and metabolic aspects. , 2000, Annual review of nutrition.

[8]  Yuanwu Bao,et al.  Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis. , 2007, Analytical biochemistry.

[9]  S. Eaton,et al.  P-selectin expression, neutrophil infiltration, and histologic injury in neonates with necrotizing enterocolitis. , 2005, Journal of pediatric surgery.

[10]  Nigel Klein,et al.  Human milk oligosaccharides reduce platelet‐neutrophil complex formation leading to a decrease in neutrophil β 2 integrin expression , 2004, Journal of leukocyte biology.

[11]  Simon C Watkins,et al.  Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. , 2000, The Journal of surgical research.

[12]  M. Karas,et al.  Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry. , 1994, Analytical biochemistry.

[13]  S. Eaton,et al.  Neurodevelopmental outcomes of neonates with medically and surgically treated necrotizing enterocolitis , 2006, Archives of Disease in Childhood - Fetal and Neonatal Edition.

[14]  Rebeccah L. Brown,et al.  Postoperative Outcomes of Extremely Low Birth-Weight Infants With Necrotizing Enterocolitis or Isolated Intestinal Perforation: A Prospective Cohort Study by the NICHD Neonatal Research Network , 2005, Annals of surgery.

[15]  D. Hackam,et al.  The development of animal models for the study of necrotizing enterocolitis , 2008, Disease Models & Mechanisms.

[16]  Lars Bode,et al.  Human milk oligosaccharides: prebiotics and beyond. , 2009, Nutrition reviews.

[17]  A. Spitzer,et al.  Characteristics of patients who die of necrotizing enterocolitis , 2011, Journal of Perinatology.

[18]  T. O'Shea,et al.  Early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants , 2007, Journal of Perinatology.

[19]  Ajit Varki,et al.  Siglecs and their roles in the immune system , 2007, Nature Reviews Immunology.

[20]  R. Holman,et al.  Necrotising enterocolitis hospitalisations among neonates in the United States. , 2006, Paediatric and perinatal epidemiology.

[21]  Elizabeth A. Phillips,et al.  Necrotizing enterocolitis in very low birth weight infants: biodemographic and clinical correlates. National Institute of Child Health and Human Development Neonatal Research Network. , 1991, The Journal of pediatrics.

[22]  B. Barlow,et al.  Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model. , 1975, Surgery.

[23]  R. Shulman,et al.  Feeding Strategies for Premature Infants: Beneficial Outcomes of Feeding Fortified Human Milk Versus Preterm Formula , 1999, Pediatrics.

[24]  J. Lowe,et al.  Leukocyte adhesion deficiency type II. , 1999, Biochimica et biophysica acta.

[25]  Lars Bode,et al.  Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides , 2004, Thrombosis and Haemostasis.

[26]  Rita Gerardy-Schahn,et al.  The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter , 2001, Nature Genetics.

[27]  R. Holman,et al.  The epidemiology of necrotizing enterocolitis infant mortality in the United States. , 1997, American journal of public health.

[28]  K. Fukuda,et al.  Determination of Sialyl and Neutral Oligosaccharide Levels in Transition and Mature Milks of Samoan Women, Using Anthranilic Derivatization Followed by Reverse Phase High Performance Liquid Chromatography , 2010, Bioscience, biotechnology, and biochemistry.

[29]  C. Sergi,et al.  Medical management of motility disorders in patients with intestinal failure: a focus on necrotizing enterocolitis, gastroschisis, and intestinal atresia. , 2011, Journal of pediatric surgery.

[30]  D. Potoka,et al.  Mechanisms of nitric oxide-mediated intestinal barrier failure in necrotizing enterocolitis. , 2005, Seminars in pediatric surgery.

[31]  M. Henry,et al.  Necrotizing Enterocolitis , 1977, Pediatric Surgery.

[32]  T. Cole,et al.  Breast milk and neonatal necrotising enterocolitis , 1990, The Lancet.

[33]  U. Kulozik,et al.  Lectin inhibition assays for the analysis of bioactive milk sialoglycoconjugates , 2011 .

[34]  Nikunj K. Chokshi,et al.  P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis , 2011, Laboratory Investigation.

[35]  A. Kobata,et al.  Structures and application of oligosaccharides in human milk , 2010, Proceedings of the Japan Academy. Series B, Physical and biological sciences.

[36]  R. Ehrenkranz,et al.  An exclusively human milk-based diet is associated with a lower rate of necrotizing enterocolitis than a diet of human milk and bovine milk-based products. , 2010, The Journal of pediatrics.

[37]  Kai Maass,et al.  High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides , 2011, Analytical and bioanalytical chemistry.

[38]  J. Hay,et al.  Costs of necrotizing enterocolitis and cost-effectiveness of exclusively human milk-based products in feeding extremely premature infants. , 2012, Breastfeeding medicine : the official journal of the Academy of Breastfeeding Medicine.