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

Abstract

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.

Figure 1

Human milk oligosaccharides (HMO) and galacto-oligosaccharides (GOS) are structurally different. (A) Fluorescence high-performance liquid chromatography (HPLC-FL) chromatogram of 2AB-labelled HMO isolated from pooled human milk. Most common HMO are annotated and listed in panel B. *Disialyllacto-N-tetraose (DSLNT), which was later identified as the necrotising enterocolitis-protective HMO. (B) Schematic representation of the most common oligosaccharides found in the isolated pooled HMO. Numbers in brackets correspond to the annotated peaks in panel A. 2′FL, 2′-fucosyllactose; 3FL, 3-fucosyllactose; 3′SL, 3′-sialyllactose; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; LNFP1, lacto-N-fucopentaose 1; LNFP2, lacto-N-fucopentaose 2; LSTb, sialyllacto-N-tetraose b; LSTc, sialyllacto-N-tetraose c. Monosaccharide key: dark circle, glucose (Glc); light circle, galactose (Gal); square, N-acetyl-glucosamine (GlcNAc); triangle, fucose (Fuc); diamond, N-acetyl-neuraminic acid (NeuAc). (C) HPLC-FL chromatogram of Vivinal GOS. Peak clusters represent structural isomers of oligosaccharides with the same degree of polymerisation and depend on the number of galactose residues per GOS molecule. Comparison of the HMO and GOS chromatograms confirmed a clear difference in the structural composition.

Figure 2

Pooled human milk oligosaccharides (HMO), but not galacto-oligosaccharides (GOS) improve survival and reduce necrotising enterocolitis (NEC) in neonatal rats. (A) Survival of neonatal rats within the first 96 h post-partum. DF, dam-fed; FF, formula-fed; FF+HMO, fed formula with HMO (10 mg/ml); FF+GOS, fed formula with GOS (8 mg/ml). (B) Macroscopic evaluation of rat intestines at 96 h post-partum. Compared to DF (left) and FF +HMO (right) animals, the intestines of FF animals (centre) were darker with patchy necrosis and evidence of haemorrhagic intestine as well as intramural gas cysts (Pneumatosis intestinalis). (C) Microscopic evaluation of H&E-stained rat ileum sections. Based on the presence or absence of histological anomalies (three examples are shown in the bottom panel), ileum sections were graded from 0 (normal) to 4 (complete destruction). (D) Ileum pathology scores at 96 h post-partum. Each intervention was tested in a total of 10–20 animals in three independent experiments. Each symbol represents the pathology score for an individual animal. Horizontal lines represent mean pathology scores. ***p<0.001.

Figure 3

Exposure to human milk oligosaccharides (HMO) in the first 24 h post-partum is required, but not sufficient to reduce necrotising enterocolitis. Neonatal rats were dam-fed (DF), fed HMO-free formula for the entire first 96 h post-partum (FF), fed HMO-free formula for the first 24 h and then switched to HMO-containing formula (10 mg/ml) for the remaining 72 h (FF+HMO End), or fed HMO-containing formula for the first 24 h and then switched to HMO-free formula (FF+HMO Start). Each intervention was tested in a total of 9–12 animals in two independent experiments. ***p<0.001.

Figure 4

A single, disialylated human milk oligosaccharide (HMO) reduces necrotising enterocolitis. (A) Ileum pathology scores in response to adding charge-fractionated HMO to formula. Anion exchange chromatography was used to fractionate pooled HMO by charge based on whether HMO contained no (0), one (−1), two (−2), three (−3) or four (−4) sialic acid residues. The −2 charged HMO fraction, containing oligosaccharides with two sialic acids (two negative charges) had the most pronounced effect. (B) HPLC-FL chromatogram of −2 charged HMO fraction. (C) MALDI-TOF mass spectra and potential composition of the four major HMO peaks in the −2 charged HMO fraction. The predicted number of hexoses (circles), hexosamines (square), N-acetylneuramic acid (NeuAc, diamond) and fucose (triangle) per molecule are listed above each mass spectrum. Loss of NeuAc during analysis reduces the mass by 291 Da. (D) Fast protein liquid chromatography (FPLC) with a gel exclusion column was used to separate the four major HMO peaks in the −2 charged HMO fraction by size. FPLC fractions containing mostly HMO peak 2 were pooled together (HMO 2). HMO peaks 3 and 4 could not be separated by gel exclusion and were pooled in one fraction (HMO 3+4). (E) Ileum pathology scores in response to adding size-fractionated HMO to formula. Each intervention was tested in a total of 11–14 animals in two independent experiments. ***p<0.001.

Figure 5

The necrotising enterocolitis-protective human milk oligosaccharide (HMO) is disialyllacto-N-tetraose (DSLNT). (A) Linkage specific neuraminidase treatment shows the presence of one α2–3- and one α2–6-linked N-acetyl-neuraminic acid (NeuAc). Fluorescence high-performance liquid chromatography (HPLC-FL) chromatogram a: protective HMO 2; b: HMO 2 after treatment with α2–3-specific neuraminidase; c: HMO 2 after treatment with linkage promiscuous neuramidase. (B) The underlying HMO backbone has a type I structure (Galβ1–3GlcNAc). HPLC-FL chromatogram d: asialo-HMO 2 (after treatment with α2–3/6 neuraminidase, product c); e: asialo-HMO 2 after treatment with β1–3-specific galactosidase; f: asialo-HMO 2 after treatment with β1–4-specific galactosidase. (C) The subterminal sugar in the HMO backbone is N-acetyl-glucosamine (GlcNAc). HPLC-FL chromatogram g: asialo-agalacto-HMO 2 (after treatment with α2–3/6 neuraminidase and β1–3 galactosidase, product e); h: asialo-agalacto-HMO 2 after treatment with GlcNAcase. (D) Gas chromatography mass spectrum (GC–MS) of partially methylated alditol acetate (PMAA) derivatives of HMO 2. (E) Schematic representation of DSLNT based on the results from sequential exoglycosidase digestion and GC–MS PMAA linkage analysis.

Figure 6

Sialic acid is required for the necrotising enterocolitis (NEC) protective effects of disialyllacto-N-tetraose (DSLNT). Ileum pathology scores in response to adding DSLNT or neuraminidase-treated DSLNT to formula. Commercially available DSLNT (300 μM) significantly reduced NEC pathology scores. Treatment with a linkage promiscuous neuraminidase (α2–3/6) or an α2–3-specific neuraminidase abolished the protective effects of DSLNT. Each intervention was tested in a total of 11–26 animals in three independent experiments. ***p<0.001.