Fecal samples with more bifidobacteria protect against intestinal permeability

• Autor(a) principal: Santos, Gilberto Maia
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/142533
• topic: alkaline phosphatase; Bifidobacteria; donor milk; intestinal permeability; own mother's milk; premature infants; Nutrição

Introduction: Ingestion of human milk during the first weeks of life leads to an increase in the amount of faecal Bifidobacteria and a concomitant increase in faecal alkaline phosphatase (ALP) activity, a proposed biomarker for necrotizing enterocolitis (NEC). However, it is necessary to confirm whether this increase in faecal ALP activity in infants fed primarily human milk is associated with the presence of more Bifidobacteria and/or their derived metabolites in faecal samples and whether this translates into an increase in intestinal permeability. Material and Methods: Faecal samples collected an average of 26 days after birth from preterm infants fed mainly on mother’s own milk (MOM), donor human milk (DHM) or formula (F) were used. Faecal suspensions were prepared by centrifugation (20,000 g x 60 min) and filtration (0.22 μm). Lipopolysaccharide (LPS) was quantified in faecal suspensions using the Chromo-Limulus Amebocyte Lysate (LAL) kit. Short chain fatty acids (SCFAs) were analyzed by High Performance Liquid Chromatography (HPLC-DAD). Caco-2 cells were incubated with faecal suspensions for 48h. Cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylethazol bromide) assay. Ecto-ALP activity was quantified by spectrophotometric measurement of p-nitrophenol at 410 nm and results are expressed as % of negative control. Cell permeability was evaluated by the ability of sulfonic acid (SA) to pass from the apical to the basolateral compartment of the transwell. Butyrate (5 mM) was used as a positive control. The Kruskal-Wallis test was used to analyze the differences between the groups and the multiple comparisons by the Dunn test. To compare human milk and formula, the Man Whitney test was used. Results: The mean amount of Bifidobacteria in the faecal samples used in this study was 2.67±0.79; 2.38±0.95 and 1.80±0.43 log10 16S rDNA gene copies, for MOM, DHM and F, respectively. MOM faecal suspensions have a significantly higher amount of LPS compared to F faecal samples (MOM: 3077.10 ± 425.84 EU/ml; DHM: 2158.65 ± 890.60 EU/ml; F: 1203.07 ± 890.60 EU/ml 193.18 EU/ml, p<0.05). The SCFAs identified in all faecal samples were: malic acids; succinic acid; malic acid; acetic acid; propionic acid. acid XX and acid YY were impossible to identify. There was absence of butyric acid and lactic acid. Succinic acid was statistically significant, being present in greater amounts in faecal samples of human milk compared to faecal samples of formula (p=0.04). Faecal suspensions prepared from these samples did not alter the cell viability of Caco-2 cells (p>0.05). MOM faecal suspensions significantly increased ALP activity compared to DHM and F faecal suspensions (MOM: 291.88 ± 98.04%; DHM: 174.32 ± 58.87%; F: 177.85 ± 48, 76%, p<0.05). Furthermore, this increase in ALP activity was greater than the increase induced by butyrate. Faecal suspensions of MOM, DHM and F altered the permeability of Caco-2 cells (MOM: 96.19 ± 3.56%; DHM: 94.79 ± 9.972%; F: 98.03 ± 8.99%; NC: 425142885 ± 356592243.3 ). Conclusions: Microbial metabolites present in faecal samples with more Bifidobacteria may be responsible for the increase in intestinal ALP activity. Increased ALP activity can result in increased dephosphorylation of LPS, preventing activation of an inflammatory cascade. These results may contribute to increase knowledge about the molecular mechanisms associated with NEC in preterm infants.