ABSTRACT
OBJECTIVES: Fat is digested in the intestine into free fatty acids (FFAs), which are detergents and therefore toxic to cells at micromolar concentration. The mucosal barrier protects cells in the adult intestine, but this barrier may not be fully developed in premature infants. Lipase-digested infant formula, but not fresh human milk, has elevated FFAs and is cytotoxic to intestinal cells, and therefore could contribute to intestinal injury in necrotizing enterocolitis (NEC), but even infants exclusively fed breast milk may develop NEC. Our objective was to determine whether stored milk and milk from donor milk (DM) banks could also become cytotoxic, especially after digestion. METHODS: We exposed cultured rat intestinal epithelial cells or human neutrophils to DM and milk collected fresh and stored at 4°C or -20°C for up to 12 weeks and then treated for 2âhours (37°C) with 0.1 or 1âmg/mL pancreatic lipase and/or trypsin and chymotrypsin. RESULTS: DM and milk stored 3 days (at 4°C or -20°C) and then digested were cytotoxic. Storage at -20°C for 8 and 12 weeks resulted in an additional increase in cytotoxicity. Protease digestion decreased, but did not eliminate cell death. CONCLUSIONS: Present storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC.
Subject(s)
Digestion , Fatty Acids, Nonesterified/metabolism , Food Storage , Lipase/metabolism , Milk, Human/metabolism , Animals , Cell Death/drug effects , Cells, Cultured , Chymotrypsin/metabolism , Epithelial Cells , Fatty Acids, Nonesterified/pharmacology , Humans , Intestinal Mucosa/cytology , Milk Banks , Milk, Human/chemistry , Neutrophils , Rats , Temperature , Time Factors , Trypsin/metabolismABSTRACT
BACKGROUND: Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than those who are breastfed, but the mechanisms of intestinal necrosis in NEC and protection by breast milk are unknown. We hypothesized that after lipase digestion, formula, but not fresh breast milk, contains levels of unbound free fatty acids (FFAs) that are cytotoxic to intestinal cells. METHODS: We digested multiple term and preterm infant formulas or human milk with pancreatic lipase, proteases (trypsin and chymotrypsin), lipase + proteases, or luminal fluid from a rat small intestine and tested FFA levels and cytotoxicity in vitro on intestinal epithelial cells, endothelial cells, and neutrophils. RESULTS: Lipase digestion of formula, but not milk, caused significant death of neutrophils (ranging from 47 to 99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula vs. milk and death from formula was significantly decreased with lipase inhibitor pretreatment, or treatments to bind FFAs. Protease digestion significantly increased FFA binding capacity of formula and milk but only enough to decrease cytotoxicity from milk. CONCLUSION: FFA-induced cytotoxicity may contribute to the pathogenesis of NEC.
