Investigations of the in Vitro Transport of Human Milk Oligosaccharides by a Caco-2 Monolayer Using a Novel High Performance Liquid Chromatography-Mass Spectrometry Technique

Abstract

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb TM column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra-and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K m for lacto-N-fucopentaose I of 1.7 ؎ 0.1 mmol/liter and for lacto-N-tetraose of 1.8 ؎ 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.Oligosaccharides are the third most abundant soluble fraction within human milk. With exception of the elephant, no other mammalian milk contains as many lactose-derived complex oligosaccharides (1). Thus far, more than 100 lactose-derived oligosaccharides have been characterized within human milk (2). HMOs 1 are postulated to be involved in immunomodulation (3, 4) by acting as soluble receptor analogues for bacterial and viral adhesion molecules in the gastrointestinal and the urogenital tract (5) and to stimulate a bifidus flora within the colon. At present little is known about the metabolism of HMOs in the breast-fed neonate. Recently, we have shown that these oligosaccharides are only minimally digested by enzymes of the upper gastrointestinal tract (6). Furthermore, neutral as well as acidic HMOs could be detected in the urine of breast-fed but not in that of formulafed infants (7,8) suggesting that they are absorbed in the intestine. These findings raise the question of how and to what extent these components pass the epithelium of the small intestine. Therefore, we performed in vitro transport studies using the human cell line Caco-2 that displays biochemical and morphological characteristics of differe...

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