Given the evolution of milk as a product of epithelial secretions nourishing mammalian offspring, the presence of non-digestible oligosaccharides would appear to be paradoxical. The question, why would milk contain indigestible material, has challenged scientists studying milk for decades. The presence and particularly the remarkable abundance of oligosaccharides in human milk as the third largest solid component have led investigators to propose biological, physiological and protective functions to these molecules [4
]. Certainly, the number and structural diversity of these molecules would allow more than one function. However, to date, the detailed structural basis of these myriad functions is not yet understood. Recently, human milk oligosaccharides (HMOs) have been demonstrated to selectively nourish the growth of highly specific strains of bifidobacteria thus establishing the means to guide the development of a unique gut microbiota in infants fed breast milk [7
]. Certain oligosaccharides derived from the mammalian epithelial cells of the mother also share common epitopes on the infant’s intestinal epithelia known to be receptors for pathogens. The presence of such structures in milk have been hypothesized to have evolved to provide a direct defensive strategy acting as decoys to prevent binding of pathogens to epithelial cells, thereby protecting infants from diseases [10
Consistent with the potential for multiple nutritional and biological functions, human milk is comprised of a complex mixture of oligosaccharides that differ in size, charge and abundance [11
]. HMOs are composed of both neutral and anionic species with building blocks of 5 monosaccharides: D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid. The basic structure of HMOs include a lactose core at the reducing end and are elongated by N-acetyllactosamine units, with greater structural diversity provided by extensive fucosylation and/or sialylation wherein fucose and sialic acid residues are added at the terminal positions [10
]. The ability to understand the diversity of biological functions of HMOs has been hindered to date in part because of the lack of detailed structural knowledge of the overall complexity of HMOs in breast milk. At present, about 200 molecular species have been identified in a pooled human milk sample consisting of mostly neutral and fucosylated oligosaccharides [12
The analytical methods that are capable of separating and characterizing the various sugar compositions and structures of oligosaccharides in human milk include high-performance liquid chromatography (HPLC), high pH anion exchange chromatography (HPAEC), capillary electrophoresis and various mass spectrometry platforms (MS) [13
]. These methods as currently used are technically cumbersome, incapable of producing large quantities of highly purified isolated molecules and, as a result, there is little information on many of the basic biological properties of this class of molecule. Even with respect to the variation in abundances and structures across the human condition, little information has been developed. As a result the variation in nourishment that is likely to occur between different infants in different breastfeeding situations is also lacking. Combining the lack of basic information on the diversity of HMOs between different lactating humans, on changes in oligosaccharide compositions and abundances during the course of lactation and on the role of genetic, dietary and physiological determinants on the structures and abundances of HMOs it is difficult to predict at present to what extend variations in health outcomes of different breastfed infants is due to variation in the oligosaccharides delivered in their milk.
To establish the various functions associated with the diverse HMO structures the details of variations in compositions and abundances of oligosaccharides among humans and during lactation need to be measured. Characterization of HMO has been accomplished using HPAEC and HPLC in combination with derivatization techniques [4
]. The identification of HMOs was based on the retention time of commercially available milk oligosaccharide standards and their quantification was relative to the amount of standards. In one study, a decrease in the total concentration of oligosaccharides was observed from the first weeks postpartum to about half the concentration after one year. In the same report, the absolute and relative concentrations of HMOs between individual donors and at different stages of lactation varied significantly [22
]. Asakuma et al. [24
] analyzed the level of several neutral oligosaccharides in human milk colostrums for 3 consecutive days from 12 Japanese women. The concentrations of 2′-fucosylactose and lactodifucotetraose on day 1 were found to be substantially higher than those on days 2 and 3. On the other hand, the lacto-N
-tetraose concentration increased from days 1 to 3. These data are compelling that variation in the structures and abundances of the various oligosaccharides in human milk are variable and it now becomes of considerable scientific and practical interest to understand the regulatory basis of these variations (genetics, diet, physiological state of the mother/infant, pathological state of the mother/infant, etc.).
The arrival of HPLC-Chip TOF/MS technology provides the analytical means to take a new strategy to routinely profile oligosaccharides in human milk [9
]. This analytical technique employs an integrated microfluidic chip coupled with a high mass accuracy time-of-flight mass analyzer. Using this analytical platform nearly daily profiles of oligosaccharides in human milk samples were determined for different individual human donors (). The levels of milk oligosaccharides and their heterogeneity were investigated both within the individual donor and among multiple donors at different stages of lactation. This approach is designed to provide basic knowledge on HMOs in normal humans as the key compositional basis to understanding the relationship between the levels of milk oligosaccharides and the specific functions these biomolecules contribute to maternal and infant health and development.
Comparison of oligosaccharide profiles of breast milk from two mothers on day 6 of lactation. Oligosaccharides analyzed by the Agilent nano-LC chip TOF system.