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Author(s): Jorgensen, Josh M; Young, Rebecca; Ashorn, Per; Ashorn, Ulla; Chaima, David; Davis, Jasmine CC; Goonatilleke, Elisha; Kumwenda, Chiza; Lebrilla, Carlito B; Maleta, Kenneth; Sadalaki, John; Totten, Sarah M; Wu, Lauren D; Zivkovic, Angela M; Dewey, Kathryn G | Abstract: BackgroundHuman milk oligosaccharides (HMOs) and bioactive proteins likely benefit infant health, but information on these relations is sparse.ObjectivesWe aimed to examine associations of milk content of HMOs and bioactive proteins with incidence and longitudinal prevalence of infant morbidity (any illness, fever, diarrhea, acute respiratory infection, and loss of appetite) and markers of inflammation [C-reactive protein (CRP) and α-1-acid glycoprotein (AGP)]. These are secondary analyses of a randomized controlled trial.MethodsBreast milk samples at 6nmo postpartum (nnn=n659) were analyzed to quantify absolute abundance of HMOs, relative abundance of fucosylated HMOs, sialylated HMOs, and 51 individual HMOs, and concentrations of 6 bioactive proteins (lactalbumin, lactoferrin, lysozyme, antitrypsin, IgA, and osteopontin). We examined associations of these constituents with infant morbidity from 6 to 7 and 6 to 12nmo, and CRP and AGP at 6 and 18nmo, considering maternal secretor status [presence or absence of the functional enzyme encoded by the fucosyltransferase 2 gene (FUT2) ] and adjusting for covariates and multiple hypothesis testing.ResultsIn secretors there were positive associations between total HMOs and longitudinal prevalence of fever (Pn=n0.032), between fucosylated HMOs and incidence of diarrhea (Pn=n0.026), and between lactoferrin and elevated CRP at 18nmo (Pn=n0.011). In nonsecretors, there were inverse associations between lactoferrin and incidence of fever (Pnn=n0.007), between osteopontin and longitudinal prevalence of lost appetite (Pnn=n0.038), and between fucosylated HMOs and incidence of diarrhea (Pn=n0.025), lost appetite (Pn=n0.019), and concentrations of AGP and CRP at 6nmo (Pn=n0.001 and 0.010); and positive associations between total HMOs and incidence of lost appetite (Pn=n0.024) and elevated CRP at 18nmo (Pnn=n0.026), between lactalbumin and incidence of diarrhea (Pn=n0.006), and between lactoferrin and elevated CRP at 18nmo (Pn=n0.015).ConclusionsCertain HMOs and bioactive proteins were associated with infant morbidity and inflammation, particularly in nonsecretors. Further research is needed to elucidate the causality of these relations.This trial was registered at clinicaltrials.gov as NCT01239693.

Author(s): Jorgensen, Josh M; Young, Rebecca; Ashorn, Per; Ashorn, Ulla; Chaima, David; Davis, Jasmine CC; Goonatilleke, Elisha; Kumwenda, Chiza; Lebrilla, Carlito B; Maleta, Kenneth; Prado, Elizabeth L; Sadalaki, John; Totten, Sarah M; Wu, Lauren D; Zivkovic, Angela M; Dewey, Kathryn G | Abstract: BackgroundHuman milk oligosaccharides (HMOs) and bioactive breast milk proteins have many beneficial properties. Information is sparse regarding associations between these milk constituents and infant growth and development in lower-income countries.ObjectivesWe aimed to examine associations of milk content of HMOs and bioactive proteins at 6 mo postpartum with infant growth and motor and cognitive development. These are secondary analyses of a randomized controlled trial in rural Malawi.MethodsBreast milk samples were analyzed at 6 mo (nn=n659) for general categories of HMOs (total HMOs, fucosylated HMOs, and sialylated HMOs), 51 individual HMOs, and 6 bioactive proteins (lactalbumin, lactoferrin, lysozyme, antitrypsin, IgA, and osteopontin). We examined associations of the relative abundances of HMOs and concentrations of bioactive proteins with infant growth from 6 to 12 mo [change in length-for-age (ΔLAZ), weight-for-age, weight-for-length, and head circumference z-scores] as well as ability to stand or walk alone at 12 mo, and motor and language skills, socioemotional development, executive function, and working memory at 18 mo. Analyses were adjusted for covariates and multiple hypothesis testing.ResultsAmong all participants, there were inverse associations of IgA and lactoferrin concentrations with motor skills (Pn=n0.018 and P =n0.044), and a positive association of lactalbumin concentration with motor skills (Pn=n0.038). Among secretors only [fucosyltransferase 2 gene (FUT2) positive], there were positive associations of absolute abundance of HMOs with ΔLAZ (Pn=n0.035), and relative abundance of fucosylated and sialylated HMOs with language at 18 mo (Pnln0.001 and Pn=n0.033, respectively), and inverse associations of osteopontin with standing and walking at 12 mo (Pn=n0.007 and 0.002, respectively). Relative abundances of several individual HMOs were associated with growth and development, mostly among secretors.ConclusionsCertain bioactive breast milk proteins and HMOs are associated with infant growth and motor and cognitive development. Further studies are needed to determine if a causal relation exists.This trial was registered at clinicaltrials.gov as NCT01239693.

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity.

Human milk oligosaccharides (HMOs) are a highly abundant constituent in human milk, and its protective and prebiotic properties have attracted considerable attention. HMOs have been shown to directly and indirectly benefit the overall health of the infant due to a number of functions including serving as a beneficial food for gut bacteria, block to pathogens, and aiding in brain development. Researchers are currently exploring whether these structures may act as possible disease and nutrition biomarkers. Because of this, rapid-throughput methods are desired to investigate biological activity in large patient sets. We have optimized a rapid-throughput protocol to analyze human milk oligosaccharides using micro-volumes of human breast milk for nutritional biomarkers. This method may additionally be applied to other biological fluid substrates such as plasma, urine, and feces. The protocol involves lipid separation via centrifugation, protein precipitation using ethanol, alditol reduction with sodium borohydride, and a final solid-phase extraction purification step using graphitized carbon cartridges. Samples are analyzed using HPLC-Chip/TOF-MS and data filtered on Agilent MassHunter using an in-house library. Individual structural identification is matched against a previously developed HMO library using accurate mass and retention time. Using this method will allow in-depth characterization and profiling of HMOs in large patient sets, and will ease the process of discovering significant nutritional biomarkers in human milk.