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New strategies for profiling and characterization of human milk oligosaccharides.

ABSTRACT: Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids, and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant, but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over non-modified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >?5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed a new light on the impact of HMOs during infant development.

SUBMITTER: Porfirio S

PROVIDER: S-EPMC7526734 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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New strategies for profiling and characterization of human milk oligosaccharides.

Porfirio Sara S Archer-Hartmann Stephanie S Moreau G Brett GB Ramakrishnan Girija G Haque Rashidul R Kirkpatrick Beth D BD Petri William A WA Azadi Parastoo P

Glycobiology 20200901 10

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is ...[more]

PMID: 32248230

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Project description:BackgroundHuman milk oligosaccharides (HMOs) in breast milk contribute to the development of the neonatal microbiota and immune system. However, longitudinal studies examining HMO profiles of Chinese mothers remain scarce.ObjectivesWe aimed to analyze HMO profiles, including their composition, concentrations, and changes during lactation, in milk of Chinese mothers.MethodsA total of 822 milk samples from 222 mothers were collected, of which 163 mothers provided single samples. Samples from the remaining 59 mothers were collected on day 3, day 7, and thereafter every 7 or 14 d until day 168. 24 HMOs were studied using high-performance anion-exchange chromatography. Secretor and nonsecretor status were determined based on Lewis blood types and a defined 2'-fucosyllactose (2'-FL) threshold.ResultsOf the 222 mothers, 77% were secretors and 23% were nonsecretors. The longitudinal study involving 59 mothers showed that the total HMOs in secretors were significantly greater than those in nonsecretors during the first 2 wk. Acidic HMOs decreased significantly during lactation and were similar between secretors and nonsecretors. Among neutral HMOs, distinctive differences were observed. Nonfucosylated and α-1-3/4-fucosylated HMOs in nonsecretors were significantly higher than those in secretors during the first month. In contrast, α-1-2-fucosylated HMOs in secretors were significantly higher than those in nonsecretors throughout 168 d. In secretors, 2'-FL concentrations peaked at (mean ± SEM) 3.02 ± 0.14 g/L (day 3) followed by significant decreases. In nonsecretors, 2'-FL concentrations were fairly low throughout 168 d. Of the 24 studied HMOs, only 3-fucosyllactose concentrations increased during lactation in both secretor and nonsecretor mothers.ConclusionsOur study showed dynamic changes of 24 HMOs in secretors and nonsecretors during lactation and revealed unique features of these HMO profiles in the milk of Chinese mothers. Interestingly, 2'-FL concentrations in secretors were found to be lower than those of Western populations but higher than those of African populations.

Project description:Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(?1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(?1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(?1-4)[Fuc(?1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(?1-4)[Fuc(?1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(?1-2)Gal(?1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(?1-3)[Gal(?1-4)GlcNAc(?1-6)]Gal(?1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species.

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New strategies for profiling and characterization of human milk oligosaccharides.

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New strategies for profiling and characterization of human milk oligosaccharides.

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