The human milk metabolome reveals diverse oligosaccharide profiles

Abstract

Breast milk delivers nutrition and protection to the developing infant. There has been considerable research on the high-molecular-weight milk components; however, low-molecular-weight metabolites have received less attention. To determine the effect of maternal phenotype and diet on the human milk metabolome, milk collected at day 90 postpartum from 52 healthy women was analyzed by using proton nuclear magnetic resonance spectroscopy. Sixty-five milk metabolites were quantified (mono-, di-, and oligosaccharides; amino acids and derivatives; energy metabolites; fatty acids and associated metabolites; vitamins, nucleotides, and derivatives; and others). The biological variation, represented as the percentage CV of each metabolite, varied widely (4-120%), with several metabolites having low variation (<20%), including lactose, urea, glutamate, myo-inositol, and creatinine. Principal components analysis identified 2 clear groups of participants who were differentiable on the basis of milk oligosaccharide concentration and who were classified as secretors or nonsecretors of fucosyltransferase 2 (FUT2) gene products according to the concentration of 2'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose I. Exploration of the interrelations between the milk sugars by using Spearman rank correlations revealed significant positive and negative associations, including positive correlations between fucose and products of the FUT2 gene and negative correlations between fucose and products of the fucosyltransferase 3 (FUT3) gene. The total concentration of milk oligosaccharides was conserved among participants (%CV = 18%), suggesting tight regulation of total oligosaccharide production; however, concentrations of specific oligosaccharides varied widely between participants (%CV = 30.4-84.3%). The variability in certain milk metabolites suggests possible roles in infant or infant gut microbial development. This trial was registered at clinicaltrials.gov as NCT01817127.

Conflict of interest statement

Author disclosures: J. T. Smilowitz, A. O’Sullivan, D. Barile, J. B. German, B. Lönnerdal, and C. M. Slupsky, no conflicts of interest.

Figures

FIGURE 1

Principal components analysis scores plot of milk samples from 52 participants (A). Those with 2′-FL concentrations at low or undetectable concentrations are labeled as “nonsecretors” and are indicated as open circles. Those with high concentrations of 2′-FL are labeled as “secretors.” Principal components are indicated [t(1) and t(2)]. A corresponding [p(1) and p(2)] loadings plot illustrating the variables responsible for separation of the observations in A is depicted in panel B. Metabolite variables contributing the most to separation are circled. LDFT, lactodifucotetraose; LNFP, lacto-N-fucopentaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; 2′-FL, 2′-fucosyllactose; 3′-FL, 3′-fucosyllactose; 3′-SL, 3′-sialyllactose; 6′-SL, 6′-sialyllactose.

FIGURE 2

Comparison of the concentration of HMOs synthesized by the product of the FUT2 (fucosyltransferase 2) gene. 2′-FL (A), LNFP I (B), and LDFT (C). Secretors (S; n = 40) and nonsecretors (NS; n = 12) are shown. ***P < 0.0005. HMO, human milk oligosaccharide; LDFT, lactodifucotetraose; LNFP, lacto-N-fucopentaose; 2′-FL, 2′-fucosyllactose.

FIGURE 3

Comparison of the concentration of sugars not produced by the product of the FUT2 (fucosyltransferase 2) gene. Fucose (A), 3′-FL (B), LNFP II (C), LNT(D), and 6′-SL (E). Secretors (S; n = 40) and nonsecretors (NS; n = 12) are shown. *P < 0.05, **P < 0.01, ***P < 0.0005. LNFP, lacto-N-fucopentaose; LNT, lacto-N-tetraose; 3′-FL, 3′-fucosyllactose; 6′-SL, 6′-sialyllactose.

FIGURE 4

Spearman correlation heatmap of human milk sugars. The size of the circle in the upper right triangle represents the magnitude of the correlation, whereas the color represents the direction of the relationship (black = positively correlated; white = negatively correlated). The values located at the bottom left are the Spearman ρ values corresponding to the pairwise correlation. LDFT, lactodifucotetraose; LNFP, lacto-N-fucopentaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; 2′-FL, 2′-fucosyllactose; 3′-FL, 3′-fucosyllactose; 3′-SL, 3′-sialyllactose; 6′-SL, 6′-sialyllactose.

FIGURE 5

Comparison of metabolites that differentiate fasting (n = 12) from fed (n = 16) states: alanine (A) and galactose (B). Values are means ± SEMs, *P < 0.01, *** P < 0.0001.