Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation

Abstract

Background: The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards.

Objective: The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers.

Methods: This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors.

Results: A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05).

Conclusions: HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective effects of milk components. The trials were registered at clinicaltrials.gov as NCT01817127 and NCT00524446.

Keywords: fucosylation; human milk oligosaccharides; lactation; mass spectrometry; secretor.

FIGURE 1

The extracted dynamic MRM chromatogram monitored for a pooled human milk sample. *Numbers stand for monosaccharide composition (e.g., 4_2_1_1: 4 Hex_2 HexNAc_1 Fucose_1 Neu5Ac). HexNAc, N-acetylhexosamine; LDFT, lacto-difucotetraose; LNFP I, lacto-N-fucopentaose I; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; MRM, multiple reaction monitoring; Neu5Ac, N-acetylneuraminic acid; 2′FL, 2′-fucosyllactose; 3FL, 3-fucosyllactose; 3′SL, 3′-sialyllactose; 6′SL, 6′-sialyllactose.

FIGURE 2

Comparison of total and MRM chromatograms of milk from secretor and nonsecretor mothers in the US study. Milk from secretors contained a much higher concentration of α(1,2)-fucosylated HMOs. Glucose (gray circles), galactose (white circles), N-acetylglucosamine (squares), fucose (triangles). HMO, human milk oligosaccharide; LDFT, lacto-difucotetraose; LNFP I, lacto-N-fucopentaose I; Resp., response; tetra-iso-LNO, tetrafucosyl-iso-lacto-N-octaose; TFLNH, trifucosyllacto-N-hexaose; 2′FL, 2′-fucosyllactose.

FIGURE 3

Changes in absolute (A) and relative (B) concentrations of HMOs in US milk samples during the course of lactation. Values are means ± SDs, n = 6 or 14. (A) Total, fucosylated, sialylated, and NF neutral HMO concentrations decreased over time (P < 0.05). (B) Percentage of fucosylation increased and percentage of sialylation and of NF neutrals decreased over time (P < 0.05). Conc., concentration; HMO, human milk oligosaccharide; NF, nonfucosylated.

FIGURE 4

Differences in HMO concentrations between secretor and nonsecretor mothers in the US study. Values are means ± SDs. (A) Day 10: secretors, n = 15; nonsecretors, n = 8. (B) Day 26: secretors, n = 18; nonsecretors, n = 8. (C) Day 71: secretors, n = 24; nonsecretors, n = 7. (D) Day 120: secretors, n = 15; nonsecretors, n = 8. *Means differ, P < 0.05. Conc., concentration; HMO, human milk oligosaccharide; NF, nonfucosylated.

FIGURE 5

Differences in absolute (A) and relative (B) concentrations of HMOs between secretor and nonsecretor mothers in the Malawi study. Values are means ± SDs, n = 69 (secretors) or 19 (nonsecretors). *Means differ, P < 0.05. Conc., concentration; HMO, human milk oligosaccharide; NF, nonfucosylated.