Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation.
ABSTRACT: The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards.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.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 ?(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.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).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.
Project description:Background:Human 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. Objectives:We aimed to analyze HMO profiles, including their composition, concentrations, and changes during lactation, in milk of Chinese mothers. Methods:A 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. Results:Of 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 ?-1-3/4-fucosylated HMOs in nonsecretors were significantly higher than those in secretors during the first month. In contrast, ?-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. Conclusions:Our 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:Rationale: Human milk oligosaccharides (HMOs) vary among mothers and genetic factors contribute to this variability. We assessed changes in HMO concentrations during the first year of lactation and the relationship with FUT2 Secretor group and FUT3 Lewis group defining genetic polymorphisms. Methods: Milk samples were collected from lactating mothers participating in the LIFE Child cohort in Leipzig, Germany. The concentrations of 24 HMOs in milk samples collected at 3 months (N = 156), 6 months (N = 122), and 12 months (N = 28) were measured using liquid chromatography. Concentrations of HMOs were compared at all time-points and were tested for their associations with FUT2 and FUT3 genetic variations by sPLS regression. Results: FUT2 SNP rs601338 was found to predominantly define the Secretor status Se-: 11.8% and it was highly correlated with 2'-fucosyllactose (2'FL, p < 0.001) and lacto-N-fucosylpentaose-I (LNFP-I, p < 0.001). FUT3 SNPs rs28362459 and rs812936 were found to define Lewis status (Le-: 5.9%) and correlated with lacto-N-fucosylpentaose-II (LNFP-II, p < 0.001). A polygenic score predicted the abundance of 2'FL levels within Secretors' milk (adj. R 2 = 0.58, p < 0.001). Mean concentrations of most of the individual HMOs, as well as the sums of the measured HMOs, the fucosylated HMOs, and the neutral HMOs were lower at 6 and 12 months compared to 3 months (p < 0.001). Conclusions: Secretor and Lewis status defined by specific FUT2 and FUT3 SNPs are confirmed to be good proxies for specific individual HMOs and milk group variabilities. The polygenic score developed here is an opportunity for clinicians to predict 2'FL levels in milk of future mothers. These results show opportunities to strengthen our understanding of factors controlling FUT2 and FUT3 functionality, the temporal changes and variability of HMO composition during lactation and eventually their significance for infant development.
Project description:Human milk oligosaccharides (HMOs) are bioactive molecules in human milk that play a critical role in infant health. Obesity and associated metabolic aberrations can negatively impact lactation and alter milk composition. Here, the relationship between maternal glucose homeostasis and HMO composition from 136 healthy women was examined. Maternal glucose homeostasis (fasting plasma glucose and insulin, homeostatic model assessment for insulin resistance, and insulin sensitivity index) was evaluated at 30 weeks of gestation in healthy women (body mass index = 18.5-35 kg/m2). Human milk samples were collected at two months postpartum. HMO concentrations were measured via high performance liquid chromatography. Women were categorized into "secretor" and "non-secretor" groups based on 2'-Fucosyllactose concentrations (<100 nmol/mL, non-secretor). Pearson's correlation analysis and linear models were used to assess the relationships between maternal glucose homeostasis and HMO concentrations. In non-secretors, third trimester fasting plasma glucose and insulin were negatively associated with total HMO-bound sialic acid and concentrations of the sialylated HMOs 3'-sialyllactose and disialylacto-N-tetraose. In secretors, difucosyllactose and lacto-N-fucopentaose-II concentrations increased and sialyllacto-N-tetraose c and sialyllacto-N-tetraose b decreased as insulin sensitivity increased. This study is the first to demonstrate a relationship between obesity-associated maternal factors and HMO composition in both secretor and non-secretor populations.
Project description:To study the variability in human milk oligosaccharide (HMO) composition of Chinese human milk over a 20-wk lactation period, HMO profiles of 30 mothers were analyzed using CE-LIF. This study showed that total HMO concentrations in Chinese human milk decreased significantly over a 20-wk lactation period, independent of the mother's SeLe status, although with individual variations. In addition, total acidic and neutral HMO concentrations in Chinese human milk decreased over lactation, and levels are driven by their mother's SeLe status. Analysis showed that total neutral fucosylated HMO concentrations in Chinese human milk were higher in the two secretor groups as compared to the nonsecretor group. On the basis of the total neutral fucosylated HMO concentrations in Chinese human milk, HMO profiles within the Se+Le+ group can be divided into two subgroups. HMOs that differed in level between Se+Le+ subgroups were 2'FL, DF-L, LNFP I, and F-LNO. HMO profiles in Dutch human milk also showed Se+Le+ subgroup division, with 2'FL, LNT, and F-LNO as the driving force.
Project description:Human milk oligosaccharide (HMO) composition varies throughout lactation and can be influenced by maternal characteristics. This study describes HMO variation up to three months postpartum and explores the influences of maternal sociodemographic and anthropometric characteristics in a Brazilian prospective cohort. We followed 101 subjects from 28-35 gestational weeks (baseline) and throughout lactation at 2-8 (visit 1), 28-50 (visit 2) and 88-119 days postpartum (visit 3). Milk samples were collected at visits 1, 2 and 3, and 19 HMOs were quantified usinghigh-performance liquid chromatography with fluorescence detection (HPLC-FL). Friedman post-hoc test, Spearman rank correlation for maternal characteristics and HMOs and non-negative matrix factorization (NMF) were used to define the HMO profile. Most women were secretors (89.1%) and presented high proportion of 2'-fucosyllactose (2?FL) at all three sample times, while lacto-N-tetraose (LNT, 2-8 days) and lacto-N-fucopentaose II (LNFPII, 28-50 and 88-119 days) were the most abundant HMOs in non-secretor women. Over the course of lactation, total HMO weight concentrations (g/L) decreased, but total HMO molar concentrations (mmol/L) increased, highlighting differential changes in HMO composition over time. In addition, maternal pre-pregnancy body mass index (BMI) and parity influence the HMO composition in healthy women in this Brazilian cohort.
Project description:There is little data on human milk oligosaccharide (HMO) composition in Sub-Saharan Africa. Iron fortificants adversely affect the infant gut microbiota, while co-provision of prebiotic galacto-oligosaccharides (GOS) mitigates most of the adverse effects. Whether variations in maternal HMO profile can influence the infant response to iron and/or GOS fortificants is unknown. The aim of this study was to determine HMO profiles and the secretor/non-secretor phenotype of lactating Kenyan mothers and investigate their effects on the maternal and infant gut microbiota, and on the infant response to a fortification intervention with 5 mg iron (2.5 mg as sodium iron ethylenediaminetetraacetate and 2.5 mg as ferrous fumarate) and 7.5 g GOS. We studied mother-infant pairs (n = 80) participating in a 4-month intervention trial in which the infants (aged 6.5-9.5 months) received daily a micronutrient powder without iron, with iron or with iron and GOS. We assessed: (1) maternal secretor status and HMO composition; (2) effects of secretor status on the maternal and infant gut microbiota in a cross-sectional analysis at baseline of the intervention trial; and (3) interactions between secretor status and intervention groups during the intervention trial on the infant gut microbiota, gut inflammation, iron status, growth and infectious morbidity. Secretor prevalence was 72% and HMOs differed between secretors and non-secretors and over time of lactation. Secretor status did not predict the baseline composition of the maternal and infant gut microbiota. There was a secretor-status-by-intervention-group interaction on Bifidobacterium (p = 0.021), Z-scores for length-for-age (p = 0.022) and weight-for-age (p = 0.018), and soluble transferrin receptor (p = 0.041). In the no iron group, longitudinal prevalence of diarrhea was higher among infants of non-secretors (23.8%) than of secretors (10.4%) (p = 0.001). In conclusion, HMO profile may modulate the infant gut microbiota response to fortificant iron; compared to infants of secretor mothers, infants of non-secretor mothers may be more vulnerable to the adverse effect of iron but also benefit more from the co-provision of GOS.
Project description:The milk glycobiome has a significant impact on the gut microbiota of infants, which plays a pivotal role in health and development. Fucosylated human milk oligosaccharides (HMOs) and N-glycans on milk proteins are beneficial for the development of healthy gut microbiota, and the fucosylation levels of these glycans can be affected by the maternal fucosyltransferase 2 gene (FUT2). Here, we present results of longitudinal research on paired milk and stool samples from 56 Chinese mothers (CMs) and their breast-fed children. Changes of HMOs and fucosylated N-glycans in milk of CMs at different lactation stages were detected, which allowed characterization of the major differences in milk glycans and consequential effects on the gut microbiome of infants according to maternal FUT2 status. Significant differences in the abundance of total and fucosylated HMOs between secretor and nonsecretor CMs were noted, especially during early lactation. Despite a tendency toward decreasing milk protein concentrations, the fucosylation levels of milk N-glycans increased during late lactation. The changes in the levels of fucosylated HMOs and milk N-glycans were highly correlated with the growth of Bifidobacterium spp. and Lactobacillus spp. in the gut of infants during early and later lactation, respectively. Enriched expression of genes encoding glycoside hydrolases, glycosyl transferases, ATP-binding cassette (ABC) transporters, and permeases in infants fed by secretor CMs contributed to the promotion of these bacteria in infants. Our data highlight the important role of fucosylated milk glycans in shaping the gut microbiome of infants and provide a solid foundation for development of "personalized" nutrition for Chinese infants. IMPORTANCE Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts.
Project description:Human milk oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear.Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis.Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes.HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.
Project description:<h4>Objective</h4>The aim of this study was to determine whether human milk oligosaccharides (HMOs) at 1 month predicted infant weight gain at 6 months and whether associations varied by HMO secretor status.<h4>Methods</h4>Participants were 157 Hispanic mother-infant pairs. Human milk samples were collected at 1 month. Nineteen individual HMOs were analyzed using high-performance liquid chromatography, and secretor status was determined by the presence of 2'-fucosyllactose or lacto-N-fucopentaose (LNFP) I. Infant weight was measured at 1 and 6 months. Path analysis was used to test effects of HMO composition on infant weight gain, adjusting for maternal age, prepregnancy BMI, and infant age, sex, and birth weight.<h4>Results</h4>In the total sample, higher LNFPII predicted lower infant weight gain (g<sub>1</sub> ?=?-4.1, P?=?0.004); this was observed in both nonsecretor (g<sub>1</sub> ?=?-3.0, P?=?0.006) and secretor groups (g<sub>1</sub> ?=?-4.7, P?=?0.014). In the nonsecretor group, higher lacto-N-neotetraose (g<sub>1</sub> ?=?7.6, P?=?0.011) and disialyllacto-N-tetraose (g<sub>1</sub> ?=?14.3, P?=?0.002) predicted higher infant weight gain. There were no other associations in the secretor group.<h4>Conclusions</h4>Our data suggest that higher LNFPII in human milk may decrease obesity risk across all infants, whereas higher lacto-N-neotetraose and disialyllacto-N-tetraose may increase obesity risk in infants of nonsecretors only.
Project description:An absolute quantitation method for measuring free human milk oligosaccharides (HMOs) in milk samples was developed using multiple reaction monitoring (MRM). To obtain the best sensitivity, the instrument conditions were optimized to reduce the source and postsource fragmentation prior to the quadrupole transmission. Fragmentation spectra of HMOs using collision-induced dissociation were studied to obtain the best characteristic fragments. At least two MRM transitions were used to quantify and identify each structure in the same run. The fragment ions corresponded to the production of singly charged mono-, di-, and trisaccharide fragments. The sensitivity and accuracy of the quantitation using MRM were determined, with the detection limit in the femtomole level and the calibration range spanning over 5 orders of magnitude. Seven commercial HMO standards were used to create calibration curves and were used to determine a universal response for all HMOs. The universal response factor was used to estimate absolute amounts of other structures and the total oligosaccharide content in milk. The quantitation method was applied to 20 human milk samples to determine the variations in HMO concentrations from women classified as secretors and nonsecretors, a phenotype that can be identified by the concentration of 2'-fucosylation in their milk.