Choline intake and genetic polymorphisms influence choline metabolite concentrations in human breast milk and plasma

Abstract

Background: Choline is essential for infant nutrition, and breast milk is a rich source of this nutrient. Common single nucleotide polymorphisms (SNPs) change dietary requirements for choline intake.

Objective: The aim of this study was to determine whether total choline intake and/or SNPs influence concentrations of choline and its metabolites in human breast milk and plasma.

Design: We gave a total of 103 pregnant women supplemental choline or a placebo from 18 wk gestation to 45 d postpartum and genotyped the women for 370 common SNPs. At 45 d postpartum, we measured choline metabolite concentrations in breast milk and plasma and assessed the dietary intake of choline by using a 3-d food record.

Results: On average, lactating women in our study ate two-thirds of the recommended intake for choline (Adequate Intake = 550 mg choline/d). Dietary choline intake (no supplement) correlated with breast-milk phosphatidylcholine and plasma choline concentrations. A supplement further increased breast-milk choline, betaine, and phosphocholine concentrations and increased plasma choline and betaine concentrations. We identified 5 SNPs in MTHFR that altered the slope of the intake-metabolite concentration relations, and we identified 2 SNPs in PEMT that shifted these curves upward. Individuals who shared sets of common SNPs were outliers in plots of intake-metabolite concentration curves; we suggest that these SNPs should be further investigated to determine how they alter choline metabolism.

Conclusion: Total intake of choline and genotype can influence the concentrations of choline and its metabolites in the breast milk and blood of lactating women and thereby affect the amount of choline available to the developing infant. This study was registered at clinicaltrials.gov as NCT00678925.

Figures

FIGURE 1

Influence of dietary choline intake on breast-milk choline metabolite concentrations. Pregnant women were enrolled and randomly assigned to receive either a choline supplement (750 mg choline/d) or a placebo (corn oil) from 18 wk gestation to 45 d postpartum. Choline and choline metabolite concentrations were measured by using liquid chromatography/mass spectrometry in breast milk collected at 45 d postpartum. Dietary intake was estimated by using 3-d food records and adding the intake from the supplement or placebo. Plots are shown for all 6 metabolites that were measured in breast milk. Regression models with metabolite as the response and total choline intake (diet plus supplement) as the predictor were fitted considering either placebo subjects, supplemented subjects, or all subjects combined (3 solid lines). The following numbers of outliers (defined in Subjects and Methods) for each metabolite were excluded: subjects who received placebo only—glycerophosphocholine (4), phosphocholine (4), phosphatidylcholine (4), sphingomyelin (3), choline (3), and betaine (2); subjects who received the supplement only—glycerophosphocholine (3), phosphocholine (1), phosphatidylcholine (2), sphingomyelin (3), choline (3), and betaine (3); and all subjects combined—glycerophosphocholine (5), phosphocholine (5), phosphatidylcholine (3), sphingomyelin (5), choline (5), and betaine (7). Weighted least-squares regression was performed in the model with breast-milk phosphocholine to overcome heteroskedasticity. To achieve normality, log transformations were made on breast-milk phosphatidylcholine, phosphocholine, betaine, and choline concentrations. Three statistical analyses were performed for each metabolite: effect of intake on concentrations for the placebo group (indicated above the line on the left), effect of intake plus supplement on concentrations for the supplemented group (indicated above the line on the right), and effect of intake plus supplement on concentrations for the combined groups (indicated above the bracket). P values are indicated when differences were significant. Group sizes for the placebo and supplement groups were 46 and 48 subjects, respectively. The R2 values for the placebo, supplement, and placebo plus supplement groups, respectively, are as follows: for phosphocholine—0.11, 0.02, and 0.16; for glycerophosphocholine—0.003, 0.0001, and 0.006; for phosphatidylcholine—0.11, 0.008, and 0.07; for sphingomyelin—0.006, 0.003, and 0.003; for choline—0.008, 0.009, and 0.08; and for betaine—0.005, 0.003, and 0.13. +, subjects randomly assigned to the placebo group; •, subjects randomly assigned to the choline supplement group.

FIGURE 2

Influence of dietary choline intake on plasma choline metabolite concentrations. Pregnant women at P values are indicated when differences were significant; group sizes for placebo and supplement groups were 46 and 48 subjects, respectively. The R2 values for placebo, supplement, and placebo plus supplement groups, respectively, are as follows: for choline—0.11, 0.15, and 0.55; for phosphatidylcholine—0.004, 0.01, and 0.0001; for betaine—0.09, 0.07, and 0.56; and for sphingomyelin—0.07, 0.02, and 0.02. +, subjects randomly assigned to the placebo group; •, subjects randomly assigned to the choline supplement group.

FIGURE 3

Outlier approach: participants with breast-milk choline metabolite concentrations that were beyond the 95% prediction limits for the mean shared common single nucleotide polymorphisms (SNPs). A: Plot of breast-milk choline concentrations compared with total choline intake in all participants. Three participants, identified with bold dots, had 5 SNPs in common: rs1076991, rs2983733, rs2987981, rs8003379, and rs17824591. All of these SNPs occurred in the methylene tetrahydrofolate dehydrogenase 1 (MTHFD1) gene. All 3 of these subjects had very high breast-milk choline concentrations, and 2 of these subjects were outliers. A linear regression model was used to compare these 3 subjects with the rest of the subjects. B: Plot of plasma choline concentrations compared with total choline intake in all participants. Five participants, identified with bold dots, had 2 SNPs in common: rs2461248 and rs7700970, both of which were in the BHMT gene. Four of the 5 subjects had lower than average plasma choline concentrations, and 2 of the subjects were outliers. A linear regression model was used to compare these 5 subjects with the rest of the subjects. +, represents participants who did not carry the common set of SNPs.