Multi-omics analysis reveals alterations of breastmilk metabolites and proteins in Hong Kong lactating mothers

Abstract

The nutritional contents of breastmilk (BM) directly participate in neonatal metabolism via breastfeeding. Currently, there is limited research on BM metabolites and proteins compositions, and their alterations during the long lactation period in Hong Kong mothers. In this study, liquid chromatography-mass spectrometry-based metabolomics, lipidomics and proteomics studies were applied to compare the compositions in BM of Hong Kong lactating mothers at the 2nd, 6th, and 12th months after delivery. Distinct metabolomics and lipidomics signatures in 6th month versus 2nd month and 12th month versus 2nd month were observed, and a total of 19 differential metabolites and 105 lipids were identified. Metabolomics study showed the significant alterations in key pathways involved in biotin metabolism, amino acid, and fatty acid-associated metabolisms. Lipidomics analysis indicated the accumulation of triglyceride and ceramide during the lactation period. The remodeling of glycerophospholipids was also observed during 12-month period. Moreover, 28 differentially expressed proteins were identified and mainly associated with GO functions and KEGG pathways of ribosome and complement and coagulation cascades, which were validated by network analysis. Our research contributes to the understanding of the BM compositions and differences during the long lactation period in postpartum women of Hong Kong.

Keywords: breastmilk; lipidomics; long lactation period; metabolomics; proteomics.

FIGURE 1

Overall study design.

FIGURE 2

Metabolic profiles of BM. PLS‐DA score plots from the indicated months in positive (A) and negative (B) modes. (C) Heatmap showed the 19 significantly changed metabolites in the indicated months. (D) The disturbed metabolic pathways showed various metabolic changes when comparing 12th month and 6th month to 2nd month. *p < .05, **p < .01.

FIGURE 3

Lipidomics profiling analysis for BM. (A) and (B) PLS‐DA score plots from the indicated months in positive (A) and negative (B) modes. (C) and (D) Volcano plots showing the relative abundances of lipids compared to the 2nd month (C: 6th month vs. 2nd month; D: 12th month vs. 2nd month). (E) Category of the significantly changed lipids in the BM.

FIGURE 4

Alternations of lipids and fatty acids in BM. (A) Heatmap showed the lipids in BM associated with the lactation period. (B) Fold changes of the related lipids and fatty acid corresponding to the 2nd month. n = 9 for fatty acid analysis between the 6th and 2nd months, n = 4 for fatty acid analysis between the 12th and 2nd months. *p < .05, **p < .01, ***p < .001, ****p < .0001.

FIGURE 5

Proposed lipid alternations in BM during the lactation period. The downregulated lipids were marked in red color and upregulated ones were marked in blue color.

FIGURE 6

Proteomics results from different lactation periods. (A) Principal components analysis (PCA) score plots for discriminating proteins from 2nd, 6th, and 12th months. (B) Volcano plots showing the relative abundances of proteins in the comparison between 12th and 2nd months. (C) Volcano plots showing the relative abundances of proteins in the comparison between 6th and 2nd months. (D) 28 shared proteins were measured by different comparisons. (E) GO annotation of differentially expressed proteins (upper) and rank KEGG pathways analysis (lower).

FIGURE 7

Proteins–proteins networks of overexpressed proteins during the lactation period. (A) Enrichment network with nodes colored by p‐value. (B) Enrichment network with nodes colored by cluster.