Metabolomic differences in breast milk throughout lactation

Human milk (HM) is considered the gold standard for the nutrition of infants, as it provides several dietary benefits to the mother-infant dyads for both the short and long term. HM comprises various types of cells including leukocytes and epithelial cells; however, data on cellular diversity and phenotypic characteristics during lactation are limited.

Study: Metabolomic Diversity of Human Milk Cells over the Course of Lactation. A Preliminary Study. Image Credit: evso /

About the study

In a recent study published in Nutrients, researchers characterize the cellular metabolome of HM throughout the course of lactation.

The study comprised 15 HM samples obtained at different postnatal periods. Cells from the HM samples were isolated through centrifugation, whereas cell proportions were assessed using immunocytochemical and cytomorphological staining. Cellular metabolites were assessed by ultra‐performance liquid chromatography-quadrupole time‐of‐flight mass spectrometry (UPLC-QqTOF‐MS) analysis using the negative and positive modes of electrospray ionization.

All lactating women who were willing to participate in the study and did not suffer from communicable diseases were included and enrolled between November 2021 and December 2021. Data were obtained on demographic parameters including the mothers’ age and infants’ gender, anthropometric measures including head circumference, length, and birth weight, as well as clinical parameters of delivery type, gestational period, post-natal age, and appearance, pulse, grimace, activity, and respiration (APGAR) scores at one, five, and ten minutes.

HM was obtained between 8:00 AM and 3:00 PM using sterilized breast pumps. HM samples were categorized as colostrum milk, transitional milk, or mature milk according to the postnatal age.

Whereas colostrum was collected one to three days following the birth of the infant, transitional milk was collected between four days and two weeks later, and mature milk was collected two weeks or more after childbirth. In total, one, three, and 11 colostrum, transitional milk, and mature milk samples were obtained, respectively.

HM cellular diversity was evaluated using the Simpson Index of Diversity, with all index values correlated with the postnatal age. Metabolomic pathway analysis was performed using the Kyoto Encyclopedia of the Genes and Genomes (KEGG) database.

The immunocytochemical profiles were assessed using hierarchical cluster analysis, whereas principal component analysis (PCA) was performed to detect the key sources of metabolomic variations in breast milk during lactation.

Study findings

Immunocytochemical analysis indicated considerable cellular variability in HM, with median values for the relative abundances of the glandular-type cells of the epithelium, keratinocytes, and leukocytes being 98%, 1%, and 1%, respectively. Significant correlations were noted between postnatal milk age, leukocyte and epithelial cell proportions, as well as total cell counts.

Hierarchical cluster analysis yielded similar results as metabolomic analysis. Colostrum differed markedly from transitional and mature HM samples based on cellular counts and leukocyte proportions.

Metabolic pathway alterations correlated with postnatal milk age and were associated with the metabolism of amino acids such as proline, arginine, phenylalanine, tryptophan, tyrosine, glutathione, and beta-alanine.

Leukocyte counts in colostrum, transitional milk, and mature milk were 18, five, and one, respectively. Keratinocyte counts in the corresponding samples were zero, one, and one, respectively, whereas glandular epithelial cell counts were 82, 94, and 98, respectively. The total cell counts in colostrum, transitional milk, and mature milk were 706, 196, and 16, respectively.

A linear and statistically significant reduction in total cell counts and leukocyte proportions with postnatal milk age was noted, with the number of glandular epithelial cells increasing linearly over the course of lactation.

A total of 63 annotated features, which corresponded to 53 distinct metabolites, were observed. Among these features, 22%, 14%, 13%, and 8% represented carboxylic acids and derivatives, organonitrogen compounds, organooxygen compounds, and fatty acyls, respectively.

Across all samples, organonitrogen compounds, especially sphinganine d16:0 and phytosphingosine, were the most abundant. Colostrum was rich in carboxylic acids and derivatives, except for pyrimidine nucleosides.

Significant and positive correlations were observed for most metabolites, especially for amino acids such as L‐phenylalanine, L‐tyrosine, L‐2‐amino‐5‐hydroxypentanoic acid, and L‐methionine. Contrastingly, for SP d16:0, significant but negative correlations were observed with other metabolites, except dihydrosphingosine d18:0, sphinganine, and phytosphingosine.

In total, 21 metabolites correlated negatively with postnatal milk age. Only SP d16:0 metabolite levels increased through the stages of lactation.

Amino acids and their analogue compounds including L‐leucine, acetylhomoserine, L‐2‐amino‐5‐hydroxypentanoic acid, creatine, L‐phenylalanine, L‐tyrosine, L‐methionine, as well as amines such as spermine, spermidine, and solamine, and purine ribonucleotides, guanosine monophosphate, and adenosine monophosphate, levels reduced with advancing postnatal milk age. HM cellularity did not significantly differ by infant gender, delivery type, and gestational age.


The current study highlights the HM metabolomic variations through different stages of lactation, from colostrum to mature milk.

Further research is needed to improve the understanding of HM cellular diversity and their metabolic components. This information will inform infant nutritionists and aid in developing novel preventive measures for health-associated hazards and diseases, particularly those that are relevant for pre-term infants.

Journal reference:

  • Ten‐Doménech, I., Cascant‐Vilaplana, M.M., Navarro‐Esteve, V., et al. (2023). Metabolomic Diversity of Human Milk Cells over the Course of Lactation. A Preliminary Study. Nutrients 15(1100). doi:10.3390/nu1505110

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