Urinary Metabolomic Differentiation of Infants Fed on Human Breastmilk and Formulated Milk

Human breastmilk is an invaluable nutritional and pharmacological resource with a highly diverse metabolite profile, which can directly affect the metabolism of infants. Application of metabolomics can discriminate the complex relationship between such nutrients and infant health. As the most common biological fluid in metabolomic study, infant urinary metabolomics may provide the physiological impacts of different nutritional resources, namely human breastmilk and formulated milk. In this study, we aimed to identify possible differences in the urine metabolome of 30 infants (1-14 days after birth) fed with breast milk (n = 15) or formulated milk (n = 15). From metabolomic analysis with gas chromatography-mass spectrometry, 163 metabolites from single mass spectrometry (GC-MS), and 383 metabolites from tandem mass spectrometry (GC-MS/MS) were confirmed in urinary samples. Various multivariate statistical analysis were performed to discriminate the differences originating from physiological/nutritional variables, including human breastmilk/formulate milk feeding, sex, and duration of feeding. Both unsupervised and supervised discriminant analyses indicated that feeding resources (human breastmilk/formulated milk) gave marginal but significant differences in urinary metabolomes, while other factors (sex, duration of feeding) did not show notable discrimination between groups. According to the biomarker analyses, several organic acid and amino acids showed statistically significant differences between different feeding resources, such as 2-hydroxyhippurate.