Recognized as the gold standard, human milk (HM) is an extremely complex yet fascinating biofluid tailored to meet an infant’s nutritional requirements throughout development. Endocannabinoids and endocannabinoid-like compounds (endocannabinoid metabolome, ECM) are endogenous lipid mediators derived from long-chain polyunsaturated fatty acids that have been identified in HM. Previous research has shown that arachidonoylglycerol might play a role in establishing the infant’s suckling response during lactation by activating the type 1 cannabinoid receptor in the infant’s brain. The mechanisms of action and the role of the ECM in HM are not fully understood. Transitional and mature milk samples were collected from lactating women (n
= 24) for ECM characterization, quantification, and to evaluate differences among the two stages. HM samples were analyzed by liquid chromatography-mass spectrometry. Identified members of the ECM were: arachidonoylethanolamine, palmitoylethanolamine, oleoylethanolamine, docosahexaenoylethanolamine, eicoapentaenoylethanolamine, eicosenoylethanolamine, arachidonoylglycerol, palmitoyglycerol, oleoylglycerol, docosahexaenoylglycerol, eicosapentaenoylglycerol, eiconenooylglycerol, arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid. Only docosahexaenoylglycerol was different across transitional and mature milk (p
≤ 0.05). Data from this cohort suggest that bioactive constituents in HM may also play a role in infant health and development. Future studies can be developed based on this study’s data to help elucidate specific roles for each ECM member in addition to understanding how the ECM modulates infant health.
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