Oxylipin profile of human milk and human milk-derived extracellular vesicles

Background: Small Extracellular Vesicles (sEVs) are nano -sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell -to -cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM -derived sEVs (HMEVs) employing an ad -hoc developed and validated ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. Results: The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semiquantitation. In 200 mu L of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM - 0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples. Significance and novelty: This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro -healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis.