Abstract
Lipids in breastmilk play a critical role in infant growth and development. However, few studies have investigated sources of variability of both high- and low-abundant milk lipids. The objective of our study was to investigate individual and morning–evening differences in the human milk lipidome. In this study, a modified two-phase method (MTBE: Methanol 7:2) was validated for the extraction of lipids from human breastmilk. This method was then applied to samples from a group of 20 healthy women to measure inter- and intra-individual (morning versus evening) variability of the breastmilk lipidome. We report here the levels of 237 lipid species from 13 sub-classes using reversed-phase liquid chromatography mass spectrometry (RP-LCMS) and direct-infusion mass spectrometry (DI-MS). About 85% of lipid species showed stable inter-individual differences across time points. Half of lipid species showed higher concentrations in the evening compared with the morning, with phosphatidylethanolamines (PEs) and triacylglycerols (TAGs) exhibiting the largest changes. In morning and evening samples, the biological variation was greater for diacylglycerols (DAGs) and TAGs compared with phospholipids and sphingolipids, and the variation in DAGs and TAGs was greater in evening samples compared with morning samples. These results demonstrate that variation in the milk lipidome is strongly influenced by individual differences and time of day.
Highlights
Milk is the primary source of nutrition for infants during early development
Lipid extraction was performed according to a modified version of the two-phase extraction reported previously by Matyash and colleagues [35]; this method is commonly used for plasma and tissues but, to the best of our knowledge, has not been previously applied to human milk samples
Using a methyl-tert-butyl ether (MTBE)/MeOH extraction method followed by RPLCMSMS and directinfusion mass spectrometry (DI-mass spectrometry (MS)), we were able to measure the concentrations of 237 lipid species from 10 μL of breastmilk
Summary
Milk is the primary source of nutrition for infants during early development. The primary contents of HM are carbohydrates (7%), lipids (5%), and proteins (0.9%), followed by lesser amounts of minerals, hormones, vitamins, and immune factors [4]. Lipids in HM are packaged in globules; these globules contain a triacylglycerol (TAG)-rich core, encapsulated by a triple-layered membrane, known as the milk fat globular membrane (MFGM). The lipid portion of the MFGM is rich in phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins (SM), phosphatidylserines (PS), phosphatidylinositols (PIs), and cholesterol, and contains small amounts of other bioactive lipids, such as gangliosides, lysophospholipids, monohexosyl(Hex1Cer), and dihexosylceramides (Hex2Cer) [9]. Sphingomyelin, together with PC, is a source of choline, which is important for development of the CNS [10]
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