Abstract
Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography–tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9–17.4 µg Crb/mL and 1.3–3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8–12.0 and 14.3–16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.
Highlights
Neutral glycosphingolipids (GSLs) are complex lipids that belong to the sphingolipid family
Typical mass spectroscopy total ion count (TIC) chromatograms of GluCer and LacCer standards separated by hydrophilic interaction liquid chromatography (HILIC) are presented in Figure 2A,E, respectively
The accumulated MS, MS2 and MS3 data were used to identify the different sphingoid bases and fatty acid moieties of the molecular species observed in both the GluCer standard and the LacCer standard
Summary
Neutral glycosphingolipids (GSLs) are complex lipids that belong to the sphingolipid family (with lipid classification codes of SP05 and SP [1]). They are amphiphilic molecules consisting of a polar head group attached to a non-polar ceramide backbone. The polar head groups typically contain sugar residues. Glucosylceramide (GluCer), galactosylceramide (GalCer) and lactosylceramide (LacCer) contain a glucose, a galactose and a lactose polar head group, respectively. The hydrophobic ceramide backbone is composed of a fatty acid chain and a sphingoid base.
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