Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis

Ceramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.

324 Milk Fat Globule Membrane from Bovine Milk on Brain Development of Early Life

The milk fat globule membrane (MFGM) is instrumental for the fat delivery system into human and bovine milk, but is typically removed during the manufacture of infant formula. MFGM contains components that may impact neurodevelopment, including sialic acid, gangliosides, sphingomyelin, choline, glycerophospholipids, proteins, and cholesterol. This presentation will review the clinical trial evidence linking MFGM supplementation to beneficial outcomes in infants and will describe potential mechanistic evidence linking MFGM with neurocognitive outcomes arising from preclinical studies in piglets. Infants fed formula supplemented with a MFGM (4% total protein) from 2 to 6 months of age had improved neurocognitive development at 1 year of age compared to infants fed standard formula. Infants fed formula with MFGM (5.0 g/L) and lactoferrin (0.6 g/L) for 1 year had an accelerated neurodevelopmental profile at 1 year and improved language subcategories at 18 months compared to infants fed a standard formula. To investigate potential mechanisms, piglets were fed a CONT formula or a TEST formula with MFGM and lactoferrin at the same concentrations from 2 to 31 days of age. Piglets underwent spatial T-maze testing to assess learning and memory, and magnetic resonance imaging to measure brain micro- and macrostructure. TEST piglets had lower radial and mean diffusivities in the internal capsule, suggesting greater myelination. The internal capsule contains motor and sensory projections from the cortex to corticospinal tract. Piglets on the CONT diet displayed shorter latency to choice in the T-maze compared to TEST piglets, potentially indicating anxiety-like behaviors or greater impulsivity. Aspects of the microbiome-gut-brain-axis (MGBA) were investigated to uncover potential mechanisms. TEST piglets had higher protein abundance of tyrosine hydroxylase and vasoactive intestinal peptide, longer villi and greater disaccharidase activity in the small intestine and differences in microbial abundances in the ascending colon and feces, suggesting potential modulation of the MGBA by MFGM.

Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling

In the present study the lysophospholipid sources for arachidonic (AA) and eicosapentaenoic acid (EPA) incorporation into and redistribution within the phospholipids of phorbol-ester-differentiated U937 cells was investigated. Initially, AA incorporated primarily into choline glycerophospholipids (PC), whereas EPA incorporated mainly into ethanolamine glycerophospholipids (PE). Bromoenol lactone (BEL), an inhibitor of the Group VI Ca2+-independent phospholipase A2 (iPLA2), diminished both lysophosphatidylcholine levels and the incorporation of AA into phospholipids. However BEL had little effect on EPA incorporation. In concanavalin A-activated cells, EPA, but not AA, incorporation was also affected by methyl arachidonyl fluorophosphonate (MAFP), suggesting an additional role for the group IV cytosolic phospholipase A2. In the activated cells AA and EPA did not compete with each other for incorporation, indicating that the pathways for AA and EPA incorporation are partially different. The AA and EPA initially incorporated into PC slowly moved to PE in a process that took several hours. The transfer of AA and EPA from PC to PE was not inhibited by BEL, MAFP or LY311727 [3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulphonic acid], raising the possibility that an as-yet-undetermined phospholipase A2 may be involved in fatty acid phospholipid remodelling. A strong candidate to be involved in these reactions is a novel Ca2+-independent phospholipase A2 that, unlike all known iPLA2s, is resistant to inhibition by BEL and also to MAFP and LY311727. The enzyme activity cleaves both PC and PE and is thus able to provide the lysoPC and lysoPE acceptors required for the fatty acid acylation reactions.