The objectives of Core H include: (1) a systems biology based identification of glycerophospholipids in primary macrophages, macrophage-like cell lines, and tissues;(2) expansion of methods for qualitative and quantitative analysis of lipids;(3) employing lipidomics to investigate macrophages and tissues under pathological conditions as disease models;and, (4) applying lipidomics to advance our understanding of the roles of lipids in metabolism. A systematic analysis continues in RAW264.7 and multiple types of primary macrophages to identify the components of the glycerophospholipid (GPL) lipidome. A variety of MSn and LC/MSn techniques identified over 850 GPL in RAW cells and we anticipate the identification of up to 1200 distinct species by the end of the second phase of this project. This tabulation includes the identification of a number of novel species (e.g., GPIns, GPA, and GPS ether-linked GPLs) and several atypical species not previously reported in macrophages. Core H developed a quantification system based on 20 odd-carbon diacyl internal standards from five GPLclasses and standard curves generated from dozens of even-carbon diacyl standards provided by the Lipid Synthesis Core. The planned addition of odd-carbon diacyl GPIns and six classes of lyso lipid internal standards, in addition to a wide variety of plasmanyl and plasmanyl-containing lipids, will greatly expand our ability to achieve absolute quantification of the vast numbers of chemically diverse phospholipid species. As described below, we also will participate in the Coordinated Studies proposed by this consortium that investigate the lipidomes of primary macrophages and tissues. In addition, a number of specialized studies will contribute to our understanding of the intersection of phospholipid metabolism with other classes of lipids and assist in the development of an integrated pathway map. We will take a leading role in defining the lipid molecular species produced during co-stimulation of TLR-4 with a variety of other inflammatory mediators (e.g., purinergic receptor subtypes), which will identify synergistic activations that are not observed upon stimulation with a single ligand. Such paradigms will provide a more physiological perspective of inflammatory pathways and identify new targets for pharmacological intervention in the treatment of diabetes, obesity, cardiovascular disease, cancer, and n neurodegeneration.
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