Abstract
A large scale profiling and analysis of glycerophospholipid species in macrophages has facilitated the identification of several rare and atypical glycerophospholipid species. By using liquid chromatography tandem mass spectrometry and comparison of the elution and fragmentation properties of the rare lipids to synthetic standards, we were able to identify an array of ether-linked phosphatidylinositols (PIs), phosphatidic acids, phosphatidylserines (PSs), very long chain phosphatidylethanolamines (PEs), and phosphatidylcholines (PCs) as well as phosphatidylthreonines (PTs) and a wide collection of odd carbon fatty acid-containing phospholipids in macrophages. A comprehensive qualitative analysis of glycerophospholipids from different macrophage cells was conducted. During the phospholipid profiling of the macrophage-like RAW 264.7 cells, we identified dozens of rare or previously uncharacterized phospholipids, including ether-linked PIs, PSs, and glycerophosphatidic acids, PTs, and PCs and PTs containing very long polyunsaturated fatty acids. Additionally, large numbers of phospholipids containing at least one odd carbon fatty acid were identified. Using the same methodology, we also identified many of the same species of glycerophospholipids in resident peritoneal macrophages, foam cells, and murine bone marrow derived macrophages.
Highlights
A large scale profiling and analysis of glycerophospholipid species in macrophages has facilitated the identification of several rare and atypical glycerophospholipid species
Ether-linked phospholipids may contain either an alkyl ether or vinyl ether bond at the sn-1 position of the glycerol backbone. These lipids are mostly found in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a variety of mammalian cell types, including macrophages [1,2,3]
On day 4, cells were washed with RPMI medium and maintained at 37°C in BMDMGM for 2 more days, after which the macrophages were plated on 100 mm tissue culture dishes at a density of 5 × 106 per plate
Summary
BMDMs were harvested from tibias and femurs of 2-month-old C57BL6 male mice. They were suspended in Bone Marrow-Derived Macrophage Growth Medium (BMDMGM), plated on 100 mm Petri dishes and maintained at 37°C in a humidified incubator for 4 days. On day 4, cells were washed with RPMI medium and maintained at 37°C in BMDMGM for 2 more days, after which the macrophages were plated on 100 mm tissue culture dishes at a density of 5 × 106 per plate. Foam cells were elicited from male mice (B6.129S7-LDLrtm Her/J) (3–5 weeks of age; Jackson Laboratory) after being placed on a high-cholesterol diet (# TD96121, Harlan Teklad) for 10 weeks. 1582 Journal of Lipid Research Volume 51, 2010 thioglycollate intraperitoneally and foam cells were harvested 4 days post injection. RAW 264.7 cells were maintained essentially as described elsewhere [12]
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