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2D-LC-MS analysis of oxidized lipid species: application to the study of cardiolipin in apoptosis and damaged tissue.

Samhan-Arias-A; Tyurin-V; Tyurina-Y; Bayir-H; Kagan-V; Amoscato-A
Toxicologist 2012 Mar; 126(Suppl 1):254
Oxidized lipid species are important signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. These oxidized lipid species can co-chromatograph with non-oxidized species making their detection extremely difficult. In this study, a normal-phase and reverse-phase two dimensional HPLC-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. We found that oxidized and nonoxidized phospholipid species can be clearly separated from in this two dimensional system. We utilized two biologically relevant model systems, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death as well as traumatic brain injury, to separate and identify cardiolipin oxidation products, critical to the cell death pathways in these tissues following cellular stress/injury. We identified oxidized linoleic acyl chains (50%) as the preferred moieties for oxidation in cardiolipin, followed by arachidonic (31%) and docosahexaenoic acyl chains (18%). The ability of the two-dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult.
Lipid-peroxidation; Phospholipids; Oxidative-phosphorylation; Oxidative-processes; Lipids; Chromatographic-analysis; Liquid-chromatography; Mass-spectrometry; Lymphocytes; Molecular-biology; Analytical-instruments; Analytical-models; Analytical-processes; Laboratory-equipment; Laboratory-techniques; Biochemical-analysis; Tissue-culture
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Grant-Number-R01-OH-008282; B04252012
Source Name
The Toxicologist. Society of Toxicology 51st Annual Meeting and ToxExpo, March 11-15, 2012, San Francisco, California
Performing Organization
University of Pittsburgh at Pittsburgh