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K. Gronert, E. Walker, R. J. Kemp, M. Laniado Schwartzman, G. Zaidman; Analyses of Lipid Autacoids in Human Corneas by LC/MS/MS-Based Lipidomics. Invest. Ophthalmol. Vis. Sci. 2007;48(13):202.
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A central role for lipid autacoids in corneal inflammation, wound healing and neovascularization is strongly supported by in vitro and animal experiments. These small molecules are some of the initial mediators released in response to injury, infection and hypoxia. However, identification of these small molecules is challenging, especially in human corneas, due to the limits of detection for direct physical analyses. Hence, we developed a sensitive and quantitative method for profiling and quantitating endogenous lipid autacoids in human corneas.
Samples (10-20 mg) from discarded corneal buttons from penetrating keratoplasty were collected within 60 min after surgery and immediately snap frozen. Frozen corneas were gently homogenized in 66% methanol, proteins precipitated and fatty acids partially purified by C18 solid phase extraction. Extracted samples were analyzed by a triple quadruple linear ion trap LC/MS/MS system (MDS SCIEX 3200 QTRAP). MS/MS analyses were carried out in negative ion mode and tissue concentration of about 20 bioactive lipid autacoids was analyzed by multiple reaction monitoring using established and specific transitions ions. Calibration curves (0.01-1000 pg) for each lipid autacoid were established. Structures of endogenous lipid autacoids were confirmed by MS/MS analyses.
Lipidomic analyses demonstrated significant concentrations (0.1-100 pg/10mg) of more than 6 arachidonic acid metabolites which included inflammatory and angiogenic lipid autacoids, PGE2, PGD2, LTB4, 5-HETE, 15-HETE, 12-HETrE. Unexpectedly, docosahexaenoic acid-derived 17-HDHA and NPD1, which are potent anti-inflammatory lipid mediators, were prominent autacoids in all human corneal samples. Lipidomic profiles were distinct in each patient sample and concentrations markedly elevated in all graft samples.
Findings from this study establish the feasibility of analyzing and profiling lipid autacoids in human corneas with limits of detection as low as 100 femtograms. Analyses revealed distinct profiles of inflammatory and angiogenic lipid autacoids in graft and patient corneas. More importantly, this is the first study to demonstrate DHA-derived anti-inflammatory lipid signals as predominant autacoids in human corneas. Collectively, data from lipidomic analyses potentially have important implications for graft survival and the underlying pathology that ultimately required corneal transplantation.
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