Analysis, physiological and clinical significance of 12-HETE: A neglected platelet-derived 12-lipoxygenase product

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease. Figure LC-MS/MS chromatogram of 104 oxylipins on a Triple Quadrupole MS system employing dynamic MRM in the Negative Ion Electrospray mode Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6226-x) contains supplementary material, which is available to authorized users.

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Human neutrophils from peripheral blood may physically interact with platelets in several settings including hemostasis, inflammation, and a variety of vascular disorders. A role for lipoxygenase (LO)-derived products has been implicated in each of these events; therefore, we investigated the formation of lipoxins during coincubation of human neutrophils and platelets. Simultaneous addition of FMLP and thrombin to coincubations of these cells led to formation of both lipoxin A4 and lipoxin B4, which were monitored by reversed-phase high pressure liquid chromatography. Neither stimulus nor cell type alone induced the formation of these products. When leukotriene A4 (LTA4), a candidate for the transmitting signal, was added to platelets, lipoxins were formed. In cell-free 100,000 g supernatants of platelet lysates, which displayed 12-LO activity, LTA4 was also transformed to lipoxins. Platelet formation of lipoxins was inhibited by the LO inhibitor esculetin and partially sensitive to chelation of Ca2+, while neither acetylsalicylic acid nor indomethacin significantly inhibited their generation. In contrast, neutrophils did not transform LTA4 to lipoxins. Cell-free 100,000 g supernatants of neutrophil lysates converted LTA4 to LTB4. These results indicate that neutrophil-platelet interactions can lead to the formation of lipoxins from endogenous sources and provide a role for platelet 12-LO in the formation of lipoxins from LTA4.