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F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the none-nzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.43 and 18.33 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted ϳ70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only ϳ30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H A diverse spectrum of signaling molecules is biosynthesized in nature from polyunsaturated fatty acids, their peroxides, and further transformations of the fatty acid peroxides. The peroxides are formed by two classes of dioxygenase enzyme. The hemoprotein dioxygenases include prostaglandin H synthase (cyclooxygenase) in animals (1), ␣-dioxygenase in plants (2), and several linoleate dioxygenases in fungi (3-5). The nonheme iron lipoxygenases are even more widespread, being almost ubiquitous among organisms that contain polyunsaturated fatty acids (6 -8). Although further biosynthetic transformation is sometimes accomplished by an additional catalytic activity of the initiating dioxygenase (e.g. leukotriene A 4 synthase (9) or aldehyde-synthesizing hydroperoxide cleaving activity (10) among the LOX 2 enzymes), commonly another distinct enzyme is used to rearrange or otherwise modify the reactive fatty acid peroxide intermediate. Two hemoprotein types are found that have become specialized for this biosynthetic role: cytochrome P450s and catalase-related enzymes.The fatty acid peroxide-metabolizing P450s are by far the better known and include CYP5 (thromboxane synthase) and CYP8A (prostacyclin synthase) in animals (11), and the entire family of CYP74 in plants (12). The individual CYP74 enzymes include allene oxide synthase (AOS), one of which catalyzes a key step in cyclopentenone synthesis in the jasmonate pathway, hydroperoxide lyase, divinyl ether synthase, and epoxyalcohol synthase (12, 13). The catalase-related enzymes are distinctive in that they have been found naturally as a fusion protein with the LOX enzyme that forms their hydroperoxide substrate (14). The known activities include AOS in Plexaura homomalla and other marine corals (with a different specificity for fatty acid hydroperoxide compared with the plant P450 AOS) (15,16), and the unique bicyclobutane synthase and other allylic epoxide synthase activities of the enzyme in the cyanobacterium Anabaena 18). Currently we are trying to understand the scope of the reactions catalyzed by this catala...

Lipoxygenases (LOX) are found in most organisms that contain polyunsaturated fatty acids, usually existing as individual genes although occasionally encoded as a fusion protein with a catalase-related hemoprotein. Such a fusion protein occurs in the cyanobacterium Acaryochloris marina and herein we report the novel catalytic activity of its LOX domain. The full-length protein and the C-terminal LOX domain were expressed in Escherichia coli, and the catalytic activities characterized by UV, HPLC, GC-MS, and CD. All omega-3 polyunsaturates were oxygenated by the LOX domain at the n-7 position and with R stereospecificity: α-linolenic and the most abundant fatty acid in A. marina, stearidonic acid (C18.4ω3), are converted to the corresponding 12R-hydroperoxides, eicosapentaenoic acid to its 14R-hydroperoxide, and docosahexaenoic acid to its 16R-hydroperoxide. Omega-6 polyunsaturates were oxygenated at the n-10 position, forming 9R-hydroperoxyoctadecadienoic acid from linoleic acid and 11R-hydroperoxy-eicosatetraenoic acid from arachidonic acid. The metabolic transformation of stearidonic acid by the full-length fusion protein entails its 12R oxygenation with subsequent conversion by the catalase-related domain to a novel allene epoxide, a likely precursor of cyclopentenone fatty acids or other signaling molecules (Gao et al, J. Biol. Chem. 284:22087-98, 2009). Although omega-3 fatty acids and lipoxygenases are of widespread occurrence, this appears to be the first description of a LOX-catalyzed oxygenation that specifically utilizes the terminal pentadiene of omega-3 fatty acids.

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