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Cyclopentenone (A2/J2) isoprostanes (IsoPs) are a group of prostaglandin (PG)-like compounds generated in vivo from the free radical-induced peroxidation of arachidonic acid. Unlike other classes of IsoPs, cyclopentenone IsoPs contain highly reactive unsaturated carbonyl moieties on the prostane ring analogous to cyclooxygenase-derived PGA2 and PGJ2 that readily adduct relevant biomolecules such as thiols via Michael addition. The purpose of this review is to summarize our knowledge of the A2/J2-IsoPs. As a starting point, we will briefly discuss the formation and biological properties of PGA2 and PGJ2. Next, we will review studies definitively showing that cyclopentenone IsoPs are formed in large amounts in vivo. This is in marked contrast to cyclopentenone PGs, for which little evidence exists that they are endogenously produced. Subsequently, we will discuss studies related to the chemical syntheses of the 15-A2-IsoP series of cyclopentenone IsoPs. The successful synthesis of these compounds provides the recent impetus to explore the metabolism and biological properties of A-ring IsoPs, particularly as modulators of inflammation, and this work will be discussed. Finally, the formation of cyclopentenone IsoP-like compounds from other fatty acids such as linolenic acid and docosahexaenoic acid will be detailed.
Free radical-initiated autoxidation of polyunsaturated fatty acids (PUFAs) has been implicated in numerous human diseases, including atherosclerosis and cancer. This review covers the free radical mechanisms of lipid oxidation and recent developments of analytical techniques to analyze the lipid oxidation products. Autoxidation of PUFAs generates hydroperoxides as primary oxidation products, and further oxidation leads to cyclic peroxides as secondary oxidation products. Characterization of these oxidation products is accomplished by several mass spectrometric techniques. Ag+ coordination ion spray mass spectrometry has proven to be a powerful tool to analyze the intact lipid peroxides. Monocyclic peroxides, bicyclic endoperoxides, serial cyclic peroxides, and a novel class of endoperoxides (dioxolane-isoprostane peroxides) have been identified from the oxidation of arachidonate. Electron capture atmospheric pressure chemical ionization mass spectrometry has been applied to study lipid oxidation products after derivatization. All eight possible diastereomeric isoprostanes are observed from the oxidation of a single hydroperoxide precursor. 5- and 15-series isoprostanes are more abundant than the 8- and 12-series because the precursors that lead to 8- and 12-series compounds can undergo further oxidation and form dioxolane-isoprostane peroxides. Furthermore, formation of isoprostanes from 15-hydroperoxyeicosatetraenoate occurs from beta-fragmentation of the corresponding peroxyl radical to generate a pentadienyl radical rather than a "dioxetane" intermediate, as previously suggested.
Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A(2), D(2), E(2), etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance.
Autoxidation of polyunsaturated fatty acids and esters leads to a complex mixture containing hydroperoxides and cyclic peroxides. Prostaglandin bicyclic endoperoxides have been detected from the autoxidation of cholesteryl arachidonate by LC-MS and GC-MS techniques. All four possible types (I-IV) of bicyclic endoperoxides have been found starting from different regioisomeric hydroperoxides of cholesteryl arachidonate. Furthermore, the stereochemistry of Type IV bicyclic endoperoxides has been determined by conversion to pentafluorobenzyl (PFB) ester, trimethylsilyl (TMS) derivatives, and comparison with synthetic standards by the use of GC-MS. All eight possible diastereomers of the derivatized isoprostanes were observed and were separated by gas chromatography. The bicyclic endoperoxides with the two alkyl chains syn on the cyclopentane ring were formed preferentially to those with anti configuration, a result anticipated from earlier work. Substantial amounts of the anti-substituted isoprostanes, including PGF(2)(alpha), were, however, observed in the product mixture.
Certain corals are rich natural sources of prostaglandins, the metabolic origin of which has remained undefined. By analogy with the lipoxygenase/allene oxide synthase pathway to jasmonic acid in plants, the presence of (8R)-lipoxygenase and allene oxide synthase in the coral Plexaura homomalla suggested a potential metabolic route to prostaglandins (Brash, A. R., Baertshi, S. W., Ingram, C.D., and Harris, T. M. (1987) J. Biol. Chem. 262, 15829-15839). Other evidence, from the Arctic coral Gersemia fruticosa, has indicated a cyclooxygenase intermediate in the biosynthesis (Varvas, K., Koljak, R., Järving, I., Pehk, T., and Samel, N. (1994) Tetrahedron Lett. 35, 8267-8270). In the present study, active preparations of G. fruticosa have been used to identify both types of arachidonic acid metabolism and specific inhibitors were used to establish the enzyme type involved in the prostaglandin biosynthesis. The synthesis of prostaglandins and (11R)-hydroxyeicosatetraenoic acid was inhibited by mammalian cyclooxygenase inhibitors (indomethacin, aspirin, and tolfenamic acid), while the formation of the products of the 8-lipoxygenase/allene oxide pathway was not affected or was increased. The specific cyclooxygenase-2 inhibitor, nimesulide, did not inhibit the synthesis of prostaglandins in coral. We conclude that coral uses two parallel routes for the initial oxidation of polyenoic acids: the cyclooxygenase route, which leads to optically active prostaglandins, and the lipoxygenase/allene oxide synthase metabolism, the role of which remains to be established. An enzyme related to mammalian cyclooxygenases is the key to prostaglandin synthesis in coral. Based on our inhibitor data, the catalytic site of this evolutionary early cyclooxygenase appears to differ significantly from both known mammalian cyclooxygenases.
The discovery of IsoPs as products of non-enzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. A drawback related to this, however, has been lack of more facile and less expensive methods than mass spectrometry for the measurement of IsoPs. On the other hand, the recent introduction of immunoassay methods for measurement of IsoPs may alleviate this problem, provided they are specific and reliable. If this is the case, immunoassay methodology will most likely lead to an expansion of the use of measurements of IsoPs to assess oxidative stress status in vivo. Another need in the field of free radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to defining the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, at least two IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Moreover, information regarding the nature of the receptor(s) that mediate the biological actions of IsoPs will be of considerable importance to the development of specific antagonists or agonists of the biological actions of IsoPs. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs, much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.
Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.
Reaction of manganese-reconstituted prostaglandin endoperoxide synthase (Mn-PGHS) with 15-hydroperoxyeicosatetraenoic acid (15-HPETE) generates two products in nearly equal amounts: 15-hydroxyeicosatetraenoic acid (15-HETE) and 15-ketoeicosatetraenoic acid (15-KETE) [Kulmacz et al. (1994) Biochemistry 33, 5428-5439]. Their proposed mechanism to explain 15-KETE formation, namely oxidation of 15-HETE by the peroxidase activity of MnPGHS, was tested and found not to occur. Instead, 15-KETE may arise by one-electron reduction of 15-HPETE followed by oxidation of an intermediate alkoxyl radical. The mechanism of hydroperoxide reduction by Mn-PGHS was investigated using 10-hydroperoxyoctadeca-8,12-dienoic acid (10-OOH-18:2), a diagnostic probe of hydroperoxide reduction pathways. Reaction of Mn-PGHS with 10-OOH-18:2 generated the two-electron reduced product, 10-hydroxyoctadeca-8,12-dienoic acid (10-OH-18:2), as well as the one-electron reduction products, 10-oxooctadeca-8,12 dienoic acid (10-oxo-18:2) and 10-oxodec-8-enoic acid (10-oxo-10:1) in relative yields of 82, 10, and 7%, respectively. The identity of the one-electron reduction products was confirmed by electrospray ionization mass spectrometry. The detection of 10-oxo-10:1 provides strong evidence for the production of an alkoxyl radical during 10-OOH-18:2 reduction by Mn-PGHS. Like 15-HPETE, reaction of Mn-PGHS with 13-hydroperoxyoctadeca-8,12-dienoic acid (13-OOH-18:2) generated two products in equal amounts: 13-hydroxyoctadeca-8,12-dienoic acid (13-OH-18:2) and the keto fatty acid 13-oxooctadeca-8,12-dienoic acid (13-oxo-18:2). Comparison of the three hydroperoxides demonstrates that 15-HPETE is a much better substrate for Mn-PGHS than 10-OOH-18:2 or 13-OOH-18:2 with 10-fold greater turnovers. The results show that Mn-PGHS catalyzes both one- and two-electron hydroperoxide reduction and that the pathway of alkoxyl radical decomposition is influenced by the protein component of Mn-PGHS and the structure of the alkoxyl radical intermediate.
Although there are many reports of epoxy alcohol synthesis from lipoxygenase products (fatty acid hydroperoxides) in mammalian tissues, there are no well-defined examples of the stereoselective synthesis of individual epoxy alcohol diastereomers. An earlier report on the metabolism of 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) in rat liver microsomes suggested such a specific reaction [Weiss, R. H., et al. (1987) Arch. Biochem. Biophys. 252, 334-338]. To characterize this reaction further, we set out to determine the precise structures and mechanism of biosynthesis of the epoxy alcohol products. We compared the products formed from 15R- and 15S-HPETE by hematin (a nonenzymatic reaction), by liver microsomes isolated from control and phenobarbital-treated rats, and by purified cytochrome P450 2B1. Eight epoxy alcohol isomers were identified by mass spectrometry and 1H NMR. In the hematin reaction, the major products are four epoxy alcohols with the epoxide in the trans configuration, diastereomers are formed in similar amounts, and the 15-HPETE enantiomers give indistinguishable patterns of products. By contrast, the liver microsomes and P450 2B1 enzyme form predominantly single diastereomers, and the configuration of the epoxide is dependent on the stereochemistry of the substrate. The main product formed from 15S-HPETE is 11S-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,12E- trienoic acid, and the amounts increase upon phenobarbital induction. The main products from 15R-HPETE are 11-hydroxy-14S,15R-epoxyeicosa-5Z,8Z,12E-t rienoic acid from microsomes from control rats and 13-hydroxy-14S,15R-cis-epoxyeicosa-5,8,11-trienoic acid in microsomes from phenobarbital-induced rats. The P450 2B1 enzyme gave products similar to those from the phenobarbital-induced microsomes. Analysis of an incubation using the 18O-labeled 15S-HPETE substrate demonstrated 97.6% retention of both hydroperoxy oxygens in the major product with progressively lower 18O retentions in the minor products (74-32%), possibly reflecting degrees of enzymatic control of these reactions. These results establish a precedent for the stereoselective synthesis of epoxy alcohols by mammalian cytochrome P450s.
A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2-4 mumol min-1 mg protein-1, Km of 10 microM and kcat of approximately 250 min-1. linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5-8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.