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Formation of F-ring Isoprostane-like Compounds (F3-Isoprostanes) in Vivo from Eicosapentaenoic Acid*

Open AccessPublished:March 28, 2006DOI:https://doi.org/10.1074/jbc.M601035200
      Eicosapentaenoic acid (EPA, C20:5, ω-3) is the most abundant polyunsaturated fatty acid (PUFA) in fish oil. Recent studies suggest that the beneficial effects of fish oil are due, in part, to the generation of various free radical-generated non-enzymatic bioactive oxidation products from ω-3 PUFAs, although the specific molecular species responsible for these effects have not been identified. Our research group has previously reported that pro-inflammatory prostaglandin F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether F2-IsoP-like compounds (F3-IsoPs) are formed from the oxidation of EPA in vivo. Oxidation of EPA in vitro yielded a series of compounds that were structurally established to be F3-IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to 8.7 + 1.0 μg/mg EPA) and greater than levels of F2-IsoPs generated from arachidonic acid. We then examined the formation of F3-IsoPs in vivo in mice. Levels of F3-IsoPs in tissues such as heart are virtually undetectable at baseline, but supplementation of animals with EPA markedly increases quantities up to 27.4 + 5.6 ng/g of heart. Interestingly, EPA supplementation also markedly reduced levels of pro-inflammatory arachidonate-derived F2-IsoPs by up to 64% (p < 0.05). Our studies provide the first evidence that identify F3-IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F3-IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA.
      Isoprostanes (IsoPs)
      The abbreviations used are: IsoP, isoprostane; PG, prostaglandin; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acid; AAPH, 2,2′-azobis(2-amidinopropane) hydrochloride; GC, gas chromatography; NICI, negative ion chemical ionization; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; CID, collision-induced dissociation; SRM, selective reaction monitoring; RT, retention time.
      2The abbreviations used are: IsoP, isoprostane; PG, prostaglandin; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acid; AAPH, 2,2′-azobis(2-amidinopropane) hydrochloride; GC, gas chromatography; NICI, negative ion chemical ionization; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; CID, collision-induced dissociation; SRM, selective reaction monitoring; RT, retention time.
      are prostaglandin (PG)-like compounds that are formed non-enzymatically from the free radical-induced oxidation of arachidonic acid. Formation of these compounds proceeds through the generation of bicyclic endoperoxide PGH2-like intermediates that are reduced to PGF2-like compounds termed F2-IsoPs (
      • Morrow J.D.
      • Hill K.E.
      • Burk R.F.
      • Nammour T.M.
      • Badr K.F.
      • Roberts II, L.J.
      ) or undergo rearrangement to PGD2 and PGE2-like compounds (D2/E2-IsoPs) (
      • Morrow J.D.
      • Minton T.A.
      • Mukundan C.R.
      • Campbell M.D.
      • Zackert W.E.
      • Daniel V.C.
      • Badr K.F.
      • Blair I.A.
      • Roberts L.J.
      ) and thromboxane-like compounds (isothromboxanes) (
      • Morrow J.D.
      • Awad J.A.
      • Wu A.
      • Zackert W.E.
      • Daniel V.C.
      • Roberts II, L.J.
      ). Unlike cyclooxygenase-derived PGs, IsoPs are formed in situ esterified to phospholipids and are subsequently released, presumably by the action of a phospholipase A2 (
      • Morrow J.D.
      • Awad J.A.
      • Boss H.J.
      • Blair I.A.
      • Roberts II, L.J.
      ). Quantification of F2-IsoPs has been shown to be one of the most reliable markers to assess oxidant stress status and lipid peroxidation in vivo (
      • Morrow J.D.
      • Roberts II, L.J.
      ,
      • Morrow J.D.
      ,
      • Roberts II, L.J.
      • Morrow J.D.
      ). In addition, F2-IsoPs have been shown to exert potent vasoconstrictive activities in the vasculature (
      • Kromer B.M.
      • Tippins J.R.
      ,
      • Cracowski J.L.
      • Stanke-Labesque F.
      • Devillier P.
      • Chavanon O.
      • Hunt M.
      • Souvignet C.
      • Bessard G.
      ), suggesting a pathogenic role for these compounds in diseases such as atherosclerosis (
      • Cracowski J.L.
      • Devillier P.
      • Durand T.
      • Stanke-Labesque F.
      • Bessard G.
      ,
      • Habib A.
      • Badr K.F.
      ).
      Eicosapentaenoic acid (C20:5 ω-3; EPA) is among the more abundant omega-3 polyunsaturated fatty acids (PUFA) present in fish oils. The other important PUFA in fish oil is docosahexaenoic acid (C22:6 ω-3; DHA). Studies in animals as well as human epidemiological studies, and more recently, clinical intervention trials, suggest that fish consumption or dietary fish oil supplementation reduces the incidence of important diseases including atherosclerosis and sudden death, neurodegeneration, and various inflammatory disorders (
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      ,
      • Ruxton C.
      ). The mechanism(s) by which these beneficial effects occurs is unknown, but it has been hypothesized that various enzymatically and non-enzymatically generated peroxidation products of EPA possess anti-inflammatory activities (
      • Hong S.
      • Gronert K.
      • Devchand P.R.
      • Moussignac R.L.
      • Serhan C.N.
      ,
      • Serhan C.N.
      • Levy B.
      ,
      • Serhan C.N.
      • Clish C.B.
      • Brannon J.
      • Colgan S.P.
      • Chiang N.
      • Gronert K.
      ,
      • Serhan C.N.
      • Hong S.
      • Gronert K.
      • Colgan S.P.
      • Devchand P.R.
      • Mirick G.
      • Moussignac R.L.
      ,
      • Sethi S.
      • Eastman A.Y.
      • Eaton J.W.
      ,
      • Sethi S.
      ).
      Previously, Änggård and colleagues (
      • Nourooz-Zadeh J.
      • Halliwell B.
      • Änggård E.
      ) provided limited evidence that F-ring IsoP-like compounds (F3-IsoPs) can be formed from the oxidation of EPA in vitro. These compounds are termed F3-IsoPs because they are structurally analogous to F2-IsoPs yet contain three double bonds. We therefore considered the possibility that IsoP-like compounds could be formed by the free radical-induced peroxidation of EPA in vivo. The rationale for undertaking these studies, in part, was based on the hypothesis that F3-IsoPs contribute to the beneficial biological effects of EPA and fish oil supplementation in that they may exert biological activities that are anti-inflammatory as compared with F2-IsoPs. Indeed, one report states that the EPA-derived IsoP, 15-F3t-IsoP, possesses activity that is different from 15-F2t-IsoP in that it does not affect human platelet shape change or aggregation (
      • Pratico D.
      • Smyth E.M.
      • Violi F.
      • FitzGerald G.A.
      ). 15-F2t-IsoP is a ligand for the thromboxane receptor and induces platelet shape change and also causes vasoconstriction (
      • Morrow J.D.
      • Hill K.E.
      • Burk R.F.
      • Nammour T.M.
      • Badr K.F.
      • Roberts II, L.J.
      ,
      • Morrow J.D.
      • Minton T.A.
      • Roberts I.I.
      ). The lack of activity of 15-F3t-IsoP is consistent with observations regarding EPA-derived PGs in that these latter compounds exert either weaker agonist effects or no effects in comparison with arachidonate-derived PGs (
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      ,
      • Balapure A.K.
      • Rexroad J.
      • Kawada K.
      • Watt D.S.
      • Fitz T.A.
      ,
      • Kulkarni P.S.
      • Srinivasan B.D.
      ).
      Based on studies of arachidonic acid oxidation, the mechanism by which F3-IsoPs could be formed is outlined in Fig. 1. There are four bis-allylic positions in EPA at carbons 7, 10, 13, and 16 as opposed to three bis-allylic positions in arachidonate where hydrogen abstraction can occur. Depending on the position of hydrogen abstraction and oxygen insertion, eight hydroperoxides are formed. These eight hydroperoxides subsequently generate six F3-IsoP regioisomers. Each regioisomer is theoretically comprised of eight racemic diastereomers for a total of 96 compounds. A nomenclature system for the IsoPs has been established and approved by the Eicosanoid Nomenclature Committee in which the different regioisomer classes are designated by the carbon number on which the side chain hydroxyl is located with the carboxyl carbon designated as C-1 (
      • Taber D.F.
      • Morrow J.D.
      • Roberts II, L.J.
      ). Thus, in accordance with this nomenclature system, the F3-IsoP regioisomers are designated as 5-, 8-, 11-, 12-, 15-, and 18-series F3-IsoPs. Herein, we present evidence that F3-IsoPs are, in fact, formed in significant amounts in vitro and in vivo from the free radical-catalyzed peroxidation of EPA.
      Figure thumbnail gr1
      FIGURE 1Pathway for the formation of F3-IsoPs by non-enzymatic peroxidation of EPA (A and B).

      EXPERIMENTAL PROCEDURES

      Materials—Eicosapentaenoic acid and d4-15-F2t-IsoP (8-iso-PGF) were purchased from Cayman Chemical Co. (Ann Arbor, MI). Dimethylformamide and undecane were obtained from Aldrich. Pentafluorobenzyl bromide and diisopropylethylamine were from Sigma. 2,2′-Azobis(2-amidinopropane) hydrochloride (AAPH) was from Eastman Kodak Co. N,O-bis (trimethylsilyl) trifluoroacetamide was from Supelco (Bellefonte, PA). [2H9]N,O-bis (trimethylsilyl) acetamide was from CDN Isotopes (Pointe-Claire, PQ). C-18 and silica Sep-Pak cartridges were from Waters Associates (Milford, MA). 60ALK6D TLC plates were from Whatman (Maidstone, UK).
      Oxidation of EPA—Five milligram of fresh EPA was dissolved in 100 μl of ethanol and added immediately to 4.9 ml of phosphate-buffered saline solution (pH 7.4) containing 10 mm AAPH. The EPA oxidation reaction mixture was incubated in a shaking water bath at 37 °C for varying amounts of time, after which it was placed immediately at –80 °C until further processing.
      Purification and Analysis of F3-IsoPs—Free and esterified F3-IsoPs were extracted using C-18 and Silica Sep-Pak cartridges, converted to a pentafluorobenzyl ester, purified by TLC, converted to a trimethylsilyl ether derivative, and quantified by stable isotope dilution gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS) with d4-15-F2t-IsoP as an internal standard using a modification of the method described for the quantification of F2-IsoPs (
      • Morrow J.D.
      • Roberts II, L.J.
      ). Instead of scraping 1 cm below to 1 cm above where PGF methyl ester migrates on TLC for analysis of F2-IsoPs, the scraped area was extended to 3.5 cm above where the PGF methyl ester migrates. This extended area of the TLC plate was determined to contain F3-IsoPs by analyzing small 5-mm cuts using approaches for their identification described below. The M−·CH2C6F5 ions were monitored for quantification (m/z 567 for F3-IsoPs and m/z 573 for d4-15-F2t-IsoP). Quantification of the total amount of F3-IsoPs was determined by integrating the peak area of material in the m/z 567 channel in comparison with the m/z 573 channel. GC/NICI/MS was carried out using an Agilent Technologies 6890N Network GC/MS system. Sample work using this method does not lead to the generation of F3-IsoPs during purification and derivatization. In some studies, F2-IsoPs were quantified as described previously (
      • Morrow J.D.
      • Roberts II, L.J.
      ).
      Analysis of F3-IsoPs by Liquid Chromatography (LC)/Electrospray Ionization (ESI)/MS/MS—F3-IsoPs generated by the in vitro and in vivo oxidation of EPA were extracted by C-18 and Silica Sep-Pak cartridge as described above. The samples were then analyzed by negative ion ESI-MS coupled with reverse-phase high pressure liquid chromatography separation using a Supelco Discovery C18 column (15 cm × 2.1 mm, 5 μm) at a flow rate of 0.2 ml/min with a linear gradient starting with 80% solvent A (2 mm ammonium acetate) to 65% in 20 min. Mobile phase B consisted of acetonitrile/methanol (95:5). The ESI source was fitted with a deactivated fused silica capillary (100 μm inner diameter). Nitrogen was used as both the sheath gas and the auxiliary gas, at 45 and 17 p.s.i., respectively. The capillary temperature was 280 °C. The spray voltage was 4.3 kV, and the tube lens voltage was 80 V. Collision-induced dissociation (CID) of the molecular ion of putative F3-IsoPs was performed from 20 to 30 eV under 1.5 millitorrs of argon. Spectra that are shown were obtained at 25 eV. Spectra were displayed by averaging scans across chromatographic peaks. Selective reaction monitoring (SRM) was performed according to characteristic fragmentation patterns of F2-IsoPs (
      • Waugh R.J.
      • Morrow J.D.
      • Roberts II, L.J.
      • Murphy R.C.
      ). The collision energy for SRM was 30 eV. LC/MS was carried out using a ThermoFinnigan TSQ Quantum 1.0 SR 1 mass spectrometer. Data acquisition and analysis were performed using Xcaliber software, version 1.3.
      Preparation of F3-IsoPs from Rodent Tissue—Mice (strain C57BL/6J(B6)) were fed with a rodent AIN-93 diet supplemented with 0, 0.28, 0.56, or 0.84% EPA (by weight). In some studies, animals were administered CCl4 (1 ml/kg) intraperitoneally to induce an oxidant stress. After 8 weeks of feeding, the mice were sacrificed. Heart and other tissues were removed and immediately flash-frozen in liquid nitrogen and stored in –80 °C. Analyzed tissue samples were homogenized in 5 ml of ice-cold chloroform:methanol (2:1, v/v) containing butylated hydroxytoluene (0.005%) to prevent ex vivo auto-oxidation. Esterified F3-IsoPs in phospholipids were hydrolyzed to liberate free F3-IsoPs. As noted previously (
      • Roberts II, L.J.
      • Montine T.J.
      • Markesbery W.R.
      • Tapper A.R.
      • Hardy P.
      • Chemtob S.
      • Dettbarn W.D.
      • Morrow J.D.
      ), the addition of various PUFAs including arachidonic acid, EPA, or DHA to tissues during workup does not increase the levels of isoprostane-like compounds in tissue extracts. F3-IsoPs in samples were then purified and analyzed as described above. F2-IsoPs were quantified as described previously (
      • Morrow J.D.
      • Roberts II, L.J.
      ). Fatty acid content in tissues was determined by GC (
      • Morrison W.R.
      • Smith L.M.
      ). For some studies, male Sprague-Dawley rats were fed a rodent AIN-93 diet supplemented with 0.56% EPA. After 8 weeks of feeding, the rats were sacrificed. F3-IsoPs in liver tissues were analyzed by LC/ESI/MS/MS as described above.

      RESULTS

      Formation of F3-IsoPs in Vitro—A representative selective ion monitoring chromatogram obtained from the AAPH-induced oxidation of EPA in vitro is shown in Fig. 2. The chromatographic peak in the lower m/z 573 ion current chromatogram represents the internal standard d4-15-F2t-IsoP. In the upper m/z 567 ion current chromatogram are a series of chromatographic peaks eluting over approximately a 1-min interval. These compounds possess a molecular mass predicted for F3-IsoPs. In addition, these compounds elute at later retention times than the deuterated 15-F2t-IsoP internal standard; it would be predicted that the retention time of F3-IsoPs on GC should be longer than that of F2-IsoPs because F3-IsoPs contain one more double bond. Furthermore, it should be noted that the retention times over which the F3-IsoPs elute may differ somewhat in the different figures because these analyses were performed on different days using different columns that vary somewhat in length.
      Figure thumbnail gr2
      FIGURE 2Selected ion current chromatograms obtained from the GC/MS analysis of F3-IsoPs generated during AAPH-induced oxidation of EPA in vitro. The series of peaks in the m/z 567 ion current chromatogram represent putative F3-IsoPs, and the single peak in the m/z 573 ion current chromatogram represents the d4-15-F2t-IsoP internal standard. Compounds are analyzed as pentafluorobenzyl ester, trimethylsilyl ether derivatives.
      Additional experimental approaches were then performed to provide further evidence that the compounds represented by the chromatographic peaks in the m/z 567 ion current chromatogram are F3-IsoPs. The m/z 566 ion current chromatogram contained no chromatographic peaks, indicating that the peaks in the 567 chromatogram are not natural isotope peaks of compounds generating an ion of less than 567. Analysis of putative F3-IsoPs as [2H9]trimethylsilyl ether derivatives resulted in a shift of the m/z 567 chromatographic peaks up 27 Da to m/z 594, indicating the presence of three hydroxyl groups (Fig. 3). Analysis of the putative F3-IsoPs following catalytic hydrogenation is shown in Fig. 4. Prior to hydrogenation, there were no chromatographic peaks present 6 Da above m/z 567 in the m/z 573 ion current chromatogram (Fig. 4A). However, following hydrogenation, intense chromatographic peaks appeared at m/z 573 with the loss of the chromatographic peaks at m/z 567 (Fig. 4B), indicating the presence of three double bonds. Collectively, these data indicate that the compounds represented by the chromatographic peaks in the m/z 567 ion current chromatogram have the functional groups and the number of double bonds predicted for the F3-IsoPs.
      Figure thumbnail gr3
      FIGURE 3Analysis of putative F3-IsoPsasa[2H9]-trimethylsilyl ether derivative. Before [2H9]trimethylsilyl ether derivatization, intense peaks are present in the m/z 567 ion current chromatogram (A) representing F3-IsoPs. There are no peaks present 27 atomic mass units higher at m/z 594. Following [2H9]trimethylsilyl ether derivatization, intense peaks appear in the m/z 594 ion current chromatogram(B), indicating that compounds representedby the peaks in the m/z 567 ion current chromatogram have three hydroxyl groups. There are no peaks present at m/z 567.
      Figure thumbnail gr4
      FIGURE 4Analysis of putative F3-IsoPs before and after catalytic hydrogenation. Compounds are analyzed as pentafluorobenzyl ester, trimethylsilyl ether derivatives. In the absence of hydrogenation, intense peaks are present in the m/z 567 ion current chromatogram (A) representing F3-IsoPs. There are no peaks present 6 atomic mass units higher at m/z 573. Following catalytic hydrogenation, intense peaks appear at m/z 573 (B), indicating that the m/z 567 compounds have three double bonds.
      Analysis of F3-IsoPs by LC/ESI/MS/MS—To provide direct evidence that the compounds analyzed by selective ion monitoring MS were F3-IsoPs, LC/ESI/MS/MS in the negative ion mode was employed. The predicted [parent molecule, H] ion, hereafter referred to as “M,” for F3-IsoPs is at m/z 351. Direct structural characterization of F3-IsoP regioisomers was obtained utilizing CID. The selective ion monitoring chromatogram of the ion at m/z 351 from this analysis is shown in Fig. 5A. As is evident, multiple chromatographic peaks are present that presumably represent different F3-IsoP stereo-isomers. All of the chromatographic peaks in Fig. 5A were analyzed by CID, and fragmentation patterns were consistent with various F3-IsoP isomers. As a representative sample, the composite CID spectrums at three retention times, including RT 7.82, 11.90, and 16.22 min, are shown in Fig. 5, B–D. CID of the ion at m/z 351 resulted in the formation of a number of relevant daughter ions that would be predicted to be common to all of the F3-IsoP regioisomers, including m/z 315 [M − 2H2O], m/z 307 [M − CO2], m/z 289 [M − H2O − CO2], m/z 271 [M − 2H2O − CO2], and m/z 245 [M − H2O − 2CO2]. Other prominent daughter ions were present that might result from fragmentation of specific F3-IsoP regioisomers. On the basis of our previous work and studies by other groups (
      • Waugh R.J.
      • Morrow J.D.
      • Roberts II, L.J.
      • Murphy R.C.
      ,
      • Kerwin J.L.
      • Torvik J.J.
      ,
      • Kerwin J.L.
      • Wiens A.M.
      • Ericsson L.H.
      ), these ions can be potentially explained as follows. In Fig. 5B (RT 7.82 min), they include m/z 233 [M − CH2 = C(OH)CH2CH3 − CH3CH2OH] and m/z 231 [M − CHO CH2CH3 − H2O − CO2] (18-series), m/z 213 [M − CHOCH2CH = CHCH2CH = CHCH2CH3] and m/z 151 [M − CHOCH2CH = CHCH2CH = CHCH2CH3 − H2O − CO2] (12-series), m/z 191 [M − CHOCH2CH = CHCH2CH3 − H2O − CO2] (15-series), and m/z 167 CH3CH = CHCH2CH = CH(CH2)3COO (11-series). These data suggest that the mass spectrum shown in Fig. 5B represents a mixture of a number of the F3-IsoP regioisomers (11-, 12-, 15-, and 18-series) that would be predicted to be formed. In Fig. 5C (RT 11.90 min), the prominent daughter ion is m/z 127, which is the characteristic fragment of 8-series −CH3CH = CH(CH2)3COO. In Fig. 5D (RT 16.22 min), the major ion m/z 115 is the characteristic fragment of 5-series, which is CHO(CH2)3COO. Taken together, these data provide direct evidence for the formation of a series of F3-IsoPs generated from the peroxidation of EPA.
      Figure thumbnail gr5
      FIGURE 5Analysis of putative F3-IsoPs as a free acid by LC/ESI/MS/MS. A, selected ion monitoring chromatogram of the [parent molecule, H] (M) ion at m/z 351 from LC/ESI/MS analysis of putative F3-IsoPs obtained from AAPH-induced oxidation of EPA in vitro. Peaks that were subjected to CID are denoted by the arrows. The m/z 351 ion was subjected to CID at 25eV, and daughter ions were scanned from 50 to 400. B, CID spectrum obtained by summing scans over the chromatographic peak at retention time 7.82 min. C, CID spectrum obtained by summing scans over the chromatographic peak at retention time 11.90 min. D, CID spectrum obtained by summing scans over the chromatographic peak at retention time 16.22 min.
      The above results utilizing CID were confirmed utilizing SRM. The major unique identifying fragments of EPA-derived F3-IsoP regioisomers predicted to be generated in SRM are shown in Fig. 6. They result primarily from cleavage either α to hydroxyl groups or across double bonds. The SRM chromatograms of putative F3-IsoP regioisomers resulting from the in vitro oxidation of EPA are shown in Fig. 7A. The peaks appearing from 7 to 20 min are putative F3-IsoPs. All six series of F3-IsoP regioisomeric characteristic fragments are detected, suggesting that all of these regioisomers are formed from in vitro oxidation of EPA. In addition, a number of F3-IsoP regioisomers, including 12-, 11-, 15-, and 18-series, elute at RT 7.8 min as denoted in the CID analysis above. However, the chromatographic peaks at RT 11.90 and 16.22 min are primarily comprised of 8- and 5-series regioisomers, respectively. Also of note, overall, the 5- and 18-series of F3-IsoP regioisomers are relatively more abundant than the other regioisomers.
      Figure thumbnail gr6
      FIGURE 6Scheme for SRM fragmentation of different F3-IsoP regioisomers. The major route of fragmentation involves cleavage α to hydroxyl groups or across double bonds.
      Figure thumbnail gr7
      FIGURE 7SRM analysis of putative F3-IsoPs obtained from AAPH-induced oxidation of EPA. A, in vitro; B, from rat livers after 8 weeks of EPA supplementation (0.56%). Please refer to for specific ions being monitored for different F3-IsoP regioisomers.
      Time Course of Formation of F3-IsoP in Vitro—Having provided significant evidence for the formation of F3-IsoPs in vitro, we next examined the time course of their formation. For those studies, EPA was again oxidized using AAPH. The results are shown in Fig. 8. As is evident, levels of F3-IsoPs increased dramatically in a time-dependent manner to a maximum of 8722.7 ± 1010.5 ng/mg of EPA from baseline levels of 29.0 ± 3.7 ng/mg of EPA (n = 6). Levels of F3-IsoPs exceeded those of F2-IsoPs by up to an order of magnitude.
      Figure thumbnail gr8
      FIGURE 8Time course of formation of F3-IsoPs during oxidation of EPA in vitro by AAPH. Data are expressed as means ± S.D. (n = 6).
      Formation of F3-IsoPs in Vivo—We then undertook experiments to determine whether F3-IsoPs are formed in vivo. Levels of EPA in tissues from animals and humans are extremely low at baseline, and F3-IsoP levels are below limits of detection (<30 pg/g of tissue). Thus, to determine whether these compounds are generated in vivo, we supplemented rodents with diets containing EPA (0.84% by weight) for 8 weeks. Subsequently, animals were sacrificed, and tissue lipids were extracted and analyzed for F3-IsoPs. In one set of studies, levels of F3-IsoPs were quantified in hearts from mice supplemented with EPA. Heart tissue was examined because supplementation of animals and humans with fish oil markedly increases levels of EPA in this organ. In addition, fish oil supplementation is associated with a marked decrease in cardiovascular disease. A representative GC/MS ion current chromatogram obtained from one of these analyses is shown in Fig. 9. The chromatographic peak in the lower m/z 573 ion current chromatogram represents the internal standard d4-15-F2t-IsoP. In the upper m/z 567 ion current chromatogram are a series of chromatographic peaks that have a molecular mass and retention times expected for the F3-IsoPs. The pattern of peaks representing F3-IsoPs is very similar to that obtained from the oxidation of EPA in vitro. In addition, as predicted, these compounds were found to contain three hydroxyl groups and three double bonds.
      Figure thumbnail gr9
      FIGURE 9Selected ion current chromatogram obtained from the GC/MS analysis for F3-IsoPs esterified in mouse hearts. The series of peaks in the m/z 567 ion current chromatogram represent putative F3-IsoPs, and the single peak in the m/z 573 ion current chromatogram represents the d4-15-F2t-IsoP internal standard.
      Analogous to LC/ESI/MS/MS studies performed in vitro, experiments were then carried out to obtain further evidence that the chromatographic peaks in the m/z 567 ion current chromatogram of Fig. 9 represent F3-IsoPs formed in vivo. For these studies, liver tissue from rats fed a diet supplemented with 0.56% EPA was utilized to obtain adequate amounts of F3-IsoPs for analysis by LC/MS. In CID experiments, all of daughter ions that would be predicted to be common to all of the F3-IsoP regioisomers, including m/z 333 [M − H2O], m/z 307 [M − CO2], m/z 289 [M − H2O − CO2], m/z 271 [M − 2H2O − CO2], and m/z 245 [M − H2O − 2CO2], were detected. In addition, all 6-series regioisomeric characteristic fragments were formed in both CID (data not shown) and SRM experiments (Fig. 7B). As is evident in Fig. 7B, the relative abundance of different regioisomers in vivo is similar to in vitro studies. Taken together, these experiments provide evidence that F3-IsoPs are formed in abundance in vivo and that the regioisomeric distribution is similar to that observed in vitro.
      We performed studies to examine the effect of different concentrations of EPA on the generation of F3-IsoPs in vivo. Mice were administered 0, 0.28, 0.56, or 0.84% EPA (by weight) in their diets for 8 weeks, and levels of EPA and F3-IsoPs were measured in heart tissue. As shown in Fig. 10, A and B, as the EPA concentration in heart tissues increases, levels of F3-IsoPs increase in a concentration-dependent manner. In addition, we also quantified the effect of EPA supplementation on arachidonate content and F2-IsoP levels in the same heart tissue. Interestingly, supplementation with EPA decreased arachidonate content (Fig. 10C). Importantly, in addition, levels of F2-IsoPs decreased dramatically by up to 64% (p < 0.05) (Fig. 10D), suggesting that EPA effectively decreases levels of proinflammatory F2-IsoPs formed from arachidonate.
      Figure thumbnail gr10
      FIGURE 10Levels of fatty acids and isoprostanes from hearts of mice after 8 weeks of EPA supplementation (0, 0.28, 0.56, 0.84% by weight). A, EPA; B, F3-IsoPs; C, arachidonate; D, F2-IsoPs. Data are expressed as means ± S.D. (n = 5). *, p < 0.05 as compared with control.
      Finally, we also determined the effect of enhanced oxidant stress on endogenous F3-IsoPs in EPA-supplemented (0.56%) mice. Levels of these compounds increased from 86.27 ± 11.01 ng/g of tissue to 311.5 ± 60.79 ng/g of tissue in livers after administration of CCl4 to induce an oxidant stress.

      DISCUSSION

      Our studies have elucidated a novel class of F2-IsoP-like compounds, F3-IsoPs, formed in vivo from the free radical-induced peroxidation of EPA. Previous studies byÄnggård and colleagues (
      • Nourooz-Zadeh J.
      • Halliwell B.
      • Änggård E.
      ) have described the formation of F3-IsoPs in vitro, although little structural information was provided, and it was not determined whether these compounds could be generated in vivo (
      • Morrow J.D.
      • Roberts II, L.J.
      ). Levels of compounds generated significantly exceed those of F2-IsoPs derived from arachidonate, perhaps because EPA contains more double bonds and is more easily oxidized. The present studies describe the formation of F-type prostane ring-containing molecules. Free radical-induced peroxidation of arachidonate results not only in the formation of F-ring IsoPs but also E/D-ring IsoPs, A/J-ring IsoPs, and thromboxane-like molecules (isothromboxanes) (
      • Morrow J.D.
      • Awad J.A.
      • Wu A.
      • Zackert W.E.
      • Daniel V.C.
      • Roberts II, L.J.
      ,
      • Morrow J.D.
      • Roberts L.J.
      ). Thus, although it remains the subject of future studies, it is likely that analogous compounds, in addition to F3-IsoPs, are also formed of products of the non-enzymatic peroxidation of EPA.
      A major impetus for undertaking the present studies regarding EPA oxidation has been the fact that supplementation of humans with fish oil containing large amounts of EPA and DHA has been shown beneficial in the prevention of important human diseases such as atherosclerosis and sudden death, neurodegeneration, and various inflammatory disorders (
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      ,
      • Ruxton C.
      ). Although the mechanism(s) by which these beneficial effects occurs is unknown, a potentially important anti-atherogenic and anti-inflammatory mechanism of ω-3 PUFAs is their interference with the arachidonic acid cascade that generates pro-inflammatory eicosanoids (
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      ,
      • Uauy R.
      • Mena P.
      • Valenzuela A.
      ). EPA not only can replace arachidonic acid in phospholipid bilayers but is also a competitive inhibitor of cyclooxygenase, reducing the production of 2-series PGs and thromboxane, in addition to the 4-series leukotrienes. As noted, the 3- and 5-series eicosanoids (respectively) derived from EPA are either less biologically active or inactive as compared with the former products and are thus considered to exert effects that are less inflammatory (
      • Calder P.C.
      ,
      • Yang P.
      • Chan D.
      • Felix E.
      • Cartwright C.
      • Menter D.G.
      • Madden T.
      • Klein R.D.
      • Fischer S.M.
      • Newman R.A.
      ). Further, Serhan and colleagues (
      • Hong S.
      • Gronert K.
      • Devchand P.R.
      • Moussignac R.L.
      • Serhan C.N.
      ,
      • Serhan C.N.
      • Levy B.
      ,
      • Serhan C.N.
      • Clish C.B.
      • Brannon J.
      • Colgan S.P.
      • Chiang N.
      • Gronert K.
      ,
      • Serhan C.N.
      • Hong S.
      • Gronert K.
      • Colgan S.P.
      • Devchand P.R.
      • Mirick G.
      • Moussignac R.L.
      ) have described a group of polyoxygenated DHA and EPA derivatives termed resolvins that are produced in various tissues. These compounds inhibit cytokine expression and other inflammatory responses in microglia, skin cells, and other cell types.
      Recently, there has been significant interest in the biological activities of non-enzymatic free radical-initiated peroxidation products of ω-3 PUFAs. Sethi et al. (
      • Sethi S.
      • Eastman A.Y.
      • Eaton J.W.
      ,
      • Sethi S.
      ) reported that EPA oxidized in the presence of Cu2+, but not native EPA, significantly inhibits human neutrophil and monocyte adhesion to endothelial cells, a process linked to the development of atherosclerosis and other inflammatory disorders. This effect was induced via inhibition of endothelial adhesion receptor expression and was modulated by the activation of the peroxisome proliferator-activated receptor-α by EPA oxidation products. In addition, oxidized EPA markedly reduced leukocyte rolling and adhesion to venular endothelium of lipopolysaccharide-treated mice in vivo, and the effect was not observed in peroxisome proliferator-activated receptor-α-deficient mice. These studies suggest that the beneficial effects of ω-3 fatty acids may be mediated, in part, by the anti-inflammatory effects of oxidized EPA. Similarly, Vallve et al. (
      • Vallve J.C.
      • Uliaque K.
      • Girona J.
      • Cabre A.
      • Ribalta J.
      • Heras M.
      • Masana L.
      ) have shown that various non-enzymatically generated aldehyde oxidation products of EPA and DHA decrease the expression of the CD36 receptor in human macrophages. Up-regulation of this receptor has been linked to atherosclerosis. Additional recent reports have suggested that other related biological effects of EPA and DHA, such as modulation of endothelial inflammatory molecules, are related to their peroxidation products (
      • Sethi S.
      • Eastman A.Y.
      • Eaton J.W.
      ,
      • Das U.N.
      ). Arita et al. (
      • Arita K.
      • Yamamoto Y.
      • Takehara Y.
      • Utsumi T.
      • Kanno T.
      • Miyaguchi C.
      • Akiyama J.
      • Yoshioka T.
      • Utsumi K.
      ) have also shown that non-enzymatically oxidized EPA enhances apoptosis in HL-60 leukemia cells, supporting the contention that oxidized ω-3 PUFAs are both anti-proliferative and anti-inflammatory. Similar findings have been reported in HepG2 (human hepatoma) cells and AH109A (rat liver cancer) cells (
      • Kiserud C.E.
      • Kierulf P.
      • Hostmark A.T.
      ,
      • Kokura S.
      • Nakagawa S.
      • Hara T.
      • Boku Y.
      • Naito Y.
      • Yoshida N.
      • Yoshikawa T.
      ). In virtually none of these reports, however, have the specific peroxidation products responsible for these effects been structurally identified. Further studies to characterize these molecules are needed. Thus, a rationale for undertaking these studies was based on the hypothesis that F3-IsoPs are formed from the peroxidation of EPA and contribute to the beneficial biological effects of EPA and fish oil supplementation in that they exert biological activities that are anti-inflammatory. Studies assessing the biological activity of 15-F3t-IsoP could test this important hypothesis but will have to await the chemical synthesis of the molecule.
      The studies reported herein have begun to systematically define the oxidation of EPA in vivo and in vitro for the first time. F3-IsoPs were identified using a variety of complementary chemical and MS approaches, including LC/ESI/MS/MS. As expected, 6-series of F3-IsoP regioisomers were identified from both in vitro and in vivo sources. The fragmentation patterns of these regioisomers are similar to F2-IsoP regioisomers, and indeed, information that we have previously acquired with F2-IsoPs was extremely useful in the present studies (
      • Waugh R.J.
      • Morrow J.D.
      • Roberts II, L.J.
      • Murphy R.C.
      ). Of note, the relative abundance of 5- and 18-series F3-IsoPs predominates over the other series. Such regioisomeric predominance has also been reported for F2-IsoP regioisomers in which 5- and 15-series compounds are formed in greater abundance than 8- and 12-series molecules (
      • Waugh R.J.
      • Morrow J.D.
      • Roberts II, L.J.
      • Murphy R.C.
      ). At least part of the reason for this is likely due to the fact that precursors of 8- and 12-series F2-IsoPs can undergo further oxidation and cyclization to yield a novel class of compounds termed dioxolane endoperoxides (
      • Yin H.
      • Morrow J.D.
      • Porter N.A.
      ). Although undetermined at present, it is likely that a similar mechanism may account for the predominance of 5- and 18-series F3-IsoPs.
      Another interesting finding from the above studies relates to the observation that EPA reduces the formation of F2-IsoPs in vivo. As mentioned, F2-IsoPs are generally considered to be pro-inflammatory molecules and have been implicated in the pathophysiological consequences of oxidative stress. It is thus intriguing to propose that part of the mechanism by which EPA prevents certain diseases associated with increased inflammation relates to its ability to decrease F2-IsoP generation.
      In summary, we report the discovery that F-ring IsoP-like compounds, termed F3-IsoPs, are formed in vivo in large amounts as products of the non-enzymatic free radical-catalyzed peroxidation of EPA, a major long chain polyunsaturated fatty acid in fish oil. Further understanding of the biological consequences of the formation of these novel compounds and factors influencing their formation and metabolism may provide valuable insights into the role of EPA in human physiology and pathophysiology.

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