FORMATION OF F-RING ISPOPROSTANE-LIKE COMPOUNDS (F 3 -ISOPROSTANES) IN VIVO FROM EICOSAPENTAENOIC ACID

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1 JBC Papers in Press. Published on March 28, 26 as Manuscript M61352 The latest version is at FORMATION OF F-RING ISPOPROSTANE-LIKE COMPOUNDS (F 3 -ISOPROSTANES) IN VIVO FROM EICOSAPENTAENOIC ACID Ling Gao, Huiyong Yin, Ginger L. Milne, Ned A. Porter#, and Jason D. Morrow From the Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, and #Department of Chemistry, Vanderbilt University, Nashville, Tennessee Running Title: Characterization of Eicosapentaenoic Acid Eicosanoids Address correspondence to: Jason D. Morrow, Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee , Tel ; Fax ; jason.morrow@vanderbilt.edu Eicosapentaenoic acid (EPA, C2:5, 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 radicalgenerated 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 (PG) F 2 -like compounds, termed F 2 -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 F 2 -IsoP-like compounds (F 3 -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 F 3 -IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to µg/mg EPA) and greater than levels of F 2 -IsoPs generated from arachidonic acid. We then examined the formation of F 3 -IsoPs in vivo in mice. Levels of F 3 -IsoPs in tissues such as heart are virtually undetectable at baseline but supplementation of animals with EPA markedly increases quantities up to ng/g heart. Interestingly, EPA supplementation also markedly reduced levels of proinflammatory arachidonate-derived F 2 -IsoPs by up to 64% (p <.5). Our studies provide the first evidence that identify F 3 -IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F 3 -IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA. Isoprostanes (IsoPs) 1 are prostaglandin (PG)-like compounds that are formed nonenzymatically from the free radical-induced oxidation of arachidonic acid. Formation of these compounds proceeds through the generation of bicyclic endoperoxide PGH 2 -like intermediates, that are reduced to PGF 2 -like compounds termed F 2 -IsoPs (1) or undergo rearrangement to PGD 2 and PGE 2 -like compounds (D 2 /E 2 -IsoPs) (2) and thromboxane-like compounds (isothromboxanes) (3). Unlike cyclooxygenase-derived PGs, IsoPs are formed in situ esterified to phospholipids and are subsequently released, presumably by the action of a phospholipase A 2 (PLA 2 ) (4). Quantification of F 2 -IsoPs has been shown to be one of the most reliable markers to assess oxidant stress status and lipid peroxidation in vivo (5-7). In addition, F 2 - IsoPs have been shown to exert potent vasoconstrictive activities in the vasculature (8,9), suggesting a pathogenic role for these compounds in diseases such as atherosclerosis (1,11). Eicosapentaenoic acid (C2:5 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 1 Copyright 26 by The American Society for Biochemistry and Molecular Biology, Inc.

2 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 (12,13). 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 antiinflammatory activities (14-19). Previously, Anggard and colleagues (2) provided limited evidence that F-ring IsoP-like compounds (F 3 -IsoPs) can be formed from the oxidation of EPA in vitro. These compounds are termed F 3 -IsoPs because they are structurally analogous to F 2 -IsoPs yet contain 3 double bonds. We therefore considered the possibility that IsoPlike 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 F 3 -IsoPs contribute to the beneficial biological effects of EPA and fish oil supplementation in that they may exert biological activities that are antiinflammatory compared to F 2 -IsoPs. Indeed, one report states that the EPA-derived IsoP, 15-F 3t -IsoP, possesses activity that is different from 15-F 2t -IsoP in that it does not affect human platelet shape change or aggregation (21). 15-F 2t -IsoP is a ligand for the Tx receptor and induces platelet shape change and also causes vasoconstriction (1,22). The lack of activity of 15-F 3t -IsoP is consistent with observations regarding EPA-derived PGs in that these latter compounds exert either weaker agonist or no effects in comparison to arachidonatederived PGs (12,23,24). Based on studies of arachidonic acid oxidation, the mechanism by which F 3 -IsoPs could be formed is outlined in Figure 1. There are four bis-allylic positions in EPA at carbons 7, 1, 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 (HpEPEs) are formed. These eight HpEPEs subsequently generate six F 3 -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 (25). Thus, in accordance with this nomenclature system, the F 3 -IsoP regioisomers are designated as 5-, 8-, 11-, 12-, 15-, and 18-series F 3 -IsoPs. Herein, we present evidence that F 3 - IsoPs are, in fact, formed in significant amounts in vitro and in vivo from the free radical-catalyzed peroxidation of EPA. EXPERIMENTAL PROCEDURES Materials. Eicosapentaenoic acid and d 4-15-F 2t -IsoP (8-iso-PGF 2α ) were purchased from Cayman Chemical Co. (Ann Arbor, MI). Dimethylformamide and undecane were obtained from Aldrich (Milwaukee, WI). Pentafluorobenzyl bromide and diisopropylethylamine were from Sigma (St. Louis, MO). 2,2 -Azobis(2- amidinopropane) hydrochloride (AAPH) was from Kodak (Rochester, NY). N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) was from Supelco (Bellefonte, PA). [ 2 H 9 ] N, O-Bis (trimethylsilyl) acetamide was from CDN Isotopes (Pointe-Claire, PQ). C-18 and silica Sep-Pak cartridges were from Waters Associatiates (Milford, MA). 6ALK6D TLC plates were from Whatman (Maidstone, UK). Oxidation of EPA. Five milligram of fresh EPA was dissolved in 1µl ethanol and added immediately to 4.9 ml of phosphate-buffered saline solution (ph 7.4) containing 1 mm AAPH. The EPA oxidation reaction mixture was incubated in a shaking water bath at 37ºC for varying amounts of time, after which it was placed immediately at 8ºC until further processing. Purification and analysis of F 3 -IsoPs. Free and esterified F 3 -IsoPs were extracted using C-18 and Silica Sep-Pak cartridges, converted to a pentafluorobenzyl ester, purified by thin layer chromatography (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 d 4-15-F 2t -IsoP as an internal standard using a modification of the method described for the quantification of F 2 - IsoPs (26). Instead of scraping 1 cm below to 1 cm above where PGF 2α methyl ester migrates on TLC 2

3 for analysis of F 2 -IsoPs, the scraped area was extended to 3.5 cm above where the PGF 2α methyl ester migrates. This extended area of the TLC plate was determined to contain F 3 -IsoPs by analyzing small 5-mm cuts using approaches for their identification described below. The M CH 2 C 6 F 5 ions were monitored for quantification (m/z 567 for F 3 -IsoPs and m/z 573 for d 4-15-F 2t - IsoP). Quantification of the total amount of F 3 - IsoPs was determined by integrating the peak area of material in the m/z 567 channel in comparison to the m/z 573 channel. GC/NICI/MS was carried out using an Agilent Technologies 689N Network GC/MS system. Sample work using this method does not lead to the generation of F 3 -IsoPs during purification and derivatization. In some studies, F 2 -IsoPs were quantified as previously described (5). Analysis of F 3 -IsoPs by Liquid Chromatography (LC)/Electrospray Ionization (ESI)/MS/MS. F 3 -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 HPLC separation using a Supelco Discovery C 18 column (15 cm x 2.1 mm, 5µ) at a flow rate of.2 ml/min with a linear gradient starting with 8% solvent A (2mM ammonium acetate) to 65% in 2 min. Mobile phase B consisted of acetonitrile/methanol (95:5). The ESI source was fitted with a deactivated fused silica capillary (1µm i.d.). Nitrogen was used as both the sheath gas and the auxiliary gas, at 45 and 17psi, respectively. The capillary temperature was 28 ºC. The spray voltage was 4.3 kv, and the tube lens voltage was 8V. Collision-induced dissociation (CID) of the molecular ion of putative F 3 -IsoPs was performed from 2 to 3 ev under 1.5 mtorr 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 F 2 -IsoPs (27). The collision energy for SRM was 3 ev. LC/MS was carried out using a ThermoFinnigan TSQ Quantum 1. SR 1 mass spectrometer. Data acquisition and analysis were performed using Xcaliber software, version 1.3. Preparation of F 3 -IsoPs from Rodent Tissue. Mice (strain C57BL/6J(B6)) were fed with a rodent AIN-93 diet supplemented with either %,.28%,.56% and.84% EPA (by weight). In some studies, animals were administered CCl 4 (1ml/kg) intraperitoneally to induce an oxidant stress. After 8 weeks feeding, the mice were sacrificed. Heart and other tissues were removed and immediately flash frozen in liquid nitrogen and stored in 8 C. Analyzed tissue samples were homogenized in 5ml of ice-cold chloroform: methanol (2:1, v/v) containing butylated hydroxytoluene (.5%) to prevent ex vivo autooxidation. Esterified F 3 -IsoPs in phospholipids were hydrolyzed to liberate free F 3 -IsoPs. As previously noted (28), the addition of various PUFAs including AA, EPA, or DHA to tissues during workup does not increase levels of isoprostane-like compounds in tissue extracts. F 3 - IsoPs in samples were then purified and analyzed as described above. F 2 -IsoPs were quantified as previously described (5). Fatty acid content in tissues was determined by GC (29). For some studies, male Sprague-Dawley rats were fed a rodent AIN-93 diet supplemented with.56% EPA. After 8 weeks feeding, the rats were sacrificed. F 3 -IsoPs in liver tissues were analyzed by LC/ESI/MS/MS as described above. RESULTS Formation of F 3 -IsoPs in vitro. A representative selective ion monitoring (SIM) chromatogram obtained from the AAPH-induced oxidation of EPA in vitro is shown in Figure 2. The chromatographic peak in the lower m/z 573 ion current chromatogram represents the internal standard d 4-15-F 2t -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 F 3 -IsoPs. In addition, these compounds elute at later retention times than the deuterated 15-F 2t -IsoP internal standard; it would be predicted that the retention time of F 3 - IsoPs on GC should be longer than that of F 2 -IsoPs because F 3 -IsoPs contain one more double bond. Furthermore, it should be noted that the retention times over which the F 3 -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. 3

4 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 F 3 -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 F 3 -IsoPs as [ 2 H 9 ]- 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 (Figure 3). Analysis of the putative F 3 - IsoPs following catalytic hydrogenation is shown in Figure 4. Prior to hydrogenation, there were no chromatographic peaks present 6 Da above m/z 567 in the m/z 573 ion current chromatogram (Figure 4A). However, following hydrogenation, intense chromatographic peaks appeared at m/z 573 with the loss of the chromatographic peaks at m/z 567 (Figure 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 F 3 - IsoPs. Analysis of F 3 -IsoPs by LC/ESI/MS/MS. To provide direct evidence that the compounds analyzed by SIM MS were F 3 -IsoPs, LC/ESI/MS/MS in the negative ion mode was employed. The predicted [parent molecule H] - ion, hereafter referred to as M -, for F 3 -IsoPs is at m/z 351. Direct structural characterization of F 3 - IsoP regioisomers was obtained utilizing CID. The SIM chromatogram of the ion at m/z 351 from this analysis is shown in Figure 5A. As is evident, multiple chromatographic peaks are present that presumably represent different F 3 -IsoP stereoisomers. All of the chromatographic peaks in Figure 5A were analyzed by CID and fragmentation patterns were consistent with various F 3 -IsoP isomers. As a representative sample, the composite CID spectrums at three retention times, including RT 7.82, 11.9 and min, are shown in Figure 5B-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 F 3 -IsoP regioisomers, including m/z 315 [M 2H 2 O] -, m/z 37 [M CO 2 ] -, m/z 289 [M H 2 O CO 2 ] -, m/z 271 [M 2H 2 O CO 2 ] -, and m/z 245 [M H 2 O 2CO 2 ] -. Other prominent daughter ions were present that might result from fragmentation of specific F 3 -IsoP regioisomers. On the basis of our previous work and studies by other groups (27,3,31), these ions can be potentially explained as follows. In Figure 5B(RT 7.82 min), they include m/z 233 [M CH 2 = C()CH 2 CH 3 CH 3 CH 2 ] - and m/z 231 [M C CH 2 CH 3 H 2 O CO 2 ] - (18-series), m/z 213 [M CCH 2 CH= CHCH 2 CH= CHCH 2 CH 3 ] - and m/z 151 [M CCH 2 CH= CHCH 2 CH= CHCH 2 CH 3 H 2 O CO 2 ] - (12-series), m/z 191 [M CCH 2 CH= CHCH 2 CH 3 H 2 O CO 2 ] - (15- series) and m/z 167 CH 3 CH= CHCH 2 CH= CH(CH 2 ) 3 COO - (11- series). These data suggest that the mass spectrum shown in Figure 5B represents a mixture of a number of the F 3 -IsoP regioisomers (11-, 12-, 15-, and 18-series) that would be predicted to be formed. In Figure 5C (RT 11.9 min), the prominent daughter ion is m/z 127, which is the characteristic fragment of 8- series ----CH 3 CH= CH(CH 2 ) 3 COO -. In Figure 5D (RT min), the major ion m/z 115 is the characteristic fragment of 5-series, which is C(CH 2 ) 3 COO -. Taken together, these data provide direct evidence for the formation of a series of F 3 -IsoPs generated from the peroxidation of EPA. The above results utilizing CID were confirmed utilizing SRM. The major unique identifying fragments of EPA-derived F 3 -IsoP regioisomers predicted to be generated in SRM are shown in Figure 6. They result primarily from cleavage either alpha to hydroxyl groups or across double bonds. The SRM chromatograms of putative F 3 -IsoP regioisomers resulting from the in vitro oxidation of EPA are shown in Figure 7A. The peaks appearing from 7-2 min are putative F 3 -IsoPs. All six series of F 3 -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 F 3 -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 11.9 and min are primarily comprised of 8- and 5-series regioisomers respectively. Also of note, overall, the 5- and 18- series of F 3 -IsoP regioisomers are 4

5 relatively more abundant than the other regioisomers. Time course of formation of F 3 -IsoP in vitro. Having provided significant evidence for the formation of F 3 -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 Figure 8. As is evident, levels of F 3 -IsoPs increased dramatically in a timedependent manner to a maximum of ± 11.5 ng/mg of EPA from baseline levels of 29.± 3.7 ng/mg of EPA (n= 6). Levels of F 3 -IsoPs exceed those of F 2 -IsoPs by up to an order of magnitude. Formation of F 3 -IsoPs in Vivo. We then undertook experiments to determine whether F 3 - IsoPs are formed in vivo. Levels of EPA in tissues from animals and humans are extremely low at baseline and F 3 -IsoP levels are below limits of detection (< 3 pg/g tissue). Thus, in order to determine whether these compounds are generated in vivo, we supplemented rodents with diets containing EPA (.84% by weight) for 8 weeks. Subsequently, animals were sacrificed and tissue lipids extracted and analyzed for F 3 -IsoPs. In one set of studies, levels of F 3 -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 Figure 9. The chromatographic peak in the lower m/z 573 ion current chromatogram represents the internal standard d 4-15-F 2t -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 F 3 -IsoPs. The pattern of peaks representing F 3 -IsoPs is very similar to that obtained from the oxidation of EPA in vitro. In addition, as predicted, these compounds were found to contain 3 hydroxyl groups and 3 double bonds. 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 Figure 9 represent F 3 -IsoPs formed in vivo. For these studies, liver tissue from rats fed a diet supplemented with.56% EPA was utilized to obtain adequate amounts of F 3 -IsoPs for analysis by LC/MS. In CID experiments, all of daughter ions that would be predicted to be common to all of the F 3 -IsoP regioisomers, including m/z 333 [M H 2 O] -, m/z 37 [M CO 2 ] -, m/z 289 [M H 2 O CO 2 ] -, m/z 271 [M 2H 2 O CO 2 ] -, and m/z 245 [M H 2 O 2CO 2 ] - were detected. In addition, all 6 series regioisomeric characteristic fragements were formed in both CID (data not shown) and SRM experiments (Figure 7B). As is evident in Figure 7B, the relative abundance of different regioisomers in vivo is similar to in vitro studies. Taken together, these experiments provide evidence that F 3 -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 F 3 -IsoPs in vivo. Mice were administered either,.28,.56, or.84% EPA (by weight) in their diets for 8 weeks and levels of EPA and F 3 -IsoPs measured in heart tissue. As shown in Figure 1A and B, as the EPA concentration in heart tissues increases, levels of F 3 -IsoPs increase in a concentration dependent manner. In addition we also quantified the effect of EPA supplementation on arachidonate content and F 2 -IsoP levels in the same heart tissue. Interestingly, supplementation with EPA decreased arachidonate content (Figure 1C). Importantly also, levels of F 2 -IsoPs decreased dramatically by up to 64% (p <.5) (Figure 1D), suggesting that EPA effectively decreases levels of proinflammatory F 2 -IsoPs formed from arachidonate. Finally, we also determined the effect of enhanced oxidant stress on endogenous F 3 -IsoPs in EPA supplemented (.56%) mice. Levels of these compounds increased from 86.27±11.1 ng/g tissue to 311.5±6.79 ng/g tissue in livers after administration of CCl 4 to induce an oxidant stress. DISCUSSION Our studies have elucidated a novel class of F 2 -IsoP-like compounds, F 3 -IsoPs, formed in vivo from the free radical-induced peroxidation of EPA. Previous studies by Anggard and colleagues have described the formation of F 3 -IsoPs in vitro although little structural information was provided 5

6 and it was not determined whether these compounds could be generated in vivo (5,2). Levels of compounds generated significantly exceed those of F 2 -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 radicalinduced peroxidation of arachidonate results not only in the formation of F-ring IsoPs but also E/Dring IsoPs, A/J-ring IsoPs and Tx-like molecules (isothromboxanes) (3,32). Thus, although it remains the subject of future studies, it is likely that analogous compounds, in addition to F 3 -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 (12,13). Although the mechanism(s) by which these beneficial effects occurs is unknown, a potentially important antiatherogenic and anti-inflammatory mechanism of ω-3 PUFAs is their interference with the arachidonic acid cascade that generates proinflammatory eicosanoids (12,33). EPA not only can replace arachidonic acid in phospholipid bilayers but is also a competitive inhibitor of COX, 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 compared to the former products and are thus considered to exert effects that are less inflammatory (34,35). Further, Serhan and colleagues 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 (14-17). Recently, there has been significant interest in the biological activities of non-enzymatic free radical-initiated peroxidation products of ω-3 PUFAs. Sethi and colleagues reported that EPA oxidized in the presence of Cu ++, 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 (18,19). This effect was induced via inhibition of endothelial adhesion receptor expression and was modulated by the activation of the peroxisome proliferator-activated receptor-α (PPAR-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 PPAR-αdeficient 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 and colleagues have shown that various non-enzymatically generated aldehyde oxidation products of EPA and DHA decrease the expression of the CD36 receptor in human macrophages (36). Upregulation 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 (18,37). Arita and colleagues have also shown that nonenzymatically oxidized EPA enhances apoptosis in HL-6 leukemia cells supporting the contention that oxidized ω-3 PUFAs are both antiproliferative and anti-inflammatory (38). Similar findings have been reported in HepG2 (human hepatoma) cells and AH19A (rat liver cancer) cells (39,4). 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 F 3 -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-F 3t -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. F 3 -IsoPs were identified using a variety of complementary 6

7 chemical and MS approaches, including LC/ESI/MS/MS. As expected, 6 series of F 3 -IsoP regioisomers were identified from both in vitro and in vivo sources. The fragmentation patterns of these regioisomers are similar to F 2 -IsoP regioisomers, and indeed, information that we have previously acquired with F 2 -IsoPs was extremely useful in the present studies (27). Of note, the relative abundance of 5- and 18-series F 3 -IsoPs predominates over the other series. Such regioisomeric predominance has also been reported for F 2 -IsoP regioisomers in which 5- and 15-series compounds are formed in greater abundance than 8- and 12-series molecules (27). At least part of the reason for this is likely due to the fact that precursors of 8- and 12-series F 2 - IsoPs can undergo further oxidation and cyclization to yield a novel class of compounds termed dioxolane-endoperoxides (41). Although undetermined at present, it is likely that a similar mechanism may account for the predominance of 5- and 18-series F 3 -IsoPs. Another interesting finding from the above studies relates to the observation that EPA reduces the formation of F 2 -IsoPs in vivo. As mentioned, F 2 -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 F 2 - IsoP generation. In summary, we report the discovery that F-ring IsoP-like compounds, termed F 3 -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. REFERENCES 1. Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F., and Roberts LJ, I. I. (199) PNAS 87, Morrow, J. D., Minton, T. A., Mukundan, C. R., Campbell, M. D., Zackert, W. E., Daniel, V. C., Badr, K. F., Blair, I. A., and Roberts, L. J. (1994) J.Biol.Chem. 269, Morrow, J. D., Awad, J. A., Wu, A., Zackert, W. E., Daniel, V. C., and Roberts, L. J., II (1996) J.Biol.Chem. 271, Morrow, J. D., Awad, J. A., Boss, H. J., Blair, I. A., and Roberts LJ, I. I. (1992) PNAS 89, Morrow JD and Roberts II, L. J. (22) Methods Mol Biol 186, Morrow JD (2) Drug Metab Rev 32, RobertsII, L. J. and Morrow, J. D. (2) Free Radical Biology and Medicine 28, Kromer, B. M. and Tippins, J. R. (1996) Br J Pharmacol 119, Cracowski, J. L., Stanke-Labesque, F., Devillier, P., Chavanon, O., Hunt, M., Souvignet, C., and Bessard, G. (2) Eur J Pharmacol 397, Cracowski JL, Devillier P, Durand T, Stanke-Labesque F, and Bessard G (21) J Vasc Res 38, Habib, A. and Badr, K. F. (24) Chem Phys Lipids 128, Kris-Etherton, P. M., Harris, W. S., Appel, L. J., and for the Nutrition Committee (22) Circulation 16, Ruxton C (24) Nurs Stand 18, Hong, S., Gronert, K., Devchand, P. R., Moussignac, R. L., and Serhan, C. N. (23) J.Biol.Chem. 278, Serhan, C. N. and Levy, B. (23) Chem Immunol.Allergy 83, Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2) J.Exp.Med. 192,

8 17. Serhan, C. N., Hong, S., Gronert, K., Colgan, S. P., Devchand, P. R., Mirick, G., and Moussignac, R. L. (22) J.Exp.Med. 196, Sethi, S., Eastman, A. Y., and Eaton, J. W. (1996) J Lab Clin Med 128, Sethi, S. (22) Redox Rep. 7, Nourooz-Zadeh, J., Halliwell, B., and anggard, E. (1997) Biochem Biophys Res Commun 236, Pratico, D., Smyth, E. M., Violi, F., and FitzGerald, G. A. (1996) J.Biol.Chem. 271, Morrow, J. D., Minton, T. A., and Roberts, I. I. (1992) Prostaglandins 44, Balapure, A. K., Rexroad, J., Kawada, K., Watt, D. S., and Fitz, T. A. (1989) Biochemical Pharmacology 38, Kulkarni, P. S. and Srinivasan, B. D. (1985) Invest.Ophthalmol.Vis.Sci. 26, Taber DF, Morrow JD, and Roberts II, L. J. (1997) Prostaglandins 53, Morrow, J. D. and Roberts II, L. J. (1999) Methods Enzymol 3, Waugh, R. J., Morrow, J. D., Roberts II, L. J., and Murphy, R. C. (1997) Free Radical Biology and Medicine 23, Roberts II, L. J., Montine, T. J., Markesbery, W. R., Tapper, A. R., Hardy, P., Chemtob, S., Dettbarn, W. D., and Morrow, J. D. (1998) J.Biol.Chem. 273, Morrison WR and Smith LM (1964) J Lipid Res 53, Kerwin, J. L. and Torvik, J. J. (1996) Analytical Biochemistry 237, Kerwin, J. L., Wiens AM, and Ericsson LH (1996) J Mass Spectrom 31, Morrow, J. D. and Roberts, L. J. (1997) Prog Lipid Res 36, Uauy, R., Mena, P., and Valenzuela, A. (1999) Eur J Clin Nutr 53 Suppl 1, S66-S Calder PC (21) Lipids 36, Yang, P., Chan, D., Felix, E., Cartwright, C., Menter, D. G., Madden, T., Klein, R. D., Fischer, S. M., and Newman, R. A. (24) J.Lipid Res. 45, Vallve, J. C., Uliaque, K., Girona, J., Cabre, A., Ribalta, J., Heras, M., and Masana, L. (22) Atherosclerosis 164, Das, U. N. (1999) Prostaglandins, Leukotrienes and Essential Fatty Acids 61, Arita, K., Yamamoto, Y., Takehara, Y., Utsumi, T., Kanno, T., Miyaguchi, C., Akiyama, J., Yoshioka, T., and Utsumi, K. (23) Free Radical Biology and Medicine 35, Kiserud, C. E., Kierulf, P., and Hostmark, A. T. (1995) Thrombosis Research 8, Kokura, S., Nakagawa, S., Hara, T., Boku, Y., Naito, Y., Yoshida, N., and Yoshikawa, T. (22) Cancer Letters 185, Yin, H., Morrow, J. D., and Porter, N. A. (24) J.Biol.Chem. 279, FOOTNOTES This work is supported by NIH Grants GM15431, ES31125, RR96, DK48831, and CA The abbreviations used are: IsoP, isoprostane; PG, prostaglandin; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acid; COX, cyclooxygenase; Tx, thromboxane; AAPH, 2,2 -azobis(2- amidinopropane) hydrochloride; GC, gas chromatography; NICI, negative ion chemical ionization; MS, mass spectrometry; TLC, thin-layer chromatography; LC, liquid chromatography; ESI, electrospray ionization; CID, collision-induced dissociation; SIM, selective ion monitoring; SRM, selective reaction monitoring. 8

9 FIGURE LEGENDS Figure 1. Pathway for the formation of F 3 -IsoPs by non-enzymatic peroxidation of EPA (A-B). Figure 2. Selected ion current chromatograms obtained from the GC/MS analysis of F 3 -IsoPs generated during AAPH-induced oxidation of EPA in vitro. The series of peaks in the m/z 567 ion current chromatogram represent putative F 3 -IsoPs, and the single peak in the m/z 573 ion current chromatogram represents the d 4-15-F 2t -IsoP internal standard. Compounds are analyzed as pentafluorobenzyl (PFB), trimethylsilyl (TMS) derivatives. Figure 3. Analysis of putative F 3 -IsoPs as a [ 2 H 9 ] trimethylsilyl ether derivative. Before [ 2 H 9 ] trimethylsilyl ether derivatization, intense peaks are present in the m/z 567 ion current chromatogram (A) representing F 3 -IsoPs. There are no peaks present 27 atomic mass units higher at m/z 594. Following [ 2 H 9 ] trimethylsilyl ether derivatization, intense peaks appear in the m/z 594 ion current chromatogram (B), indicating that compounds represented by the peaks in the m/z 567 ion current chromatogram have three hydroxyl groups. There are no peaks present at m/z 567. Figure 4. Analysis of putative F 3 -IsoPs before and after catalytic hydrogenation. Compounds are analyzed as pentafluorobenzyl (PFB), trimethylsilyl (TMS) derivatives. In the absence of hydrogenation, intense peaks are present in the m/z 567 ion current chromatogram (A) representing F 3 -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. Figure 5. Analysis of putative F 3 -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 F 3 - 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 5 to 4. (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 11.9 min. (D) CID spectrum obtained by summing scans over the chromatographic peak at retention time min. Figure 6. Scheme for SRM fragmentation of different F 3 -IsoP regioisomers. The major route of fragmentation involves cleavage alpha to hydroxyl groups or across double bonds. Figure 7. SRM analysis of putative F 3 -IsoPs obtained from AAPH-induced oxidation of EPA. In vitro. (A); from rat livers after 8 weeks of EPA supplementation (.56%) (B). Please refer to Figure 6 for specific ions being monitored for different F 3 -IsoP regioisomers. Figure 8. Time course of formation of F 3 -IsoPs during oxidation of EPA in vitro by AAPH. Data are expressed as means ± the standard deviation of the mean (n= 6). Figure 9. Selected ion current chromatogram obtained from the GC/MS analysis for F 3 -IsoPs esterified in mouse hearts. The series of peaks in the m/z 567 ion current chromatogram represent putative F 3 -IsoPs, and the single peak in the m/z 573 ion current chromatogram represents the d 4-15-F 2t -IsoP internal standard. Figure 1. Levels of fatty acids and isoprostanes from hearts of mice after 8 weeks of EPA supplementation (,.28,.56,.84% by weight (A) EPA. (B) F 3 -IsoPs. (C) arachidonate. (D) F 2 -IsoPs. Data are expressed as means ± the standard deviation of the mean (n= 5). p<.5 compared to control. 9

10 Figure 1 A CO EPA CO CO O O CO O O CO O O CO 5 CO 8 CO CO 12 5-series 8-series 12-series B CO EPA CO CO CO CO CO O O O O O O 11 CO 15 CO 18 CO 11-series 15-series 18-series 1

11 Figure 2 RELATIVE INTENSITY 5 24 m/z 567 F 3 -IsoPs m/z 573 d 4-15-F 2t -IsoP RETENTION TIME (MIN) 11

12 Figure 3 8 A 5 B m/z 567 m/z 567 RELATIVE INTENSITY 8 m/z m/z RETENTION TIME (MIN) 12

13 Figure 4 5 A 3 B m/z 567 m/z 567 RELATIVE INTENSITY 5 m/z m/z RETENTION TIME (MIN) 13

14 Figure Relative Abundance Relative Abundance Time (min) RT 7.82min [M-H 2 O-2CO 2 ] - [M-2H 2 O-CO 2 ] B [M-CO 8 2 ] M [M-H 2 O-CO 2 ] [M-2H O] m/z A Relative Abundance Relative Abundance RT 11.9min RT 16.22min [M-H 2 O-2CO 2 ] - C 245 M m/z [M-H 2 O-2CO 2 ] - D 271 M m/z 245 [M-2H 2 O-CO 2 ] - [M-CO 2 ] - 14

15 Figure 6 SRM 5-series 5 (CH 2 ) 3 CO series 8 (CH 2 ) 3 CO series 11 (CH 2 ) 3 CO series (CH 2 ) 3 CO series 15 (CH 2 ) 3 CO series 18 (CH 2 ) 3 CO

16 Figure series F 3 -IsoP A NL: 4.31E4 1 5-series F 3 -IsoP B NL: 2.26E series F 3 -IsoP NL: 4.32E series F 3 -IsoP NL: 2.5E4 Relative Abundance series F 3 -IsoP series F 3 -IsoP NL: 2.59E3 NL: 3.18E3 Relative Abundance series F 3 -IsoP series F 3 -IsoP NL: 1.47E4 NL: 2.16E series F 3 -IsoP 1.47 NL: 2.9E series F 3 -IsoP NL: 1.2E series F 3 -IsoP 1.54 NL: 2.87E series F 3 -IsoP 1.72 NL: 2.72E Time (min) Time (min) 16

17 Figure 8 ng F 3 -IsoPs (per mg EPA) Time (hr) 17

18 Figure 9 RELATIVE INTENSITY 2 8 m/z 567 F 3 -IsoPs m/z 573 d 4-15-F 2t -IsoP RETENTION TIME (MIN) 18

19 Figure 1 EPA (nmol/mg tissue) Control.28% EPA A.56% EPA.84% EPA F 3 -IsoPs (ng/g) Control.28% EPA B.56% EPA.84% EPA C D AA (nmol/mg tissue) Control.28% EPA.56% EPA.84% EPA F 2 -IsoPs (ng/g) Control.28% EPA.56% EPA.84% EPA 19