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J. Biol. Chem., Vol. 282, Issue 41, 29890-29901, October 12, 2007

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Identification of Novel Autoxidation Products of the -3 Fatty Acid Eicosapentaenoic Acid in Vitro and in Vivo^*

Huiyong Yin, Joshua D. Brooks, Ling Gao¹, Ned A. Porter, and Jason D. Morrow²

From the Departments of Medicine and Pharmacology and Chemistry, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, April 12, 2007 , and in revised form, August 20, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Increased intake of fish oil rich in the -3 fatty acids eicosapentaenoic acid (EPA, C20:5 -3) and docosahexaenoic acid (DHA, C22:6 -3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-of-the-art mass spectrometric techniques including Ag⁺ coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides. Systematic study of the peroxidation of EPA provides the basis to examine the role of specific oxidation products as mediators of the biological effects of fish oil.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Eicosapentaenoic acid (C20:5 -3; EPA)³ and docosahexaenoic acid (C22:6, -3; DHA) are two major polyunsaturated fatty acids (PUFAs) in fish oil (Scheme 1). Experiments in animals, as well as human epidemiological studies and recent clinical intervention trials, suggest that fish consumption or dietary supplementation with fish oil reduces the incidence of important human disorders including atherosclerotic cardiovascular disease, cardiac sudden death, stroke, and asthma, among others (1–3). Dietary supplementation with fish oil also shows promise for slowing the progression of neurodegenerative diseases, psychiatric disorders, and AIDS (4–9). Identifying the mechanism(s) responsible for the beneficial effects of -3 fatty acids is of significant importance although it is not currently understood. One hypothesis to account for the anti-atherogenic and anti-inflammatory properties of -3 PUFAs is via interference with the arachidonic acid cascade that generates pro-inflammatory eicosanoids. EPA, a structurally close analogue of arachidonic acid, not only can replace arachidonic acid in phospholipid bilayers but is also a substrate for the cyclooxygenase (COX) and lipoxygenase enzymes, generating 3-series prostaglandins (PGs) and thromboxane and 5-series leukotrienes (10). In addition, EPA reduces the production of 2-series PGs and thromboxane and 4-series leukotrienes from arachidonate. These latter compounds are known to possess potent pro-inflammatory activities (11, 12). On the other hand, eicosanoids derived from EPA are either less biologically active or inactive compared with those from arachidonate and are thus considered to exert effects that are less inflammatory (13). Of particular interest, Serhan et al. (14) have more recently reported that lipid mediators derived from EPA via COX-2 after aspirin treatment show potent anti-inflammatory actions. These compounds are termed Resolvins because they were first identified in resolving inflammatory exudates (15).

Several studies have also reported that -3 PUFAs can be oxidized non-enzymatically and that the products are both anti-inflammatory and anti-proliferative (16, 17). However, structural characterization of these biologically active peroxidation products of EPA and DHA has never been undertaken. EPA and DHA are readily oxidized under free radical conditions because they have additional carbon-carbon double bonds compared with arachidonate and other PUFAs (18–20). Thus, studies to rationally define the peroxidation of EPA and DHA may help to identify those products that contribute to the biology of -3 PUFA consumption.

Free radical-induced oxidation of arachidonic acid has been well studied and a unified mechanism that leads to the formation of various oxidation products has been proposed (Scheme 2) (21–23). The oxidation proceeds via hydrogen atom abstraction at one of three bis-allylic positions at carbon 7, 10, and 13 to form a pentadienyl radical that can be trapped by molecular oxygen to form a peroxyl radical. A regiosomeric mixture of peroxyl radicals can be formed and the 11-peroxyl radical 2b is taken as an example. This peroxyl radical can lead to the formation of 11-hyperoxyeicosatetraenoic acid (HpETE), 2c and the corresponding alcohol 11-hydroxyeicosatetraenoic acid (11-HETE), 2d. On the other hand, further cyclization of the peroxyl radical 2b will generate bicyclic endoperoxides 2g via two consecutive steps of 5-exo cyclization. Reduction of 2g gives rise to one of the major classes of oxidation products, F₂-isoprostanes (IsoPs) (24, 25). These compounds are analogues of PGs but are formed non-enzymatically from the autooxidation of arachidonic acid. Quantification of F₂-IsoPs by gas chromatography/mass spectrometry (GC-MS) has been shown to be one of the most reliable markers to assess oxidant stress status and lipid peroxidation in vivo (26–28). In addition to the IsoPs, monocyclic peroxides 2j, serial cyclic peroxides 2k, and dioxolane-isoprostane peroxides 2l have also been identified. Thus, the major oxidation products from arachidonate can be predicted based on a unified free radical mechanism and this mechanism has been confirmed experimentally.

View larger version (7K): [in this window] [in a new window]   SCHEME 1

View larger version (27K): [in this window] [in a new window]   SCHEME 2

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Eicosapentaenoic acid methyl ester was purchased from Nu Chek Prep (Elysian, MN) and was of the highest purity (>99+%). [²H₄] 15-F_2t-IsoP (8-iso-PGF₂) was purchased from Cayman Chemical Co. (Ann Arbor, MI). Pentafluorobenzyl (PFB) bromide and diisopropylethylamine (DIEA) were from Sigma. N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) was from Supelco (Bellefonte, PA). C-18 and silica Sep-Pak cartridges were purchased from Waters Associates (Milford, MA). HPLC grade solvents, methanol, chloroform, hexane, isopropyl alcohol, and ethanol are purchased from Burdick and Jackson brand (VWR Scientific Inc., McGraw Park, IL). Other chemicals were from Aldrich (Milwaukee, WI) and were of the highest purity.

Detection and Separation of Lipid Oxidation Products—Reactions involving hydroperoxides were visualized by thin layer chromatography (TLC) using a stain of 1.5 g of N,N'-dimethyl-p-phenylenediamine dihydrochloride/25 ml H₂O/125 ml of MeOH/1 ml of acetic acid. Hydroperoxides yield an immediate pink color, while cyclic peroxides exhibit a pink color after mild charring. General TLC staining was accomplished by use of a phosphomolybdic acid stain prepared as a 20% (w:v) solution in ethanol. In general, hydroperoxides were stored as dilute solutions with 1 mol% BHT in either hexanes or benzene at –78 °C and were never exposed to temperatures >40 °C. Flash column chromatography was performed using silica gel 60 (EMD, NJ) and eluted with hexane:ethyl acetate (90:10). TLC was performed using 0.2-mm layer thickness silica gel-coated aluminum (60 F₂₅₄, EM Industries, Industry, CA), and TLC plates were analyzed using UV light (254 nm) with a Mineralight UVSL-25 hand lamp.

Analytical HPLC was performed using a Waters Model 600E pump with a Waters 996 photodiode array detector. Millenium32 chromatography software (Waters Corp., Milford, MA) was utilized to control the array detector and to collect and process data. Semi-preparative HPLC was carried out using a Dynamax-60 Å 8 µm (83–121-C) silica column (21.4 mm x 25 cm) with a flow rate of 10 ml/min. For reverse phase HPLC analysis of PFB esters, a Phenomenex Ultracarb ODS 5 µm 4.6 mm x 25 cm column (Torrence, CA) was used at 1 ml/min flow rate with a gradient starting with 50% solvent A (95:5, water: MeOH) and 50% solvent B (5: 95, water: MeOH), holding for 2 min and increasing to 100% B in 20 min and holding for 10 min. PFB esters of F₃-IsoPs were also analyzed by normal phase HPLC using 12% IPA in hexanes and a analytical Beckman Ultrasphere 5µ (4.6 mm x 25 cm) silica column or narrowbore Beckman Ultrasphere 5 µm (2.0 mm x 25 cm) Si column (the latter column coupled to mass spectrometry) A flow rate of 1 ml/min was used for analytical normal phase HPLC whereas 0.2 ml/min was employed for narrowbore columns.

Mass Spectrometry—On-line reverse phase liquid chromatography (LC) was carried out using the ThermoFinnigan Surveyor MS Pump 2.0 equipped with the columns mentioned above. Samples were analyzed using a ThermoFinnigan TSQ Quantum Ultra instrument. The atmospheric pressure chemical ionization (APCI) source was fitted with a deactivated fused silica capillary (100 µm i.d.). The mass spectrometer was operated in negative ion mode with a capillary temperature of 300 °C, vaporizer temperature of 300 °C. The spray voltage, ion transfer tube voltage, and skimmer voltage were optimized to achieve maximal response. Nitrogen was used as both the sheath gas and the auxiliary gas. For tandem mass spectrometry (MS/MS) experiments, collision energies were optimized under 1.5 mTorr of argon. Data acquisition and analysis were performed using Xcaliber software, version 2.0 Service Release-2 (SR-2). Samples were introduced either by direct liquid infusion (DLI) or by HPLC. For normal phase HPLC, samples were introduced by a Hewlett-Packard 1090 HPLC system.

Oxidation of EPA Methyl Ester to Primary Hydroperoxides and More Complex Oxidation Products—EPA methyl ester (500 mg, 1.59 mmol) was dissolved in 1.5 ml of acetonitrile to make a solution of 1.06 M. To the mixture were added 44 mg of N-methyl benzohydoxamic acid (NMBHA), 2.92 mmol and 0.05 equivalent in benzene of free radical initiator di-tert-butyl hyponitrite (DTBN) (29). The reaction was stirred under oxygen at 37 °C for 24 h. The reaction was quenched by adding 2 mg of BHT and NMBHA and DTBN were removed by flash chromatography on silica gel using hexane:ethyl acetate (90: 10). The hydroperoxides were analyzed and separated by analytical and semi-preparative HPLC. The separated fractions of primary hydroperoxides were converted to more highly oxidized peroxides according to our previous procedure (30).

Ag⁺ Coordination Ionspray (CIS)-MS Analysis of Oxidized EPA Methyl Esters—The Ag⁺ CIS-MS experiments were carried out following the methods reported previously (21, 30–33). A narrowbore C18 column was used with a flow rate of 0.2 ml/min employing 0.15 mM AgBF₄ in a mixture of isopropyl alcohol (50%) and water (50%).

Analysis of Oxidized EPA by LC/APCI-MS—Oxidized lipids derived from 100 µg of lipid hydroperoxides were reduced by adding an excess amount of triphenylphosphine (PPh₃). The reduced lipid was subjected to basic hydrolysis using 1.0 M KOH for 60 min at 37 °C. After adjusting to pH 3, the mixture was loaded onto a C18 Sep-Pak cartridge that was preconditioned with 5 ml of methanol and then 5 ml of water. The column was washed with 5 ml of water and 5 ml of heptane. The eluent of 10 ml of ethyl acetate was collected. The solution was dried over Na₂SO₄. After evaporation of the solvent, the residue was dissolved in 20 µl of CH₃CN. To the resulting solution was added 20 µl of 10% (v:v) PFB bromide in acetonitrile and 10 µl of 10% (v:v) N,N-diisopropylethylamine (DIEA) in acetonitrile and the mixture was kept at room temperature for 30 min. The reagent was dried under nitrogen, and the residue was subjected to TLC separation (60ALK6D plates, Whatman, Maidstone UK). The PFB esters, after TLC separation, were either directly analyzed by LC/APCI-MS or were further derivatized to TMS derivatives. For the latter, after evaporation of the ethyl acetate, 20 µl of BSTFA and 10 µl of dimethylformamide were added to the residue, and the mixture was incubated at 37 °C for 20 min.

Identification of Oxidation Products of EPA in Mice Tissue— Mice (strain C57BL/6J(B6)) were fed with a rodent American Institute of Nutrition (AIN)-93 M (maintenance) (Dyets Inc., Bethlehem, PA) diet supplemented with either 0% (control diet) or 0.56% EPA (EPA diet) (by weight). The formulation of AIN-93 diets is an improvement to replace the previous version of AIN-76 (34). The EPA in this modified AIN-93 diet is stable employing the feeding conditions used in the studies as noted under "Results." In particular, experiments were carried out to ensure that complex oxidation products of EPA were present at minimal levels in the diet when freshly obtained, at the end of the study, and after being stored at room temperature in the same conditions used in the feeding studies. After 8 weeks of feeding, mice were administered CCl₄ (2 ml/kg in corn oil). After 2 h, the animals were anesthetized with pentobarbital (60 mg/kg) intraperitoneally, sacrificed, and the livers were removed. Approximately 1.0 g of tissue was immediately homogenized and extracted in 20 ml of Folch solution (MeOH: CHCl₃ = 1:2 containing BHT 0.005%) and 4 ml of 0.9% NaCl solution was added to obtain a crude phospholipid extract (35). The aqueous layer was not acidified prior to the extraction. It should be noted that we have previously shown that this extraction approach does not lead to oxidation of arachidonate or EPA ex vivo (19, 25). Basic hydrolysis was carried out in 0.5 ml of MeOH and 0.5 ml of 15% KOH at 37 °C for 30 min. The pH of the solution was adjusted to 3 by addition of 1 N HCl. 10 µl of 0.1 ng/µl internal standard 15-F_2t-IsoP-d₄ ethanol was added to the mixture to correct the sample loss. Then the mixture was further purified by C18 as mentioned above and an additional silica Sep-Pak solid phase extraction was carried out. The silica Sep-Pak was preconditioned with 5 ml of ethyl acetate. Afterward, it was washed with 5 ml of ethyl acetate and the samples were eluted with 5 ml of 1:1 (ethyl acetate:methanol). PFB esterification was carried out, and TLC was performed using chloroform: ethanol (93:7). A wide TLC cut was chosen to include all the possible oxidation products. After extraction of the compounds from the scraped silica gel by ethyl acetate, the TMS derivatives were obtained by the procedures described above. LC/APCI-MS analyses were carried out using a narrowbore reverse phase column using methanol as a solvent. Analysis of F₃-IsoPs as PFB esters was performed using the same protocol as above employing normal phase LC with a solvent system of 12% isopropyl alcohol in hexanes (32). For quantitative studies involving the measurement of F₃-IsoPs in mouse tissues, a highly accurate gas chromatography/MS assay using stable isotope dilution methods was employed as described (19).

View larger version (14K): [in this window] [in a new window]   SCHEME 3

View larger version (23K): [in this window] [in a new window]   SCHEME 4

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A number of MS techniques were developed and applied to identify these novel oxidation products (Scheme 5). Ag⁺ CIS-MS has proven powerful to characterize the intact peroxides of PUFAs based on the unique fragmentation patterns induced by Ag⁺ coordination and was initially employed in studies discussed below. Oxidation mixtures were then reduced by PPh₃ and derivatized to PFB esters and APCI-MS was employed to further confirm the structure of the oxidation products. APCI-MS was also utilized to provide additional information about EPA oxidation products after trimethylsilyl (TMS) derivatization.

Formation of Eight Regioismeric Hydroperoxides from the Oxidation of EPA—Primary hydroperoxides of EPA can be obtained from the oxidation of EPA in the presence of good hydrogen atom donors such as -tocopherol, methyl trolox, or cyclohexadiene although a catalytic system based on the hydrogen atom donor NMBHA gives much higher yield of hydroperoxides from polyunsaturated fatty acids (29). The eight hydroperoxide regioisomers of HpEPA Me were prepared using the latter reagent and separated by HPLC (Fig. 1a). Only 5- and 15-HpEPA Me can be separated from other hydroperoxides whereas 12 and 14-HpEPA, 11- and 18-HpEPA, and 8-HpEPA and 9-HpEPA are collected as mixtures. The structures of these different hydroperoxides were confirmed using Ag⁺ CIS-MS based on the specific Hock cleavage fragments of each hydroperoxides (data not shown). It is interesting that once the hydroperoxides are reduced to corresponding alcohols (hydroxyeicosapentaenoic acids, HEPAs), all regioisomers can be separated as eight distinct peaks and their structures can be identified by APCI-MS after PFB esterification (Fig. 1b). The elution order of the eight alcohols is 15-, 12-, 14-, 11-, 18-, 8-, 9-, and 5-HEPA employing normal phase HPLC.

View larger version (18K): [in this window] [in a new window]   SCHEME 5

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Separation of HpEPA Me and HEPA Me by normal phase HPLC using a semi-preparative column with a flow rate of 10 ml/min and 0.7% IPA in hexane. A UV spectrometer is used as a detector to monitor conjugated dienes with a wavelength of either 234 nm or 207 nm for isolated double bonds, respectively. a, HpEPA; b, HEPA. The number indicates the location of the hydroperoxyl or hydroxyl group in the molecules.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Direct liquid infusion analysis of an oxidized mixture of 5-HpEPA Me using Ag+ CIS-MS and the representative structures of predicted oxidation products. The oxidation mixture was dissolved in 50 ng/µl AgBF4 in IPA and a syringe pump was used to deliver 10 µl/min of the mixture. A mass range of m/z 100 to 1000 was monitored in the scan mode.

As previously noted, the oxidation profile of different hydroperoxyl radical precursors will vary depending on the starting compound. Formation of dioxolane-IsoP peroxides and bis-dioxolane-IsoP peroxides is predicted from 15-HpEPA and generation of these compounds is believed to be responsible for the regioisomeric selectivity of different F₃-IsoPs. Identification of these novel IsoPs from EPA oxidation would confirm this hypothesis. Thus, 15-HpEPA Me was oxidized under free radical conditions and the oxidation mixture was examined in a manner similar to that for 5-HpEPA. The CID spectra of m/z 519 (dioxolane-IsoP) and 551 (bis-dioxolane-IsoP are shown in Fig. 5. In addition to dehydration from the precursor ion that leads to the formation of m/z 501, there are characteristic fragments that are useful to identify the presence of dioxolane-IsoP peroxide 4d (Fig. 5a). The peak with m/z 305 is consistent with the cleavage of a dioxolane moiety to generate an aldehyde and dehydration from the hydroperoxide. The other half of the fragment with an epoxide will have an m/z 303. The cleavage of the cyclic peroxide bond to form an aldehyde toward the end of the molecule can give rise to a fragment with m/z 289. Fragments derived from the cleavage of the hydroperoxide will generate aldehydes m/z 305 and 361 respectively. Overall, the major fragments derived from the CID of m/z 519 from 15-HpEPA Me oxidation are consistent with the presence of the dioxolane-IsoP peroxides 4d.

Further cyclization of the peroxyl radical from 4d will lead to the formation of the bis-dioxolane-IsoP peroxide 4e. The CID of m/z 551 is shown in Fig. 5b. Some similar fragments are observed as those from 4d, for example, m/z 289, 303, and 361 but there are some unique fragments that are not present in 4d. Peak m/z 433 and 237 may be derived from the cleavage of hydroperoxide at carbon C5.

Identification of Novel Oxidation Products from EPA Oxidation by LC/APCI-MS after Derivatization—Ag⁺ CIS-MS has been successfully employed to identify the intact novel oxidation products from EPA as predicted based on free radical mechanisms. Besides the hydroperoxides, HpEPAs and corresponding alcohols HEPAs, monocyclic peroxides, bicyclic endoperoxides (precursor to 3-series IsoPs), bicyclic peroxide, tricyclic peroxides, dioxolane-IsoP peroxides and bis-dioxolane-IsoP peroxides have also been identified from the oxidation of EPA. To further confirm the identification of these novel products, we employed various MS methods after compounds were reduced to alcohols with PPh₃ and/or derivatized to PFB esters/TMS ether derivatives. The protocols and MS methods that were used to confirm the structures are summarized in Scheme 5. The dioxolane-IsoP peroxide 4d is taken as an example. PPh₃ reduces the bicyclic endoperoxide to a 1,3-diol on the cyclopentane ring and the hydroperoxide to alcohol whereas the dioxolane moiety is not reduced. After basic hydrolysis, the free acid can be derivatized to the PFB ester which makes it amenable to APCI-MS analysis. TMS derivatization caps the hydroxyl groups on the molecule and makes them amenable for further APCI-MS analysis.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. CID analysis of oxidation products of 5-HpEPA Me analyzed by LC/Ag+ CIS-MS (a) CID of starting material 5-HpEPA Me (m/z 455). b, monocyclic peroxides 4g (m/z 487). c, bicyclic endoperoxides 4h (m/z 487). d, bicyclic peroxides 4j (m/z 519). e, tricyclic peroxides 4i (m/z 551). LC separation was carried out using 0.15 mM of AgBF4 in IPA and water (50:50) and a flow rate of 0.2 ml/min on a phenomenex Luna ODS (5 µm, 4.5 x 250 mm) column. Only adducts with Ag 107 isotope were chosen for CID experiments. Mass ranges of m/z 100 to 700 were monitored.

Identification of Novel Oxidation Products from EPA in Vivo— After identification of novel EPA oxidation products in vitro using various MS methods, we undertook studies to detect these compounds in vivo in mice.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Selective reaction monitoring (SRM) of the oxidation products from 5-HpEPA Me by LC/Ag+ CIS-MS. a, starting material 4f (5-HpEPA). b, bicyclic endoperoxides 4h. c, monocyclic peroxide 4g. d, bicyclic peroxides 4j. e, tricyclic peroxides 4i. Similar LC and MS conditions were used as described in the legend to Fig. 3 and only adducts with Ag 107 are shown.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. CID of dioxolane-IsoP peroxides 4d and bis-dioxolane-IsoP peroxides 4e from 15-HpEPA by LC/Ag+ CIS-MS and the major fragments. a, dioxolane-IsoP peroxides. b, bis-dioxolane-IsoP peroxides. Similar LC and MS conditions were used as described in the legend to Fig. 3, and only adducts with Ag 107 were indicated.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. LC/APCI-MS analysis of derivatives from oxidation products of 5-HpEPA Me. a, selective ion monitoring (SIM) of the oxidation products as PFB esters. LC conditions: C18 analytical column, flow rate of 1.0 ml/min, and a solvent mixture of methanol and water with a gradient of 25–100% methanol in 20 min. b, SIM of PFB/TMS derivatives by LC/APCI-MS. LC conditions: C18 analytical column, isocratic 1.0 ml/min of 100% methanol. APCI-MS was carried out in negative mode with all the parameters optimized to achieve maximal response. Representative structures of the various oxidation products are shown.

View larger version (27K): [in this window] [in a new window]   SCHEME 6

View larger version (17K): [in this window] [in a new window]   FIGURE 7. LC/APCI-MS detection of novel oxidation products of EPA from livers of mice fed a diet supplemented with 0.56% EPA for 8 weeks. Compounds were analyzed as PFB/TMS derivatives. LC conditions: C18 analytical column, isocratic 1.0 ml/min of 100% methanol. MS conditions are the same as described in the legend to Fig. 6. SRM: HEPA m/z 389 to 299 @ 25 eV; DiHO-EPA, m/z 477 to 297 @ 25 eV; bicyclic peroxides, m/z 453 to 363 @ 25 eV; monocyclic peroxides, m/z 421 to 331@ 25 eV; F3-IsoPs, m/z 567 to 297 @ 25 eV; dioxolane-IsoPs, m/z 599 to 329 @ 25 eV; bis-Dioxolane-IsoPs, m/z 631 to 361 @ 25 eV.

View larger version (8K): [in this window] [in a new window]   FIGURE 8. Levels of F3-IsoPs in liver tissue from mice fed an AIN-93 diet containing 0.56% EPA who either received CCl4 (1 ml/kg intraperitoneally) or vehicle. F3-IsoPs were quantified using stable isotope dilution methodology employing gas chromatography/MS. n = 4 animals per group; ***, p = 0.0003.

View larger version (12K): [in this window] [in a new window]   FIGURE 9. LC/APCI-MS analysis of regioisomers and diastereomers of F3-IsoPs in vitro (a) and in vivo (b). The intensity is normalized to 5-series of F3-IsoP. Compounds were analyzed as PFB derivatives. LC is carried out on an analytical column with 12% IPA in hexane with a flow rate of 1 ml/min. SRM: 5-series, m/z 351 to 115 @ 30 eV; 12-series, m/z 351 to 151 @ 30 eV; 8-series, m/z 351 to 127 @ 30 eV; 11-series, m/z 351 to 167 @ 30 eV; 15-series, m/z 351 to 193 @ 30 eV; 18-series, m/z 351 to 233 @ 30 eV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The systematic identification of novel oxidation products from EPA is of potential biological importance. A number of studies have shown that supplementation of humans with fish oil rich in EPA and DHA has beneficial effects on the prevention of important human diseases such as atherosclerosis and sudden death, neurodegeneration, and various inflammatory disorders (2, 3, 6–8, 37). A recent Japanese study on effects of EPA on major coronary events in hypercholesterolemia (JELIS) showed a 19% reduction in major coronary events after 4.6 years of EPA intervention in 18,645 hypercholesterolemic patients (3). Although the mechanism(s) by which these beneficial effects occurs is unknown, a potentially important aspect related to the biological activity of -3 PUFAs is their ability to interfere with the arachidonic acid cascade that generates proinflammatory eicosanoids (2, 38). 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 (11, 12). Recent studies have also shown that 3- and 5-series eicosanoids derived from EPA are either less biologically active or inactive compared with the former products and are thus considered to exert effects that are less inflammatory (10, 13, 39, 40). Furthermore, Serhan (14, 15, 39, 41, 42) and co-workers have described a group of polyoxygenated DHA and EPA derivatives termed Resolvins that are produced enzymatically in various tissues. These compounds inhibit cytokine expression and other inflammatory responses in microglia, skin cells, and other cell types.

There has also been significant interest in the biological activities of non-enzymatic free radical-initiated peroxidation products of -3 PUFAs (18–20, 43). Sethi et al. (16, 17) recently 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- (PPAR-) 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--deficient mice. These studies suggest that the beneficial effect of -3 fatty acids may be mediated, in part, by the anti-inflammatory effects of oxidized EPA. Similarly, Vallve et al. (44) 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. Arita et al. (45) have also shown that non-enzymatically oxidized EPA enhances apoptosis in HL-60 leukemia cells supporting the contention that oxidized -3 PUFAs are both anti-proliferative and anti-inflammatory. In virtually none of these reports, however, have the specific peroxidation products responsible for these effects been identified.

Our studies reported herein have systematically defined the oxidation of EPA in vitro and in vivo for the first time. The vast majority of oxidation products are peroxides and because of their stability, detection is an important issue for any analytical technique employed. As reported, Ag⁺ CIS-MS has proven powerful to identify these intact peroxides generated in vitro. The unique fragmentation patterns induced by silver ion coordination were employed to characterize these novel compounds. However, the poor sensitivity of this technique makes it difficult to be used to study these oxidation products in vivo (46). Furthermore, the stability of these peroxides in vivo is virtually unknown. Other MS techniques such as APCI-MS afford significant sensitivity and can thus be complimentary to silver ion CIS-MS even though compound derivatization is needed. We successfully utilized this latter approach to indeed show that the oxidation of EPA can be rationally defined both in vitro and in vivo. Nonetheless, it is noteworthy that accurate quantification of EPA oxidation products in vivo is difficult because the stability and metabolism of these compounds are unknown. Furthermore, internal standards for each class of oxidation products are lacking (except for F₃-IsoPs), which poses another challenge to accurately quantifying these compounds.

Systematic identification of the oxidation products of EPA and other PUFAs provides the rationale for studying the biology of these compounds. For example, we reported recently that a novel class of J₃-IsoPs derived from EPA activates the master transcription factor, Nrf₂ by destabilizing the association between Keap1 and Cullin 3 (18). Nrf₂ activation leads to antioxidant gene expression that regulates detoxification of reactive oxygen species. This pathway represents a novel and potentially beneficial aspect of fish oil supplementation and studies are further ongoing to examine the role of EPA oxidation products to regulate this response to oxidant stress in vivo.

Another rationale for undertaking these studies was based on the hypothesis that oxidation products contribute to the beneficial biological effects of EPA and fish oil supplementation in that they exert biological activities that are less pro-inflammatory than oxidation products derived from arachidonic acid. Indeed, as noted, one limited report noted that the EPA-derived IsoP, 15-F_3t-IsoP, possesses activity that is significantly different from 15-F_2t-IsoP in that it does not affect human platelet shape change or aggregation (36). 15-F_2t-IsoP is a ligand for the Tx receptor and induces platelet shape change and also causes vasoconstriction. 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 arachidonate-derived PGs. Future studies will need to further explore these observations.

In summary, we report, for the first time, systematic identification of novel oxidation products of EPA by various MS techniques. The same oxidation products generated in vitro can be detected in vivo. We also observed a strikingly similar pattern of product distribution of F₃-IsoPs in vivo and in vitro, suggesting that the same free radical mechanisms operative in vitro occur in vivo. Further understanding of the biological consequences of the formation of these novel compounds and factors influencing their formation and metabolism will provide valuable insights into the role of EPA in human physiology and pathophysiology.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants ES13125, ES000267, GM15431, ES31125, RR00096, DK48831, and CA77839. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Pharmaceutical Candidate Optimization, Discovery Metabolism, and Pharmacokinetics, Bristol-Myers Squibb, Route 206 and Provinceline Rd., Princeton, NJ 08540.

² To whom correspondence should be addressed: Division of Clinical Pharmacology, Depts. of Medicine and Pharmacology, Vanderbilt University School of Medicine, 532 RRB, 23^rd and Pierce Ave., Nashville, TN 37232-6602. Tel.: 615-343-1124; Fax: 615-322-3669; E-mail: Jason.morrow{at}vanderbilt.edu.

³ The abbreviations used are: EPA, eicosapentaenoic acid; AIN-93, American Institute of Nutrition-93; DHA, docosahexaenoic acid; PUFA, polyunsaturated fatty acid; COX, cyclooxygenase; PG, prostaglandin; IsoP, isoprostane; PFB, pentafluorobenzyl; DTBN, di-tert-butyl hyponitrite; BSTFA, N,O-bis(trimethylsilyl) trifluoroacetamide; DIEA, diisopropylethylamine; HPLC, high pressure liquid chromatography; APCI, atmospheric pressure chemical ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NMBHA, N-methyl benzohydroxamic acid; IPA, isopropyl alcohol; IsoP, isoprostane; TLC, thin layer chromatography; CIS, coordination ionspray; HpEPA, hydroperoxyeicosapentaenoic acid; HEPA, hydroxyeicosapentaenoic acid; TMS, trimethylsilyl; CID, collision-induced dissociation; SRM, selective reaction monitoring; SIM selective ion monitoring; PPh₃, triphenylphosphine.

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