Dietary fish oil exerts hypolipidemic effects in lean and insulin sensitizing effects in obese LDLR-/- mice.

Obesity is often associated with dyslipidemia, insulin resistance, and hypertension. Together, these metabolic perturbations greatly increase the risk of developing cardiovascular disease and diabetes. Although fish oil is a well-established hypolipidemic agent, the mechanisms by which it mediates its lipid-lowering effects are not clear. In addition, it has not been established whether dietary fish oil has different effects in lean and obese mice. LDL receptor deficient (LDLR-/-) and leptin deficient mice on a LDLR-/- background (ob/ob;LDLR-/-) were fed a high fat diet (39% total fat) supplemented with 6% olive oil or fish oil for 6 wk. Fish oil supplementation resulted in lower concentrations of plasma total cholesterol (P < 0.01), triglycerides (P < 0.01), and free fatty acids (P < 0.001) in lean LDLR-/- mice, but not in ob/ob;LDLR-/- mice. In contrast, a fish oil diet did not modulate insulin sensitivity in lean LDLR-/- mice, but it improved insulin sensitivity in ob/ob;LDLR-/- mice (P < 0.05) compared with olive oil fed ob/ob;LDLR-/- mice. Interestingly, plasma adiponectin concentrations were significantly higher and hepatic steatosis was reduced in both mouse models upon fish oil feeding. Finally, fish oil fed LDLR-/- mice exhibited higher hepatic AMP activated protein kinase (AMPK) phosphorylation (P < 0.05), whereas AMPK phosphorylation was not elevated by fish oil feeding in ob/ob;LDLR-/- mice. Taken together, our data suggest that fish oil reduces hepatic steatosis in both lean and obese mice, has potent plasma lipid lowering effects in lean mice, and exerts insulin sensitizing effects in obese mice.

Dietary supplementation of omega-3 fatty acid-containing fish oil suppresses F2-isoprostanes but enhances inflammatory cytokine response in a mouse model of ovalbumin-induced allergic lung inflammation.

Epidemiological and clinical evidence has suggested that increased dietary intake of fish oil containing omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may be associated with a reduced risk of asthma. However, interventional studies on these effects have been equivocal and controversial. Free radical oxidation products of lipids and cyclooxygenases-derived prostaglandins are believed to play an important role in asthma, and fish oil supplementation may modulate the levels of these critical lipid mediators. We employed a murine model of allergic inflammation produced by sensitization to ovalbumin (OVA) to study the effects of fish oil supplementation on airway inflammation. Our studies demonstrated that omega-3 fatty acids were dose dependently incorporated into mouse lung tissue after dietary supplementation. We examined the oxidative stress status by measuring the levels of isoprostanes (IsoPs), the gold standard for oxidative stress in vivo. OVA challenge caused significant increase of F(2)-IsoPs in mouse lung, suggesting an elevated level of oxidative stress. Compared to the control group, fish oil supplementation led to a significant reduction of F(2)-IsoP (from arachidonic acid) with a concomitant increase of F(3)-IsoPs (from EPA) and F(4)-IsoPs (from DHA). Surprisingly, however, fish oil supplementation enhanced production of proinflammatory cytokine IL-5 and IL-13. Furthermore, fish oil supplementation suppressed the production of pulmonary protective PGE(2) in the bronchoalveolar lavage (BAL) while the level of urinary metabolites of the PGE(2) was increased. Our data suggest that augmented lung inflammation after fish oil supplementation may be due to the reduction of PGE(2) production in the lung and these dichotomous results bring into question the role of fish oil supplementation in the treatment of asthma.

The enteropathy of prostaglandin deficiency.

BACKGROUND: Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. METHODS: Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. RESULTS: We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45-year-old man who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls (P<0.006), and serum 12-HETE was 1.3% of controls (P<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore-activated leukocytes was only 3% of controls, and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-alpha (cPLA2-alpha), which regulates cyclooxygenase- and lipoxygenase-mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-alpha cDNA demonstrated two heterozygous nonsynonymous single-base-pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known single nucleotide polymorphism (SNP), Lys651Arg (K651R). CONCLUSIONS: Characterization of this cPLA2-alpha deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers.

Thromboxane and isoprostanes as inflammatory and vasoactive mediators in black walnut heartwood extract induced equine laminitis.

Inflammation and vascular dysfunction occur concurrently during the prodromal stages of equine laminitis. The aim of this study was to provide insights into the role that thromboxane and isoprostanes may play in the development of black walnut heartwood extract (BWHE)-induced laminitis. Horses were divided into two groups, either control or BWHE-administered horses. Plasma concentrations of thromboxane increased transiently after administration of BWHE and coincided with the nadir in white blood cell counts, whereas plasma concentrations of iso-prostaglandin PGF(2alpha) (iso-PGF(2alpha)) did not change in either group. At 12h (for the control group) or Obel grade 1 laminitis (for the BWHE group) the horses were euthanized and laminar tissue collected. Laminar arteries and veins were used in functional studies with vasoconstrictor substances and tissue samples were used for the determination of laminar iso-PGF(2alpha) concentrations. Laminar tissue concentrations of iso-PGF(2alpha) were significantly greater in BWHE horses when compared to control horses. In parallel studies concentrations of iso-PGF(2alpha) in laminar tissue samples obtained 1.5 and 3h after administration of BWHE were indistinguishable from those for control horses at 3 or 12h after administration of an equal volume of water. Laminar vessel constrictor responses to either a thromboxane mimetic (U46619), iso-prostaglandin PGE(2) (iso-PGE(2)) or iso-PGF(2alpha) were determined using small vessel myographs. In some vessels, the effects of putative prostanoid and thromboxane receptor antagonists, SQ 29,548, SC-19220 and AH 6809, upon contractile responses were determined. In control horses, U46619, iso-PGF(2alpha) and iso-PGE(2) more potently and efficaciously constricted laminar veins when compared to laminar arteries. Responses of laminar veins from BWHE horses to iso-PGE(2) were similar to those of laminar veins from control horses, whereas iso-PGF(2alpha) elicited significantly greater responses in laminar veins from BWHE horses when compared to controls. In contrast, responses to U46619 were smaller in laminar veins isolated from BWHE horses when compared to those in laminar veins from control horses. In the presence of SQ 29,548, iso-PGF(2alpha) elicited a small dilation in laminar veins from control horses, which was not apparent in laminar veins from BWHE horses. These results are consistent with both systemic and local inflammatory events occurring during the prodromal stages of BWHE-induced laminitis. Because laminar veins are sensitive to thromboxane and isoprostanes, these substances may act as conduits between the inflammatory and vascular events occurring in laminitis and may be therapeutic targets for this crippling condition.

Supplementation with orange and blackcurrant juice, but not vitamin E, improves inflammatory markers in patients with peripheral arterial disease.

Inflammation and endothelial activation are associated with an increased risk of CVD and epidemiological evidence suggests an association between levels of markers of inflammation or endothelial activation and the intake of fruit. Also, vitamin E, a fat-soluble antioxidant, has anti-inflammatory properties. We performed a randomised 2 x 2 factorial, crossover trial to determine the effect of orange and blackcurrant juice (500 ml/d) and vitamin E (15 mg RRR-alpha-tocopherol/d) supplementation on markers of inflammation and endothelial activation in forty-eight patients with peripheral arterial disease. Patients were randomly allocated to two dietary supplements from the four possible combinations of juice and vitamin E: juice+vitamin E; juice+placebo; reference beverage (sugar drink)+vitamin E; and reference beverage+placebo. The supplementations were given for 28 d, separated by a 4-week wash-out period. Analysis of main effects showed that juice decreased C-reactive protein (CRP) by 11% and fibrinogen by 3% while the reference drink increased CRP by 13% and fibrinogen by 2% (P<0.008 and P<0.002, respectively). No significant differences were measured for IL-6 and the endothelial activation markers von Willebrand factor, tissue-plasminogen activator and plasmin activator inhibitor-1. Vitamin E supplementation had no significant effects on the various markers. We observed no significant interaction between juice and vitamin E. In this study, orange and blackcurrant juice reduced markers of inflammation, but not markers of endothelial activation, in patients with peripheral arterial disease, relative to sugar drinks.

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.

Formation of highly reactive cyclopentenone isoprostane compounds (A3/J3-isoprostanes) in vivo from eicosapentaenoic acid.

Omega-3 (omega-3) polyunsaturated fatty acids (PUFAs) found in marine fish oils are known to suppress inflammation associated with a wide variety of diseases. Eicosapentaenoic acid (EPA) is one of the most abundant omega-3 fatty acids in fish oil, but the mechanism(s) by which EPA exerts its beneficial effects is unknown. Recent studies, however, have demonstrated that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and anti-proliferative effects. Very few studies to date have investigated which EPA oxidation products are responsible for this bioactivity. Our research group has previously reported that anti-inflammatory prostaglandin A(2)-like and prostaglandin J(2)-like compounds, termed A(2)/J(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 cyclopentenone-IsoP compounds are formed from the oxidation of EPA in vivo. Herein, we report the formation of cyclopentenone-IsoP molecules, termed A(3)/J(3)-IsoPs, formed in abundance in vitro and in vivo from EPA peroxidation. Chemical approaches coupled with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to structurally characterize these compounds as A(3)/J(3)-IsoPs. We found that levels of these molecules increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 +/- 0.4 ng/mg EPA to 196 +/- 23 ng/mg EPA after 36 h. We also detected these compounds in significant amounts in fresh liver tissue from EPA-fed rats at basal levels of 19 +/- 2 ng/g tissue. Amounts increased to 102 +/- 15 ng/g tissue in vivo in settings of oxidative stress. These studies have, for the first time, definitively characterized novel, highly reactive A/J-ring IsoP compounds that form in abundance from the oxidation of EPA in vivo.

Oral quercetin supplementation and blood oxidative capacity in response to ultramarathon competition.

Previous research indicates that ultramarathon exercise can result in blood oxidative stress. The purpose of this investigation was to examine the efficacy of oral supplementation with quercetin, a naturally occurring compound with known antioxidant properties, as a potential countermeasure against blood oxidative stress during an ultramarathon competition. In double-blind fashion, 63 participants received either oral quercetin (250 mg, 4x/day; 1,000 mg/day total) or quercetin-free supplements 3 weeks before and during the 160-km Western States Endurance Run. Blood drawn before and immediately after (quercetin finishers n = 18, quercetin-free finishers n = 21) the event was analyzed for changes in blood redox status and oxidative damage. Results show that quercetin supplementation did not affect race performance. In response to the ultramarathon challenge, aqueous-phase antioxidant capacity (ferric-reducing ability of plasma) was similarly elevated in athletes in both quercetin and quercetin-free treatments and likely reflects significant increases in plasma urate levels. Alternatively, trolox-equivalent antioxidant capacity was not altered by exercise or quercetin. Accordingly, neither F2-isoprostances nor protein carbonyls were influenced by either exercise or quercetin supplementation. In the absence of postrace blood oxidative damage, these findings suggest that oral quercetin supplementation does not alter blood plasma lipid or aqueous-phase antioxidant capacity or oxidative damage during an ultramarathon challenge.

The relationship between dose of vitamin E and suppression of oxidative stress in humans.

The oxidation hypothesis of atherogenesis has been the focus of much research over the past 2 decades. However, randomized placebo-controlled trials evaluating the efficacy of vitamin E in preventing cardiovascular events in aggregate have failed to show a beneficial effect. Implicit in these trials is that the dose of vitamin E tested effectively suppressed oxidative stress status but this was never determined. We defined the dose-dependent effects of vitamin E (RRR-alpha-tocopherol) to suppress plasma concentrations of F2-isoprostanes, a biomarker of free radical-mediated lipid peroxidation, in participants with polygenic hypercholesterolemia and enhanced oxidative stress, a population at risk for cardiovascular events. A time-course study was first performed in participants supplemented with 3200 IU/day of vitamin E for 20 weeks. A dose-ranging study was then performed in participants supplemented with 0, 100, 200, 400, 800, 1600, or 3200 IU/day of vitamin E for 16 weeks. In the time-course study, maximum suppression of plasma F2-isoprostane concentrations did not occur until 16 weeks of supplementation. In the dose-ranging study there was a linear trend between the dosage of vitamin E and percentage reduction in plasma F2-isoprostane concentrations which reached significance at doses of 1600 IU (35+/-2%, p<0.035) and 3200 IU (49+/-10%, p<0.005). This study provides information on the dosage of vitamin E that decreases systemic oxidant stress in vivo in humans and informs the planning and evaluation of clinical studies that assess the efficacy of vitamin E to mitigate disease.

F(2)-isoprostanes as biomarkers of late-onset Alzheimer's disease.

Alzheimer's disease (AD) is a syndrome caused by a few uncommon mutations that lead to early-onset disease, occurs in adults with Down's syndrome, but is by far most commonly seen as a late-onset disease with multiple risk factors but no causative factors yet identified. Emerging data suggests a chronic disease model for AD with latency, prodrome, and dementia stages together lasting decades. Free radical damage to lipids in brain is one pathogenic process of AD that may be quantified with F(2)-isoprostanes (IsoPs). Whereas brain and cerebrospinal fluid (CSF) F(2)-IsoPs are reproducibly elevated in AD patients at both dementia and prodromal stages of disease, plasma and urine F(2)-IsoPs are not reproducibly increased in AD patients. CSF F(2)-IsoPs may be used to assist in diagnosis and aid in objective assessment of disease progression and response to therapeutics in patients with AD.

Identification of novel autoxidation products of the omega-3 fatty acid eicosapentaenoic acid in vitro and in vivo.

Increased intake of fish oil rich in the omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5 omega-3) and docosahexaenoic acid (DHA, C22:6 omega-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.

Effects of vitamin E on oxidative stress and atherosclerosis in an obese hyperlipidemic mouse model.

Vitamin E is a natural antioxidant that has been used in animal and human studies to determine its potential in reducing cardiovascular risk; however, a detailed study in an established obese model of atherosclerosis has yet to be performed. In our current study, we show that obesity and hyperlipidemia cause a synergistic, age-related increase in urinary isoprostane levels in mice deficient in both leptin and low-density lipoprotein receptor (ob/ob;LDLR-/-). Based upon this observation, we hypothesized that vitamin E supplementation would induce potent antiatherogenic effects in this model. Lean and obese LDLR-/- mice were provided vitamin E (2000 IU/kg) in a Western-type high-fat diet for 12 weeks. Plasma lipid parameters, such as total cholesterol (TC), triglyceride (TG) and free fatty acid, were significantly higher in obese mice compared to lean mice at baseline (P<.001). Western-type diet (WD) feeding caused an increase in TC levels in all groups (P<.001); however, TG (P<.001) and free fatty acid (P<.01) were elevated only in lean mice following WD feeding. Vitamin E supplementation neither influenced any of these parameters nor reduced urinary isoprostanes in lean or obese mice. Vitamin E supplementation in ob/ob;LDLR-/- mice resulted in a trend toward a reduction in atherosclerotic lesion area (P=.10), although no differences in lesion area were noted in lean LDLR-/- animals. These data provide evidence that vitamin E supplementation is not sufficient to reduce extreme elevations in systemic oxidative stress due to hyperlipidemia and obesity and, thus, may not be cardioprotective in this setting.

In vivo and in vitro lipid peroxidation of arachidonate esters: the effect of fish oil omega-3 lipids on product distribution.

The effect of lipid composition on the distribution of free radical oxidation products derived from arachidonic acid (20:4) esters has been studied in vitro and in vivo. Pro-inflammatory prostaglandin (PG) F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo and in vitro by the free radical-catalyzed peroxidation of arachidonic acid. Controlled free radical oxidation of mixtures of fatty acid esters in vitro showed that the formation of IsoPs from arachidonate is dramatically influenced by the presence of other fatty acid esters in the reaction mixture. Thus, three lipid mixtures containing the same arachidonate concentration but different amounts of other fatty esters (16:0; 18:1; 18:2; 20:5, and 22:6) were oxidized, and the product yields were determined by GC and LC/MS/MS analysis. The yield of F2-IsoP formed after 1 h of oxidation was 18% (based on arachidonate consumed) for mixtures containing arachidonate as the only oxidizable PUFA, but yields of these biologically active compounds dropped to 6% in polyunsaturated fatty acid (PUFA) mixtures typical of those found in tissues of fish oil-fed animals. F2-IsoP levels were also monitored in the livers of mice on diets supplemented with eicosapentaenoic acid (C20:5 omega-3; EPA), the PUFA most abundant in fish oil. While the level of arachidonic acid present in livers was not significantly different from that in control animals, levels of IsoPs in the liver were reduced in the EPA-fed mice compared to those in controls under conditions of oxidative stress (60 +/- 25% reduction, n = 5) or at baseline (48 +/- 14% reduction, n = 5). These results suggest that dietary omega-3 PUFAs may influence the formation of bio-active peroxidation products derived from omega-6 PUFAs by channeling the free radical pathway away from the F2-IsoPs.

Nitric oxide deficiency promotes vascular side effects of cyclooxygenase inhibitors.

The cardiovascular safety of COX-2 selective and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) has recently been called into question. The factors that predispose to adverse events by NSAIDs are unknown. Because patients with arthritis have decreased nitric oxide (NO) bioavailability, the in vivo effects of NSAIDs on murine vascular tone and platelet activity in the presence or absence of NO were examined. Here, we show that acute hypertensive and prothrombotic activities of the COX-2-selective inhibitor celecoxib are revealed only after in vivo inhibition of NO generation. The nonselective NSAID indomethacin was hypertensive but antithrombotic when NO was absent. In vitro myography of aortic rings confirmed that vasoconstriction required inhibition of both NOS and COX-2 and was abolished by supplementation with exogenous NO. These data indicate that NO suppresses vascular side effects of NSAIDs, suggesting that risk will be greatest in patients with impaired vascular function associated with decreased NO bioavailability.

Brassica vegetable consumption reduces urinary F2-isoprostane levels independent of micronutrient intake.

Isothiocyanates and indoles (e.g. indole-3-carbinol) from Brassica vegetables (e.g. broccoli) induce Phase I and Phase II enzymes responsible for the oxidation, reduction and metabolism of endogenous and exogenous carcinogens. Brassica vegetables also contain micronutrients that may provide additional DNA protection from reactive oxygen species. This randomized crossover trial (n = 20) compares the effects of a Brassica Vegetable (BV) intervention against a Micronutrient and Fiber Supplementation (M+F) intervention on urinary F2-isoprostane levels (F2-iP), a stable biomarker of systemic oxidative stress. Brassica intake was monitored by repeated 24 h recalls, urinary ITC levels and questionnaire. Urinary F2-iP levels were measured by mass spectrometry from first-morning urine samples collected at Baseline and after each intervention, and change in natural log transformed urinary F2-iP levels were analyzed using repeated measures regression. Brassica consumption increased from 2 grams/day (g/d) during the Baseline or M+F intervention periods to 218 g/d during the BV intervention, whereas exposure to most antioxidant vitamins and minerals was greatest during the M+F intervention. F2-iP levels significantly decreased by 22.0 or 21.8% during the BV intervention compared with Baseline or the M+F intervention (P = 0.05, P = 0.05, respectively). Urinary F2-iP levels did not significantly differ between Baseline and the M+F intervention (difference = 0.2%; P = 0.98). Brassica intake has been associated with reduced risk of colon, lung, bladder, breast, prostate and other cancers. Our results suggest that Brassica consumption reduces systemic oxidative stress independent of the vitamin and mineral content of these vegetables.

Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid.

Eicosapentaenoic acid (EPA, C20:5, omega-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 omega-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 microg/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.

Effect of alpha-tocopherol supplementation on plasma homocysteine and oxidative stress in highly trained athletes before and after exhaustive exercise.

The interrelationship between physical exercise, antioxidant supplementation, oxidative stress and plasma levels of homocysteine (Hcy) has not been adequately examined. The purpose of this study was to examine the effect of 2 months of vitamin E supplementation (800 IU/day alpha-tocopherol) (E) or placebo (P) in 38 triathletes on plasma Hcy concentrations, antioxidant potential and oxidative stress. It was hypothesized that vitamin E supplementation would reduce plasma Hcy and oxidative stress markers compared to placebo. Blood samples were collected 1 day prior to the race, immediately postrace and 1.5 h postrace. Plasma alpha-tocopherol was 75% higher (P<.001) in E versus P prerace (24.1+/-1.1 and 13.8+/-1.1 micromol/L, respectively), and this group difference was maintained throughout the race. Cortisol was significantly increased in both E and P (P<.001), but there was no difference in the pattern of change. There were no significant time, group or interaction effects on plasma Hcy concentrations between E and P. Plasma F(2)-isoprostanes increased 181% versus 97% during the race in E versus P, and lipid hydroperoxides were significantly elevated (P=.009) 1.5 h postrace in E versus P. Plasma antioxidant potential was significantly higher 1.5 h postrace in E versus P (P=.039). This study indicates that prolonged large doses of alpha-tocopherol supplementation did not affect plasma Hcy concentrations and exhibited pro-oxidant characteristics in highly trained athletes during exhaustive exercise.

In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets.

Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.

Antioxidant supplementation prevents exercise-induced lipid peroxidation, but not inflammation, in ultramarathon runners.

To determine if 6 weeks of supplementation with vitamins E and C could alleviate exercise-induced lipid peroxidation and inflammation, we studied 22 runners during a 50 km ultramarathon. Subjects were randomly assigned to one of two groups: (1) placebos (PL) or (2) antioxidants (AO: 1000 mg vitamin C and 300 mg RRR-alpha-tocopheryl acetate). Blood samples were obtained prior to supplementation (baseline), after 3 weeks of supplementation, 1 h pre-, mid-, and postrace, 2 h postrace and for 6 days postrace. Plasma levels of alpha-tocopherol (alpha-TOH), ascorbic acid (AA), uric acid (UA), F2-isoprostanes (F2-IsoPs), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and C-reactive protein (CRP) were measured. With supplementation, plasma alpha-TOH and AA increased in the AO but not the PL group. Although F2-IsoP levels were similar between groups at baseline, 28 +/- 2 (PL) and 27 +/- 3 pg/ml (AO), F2-IsoPs increased during the run only in the PL group (41 +/- 3 pg/ml). In PL women, F2-IsoPs were elevated postrace (p <.01), but returned to prerace concentrations by 2 h postrace. In PL men, F2-IsoP concentrations were higher postrace, 2 h postrace, and 1, 2, 3, 4, and 6 days postrace (PL vs. AO group, each p <.03). Markers of inflammation were increased dramatically in response to the run regardless of treatment group. Thus, AO supplementation prevented endurance exercise-induced lipid peroxidation but had no effect on inflammatory markers.