Effect of fish and fish oil-derived omega-3 fatty acids
on lipid oxidation
School of Medicine and Pharmacology, The University of Western Australia; Cardiovascular Research Centre; and The Western Australian Institute for Medical Research, Perth, Western Australia, Australia There is evidence that omega-3 (ω3) fatty acids derived from fish and fish oils reduce the risk ofcardiovascular disease via mechanisms underlying atherosclerosis, thrombosis and inflammation.
Despite these benefits, there has been concern that these fatty acids may increase lipid peroxidation.
However, the in vivo data to date are inconclusive, due in part to limitations in the methodologies. Inthis regard, our findings using the measurement of F -isoprostanes, a reliable measure of in vivo lipid peroxidation and oxidant stress, do not support adverse effects of ω3 fatty acids on lipidperoxidation.
Keywords: Fish oil, omega-3 fatty acids, lipid oxidation, F -isoprostanes There is now considerable evidence that a diet rich in ω3 dation and oxidative stress in the pathogenesis of cardio- fatty acids derived from fish and fish oils, specifically vascular disease.8 It is thought that oxidative stress and eicosapentaenoic acid (EPA, 20:5 ω3) and docosa- oxidized lipids play a critical part in the genesis and pro- hexaenoic acid (DHA, 22:6 ω3), protects against athero- gression of the atherosclerotic lesion.8 The hypothesis that sclerotic heart disease, myocardial infarction and sudden increased intake of ω3 fatty acids may lead to increased death.1 ω3-Fatty acids have a wide range of biological lipid peroxidation is based on the premise that fatty acid effects, including benefits on lipoprotein metabolism, oxidizability increases with an increase in the number of platelet and endothelial function, blood pressure, vascu- double bonds in the fatty acid chain.9 Whilst this may be lar reactivity, cytokine production, coagulation and fibri- true of in vitro studies of lipid peroxidation in homoge- nolysis.1–3 Recent evidence also has demonstrated that, neous solutions, in vivo systems are more complicated and in humans, EPA and DHA have differential effects on influenced by additional factors. In support of this, Visioli lipids and lipoproteins,4 blood pressure5 and heart rate,5 et al.10 showed that, under equal conditions of oxidative Published by Maney Publishing (c) W. S. Maney & Son Limited stress, fatty acids oxidized at different rates and generated Despite the benefits associated with increased ω3 fatty different oxidation products, in a manner that was unre- acid consumption, there remains a theoretical concern that lated to their degree of unsaturation. Lipid peroxide levels these fatty acids may increase the unsaturation index, con- were in fact highest following oxidation of linoleic acid sequent to the incorporation of EPA and DHA into mem- (18:2 ω6).10 Arachidonic acid (20:4 ω6) and DHA gener- branes and lipoproteins, leading to increased lipid ated lower levels of lipid peroxides, with lowest levels peroxidation.7 The significance of this relates to the fact arising from oxidation of EPA. Formation of conjugated that there is much evidence supporting a role for lipid oxi- dienes was also maximal for 18:2 ω6. In particular, theproduction of conjugated dienes from 20:4 ω6 and EPAwas approximately 25% of that of 18:2 ω6. DHA oxida- tion yielded only 10% of the conjugated dienes relative to Correspondence to: Dr Trevor A. Mori, School of Medicine and The data relating to the effects of ω3 fatty acids on lipid Pharmacology, Medical Research Foundation Building, Box X 2213 peroxidation and oxidative stress in vivo are contradictory.7 GPO, Perth, Western Australia 6847, AustraliaTel: +61 8 9224 0273; Fax: +61 8 9224 0246; These inconsistencies may be related to differences in the populations studied, the quantity and presentation of the gas chromatography mass spectrometry. In addition,plasma TBARS were significantly elevated following ω3 fatty acids, whereas MDA was reduced. Whenplasma MDA levels were normalized to plasma polyun-saturated fatty acid concentrations, significant differ-ences were eliminated.25 Other measures of oxidative stress include electron spin resonance detecting free radical species,24 measure-ment of antibodies to oxidized LDL31 and breath excre-tion of ethane and pentane.24 The latter assay, however,has yielded variable results in animals32 and humans27supplemented with ω3 fatty acids. Other literaturereports have shown that ω3 fatty acids had no adverseeffects on plasma protein oxidation26 and rendered ery-throcytes more resistant to haemolysis following oxida-tive challenge.33 On the basis of studies reporting adverse effects on Fig. 1. Change in urinary F -isoprostane excretion from baseline to post-
intervention by intervention group. Mean ± SEM. P < 0.013 for the main lipid peroxidation, some researchers have suggested that effect of fish after adjustment for baseline values, using general linear ω3 fatty acids may affect the antioxidant status and, models (GLM). Reproduced from Mori et al.41 with permission.
therefore, should be taken in conjunction with vitaminsupplementation. The data from such studies, however,are inconclusive.16,27,34–37 ω3 fatty acids (fish versus encapsulated fish oils), the Although most of the above-mentioned methods rep- duration of the study, the study design, the antioxidant resent different aspects of lipid oxidation and collec- content of the supplement, or the composition of the tively provide some knowledge of oxidative damage, background diet. It also has also been suggested that the none is considered a reliable measure of oxidative stress.
total concentration of polyunsaturates, rather than the In this regard, the F -isoprostanes are prostaglandin-like unsaturation index, may be more important in determin- metabolites of free radical peroxidation of arachidonic ing lipid peroxidation.11 However, the most plausible acid38 and there is now good evidence that they provide a explanation for the inconsistency between studies is dif- reliable measure of in vivo oxidative stress.39,40 In sup- ferences in the methodologies employed to assess lipid port of this, elevated F -isoprostanes have been reported in animal models of free radical injury, in human condi- Much of the literature regarding the effect of ω3 fatty tions associated with increased oxidative stress, and in acids on lipid peroxidation is based on indirect and/or non-specific assays. Studies utilizing the oxidizability of Using F -isoprostanes, measured by gas chromatogra- LDL have shown either enhanced,12–17 or reduced, or no phy–mass spectrometry, we have demonstrated that effect,11,18–22 of ω3 fatty acids. In this assay, LDL is iso- these metabolites are significantly reduced following lated from plasma and then subjected to oxidative condi- consumption of ω3 fatty acids taken as fish oils or in fish tions. It is feasible that in patients on various meals. We showed that fish meals providing approxi- Published by Maney Publishing (c) W. S. Maney & Son Limited medications, LDL oxidizability could be affected by par- mately 3.6 g/day of ω3 fatty acids for 8 weeks to Type 2 tition of drugs into the LDL. Indeed, a divergence has diabetic patients, significantly (P = 0.013) reduced uri- been shown between the measurement of LDL oxidative nary F -isoprostanes by 20% (Fig. 1).41 Relative to a susceptibility and urinary F -isoprostanes as a measure control group, urinary F -isoprostanes were reduced by 0.83 nmol/24-h. This effect was independent of age, One of the most common methods for assessment of gender, body weight change and the increase in ω3 fatty lipid peroxidation measures thiobarbituric acid reactive acids or the fall in ω6 fatty acids in plasma, platelets and substances (TBARS) by a colorimetric assay. This assay has been widely criticized on the basis of its lack of We recently have shown that cord plasma and urinary specificity and results need to be interpreted with care.24 F -isoprostanes were reduced in infants whose mother Studies of ω3 fatty acids using this methodology have received fish oil during pregnancy.42 Pregnant atopic shown either elevated levels16,17,25–28 or no change29,30 in women received 4 g daily fish oil or olive oil from 20 TBARS. The limitation of this assay was exemplified by weeks’ gestation. Cord plasma F -isoprostanes were sig- Higdon et al.25 who showed that, in post-menopausal nificantly lower (P < 0.001) in the offspring of women women given ω3 fatty acids, plasma TBARS were 10- who had taken fish oil during pregnancy compared with fold higher than malondialdehyde (MDA) measured by those who took olive oil (Fig. 2). These differences were Effect of fish and fish oil-derived omega-3 fatty acids on lipid oxidation mildly hyperlipidaemic men, supplementation with 4 gdaily of purified EPA or DHA for 6 weeks decreased post-intervention urinary F -isoprostane levels by 27% follow- ing EPA (1.24 nmol/24-h, P < 0.0001) and 26% followingDHA (1.20 pmol/24-h, P < 0.0001), relative to an olive oilcontrol group, after adjusting for baseline values (Fig.
3A).44 In a study of similar design in treated hypertensiveType 2 diabetic patients, we showed that post-interventionurinary F -isoprostanes were reduced 19% by EPA (P = 0.017) and 20% by DHA (P = 0.014), relative to an oliveoil group (Fig. 3B).45 In each of these studies,41,42,44,45 the changes in F -iso- prostanes were unrelated to changes in EPA, DHA, 20:4 ω6, total ω3 or ω6 fatty acids. This lack of associationwith changes in fatty acids is noteworthy, in view of the Fig. 2. Cord plasma F -isoprostane excretion in neonates whose mothers
fact that F -isoprostanes are derived from free radical were supplemented with fish oil or olive oil during pregnancy. Mean ± oxidation of arachidonic acid, which is significantly SEM. Between group differences were assessed using GLM. *P < 0.001 reduced following ω3 fatty acids. Therefore, the changes after adjustment for red cell 20:4 ω6. Reproduced from Barden et al.42 in F -isoprostanes most likely reflect a true reduction in oxidative stress, rather than as result of a reduction in thesupply of substrate.
independent of red cell 20:4 ω6 levels. Urinary F -iso- How F -isoprostanes are reduced following ω3 fatty prostanes corrected for creatinine excretion tended to be acid supplementation remains unresolved. We suggested lower in infants whose mother had taken fish oil (P = 0.06).
this might be explained, at least in part, by the anti- Our findings are in accordance with several other studies inflammatory effects of ω3 fatty acids and a reduction in in which ω3 fatty acids have been supplemented. Quaggiotto leukocyte free radical formation. Activated leukocytes et al.43 showed that, compared to beef tallow, high doses of generate powerful oxidants during phagocytosis46 and ω3 fatty acids reduced plasma F -isoprostanes after coronary cytokines such as TNF-α and IL-6 stimulate leukocytes occlusion in pigs. Similarly, Higdon et al.25 demonstrated a and endothelial cells to generate free radicals, further fall in plasma F -isoprostanes in post-menopausal women propagating the pro-oxidant condition. In support of this given ω3 fatty acids compared with diets enriched in oleate hypothesis, we showed that the changes in urinary F - or linoleate. In the latter study, however, significant differ- isoprostanes were significantly positively associated ences were eliminated when F -isoprostanes were adjusted with changes in TNF-α concentration.45 Numerous stud- for plasma arachidonic acid concentrations.25 ies have demonstrated anti-inflammatory actions of ω3 We have demonstrated in two trials that both EPA and fatty acids, with falls in cytokines most often observed DHA equally reduced F -isoprostanes.44,45 In overweight, following leukocyte stimulation.47 ω3-Fatty acids also Published by Maney Publishing (c) W. S. Maney & Son Limited Fig. 3. Change in urinary F -isoprostanes in (A) overweight, hyperlipidaemic men and (B) hypertensive Type 2 diabetic patients. Mean ± SEM. *P < 0.01
for a treatment effect (GLM). Reproduced from Mori et al.44,45 with permission.
have been shown to suppress production of reactive oxy- Simopoulos AP, Kifer RR, Martin RE, Barlow SM. (eds) World gen species (superoxide and hydrogen peroxide) in stim- Review of Nutrition and Dietetics, vol. 66. Basel: Karger, 1991; 1–592.
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clear leukocytes, as well as in human monocytes.48,53 Mori TA, Burke V, Puddey IB et al. Purified eicosapentaenoic and Other potential mechanisms may relate to the assembly docosahexaenoic acids have differential effects on serum lipids and of ω3 fatty acids in membrane lipids and lipoproteins lipoproteins, LDL particle size, glucose, and insulin in mildlyhyperlipidemic men. making the double bonds less susceptible to free radical Mori TA, Bao DQ, Burke V, Puddey IB, Beilin LJ. Docosahexaenoic attack,55 inhibition of the pro-oxidant enzyme phospholi- acid but not eicosapentaenoic acid lowers ambulatory blood pressure pase A ,56 and stimulation of antioxidant enzymes.57,58 In this regard, there is evidence that ω3 fatty acids up-regu- Mori TA, Watts GF, Burke V et al. Differential effects of late gene expression of antioxidant enzymes and down- eicosapentaenoic acid and docosahexaenoic acid on forearm vascularreactivity of the microcirculation in hyperlipidaemic, overweight men.
regulate genes associated with production of reactive Nenseter MS, Drevon CA. Dietary polyunsaturates and peroxidation Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL.
Beyond cholesterol. Modifications of low-density lipoprotein thatincrease its atherogenicity. Liu J, Yeo HC, Doniger SJ, Ames BN. Assay of aldehydes from lipid There is no evidence for a pro-oxidant effect of ω3 fatty peroxidation: gas chromatography-mass spectrometry compared to acids. Our findings and the recent literature demonstrate that ω3 fatty acids do not adversely affect, and indeed 10. Visioli F, Colombo C, Galli C. Oxidation of individual fatty acids may attenuate, oxidative stress. The results clearly high- yields different profiles of oxidation markers. light the need for caution in choosing methodologies for 11. Brude IR, Drevon CA, Hjermann I et al. Peroxidation of LDL from the assessment of oxidative stress. Further studies are combined-hyperlipidemic male smokers supplied with omega-3 fatty also required to explore potential mechanisms for the observation of an association between oxidative stress, markers of inflammation and atherosclerosis following 12. Suzukawa M, Abbey M, Howe PR, Nestel PJ. Effects of fish oil fatty acids on low density lipoprotein size, oxidizability, and uptake by 3 fatty acids. Nonetheless, there appears no reason why 3 fatty acids should not be taken either as fish meals or 13. Wander RC, Du SH, Ketchum SO, Rowe KE. Effects of interaction of fish oils capsules, in view of their overall favourable RRR-alpha-tocopheryl acetate and fish oil on low-density-lipoprotein effects on cardiovascular risk reduction.
oxidation in postmenopausal women with and without hormone-replacement therapy 14. Hau MF, Smelt AH, Bindels AJ et al. Effects of fish oil on oxidation resistance of VLDL in hypertriglyceridemic patients. Arterioscl Thromb Vasc Biol 1996; 16): 1197–1202.
15. Lussier-Cacan S, Dubreuil-Quidoz S, Roederer G et al. Influence of The studies described were supported by grants from the probucol on enhanced LDL oxidation after fish oil treatment of National Health and Medical Research Council of Australia, the West Australian Health Promotion 16. Harats D, Dabach Y, Hollander G et al. Fish oil ingestion in smokers Foundation (Healthway) and the Royal Perth Hospital Published by Maney Publishing (c) W. S. Maney & Son Limited and nonsmokers enhances peroxidation of plasma lipoproteins Medical Research Foundation. Purified eicosapen- taenoic and docosahexaenoic acids and olive oil cap- 17. Stalenhoef AF, de Graaf J, Wittekoek ME, Bredie SJ, Demacker PN, sules were kindly provided by the Fish Oil Test Kastelein JJ. The effect of concentrated n-3 fatty acids versusgemfibrozil on plasma lipoproteins, low density lipoprotein Materials Program and the US National Institutes of heterogeneity and oxidizability in patients with hypertriglyceridemia.
Health. I would like to acknowledge my collaborators, Assoc. Prof. Kevin Croft, Prof. Ian Puddey, Prof. Lawrie 18. Higgins S, Carroll YL, McCarthy SN et al. Susceptibility of LDL to Beilin, Dr Valerie Burke, Dr Anne Barden, Assoc. Prof.
oxidative modification in healthy volunteers supplemented with low Susan Prescott, Dr David Dunstan, Dr Richard doses of n-3 polyunsaturated fatty acids. 19. Bonanome A, Biasia F, De Luca M et al. n-3 fatty acids do not Woodman and Dr Jan Dunstan, and the technical assis- enhance LDL susceptibility to oxidation in hypertriacylglycerolemic 20. Nenseter MS, Rustan AC, Lund-Katz S et al. Effect of dietary supplementation with n-3 polyunsaturated fatty acids on physicalproperties and metabolism of low density lipoprotein in humans.
21. Frankel EN, Parks EJ, Xu R, Schneeman BO, Davis PA, German JB.
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F isoprostane levels are lowered in pigs fed an (n-3) polyunsaturated fatty acid supplemented diet following occlusion of the left anterior 25. Higdon JV, Liu JK, Du SH, Morrow JD, Ames BN, Wander RC.
Supplementation of postmenopausal women with fish oil rich in 44. Mori TA, Puddey IB, Burke V et al. Effect of ω3 fatty acids on eicosapentaenoic acid and docosahexaenoic acid is not associated with oxidative stress in humans: GCMS measurement of urinary F2- greater in vivo lipid peroxidation compared with oils rich in oleate and linoleate as assessed by plasma malondialdehyde and F -isoprostanes.
45. Mori TA, Woodman RJ, Burke V, Puddey IB, Croft KD, Beilin LJ.
Effect of eicosapentaenoic acid and docosahexaenoic acid on 26. Wander RC, Du SL. Oxidation of plasma proteins is not increased oxidative stress and inflammatory markers, in treated-hypertensive after supplementation with eicosapentaenoic and docosahexaenoic 46. Babior BM. Phagocytes and oxidative stress. 27. Allard JP, Kurian R, Aghdassi E, Muggli R, Royall D. Lipid peroxidation during n-3 fatty acid and vitamin E supplementation in 47. Calder PC. N-3 polyunsaturated fatty acids, inflammation and 28. Palozza P, Sgarlata E, Luberto C et al. n-3 fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration 48. Hiramatsu K, Arimori S. Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes.
29. Hansen JB, Berge RK, Nordoy A, Bonaa KH. Lipid peroxidation of isolated chylomicrons and oxidative status in plasma after intake of 49. Thompson PJ, Misso NL, Passarelli M, Phillips MJ. The effect of eicosapentaenoic acid consumption on human neutrophil 30. Ando K, Nagata K, Beppu M et al. Effect of n-3 fatty acid 50. Varming K, Schmidt EB, Svaneborg N et al. The effect of n-3 fatty supplementation on lipid peroxidation and protein aggregation in rat 31. Uusitupa MI, Niskanen L, Luoma J et al. Autoantibodies against 51. Fisher M, Levine PH, Weiner BH et al. Dietary n-3 fatty acid oxidized LDL do not predict atherosclerotic vascular disease in non- supplementation reduces superoxide production and chemiluminescence in a monocyte-enriched preparation of leukocytes.
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Published by Maney Publishing (c) W. S. Maney & Son Limited 35. Haglund O, Luostarinen R, Wallin R, Wibell L, Saldeen T. The effects 55. Applegate KR, Glomset JA. Computer-based modeling of the of fish oil on triglycerides, cholesterol, fibrinogen and conformation and packing properties of docosahexaenoic acid. malondialdehyde in humans supplemented with vitamin E. J Nutr 1991; 121: 165–169.
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