Four decades ago, Danish medical students Jørn Dyerberg and Hans Olaf Bang traveled west across the Greenland ice sheet on dogsleds to test a theory. For many years prior to their journey, there had been anecdotal reports that Greenland Eskimos had an extremely low incidence of heart disease, and Dyerberg and Bang speculated that this was linked to the high levels of polyunsaturated fatty acids (PUFAs) in the fish the native people consumed on a daily basis. After collecting and analyzing scores of blood samples, their hypothesis was borne out, and ever since, the medical and scientific community has been on a quest to determine exactly how PUFAs impart protective effects, and what amount must be ingested in order to achieve such benefits. Nearly 40 years and thousands of published studies later, however, these questions remain largely unanswered.

The PUFA mystery

Cardiovascular disease can affect any part of the circulatory system, from the heart and major arteries to veins and capillaries. Its causes are diverse, as are its treatments, which include compounds that exert vasodilating, anti-inflammatory, anti-thrombotic (reducing the formation of blood clots), anti-arrhythmic (suppressing abnormal heart rhythms), and heart rate–lowering effects. PUFAs from PUFA-rich foods and dietary supplements have shown therapeutic promise in virtually all of these areas. One of the more intriguing therapeutic potentials for n-3 PUFAs is in the treatment and prevention of heart failure.1 PUFAs in fish oil, in particular docosahexaenoic acid (DHA), have been shown in several animal models of heart failure to improve cardiac function and the efficiency with which the organ pumps blood. These findings have been supported recently by placebo-controlled clinical trials showing that daily intake of DHA and another fish-oil PUFA, eicosapentaenoic acid (EPA), for at least a year improved left ventricular function and exercise capacity in patients with established heart failure.2 Furthermore, clinically significant changes in left ventricular function have been reported as early as 3 months after initiating n-3 PUFA treatment.3

The results of large-scale meta-analyses and clinical trials involving PUFAs and heart-dis­ease risk have been mixed, raising concerns that initial evidence regarding their effectiveness was misleading.

Despite these promising outcomes, the results of large-scale meta-analyses and clinical trials involving PUFAs and heart-disease risk have been mixed, raising concerns that initial evidence regarding their effectiveness was misleading. A review published in September in The Journal of the American Medical Association, for example, found that increased PUFA intake failed to reduce the risk of stroke, heart attack, or death.4 Reasons for this disparity can be attributed to a variety of factors, including studies that lack sufficient statistical power and the use of differing methodologies to determine serum and tissue levels of PUFAs. Probably the greatest limitation to properly evaluating the results of many clinical trials is that they varied so widely in the type of n-3 PUFA given, dose, formulation (e.g. capsules or oil), and duration of intake. This widespread variation reflects the paucity of understanding regarding mechanism. If we better understood exactly how these compounds act in the body, then clinical trials regarding their use could be vastly improved and designed to be more reproducible. What researchers have learned about mechanisms of n-3 PUFA therapy has led us to propose a novel hypothesis that may help reconcile the controversy, uniting well-characterized n-3 PUFA effects with as-yet unresolved questions.

© THOM GRAVES

Metabolic fate of PUFAs

Because long-chain fatty acids are subjected to many different pathways of enzymatic and nonenzymatic metabolism, the identification of exactly how PUFAs are therapeutically effective for cardiovascular ailments has been particularly elusive. Until recently, researchers have focused most of their attention on the incorporation of EPA and DHA into membrane phospholipids, and the enzymatic oxidation of these compounds into signaling molecules called eicosanoids. (See illustration on opposite page.) Experimental evidence suggests that increasing levels of EPA/DHA in membranes reduces platelet aggregation and lowers expression of genes that promote inflammation and the accumulation of fatty acids in the arteries.

Further support for eicosanoid-mediated effects of n-3 PUFAs comes from the discovery by Charles Serhan of Harvard Medical School and colleagues of an exciting new family of eicosanoids derived from EPA and DHA.5 This group of compounds has potent properties that help put out the “fire” of inflammation, leading to their designation as resolvins, and they are currently being studied for their pain-relief potential, among other therapeutic effects. (See “Resolving Chronic Pain,” The Scientist, January 2012.) These benefits aside, burgeoning experimental evidence suggests that attributing the benefits of n-3 PUFA therapy for treatment of cardiovascular disease solely to eicosanoid-mediated effects is grossly oversimplistic.

The physiological consequences of n-3 PUFAs becoming incorporated into membrane phospholipids is particularly important in highly oxidative, excitable tissues such as the heart. Here, phospholipid composition is critical for proper membrane structure, thereby ensuring the maintenance of ion channel activity, charge separation, and energy conservation—all necessary for proper cardiac function. Several studies have proposed that EPA and DHA directly modify the exchange of ions through the cardiomyocyte plasma membrane, which may partially explain the PUFAs’ anti-arrhythmic properties.

© THOM GRAVESPUFAs have also been shown to become incorporated into organelle membranes inside the cell. One possible PUFA incorporation site is cardiolipin, a phospholipid unique to mitochondria that helps maintain the electrochemical gradient necessary for oxidative phosphorylation, which generates energy to fuel the cell. Recent findings in animal models of heart failure have demonstrated that altering cardiolipin structure results in increased left ventricular function similar to the effect seen with EPA/DHA treatment. However, it is not yet clear how altering cardiolipin structure by EPA/DHA incorporation would enhance mitochondrial function and improve cardiac energetics.

Finally, aside from eicosanoid synthesis and other well-characterized enzymatic pathways in which PUFAs are known to become oxidized, these compounds are prone to nonenzymatic, spontaneous oxidation because of their highly unsaturated structure. (See illustration above, left.) Carbon-carbon double bonds in PUFAs present prime targets for oxygen or lipid radicals. These reactions ultimately form lipid peroxides, reactive aldehydes, and other electrophilic lipids that have very diverse biological effects.

Of all the PUFAs, DHA is the most susceptible to spontaneous oxidation, due to its chemical structure. To date, such nonenzymatic oxidation pathways and their products have been largely ignored by investigators, partly due to the suspicion that the rate of nonenzymatic n-3 PUFA oxidation in vivo is negligible. Also, because the dogma regarding lipid peroxides has always been that they are toxic and undesirable, the possibility that they may be involved in mediating the beneficial effect of n-3 PUFAs is counterintuitive.

But accumulating evidence suggests that spontaneously oxidized PUFAs can be beneficial in many contexts, due largely to the fact that they are highly reactive agonists for certain receptors. For example, recent reports demonstrate that oxidized DHA has a high affinity for the peroxisome proliferator-activated receptor (PPAR) family of transcription factors, which regulate cellular differentiation, development, metabolism, and tumorigenesis. Moreover, oxidized DHA has a greater PPAR-activating effect than any other PPAR ligand tested.6 These findings could have broad clinical implications because they indicate that DHA peroxidation in vivo could greatly enhance its potency as a PPAR agonist—a class of widely prescribed drugs that treat a variety of ailments, from high cholesterol to type 2 diabetes.

Additionally, beyond the beneficial roles for the oxidized PUFAs themselves, the by-products of PUFA-derived “lipoxidative stress”—the very molecules that are thought to induce harm—could, in fact, be doing the body good.

© MARCELA BARSSE/ISTOCKPHOTO.COM

PUFAs and stress

Mithridates VI, king of Pontus and Armenia Minor in northern Anatolia (now Turkey) from about 120 BC to 63 BC, was a forward-thinking and perceptive individual who understood that a little bit of stress can be a good thing. Terrified of succumbing to the same fate as his father, who was assassinated by poisoning at his own banquet, Mithridates began ingesting sublethal doses of poisons to develop immunity to them, a real-life example of The Princess Bride’s Westley.

The benefit of this practice, which in modern times is known as “hormesis,” is believed to stem from the fact that in low, subtoxic amounts, poisons, toxins, and other types of stress will upregulate antioxidants and detoxification enzymes in the liver, heart, and other major organs, thereby augmenting the natural ability of the body to detoxify and protect itself against future exposure to those same toxins. Could that be what’s happening with n-3 PUFAs in the heart? Could the highly reactive oxidized products generated from PUFA oxidation cause adaptations in the heart—such as biochemical/biophysical alterations in membranes and the upregulation of cardio-protective genes—that subsequently protect the vital organ against disease and stress?

It has long been known that 4-hydroxynonenal, an aldehyde formed from the oxidation of n-6 PUFAs (primarily found in corn and vegetable oils and a huge part of our diet), can both help and harm the heart. At subtoxic concentrations (≤10µM), 4-hydroxynonenal exerts a beneficial “hormetic” effect that activates antioxidant response gene pathways. At higher concentrations, however, 4-hydroxynonenal causes cell death. Yan Zhang of the Keio University School of Medicine in Tokyo and colleagues recently showed that treating cardiomyocytes with small, subtoxic doses (5µM) of 4-hydroxynonenal offers protection from subsequent exposure to toxic doses (≥20µM).7 This group further showed the physiological relevance of this effect by pretreating mice with 4-hydroxynonenal prior to restriction of their coronary blood supply, and showed that treated mice fared better: less of the ventricular tissue died following the simulated heart attack.

Scientists are only just beginning to elucidate the molecular mediators of these positive responses to hormesis, but recent studies have pointed to a number of possibilities, including the upregulation of amino acid biosynthesis, increased expression of antioxidant/anti-inflammatory genes, and mitochondrial biogenesis. Some studies, including the one just described by Zhang and colleagues, have specifically implicated the involvement of the transcription factor NF-E2-related factor-2 (Nrf2) in upregulating detoxification and antioxidant genes in response to PUFA oxidation. Furthermore, a recent study in our laboratory showed that mice fed a high-fat diet enriched with n-3 PUFAs showed an increase in n-3 PUFA-derived 4-hydroxyhexenal and upregulation of Nrf2-mediated enzymes in the heart.8 The net result was a large increase in antioxidant enzyme activity, decreased production of mitochondrial reactive oxygen species (ROS), and augmented levels of the antioxidant glutathione and related enzymes.

A number of other studies in animals have similarly reported increased expression of antioxidant/anti-inflammatory enzymes in the heart following n-3 PUFA dietary supplementation, and clinical studies have shown similar effects in humans. Several trials have reported marked increases in antioxidant enzyme levels in the blood of patients taking n-3 PUFAs, and in some cases, lipid peroxidation was shown to precede the elevation in these enzymes. Furthermore, a small clinical trial conducted by Rodrigo Castillo and colleagues at the University of Chile recently showed that 2 weeks of fish oil and vitamin E treatment before cardiac revascularization surgery augmented antioxidant activity and suppressed the activation of pro-inflammatory transcription factor NFκB in the myocardium, leading to reduced inflammation and oxidative stress during the procedure.9

Could the highly reactive oxidized products generated from PUFA oxidation cause adaptations in the heart that subsequently protect the vital organ against disease

and stress?

It is further possible that a large part of the electrophysiological effects attributed to n-3 PUFAs may be dependent on their oxidation. An interesting study led by Sébastien Judé of Nutrition, Croissance et Cancer in France showed that the electrophysiological effects of DHA on the transient outward current in cardiomyocytes were only present when the DHA was oxidized with a small amount of hydrogen peroxide; DHA on its own was much less effective.10 This finding led the authors to speculate that perhaps it is oxidized derivatives of DHA that are responsible for many of the electrophysiological effects of DHA observed to date—primarily in culture dishes, where DHA is exposed to room air and thus likely to be oxidized.

These results point to the idea that the products formed during PUFA oxidation contribute to the cardiac benefits observed so far. If true, this mechanism would be very important from a clinical perspective: low levels of oxidative stress could theoretically protect the heart from a broad array of other stressors, including metabolic disease, infection, aging, and ischemia. So, it seems, Mithridates may not have been original in his idea to build up tolerance by exposing himself to sublethal levels of poison; the natural biological processes at work in his own body may have already been employing the same strategy.

Future directions

If n-3 PUFA-derived lipoxidative products are indeed at least partially responsible for the therapeutic effects observed, it would follow that any physiological state resulting in sustained oxidative stress—such as cardiovascular or metabolic disease—would drive increased lipoxidative product formation in the presence of EPA and DHA. This hypothesis is controversial because it contradicts existing paradigms regarding the relationship between oxidative stress and disease, but we believe that the evidence presented above certainly seems to challenge those paradigms.

It must be emphasized, however, that 4-hydroxyhexenal, and indeed all n-3 PUFA-derived lipoxidative products, will affect tissues and organ systems differently depending on their ability to adapt positively to mild lipoxidative stress. Thus, the protective effects of these compounds would be manifested to the greatest extent in organs with large antioxidant capacities, such as the heart. Moreover, it is expected that this adaptation would require several days or weeks to become optimal, a fact that could explain why long-term intake of PUFAs appears to be the most beneficial. These considerations could inform the development of relevant and proper dosing strategies for n-3 PUFA use in clinics. Consequently, the notion that many of the broad effects of n-3 PUFAs in cardiovascular disease can be explained by lipoxidative products derived from them is deserving of rigorous evaluation.



Ethan J. Anderson is an assistant professor and David A. Taylor is a professor and chairman in the Department of Pharmacology and Toxicology at East Carolina University (ECU) in Greenville, North Carolina. Anderson is also an affiliate member of the East Carolina Diabetes and Obesity Institute at ECU.

This article is adapted from a review in F1000 Medicine Reports, DOI:10.3410/M4-13 (open access).