Long chain n–3 PUFAs (polyunsaturated fatty acids) are found in fatty fish and in fish oils. Substantial evidence from epidemiological and case-control studies indicates that consumption of fish, fatty fish and long-chain n–3 PUFAs reduces the risk of cardiovascular mortality. Secondary prevention studies using long-chain n–3 PUFAs in patients post-myocardial infarction have shown a reduction in total and cardiovascular mortality, with an especially potent effect on sudden death. Long-chain n–3 PUFAs have been shown to decrease blood triacylglycerol (triglyceride) concentrations, to decrease production of chemoattractants, growth factors, adhesion molecules, inflammatory eicosanoids and inflammatory cytokines, to lower blood pressure, to increase nitric oxide production, endothelial relaxation and vascular compliance, to decrease thrombosis and cardiac arrhythmias and to increase heart rate variability. These mechanisms most likely explain the primary and secondary cardiovascular protection afforded by long-chain n–3 PUFA consumption. A recent study suggests that long-chain n–3 PUFAs might also act to stabilize advanced atherosclerotic plaques, perhaps through their anti-inflammatory effects. As a result of the robust evidence in their favour, a number of recommendations to increase intake of long-chain n–3 PUFAs have been made.
- cardiovascular disease
- docosahexaenoic acid
- eicosapentaenoic acid
- fish oil
- long-chain n–3 fatty acid
- polyunsaturated fatty acid
n–3 FATTY ACIDS: NAMING, SOURCES AND METABOLISM
n–3 (or omega-3) Fatty acids are a family of naturally occurring PUFAs (polyunsaturated fatty acids). It is the position of the double bonds within their hydrocarbon chain that gives n–3 fatty acids their name and also their physical, and physiological, properties. In n–3 fatty acids, the terminal double bond (i.e. that closest to the methyl end of the hydrocarbon chain) is on C3 (Figure 1). In this respect, they are structurally distinct from the more commonly encountered n–6 (or omega-6) family of PUFAs (Figure 1). The simplest members of the n–6 and n–3 fatty acid families are linoleic (18:2n–6) and α-linolenic (18:3n–3) acids respectively. Each of these fatty acids has 18 carbons in its hydrocarbon chain (Figure 1). The n–6 and n–3 double bonds cannot be inserted into fatty acids by animal enzymes. Only plant enzymes (Δ12- and Δ15-desaturase respectively) can do this and so this means that linoleic and α-linolenic acids can only be formed in plants. It also means that linoleic and α-linolenic acids, and indeed n–6 and n–3 fatty acids in general, cannot be interconverted in animals. Although linoleic and α-linolenic acids cannot be synthesized in animals, mammals have a requirement for them, and experiments as long ago as the late 1920s identified symptoms of ‘essential fatty acid deficiency’ that are similar to other essential nutrient deficiencies [1,2]. It is estimated that minimum human requirements for these fatty acids are 1 and 0.2% of daily energy intake respectively . Because they are synthesized by plants, plant tissues and oils tend to be good sources of linoleic and α-linolenic acids. For example, green plant tissues are especially rich in α-linolenic acid, which typically comprises approx. 55% of the fatty acids present. However, such tissues are not rich in fat and therefore this source does not make a major contribution to intake of this fatty acid in humans. In contrast, plant oils used in cooking (e.g. corn oil, sunflower oil and rapeseed oil) and margarines made from such oils make a significant contribution to the intakes of these fatty acids. Most plant oils are much richer in linoleic than α-linolenic acid (indeed, many plant oils contain relatively little α-linolenic acid) and so intakes of the former are much greater in most Western populations. On average, adult men in the U.K. consume about 13.5 and 1.7 g of linoleic and α-linolenic acids respectively, per day, whereas adult women consume about 9.3 and 1.2 g of linoleic and α-linolenic acids respectively, per day [4,5]. These intakes equate to approx. 5 and 0.6% of dietary energy respectively, and are typical of intakes in Western countries . Plant oil and margarine consumption has increased greatly over the last 35 years or so. Thus the intake of linoleic acid in particular has increased markedly over this period. For example, in the U.K. average daily intake of linoleic acid among adults increased from approx. 11 g in 1970 to approx. 14 g in 1990 . A role for linoleic acid in cardiovascular health is in lowering of total and LDL (low density lipoprotein)-cholesterol concentrations when it replaces either saturated fatty acids or carbohydrate in the diet .
Although mammalian cells cannot synthesize linoleic and α-linolenic acids, they can metabolize them by further desaturation and elongation; desaturation occurs at carbon atoms below C9 (counting from the carboxyl carbon). Linoleic acid can be converted into γ-linolenic acid (18:3n–6) by Δ6-desaturase and then γ-linolenic can be elongated to dihomo-γ-linolenic acid (20:3n–6; Figure 2). Dihomo-γ-linolenic acid can be desaturated further by Δ5-desaturase to yield arachidonic acid (20:4n–6) (Figure 2). Using the same series of enzymes as used to metabolize n–6 PUFAs, α-linolenic acid is converted into EPA (eicosapentaenoic acid; 20:5n–3; Figure 2). In mammals, the pathway of desaturation and elongation occurs mainly in the liver. It is evident from the pathway shown in Figure 2 that there is competition between the n–6 and n–3 fatty acid families for metabolism. The Δ6-desaturase reaction is rate limiting in this pathway . Although the preferred substrate for Δ6-desaturase is α-linolenic acid , because linoleic acid is much more prevalent in most human diets than α-linolenic acid, metabolism of n–6 fatty acids is quantitatively the more important.
Further conversion of EPA into DHA (docosahexaenoic acid; 22:6n–3) involves addition of two carbons to form DPA (docosapentaenoic acid; 22:5n–3), two further carbons to produce 24:5n–3 and desaturation at the Δ6 position to form 24:6n–3  (Figure 2). Then two carbons are removed from 24:6n–3 by limited β-oxidation to yield DHA (Figure 2). Arachidonic acid can be metabolized by the same series of enzymes to yield, in turn, 22:4n–6, 24:4n–6, 24:5n–6 and 22:5n–6.
The long-chain, more unsaturated, derivatives of linoleic and α-linolenic acids have potent biological activities. For example, arachidonic acid is the substrate for synthesis of prostaglandins, thromboxanes and leukotrienes that have important roles in regulation of smooth muscle contraction, platelet aggregation, inflammation, immune function and cell proliferation [4,5]. However, the longer chain highly unsaturated fatty acids are consumed in relatively small amounts in most Western diets. The main dietary source of arachidonic acid is meat and meat products and estimates of intake of this fatty acid range from 50 and 300 mg/day for adults. A good dietary source of longer chain n–3 fatty acids is fish. Fish can be classified into lean fish that store fat as triacylglycerols (triglycerides) in the liver (e.g. cod) or ‘fatty’ (‘oily’) fish that store fat as triacylglycerols in the flesh (e.g. mackerel, herring, salmon and tuna). The oil obtained from fatty fish flesh or lean fish livers is termed ‘fish oil’ and it has the distinctive characteristic of being rich in long-chain n–3 fatty acids. Different oily fish (and so different fish oils) contain different amounts of n–3 fatty acids (Table 1). This relates to the dietary habits and metabolic characteristics of the fish as well as to season, water temperature, phase in the breeding cycle and so on. Average intake of the long-chain n–3 fatty acids in the U.K. is estimated at <250 mg/day . In the absence of fatty fish or fish oil consumption, α-linolenic acid is the by far the major dietary n–3 fatty acid. Recent studies have revealed that conversion of α-linolenic acid into its longer chain derivatives is not at all efficient in adult humans, especially males [8–11]. This means that consuming the long-chain derivatives (i.e. EPA and DHA) themselves is by far the easiest way to increase the amounts of those fatty acids in human tissues.
n–3 FATTY ACIDS AND LOWER CARDIOVASCULAR DISEASE RISK: EVIDENCE EXPLAINED
Some years ago it was documented that Inuit populations in Greenland, Northern Canada and Alaska consuming their traditional diet had much lower cardiovascular mortality than predicted, despite their high fat intake [12–15]. Typically, the rate was <10% of that predicted (for example, see ). The protective component was suggested to be the long-chain n–3 fatty acids consumed in very high amounts as a result of the regular intake of seal meat and whale blubber . Intake of these fatty acids was estimated to average as much as 5–15 g/day among such populations . The Japanese also exhibit a low cardiovascular mortality , and the traditional Japanese diet is rich in seafood, including oily fish, which contain significant amounts of EPA and DHA. Substantial evidence from epidemiological and case-control studies has now accumulated indicating that consumption of fish or of long-chain n–3 fatty acids reduces the risk of cardiovascular mortality in Western populations ([18–31]; see Table 2). Data from China also provides strong evidence for a protective effect of fish and n–3 fatty acid consumption towards cardiovascular disease . Although not all studies agree (for example, see ; Table 2), the protective effects of the fish and/or long-chain n–3 fatty acids have been confirmed by a number of recent studies ([34–43]; Table 2). These studies have been summarized and discussed in detail recently [44,45].
A secondary prevention study providing long-chain n–3 fatty acids in the form of oily fish or fish oil capsules to patients who had already suffered an MI (myocardial infarction) demonstrated a significant reduction (29%) in mortality compared with the control group . More recently, the GISSI Prevenzione study  investigated the effect of n–3 fatty acids (0.885 g of EPA+DHA/day) on 3.5-year mortality outcomes in post-MI patients in a placebo-controlled study also investigating vitamin E and involving approx. 11000 patients in Italy. The major findings are shown in Table 3, the most remarkable of which is the approx. 45% reduction in risk of sudden death. Although the effects observed in the GISSI Prevenzione study apparently occurred in the absence of lipid-lowering , the real impact of altered blood lipid concentrations on the outcomes is somewhat masked by the progressive introduction of lipid-lowering medication (especially statins) during the course of the study. Nevertheless, the reduction in risk of sudden death at 3.5 years in those patients consuming long-chain n–3 fatty acids was already apparent at 4 months and the reductions in risk of cardiovascular mortality and CHD (coronary heart disease) mortality were apparent within 6–8 months of initiating n–3 fatty acid treatment .
n–3 FATTY ACIDS AND LOWER CARDIOVASCULAR DISEASE RISK: MECHANISMS EXPLORED
Long-chain n–3 fatty acid consumption may protect against both the pathological processes leading to the cardiovascular disease (i.e. atherosclerosis) and the processes that ultimately cause death (e.g. MI and stroke). Long-chain n–3 fatty acids favourably affect a number of factors involved in the development of atherosclerosis (Table 4), indicating that they most likely slow the progression of the disease. For example, elevated fasting and post-prandial plasma triacylglycerol concentrations are now recognized to increase the risk of cardiovascular disease, and long-chain n–3 PUFAs lower both (for reviews, see [49–51]). Typically, a 25–30% lowering of fasting triacylglycerol concentrations could be expected from an intake of more than 2 g of EPA+DHA/day. Long-chain n–3 fatty acids also decrease chemoattractant [52–54], growth factor [54,55] and adhesion molecule [56,57] production and so could down-regulate processes leading to leucocyte and smooth muscle migration into the vessel wall intima. Long-chain n–3 fatty acids are also anti-inflammatory [58,59] and so could decrease inflammatory processes within the vessel wall, which are now recognized to be a major contributory factor in the atherosclerosis [60–62]. Long-chain n–3 PUFAs also have a small, but significant, hypotensive effect in both normotensive and hypertensive individuals, as confirmed in a recent meta-analysis . Finally, these fatty acids cause endothelial relaxation and promote arterial compliance [64–67], which might be related to altered nitric oxide production . Thus long-chain n–3 fatty acids exert effects at many steps involved in the process of atherosclerosis and so they might be expected to decrease or slow this disease. Indeed, including long-chain n–3 PUFAs in the diet has been demonstrated to decrease atherosclerosis in a variety of animal models [69–74].
Despite the potential for protection against atherosclerosis, much interest has been focussed on the potent protective effect of n–3 fatty acids towards fatal MI [25,27,46,47], and particularly towards sudden death [28,34,35,47], suggesting that they influence acute events. Several studies also report protection against non-fatal MI [25,30,35,38], which is consistent with a lowered risk of acute events be they non-fatal or fatal.
Two mechanisms are considered to contribute to the strong protective effect of long-chain n–3 fatty acids towards acute cardiovascular events, especially those that are fatal. The first is an anti-thrombotic effect of long-chain n–3 fatty acids. This is mediated largely through changes in eicosanoid generation from the n–6 fatty acid arachidonic acid. Arachidonic acid is released from cell membrane phospholipids by the increased activity of PLA2 (phospholipase A2) following stimulation of platelets and endothelial cells. Metabolism of free arachidonic acid by COX-2 (cyclo-oxygenase-2) gives rise to TXA2 (thromboxane A2), a potent promoter of platelet aggregation, and to PGI2 (prostacyclin I2), a potent inhibitor of platelet aggregation. One of the characteristic features of increased availability of long-chain n–3 fatty acids, especially EPA, is a reduction in the content of arachidonic acid in membrane phospholipids in platelets [75–77] and, presumably, also endothelial cells, thus decreasing the; amount of substrate available for eicosanoid synthesis. Therefore n–3 fatty acids are associated with a decrease in production of TXA2 and PGI2. Furthermore, EPA, which is readily incorporated into cell membrane phospholipids, is released by the action of PLA2 and also acts as a substrate for COX-2. The products produced (e.g. TXA3 and PGI3) have a different structure from those produced from arachidonic acid and this can affect their biological potency. TXA3 has a weaker pro-aggregatory effect than does TXA2. In contrast PGI2 and PGI3 have similar anti-aggregatory potencies. Therefore the effect of long-chain n–3 fatty acids is to promote a less thrombotic environment .
The second mechanism that might be important is an anti-arrhythmic action of n–3 fatty acids. Studies in rats, dogs and marmosets suggest that long-chain n–3 fatty acids from fish oil have anti-arrhythmic effects [78–80]. These effects can be mimicked in cultured cardiomyocytes [81,82]. The presence of n–3 fatty acids in cardiomyocyte membrane phospholipids decreases electrical excitability and modulates the activity of ion (e.g. sodium, potassium and calcium) channels [83,84], effects that are claimed to promote electrical stability in the cell and prevent arrhythmias. In addition to anti-arrhythmic actions due to their effects on ion channels, long-chain n–3 fatty acids might influence heart rate variability (low heart rate variability is believed to be associated with increased mortality post-MI) and this might have an anti-arrhythmic effect via the autonomic nervous system. Christensen et al.  reported a positive correlation between the n–3 fatty acid content of platelets and heart rate variability in patients with Type I diabetes mellitus. These authors  also reported increased heart rate variability in MI survivors given 5.2 g of EPA+DHA/day for 12 weeks. Although this dose is substantially higher than those given in the secondary prevention studies [46,47], these studies are suggestive of two potential ways by which long-chain n–3 fatty acids could affect cardiac arrhythmias: via modulation of ion channels and by increased heart rate variability. A very recent study  has indicated that long-chain n–3 fatty acids may be able to alter arrhythmias acutely. In the study , patients with ventricular tachycardia and with implanted defibrillators were infused with a solution containing 3.8 g of long-chain n–3 fatty acids. The infusion rendered five out of seven patients in this uncontrolled and unblinded study non-responsive to the induction of sustained monomorphic ventricular tachycardia.
Recently, a third mechanism has been suggested to play a role in the protective effects of long-chain n–3 fatty acids towards acute cardiovascular events: the well-documented anti-inflammatory effects of n–3 fatty acids may be important. Inflammation is recognized to play a key role in the progression of atherosclerosis [60,61], and so decreased inflammatory activity as a result of dietary exposure to n–3 fatty acids could alter the progression of the disease. However, the rupture of an atherosclerotic plaque, which is the acute event that exposes the plaque contents to the highly pro-thrombotic environment of the vessel lumen, is, essentially, an inflammatory event [62,88]. The characteristics of an atherosclerotic plaque that make it vulnerable to rupture include a thin fibrous cap and increased numbers of inflammatory cells, such as macrophages [88–90]. Long-chain n–3 fatty acids might act to stabilize atherosclerotic plaques by decreasing infiltration of inflammatory and immune cells (e.g. monocyte/macrophages and lymphocytes) into the plaques and/or by decreasing the activity of those cells once in the plaque. A recent study  showed that long-chain n–3 fatty acids are readily incorporated from dietary fish oil supplements (providing 1.4 g of EPA+DHA/day) into advanced atherosclerotic plaques and that this incorporation is associated with structural changes consistent with increased plaque stability (Table 5). The morphology of carotid plaque sections was characterized according to the American Heart Association (AHA) classification . Plaques from patients treated with fish oil were more likely to be Type IV (fibrous cap atheromas: well-formed necrotic core with an overlaying thick fibrous cap) than those from the placebo group (odds ratio 1.19; Table 5). Conversely, plaques from patients treated with fish oil were less likely to be Type V (thin fibrous capo atheromas: thin fibrous cap infiltrated by macrophages and lymphocytes) than those from the placebo group (odds ratio, 0.52). Thus there were more plaques with a well-formed fibrous cap, rather than a thin inflamed cap, in the fish oil group. Infiltration by macrophages was investigated using immunohistochemistry. It was found that plaques from patients given fish oil were more likely to be less heavily infiltrated with macrophages than those in the placebo group (Table 5).
Since it is the vulnerability of the plaque to rupture rather than the degree of atherosclerosis that is the primary determinant of thrombosis-mediated acute cardiovascular events , it is likely that the findings of Thies et al.  are clinically relevant. If carotid plaques really are stabilized by fish oil, then the risk of neurological events in patients with advanced carotid atherosclerosis may be reduced. If a similar stabilizing effect of n–3 PUFAs occurs in coronary plaques then these too might be stabilized. This might explain the significant protective effects of n–3 PUFAs towards both fatal and non-fatal cardiovascular events (see above for references), which are so far not fully explained.
The observations made by Thies et al.  suggest that the primary effect of n–3 PUFAs might be on macrophages. Macrophage numbers within the plaque might be decreased due to fewer monocyte/macrophages entering the plaque as a result of decreased adhesion molecule expression on endothelial cells and/or the monocyte/macrophage itself, which would act to limit movement of monocyte/macrophages into the plaque. Cell culture studies have shown that n–3 PUFAs can decrease the expression of ICAM-1 (intercellular cell-adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) on the surface of endothelial cells [92–94] and monocytes . Furthermore, feeding fish oil decreased the expression of several adhesion molecules, including ICAM-1, on the surface of rat lymphocytes , mouse macrophages  and human monocytes . However, Thies et al.  reported no reduction in staining of ICAM-1 or VCAM-1 in plaques following fish oil treatment, suggesting that this is not the mechanism by which the reduction in macrophage numbers occurs. A second mechanism by which monocyte/macrophage entry into the plaque might be decreased is through decreased generation of chemoattractants. There is evidence that dietary fish oil decreases the production of a range of chemoattractants including leukotriene B4 [52,53], PDGF (platelet-derived growth factor) , PAF (platelet-activating factor)  and MCP-1 (monocyte chemoattractant protein-1) . This mechanism was not investigated by Thies et al.  and cannot be excluded. An alternative means by which macrophage numbers within the plaque could be decreased is by an increased rate of cell death by either apoptosis or necrosis. Although feeding fish oil to mice was shown to increase the level of Fas expression on lymphocytes  and to increase lymphocyte apoptosis , there is little published information about dietary n–3 PUFAs and monocyte/macrophage apoptosis. However, both EPA and DHA have been shown to increase apoptosis of human monocytes and monocytic cell lines in culture [100,101]. Activation of PPARγ (peroxisome-proliferator-activated receptor γ) has been demonstrated to result in monocyte/macrophage apoptosis , and studies suggest that n–3 PUFAs can induce PPARγ activation. PPARγ is found in atherosclerotic plaques  and it is tempting to speculate that dietary fish oil might result in activation of PPARγ in plaque monocyte/macrophages, driving them towards apoptosis. Cell culture studies indicate that activation of PPARγ in human monocytes also results in inhibition of production and activity of matrix metalloproteinase 9 . Since matrix metalloproteinases are a major contributor to plaque instability , this might provide a mechanism by which n–3 PUFAs improve plaque stability. Future studies should examine the relationship between n–3 PUFA incorporation into plaques, PPARγ and inflammatory mediator expression in the plaque, plaque monocyte/macrophage apoptosis and plaque morphology.
DIETARY RECOMMENDATIONS FOR INTAKE OF LONG-CHAIN n–3 PUFAS
It is clear from the forgoing discussion that long-chain n–3 fatty acids have been proven to be effective in secondary prevention of MI, with a particularly marked effect on sudden death. Thus it would be prudent to advise post-MI patients to increase long-chain n–3 PUFA consumption. Epidemiological studies, studies investigating effects on classic and emerging risk factors and mechanistic studies indicate that long-chain n–3 fatty acids also play a key role in primary prevention. This is supported by studies in animal models, including monkeys. Thus long-chain n–3 fatty acid consumption should be promoted for all individuals especially those at risk of developing cardiovascular disease. This is the reason why a number of organizations have now made recommendations relating to the intake of fatty fish (for example ) and of long-chain n–3 PUFAs (Table 6). It is clear that there is a wide gap between current intakes of long-chain n–3 PUFAs and many of these recommendations (Table 6). To meet these recommendations strategies other than increased consumption of fatty fish may be required.
Abbreviations: CHD, coronary heart disease; COX-2, cyclo-oxygenase-2; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; ICAM-1, intercellular cell-adhesion molecule-1; MI, myocardial infarction; PGI, prostacyclin I; PLA2, phospholipase A2; PPARγ, peroxisome-proliferator-activated receptor γ; PUFA, polyunsaturated fatty acid; TXA, thromboxane A; VCAM-1, vascular cell adhesion molecule-1
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