ω-3 Index and cardiovascular health

A widely accepted method to assess an individual's medium-term dietary intakes of LC n-3 PUFA, is by measuring the percentage of erythrocyte membrane fatty acids (erythrocyte phospholipids) that are EPA and DHA, which correlates closely with the EPA + DHA content in cardiac tissue⁽ Reference Harris, Sands and Windsor ^{33 )}. Proportions of EPA + DHA in erythrocyte phospholipids have been identified as an independent predictor of CVD risk⁽ Reference Harris and Von Schacky ^{34 ,} Reference Kleber, Delgado and Lorkowski ^{35 )}. Erythrocyte EPA + DHA as a % of total erythrocyte fatty acids is termed the ω-3 index, with ≥8 % considered to be associated with the maximum protective effect⁽ Reference Harris and Von Schacky ^{34 )}. Incorporation of supplemented EPA + DHA into erythrocyte membranes occurs in a dose–response fashion and significant increases can be measured over a supplementation period of 2–12 months at relatively low doses akin to intake of 1–4 portions of oily fish per week⁽ Reference Browning, Walker and Mander ^{36 )}. The effects of age and sex on the efficiency of incorporation of supplemental EPA and DHA in blood cells and plasma fractions seem to be relatively minor⁽ Reference Walker, Browning and Mander ^{37 )}, but there may be an increased efficiency of uptake in individuals with lower baseline intakes of LC n-3 PUFA⁽ Reference Cao, Schwichtenberg and Hanson ^{38 )} and potentially at times of greater need such as during pregnancy⁽ Reference Dunstan, Mori and Barden ^{39 )}. Reported average ω-3 indices vary widely in human populations that are identified as being omnivorous, most likely representing the fact that meat-consumers who eat very little fish will have much lower proportions of EPA and DHA in their membrane lipids than meat-consumers who also regularly consume fish, whereas reported ω-3 indices in vegans are distinctly lower compared with meat and fish-eaters (Table 1). Observational evidence suggests that low whole blood or erythrocyte membrane levels of EPA and DHA are associated with a greater risk of sudden cardiac death, primary cardiac arrest, acute coronary syndrome⁽ Reference Albert, Campos and Stampfer ^{14 ,} Reference Siscovick, Raghunathan and King ^{40 ,} Reference Block, Harris and Reid ^{41 )} and a meta-analysis of prospective cohort studies that measured circulating fatty acids showed associations between higher EPA and DHA and lower relative risks of coronary outcomes⁽ Reference Chowdhury, Warnakula and Kunutsor ^{21 )}.

Table 1. Erythrocyte EPA and DHA (% or absolute concentrations) of meat- and fish-consumers (omnivores), vegetarians and vegans

Wt%, weight percentage of the sum of fatty acids; mol%, mole percentage of the sum of fatty acids.

* A vegetarian was defined as someone who ate no meat and not more than one fish meal a month.

Cardiovascular health of populations with low ω-3 index: vegetarians and vegans

Evidence from prospective cohort studies with a high proportion of vegetarians and vegans suggests that they are less likely to develop CHD than meat- and fish-consumers. For example, a 1999 meta-analysis of five prospective studies (USA, UK, Germany) reported that vegetarians had 24 % lower mortality from CHD (specific to those who followed diet for >5 years)⁽ Reference Key, Fraser and Thorogood ^{42 )}. Additional prospective cohorts included in more recent meta-analyses agreed with earlier findings⁽ Reference Huang, Yang and Zheng ^{43 ,} Reference Kwok, Umar and Myint ^{44 )}. Reduced risk of CHD appears to be driven by lower blood LDL cholesterol, blood pressure and possibly lower BMI in vegans/vegetarians⁽ Reference Key, Appleby and Rosell ^{45 –} Reference Appleby, Davey and Key ^{47 )}. Taking this evidence at face value it appears that a vegetarian/vegan diet may be a cardio-protective diet. It has been suggested that there seems to be a minimum basal rate of conversion of ALA to DHA, which is sufficient to maintain cardiovascular health where dietary sources of pre-formed LC n-3 PUFA are lacking⁽ Reference Sanders ^{48 ,} Reference Sanders ^{49 )}. Furthermore, authors of an observational study, using statistical estimates of ALA to longer chain n-3 PUFA conversion rates, suggested that conversion might actually be increased in non-fish eaters compared with fish-eaters. This would account for the relatively low margin of difference in ω-3 indices of fish-eaters compared with non-fish eaters⁽ Reference Welch, Shakya-Shrestha and Lentjes ^{50 )}, although conversion rates to DHA specifically were not clear.

Closer inspection of the observational evidence for coronary risk in vegetarians/vegans, raises the question of whether it is valid to translate these findings to the wider population. Vegetarian/vegan cohorts vary in their motivations for dietary choice (religious, health, animal welfare), which may converge with other distinct lifestyle behaviours that may affect cardiovascular health (physical exercise, practising mindfulness/meditation/prayer, prevalence of alcohol and drug use). This concern seems particularly pertinent to the Seventh Day Adventist cohorts. Kwok et al. applied sub-group testing in their meta-analysis of seven prospective cohort studies that evaluated mortality and clinical cardiovascular outcomes in vegetarian populations compared with non-vegetarian controls and revealed that the overall pooled difference in risk of cardiac events was mainly driven by the Seventh Day Adventist cohorts⁽ Reference Kwok, Umar and Myint ^{44 )}, suggesting that the results from this particular population might not be generalisable to other populations. Furthermore, the definition of vegetarian in Seventh Day Adventist cohorts tends to be inconsistent with the generally accepted definition of eating no meat or fish. Seventh Day Adventist cohorts incorporated into the pooled analyses⁽ Reference Key, Fraser and Thorogood ^{42 ,} Reference Kwok, Umar and Myint ^{44 )} included those who ate meat <1/week or less with no mention of what their fish intakes were⁽ Reference Snowdon ^{51 ,} Reference Beeson, Mills and Phillips ^{52 )}, or were otherwise not clearly defined⁽ Reference Berkel and de Waard ^{53 )}. It is therefore possible that the Adventist cohorts were also fish-consumers and may have had higher ω-3 indices than ‘true’ vegetarians/vegans who do not eat any fish. The more recent Adventist Health Study 2 clearly defined participants as vegans, lacto-ovo vegetarians, pesco-vegetarians, semi-vegetarians and non-vegetarians⁽ Reference Orlich, Singh and Sabaté ^{54 )}. The results of Adventist Health Study 2 showed that the reduced risk in CHD mortality was not quite statistically significant (hazard ratio 0·81, 95% CI 0·64, 1·02) for all vegetarians combined. Further analysis of dietary sub-groups revealed that the reduced risk was significant in pesco-vegetarians when both sexes were combined (hazard ratio 0·65, 95% CI 0·43, 0·97) but not vegans (hazard ratio 0·90, 95% CI 0·60, 1·33) or lacto-ovo vegetarians (hazard ratio 0·90, 95% CI 0·76, 1·06).

The UK vegetarian/vegan cohorts might be considered more generalisable to the broader population since they are not affiliated with a religious faith and may be more diverse in terms of demographics and lifestyle behaviours. Furthermore, vegetarians and vegans were clearly defined as not eating meat or fish⁽ Reference Appleby, Davey and Key ^{47 ,} Reference Appleby, Crowe and Bradbury ^{55 ,} Reference Crowe, Appleby and Travis ^{56 )} and the vegetarian and vegan men from the EPIC-Oxford cohort were reported to have lower EPA and DHA proportions of total plasma fatty acids compared with omnivores, providing an objective biomarker of low fish intake⁽ Reference Rosell, Lloyd-Wright and Appleby ^{57 )}. Although vegetarians/vegans had a lower incidence of CHD in the UK EPIC-Oxford cohort⁽ Reference Crowe, Appleby and Travis ^{56 )}, there was no difference in CHD mortality compared with controls who ate meat and fish (death rate ratio 0·83, 95% CI 0·59, 1·18)⁽ Reference Key, Appleby and Spencer ^{58 )}. The latter study may have been statistically underpowered, but a more recent combined analysis of mortality rates (total 644 CHD deaths) reported that there was no significant difference in CHD mortality in UK vegans/vegetarians up to 90 year (nor 75 year) compared with comparable regular meat-eaters (hazard ratio 0·99, 95% CI 0·79, 1·23)⁽ Reference Appleby, Crowe and Bradbury ^{55 )}, who consumed meat ≥5 times/week as well as fish.

In translating these observations to the question of the cardiovascular health impact of low EPA + DHA tissue status, it is impossible to cleanly dissect the influence of dietary LC n-3 fatty acid intake from other dietary influences. For example, vegetarian/vegan diets are lower in SFA, higher in fibre, but can also be deficient in bioavailable iron, vitamin B₁₂ and vitamin D. Low serum 25-hydroxyvitamin D concentrations are also associated with increased CVD mortality⁽ Reference Schöttker, Jorde and Peasey ^{59 )}. Furthermore, the nature of the comparator group is an important consideration, particularly as health-conscious participants were recruited to UK cohorts who tended to have lower intakes of dietary animal protein compared with Adventist non-vegetarian/non-vegans⁽ Reference Crowe, Appleby and Travis ^{56 )}. However, equivalent CHD mortality rates could alternatively signify that the vegetarian/vegan diet is also associated with other raised CHD risk factors (e.g. low ω-3 index) that might negate the otherwise cardio-protective effects of a vegetarian/vegan diet. It is hypothesised here that long-term low tissue LC n-3 PUFA levels may counterbalance the athero-protective qualities of vegetarian/vegan diets in the sum effect on overall CHD mortality risk. The implications of this for the sizeable non-fish-eating, non-vegetarian/non-vegan population are more serious. Low tissue EPA and DHA levels in a less health-conscious meat-eating population might have a more deleterious effect on cardiovascular health, such as increasing risk of cardiac arrhythmia, against the background of higher LDL-cholesterol, blood pressure and BMI. Fig. 1 summarises the counterbalance of dietary factors that may positively or negatively influence risk of CHD mortality in non-fish eaters. The next sections will address the cardiovascular risk factors that may mediate increased risk of CHD mortality in populations with very low LC n-3 PUFA intakes, including low HR variability (HRV) and a reduced capacity to regulate inflammatory responses.

Fig. 1. Theoretical schematic showing how low long chain n-3 PUFA intakes may oppose the cardioprotective effects of vegetarian/vegan diets, resulting in an equivalent risk of CHD mortality (A). The majority of the UK population eats little or no fish and may be at risk of low ω-3 status. Without the counterbalancing cardio-protective qualities of a vegetarian/vegan diet, this could lead to an increased risk of CHD mortality mediated by arrhythmia (B). Picture of artery attributed to ‘Blausen gallery 2014’^{( 136 )}.

Heart rate variability and risk of arrhythmia

Low HRV indicates a reduced capacity to regulate HR in response to internal and external stressors and demands, and is associated with mortality after a myocardial infarction⁽ Reference Vaishnav, Stevenson and Marchant ^{60 – 62 )} and risk of cardiac events in the general population⁽ Reference Tsuji, Larson and Venditti ^{63 )}. Low HRV is considered to be a predictor of sudden cardiac death⁽ Reference Algra, Tijssen and Roelandt ^{64 )}. Therefore, measurement of parameters of short- and long-term variability in heart beat intervals (RR intervals, or interbeat intervals) using Holter monitors or less intrusive HRV/accelerometry integrated chest-worn monitors (e.g. Actiheart, eMotion Faros), can be a useful non-invasive method to assess the adaptability/resilience of the heart. HRV is partly under the control of the autonomic nervous system, which receives afferent impulses from, and exerts efferent activity to, the intrinsic cardiac nervous system, which interacts with the sino-atrial node. Consequently, variability in HR reflects the sum effect of sympathetic and parasympathetic outflow. Low HRV is associated with a high degree of sympathetic activity (which raises HR thus reducing the capacity to self-regulate in response to demand) and suppressed parasympathetic activity (vagal activity slows HR). Higher LC n-3 PUFA tissue status or fish consumption has been positively associated with HRV⁽ Reference Christensen ^{65 ,} Reference Mozaffarian, Stein and Prineas ^{66 )}. Mixed results have been obtained in previous investigations into the effect of n-3 PUFA on HRV in healthy subjects⁽ Reference Mozaffarian, Stein and Prineas ^{66 –} Reference Christensen, Christensen and Dyerberg ^{69 )}, haemodialysis patients⁽ Reference Svensson, Schmidt and Jorgensen ^{70 )}, subjects with previous myocardial infarction⁽ Reference Christensen, Korup and Aaroe ^{71 –} Reference Hamaad, Kaeng Lee and Lip ^{74 )} and patients with epilepsy⁽ Reference DeGiorgio, Miller and Meymandi ^{75 )}. These studies were typically only 12 weeks long, the durations of HR monitoring differed widely and relatively high doses of n-3 PUFA ranged from 1 to 6·6 g. Furthermore, comparisons between studies are difficult due to different HRV parameters being reported, varying study designs, and methodological inconsistencies. Nevertheless, fish oil supplementation has been shown to increase a parameter of beat-to-beat (vagally regulated) HRV in a meta-analysis of randomised controlled trials⁽ Reference Xin, Wei and Li ^{76 )}, supporting the role of adequate tissue EPA + DHA status in preventing arrhythmic events. Thus autonomic balance may be improved by increased EPA and DHA membrane levels, although a direct effect on pacemaker activity independently of the autonomic nervous system may also occur. Increasing LC n-3 PUFA content in rabbit cardiomyocyte membranes decreases HR in isolated hearts and reduces pacemaker activity and pacemaker current in sinoatrial node cells⁽ Reference Verkerk, den Ruijter and Bourier ^{77 ,} Reference Billman ^{78 )}. In accord with its effects on HRV in clinical trials, fish oil supplementation also reduces HR in human studies⁽ Reference Mozaffarian, Geelen and Brouwer ^{79 )}.

In summary, fish consumption and to some extent fish oil supplementation, is associated with reductions in HR and increased HRV, which would be predicted to reduce the risk of arrhythmia. However, the impact on HRV of consuming a LC n-3 PUFA-free diet was unexplored until recently, when we addressed this question in a cross-sectional study in vegans and age- and BMI-matched omnivores, measuring the average duration of interbeat intervals (the reciprocal of HR), HRV, erythrocyte phospholipid fatty acids, plasma fatty acids and oxygenated PUFA metabolites⁽ Reference Pinto, Sanders and Kendall ^{80 )}. Vegans had lower day-time beat-to-beat HRV and shorter day-time interbeat intervals compared with omnivores, adjusted for age, sex, BMI and physical activity levels by accelerometry. Twenty-four hour HRV was higher in vegans due to the greater night–day differences in vegans compared with omnivores, which was mainly due to the relatively lower HRV during the day rather than higher HRV at night in vegans; no differences between groups during nocturnal sleep were observed. This could indicate a greater predominance of sympathetic regulation (associated with reduced parasympathetic activity) during waking hours due to an exaggerated response to stimuli, or a direct effect of low LC n-3 PUFA membrane concentrations in cardiac cells on the pacemaker that manifested in altered cardiac function under conditions of stimulation. There were no differences in serum vitamin D or B₁₂ status between the groups, factors which may also have impacted on HRV. Although these observations only demonstrate an associative link, n-3 PUFA tissue status is implicated as a strong candidate for being the key factor in determining the disparate HRV patterns during waking hours observed in vegans and omnivores.

Dampening and resolving inflammation

Both n-6 and n-3 PUFA in cell membranes can be metabolised into an array of pro- and anti-inflammatory metabolites that are likely to influence prevention of arrhythmia by influencing myocyte cell-signalling and also autonomic function. Chronic inflammation is the key underlying factor in the lifelong accrual of vascular lesions that eventually leads to atherosclerosis and CHD, and LC n-3 PUFA supplementation appears to improve plaque stability⁽ Reference Thies, Garry and Yaqoob ^{81 )}. Although the role of inflammation in initiating and propagating atherosclerosis is at the crux of CHD, chronic inflammation probably also increases coronary risk independently of atherosclerosis by impairing neuronal function⁽ Reference Dyall and Michael-Titus ^{82 )} and myocyte membrane properties⁽ Reference Papaioannou, Verkerk and Amin ^{83 )}, thereby having a direct influence on cardiac electrophysiology and risk of arrhythmia⁽ Reference Lanza, Sgueglia and Cianflone ^{84 ,} Reference Malave, Taylor and Nattama ^{85 )}. LC n-3 PUFA levels in plasma and erythrocytes are inversely associated with circulating markers of inflammation (C-reactive protein, IL-6, IL-1 receptor antagonist, TNF receptor 2, transforming growth factor-β)⁽ Reference Farzaneh-Far, Harris and Garg ^{86 –} Reference Fontes, Rahman and Lacey ^{88 )}. LC n-3 PUFA may lower inflammatory burden in a number of ways: by inhibiting the conversion of LA to more pro-inflammatory eicosanoids by substrate competition, being converted themselves to less potent pro-inflammatory eicosanoids, modulation of transcription factor activation, inhibiting the expression of vascular cell adhesion molecules and cytokines, and through the inflammation-resolving properties of their oxygenated metabolites⁽ Reference Calder ^{11 )}. The lack of sensitivity of measurements of circulating inflammatory biomarkers, and also the large variability due to hyper-responsivity to short-term infections, has up to now hindered efforts to build a body of evidence for a role of LC n-3 PUFA in modulating chronic, low-grade inflammation in the context of CVD⁽ Reference Skulas-Ray ^{89 )}. The relatively recent detection of a diverse array of oxygenated PUFA metabolites presents a whole new line of investigation with respect to the anti-inflammatory effects of marine fatty acids. Some of the lipid mediators derived from LC n-3 PUFA, termed specialised pro-resolving mediators (SPM), act in an autacoid manner to resolve inflammation (Fig. 2). SPM play a functional role in ending acute inflammatory events by inhibition of neutrophil influx to the site of trauma, counter-regulating pro-inflammatory cytokines and stimulating resolving macrophages to clear the products of the inflammatory response, thereby allowing the injured area to heal⁽ Reference Serhan ^{90 )}. Protectin-D1 (originally termed neuroprotectin) is thought to be important in inhibiting proinflammatory gene expression and promoting nerve regeneration in neural tissue, thereby protecting neurons from inflammation-related injury in a DHA-dependent manner⁽ Reference Bazan, Molina and Gordon ^{12 ,} Reference Bazan, Musto and Knott ^{91 )}. DHA-derived neuroprostanes, F₄ isoprostane-like compounds formed non-enzymatically through free radical-catalysed reactions, are also thought to be significant markers of oxidative stress in neural tissue⁽ Reference Galano, Lee and Gladine ^{92 )}; furthermore, hepatic F₄-neuroprostanes are negatively associated with the extent of atherosclerotic plaque⁽ Reference Gladine, Newman and Durand ^{93 )}, inhibit inflammatory cell signalling in macrophages⁽ Reference Gladine, Laurie and Giulia ^{94 )} and may have cardiac arrhythmic effects⁽ Reference Roy, Oger and Thireau ^{95 )}.

Fig. 2. Overview of the hypothesised role of membrane PUFA profiles in the production of pro-inflammatory and inflammation-resolving oxygenated lipid mediators. In addition to inhibition of arachidonic acid (ARA)-derived pro-inflammatory eicosanoid production, higher proportions of membrane phospholipid long chain (LC) n-3 PUFA may increase availability of LC n-3 PUFA available for enzymic oxygenation to lipid mediators that contribute to the resolution of an acute inflammatory response⁽ Reference Serhan ^{90 )}. ALA, α-linolenic acid; COX, cyclooxygenase; CYP450, cytochrome P450; HDHA, hydroxyl-DHA; HEPE, hydroxyeicosapentaenoic acid; LA, linoleic acid; LOX, lipoxygenase; PLA₂, phospholipase A2; SPM, specialised pro-resolving mediators.

SPM, namely E- and D- series resolvins (RvD1 and RvE1), the DHA-derived (neuro)protectin-D1 and maresin (MaR1), are released in nano- and pico-molar concentrations at sites of local inflammation, can be detected in ex vivo cultured mononuclear cells following EPA + DHA supplementation⁽ Reference Wang, Hjorth and Vedin ^{96 )} and sometimes resolvins can be detected in human plasma although concentrations are not responsive to supplementation⁽ Reference Mas, Croft and Zahra ^{97 –} Reference Mas, Barden and Burke ^{100 )}. Large changes in other postprandial and fasting oxygenated PUFA metabolites have been reported in response to fish oil supplementation⁽ Reference Barden, Mas and Croft ^{99 ,} Reference Schuchardt, Schmidt and Kressel ^{101 )}. But the lipid mediator profiles of unsupplemented populations remain undetermined, particularly those who have very low or zero habitual dietary LC n-3 PUFA intakes. We investigated whether the vegans in our cross-sectional study also had lower circulating concentrations of oxygenated EPA and DHA metabolites, since this might have implications for their ability to resolve acute and chronic inflammation. It was observed that vegans had significantly lower plasma concentrations of EPA- and DHA-derived lipid mediators compared with omnivores, including 18-HEPE, 17-HDHA and 14-HDHA, likely precursor markers for RvE1, RvD1 and MaR1 availability, respectively⁽ Reference Pinto, Sanders and Kendall ^{80 )}. Although circulating plasma concentrations of lipid mediators are likely to be an insensitive marker of capacity for autacoid release and activity of SPM in specific inflamed sites in the nervous and cardiovascular systems, higher concentrations of precursor markers of SPM bioavailability (18-HEPE, 17-HDHA and 14-HDHA) may indicate greater capacity for conversion to SPM at times of need, with clear functional implications for populations with low tissue EPA and DHA stores.

The accumulating evidence suggests that tissue LC n-3 PUFA are crucial in moderating inflammatory responses in the cardiovascular system. Consequently individuals with no dietary intake of marine n-3 PUFA may be at risk of incurring inflammatory-related neuronal and cardiovascular damage at a greater rate than fish-consumers. An adverse LC n-3 PUFA-derived lipid mediator profile may also be implicated in impaired haemostatic function in vegetarians⁽ Reference Mezzano, Muñoz and Martínez ^{102 )}. However, presently these ideas remain strictly hypothetical. Health-conscious non-fish-consuming populations may also have a greater bioavailability of ALA- and linoleic acid (LA; 18 : 2n-6)-derived lipid mediators that may have cytoprotective effects⁽ Reference Ramsden, Ringel and Feldstein ^{103 ,} Reference Kumar, Gupta and Anilkumar ^{104 )}, although this may not be applicable to the majority of the non-fish-eating population. Few of these lipid mediators have been fully characterised regarding their functional effects, but evidence in animal and cell models to date suggests that plant PUFA-derived oxygenated lipid mediators comprise a complex array of diverse bioactive molecules that may also induce a range of physiological effects in various tissues⁽ Reference Choque, Catheline and Rioux ^{105 –} Reference Nicolaou, Mauro and Urquhart ^{107 )}.

Alternative sources for non-fish consumers?

Soya and rapeseed oils are particularly rich in ALA, with the main dietary sources of ALA being cereal-based products, cooking oils and spreading fats, and vegetables. The high concentrations in oilseed crops presents a potentially easy, sustainable and cheap alternative source of n-3 PUFA, but the evidence for a protective effect against CVD is weaker in comparison with marine n-3 fatty acids⁽ Reference Fleming and Kris-Etherton ^{108 )}. Populations in Europe and the USA consume 3–9 % of energy as LA^{( 16 , 109 )}. Dietary intake of ALA has proven difficult to assess due to methodological limitations associated with accurate fatty acid composition data in food databases, but intakes are estimated to be much lower (0·3–0·8 % energy)^{( 16 , 109 )} than LA intakes, which would limit the amount of endogenous LC n-3 PUFA production from ALA⁽ Reference Rosell, Lloyd-Wright and Appleby ^{57 )}. Reports are conflicting as to whether vegans/vegetarians consume more ALA than omnivores/fish-eaters⁽ Reference Sanders ^{49 ,} Reference Welch, Shakya-Shrestha and Lentjes ^{50 ,} Reference Kornsteiner, Singer and Elmadfa ^{110 )}. Dietary LA intakes on the other hand tend to be higher in vegetarians/vegans than omnivores⁽ Reference Sanders ^{49 ,} Reference Welch, Shakya-Shrestha and Lentjes ^{50 )}, which may also limit conversion of ALA to EPA through substrate competition⁽ Reference Liou, King and Zibrik ^{111 )}. Furthermore, supplementation with additional ALA in vegans appears to be ineffective in increasing conversion to LC n-3 PUFA⁽ Reference Fokkema, Brouwer and Hasperhoven ^{112 )} and supplementation with pre-formed EPA and DHA may be necessary for cardioprotective benefit in non-fish-eating populations.

GM crops that can yield EPA and DHA are well-advanced along the experimental process⁽ Reference Napier, Usher and Haslam ^{113 )}, although the first human trial using LC n-3 PUFA-rich Camelina sativa oil has only just commenced at the time of writing. The main impetus for this bioengineering research was to find a way of producing EPA- and DHA-rich oil to replace fish oils to feed farmed fish⁽ Reference Betancor, Sprague and Usher ^{114 )}, but the development of transgenic oilseed crops, using genes from microalgae to synthesise EPA and DHA from shorter chain PUFA, could provide an alternative to fish oil for human supplementation and food fortification. The other, already commercially available, option is DHA-only and DHA + EPA-rich microalgal oil⁽ Reference Sarter, Kelsey and Schwartz ^{115 )}. These LC n-3 PUFA rich algal oils have been demonstrated to increase the DHA content of phospholipids efficiently and to have TAG- and blood pressure-lowering properties⁽ Reference Maki, Yurko-Mauro and Dicklin ^{116 –} Reference Theobald, Goodall and Sattar ^{120 )}. Microalgal oils are also already being used to supplement infant formula and a limited range of adult foods but the limited scale of production and high costs presently precludes any efforts to apply to large populations⁽ Reference Chauton, Reitan and Norsker ^{121 )}. Furthermore, little is known about what dosage and which EPA : DHA ratio is likely to be cardio-protective in disease-free populations with low ω-3 indices⁽ Reference Cottin, Sanders and Hall ^{122 )}. High-DHA purified fish oils are slightly more effective than high-EPA equivalents in lowering blood pressure⁽ Reference Mori, Bao and Burke ^{123 )}. DHA-only algal oil consumed in combination with a fatty meal appears to avoid the less desirable effects of increasing postprandial concentrations of plasma F₂ isoprostanes (markers of oxidative stress produced from arachidonic acid following non-enzymatic reaction with reactive oxygen species) compared with EPA + DHA containing fish oil⁽ Reference Purcell, Latham and Botham ^{124 )}. However, although retroconversion of DHA to EPA is estimated at between 8 and 14 %⁽ Reference Conquer and Holub ^{125 )}, the functional consequences of the lack of dietary EPA against the background of plentiful dietary DHA are not known and may be significant if EPA-derived lipid mediators have any tissue specific roles in cytoprotection. It is equally possible that EPA supplementation is less effective and that DHA-only oils are sufficient to reduce risk of lethal arrhythmia. On the whole, fish oil supplementation studies to date have not considered baseline ω-3 status, which in hindsight has hindered progress towards a robust body of evidence that can be used to formulate dietary guidelines⁽ Reference Dwyer, Rubin and Fritsche ^{26 )} and consequently necessitates a fresh approach to gathering new evidence on whether non-fish-consuming populations would be at reduced risk of CHD if they had access to alternative dietary sources of EPA and DHA.