A review of the effect of omega-3 polyunsaturated fatty acids on blood triacylglycerol levels in normolipidemic and borderline hyperlipidemic individuals

Circulating levels of triacylglycerol (TG) is a recognized risk factor for developing cardiovascular disease, a leading cause of death worldwide. The Institute of Medicine and the American Heart Association both recommend the consumption of n-3 polyunsaturated fatty acids (PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to reduce serum TG in hyperlipidemic individuals. Additionally, a number of systematic reviews have shown that individuals with any degree of dyslipidemia, elevated serum TG and/or cholesterol, may benefit from a 20-30 % reduction in serum TG after consuming n-3 PUFA derived from marine sources. Given that individuals with serum lipid levels ranging from healthy to borderline dyslipidemic constitute a large portion of the population, the focus of this review was to assess the potential for n-3 PUFA consumption to reduce serum TG in such individuals. A total of 1341 studies were retrieved and 38 clinical intervention studies, assessing 2270 individuals, were identified for inclusion in the current review. In summary, a 9-26 % reduction in circulating TG was demonstrated in studies where ≥ 4 g/day of n-3 PUFA were consumed from either marine or EPA/DHA-enriched food sources, while a 4-51 % reduction was found in studies where 1–5 g/day of EPA and/or DHA was consumed through supplements. Overall, this review summarizes the current evidence with regards to the beneficial effect of n-3 PUFA on circulating TG levels in normolipidemic to borderline hyperlipidemic, otherwise healthy, individuals. Thus demonstrating that n-3 PUFA may play an important role in the maintenance of cardiovascular health and disease prevention.

The 2002 Institute of Medicine (IOM) report on Dietary Reference Intakes for various macronutrients, including fat, states that, "Supplementation with fish oil, which is high in EPA and DHA, reduces triacylglycerol concentrations; low density lipoprotein cholesterol and high density lipoprotein cholesterol concentrations are either increased or unchanged" [11]. Food and supplement based studies assessing the lipid-lowering effects of n-3 PUFA have utilized a variety of oils comprised of either flaxseed-derived alphalinolenic acid (18:3 n-3, ALA), algal-derived pure eicosapentaenoic acid (20:5 n-3, EPA) or docosahexaenoic acid (22:6 n-3, DHA), or some combination of these n-3 PUFA through the consumption of fish or fish oils. It is therefore important to determine which forms of n-3 PUFA, or combination of forms, are bioactive in affecting serum TG levels and to detect differences in efficacy among various forms.
Systematic reviews by Wei and Jacobson, Bernstein et al., and Eslick et al. highlight the lipid-lowering capacity of different sources and forms of n-3 PUFA in populations that range from normolipidemic to hyperlipidemic [20][21][22]. Wei and Jacobson compared the efficacy of DHA to EPA and found that individuals who consumed either DHA or EPA experienced reductions in serum TG of 18.5 % and 32 %, respectively. Individuals also exhibited significantly elevated serum low density lipoprotein cholesterol (LDL-c) levels by 5 % after DHA consumption, while those consuming EPA had a non-significant reduction in serum LDL-c of 1 % [20]. Additionally, Bernstein et al. concluded that DHA from algal oil plays a role in reducing serum TG while elevating serum LDL-c [21]. The results of a systematic review by Eslick et al. showed that 3.25 g/day of fish oil (1.9 g of EPA and 1.35 g of DHA) reduced serum TG by 14 %, yet cholesterol levels were not altered beyond a clinically insignificant increase in LDL-c [22]. Similar results were obtained in systematic reviews by Balk et al. and Mori et al., both of which assessed studies in which individuals consumed either fish, algal EPA or algal DHA oils [23,24]. While results from the previously identified systematic reviews indicate that DHA contributes to slight increases in LDL-c and EPA contributes to minor reductions in LDL-c, both n-3 PUFA were established to be efficacious in significantly lowering serum TG levels.
The previously discussed meta-analyses and systematic reviews concur with the observation that EPA and DHA, derived from fish or algal oils, can reduce serum lipids, most notably TG. However, these analyses primarily focused on hyperlipidemic individuals, a population likely to achieve the most drastic reduction in TG levels upon n-3 PUFA consumption, and may conceal a lack of response in normolipidemic subjects, whom with they were pooled [18]. The purpose of the current review is to provide new knowledge with regards to our understanding of the effect of dietary and supplemental n-3 PUFA intake on blood lipid profiles in healthy individuals, using the 2002 IOM report as a reference point. A focus will be placed on the TG-lowering ability of n-3 PUFA in normal to borderline hyperlipidemic populations as defined by AHA guidelines.

Search strategy
Using the PubMed search engine, clinical trials and observational studies relating to the effects of n-3 PUFA on serum biomarkers of CVD were collected. Search terms (described below) were used to gather studies published from January 1, 2000 -October 1, 2013 as they were not captured within the reference point of the 2002 IOM report. Search terms relating to cholesterol, C-reactive protein (CRP) and chronic illnesses were used to capture the entirety of literature analyzing blood lipids as such studies would likely have included TG measurements ( Fig. 1 summarizes the search strategy).

Exclusion criteria
Participants that fulfilled the inclusion criteria were analyzed with participants that were explicitly stated to have not met the inclusion criteria Endpoints were analyzed ex vivo Low quality studies** *Studies that did not report a baseline profile for total-c, LDL-c and/or TG were included in analysis if they fulfilled all remaining inclusion and exclusion criteria described above.
**Study quality was assessed by the Appraisal guide for intervention/experimental studies provided by Health Canada [26], if studies scored below a threshold of seven they were excluded from the current review (a single study scored below this threshold).
Applying these parameters yielded 38 clinical trials that were further stratified into dietary interventions (16 studies) and supplementation trials (22 studies).

Dietary interventions
Using the indicated selection criteria, 16 dietary intervention studies (633 subjects in total) were deemed appropriate for the assessment of the impact of n-3 PUFA consumption on circulating TG and cholesterol levels within normolipidemic and borderline hyperlipidemic (otherwise healthy) adults (Table 1). It is noteworthy that two of the 16 studies [27,28] were subsequent analyses of formerly identified interventions [29,30]. Therefore, they are presented as single experiments in this review. As a result, a final list of 14 unique dietary intervention studies were assessed (Table 1). Among the 14 dietary intervention studies, four studies utilized increased fish consumption [31][32][33][34]; five studies examined the benefits of increasing EPA/DHA intake by enriching foods to contain a higher content of n-3 PUFA [27,29,[35][36][37][38]; three studies measured the effects of increasing ALA intake [28,30,39,40]; and 2 studies altered the n-6:n-3 PUFA ratio while maintaining a constant amount of n-3 PUFA consumption [41,42]. Alterations from baseline plasma TG and cholesterol values were assessed in all 14 intervention studies in the current review. Overall, of the 24 experimental arms within the 14 studies, 15 showed reduced serum TG levels (five were statistically significant reductions; Table 2) and eight of the 14 studies showed reduced serum total-c, LDL-c or both (five were statistically significant reductions; Table 1). The three dietary interventions that provided ≥ 4 g/day of EPA and/or DHA noted significant reductions in TG of 9-26 % [27,29,31,33], while six out of seven experimental arms providing 2.3-3.4 g/day of marine based n-3 PUFA produced non-significant reductions in TG of 3-14 % [32,34,42], and the effect of n-3 PUFA provided from flaxseed, flaxseed oil or ≤ 2 g/day of EPA and/or DHA remains ambiguous [28,30,[35][36][37][38][39][40].
TG and LDL-c were only significantly lowered in interventions providing more than 4 g/day of n-3 PUFA through increased fish consumption. Two trials, of 4 weeks [31] and 8 weeks in duration [32], showed that fish consumption of 125-150 g/day (3.4-5.4 g/day of n-3 PUFA) reduced TG levels by 14-15 %. The 4-week intervention also showed a statistically non-significant 7 % reduction in LDL-c [31]. Another study demonstrated that switching from a Swedish diet to a Mediterranean diet (2.3 g vs. 4.1 g/day of n-3 PUFA) for 4 weeks resulted in statistically significant 17 %, 23 % and 17 % reductions in serum total-c, LDL-c and TG, respectively [33]. Finally, participants consuming an oily fish diet (2.6-3.0 g/day of n-3 PUFA) for 8 weeks experienced non-significant reductions in total-c and LDL-c by 2.3 % and 7.5 %, respectively, and a trend towards lowered TG levels (by 3.1 %) was observed when compared to a red meat diet (1.2-1.4 g/day of n-3 PUFA) [34].
There were five studies designed to increase EPA and/ or DHA consumption through enriched baked goods [27,29], drinks [35][36][37], or pork derived from animals consuming marine based n-3 PUFA-enriched food [38]. Two 12 week studies, one providing 0.86 g/day of n-3 PUFA from enriched milk and the other providing 0.185 g/day of n-3 PUFA from enriched pork, produced significant reductions in serum TG of 17 % and 27 %, respectively, while not significantly altering serum cholesterol levels [37,38]. One 8-week intervention providing 0.33 g/day of n-3 PUFA from enriched milk showed a reduction in total-c and LDL-c of 6 % and 16 %, respectively [35]; while a second 8-week study providing 4 g/day of n-3 PUFA from enriched baked goods produced a statistically significant 26 % reduction in TG levels [27,29]. In contrast, a 2-week study providing 0.5 g/day of n-3 PUFA from EPA/ DHA enriched tomato juice did not report any changes in TG or cholesterol levels, however, participants show significant reductions in circulating homocysteine, VCAM-1 and ICAM-1 [36].
Studies assessing an increased consumption of ALA utilized 30-40 g/day of flaxseed (5.74-8.42 g/day of ALA) [28,30,39,40], yet the results from these trials produced inconsistent effects on lipid levels. For instance, a 4-week intervention with 32.7 g/day of ground flaxseed led to a 41 % increase in TG levels [39]. In contrast, a 12-month intervention, providing 40 g/day of flaxseed, resulted in the maintenance of cholesterol and TG levels, while the placebo significantly elevated plasma total-c and LDL-c levels [28,30]. Additionally, one 4-week intervention that provided participants with 5.74-6.5 g/day of ALA found nonsignificant reductions in serum lipids [40]. Interestingly, although non-significant, the consumption of ground flaxseed or flaxseed oil (each providing an equal amount of ALA) resulted in 11 % and 20 % reductions in TG levels, respectively, in individuals 18-29 years of age; ground flaxseed also led to 7 % and 12 % reductions in total-c and LDL-c, respectively [40].
All of the previously mentioned studies reduced an individual's dietary n-6:n-3 PUFA ratio by elevating n-3 PUFA consumption. However, two studies investigated the effects of altering the n-6:n-3 PUFA dietary ratio by increasing n-6 PUFA intake while maintaining a constant n-3 PUFA intake. This allowed for the investigation into whether a reduced n-6:n-3 PUFA ratio alone is more beneficial than an absolute increase in n-3 PUFA consumption as well as a decreased dietary n-6:n-3 PUFA ratio, as evaluated in the previous studies. One study accomplished this by replacing monounsaturated fatty acids with n-6 PUFA; participants either consumed a moderate or high ratio of n-6:n-3 PUFA consisting of 15 g or 26 g/day, respectively, of n-6 PUFA while consistently consuming 2 g/day of n-3 PUFA for 6 weeks [41]. While there were no statistically significant     differences between treatments, the diet providing 15 g of n-6 PUFA produced a trend towards reduced total-c and LDL-c by 3 % and 8 %, respectively, and a trend towards elevated HDL-c by 8 % [41]. During a 10-week cross-over study, participants consumed 90 g/day of fishmeal-or plant protein-fed gilthead sea bream that provided either 1 g or 2 g/day of n-6 PUFA, respectively, while consistently providing 2.3 g/day of n-3 PUFA [42]. This study showed that individuals who first consumed the fishmeal-fed gilthead sea bream had a reduction in total-c, LDL-c and TG by 29.3 %, 21.6 % and 11.7 %, respectively, prior to rebounding after 10 weeks of consuming plant protein-fed fish [42]. However, participants who first consumed the plant protein-fed fish did not experience reductions in any lipid markers following either dietary intervention, except for a 5 % reduction in total-c following the cross over period [42].

B) 1 g of FO and 8 g of sunflower oil
Vs.
Total-c and LDL-c was elevated by 2-8 % in Groups A, B, C, E (non-sig) and by 7 % and 13 % in Group D, respectively (sig) C) 2 g of FO and 8 g of sunflower oil Vs.
HDL-c was elevated by 4-6 % in Groups A, C, D and reduced by 10 % and 3 % in Groups B, E, respectively (non-sig)

D) 4 g of FO and 8 g of sunflower oil
Vs.
After 12 weeks : TG was reduced by 12 %, 12 %, 2 %, 5 % in Groups A, B, D, E respectively, and elevated by 1 % in Groups C (non-sig) Total-c was elevated by 4 % in Groups A, D, and reduced by 1 %, 4 % and 10 % in groups B, C, E, respectively (only Group E was sig)

E) 8 g of sunflower oil
LDL-c was elevated by 3-7 % in Groups A, B, D (non-sig) and reduced by 5 % and 13 % in Group C, E, respectively (only Group E was sig) HDL-c was elevated by 6 %, 2 % in Groups A, D, respectively, and reduced by 5-7 % in Groups B, C, E, (non-sig)    Flaxseed oil produced a 4 % and 12 % increase in total-c and LDLc, respectively No significant change in platelet aggregation stimulated by thrombin or collagen Hempseed oil produced a 4 % increase in both total-c and LDLc and an 18 % increase in TG Vs.
Hempseed oil (0.372 g of ALA,1.14 g of LA) Vs.   Table 4, fasting TG levels were reduced following supplementation with EPA and/or DHA exclusively in 30/34 experimental arms within the 22 studies (i.e. 88 % of the interventions evaluated, without taking statistical significance into account) and post-supplementation TG levels were significantly lowered from baseline in 23 of the 34 aforementioned experimental arms (i.e. 68 % of the interventions; however, one study did not analyze for an ingroup difference in serum lipid levels from baseline to postsupplementation [62]). The magnitude of the n-3 PUFA-mediated TG-lowering effect varied depending on the supplementation dose and the study duration. Low doses such as 0.3-0.9 g/day of n-3 PUFA for 12-52 weeks did not consistently produce a significant reduction in TG levels [43][44][45]63]. While studies solely providing n-3 PUFA in doses < 1 g/day (EPA, DHA or both) significantly lowered fasting TG by 8-38 % [44-55, 59, 61, 62, 64]. In a dose-response study, 1.8 g/ day of EPA and DHA for 52 weeks was sufficient to lower TG levels by 16.5 %, whereas 0.45 and 0.9 g/day did not affect fasting TG levels [44]. A second study demonstrated a significant dose-response effect whereby 1 g/day of marine derived n-3 PUFA produced an elevation in TG levels by 9 %, while 2 and 4 g/day of n-3 PUFA for 12 weeks produced decreases in TG levels of 15 % and 20 %, respectively [54]. However, none of the aforementioned doses produced a significant change from baseline values [54].
Two studies investigated the effect of fish oil based n-3 PUFA supplementation combined with a background diet that was either high or moderate in n-6 PUFA, specifically linoleic acid (18:2 n-6, LA) [56,57]. In an 8 week study, 3.1 g/day of n-3 PUFA resulted in a 19 % and a 51 % reduction in TG levels while consuming either a high or moderate n-6 PUFA background diet, respectively [56]. Similarly, participants of a 6 week study, stratified to a high or moderate LA background diet found that 2.5 g/day of EPA and DHA benefited both groups with similar reductions in TG levels of 20 % and 25 %, respectively [57].
Blood cholesterol levels were also examined in the 22 supplementation studies within the current review, and only two studies [48,60] observed modest n-3 PUFAinduced increases in HDL-c levels that were significantly different from baseline. Thus, total-c, LDL-c, and HDL-c remained largely unchanged with n-3 PUFA supplementation. A modest reduction in total-c occurred with 4.9 g/day of DHA, yet this difference was not statistically significant [59]. Additionally, only 1 study produced a significant 11 % reduction in LDL-c levels following 4 weeks of supplementation with 4 g/ day of n-3 PUFA, however, this effect was produced with an additional 2 g/day supplement of gammalinolenic acid (18:3 n-6, GLA) [51].

Discussion
This review indicates that the established TG-lowering effect of n-3 PUFA in hyperlipidemic individuals is maintained within populations who are normolipidemic to borderline hyperlipidemic. Studies in which participants consumed EPA and/or DHA or fish consistently reported lower blood TG levels in comparison to those studies in which participants consumed plant-based sources of n-3 PUFA. Overall, the studies involving dietary interventions evaluated within the current review suggest that a TG-lowering effect of marine based n-3 PUFA is produced in healthy individuals upon the consumption of ≥ 4 g/day of n-3 PUFA. In contrast, the supplementation studies assessed within the current review  suggest that a minimum of 1 g/day of EPA and/or DHA (derived from either fish or algal oil) is required to confer a similar benefit as observed in the dietary interventions.
Within the past 5 years, the European Food Safety Authority (EFSA), the AHA and the Food Standards -Australia and New Zealand (FSANZ) organization have all recognized n-3 PUFA as a preventative measure against the development of CVD, primarily by reducing risk factors for CVD, including elevated blood TG levels [12][13][14][15]. The EFSA established and substantiated a health claim in 2010 indicating the consumption of 2 g of EPA/DHA per day has the ability to maintain normal blood TG concentrations [12,13]. Furthermore, the AHA released a statement in 2011 indicating that a daily dosage of 2-4 g of n-3 PUFA, specifically EPA and DHA, confers a 25-30 % decrease in serum TG levels [14].
The health claim by the EFSA and the statement by the AHA are largely based on the findings of a systematic review by W.S. Harris [19]. This review stratified studies comparing participants with either healthy (serum levels < 177 mg/dL) or elevated levels (serum levels ≥ 177 mg/dL) of serum TG; relative to the upper limit for serum TG of 200 mg/dL utilized in the current review [19]. However, in the review by Harris, a quarter of the studies assessing participants with healthy baseline TG levels contained a hypercholesterolemic population (serum TC ≥ 240 mg/dL). Nonetheless, the results indicated that 3-4 g/day of EPA and/or DHA produced a 25-34 % decrease in serum TG levels [19]. This finding is supported by a later systematic review which reported that individuals with either borderline high or high levels of serum TG (according to AHA guidelines) experienced reductions in TG levels of~20 % and~30 %, respectively, when consuming 4 g/day of EPA and/or DHA [18].
The previous reviews indicate that marine based n-3 PUFA can reduce serum TG levels; however, they primarily focused on supplementation studies within dyslipidemic populations. An earlier review by W. S. Harris, than the 1997 study previously discussed, included dietary intervention studies and produced findings similar to those of the current review. Overall, consuming n-3 PUFA through dietary forms primarily showed a trend in TG reductions, while a significant effect was only observed when large amounts of n-3 PUFA were consumed (as observed in Table 2, ≥ 4 g/day of n-3 PUFA) and the effects of ALA supplementation were highly variable [17]. W. S. Harris concluded that this inconsistency in the ability of n-3 PUFA to reduce serum TG levels during dietary interventions was likely due to the manipulation of multiple variables as the food source was not highly controlled between studies [19]. The lack of a consistent study design for elevating n-3 PUFA consumption through dietary modifications continues to be a limitation for the field. This constraint reduces the ability to evaluate an exact dosage and source of EPA and/or DHA required to significantly, and routinely, lower serum TG levels across all populations.
Several reviews have repeatedly shown an effect of EPA and/or DHA in lowering TG levels during supplementation trials [17,[19][20][21][22][23][24]. Based on the current review, when ≥ 1 g/day of EPA and/or DHA is consumed by individuals, without any other increases in dietary fat intake, an 8-40 % reduction in TG levels can be observed, as shown in Table 4. The apparent presence of a lipid-lowering dose-response to marine derived n-3 PUFA intake, demonstrated in the study by Di Stasi et al. [54], suggests additional benefits are attainable when supplementation is raised as high as 4.9 g/day [54]. Figure 2 summarizes the TG-lowering effects of individuals consuming ALA, EPA, DHA or some combination of these n-3 PUFA Fig. 2 The percent change in serum TG levels from baseline values in normolipidemic and borderline hyperlipidemic subjects receiving n-3 PUFA either through the diet or supplemental forms. Shaded markers indicate changes from baseline that are statistically significant (p < 0.05) within the studies analyzed in this review. This figure highlights the consistent lipid-lowering effect of EPA and DHA; studies providing ALA remain inconclusive. Additionally, Fig. 2 indicates that as the dose of EPA and/or DHA increases, in both supplementation and dietary intervention studies, a concurrently larger reduction in TG levels is obtained. Furthermore, Fig. 2 shows neither EPA and/or DHA significantly raised TG levels from baseline in either supplementation or dietary intervention studies.
Based on the present review, the beneficial effects of n-3 PUFA, specifically EPA and DHA, which have been substantiated in hyperlipidemic individuals, extend to individuals with normal to borderline high levels of serum lipids. In summary, using select search terms and criteria, our review of the existing evidence has shown that consumption of ≥ 4 g/day of n-3 PUFA through marine and EPA and/or DHA-enriched food sources, or 1-5 g/ day of EPA and/or DHA in supplement form, has the ability to reduce serum TG by 9-26 % and 4-51 %, respectively, in normolipidemic to borderline hyperlipidemic and otherwise healthy individuals. This provides evidence that the consumption of marine based n-3 PUFA is not only extremely useful to treat dyslipidemia, but is also beneficial for otherwise healthy populations in the prevention of hyperlipidaemia and may subsequently reduce the risk of developing CVD.