Thematic Review Section: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases| Volume 53, ISSUE 12, P2525-2545, December 01, 2012

Omega-3 fatty acid supplementation and cardiovascular disease

Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases
      Epidemiological studies on Greenland Inuits in the 1970s and subsequent human studies have established an inverse relationship between the ingestion of omega-3 fatty acids [C20–22ω 3 polyunsaturated fatty acids (PUFA)], blood levels of C20–22ω 3 PUFA, and mortality associated with cardiovascular disease (CVD). C20–22ω 3 PUFA have pleiotropic effects on cell function and regulate multiple pathways controlling blood lipids, inflammatory factors, and cellular events in cardiomyocytes and vascular endothelial cells. The hypolipemic, anti-inflammatory, anti-arrhythmic properties of these fatty acids confer cardioprotection. Accordingly, national heart associations and government agencies have recommended increased consumption of fatty fish or ω 3 PUFA supplements to prevent CVD. In addition to fatty fish, sources of ω 3 PUFA are available from plants, algae, and yeast. A key question examined in this review is whether nonfish sources of ω 3 PUFA are as effective as fatty fish-derived C20–22ω 3 PUFA at managing risk factors linked to CVD. We focused on ω 3 PUFA metabolism and the capacity of ω 3 PUFA supplements to regulate key cellular events linked to CVD. The outcome of our analysis reveals that nonfish sources of ω 3 PUFA vary in their capacity to regulate blood levels of C20–22ω 3 PUFA and CVD risk factors.

      Abbreviations:

      ALA
      α -linolenic acid
      ARA
      arachidonic acid
      CHD
      coronary heart disease
      ChREBP
      carbohydrate regulatory element binding protein
      CVD
      cardiovascular disease
      DHA
      docosahexaenoic acid
      DNL
      de novo lipogenesis
      DPA
      docosapentaenoic acid
      Elovl
      fatty acid elongase
      EPA
      eicosapentaenoic acid
      FADS
      fatty acid desaturase
      GPR
      G-protein receptor
      HDL-C
      HDL-cholesterol
      HNF4 α
      hepatic nuclear protein 4 α
      ICD
      implantable cardioverter defibrillator
      LA
      linoleic acid
      LDL-C
      LDL-cholesterol
      MetS
      metabolic syndrome
      MI
      myocardial infarction
      MLX
      max-like factor X
      NF κB
      nuclear factor κB
      PC
      prospective cohort
      PPARα
      peroxisome proliferator activated receptor α
      RBC
      red blood cell
      RCT
      randomized clinical trial
      SCD
      sudden cardiac death
      SDA
      stearidonic acid
      SREBP-1
      sterol regulatory element binding protein-1
      T1DM
      type 1 diabetes mellitus
      T2DM
      type 2 diabetes mellitus
      Omega-3 ( ω 3) polyunsaturated fatty acids (PUFA) represent one of two major classes of long chain highly unsaturated fatty acids encountered in the diet. Dietary ω 3 PUFA include α -linolenic acid (ALA, 18:3, ω 3); stearidonic acid (SDA, 18:4, ω 3); eicosapentaenoic acid (EPA, 20:5, ω 3); docosapentaenoic acid (DPA, 22:5, ω 3); and docosahexaenoic acid (DHA, 22:6, ω 3) (Fig. 1). The major dietary ω 6 PUFA is linoleic acid (LA, 18:2, ω 6). LA and ALA are essential fatty acids; they cannot be synthesized de novo in humans and are required for good health (
      • Spector A.A.
      Essentiality of fatty acids.
      ). These two fatty acids are precursors for C20–22ω 3 and ω 6 PUFA found throughout the body.
      Figure thumbnail gr1
      Fig. 1Structures of dietary ω 3 and ω 6 polyunsaturated fatty acids. A: C18ω 3 and ω 6 PUFA. B: C20–22ω 3 and ω 6 PUFA.
      Considerable interest in the health benefits of very long chain C20–22ω 3 PUFA arose in the 1970s when epidemiological studies on Greenland Inuits established that this population had reduced rates of myocardial infarction (MI) compared with individuals in Western countries (
      • Bang H.O.
      • Dyerberg J.
      • Nielsen A.B.
      Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos.
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ). These observations were linked to the high dietary intake of C20–22ω 3 PUFA, enrichment of blood lipids with C20–22ω 3 PUFA, and reduced fasting triglycerides (
      • Bang H.O.
      • Dyerberg J.
      • Nielsen A.B.
      Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos.
      ,
      • Dyerberg J.
      • Bang H.O.
      • Hjorne N.
      Fatty acid composition of the plasma lipids in Greenland Eskimos.
      ,
      • Dyerberg J.
      • Bang H.O.
      • Stoffersen E.
      • Moncada S.
      • Vane J.R.
      Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis?.
      ). The potential health benefits of ω 3 PUFA stimulated considerable research interest, resulting in over 500 clinical trials on ω 3 PUFA (Table 1). Over 250 clinical studies have examined the impact of ω 3 PUFA on cardiovascular disease (CVD) or risk factors linked to CVD, such as metabolic syndrome (MetS), diabetes, obesity, inflammation, dyslipidemia, and hypertension. Other equally important areas of ω 3 PUFA human research include visual acuity, cognitive development and decline, cancer prevention, and total mortality (
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ).
      TABLE 1Clinical trials on ω 3 fatty acids
      Number of Trials
      Cardiovascular disease and stroke2340
      Omega 3 fatty acids534
      Omega 3 fatty acids and:
        CVD or stroke110
        Metabolic syndrome16
        Diabetes or obesity12
        Inflammation97
        Dyslipidemia55
        Hypertension10
        Total290
      Queries: Cardiovascular disease and stroke; omega 3 fatty acids.
      This review examines current recommendations for ω 3 PUFA intake, ω 3 PUFA metabolism, and their effects on physiological processes relevant to CVD. We also examine several prospective cohort (PC) studies (Table 2) and randomized clinical trials (RCT) (Table 3) that report on the benefit or lack of benefit of ω 3 PUFA on cardiovascular health. Finally, we examine various sources of ω 3 PUFA for their capacity to regulate risk factors relevant to CVD. Our goal is to provide up-to-date evidence-based information on the benefits and limitations of dietary ω 3 PUFA in the management of cardiovascular health.
      TABLE 2Prospective cohort studies on ω 3 PUFA and clinical cardiovascular events
      Year (Ref)PopulationDesignFollow-up YearsKey Outcome
      2008 (
      • Yamagishi K.
      • Nettleton J.A.
      • Folsom A.R.
      ARIC study investigators: plasma fatty acid composition and incident heart failure in middle-adge adults: the atherosclerosis risk in communities (ARIC) study.
      )
      Atherosclerosis Risk in Communities (ARIC) StudyAssociation of plasma cholesterol ester fatty acids with incident HF14.3110 men and 87 women developed HF
      USA (Minneapolis); 3,592 white men and women (ages 45–64)Hazard ratio (95% CI); [P for trend]:
      LA status and HF: 0.54; [0.001]
      ALA status and HF: 0.99; [0.81]
      ARA status and HF: men: 1.37; [0.28]; women: 0.43 [0.02]
      EPA status and HF: 1.37; [0.26]
      DHA status and HF: men: 1.3, [0.47]; women: 0.21 [0.001]
      ARA and DHA may reduce risk of HF, particularly in women.
      2009 (
      • Virtanen J.K.
      • Mursu J.
      • Voutilainen S.
      • Tuomainen T.P.
      Serum long-chain n-3 polyunsaturated fatty acids and risk of hospital diagnosis of atrial fibrillation in men.
      )
      Kuopio Ischemic Heart Disease Risk Factor StudyAssociation of serum ω 3 PUFA with incident atrial fibrillation17.7240 men developed atrial fibrillation (AF)
      Finland; 2,174 men (ages 42–60)eduction of AF; [P for trend]:
      EPA + DPA + DHA: 35% [0.07] versus lowest quartile
      ALA: Serum ALA was not associated with the incidence of AF
      Serum C20–22 ω 3 PUFA, but not ALA, was inversely associated with AF.
      2010 (

      Pottala, J. V., Garg, S., Cohen, B. E., Whooley, M. A., Harris, W. S., . 2010. Blood eicosapentaenoic and docosahexaenoic acids predict all-cause mortality in patients with stable coronary heart disease: the Heart and Soul study. Circ. Cardiovasc. Qual. Outcomes. 3: 406–412.

      )
      Heart and Soul StudyAssociation of CHD with blood levels of EPA + DHA; Median blood level of EPA + DHA = 3.6%5.9237 deaths among 956 patients
      USA (San Francisco); 956 patients (ages 65–69) with a history of CHDBelow median: <3.6% EPA + DHA (N = 478)
      Above median: >3.6% EPA + DHA (N = 478)
      Below median versus above median (Hazard Ratio; 95% CI)
      27% reduced risk of CHD [HR = 0.73; 95% CI = 0.56–0.94]
      Blood EPA + DHA levels were inversely associated with total mortality in patients with stable CHD.
      2011 (
      • de Goede J.
      • Verschuren W.M.
      • Boer J.M.
      • Kromhout D.
      • Geleijnse J.M.
      Alpha-linolenic acid intake and 10-year incidence of coronary heart disease and stroke in 20,000 middle-aged men and women in the Netherlands.
      )
      The Netherlands; 20,069 generally healthy men and women (ages 20–65)Association of ALA intake (FFQ) with incident CHD (CVD and stroke)8–13280 CHD events (19% fatal); 221 strokes (4% fatal)
      Intake of energy-adjusted ALA in quintiles
      ALA intake range Q1: 1.0 g/day to Q5: 1.9 g/day
      For incident CHD: HR 0.89–1.01 in Q2-Q5 versus Q1
      For incident stroke: HR: 0.65, 0.49, 0.53 and 0.65 for Q2–Q5, respectively
      ALA was not associated with incident CHD.
      2011 (
      • Mozaffarian D.
      • Lemaitre R.N.
      • King I.B.
      • Song X.
      • Spiegelman D.
      • Sacks F.M.
      • Rimm E.B.
      • Siscovick D.S.
      Circulating long-chain omega-3 fatty acids and incidence of congestive heart failure in older adults: the cardiovascular health study: a cohort study.
      )
      Cardiovascular Health StudyPlasma phospholipid profiles and CHF14555 cases of CHF during 26,490 person-years
      USA (4 communities); 2,735 adults (age > 65) free of prevalent heart diseaseHighest versus lowest quartile hazard ratio (95% CI)
      EPA: 0EPA: 0.52 (0.38 - 0.72; P-trend = 0.001)
      DPA: 0.76 (0.56 - 1.04; P-trend = 0.057)
      DHA: 0.84 (0.58 - 1.21; P-trend = 0.38)
      Total ω 3 PUFA: 0.7 (0.49 – 0.99; P-trend = 0.062)
      Circulating individual and total ω 3 PUFA were associated with lower incidence of CHF in older (>65 yrs) adults.
      2010 (
      • Heine- Bröring R.C.
      • Brouwer I.A.
      • Proenca R.V.
      • van Rooij F.J.
      • Hofman A.
      • Oudkerk M.
      • Witteman J.C.
      • Geleijnse J.M.
      Intake of fish and marine n-3 fatty acids in relation to coronary calcification: the Rotterdam Study.
      )
      Rotterdam StudyDietary ω 3 fatty acid via FFQ and coronary calcification7Prevalence ratio (PR) for coronary calcification versus fish intake
      1,570 men and women (ages 62–85) asymptomatic for CVDFor fish intake > 19 g/day versus no fish intake
      Mild/moderate calcification: PR, 0.87 (95% CI = 0.78)
      Severe calcification: PR: 0.88 (95% CI = 0.74)
      Weak inverse association between fish intake and coronary calcification.
      CHF, congestive heart failure; CI, confidence interval; FFQ, food frequency questionnaire; HF, heart failure; HR, hazard ratio; PR, prevalence ratio; Q, quintile.
      TABLE 3Randomized clinical trials of ω 3 PUFA and clinical cardiovascular events
      Trial [Year] (Ref)GroupsInterventionYears of Follow-upBlood EPA and/or DHA ω 3 PUFA versus PlaceboEvents ω 3 PUFA versus Placebo
      DART-1 [1989] (
      • Burr M.L.
      • Fehily A.M.
      • Gilbert J.F.
      • Rogers S.
      • Holliday R.M.
      • Sweetnam P.M.
      • Elwood P.C.
      • Deadman N.M.
      Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART).
      ,
      • Burr M.L.
      Secondary prevention of CHD in UK men: the Diet and Reinfarction Trial and its sequel.
      )
      1,015 males, fish adviceAdvice: Increase fatty fish consumption or take fish oil supplement versus usual diet2EPA: 0.59 versus 0.46 mol%All deaths: N = 94 (9.3%) versus N = 130 (12.8%); P < 0.05
      1,018 males, no fish adviceIHD deaths: N = 78 (7.7%) versus N = 116 (11.4%), P < 0.01
      Recovered from MINonfatal MI: N = 49 (4.8%) versus N = 33 (3.2%), P = NS
      100% male, mean age <56.5
      DART-2 [2003] (
      • Burr M.L.
      Secondary prevention of CHD in UK men: the Diet and Reinfarction Trial and its sequel.
      ,
      • Burr M.L.
      • Ashfield-Watt P.A.
      • Dunstan F.D.
      • Fehily A.M.
      • Breay P.
      • Ashton T.
      • Zotos P.C.
      • Haboubi N.A.
      • Elwood P.C.
      Lack of benefit of dietary advice to men with angina: results of a controlled trial.
      )
      1,571 males, fish adviceAdvice: Increase fatty fish consumption or take fish oil supplement (3 g max EPA/day) versus usual diet3–9EPA: 45.8 versus 30.3 μ g/mlAll deaths: N = 283 (18.0%) versus N = 242 (15.7%); P = 0.8
      1,543 males, no fish adviceCardiac deaths: N = 180 (11.5%) versus N = 139 (9.0%); P = 0.02
      Recovered from MISudden cardiac deaths: N = 73 (4.6%) versus N = 47 (3.0%); P = 0.02
      100% male, age <70 yr
      GISSI [1999] (
      • GISSI-Prevenzione Investigators
      Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico.
      )
      5,666 in ω 3 PUFA group (85% male)EPA + DHA ethyl ester3.5NDCardiovascular deaths [RR; 95% CI)]: N = 291 (5.1%) versus N = 348 (6.2%) [0.83; 0.71–0.097]
      5,668 in placebo group (85% male)EPA + DHA (1:2)Coronary deaths: N = 214 (3.8%) versus N = 265 (4.7%), [0.80; 0.67–0.96]
      Prior MI ≤ 3 months850–882 mg/day versus placeboSudden cardiac deaths: N = 122 (2.2%) versus N = 164 (2.9%), [0.74; 0.58–0.93]
      (±300 mg/day Vitamin E)
      JELIS [2007] (
      • Yokoyama M.
      • Origasa H.
      • Matsuzaki M.
      • Matsuzawa Y.
      • Saito Y.
      • Ishikawa Y.
      • Oikawa S.
      • Sasaki J.
      • Hishida H.
      • Itakura H.
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ,
      • Itakura H.
      • Yokoyama M.
      • Matsuzaki M.
      • Saito Y.
      • Origasa H.
      • Ishikawa Y.
      • Oikawa S.
      • Sasaki J.
      • Hishida H.
      • Kita T.
      • et al.
      Relationships between plasma fatty acid composition and coronary artery disease.
      )
      9,326 in EPA + statin group (mean age 61; 32% male)EPA ethyl ester (1.8 g/day) ± statin4.6EPA: 170 versus 95 μ g/mlMajor coronary events: N = 262 (2.8%) versus N = 324 (3.5%); (P = 0.011)
      9,319 in statin-only group (mean age 61; 31% male)Blood cholesterol ≥ 6.5 mmol/lDHA: 154 versus 165 μ g/mlUnstable angina: N = 147 (1.6%) versus N = 193 (2.1%); (P = 0.014)
      Nonfatal coronary events: N = 240 (2.6%) versus N = 297 (3.2%); (P = 0.015)
      GISSI-HF [2008] (
      • Gissi-HF Investigators
      • Tavazzi L.
      • Maggioni A.P.
      • Marchioli R.
      • Barlera S.
      • Franzosi M.G.
      • Latini R.
      • Lucci D.
      • Nicolosi G.L.
      • Porcu M.
      • et al.
      Effect of n-3 polyunsaturated fatty acids in patients with chronic heart failure (the GISSI-HF trial): a randomized, double-blind, placebo-controlled trial.
      )
      3,494 in ω 3 PUFA group (85% male)EPA + DHA ethyl ester3-9NDCardiovascular deaths [HR; 95% CI]: N = 712 (20.4%) versus N = 765 (22.0%) [0.90; 0.81–0.99]
      3,481 in placebo group (85% male)EPA + DHA (1:2)Sudden cardiac deaths: N = 307 (8.8%) versus N = 325 (9.3%) [0.93; 0.79–1.08]
      850-882 mg/day versus placeboFatal and nonfatal stroke: N = 122 (3.5%) versus N = 103 (3.0%) [1.16; 0.89–1.51]
      Omega [2010] (
      • Rauch B.
      • Schiele R.
      • Schneider S.
      • Diller F.
      • Victor N.
      • Gohlke H.
      • Gottwik M.
      • Steinbeck G.
      • Del Castillo U.
      • Sack R.
      • et al.
      OMEGA, a randomized, placebo-controlled trial to test the effect of highly purified omega-3 fatty acids on top of modern guideline-adjusted therapy after myocardial infarction.
      )
      1,919 in Omega group (mean age 64; 75% male)EPA + DHA (1:1.2)1NDTotal mortality: N = 88 (4.6%) versus N = 70 (3.7%); (P = 0.18)
      1,885 in control group (mean age 64; 74% male)850 mg/day of ethyl ester versus 1 g/day of olive oilSudden cardiac deaths: N = 28 (1.5%) versus N = 29 (1.5%); (P = 0.84)
      Recent MI ≤ 2 weeks priorMajor adverse cerebro- or cardiovascular events: N = 182 (10.4%) versus N = 149 (8.8%); (P = 0.1)
      ICD-terminated ventricular tachycardia or fibrillation in survivor: N = 9 (0.5%) versus N = 2 (0.1%); (P = 0.06)
      Alpha-Omega [2010] (
      • Kromhout D.
      • Giltay E.J.
      • Geleijnse J.M.
      n-3 fatty acids and cardiovascular events after myocardial infarction.
      )
      2,404 in EPA-DHA group (mean age 69; 78% male)EPA-DHA group: margarine supplemented with EPA + DHA; targeted daily intake of ∼ 400 mg EPA + DHA3.3NDEPA-DHA group versus placebo or ALA group
      2,433, ALA or placebo group (mean age 69; 78% male)Placebo group: ±2 g ALA per dayTotal mortality: N = 186 (7.7%) versus N = 184 (7.6%); (P = 0.92)
      MI within prior 10 yearsPatients were also on state-of-the-art antihypertensive, antithrombotic, lipid-lowering therapyMajor cardiovascular events: N = 336 (14%) versus N = 335 (13.8%); (P = 0.93)
      Death from cardiovascular events: N = 80 (3.3%) versus N = 82 (3.4%); (P = 0.75)
      Death from CHD: N = 67 (2.8%) versus N = 71 (2.9%); (P = 0.75)
      Incident CVD: N = 67 (2.8%) versus N = 71 (2.9%); (P = 0.75)
      Ventricular-arrhythmia-related events: N = 67 (2.8%) versus N = 74 (3.0%); (P = 0.55)
      Su.Fol.Om3 [2010] (
      • Galan P.
      • Kesse-Guyot E.
      • Czernichow S.
      • Braincon S.
      • Blacher J.
      • Hercberg S.
      Effects of B vitamins and omega 3 fatty acids on cardiovascular diseases: a randomized placebo controlled trial.
      )
      1,253 in EPA + DHA group (mean age 60; 79% male)EPA + DHA (2:1)4.7EPA (2.1 versus 1.2% of total)Total mortality: N = 58 (4.7%) versus N = 59 (4.7%); (P = 0.33)
      Recent history of prior coronary or cerebral ischemic event600 mg/day versus Vitamin B group or placeboDHA (3.1 versus 2.7% of total)Major cardiovascular events: N = 81 (6.5%) versus N = 76 (6.1%); (P = 0.64)
      DART, Diet and Reinfarction Trial; GISSI, Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardio; HR, hazard ratio; IHD, ischemic heart disease; JELIS, Japanese EPA Lipid Intervention Study; ND, not determined; OM3FA, omega-3 fatty acid; RR, relative risk; Su.Fol.Om3, Supplementation with Folate, Vitamins (B6 and B12) and/or ω 3 Fatty Acids (B vitamins: 5-methyltetrahydrofolate, 560 μ g; vitamin B6, 3 mg; vitamin B12, 20 μ g).

      CURRENT RECOMMENDATIONS FOR ESSENTIAL FATTY ACID CONSUMPTION

      ALA and LA are precursors to ω 3 and ω 6 C20–22 PUFA, i.e., DHA (22:6, ω 3) (Fig. 2) and arachidonic acid (ARA, 20:4, ω 6), respectively. These fatty acids play structural roles in cells and serve as substrates for β -oxidation and energy production. They also regulate many physiological processes impacting human health, as nonesterified fatty acids, esterified (membrane-associated) fatty acids, or oxidized fatty acids.
      Figure thumbnail gr2
      Fig. 2Pathways for C18 PUFA conversion to C20–22 PUFA synthesis. The pathway illustrates the conversion of the essential fatty acid ALA (18:3, ω 3) to the end product DHA (22:6, ω 3). The enzymes involved in this pathway include two fatty acid desaturases (FADS1 and FADS2), two fatty acid elongases (Elovl2 and Elovl5), and peroxisomal β -oxidation (p- β Ox). Nonesterified fatty acids are converted to fatty acyl-CoAs by fatty acyl CoA synthetases; the fatty acids progress through the pathway as fatty acyl CoAs. Humans and rodents ingesting essential fatty acid-sufficient diets accumulate DHA in blood and tissues. Sources of dietary ω 3 PUFA [ALA, SDA (18:4, ω 3), EPA (20:5, ω 3), and DHA] include plants, fish, yeast, and algae. Details of the pathway are presented in the text.
      The American Heart Association has recommended the consumption of no more than 30% of total energy as fat and 5–10% of total energy as ω 6 PUFA (
      • Harris W.S.
      • Mozaffarian D.
      • Rimm E.
      • Kris-Etherton P.
      • Rudel L.L.
      • Appel L.J.
      • Engler M.M.
      • Engler M.B.
      • Sacks F.
      Omega-6 fatty acids and risk for cardiovascular disease: a science advisory from the American Heart Association Nutrition Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council on Cardiovascular Nursing; and Council on Epidemiology and Prevention.
      ,
      • Danaei G.
      • Ding E.L.
      • Mozaffarian D.
      • Taylor B.
      • Rehm J.
      • Murray C.J.
      • Ezzati M.
      The preventable causes of death in the United States: comparative risk assessment of dietary, lifestyle, and metabolic risk factors.
      ). Replacing saturated fat with PUFA ( ω 3 and ω 6) has a strong health benefit (
      • Mozaffarian D.
      • Clarke R.
      Quantitative effects on cardiovascular risk factors and coronary heart disease risk of replacing partially hydrogenated vegetable oils with other fats and oils.
      ,
      • Kris-Etherton P.
      • Flemming J.
      • Harris W.S.
      The debate about n-6 polyunsaturated fatty acid recommendatons for cardiovascular health.
      ). The amount of ALA required to prevent deficiency symptoms is ∼ 10% of LA, i.e., 0.6–1.2% of total energy [1.6 g for men and 1.1 g. for women per day (
      • Han S.N.
      • Leka L.S.
      • Lichtenstein A.H.
      • Ausman L.M.
      • Schaefer E.J.
      • Meydani S.N.
      Effect of hydrogenated and saturated, relative to polyunsaturated, fat on immune and inflammatory responses of adults with moderate hypercholesterolemia.
      )]. Consumption of LA and ALA in the United States is estimated to be ≤ 10% and ≤ 1% total energy, respectively (
      • Harris W.S.
      • Klurfeld D.M.
      Twentieth-century trends in essential fatty acid intakes and the predicted omega-3 index: evidence versus estimates.
      ). Up to 10% of the dietary ω 3 PUFA requirement can be provided by EPA or DHA (
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ,
      • Gebauer S.K.
      • Psota T.L.
      • Harris W.S.
      • Kris-Etherton P.M.
      n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits.
      ,
      • Craig W.J.
      • Mangels A.R.
      Position of the American Dietetic Association: vegetarian diets.
      ). Food sources of ALA include flaxseed, walnuts, canola oil, and chia seeds, while food sources of EPA and DHA include fatty fish, like salmon and anchovies, or oils derived from fatty fish and krill. Consumption of ∼ 500 mg/day of EPA and DHA (combined) is recommended to lower the risk for CVD. This level can be achieved by the consumption of two 3 ounce portions of fatty fish per week or the consumption of dietary fish or krill oil supplements. The level of ω 3-PUFA consumption should be increased to 1 g/day if CVD is present (
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ,
      • Gebauer S.K.
      • Psota T.L.
      • Harris W.S.
      • Kris-Etherton P.M.
      n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits.
      ,
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease.
      • Saravanan P.
      • Davidson N.C.
      • Schmidt E.B.
      • Calder P.C.
      Cardiovascular effects of marine omega-3 fatty acids.
      ).

      OMEGA-3 INDEX

      In 2004, Harris and Von Schacky (
      • Harris W.S.
      • Von Schacky C.
      The Omega-3 Index: a new risk factor for death from coronary heart disease?.
      ) introduced a new risk factor for death from coronary heart disease (CHD), the omega-3 index. The omega-3 index is defined as the percentage of whole-blood fatty acids that are the sum of EPA (20:5, ω 3), DPA (22:5, ω 3), and DHA (22:6, ω 3). An omega-3 index of less than 4% is associated with low cardioprotection, whereas an index of 8% or more is associated with high cardioprotection. The rationale for this measure is that fatty acid composition in whole-blood and red blood cell (RBC) phospholipids parallels fatty acid composition in cardiac muscle phospholipids (
      • Harris W.S.
      • Sands S.A.
      • Windsor S.L.
      • Ali H.A.
      • Stevens T.L.
      • Magalski A.
      • Porter C.B.
      • Borkon A.M.
      Omega-3 fatty acids in cardiac biopsies from heart transplantation patients: correlation with erythrocytes and response to supplementation.
      ).
      The first evidence in support of the relationship between RBC and cardiac muscle fatty acid content was based on studies with heart transplant patients before and after fish oil supplementation. The mol% of ω 3 and ω 6 PUFA in cardiac muscle prior to fish oil supplementation was LA (9.1%), ALA (0.3%), ARA (9.1%), EPA (0.18%), DPA (0.81%), and DHA (1.5%). After fish oil supplementation (1 g/day of EPA plus DHA for 6 months), the mol% of LA and ARA decreased by ∼ 15%, while ALA, EPA and DHA increased by 33, 333 and 53%, respectively. Changes in cardiac fatty acid profiles paralleled changes in RBC and plasma fatty acid profiles. In a subsequent report, Metcalf et al. (
      • Metcalf R.G.
      • James M.J.
      • Gibson R.A.
      • Edwards J.R.
      • Stubberfield J.
      • Stuklis R.
      • Roberts-Thomson K.
      • Young G.D.
      • Cleland L.G.
      Effects of fish-oil supplementation on myocardial fatty acids in humans.
      ) examined the accumulation of fatty acids in heart tissue after nontransplantation patients consumed 10 ml/day of flaxseed oil ( ∼ 59% ALA), olive oil ( ∼ 0.5% ALA), or fish oil (1.8% ALA, 32% EPA, 3.4% DPA, and 31.2% DHA). The time course studies (0–65 days) showed a significant curvilinear increase in EPA + DHA in atrial myocardial phospholipids, beginning as early as 7 days and continuing up to 30 days. Moreover, changes in RBC phospholipid C20–22ω 3 PUFA content paralleled C20–22ω 3 PUFA myocardial phospholipid. ARA declined in both myocardial and RBC phospholipids in patients receiving fish oil. Patients receiving the flaxseed or olive oil supplement had no significant increase in C20–22ω 3 PUFA in myocardial or RBC phospholipids. Both studies established that EPA shows the greatest fold increase in RBC and cardiac muscle phospholipids (
      • Harris W.S.
      • Sands S.A.
      • Windsor S.L.
      • Ali H.A.
      • Stevens T.L.
      • Magalski A.
      • Porter C.B.
      • Borkon A.M.
      Omega-3 fatty acids in cardiac biopsies from heart transplantation patients: correlation with erythrocytes and response to supplementation.
      ,
      • Metcalf R.G.
      • James M.J.
      • Gibson R.A.
      • Edwards J.R.
      • Stubberfield J.
      • Stuklis R.
      • Roberts-Thomson K.
      • Young G.D.
      • Cleland L.G.
      Effects of fish-oil supplementation on myocardial fatty acids in humans.
      ) following fish oil supplementation. DHA, however, remains ∼ 10-fold more abundant than EPA in RBC and myocardial phospholipids. Thus, dietary supplementation with fish oil (C20–22ω 3 PUFA), but not dietary flaxseed (ALA), significantly increases cardiac muscle C20–22ω 3 PUFA content.
      Such studies suggest that the omega-3 index may be a reasonable approach to assess cardiac ω 3 PUFA content and predict future CVD events (
      • Siscovick D.S.
      • Raghunathan T.E.
      • King I.
      • Weinmann S.
      • Wicklund K.G.
      • Albright J.
      • Bovbjerg V.
      • Arbogast P.
      • Smith H.
      • Kushi L.H.
      • et al.
      Dietary intake and cell membrane levels of long-chain n-3 polyunsaturated fatty acids and the risk of primary cardiac arrest.
      ,
      • Aarsetoey H.
      • Ponitz V.
      • Grundt H.
      • Staines H.
      • Harris W.S.
      • Nilsen D.W.
      (n-3) Fatty acid content of red blood cells does not predict risk of future cardiovascular events following an acute coronary syndrome.
      ). Sudden cardiac death (SCD), for example, is estimated to account for 50% of all deaths from CHD (
      • Myerburg R.J.
      • Interian Jr, A.
      • Mitrani R.M.
      • Kessler K.M.
      • Castellanos A.
      Frequency of sudden cardiac death and profiles of risk.
      • Myerburg R.J.
      • Junttila J.
      Sudden cardiac death caused by coronary heart disease.
      ). An epidemiologic study reported that the risk of SCD was lower in individuals with long-term fish consumption (
      • Streppel M.T.
      • Ocke M.C.
      • Boshuizen H.C.
      • Kok F.J.
      • Kromhout D.
      Long-term fish consumption and n-3 fatty acid intake in relation to (sudden) coronary heart disease death: the Zutphen study.
      ). This finding is consistent with studies reporting that a low omega-3 index increased the risk of ventricular fibrillation during acute ischemic phase of a MI and SCD (
      • Aarset⊘y H.
      • Pönitz V.
      • Nilsen O.B.
      • Grundt H.
      • Harris W.S.
      • Nilsen D.W.
      Low levels of cellular omega-3 increase the risk of ventricular fibrillation during the acute ischaemic phase of a myocardial infarction.
      ,
      • Aarsetoey H.
      • Aarsetoey R.
      • Lindner T.
      • Staines H.
      • Harris W.S.
      • Nilsen D.W.
      Low levels of the omega-3 index are associated with sudden cardiac arrest and remain stable in survivors in the subacute phase.
      ).
      Several factors, such as baseline values for the omega-3 index and health status, affect the capacity of dietary ω 3 PUFA to alter the omega-3 index and cardiac myocardial phospholipid content. For example, the average omega-3 index in Western countries is ∼ 5% and the incidence of SCD is 150/100,000 person-years. In Japan, a country with a high fish consumption, the omega-3 index is greater than 9% and the incidence of SCD is 7.8/100,000 person-years in the general population (
      • Von Schacky C.
      Omega-3 fatty acids vs. cardiac disease-the contribution of the omega-3 index.
      ). Most sudden deaths are caused by ventricular arrhythmias in patients with structural heart disease and impaired left-ventricular function (
      • Huikuri H.V.
      • Castellanos A.
      • Myerburg R.J.
      Sudden death due to cardiac arrhythmias.
      ). Primary prevention trials have established a survival benefit in high-risk patients who receive implantable cardioverter defibrillators (ICD) (
      • Wilhelm M.
      • Tobias R.
      • Asskali F.
      • Kraehner R.
      • Kuly S.
      • Klinghammer L.
      • Boehles H.
      • Daniel W.G.
      Red blood cell omega-3 fatty acids and the risk of ventricular arrhythmias in patients with heart failure.
      ). Omega-3 PUFA supplementation trials in ICD patients, however, have produced conflicting results: ω 3 PUFA supplementation induced anti-arrhythmic, pro-arrhythmic or no response (
      • Wilhelm M.
      • Tobias R.
      • Asskali F.
      • Kraehner R.
      • Kuly S.
      • Klinghammer L.
      • Boehles H.
      • Daniel W.G.
      Red blood cell omega-3 fatty acids and the risk of ventricular arrhythmias in patients with heart failure.
      ,
      • Von Schacky C.
      Omega-3 fatty acids: anti-arrhythmic, pro-arrhythmic or both?.
      • Brouwer I.A.
      • Raitt M.H.
      • Dullemeijer C.
      • Kraemer D.F.
      • Zock P.L.
      • Morris C.
      • Katan M.B.
      • Connor W.E.
      • Camm J.A.
      • Schouten E.G.
      • et al.
      Effect of fish oil on ventricular tachyarrhythmia in three studies in patients with implantable cardioverter defibrillators.
      ). In patients with idiopathic cardiomyopathy, cardiac muscle fatty acid uptake and oxidation decrease, while glucose metabolism increases (
      • Dávila-Román V.G.
      • Vedala G.
      • Herrero P.
      • de las Fuentes L.
      • Rogers J.G.
      • Kelly D.P.
      • Gropler R.J.
      Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy.
      ). Patients with type 1 and type 2 diabetes, major risk factors for CVD, have increased myocardial fatty acid uptake and oxidation and reduced glucose oxidation (
      • Herrero P.
      • Peterson L.R.
      • McGill J.B.
      • Matthew S.
      • Lesniak D.
      • Dence C.
      • Gropler R.J.
      Increased myocardial fatty acid metabolism in patients with type 1 diabetes mellitus.
      • Goldberg I.J.
      • Trent C.M.
      • Schulze P.C.
      Lipid metabolism and toxicity in the heart.
      ). Such changes in cardiac fatty acid uptake and metabolism reflect the plasticity of cardiac muscle metabolism for fuel utilization for energy production (
      • Goldberg I.J.
      • Trent C.M.
      • Schulze P.C.
      Lipid metabolism and toxicity in the heart.
      ). Such findings also suggest that patients with chronic metabolic disease may have altered cardiac muscle fatty acid composition that may not be reflected in RBC phospholipids. As such, the omega-3 index in certain pathophysiological states may not accurately predict cardiac ω 3 PUFA content or health benefit. Factors governing the availability of fatty acids, glucose, lactate, or ketone bodies for cardiac muscle energy metabolism (ATP generation) depend on the nutritional supply and hormonal and health status (
      • Goldberg I.J.
      • Trent C.M.
      • Schulze P.C.
      Lipid metabolism and toxicity in the heart.
      ). Although there has been considerable interest in cardiac lipid metabolism, lipid storage, and lipotoxicity, much less attention has focused on cardiac muscle PUFA metabolism.

      CONVERSION OF C18 ESSENTIAL FATTY ACIDS TO C20ndash22 PUFA

      In addition to dietary sources, heart and blood levels of fatty acids depend on essential fatty acid metabolism. The pathway for conversion of the essential fatty acids LA and ALA to C20–22ω 3 and ω 6 PUFA involves two fatty acid desaturases (FADS1 and FADS2) and two fatty acid elongases (Elovl2 and Elovl5). The final step in DHA (22:6, ω 3) synthesis requires peroxisomal β -oxidation of 24:6, ω 3 (
      • Jump D.B.
      N-3 polyunsaturated fatty acid regulation of hepatic gene transcription.
      ). The conversion of ALA to DHA is illustrated in Fig. 2; intermediates in the ω 3 PUFA pathway include SDA, EPA, DPA, and C24ω 3 PUFA. DHA is the major product of this pathway and the major ω 3 PUFA accumulating in cells of all tissues, including cardiac muscle and vascular endothelial cells. The intermediates of the pathway are found in cells but at levels well below DHA. The abundance of DHA in the heart is not the sole factor in cardioprotection; DHA and other ω 3 PUFA are converted to bioactive fatty acids that affect multiple signaling mechanisms controlling cardiac and vascular function.
      Non-esterified fatty acids (NEFA) are transported into cells and rapidly converted to fatty acyl-CoAs by fatty acid CoA synthetases. These fatty acyls are either assimilated into neutral lipids (triglycerides and cholesterol esters) or phospholipids, or they enter metabolic pathways involved in fatty acid oxidation, elongation, or desaturation. The desaturases and elongases involved in converting C18 PUFA-CoAs to C20–22 fatty acyl-CoAs are associated with the endoplasmic reticulum (microsome) (Fig. 2). The products and some intermediates of the pathway are typically assimilated into complex lipids as phospholipids used in cell membranes. Both FADS1 and FADS2 are required for C20–22 PUFA synthesis, and FADS2 is considered the rate-limiting enzyme in the pathway (
      • Park W.J.
      • Kothapalli K.S.
      • Lawrence P.
      • Tyburczy C.
      • Brenna J.T.
      An alternate pathway to long chain polyunsaturates: the FADS2 gene product Delta8-desaturates 20:2n-6 and 20:3n-3.
      ,
      • Gregory M.K.
      • Gibson R.A.
      • Cook-Johnson R.J.
      • Cleland L.G.
      • James M.J.
      Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis.
      ). Fatty acid desaturation requires additional factors, including i) fatty acyl CoA substrate, ii) NADH, iii) cytochrome B5, and iv) cytochrome B5 reductase. Ablation of FADS1 or FADS2 expression in mice decreases ARA and DHA formation, but it increases the formation of C20 elongation products derived from LA or ALA, i.e., 20:2, ω 6 and 20:3, ω 3 (
      • Stroud C.K.
      • Nara T.Y.
      • Roqueta-Rivera M.
      • Radlowski E.C.
      • Lawrence P.
      • Zhang Y.
      • Cho B.H.
      • Segre M.
      • Hess R.A.
      • Brenna J.T.
      • et al.
      Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.
      • Fan Y.Y.
      • Monk J.M.
      • Hou T.Y.
      • Callway E.
      • Vincent L.
      • Weeks B.
      • Yang P.
      • Chapkin R.S.
      Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model.
      ). Eicosadienoic acid (20:2, ω 6) is a pro-inflammatory fatty acid (
      • Huang Y.S.
      • Huang W.C.
      • Li C.W.
      • Chuang L.T.
      Eicosadienoic acid differentially modulates production of inflammatory modulators in murine macrophages.
      ).
      Seven fatty acid elongase (Elovl) subtypes are expressed in humans and rodents (
      • Jump D.B.
      • Torres-Gonzalez M.
      • Olson L.K.
      Soraphen A, a inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.
      ,
      • Jakobsson A.
      • Westerberg R.
      • Jacobsson A.
      Fatty acid elongases in mammals: their regulation and role in metabolism.
      ,
      • Guillou H.
      • Zadravec D.
      • Martin P.G.
      • Jacobsson A.
      The key roles of elongases and desaturases in mammalian fatty acid metabolism: insights from transgenic mice.
      ). Expression of these enzymes is regulated by tissue-specific and developmental factors as well as diet and hormones (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      • Tripathy S.
      • Torres-Gonzalez M.
      • Jump D.B.
      Elevated hepatic fatty acid elongase-5 (Elovl5) activity corrects dietary fat induced hyperglycemia in obese C57BL/6J mice.
      ). Two of these enzymes, Elovl2 and Elovl5, are involved in the conversion of C18ω 3 and ω 6 PUFA to the corresponding C20–22ω 3 and ω 6 PUFA (Fig. 2). Like fatty acid desaturases, fatty acid elongation occurs in a multi-enzyme complex in the endoplasmic reticulum. The enzymes required for fatty acid elongation include i) 3-keto acyl CoA reductase (Elovl), ii) 3-ketoacyl CoA reductase, iii) 3-hydroxy acyl CoA dehydratase, and iv) trans-2,3-enoyl CoA reductase. Fatty acid elongation is similar to the synthesis of fatty acids carried out by fatty acid synthase and involves the 2-carbon addition to the acyl chain. The key substrates include fatty acyl CoA, malonyl CoA, and NADPH. Factors controlling cellular fatty acyl CoA, malonyl CoA, or NADPH levels affect the pace of fatty acid elongation (
      • Jump D.B.
      • Torres-Gonzalez M.
      • Olson L.K.
      Soraphen A, a inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.
      ). The elongase (Elovl, 3-keto acyl CoA synthase) establishes substrate specificity and is rate limiting. For example, Elovl2 condenses malonyl CoA and C20 PUFA to form the C22 and C24 PUFA, whereas Elovl5 condenses malonyl CoA and C18 PUFA or C20 PUFA to form C20 and C22 PUFA. Elovl5 does not convert C22 PUFA to C24 PUFA (
      • Gregory M.K.
      • Gibson R.A.
      • Cook-Johnson R.J.
      • Cleland L.G.
      • James M.J.
      Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis.
      ,
      • Jakobsson A.
      • Westerberg R.
      • Jacobsson A.
      Fatty acid elongases in mammals: their regulation and role in metabolism.
      ,
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ). Elovl2 is a key enzyme in DHA synthesis, and its cellular abundance determines the capacity of cells to generate DHA. In rodents, cardiac Elovl2 mRNA (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      ,
      • Igarashi M.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Rat heart cannot synthesize docosahexaenoic acid from circulating alpha-linolenic acid because it lacks elongase-2.
      ) and the capacity of the heart to convert ALA to DHA is low compared with other tissues like the liver (
      • Rapoport S.I.
      • Igarashi M.
      • Gao F.
      Quantitative contributions of diet and liver synthesis to docosahexaenoic acid homeostasis.
      ).
      Elovl5 participates not only in PUFA synthesis but also in MUFA synthesis, e.g., conversion of 16:1, ω 7 to 18:1, ω 7 (
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ,
      • Green C.D.
      • Ozguden-Akkoc C.G.
      • Wang Y.
      • Jump D.B.
      • Olson L.A.
      Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species.
      ). Recent studies suggest that fatty acids derived from the de novo lipogenesis (DNL), MUFA synthesis, and β -oxidation of 18:1, ω 9 and appearing in plasma phospholipids, i.e., 18:1 ω 7, 16:1 ω 9, are associated with the elevated risk of SCD but not of other CVD events (
      • Wu J.H.
      • Lemaitre R.N.
      • Imamura F.
      • King I.B.
      • Song X.
      • Spiegelman D.
      • Siscovick D.S.
      • Mozaffarian D.
      Fatty acids in the de novo lipogenesis pathway and risk of coronary heart disease: the Cardiovascular Health Study.
      ). These fatty acids are in low abundance in the diet, but they are generated from palmitate (16:0) by the action of fatty acid desaturases (stearoyl CoA desaturase) and elongases (Elovl5 and Elovl6). The expression of all three enzymes is regulated by C20–22ω 3 PUFA through the control of two transcription factors, peroxisome proliferator activated receptor- α (PPAR α ) and sterol regulatory element binding protein-1 (SREBP1), in rodents (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      ,
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ,
      • Matsuzaka T.
      • Shimano H.
      • Yahagi N.
      • Amemiya-Kudo M.
      • Yoshikawa T.
      • Hasty A.H.
      • Tamura Y.
      • Osuga J.
      • Okazaki H.
      • Iizuka Y.
      • et al.
      Dual regulation of mouse Delta(5)- and Delta(6)-desaturase gene expression by SREBP-1 and PPARalpha.
      ,
      • Matsuzaka T.
      • Shimano H.
      Elovl6: a new player in fatty acid metabolism and insulin sensitivity.
      ). As such, the expression of these enzymes is sensitive to regulation by changes in blood insulin, a key regulator of SREBP1 nuclear abundance, and factors controlling PPAR α activity, i.e., fibrates and fatty acids.

      RETROCONVERSION OF C22 to C20 PUFA

      C22 PUFA are retroconverted to C20 PUFA (
      • Sprecher H.
      Metabolism of highly unsaturated n-3 and n-6 fatty acids.
      ). Primary rat hepatocytes treated with EPA accumulate 22:5, ω 3 due to fatty acid elongation. These cells, however, do not accumulate DHA because of low FADS2 activity. In contrast, rat primary hepatocytes treated with DHA accumulate DHA, DPA, and EPA (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      ). The accumulation of EPA is due to retroconversion of DHA; a pathway that operates in rodents and humans (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      • Plourde M.
      • Chouinard-Watkins R.
      • Vandal M.
      • Zhang Y.
      • Lawrence P.
      • Brenna J.T.
      • Cunnane S.C.
      Plasma incorporation, apparent retroconversion and beta-oxidation of 13C-docosahexaenoic acid in the elderly.
      ). The reaction involves peroxisomal β -oxidation and the reduction of double bond to generate EPA from DHA (Fig. 2) (
      • Sprecher H.
      Metabolism of highly unsaturated n-3 and n-6 fatty acids.
      ). This reaction is not trivial: it increases plasma and cellular levels of EPA in animals fed DHA (
      • Ryan A.S.
      • Bailey-Hall E.
      • Nelson E.B.
      • Salem Jr, N.
      The hypolipidemic effect of an ethyl ester of algal-docosahexaenoic acid in rats fed a high-fructose diet.
      ), DHA retroconversion to EPA is sufficient to activate PPAR α in primary hepatocytes (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      ). DPA is formed after retroconverted EPA is elongated by either Elovl2 or Elovl5. The retroconversion of DHA to EPA and DPA in humans (
      • Plourde M.
      • Chouinard-Watkins R.
      • Vandal M.
      • Zhang Y.
      • Lawrence P.
      • Brenna J.T.
      • Cunnane S.C.
      Plasma incorporation, apparent retroconversion and beta-oxidation of 13C-docosahexaenoic acid in the elderly.
      ), rodents, and cells (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      ) confounds efforts to establish cause and effect relationships between dietary EPA, DPA, or DHA and specific physiological or biochemical outcomes.

      REGULATION OF PUFA SYNTHESIS

      Although DNL is a highly regulated pathway that is sensitive to changes in dietary carbohydrate and hormonal status (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ), hepatic enzymes involved in PUFA synthesis are less sensitive to fasting-refeeding or dietary carbohydrate (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      ,
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ,
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ). Enzymes involved in DNL, MUFA, and PUFA synthesis, however, are regulated by the type and quantity of dietary fat ingested (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      • Tripathy S.
      • Torres-Gonzalez M.
      • Jump D.B.
      Elevated hepatic fatty acid elongase-5 (Elovl5) activity corrects dietary fat induced hyperglycemia in obese C57BL/6J mice.
      ,
      • Gao F.
      • Kim H.W.
      • Igarashi M.
      • Kiesewetter D.
      • Chang L.
      • Ma K.
      • Rapoport S.I.
      Liver conversion of docosahexaenoic and arachidonic acids from their 18-carbon precursors in rats on a DHA-free but alpha-LNA-containing n-3 PUFA adequate diet.
      • Mohrhauer H.
      • Holman R.T.
      The effect of dose level of essential fatty acids upon fatty acid composition of the rat liver.
      ). In rodents, the mRNAs encoding FADS1, FADS2, Elovl2, and Elovl5 are regulated by SREBP1 and PPAR α (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      ,
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ,
      • Matsuzaka T.
      • Shimano H.
      • Yahagi N.
      • Amemiya-Kudo M.
      • Yoshikawa T.
      • Hasty A.H.
      • Tamura Y.
      • Osuga J.
      • Okazaki H.
      • Iizuka Y.
      • et al.
      Dual regulation of mouse Delta(5)- and Delta(6)-desaturase gene expression by SREBP-1 and PPARalpha.
      ,
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ,
      • Qin Y.
      • Dalen K.T.
      • Gustafsson J.A.
      • Nebb H.I.
      Regulation of hepatic fatty acid elongase 5 by LXRalpha-SREBP-1c.
      ); both transcription factors are well-established targets of regulation by dietary PUFA (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ). Substrate availability also plays a major role in controlling product (C20–22 PUFA) formation (
      • Tu W.C.
      • Cook-Johnson R.J.
      • James M.J.
      • Muhlhausler B.S.
      • Gibson R.A.
      Omega-3 long chain fatty acid synthesis is regulated more by substrate levels than gene expression.
      ). In addition to fatty acyl-CoAs, factors controlling cellular malonyl CoA affect both DNL and PUFA synthesis. Malonyl CoA is synthesized by acetyl CoA carboxylase (ACC)1 and ACC2 and degraded by malonyl CoA decarboxylase (
      • Saggerson D.
      Malonyl-CoA, a key signaling molecule in mammalian cells.
      ). Malonyl CoA is utilized for DNL and fatty acid elongation; it also inhibits carnitine palmitoyl transferase-1. Enzymes that generate (ACC1 and ACC2) and degrade malonyl CoA (malonyl CoA decarboxylase) are regulated by insulin. Low levels of circulating insulin in type 1 diabetes lowers ACC1 expression, leading to decreased malonyl CoA for DNL and fatty acid elongation (
      • Saggerson D.
      Malonyl-CoA, a key signaling molecule in mammalian cells.
      ,
      • Muoio D.M.
      • Newgard C.B.
      Fatty acid oxidation and insulin action: when less is more.
      ). Moreover, the ACC inhibitor soraphen A inhibits DNL and fatty acid elongation (IC50∼ 5 nM) by lowering cellular malonyl CoA. Soraphen A, however, does not block fatty acid desaturation (
      • Jump D.B.
      • Torres-Gonzalez M.
      • Olson L.K.
      Soraphen A, a inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.
      ). The ratio of 20:4, ω 6 to 18:2, ω 6 in plasma or RBC membrane lipids is often used as a surrogate marker for PUFA conversion in vivo. Suppressing fatty acid elongation in the prostate cancer cell line LnCAP inhibits LA conversion to ARA, resulting in a decrease in the 20:4, ω 6 to 18:2, ω 6 ratio and an increase in the 18:3, ω 6 to 18:2, ω 6 ratio. Thus, changes in 20:4, ω 6 to 18:2, ω 6 ratio can be due to changes in activity of either the desaturases (FADS1, FADS2) or elongases (Elovl2, Elovl5).
      Evidence that PUFA synthesis may be regulated in chronic disease comes from human and animal studies. In 301 Swedish men (60 years of age), for example, FADS1 products (20:4, ω 6/20:3, ω 6) in adipose tissue and plasma phospholipids were inversely correlated with obesity and insulin resistance, whereas FADS2 products (18:3, ω 6/18:2, ω 6) showed a positive association (
      • Warensjö E.
      • Rosell M.
      • Hellenius M.L.
      • Vessby B.
      • De Faire U.
      • Riserus U.
      Associations between estimated fatty acid desaturase activities in serum lipids and adipose tissue in humans: links to obesity and insulin resistance.
      ). A study involving 97 Japanese men ( ∼ 50 years of age) showed reduced 20:4, ω 6/20:3, ω 6 (FADS1 product), but higher levels of 18:3, ω 6/18:2, ω 6 (FADS2 product) in obese and MetS patients versus control (
      • Kawashima A.
      • Sugawara S.
      • Okita M.
      • Akahane T.
      • Fukui K.
      • Hashiuchi M.
      • Kataoka C.
      • Tsukamoto I.
      Plasma fatty acid composition, estimated desaturase activities and intakes of energy and nutrient in Japanese men with abdominal obesity or metabolic syndrome.
      ). Feeding C57BL/6J mice a high-fat diet (60% energy as fat) induces obesity, insulin resistance, and hepatosteatosis (fatty liver) and suppresses the plasma and hepatic 20:4, ω 6 to 18:2, ω 6 ratio by ∼ 60% compared with lean euglycemic mice fed a low-fat diet (10% energy as fat). The decline in the 20:4, ω 6/18:2, ω 6 ratio in this mouse model was linked to suppressed hepatic expression and activity of Elovl5 and SREBP1 nuclear abundance. Other enzymes involved in PUFA synthesis, like Elovl2, FADS1, or FADS2, were not affected by the high-fat diet (
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ). Changes in hepatic PUFA metabolism alter both hepatic and plasma PUFA composition. The conversion of ALA to DHA is low in the heart versus the liver (
      • Rapoport S.I.
      • Igarashi M.
      • Gao F.
      Quantitative contributions of diet and liver synthesis to docosahexaenoic acid homeostasis.
      ); as such, the liver likely plays an important role in providing DHA to the heart.
      In lean rats and obese mice, dietary C20–22ω 3 PUFA suppress the expression of Elovl5 and FADS1 (
      • Wang Y.
      • Botolin D.
      • Christian B.
      • Busik J.
      • Xu J.
      • Jump D.B.
      Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases.
      ,
      • Depner C.M.
      • Torres-Gonzalez M.
      • Tripathy S.
      • Milne G.
      • Jump D.B.
      Menhaden oil decreases high-fat diet-induced markers of hepatic damage, steatosis, inflammation, and fibrosis in obese ldlr-/- mice.
      ). The decline in expression of these enzymes correlates with a decrease ( ≥ 50%) ratio of 20:4, ω 6 to 18:2, ω 6 in liver and plasma. Dietary C20–22ω 3 PUFA are feedback inhibitors of endogenous C20–22ω 3 and ω 6 PUFA synthesis, as well as robust inhibitors of DNL (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ). Recent studies by Pachikian et al. (
      • Pachikian B.D.
      • Essaghir A.
      • Demoulin J.B.
      • Neyrinck A.M.
      • Catry E.
      • De Backer F.C.
      • Dejeans N.
      • Dewulf E.M.
      • Sohet F.M.
      • Portois L.
      • et al.
      Hepatic n-3 polyunsaturated fatty acid depletion promotes steatosis and insulin resistance in mice: genomic analysis of cellular targets.
      ) establish the impact of dietary ω 3 PUFA on whole-body metabolism. In this study, all ω 3 PUFA were removed from the diet, but ω 6 PUFA were maintained at essential fatty acid-sufficient levels. After 3 months on the ω 3 PUFA-deficient diet, mice developed hepatosteatosis and insulin resistance. The mechanism was linked to a major decline in hepatic ALA, EPA, and DHA, but no change in hepatic LA and ARA levels. Depletion of hepatic ω 3 PUFA lowered fatty acid oxidation, a PPAR α -regulated mechanism, and increased fatty acid synthesis and triglyceride accumulation, SREBP1- and ChREBP/MLX-regulated mechanisms. PPAR α , SREBP1, and the ChREBP/MLX heterodimer are well-established targets of C20–22ω 3 PUFA control (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ). These in vivo studies illustrate how ω 3 PUFA deficiency alone regulates a series of transcriptional regulatory mechanisms that alter hepatic and systemic lipid metabolism.

      GENE POLYMORPHISMS, BLOOD PUFA, AND CVD

      Genes encoding fatty acid desaturases and elongases involved in PUFA synthesis display single nucleotide polymorphisms (SNP). The FADS gene cluster has been most extensively studied; it consists of FADS1, FADS2, and FADS3 on chromosome 11 (
      • Glaser C.
      • Lattka E.
      • Rzehak P.
      • Steer C.
      • Koletzko B.
      Genetic variation in polyunsaturated fatty acid metabolism and its potential relevance for human development and health.
      ). Although the enzymatic functions of FADS1 and FADS2 are well described, FADS3 remains a gene without a clearly defined enzymatic function (
      • Blanchard H.
      • Legrand P.
      • Pedrono F.
      Fatty Acid Desaturase 3 (Fads3) is a singular member of the Fads cluster.
      ). More than 20 SNPs have been identified in the FADS gene cluster. One (rs174546) is found in the 3 ′ UTR of FADS1, while 12 SNPs are found in or near the FADS2 gene. Much emphasis has been placed on associating specific SNPs with changes in blood C18–22ω 3 and ω 6 PUFA content (
      • Sergeant S.
      • Hugenschmidt C.E.
      • Rudock M.E.
      • Ziegler J.T.
      • Ivester P.
      • Ainsworth H.C.
      • Vaidya D.
      • Douglas Case L.
      • Langefeld C.D.
      • Freedman B.I.
      • et al.
      Differences in arachidonic acid levels and fatty acid desaturase (FADS) gene variants in African Americans and European Americans with diabetes or the metabolic syndrome.
      ,
      • Lattka E.
      • Illig T.
      • Koletzko B.
      • Heinrich J.
      Genetic variants of the FADS1 FADS2 gene cluster as related to essential fatty acid metabolism.
      ) (
      • Glaser C.
      • Heinrich J.
      • Koletzko B.
      Role of FADS1 and FADS2 polymorphisms in polyunsaturated fatty acid metabolism.
      • Mathias R.A.
      • Vergara C.
      • Gao L.
      • Rafaels N.
      • Hand T.
      • Campbell M.
      • Bickel C.
      • Ivester P.
      • Sergeant S.
      • Barnes K.C.
      • et al.
      FADS genetic variants and omega-6 polyunsaturated fatty acid metabolism in a homogeneous island population.
      ). A meta-analysis of genome-wide associated studies (GWAS) from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium found that genetic variations in FADS1 and FADS2 were associated with high levels of ALA versus EPA and DPA (
      • Lemaitre R.N.
      • Tanaka T.
      • Tang W.
      • Manichaikul A.
      • Foy M.
      • Kabagambe E.K.
      • Nettleton J.A.
      • King I.B.
      • Weng L.C.
      • Bhattacharya S.
      • et al.
      Genetic loci associated with plasma phospholipid n-3 fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium.
      ). Of the SNPs identified in the FADS gene cluster, all are found in introns, except rs968567; which is located in the 5 ′ UTR of FADS2 (
      • Bokor S.
      • Dumont J.
      • Spinneker A.
      • Gonzalez-Gross M.
      • Nova E.
      • Widhalm K.
      • Moschonis G.
      • Stehle P.
      • Amouyel P.
      • De Henauw S.
      • et al.
      Single nucleotide polymorphisms in the FADS gene cluster are associated with delta-5 and delta-6 desaturase activities estimated by serum fatty acid ratios.
      ). This SNP is linked to changes in FADS2 promoter activity through binding of Elk1, an ETS domain transcription factor (
      • Lattka E.
      • Eggers S.
      • Moeller G.
      • Heim K.
      • Weber M.
      • Mehta D.
      • Prokisch H.
      • Illig T.
      • Adamski J.
      A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1.
      ).
      In contrast to the FADS, fatty acid elongase genes are not clustered on a single chromosome; they are encoded from 5 different chromosomes: Elovl1, chromosome (Ch) 1; Elovl2, Ch 6; Elovl3, Ch 10; Elovl4, Ch 6; Elovl5, Ch 5; Elovl6, Ch 4; and Elovl7, Ch 5. Genetic variation in the Elovl2 gene was associated with higher levels of EPA and DPA and lower levels of DHA (
      • Lemaitre R.N.
      • Tanaka T.
      • Tang W.
      • Manichaikul A.
      • Foy M.
      • Kabagambe E.K.
      • Nettleton J.A.
      • King I.B.
      • Weng L.C.
      • Bhattacharya S.
      • et al.
      Genetic loci associated with plasma phospholipid n-3 fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium.
      ). While Elovl2 and Elovl5 convert C20 to C22 PUFA, only Elovl2 converts C22 to C24 PUFA, a requirement for DHA synthesis (
      • Wang Y.
      • Botolin D.
      • Xu J.
      • Christian B.
      • Mitchell E.
      • Jayaprakasam B.
      • Nair M.G.
      • Peters J.M.
      • Busik J.V.
      • Olson L.K.
      • et al.
      Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.
      ,
      • Ohno Y.
      • Suto S.
      • Yamanaka M.
      • Mizutani Y.
      • Mitsutake S.
      • Igarashi Y.
      • Sassa T.
      • Kihara A.
      ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis.
      ) (Fig. 2). Although these outcomes agree favorably with the previous INCHIANTI study (
      • Tanaka T.
      • Shen J.
      • Abecasis G.R.
      • Kisialiou A.
      • Ordovas J.M.
      • Guralnik J.M.
      • Singleton A.
      • Bandinelli S.
      • Cherubini A.
      • Arnett D.
      • et al.
      Genome-wide association study of plasma polyunsaturated fatty acids in the InCHIANTI Study.
      ), Aslibekyan et al. (
      • Aslibekyan S.
      • Jensen M.K.
      • Campos H.
      • Linkletter C.D.
      • Loucks E.B.
      • Ordovas J.M.
      • Deka R.
      • Rimm E.B.
      • Baylin A.
      Genetic variation in fatty acid elongases is not associated with intermediated cardiovascular phenotypes or myocardial infarction.
      ) concluded that genetic variation in Elovl2 and Elovl5 was not associated with serum lipids, biomarkers of systemic inflammation, or the risk of MI.
      Studies on gene polymorphisms in desaturase and elongase genes are associated with changes in blood C20–22ω 3 and ω 6 PUFA. With the exception of studies on the polymorphism in the FADS2 promoter (
      • Lattka E.
      • Eggers S.
      • Moeller G.
      • Heim K.
      • Weber M.
      • Mehta D.
      • Prokisch H.
      • Illig T.
      • Adamski J.
      A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1.
      ), few studies have established that changes in blood fatty acid profiles are due to an effect on the expression or activity of the enzymes involved in the pathway. Many of the FADS SNPs are in introns or 3 ′ flanking regions. As such, cause and effect relationships between SNPs and activities of specific enzymes remain to be established.

      OVERVIEW OF C20ndash22 omega 3 PUFA REGULATION OF CELL FUNCTION

      Studies on essential fatty acid deficiency and null mutations in the FADS1, FADS2, and Elovl5 genes have established the requirement for this pathway to convert dietary LA and ALA to C20–22ω 3 and ω 6 PUFA and to regulate multiple physiological processes (
      • Stroud C.K.
      • Nara T.Y.
      • Roqueta-Rivera M.
      • Radlowski E.C.
      • Lawrence P.
      • Zhang Y.
      • Cho B.H.
      • Segre M.
      • Hess R.A.
      • Brenna J.T.
      • et al.
      Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.
      ,
      • Fan Y.Y.
      • Monk J.M.
      • Hou T.Y.
      • Callway E.
      • Vincent L.
      • Weeks B.
      • Yang P.
      • Chapkin R.S.
      Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model.
      ,
      • Stoffel W.
      • Holz B.
      • Jenke B.
      • Binczek E.
      • Gunter R.H.
      • Kiss C.
      • Karakesisoglou I.
      • Thevis M.
      • Weber A.A.
      • Arnhold S.
      • et al.
      Delta-6 desaturase (FADS2) deficiency unveils the role of omega-3 and omega-6 polyunsaturated fatty acids.
      • Moon Y.A.
      • Hammer R.E.
      • Horton J.D.
      Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice.
      ). FADS2− / − mice or cells expressing low FADS2 do not desaturate LA or ALA. Instead, the elongation products, 20:2, ω 6 and 20:3, ω 3, are formed (
      • Park W.J.
      • Kothapalli K.S.
      • Lawrence P.
      • Tyburczy C.
      • Brenna J.T.
      An alternate pathway to long chain polyunsaturates: the FADS2 gene product Delta8-desaturates 20:2n-6 and 20:3n-3.
      ,
      • Jump D.B.
      • Torres-Gonzalez M.
      • Olson L.K.
      Soraphen A, a inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.
      ). The decline in blood and hepatic ARA and DHA in Elovl5− / − mice promotes hepatosteatosis, which correlates with increased nuclear content of SREBP1 (
      • Moon Y.A.
      • Hammer R.E.
      • Horton J.D.
      Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice.
      ). Health status, hormones, signaling pathways, diet, drugs, and possibly gene polymorphisms regulate the expression and/or activity of enzymes involved in hepatic PUFA synthesis. Mechanisms controlling cardiac PUFA metabolism are less well defined. Cellular C20–22ω 3 PUFA content regulates membrane composition, cell signaling pathways originating from cell membranes, and gene expression (Fig. 3) (
      • Jump D.B.
      The biochemistry of n-3 polyunsaturated fatty acids.
      ). Below is a brief overview of how changes in cellular content of these fatty acids control cell functions relevant to CVD.
      Figure thumbnail gr3
      Fig. 3Overview of C20–22ω 3 PUFA regulation of cell function.

       Membrane effects of omega 3 PUFA

      Assimilation of ω 3 PUFA into plasma membrane phospholipids has major effects on cell signaling by altering membrane fluidity, lipid raft structure, and substrate availability for the synthesis of bioactive oxidized fatty acids (
      • Jump D.B.
      The biochemistry of n-3 polyunsaturated fatty acids.
      ). DHA plays a key structural role in membrane architecture; this highly unsaturated fatty acid alters membrane fluidity, membrane cholesterol content, and lipid raft organization (
      • Wassall S.R.
      • Stillwell W.
      Polyunsaturated fatty acid-cholesterol interactions: domain formation in membranes.
      ,
      • Soni S.P.
      • LoCascio D.S.
      • Liu Y.
      • Williams J.A.
      • Bittman R.
      • Stillwell W.
      • Wassall S.R.
      Docosahexaenoic acid enhances segregation of lipids between: 2H-NMR study.
      ). Some receptor systems affected by ω 3 PUFA include the toll-like receptors (TRL2 and TRL4) (
      • Hsueh H.W.
      • Zhou Z.
      • Whelan J.
      • Allen K.G.
      • Moustaid-Moussa N.
      • Kim H.
      • Claycombe K.J.
      Stearidonic and eicosapentaenoic acids inhibit interleukin-6 expression in ob/ob mouse adipose stem cells via Toll-like receptor-2-mediated pathways.
      • Lee J.Y.
      • Plakidas A.
      • Lee W.H.
      • Heikkinen A.
      • Chanmugam P.
      • Bray G.
      • Hwang D.H.
      Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids.
      ) and Src-family kinases (Fyn, c-Yes) (
      • Stulnig T.M.
      • Berger M.
      • Sigmund T.
      • Raederstorff D.
      • Stockinger H.
      • Waldhausl W.
      Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains.
      • Chen W.
      • Jump D.B.
      • Esselman W.J.
      • Busik J.V.
      Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid.
      ). Disruption of raft structure alters downstream signaling events, like NF κ B, a key transcription factor controlling the expression of cyclooxygenase-2 (COX2), and multiple genes encoding cytokines and adhesion molecules. The attenuation of expression of these genes represents one mechanism for the anti-inflammatory actions of ω 3 PUFA.
      Fatty acids also bind G-protein-coupled receptors (
      • Nilsson N.E.
      • Kotarsky K.
      • Owman C.
      • Olde B.
      Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids.
      ,
      • Brown A.J.
      • Jupe S.
      • Briscoe C.P.
      A family of fatty acid binding receptors.
      ). These receptors control cellular levels of second messengers (cAMP and intracellular Ca+2) and signaling pathways and are expressed in a tissue-specific fashion. One receptor that has gained attention recently is GRP120; it binds ALA, EPA, and DHA (
      • Oh D.Y.
      • Talukdar S.
      • Bae E.J.
      • Imamura T.
      • Morinaga H.
      • Fan W.
      • Li P.
      • Lu W.J.
      • Watkins S.M.
      • Olefsky J.M.
      GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.
      • Burns R.N.
      • Moniri N.H.
      Agonism with the omega-3 fatty acids alpha-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor.
      ). GRP120 is expressed as a long and short form; its activity is regulated through agonist-induced receptor phosphorylation. GRP120 is expressed in macrophages and fat cells; its activation inhibits inflammatory cascades in macrophages, including the control of NF κ B and JNK, and reverses insulin resistance in obese mice (
      • Oh D.Y.
      • Talukdar S.
      • Bae E.J.
      • Imamura T.
      • Morinaga H.
      • Fan W.
      • Li P.
      • Lu W.J.
      • Watkins S.M.
      • Olefsky J.M.
      GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.
      ,
      • Talukdar S.
      • Olefsky J.M.
      • Osborn O.
      Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases.
      ,
      • Oh D.Y.
      • Lagakos W.S.
      The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity.
      ,
      • Saltiel A.R.
      Fishing out a sensor for anti-inflammatory oils.
      ).

       Oxidation of omega 3 PUFA

      Although C20–22ω 3 PUFA are β -oxidized in peroxisomes and mitochondria (
      • Sprecher H.
      Metabolism of highly unsaturated n-3 and n-6 fatty acids.
      ), other oxidation mechanisms convert these fatty acids to bioactive mediators of cell function. C20–22ω 3 PUFA compete with ARA for assimilation into the sn2 position of membrane phospholipids. Dietary C20–22ω 3 PUFA-mediated suppression of ARA synthesis also contributes to the decline in cellular phospholipid ARA levels. Fatty acids in the sn-2 position are excised from phospholipids by cellular phospholipase-A2 (cPLA2); the excised NEFA is a substrate for cyclooxygenases [COX1 (constitutive) and COX2 (inducible)], lipoxygenases (LOX-5, LOX-12, LOX-15), and cytochrome P450 enzymes.
      Eicosanoids derived from the COX and LOX pathways (
      • Wada M.
      • DeLong C.J.
      • Hong Y.H.
      • Rieke C.J.
      • Song I.
      • Sidhu R.S.
      • Yuan C.
      • Warnock M.
      • Schmaier A.H.
      • Yokoyama C.
      • et al.
      Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products.
      ) regulate G-protein-coupled receptors that control signaling pathways in response to changes in intracellular second messengers (cAMP and Ca+2). Although EPA and DHA are poor substrates for COX and LOX, these enzymes generate series 3 and series 5 eicosanoids from EPA, respectively. These products have weak agonist activity compared with the ω 6 PUFA products of COX and LOX (
      • Wada M.
      • DeLong C.J.
      • Hong Y.H.
      • Rieke C.J.
      • Song I.
      • Sidhu R.S.
      • Yuan C.
      • Warnock M.
      • Schmaier A.H.
      • Yokoyama C.
      • et al.
      Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products.
      • Calder P.C.
      n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases.
      ). Resolvins and protectins have attracted considerable attention because of their capacity to protect against prolonged inflammation in animals models (
      • Spite M.
      • Serhan C.N.
      Novel lipid mediators promote resolution of acute inflammation: impact of aspirin and statins.
      ). The E-series resolvins (from EPA) and D-series resolvins (from DHA) are formed by the action of COX2 plus aspirin, whereas neuroprotectin-D1 is formed by the action of 5-LOX. Resolvins and neuroprotectins regulate inflammatory mechanisms (
      • Serhan C.N.
      • Yang R.
      • Martinod K.
      • Kasuga K.
      • Pillai P.S.
      • Porter T.F.
      • Oh S.F.
      • Spite M.
      Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions.
      ,
      • Zhang C.
      • Bazan N.G.
      Lipid-mediated cell signaling protects agains injury and neurodegeneration.
      ).
      Nonesterified C20–22ω 3 and ω 6 PUFA are substrates for cytochrome-P450 enzymes (e.g., CYP2C and CYP4A/4F). This is a major mechanism for generating oxidized fatty acids in cells lacking cyclooxygenase or lipoxygenase activity, like hepatic parenchymal cells. CYP2C/2J, for example, synthesizes regioisomeric epoxides of ARA, EPA, and DHA, i.e., 14,15-epoxyeicosatrienoic acid, 17,18-epoxy-eicosatetraenoic acid, and 19,20-epoxydocosapentaenoic acid, respectively (
      • Arnold C.
      • Markovic M.
      • Blossey K.
      • Wallukat G.
      • Fisher R.
      • Dechend R.
      • Konkel A.
      • Von Schaky C.
      • Luft F.C.
      • Muller D.N.
      • et al.
      Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of omega-3 fatty acids.
      ,
      • Arnold C.
      • Konkel A.
      • Fischer R.
      • Schunck W-H.
      Cytochrome P450-dependent metabolism of omega-6 and omega-3 long-chain polyunsaturated fatty acids.
      ). These epoxides are converted to di-hydroxy fatty acids (diols) by epoxide hydrolases. The EPA-derived epoxide, 17(R),18(S)-epoxyeicosatetraenoic acid, has anti-arrhythmic effects; it rapidly and with high potency (EC50∼ 1–2 nM) protects neonatal cardiomyocytes against Ca+2 overload (
      • Falck J.R.
      • Wallukat G.
      • Puli N.
      • Goli M.
      • Arnold C.
      • Konkel A.
      • Rothe M.
      • Fischer R.
      • Muller D.N.
      • Schunck W.H.
      17(R),18(S)-epoxyeicosatetraenoic acid, a potent eicosapentaenoic acid (EPA) derived regulator of cardiomyocyte contraction: structure-activity relationships and stable analogues.
      ). CYP4A/4F generates ω - and ω -1 hydroxylation products of ARA, EPA, and DHA (
      • Arnold C.
      • Markovic M.
      • Blossey K.
      • Wallukat G.
      • Fisher R.
      • Dechend R.
      • Konkel A.
      • Von Schaky C.
      • Luft F.C.
      • Muller D.N.
      • et al.
      Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of omega-3 fatty acids.
      ,
      • Spector A.A.
      Arachidonic acid cytochrome P450 epoxygenase pathway.
      ). In this case, inhibition of 20-hydroxyeicosatrienoic acid (20-HETE) synthesis protects the kidney from ischemia/reperfusion damage (
      • Hoff U.
      • Lukitsch I.
      • Chaykovska L.
      • Ladwig M.
      • Arnold C.
      • Manthati V.L.
      • Fuller T.F.
      • Schneider W.
      • Gollasch M.
      • Muller D.N.
      • et al.
      Inhibition of 20-HETE synthesis and action protects the kidney from ischemia/reperfusion injury.
      ). These CYP450-dependent metabolites of EPA and DHA are found in the heart, and they may play a role in C20–22ω 3 PUFA-linked cardioprotection (
      • Arnold C.
      • Konkel A.
      • Fischer R.
      • Schunck W-H.
      Cytochrome P450-dependent metabolism of omega-6 and omega-3 long-chain polyunsaturated fatty acids.
      ).
      ARA, EPA, and DHA are oxidized to F2-isoprostanes, F3-isoprostanes, and F4-neuroprostanes, respectively, by nonenzymatic processes (
      • Milne G.L.
      • Yin H.
      • Morrow J.D.
      Human biochemistry of the isoprostane pathway.
      • Milne G.L.
      • Yin H.
      • Hardy K.D.
      • Davies S.S.
      • Roberts 2nd, L.J.
      Isoprostane generation and function.
      ). C20–22 PUFA are susceptible to free radical attack when oxidative stress is increased in cells. Lipid peroxidation is a hallmark of oxidative stress; excessive production of lipid peroxides has been implicated in the pathogenesis of human diseases. The free radical-mediated (hydroxy-, alkoxyl-, peroxyl-radicals or peroxynitrate) lipid peroxidation chain reaction generates oxidized fatty acids found in membrane lipids and as NEFA in cells, blood, and urine. Formation of these oxidized lipids in membranes likely affects membrane fluidity and the function of membrane-associated proteins. Feeding Ldlr− / − mice a high-fat, high-cholesterol diet promotes hepatic oxidative stress. Including fish oil in this high-fat, high-cholesterol diet stimulates the production of F2-isoprostanes, F3-isoprostanes, and F4 neuroprostanes that appear in liver and urine (
      • Depner C.M.
      • Torres-Gonzalez M.
      • Tripathy S.
      • Milne G.
      • Jump D.B.
      Menhaden oil decreases high-fat diet-induced markers of hepatic damage, steatosis, inflammation, and fibrosis in obese ldlr-/- mice.
      ,
      • Saraswathi V.
      • Morrow J.D.
      • Hasty A.H.
      Dietary fish oil exerts hypolipidemic effects in lean and insulin sensitizing effect in obese LDLR -/- mice.
      ,
      • Saraswathi V.
      • Gao L.
      • Morrow J.D.
      • Chait A.
      • Niswender K.D.
      • Hasty A.H.
      Fish oil increases cholesterol storage in white adipose tissue with concomitant decreases in inflammation, hepatic steatosis, and atherosclerosis in mice.
      ). F2-isoprostanes activate thromboxane and PGF2 α receptors and platelets; they also induce vasoconstriction in vascular smooth muscle cells. In contrast, F3-isoprostanes do not regulate these receptors, platelets, or smooth muscles cells (
      • Milne G.L.
      • Yin H.
      • Hardy K.D.
      • Davies S.S.
      • Roberts 2nd, L.J.
      Isoprostane generation and function.
      ,
      • Song W.L.
      • Paschos G.
      • Fries S.
      • Reilly M.P.
      • Yu Y.
      • Rokach J.
      • Chang C.T.
      • Patel P.
      • Lawson J.A.
      • Fitzgerald G.A.
      Novel eicosapentaenoic acid-derived F3-isoprostanes as biomarkers of lipid peroxidation.
      ). Whether the F3-isoprostanes and F4-neuroprostanes have anti-inflammatory properties like series 3 and series 5 eicosanoids remains to be established.

       Nuclear effects of omega 3 PUFA

      Non-esterified fatty acids bind to and regulate the activity of several nuclear receptors including, PPAR ( α , β / δ , γ ), LXR ( α , β ), RXR and HNF4 α (
      • Jump D.B.
      N-3 polyunsaturated fatty acid regulation of hepatic gene transcription.
      ,
      • Jump D.B.
      • Botolin D.
      • Wang Y.
      • Xu J.
      • Demeure O.
      • Christian B.
      Docosahexaenoic acid (DHA) and hepatic gene transcription.
      ). Of these, fatty-acid regulation of the PPAR family has been most extensively studied. In primary rat hepatocytes, EPA significantly induces PPAR α -regulated target genes. While DHA modestly induces these same genes, ALA and DPA are ineffective. Analysis of hepatocyte NEFA following fatty acid treatment revealed that addition of DHA to cells increases esterified and nonesterified EPA through retroconversion (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      ). Cocrystals of PPAR β / δ -EPA, but not PPAR-DHA, have been described (
      • Xu H.E.
      • Lambert M.H.
      • Montana V.G.
      • Parks D.J.
      • Blanchard S.G.
      • Brown P.J.
      • Sternbach D.D.
      • Lehmann J.M.
      • Wisely G.B.
      • Willson T.M.
      • et al.
      Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.
      ). In vitro, EPA binds PPAR α (Kd∼ 1 μ M); in primary rat hepatocytes, EPA is the most potent ω 3 PUFA activator of PPAR α.
      PUFA suppress the nuclear abundance of several transcription factors involved in carbohydrate and lipid metabolism, including SREBP1, ChREBP, and MLX (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ,
      • Jump D.B.
      The biochemistry of n-3 polyunsaturated fatty acids.
      ,
      • Horton J.D.
      • Goldstein J.L.
      • Brown M.S.
      SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.
      ,
      • Postic C.
      • Dentin R.
      • Denechaud P.D.
      • Girard J.
      ChREBP, a transcriptional regulator of glucose and lipid metabolism.
      ). PUFA (both ω 3 and ω 6 PUFA) control the nuclear abundance of SREBP1 by regulating transcription of the SREBP1c gene and the turnover of the SREBP1 mRNA. DHA, however, is the only PUFA that induces proteasomal degradation of nuclear SREBP1 (
      • Botolin D.
      • Wang Y.
      • Christian B.
      • Jump D.B.
      Docosahexaenoic acid (22:6,n-3) regulates rat hepatocyte SREBP-1 nuclear abundance by Erk- and 26S proteasome-dependent pathways.
      ). Molecular mechanisms for PUFA control of ChREBP and MLX nuclear abundance remain undefined. C20–22ω 3 PUFA control of these transcription factors represents a mechanism for C20–22ω 3 PUFA suppression of DNL and triglyceride synthesis.
      NF κ B is a major transcription factor regulating expression of genes encoding proteins involved in inflammation (
      • Ben-Neriah Y.
      • Karin M.
      Inflammation meets cancer, with NF-kappaB as the matchmaker.
      ); some target genes include COX2, cytokines (e.g., TNF α ), and chemokines (e.g., MCP1). Omega-3 PUFA suppress the nuclear levels of NF κ B in several model systems (
      • Chen W.
      • Esselman W.J.
      • Jump D.B.
      • Busik J.V.
      Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells.
      ,
      • Jump D.B.
      Fatty acid regulation of gene transcription.
      ). NF κ B nuclear abundance is typically regulated by controlling the interaction of NF κ B subunits (p50 and/or p65) with I κ B subtypes ( α , β , ∊ , ζ ). I κ B sequesters p50/p65 in the cytosol; phosphorylation of I κ B by I κ B-kinase promotes I κ B dissociation from p50/p65, I κ B is degraded in the proteasome, and p50/p65 accumulates in nuclei. I κ B-kinase activity is regulated by its phosphorylation status; two kinases controlling I κ B-kinase phosphorylation status include Akt and TAK1. The NF κ B subunits bind promoters as heterodimers of p50/p65 and homodimers of p50; p65 can heterodimerize with other transcription factors, e.g., c/EBP α. Recent studies using the Ldlr− / − mouse model of high-fat, high-cholesterol diet-induced nonalcoholic fatty liver disease established that NF κ B-p50 is more vulnerable to ω 3 PUFA control than NF κ B-p65 (
      • Depner C.M.
      • Torres-Gonzalez M.
      • Tripathy S.
      • Milne G.
      • Jump D.B.
      Menhaden oil decreases high-fat diet-induced markers of hepatic damage, steatosis, inflammation, and fibrosis in obese ldlr-/- mice.
      ). Although the mechanism for this selective control remains to be established, this outcome suggests that ω 3 PUFA controls a subset of NF κ B-regulated genes.

      TARGETS OF omega 3 PUFA REGULATION IN CVD

      The pleiotropic effects of ω 3 PUFA on cell function are well established (
      • Jump D.B.
      The biochemistry of n-3 polyunsaturated fatty acids.
      ). In the cardiovascular system, the type and quantity of ω 3 fatty acid ingested and cellular ω 3 and ω 6 PUFA content affect blood lipids, inflammation, and endothelial cell and cardiomyocyte function (Fig. 4). The underlying mechanisms for these effects are described below.
      Figure thumbnail gr4
      Fig. 4Overview of dietary C18-22ω 3 PUFA effects on cardiovascular disease.

       Dyslipidemia

      Fasting and nonfasting triglycerides have long been associated with CVD (
      • Austin M.A.
      • Hokanson J.E.
      • Edwards K.L.
      Hypertriglyceridemia as a cardiovascular risk factor.
      • Sarwar N.
      • Danesh J.
      • Eiriksdottir G.
      • Sigurdsson G.
      • Wareham N.
      • Bingham S.
      • Boekholdt S.M.
      • Khaw K.T.
      • Gudnason V.
      Triglycerides and the risk of coronary heart disease: 10,158 incident cases among 262,525 participants in 29 Western prospective studies.
      ). Monitoring fasting blood triglycerides represents a well-established marker for ω 3-PUFA action. The association between blood triglycerides with CVD, however, has been the subject of debate (
      • Goldberg I.J.
      • Eckel R.H.
      • McPherson R.
      Triglycerides and heart disease: still a hypothesis?.
      ). Miller and colleagues (
      • Miller M.
      • Stone N.J.
      • Ballantyne C.
      • Bittner V.
      • Criqui M.H.
      • Ginsberg H.N.
      • Goldberg A.C.
      • Howard W.J.
      • Jacobson M.S.
      • Kris-Etherton P.M.
      • et al.
      Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association.
      ) recently reviewed the evidence linking triglycerides to CVD. While triglycerides are not directly atherogenic, they represent an important biomarker for CVD risk. Plasma triglycerides are part of the atherogenic triad consisting of elevated plasma triglycerides and LDL-cholesterol with low HDL-cholesterol. Triglycerides are associated with atherogenic remnant particles derived from chylomicrons and VLDL. Of these, fasting triglycerides are the most responsive to changes in blood and tissue levels of C20–22ω 3 PUFA. Fish oil has little or no effect on total blood cholesterol, but it affects LDL-C, LDL particle size, and HDL-C; these effects are variable and depend on dose and population studied (
      • Cottin S.C.
      • Sanders T.A.
      • Hall W.L.
      The differential effects of EPA and DHA on cardiovascular risk factors.
      ,
      • Mozaffarian D.
      • Wu J.H.
      (n-3) Fatty acids and cardiovascular health: are effects of EPA and DHA shared or complementary?.
      ). When tested alone, DHA supplements increased HDL-C and LDL particle size, whereas EPA decreased HDL3 (
      • Cottin S.C.
      • Sanders T.A.
      • Hall W.L.
      The differential effects of EPA and DHA on cardiovascular risk factors.
      • Jacobson T.A.
      • Glickstein S.B.
      • Rowe J.D.
      • Soni P.N.
      Effects of eicosapentaenoic acid and docosahexaenoic acid on low-density lipoprotein cholesterol and other lipids: a review.
      ).
      Both C18ω 3 and C20–22ω 3 PUFA lower triglycerides. However, the effect seen with ALA was equivalent to the effect seen with dietary LA. ALA, however, is not equivalent to C20–22ω 3 PUFA in controlling serum lipoproteins in humans (
      • Harris W.S.
      n-3 fatty acids and serum lipoproteins: human studies.
      ). Accordingly, pharmaceutical control of blood triglycerides uses C20–22ω 3 PUFA, not ALA. A 1 g capsule of Lovaza™ (GlaxoSmithKline) contains 465 mg of EPA and 375 mg of DHA as fatty acid ethyl esters (
      • Davidson M.H.
      • Stein E.A.
      • Bays H.E.
      • Maki K.C.
      • Doyle R.T.
      • Shalwitz R.A.
      • Ballantyne C.M.
      • Ginsberg H.N.
      Efficacy and tolerability of adding prescription omega-3 fatty acids 4 g/d to simvastatin 40 mg/d in hypertriglyceridemic patients: an 8-week, randomized, double-blind, placebo-controlled study.
      ). Lovaza™ is prescribed at ∼ 4 g/day for patients with hypertriglyceridemia (
      • Bays H.E.
      • Maki K.C.
      • Doyle R.T.
      • Stein E.
      The effect of prescription omega-3 fatty acids on body weight after 8 to 16 weeks of treatment for very high triglyceride levels.
      ). Amarin Pharma recently developed Amr101™, a preparation of EPA as an ethyl ester. At a 4 g/day dose, AMR101 is well tolerated by patients; this dose significantly reduces non-HDL-C, apolipoprotein B, lipoprotein-associated phospholipase A2, VLDL-C, and total cholesterol in patients with hypertriglyceridemia (
      • Bays H.E.
      • Ballantyne C.M.
      • Kastelein J.J.
      • Isaacsohn J.L.
      • Braeckman R.A.
      • Soni P.N.
      Eicosapentaenoic acid ethyl ester (AMR101) therapy in patients with very high triglyceride levels (from the Multi-center, plAcebo-controlled, Randomized, double-blInd, 12-week study with an open-label Extension [MARINE] trial).
      ). EPA and DHA appear to be equally effective at reducing blood triglyceride levels (
      • Cottin S.C.
      • Sanders T.A.
      • Hall W.L.
      The differential effects of EPA and DHA on cardiovascular risk factors.
      ).
      The hypolipemic effect of C20–22ω 3 PUFA is complex and involves control of hepatic VLDL production and clearance of triglyceride-rich lipoprotein (chylomicrons and VLDL) (
      • Pan M.
      • Cederbaum A.I.
      • Zhang Y.L.
      • Ginsberg H.N.
      • Williams K.J.
      • Fisher E.A.
      Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production.
      • Qi K.
      • Fan C.
      • Jiang J.
      • Zhu H.
      • Jiao H.
      • Meng Q.
      • Deckelbaum R.J.
      Omega-3 fatty acid containing diets decrease plasma triglyceride concentrations in mice by reducing endogenous triglyceride synthesis and enhancing the blood clearance of triglyceride-rich particles.
      ). A major driver for hepatic VLDL production during fasting is the availability of NEFA for esterification to form triglycerides. In humans and rodents, hepatic NEFA for triglyceride synthesis are derived from DNL, mobilization of adipose tissue lipid, and the portal circulation. In fasting, when VLDL secretion is greatest, the major fraction of NEFA entering VLDL is derived from adipose tissue, as DNL is suppressed (
      • Parks E.J.
      • Hellerstein M.K.
      Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques.
      ). Dietary C20–22ω 3 PUFA lower fasting blood levels of NEFA; dietary C20–22ω 3 PUFA also reduce adipose tissue lipolysis (
      • Puglisi M.J.
      • Hasty A.H.
      • Saraswathi V.
      The role of adipose tissue in mediating the beneficial effects of dietary fish oil.
      ). The mechanism for this effect is linked to activation of PPAR γ and suppression of inflammation; it may also involve the GPR120 receptors mentioned earlier. In rodent liver and primary hepatocytes, C20–22ω 3 PUFA inhibit DNL and induce fatty acid oxidation (
      • Jump D.B.
      Fatty acid regulation of hepatic gene expression.
      ). In addition to effects of NEFA supply, elevated hepatic DHA induces oxidative stress mechanisms that promote ApoB degradation, leading to a decline in VLDL assembly and secretion (
      • Andreo U.
      • Elkind J.
      • Blachford C.
      • Cederbaum A.I.
      • Fisher E.A.
      Role of superoxide radical anion in the mechanism of apoB100 degradation induced by DHA in hepatic cells.
      ).
      ApoCIII is a key apolipoprotein involved in controlling VLDL-triglyceride clearance. ApoCIII inhibits lipoprotein lipase, an enzyme involved in triglyceride hydrolysis (
      • McConathy W.J.
      • Gesquiere J.C.
      • Bass H.
      • Tartar A.
      • Fruchart J.C.
      • Wang C.S.
      Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III.
      ,
      • Ginsberg H.N.
      • Le N.A.
      • Goldberg I.J.
      • Gibson J.C.
      • Rubinstein A.
      • Wang-Iverson P.
      • Norum R.
      • Brown W.V.
      Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.
      ). Animal studies established that hepatic ApoCIII expression was suppressed by PPAR α; PPAR α is activated by fibrates and EPA (
      • Pawar A.
      • Jump D.B.
      Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.
      ,
      • Schoonjans K.
      • Staels B.
      • Auwerx J.
      Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression.
      ,
      • Ren B.
      • Thelen A.P.
      • Peters J.M.
      • Gonzalez F.J.
      • Jump D.B.
      Polyunsaturated fatty acid suppression of hepatic fatty acid synthase and S14 gene expression does not require peroxisome proliferator-activated receptor alpha.
      ). Human studies have established that fatty fish consumption or increased RBC C20–22ω 3 PUFA content is linked to lower triglycerides and plasma ApoCIII (
      • Zhang J.
      • Wang C.
      • Li L.
      • Man Q.
      • Song P.
      • Meng L.
      • Du Z.Y.
      • Froyland L.
      Inclusion of Atlantic salmon in the Chinese diet reduces cardiovascular disease risk markers in dyslipidemic adult men.
      • Olivieri O.
      • Martinelli N.
      • Sandri M.
      • Bassi A.
      • Guarini P.
      • Trabetti E.
      • Pizzolo F.
      • Girelli D.
      • Friso S.
      • Pignatti P.F.
      • et al.
      Apolipoprotein C–III, n-3 polyunsaturated fatty acids, and “insulin-resistant” T-455C APOC3 gene polymorphism in heart disease patients: example of gene-diet interaction.
      ). Interestingly, patients homozygous for the T-455C variant in the ApoCIII promoter are poorly responsive to the ApoCIII-lowering effect of ω 3 PUFA (
      • Olivieri O.
      • Martinelli N.
      • Sandri M.
      • Bassi A.
      • Guarini P.
      • Trabetti E.
      • Pizzolo F.
      • Girelli D.
      • Friso S.
      • Pignatti P.F.
      • et al.
      Apolipoprotein C–III, n-3 polyunsaturated fatty acids, and “insulin-resistant” T-455C APOC3 gene polymorphism in heart disease patients: example of gene-diet interaction.
      ).

       Effects of omega 3 PUFA on endothelial cells and inflammation

      Vascular endothelial cells represent a barrier between the blood and the underlying intima and smooth muscle cells in coronary arteries and the peripheral arterial vascular system. Endothelial cells produce several vasoactive substances, including factors that promote vascular smooth cell contraction [endothelins (ET1), thromboxanes (TXA2), and prostaglandins (PGH2)] and relaxation [prostacyclins (PGI2) and nitric oxide (NO)] (
      • Flammer A.J.
      • Luscher T.F.
      Human endothelial dysfunction: EDRFs.
      ). Dysfunctional coronary and peripheral endothelial cells are linked to CVD, myocardial infarction, stroke, and diabetic retinopathy (
      • Egert S.
      • Stehle P.
      Impact of n-3 fatty acids on endothelial function: results from human interventions studies.
      • Stebbins C.L.
      • Stice J.P.
      • Hart C.M.
      • Mbai F.N.
      • Knowlton A.A.
      Effects of dietary docosahexaenoic acid (DHA) on eNOS in human coronary artery endothelial cells.
      ). Atherosclerosis is an inflammatory disease of the vascular system (
      • Lusis A.J.
      Atherosclerosis.
      ,
      • Ross R.
      Atherosclerosis: an inflammatory disease.
      ). Cytokine-mediated induction of adhesion molecules in endothelial cells is an early step in monocyte binding to endothelial cell membranes that enable monocytes to move into vascular intima where they differentiate to macrophages and promote inflammation, culminating in plaque formation (
      • Lusis A.J.
      Atherosclerosis.
      ,
      • Shibata N.
      • Glass C.K.
      Regulation of macrophage function in inflammation and atherosclerosis.
      ,
      • Olefsky J.M.
      • Glass C.K.
      Macrophages, inflammation and insulin resistance.
      ). Controlling endothelial cell expression of adhesion molecules, like ICAM and VCAM, is central to controlling the onset of the inflammatory events linked to atherosclerosis. As noted earlier, C20–22ω 3 PUFA have anti-inflammatory capacity through effects on membrane lipid raft structure, NF κ B, and COX2- and CYP450-generated eicosanoids and docosanoids (
      • Saravanan P.
      • Davidson N.C.
      • Schmidt E.B.
      • Calder P.C.
      Cardiovascular effects of marine omega-3 fatty acids.
      ,
      • Serhan C.N.
      • Yacoubian S.
      • Yang R.
      Anti-inflammatory and proresolving lipid mediators.
      ).
      In human coronary endothelial cells, DHA regulates eNOS function through Akt activation (
      • Stebbins C.L.
      • Stice J.P.
      • Hart C.M.
      • Mbai F.N.
      • Knowlton A.A.
      Effects of dietary docosahexaenoic acid (DHA) on eNOS in human coronary artery endothelial cells.
      ). In mouse coronary and human retinal endothelial cells, C20–22ω 3 PUFA suppresses ICAM1 and VCAM1 expression by interfering with NF κ B signaling (
      • Chen W.
      • Esselman W.J.
      • Jump D.B.
      • Busik J.V.
      Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells.
      ,
      • Chen W.
      • Jump D.B.
      • Esselman W.J.
      • Busik J.V.
      Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid.
      ,
      • Yamada H.
      • Yoshida M.
      • Nakano Y.
      • Suganami T.
      • Satoh N.
      • Mita T.
      • Azuma K.
      • Itoh M.
      • Yamamoto Y.
      • Kamei Y.
      • et al.
      In vivo and in vitro inhibition of monocyte adhesion to endothelial cells and endothelial adhesion molecules by eicosapentaenoic acid.
      ,
      • Chen W.
      • Jump D.B.
      • Grant M.B.
      • Esselman W.J.
      • Busik J.V.
      Dyslipidemia, but not hyperglycemia, induces inflammatory adhesion molecules in human retinal vascular endothelial cells.
      ). In freshly isolated human retinal endothelial cells, phospholipid DHA levels are very low ( ∼ 1 mol%). Treating these primary cells with DHA significantly increases phospholipid DHA levels, leading to changes in membrane cholesterol content, lipid raft organization, and interaction of Src-kinases (Fyn and cYes) with lipid rafts. The net effect of elevated membrane DHA content is the disruption of NF κ B signaling and the downregulation acid sphingomyelinase. The suppression of sphingomyelinase activity is associated with a decline in ceramide, an inflammatory and pro-apoptotic lipid (
      • Opreanu M.
      • Lydic T.A.
      • Reid G.E.
      • McSorley K.M.
      • Esselman W.J.
      • Busik J.V.
      Inhibition of cytokine signaling in human retinal endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid.
      ).
      Although cell culture studies have established a strong case for DHA control of inflammatory events in vascular endothelial cells, human studies are less convincing. Egert and Stehle recently reviewed the evidence for ω 3 PUFA regulation of endothelial cells in humans (
      • Egert S.
      • Stehle P.
      Impact of n-3 fatty acids on endothelial function: results from human interventions studies.
      ). Their analysis covered 33 intervention trials on fasting and/or postprandial endothelial function and included individuals receiving EPA + DHA or ALA supplementation. While dietary ω 3 PUFA may improve endothelial function in individuals with CVD risk factors, including overweight, dyslipidemia, and type 2 diabetes, the strength of the evidence for clinical efficacy was not sufficiently strong to make recommendations. A key feature for ω 3 PUFA control of endothelial cell function is the capacity of dietary ω 3 PUFA to increase endothelial cell EPA + DHA content. Although the omega-3 index parallels changes in C20–22ω 3 PUFA in cardiac muscle, it is unclear if the omega-3 index parallels changes in coronary and vascular endothelial cells.

       omega 3 PUFA regulation of cardiomyocyte function

      Human studies have established that SCD is reduced in patients consuming fish oil (
      • Burr M.L.
      • Fehily A.M.
      • Gilbert J.F.
      • Rogers S.
      • Holliday R.M.
      • Sweetnam P.M.
      • Elwood P.C.
      • Deadman N.M.
      Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART).
      • London B.
      • Albert C.
      • Anderson M.E.
      • Giles W.R.
      • Van Wagoner D.R.
      • Balk E.
      • Billman G.E.
      • Chung M.
      • Lands W.
      • Leaf A.
      • et al.
      Omega-3 fatty acids and cardiac arrhythmias: prior studies and recommendations for future research: a report from the National Heart, Lung, and Blood Institute and Office of Dietary Supplements Omega-3 Fatty Acids and their Role in Cardiac Arrhythmogenesis Workshop.
      ). The consumption of EPA and DHA from fish oil increases atrial and ventricular EPA and DHA in membrane phospholipids (
      • Harris W.S.
      • Sands S.A.
      • Windsor S.L.
      • Ali H.A.
      • Stevens T.L.
      • Magalski A.
      • Porter C.B.
      • Borkon A.M.
      Omega-3 fatty acids in cardiac biopsies from heart transplantation patients: correlation with erythrocytes and response to supplementation.
      ,
      • Metcalf R.G.
      • James M.J.
      • Gibson R.A.
      • Edwards J.R.
      • Stubberfield J.
      • Stuklis R.
      • Roberts-Thomson K.
      • Young G.D.
      • Cleland L.G.
      Effects of fish-oil supplementation on myocardial fatty acids in humans.
      ,
      • Billman G.E.
      Effect of dietary omega-3 polyunsaturated fatty acids on heart rate and heart rate variability in animals susceptible or resistant to ventricular fibrillation.
      ). Several clinical trials have reported that ω 3 PUFA were able to i) prevent SCD, ii) prevent ventricular arrhythmia in patients with implantable cardioverter, iii) lower occurrences of premature ventricular contractions, iv) reduce heart rate, and v) prevent atrial fibrillation (
      • Billman G.E.
      Effect of dietary omega-3 polyunsaturated fatty acids on heart rate and heart rate variability in animals susceptible or resistant to ventricular fibrillation.
      ,
      • Richardson E.S.
      • Iaizzo P.A.
      • Xiao Y-F.
      Electrophysiological mechanisms and the anti-arrhythmic effects of omega-3 fatty acids.
      ). Studies in dogs showed anti-arrhythmic effects of EPA or DHA when administered during ischemia (
      • Billman G.E.
      • Kang J.X.
      • Leaf A.
      Prevention of ischemia-induced cardiac sudden death by n-3 polyunsaturated fatty acids in dogs.
      ).
      Approximately 80–90% of sudden cardiac deaths are linked to ventricular arrhythmias, and arrhythmias are linked to control of heart contraction, a process involving electrophysiological mechanisms controlling muscle contraction (
      • Billman G.E.
      Effect of dietary omega-3 polyunsaturated fatty acids on heart rate and heart rate variability in animals susceptible or resistant to ventricular fibrillation.
      ,
      • Richardson E.S.
      • Iaizzo P.A.
      • Xiao Y-F.
      Electrophysiological mechanisms and the anti-arrhythmic effects of omega-3 fatty acids.
      ,
      • Stanley W.C.
      • Khairallah R.J.
      • Dabkowski E.R.
      Update on lipids and mitochondrial function: impact of dietary n-3 polyunsaturated fatty acids.
      ,
      • Leaf A.
      • Kang J.X.
      • Xiao Y.F.
      • Billman G.E.
      Dietary n-3 fatty acids in the prevention of cardiac arrhythmias.
      ). As such, studies with cardiomyocytes have focused on ω 3 PUFA regulation of Na+-, Ca+2-, K+-channels, ion pumps, and the Na+/Ca+2 exchangers (
      • London B.
      • Albert C.
      • Anderson M.E.
      • Giles W.R.
      • Van Wagoner D.R.
      • Balk E.
      • Billman G.E.
      • Chung M.
      • Lands W.
      • Leaf A.
      • et al.
      Omega-3 fatty acids and cardiac arrhythmias: prior studies and recommendations for future research: a report from the National Heart, Lung, and Blood Institute and Office of Dietary Supplements Omega-3 Fatty Acids and their Role in Cardiac Arrhythmogenesis Workshop.
      ,
      • Richardson E.S.
      • Iaizzo P.A.
      • Xiao Y-F.
      Electrophysiological mechanisms and the anti-arrhythmic effects of omega-3 fatty acids.
      ). The impact on intracellular calcium handling is particularly noteworthy. Stanley et al. (
      • Stanley W.C.
      • Khairallah R.J.
      • Dabkowski E.R.
      Update on lipids and mitochondrial function: impact of dietary n-3 polyunsaturated fatty acids.
      ,
      • Khairallah R.J.
      • Sparagna G.C.
      • Khanna N.
      • O'Shea K.M.
      • Hecker P.A.
      • Kristian T.
      • Fiskum G.
      • Des Rosiers C.
      • Polster B.M.
      • Stanley W.C.
      Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition.
      ,
      • O'Shea K.M.
      • Khairallah R.J.
      • Sparagna G.C.
      • Xu W.
      • Hecker P.A.
      • Robillard-Frayne I.
      • Des Rosiers C.
      • Kristian T.
      • Murphy R.C.
      • Fiskum G.
      • et al.
      Dietary omega-3 fatty acids alter cardiac mitochondrial phospholipid composition and delay Ca2+-induced permeability transition.
      ) recently identified the mitochondrial permeability transition pore (MPTP) as a target for ω 3 PUFA regulation. MPTP is a large-diameter, high-conductance, voltage-dependent channel that allows passage of water, ions, and molecules up to 1.5 kDa. Mitochondrial accumulation of Ca+2 induces MPTP opening, whereas Ca+2 chelation closes the channel. Opening of MPTP is linked to cell death and has been implicated in ischemic injury and heart failure (
      • Stanley W.C.
      • Khairallah R.J.
      • Dabkowski E.R.
      Update on lipids and mitochondrial function: impact of dietary n-3 polyunsaturated fatty acids.
      ,
      • Duda M.K.
      • O'Shea K.M.
      • Lei B.
      • Barrows B.R.
      • Azimzadeh A.M.
      • McElfresh T.E.
      • Hoit B.D.
      • Kop W.J.
      • Stanley W.C.
      Dietary supplementation with omega-3 PUFA increases adiponectin and attenuates ventricular remodeling and dysfunction with pressure overload.
      ). Dietary supplementation with DHA, but not dietary EPA, dramatically increases cardiac mitochondrial phospholipid DHA levels. The accumulation of cardiac mitochondrial membrane DHA content correlates with the suppression of Ca+2-induced opening of MPTP. While other ω 3 PUFA, like ALA and EPA, modify mitochondrial membrane phospholipid composition, they do so to a lesser extent and have less effect on mitochondrial function (
      • Stanley W.C.
      • Khairallah R.J.
      • Dabkowski E.R.
      Update on lipids and mitochondrial function: impact of dietary n-3 polyunsaturated fatty acids.
      ). Thus, DHA control of MPTP may be one mechanism for ω 3 PUFA-mediated protection against heart failure. Other mechanisms include the control of oxidized lipids and inflammatory processes mentioned previously.

      PROSPECTIVE COHORT STUDIES AND RANDOMIZED CLINICAL TRIALS ON DIETARY omega 3 PUFA AND CHD

      Several PCs and RCTs have examined the effect of dietary ω 3 PUFA on cardiovascular health. Representative PCs and RCTs are presented in TABLE 2, TABLE 3. The PC studies examine associations between food consumption (data obtained by a food frequency questionnaire), blood levels of ω 3 PUFA, and cardiovascular events, such as heart failure, atrial fibrillation, CHD, stroke, and coronary calcification (Table 2). The six PC studies described in Table 2 consist of an overall patient population of 31,096 men and women, ranging from 20 to 85 years of age; the follow-up period ranged from 5.9 to 17.7 years (
      • Yamagishi K.
      • Nettleton J.A.
      • Folsom A.R.
      ARIC study investigators: plasma fatty acid composition and incident heart failure in middle-adge adults: the atherosclerosis risk in communities (ARIC) study.
      • Mozaffarian D.
      • Lemaitre R.N.
      • King I.B.
      • Song X.
      • Spiegelman D.
      • Sacks F.M.
      • Rimm E.B.
      • Siscovick D.S.
      Circulating long-chain omega-3 fatty acids and incidence of congestive heart failure in older adults: the cardiovascular health study: a cohort study.
      ). Most patients in these studies were free of CVD. The outcome of these studies was that elevated blood levels of C20–22ω 3 PUFA were associated with reduced risk of cardiovascular events. In contrast, blood levels of ALA were not associated with a reduced risk of CVD. The PC studies agree favorably with several epidemiological studies (
      • Bang H.O.
      • Dyerberg J.
      • Nielsen A.B.
      Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos.
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ) and suggest that dietary C20–22ω 3 PUFA are useful in primary prevention of CVD.
      The RCTs listed in Table 3 are secondary prevention trials (
      • Burr M.L.
      • Fehily A.M.
      • Gilbert J.F.
      • Rogers S.
      • Holliday R.M.
      • Sweetnam P.M.
      • Elwood P.C.
      • Deadman N.M.
      Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART).
      ,
      • GISSI-Prevenzione Investigators
      Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico.
      ,
      • Burr M.L.
      Secondary prevention of CHD in UK men: the Diet and Reinfarction Trial and its sequel.
      • Galan P.
      • Kesse-Guyot E.
      • Czernichow S.
      • Braincon S.
      • Blacher J.
      • Hercberg S.
      Effects of B vitamins and omega 3 fatty acids on cardiovascular diseases: a randomized placebo controlled trial.
      ). Each trial includes at least 1,000 patients (mostly males; age >50 years) with prior CVD, e.g., MI. The first Diet and Reinfarction Trial (DART) included patients (2,033 males <56.6 years of age) who had prior MI; these patients were advised to eat or not to eat at least two portions of fatty fish/week or take dietary EPA + DHA supplements (
      • Burr M.L.
      • Fehily A.M.
      • Gilbert J.F.
      • Rogers S.
      • Holliday R.M.
      • Sweetnam P.M.
      • Elwood P.C.
      • Deadman N.M.
      Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART).
      ). Men assigned to the fish advice group had increased blood levels of EPA and a 27% and 32% reduction in total mortality and ischemic heart disease, but they had a nonsignificant increase in nonfatal MI. The second DART trial used a similar design, but it involved an older male population (<70 years of age). As with the previous study, blood levels of EPA increased in the group advised to consume fish. In contrast to the previous study, all deaths were not significantly different between the two groups; but cardiac deaths and SCD increased significantly by 28% and 53%, respectively, in the ω 3-PUFA group (
      • Burr M.L.
      Secondary prevention of CHD in UK men: the Diet and Reinfarction Trial and its sequel.
      ,
      • Burr M.L.
      • Ashfield-Watt P.A.
      • Dunstan F.D.
      • Fehily A.M.
      • Breay P.
      • Ashton T.
      • Zotos P.C.
      • Haboubi N.A.
      • Elwood P.C.
      Lack of benefit of dietary advice to men with angina: results of a controlled trial.
      ).
      The Gruppo Italiano per la Sperimentazione della Streptochinasi nell'Infarto Miocardio Prevenzione (GISSI) trial (
      • GISSI-Prevenzione Investigators
      Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico.
      ) was the first large-scale RCT (11,334 patients) that had patients consume EPA + DHA supplements (850–882 mg/day, as ethyl esters) or a placebo. Like the DART trials, the GISSI patients had a history of heart disease, i.e., (MI ≤ 3 months). Patients assigned to the EPA + DHA supplement group had a significant reduction in cardiovascular death (18%), coronary death (20%), and sudden cardiac death (24%) compared with the placebo group. These outcomes support the use of C20–22ω 3 PUFA to reduce the incidence of cardiovascular-related deaths, including SCD and arrhythmias (
      • London B.
      • Albert C.
      • Anderson M.E.
      • Giles W.R.
      • Van Wagoner D.R.
      • Balk E.
      • Billman G.E.
      • Chung M.
      • Lands W.
      • Leaf A.
      • et al.
      Omega-3 fatty acids and cardiac arrhythmias: prior studies and recommendations for future research: a report from the National Heart, Lung, and Blood Institute and Office of Dietary Supplements Omega-3 Fatty Acids and their Role in Cardiac Arrhythmogenesis Workshop.
      ).
      The Japanese EPA Lipid Intervention Study (JELIS) was the first large clinical study to examine the impact of EPA on CVD (
      • Yokoyama M.
      • Origasa H.
      • Matsuzaki M.
      • Matsuzawa Y.
      • Saito Y.
      • Ishikawa Y.
      • Oikawa S.
      • Sasaki J.
      • Hishida H.
      • Itakura H.
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ). This trial enrolled 18,645 Japanese subjects with hypercholesterolemia; 14,981 had no history of CVD. The subjects received 1.8 g/day of purified EPA-ethyl esters in combination with statins or statins alone (control group). After 5 years, the EPA group had a 26%, 24%, and 19% reduction in major coronary events, unstable angina, and nonfatal coronary events, respectively (
      • Jacobson T.A.
      Beyond lipids: the role of omega-3 fatty acids from fish oil in the prevention of coronary heart disease.
      ). Patients receiving EPA-ethyl esters showed an increase in plasma EPA, from ∼ 95 μ g/ml to 170 μ g/ml, while both AA and DHA decreased ∼ 9% (
      • Itakura H.
      • Yokoyama M.
      • Matsuzaki M.
      • Saito Y.
      • Origasa H.
      • Ishikawa Y.
      • Oikawa S.
      • Sasaki J.
      • Hishida H.
      • Kita T.
      • et al.
      Relationships between plasma fatty acid composition and coronary artery disease.
      ). The decline in AA and DHA likely reflects the feedback inhibition of PUFA synthesis by elevated EPA ingestion.
      The Omega (
      • Rauch B.
      • Schiele R.
      • Schneider S.
      • Diller F.
      • Victor N.
      • Gohlke H.
      • Gottwik M.
      • Steinbeck G.
      • Del Castillo U.
      • Sack R.
      • et al.
      OMEGA, a randomized, placebo-controlled trial to test the effect of highly purified omega-3 fatty acids on top of modern guideline-adjusted therapy after myocardial infarction.
      ), Alpha-Omega (
      • Kromhout D.
      • Giltay E.J.
      • Geleijnse J.M.
      n-3 fatty acids and cardiovascular events after myocardial infarction.
      ), and Su.Fol.OM3 (
      • Galan P.
      • Kesse-Guyot E.
      • Czernichow S.
      • Braincon S.
      • Blacher J.
      • Hercberg S.
      Effects of B vitamins and omega 3 fatty acids on cardiovascular diseases: a randomized placebo controlled trial.
      ) RCTs involved 3,704, 4,837, and 2,606 patients, respectively, with recent cardiovascular events, e.g., MI, coronary, or cerebral ischemic events. The Omega trial had patients consume 850 mg/day EPA + DHA ethyl esters, the Alpha-Omega trial had patients consumed margarine supplemented with EPA + DHA without and with ALA. The target consumption was ∼ 400 mg/day of EPA + DHA combined and 1.9 g ALA/day. The Su.Fol.OM3 trial had patients consume 600 mg/day EPA + DHA ethyl esters. The follow-up period for the Omega, Alpha-Omega and Su.Fol.Om3 trials was 1 year, 3.3 years, and 4.7 years, respectively. The outcome of these trials indicated that C20–22ω 3 PUFA consumption provided no significant benefit to cardiovascular health.
      The outcome of these RCTs has lead several investigators to question whether increasing dietary C20–22ω 3 PUFA significantly benefits cardiovascular health in secondary prevention (
      • Burr M.L.
      • Dunstan F.D.
      • George C.H.
      Is fish oil good or bad for heart disease? Two trials with apparently conflicting results.
      • Kwak S.M.
      • Myung S.-K.
      • Lee Y.J.
      • Seo H.G.
      Efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease. A meta-analysis of randomized, double-blind, placebo-control trials.
      ). A meta-analysis of 14 randomized, double-blind, placebo-controlled trials involving 20,485 patients with a history of CVD indicated that supplementation with ω 3 PUFA did not reduce the risk of cardiovascular events (
      • Kwak S.M.
      • Myung S.-K.
      • Lee Y.J.
      • Seo H.G.
      Efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease. A meta-analysis of randomized, double-blind, placebo-control trials.
      ). This report, however, was criticized for including small short-term analyses that were not designed to detect end points of CVD (
      • Hu F.B.
      • Manson J.E.
      Omega-3 fatty acids and secondary prevention of cardiovascular disease–is it just a fish tail?.
      ).
      A number of factors may have a bearing on the outcome of RCTs in CVD treatment. First, each of the RCTs (Table 2) is a secondary prevention trial. As such, dietary EPA + DHA were used to treat or prevent recurrence of preexisting CVD. Second, the background levels blood EPA and/or DHA were high before beginning the trial; this was particularly the case in the JELIS trial. The Japanese population had higher fish consumption, higher blood levels of EPA and DHA, and lower incidence of CVD than Western societies (
      • Von Schacky C.
      Omega-3 fatty acids vs. cardiac disease-the contribution of the omega-3 index.
      ,
      • Von Schacky C.
      Omega-3 fatty acids: anti-arrhythmic, pro-arrhythmic or both?.
      ). Third, the follow-up for certain trials, like the Omega trial, may have been too short to measure significant effects. Fourth, several of the RCTs included state-of-the-art therapies for CVD in the design, e.g., statins in the JELIS trial and anti-hypertensive, anti-thrombotic, and lipid-modifying therapy in the Alpha-Omega trial. In a recent follow-up analysis of the Alpha-Omega trial, the investigators reported that MI patients who were not treated with statins but who received ω 3 PUFA supplementation might have reduced major cardiovascular events. Statin treatment appears to modify effects of ω 3 PUFA on the incidence of major cardiovascular events (
      • Eussen S.R.
      • Geleijnse J.M.
      • Giltay E.J.
      • Rompelberg C.J.
      • Klungel O.H.
      • Kromhout D.
      Effects of n-3 fatty acids on major cardiovascular events in statin users and non-users with a history of myocardial infarction.
      ). Such studies suggest there may be limitations to the use of ω 3 PUFA supplementation in secondary prevention of CVD, particularly in combination with state-of-the-art therapies.
      Finally, the Vitamin D and Omega-3 Fatty Acid (VITAL) trial is an ongoing RCT enrolling 20,000 participants (men ≥ 50 years of age and women ≥ 55 years of age). This double-blind, placebo-controlled primary prevention trial will be randomized to receive vitamin D (2000 IU/day), C20–22ω 3 PUFA (840 mg/day), both supplements, or neither supplement. The study is powered to examine major cardiovascular events, as well as CVD and stroke individually (
      • Manson J.E.
      • Bassuk S.S.
      • Lee I.M.
      • Cook N.R.
      • Albert M.A.
      • Gordon D.
      • Zaharris E.
      • Macfadyen J.G.
      • Danielson E.
      • Lin J.
      • et al.
      The VITamin D and OmegA-3 TrialL (VITAL): rationale and design of a large randomized controlled trial of vitamin D and marine omega-3 fatty acid supplements for the primary prevention of cancer and cardiovascular disease.
      ). The outcome of the VITAL trial may clarify the utility of C20–22ω 3 PUFA in primary prevention of CVD.

      CAPACITY OF DIETARY C18-22 omega 3 PUFA TO MANAGE CVD RISK FACTORS

      National health associations and government agencies recommend the consumption of fatty fish (two 3.5 oz portions/week) or ω 3 fatty acid supplements (EPA + DHA, 200–2,000 mg/day) to lower the risk for CVD (
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ,
      • Gebauer S.K.
      • Psota T.L.
      • Harris W.S.
      • Kris-Etherton P.M.
      n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits.
      ,
      • Kris-Etherton P.M.
      • Harris W.S.
      • Appel L.J.
      Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease.
      • Saravanan P.
      • Davidson N.C.
      • Schmidt E.B.
      • Calder P.C.
      Cardiovascular effects of marine omega-3 fatty acids.
      ,
      • Mozaffarian D.
      • Wu J.H.
      (n-3) Fatty acids and cardiovascular health: are effects of EPA and DHA shared or complementary?.
      ). A recent meta-analysis suggests that a daily intake of combined EPA + DHA (200–300 mg/day) is sufficient to provide cardioprotection (

      Trikalinos, T. A., Lee, J., Moorthy, D., Yu, W. W., Lau, J., Lichtenstein, A. H., Chung, M., . 2012. Effects of Eicosapentanoic Acid and Docosahexanoic Acid on Mortality Across Diverse Settings: Systematic Review and Meta-Analysis of Randomized Trials and Prospective Cohorts. Nutritional Research Series, Vol. 4. Report No. 12-EHC040-EF. US Agency for Healthcare Research and Quality, Rockville, MD.

      ). Dietary sources of ω 3 PUFA are in the form of whole foods or supplements and are derived from cold-water seafood like salmon (wild and farm raised), anchovy, menhaden, or krill, as well as plants, yeast, and algae. Most human studies have used fish, fish oil, or a mix of EPA + DHA ethyl esters to examine associations between ω 3-PUFA consumption and CVD (
      • Weitz D.
      • Weintraub H.
      • Fisher E.
      • Schwartzbard A.Z.
      Fish oil for the treatment of cardiovascular disease.
      ,
      • Burr M.L.
      • Fehily A.M.
      • Gilbert J.F.
      • Rogers S.
      • Holliday R.M.
      • Sweetnam P.M.
      • Elwood P.C.
      • Deadman N.M.
      Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART).
      ,
      • GISSI-Prevenzione Investigators
      Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico.
      ,
      • Rauch B.
      • Schiele R.
      • Schneider S.
      • Diller F.
      • Victor N.
      • Gohlke H.
      • Gottwik M.
      • Steinbeck G.
      • Del Castillo U.
      • Sack R.
      • et al.
      OMEGA, a randomized, placebo-controlled trial to test the effect of highly purified omega-3 fatty acids on top of modern guideline-adjusted therapy after myocardial infarction.
      ,
      • Burr M.L.
      • Fehily A.M.
      • Rogers S.
      • Welsby E.
      • King S.
      • Sandham S.
      Diet and reinfarction trial (DART): design, recruitment, and compliance.
      ,
      • Burr M.L.
      • Holliday R.M.
      • Fehily A.M.
      • Whitehead P.J.
      Haematological prognostic indices after myocardial infarction: evidence from the diet and reinfarction trial (DART).
      ). Only one large-scale clinical trial, i.e., JELIS, assessed the capacity of EPA in cardioprotection (
      • Yokoyama M.
      • Origasa H.
      • Matsuzaki M.
      • Matsuzawa Y.
      • Saito Y.
      • Ishikawa Y.
      • Oikawa S.
      • Sasaki J.
      • Hishida H.
      • Itakura H.
      • et al.
      Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.
      ,
      • Yokoyama M.
      • Origasa H.
      Effects of eicosapentaenoic acid on cardiovascular events in Japanese patients with hypercholesterolemia: rationale, design, and baseline characteristics of the Japan EPA Lipid Intervention Study (JELIS).
      ). This section examines various sources of dietary ω 3 PUFA for their capacity to control CVD risk factors, including the omega-3 index and inflammatory cytokines.

       omega 3 PUFA from farm-raised and wild-caught fish

      The original observations by Dyerberg and colleagues on the cardioprotective effects of ω 3 PUFA were based on Greenland Inuits ingesting wild-caught fish enriched in C20–22ω 3 PUFA (
      • Bang H.O.
      • Dyerberg J.
      • Nielsen A.B.
      Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos.
      • Harris W.S.
      • Mozaffarian D.
      • Lefevre M.
      • Toner C.D.
      • Colombo J.
      • Cunnane S.C.
      • Holden J.M.
      • Klurfeld D.M.
      • Morris M.C.
      • Whelan J.
      Towards establishing dietary reference intakes for eicosapentaenoic and docosahexaenoic acids.
      ). Fish, fish oil supplements, or EPA + DHA ethyl esters have been used in most CVD prevention trials (TABLE 2, TABLE 3).
      Although farm-raised fish provide an alternative source of C20–22ω 3 PUFA, levels of these fatty acids in farm-raised fish are not as high as in wild-caught fish (
      • Jenkins D.J.
      • Sievenpiper J.L.
      • Pauly D.
      • Sumaila U.R.
      • Kendall C.W.
      • Mowat F.M.
      Are dietary recommendations for the use of fish oils sustainable?.
      ,
      • Miller M.R.
      • Bridle A.R.
      • Nichols P.D.
      • Carter C.G.
      Increased elongase and desaturase gene expression with stearidonic acid enriched diet does not enhance long-chain (n-3) content of seawater Atlantic salmon (Salmo salar L.).
      ). Efforts to increase C20–22ω 3 PUFA in farm-raised Atlantic Salmon (Salmo salar L.) involve feeding fish ω 3-PUFA supplements (
      • Miller M.R.
      • Bridle A.R.
      • Nichols P.D.
      • Carter C.G.
      Increased elongase and desaturase gene expression with stearidonic acid enriched diet does not enhance long-chain (n-3) content of seawater Atlantic salmon (Salmo salar L.).
      ). For example, farm-raised salmon were fed diets enriched in fish oil, canola oil (enriched in ALA), or echium oil (Echium plantagineum). E. plantagineum oil seeds are enriched in ALA (18:3, ω 3), γ -linolenic acid (18:3, ω 6), and SDA (18:4, ω 3). Compared with farm-raised salmon fed fish oil diets, fish fed the diets enriched in either canola oil (enriched in ALA) or echium oil (enriched in SDA) did not accumulate comparable levels of DHA. Fish, like humans, require a dietary source of DHA to significantly increase cellular DHA.
      Whereas ω 3 PUFA in fish or plants are supplied as triglycerides, ω 3 PUFA in supplements are supplied as free fatty acids, fatty acids-ethyl esters, triglycerides, or reesterified triglycerides, most commonly in gel capsules (
      • Dyerberg J.
      • Madsen P.
      • Moller J.M.
      • Aardestrup I.
      • Schmidt E.B.
      Bioavailability of marine n-3 fatty acid formulations.
      ,
      • Gillies P.J.
      • Harris W.S.
      • Kris-Etherton P.M.
      Omega-3 fatty acids in food and pharma: the enabling role of biotechnology.
      ). The US Food and Drug Administration requires the dietary supplement industry to list the contents of the capsule and the source of the omega-3 fatty acid. The percentage of ω 3 PUFA in supplements relative to other fatty acids and the ratio of EPA to DHA in these preparations are highly variable. The information contained on the labels is helpful in determining one's daily consumption of ω 3-PUFA.

       omega 3 PUFA from plants, algae, or yeast

      ALA is an essential fatty acid and it is the major plant-based ω 3 PUFA. Dietary supplements of ALA are derived from flax or chia seeds or oils produced from these sources. Chia seeds (Salvia hispanica) have high levels of ALA; ∼ 50–60% of the plant oil is ALA. The amount of ALA required to prevent deficiency symptoms is ∼ 10% of LA, i.e., 0.6–1.2% of total energy (1.6 g for men and 1.1 g. for women per day) (
      • Han S.N.
      • Leka L.S.
      • Lichtenstein A.H.
      • Ausman L.M.
      • Schaefer E.J.
      • Meydani S.N.
      Effect of hydrogenated and saturated, relative to polyunsaturated, fat on immune and inflammatory responses of adults with moderate hypercholesterolemia.
      ). ALA intakes higher than 1.5 g/day are important for human health (
      • Gebauer S.K.
      • Psota T.L.
      • Harris W.S.
      • Kris-Etherton P.M.
      n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits.
      ). Humans, however, convert 0.2–9% of ingested ALA to DHA (
      • Rapoport S.I.
      • Igarashi M.
      • Gao F.
      Quantitative contributions of diet and liver synthesis to docosahexaenoic acid homeostasis.
      ,
      • Brenna J.T.
      Efficiency of conversion of alpha-linolenic acid to long chain n-3 fatty acids in man.
      • Hussein N.
      • Ah-Sing E.
      • Wilkinson P.
      • Leach C.
      • Griffin B.A.
      • Millward D.J.
      Long-chain conversion of [13C]linoleic acid and alpha-linolenic acid in response to marked changes in their dietary intake in men.
      ). The conversion of ALA to DHA in men is lower ( ∼ 0.1%) than in women ( ∼ 9%). This may relate to the decreased β -oxidation of ALA in women or increased expression of FADS2 (
      • Williams C.M.
      • Burdge G.
      Long-chain n-3 PUFA: plant v. marine sources.
      ). Despite its low level of conversion to DHA, ALA does not accumulate in blood or most cells; ALA is channeled to β -oxidization or adipose storage (
      • Brenna J.T.
      Efficiency of conversion of alpha-linolenic acid to long chain n-3 fatty acids in man.
      ,
      • Pawlosky R.J.
      • Hibbeln J.R.
      • Novotny J.A.
      • Salem Jr, N.
      Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans.
      ).
      ALA is added as a supplement to several foods, like margarine. A recent study compared the capacity of margarines supplemented with ALA, EPA, or DHA ethyl esters to affect the omega-3 index. Seventy-four health men and women were randomly assigned to have a total intake of 4.4, 2.2, or 2.3 g/day of ALA, EPA, or DHA, respectively (
      • Egert S.
      • Lindenmeier M.
      • Harnack K.
      • Krome K.
      • Erbersdobler H.F.
      • Wahrburg U.
      • Somoza V.
      Margarines fortified with alpha-linolenic acid, eicosapentaenoic acid, or docosahexaenoic acid alter the fatty acid composition of erythrocytes but do not affect the antioxidant status of healthy adults.
      ). In contrast to the group consuming the EPA- and DHA-supplemented margarines, the group consuming the ALA-supplemented margarine diet did not have an increased omega-3 index. Earlier studies (
      • Metcalf R.G.
      • James M.J.
      • Gibson R.A.
      • Edwards J.R.
      • Stubberfield J.
      • Stuklis R.
      • Roberts-Thomson K.
      • Young G.D.
      • Cleland L.G.
      Effects of fish-oil supplementation on myocardial fatty acids in humans.
      ) established that dietary ALA supplementation (10 mg/day, flaxseed oil ∼ 60% ALA) did not significantly affect the omega-3 index or myocardial EPA or DHA content.
      Geleijnse et al. (
      • Geleijnse J.M.
      • de Goede J.
      • Brouwer I.A.
      Alpha-linolenic acid: is it essential to cardiovascular health?.
      ) recently reviewed the clinical evidence on the capacity of dietary ALA supplementation to impact cardiovascular health. Short-term trials (6–12 weeks) in healthy patients showed no consistent effects of dietary ALA supplementation on blood lipids, LDL protein oxidation, Lp(a), ApoA1, or ApoB. Observational studies provide evidence for a protective effect against nonfatal MI. However, no protective association was observed between ALA status and the risk of heart failure, atrial fibrillation, or SCD. Effects of ALA on inflammatory markers linked to CVD were inconsistent (
      • Zhao G.
      • Etherton T.D.
      • Martin K.R.
      • West S.G.
      • Gillies P.J.
      • Kris-Etherton P.M.
      Dietary alpha-linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hypercholesterolemic men and women.
      ,
      • Dewell A.
      • Marvasti F.F.
      • Harris W.S.
      • Tsao P.
      • Gardner C.D.
      Low- and high-dose plant and marine (n-3) fatty acids do not affect plasma inflammatory markers in adults with metabolic syndrome.
      ). A prospective cohort analysis (Table 2) examined the association of ALA intake with 10-year incidence of coronary heart disease and stroke in the Netherlands. The analysis involved over 20,000 men and women between the ages of 20 and 65. ALA intake was not associated with the incidence of coronary heart disease, but low ALA intake may be a risk factor for stroke (
      • de Goede J.
      • Verschuren W.M.
      • Boer J.M.
      • Kromhout D.
      • Geleijnse J.M.
      Alpha-linolenic acid intake and 10-year incidence of coronary heart disease and stroke in 20,000 middle-aged men and women in the Netherlands.
      ). The Alpha-Omega trial (
      • Kromhout D.
      • Giltay E.J.
      • Geleijnse J.M.
      n-3 fatty acids and cardiovascular events after myocardial infarction.