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Low dietary fish-oil threshold for myocardial membrane n-3 PUFA enrichment independent of n-6 PUFA intake in rats

2010; Elsevier BV; Volume: 51; Issue: 7 Linguagem: Inglês

10.1194/jlr.m004069

ISSN

1539-7262

Autores

Emily L. Slee, Peter L. McLennan, Alice Owen, Mandy L. Theiss,

Tópico(s)

Antioxidant Activity and Oxidative Stress

Resumo

Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil. Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil. Regular consumption of long chain n-3 polyunsaturated fatty acids (PUFA) from fish is associated with a low incidence of premature mortality from cardiovascular disease. The greatest reductions in relative risk for premature mortality are achieved through regular intake of 1–2 meals of n-3 PUFA–rich fatty fish per week. Only small incremental advantages are evident as intakes increase (1.Kromhout D. Bosschieter E.B. Coulander C.D. The inverse relation between fish consumption and 20-year mortality from coronary heart-disease.N. Engl. J. Med. 1985; 312: 1205-1209Crossref PubMed Scopus (1829) Google Scholar, 2.Kris-Etherton P.M. Harris W.S. Appel L.J. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease.Circulation. 2002; 106: 2747-2757Crossref PubMed Scopus (2877) Google Scholar, 3.Albert C.M. Hennekens C.H. O'Donnell C.J. Ajani U.A. Carey V.J. Willett W.C. Ruskin J.N. Manson J.E. Fish consumption and risk of sudden cardiac death.JAMA. 1998; 279: 23-28Crossref PubMed Scopus (863) Google Scholar, 4.Hu F.B. Bronner L. Willett W.C. Stampfer M.J. Rexrode K.M. Albert C.M. Hunter D. Manson J.E. 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Fish intake and risk of incident atrial fibrillation.Circulation. 2004; 110: 368-373Crossref PubMed Scopus (393) Google Scholar). These can all be attributed to direct effects in the heart on the basis of experimental observations (14.McLennan P.L. Abeywardena M.Y. Membrane basis for fish oil effects on the heart: linking natural hibernators to prevention of human sudden cardiac death.J. Membr. Biol. 2005; 206: 85-102Crossref PubMed Scopus (52) Google Scholar). Animal studies show that the protective effects of dietary fish oil are achieved by n-3 PUFA incorporation into myocardial cell membrane phospholipids (15.McLennan P.L. Myocardial membrane fatty acids and the antiarrhythmic actions of dietary fish oil in animal models.Lipids. 2001; 36: S111-S114Crossref PubMed Scopus (114) Google Scholar, 16.McLennan P.L. Abeywardena M.Y. Charnock J.S. Dietary fish oil prevents ventricular fibrillation following coronary artery occlusion and reperfusion.Am. Heart J. 1988; 116: 709-717Crossref PubMed Scopus (299) Google Scholar). Several biologically plausible mechanisms of n-3 PUFA action on cardiac function have been proposed (17.Matthan N.R. Jordan H. Chung M. Lichtenstein A.H. Lathrop D.A. Lau J. A systematic review and meta-analysis of the impact of omega-3 fatty acids on selected arrhythmia outcomes in animal models.Metabolism. 2005; 54: 1557-1565Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Effects observed using hearts ex vivo after prior dietary intervention (but free of neural or hormonal input or circulating n-3 PUFA at time of study) support an obligatory role for myocardial membrane incorporation of docosahexaenoic acid (22:6n-3) (18.Pepe S. McLennan P.L. Cardiac membrane fatty acid composition modulates myocardial oxygen consumption and post-ischemic recovery of contractile function.Circulation. 2002; 105: 2303-2308Crossref PubMed Scopus (178) Google Scholar). However, extrapolation from animal to human must consider that n-3 PUFA presentation in animal diets and infusions are often well above the equivalent intakes found beneficial in clinical or epidemiological studies (17.Matthan N.R. Jordan H. Chung M. Lichtenstein A.H. Lathrop D.A. Lau J. A systematic review and meta-analysis of the impact of omega-3 fatty acids on selected arrhythmia outcomes in animal models.Metabolism. 2005; 54: 1557-1565Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The membrane incorporation of low, supplemental intake has not been examined. Furthermore, contemporary Western diets are exceedingly rich in n-6 PUFA, and competition between n-3 PUFA and n-6 PUFA for incorporation into the heart may be of substantial importance (19.Hu F.B. Manson J.E. Willett W.C. Types of dietary fat and risk of coronary heart disease: a critical review.J. Am. Coll. Nutr. 2001; 20: 5-19Crossref PubMed Scopus (695) Google Scholar, 20.Cordain L. Eaton S.B. Sebastian A. Mann N. Lindeberg S. Watkins B.A. O'Keefe J.H. Brand-Miller J. Origins and evolution of the Western diet: health implications for the 21st century.Am. J. Clin. Nutr. 2005; 81: 341-354Crossref PubMed Scopus (1605) Google Scholar), posing the dietary ratio of n-6:n-3 PUFA as a potential determinant of cardiac effects (21.Wijendran V. Hayes K.C. Dietary n-6 and n-3 fatty acid balance and cardiovascular health.Annu. Rev. Nutr. 2004; 24: 597-615Crossref PubMed Scopus (461) Google Scholar). The present study addressed two challenges to linking human disease risk to the biological mechanisms of n-3 PUFA action derived from animal studies. First, it aimed to extend the concentration-effect relationship for membrane incorporation of n-3 PUFA to dietary concentrations within the range of human intake (22.Siscovick D.S. Lemaitre R.N. Mozaffarian D. The fish story: a diet-heart hypothesis with clinical implications: n-3 polyunsaturated fatty acids, myocardial vulnerability, and sudden death.Circulation. 2003; 107: 2632-2634Crossref PubMed Google Scholar) and to estimate a threshold for incorporation. As concurrent intake of n-6 PUFA may predicate against n-3 PUFA effectively modulating myocardial composition, a second aim of the study was to quantify the effect of dietary n-6 PUFA on membrane incorporation of long chain n-3 PUFA. Ten-week old Sprague-Dawley rats were fed for two weeks on a control, fabricated diet containing olive oil as the fat source, followed by four weeks on a diet containing one of the following concentrations of fish oil (0.16, 0.31, 0.63, 1.25, 5.0%; n = 4 per diet) (high DHA tuna fish oil, NuMega Ingredients, Australia). Control diets were prepared containing no fish oil (n = 4 per diet). These diets contained either low n-6 PUFA (fish oil with remainder as olive oil; n = 24) or high n-6 PUFA (fish oil plus 5% sunflower seed oil with remainder as olive oil; n = 24) providing a range of dietary n-6:n-3 PUFA ratios manipulated by independently changing either n-3 PUFA content or n-6 PUFA content (Fig. 1). Fabricated diets were based on the AIN-93 M diet (23.Reeves P.G. Components of the AIN-93 diets as improvements in the AIN-76A diet.J. Nutr. 1997; 127: 838S-841SCrossref PubMed Google Scholar) containing (% dry weight) 57% cornstarch, 10% sucrose, 9% casein, 5% gelatin, 5% cellulose, 10% oil, 3.5% mineral mix, and 1% vitamin mix (24.Owen A.J. Peter-Przyborowska B.A. Hoy A.J. McLennan P.L. Dietary fish oil dose- and time-response effects on cardiac phospholipid fatty acid composition.Lipids. 2004; 39: 955-961Crossref PubMed Scopus (87) Google Scholar). The diet provided 64.6% of energy (%en) as carbohydrate, 13.6%en as protein, and 22%en as fat. The fatty acid profile of each diet is shown in Table 1. Experiments were conducted according to the NHMRC Australia, Guidelines for the Use of Experimental Animals.TABLE 1Dietary fatty acid composition for diets with different concentrations of fish oil and n-6 PUFALow n-6 PUFA Background0% FO0.16% FO0.31% FO0.63% FO1.25% FO5.0% FO10% OO9.84% OO9.69% OO9.37% OO8.75% OO5.0% OOFatty Acid (% total fat)14:000.050.090.190.381.5216:010.4210.5710.7211.0111.6115.1618:02.822.862.902.973.124.0218:1 (OA)75.7974.8573.9072.0268.2445.6018:2 n-6 (LA)8.328.218.117.897.464.8818:3 n-3 (LNA)0.520.520.520.520.530.5520:4 n-6 (AA)0.120.150.170.230.330.9720:5 n-3 (EPA)00.110.220.430.873.4822:5 n-3 (DPA)00.020.030.070.140.5522:6 n-3 (DHA)00.450.901.803.6114.43Σ n-6 PUFA8.658.578.498.338.006.05Σ n-3 PUFA0.521.101.682.835.1419.00Ratio n-6:n-316.637.795.052.941.560.32LA %en1.831.811.781.741.641.07EPA %en00.020.050.100.190.76DHA %en00.100.200.400.793.15High n-6 PUFA Background0% FO0.16% FO0.31% FO0.63% FO1.25% FO5.0% FO5.0% OO4.84% OO4.69% OO4.37% OO3.75% OO0% OO5.0% SSO5.0% SSO5.0% SSO5.0% SSO5.0% SSO5.0% SSOFatty Acid (% total fat)14:000.050.090.190.381.5216:08.088.238.388.679.2712.8218:03.633.663.703.783.934.8318:1 (OA)49.9749.0348.0846.2042.4219.7818:2 n-6 (LA)36.0035.8935.7835.5735.1432.5618:3 n-3 (LNA)0.490.490.490.490.500.5220:4 n-6 (AA)0.060.090.110.170.270.9120:5 n-3 (EPA)00.110.220.430.873.4822:5 n-3 (DPA)00.020.030.070.140.5522:6 n-3 (DHA)00.450.901.803.6114.43Σ n-6 PUFA36.1636.0836.0035.8435.5133.56Σ n-3 PUFA0.491.071.652.805.1118.97Ratio n-6:n-373.833.7221.8212.806.951.77LA %en7.927.907.877.837.737.16EPA %en00.020.050.100.190.76DHA %en00.100.200.400.793.15Values are expressed as percentage of total lipids by weight. Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA docosapentaenoic acid; EPA, eicosapentaenoic acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid; SSO, sunflower seed oil; %en, % of dietary metabolizable energy. Open table in a new tab Values are expressed as percentage of total lipids by weight. Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA docosapentaenoic acid; EPA, eicosapentaenoic acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid; SSO, sunflower seed oil; %en, % of dietary metabolizable energy. At completion of the feeding period, animals were anesthetized (pentobarbitone sodium 60 mg/kg ip), exsanguinated via the abdominal aorta, and the heart was removed. The heart was dissected free, ventricles were rinsed in ice-cold saline (0.9% NaCl), blotted dry, snap frozen, and stored at −80°C. Total lipids were extracted from 100–200 mg samples of myocardium using a modification of the Folch method (25.Folch J. Lees M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Phospholipids were isolated from the total muscle lipid by solid phase extraction using silica Sep-pak™ cartridges (Waters, Australia). Fatty acid methyl esters were prepared by direct transesterification (26.Lepage G. Roy C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-121Abstract Full Text PDF PubMed Google Scholar) and analyzed by gas chromatography using a Shimadzu GC-17A with flame ionization detection using a 30 m × 0.25 mm, 0.25 μm FAMEWAX column (J and W Scientific, US) with hydrogen as carrier gas and a step temperature program rising from 150°C to 260°C over 27 min and held for 6 min. Individual fatty acids were identified from authentic fatty acid methyl ester standards (Sigma-Aldrich, Australia) and expressed as a percentage of total fatty acids. A factorial experimental design was used to investigate the effect of dietary n-3 PUFA concentration and n-6 PUFA background on tissue fatty acid composition, analyzed by two-way ANOVA with FO dose and n-6 PUFA as the main effects and for interactions between the main effects. Tukey HSD post hoc analyses were used to compare individual means of dietary groups. Power calculation showed that n = 4 per group would have 80% power to detect a 12.5% change in the DHA concentration of myocardial membranes from a control concentration of 10.34 ± 0.72% (mean ± SD). A previous study showed 1.25% FO feeding over four weeks increased DHA content by 100% (to 20.06 ± 2.98%; mean ± SD) (24.Owen A.J. Peter-Przyborowska B.A. Hoy A.J. McLennan P.L. Dietary fish oil dose- and time-response effects on cardiac phospholipid fatty acid composition.Lipids. 2004; 39: 955-961Crossref PubMed Scopus (87) Google Scholar). Statistical analyses were performed using Statistix software, version 8 (Analytical Software, US). Linear regression analysis with Pearson's correlation was performed to determine linear associations between diet and heart n-3 PUFA concentrations using GraphPad Prism (version 4.03) for Windows (GraphPad Software, US). Data were expressed as mean ± SEM. Statistical significance was accepted at P < 0.05. Energy intake, body weight, body weight gain, and liver and heart weights did not differ between diets (data not shown). Myocardial membranes of rats fed the low n-6 PUFA control diet contained 31.6% saturated fat, 12.6% monounsaturated fatty acids (MUFA), and 54.7% PUFA (Table 2). The main membrane PUFA were arachidonic acid (AA) (20:4n-6); linoleic acid (LA) (18:2n-6); and n-3 PUFA DHA. Hearts from rats fed the high n-6 PUFA control diet had significantly less MUFA, significantly more total PUFA, and slightly (but significantly) more saturated fatty acids (all P < 0.001) (Table 3). The high n-6 PUFA control diet increased myocardial LA (P < 0.05), but AA was unchanged (P = 0.72).TABLE 2Fatty acid composition of heart membranes as percentage of total fatty acids for various FO dosesLow n-6 PUFA Background0% FO0.16% FO0.31% FO0.63% FO1.25% FO5% FO10% OO9.84% OO9.69% OO9.37% OO8.75% OO5% OOFatty Acid16:009.27 ± 0.10a10.18 ± 0.11a,b10.70 ± 0.36b11.07 ± 0.18b10.99 ± 0.16b10.65 ± 0.32b18:0021.77 ± 0.14a,b21.06 ± 0.22a,b21.01 ± 0.25a,b20.77 ± 0.31b20.63 ± 0.22b22.00 ± 0.32a18:1n-9 (OA)8.11 ± 0.14a6.68 ± 0.55b6.83 ± 0.23b6.74 ± 0.19b6.56 ± 0.23b6.51 ± 0.14b18:1n-74.08 ± 0.074.14 ± 0.103.97 ± 0.303.95 ± 0.193.77 ± 0.093.50 ± 0.0518:2 n-6 (LA)17.95 ± 0.94a17.00 ± 1.08a18.39 ± 0.70a16.98 ± 0.10a17.32 ± 0.30a10.44 ± 0.63b18:3 n-3 (LNA)0.01 ± 0.010.05 ± 0.010.03 ± 0.010.03 ± 0.010.03 ± 0.010.03 ± 0.0220:4 n-6 (AA)24.79 ± 0.39a22.44 ± 0.32b19.59 ± 0.53c18.12 ± 0.33c,d16.78 ± 0.48d,e16.09 ± 0.41e20:5 n-3 (EPA)0.01 ± 0.01a0.08 ± 0.01a.b0.18 ± 0.01b,c0.22 ± 0.02c0.39 ± 0.04d1.13 ± 0.03e22:5 n-3 (DPA)0.79 ± 0.04a0.88 ± 0.05a,b0.92 ± 0.07a,b0.88 ± 0.08a,b1.04 ± 0.05b1.05 ± 0.03b22:6 n-3 (DHA)7.69 ± 0.55a12.75 ± 0.57b14.91 ± 0.38b17.90 ± 0.44c19.29 ± 0.41c24.54 ± 1.01dΣ n-6 PUFA46.18 ± 0.74a41.52 ± 0.68b39.61 ± 0.49b36.43 ± 0.31c35.33 ± 0.47c27.15 ± 0.98dΣ n-3 PUFA8.55 ± 0.58a13.79 ± 0.60b16.05 ± 0.44b19.04 ± 0.49c20.76 ± 0.34c26.78 ± 0.98dΣ PUFA54.73 ± 0.22a55.31 ± 0.14a,b55.66 ± 0.20a,b55.48 ± 0.30a,b56.08 ± 0.16b54.70 ± 0.41aΣ MUFA12.56 ± 0.13a11.37 ± 0.50a,b11.28 ± 0.29b11.11 ± 0.19b10.72 ± 0.15b10.63 ± 0.22bΣ Saturated fat31.65 ± 0.13a31.86 ± 0.23a,b32.27 ± 0.24a,b32.44 ± 0.15b32.29 ± 0.14a,b33.64 ± 0.11cRatio n-6:n-35.50 ± 0.45a3.03 ± 0.18b2.48 ± 0.10b,c1.92 ± 0.07c,d1.71 ± 0.05c,d1.05 ± 0.08dDietary oils (FO and OO) are percentage of diet dry weight. Values are mean ± SEM of FA as percentage of total FA measured (n = 4). Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; MUFA, monounsaturated fatty acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid. Within rows, values not sharing a common superscript are significantly different from one another, P < 0.05 (two-way ANOVA with Tukey's posthoc test for multiple comparison of means). Open table in a new tab TABLE 3Fatty acid composition of heart membranes as percentage of total fatty acids for various FO dosesHigh n-6 PUFA Background0%FO0.16% FO0.31% FO0.63% FO1.25% FO5% FO5% OO4.84% OO4.69% OO4.37% OO3.75% OO5% SSO5% SSO5% SSO5% SSO5% SSO5% SSOFatty Acid16:009.38 ± 0.21a9.56 ± 0.19a,b10.31 ± 0.27a,b10.42 ± 0.24a,b10.32 ± 0.40a,b10.69 ± 0.25b18:0022.11 ± 0.3222.45 ± 0.3221.55 ± 0.2021.86 ± 0.3121.69 ± 0.2821.93 ± 0.3118:1n-9 (OA)5.60 ± 0.10a4.86 ± 0.20a,b4.96 ± 0.09a,b4.34 ± 0.11b4.79 ± 0.35b3.09 ± 0.06c18:1n-73.36 ± 0.05a3.30 ± 0.03a,b3.27 ± 0.14a,b3.18 ± 0.18a,b3.18 ± 0.20a,b2.75 ± 0.06b18:2 n-6 (LA)21.13 ± 0.5018.76 ± 1.0620.70 ± 0.7019.87 ± 0.7818.93 ± 1.0818.86 ± 0.5918:3 n-3 (LNA)0.04 ± 0.000.03 ± 0.000.03 ± 0.000.03 ± 0.000.04 ± 0.010.03 ± 0.0020:4 n-6 (AA)23.88 ± 0.33a22.40 ± 0.27a20.05 ± 0.45b18.36 ± 0.54b,c16.64 ± 0.27c14.42 ± 0.64d20:5 n-3 (EPA)0.02 ± 0.01a0.05 ± 0.00a0.09 ± 0.01a,b0.15 ± 0.02b0.25 ± 0.01c0.79 ± 0.04d22:5 n-3 (DPA)0.67 ± 0.040.88 ± 0.220.78 ± 0.070.86 ± 0.041.02 ± 0.061.02 ± 0.0722:6 n-3 (DHA)6.70 ± 0.21a13.00 ± 0.82b14.29 ± 0.52b,c17.16 ± 1.09c,d19.36 ± 0.95d,e22.25 ± 0.35eΣ n-6 PUFA49.85 ± 0.14a43.54 ± 0.76b42.56 ± 0.40b,c39.73 ± 1.13c,d36.79 ± 1.24d,e34.56 ± 0.49eΣ n-3 PUFA7.47 ± 0.23a13.99 ± 0.97b15.22 ± 0.50b,c18.23 ± 1.06c,d20.68 ± 0.95d24.12 ± 0.33eΣ PUFA57.32 ± 0.16a57.53 ± 0.21a,b57.78 ± 0.24a,b57.96 ± 0.09a,b57.47 ± 0.49a,b58.68 ± 0.33bΣ MUFA9.46 ± 0.12a8.57 ± 0.23a,b8.62 ± 0.12a,b7.98 ± 0.24b8.45 ± 0.266.40 ± 0.18cΣ Saturated fat32.21 ± 0.17a32.82 ± 0.15a32.59 ± 0.13a33.06 ± 0.18a,b32.89 ± 0.41a,b33.79 ± 0.07bRatio n-6:n-36.69 ± 0.22a3.17 ± 0.27b2.81 ± 0.11b,c2.21 ± 0.18c,d1.80 ± 0.13d1.43 ± 0.04dDietary oils (FO, OO, and SSO) are percentage of diet dry weight. Values are mean ± SEM of FA as percentage of total FA measured (n = 4). Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA, doco­sapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; MUFA, monounsaturated fatty acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid; SSO, sunflower seed oil. Within rows, values not sharing a common superscript are significantly different from one another, P < 0.05 (two-way ANOVA with Tukey's posthoc test for multiple comparison of means). Open table in a new tab Dietary oils (FO and OO) are percentage of diet dry weight. Values are mean ± SEM of FA as percentage of total FA measured (n = 4). Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; MUFA, monounsaturated fatty acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid. Within rows, values not sharing a common superscript are significantly different from one another, P < 0.05 (two-way ANOVA with Tukey's posthoc test for multiple comparison of means). Dietary oils (FO, OO, and SSO) are percentage of diet dry weight. Values are mean ± SEM of FA as percentage of total FA measured (n = 4). Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; DPA, doco­sapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FO, fish oil; LA, linoleic acid; LNA, α-linolenic acid; MUFA, monounsaturated fatty acid; OA, oleic acid; OO, olive oil; PUFA, polyunsaturated fatty acid; SSO, sunflower seed oil. Within rows, values not sharing a common superscript are significantly different from one another, P < 0.05 (two-way ANOVA with Tukey's posthoc test for multiple comparison of means). Myocardial n-3 PUFA concentration was significantly different at every dietary concentration of fish oil (Tables 2, 3). Incorporation of DHA was dose-related and hyperbolic in nature (r2 = 0.937) with half maximal incorporation associated with a FO concentration in the diet of 0.37% (95% CI; 0.29%–0.45%), equal to EPA+DHA 0.29%en (95% CI; 0.23%–0.35%) (Fig. 2A). There was no significant effect of background dietary n-6 PUFA on the incorporation of DHA into cardiac cell membranes (P = 0.65). Log transformation of the fish-oil dose revealed a linear relationship between [fish oil]log10 and myocardial membrane DHA (slope P < 0.001; r2 = 0.88) (Fig. 2B). The regression line extrapolated to intersect with the residual DHA concentration of control hearts at a fish -oil dose of 0.027% (95% CI; 0.013%–0.044%) equal to EPA+DHA 0.021%en (95% CI; 0.010%–0.034%). Interpolation of the regression line predicted that a fish-oil dose of 0.31 ± 0.08% (EPA+DHA: 0.24 ± 0.01%en) would double the myocardial membrane content of DHA. A statistically significant concentration-related increase in EPA was observed with FO feeding, but it never exceeded 1.25% of membrane fatty acids (Fig. 2B and Tables 2, 3). Total membrane n-6 PUFA decreased with FO feeding (Tables 2, 3). Myocardial AA decreased significantly with each fish-oil dose (P < 0.001) in a log-linear fashion (low n-6 PUFA: r2 = 0.79, P < 0.0001 for slope; high n-6 PUFA: r2 = 0.88, P < 0.0001 for slope) (Tables 2, 3). Myocardial LA content was unchanged by fish oil (low n-6 PUFA: r2 = 0.002, P = 0.88 for slope; high n-6 PUFA: r2 = 0.022, P = 0.53 for slope), except for a significant lowering with the 5% dietary FO concentration in the low n-6 PUFA diet (P < 0.001) (Table 2). Myocardial total n-3 PUFA increased and total n-6 PUFA decreased significantly with FO feeding, resulting in a significant FO concentration-related decrease in the membrane n-6:n-3 PUFA ratio. The dietary n-6:n-3 PUFA ratio decreased with increasing dietary FO concentration (Fig. 1), and there was an inverse relationship between dietary ratio and the incorporation of EPA+DHA in myocardial membranes within both the low and the high n-6 PUFA diets (Fig. 3). A plot of dietary ratio against the myocardial membrane ratio revealed a linear relationship (Fig. 3, inset). The background concentration of n-6 PUFA in the diet significantly influenced the slope of the relationship (low n-6 PUFA diet: r2 = 0.936; high n-6 PUFA: r2 = 0.956; difference in slopes: P < 0.0001). However, neither the myocardial membrane EPA+DHA concentration nor its n-6:n-3 PUFA ratio differed between high and low n-6 PUFA diets at any individual dietary FO concentration (Fig. 3) despite large differences in dietary ratio. Only for the FO-free control diets was there a significant difference in the membrane ratio between high and low n-6 PUFA. There was a direct relationship between the n-6:n-3 PUFA ratio in the diet and AA concentrations in the heart. However, at each fish-oil dose, AA was displaced from heart membranes independently of the dietary ratio of n-6:n-3 PUFA (Tables 2, 3). This study found that very small intakes of fish oil can markedly increase the myocardial membrane long chain n-3 PUFA concentration. We identified a dietary threshold for the myocardial membrane incorporation of long chain n-3 PUFA, predicting that increased incorporation can be achieved when fish-oil intake exceeds 0.019% of dietary metabolizable energy as EPA+DHA (0.027% of the diet by weight as FO). This is similar to the intake (0.013%en) of EPA+DHA that marginally but significantly raises heart DHA concentration in n-3 PUFA–­deficient rats (27.Bourre J.M.E. Dumont O.L. Piciotti M.J. Clement M.E. Durand G.A. Comparison of vegetable and fish oil in the provision of N-3 polyunsaturated fatty acids for nervous tissue and selected organs.J. Nutr. Biochem. 1997; 8: 472-478Crossref Scopus (11) Google Scholar). In terms of relative human intake, we calculated this estimated threshold to be equivalent to a single 1 g capsule of fish oil per week (Table 4). Moreover, long chain n-3 PUFA intake in the rat, equivalent in humans to only two meals of fatty fish per week, was sufficient to double the myocardial concentration of DHA, the principle myocardial n-3 fatty acid in the heart of many species (14.McLennan P.L. Abeywardena M.Y. Membrane basis for fish oil effects on the heart: linking natural hibernators to prevention of human sudden cardiac death.J. Membr. Biol. 2005; 206: 85-102Crossref PubMed Scopus (52) Google Scholar). This equates to an n-3 PUFA intake consistently associated with low risk of cardiovascular disease mortality (28.Mozaffarian D. Rimm E.B. Fish intake, contaminants, and human health - Evaluating the risks and the benefits.JAMA. 2006; 296: 1885-1899Crossref PubMed Scopus (1541) Google Scholar, 29.Psota T.L. Gebauer S.K. Kris-Etherton P.M. Dietary Omega-3 Fatty Acid Intake and Cardiovascular Risk.Am. J. Cardiol. 2006; 98: 3-18Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar).TABLE 4How much is that for human consumption?RatHumanFish oil % weightEPA+DHA mg per 100 gEPA+DHA % energyEPA+DHA g per dayaBased on human energy intake of 8700 kJ per day.100 g salmon per weekbBased on salmon n-3 content of 1.9 g per 100 g.Fish oil per daycBased on typical fish oil capsule content of 330 mg EPA+DHA.5.017913.919.133281.254480.982.386.80.632260.491.243.50.311110.240.5721.70.16570.130.281<10.027dExtrapolated threshold intake of fish oil from Fig. 2B.100.0190.040.150.12Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.a Based on human energy intake of 8700 kJ per day.b Based on salmon n-3 content of 1.9 g per 100 g.c Based on typical fish oil capsule content of 330 mg EPA+DHA.d Extrapolated threshold intake of fish oil from Fig. 2B. Open table in a new tab Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid. Our previous identification of a linear relationship between fish-oil intake and myocardial n-3 PUFA incorporation (24.Owen A.J. Pet

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