AJCN. First published ahead of print August 24, 2011 as doi: 10.3945/ajcn.111.018036.
Effect of low doses of long-chain n23 PUFAs on endothelial function and arterial stiffness: a randomized controlled trial1–3 Thomas AB Sanders, Wendy L Hall, Zoitsa Maniou, Fiona Lewis, Paul T Seed, and Philip J Chowienczyk ABSTRACT Background: The dietary intake of n23 (omega-3) long-chain PUFAs (LC-PUFAs) from fish may improve endothelial function and arterial stiffness. Objective: The objective was to test the hypothesis that increasing intakes of n23 LC-PUFAs—equivalent to the consumption of 1, 2, or 4 portions of oily fish per week—favorably affects endothelial function and arterial stiffness. Design: A parallel-design, randomized, double-blind study compared daily doses of 0.45, 0.9, and 1.8 g n23 LC-PUFAs (EPA: DHA ratio of 1.51:1) with placebo (refined olive oil). The primary and secondary outcomes were changes in flow-mediated dilatation (FMD) of the brachial artery, arterial stiffness, and blood pressure. Nonsmoking men (n = 142) and women (n = 225) aged 45–70 y were randomly assigned to treatment for 12 mo; 312 subjects completed the intervention. Results: Compliance with the intervention was corroborated by significant dose-dependent increases in the proportions of EPA and DHA in erythrocyte lipids and a 16.5% reduction in serum triacylglycerol concentrations with 1.8 g n23 LC-PUFAs/d. FMD was lower in men than in women (P , 0.0001) and decreased with age (q = 0.270, P , 0.001) but was not significantly (P = 0.781) related to n23 LC-PUFA intake. The mean changes in FMD (95% CIs) compared with placebo were 0.1% (20.9%, 1.1%), 20.3% (21.3%, 0.6%), and 20.3% (21.3%, 0.7%) on daily intakes of 0.45, 0.9, and 1.8 g n23 LC-PUFAs, respectively. No significant treatment effects were noted for arterial stiffness and central mean or 24-h ambulatory blood pressure. Conclusion: Intakes of n23 LC-PUFAs 1.8 g/d do not improve endothelial function in healthy adults. The trial is registered at controlled-trials.com as ISRCTN66664610. Am J Clin Nutr doi: 10.3945/ajcn.111.018036.
INTRODUCTION
Favorable effects on vascular function may be exerted by dietary n23 LC-PUFAs,4 principally EPA (20:5n23) and DHA (22:6n23). A large prospective cohort study from Japan found a strong inverse relation between n23 LC-PUFA intake and subsequent cardiovascular disease (1). Other studies from Japan (2, 3) suggest that differences in arterial stiffness exist between communities that eat high amounts of fish and those that eat small amounts of fish. Prospective cohort studies in other populations also show an association between n23 intake from fish and decreased cardiovascular disease (4). It is well established that high intakes (15 g/d) can alter the balance between pro- and
antithrombotic eicosanoids (5), but these are pharmacologic quantities much greater than the range of intakes seen in most populations (2 g/d). More recently, attention has been drawn to other mechanism (6) that may operate at low intakes; furthermore, recently discovered metabolites derived from EPA and DHA, known as resolvins and neuroprotectins, may mitigate damage to tissues during ischemic episodes. The mechanisms by which protection is afforded remain elusive, but may operate by helping to conserve normal endothelial function. Nitric oxide produced by the vascular endothelium plays a central role in regulating endothelial function. Nitric oxide appears to have both antithrombotic and antiatherosclerotic properties. The capacity of the endothelium to synthesize nitric oxide can be assessed in vivo by measuring FMD of the brachial artery (7). A higher proportion of DHA in erythrocyte lipids has been associated with improved endothelial function measured by the FMD technique (8). Previous intervention studies (9) that measured the effects of n23 LC-PUFAs on endothelial function have been statistically underpowered, used disparate groups of subjects, and have suffered from significant operator variability in the measurement of FMD. To the best of our knowledge, this was the first randomized controlled trial with endothelial function as the primary outcome that was designed with sufficient statistical power to compare increases in the intake of n23 LCPUFAs in the range likely to be encountered in the human diet, ie, equivalent to intakes of 1, 2, or 4 servings of oily fish per week in older adults at moderate risk of cardiovascular disease. 1
From the Diabetes and Nutritional Sciences Division (TABS, WLH, ZM, and FL), the Division of Women’s Health (PTS), and the Cardiovascular Sciences Division (PJC), School of Medicine, King’s College London, London, United Kingdom. 2 Supported by a grant from the Food Standards Agency (United Kingdom) and the Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London (project code N02041). The study oils were kindly supplied by Croda Chemicals Europe Ltd. 3 Address correspondence to TAB Sanders, Diabetes and Nutritional Sciences Division, School of Medicine, King’s College London, FranklinWilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom. E-mail:
[email protected]. 4 Abbreviations used: BP, blood pressure; FMD, flow-mediated dilatation; GTN, glycerol trinitrate; LC-PUFAs, long-chain PUFAs; PWVc-f, carotid to femoral pulse wave velocity. Received April 22, 2011. Accepted for publication July 5, 2011. doi: 10.3945/ajcn.111.018036.
Am J Clin Nutr doi: 10.3945/ajcn.111.018036. Printed in USA. Ó 2011 American Society for Nutrition
Copyright (C) 2011 by the American Society for Nutrition
1 of 8
2 of 8
SANDERS ET AL
SUBJECTS AND METHODS
Subjects The MARINA trial (ISRCTN66664610) was a single-center dietary intervention study conducted at King’s College London in the United Kingdom between April 2008 and October 2010. Ethical approval was obtained from the relevant research ethics committees in the United Kingdom (NREC 08/H0802/3), and written informed consent was provided by the participants. Advertisements in newspapers and on the study website and a circular e-mail within King’s College London were used for recruitment. A participant information sheet was provided to subjects who expressed interest. Men and women aged 45–70 y attended a screening visit in the fasting state for measurement of height, weight, waist circumference, seated BP, liver function, glucose, lipids, and hematologic indicators. A spot urine sample was provided to check absence of albumin and to confirm nonsmoking status by measuring cotinine (a metabolite of cigarette smoke that indicates a smoking habit). Exclusion criteria for this study included a medical history of cardiovascular disease; overall risk of cardiovascular disease .20% over the next 10 y; cancer (excluding basal cell carcinoma) in the previous 5 y; type 1 diabetes mellitus; uncontrolled type 2 diabetes (fasting plasma glucose .7 mmol/L); chronic renal, liver, or inflammatory bowel disease; history of substance abuse or alcoholism; pregnancy; weight change of .3 kg in preceding 2 mo; and BMI (in kg/m2) ,20 and .35. A small remuneration was given for participation in the study. Once eligibility of the participants was established, the participants were asked to complete a food-frequency and lifestyle questionnaire (10).
The participants were supplied at intervals with new batches of capsules, and the unused capsules that were returned by the participants were counted and recorded. Compliance with the intervention was also assessed by measurement of the proportion of EPA and DHA in erythrocyte lipids at baseline, 6 mo, and 12 mo. Power calculations were based on 72 subjects/group completing the study to detect a 20% difference between means of FMD at 90% power and 1% significance. To allow for a noncompletion rate of 20%, the aim was to recruit 360 participants (292 completing); estimates were based on a previous study in which the mean FMD was measured under similar conditions in adults without overt cardiovascular disease (11). The mean value was 6.7% with a common SD of 2% and a within-subject SD of 1.1. We presumed that this size change in FMD was the smallest difference that was likely to be clinically significant and detectable. Prespecified secondary outcomes included changes in arterial stiffness measured as PWVc-f and 24-h ambulatory BP. Methods Measurements of endothelial function and arterial stiffness were measured at baseline and after 12 mo of treatment. On the day before each study visit involving vascular measurements,
Study design A randomized parallel design was used to compare the effects of 3 dose levels of DHA and EPA with those of placebo. The dietary intervention involved supplementation with encapsulated n23 LC-PUFAs at 3 different doses (0.45, 0.9, and 1.8 g/d), and the placebo consisted of olive oil (BP specification). Each treatment consisted of a run-in period (3 placebo capsules/d for 1 mo) and a 12-mo intervention phase (3 capsules/d). The random allocation sequence was generated with a computer program by using the process of minimization to balance age, sex, and ethnicity between treatment groups. We enrolled eligible participants, and the study database program allocated a series of capsules to the participant. The treatments associated with the capsule codes were concealed from all investigators and associated clinical staff until the data analysis was complete. The code breaker was an employee of MedSciNet who constructed the trial database (MedSciNet AB and MedSciNet UK Ltd). The experimental oils were supplied by Croda Chemicals Europe Ltd and encapsulated by Powerhealth Ltd (Pocklington) in gelatin capsules (1 g oil/capsule). The oils supplied consisted of blends of the test fat with 0.1 wt% peppermint oil to disguise the fish taste of the EPA and DHA. The placebo capsules contained refined olive oil plus 0.1% peppermint oil. The EPA and DHA blends consisted of blends of an EPA concentrate (TG7010, code SF06396, batch 213000) and a DHA concentrate (DHA700TG, code SF06405, batch 213003) and refined olive oil (batches 10813 and 14346). These were blended to provide 1.8, 0.9, and 0.45 g EPA+DHA/d (with an EPA/DHA ratio of 1.51).
FIGURE 1. Consort diagram of the flow of participants throughout the study.
3 of 8
N3 LC-PUFAS AND ENDOTHELIAL FUNCTION
participants were requested to abstain from strenuous exercise up to the time of the visit and to avoid consuming caffeinecontaining beverages or alcohol on the previous day from 1400. In addition, participants were advised to avoid consuming foods high in fat on the day before the visit and were asked to consume a low-fat frozen meal (,10 g fat, 3 MJ) in the evening (before 2200) and to fast overnight. Participants reported to the St Thomas Hospital clinical research facility between 0800 and 1000, measurements of weight and body composition were made, and the food-frequency and lifestyle questionnaire was checked for completeness. Seated BP was measured, and a venous blood sample was collected. After 15 min of rest in the supine position, measurement of arterial stiffness and supine BP were made. After a further 30-min supine rest, FMD of the brachial artery was measured to assess endothelium-dependent vascular reactivity with a high-resolution ultrasound system with a 7–10-MHz linear array transducer (Acuson Aspen; Acuson Corporation). Measurements were made by a trained vascular ultrasonographer according to the recommendations of the International Brachial Artery Reactivity Task Force Guidelines for the ultrasound assessment of endothelial-dependent FMD vasodilatation of the brachial artery (12). A longitudinal section of the brachial artery, 2–15 cm above the elbow, was initially scanned with a probe holder and clamp to help secure the position of the transducer. Reactive hyperemia was then induced
by inflation of a pneumatic tourniquet placed around the forearm (distal to the arterial segment being scanned) to a pressure of 250 mm Hg for 5 min, which was followed by the release of pressure on deflation of the cuff. The images were recorded continuously for 1 min in the baseline state, for 5 min during cuff inflation, and 5 min postcuff release. After a 10-min break to allow vessel recovery, another resting scan was taken before sublingual GTN (25 lg as an endothelium-independent control) was administered, and a continuous scan was performed 5 min later. Images were digitized for subsequent blinded analysis; the diameter of the brachial artery was determined by using computer-assisted edge detection software (Brachial Analyzer; Medical Imaging Applications, LCC). FMD is expressed as the percentage increase in brachial artery diameter from baseline to maximal dilation, which occurs 30–90 s after release of the cuff. The CV for repeat measures on the same day is 7% and between days is 18% in the same individual in our laboratory. Dilatation to GTN is expressed as the percentage increase in brachial artery diameter from baseline to maximal dilatation after GTN. Arterial stiffness was estimated by measuring PWVc-f with the SphygmoCor VW apparatus with SphygmoCor analysis software (SphygmoCor version 7.01 AtCor Medical Pty). PWVc-f was computed from the time delay between the upstroke of the arterial pressure wave at the carotid and femoral arteries and the anatomic carotid to femoral distance. Measurements were made
TABLE 1 Details of the study participants at run-in by randomly assigned treatment group1 Placebo (n = 88) Age (y) Male [n (%)] Female [n (%)] Postmenopausal [n (%)] Ethnicity [n (%)] White Black Asian3 Far Eastern Other BMI (kg/m2) Women Men Waist (cm) Women Men SBP (mm Hg) DBP (mm Hg) Glucose (mmol/L) Total:HDL cholesterol 10-y risk of CVD (%) Women Men Medication [n (%)] Statins Antihypertensive HRT Thyroxine
2
0.45 g/d (n = 94)
0.9 g/d (n = 93)
55 34 54 39
(54, 57) (38.6) (61.4) (72.2)
55 36 58 41
(53, 56) (38.3) (61.7) (70.6)
55 36 57 38
68 9 6 2 3
(77.3) (10.2) (6.8) (2.3) (3.4)
76 4 6 4 4
(80.9) (4.3) (6.4) (4.3) (4.3)
26 (25, 27) 27 (26, 28) 85 96 120 77 5.4 3.6
(82, 88) (93, 99) (117, 124) (75, 79) (5.3, 5.5) (3.4, 3.8)
6 (4.6, 7.3) 9.8 (7.9, 12) 3 4 1 1
(3) (5) (1) (1)
25 (24, 26) 26 (25, 27) 84 94 121 77 5.4 3.5
(81, 87) (91, 97) (118, 124) (75, 79) (5.3, 5.6) (3.4, 3.7)
6 (4.6, 7.3) 8.7 (6.8, 11) 4 4 1 4
(4) (4) (1) (4)
55 36 56 42
(54, 57) (39.1) (60.9) (75.0)
73 (78.5) 6 (6.5) 10 (10.8) 0 (0.0) 4 (4.3)
79 1 2 4 6
(85.9) (1.1) (2.2) (4.3) (6.5)
26 (24, 27) 27 (26, 28)
25 (24, 26) 26 (25, 27)
85 96 122 78 5.4 3.4
(54, 56) (38.7) (61.3) (66.6)
1.8 g/d (n = 92)
(82, 87) (93, 100) (119, 125) (76, 80) (5.3, 5.5) (3.3, 3.6)
6 (4.6, 7.3) 8.6 (6.8, 11) 10 9 4 4
(11) (10) (4) (4)
84 96 121 76 5.3 3.47
(82, 87) (92, 99) (117, 124) (75, 78) (5.2, 5.4) (3.3, 3.7)
6 (4.6 ,7.3) 9.6 (7.6, 12) 1 2 3 2
(1) (2) (3) (2)
1 No significant differences were found between treatment groups. CVD, cardiovascular disease; DBP, seated diastolic blood pressure; HRT, hormone replacement therapy; SBP, seated systolic blood pressure. 2 Mean; 95% CI in parentheses (all such values). 3 South Asian, Southeast Asian, and Middle Eastern.
4 of 8
SANDERS ET AL
after a 15-min supine rest, and BP was recorded with an automated sphygmomanometer (Omron 70CP).) Central supine BP, peripheral and central augmentation indexes were also determined with the SphygmoCor device by analysis of the radial arterial waveform obtained by tonometry. Measurements of 24-h ambulatory BP were made by using A&D TM-2430 devices (A&D Instruments) in accordance with
UK guidelines (13). Participants were fitted with the monitors at the end of the clinic visit, and the monitors were programmed to take measurements every 30 min during the day (0700–2200) and hourly at night (2200–0700). Ambulatory BP measurements were made at the end of the run-in period, after 6 mo of intervention, and after 12 mo of intervention. The first 3 readings obtained after fitting the monitor were discarded. The data were analyzed by
TABLE 2 Dietary intakes, physical activity, and serum lipids before and after treatment with n23 long-chain PUFAs in study participants by randomly assigned treatment group1 Placebo (n = 71)
0.45 g/d (n = 80)
0.9 g/d (n = 81)
1.8 g/d (n = 80)
8.36 (7.57, 8.98) 7.79 (6.92, 8.67)
7.81 (7.16, 8.46) 7.05 (6.46, 7.64)
8.12 (7.56, 8.68) 7.98 (7.28, 8.68)
7.82 (7.24, 8.40) 7.60 (6.99, 8.22)
15.8 (15.2, 16.5) 15.8 (15.1, 16.6)
16.0 (15.3, 16.6) 16.1 (15.4, 16.8)
15.8 (15.3, 16.3) 16.3 (15.7, 16.9)
16.7 (16.0, 17.4) 16.4 (15.7, 17.2)
48.2 (46.3, 50.0) 49.5 (47.4, 51.6)
46.9 (45.1, 48.6) 47.1 (45.2, 48.9)
47.1 (45.6, 48.6) 46.6 (44.9, 48.3)
46.0 (44.5, 47.5) 45.7 (44.0, 47.4)
32.1 (30.3, 33.8) 30.8 (28.9, 32.6)
33.6 (31.9, 35.2) 33.1 (31.4, 34.7)
33.7 (32.3, 35.1) 34.0 (32.4, 35.5)
33.9 (32.5, 35.4) 33.8 (32.1, 35.4)
11.2 (10.5,11.9) 11.1 (10.3, 11.9)
12.0 (11.2,12.8) 11.8 (11.1, 12.6)
12.1 (11.4,12.8) 12.1 (11.5, 12.8)
12.1 (11.4,12.8) 12.1 (11.4,12.8)
6.2 (5.8, 6.6) 5.7 (5.3, 6.1)
6.4 (5.9, 6.9) 6.1 (5.7, 6.5)
6.3 (6.0, 6.6) 6.4 (6.0, 6.8)
6.4 (5.9, 6.9) 6.2 (5.8, 6.7)
76.3 (72.6, 80.1) 75.9 (72.1, 79.6)
70.8 (67.8, 73.8) 71.3 (68.3, 74.3)
76.1 (72.6, 79.6) 76.6 (73.1, 80.2)
72.4 (69.4, 75.4) 72.4 (69.5, 75.4)
42.7 (41.5, 44.0) 41.4 (40.1, 42.7)
42.6 (41.2, 44.0) 41.6 (40.0, 43.1)
42.8 (41.6, 43.9) 42.1 (40.6, 43.5)
43.7 (41.8, 45.5) 42.2 (40.5, 44.0)
5.5 (5.2, 5.7) 5.6 (5.4, 5.7)
5.4 (5.1, 5.6) 5.7 (5.5, 5.9)
5.4 (5.2, 5.6) 5.5 (5.3, 5.7)
5.4 (5.2, 5.6) 5.6 (5.4, 5.8)
3.3 (3.1, 3.5) 3.4 (3.3, 3.6)
3.2 (3.0, 3.4) 3.5 (3.3, 3.7)
3.2 (3.0, 3.4) 3.3 (3.1, 3.5)
3.2 (3.0, 3.3) 3.4 (3.3, 3.6)
1.6 (1.5, 1.7) 1.5 (1.5, 1.6)
1.6 (1.5, 1.6) 1.6 (1.5, 1.7)
1.7 (1.5, 1.8) 1.6 (1.5, 1.7)
1.7 (1.6, 1.8) 1.7 (1.6, 1.8)
3.53 (3.33, 3.75) 3.67 (3.46, 3.89)
3.49 (3.31, 3.69) 3.62 (3.43, 3.83)
3.34 (3.16, 3.52) 3.45 (3.26, 3.64)
3.34 (3.16, 3.53) 3.41 (3.22, 3.60)
1.16 (1.05, 1.29) 1.19 (1.09, 1.31)
1.14 (1.03, 1.26) 1.14 (1.04, 1.24)
1.13 (1.02, 1.25) 1.12 (1.03, 1.22)
1.10 (1.00, 1.22) 0.96 (0.88, 1.05)6
0.8 (0.3, 1.4) 1.0 (0.4, 1.7)
0.5 (0.3, 1.8) 0.5 (0.2, 1.4)
0.6 (0.3, 1.8) 0.9 (0.3, 1.9)
2
Energy (MJ/d) Baseline After treatment Protein (% of energy)2 Baseline After treatment Carbohydrates (% of energy)2 Baseline After treatment Fat (% of energy)2 Baseline After treatment Saturated fatty acids (% of energy)2 Baseline After treatment PUFAs (% of energy)2 Baseline After treatment Body weight (kg)2,3 Run-in After treatment Physical activity (MET/d)2 Baseline After treatment Serum cholesterol (mmol/L)2 Run-in After treatment LDL cholesterol (mmol/L)2,3 Run-in After treatment HDL cholesterol (mmol/L)2,3 Run-in After treatment Total:HDL cholesterol4 Run-in After treatment Triacylglycerol (mmol/L)4,5 Run-in After treatment C-reactive protein (mg/dL)3,7 Run-in After treatment
0.5 (0.2, 1.5) 0.6 (0.2, 1.7)
1 Data were analyzed by ANCOVA with the value on treatment regressed against BMI, age, sex, and the baseline value. MET, metabolic equivalent task as a ratio of resting metabolic rate. 2 Values are means; 95% CIs in parentheses. 3 Values are the means of measures at 6 and 12 mo of treatment. 4 Values are geometric means; 95% CIs in parentheses. Data were log-transformed before the analysis. 5 P-trend = 0.014. 6 Change from run-in significantly different from placebo, 0.45 g/d, and 0.9 g/d: P = 0.002, P = 0.005, and P = 0.045, respectively. 7 Values are medians; interquartile ranges in parentheses. Subjects with values .10 mg/L were excluded from the analysis.
N3 LC-PUFAS AND ENDOTHELIAL FUNCTION
using TM-2430–13 Doctor Pro Software and were checked for accuracy. If inaccurate measurements were obtained, the participants were requested to repeat the 24-h BP monitoring. Serum lipid and C-reactive protein concentrations and erythrocyte lipid composition were determined in blood collected at the end of the run-in period and after 6 and 12 mo of the intervention. The methods used to measure serum lipids and C-reactive protein were described previously (14). Interassay CVs for total cholesterol were 1.1%, 1.5%, and 1.0% at 3.9, 5.1 and 5.7 mmol/L respectively; for HDL cholesterol were 2.2%, 2.1%, and 2.5% at concentrations of 0.91, 1.39 and 1.95 mmol/L respectively; and for triacylglycerols were 2.5% and 1.5% at concentrations of 1.32 and 2.36 mmol/L, respectively. Erythrocyte lipids were extracted from washed red blood cells within 3 d of blood collection in the presence of butylated hydroxyl toluene (50 mg/L), as previously described (15). The lipid extract was stored at 220°C until derivatized for analysis. The phosphoglycerides were transesterified with sodium methoxide to yield fatty acid methyl esters, which were separated and quantified by gas chromatography (Agilent 7890A GC; Agilent Technologies) with a BPX70 column (25 m · 220 lm · 0.25 lm; SGE Analytic Science). Mean interassay and intraassay CVs were 3% and 2%, respectively. To increase the precision of the analysis, all of the samples for a given individual were performed within the same run. Statistical analyses Data were analyzed on an intention-to-treat basis by ANCOVA with adjustment for baseline values, age, sex, BMI, and ethnicity by using STATA 11 software (StataCorp LP). When multiple measurements were available (BP, serum lipids, C-reactive protein, and erythrocyte lipids), the mean value during treatment was used in the analysis. Standard distributional checks were made, and, where appropriate, the analyses were attempted after log transformation or other transformation. After log transformation, geometric means and ratios are presented. Significance tests for comparisons between groups were done by using means of trend tests, based on a weighted combination (0:1:2:4) of the treatment effects expected in the 4 groups. Spearman’s rank correlation test was used to examine associations between variables.
5 of 8
menopausal. About one-fifth of the study population was nonwhite, with similar proportions of Asian and black participants. The average BMI was above the desirable range (20–25), and the mean waist circumferences were greater than the cutoffs used to indicate risk of metabolic syndrome (94 cm in men and 80 cm in women). Female and male participants had a 6% and 10% risk of having a clinical cardiovascular event (nonfatal and fatal) over the next 10 y on the basis of the QRISK2 algorithm (www.qrisk.org). Body weights were relatively stable, and no significant differences in changes in body weight were found between treatments. The proportion of energy derived from total fatty acids, saturated fatty acids, and PUFAs (Table 2) was similar to that of the general population as reported in the rolling UK National Dietary and Nutritional Surveys (16, 17), and physical activity levels were low and did not change over the study period. The study, therefore, achieved the objective of recruiting nonsmoking participants who were representative of the UK general population and at moderately increased risk of developing cardiovascular disease over the next decade. Capsule counts indicated that 88.5% of the participants consumed .90% of the capsules provided. No statistically significant differences in reported capsule intakes were found between treatment groups. The proportions of EPA and DHA in erythrocyte lipids increased in a dose-dependent manner compared with placebo, which indicated long-term compliance with the intervention Figure 2. There was a significant trend for dose of n23 LC-PUFAs and serum triacylglycerol concentration (P = 0.014). Serum triacylglycerol concentrations were significantly lower after the highest dose (1.8 g/d) than after the placebo and lower doses (Table 2). The results for the primary and secondary outcomes are shown in Table 3. Approximately 45% of men and 33% of women had FMD values of 4%, which is considered in our laboratory to indicate impaired endothelial function, at baseline, and these proportions did not change on follow-up. No statistically significant differences were found between treatment groups in baseline brachial artery diameter or FMD- or GTN-induced dilatation, and this conclusion was not altered by excluding data
RESULTS
Recruitment into the study commenced in May 2008, and the study was completed in September 2010. Of 367 participants randomly assigned to treatment, a total of 25 randomly assigned participants withdrew from the study in the run-in period, and 6 withdrew after the baseline measures because of an unwillingness to comply with the study protocol and to attend follow-up visits. An additional 19 subjects withdrew their consent for personal reason, 1 withdrew for medical reasons, and an additional 4 participants did not attend their final appointment (Figure 1). Data were available for the analysis of 312 participants, and primary endpoints were available for 310 participants. Details of all randomly assigned participants are shown in Table 1. The number of participants allocated to each of the 4 groups did not significantly differ, nor did dropout rates (Figure 1). There were more women than men and most of the women were post-
FIGURE 2. Mean changes (and 95% CIs) compared with placebo (n = 80) in the proportions of EPA (20:5n23) and DHA (22:6n23) in erythrocyte lipids after 6 and 12 mo of treatment with different doses of n23 long-chain PUFAs: 0.45 g/d (n = 82), 0.9 g/d (n = 81), and 1.8 g/d (n = 82). The probability for trend test is based on a weighted combination (0:1:2:4) of the treatment effects expected in the 4 groups derived from ANCOVA from the value on treatment regressed against age, sex, BMI, ethnicity, and the value at baseline. Mean placebo values at baseline (and 95% CIs) were as follows: 1.3% (1.2%, 1.4%) for EPA and 6.5% (6.2%, 6.9%) for DHA.
6 of 8
SANDERS ET AL
TABLE 3 Changes in vascular function indexes at run-in and after treatment with increasing dose of n23 long-chain PUFAs compared with placebo1 Placebo (n = 71) BAD (mm)3 Run-in After treatment FMD (%)3 Run-in After treatment GTN (%)3 Run-in After treatment PWVc-f (m/s)3 Run-in After treatment Aortic AIX (%)4 Run-in After treatment Peripheral AIX (%) Run-in After treatment MAP (mm Hg)4 Run-in After treatment 24-h SBP (mm Hg)3,5 Run-in After treatment 24-h DBP (mm Hg)3,5 Run-in After treatment
0.45 g/d (n = 80)
0.9 g/d (n = 79)
1.8 g/d (n = 80)
P value2
3.6 (3.4, 3.7) 3.6 (3.4, 3.7)
3.3 (3.2, 3.5) 3.4 (3.3, 3.5)
3.4 (3.3, 3.6) 3.4 (3.3, 3.6)
3.3 (3.2, 3.5) 3.3 (3.2, 3.5)
0.84
4.8 (4.2, 5.5) 5.0 (4.2, 5.7)
5.9 (5.3, 6.5) 5.7 (5.0, 6.4)
4.9 (4.3, 5.5) 4.9 (4.2, 5.7)
5.2 (4.6, 5.8) 4.8 (4.1, 5.6)
0.781
11.2 (10.2, 12.2) 10.7 (9.8, 11.5)
13.2 (12.3, 14.2) 11.4 (10.6, 12.2)
11.3 (10.4, 12.3) 11.4 (10.6, 12.2)
12.1 (11.1, 13.1) 11.5 (10.7, 12.2)
0.337
8.96 (8.60, 9.35) 8.80 (8.40, 9.22)
8.75 (8.38, 9.14) 8.36 (8.02, 8.72)
8.87 (8.55, 9.20) 8.83 (8.53, 9.14)
8.74 (8.36, 9.13) 8.62 (8.28, 8.96)
0.683
27.6 (25.1, 30.2) 27.9 (24.9, 31.0)
28.8 (26.6, 31.1) 28.2 (26.0, 30.4)
29.2 (26.7, 31.7) 28.3 (25.8, 30.8)
28.9 (26.4, 31.4) 28.8 (26.1, 31.4)
0.580
78.7 (74.9, 82.7) 81.4 (76.9, 86.2)
80.5 (76.9, 84.3) 80.6 (77.3, 84.0)
81.2 (77.7, 84.9) 81.3 (77.9, 84.8)
80.7 (76.9, 84.7) 80.9 (77.4, 84.6)
0.112
93 (90, 95) 90 (88, 93)
93 (90, 95) 90 (88, 93)
94 (91, 96) 92 (90, 94)
91 (88, 93) 89 (87, 91)
0.805
122.6 (120.0, 125.2) 122.1 (119.2, 125.1)
122.6 (120.0, 125.2) 122.1 (119.2, 125.1)
123.5 (120.4, 126.6) 122.2 (119.7, 124.8)
119.1 (116.1, 122.0) 118.3 (115.6, 121.0)
0.669
74.1 (72.6, 75.7) 72.9 (71.4, 74.4)
71.2 (69.8, 72.7) 71.2 (69.8, 72.6)
73.9 (72.3, 75.5) 73.3 (72.0, 74.6)
71.8 (70.4, 73.2) 71.2 (69.9, 72.5)
0.624
1
AIX, augmentation index; BAD, brachial artery diameter; DBP, diastolic blood pressure; FMD, flow-mediated dilatation of the brachial artery; GTN, glycerol trinitrate (25 lg)–mediated dilatation of the brachial artery; MAP, central supine mean arterial blood pressure measured in clinic; PWVc-f, carotid to femoral pulse wave velocity; SBP, systolic blood pressure. 2 The probability is for trend test based on a weighted combination (0:1:2:4) of the treatment effects expected in the 4 groups derived from ANCOVA of the value on treatment regressed against age, sex, BMI, ethnicity, and the value at run-in. 3 Values are means; 95% CIs in parentheses. 4 Values are geometric means; 95% CIs in parentheses. Data were log-transformed before the analysis. 5 Values are the means of measures made at 6 and 12 mo of treatment.
from participants who were considered poor compliers on the basis of capsule counts. No evidence suggested that menopausal status or use of hormone replacement therapy influenced the results or that basal vessel diameter changed during the study. Multiple regression analysis of the FMD at baseline with age, sex, ethnicity, BMI, and the n23 index (% EPA+DHA in erythrocyte) did not show any statistically significant relation between the omega-3 index and FMD. Mean (6SD) values for FMD differed between sexes (P , 0.0001) and were 4.6 6 3.1% and 5.9 6 2.5% in men and women, respectively. FMD also decreased with increasing age (q = 0.270, P , 0.001) and was weakly negatively correlated with PWVc-f (q = 20.138, P = 0.016). No significant changes were found in PWVc-f, peripheral or central augmentation indexes, central supine BP, or 24-h ambulatory BP (Table 3). Exclusion of participants whose weight fluctuated by .3 kg over the year did not alter any of the above conclusions. No other significant differences were noted. DISCUSSION
Egert and Stehle (9) and Hall (18) reviewed the effects of n23 LC-PUFAs (EPA plus DHA) on endothelial function as de-
termined by the FMD technique (9, 18) and concluded that evidence was insufficient to support firm recommendations. More recently, Skulas-Ray et al (19) compared a nutritional dose of n23 LC-PUFAs (0.85 g/d) with a pharmacologic dose (3.4 g/d) taken for 6 wk by 23 men and 3 postmenopausal women according to a placebo-controlled crossover design. The higher dose (3.4 g/d) of EPA+DHA significantly lowered triacylglycerol, but neither dose improved endothelial function. Haberka et al (20) compared the effects of 1 g n23 LC-PUFAs in 40 patients who had experienced an acute myocardial infarction and had been treated by angioplasty. Half of the patients received the n23 LC-PUFAs and the other group received usual care. The authors claim that there was an improvement after the n23 LC-PUFAs, from 7.4 6 6.4% to 15.5 6 10.5%. However, closer inspection showed significant differences between groups at baseline, and the differences in the changes in FMD between groups were not statistically significant. The measurement of FMD is subject to considerable variability, particularly if the method is not well standardized or personnel are not appropriately trained. Our study was the first randomized controlled trial to be sufficiently statistically powered to test this hypothesis. We used a small dose (3 g) of refined olive oil as placebo, which has been shown not to influence endothelial function (21). We used
N3 LC-PUFAS AND ENDOTHELIAL FUNCTION
a standardized protocol for measurement of FMD that requires participant to avoid foods high in fat, caffeine, or alcohol on the previous day and to avoid strenuous exercise. Measurements were made after an overnight fast at the time of day by an experienced vascular ultrasonographer. The findings of the current study indicate that, in healthy adults without clinically overt coronary heart disease, no effects of n23 LC-PUFA intakes in the range of 0.45 to 1.8 g/d on endothelial function were found. These findings are consistent with those of a recent crosssectional survey in 3045 adults (22), which found no significant relation between n23 LC-PUFA intake or dietary nonfried fish intake with FMD. FMD was measured at rest, and the current study cannot rule out a beneficial effect of changes induced in endothelial function after a “stress” induced by, for example, a fat load or psychological stress. Arterial stiffness is a powerful predictor of cardiovascular events and mortality (22, 23). Tomiyama et al (24) reported a beneficial effect on arterial stiffness over 12 mo with 1.8 g EPA/d in an open study. We were, however, unable to demonstrate any effect at this level of intake or below. Arterial stiffening occurs over many years, but accelerates beyond the age of 60 y, it may be that a much longer period of intervention (up to 5 y) may be necessary to detect subtle differences in the rate of arterial stiffening. A meta-analysis (25) of randomized controlled trials indicate that intakes .3 g n23 LC-PUFAs/d as fish-oil supplements lower both systolic and diastolic BP, particularly in subjects aged .45 y; the average fall in systolic/diastolic BP was 2.3/1.5 mm Hg. The current study was unable to show any significant changes in BP, and this would be consistent with the epidemiologic observations from the INTERMAP Study (26), which concluded that any effect of n23 LC-PUFAs from fish is small (;1 mm Hg lower) on the basis of the relation between fatty acid intake and BP by using four 24-h dietary recalls to assess dietary intake and 8 clinic BP measurements over 3 wk in 17 populations in Japan, China, the United Kingdom, and the United States (n = 4680). Recent reviews of the evidence (27, 28) regarding n23 LCPUFAs and the risk of CVD have advocated population intakes ranging from 0.25 to 2 g/d in the average diet. The evidence supporting these recommendations is mainly derived from metaanalyses of observational data from prospective cohort studies, which show an association between the intake of n23 LCPUFAs from fish and decreased cardiovascular disease risk (4) rather than from effects on surrogate risk markers. The current study showed no effect of intakes up to 1.8 g n23 LC-PUFAs on BP or total:HDL cholesterol, which is regarded as the most robust lipid metric of cardiovascular disease risk (29). A limitation of our study was that it was conducted in adults at mildto-moderate risk of cardiovascular disease rather than in those with clinically evident disease. Furthermore, the participants of the current study were nonsmokers. This indicates that n23 LCPUFA intake might not have any added cardiovascular health benefit in a healthy population; in fact, no clear evidence of benefit (ie, decreased incidence of cardiovascular events or mortality) has been shown in secondary prevention cohort and randomized controlled trials (30–34). The findings of the current study contribute evidence to the debate about whether preformed n23 LC-PUFAs are required in adults to prevent cardiovascular disease. The need for preformed n23 LC-PUFAs has been questioned because the risk of cardiovascular disease in
7 of 8
vegetarians, whose diet is virtually devoid of these fatty acids, is lower than that of the general population (29, 35–38). However, serum total and LDL-cholesterol concentrations are lower in vegetarians, especially vegans, than in meat eaters because of their lower intakes of saturated fatty acids and correspondingly higher intakes of PUFAs and lower BMIs. Vegetarians depend on an intake of a-linolenic acid to meet their requirements for n23 PUFAs. Dietary a-linolenic acid is not readily converted to DHA and has different effects from those of n23 LC-PUFAs on several surrogate risk factors for cardiovascular disease (39, 40). However, evidence from prospective cohort and case-control studies (41, 42) indicates that the intake of a-linolenic acid is associated with a decreased risk of sudden cardiac death. Thus, it is likely that the postulated role of n23 PUFAs in reducing risk of CVD may be met by a-linolenic acid and by the small amount amounts of n23 LC-PUFAs present in diets containing fish, meat, and eggs (28). In the United Kingdom, the dietary recommendation to consume 2 portions of fish per week, 1 of which should be oily fish, is based on a risk-benefit analysis of fish consumption rather than on a specified dietary requirement for n23 LC-PUFAs (43). In conclusion, the randomized controlled trial indicated that intakes of n23 LC-PUFAs 1.8 g/d do not improve endothelial function in nonsmoking healthy adults with a mild-to-moderate increased risk of cardiovascular disease. We are grateful to Malcolm Law, Lee Hooper, and Kennedy Cruickshank for agreeing to be on the Data Monitoring Committee; to CRODA Europe Ltd for formulating and supplying the oils for the intervention; and particularly to the participants of the study. The assistance of Paula Darroch (manager of the St Thomas’ Hospital Clinical Research Centre), Robert Gray, Karen McNeill, Benyu Jiang, Kenneth Connell, Roy Sherwood, Ann Donald, and Laura O’Sullivan is gratefully acknowledged. The authors’ responsibilities were as follows—TABS, WLH, and PJC: conceived, devised, and helped conduct the study; ZM and FL: organized and conducted the study; and PTS: devised the data analysis plan and conducted the statistical analyses. All authors contributed to the preparation of the manuscript. None of the authors had a financial or commercial interest in any company or organization sponsoring the research.
REFERENCES 1. Iso H, Kobayashi M, Ishihara J, Sasaki S, Okada K, Kita Y, Kokubo Y, Tsugane S; JPHC Study Group. Intake of fish and n3 fatty acids and risk of coronary heart disease among Japanese: the Japan Public Health Center-Based (JPHC) Study Cohort I. Circulation 2006;113:195–202. 2. Hamazaki T, Urakaze M, Sawazaki S, Yamazaki K, Taki H, Yano S. Comparison of pulse wave velocity of the aorta between inhabitants of fishing and farming villages in Japan. Atherosclerosis 1988;73:157–60. 3. Yamada T, Strong JP, Ishii T, Ueno T, Koyama M, Wagayama H, Shimizu A, Sakai T, Malcom GT, Guzman MA. Atherosclerosis and omega-3 fatty acids in the populations of a fishing village and a farming village in Japan. Atherosclerosis 2000;153:469–81. 4. Skeaff CM, Miller J. Dietary fat and coronary heart disease: summary of evidence from prospective cohort and randomised controlled trials. Ann Nutr Metab 2009;55:173–201. 5. Knapp HR, Fitzgerald GA. The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension. N Engl J Med 1989;320:1037–43. 6. Saravanan P, Davidson NC, Schmidt EB, Calder PC. Cardiovascular effects of marine omega-3 fatty acids. Lancet 2010;376:540–50. 7. Donald AE, Halcox JP, Charakida M, Storry C, Wallace SM, Cole TJ, Friberg P, Deanfield JE. Methodological approaches to optimize reproducibility and power in clinical studies of flow-mediated dilation. J Am Coll Cardiol 2008;51:1959–64. 8. Leeson CP, Mann A, Kattenhorn M, Deanfield JE, Lucas A, Muller DP. Relationship between circulating n23 fatty acid concentrations and endothelial function in early adulthood. Eur Heart J 2002;23:216–22.
8 of 8
SANDERS ET AL
9. Egert S, Stehle P. Impact of n23 fatty acids on endothelial function: results from human interventions studies. Curr Opin Clin Nutr Metab Care 2011;14:121–31. 10. Bingham S, Luben R, Welch A, Low YL, Khaw KT, Wareham N, Day N. Associations between dietary methods and biomarkers, and between fruits and vegetables and risk of ischaemic heart disease, in the EPIC Norfolk Cohort Study. Int J Epidemiol 2008;37:978–87. 11. Berry SE, Mulla UZ, Chowienczyk PJ, Sanders TA. Increased potassium intake from fruit and vegetables or supplements does not lower blood pressure or improve vascular function in UK men and women with early hypertension: a randomised controlled trial. Br J Nutr 2010; 104:1839–47. 12. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39:257–65. 13. O’Brien E, Asmar R, Beilin L, Imai Y, Mancia G, Mengden T, Myers M, Padfield P, Palatini P, Parati G, et al. Practice guidelines of the European Society of Hypertension for clinic, ambulatory and self blood pressure measurement. J Hypertens 2005;23:697–701. 14. Jebb SA, Lovegrove JA, Griffin BA, Frost GS, Moore CS, Chatfield MD, Bluck LJ, Williams CM, Sanders TA. Effect of changing the amount and type of fat and carbohydrate on insulin sensitivity and cardiovascular risk: the RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) trial. Am J Clin Nutr 2010;92:748–58. 15. Sanders TA, Lewis F, Slaughter S, Griffin BA, Griffin M, Davies I, Millward DJ, Cooper JA, Miller GJ. Effect of varying the ratio of n26 to n23 fatty acids by increasing the dietary intake of alphalinolenic acid, eicosapentaenoic and docosahexaenoic acid, or both on fibrinogen and clotting factors VII and XII in persons aged 45-70 y: the OPTILIP study. Am J Clin Nutr 2006;84:513–22. 16. Hoare J, Henderson L, Bates CJ, Prentice A, Birch M, Swan G, Farron M. The National Diet and Nutritional Survey: adults aged 19-64 years. Summary report. Vol 5. London, United Kingdom: TSO, 2004. 17. Nelson M, Erens B, Bales B, Church S, Boshier T. Low income diet and nutrition survey, Vol 2: Food consumption and nutrient intake. Norwich, United Kingdom: The Stationery Office, 2007. 18. Hall WL. Dietary saturated and unsaturated fats as determinants of blood pressure and vascular function. Nutr Res Rev O’Brien E, Asmar R, Beilin L, Imai Y, Mancia G, Mengden T, Myers M, Padfield P, Palatini P, Parati G 2009;22:18–38. 19. Skulas-Ray AC, Kris-Etherton PM, Harris WS, Vanden Heuvel JP, Wagner PR, West SG. Dose-response effects of omega-3 fatty acids on triglycerides, inflammation, and endothelial function in healthy persons with moderate hypertriglyceridemia. Am J Clin Nutr 2011;93:243–52. 20. Haberka M, Mizia-Stec K, Mizia M, Janowska J, Gieszczyk K, Chmiel A, Zahorska-Markiewicz B, Ga˛sior Z. N-3 polyunsaturated fatty acids early supplementation improves ultrasound indices of endothelial function, but not through NO inhibitors in patients with acute myocardial infarction N-3 PUFA supplementation in acute myocardial infarction. Clin Nutr 2011;30:79–85. 21. Theobald HE, Goodall AH, Sattar N, Talbot DC, Chowienczyk PJ, Sanders TA. Low-dose docosahexaenoic acid lowers diastolic blood pressure in middle-aged men and women. J Nutr 2007;137:973–8. 22. Anderson JS, Nettleton JA, Herrington DM, Johnson WC, Tsai MY, Siscovick D. Relation of omega-3 fatty acid and dietary fish intake with brachial artery flow-mediated vasodilation in the Multi-Ethnic Study of Atherosclerosis. Am J Clin Nutr 2010;92:1204–13. 23. Terai M, Ohishi M, Ito N, Takagi T, Tatara Y, Kaibe M, Komai N, Rakugi H, Ogihara T. Comparison of arterial functional evaluations as a predictor of cardiovascular events in hypertensive patients: the Non-Invasive Atherosclerotic Evaluation in Hypertension (NOAH) study. Hypertens Res 2008;31:1135–45. 24. Tomiyama H, Takazawa K, Osa S, Hirose K, Hirai A, Iketani T, Monden M, Sanoyama K, Yamashina A. Do eicosapentaenoic acid supplements attenuate age-related increases in arterial stiffness in patients with dyslipidemia? A preliminary study. Hypertens Res 2005;28: 651–5. 25. Geleijnse JM, Giltay EJ, Grobbee DE, Donders AR, Kok FJ. Blood pressure response to fish oil supplementation: metaregression analysis of randomized trials. J Hypertens 2002;20:1493–9.
26. Ueshima H, Stamler J, Elliott P, Chan Q, Brown IJ, Carnethon MR, Daviglus ML, He K, Moag-Stahlberg A, Rodriguez BL, et al. Food omega-3 fatty acid intake of individuals (total, linolenic acid, longchain) and their blood pressure: INTERMAP study. Hypertension 2007;50:313–9. 27. EFSA Panel on Dietetic Products, Nutrition and Allergies. Scientific opinion on the dietary references values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids and cholesterol. EFSA J 2010;8:1461–568. 28. WHO/FAO. Fats and fatty acids in human nutrition. Report of an expert consultation. Rome, Italy: Food and Agricultural Organisation, 2010. Available from: http://www.fao.org (cited 2011). 29. Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, Halsey J, Qizilbash N, Peto R, Collins R. Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths. Lancet 2007;370:1829–39. 30. Hooper L, Thompson RL, Harrison RA, Summerbell CD, Ness AR, Moore HJ, Worthington HV, Durrington PN, Higgins JP, Capps NE, et al. Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review. BMJ 2006;332:752–60. 31. GISSI-HF Investigators. Effect of n23 polyunsaturated fatty acids in patients with chronic heart failure (the GISSI-HF trial): a randomised, double-blind, placebo-controlled trial. Lancet 2008;372:1223–39. 32. Marchioli R, Barzi F, Bomba E, Chieffo C, Di Gregorio D, Di Mascio R, Franzosi MG, Geraci E, Levantesi G, Maggioni AP, et al. Early protection against sudden death by n23 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI)-Prevenzione. Circulation 2002;105:1897–903. 33. 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. Lancet 2007;369:1090–8. 34. Kromhout D, Giltay EJ, Geleijnse JM. n23 Fatty acids and cardiovascular events after myocardial infarction. N Engl J Med 2010;363: 2015–26. 35. Key TJ, Appleby PN, Spencer EA, Travis RC, Roddam AW, Allen NE. Mortality in British vegetarians: results from the European Prospective Investigation into Cancer and Nutrition (EPIC-Oxford). Am J Clin Nutr 2009;89(suppl):1613S–9S. 36. O’Keefe JH Jr, Abuissa H, Sastre A, Steinhaus DM, Harris WS. Effects of omega-3 fatty acids on resting heart rate, heart rate recovery after exercise, and heart rate variability in men with healed myocardial infarctions and depressed ejection fractions. Am J Cardiol 2006;97: 1127–30. 37. Rosell MS, Lloyd-Wright Z, Appleby PN, Sanders TA, Allen NE, Key TJ. Long-chain n23 polyunsaturated fatty acids in plasma in British meat-eating, vegetarian, and vegan men. Am J Clin Nutr 2005;82: 327–34. 38. Welch AA, Shakya-Shrestha S, Lentjes MA, Wareham NJ, Khaw KT. Dietary intake and status of n23 polyunsaturated fatty acids in a population of fish-eating and non-fish-eating meat-eaters, vegetarians, and vegans and the precursor-product ratio of alpha-linolenic acid to longchain n23 polyunsaturated fatty acids: results from the EPIC-Norfolk cohort. Am J Clin Nutr 2010;92:1040–51. 39. Finnegan YE, Minihane AM, Leigh-Firbank EC, Kew S, Meijer GW, Muggli R, Calder PC, Williams CM. Plant- and marine-derived n23 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects. Am J Clin Nutr 2003;77:783–95. 40. Sanders TA. Fat and fatty acid intake and metabolic effects in the human body. Ann Nutr Metab 2009;55:162–72. 41. Albert CM, Oh K, Whang W, Manson JE, Chae CU, Stampfer MJ, Willett WC, Hu FB. Dietary alpha-linolenic acid intake and risk of sudden cardiac death and coronary heart disease. Circulation 2005;112: 3232–8. 42. Campos H, Baylin A, Willett WC. Alpha-linolenic acid and risk of nonfatal acute myocardial infarction. Circulation 2008;118:339–45. 43. Standing Advisory Committee on Nutrition. Advice on fish consumption: benefits and risks. London, United Kingdom: TSO, 2004.
