Nutrition and Disease

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1 Nutrition and Disease Flaxseed Oil Increases the Plasma Concentrations of Cardioprotective (n-3) Fatty Acids in Humans 1 Charles R. Harper,* 2 Megan J. Edwards, y Andrew P. DeFilipis,* and Terry A. Jacobson* ** *Department of Medicine, y Emory University School of Public Health, and **Office of Health Promotion and Disease Prevention, Emory University, Atlanta, GA ABSTRACT a-linolenic acid (ALA) is a major dietary (n-3) fatty acid. ALA is converted to longer-chain (n-3) PUFA, such as eicosapentaenoic acid (EPA) and possibly docosahexaenoic acid (DHA). EPA and DHA are fish-based (n-3) fatty acids that have proven cardioprotective properties. We studied the effect of daily supplementation with 3 g of ALA on the plasma concentration of long-chain (n-3) fatty acids in a predominantly African-American population with chronic illness. In a randomized, double-blind trial, 56 participants were given 3 g ALA/d from flaxseed oil capsules (n ¼ 31) or olive oil placebo capsules (n ¼ 25). Plasma EPA levels at 12 wk in the flaxseed oil group increased by 60%, from to mmol/l (P ¼ 0.004), whereas no change occurred in the olive oil group. Plasma docosapentaenoic acid (DPA) levels in the flaxseed oil group increased by 25% from to mmol/l (P ¼ 0.03) with no change in the olive oil group. Plasma DHA levels did not change in either group. This study demonstrates the efficacy of the conversion of ALA to EPA and DPA in a minority population with chronic disease. ALA may be an alternative to fish oil; however, additional clinical trials with ALA are warranted. J. Nutr. 136: 83 87, KEY WORDS: fatty acids prevention coronary disease Several clinical trials have now demonstrated that the long chain (n-3) PUFA, eicosapentaenoic acid (EPA) 3 and docosahexaenoic acid (DHA), reduce the risk of cardiac death (1 4). Although the AHA recently made recommendations for the general public to consume fish rich in EPA and DHA 2 times/wk, the public is still slow to adopt these guidelines due to concerns about toxins in fish (methyl mercury), taste preferences, and cost (5). The precursor to long-chain (n-3) fatty acids is a-linolenic acid (ALA), which is converted to EPA and possibly DHA in the body. The conversion of ALA to EPA in the body is limited, but may be physiologically and clinically important. Studies evaluating the efficiency of the conversion of ALA to its longerchain metabolite EPA have had highly variable results (6). Doses of ALA in these studies ranged from 5 to 40 g/d and the duration of the studies varied widely. Several factors are thought to influence the conversion to EPA. The (n-6) fatty acid, linoleic acid [LA; 18:2(n-6)] is thought to decrease the conversion of ALA to longer-chain n-3 PUFA by competing with ALA for the rate-limiting enzyme D6-desaturase (7). In addition, other dietary factors such as the polyunsaturated: 1 Supported in part by a grant (M01-RR00039) from the Emory University General Clinical Research Center (Grady Health System) and in part by a grant from the Emory Medial Care Foundation. 2 To whom correspondence should be addressed. charper@emory. edu. 3 Abbreviations used: ALA, a-linolenic acid; CHD, coronary heart disease; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FORCE, flaxseed oil to reduce intermediate cardiac endpoints; LA, linoleic acid; MI, myocardial infarction. saturated fat ratio, the amount of dietary EPA and trans fatty acids consumed, and amount and type of protein consumed have all been implicated in effecting this conversion (8 10). The efficiency with which this conversion occurs and the factors that may modify it could have important public health implications. Because of limitations in increasing the public s consumption of fish, the use of the plant-based (n-3) PUFA, ALA, may be an important alternative for providing optimal EPA and DHA concentrations in the plasma and cell membranes. Unlike its longer-chain metabolites, ALA can be obtained from several types of nuts, seeds, and seed oils, including flaxseed oil, English walnuts, canola oil, and soybean oil. Flaxseed meal or flaxseed oil can easily be incorporated into common dietary items such as breads, rolls, cereals, muffins, margarines, and salad dressings. ALA is generally regarded as safe for public consumption in doses up to 3 g/d by the FDA (11). In the FORCE trial (Flaxseed Oil to Reduce intermediate Cardiac Endpoints), we utilized a randomized, placebocontrolled study design to analyze the effect a 3-g daily supplement of ALA, in the form of flaxseed oil capsules, on plasma long-chain (n-3) PUFA (EPA, DHA) levels vs. an olive oil placebo. The study population was predominantly African- American patients with multiple chronic diseases. SUBJECTS AND METHODS Subjects. A total of 56 patients (49 women, 7 men) were enrolled in the study. Patients without known coronary heart disease were /06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 22 July Initial review completed 22 August Revision accepted 27 October

2 84 HARPER ET AL. recruited predominantly from an academic general medicine clinic affiliated with a large urban public hospital. Participants were screened and excluded if they reported taking multivitamins, antioxidants, and fish oil or (n-3) fatty acid supplements. Women of child-bearing age not using contraception and those who consumed.2 servings of fish/wk were also excluded. The experimental protocol was reviewed and approved by the IRB at Emory University and the Grady Research Oversight Committee. Informed consent was obtained from each subject before the start of the study. Experimental design. Potential participants were evaluated during an initial screening visit that included a review of the eligibility and exclusion criteria. Participants who met the eligibility criteria were scheduled for 2 run-in visits at 8 and 4 wk before randomization. All study visits took place in the Emory General Clinical Research Center. During these run-in visits, the research dietician instructed the participants on an AHA Step-I diet. Participants were taught to complete 3-d food records, which were analyzed for compliance with the diet using a standardized food record rating (Nutritionist V). After 8 wk of following the AHA diet, participants were randomly assigned to either a treatment group administered 3.0 g ALA/d (5.2 g of flaxseed oil) in the form of flaxseed oil capsules (Rx vitamins) or a control group administered 5.2 g olive oil/d in the form of olive oil capsules (Oleomed) (Table 1). The study biostatistician developed the randomization procedure. A randomization list was prepared using computer-generated random numbers. Random permuted blocks (size 2 or 4) were used to help ensure balance between the numbers of patients assigned to each treatment. The randomization scheme was provided to the data manager as a set of 60 sealed, sequenced, opaque envelopes containing the treatment assignment. As a patient entered the trial, the patient received the next envelope in the sequence. At the randomization visit, baseline plasma fatty acid levels were measured and dietary diaries were collected. Participants returned for visits at 12 and 26 wk and were instructed to fast for 12 h before each visit for laboratory tests. During these visits, plasma fatty acid levels were measured and pill counts were obtained to monitor compliance. The research dietician reinforced the AHA Step-I diet throughout the study and analyzed patient 3-d food diaries. Medication compliance was assessed by both pill count and patient self-report throughout the study. A priori, noncompliance with therapy was defined as consuming,80% of medications by pill count or by self-report. Blood collection and analytic methods. Blood samples were collected into 6-mL lavender-top EDTA glass tubes (Vacutainer tube, Becton Dickinson). The tubes were inverted 10 times and then centrifuged for 10 min at g. Plasma (3 ml) was pipetted into a transfer tube and stored in a 2708C freezer until the end of the study, when all samples were analyzed at the same time. Plasma fatty acids were determined using GC (Metametrix Laboratories). Total fatty acids were esterified using direct transesterification with an acetyl chloride methanol:iso-octane mixture. The FAME were than separated by GC using an HP-23 Cis/Trans FAME Capillary Column. The sample fatty acids were identified and quantified against a standard mixture of known fatty acids using an HP 5793 MS (12). Plasma fatty acid measurements were reported in mmol/l. Blood samples were sent to Atherotec Laboratories (Birmingham, Alabama) to be assayed. Plasma lipids were measured utilizing nonequilibrium density gradient ultracentrifugation (600 3 g; 10 min) to separate lipoproteins; ultracentrifugal separation was followed by enzymatic determination of cholesterol in all lipoprotein TABLE 1 Fatty acid composition of flaxseed oil and olive oil capsules Fatty acid Flaxseed oil Olive oil g/100 g a-linolenic acid [18:3(n-3)] 58 0 Linoleic acid [18:2(n-6)] 17 8 Oleic acid [18:1(n-9)] Palmitic acid (16:0) 6 0 Stearic acid (18:0) 3 16 fractions with ;400 sequential spectrophotometric measurements per sample. The final stage of analysis employs proprietary deconvolution software to determine subfractions of HDL, LDL, and VLDL. All VAP testing was performed at Atherotech, a CDC-National Heart, Lung, and Blood Institute standardized cholesterol testing laboratory. Statistical Analysis. Statistical analyses were performed using SAS 6.12 software. Fatty acid measurements were analyzed 4 wk before study randomization, at baseline, and at 12 and 26 wk after the start of the study. Baseline fatty acid levels were determined by averaging the measured fatty acid levels 4 wk before randomization with those obtained at randomization (wk 0) to minimize subject variability. The change in plasma fatty acid levels from baseline to 12 wk was calculated by subtracting the baseline value from the 12-wk value. Similar calculations were done to determine the change from baseline to 26 wk for each patient. Continuous variables were analyzed by Student s t test to determine the difference in means between groups. The relative risks and 95% CI of dichotomous variables such as gender, smoking status, diabetes, CHD, and hypertension were calculated by Cochran-Mantel-Haenszel statistics. For serial changes in fatty acids, we used a repeated-measures 2-way ANOVA, a 2-factor ANOVA with a repeated measure on 1 factor (time: 0, 12, and 26 wk). When there was a significant interaction by randomization group 3 time, within-group analysis was performed. In all cases, two-sided significance was determined at the P, 0.05 level. RESULTS Subjects and diet. The trial was successfully completed by 27 of the 31 subjects randomized to flaxseed oil and by 22 of the 25 given olive oil; these subjects were included in the final analysis. Of the 7 patients excluded, 1 participant was excluded after records indicated consumption of.4 fish meals/wk. The 6 other participants were excluded from the final analysis because of low compliance rates. The excluded patients and the remainder of the cohort did not differ in terms of demographics or baseline plasma fatty acids. During the study, only 5 participants reported any symptoms that they related to the fatty acid supplements, and both flaxseed oil and olive oil capsules were well tolerated. Symptoms were minimal and included dry mouth (3%), change in bowel habits (3%), and dyspepsia (3%). Randomization was successful in that the groups did not differ at baseline (Table 2). Participants were predominantly African-American (80%) women (87%) with a mean age of 51 y. Participants had multiple chronic illnesses including hypertension (55%) and Type II diabetes (12%). Most participants were overweight or obese with a mean BMI of 37.4 kg/m 2. Of the participants, 24% were taking a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, 26% were taking daily aspirin, and 53.7% were taking antihypertensives. Compliance with the AHA Step-1 Diet was monitored from 3-d food records obtained 4 wk before randomization and at wk 0, 12 and 26. The total energy intake and the percentage of energy obtained from fat, protein, carbohydrate, and alcohol did not differ between the groups at baseline (Table 3) and remained the same at follow-up dietary assessment. Despite pretrial education on an AHA Step-1 diet, participants dietary patterns at wk 0 reflected high amounts of total fat (32% of total energy) and saturated fat (15% of total energy). Weight measurements remained unchanged within and between groups through wk 26. Plasma fatty acids. At the start of the trial the groups did not differ in plasma concentrations of saturated, monounsaturated, polyunsaturated, (n-3), and (n-6) fatty acids. Within 12 wk, the group receiving flaxseed oil capsules demonstrated a 70% increase in plasma ALA concentration, whereas that in the olive oil group was unaffected (Table 4). The plasma EPA

3 INCREASING PLASMA EPA LEVELS WITHOUT FISH 85 Variable TABLE 2 Baseline characteristics of study participants 1 Flaxseed oil n =27 Olive oil n =22 Age, y Sex, % female Race, % African-American Caucasian Other Education, % None up to some high school High school/technical school College or higher CHD risk factors Current smoking, % Diabetes, % Hypertension, 2 % BMI, kg/m Weight, kg Medicines, % Statins NSAID Aspirin Plasma lipids, mmol/l Total cholesterol HDL cholesterol LDL cholesterol Triglycerides Values are means 6 SD or %. 2 Hypertension is defined as taking blood pressure medication or systolic $ 140 mm/hg or diastolic $ 90 mm/hg. 3 Statin, 3-hydroxy-3-methylglutaryl CoA reductase inhibitor. 4 NSAID, nonsteroidal anti-inflammatory drug. level increased by 60% at 12 wk in the flaxseed oil group, and docosapentaenoic acid (DPA) by 34% in the flaxseed oil group (Table 4). EPA and DPA did not change significantly in the olive oil group, whereas plasma DHA was unaffected at wk 12 and decreased at wk 26. The change in DHA did not differ between the groups. The (n-6):(n-3) ratio decreased significantly in the flaxseed oil group and was not affected in the olive oil group. DISCUSSION Our results clearly demonstrate the efficacy of increasing plasma EPA levels by providing its precursor, ALA, in the form TABLE 3 Composition of baseline diets 1 Flaxseed oil Olive oil n Energy, kj/d Fat, % energy Saturated fat, g/d Monounsaturated fat, g/d Polyunsaturated fat, g/d Protein, % energy Carbohydrate, % energy Alcohol, % energy Values are means 6 SD. TABLE 4 Plasma fatty acid concentrations in chronically ill subjects at baseline and after 12 and 26 wk of flaxseed oil or olive oil supplementation 1,2 Between-group P-values Olive oil (n = 22) Flaxseed oil (n = 27) 0 26 wk wk 12 wk wk 0 26 wk wk 0 wk 12 wk wk Fatty acid wk 0 wk 12 wk 26 mmol/l mmol/l ALA EPA DPA DHA OA LA AA Trans fat S (n-3) S (n-6) (n-6):(n-3) Values are means 6 SD. 2 Abbreviations: OA, oleic acid; AA, arachadonic acid.

4 86 HARPER ET AL. of a dietary supplement (flaxseed oil capsules). Earlier trials with ALA yielded mixed results. Some of these trials used doses of ALA that would not be easily obtainable in a reasonable diet (10 40 g/d) (13 17). These studies were also limited by their shorter duration (2 8 wk) and recruitment of predominantly healthy subjects. Our finding of a significant increase in EPA and DPA but not DHA in plasma lipids is similar to earlier reports of ALA supplementation in adults; nevertheless, the results of the FORCE trial were obtained with much lower doses of ALA (18,19). The results suggest that a significant increase in plasma EPA levels can be obtained with a relatively low daily dose of ALA (3 g/d). Individuals consuming a reasonable diet can obtain 2 3 g of ALA without having to resort to dietary supplements (20). This can be achieved by consuming ALArich nuts, cereals, oils, and fortified breads. As expected, the olive oil group did not have an increase in EPA or DPA levels; however, the DHA levels decreased somewhat from wk 0 to 26. When we analyzed between-group differences in plasma DHA changes, there was no difference at wk 12 (P ¼ 0.24) or wk 26 (P ¼ 0.06). We did not find any earlier studies describing a decrease in DHA levels with olive oil. This change may have been due to dietary changes in the olive oil control group not detected by the 3-d food record ratings. The conversion of ALA to its longer-chain metabolite, EPA, is a complicated process that is thought to be modified by several factors. Early studies demonstrated that high dietary concentrations of (n-6) PUFA, particularly LA, cause a decrease in the conversion of ALA to long-chain (n-3) PUFA (21). Data from more recent trials suggest that consumption of long-chain (n-3) PUFA may reduce the conversion of ALA to EPA and DHA due to downregulation of desaturase enzymes (22). Evidence also exists from studies suggesting that populations with omnivorous or carnivorous diets have reduced ability to convert ALA to long-chain (n-3) PUFA compared with vegan populations (23). Meats, eggs, and cheeses contain small amounts of long-chain (n-3) PUFA, which are thought to downregulate desaturase enzymes, whereas a vegan diet, which does not contain any long-chain (n-3) PUFA, is thought to upregulate desaturase enzymes. Our study population consumed relatively high amounts of saturated fat and polyunsaturated fat, both factors that are thought to inhibit the conversion of ALA to EPA and DHA; nevertheless, our study demonstrated a significant 60% increase in EPA levels (P ¼ ). Whether the race or health status of our study participants played a role in this conversion is uncertain. Before broad-based dietary recommendations and governmental guidelines concerning ALA consumption are established in a population as heterogeneous as that in the United States, studies on ALA metabolism and ultimately clinical studies should be conducted in patients with a variety of ethnic and medical backgrounds. A unique feature of the FORCE Trial was the inclusion of a relatively understudied population. Unlike earlier trials, our population comprised predominantly overweight African-American women with chronic medical problems. Although the results from the FORCE trial are noteworthy, key limitations exist. First, the participants were largely noncompliant with the AHA diet and continued to consume a diet high in (n-6) polyunsaturated fats and saturated fats. This type of diet may have reduced the conversion of ALA to EPA. Second, noncompliers were not included in the final analysis; thus this was not an intention-to-treat analysis. Third, the 3- d food record rating used to measure dietary variables is not a precise tool, and other significant changes in dietary fatty acid consumption may have occurred. Fourth, it appears that there was underreporting of energy intake because the amount reported is well below what it would take to maintain the weight of the subjects. In addition, the study used an ALA supplement instead of whole foods, and the palatability and use of ALA-enriched foods has to be evaluated in this population. In a more recent trial, the Mediterranean Alpha-linolenic Enriched Groningen Dietary Intervention (MARGARIN) study, investigators analyzed the effect of increased intakes of ALA in the form of an ALA-rich margarine (24). In the MARGARIN study, participants receiving the enriched margarine, consumed an average of 6.3 g ALA/d resulting in a 37% increase in EPA serum fatty acid levels (P, 0.01). The Lyon Diet Heart Study is the only clinical trial completed with ALA supplementation designed to analyze hard cardiac endpoints in which the investigators reported plasma fatty acid levels (25). In the Lyon Study, ALA supplementation in the form of canola-enriched margarine was given to patients recently recovering from a myocardial infarction (MI); the ALA group experienced a 65% relative risk reduction in cardiac death and nonfatal MI (P ¼ 0.001). Coinciding with this large reduction in cardiac events, participants in the ALA arm of the Lyon study demonstrated a 35% increase in EPA levels (expressed as a percentage of total fatty acids). The results from the Lyon Diet Heart Study suggest that the increases in plasma EPA levels demonstrated in our trial may be clinically relevant. The Lyon Diet Heart Study is limited, however, by the fact that multiple changes in the diet were made in the experimental group including increased intake of nuts, fruits, and fish as well as favorable changes in saturated, monounsaturated, and (n-6) fatty acids. Several key questions remain unanswered concerning ALA supplementation: Will dietary supplementation with ALA result in cardiovascular benefit similar to that seen with EPA and DHA? If increasing consumption of ALA results in reduced cardiac risk, is it because of its conversion to EPA or another mechanism unrelated to EPA (i.e., improvements in endothelial function, inflammation, lipid changes, or antiarrhythmic effect)? The answers to these questions may have important clinical and public health implications because many people do not consume fish or do not have access to fish rich in EPA. The FORCE trial demonstrates the feasibility of increasing EPA levels by supplementing the diet with ALA in a high risk population and underscores the need for a larger more definitive clinical trial with coronary endpoints to determine whether ALA is indeed cardioprotective. ACKNOWLEDGMENTS We thank Mrs. Betty Webb for her help in preparing this manuscript and Susan Logan for statistical support. LITERATURE CITED 1. Burr ML, Fehily AM, Gilbert JF, Rogers S, Holliday RM, Sweetnam PM, Elwood PC, Deadman NM. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet. 1989;2: Singh RB, Niaz MA, Sharma JP, Kumar R, Rastogi V, Moshiri M. Randomized, double-blind, placebo-controlled trial of fish oil and mustard oil in patients with suspected acute myocardial infarction: the Indian experiment of infarct survival-4. Cardiovasc Drugs Ther. 1997;11: De Lorgeril M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. 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