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American Journal of Clinical Nutrition, Vol. 88, No. 3, 801-809, September 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Flaxseed oil and fish-oil capsule consumption alters human red blood cell n–3 fatty acid composition: a multiple-dosing trial comparing 2 sources of n–3 fatty acid1,2,3

Gwendolyn Barceló-Coblijn, Eric J Murphy, Rgia Othman, Mohammed H Moghadasian, Tarek Kashour and James K Friel

1 From the Departments of Pharmacology, Physiology, and Therapeutics (GB-C and EM) and Chemistry (EJM), University of North Dakota, Grand Forks, ND, and the Departments of Human Nutritional Sciences (MHM, RO, and JKF) and of Medicine and Biochemistry and Medical Genetics (TK), University of Manitoba, Winnipeg, Canada

2 Supported by Center of Biomedical Research Excellence grant no. 1P20 RR117699-01 from the National Institutes of Health (to EJM), a grant from the Canadian Flax Council and Flax Canada (to EJM, JKF, and MHM), and an operating grant from the Canadian Institutes for Health Research (to JKF).

3 Reprints not available. Address correspondence to JK Friel, Room 511 Duff Roblin Building, Department of Human Nutritional Sciences, 190 Dysart Road, University of Manitoba, Winnipeg, MB, Canada R3T 2N2. E-mail: frielj{at}ms.umanitoba.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: An increase in plasma n–3 fatty acid content, particularly eicosapentaenoic acid (20:5n–3; EPA) and docosahexaenoic acid (22:6n–3; DHA), is observed after consumption of fish oil–enriched supplements. Because {alpha}-linolenic acid (18:3n–3; ALA) is the direct precursor of EPA and DHA, ALA-enriched supplements such as flax may have a similar effect, although this hypothesis has been challenged because of reported low conversion of ALA into DHA.

Objective: To address this question, we designed a clinical trial in which flax oil, fish-oil, and sunflower oil (placebo group) capsules were given to firefighters (n = 62), a group traditionally exposed to cardiovascular disease risk factors.

Design: Firefighters were randomly divided into 6 experimental groups receiving 1.2, 2.4, or 3.6 g flax oil/d; 0.6 or 1.2 g fish oil/d; or 1 g sunflower oil/d for 12 wk. Blood was drawn every 2 wk, and the total phospholipid fatty acid composition of red blood cells was determined.

Results: As expected, fish oil produced a rapid increase in erythrocyte DHA and total n–3 fatty acids. The consumption of either 2.4 or 3.6 g flax oil/d (in capsules) was sufficient to significantly increase erythrocyte total phospholipid ALA, EPA, and docosapentaenoic acid (22:5n–3) fatty acid content. There were no differences among groups in plasma inflammatory markers or lipid profile.

Conclusions: The consumption of ALA-enriched supplements for 12 wk was sufficient to elevate erythrocyte EPA and docosapentaeoic acid content, which shows the effectiveness of ALA conversion and accretion into erythrocytes. The amounts of ALA required to obtain these effects are amounts that are easily achieved in the general population by dietary modification.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The beneficial effects of eicosapentaenoic acid (EPA; 20:5n–3) and docosahexaenoic acid (DHA; 22:6n–3) on human health, particularly on cardiovascular disease (CVD), are widely accepted (1-8). Currently, the international recommendations for the daily consumption of long-chain n–3 polyunsaturated fatty acids (PUFAs) vary depending on the organization, but they are in a range of 200 mg/d to 1 g/d (9). However, there are several reasons that it may be difficult to fulfill these daily requirements over a prolonged period. There is a limited number of natural EPA- and DHA-enriched sources, which are basically limited to oily fish and a few other types of seafood (9). In addition, these sources are rapidly diminishing at the same time that there are increasing concerns about the toxins that fish may contain—eg, methyl mercury (10). Although it is becoming popular to consume fish oil–enriched capsules, this practice has some difficulties because the bioavailability of fish oil from those capsules is different from that from food-derived n–3 long-chain PUFAs (11). In addition, low compliance over long periods, issues with palatability, and other deleterious side effects are a concern. Therefore, it is necessary to find alternative sources of n–3 fatty acid (FA) that are readily available in large quantities and are renewable.

A good alternative would be plant-derived sources that contain high amounts of {alpha}-linolenic acid (ALA; 18:3n–3), the essential FA precursor of the n–3 FA family. Sources of ALA are widely available, and they range from vegetable oils derived from flaxseed, canola, and soybeans, to nuts such as walnuts and almonds, and to leafy vegetables. Another way of getting n–3 PUFAs into dietary sources is to enrich foods with these FAs. For instance, by supplementing feed rations with ALA-containing oil seeds, beef can be enriched in ALA, EPA, and docosapentaenoic acid (DPA; 22:5n–3) without adverse effects on organoleptic properties (12-15).

Although there are some concerns regarding the efficiency of the ALA conversion (16, 17), we have shown that feeding ALA-enriched supplements elevates tissue EPA, DPA, and DHA concentrations in rats in a tissue-dependent manner (18) and that ALA-supplemented rations increase EPA and DPA content in cattle (13, 19). In addition, others have shown that fetal baboons convert ALA into DHA (20), although that same conversion in humans seems to be less than that in baboons (16, 17). It is important to note that plasma was used in the human studies, which may not reflect potential elongation seen in tissue compartments such as brain (18). Human clinical studies show that an increase in dietary ALA leads to significant increases in ALA, EPA, and DPA in the blood (21-25), and yet many of these studies are carried out using very large daily intakes, often >5 g ALA.

The beneficial effects of ALA-enriched diets or supplements on preventing CVD are less well documented than those of EPA or DHA. Whereas some clinical trials indicate that a greater ALA consumption is associated with a lower risk of myocardial infarction (MI) and fatal ischemic heart disease (IHD) in women and in men (6, 8, 26), other studies show no benefit on adverse cardiovascular events (27). It is interesting that it is still not clear whether all n–3 FAs may have beneficial effects in CVD or whether the benefit is exclusively related to EPA and DHA.

To begin to address this question, we designed a trial to examine the optimal dosing of ALA required to elicit a maximum response in the accretion of ALA and its elongation and desaturation products in erythrocyte (red blood cell; RBC) phospholipids. In addition, we compared the accretion of n–3 FAs in the RBCs during ALA feeding with that obtained by using EPA or DHA containing fish oil.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this trial was to ascertain, by using RBC phospholipids, the optimal daily dose of ALA required for optimal elongation and desaturation. This information is required for an anticipated trial with many more subjects per group that will examine n–3 FAs on risk factors associated with CVD.

Subjects
The Firefighter-paramedic, Flax and Fish study cohort consists of noninstitutionalized men and women recruited in the greater Winnipeg area. Firefighters were approached because, as a profession, they exhibit traditional risk factors for coronary heart disease (CHD), including elevated stress levels (often in response to acute events), high dietary fat intake combined with limited exercise while in the fire hall, and age >40 y (28). Firefighters were excluded if they were consuming supplements of any kind, if they were consuming lipid-lowering medication, or if they had been diagnosed with diabetes. The participants in this study (n = 62) were randomly assigned by using random-number tables to 1 of 6 treatment groups with 9–12 participants per group. These groups received either 1.2 g (2 capsules), 2.4 g (4 capsules), or 3.6 g (6 capsules) flax oil/d; 0.6 (1 capsule) or 1.2 (2 capsules) fish oil g/d; or placebo (2 capsules) containing 1 g sunflower oil/d. The supplement phase lasted 12 wk. The different doses were selected according to recommendations of the International Society for the Study of Fatty acids and Lipids for ALA and EPA or DHA intakes and were based on 0.6 g/d for EPA or DHA and 1.2 g/d for ALA (Internet: http://www.issfal.org.uk/Welcome/PolicyStatement3.asp).

Participants were assessed in fire halls where demographic data, anthropometric data, blood pressure, 3-d dietary intakes, and a medical history were collected by trained personnel, a group that included 2 physicians. The subjects' weight, height, and body mass index (BMI; in kg/m2) were measured. Dietary fat intakes were calculated by using FOOD FOCUS software (version 3.3; Food Focus, Winnipeg, Canada) that was based on the Canadian Nutrient File (Internet: http://www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/cnf_aboutus-aproposdenous_fcen-eng.php). Systolic pressures and pulse were also measured at the beginning and end of the trial. Blood was collected before the first dose and every 2 wk thereafter for 12 wk. Because subjects were working different shifts and because firefighters are naturally subjected to interruptions of normal eating schedules, measurements were obtained under fasting conditions only at the 12-wk time-point.

All clinics were held in various fire halls throughout the greater Winnipeg area from September through December 2005. A team of ≥1 physician, 1 nurse, and a research assistant attended every clinic. Each clinic usually lasted 8 h. Blood samples were collected at a central laboratory, separated into plasma and RBCs, aliquoted, and picked up by study staff for delivery to the University of Manitoba, where they were stored at –80 °C until use. Samples were shipped on dry ice to the University of North Dakota for analysis of RBC phospholipid FA composition.

All patients gave written informed consent before participating in the trial. The study protocol was approved by the institutional review boards at the University of North Dakota and the University of Manitoba.

Capsules
Flax oil and sunflower oil capsules were obtained from Bioriginal Food & Science Corp (Saskatoon, Canada). Fish-oil capsules were obtained from Ocean Nutrition Canada (Dartmouth, Canada). The FA content of the capsules is shown in Table 1Go. The capsules were well tolerated by almost all participants; one subject in the 6 capsules/d fish-oil group developed abdominal discomfort and was dropped from the study.


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  		TABLE 1 Fatty acid composition of the capsules used1

 
Blood collection
Blood was collected in standard evacuated tubes using EDTA as an anticoagulant. Whole blood was centrifuged for 15 min at 1400 x g, and plasma was aliquoted into microcentrifuge tubes. The buffy layer of white blood cells was removed by using a pasture pipette, and then the RBCs were aliquoted into separate microcentrifuge tubes. Blood was also collected into one tube without EDTA to provide serum. All of the tubes were stored at –80 °C until they were analyzed.

Red blood cell fatty acid analysis
Packed RBCs ({approx}500 µL)were diluted in 500 µL water and thoroughly mixed by vortex, and the mixture was kept on ice for 15 min. To initiate the lipid extraction, isopropanol (4 mL) was added, and the samples were vigorously mixed by vortex and kept for 1 h with frequent mixing. Then hexane (6 mL) was added, and the mixture was mixed frequently for another hour (29, 30). The samples were subjected to centrifugation at 1200 x g for 10 min, and the lipid-containing organic fraction was removed. A portion of this extract was then subjected to base-catalyzed transesterification, which converted the phospholipid acyl chains to FA methyl esters (FAME). To each fraction, 2 mL of 0.5 mol KOH/L dissolved in anhydrous methanol was added (31). FAME were extracted from the methanol by using 3 mL of n-hexane, and the n-hexane phase containing the FAME was removed. The lower phase was extracted 2 more times with 3 mL n-hexane, and these washes were combined with the original aliquot.

Individual FAs were separated by gas-liquid chromatography (GLC) by using an SP-2330 column (0.32-mm ID x 30-m length; Supelco, Bellefonte, PA) and a Trace GLC equipped with dual autosamplers and dual flame ionization detectors (FIDs) (all: ThermoElectron, Austin, TX). FAs were quantified by using a standard curve from commercially purchased standards (NuChek Prep, Elysian, MN), and 17:0 was used as the internal standard.

Inflammatory markers
Plasma concentrations of inflammatory markers related to CVD including C-reactive protein (CRP), tumor necrosis factor-{alpha} (TNF-{alpha}), and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured by using commercially available kits. CRP concentrations were measured by using enzyme-linked immunosorbent assay (ELISA) kits (Alpco Diagnostics, Windham, NH), and TNF-{alpha} and sVCAM-1 concentrations were measured by using immunoassay kits (R&D Systems Inc, Minneapolis, MN).

Plasma lipid profile
Plasma samples at weeks 0, 6, 10, and 12 were used for measurement of the concentrations of total cholesterol (TC), triacylglycerol (TAG), HDL cholesterol, and calculated non-HDL-cholesterol. Briefly, plasma TC and TAG concentrations were measured by using standard enzymatic assays (Diagnostic Chemicals, Montreal, Canada) as described previously (19, 32, 33). HDL-cholesterol concentrations were measured by using the same enzymatic assay according to standard precipitation techniques (19, 32, 33). Serum control (Wako Chemicals GmbH, Neuss, Germany) was used as the quality control in all of the abovementioned assays. Because the application of Friedewald's formula was not appropriate for estimating LDL cholesterol in some cases, we calculated non-HDL-cholesterol concentrations by subtracting HDL-cholesterol concentrations from TC values (34, 35). The ratio of concentrations of total to HDL cholesterol (total:HDL) was calculated mathematically for each subject and reported as means ± SDs for each treatment group (35).

Statistical analysis
Group results are expressed as means ± SDs. All analyses were performed according to the intention-to-treat principle, and data for each patient were analyzed according to the original randomized treatment assignment. Repeated-measures analysis of variance (ANOVA) was used to compare treatment effects over time on each FA. When the treatment x time interactions were statistically significant, Tukey's pairwise contrasts were used to analyze group means. All repeated-measures ANOVAs and Tukey's contrast were performed with the use of the PROC MIXED procedure in SAS/STAT software (version 9.1; SAS Institute Inc, Cary, NC). Cross-sectional analyses were performed to test between-treatment effects at each time-point. Lipid profile values and inflammatory markers obtained at week 12 were analyzed with the use of SAS/STAT software (version 9.1), and statistical significance was assessed by using the 2-tailed, unpaired Student's t test. For all tests, P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anthropometric measurements, blood pressure, and fat intake
There were no significant differences between groups in any of the anthropometric measures or in blood pressure at the beginning or end of the trial (Table 2Go). Subjects received either 2, 4, or 6 capsules of fish, flax, or sunflower oil supplements to achieve the intended quantity of FAs. The difference in fat intake between 2 and 6 capsules was 4 g/d in 10 subjects; otherwise, the difference was 2 g/d. This difference was insignificant compared with the average daily fat intake of all subjects of 98 ± 42 g/d.


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  		TABLE 2 Characteristics of treatment groups at the baseline1

 
Red blood cell phospholipid fatty acid composition
The first aims of this trial were to establish the dose of ALA (1.2, 2.4, or 3.6 g flax oil/d) that would provide the largest increase in RBC phospholipid n–3 FAs and to compare the dose with changes in groups receiving fish-oil capsules (0.6 or 1.2 g fish oil/d). The second aim was to determine the time course over which these changes occurred. We analyzed the total phospholipid FA composition of RBCs because it is a good indicator of long-term dietary changes and it has been widely used in human clinical trials (36-39).

Supplement-induced changes in red blood cell phospholipid n–3 content
At the beginning of the study, the total phospholipid FA composition of RBCs did not differ significantly between groups (Table 3Go and Table 4Go). Although the complete FA profile was obtained for each participant, for simplicity's sake, only alterations in n–3 and n–6 FAs are presented herein, because no significant changes were observed between groups in total saturated FAs, total monounsaturated FAs, or total PUFAs (data not shown).


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  		TABLE 3 Changes in n–3 fatty acid content in erythrocyte membrane1

 

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  		TABLE 4 Changes in n–6 fatty acid content in erythrocyte membrane1

 
The ALA concentrations in the group receiving 3.6 g flax oil/d increased significantly compared with the control group at weeks 2, 4, 6, and 8. The ALA concentrations in the group receiving 2.4 g flax oil/d also increased significantly compared with the control group at weeks 8 and 10. In general, within the flax oil groups, ALA content increased significantly in groups consuming 2.4 and 3.6 g flax oil/d in capsules, whereas ALA content was not altered in the fish-oil groups (Table 3Go). In the group receiving 2.4 g flax oil/d, the increase in ALA became significant at week 4, when ALA content increased 1.7-fold from baseline. ALA content increased to 0.8% of the total FA content at week 10 and decreased to 0.5% of the total FA content at week 12. This decrease may be an indicator of compliance in the ALA groups. In the group receiving 3.6 g flax oil/d, the ALA concentration was significantly (2-fold) greater at week 2. This value remained constant throughout the study, although, at the end of the trial, there was a slight tendency for ALA content to decrease, which may represent conversion of ALA to EPA and then to DPA.

In contrast, EPA content increased significantly in groups consuming 2.4 and 3.6 g flax oil/d and in both fish-oil groups. In the group receiving 2.4 g flax oil/d, EPA increased 1.4-fold at week 2, a concentration that remained constant until week 12. In the group receiving 3.6 g flax oil/d, a significant (1.3-fold) increase in EPA occurred at week 6. In contrast, EPA significantly (1.6-fold) increased at week 2 in the groups receiving 0.6 and 1.2 g fish oil/d. In the group receiving 0.6 g fish oil/d, EPA content did not increase further, whereas, in the group receiving 1.2 g fish oil/d, the concentration of EPA steadily increased in a time-dependent manner, to be 2.1-fold at week 12. The EPA concentration was higher than the highest concentration achieved in the flax oil groups by 6 wk in the group receiving 1.2 g fish oil/d and by 10 wk in the group receiving 0.6 g fish oil/d. At 10 wk, EPA content was significantly higher in the group receiving 1.2 g fish oil/d than in the group receiving 0.6 g fish oil/d. Compared with the EPA content in the control group, that in the group receiving 1.2 g fish oil/d increased significantly at weeks 4, 6, 10, and 12, whereas that in the group receiving 0.6 g fish oil/d increased significantly at weeks 6, 10, and 12.

DPA content increased significantly in groups consuming 2.4 g flax oil/d and in those consuming fish-oil capsules. In the group receiving 2.4 g flax oil/d, the increase in DPA was significant at week 12, whereas in the group receiving 3.6 g flax oil/d, there was no significant increase in DPA concentrations. In the group receiving 0.6g fish oil/d, the increase in DPA content was significant at 6 wk, whereas, in the group receiving 1.2 g fish oil/d, it was already significant at week 4. Contrary to the changes observed in EPA content, DPA content was similar in both fish-oil groups, and that in both groups differed significantly from that in the control group at week 10.

DHA content increased only in groups consuming fish-oil capsules. Changes in DHA content were very similar in both fish-oil groups, and there were no statistical differences between groups at any time-point in the trial. DHA content was significantly higher in fish-oil groups at week 4 than at baseline. At the end of the study, the DHA concentration in the group consuming 0.6 g fish oil/d was 1.2-fold that at baseline, whereas that in the group receiving 1.2 g fish oil/d was 1.4-fold that at baseline. The lack of elevated DHA in the flax oil groups strongly indicates that the conversion of ALA to DHA and subsequent incorporation into RBC phospholipids was very low over the 12-wk study.

Increases in total n–3 content were significantly different between the groups consuming 2.4 g flax oil/d and the fish-oil groups with respect to time (Figure 1Go). In the group receiving 2.4 g flax oil/d, the increase was statistically significant at week 6 of treatment, whereas, in both fish-oil groups, it was statistically significant at week 2. With respect to treatment groups, total n–3 values at weeks 6–12 in the group receiving 1.2 g fish oil/d were significantly different from those in the group receiving 1.2 g flax oil/d and the control group. At week 10, total n–3 FAs in the group receiving 0.6 g fish oil/d were significantly greater than those in the group receiving 1.2 g flax oil/d and the control group, but this significance was not observed at week 12. At week 12, total n–3 values in the group receiving 1.2 g fish oil/d were significantly greater than those in all of the other groups.


Figure 1
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  		FIGURE 1. Mean changes in the total n–3 fatty acid composition of red blood cells (RBCs) was determined over the 12-wk treatment period for each group. n = 9–12/group. Repeated-measures ANOVA was used to compare treatment effects over time on each fatty acid. When the treatment x time interactions were statistically significant, Tukey's pairwise contrasts were used to analyze group means. The means at baseline did not differ significantly between any groups. In the group receiving 0.6 g fish oil/d, values at weeks 2–12 were significantly different from baseline. In the group receiving 1.2 g fish oil/d, values at weeks 2–12 were significantly different from baseline, values at 4–12 wk were significantly different from values at 2 wk, and values at 12 wk were significantly different from values at 4 wk. In the group receiving 1.2 g flax oil/d, there was no significant difference between any weeks. In the group receiving 2.4 g flax oil/d, values at 6–12 wk were significantly different from baseline. In the group receiving 3.6 g flax oil/d, there were no significant differences between any weeks. There were no between-group differences in total n–3 fatty acid content at baseline, week 2, and week 4. At weeks 6 and 8, values in the group receiving 1.2 g fish oil/d differed significantly from those in the group receiving 1.2 g flax oil/d and the control group. At 10 wk, values in the groups receiving 0.6 and 1.2 g fish oil/d differed significantly from those in the group receiving 1.2 g flax oil/d and the control group. At 12 wk, values in the group receiving 1.2 g fish oil/d differed significantly from those in the groups receiving 1.2, 2.4, and 3.6 g flax oil/d and the control group. All concentrations of n–3 with flax were similar to the control treatment.

 
Supplement-induced changes in red blood cell phospholipid n–6 content
Arachidonic acid (ARA) and adrenic acid (22:4n–6) concentrations decreased significantly (≤15%) at weeks 10 and 12 in the group receiving 1.2 g fish oil/d with respect to time (0 wk), but there were no significant differences between treatment groups. Total n–6 FAs decreased in the groups receiving 0.6 and 1.2 g fish oil/d at weeks 10 and 12 compared with week 0 (both groups) and week 2 (1.2 g fish oil/d group). As with individual n–6 FAs, there were no significant differences between treatment groups. The ratio of n–3 to n–6 (n–3:n–6) increased significantly in the 2 fish-oil groups with increasing time. With respect to treatment groups, n–3:n–6 at week 8 differed significantly between the group receiving 1.2 g fish oil/d and the group receiving 1.2 g flax oil/d. By week 10, n–3:n–6 in the group receiving 0.6 g fish oil/d was significantly greater than that in the group receiving 1.2 g flax oil/d, and n–3:n–6 in the group receiving 1.2 g fish oil/d was significantly greater than that in the group receiving 1.2 g flax oil/d and the control group. At week 12, n–3:n–6 in the group receiving 1.2 g fish oil/d was significantly greater than the ratio in all other groups except the other fish-oil group.

Plasma inflammatory marker content
Because diets enriched in n–3 FAs from either vegetable or animal sources decrease the content of plasma inflammatory markers (21, 40), we analyzed the effects of flax oil and fish-oil supplements on CRP, sVCAM-1, and TNF-{alpha} plasma content (data not shown). Our results showed that the doses given in the present study were not sufficient to significantly alter CRP, sVCAM-1, or TNF-{alpha} content in plasma and that the concentrations in these groups were not high enough to clinically indicate a proinflammatory state.

Plasma lipid profile
Because clinical trials showed that diets enriched with n–3, particularly fish oil, reduce plasma triacylglycerol content (41, 42), we determined the effects of flax oil and fish-oil capsule consumption on TC, TAG, and HDL-cholesterol content. We did not observe any significant differences between groups in the plasma content of TC, TAG, or HDL cholesterol (data not shown).

Summary
As we expected, the consumption of fish-oil capsules significantly increased EPA, DPA, and DHA concentrations. The lowest dose of flax oil (1.2 g/d) did not alter the total phospholipid FA composition of RBCs, whereas the groups consuming higher doses of flax oil (2.4 and 3.6 g/d) had significantly greater total n–3 concentrations, primarily as a result of elevated ALA, EPA, and DPA concentrations (2.4 g flax oil/d group only). These changes occurred at week 2, and they tended to plateau by week 8. In addition, none of the supplements enriched with n–3 significantly altered plasma inflammatory biomarker content or the plasma lipid profile.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An increasing number of clinical studies indicate that the consumption of ALA has beneficial effects on human health, particularly on CVD. ALA consumption is protective against cardiac death and nonfatal MI in prospective (7, 8) and intervention (3, 4) trials. In addition, in a cross-sectional study, women and men consuming higher amounts of ALA had a lower incidence of coronary artery disease (CAD) (6). In contrast, another study showed no correlation between increased ALA intake and a reduction in CVD (27). The mechanism or mechanisms of action by which n–3 FAs appear to have a benefit for heart health are unknown. Currently, there are ≥4 possible mechanisms that may account for the beneficial effects of n–3 FAs. The first possibility is that the n–3 FA accumulation accounts for the benefits, because these FAs have antiarrhythmic properties (43, 44), influence gene expression (45), and alter membrane biophysical properties (46, 47). The second possibility is that n–3 FAs may alter the plasma lipid profile by increasing HDL-cholesterol content and decreasing plasma TAG and LDL-cholesterol content (48). The third possibility is that an increase in the blood n–3 FA content is associated with decreases in inflammatory markers, such as TNF-{alpha}, interleukin-β6, and sVCAM-1 (21, 40, 49), which leads to a reduction in cardiovascular events. And the fourth possibility is that an increase in n–3 FAs lowers plasma ARA concentrations, thus decreasing the synthesis of inflammatory prostaglandins, as seen in the present study with reduced ARA in RBC total phospholipids. In the present study, we focused on the effect that different doses of plant- and marine-derived n–3 FAs had on RBC phospholipid FA composition, inflammatory markers, and plasma lipid profile.

The present study was designed to investigate 2 important aspects of ALA metabolism in humans. Our first aims were to establish the optimal dose of ALA needed to obtain the largest increase in RBC n–3 FA content and to compare those changes with the alterations observed in the fish-oil groups. These doses were 2.4 g flax oil/d and 1.2 g fish oil/d, which could be used in a larger trial with a longer follow-up that would be focused on determining the potential of these FAs to reduce cardiovascular morbidity and mortality. The second aim was to establish the time course by which these alterations take place. In general, the optimal doses had a maximal effect after 6–8 wk of continuous intake of the FAs.

We are aware of no other study examining the direct changes of flax oil and fish-oil supplements given at modest doses on RBC phospholipids in a dose-dependent manner and in an occupational group at risk of heart disease. Our results clearly indicate that an increase in the consumption of flax oil, which was highly enriched in ALA, led to an increase in n–3 FA content in the groups receiving 2.4 and 3.6 g flax oil/d and that the consumption of fish oil in the groups receiving 0.6 and 1.2 g fish oil/d led to an increase in n–3 FA content compared with the placebo group. As we expected, the fish-oil groups had significant increases in EPA and DHA content, whereas the flax oil groups had significant increases in ALA, EPA, and DPA content (in the 2.4 g flax oil/d group only). Conversion of ALA into longer-chain FAs was previously reported in several clinical trials (1-6, 8, 26, 50); however, it is important to note that the doses used in the present trial were significantly less than those used in these other trials. In addition, we show that a dose of 2.4 g ALA/d is sufficient to obtain alterations in RBC phospholipid n–3 FAs and thus presumably similar or greater changes in various tissues, depending on the needs of those tissues for EPA or DHA (18). It is important to note that this amount is quite obtainable by regular consumption of oil seeds, nuts, and vegetables containing n–3 FAs.

Increased concentrations of n–3 FAs are associated with reductions in the concentrations of circulating inflammatory markers in the plasma lipid profile, which may account for the beneficial effects of diets enriched with n–3 FAs. Other human clinical studies showed a negative correlation between n–3 FAs and inflammatory TNF-{alpha}, sVCAM-1, or CRP concentrations in plasma (21, 40, 49). The consumption of supplements enriched with n–3 is also related to a decrease in plasma TAG concentrations and an increase in HDL-cholesterol concentrations (41). However, it is important to note that not all of these changes are shown in other clinical studies (3, 25, 51). We did not observe a significant reduction in the present trial with either ALA or EPA and DHA; however, the values in our subjects were within the healthy range, and hence it is difficult to ascertain a positive effect of n–3 FAs on these concentrations. In addition, the aim of this pilot study was to establish the optimal dose of flax oil needed to obtain the largest increase in n–3 FA content, and only a small number of subjects per group were needed to achieve this goal. The n–3 FA content in the control group was unusually high, so that relatively few differences were found.

Finally, the possible interactions of the background diet were not considered in the present study on the elongation and desaturation of ALA and the downstream effect on plasma inflammatory markers and lipid profiles. The proportion of saturated, monounsaturated, and polyunsaturated fat content may have an effect not only on the ALA conversion into EPA and DHA, but also on the concentrations of inflammatory markers and on the plasma lipid profile (42, 52).

In summary, our study clearly shows that plant-derived n–3 FAs can increase the phospholipid n–3 FA composition of RBCs in a dose- and time-dependent manner. In groups receiving 2.4 and 3.6 g flax oil/d, 2 wk of treatment was sufficient to observe a significant increase in ALA or EPA n–3 content in RBC phospholipids. On the other hand, as expected, fish-oil groups also had increases in the n–3 FA content of RBC phospholipids, including an increase in DHA. Nevertheless, our results clearly indicate that 2.4 g flax oil/d is sufficient to significantly increase RBC phospholipid n–3 FAs, which suggests that a similar effect may be occurring in other tissues. The similarity between the greater concentrations of EPA and DPA in the group receiving 2.4 g flax oil/d than in either fish-oil group suggests that dietary ingestion of plant-derived n–3 FAs is sufficient to meet the dietary needs of humans.


   ACKNOWLEDGMENTS
 
We thank LuAnn Johnson for conducting the statistical analysis, Carole Haselton for technical assistance in making these measurements, Soheila Najafi and Lisa Maximus for clinical assessments, and Cindy Murphy for the preparing the final version of the manucript.

The authors' responsibilities were as follows—EJM, MHM, and JKF: designed the project and sought financial support; TK and MHM: assisted in the carrying out of the protocol and the submission for ethical approval; GB-C: conducted most of the laboratory analysis and wrote the draft manuscript; RO: helped with lipid analysis; and all authors: contributed to revisions of the manuscript, contributed intellectually to the manuscript, and reviewed the final version. At the time of the study, none of the authors had a financial interest in the companies that sponsored or provided supplies for this research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Connor WE. Importance of n–3 fatty acids in health and disease. Am J Clin Nutr 2000;71(suppl):171S-5S.[Abstract/Free Full Text]
  2. Albert CM, Oh K, Whang W, et al. Dietary alpha-linoleic acid intake and risk of sudden cardiac death and coronary heart disease. Circulation2005;112:3232–8.[Medline]
  3. de Lorgeril M, Renaud S, Mamelle N, et al. Mediterranean alpha-linoleic acid-rich diet in secondary prevention of coronary heart disease. Lancet 1994;343:1454–9. (Published erratum appears Lancet 1995;345:738.)[Medline]
  4. de Lorgeril M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation1999;99:779–85.[Medline]
  5. Lemaitre RN, King IB, Mozaffarian D, Kuller LH, Tracy RP, Siscovick DS. n–3 Polyunsaturated fatty acids, fatal ischemic heart disease, and nonfatal myocardial infarction in older adults: the Cardiovascular Health Study. Am J Clin Nutr2003;77:319–25.[Abstract/Free Full Text]
  6. Djoussé L, Pankow JS, Eckfeldt JH, et al. Relation between dietary linolenic acid and coronary artery disease in the National Heart, Lung, and Blood Institute Family Heart Study. Am J Clin Nutr2001;74:612–9.[Abstract/Free Full Text]
  7. Hu FB, Stampfer MJ, Manson JE, et al. Dietary intake of alpha-linoleic acid and risk of fatal ischemic heart disease among women. Am J Clin Nutr1999;69:890–7.[Abstract/Free Full Text]
  8. Ascherio A, Rimm EB, Giovannucci EL, Spiegelman D, Stampfer M, Willett WC. Dietary fat and risk of coronary heart disease in men: cohort follow up study in the United States. BMJ1996;313:84–90.[Abstract/Free Full Text]
  9. Gebauer SK, Psota TL, Harris WS, Kris-Etherton PM. n–3 Fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits. Am J Clin Nutr 2006;83(suppl):1526S-35S.[Medline]
  10. Guallar E, Sanz-Gallardo MI, van't Veer P, et al. Mercury, fish oils, and the risk of myocardial infarction. N Engl J Med2002;347:1747–54.[Abstract/Free Full Text]
  11. Visioli F, Rise P, Barassi MC, Marangoni F, Galli C. Dietary intake of fish vs. formulations leads to higher plasma concentrations of n–3 fatty acids. Lipids2003;38:415–8.[Medline]
  12. Maddock TD, Bauer ML, Koch K, et al. Effect of processing flax in beef feedlot diets on performance, carcass characteristics, and trained sensory panel ratings. J Anim Sci2006;84:1544–51.[Abstract/Free Full Text]
  13. Kronberg S, Barceló-Coblijn G, Golovko MY, Berger J, Weinhofer I, Murphy EJ. Flax finishing enriches meat n–3 fatty acids levels in the cattle. Lipids2006;41:1059–68.[Medline]
  14. Demirel G, Wachira AM, Sinclair LA, Wilkinson RG, Wood JD, Enser M. Effects of dietary n–3 polyunsaturated fatty acids, breed and dietary vitamin E on the fatty acids of lamb muscle, liver and adipose tissue. Br J Nutr2004;91:551–65.[Medline]
  15. Scollan ND, Choi NJ, Kurt E, Fisher AV, Enser M, Wood JD. Manipulating the fatty acid composition of muscle and adipose tissue in beef cattle. Br J Nutr2001;85:115–24.[Medline]
  16. Burdge GC, Jones AE, Wooton SA. Eicosapentaenoic and docosapentaenoic acids are the principal products of {alpha}-linolenic acid metabolism in young men. Br J Nutr2002;88:355–63.[Medline]
  17. Pawlosky RJ, Hibbeln JR, Novotny JA, Salem N Jr. Physiological compartmental analysis of {alpha}-linolenic acid metabolism in adult humans. J Lipid Res2001;42:1257–65.[Abstract/Free Full Text]
  18. Barceló-Coblijn G, Collison LW, Jolly CA, Murphy EJ. Dietary {alpha}-linolenic acid increases brain but not heart and liver docosahexaenoic acid levels. Lipids2005;40:787–98.[Medline]
  19. Moghadsian MH, McManus BM, Nguyen LB, et al. Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans. FASEB J2001;15:2623–30.[Abstract/Free Full Text]
  20. Su HM, Huang MC, Saad NM, Nathanielsz PW, Brenna JT. Fetal baboons convert 18:3n–3 to 22:6n–3 in vivo. A stable isotope tracer study. J Lipid Res2001;42:581–6.[Abstract/Free Full Text]
  21. Zhao G, Etherton TD, Martin KR, West SG, Gillies PJ, Kris-Etherton PM. Dietary {alpha}-linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hypercholesterolemic men and women. J Nutr2004;134:2991–7.[Abstract/Free Full Text]
  22. Makrides M, Neumann MA, Jeffrey B, Lien EL, Gibson RA. A randomized trial of different ratios of linoleic to alpha-linoleic acid in the diet of term infants: effects on visual function and growth. Am J Clin Nutr2000;71:120–9.[Abstract/Free Full Text]
  23. Mantzioris E, James MJ, Gibson RA, Cleland LG. Dietary substitution with an {alpha}-linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentrations in tissues. Am J Clin Nutr1994;59:1304–9.[Abstract/Free Full Text]
  24. Harper CR, Edwards MJ, DeFilipis AP, Jacobson TA. Flaxseed oil increases the plasma concentrations of cardioprotective (n–3) fatty acids in humans. J Nutr2006;136:83–7.[Abstract/Free Full Text]
  25. Wallace FA, Miles EA, Calder PC. Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. Br J Nutr2003;89:679–89.[Medline]
  26. Hu FB, Bronner L, Willett WC, et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA2002;287:1815–21.[Abstract/Free Full Text]
  27. Oomen CM, Ocke MC, Feskens EJ, Kok FJ, Kromhout D. {alpha}-Linolenic acid intake is not beneficially associated with 10-y risk of coronary artery disease incidence: the Zutphen Elderly Study. Am J Clin Nutr2001;74:457–63.[Abstract/Free Full Text]
  28. Horowitz MR, Montgomery DL. Physiological profile of firefighters compared to norms for the Canadian population. Can J Public Health1993;84:50–2.[Medline]
  29. Hara A, Radin NS. Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem1978;90:420–6.[Medline]
  30. Rose HG, Oklander M. Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res1965;6:428–31.[Abstract]
  31. Brockerhoff H. Determination of the positional distribution of fatty acids in glycerolipids. Methods Enzymol1975;35:315–25.[Medline]
  32. Nashed B, Yeganeh B, HayGlass KT, Moghadasian MH. Antiatherogenic effects of dietary plant sterols are associated with inhibition of proinflammatory cytokine production in Apo E-KO mice. J Nutr2005;135:2438–44.[Abstract/Free Full Text]
  33. Yeganeh B, Moshtaghi-Kashanian GR, Declercq V, Moghadasian MH. Combination of dietary phytosterols plus niacin or fenofibrate: effects on lipid profile and atherosclerosis in apo E-KO mice. J Nutr Biochem2005;16:222–8.[Medline]
  34. de Mello VD, Zelmanovitz T, Perassolo MS, Azevedo MJ, Gross JL. Withdrawal of red meat from the usual diet reduces albuminuria and improves serum fatty acid profile in type 2 diabetes patients with macroalbuminuria. Am J Clin Nutr2006;83:1032–8.[Abstract/Free Full Text]
  35. Ballantyne CM, Bertolami M, Hernandez Garcia HR, et al. Achieving LDL cholesterol, non-HDL cholesterol, and apolipoprotein B target levels in high-risk patients: Measuring Effective Reductions in Cholesterol using Rosuvastatin therapY (MERCURY) II. Am Heart J 2006;151:975e. 1-975e.1-9.
  36. Jimenez J, Boza J Jr, Suarez MD, Gil A. Changes in fatty acid profiles of red blood cell membranes mediated by dietary nucleotides in weanling rats. J Pediatr Gastroenterol Nutr1992;14:293–9.[Medline]
  37. Palozza P, Sgarlata E, Luberto C, et al. n–3 Fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration of dietary supplementation. Am J Clin Nutr1996;64:297–304.[Abstract/Free Full Text]
  38. Calviello G, Palozza P, Franceschelli P, Bartoli GM. Low-dose eicosapentaenoic or docosahexaenoic acid administration modifies fatty acid composition and does not affect susceptibility to oxidative stress in rat erythrocytes and tissues. Lipids1997;32:1075–83.[Medline]
  39. Ho M, Maple C, Bancroft A, McLaren M, Belch JJ. The beneficial effects of omega-3 and omega-6 essential fatty acid supplementation on red blood cell rheology. Prostaglandins Leukot Essent Fatty Acids1999;61:13–7.[Medline]
  40. Lopez-Garcia E, Schulze MB, Manson JE, et al. Consumption of (n–3) fatty acids is related to plasma biomarkers of inflammation and endothelial activation in women. J Nutr2004;134:1806–11.[Abstract/Free Full Text]
  41. Mori TA, Burke V, Puddey IB, et al. Purified eicosapentaenoic and docosahexaenoic acids have differential effects on serum lipids and lipoproteins, LDL particle size, glucose, and insulin in mildly hyperlipidemic men. Am J Clin Nutr2000;71:1085–94.[Abstract/Free Full Text]
  42. Layne KS, Goh YK, Jumpsen JA, Ryan EA, Chow P, Clandinin MT. Normal subjects consuming physiological levels of 18:3(n–3) and 20:5(n–3) from flaxseed or fish oils have characteristic differences in plasma lipid and lipoprotein fatty acid levels. J Nutr1996;126:2130–40.[Abstract/Free Full Text]
  43. Pepe S, McLennan PL. Cardiac membrane fatty acid composition modulates myocardial oxygen consumption and postischemic recovery of contractile function. Circulation2002;105:2303–8.[Medline]
  44. Leaf A, Kang JX, Xiao Y-F, Billman GE. Clinical prevention of sudden cardiac death by n–3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n–3 fish oils. Circulation2003;107:2646–52.[Medline]
  45. Jump DB. Dietary polyunsaturated fatty acids and regulation of gene transcription. Curr Opin Lipidol2002;13:155–64.[Medline]
  46. Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta1984;779:89–137.[Medline]
  47. Clamp AG, Ladha S, Clark DC, Grimble RF, Lund EK. The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyte plasma membrane. Lipids1997;32:179–84.[Medline]
  48. Mori TA, Beilin LJ. Long-chain omega 3 fatty acids, blood lipids and cardiovascular risk reduction. Curr Opin Lipidol2001;12:11–7.[Medline]
  49. Caughey GE, Mantzioris E, Gibson RA, Cleland LG, James MJ. The effect on human tumor necrosis factor alpha and interleukin 1 beta production of diets enriched in n–3 fatty acids from vegetable oil or fish oil. Am J Clin Nutr1996;63:116–22.[Abstract/Free Full Text]
  50. Finnegan YE, Minihane AM, Leigh-Firbank EC, et al. Plant- and marine-derived n–3 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 Nutr2003;77:783–95.[Abstract/Free Full Text]
  51. Danesh J, Wheeler JG, Hirschfield GM, et al. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med2004;350:1387–97.[Abstract/Free Full Text]
  52. Paschos GK, Rallidis LS, Liakos GK, et al. Background diet influences the anti-inflammatory effect of alpha-linolenic acid in dyslipidaemic subjects. Br J Nutr2004;92:649–55.[Medline]
Received for publication March 20, 2008. Accepted for publication May 16, 2008.





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