European Journal of Clinical Nutrition (2003) 57, 1605–1612

& 2003 Nature Publishing Group All rights reserved 0954-3007/03 $25.00
www.nature.com/ejcn

ORIGINAL COMMUNICATION
A practical approach to increasing intakes of n-3
polyunsaturated fatty acids: use of novel foods
enriched with n-3 fats
RG Metcalf1*, MJ James1, E Mantzioris1 and LG Cleland1

1
Department of Rheumatology, Royal Adelaide Hospital, Adelaide, SA, Australia

Objectives: To assess the effects of providing a wide range of foodstuffs containing n-3 polyunsaturated fatty acids (PUFA),
occurring naturally or from fortification, on intake and blood and tissue proportions of n-3 PUFA.
Design: Before/after dietary intervention study.
Setting: Adelaide, Australia.
Subjects: 16 healthy males recruited from the community.
Interventions: Subjects were provided with a range of foodstuffs naturally containing n-3 PUFA (fresh fish, canned fish, flaxseed
meal, canola oil) and items fortified with fish oil (margarine spread, milk, sausages, luncheon meat, french onion dip). Food
choices were left to the discretion of each subject. Intake was estimated by diet diary. Blood was collected atF2, 0, 2, and 4
weeks for fatty acid analysis.
Main outcome measures: Dietary intakes; plasma, platelet, and mononuclear cell phospholipid fatty acids.
Results: Consumption of n-3 PUFA increased significantly: a-linolenic acid (ALA) from 1.4 to 4.1 g/day (Po0.001),
eicosapentaenoic acid (EPA) from 0.03 to 0.51 g/day (Po0.001), and docosahexaenoic acid (DHA) from 0.09 to 1.01 g/day
(Po0.001). Linoleic acid (LA) intake decreased from 13.1 to 9.2 g/day (Po0.001). The proportions of EPA and DHA increased
significantly in all phospholipid pools examined; plasma EPA from 1.13% of total fatty acids to 3.38% (Po0.001) and DHA from
3.76 to 7.23% (Po0.001); mononuclear cell EPA from 0.40 to 1.25% (Po0.001) and DHA from 2.33 to 4.08% (Po0.001);
platelet EPA from 0.41 to 1.2% (Po0.001) and DHA from 1.64 to 3.07% (Po0.001).
Conclusion: Incorporating fish oil into a range of novel commercial foods provides the opportunity for wider public
consumption of n-3 PUFA with their associated health benefits.
Sponsorship: Dawes Scholarship, Royal Adelaide Hospital.
European Journal of Clinical Nutrition (2003) 57, 1605–1612. doi:10.1038/sj.ejcn.1601731

Keywords: fish oils; fatty acids; omega-3; food; dietary fats

Introduction significant health benefits in cardiovascular disease (Hu et al,

There is a considerable body of evidence demonstrating that
the regular consumption of n-3 polyunsaturated fatty acids
(PUFA) as fish, fish oil, or vegetable oil, is associated with
*Correspondence: RG Metcalf, Department of Rheumatology, Royal
Adelaide Hospital, North Tce, Adelaide, SA 5000, Australia.
E-mail: robert.metcalf@adelaide.edu.au
Guarantor: MJ James
Contributors: RGM participated in study design, recruitment, labora-
tory analyses, data collection, data analysis, writing and editing of the
report. EM participated in study design and advice on study
management. MJJ and LGC participated in study design, obtaining
funding and editing of the report.
Received 24 October 2002; revised 18 December 2002;
accepted 25 December 2002
2001) and rheumatoid arthritis (Cleland & James, 2000). Fish
oil contains the long-chain n-3 PUFA (LC n-3 PUFA),
eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA). The n-3 containing vegetable oils, primarily flax,
canola, and soy, contain the shorter chain n-3 PUFA,
a-linolenic acid (ALA).
Increased consumption of n-3 fatty acids has been
recommended by health authorities in Canada (Scientific
Review Committee, 1990) and the United Kingdom (The
British Nutrition Foundation, 1992), and daily intakes of
about 200–400 mg of LC n-3 PUFA have been recommended
in Europe and the US (de Deckere et al, 1998; Simopoulos
et al, 2000).
 Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1606
Despite this, Western societies consume little fish, and participants is detailed in Table 1. The fish oil was fortified
increasing consumption to recommended levels would with 1000 p.p.m. dl-a-tocopherol.
involve major alterations in dietary habits. To date, the only Subjects were not asked to consume set quantities of each
alternative strategy has been a regular consumption of food, but rather were instructed to incorporate these foods
encapsulated fish oil, an approach that is unsuitable over a into their normal diet in desired quantities, and advice was
prolonged period for many people. An alternative strategy provided on using the novel foodstuffs. Foods were provided
for enhancing the long-term increase in n-3 PUFA intake in quantities sufficient for the whole family, to avoid
may be to provide a wide range of commercial food products separate meal preparation being required for participants.
and ingredients fortified with fish oil or ALA, which can be To maximize the incorporation of n-3 PUFA, dietary advice
incorporated into an existing diet. In this study, we have was provided on reducing their intake of foods which are
provided a number of novel foods fortified with fish oil, and high in LA, for example, nuts, cooking oils, and commercial
products high in ALA, and allowed the subjects to incorpo- food products high in LA-rich vegetable oils.
rate them into their domestic food preparation in accor- Subjects were instructed to complete weighed food
dance with their own tastes and preferences. The aims were records for 3 days/week (2 days during the week, and 1 day
to examine intakes of individual n-3 PUFA, together with the on the weekend) for 2 weeks prior to the dietary intervention
dietary preferences of the subjects and the proportion of LC period to provide data on baseline fatty acid intakes, and
n-3 PUFA which were achieved in blood and tissue by this for the 4-week dietary intervention period. Diet diaries
approach. were analyzed using the Diet-1 software package (v 4.20,
Xyris Software, Brisbane, Australia), which is based on the
Australian Nutrient Data Table NUTTAB95 (Australian
Government Publishing Service, Canberra, Australia),
Materials and methods
modified to include the fatty acid content of Australian
Subject selection
foods and ingredients, as well as the novel fish-oil fortified
In all, 16 healthy male subjects were recruited through
foods. Participants were provided with digital kitchen scales
advertisements for volunteers to participate in dietary
(2 g increments).
intervention studies. The details of the study were explained
and written, informed consent was obtained from all
participants. The study was approved by the Research Ethics
Analytic methods
Committee of the Royal Adelaide Hospital.
Fasting peripheral blood was sampled at
2, 0, 2, and 4
weeks, separated into plasma, mononuclear cell and platelet
fractions, and fatty acids were extracted and analyzed as
Diets described previously (Mantzioris et al, 2000). Briefly, 20 ml of
Subjects were provided with a range of products designed to peripheral blood was added to 4 ml of 4.5% EDTA in water,
increase their intake of n-3 PUFA, and decrease their intake and 4 ml of 6% dextran in normal saline (pH 7.0), and
of n-6 PUFA. This was achieved by providing a range of incubated in a water bath at 371C for 30 min to allow the
products (a) rich in ALA, (b) rich in EPA and DHA, and (c) erythrocytes to sediment under gravity. The remaining
olive oil-based products high in monounsaturated fatty acids leukocyte and platelet-rich plasma was loaded onto a
to replace linoleic acid (LA) in the background diet. Products Ficoll–Paque gradient (Lymphoprep, Nycomed Pharma,
provided which were high in ALA were canola oil (Meadow
Lea Foods Pty Ltd, Sydney, Australia), and flaxseed-based
products (stabilized flaxseed meal, and Alena Energy Drink, Table 1 Selected fatty acid content of the foods provided
both provided by ENRECO, Manitowoc, WI, USA). Foods
Food 18:1n-9 18:2n-6 18:3n-3 20:5n-3 22:6n-3
which were high in EPA and DHA were fresh fish (mullet and
deep sea bream), canned fish (pink salmon and sardines;
Canola oil 59.3a 19.2 7.9 F F
Safcol Holdings, Adelaide, Australia), and a number of items Margarine spread 50.6 5.2 0.34 0.36 0.52
fortified with fish oil (Ropufa-30; Roche Vitamins, Sydney, Salad dressing 10.0 1.1 0.1 F F
Australia), including sausages, luncheon meat (Conroys Mayonnaise 12.5 11.2 0.6 F F
Milk 0.4 0.05 0.01 0.03 0.04
Smallgoods, Adelaide, Australia), long-life milk (Pauls Ltd,
Flaxseed products 5.3 4.3 14.2 F F
Brisbane, Australia), olive oil-based margarine spread (Mea- Luncheon meat 4.9 0.8 0.12 0.10 0.15
dow Lea Foods Pty Ltd, Sydney, Australia), and French onion Sardines 0.1 0.05 0.02 0.12 0.57
dip. Subjects were also provided with olive oil-based salad Pink salmon 2.3 0.22 0.13 0.67 1.96
Deep sea bream 0.2 0.01 0.00 0.04 0.23
dressing and mayonnaise (Meadow Lea Foods Pty Ltd,
Mullet 0.3 0.03 0.02 0.40 0.38
Sydney, Australia), and muffin mix (White Wings Foods, Sausages 6.0 0.38 0.08 0.22 0.34
Sydney, Australia) from which shortening was omitted, so French onion dip 6.2 0.49 0.22 0.33 0.46
that canola oil could be added during preparation. The fatty
a
acid composition of the n-3 enriched foods provided to the g/100 g edible portion.

European Journal of Clinical Nutrition


Oslo, density 1.077 kg/l), and centrifuged at 110  g for
10 min at 251C. The resulting top layer containing the
platelet-rich plasma was removed into a separate tube and
was centrifuged at 200  g for 25 min. The plasma fraction
was removed from the platelet pellet and stored at
201C.
The platelet pellet was resuspended in 4 ml of 0.2% saline for
30 s to lyse any remaining erythrocytes, followed by addition
of 4 ml of 1.6% saline and centrifuged at 320  g for 10 min
and resuspended in 1.5 ml of normal saline. The remaining
density gradient containing the mononuclear and polymor-
phonuclear cells was centrifuged at 200  g for 25 min. The
mononuclear cell layer was removed from the gradient
interface, washed once with PBS, centrifuged at 320  g
for 10 min and resuspended in 1.5 ml of normal saline.
The remainder of the density gradient containing the
neutrophils was discarded.
Total lipids were extracted by vortex mixing the cell
fraction with 2 ml of methanol, followed by 4 ml of chloro-
form, and centrifuging at 320  g for 10 min. The chloroform
layer was removed and evaporated to dryness in a centrifugal
Speed Vac Concentrator (Savant Instruments Inc, Farming-
dale, NY, USA). Samples were then resuspended in 2 ml of
chloroform/methanol (9:1 v/v) and stored at
201C.
Phospholipid fractions were separated from total lipid
extracts by activated silica thin layer chromatography
developed in petroleum spirit/acetone (3:1 v/v), and trans-
esterified by methanolysis (1% H2SO4 in methanol at 701C
for 3 h). The resultant fatty acid methyl esters were separated
and quantified by GLC (Hewlett-Packard 5890, Hewlett-
Packard, Palo Alto, CA, USA), with identification based on
retention times of authentic standards (NuCheck Prep,
Elysian, MN, USA).
Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1607
Statistics
Differences in dietary intakes and blood fatty acids between
visits were compared by repeated measures analysis of
variance (ANOVA), with post hoc analysis by Newman–Keuls
multiple comparisons method. Statistical analyses were
performed using Winks v. 4.5 Professional Edition (TexaSoft,
Cedar Hill, TX, USA).
Results
Subjects
In all, 16 participants completed the study, and there were
no withdrawals. The participants had a mean age of
39.274.4 y, and a mean body mass index (BMI) of
25.872.6 kg/m2 at the commencement of the study. There
was a small but significant increase in BMI during the
first 2-week dietary intervention period to 26.072.6 kg/m2
(Po0.01), followed by a nonsignificant increase to
26.272.6 kg/m2 at the end of 4 weeks of dietary interven-
tion.
Dietary intakes
Macronutrient intakes were estimated from the diet diaries,
and are provided in Table 2. There was a significant increase
in the amount of protein in the diet during the first dietary
intervention period compared to baseline, both in terms of
grams per day, and as percentage of dietary energy. There was
no further increase during the second dietary intervention
period. There were no changes in total fat or carbohydrate
intake during the study.

Table 2 The estimated daily macronutrient intake during the baseline and dietary intervention periods

Dietary intervention

Macronutrient Baseline 2 weeks 4 weeks

Energy
MJ/day 10.071.6 (8.3–13.1)* 10.271.8 (7.7–13.5) 10.572.1 (8.0–14.7)
kcal/day 23897383 (1981–3127) 24487425 (1845–3225) 25087507 (1913–3507)
Protein
g/day 102718a (79–144) 116719b (83–157) 121717b (92–164)
% of energy 17.572.3a (14.6–22.6) 19.472.4b (15.0–23.5) 19.972.2b (15.7–24.8)
Fat
g/day 91720 (61–118) 95720 (60–129) 98729 (53–151)
% of energy 33.775.5 (26.3–44.9) 34.174.1 (27.0–39.9) 34.175.6 (24.5–42.7)
Carbohydrate
g/day 283767 (199–425) 280769 (171–417) 281767 (200–406)
% of energy 45.276.6 (34.5–54.2) 43.576.0 (34.6–53.4) 42.975.8 (33.3–52.0)
Alcohol
g/day 12.1713.4 (0–40) 10.2712.4 (0–39) 11.1712.3 (0–43.3)
% of energy 3.574.0 (0–12.7) 3.073.9 (0–11.8) 3.173.8 (0–15.1)

*x7s.d.; range in parentheses.

Values within a row with different superscript letters indicate significant differences at P=0.05 (repeated measures ANOVA, post hoc analysis by Newman–Keuls
multiple comparisons method).

European Journal of Clinical Nutrition

 Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1608
Fatty acids. There were significant increases in the intake
of oleic acid, ALA, EPA, and DHA, and a significant decrease
in LA intake during the dietary intervention period com-
pared to baseline intakes (Table 3).
The contribution of each provided food item to the total
n-3 intake is provided in Table 4. All subjects consumed the
cooking oil, flaxseed products and margarine spread at some
time during the study period, while only 50% of subjects ate
sardines on at least one of the recording days. Fish (canned
and fresh) provided the largest contribution to the LC n-3
PUFA intake, with canned salmon providing about 24% of
the EPA and 34% of the DHA intakes of those eating salmon.
Fresh fish (deep sea bream and mullet) together provided
about 19% each of the EPA and DHA intake of those subjects
that ate fresh fish.
Flaxseed products provided about 57% of the total ALA
intake, with canola cooking oil providing a further 19%. The
margarine spread was primarily olive oil based, and therefore
contributed only a very small proportion of the ALA
intake. Of the novel fish oil enriched products, the
margarine spread was the most popular, with all subjects
consuming it. The mean intake was 18 g/day, which
provided about 160 mg/day of EPA þ DHA, which repre-
sented about 10% of the total EPA þ DHA in the diet. Milk

Table 3 Estimated daily intake of selected fatty acids

Dietary intervention

Fatty acids Baseline 2 weeks 4 weeks

18:1 n-9
g/day 29.676.9a (18.3–37.9)* 35.877.5b (23.6–55.2) 35.5710.4b (18.5–55.4)
% of energy 10.872.4a (7.6–14.7) 12.972.2b (9.1–17.4) 12.472.6b (8.1–18.0)
18:2 n-6
g/day 13.175.4a (6.7–28.8) 9.372.1b (5.8–14.1) 9.072.8b (4.7–17.0)
% of energy 4.872.1a (2.7–11.3) 3.370.5b (2.7–4.3) 3.170.6b (2.1–4.4)
18:3 n-3
g/day 1.470.6a (0.5–2.6) 4.271.8b (2.1–8.6) 4.072.3b (0.9–10.2)
% of energy 0.5170.24a (0.22–1.05) 1.5070.60b (0.84–3.22) 1.370.7b (0.4–3.4)
20:5 n-3
g/day 0.0370.03a (0.00–0.08) 0.5270.19b (0.21–0.86) 0.5070.25b (0.11–1.07)
% of energy 0.0170.01a (0.00–0.03) 0.1970.07b (0.06–0.32) 0.1770.07b (0.04–0.35)
22:6 n-3
g/day 0.0970.07a (0.00–0.23) 1.0170.38b (0.41–1.88) 1.0170.42b (0.27–1.79)
% of energy 0.0470.03a (0.00–0.10) 0.3370.14b (0.12–0.68) 0.3570.13b (0.10–0.54)
Total n-3
g/day 1.570.8a (0.7–2.8) 5.972.1b (3.3–11.2) 5.772.9b (2.1–12.6)
% of energy 0.5670.26a (0.28–1.19) 2.170.7b (1.3–4.2) 1.970.8b (0.9–4.1)

*x7s.d.; range in parentheses.

Values within a row with different superscript letters indicate significant differences at P=0.05 (repeated measures ANOVA, post hoc analysis by Newman–Keuls
multiple comparisons method).

Table 4 Subject consumption of the foods provided, and estimates of the contribution of those foods to the overall intake of n-3 PUFAs

ALA EPA DHA

No. of subjects Mean intake % of total % of total % of total

Food consuming (g/day) g/day ALA mg/day EPA mg/day DHA

Canola oil 16 8.173.7 0.6470.29*,w 19.1711.0 F F F F

Flaxseed products 16 18.1713.4 2.672.0 56.6719.7 F F F F
Margarine spread 16 18.1711.5 0.0670.04 1.670.9 63740 12.374.5 93759 9.274.0
Milk 15 2857175 o1 83751 16.076.4 117772 11.374.7
Fresh fish 14 65.5735.3 o1 101780 19.4713.0 1787102 18.9711.1
Sausages 14 42.8732.1 o1 92770 16.777.6 1387104 12.676.5
Pink salmon 13 26.9716.6 o1 115771 23.8713.9 3387208 33.8717.5
Luncheon meat 9 32.8731.4 o1 32731 5.674.7 50747 4.573.4
French onion dip 9 15.3710.2 o1 51734 11.6710.5 71747 8.378.3
Sardines 8 36.6723.9 o1 54735 9.776.4 2607170 21.9712.3

*x7s.d.
w
The mean quantity and percentage of the n-3 PUFA contributed by the food in only those subjects consuming that particular food.

European Journal of Clinical Nutrition


was the second most popular food, being consumed by 15/16
subjects, with the mean intake amongst consumers of
about 275 ml/day. This contributed about 200 mg/day
(12.8% of total intake) of EPA þ DHA. The least popular
of the novel foods were the luncheon meat and the dip,
with only 9/16 subjects consuming these items on any
recording day. Among consumers, the luncheon meat
and dip contributed a mean daily intake of EPA þ DHA of
122 and 82 mg, respectively. However, the sausages provided
a similar contribution to EPA þ DHA intake as that of
fresh fish.
Plasma and cellular fatty acids
There were significant decreases in the proportions of n-6
PUFAs in plasma phospholipids following 2 weeks of dietary
modification with falls of 15, 9, and 15% for LA, AA and total
n-6, respectively (Table 5). There were no further decreases
recorded following a further 2 weeks of dietary modification.
There were significant increases in the proportion of ALA
(56%), EPA (174%), DHA (80%), and total n-3 (79%) in the
plasma phospholipid fraction at 2 weeks, with a small
decrease in the ALA content being the only significant
change between 2 and 4 weeks.
As with plasma phospholipids, there were significant
decreases in the proportions of n-6 PUFAs in mononuclear
cell phospholipids after 2 weeks of diet modification, with
falls of 5, 11, and 12% for LA, AA and total n-6, respectively
(Table 6). During the final 2 weeks of dietary modification,
the LA content rose back to those recorded at baseline. The
proportions of EPA, DHA, and total n-3 PUFA increased
significantly during the first 2 weeks, with rises of 173, 57,
and 38%, respectively. All three continued to increase during
Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1609
the final 2 weeks, with further rises of 15, 12, and 8%,
respectively.
Changes in platelet phospholipid fatty acids were qualita-
tively similar to those with mononuclear cells (Table 7).
There were significant decreases of 10, 3, and 6% for LA, AA
and total n-6 PUFA, respectively, during the first 2 weeks of
dietary intervention, with further significant falls of 3 and
2% for AA and total n-6 PUFA, respectively, during the
second period, whereas LA increased during this period by
5%. The proportions of ALA, EPA, DHA, and total n-3 PUFA
increased significantly during the first 2 weeks, with rises of
63, 166, 70, and 49%, respectively. EPA, DHA and total n-3
PUFA continued to increase during the final 2 weeks, with
further rises of 10, 10, and 7%, respectively.
The correlation coefficients (r) between platelet and
mononuclear cell EPA, DHA, and total n-3 PUFA for all time
points were 0.99, 0.95, and 0.94, respectively. Similarly, the
correlation coefficients between plasma phospholipid and
mononuclear cell EPA, DHA, and total n-3 PUFA were 0.94,
0.92, and 0.91, respectively, while for plasma phospholipid
and platelets, the correlation coefficients were 0.93, 0.87,
and 0.88, respectively.
Discussion
In this study, we have demonstrated that by providing
subjects with everyday food items fortified with fish oil, LC
n-3 PUFA intakes of the order of 200–400 mg/day recom-
mended for community-wide intakes (Scientific Review
Committee, 1990; The British Nutrition Foundation, 1992;
de Deckere et al, 1998; Simopoulos et al, 2000) and intakes of
about 1 g/day as recommended for people with documented
CHD (Kris-Etherton et al, 2002) are readily achievable.

Table 5 Fatty acid composition of plasma phospholipids

Baseline Dietary intervention period

Fatty acid
2 weeks 0 weeks 2 weeks 4 weeks

Total Saturated 42.770.8a,*,w 42.270.9b 42.071.2b 42.370.9b

18:1 n-9 9.971.0 9.870.9 10.170.9 9.970.7
Total Monounsaturated 13.471.1 13.471.1 14.070.9 13.770.7
n-6
18:2 20.973.3a 20.973.2a 17.873.6b 17.673.4b
20:3 3.8070.85a 3.8270.78a 2.7770.63b 2.7670.76b
20:4 10.672.1ab 11.072.3a 10.071.8bc 9.671.7c
Total 36.571.5a 36.971.4a 31.571.9b 30.872.1b
n-3
18:3 0.2070.07a 0.2370.09a 0.3670.19b 0.2970.14ab
20:5 1.0470.34a 1.1370.34a 3.1070.86b 3.3870.88b
22:5 1.4170.29 1.3970.21 1.4370.28 1.3870.23
22:6 3.6470.70a 3.7670.81a 6.7671.04b 7.2371.16b
Total 6.370.9a 6.571.0a 11.671.6b 12.372.0b

*x7s.d.
w
Values represent % of total fatty acids.
Values within a row with different superscript letters indicate significant differences at P=0.05 (repeated measures ANOVA, post hoc analysis by Newman–Keuls
multiple comparisons method).

European Journal of Clinical Nutrition

 Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1610
Table 6 Fatty acid composition of mononuclear cell phospholipids

Baseline Dietary intervention period

Fatty acid
2 weeks 0 weeks 2 weeks 4 weeks

Total Saturated 43.071.3a,*w 42.270.6b 43.570.8a 43.170.6a

18:1 n-9 12.070.7a 12.270.6a 12.970.8b 12.970.9b
Total Monounsaturated 15.770.9a 15.870.8a 16.770.9b 16.770.9b
n-6
18:2 5.3970.99ab 5.6070.87a 5.3370.89b 5.6170.94a
20:3 1.9170.41a 1.9370.38a 1.7370.34b 1.8270.43c
20:4 23.471.0a 23.971.1b 21.371.0c 21.271.0c
Total 34.771.1a 35.470.8b 31.270.7c 31.070.7c
n-3
18:3 ndz nd nd nd
20:5 0.4070.09a 0.4070.10a 1.0970.30b 1.2570.34c
22:5 2.7970.38 2.7970.25 2.8870.32 2.9170.37
22:6 2.2270.33a 2.3370.36a 3.6570.38b 4.0870.28c
Total 5.4870.48a 5.5270.39a 7.6270.70b 8.2670.70c

*x7s.d.
w
Values represent % of total fatty acids.
Values within a row with different superscript letters indicate significant differences at P=0.05 (repeated measures ANOVA, post hoc analysis by Newman–Keuls
multiple comparisons method).
z
Not detected.

Table 7 Fatty acid composition of platelet phospholipids

Baseline Dietary intervention period

Fatty acid
2 weeks 0 weeks 2 weeks 4 weeks

Total Saturated 43.171.1a,*,w 43.470.6a 42.470.8b 42.370.6b

18:1 n-9 14.570.7a 14.670.7a 15.570.7b 15.870.8c
Total Monounsaturated 18.271.0a 18.270.9a 19.270.9b 19.670.9c
n-6
18:2 5.0670.75a 5.0470.69a 4.5370.81b 4.7670.72c
20:3 1.6470.35a 1.5970.33b 1.4470.31c 1.5370.32d
20:4 23.471.0a 23.571.0a 22.971.3b 22.171.0c
Total 33.371.0a 33.270.8a 31.370.9b 30.770.9c
n-3
18:3 0.05670.019a 0.05970.021a 0.09670.036b 0.08370.030b
20:5 0.4270.09a 0.4170.10a 1.0970.26b 1.2070.31c
22:5 2.1370.32a 1.9770.22b 2.1470.22a 2.1570.29a
22:6 1.6970.32a 1.6470.29a 2.7870.35b 3.0770.45c
Total 4.3370.45a 4.0970.39a 6.1170.56b 6.5170.77c

*x7s.d.
z
Values represent % of total fatty acids.
Values within a row with different superscript letters indicate significant differences at p=0.05 (repeated measures ANOVA, post hoc analysis by Newman–Keuls
multiple comparisons method).

Lovegrove et al (1997) fortified some everyday items
with fish oil such as bread and spread, along with a
number of sweet foods, including biscuits, cake, ice
cream, and milkshake powder, and used a portion exchange
system with the aim of achieving a daily intake of 1.8 g
EPA þ DHA. Likewise, Mantzioris et al (2000) used a
similar dietary approach with fish-oil-fortified savoury
products like sausages and dip, and had a large
emphasis on ingestion of fresh fish, and set a target
EPA content in mononuclear cell phospholipids of
1.5% of total fatty acids. Subjects achieved mean daily
intakes of EPA þ DHA of 1.4 and 1.8 g, respectively.
However, in both studies, the investigators gave no
indication of the popularity of each food item, or the
contribution of each food item to the total LC n-3
PUFA intake.
In the present study, we have used similar foods to those
used by Mantzioris et al (2000) with the addition of fish-oil-
fortified margarine spread and long-life milk. This is the first
study to examine the food preferences of the subjects, and
the contribution of the individual food items to the overall
intakes of EPA and DHA.

European Journal of Clinical Nutrition


A combination of the mean daily intakes of margarine
spread (18 g/day providing 156 mg of EPA þ DHA), milk
(285 ml/day providing 200 mg of EPA þ DHA), sausages
(4/week providing an average daily intake of about
250 mg EPA þ DHA), 15 g of dip provided about 120 mg of
EPA þ DHA and 32 g of luncheon meat providing about
80 mg of EPA þ DHA gives a total daily intake of about
800 mg. This approaches the mean daily intake of 1 g of
EPA þ DHA as recommended for people with documented
CHD (Kris-Etherton et al, 2002).
A number of other studies have examined the effects of
incorporating fish-oil enriched products in the diet. Saldeen
et al (1998) added fish oil to bread and instructed subjects to
consume 125 g/day, which provided a daily LC n-3 PUFA
intake of 300 mg. They observed increases in plasma
phospholipid EPA and DHA of 32 and 18%, respectively,
after 2 weeks. When 500 ml/day of fish oil fortified, partially
fat reduced milk, also providing 300 mg EPA þ DHA daily,
was consumed for 6 weeks, plasma phospholipid EPA and
DHA both increased by 31% (Visioli et al, 2000). Wallace et al
(2000) used microencapsulated fish oil incorporated into
bread, biscuits and soup to achieve a daily intake of 900 mg
of LC n-3 PUFA/day, and reported that subjects expressed a
preference for the bread as it was easier to incorporate into
the normal diet. Increases in platelet EPA and DHA of 82 and
44%, respectively, were observed after 4 weeks. In the current
study, both mononuclear cell and platelet EPA increased by
about 170% in the first 2 weeks, with further small but
significant increases during the next 2 weeks, however, only
two subjects achieved mononuclear cell EPA levels of 1.5% of
total fatty as proposed by Mantzioris et al (2000) as a
potential target for anti-inflammatory benefits.
A major hurdle to the generalized worldwide enrichment
of foodstuffs with fish oil is the limited availability of fish
stocks, and the possibility of decreased supply and increased
cost of fish oil (Naylor et al, 2000). The use of renewable
terrestrial sources of n-3 PUFA therefore becomes important.
While the efficiency of the conversion of ALA to the longer
chain n-3 metabolites remains controversial (Gerster, 1998),
there is clearly an indication for promoting an increase in
ALA consumption. Increasing ALA consumption to about
9 g/day through the addition of flaxseed to the diet as
margarine spread (Mantzioris et al, 1994) or baked in muffins
(Cunnane et al, 1995) significantly increased plasma phos-
pholipid proportions of EPA and DPA. Previous research
suggests that minimizing the background intake of LA (the
predominant n-6 PUFA) is important in maximizing the
uptake of LC n-3 PUFA (Cleland et al, 1992) and the
conversion of ALA to EPA (Emken et al, 1994).
Irrespective of elevation in tissue LC n-3 PUFA, ALA
consumption itself has been associated with reduced risk of
cardiovascular disease. The Health Professionals Follow-up
Study (Ascherio et al, 1996) found a 59% reduction in the risk
of MI associated with an increase in ALA consumption of 1%
of dietary energy. In a 10 MJ diet, a 1% increase in energy
equates to 2.7 g ALA, an amount found in about two
Increasing consumption of n-3 fats using novel foods
RG Metcalf et al
1611
tablespoons of ground flaxseed meal. A similar quantity of
ALA supplementation (2.9 g/day in the form of mustard oil)
was given in the Indian Infarct Survival study (Singh et al,
1997), a secondary prevention study among survivors of
suspected acute MI, which found a significant reduction in
the risk of total cardiac events and nonfatal MI compared to
placebo (28 vs 34.7% and 15.0 vs 25.4%, respectively) after 12
months of follow-up.
Significant reductions in risk of all-cause mortality (31%)
and cardiovascular disease mortality (37%) were associated
with the highest quintile of ALA consumption compared to
the lowest quintiles (mean intakes, 2.80 and 0.87 g/day,
respectively) in the MRFIT Study (Dolecek & Granditis,
1991), while in the Nurse’s Health Study (Hu et al, 1999),
there was a 45% reduction in coronary heart disease
mortality from the lowest (0.71 g/day) to the highest
(1.36 g/day) quintiles of ALA consumption. The Lyon Diet
Heart Study (de Lorgeril et al, 1994) found a significant
reduction in risk of both cardiac death and nonfatal MI
associated with increased consumption of ALA.
In the study presented here, subjects consumed a mean
intake of 4.1 g ALA/day, of which over 50% was derived from
flaxseed meal. In this study, the margarine spread was olive
oil-based, whereas had a canola oil-based margarine spread
been used, the mean ALA intake would have increased by
about 1 g/day.
Having a wide range of n-3 PUFA-enriched products
enables those people who do not eat fish habitually or who
never eat fish, to benefit from the increased intake of
n-3 PUFA without radical modification of their normal
dietary habits. Increased daily consumption of n-3 PUFA
from an early age may help prevent the onset of a variety
of so-called ‘lifestyle’ diseases, such as atherosclerosis,
diabetes, essential hypertension, and obesity (Das, 2000),
thereby reducing the need for high therapeutic doses of n-3
PUFA, which have been the hallmark of most intervention
studies to date.
Acknowledgements
We acknowledge Meadow Lea Foods Ltd (Sydney, Australia)
for supplying the margarine spread, canola oil, salad
dressing, and mayonnaise, Pauls Ltd (Brisbane, Australia)
for supplying the milk, Safcol Ltd (Adelaide, Australia) for
supplying the canned salmon and sardines, Conroys
Smallgoods Pty Ltd (Adelaide, Australia) for supplying the
luncheon meat, Enreco Inc (Manitowoc, WI, USA) for
supplying the flax products and Roche Vitamins (Sydney,
Australia) for supplying the fish oil. This study was supported
in part by the National Heart Foundation of Australia.
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