Content uploaded by Hayati Yusof
Author content
All content in this area was uploaded by Hayati Yusof on Dec 05, 2017
Content may be subject to copyright.
Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228
Influence of very long-chain n-3 fatty acids on plasma markers of
inflammation in middle-aged men
Hayati M. Yusof
, Elizabeth A. Miles, Philip Calder
Institute of Human Nutrition, School of Medicine, University of Southampton, Tremona Road, Southampton SO16 6YD, UK
Received 26 November 2007; received in revised form 20 February 2008; accepted 28 February 2008
Abstract
This study investigated the effects of a moderate dose of long-chain n-3 polyunsaturated fatty acids (1.8 g eicosapentaenoic acid
(EPA) plus 0.3 g docosahexaenoic acid (DHA) per day) given for 8 weeks to healthy middle-aged males on cardiovascular risk
factors, particularly plasma lipids and inflammatory markers. The study was double-blind and placebo-controlled. The proportion
of EPA was significantly increased in plasma phosphatidylcholine (from 1.4% to 5.0% of total fatty acids; Po0.001), cholesteryl
esters (from 1.2% to 4.5%; Po0.001) and triacylglycerols (from 0.3% to 1.8%; Po0.001). In contrast, the more modest increases in
DHA in these lipid fractions were not significant. There was very little effect of n-3 fatty acids on the risk factors measured, apart
from a reduction in plasma soluble intercellular adhesion molecule (sICAM)-1 concentration compared with placebo (P¼0.05).
The change in plasma sICAM-1 concentration was significantly inversely related to the change in DHA in plasma
phosphatidylcholine (r¼0.675; P¼0.001), but less so to the change in EPA (r¼0.406; P¼0.076). Data from the present
study suggest that marine oil providing 1.8 g of EPA plus 0.3 g DHA/day is not sufficient to demonstrate marked effects on
cardiovascular risk factors (plasma lipids and inflammatory markers) in healthy middle-aged men, although there may be a slight
anti-inflammatory effect as indicated by the decrease in sICAM-1. The stronger association between changes in DHA than EPA and
sICAM-1 concentrations suggest that DHA may be more anti-inflammatory than EPA. Thus, one reason why only limited effects
were seen here may be that the dose of DHA provided was insufficient.
r2008 Elsevier Ltd. All rights reserved.
1. Introduction
Classic risk factors for atherosclerosis and cardiovas-
cular disease include elevated plasma lipids, including
triacylglycerols (TAGs) and total and low-density lipo-
protein (LDL) cholesterol, high blood pressure and
insulin resistance [1]. However, it is now recognised that
atherosclerosis is an inflammatory process involving
movement of leucocytes, especially monocytes and T
lymphocytes, from the bloodstream into the intima of
the blood vessel wall and subsequent release of inflam-
matory mediators that contribute to plaque growth and
development and ultimately to its rupture [1–3]. Prior to
entry into the intima, blood leucocytes interact with
the endothelial cells lining the vessel wall. These
interactions are largely mediated by ligand–ligand inter-
actions between proteins termed adhesion molecules.
These interactions serve to slow and then tether the
flowing blood leucocytes. Most important among the
adhesion molecules involved are intercellular adhesion
molecule (ICAM)-1, vascular cell adhesion molecule
(VCAM)-1 and E-selectin. The endothelial expression
of these molecules is up-regulated by inflammatory
stimuli and they may then be cleaved from the surface
of the endothelial cells [4].Invivothisresultsinnon-
surface bound forms of the adhesion molecules circulat-
ing in the bloodstream; these are termed soluble adhesion
molecules (e.g. soluble ICAM-1 (sICAM-1)). It has been
demonstrated that elevated plasma concentrations of
ARTICLE IN PRESS
www.elsevier.com/locate/plefa
0952-3278/$ - see front matter r2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plefa.2008.02.002
Corresponding author. Tel.: +44 2380 795250;
fax: +442380 795255.
E-mail addresses: hmy1@soton.ac.uk (H.M. Yusof),
p.c.calder@soton.ac.uk (P. Calder).
soluble adhesion molecules and other inflammatory
proteins such as interleukin-6 (IL-6) and C-reactive
protein (CRP) are associated with increased cardiovas-
cular risk and are higher in individuals with diagnosed
cardiovascular disease compared with those without
[5–8]. Thus, a reduction in plasma concentrations of
these inflammatory markers, which may reflect a reduc-
tion in inflammatory processes at and within the vessel
wall, would be interpreted as a lowering of cardiovascular
risk.
There is significant epidemiological evidence that
consumption of fish, especially oily fish, is protective
against cardiovascular morbidity and mortality [9–17].
This is believed to be due to the long-chain n-3
polyunsaturated fatty acids (n-3 PUFAs) found in oily
fish, since both long-chain n-3 PUFA consumption in
the diet [13–15,18,19] and blood and tissue concentra-
tions [20–22] have been shown to be protective against
cardiovascular disease morbidity and mortality. The
long-chain n-3 PUFAs found in oily fish, eicosapentae-
noic acid (EPA) and docosahexaenoic acid (DHA), are
found in fish oils and similar preparations. Studies using
fish oils have demonstrated that long-chain n-3 PUFAs
influence many cardiovascular risk factors in a manner
that could contribute to cardiovascular protection
[23–25]. There is now much evidence that these fatty
acids are anti-inflammatory [26–29] and this is believed
to be important in the context of cardiovascular disease
[24]. However, many supplementation studies in humans
have used very high doses of long-chain n-3 PUFAs,
which greatly exceed those that could be achieved
through fish consumption and so have limited relevance
to explaining the epidemiology. Furthermore, in the
context of the very important adhesive interactions
between leucocytes and the endothelium there is fairly
limited information. In vitro studies have shown that
EPA and DHA can inhibit inflammation-induced up-
regulation of VCAM-1 on human endothelial cells
[30–33] and of ICAM-1 on human monocytes [34].
Feeding studies in laboratory rodents report that fish oil
lowers ICAM-1 expression on the surface of macro-
phages [35] and T lymphocytes [36], while fish oil
supplementation was shown to decrease ICAM-1 on the
surface of human monocytes [37]. With regard to
soluble forms of adhesion molecules, reports in the
literature are mixed with some studies showing a
reduction in some of these molecules but not in others
[38–41]. Long-chain n-3 PUFA dose, duration of
exposure, differences between the subjects studied and
differences in experimental design might contribute to
the different findings of these studies.
Most studies of fish oil and inflammation have used
high doses of long-chain n-3 PUFAs [29]. In the UK, the
guideline range for oily fish intake among males and
adult women not of childbearing age is 1–4 portions per
week [42]. The long-chain n-3 PUFA content of a
portion of oily fish ranges from about 1.5 to 3.5 g [43].
Thus, 1–4 portions per week could provide between 1.5
and 14.0 g long-chain n-3 PUFAs. This equates to an
average daily long-chain n-3 PUFA intake of 0.2–2 g. It
is important to know the effect of consumption of long-
chain n-3 PUFAs within this guideline intake range on
risk factors for cardiovascular disease. Thus, in this
study a dose of EPA plus DHA at the upper end of the
guideline intake range was used to investigate the effects
on selected cardiovascular risk factors, with a focus on
plasma inflammatory markers.
2. Materials and methods
2.1. Subjects and study design
Ethical approval (05/Q1704/151) was obtained from
the Southampton and South West Hampshire Joint
Ethics Committee. Volunteers were invited to partici-
pate in the study by advertisement and their eligibility
was screened using a ‘‘health and lifestyle question-
naire’’. Volunteers were identified as eligible to partici-
pate in the study if they were male, aged 35–60 years,
had a body mass index (BMI) of 18.5–29.9 kg/m
2
, were
not on drug treatment for hyperlipidaemia or inflam-
matory conditions or a regular (daily) aspirin user, were
not suffering from any gastrointestinal disorder, dia-
betes mellitus or other endocrine disorder, were not
taking any dietary supplements including fatty acids and
vitamins, were not vegetarian or vegan, did not consume
more than one serving of oily fish per month, were not a
heavy smoker (X10 cigarettes per day), were not a
vigorous exerciser (did not engage in more than
330 min vigorous sessions per week), were not
planning to lose weight, were not a blood donor and
had not participated in a clinical trial in the previous 3
months. Twenty-one subjects were recruited into the
study; one subject withdrew during the study. Written
informed consent was taken from each recruit and their
general practitioner was informed of their participation
in the trial.
The study was a randomised, double-blind and
placebo-controlled trial of 8 weeks duration and ran
from March until June 2006. Subjects attended the
Welcome Trust Clinical Research Facility at South-
ampton General Hospital on two occasions (at study
entry (‘‘baseline’’) and at end of the 8-week interven-
tion). On both occasions, they were in the fasted state
(412 h without food and drink apart from water) and
gave 20 ml blood samples. Weight, height (only for the
first visit) and blood pressure were also taken at each
visit. Weight and height were taken to the nearest 0.1 kg
and 0.1 cm, respectively. BMI (kg/m
2
) was calculated.
Two blood pressure measurements were obtained in the
supine position from the non-dominant side arm using a
ARTICLE IN PRESS
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228220
Marrquette
s
blood pressure monitor; an additional
reading was done if the values from the two measure-
ments were more than 10 mm Hg apart.
Subjects were randomly assigned in a double blind
manner to either fish oil (67% of fatty acids as EPA and
11% as DHA) or placebo (97% w/w coconut oil rich in
medium-chain saturated fatty acids). The capsules used
were a gift from Equazen, UK, Ltd. and contained 0.5 g
of oil in a gelatine coating. The placebo capsules
contained 3% fish oil in order that both capsules had
a ‘‘fishy’’ flavour. Coconut oil was selected as placebo
because medium-chain fatty acids (MCFAs) are readily
oxidised in the liver and so are expected to have little
impact on human health related biomarkers. Capsules
were received from the suppliers packed in sealed pots
that bore the subject number and a treatment code;
the key to the code was retained by the supplier and
not divulged to the researchers until after all analyses
(biochemical and statistical) had been completed.
Table 1 shows the fatty acid composition of the capsules
used in the study. Subjects consumed six capsules (i.e.
3 g oil) per day. Thus, subjects in the fish oil group
consumed 2.1 g/day of EPA and DHA (1.8 g EPA and
0.3 g DHA) from the capsules, while subjects in the
placebo group consumed 2.6 g/day of medium-chain
saturated fatty acids from the capsules. Subjects were
provided with more capsules than needed for the period
of the study. Pots were returned at the end of the study;
all subjects returned their pots and based on the
counting of the returned capsules, compliance was
90.174.1% and 93.372.2% for the fish oil and placebo
groups, respectively. Compliance was not significantly
different between groups (P¼0.084). Compliance to
fish oil was also confirmed by an increase in the
proportions of EPA in plasma lipids in the fish oil
group (see Section 3).
Blood was collected into tubes containing lithium–
heparin or fluoride oxalate at study entry and at the end
of 8 weeks supplementation with the capsules. Blood
samples were collected between 0800 and 1030 h after an
overnight fast of at least 12 h. Plasma was obtained by
centrifugation at 3000 rpm for 10 min at 4 1C. Aliquots
of plasma were kept frozen at 80 1C until analysis.
Apart from glucose concentration, which was measured
in plasma from blood collected into fluoride oxalate, all
measurements were made on plasma from blood
collected into heparin.
2.2. Analysis of plasma fatty acid composition
Lipids were extracted from plasma with chloroform:-
methanol (2:1, v/v) containing butylated hydroxytoluene
(50 mg/l) as antioxidant. Lipid classes (phosphatidylcho-
line (PC), TAGs and cholesteryl esters (CEs)) were then
separated and isolated by solid phase extraction (see
[44]). Fatty acids were subsequently methylated by
incubation with methylation reagent (methanol contain-
ing 2% v/v H
2
SO
4
)at501C for 2 h. Fatty acid methyl
esters were separated and identified using a Hewlett
Packard 6890 gas chromatograph (Hewlett Packard,
Avondale, PA), fitted with a 30 m 32 mm BPX 70
capillary column, film thickness 0.25mm. Helium, at
the initial flow of 1.0 ml/min was used as the carrier
gas. The split ratios for TAGs, CEs and PC were 100:1,
100:1 and 50:1, respectively. Injector and detector
temperature were 275 1C and the column oven tempera-
ture was maintained at 170 1C for 12 min after sample
injection. The oven temperature was programmed to
increase from 170 to 210 1Cat51C/min. Fatty acid
methyl esters were identified by comparison with
authentic standards run previously. Peak areas were
quantified using ChemStation software (Hewlett Pack-
ard, Avondale, PA). Each fatty acid is expressed as wt%
of total fatty acids present.
2.3. Measurement of plasma insulin concentrations
Plasma insulin concentrations were determined using
an ELISA kit from Biosource Europe (Nivelles,
Belgium). The assay was performed according to the
manufacturer’s instructions and the absorbance was
read at 450 nm with a reference filter of 650 nm. Results
were then calculated based on the standard curve
plotted of optical density against standard concentra-
tions. The sensitivity of the assay was o0.15 mIU/ml.
2.4. Measurement of plasma glucose and lipid
concentrations
Plasma TAG, total cholesterol, LDL cholesterol,
HDL cholesterol and glucose concentrations were
measured using a commercial kit from Konelab
TM
ARTICLE IN PRESS
Table 1
Fatty acid compositions of the capsules used
Fatty acid Placebo Fish oil
Caprylic acid, 8:0 20.8 –
Capric acid, 10:0 73.9 –
Lauric acid, 12:0 2.2 –
Palmitic acid, 16:0 1.5 0.7
Palmitoleic acid, 16:1n-7 – 0.5
Oleic acid, 18:1n-9 – 2.2
Elaidic acid, t18:1n-9 – 0.6
Linoleic acid, 18:2n-6 – 1.1
g-Linolenic acid, 18:3n-6 – 0.7
a-Linolenic acid, 18:3n-3 – 0.9
Eicosanoic acid, 20:1n-9 – 9.2
Arachidonic acid, 20:4n-6 – 4.0
Erucic acid, 22:1n-9 – 2.5
Eicosapentaenoic acid, 20:5n-3 1.8 66.8
Docosahexaenoic acid, 22:6n-3 – 10.8
Data are expressed as g/100 g total fatty acid and are mean from five
separate determinations, each on a different capsule.
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219 –228 221
(Vantaa, Finland). The results were calculated auto-
matically by the Konelab
TM
analyser using a calibra-
tion curve whereby the absorbance at 500–550 nm was
directly proportional to the concentration measured
in the plasma sample. Plasma non-esterified fatty
acid (NEFA) concentrations were measured using a
commercial kit from Wako (Neuss, Germany). The
intensity of the colour at 550 nm was proportional
to the concentration of NEFAs in the sample. Homo-
eostatic model assessment (HOMA) was calculated
as follows:
Glucose concentration ðmg=dlÞinsulin concentrationðmIU=mlÞ
405 .
2.5. Measurement of plasma inflammatory marker
concentrations
The plasma concentrations of IL-6, sE-selectin,
sICAM-1, sVCAM-1 and CRP were measured using
ELISA kits that use a biotin–avidin enhanced immu-
noassay. sE-selectin, sICAM-1 and sVCAM-1 kits were
purchased from Biosource Europe (Nivelles, Belgium),
the IL-6 kit was from R&D Systems (Abingdon, UK),
and the high sensitivity (hs)-CRP kit was from
Diagnostic System Laboratories (Webster, TX). For
all assays, the manufacturer’s instructions were followed
and the absorbance of each assay was read at 450 nm as
the primary wavelength and 610–650 as the reference
wavelength. The sensitivities of the assays were
o0.039 pg/ml (IL-6), o0.5 ng/ml (sE-selectin), 0.5 ng/
ml (sICAM-1), 0.9 ng/ml (sVCAM-1) and 1.6 ng/ml
(hs-CRP), respectively.
2.6. Platelet reactivity assay
Plasma sP-selectin concentration was measured using
an ELISA kit from Biosource Europe (Nivelles,
Belgium) according to the manufacturer’s instructions.
The sensitivity of the assay was o1.3 ng/ml.
2.7. Statistical analysis
Sample size was based upon previous studies indicat-
ing that a fish oil supplement providing about 2 g EPA/
day would be expected to increase the EPA content of
plasma phospholipids from approximately 1% to 4% of
total fatty acids [45]. Using standard deviations for EPA
contents of plasma phospholipids from previous studies
[38,45,46], it was estimated that a sample size of seven
would give 80% power of detecting this effect as
statistically significant with Pset at 0.01. To allow for
drop-outs, it was decided to recruit 10 subjects per
group.
The Kolmogorov–Smirnov and Shapiro–Wilk tests
were applied to assess normality of data. Data for
continuous variables that were normally distributed are
presented as mean values and their standard errors
(SEM) while non-normally distributed data are pre-
sented as medians and 10th and 90th percentiles.
Comparison of normally distributed data between
groups was performed using the unpaired Student’s t-
test and within a group using the paired Student’s t-test.
Not-normally distributed data were compared using the
Wilcoxan signed ranks and Mann–Whitney U-tests.
Relationships between variables were evaluated using
Pearson’s correlation coefficient. In all cases, a value for
Pp0.05 was taken to indicate a significant effect. SPSS
version 14.02 (SPSS Inc., Chicago, IL) was used for all
statistical analyses.
3. Results
3.1. Subject characteristics
Ten subjects were randomised to fish oil and 11 to
placebo. One subject in the fish oil group withdrew from
the study. Baseline characteristics of the subjects who
completed the study are shown in Table 2. There were
no differences between the groups at baseline apart from
HDL-cholesterol concentration, which was lower
(P¼0.045) in the placebo group.
ARTICLE IN PRESS
Table 2
Characteristics of the subjects at study entry
Variable Placebo Fish oil
Age (years) 44.772.0 43.772.3
Weight (kg) 81.673.0 80.173.4
Height (m) 1.7670.02 1.7670.02
BMI (kg/m
2
) 26.571.0 25.770.8
Systolic blood pressure (mmHg)
z
110 (109, 143) 122 (102, 140)
Diastolic blood pressure (mmHg)
z
67 (64, 93) 72 (60, 90)
Plasma total cholesterol (mmol/l) 4.7870.31 5.1970.37
Plasma LDL-cholesterol (mmol/l) 3.0170.25 3.0970.30
Plasma HDL-cholesterol (mmol/l) 1.0870.06 1.3870.13
Plasma TAGs (mmol/l) 1.1470.14 1.3070.19
Plasma total: HDL cholesterol 4.5170.29 4.0570.47
Plasma LDL:HDL cholesterol 2.7670.25 2.4570.36
Plasma total NEFAs (mmol/l)
z
334 (248, 602) 396 (209, 458)
Plasma glucose (mmol/l) 5.7470.13 5.9070.11
Plasma insulin (mIU/ml)
z
4.7 (4.1, 6.2) 5.5 (1.3, 28.4)
HOMA
z
1.20 (0.97, 1.72) 1.33 (0.34, 7.45)
Data are mean 7SEM (except for variables marked
z
, which are
median (10th, 90th percentile)) for nine subjects in the fish oil group
and 11 in the placebo group.
Abbreviations: BMI, body mass index; LDL, low-density lipoprotein;
HDL, high-density lipoprotein; NEFAs, non-esterified fatty acids;
HOMA, homeostatic model assessment.
Significantly different from placebo group (P¼0.045).
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228222
3.2. Fatty acid composition of blood lipids
The fatty acid compositions of the plasma lipid
fractions were very similar between the two groups at
baseline and were not affected by the placebo treatment
(Tables 3–5). In contrast, fish oil induced significant
changes in the fatty acid composition of plasma PC, CEs
and TAGs (Tables 3–5). In all three fractions, there was
a marked increase in the proportion of EPA (by 260%,
275% and 620%, respectively). The proportion of DPA
increased in plasma PC (Table 3). Thus, at the end of the
supplementation period plasma lipids from subjects in
the fish oil group had greater proportions of EPA, and
in the case of PC of DPA as well, compared with those
from subjects in the placebo group (Tables 3–5). The
increase in n-3 PUFAs in plasma PC was accompanied
by decreases in the proportions of linoleic and dihomo-
g-linolenic acids (Table 3), while in plasma CEs only
linoleic acid was significantly decreased (Table 4).
Changes in the proportion of DHA were more modest
than for EPA (14%, 66% and 36% for plasma PC, CEs
and TAGs, respectively) and DHA was not significantly
different between time points in the fish oil group
or between groups at the end of supplementation
(Tables 3–5).
3.3. Blood pressure, plasma lipids, glucose and insulin
Fish oil did not significantly affect blood pressure or
the plasma concentrations of LDL cholesterol, HDL
cholesterol, TAGs, NEFAs, glucose or insulin or
HOMA (data not shown). In contrast, in the placebo
group the plasma concentrations of total, LDL- and
HDL-cholesterol were significantly increased (all
Po0.001); the increases were 0.8470.11, 0.4970.07
and 0.2170.03 mmol/l, respectively. Each of these
increases was approximately 18% from baseline, so that
the ratios of total to HDL cholesterol and of LDL to
ARTICLE IN PRESS
Table 3
Fatty acid composition of plasma PC at baseline and after 8 weeks
treatment with placebo or fish oils
Fatty acid Plasma PC
Placebo Fish oil
Baseline 8 weeks Baseline 8 weeks
14:0 0.3270.02 0.3070.04 0.470.03 0.3970.04
16:0 31.170.3 32.070.7 31.470.4 31.270.5
16:1n-7 0.3370.04 0.4170.07 0.4270.09 0.4870.11
18:0 13.770.3 14.770.9 13.970.3 14.170.5
18:1n-9 10.670.4 10.370.5 10.270.5 9.970.7
18:2n-6 23.470.8 22.271.3 24.071.1 20.470.8
18:3n-3 0.2170.04 0.2570.04 0.2070.07 0.2770.05
20:3n-6 3.570.2 3.370.3 3.470.3 2.670.3
20:4n-6 8.970.5 9.070.5 8.670.6 8.070.4
20:5n-3 1.370.2 1.470.4 1.470.2 5.070.3
,z
22:5n-3 0.9570.13 0.8270.09 0.8470.07 1.8170.11
,z
22:6n-3 3.370.3 3.870.3 3.670.3 4.170.3
Data are mean 7SEM for nine subjects in the fish oil group and 11 in
the placebo group.
Significantly different from baseline (Po0.001).
z
Significantly different from placebo group at the same time point
(Po0.001).
Table 4
Fatty acid composition of plasma CEs at baseline and after 8 weeks
treatment with placebo or fish oils
Fatty acid Plasma CEs
Placebo Fish oil
Baseline 8 weeks Baseline 8 weeks
14:0 0.7070.04 0.7370.07 0.7370.07 0.9570.08
16:0 11.670.2 11.770.2 11.470.2 12.270.2
16:1n-7 2.470.3 2.670.4 2.870.4 2.970.5
18:0 1.170.1 0.970.1 1.070.1 1.070.1
18:1n-9 19.670.4 19.470.5 19.170.7 18.671.1
18:2n-6 52.671.1 52.371.4 53.971.8 49.871.8
18:3n-3 0.670.1 0.770.1 0.570.1 0.770.1
20:3n-6 0.770.1 0.870.1 0.770.1 0.670.1
20:4n-6 6.670.5 6.970.5 6.170.4 6.170.3
20:5n-3 1.170.2 1.270.2 1.270.2 4.570.3
,z
22:6n-3 0.970.2 0.570.1 0.370.1 0.570.1
Data are mean 7SEM for nine subjects in the fish oil group and 11 in
the placebo group.
Significantly different from baseline (Po0.02).
Significantly different from baseline (P¼0.01).
Significantly different from baseline (Po0.001).
z
Significantly different from placebo group at the same time point
(Po0.001).
Table 5
Fatty acid composition of plasma TAGs at baseline and after 8 weeks
treatment with placebo or fish oils
Fatty acid Plasma TAGs
Placebo Fish oil
Baseline 8 weeks Baseline 8 weeks
14:0 1.970.2 2.070.3 2.470.4 2.370.3
16:0 26.270.6 24.572.2 28.371.7 25.273.5
16:1n-7 3.470.3 6.772.9 3.670.3 3.570.4
18:0 3.470.1 3.970.3 3.870.2 4.170.4
18:1n-9 41.871.0 38.571.0 38.871.3 36.271.4
18:2n-6 16.171.1 15.071.2 16.272.0 15.971.6
18:3n-3 1.470.2 1.270.1 1.270.1 1.770.5
20:3n-6 0.1770.07 0.1270.05 0.1270.05 0.1770.06
20:4n-6 1.070.1 1.370.1 1.170.2 1.470.2
20:5n-3 0.2970.13 0.5070.37 0.2570.14 1.8070.47
,z
22:5n-3 0.3970.12 0.6970.28 0.52 70.13 0.7770.17
22:6n-3 1.070.2 1.670.9 1.1 70.2 1.570.4
Data are mean 7SEM for nine subjects in the fish oil group and 11 in
the placebo group.
Significantly different from baseline (P¼0.025).
z
Significantly different from placebo group at the same time point
(Po0.05).
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219 –228 223
HDL cholesterol were not significantly changed (data
not shown). Fish oil did not exert a TAG lowering
effect. Plasma concentrations of TAGs, NEFAs and
glucose were not significantly affected in the placebo
group (data not shown), but plasma insulin was
increased (P¼0.013) by 1.370.4 mIU/ml (27%) result-
ing in an increased (P¼0.016) HOMA (by 0.3570.12;
31%).
3.4. Inflammatory markers
Plasma inflammatory marker concentrations are
shown in Table 6. There were no differences between
the two groups at study entry or at the end of the
supplementation period. Neither placebo nor fish oil
had a significant effect on the plasma inflammatory
markers measured, although fish oil tended to lower
sICAM-1 concentrations. When data were analysed as
% change from baseline concentration, the two groups
were significantly different for sICAM-1 (P¼0.05),
with an approximately 10% decrease from baseline in
the fish oil group.
3.5. Correlations between changes in individual fatty
acids and changes in inflammatory markers
If fatty acids are causally associated with inflamma-
tion, as determined by plasma inflammatory markers,
then changes in status of the fatty acids involved should
be associated with changes in levels of the inflammatory
markers. Therefore, the changes in the proportions of
arachidonic acid, EPA and DHA in the different plasma
pools were related to the changes in inflammatory
marker concentrations; pooled subjects were used in this
analysis. There were no significant relationships ob-
served for sE-selectin, sP-selectin, sVCAM-1 or CRP
(data not shown). There was a significant positive
association between the change in the amount of EPA
in plasma PC and the change in plasma IL-6 concentra-
tion (r¼0.451; P¼0.046), with a trend for a positive
association for change in DHA in plasma PC and
change in IL-6 (r¼0.424; P¼0.063). There was a
highly significant inverse association (r¼0.675;
P¼0.001) between the change in the amount of DHA
in plasma PC and change in plasma sICAM-1 concen-
tration, with a trend for a similar association
(r¼0.406; P¼0.076) between changes in plasma
PC EPA and plasma IL-6. When data were expressed as
% changes from baseline, the inverse association
between the change in DHA in plasma PC and change
in plasma sICAM-1 concentration remained highly
significant (r¼0.474; P¼0.008).
4. Discussion
This study investigated the effect of moderate dose
fish oil providing 1.8 g EPA plus 0.3 g DHA/day on a
range of cardiovascular risk factors, particularly plasma
lipids and inflammatory markers. The subjects were
healthy middle-aged males and the supplementation
period was 8 weeks. The dose of long-chain n-3 PUFAs
was chosen to approximate the upper limit of the
current UK guideline range, based upon oily fish
consumption [42] and the typical n-3 PUFA content of
oily fish [43]. Many studies investigating the effects of
long-chain n-3 PUFAs on cardiovascular risk factors
have used higher doses, sometimes much higher, of these
fatty acids than used here (see below). A medium-chain
triglyceride (MCT)-rich oil (providing 0.6 g of caprylic
acid (8:0) and 2 g of capric acid (10:0) per day) was used
as the placebo. The MCT oil was used because these
fatty acids are considered to be physiologically neutral
and were not expected to change the blood (or cell) fatty
acid profiles. This is because the MCFAs are absorbed
directly into the hepatic portal vein and subsequently
ARTICLE IN PRESS
Table 6
Inflammatory markers at baseline and after 8 weeks treatment with placebo or fish oils
Variable Placebo Fish oil
Baseline 8 weeks % Change Baseline 8 weeks % Change
Plasma sICAM-1 (ng/ml) 224716 240715 9.976.1 257728 219720 9.576.9
Plasma sVCAM-1 (ng/ml)
z
649 (437, 957) 636 (431, 1024) 2.376.5 456 (61, 1375) 448 (82, 1345) 2.176.7
Plasma sE-selectin (ng/ml) 84.3713.2 69.379.3 2.5716.3 85.1716.1 94.4720.5 18.1717.4
Plasma sP-selectin (ng/ml) 33.574.0 32.373.8 4.379.0 36.3710.3 37.6710.2 17.2710.6
Plasma IL-6 (pg/ml)
z
1.24 (0.81, 3.87) 1.13 (0.80, 1.74) 2.9 (65.7, 16.3) 1.35 (0.86, 2.75) 1.27 (0.70, 2.17) 17.6 (44.0, 104.4)
Plasma CRP (mg/l)
z
2.0 (1.9, 9.3) 2.0 (1.9, 4.0) 0 (72.3, 14.4) 2.0 (1.9, 4.0) 2.0 (1.9, 2.0) 0 (50.3, 0)
Data are mean 7SEM (except for variables marked
z
which are median (10th, 90th percentile)) for nine subjects in the fish oil group and 11 in the
placebo group.
Abbreviations: sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cellular adhesion molecule-1; sE, soluble endothelial;
sP, soluble platelet; IL-6, interleukin-6; CRP, C-reactive protein.
Significantly different from placebo group (P¼0.05).
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228224
oxidised by the liver providing no opportunity for them
to enter the systemic circulation. Indeed, there was no
appearance of MCFAs in any of the plasma lipid
fractions studied here. This agrees with the study by
Tholstrup et al. [47], which showed no appearance of 8:0
or 10:0 in plasma PC, TAGs or CEs after consumption
of a diet containing increased amounts of these fatty
acids.
Fish oil induced significant changes in the fatty acid
composition of plasma lipids; there was a marked
increase in the proportion of EPA in PC, CEs and
TAGs. The proportion of DPA was increased in plasma
PC. The increase in EPA and DPA in plasma PC was
accompanied by decreases in the proportions of linoleic
and dihomo-g-linolenic acids, while in plasma CEs only
linoleic acid was significantly decreased. Changes in the
proportion of DHA were more modest than for EPA
and DHA was not significantly increased in the fish oil
group. This most likely reflects the relatively low dose of
DHA provided. Interestingly, and of importance when
considering potential functional effects of n-3 PUFAs,
arachidonic acid was not significantly altered by fish oil
in any of the plasma lipid fractions investigated.
Fish oil did not significantly affect blood pressure.
Although there are reports of modest hypotensive effects
of fish oil (see [48,49] for reviews), these most often are
seen in hypertensive and/or elderly subjects. The
subjects studied here were middle-aged and generally
normotensive and this may explain the lack of effect.
Furthermore, the effect of fish oil on blood pressure,
where observed, is of the order of a 1.5–5% lowering
[48,49]; the current study was not sufficiently powered to
detect an effect of this magnitude.
Fish oil did not influence plasma total, LDL or HDL
cholesterol concentrations. This is consistent with the
now recognised limited effect of long-chain n-3 PUFAs
on cholesterol metabolism when given at modest doses
(see [50,51] for reviews). In contrast, the MCT placebo
oil increased total, LDL and HDL concentrations by
about 18%. This observation contrasts with early
studies, which reported that saturated fatty acids with
a chain length o12 carbons failed to raise serum
cholesterol [50], although it has been recognised that
the effects of these fatty acids have been under-studied
[52]. In fact, it was also documented that, compared
with oleic acid, MCFAs (8:0 plus 10:0, as used in the
current study) result in 11% higher plasma total
cholesterol and 12% higher plasma LDL cholesterol
[47]. Furthermore, Cater et al. [53] described that MCTs
act similar to palmitic acid as compared with oleic acid.
Thus, the findings of the current study agree with those
of Cater et al. [53] and Tholstrup et al. [47].
Fish oil did not cause plasma TAG lowering, as was
expected [51,54]. However, most studies reporting
significant reductions in TAG concentration have used
long-chain n-3 PUFA doses of 4 g/day or more [55–59]
and there are reports that lower doses are much less
effective. For example, Yamamoto et al. [60] reported
that 1.8 g EPA/day was ineffective at lowering TAG
concentrations in patients with angina and elevated
TAG levels. The results of the current study are
consistent with this lack of effect of modest doses of
LC n-3 PUFAs. There is one other important factor to
consider: the oil used in the current study was rich in
EPA and relatively poor in DHA. It is now believed that
DHA is more important for TAG lowering than EPA
[57]. Even so, it has been documented that TAG levels
were unchanged in studies using DHA up to 2 g/day [61]
and in subjects with normal TAG levels at baseline [55].
Thus, reasons for a lack of TAG lowering in the current
study may be too low an intake of long-chain n-3
PUFAs, not enough DHA or the use of normotrigly-
ceridemic subjects.
In the present study, positive effects of long-chain n-3
PUFAs on inflammatory markers were not observed,
except for a significant difference between fish oil and
placebo in terms of percent change from baseline for
sICAM-1. There were no effects of fish oil on the plasma
concentrations of other soluble adhesion molecules, the
cytokine IL-6 or the acute phase protein CRP. Previous
studies have investigated effects of n-3 PUFAs on
adhesion molecule expression on cultured endothelial
cells [30–33], on soluble adhesion molecule [38–41] and
CRP concentrations [62–65], and on inflammatory
cytokine production by isolated cells studied ex vivo
(see [26–29] for references). Some, though not all, of
these studies report anti-inflammatory effects of long-
chain n-3 PUFAs [26–29]. De Caterina et al. [30] found
reduced cytokine-induced expression of VCAM-1,
ICAM-1 and sE-selectin on cultured human endothelial
cells exposed to DHA, but not EPA, although Collie-
Duguid and Wahle [33] reported reduced expression of
both VCAM-1 and ICAM-1 with both EPA and DHA.
Likewise, Hughes et al. [34] found that both EPA and
DHA decreased cytokine-induced ICAM-1 expression
on cultured human monocytes. Miles et al. [38] reported
decreased plasma sVCAM-1 after providing 0.8 g EPA
plus 0.3 g DHA/day to elderly subjects, but not to young
male subjects, for 12 weeks, and there were no effects on
sICAM-1 or sE-selectin concentrations in either young
or elderly subjects. On the other hand, Abe et al. [40]
demonstrated reduction of sICAM-1 concentration after
7 months supplementation with 2.2 g EPA plus 1.8 g
DHA/day in hypertriglyceridemic men. They also found
that more pronounced reduction in sE-selectin with n-3
PUFAs was seen in diabetics than in healthy subjects
and that n-3 PUFAs reduced sVCAM-1 concentrations
in diabetics but not in healthy subjects [40]. Berstad
et al. [66] reported that supplementation with 1.8 g EPA
plus 0.6 g DHA/day decreased sICAM-1 concentrations
in elderly subjects, while Hjerkinn et al. [67] found that
supplementation with 1.5 g EPA plus 0.9 g DHA/day, in
ARTICLE IN PRESS
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219 –228 225
combination with dietary counselling, for 3 years
decreased sICAM-1 concentrations in elderly subjects.
The finding of the current study of an effect of fish oil on
sICAM-1 is in general accordance with some of these
previous studies, although putting all available data
together would suggest that effects are greater with
higher doses of long-chain n-3 PUFAs than used here
and in elderly, hypertriglyceridemic or diabetic subjects.
Studies of fish oil and inflammatory cytokine production
and on CRP concentrations are also equivocal with only
some studies showing effects on IL-6 production
[46,68,69] and on CRP concentrations [62]. Other
studies report no effect of long-chain n-3 PUFAs on
these outcomes [26–29,63–65].
Thus overall, strong anti-inflammatory effects of fish
oil were not seen in the current study although there was
a small decrease in plasma sICAM-1 concentration.
sICAM-1 has been demonstrated to be predictive of
future myocardial infarction [8], and so even the small,
10% decrease observed here may be of clinical
relevance. The dose of total and of individual n-3
PUFAs and subject characteristics seem likely to be
important in determining whether an anti-inflammatory
effect of long-chain n-3 PUFAs is seen or not. More
research is required in order to identify whether EPA or
DHA is the more potent anti-inflammatory n-3 PUFA,
although the stronger negative correlation between
changes in DHA in plasma PC and sICAM-1 concen-
tration, with only a very weak correlation with change in
EPA status, are suggestive that DHA may be more
important than EPA in this regard. In support of this,
Yli-Jama et al. [70] reported that the inverse correlation
between sVCAM-1 concentrations was more prominent
for DHA than for EPA.
In summary, findings from the present study are in
general accordance with previous studies that suggest
that a dose of 2.1 g/day of EPA plus DHA, with the
majority of this being in the form of EPA, is not
sufficient to demonstrate strong effects on cardiovascu-
lar risk factors including inflammatory markers,
although there is a small effect of this dose of n-3
PUFAs on sICAM-1 concentration. There is a need
to establish dose-dependent effects of EPA and
DHA separately and in different population groups. If
findings from this study are applicable to consumption
of fish, then intake at the upper level of the current UK
guideline range [42] may not influence cardiovascular
risk factors in fairly healthy, normolipidemic and
middle-aged males.
Acknowledgements
This research was supported by a grant to PCC from
Heart UK. Capsules were supplied by Equazen Ltd.
References
[1] British Nutrition Foundation, Task Force Report—Cardiovas-
cular Disease: Diet, Nutrition and Emerging Risk Factors,
Blackwell, Oxford, 2005.
[2] R. Ross, Atherosclerosis—an inflammatory disease, N. Engl.
J. Med. 340 (1999) 115–126.
[3] C.K. Glass, J.L. Witztum, Atherosclerosis: the road ahead, Cell
104 (2001) 503–516.
[4] A.J.H. Gearing, W. Newman, Circulating adhesion molecules in
disease, Immunol. Today 14 (1993) 506–512.
[5] N. Morisaki, I. Saito, K. Tamura, J. Tashiro, M. Masuda,
T. Kanzaki, S. Watanabe, Y. Masuda, Y. Saito, New indices of
ischemic heart disease and aging: studies on the serum levels of
soluble intercellular adhesion molecule-1 (ICAM-1) and soluble
vascular cell adhesion molecule-1 (VCAM-1) in patients with
hypercholesterolemia and ischemic heart disease, Atherosclerosis
131 (1997) 43–48.
[6] W.H. Haught, M. Mansour, R. Rothlein, T.K. Kishimoto, E.A.
Mainolfi, J.B. Hendricks, C. Hendricks, J.L. Mehta, Alterations
in circulating intercellular adhesion molecule-1 and L-selectin:
further evidence for chronic inflammation in ischemic heart
disease, Am. Heart J. 132 (1996) 1–8.
[7] P.M. Ridker, C.H. Hennekens, B. Roitman-Johnson, M.J.
Stampfer, J. Allen, Plasma concentration of soluble intercellular
adhesion molecule 1 and risks of future myocardial infarction in
apparently healthy men, Lancet 351 (1998) 88–92.
[8] G.J. Blake, P.M. Ridker, Novel clinical markers of vascular wall
inflammation, Circ. Res. 89 (2001) 763–771.
[9] D. Kromhout, E.B. Bosschieter, C.L. Coulander, The inverse
relationship between fish consumption and 20-year mortality from
coronary heart disease, N. Engl. J. Med. 312 (1985) 1205–1209.
[10] R.B. Shekelle, L. Missell, O. Paul, A.M. Shryock, J. Stamler, Fish
consumption and mortality from coronary heart disease, N. Engl.
J. Med. 313 (1985) 820.
[11] S. Norell, A. Ahlbom, M. Feychling, N.L. Pedersen, Fish
consumption and mortality from coronary heart disease, Br.
Med. J. 293 (1986) 426.
[12] M.L. Daviglus, J. Stamler, A.J. Orencia, D. Morris, R.B.
Shekelle, Fish consumption and the 30-year risk of fatal
myocardial infarction, N. Engl. J. Med. 336 (1997) 1046–1053.
[13] C.M. Albert, C.H. Hennekens, C.J. O’Donnell, U.A. Ajani, V.J.
Carey, W.C. Willett, Fish consumption and risk of sudden cardiac
death, J. Am. Med. Assoc. 279 (1998) 23–28.
[14] F.B. Hu, L. Bronner, W.C. Willett, M.J. Stampfer, K.M.
Rexrode, C.M. Albert, D. Hunter, J.E. Manson, Fish and
omega-3 fatty acid intake and risk of coronary heart disease in
women, J. Am. Med. Assoc. 287 (2002) 1815–1821.
[15] H. Iso, K.M. Rexrode, M.J. Stampfer, J.E. Manson, G.A.
Colditz, F.E. Speizer, C.H. Hennekens, W.C. Willett, Intake of
fish and omega-3 fatty acids and risk of stroke in women, J. Am.
Med. Assoc. 285 (2001) 304–312.
[16] K. He, E.B. Rimm, A. Merchant, B.A. Rosner, M.J. Stampfer,
W.C. Willett, A. Ascherio, Fish consumption and risk of stroke in
men, J. Am. Med. Assoc. 288 (2002) 3130–3136.
[17] R.F. Gillum, M.E. Mussolino, J.H. Madans, The relationship
between fish consumption and stroke incidence: The NHANES I
Epidemiologic Follow-up Study, Arch. Intern. Med. 156 (1996)
537–542.
[18] T.A. Dolecek, Epidemiologic evidence of relationships between
dietary polyunsaturated fatty acids and mortality in the multiple
risk factor intervention trial, Proc. Soc. Exp. Biol. Med. 200
(1982) 177–182.
[19] D.S. Siscovick, T.E. Raghunathan, I. King, S. Weinmann, K.G.
Wicklund, G.J. Albright, V. Bovbjerg, P. Arbogast, H. Smith,
L.H. Kushi, L.A. Cobb, M.K. Copass, B.M. Psaty, R. Lemaitre,
ARTICLE IN PRESS
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228226
B. Retzlaff, M. Childs, R.H. Knopp, Dietary intake and cell
membrane levels of long-chain n-3 polyunsaturated fatty acids
and the risk of primary cardiac arrest, J. Am. Med. Assoc. 274
(1995) 1363–1367.
[20] J.I. Pedersen, J. Ringstad, K. Almendingen, T.S. Haugen, I.
Stensvold, D.S. Thelle, Adipose tissue fatty acids and risk of
myocardial infarction—a case-control study, Eur. J. Clin. Nutr.
54 (2000) 618–625.
[21] C.M. Albert, H. Campos, M.J. Stampfer, P.M. Ridker, J.A.
Manson, W.C. Willett, J. Ma, Blood levels of long-chain n-3 fatty
acids and the risk of sudden death, N. Engl. J. Med. 346 (2002)
1113–1118.
[22] R.N. Lemaitre, I.B. King, D. Mozaffarian, L.H. Kuller, R.P.
Tracy, D.S. Siscovick, n-3 Polyunsaturated fatty acids, fatal
ischemic heart disease, and nonfatal myocardial infarction in
older adults: The Cardiovascular Health Study, Am. J. Clin. Nutr.
77 (2003) 319–325.
[23] P.M. Kris-Etherton, W.S. Harris, L.J. Appel, Fish consumption,
fish oil, omega-3 fatty acids, and cardiovascular disease. American
Heart Association Nutrition Committee, Circulation 106 (2002)
2747–2757.
[24] P.C. Calder, N-3 fatty acids and cardiovascular disease: evidence
explained and mechanisms explored, Clin. Sci. 107 (2004) 1–11.
[25] C. von Schacky, Omega-3 fatty acids and cardiovascular disease,
Curr. Opin. Clin. Nutr. Metab. Care 7 (2004) 131–136.
[26] P.C. Calder, Polyunsaturated fatty acids, inflammation, and
immunity, Lipids 36 (2001) 1007–1024.
[27] P.C. Calder, N-3 polyunsaturated fatty acids and inflammation:
from molecular biology to the clinic, Lipids 38 (2003) 342–352.
[28] P.C. Calder, N-3 polyunsaturated fatty acids, inflammation, and
inflammatory diseases, Am. J. Clin. Nutr. 83 (2006) 1505S–1519S.
[29] P.C. Calder, Polyunsaturated fatty acids and inflammation, Prost.
Leuk. Essent. Fatty Acids 75 (2006) 197–202.
[30] R. De Caterina, M.I. Cybulsky, S.K. Clinton, M.A. Gimbrone,
P. Libby, The omega-3 fatty acid docosahexaenoate reduces
cytokine-induced expression of proatherogenic and proinflamma-
tory proteins in human endothelial cells, Arterioscler. Thromb. 14
(1994) 1829–1836.
[31] B. Khalfoun, G. Thibault, P. Bardos, Y. Lebranchu, Docosahex-
aenoic and eicosapentaenoic acids inhibit in vitro human
lymphocyte-endothelial cell adhesion, Transplantation 62 (1996)
1649–1657.
[32] C. Weber, W. Erl, A. Pietsch, U. Danesch, P.C. Weber,
Docosahexaenoic acid selectively attenuates induction of vascular
cell adhesion molecule-1 and subsequent monocytic cell adhesion
to human endothelial cells stimulated by tumor necrosis factor-
alpha, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 622–628.
[33] E.S.R. Collie-Duguid, K.W.J. Wahle, Inhibitory effect of fish oil
n-3 polyunsaturated fatty acids on the expression of endothelial
cell adhesion molecules, Biochem. Biophys. Res. Commun. 220
(1996) 969–974.
[34] D.A. Hughes, S. Southon, A.C. Pinder, (n-3) Polyunsaturated
fatty acids modulate the expression of functionally associated
molecules on human monocytes in vitro, J. Nutr. 126 (1996)
603–610.
[35] E.A. Miles, F.A. Wallace, P.C. Calder, Dietary fish oil reduces
intercellular adhesion molecule 1 and scavenger receptor expres-
sion on murine macrophages, Atherosclerosis 152 (2000) 43–50.
[36] P. Sanderson, P.C. Calder, Dietary fish oil diminishes lymphocyte
adhesion to macrophage and endothelial cell monolayers,
Immunology 94 (1998) 79–87.
[37] D.A. Hughes, A.C. Pinder, Z. Piper, I.T. Johnson, E.K. Lund,
Fish oil supplementation inhibits the expression of major
histocompatibility complex class II molecules and adhesion
molecules on human monocytes, Am. J. Clin. Nutr. 63 (1996)
267–272.
[38] E.A. Miles, F. Thies, F.A. Wallace, J.R. Powell, T.L. Hurst, E.A.
Newsholme, P.C. Calder, Influence of age and dietary fish oil on
plasma soluble adhesion molecule concentrations, Clin. Sci. 100
(2001) 91–100.
[39] I. Seljeflot, H. Arnesen, I.R. Brude, M.S. Nenseter, C.A. Drevon,
I. Hjermann, Effects of omega-3 fatty acids and/or antioxidants
on endothelial cell markers, Eur. J. Clin. Invest. 28 (1998)
629–635.
[40] Y. Abe, B. El-Masri, K.T. Kimball, H. Pownall, C.F. Reilly,
K. Osmundsen, C.W. Smith, C.M. Ballantyne, Soluble cell
adhesion molecules in hypertriglyceridemia and potential sig-
nificance on monocyte adhesion, Arterioscler. Thromb. Vasc.
Biol. 18 (1998) 723–731.
[41] O. Johansen, I. Seljeflot, A.T. Hostmark, H. Arnesen, The effect
of supplementation with omega-3 fatty acids on soluble
markers of endothelial function in patients with coronary
heart disease, Arterioscler. Thromb. Vasc. Biol. 19 (1999)
1681–1686.
[42] Scientific Advisory Committee on Nutrition/Committee on
Toxicity, Advice on Fish Consumption; Benefits and Risks,
TSO, London, 2004.
[43] British Nutrition Foundation, N-3 Fatty Acids and Health,
British Nutrition Foundation, London, 1999.
[44] G.C. Burdge, P. Wright, A.E. Jones, S.A. Wootton, A method for
separation of phosphatidylcholine, triacylglycerol, non-esterified
fatty acids and cholesterol esters from plasma by solid-phase
extraction, Br. J. Nutr. 84 (2000) 781–787.
[45] P. Yaqoob, H.S. Pala, M. Cortina-Borja, E.A. Newsholme, P.C.
Calder, Encapsulated fish oil enriched in a-tocopherol alters
plasma phospholipid and mononuclear cell fatty acid composi-
tions but not mononuclear cell functions, Eur. J. Clin. Invest. 30
(2000) 260–274.
[46] F.A. Wallace, E.A. Miles, P.C. Calder, Comparison of the effects
of linseed oil and different doses of fish oil on mononuclear
cell function in healthy human subjects, Br. J. Nutr. 89 (2003)
679–689.
[47] T. Tholstrup, C. Ehnholm, M. Jauhiainen, M. Petersen, C.E.
Hoy, P. Lund, B. Sandstrom, Effects of medium-chain fatty acids
and oleic acid on blood lipids, lipoproteins, glucose, insulin,
and lipid transfer protein activities, Am. J. Clin. Nutr. 79 (2004)
564–569.
[48] L.J. Appel, E.R. Miller, A.J. Seidler, P.K. Whelton, Does
supplementation of diet with ‘fish oil’ reduce blood pressure? A
meta-analysis of controlled clinical trials, Arch. Intern. Med. 153
(1993) 1429–1438.
[49] J.M. Geleijnse, E.J. Giltay, D.E. Grobbee, A.R. Donders, F.J.
Kok, Blood pressure response to fish oil supplementation:
metaregression analysis of randomized trials, J. Hypertens. 20
(2002) 1493–1499.
[50] M.B. Katan, P.L. Zock, R.P. Mensink, Dietary oils, serum
lipoproteins, and coronary heart disease, Am. J. Clin. Nutr. 61
(1995) 1368S–1373S.
[51] W.S. Harris, N-3 fatty acids and serum lipoproteins: human
studies, Am. J. Clin. Nutr. 65 (1997) 1645S–1654S.
[52] P.M. Kris-Etherton, S. Yu, Individual fatty acid effects on plasma
lipids and lipoproteins: human studies, Am. J. Clin. Nutr. 65
(1997) 1628S–1644S.
[53] N.B. Cater, H.J. Heller, M.A. Denke, Comparison of the effects
of medium-chain triacylglycerols, palm oil, and high oleic acid
sunflower oil on plasma triacylglycerol fatty acids and lipid and
lipoprotein concentrations in humans, Am. J. Clin. Nutr. 65
(1997) 41–45.
[54] H.M. Roche, Unsaturated fatty acids, Proc. Nutr. Soc. 58 (1999)
397–401.
[55] T. Hamazaki, S. Sawazaki, E. Asaoka, M. Itomura, Y. Mizushima,
K. Yazawa, T. Kuwamori, M. Kobayashi, Docosahexaenoic
ARTICLE IN PRESS
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219 –228 227
acid-rich fish oil does not affect serum lipid concentrations of
normolipidemic young adults, J. Nutr. 126 (1999) 2784–2789.
[56] S. Grimsgaard, K.H. Bonaa, J.B. Hansen, E.S. Myhre, Effects of
highly purified eicosapentaenoic acid and docosahexaenoic
acid on hemodynamics in humans, Am. J. Clin. Nutr. 68 (1998)
52–59.
[57] T.A. Mori, V. Burke, I.B. Puddey, G.F. Watts, D.N. O’Neal, J.D.
Best, L.J. Beilin, 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. Nutr. 71 (2000) 1085–1094.
[58] R.J. Woodman, T.A. Mori, V. Burke, I.B. Puddey, G.F. Watts,
L.J. Beilin, Effects of purified eicosapentaenoic and docosahex-
aenoic acids on glycemic control, blood pressure, and serum lipids
in type 2 diabetic patients with treated hypertension, Am. J. Clin.
Nutr. 76 (2002) 1007–1015.
[59] R. Buckley, B. Shewring, R. Turner, P. Yaqoob, A.M. Minihane,
Circulating triacylglycerol and apoE levels in response to EPA
and docosahexaenoic acid supplementation in adult human
subjects, Br. J. Nutr. 92 (2004) 477–483.
[60] H. Yamamoto, H. Yoshimura, M. Noma, S. Suzuki, H. Kai, T.
Tajimi, M. Sugihara, Y. Kikuchi, Improvement of coronary
vasomotion with eicosapentaenoic acid does not inhibit acetylcho-
line-induced coronary vasospasm in patients with variant angina,
Jpn. Circ. J. 59 (1995) 608–616.
[61] J.A. Conquer, B.J. Holub, Effect of supplementation with
different doses of DHA on the levels of circulating DHA as
non-esterified fatty acid in subjects of Asian Indian background,
J. Lipid Res. 39 (1998) 286–292.
[62] I. Ciubotaru, Y.S. Lee, R.C. Wander, Dietary fish oil decreases
C-reactive protein, interleukin-6, and triacylglycerol to HDL-
cholesterol ratio in postmenopausal women on HRT, J. Nutr.
Biochem. 14 (2003) 513–521.
[63] A. Geelen, I.A. Brouwer, E.G. Schouten, C. Kluft, M.B. Katan,
P.L. Zock, Intake of n-3 fatty acids from fish does not lower
serum concentrations of C-reactive protein in healthy subjects,
Eur. J. Clin. Nutr. 58 (2004) 1440–1442.
[64] T.A. Mori, R.J. Woodman, V. Burke, I.B. Puddey, K.D. Croft,
L.J. Beilin, Effect of eicosapentaenoic acid and docosahexaenoic
acid on oxidative stress and inflammatory markers in treated-
hypertensive type 2 diabetic subjects, Free Radic. Biol. Med. 35
(2003) 772–781.
[65] T. Madsen, J.H. Christensen, M. Blom, E.B. Schmidt, The effect
of dietary n-3 fatty acids on serum concentrations of C-reactive
protein: a dose–response study, Br. J. Nutr. 89 (2003) 517–522.
[66] P. Berstad, I. Seljeflot, M.B. Veierod, E.M. Hjerkinn, H. Arnesen,
J.I. Pedersen, Supplementation with fish oil affects the association
between very long-chain n-3 polyunsaturated fatty acids in serum
non-esterified fatty acids and soluble vascular cell adhesion
molecule-1, Clin. Sci. 105 (2003) 13–20.
[67] E.M. Hjerkinn, I. Seljeflot, I. Ellingsen, P. Berstad, I. Hjermann,
L. Sandvik, H. Arnesen, Influence of long-term intervention with
dietary counseling, long-chain n-3 fatty acid supplements, or both
on circulating markers of endothelial activation in men with long-
standing hyperlipidemia, Am. J. Clin. Nutr. 81 (2005) 583–589.
[68] R. Abbate, A.M. Gori, F. Martini, T. Brunelli, M. Filippini, I.
Francalanci, R. Paniccia, D. Prisco, G.F. Gensini, G.G. Neri
Serneri, n-3 PUFA supplementation, monocyte PCA expression
and interleukin-6 production, Prost. Leukot. Essent. Fatty Acids
54 (1996) 439–444.
[69] T. Trebble, N.K. Arden, M.A. Stroud, S.A. Wootton, G.C.
Burdge, E.A. Miles, A.B. Ballinger, R.L. Thompson, P.C. Calder,
Inhibition of tumour necrosis factor-alpha and interleukin 6
production by mononuclear cells following dietary fish-oil
supplementation in healthy men and response to antioxidant co-
supplementation, Br. J. Nutr. 90 (2003) 405–412.
[70] P. Yli-Jama, I. Seljeflot, H.E. Meyer, E.M. Hjerkinn, H. Arnesen,
J.I. Pedersen, Serum non-esterified very long-chain PUFA are
associated with markers of endothelial dysfunction, Atherosclero-
sis 164 (2002) 275–281.
ARTICLE IN PRESS
H.M. Yusof et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 219–228228
