Atherosclerosis 204 (2009) 476–482
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journal homepage: www.elsevier.com/locate/atherosclerosis
Anti-inflammatory and cardioprotective effects of n-3 polyunsaturated fatty
acids and plant sterols in hyperlipidemic individuals
Michelle A. Micallefa, Manohar L. Garga,b,∗
aNutraceuticals Research Group, School of Biomedical Sciences, Faculty of Health, University of Newcastle, Callaghan, NSW, Australia
bHunter Medical Research Institute, John Hunter Hospital, New Lambton, NSW, Australia
a r t i c l ei n f o
Received 20 May 2008
Received in revised form 22 June 2008
Accepted 11 September 2008
Available online 27 September 2008
Omega-3 fatty acids
a b s t r a c t
Background: Risk factors of cardiovascular disease such as lipid aberrations, hypertension, abdomi-
nal adiposity and elevations in systemic inflammation, are prominent aetiologies in hyperlipidemia.
Supplementation with n-3 PUFA is associated with a reduction in cardiovascular events through its
holesterolemic properties, although their effect on the inflammatory cascade is uncertain. This study
investigated the effect of combined supplementation with n-3 PUFA and plant sterols on cardiovascu-
lar risk factors, blood pressure, body composition, markers of systemic inflammation and overall risk, in
Methods: The study was a 3-week randomised, double-blind, placebo-controlled, 2×2 factorial design,
in four parallel groups. Sixty hyperlipidemic participants were randomised to recieve either sunola oil or
1.4g/d n-3 PUFA capsules with or without 2g plant sterols per day.
tivity C-reactive protein (hs-CRP) was reduced by 39% (P=0.009), tumor necrosis factor-? (TNF-?) by 10%
was increased by 29.5% (P=0.05). Overall cardiovascular risk was reduced by 22.6% (P=0.006) in the
Conclusion: We have demonstrated, for the first time that dietary intervention with n-3 PUFA and plant
sterols reduces systemic inflammation in hyperlipidemic individuals. Furthermore, our results suggest
that reducing inflammation provides a potential mechanism by which the combination of n-3 PUFA and
plant sterols are cardioprotective.
© 2008 Elsevier Ireland Ltd. All rights reserved.
The cardioprotective effects of n-3 polyunsaturated fatty acids
greater fish oil consumption. Several mechanisms have been pro-
implicated in the pathogenesis of atherosclerosis and thrombotic
disease. These include improving vascular reactivity, decreasing
platelet aggregation, lowering plasma triglycerides, decreasing
blood pressure, preventing arrhythmias and reducing inflamma-
Current evidence supports a central role for inflammation in
all phases of the development of atherosclerosis . Circulat-
∗Corresponding author at: Nutraceuticals Research Group, School of Biomedical
Sciences, The University of Newcastle, 305C Medical Sciences Building, Callaghan,
NSW 2308, Australia. Tel.: +61 2 49215647; fax: +61 2 49212028.
E-mail address: firstname.lastname@example.org (M.L. Garg).
ing markers of inflammation, such as C-reactive protein (CRP),
tumor necrosis factor-? (TNF-?), and some interleukins (IL-6, IL-
1) correlate with propensity to develop cardiovascular events. The
relationship between inflammation and plasma lipid aberrations,
as seen in hyperlipidemia, is not as yet known. The tendency of
cardiovascular risk factors to cluster in hyperlipidemic individuals,
this relationship, we examined the changes in inflammatory
markers following plasma lipid modification, by supplementing
the diet with n-3 PUFA and/or plant sterols in hyperlipidemic
Fish oils, rich in the long-chain n-3 PUFA, eicosapentaenoic
acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3),
have hypotriglyceridemic and anti-arrhythmic properties . An
increased consumption of n-3 PUFA results in increased propor-
tions of those fatty acids in immune cell phospholipids, partly at
the expense of arachidonic acid . The functional significance of
this, is that mediators formed from n-3 PUFA are deemed to inhibit
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
M.A. Micallef, M.L. Garg / Atherosclerosis 204 (2009) 476–482
early atherogenic events, by reducing cytokine-induced expres-
sion of pro-atherogenic/inflammatory proteins in the endothelium
. Epidemiological evidence shows the consumption of n-3 PUFA
protects against CVD within Western populations  and has the
vated blood pressure, plasma lipid profile, platelet aggregation and
A recently renewed interest in plant sterols as a ‘nutraceutical’,
stems from its efficacious lipid-lowering property and heightened
by its commercial availability, now readily added to fat spreads,
lower plasma total and low-density lipoprotein (LDL) cholesterol,
by competing with dietary and biliary cholesterol for intestinal
absorption, but research on their effect in the inflammatory pro-
cess, is scarce . Given that patients with elevated inflammatory
levels are at an increased risk of developing diabetes, hypertension
and CVD, the effect of lipid-lowering foods on circulating inflam-
matory markers warrants investigation. The possibility that plant
sterols may improve cardiovascular risk factors is speculative, but
deserves further consideration.
The present study was conducted to evaluate the cardioprotec-
tive effects as measured by improvements in blood pressure, body
composition, markers of systemic inflammation and overall CVD
tation of n-3 PUFA and plant sterols in hyperlipidemic individuals.
2. Subjects and methods
A detailed methodology of this study has previously been
described, along with participant demographics, plasma fatty acid
concentration and plasma lipid profiles .
2.1. Study design and intervention
muted block randomisation, stratified for gender. Two groups took
4g placebo (sunola oil, SO) capsules per day and two groups took
4g tuna oil capsules (NuMega Ingredients Pty Ltd., QLD, Australia)
per day, providing 80mg 20:5n-3 and 280mg 22:6n-3 in each 1g
capsule, in a triacylglycerol form. One of the groups assigned to
each oil treatment also consumed 25g/d of a plant sterol-enriched
spread (Logicol®original) containing 2g/d plant sterols. The four
groups were identified as: placebo (SO; n=15), fish oil (FO; n=15),
plant sterol (SOP; n=15) and fish oil plus plant sterol combi-
nation (FOP; n=15). The study period was 3 consecutive weeks
and participants were instructed to maintain their habitual diet
Capsule containers were labelled with a blind code, so that nei-
being consumed. Participants were instructed to take four capsules
daily with their main meals. The plant sterol spread was provided
as individually portioned tubs (25g each), comprising predomi-
nantly of ?-sitosterol, campesterol and stigmasterol. Participants
ual margarine/butter consumption. Compliance was monitored by
weighing of tubs and capsule count-back before and after the trial
period, regular telephone contact, evaluation of dietary records
(analysed with FoodWorks, Xyris®, QLD, Australia) and analysis of
plasma fatty acid composition.
Sixty participants (male n=27 and female n=33) with
established combined hyperlipidemia, aged 35–70 years were
enrolled from the general community of Newcastle, Australia.
Primary inclusion criteria included plasma total cholesterol
concentration≥6.0mmol/L (231mg/dL), triacylglycerol concen-
events, diabetes mellitus, chronic inflammatory disease, hyper-
tension (≥140/95mmHg) or liver/renal disease, not taking
anti-inflammatory or hypolipidemic medication, not consuming a
plant sterol-enriched spread and/or fish oil supplements, no strong
aversion or any known allergies/intolerances to the foods involved
in the study, and a usual weekly consumption of no more than two
fatty fish meals per week. Nutrient composition of the plant sterol-
enriched spread and participant dietary intake has been reported
Written informed consent was obtained from all participants
prior to commencement. Ethical approval was obtained from the
Human Research Ethics Committee, University of Newcastle and
registered with the Australian New Zealand Clinical Trials Registry
(trial # 00081597).
2.3. Clinical assessments
intervention) where anthropometric measures, cardiovascular
data and fasting blood samples were collected.
2.3.1. Anthropometry and body composition
All anthropometric measures were made with participants
wearing light clothing and no shoes. Body mass index (BMI) was
calculated as weight in kilograms (kg) divided by the square root of
height in meters (m) to the nearest 0.1 using a calibrated balance
beam scale (PCS Measurement, NSW, Australia), and waist to hip
ratio was calculated as waist girth in centimeters (cm) divided by
hip girth (cm). Single frequency bioelectrical impedance (BIA) was
fast and refrained from physical activity and alcohol consumption
24h prior to testing. Body fat mass and fat free mass were recorded
as percentage of total body mass.
2.3.2. Blood pressure and heart rate
Blood pressure and heart rate were measured using an auto-
mated monitor (Microlife BP 3AD1-A, Heerbrugg, Switzerland)
(pressure±3mmHg; pulse±5%) from the supported left arm of
the rested (10min), seated participant. Systolic blood pressure and
diastolic blood pressure were based on the average of two separate
measurements, taken by the investigating researcher.
2.3.3. Cardiovascular disease risk analysis
Ten-year risk of coronary artery disease was measured using
the US National Cholesterol Education Program Adult Treatment
Panel-III algorithm [11,12]. This risk factor sum model is an adap-
tation of the Framingham Study risk equations based on 10-year
risk of hard cardiac points, including coronary artery disease and
myocardial infarction in patients without diabetes mellitus or clin-
ically evident cardiovascular disease. The prediction equation has
taken the form of gender-specific equations, using continuous vari-
ables such as age, total cholesterol level (mmol/L), HDL-cholesterol
level (mmol/L) and systolic blood pressure (mmHg) and smoking
as a dichotomous variable (yes/no). The model is age-adjusted for
cholesterol and smoking status and corrects for treatment of blood
pressure. A risk factor weighting approach is assigned to each vari-
able and when totalled, they correspond to estimates of absolute
10-year risk % .
M.A. Micallef, M.L. Garg / Atherosclerosis 204 (2009) 476–482
Participant characteristics at baseline in the sunola oil (SO), fish oil (FO), SO and plant sterol (SOP), and FO and plant sterol (FOP) groupsa.
SO (n=15) FO (n=15) SOP (n=15) FOP (n=15)
Male, n (%)
Body mass index (kg/m2)
Body fat (%)
Fat free mass (%)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Heart rate (bpm)
26.6 ± 1.0
0.94 ± 0.03
33.6 ± 1.5
66.3 ± 1.5
137 ± 3.9
84.2 ± 2.6
71.0 ± 2.5
2.1 ± 0.1
15.1 ± 2.7
3.2 ± 1.0
3.8 ± 0.4
1.8 ± 0.4
20.3 ± 5.2
26.4 ± 1.3
0.90 ± 0.02
32.3 ± 2.4
67.6 ± 2.4
133 ± 4.0
83.3 ± 2.6
71.4 ± 2.3
1.5 ± 0.2
19.1 ± 5.2
2.1 ± 0.4
3.0 ± 0.2
1.7 ± 0.1
27.2 ± 3.5
27.4 ± 1.4
0.91 ± 0.02
33.1 ± 2.5
66.6 ± 2.5
134 ± 3.7
83.4 ± 2.4
68.6 ± 2.8
1.9 ± 0.1
14.4 ± 3.2
2.9 ± 0.9
2.6 ± 0.1
1.4 ± 0.2
24.9 ± 3.0
27.3 ± 0.9
0.96 ± 0.01
34.2 ± 1.7
65.8 ± 1.7
131 ± 3.8
82.8 ± 2.8
66.5 ± 2.3
1.7 ± 0.2
19.7 ± 4.7
2.9 ± 0.5
3.8 ± 0.5
1.7 ± 0.2
19.6 ± 3.4
hs-CRP: high sensitivity C-reactive protein; TNF-?: tumor necrosis factor-?; IL-6: interleukin-6; LTB4: leukotriene B4.
aValues are reported as means±S.E.M. P-values refer to the variance between treatment regimens (two-way ANOVA).
2.4. Biochemical analysis
2.4.1. Blood sample collection
Fasting (≥10h) blood samples were collected into tubes
pre-coated with EDTA by venipuncture at baseline and post-
intervention. Samples were prepared by centrifuging (Heraeus
Biofuge Stratos) for 10min 3000×g at 4◦C. Plasma, buffy coat and
red blood cell sub-fractions were collected and stored at −80◦C
until further analysis.
2.4.2. Determination of inflammatory markers
Adiponectin was quantitated by sandwich enzyme-linked
immunosorbent assay (ELISA) (SPI-bio, Montigny le Bretonneux,
France). This assay measures total circulating concentration of
adiponectin in the presence of an adiponectin-specific antibody
. Intra- and inter-assay coefficients of variation (CV) were 6.4%
and 7.3%, respectively, and detection range was 0.1–10.0?g/mL.
Plasma leptin levels were determined using a commercial double-
antibody enzyme immunometric assay (EIA) (Cayman Chemical
Company, Ann Arbor, MI, USA). This assay measures the total
amount of leptin present in the sample, independent of the pres-
ence of leptin-binding proteins. Intra- and inter-assay CV were
3.5% and 6.4%, respectively, with a detection limit of 1.0ng/mL.
High sensitivity tumor necrosis factor-? (TNF-?) and interleukin-
6 (IL-6) ELISA kits (R&D Systems, Minneapolis, MN, USA) were
employed with a minimal detectable concentration of 0.106pg/mL
and 0.039pg/mL, respectively, and an intra- and inter-assay CV
of <9%. High sensitivity C-reactive protein (hs-CRP) analysis was
carried out using an immunoturbidimetric method (Hunter Area
Pathology Service, Newcastle, NSW, Australia). Plasma leukotriene
B4 (LTB4) levels were determined by an EIA kit based on the
competition between LTB4and its conjugate (Cayman Chemical
Company, Ann Arbor, MI, USA) . The intra- and inter-assay
CV were 8.3% and 9.7%, respectively, with a detection limit
of 13.0pg/mL. For all variables, samples from one participant
were determined in the same series, to avoid bias due to assay
2.4.3. Plasma fatty acid analysis
The fatty acid composition of plasma lipids was determined
according to a modification in the method of Lepage and Roy ,
using an acetyl chloride methylation procedure, as detailed previ-
ously . Fatty acid methyl esters were quantified using a Hewlett
Packard 6890 gas chromatograph and quantified by comparison
with fatty acid methyl ester standards (Nu Check Prep).
2.5. Statistical methods
Statistical analysis was performed using SPSS, Version 15.0 for
Windows (SPSS, Inc., Chicago). Based on previous estimates of
variance in plasma total cholesterol concentration, 60 participants
provide 80% power at P<0.05 for detection of a 0.60mmol/L (10%)
Significance was set at P-value<0.05. Baseline characteristics
of each group were compared using two-way ANOVA to test
for homogeneity of variance violation. Changes from baseline
were determined using paired samples t-test. The effect of each
treatment on the percentage change on the dependent variable
explored using two-way ANOVA (i.e., SO compared with FO com-
honestly significant difference) were used when significance was
found. This analysis was also used to determine whether there was
a significant main effect of each independent variable (n-3 PUFA or
tion effect was tested between the two independent variables (n-3
PUFA×plant sterol) in their effect on the dependent variable.
3. Experimental results
3.1. Baseline assessment
The average participant age was 55.4±1.0 y, with a BMI of
26.9±0.5kg/m2and a waist-to-hip ratio of 0.93±0.01. No dif-
ferences were observed within and between the four groups at
baseline and post-intervention for all anthropometric measures
3.2. Effect of plant sterol and fish oil intervention
3.2.1. Body composition and blood pressure
Measures of body composition, blood pressure and heart rate
were assessed in all 60 subjects (Table 1). Body composition did
not differ among the groups at baseline, with an average body fat
mass of 26.0±1.2kg (33.3±1.0%) and fat free mass of 50.8±1.2kg
(66.6±1.0%). Participants had an average systolic blood pressure
of 134±1.9mmHg, a diastolic blood pressure of 82.1±1.7mmHg,
and a heart rate of 69.4±1.2bpm at baseline. Consistent with cur-
rent literature, those treated with n-3 PUFA (FO and FOP groups)
tended to exhibit reductions in systolic (3.1±1.7% and 1.5±1.3%)
and diastolic blood pressure (4.13±1.2% and 3.56±1.7%) over the 3
weeks; however, this failed to reach significance. Further analysis
M.A. Micallef, M.L. Garg / Atherosclerosis 204 (2009) 476–482
Fig. 1. Effect of dietary intervention with 4g sunola oil/d (SO), 4g fish oil/d (FO), SO and 2g plant sterols/d (SOP), or FO and 2g plant sterols/d (FOP) on selected inflammatory
markers; (A) CRP, (B) TNF-?, (C) IL-6, (D) LTB4, (E) adiponectin, and (F) leptin. Bars represent percentage change from baseline (means±S.E.M.), following 3 weeks of dietary
supplementation. Statistical analyses were performed using paired samples t-test: *P<0.05,†P<0.01,‡P<0.001 vs. baseline. Between-group differences were analysed using
two-way ANOVA. Where significance was found, Tukey’s HSD post hoc analysis was used for multiple comparisons. Bars without a common letter differ, P<0.05.
for any of these cardiovascular risk factors.
3.2.2. Markers of inflammation
There were no significant differences in inflammatory markers
between each of the four groups at baseline (Table 1). Percentage
change from baseline was examined for each group (Fig. 1A–F).
Plasma inflammatory markers did not significantly change in the
SO group. In the FO group, hs-CRP and TNF-? were significantly
decreased from baseline (2.1±0.4?g/mL to 1.7±0.3?g/mL,
P=0.02 and 3.0±0.2pg/mL to 2.8±0.2pg/mL, P=0.002, respec-
tively). IL-6, LTB4
and leptin decreased (1.7±0.1pg/mL to
whilst adiponectin increased (1.5±0.2?g/mL to 1.7±0.2?g/mL;
were no significant changes in any of the inflammatory markers
in the SOP group. Conversely, for the FOP group there was a sig-
nificant reduction in hs-CRP (2.9±0.5?g/mL to 2.6±0.5?g/mL,
P=0.14;27.2±3.5pg/mL to 24.2pg/mL±3.3
P=0.009), TNF-? (3.8±0.5pg/mL to 3.4±0.5pg/mL, P=0.02),
IL-6 (1.7±0.2pg/mL to 1.5±0.2pg/mL, P=0.009), and LTB4
(19.6±3.4pg/mL to 16.2±2.6pg/mL, P=0.01) and an increase in
adiponectin (1.7±0.2?g/mL to 1.9±0.1?g/mL, P=0.05). There
was no change in leptin.
The change in hs-CRP in the FO and FOP groups were signifi-
cantly different to the SO group (P=0.007, 95% CI, 0.76–100.1 and
P=0.004, 95% CI, −1.6 to 82.7, respectively). No other between-
group differences were found. Post hoc analysis found a significant
main effect of fish oil for hs-CRP and TNF-? (P<0.0001 and P=0.05,
3.2.3. Plasma fatty acid concentration
Percentage fatty acid composition of plasma lipids was anal-
ysed at baseline and post-intervention. There were no differences
at baseline between groups. Total saturated and monounsaturated
(data not shown). Plasma C20:5n-3 was significantly increased in
the FO (1.1±0.1 to 1.5±0.1, P=0.002) and FOP (1.2±0.1 to 2.0±0.1,
M.A. Micallef, M.L. Garg / Atherosclerosis 204 (2009) 476–482
Fig. 2. Percentage change in C20:5n-3 (A) and C22:6n-3 (B) composition of plasma
phospholipids with supplementation of 4g sunola oil/d (SO), 4g fish oil/d (FO), SO
and 2g plant sterol/d (SOP) and FO and 2g plant sterol/d (FOP). Bars represent
percentage change from baseline (means±S.E.M.), following 3 weeks of dietary
supplementation. Analysis was performed using paired samples t-test: *P<0.05,
†P<0.01,‡P<0.001 vs. baseline. Between-group differences were analysed using
two-way ANOVA. Tukey’s HSD post hoc analysis was used for multiple compar-
C22:6n-3, docosahexaenoic acid.
P=0.003) groups (Fig. 2A). In the n-3 PUFA supplemented groups,
a significant increase in C22:6n-3 was observed (FO=2.3±0.2
to 3.5±0.2, P=0.02 and FOP=2.2±0.1 to 4.7±0.2, P<0.0001)
(Fig. 2B). These respective changes demonstrate participant com-
pliance to the n-3 PUFA supplementation.
Furthermore, an exploration of treatment effects on C20:5n-
3 and C22:6n-3 concentration between-groups was undertaken.
Changes in C20:5n-3 in the FOP group was significantly different to
95% CI, 13.7–115.0) groups. Also, changes in C20:5n-3 in the FO
group was significantly different to the SO group (P=0.001, 95% CI,
26.4–127.8). Changes in C22:6n-3 in the FOP group were signifi-
cantly different to that of the SO (P<0.0001, 95% CI, 64.8–160.2),
and SOP (P<0.0001, 95% CI, 49.9–145.4) groups. Changes seen in
the FO group were significantly different to the SO (P=0.001, 95%
CI, 26.9–122.4) and SOP (P=0.008, 95% CI, 12.1–107.6) groups.
Post hoc analysis showed a significant main effect of n-3 PUFA
supplementation on C20:5n-3 and C22:6n-3 plasma concentration
(P<0.0001). Plant sterol consumption also showed a significant
main effect on C22:6n-3 plasma concentration (P=0.04).
3.2.4. Cardiovascular disease risk
Cardiovascular risk, as determined by the NCEP ATP-III model,
showed no significant changes from baseline among the SO
(P=0.08), FO (P=0.10) and SOP (P=0.06) groups (Fig. 3). However,
a significant risk reduction (22.6±5.1%, P=0.006) was found in the
Fig. 3. Effect of dietary intervention with 4g sunola oil/d (SO), 4g fish oil/d (FO), SO
and 2g plant sterols/d (SOP), or FO and 2g plant sterols/d (FOP) on cardiovascular
risk. Bars represent percentage change from baseline (means±S.E.M.), following 3
samples t-test: *P<0.05,†P<0.01,‡P<0.001 vs. baseline.
ferences in percentage change from baseline and post hoc analysis
did not find any fish oil×plant sterol interactions for overall risk.
n-3 PUFA and plant sterols significantly reduces the inflammatory
markers hs-CRP, TNF-?, IL-6 and LTB4and significantly increases
adiponectin in hyperlipidemic individuals. More importantly, the
data demonstrates a 22.6% reduction in overall cardiovascular risk.
Together, these findings suggest that reducing systemic inflam-
mation in hyperlipidemia represents an important mechanism by
which n-3 PUFA and plant sterols confer their putative cardiovas-
We demonstrated a 39% reduction in circulating hs-CRP, 10%
reduction in TNF-?, 10.7% reduction in IL-6, 15.3% reduction in
LTB4and a 29.5% increase in adiponectin levels, in response to
combined n-3 PUFA and plant sterol supplementation for 3 weeks.
C20:5n-3, inhibit early atherogenic events by reducing cytokine
expression of pro-inflammatory proteins in the endothelium, how-
ever, it is not yet clear whether this effect is associated with
individual or combined effects of the two fatty acids . In a
study by Thies et al.  comparing the effects of supplementation
with fish oil (1g/d C20:5n-3+C22:6n-3), highly purified C22:6n-
3 (720mg/d) and a placebo oil on lymphocyte proliferation in
healthy subjects, it was shown that the fish oil combination sig-
nificantly reduced lymphocyte proliferation, while C22:6n-3 alone
had no effect. Another study by Halvorsen et al.  compared
the effects of 3.8g/d C20:5n-3 and 3.6g/d C22:6n-3 on phagocytic
activity of monocytes, reporting no effect. These findings may be
taken to suggest that neither C20:5n-3 nor C22:6n-3 is responsi-
ble for an immunomodulatory effect alone, but required together
for effective treatment. In a more recent study  on purified
fatty acid affected monocyte nor neutrophil phagocytosis, how-
ever, C22:6n-3 did appear to decrease the production of some
inflammatory cytokines. In our study, we supplemented hyper-
lipidemic participants with a C22:6n-3 rich tuna oil, which was
effective in reducing several inflammatory markers. Interestingly,
when plant sterols were combined with n-3 PUFA, a greater anti-
inflammatory effect was seen, whereas plant sterols alone had no
M.A. Micallef, M.L. Garg / Atherosclerosis 204 (2009) 476–482
Although it is hard to speculate on the exact mechanism by
which plant sterols are anti-inflammatory, it should be noted that
plant sterol supplementation did have a significant main effect on
used in this study provided an additional 1.5g/d of n-3 PUFA as
C18:3n-3. The authors speculate that the conversion of C18:3n-3
to C22:6n-3 may be a possible mechanism by which the combina-
tion of the two supplements elicits anti-inflammatory effects. We
acknowledge the relatively short duration of this intervention trial,
and plant sterol supplementation have been highly responsive.
Our current knowledge of the interaction between plant sterols
and inflammatory markers is poor. In a recent study by Clifton et
weeks, compared to a control. In this study, no significant changes
in hs-CRP were found, although there was a modest reduction
trend (P=0.07). In a similar study with hypercholesterolemic men,
a 4-week supplementation period with a plant sterol-enriched
spread (2.0g/d) did not significantly change CRP levels . More
recently, De Jong et al.  provided statin treatment patients with
2.5g/d of plant sterols as a margarine for 16 weeks. No effects were
found for soluble adhesion molecules, CRP or monocyte chemotac-
tic protein-1 concentrations. These studies support our findings of
a non-significant reduction in hs-CRP with 2g/d plant sterols for 3
weeks. Conversely, a study by Devaraj et al.  a median reduc-
tion in hs-CRP of 12% (P=0.02) was found with 2g/d plant sterol
supplementation provided as a reduced-calorie orange juice bev-
erage, perhaps due to large inter-individual variations. The role of
plant sterols in the inflammatory process is largely unknown, how-
and efflux at the phospholipid membrane, there is some degree of
interaction with receptor activity. Potentially these could influence
gene expression of COX-2 and IL-6, thereby having a direct impact
on plasma markers of inflammation .
The relationship between plasma lipids and inflammatory
cytokines, suggests that hyperlipidemia and enhanced inflam-
mation are separate but interactive processes . We have
exploration of the data found no correlation between reductions
in inflammatory markers and reductions in plasma lipid profile
(data not shown), suggesting that the anti-inflammatory effects of
combined n-3 PUFA and plant sterol.
To a large extent, reducing the inflammatory milieu to provide
benefit among cardiovascular risk factors is yet to be fully under-
stood in the context of hyperlipidemia.
It is evident that markers of systemic inflammation such as
hs-CRP, TNF-?, IL-6 and several adipokines are elevated in hyper-
lipidemic individuals . The relevance of this remains in the
setting of primary prevention, as many large-scale studies have
shown baseline levels of such markers can independently predict
all Framingham covariates .
An additional objective of this study was to attenuate a possible
indicate an association between the consumption of fish and rel-
ative risk of sudden death. In the US Physicians Health Study, an
inverse relationship between plasma levels of n-3 PUFA and risk of
sudden death in men without a history of CVD was found . In
the Diet and Reinfarction Trial (DART) a 29% reduction in all-cause
mortality over 2 years in male myocardial infarction survivors was
found, following an increase of oily fish intake (200–400g/week)
. In one of the largest randomised controlled trials, the GISSI-
Prevention Study with 11,324 patients with pre-existing coronary
heart disease randomised to 300mg vitamin E, 850mg n-3 PUFA,
myocardial infarction and non-fatal stroke (P<0.02), compared
with the control.
In our study we showed a 22.6% (P=0.006) reduction in overall
cardiovascular risk using the combination of n-3 PUFA and plant
sterols. To date, this is the first study to investigate the combined
cardioprotective effects of these two functional foods in hyper-
lipidemic individuals with no history of cardiovascular events. We
speculate that the improvement in cardiovascular risk is primarily
representative of subsequent improvements in plasma lipid pro-
file, given we did not see significant changes in blood pressure ,
a risk factor usually affected by n-3 PUFA supplementation .
Dietary supplementation longer than 3 weeks in duration may be
needed to significantly influence other cardiovascular risk factors
such as blood pressure, which merits further investigation.
Since cardiovascular risk factors such as increased inflamma-
tion and elevated plasma lipid levels rarely occur in isolation,
combined therapy with anti-inflammatory agents such as n-3
PUFA and lipid-lowering agents such as plant sterols may pro-
vide greater risk reduction compared to either of the supplements
alone. Collectively, our data is supportive of the cardioprotective
benefits of combined n-3 PUFA and plant sterol supplementation
for hyperlipidemic individuals. It is the first study to demon-
strate overall lipid-lowering benefits, reduce markers of systemic
inflammation and reduce overall cardiovascular risk, using a non-
pharmacological dietary approach. This makes the combined n-3
PUFA and plant sterol therapy an ideal alternative or adjunct to
pharmacological treatment, for maximum cardioprotection in high
Conflict of interest
sible for the conduct of the clinical trial, biochemical analyses, data
collection, statistical analysis and manuscript preparation; MLG
was involved in obtaining funding, the concept development and
planning of the study, the overall supervision of the trial and inter-
pretation and writing of the final draft of the paper. None of the
authors had a personal or financial conflict of interest.
This study was funded in part by a University of Newcastle Pilot
Grant. We would like to thank Miss Melinda Phang (Nutraceuticals
Research Group, University of Newcastle, Australia) for assistance
in the analysis of plasma fatty acids.
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