Fish oil supplementation augments post-exercise immune function in young males.
Gray,Patrick.1; Gabriel,Brendan.1; Thies, Frank.1; Gray, Stuart.R.1;
1
Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD
Corresponding Author:
Dr Stuart Gray
Health Sciences Building
Institute of Medical Sciences
Foresterhill
University of Aberdeen
AB25 2ZD
Tel: 01224 438026
Fax: 01224 437465
Email: s.r.gray@abdn.ac.uk
CONFLICT OF INTEREST STATEMENT
All authors declare that there are no conflicts of interest.
ABSTRACT
Purpose: Fish oils and related fatty acid components have anti-inflammatory properties, with
beneficial effects against various disorders such as cardiovascular disease. A single bout of
exercise can alter immune function. However, the effects of fish oil on immune function after
a single bout of exercise are currently unknown. This study investigated the effect of
supplementation with fish oil on the immune response to an acute bout of endurance exercise.
Methods: Sixteen male subjects underwent a 6 week double blind randomised placebo
controlled supplementation trial involving two groups (fish oil or placebo oil, 3g/day). They
attended for two visits, the first involving a maximal exercise test and the second involving a
.
1-hour bout of endurance exercise on a cycle ergometer at 70% VO2peak. Blood samples were
taken pre-supplementation, pre-exercise (post-supplementation), immediately, 1h and 3h
post-exercise. Samples were analysed for plasma IL-6, eicosapentaenoic acid (EPA),
docosahexaenoic acid (DHA) and cortisol; peripheral blood mononuclear cell (PBMC) IL-2,
IL-4 and IFN-γ production; neutrophil phagocytosis/oxidative burst; and natural killer (NK)
cell cytotoxic activity. Results: Post-supplementation EPA concentration was increased
(p=0.0127) in the fish oil group. At 3h post-exercise PBMC IL-2 (p=0.0067) and NK cell
activity (p=0.0163) was greater in the fish oil compared with the control group. However,
PBMC IL-4 and IFN-γ productions, plasma IL-6 and cortisol concentrations, as well as
neutrophil activity were unaffected by fish oil supplementation. Conclusion: The current
study demonstrates that fish oil supplementation increases PBMC IL-2 production and NK
cell cytotoxic activity in the recovery period after exercise.
Key words: exercise, eicosapentaenoic acid, docosahexaenoic acid, NK cells, neutrophils
INTRODUCTION
Long chain n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA), abundant in oily fish, are beneficial against chronic
inflammatory disease, such as cardiovascular disease (Lemaitre et al., 2003) or diabetes
mellitus (Nettleton and Katz, 2005), as well as neurological disorders, such as Alzheimer’s
disease and dementia (Samieri et al., 2008). Amongst the possible mechanisms of action
through which dietary fish can protect against cardiovascular disease, their anti-inflammatory
properties seem to play a major role (Thies et al., 2003).
PUFAs are essential constituents of cell membranes of immune cells and are precursors of
inflammatory mediators, such as prostaglandins and leukotrienes (for review see (Calder,
2006)). As leukocytes typically contain a high proportion of the n-6 PUFA arachidonic acid
(AA), and low proportions of other PUFAs, AA is normally the major substrate for
eicosanoid synthesis. However, increasing the consumption of EPA or DHA leads to an
alteration of cell membrane composition, with increased incorporation of these fatty acids at
the expense of AA (Yaqoob et al., 2000). Consequently, a reduced amount of proinflammatory AA-derived mediators are generated while a greater amount of, the less potent,
EPA/DHA-derived alternative inflammatory mediators are produced (Calder, 1997).
Evidence from epidemiological studies has revealed an inverse relationship between dietary
fish (rich in EPA and DHA) consumption and systemic C-Reactive Protein levels (Niu et al.,
2006). Furthermore, increased n-3 PUFA consumption has been shown to decrease
lymphocyte PGE-2 production with concurrent increases in IFN-γ production, lymphocyte
proliferation (Trebble et al., 2003) and altered neutrophil and NK cell function (e.g. (Thies et
al., 2001, Varming et al., 1995)). Consequently, Simopoulos et al. (Simopoulos, 2007) has
recommended that athletes should consume approximately 2.0g fish oil daily, although the
immunomodulatory effects of fish oil during, or after, exercise still remains to be fully
elucidated.
Depending on its intensity and duration a single bout of exercise can itself have
immunomodulatory effects, which may provide an opportunity for infectious agents to enter
the body and take hold in the recovering individual (Nieman, 1995). Numerous alterations in
immune function have been noted after exercise, including an increased cytokine production,
a suppression of PBMC IL-2 and IFN-γ production, and variable effects on neutrophil and
NK cell function (for review see (Gleeson et al., 2011)). Post exercise production of
cytokines such as IL-6 can be diminished by increasing n-3 PUFA intake (Meydani, 1991),
although others find no such effect (Nieman et al., 2009, Toft et al., 2000). Furthermore,
higher ex vivo activated peripheral blood mononuclear cell proliferation was reported in fish
oil supplemented swimmers (Andrade et al., 2007). Little else is currently known regarding
the combined effects of fish oil and exercise. Considering the scarcity of such studies
elucidating information on the effect of fish oil on immune function after a single bout of
endurance exercise remains an area requiring investigation.
The aim of the present study was to investigate the effects of six weeks’ supplementation
with EPA-rich fish oil, on markers of immune function (neutrophil function, natural killer
cell cytotoxicity and PBMC Th1/Th2 cytokine production) following an acute bout of
endurance exercise.
METHODS
Subjects
Sixteen males (aged 24 ± 3.8 years, height 182 ± 8.3 cm, weight 79 ± 7.9 kg; mean ± S.D.)
volunteered to participate in the study. All participants were recreationally active but none
were specifically trained. The study was approved by the University of Aberdeen College of
Life Sciences and Medicine Ethics Review Board and participants were made aware of the
aims, risks and potential discomfort associated with the study before providing written
informed consent.
Supplementation
Participants were randomly assigned to either a placebo (n=8) or fish oil (n=8) group.
Capsules were closely matched for both colour and shape and both participants and
investigators were blind to supplementation group. After the preliminary maximal exercise
test, participants in the placebo group consumed 3g of olive oil daily while those in the fish
oil group consumed 3g of fish oil (in triglyceride form: 1.3g EPA, 0.3g DHA and 45I.U. d-α
tocopherol) daily for a six week period. Fish oil capsules were provided by Nordic Naturals.
Olive oil was chosen as the placebo as oleic acid, the main fatty acid present in olive oil, is
ubiquitous. On average, British adults eat between 22-30 g/day of monounsaturated fatty
acids (Department of Health, 2011), mainly as oleic acid, so the supplementation of 3g/day
olive oil would represent a maximum increase of 10% in oleic acid intake. Furthermore, the
plasma fatty acid profile was similar before and after intervention with the placebo. There is
also insufficient evidence indicating that oleic acid has potent anti-inflammatory properties
(Galli and Calder, 2009) which further justify our choice of placebo. However, several
studies, have suggested some anti-inflammatory properties of olive oil, effects likely related
to the relatively high concentration of polyphenolic compounds. These results were mostly
obtained in vitro, or from poorly designed/insufficiently powered in vivo studies using high
levels of supplementation with olive oil extracts or extra virgin olive oil, which was not used
in the current study.
Maximal Exercise Test
Participants performed a maximal exercise test in order to determine workload for the main
trial. For the 24h period prior to each visit, participants were instructed to refrain from the
consumption of alcohol and strenuous exercise. For the same period prior to the maximal
exercise test, participants were asked to record their dietary intake and replicate this prior to
the main trial. A fasting blood sample was collected prior to performing an incremental
maximal exercise test on a cycle ergometer (Lode, Netherlands). Participants cycled at 70
revolutions per minute (rpm) with workload increasing by 30 Watts every minute until
volitional exhaustion. Gas exchange (Medical Graphics, UK) and heart rate (Polar, Finland)
.
.
were monitored throughout the test. VO2peak was taken as the highest VO2 measured over a 30
.
second period and a workload estimated to elicit 70% VO2peak calculated to be used in the
main trial.
Main Trial
After 6 week’s supplementation, fasted blood samples were taken and gas exchange
.
monitored for a 5 minute period. Participants then cycled for 1 hour at 70% of their VO2peak
at a pedal rate of 70 rpm with gas exchange and heart rate monitored every 15 minutes for 5
minute intervals. Immediately on cessation of exercise a post-exercise blood sample was
drawn. During a three hour recovery period, participants remained fasted, and further blood
samples were taken 1h and 3h post-exercise. Throughout the main trial participants were
permitted to consume water ad libitum.
Blood Sampling
Blood samples were drawn from an antecubital vein using a butterfly needle (21G). Blood
was collected in vacutainers coated with Lithium-Heparin (BD, Oxford, UK), for neutrophil
measures and peripheral blood mononuclear cells (PBMC) isolation, and K+EDTA (BD,
Oxford, UK), for separation of plasma. The K+EDTA vacutainer was centrifuged at 800g for
10 minutes at 4°C and plasma stored at -80°C until analysis.
PBMC Isolation
Twenty ml whole blood was mixed with 10ml PBS and layered onto 20ml histopaque
(Sigma-Aldrich, St Louis, USA) before being centrifuged at 400g for 30 minutes at room
temperature. The PBMC layer was collected and mixed with 40ml PBS and centrifuged at
250g for 10 minutes. The supernatant was then removed and the cell pellet reconstituted with
20ml PBS. This washing phase was then repeated. The final pellet was reconstituted with 1ml
RPMI medium and cell numbers determined. 1.5x106 cells, in duplicate, were then incubated
at 37°C and 5% CO2 for 24h with 2mM glutamine, antibiotics, 2.5% autologous plasma and
10 mg/L Con A or RPMI. At the end of incubation, each well was centrifuged at 250g for 10
minutes, the supernatant removed and frozen at -80°C for analysis of PBMC cytokine
production.
PBMC Cytokine Production
All measurements were performed using commercially available multiplex assay kits (R&D
Systems, UK) according to the manufacturer’s instructions.
Neutrophil Function
Neutrophil function was investigated in whole blood using the PHAGOTEST and
BURSTTEST kits (Opregen Pharma, Heidelberg, Germany) according to the manufacturer’s
instructions. For PHAGOTEST, 20μl E. coli bacteria was added to each whole blood sample.
Control samples were placed on ice and test samples incubated in water for 10 minutes.
Immediately following 10 minutes incubation, samples were removed from water bath to stop
phagocytosis. 100μl quenching solution was added to each sample. 3ml washing solution was
added and tubes were centrifuged at 250g for 5 minutes at 4°C. Washing stage was repeated
once. Whole blood was then lysed and fixed by adding 2ml lysing solution and incubated for
20 minutes at room temperature. Tubes were centrifuged again at 250g for 5 minutes at 4°C.
Washing stage was repeated once more. Then 200μl DNA staining solution was added and
tubes incubated for 10 minutes at room temperature and protected from light. Cells were then
analysed within 60 minutes. For BURSTTEST, heparinised whole blood samples were left on
ice to cool. After 5 minutes, 20μl wash solution was added to tube 1 as the negative control,
20μl E. coli bacteria added to tube 2, 20μl of the chemotactic peptide N-formyl-MetLeuPhe
(fMLP)
added to tube 3 as the low control, 20μl phorbol 12-myristate 13-acetate (PMA)
added to tube 4 as high control and all tubes incubated at 37°C for 10 minutes. Subsequently
20μl substrate solution was added to each tube to induce the oxidation phase and tubes were
again incubated at 37°C for 10 minutes. The reaction was stopped by adding 2ml lysis
solution and incubating for a further 20 minutes. Samples were all washed and stained using
200μl DNA staining solution before analysis. Samples were analysed using a FACS calibur
(BD, Oxford, UK) and the data processed using FlowJo software. A gate was set in the red
fluorescence histogram to identify cells with DNA content and neutrophils were identified
using forward and side scatter plots. Percentage phagocytosis and oxidative burst was
recorded as the percentage positive cells on the green fluorescence histogram with mean
fluorescence intensity (Geo Mean) also measured to indicate the number of bacteria that have
undergone phagocytosis or oxidation quantity per leukocyte. For both assays 50,000 cells
were analysed for each sample, allowing us to determine the effects of exercise and fish oil
on a per cell basis.
Natural Killer Cell Cytotoxic Activity
Natural killer cell cytotoxic activity was measured in PBMCs using NKTEST kits (Opregen
Pharma, Heidelberg, Germany) according to the manufacturer’s instruction. Briefly PBMCs
were mixed with pre stained K562 target cells in ratios of 50:1 and 25:1. Tubes were
centrifuged for 3 mins at 120g and then incubated for 2 hours in a CO2 incubator at 37°C. To
the cells 50μl of DNA staining solution was added and cells were incubated in the dark on ice
for 5 mins before analysis by flow cytometry. 2,500 cells were analysed for each sample,
allowing us to determine the effects of exercise and fish oil on a per cell basis. To
discriminate target and effector cells a gate was set in the green fluorescence histogram. The
percentage of dead cells was then determined using the red fluorescence histogram and the
percentage specific cytotoxicity determined by subtracting the percentage dead cells in a tube
containing K562 cells alone from the percentage of dead target cells in the samples.
Plasma IL-6 and Cortisol
All measurements were performed using a commercially available (high sensitivity for IL-6)
ELISA kits (R&D Systems, UK) according to the manufacturer’s instructions.
EPA/DHA Analysis
Plasma total lipids were extracted as described by Bligh & Dyer (Bligh and Dyer, 1959)
before analysis by gas chromatography.
Statistical Analysis
Data analysis was carried out using Prism version 5 software. All data are expressed as mean
± SD. Baseline characteristic data were compared between groups using independent t-test’s.
All other data were analysed using a two way (group and time) repeated measures ANOVA
with bonferroni post hoc tests. Statistical significance was accepted at P <0.05. Data are
presented as mean ± SD.
RESULTS
Baseline characteristics and Performance measures
Subject baseline characteristics are summarised in Table 1. Baseline physical characteristics
.
were similar between the groups. Heart rate and VO2 throughout the 1 hour exercise bout of
the main trial were also similar between the groups.
Plasma EPA and DHA concentrations
The ANOVA to analyse plasma EPA revealed a time (F(3,42)=14.96, P<0.05), and interaction
(F(3,42)=9.61, P<0.05) effect, but no effect of group (F(1,14)=2.55, P=0.14). Post-hoc analysis
demonstrated that plasma EPA increased (P<0.05) in the fish oil group only and was higher
(P<0.05) than in the control group after supplementation. Analysis of plasma DHA showed
an effect of time (F(3,42)=11.72, P<0.05), with post-hoc analysis showing that DHA was
higher (P<0.05) post supplementation in both groups. No interaction (F(3,42)=0.21, P=0.66) or
group (F(1,14)=1.9, P=0.20) effects were observed (Figure 1).
Cytokines and Cortisol
The analysis of plasma IL-6 showed no time (F(3,42)=1.19, P=0.33), group (F(1,14)=0.06,
P=0.80) or interaction (F(3,42)=0.40, P=0.75) effect. There was also no group (F(1,14)=0.17,
P=0.68) or interaction (F(3,42)=0.68, P=0.57) effect with plasma cortisol. There was an effect
of time (F(3,42)=17.42, P<0.05) observed, with post-hoc analysis showing that plasma cortisol
was lower (P<0.05), than baseline, 3h post exercise in both groups (Figure 2).
The ANOVA revealed an interaction (F(3,42)=3.58, P<0.05) and time effect (F(3,42)=10.20,
P<0.05) for PBMC IL-2. Post-hoc analysis revealed that PBMC IL-2 was lower (P<0.05),
than baseline, at 1h post exercise in the control group and higher (P<0.05), than baseline, at
3h post-exercise in the fish oil group. Furthermore 3h post-exercise PBMC IL-2 was higher
(P<0.05) in the fish oil compared to the control group. No group effect (F(1,14)=3.24, P=0.09)
was observed for PBMC IL-2 (Figure 3). Analysis revealed a time effect for PBMC IL-4
(F(3,42)=2.99, P<0.05) with post hoc analysis showing that PBMC IL-4 was greater (P<0.05)
at 3h post exercise, compared with baseline, in the fish oil group (Figure 3). PBMC IL-4
showed no interaction (F(3,42)=0.29, P=0.83) nor group effect (F(1,14)=0.02, P=0.89). The
ANOVA revealed an effect of time for PBMC IFN-γ (F(3,42)=5.76, P<0.05) with post-hoc
analysis revealing that PBMC IFN-γ was lower (P<0.05) 1h post exercise compared with
baseline in both groups. No group (F(1,14)=0.04, P=0.85) or interaction (F(3,42)=0.36), P=0.78)
effect was observed for PBMC IFN-γ.
Neutrophil and NK cell function
The ANOVA revealed no differences in neutrophil phagocytic activity between groups
(F(1,14)=0.07, P=0.78), time (F(3,42)=0.23, P=0.88) and no interaction effect (F(3,42)=1.39,
P=0.25) (Figure 4, upper panel). Similarly, analysis revealed no differences in neutrophil
oxidative burst activity between groups (F(1,14)=0.02, P=0.89), with time (F(3,42)=0.4, P=0.75)
and no interaction effect (F(3,42)=0.31, P=0.82) was observed (Figure 4, lower panel).
Analysis of natural killer cell cytotoxic activity revealed a significant interaction effect at
both 50:1 (F(3,42)=4.48, P=0.0163) and 25:1 (F(3,42)=3.93, P=0.0256) effector:target cell ratios.
Post hoc analysis revealed that at 3h post-exercise NK cell cytotoxic activity was greater in
the fish oil group, compared with the control group, for the effector:target ratio of 50:1
(P<0.05), with a similar trend was observed for 25:1 ratio (P=0.07) (Figure 5). In addition,
there was a trend for a time effect (F(3,42)=2.9, P=0.06) in NK cells at the 50:1 ratio, with NK
cell cytotoxic activity being greater (P<0.05) at 3h post-exercise compared to baseline in the
fish oil group. There was no effect of group for NK cell cytotoxic activity (F(1,14)=0.65,
P=0.45).
DISCUSSION
Our results showed that consumption of 3.0g/day EPA-rich fish oil for six weeks increased
PBMC IL-2 production and NK cell cytotoxic activity in the recovery period after exercise,
compared with the control group. Our supplementation regimen was successful in resulting in
a ~2.5 fold greater plasma EPA concentration in the fish oil group post supplementation.
Unexpectedly, we also found a small ~0.6 fold increase in plasma DHA in both groups after
the supplementation period. There is no clear reason for this increase but due to its relatively
small magnitude, in comparison to the rise in EPA, and its similarity between groups it is
unlikely to have any bearing on our findings of between group differences.
Previous studies have shown that NK cell cytotoxic activity immediately increases after a
bout of endurance exercise, but then quickly decreases below basal levels during the 2 hours
following exercise (Pedersen et al., 1990, Pedersen et al., 1988). These responses were likely
due to changes in NK cell numbers and not to functional changes on a per cell basis (Walsh et
al., 2011), which supports the current findings of no changes in NK cell function measured on
a per cell basis in a fixed number of cells. It has previously been shown that PGE-2 retards
NK cell cytotoxic activity (Hall et al., 1983) in human peripheral mononuclear cells,
collected at rest, and this may explain, at least partly, the beneficial effects of fish oil with
regards to NK cell function observed in the current study. Increasing EPA membrane content
would result in a reduction in PGE-2 production, thus relieving its suppression of NK cell
function. However, previous research investigating the effects of fish oil on NK cell cytotoxic
activity has shown mixed results.
In vitro studies found a reduction of NK cell cytotoxic activity after EPA or DHA treatment
(Purasiri et al., 1997, Yamashita et al., 1986). Furthermore, supplementation of healthy
subjects over 55 years of age with 1.2 g/day EPA plus DHA for 12 weeks decreased NK cell
cytotoxic activity (Thies et al., 2001), an effect also observed by Kelley et al. after
supplementation with 6g/day DHA in young men (Kelley et al., 1999). However, similarly to
our results, NK cell cytotoxic activity tended to increase in healthy young men after
supplementation with increasing amounts of EPA (Miles et al., 2006), suggesting a beneficial
effect in such a population. Taken together with the current data, this suggests that NK cell
function can be suppressed by fish oil in older people and at high doses, with a beneficial
effect at low doses in younger individuals. Further study is, however, warranted to directly
test this assertion.
NK cell activity is stimulated by IL-2 (Henney et al., 1981), with investigations using a rat
model showing that IL-2 stimulates the production of inducible nitric oxide synthase in NK
cells, a major mechanism by which NK cells achieve target cell lysis (Cifone et al., 1999).
Another important finding of the current study was that fish oil supplemented subjects
showed higher PBMC IL-2 production, after exercise, compared with the control subjects.
This increase in IL-2 may also contribute to the greater NK cell cytotoxic activity observed in
the current study. The importance of IL-2 in determining NK cell cytotoxic activity during
and after exercise has previously been highlighted (Shephard et al., 1994)(McFarlin et al.,
2004). Whilst the current study demonstrated that fish oil consumption enhances post
exercise NK cell cytotoxic activity and PBMC IL-2 production, further work is required to
establish a causative link between these two observations.
Previous work has demonstrated that exercise results in a decrease in PBMC Th1 cytokines
(e.g. IL-2 and IFN-γ) with little or no effect on PBMC Th2 cytokines (e.g. IL-4) (Lancaster et
al., 2005, Starkie et al., 2001, Baum et al., 1997). Lancaster et al. (Lancaster et al., 2005)
found that carbohydrate consumption, which amongst other effects suppresses cortisol
production, attenuated the decrease in Th1 cytokine production. As adrenergic stimulation
has been ruled out as the mechanism behind the exercise induced suppression of Th1
cytokines (Starkie et al., 2001) and as cortisol has previously been found to suppress IL-2 and
IFN-γ but not IL-4 (Moynihan et al., 1998) cortisol is currently the most accepted candidate
for exercise induced Th1 cytokine suppression. The current investigation supports these
findings of a Th1 suppression as IL-2 and IFN-γ productions were decreased 1 hour after
exercise. However, in contrast with previous work, we found that PBMC IL-4 production was
slightly greater 3 hours after exercise, indicating a possible Th2 enhancement alongside the
Th1 suppression.
Fish oil consumption had no effect on IL-4 or IFN-γ production but did result in a marked
increase in PBMC IL-2 three hours after exercise, with no differences in cortisol
concentration between the groups. Plasma cortisol concentration at 3h post-exercise did fall
below baseline levels, in both groups, and it is likely due to several factors such as increased
tissue uptake in the post exercise period (Few, 1974) and circadian rhythms (Bailey, 2001). It
has been demonstrated that PGE-2 can suppress the production of Th1 cytokines and
stimulate the production of Th2 cytokines (Fedyk et al., 1997). Therefore a fish oil-induced
reduction of PGE-2 production could be responsible for increasing Th1 and decreasing Th2
responses. Indeed fish oil consumption has previously been shown to increase PBMC IFN-γ
production with no affect on IL-4 (Trebble et al., 2003). On the other hand, contradictory
research by Miles et al. (Miles et al., 2006) found no effect of 2.0 g/day fish oil consumption
on PBMC production of IL-2 or IFN-γ but did report an increase in IL-4. The reason behind
these mixed results is not clear but what is evident from the current investigation is that when
combined with an acute bout of exercise fish oil consumption increases post-exercise PBMC
IL-2 production to levels greater than those seen at baseline.
Plasma IL-6 and neutrophil function were unaffected by the treatment and these findings
agree with previous studies that have shown that fish oil consumption does not affect
neutrophil phagocytosis or oxidative burst in healthy young men (Rees et al., 2006, Kew et
al., 2004, Miles, 2004), although a dose dependent decrease in neutrophil oxidative burst has
been seen in older men (Rees et al., 2006). Fish oil consumption also had no effect on plasma
cytokine concentrations, including IL-6, after a marathon (Toft et al., 2000) or 3 days of
intensive exercise (Nieman et al., 2009).
The current study has demonstrated that the consumption of fish oil can result in an increase
in PBMC IL-2 production and NK cell cytotoxic activity, but what remains to be established
is the clinical significance of these findings. Within the athletic population it has previously
been shown that periods of intense training can increase the incidence of upper respiratory
tract infections (URTI), with the possibility that regular moderate intensity exercise can
reduce URTI incidence (Nieman, 1995). The increase in PBMC IL-2 and NK cell activity
that we have found after fish oil consumption may result in an improved antiviral defence, to
pathogens such as rhinoviruses, and provide some protection against the development of
URTI’s. Indeed it has previously been demonstrated that the severity of respiratory
symptoms, after rhinovirus infection, is associated with a weak Th1 response (Gern et al.,
2000) and that the frequency of the common cold is related to NK cells cytotoxic activity (Xu
et al., 2000). Such a beneficial effect could allow athletes to remain at peak health during
intense training periods. Furthermore the beneficial effects of moderate intensity exercise,
with respect to URTI incidence, may even be further enhanced by fish oil consumption. This
could be of great importance as URTI’s are one of the main causes of GP visits (Graham,
1990) with every adult, on average, suffering between 2 and 5 colds every year (Heath et al.,
1991), making the socioeconomic cost (e.g. lost work days and medical expenses) of these
illnesses quite considerable. Such a hypothesis, however, remains to be tested and
determining the long terms effects of exercise and fish oil on the incidence of URTI therefore
requires further study.
The findings in the current study demonstrate that supplementation with fish oil rich in n-3
PUFAs can enhance aspects of the innate and acquired immune system after exercise and it
would be of clear interest, therefore, to determine whether over the longer term fish oil could
decrease the incidence of URTI associated with intense exercise training.
A possible limitation of the present study may be the inability to directly monitor
participant’s exercise habits during the six week supplementation period. Participants were
asked to maintain ordinary levels of physical activity during this time and they all reported
that they did not change their exercise habits, although there may be errors in such self
reported measures of exercise habits. Furthermore participants were also asked to record and
hand in logs of their pre-trial diet and physical activity and replicate this, but as mentioned
previously this would also be subject to a level of reporting error. To limit any dietary
influences the overnight fast before the main trial was employed.
In conclusion, the current study has shown that six weeks supplementation with fish oil, rich
in EPA and DHA, can increase production of the Th1 cytokine IL-2 and increases natural
killer cell cytotoxic activity, 3h post-exercise, compared with control participants.
ACKNOWLEDGEMENTS
We thank the technical support of Denise Tosh and the Institute of Medical Sciences Flow
Cytometry Facility.
CONTRIBUTORSHIP
SG and FT conceived and designed the study.
PG, SG and BG performed sample collection and analysis.
PG and SG wrote the manuscript.
BG and FT reviewed and amended the manuscript.
FUNDING
This work was supported by a grant from Tenovus Scotland.
TABLE AND FIGURE LEGENDS
Table 1 – Baseline characteristics of subjects in control and fish oil groups. Values are mean
± SD.
Figure 1 – The effect of 6 weeks fish oil or placebo supplementation on plasma EPA and
DHA concentration. * denotes a significant difference compared to the control group
Figure 2 – The effect of 6 weeks fish oil/placebo supplementation on plasma IL-6 can
cortisol before and after exercise. * denotes a significant difference from baseline in both
groups
Figure 3 – The effect 6 weeks fish oil/placebo supplementation on exercise induced Th1/Th2
cytokine production by PBMCs stimulated with Con-A. * indicates significant difference
from baseline values. † indicates significant difference between groups.
Figure 4 – The effect of 6 weeks fish oil/placebo supplementation on phagocytic and
oxidative burst activity of neutrophils before and after exercise.
Figure 5 – The effect of 6 weeks fish oil/placebo supplementation on NK cell cytotoxic
activity at 50:1 and 25:1 effector:target cell ratio, before and after exercise. * indicates
significant difference between groups.
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