Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior
journal homepage: www.elsevier.com/locate/pharmbiochembeh
Omega-3 fatty acids and stress-induced changes to mood and cognition
in healthy individuals
Grace E. Giles ⁎, Caroline R. Mahoney, Heather L. Urry, Tad T. Brunyé, Holly A. Taylor, Robin B. Kanarek
Department of Psychology, Tufts University, Medford, MA 02155, USA
a r t i c l e
i n f o
Article history:
Received 10 November 2014
Received in revised form 23 January 2015
Accepted 19 February 2015
Available online 27 February 2015
Keywords:
Omega-3
Fatty acids
Mood
Emotion
Cognition
Stress
a b s t r a c t
Omega-3 fatty acid (n-3 PUFA) intake is associated with improved mood and cognition, but randomized
controlled trials addressing the causal nature of such relationships are less clear, especially in healthy, young
adults. Stress is one potential mechanism by which n-3 PUFAs may influence mood. Thus the present aim is to
evaluate the influence of n-3 PUFA supplementation on stress-induced changes to mood, cognition, and physiological stress markers in healthy, young adults. Using a double-blind, placebo-controlled design, 72 young adults
were randomized to receive 2800 mg/day fish oil (n = 36, 23 females) or olive oil control (n = 36, 22 females) for
35 days. Subjects completed measures of mood and cognition before supplementation, and two times after
supplementation: following an acute stressor or non-stressful control task. The stress induction was effective
in that the stressor impaired mood, including augmenting feelings of tension, anger, confusion and anxiety,
reduced accuracy on a cognitive task measuring attentional control and the ability to regulate emotion, and
increased salivary cortisol and pro-inflammatory cytokine interleukin-1β (IL-1β). Rated anger and confusion
increased with stress in the olive oil group, but remained stable in the fish oil group. However, fish oil had no
further effects on mood, cognitive function, cortisol, or IL-1β. Fish oil exerted few effects in stressful and nonstressful situations, consistent with findings showing little influence of n-3 PUFA supplementation on mood
and cognition in young, healthy individuals. Potential target populations who would more likely benefit from
increased n-3 PUFA intake are discussed.
Published by Elsevier Inc.
1. Introduction
Polyunsaturated fatty acid (PUFA) levels play a role in neurological
and psychological functions (Dacks et al., 2013; Parker et al., 2006).
Omega-3 (n-3) and omega-6 (n-6) fatty acids are the main constituents
of the PUFA family. n-6 PUFAs include linoleic acid (LA) and arachidonic
acid (ARA) and n-3 PUFAs include alpha linolenic acid (ALA),
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Parker
et al., 2006).
1.1. n-3 PUFAs and mood and cognition
Extant evidence investigating the potential antidepressant effects of
n-3 PUFA supplementation is generally split between studies finding a
beneficial influence in individuals with major depressive disorder and
those reporting null results (for reviews, see Parker et al., 2006;
Martins, 2009; Lin et al., 2010; Giles et al., 2013). Only a few published
empirical studies have assessed the effect of n-3 PUFA supplementation
⁎ Corresponding author.
E-mail addresses: [email protected] (G.E. Giles), [email protected]
(C.R. Mahoney), [email protected] (H.L. Urry), [email protected] (T.T. Brunyé),
[email protected] (H.A. Taylor), [email protected] (R.B. Kanarek).
http://dx.doi.org/10.1016/j.pbb.2015.02.018
0091-3057/Published by Elsevier Inc.
on mood in young, healthy populations. Fontani et al. (2005a,b)
assessed the influence of 2.8 g/day n-3 PUFAs (2:1 EPA:DHA) on mood
using the Profile of Mood States (POMS). They found increased feelings
of vigor and reduced feelings of anger, anxiety, fear, depression, and
confusion in the n-3 PUFA group compared to placebo (Fontani et al.,
2005a,b). However, a more recent study found that 2.3 g/day n-3
PUFA (approximately 7:1 EPA:DHA) reduced feelings of POMS fatigue
only (Antypa et al., 2009).
The majority of research into cognitive effects of n-3 PUFA supplementation has occurred at either end of the lifespan: in infants
and older adults (Giles et al., 2014b). However, less work has examined
n-3 PUFAs and cognition in the middle of the lifespan, when dietary
supplement use may be highest (e.g., Briefel and Johnson, 2004), and
the results are equivocal. In young adults, some studies show that n-3
PUFA supplementation enhanced verbal learning (Karr et al., 2012)
and episodic and working memory (Stonehouse et al., 2013). However,
another study suggests that n-3 PUFA supplementation did not influence cognitive performance across a range of tasks measuring response
inhibition, facial expression recognition, and immediate and delayed recall (Antypa et al., 2009). Further, n-3 PUFA supplementation improved
response inhibition and sustained attention in one experiment (Fontani
et al., 2005b), but not others (Antypa et al., 2009; Hamazaki et al., 1996,
1999; Karr et al., 2012; Stonehouse et al., 2013).
G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
Perhaps contributing to the mood and cognitive effects described
above, additional evidence suggests that n-3 PUFA intake may influence
neural function. In one study of healthy 8–10 year old boys, DHA supplementation increased functional activation in the dorsolateral prefrontal
cortex (DLPFC) during a sustained attention task (McNamara et al.,
2010). In addition, erythrocyte DHA composition was positively correlated with DLPFC activation. The DLPFC plays a role in working memory
(Cohen et al., 1997; Miller et al., 1996) and task switching (Dove et al.,
2000; Konishi et al., 2003) and previous neuroimaging studies indicate
hypo-activation in the DLPFC in individuals with depression compared
to healthy controls (for review, see Koenigs and Grafman, 2009). Thus
n-3 PUFA supplementation may influence DLPFC-associated cognitive
function, including mood and emotion-related cognitive processing.
1.2. n-3 PUFAs and stress
Mixed findings for mood and cognitive effects of n-3 PUFA supplementation may be due, in part, to variations in the degree to which
subjects experienced stress. While some studies evaluated postsupplementation measures during periods of stress (Hamazaki et al.,
1996, 1999), others did not manipulate stress (Antypa et al., 2009;
Fontani et al., 2005a,b). Stress may be one pathway by which n-3
PUFA levels modulate mood and cognition. Stress and depression, as
well as dietary composition akin to the Western diet with high n-6 to
n-3 PUFA ratio, have been shown to influence inflammation through
the same pathways (Kiecolt-Glaser, 2010). For instance, stress and
depression increase pro-inflammatory cytokine production, a broad
class of polypeptide mediators that are secreted by cells of the body,
often as part of an immune response, including interleukin (IL)-1, IL-6,
and tumor necrosis factor (TNF) (O'Brien et al., 2004). In healthy
young adults, three weeks of n-3 PUFA supplementation eliminated
stress-induced cortisol increase and dampened epinephrine increase
in response to an experimentally-induced stressor (Delarue et al.,
2003). To our knowledge, this is the only study to measure the influence
of n-3 PUFA on physiological responses to stress, and suggests that n-3
PUFA supplementation may influence stress-induced changes to mood
in healthy individuals.
Animal studies suggest that n-3 fatty acids mitigate stress-induced
cognitive impairments. n-3 PUFA supplementation in γ-irradiationand cerebral ischemia-damaged rats reduced levels of reactive oxidative
species and number of apoptotic neurons in the hippocampus (Su,
2010). Further, an n-3 deficient diet is associated with learning deficits
and heightened anxiety (Heinrichs, 2010) and n-3 PUFA supplementation in rats prevented anxiety- and depressive-like behaviors and learning and memory deficits induced by stress (Ferraz et al., 2011).
Similarly, cod liver oil-containing DHA and EPA reduced rats' restraint
stress induced impairments on recall and spatial memory (Trofimiuk
and Braszko, 2011). However it should be noted that the relative dosage
in animal studies which is generally higher than that in humans, e.g.,
525–900 mg/kg/day EPA+ DHA in rats (Ferraz et al., 2011; Trofimiuk
and Braszko, 2011) would equal approximately 35.7–61.2 g/day
EPA+DHA in a 68 kg human, well above the recommended .3–.5 g/day
EPA+DHA (Kris-Etherton et al., 2003).
11
The primary objective was to evaluate the influence of n-3 PUFA
supplementation on stress-induced changes in mood. Based on available evidence that n-3 PUFA supplementation improves mood
(Antypa et al., 2009; Fontani et al., 2005a,b) and ameliorates physiological stress responses (Delarue et al., 2003), we expected that n-3 PUFA
supplementation would result in enhanced mood relative to control
supplementation, and that stress would impair mood but that n-3
PUFA would reduce this impact relative to control supplementation.
Preliminary evidence suggests that n-3 PUFA supplementation influences cognitive performance (Fontani et al., 2005b; Karr et al., 2012;
Stonehouse et al., 2013) and associated brain regions, i.e., the DLPFC
(McNamara et al., 2010), therefore a second objective was to determine
the impact of n-3 PUFA supplementation on emotion-associated
cognitive processing. We chose two tasks to evaluate a range of such
processing, including the Emotional Interference Task (EIT; Dolcos and
McCarthy, 2006) and Morphed Faces Task (MFT; Brunyé et al., 2013;
Joormann and Gotlib, 2006; Young et al., 1997), which have been
shown to activate brain regions similar to those influenced by n-3
PUFA supplementation, including the DLPFC (Dolcos and McCarthy,
2006). Given that DHA supplementation enhanced prefrontal activation
associated with cognitive processing, we hypothesized that n-3 PUFA
supplementation would enhance performance on these tasks.
The present study also sought to quantify the interactive effects of
n-3 PUFA and stress on salivary cortisol, a biomarker of arousal
(Hellhammer and Schubert, 2012) and the pro-inflammatory cytokine
interleukin 1β (IL-1β), a biomarker of inflammation (Calder, 2010).
We expected that stress would increase both salivary cortisol and
IL-Iβ, but that this effect would be less pronounced following n-3
PUFA than control supplementation.
2. Methods
Seventy two individuals participated for monetary compensation.
All the participants were in good health, and did not use nutritional supplements or prescription medication other than oral contraceptives.
Written informed consent was obtained, and all procedures were
approved by the Tufts University Institutional Review Board. This
study used a double-blind, mixed-factor, repeated measures design
with Treatment (5 weeks fish oil “FO” or olive oil “OO”) as a betweensubjects factor and Stress (stress; no stress) and Time (pre-; postsupplementation) as within-subjects factors.
2.1. Omega-3 administration
In order to control for taste, FO and OO were administered in capsule
form (developed by Dr. Michael Roberge, RPh, Compounded Solutions,
Monroe, CT). Capsules contained a total of either 2800 mg FO
(1680 mg EPA, 1120 mg DHA) or 2800 mg OO (control), divided into
7 capsules per day. All capsules appeared identical in shape and size.
At the end of the final test session, subjects were asked which treatment
they thought they had received (answer choices: “Fish Oil,” “Olive Oil,”
or “Not sure”). They were then asked if they experienced any side effects
from their treatment (answer choices: “Yes” or “No”), and if “Yes”, to list
these side effects.
1.3. The present study
2.2. Diet record
Preliminary evidence exists for the anti-depressive actions of n-3
PUFA (Giles et al., 2013; Martins, 2009; Parker et al., 2006) and antistress actions in humans (Delarue et al., 2003) and rodents (Ferraz
et al., 2011). However, to date, there have been no published empirical
studies examining the effects of n-3 PUFA supplementation on stressinduced changes in mood and cognitive behavior in young, healthy individuals. Given that the Western diet has shifted towards greater intake
of n-6 PUFAs at the expense of intake of n-3 PUFAs (Kiecolt-Glaser,
2010; Parker et al., 2006), these questions warrant further empirical
study.
Participants reported their dietary intake of foods that contain n-3
and n-6 PUFAs once per week throughout the duration of the study
(Tufts University School of Medicine, Nutrition Infection Unit, 2002).
Foods included fish (e.g., salmon, sardines, tuna), nuts and seeds (e.g.,
almonds, walnuts, flax seed), oils (e.g., walnut oil, soybean oil, flax
seed oil), grains and beans (e.g., soybean oil, tofu) and greens (e.g., spinach, kale, collard greens). One serving size equaled 4 oz (fish and tofu),
1 oz (nuts), 1 tablespoon (oils), and 1/2 cup (soybeans and greens). The
subjects were asked to report the number of servings of each food that
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G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
they had eaten in the past week, and were given examples of serving
sizes for reference (e.g., 4 oz equals a bar of soap; Sugar, 2007).
2.3. Mood measures
2.3.1. Profile of Mood States (POMS) Questionnaire
The POMS is an inventory of subjective mood and arousal states
(McNair et al., 1971). Participants rate a series of 65 mood-related
adjectives on a five point scale, which factor into six subscales: tension,
depression, anger, vigor, fatigue, and confusion as well as a composite
score of total mood disturbance (Lieberman et al., 1996).
displayed the target emotion (during onset trials) or a neutral expression (during offset trials). Upon spacebar press, the face paused on the
current frame, and participants rated the emotional intensity of the
paused face on a scale from 1–7. Half of the trials depicted emotional
expression onset, and half offset. Dependent measures included (1) a
stop frame at which the participant considered the face to express the
target emotion and (2) rating of emotional intensity of stopped face.
Onset and offset trials were equated by subtracting offset stop frames
from 100, such that lower stop frames signify higher sensitivity to the
target emotion.
2.7. Procedure
2.6.2. Morphed Faces Task (MFT)
The MFT involves the dynamic onset and offset of six depicted facial
emotions: anger, disgust, happiness, fear, sadness, and surprise
(i.e., Brunyé et al., 2013; Joormann and Gotlib, 2006; Young et al.,
1997). During each trial, 100-frame animations (at approximately 50
frames per millisecond) of faces either depicted the gradual onset
(from neutral) or the gradual offset (to neutral) of an emotional expression. Participants pressed the spacebar when they believed that the face
0
5
30
Saliva
Time (min)
MFT
2.6.1. Emotional Interference Task (EIT)
The EIT task is a modified Sternberg item recognition paradigm
using visuospatial stimuli of abstract shapes (Erk et al., 2007; Wolf
and Walter, 2005), which measures attentional control and the ability
to regulate emotion. Participants complete a delayed match-to-sample
task, with distracters presented during the delay period (Dolcos and
McCarthy, 2006). During each trial, three visuospatial stimuli appear
during the initial stimulus presentation, and then two scrambled, neutral or negative distracter images are presented immediately prior to a
recognition task. Dependent measures include accuracy and response
time in identifying whether the target visuospatial stimuli presented
after the distracter matches one of the three initial visuospatial stimuli.
EIT
2.6. Cognitive tasks
Saliva
The TSST is a 20-minute psychosocial stress task consisting of 3
stages: (1) 10-minute preparatory stage, (2) 5-minute public speaking
task, and (3) 5-minute mental arithmetic task (Kirschbaum et al.,
1993). It is a commonly used method of effectively, experimentally inducing psychosocial stress (Giles et al., 2014a). The control condition
consisted of a 5 minute speech (about a movie or a book) and 5 min
of mental arithmetic, both completed in an empty room. This control
condition is relatively similar in physical and mental workload but
lacks the stress-inducing components of the TSST (i.e., social evaluative
threat and uncontrollability; Kuhlmann et al., 2005). To ensure the
efficacy of the stress manipulation, subjects were blinded to its true purpose, i.e., to induce stress, and were told that the task was intended to
assess verbal communication ability. Participants were fully debriefed
as to the true intention of the TSST upon completion of the study.
POMS, STICSA
2.5. Stress manipulation: Trier Social Stress Test (TSST)
TSST
Saliva was collected for analyses of salivary cortisol and the proinflammatory cytokine interleukin-1β (IL-1β) through passive drool.
Samples were analyzed in duplicate in an independent laboratory
(Salimetrics LLC, State College, Pennsylvania).
Saliva
2.4. Salivary cortisol and interleukin-1β
Participants completed four sessions on separate days: one practice
session 2–7 days before beginning supplementation to become
familiarized with the experimental procedure and tasks, and three test
sessions, one before supplementation (Day 0) and two after supplementation (Days 34 and 35). The practice and test sessions took place
in the morning; beginning between 0700–0900 h. Start times were
consistent within each participant.
During test sessions, participants completed baseline measures of
the POMS and STICSA-S and provided a saliva sample for salivary cortisol and IL-1β analysis. During the first test session, the participants then
completed the EIT and MFT, followed by a second set of questionnaires
and provision of a saliva sample. After the first test session, participants
consumed FO or OO every day for 35 days. To promote treatment adherence, participants came to the lab each weekday and took the capsules
in front of the experimenter. On weekends, participants took the
capsules on their own, and were required to send a text message to
the experimenter when they did so. Of the 72 subjects, five subjects in
the FO group and six subjects in the OO group missed one day of supplementation over the course of the five-week study, and one subject in the
OO group missed three days, resulting to compliance rates of 99.6% in
the FO group and 99.3% in the OO group.
The final two test sessions took place on the 34th and 35th days
of supplementation and were identical to the first session with the
addition of the stressful or non-stressful TSST, in counterbalanced
order, and a third saliva sample 60 min after the TSST (see Fig. 1 for a
schematics of the procedure). Participants also completed a Cognitive
Reappraisal Task (Urry, 2009) after the EIT and MFT on days 34 and
35, but data will not be reported due to technical problems with the
task. The Cognitive Reappraisal Task involves viewing a series of neutral
and unpleasant images while attempting to either reappraise or maintain their thoughts of the images.
POMS, STICSA
2.3.2. State–Trait Inventory for Cognitive and Somatic Anxiety (STICSA)
The STICSA consists of independent State and Trait scales, each
composed of 21 self-report items (Ree et al., 2008). The scale asks
how participants feel in response to a number of somatic (e.g., dizziness,
fast breathing, clammy palms) and cognitive anxiety symptoms
(e.g., lack of concentration, worry, trouble remembering).
Post-supplementation-only
Fig. 1. Schematic representation of the schedule for each study session. During the study
sessions, participants first completed baseline measures of the Profile of Mood States
(POMS) and State–Trait Inventory for Cognitive and Somatic Anxiety (STICSA-S) and
provided saliva samples for analysis of cortisol and the pro-inflammatory cytokines interleukin-1β (IL-1β). They completed the stressful or non-stressful Trier Social Stress Test
(TSST), followed by the POMS, STICSA-S, and saliva (post-supplementation on days 34
and 35 only). They then completed the Emotional Interference Task (EIT) and Morphed
Faces Task (MFT) and the final saliva sample.
G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
2.8. Statistical methods
3. Results
First, to determine whether the FO and OO groups differed on any
measures prior to supplementation, all pre-supplementation measures
were subjected to univariate analysis of variance (ANOVA)s with
supplementation 2(FO, OO) as the between-subjects factor and alpha
levels adjusted using the Bonferroni correction. The two POMS and
STICSA time-points pre-supplementation were averaged and then
compared.
Change scores were then calculated for each POMS and STICSA
subscale on both the stressful and non-stressful TSST sessions (postTSST − pre-TSST), such that positive values indicate an increase from
pre-TSST and negative values indicated a decrease from pre-TSST. Calculated in this way, change scores account for pre-treatment differences
in the STICSA-S. The POMS and STICSA as well as IL-1β, EIT and MFT
were then analyzed to determine the difference between the stressful
and control post-supplementation sessions using repeated measures
ANOVAs with group 2(FO, OO) as the between subjects factor and stress
2(Stress, Control) as the within subjects factor.
Cortisol was analyzed in a similar manner, with the additional
between-subjects factor of time 3(pre-TSST, 10 min post-TSST,
60 min post-TSST). Because similar studies found that salivary cortisol concentrations had skewed distributions (Schoofs et al., 2008),
we tested for normality using the Lilliefors procedure. Cortisol and
IL-1β data showed a positively skewed distribution, and therefore
were log-transformed. The ANOVAs were performed with the transformed data.
Given that previous studies found inverse relationships between fish
intake and depression in women only (Colangelo et al., 2009;
Sanchez-Villegas et al., 2007; Tanskanen et al., 2001; Timonen et al.,
2004), exploratory analyses also included gender 2(Male, Female) as a
between subjects factor. Unless otherwise noted (see the Salivary
cortisol and interleukin-1β and Morphed Faces Task (MFT) sections),
no gender differences were found (all ps N .13).
An effect was deemed statistically significant if the likelihood of its
occurrence by chance was p b 0.05. When sphericity was violated,
Greenhouse–Geisser corrected p-values were used. When an ANOVA
yielded a significant main effect, post-hoc tests using the Bonferroni
correction were conducted. All statistical analyses were performed
using SPSS 17.0.
3.1. Preliminary analyses
13
3.1.1. Demographic Information
Table 1 shows demographic information, dietary n-3 and n-6 PUFA
intake, and trait cognitive and somatic anxiety for the FO and OO groups.
Groups did not differ on any of the measures (all ps N .14).
3.1.2. Blinding and side effects
Of the 36 subjects in each group, 30 (83%) subjects in the FO and 16
(44%) subjects in the OO group correctly guessed their treatment assignment. Thus, subjects given FO were able to guess their treatment
group above chance. Side effects included fishy burps and aftertaste
(n = 6 FO, n = 0 OO), queasiness, upset stomach and bloating (n = 1
FO, n = 4 OO), loose or discolored stools (n = 0 FO, n = 2 OO) and
headaches (n = 1 FO, n = 1 OO).
3.1.3. Baseline group differences
Table 2 shows baseline between group differences for all mood,
cognitive and physiological measures. No single comparison reached
full or marginal significance with one exception: STICSA state cognitive
anxiety was higher in the FO than that in the OO group.
3.1.4. Stress effects on mood
POMS data reflects 71 subjects (n = 36 FO, 35 OO) as one subject
failed to complete instrumentation following the stressful TSST
(Table 3). Stress increased POMS subscales for tension F(1,70) =
33.468, p b .001 (η2 = .239), depression F(1,70) = 6.838, p b .05
(η2 = .049), anger F(1,70) = 10.865, p b .01 (η2 = .095), confusion
F(1,70) = 17.817, p b .001 (η2 = .150), and total mood disturbance.
More specifically, rated tension increased pre- to post-stressful TSST
t(71) = 5.355, p b .001 (d = .631) but decreased pre- to post-nonstressful TSST t(71) = − 2.308, p b .05 (d = .272) (Stress = 3.65 ±
.68, Non-Stress = − 1.03 ± .44). Rated depression did not change
pre- to post-stressful TSST (p N .51) but decreased pre- to post-nonstressful TSST t(71) = −2.794, p b .01 (d = .329) (Stress = .41 ± .59,
Control = − 1.28 ± .46). Rated anger increased pre- to post-stressful
TSST t(71) = 2.644, p b .05 (d = .312) but decreased pre- to postnon-stressful TSST t(71) = − 2.144, p b .05 (d = .253) (Stress =
Table 1
Mean ± standard deviations or number of participants (n) in each group. p values denote significance using univariate analysis of variance (ANOVA) for continuous variables.
FO
Mean
n (female)
36 (23)
Oral contraceptives (n)
15
Age (years)
BMI (kg/m2)
Exercise (hours per week)
n-3 intake (g/week)
20.80
20.45
3.35
14.62
11.86
10.41
10.35
9.93
8.44
45.23
36.64
35.49
34.86
32.58
35.79
1.45
1.74
n-6 intake (g/week)
STICSA-T
Pre-supplementation
Week 1
Week 2
Week 3
Week 4
Week 5
Pre-supplementation
Week 1
Week 2
Week 3
Week 4
Week 5
Somatic
Cognitive
OO
SD
Mean
SD
p
1.704
.73
.39
13.37
13.61
10.04
12.59
9.07
7.54
31.40
26.28
22.07
23.49
27.16
24.10
0.06
0.10
.528
.243
.296
.445
.805
.568
.767
.894
.874
.143
.341
.149
.408
.484
.253
.145
.735
36 (22)
9
2.386
.73
.39
12.73
10.45
10.10
9.49
10.88
8.30
44.81
33.26
29.22
42.07
43.58
42.61
0.06
0.10
20.49
21.67
2.76
12.22
11.13
9.02
11.16
9.60
8.12
31.60
29.73
24.33
27.97
26.36
25.63
1.34
1.69
No differences in age, body mass index (BMI), habitual exercise, weekly n-3 or n-6 PUFA intake or trait anxiety between the fish oil (FO) and olive oil (OO) groups pre-supplementation.
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G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
Table 2
Between-group (fish oil “FO” versus olive oil “OO”) pre-supplementation differences for all mood, physiological and cognitive measures.
FO
POMS
Tension
Depression
Anger
Vigor
Fatigue
Confusion
Total mood disturbance
Somatic anxiety
Cognitive anxiety
(μg/dL)
(pg/mL)
Negative
Neutral
Scrambled
Negative
Neutral
Scrambled
Happy
Anger
Sad
Surprise
Fear
Disgust
Happy
Anger
Sad
Surprise
Fear
Disgust
STICSA-S
Cortisol
IL-1β
EIT accuracy
EIT reaction time
Morphed Faces intensity rating
Morphed Faces stop frames
OO
M
SD
M
SD
αcritical
p
2.68
2.92
2.61
8.28
9.40
3.21
12.92
14.89
16.57
0.35
212.36
0.87
0.77
0.80
1452.66
1482.18
1407.031
3.10
5.13
2.91
3.12
4.47
5.26
13.84
68.34
90.04
19.44
78.40
11.78
4.13
0.74
0.54
1.07
0.91
0.59
3.11
0.55
0.76
0.04
37.61
0.04
0.03
0.03
51.20
53.27
59.95
0.24
0.15
0.15
0.22
0.14
1.59
2.04
2.71
1.83
2.29
2.80
1.99
1.21
3.49
3.11
9.76
7.76
1.37
7.19
13.92
13.92
0.42
220.04
0.84
0.79
0.76
1457.41
1488.90
1431.13
3.15
5.14
2.61
3.03
4.34
3.02
17.05
69.42
89.40
21.95
77.59
13.70
3.69
0.75
0.55
1.09
0.93
0.59
3.15
0.55
0.76
0.04
37.61
0.04
0.03
0.27
51.20
53.27
59.95
0.23
0.15
0.14
0.22
0.14
1.59
2.04
2.71
1.83
2.29
2.80
1.99
.007
.120
.853
.514
.337
.210
.031
.199
.213
.016⁎
.341
.337
.596
.685
.276
.948
.929
.777
.880
.943
.144
.781
.484
.323
.270
.778
.805
.440
.839
.498
.025
.05
.05
.017
.017
.008
.008
⁎ No single comparison reached full (αcritical = .050/# subscales) or partial significance (αcritical = .025/# subscales), with the exception of the state cognitive anxiety subscale of the
STICSA, in which rated cognitive anxiety was higher in the fish oil (FO) than that in the olive oil (OO) group.
2.04 ± .75, Control = − .89 ± .42). Rated confusion increased pre- to
post-stressful TSST t(71) = 4.088, p b .001 (d = .482) but did not
change pre- to post-non-stressful TSST (p N .07) (Stress = 1.98 ± .48,
Control = − .59 ± .31). The total mood disturbance composite score
increased pre- to post-stressful TSST t(71) = 2.829, p b .01 (d = .333)
but decreased pre- to post-non-stressful TSST t(71) = −3.005, p b .01
(d = .354) (Stress = 6.87 ± 2.39, Non-stress = − 5.06 ± 1.70).
No main effects or interactions were found for rated vigor or fatigue
(all ps N .38).
Stress also increased STICSA somatic F(1,70) = 14.989, p b .001
(η2 = .108) and cognitive anxiety F(1,70) = 4.924, p b .05 (η2 =
.045). Somatic anxiety increased pre- to post-stressful TSST t(71) =
2.299, p b .05 (d = .271) and decreased pre- to post-non-stressful
TSST t(71) = −2.712, p b .01 (d = .320) (Stress = 0.67 ± 0.29, Nonstress = − 0.78 ± 0.27; Table 4). Cognitive anxiety did not change
pre- to post-stressful TSST (p N .35) but decreased pre- to post-nonstressful TSST t(71) = − 3.041, p b .01 (d = .358) (Stress = 0.46 ±
0.49, Non-stress = −0.78 ± 0.26).
3.1.5. Stress effects on cognitive performance
Accuracy on the EIT was lower following the stressful than nonstressful TSST F(1,70) = 5.117, p b .05 (η2 = .013) (Stress = 0.88 ±
0.02, Control = 0.91 ± 0.01). Stress did not influence response time
(p N .2).
Analysis of rated intensity on the MFT revealed an Emotion by Stress
by Gender interaction F(5,330) = 3.838, p b .01 (η2 = .002). For happy
faces F(1,66) = 14.253, p b .001 (η2 = .178) females rated happy faces
as more intense following the stressful TSST (p b .01) whereas males
rated happy faces as more stressful during the non-stressful TSST
(p b .05). No differences were found for other emotional expressions
Table 3
Profile of Mood States (POMS) change score (post–pre-Trier Social Stress Test) means (SEM) for each treatment combination (n = 71; 36 FO, 35 OO).
Stress
Non-stress
FO
Tension
Depression
Anger
Vigor
Fatigue
Confusion
Total mood disturbance
OO
FO
OO
M
SEM
M
SEM
p
M
SEM
M
SEM
p
3.41
−0.38
0.70
−0.68
−1.76
1.32
3.97
0.95
0.83
1.04
0.80
0.65
0.66
3.34
−1.11
1.20
3.37
−.51
−.74
2.63
9.77
0.63
0.85
1.07
0.75
0.66
0.68
3.43
.727
.187
.078
.277
.280
.175
.230
−0.89
−1.622
−1.378
−0.24
−1.70
0.08
−5.32
0.61
0.65
0.60
.727
.607
.433
2.37
−1.11
−.94
−.40
.57
−1.66
−1.20
−4.80
0.63
0.66
0.60
0.82
0.62
0.45
2.44
.800
.467
.248
.958
.796
.052
.878
Stress increased rated tension (p b .001), anger (p b .01), confusion (p b .001), and total mood disturbance (p b .001). Rated anger remained stable in the fish oil (FO) and increased in
the olive oil (OO) group (p b .05) and rated confusion remained stable in the FO group across both stressful and non-stressful TSST but increased in the OO group during the stressful
TSST (p b .05; see Fig. 2). No other main effects or interactions were found between FO and OO group, and no effects were found for rated vigor or fatigue (all ps N .38).
G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
Table 4
State–Trait Inventory of Cognitive and Somatic Anxiety (STICSA) state subscale change
scores (post–pre-Trier Social Stress Test) means (SEM) for each treatment combination
(n = 72; 36 FO, 36 OO).
Stress
Somatic
Cognitive
Table 6
Salivary interleukin-1β (IL-1β) in pg/mL means (SEM) for each treatment combination
(n = 72; 36 FO, 36 OO).
FO
Non-stress
FO
OO
FO
OO
M
SEM
M
SEM
p
M
SEM
M
SEM
p
0.86
0.72
0.41
0.69
0.47
0.19
0.41
0.69
.506
.002
−1.64
−1.00
0.38
0.36
0.08
−0.56
0.38
0.36
.591
.389
Stress increased rated state somatic (p b .001) and cognitive (p b .05) anxiety. Rated state
somatic anxiety increased during the stressful and decreased during the non-stressful
TSST in the fish oil (FO) but not in the olive oil (OO) group (p b .01; see Fig. 3). No other
main effect effects or interaction were found (all ps N .11).
(all ps N .12). For stop frames, i.e., the number of frames passed before
pressing the spacebar, an Emotion by Gender interaction F(5,340) =
6.099, p b .001 (η2 = .012) showed lower stop frames, i.e., higher
sensitivity to emotional expression, to fearful (p b .05) and angry
(p b .01) faces in females than males, and higher stop frames to
surprised (p b .01) and disgusted (p b .05) faces in females than
males. No differences were found for happy (p N .08) or sad (p N .05)
faces. No main effects of Stress or Stress by Emotion interactions were
found (all ps N .30).
3.1.6. Stress effects on cortisol and IL-Iβ
Cortisol data reflects 71 subjects (n = 36 FO, 35 OO) as one subject
failed to provide sufficient saliva (Table 5). Cortisol was higher during
the stressful than non-stressful test session F(1,69) = 9.803, p b .01
(η2 = . 024) (Stress = .38 ± .03, Non-stress = .32 ± .02). Further, cortisol was higher 10-min after the TSST than before or 60-min after the
TSST F(2,138) = 33.653, p b .001 (η2 = . 154) (pre-TSST = .37 ± .03,
10 min post-TSST = .40 ± .02, 60 min post-TSST = .26 ± .02). A
main effect of Gender F(1,67) = 9.614, p b .01 (η2 = .024) showed
that cortisol levels were higher in females than those in males
(Female = .40 ± .03, Male = .27 ± .06). No Time × Stress interactions
were found for cortisol (p N .1).
IL-Iβ (in pg/mL) was higher during the stressful than the non-stressful
test session F(1,70) = 4.140, p b .05 (η2 = . 011) (Stress = 190.96 ±
24.09, Non-stress = 227.27 ± .74.22; Table 6).
3.2. Primary analyses
3.2.1. Does n-3 PUFA supplementation improve mood and reduce the effect
of stress on mood relative to control supplementation?
On the POMS, a main effect of diet Group was found for anger
F(1,70) = 1.949, p b .05 (η2 = .018), in which rated anger did not
change pre- to post-TSST in the FO (p N .50) but increased pre- to
post-TSST in the OO group t(36) = 2.243, p b .05 (d = .379)
(FO = −.34 ± .57, OO = 1.49 ± .59). A Stress × Group interaction for
confusion F(1,70) = 4.345, p b .05 (η2 = .037) showed that feelings of
confusion increased pre- to post-stressful TSST t(34) = 3.866, p b .001
(d = .653) and decreased pre- to post-non-stressful TSST in the OO
15
Stress
Non-stress
OO
M
SEM
M
SEM
p
178.01
305.15
34.08
105.96
203.91
149.40
34.08
104.96
.593
.298
Although ANOVAs were performed on log-transformed data, the table shows raw data.
Salivary IL-Iβ was higher during the stressful than the non-stressful test session (p b .05).
No effects were found for Group (p N .96) or Stress × Group (p N .19).
group t(34) = 4.088, p b .001 (d = .410) but did not change pre- to
post-stressful or non-stressful TSST in the FO (ps N .05) group (Fig. 2).
No other main effects or interactions for diet Group were found for
POMS subscales (all ps N .34).
On the STICSA, Stress × Group interaction for somatic anxiety
F(1,70) = 8.004, p b .01 (η2 = .058) showed that in the FO group,
rated somatic anxiety increased pre- to post-stressful TSST t(35) =
2.069, p b .05 (d = .345) and decreased pre- to post-non-stressful
TSST t(35) = − 3.784, p b .01 (d = .631) but in the OO group did not
change pre- to post-stressful or non-stressful TSST (ps N .25) group
(Fig. 3). No other main effects or interactions were found for diet
Group for somatic or cognitive anxiety (all ps N .11).
3.2.2. Does n-3 PUFA supplementation improve cognition and reduce the
effect of stress on cognition relative to control supplementation?
No effects for diet Group or Stress × Group were found on the EIT or
MFT (all ps N .12).
3.2.3. Does n-3 PUFA supplementation reduce cortisol and IL-1β and reduce
the effect of stress on salivary cortisol and IL-1β relative to control
supplementation?
No effects for diet Group, Time × Group, Stress × Group or
Stress × Time × Group were found for cortisol or IL-Iβ (all ps N .07).
4. Discussion
We evaluated the influence of n-3 PUFA supplementation on stressinduced changes to mood and emotion-related cognitive processing in
healthy young adults. We found that the stress manipulation exerted effects consistent with the stress literature, including impaired mood and
anxiety and increased cortisol and IL-1β. However we detected limited
effects of n-3 PUFA supplementation on mood, cognition, or physiological responses either as a main effect or in interaction with stress.
4.1. n-3 PUFA influence on mood
Stress impaired mood, including augmenting feelings of tension,
anger, confusion and cognitive and somatic anxiety. Rated anger and
confusion increased with stress in the OO group, but remained relatively
stable in the FO group. However, the opposite was true of somatic
anxiety, as rated somatic anxiety increased with stress in the FO but
Table 5
Salivary cortisol means in μg/dL (SEM) for each treatment combination (n = 71; 36 FO, 35 OO).
Stress
Non-stress
FO
Pre-TSST
10 min post-TSST
60 min post-TSST
OO
FO
OO
M
SEM
M
SEM
p
SEM
M
SEM
M
p
0.30
0.39
0.24
0.06
0.05
0.04
0.49
0.53
0.32
0.06
0.05
0.04
.135
.366
.512
0.29
0.31
0.24
0.04
0.03
0.03
0.42
0.38
0.25
0.04
0.03
0.03
.264
.495
.999
Although ANOVAs were performed on log-transformed data, the table shows raw data. Salivary cortisol was higher during the stressful than the non-stressful test session (p b .01) and
higher 10-min after the TSST than before or 60-min after the TSST (p b .001). No differences were found for Group (p N .10), Stress × Time (p N .10), Stress × Group (p N .49), Time ×
Group (p N .07), or Stress × Time × Group (p N .85).
16
G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
Post-Pre
Trier Social Stress Test
4
3
2
1
Fish oil
0
Olive Oil (Control)
-1
-2
-3
-4
Stress
Non-Stress
Fig. 2. Confusion as a function of Stress and Group. Rated confusion increased during the
stressful and decreased during the non-stressful TSST in the olive oil (OO) group
(p b .001) but remained relatively stable in the fish oil (FO) (p = .100) group.
tasks relative to OO supplementation based on previous findings that
n-3 supplementation benefited other cognitive tasks including response
inhibition, sustained attention, and verbal learning (Fontani et al.,
2005b; Karr et al., 2012). However other studies support our null results
finding that n-3 PUFA supplementation had no influence on cognitive
processes on response inhibition, facial expression recognition,
and memory (Antypa et al., 2009; Karr et al., 2012). Unlike mood, inconsistent effects on cognition may be due to differences in dose as well as
the EPA to DHA ratio in fish oil, as fish oil relatively high in DHA content,
e.g., 1:5 EPA:DHA, enhanced performance on the Stroop task measuring
selective attention as response inhibition (Jackson et al., 2012a) and
oxygenation in the prefrontal cortex during the Stroop task (Jackson
et al., 2012b,c). Our 1.5:1 EPA:DHA ratio was chosen based on previous
reviews that suggested that EPA has greater antidepressant efficacy
than DHA (Martins, 2009; Parker et al., 2006), but it may be the case
that our DHA dose was insufficient to produce cognitive benefits.
4.3. n-3 influence on physiological stress response
not the OO group. Thus overall n-3 PUFA supplementation exerted
limited effects on mood and did not support our hypothesis that FO
would improve mood, in stressful or non-stressful circumstances.
These results are generally in line with previous epidemiological
studies and randomized controlled trials. For instance, prospective and
cross-sectional studies assessing the association between n-3 PUFA
intake and depressive mood within the general population are quite
mixed, with some studies finding an inverse association between fish
or n-3 PUFA intake and depressive symptoms (Appleton et al., 2007;
Astorg et al., 2008; Panagiotakos et al., 2010), but others finding no
relationship between n-3 PUFA intake and depressive symptoms
(Hakkarainen et al., 2004; Murakami et al., 2008). Similar to results
from epidemiological studies, those from randomized controlled trials
assessing the influence of PUFA supplementation on depressive symptoms are divided between those reporting beneficial effects (da Silva
et al., 2008; Lesperance et al., 2011; Lucas et al., 2009; Nemets et al.,
2002; Peet and Horrobin, 2002; Su et al., 2003) and no influence
(Carney et al., 2009; Grenyer et al., 2007; Marangell et al., 2003;
Rogers et al., 2008; Silvers et al., 2005). Thus the present study supports
a number of previous findings across epidemiological studies as well as
randomized controlled trials showing limited effects of n-3 PUFA
supplementation on mood in stressful or non-stressful situations.
4.2. n-3 PUFA influence on cognition
Likewise, n-3 PUFA supplementation had no effects on emotionrelated cognitive processing. n-3 PUFA supplementation did not affect
performance on the Emotional Interference Task or Morphed Faces
Task. We predicted that FO would improve performance on these
Rated Somatic Anxiety
4
3
2
1
Fish Oil
0
Olive Oil (Control)
-1
-2
-3
-4
Stress
Non-Stress
Fig. 3. Somatic anxiety as a function of Stress and Group. Rated somatic anxiety increased
during the stressful and decreased during the non-stressful TSST in the fish oil (FO)
(p b .001) but not in the olive oil (OO) group (p N .42).
Previous evidence that n-3 PUFA intake may influence mood via a
mechanism involving inflammation (Sinclair et al., 2007) suggests that
n-3 PUFA supplementation may mediate stress induced changes to
mood, cognition, and physiological stress response. To this end, we
found increased salivary cortisol and IL-1β (Kirschbaum et al., 1993;
Steptoe et al., 2001) but contrary to extant evidence, n-3 supplementation did not influence stress-induced effects on cortisol or IL-1β (Calder,
2006; Delarue et al., 2003). As discussed in the following section, selection of acute rather than chronic stress as well as high baseline n-3 PUFA
consumption may at least partially account for limited n-3 PUFA effects
on HPA activation and inflammation.
4.4. Limitations
A primary limitation to the present study is the lack of erythrocyte
fatty acid analysis. Erythrocyte PUFA levels are associated with, and
may influence, mood changes including depressive symptoms. For instance, higher erythrocyte DHA + EPA and lower AA:EPA levels have
been associated with improved depressive symptoms (Sinn et al.,
2011). This is consistent with recent work from Meyer et al. (2013)
who found that erythrocyte DHA and DHA + EPA, but not EPA alone,
were associated with improved depressive symptoms, in the absence
of a between-group effect of n-3 PUFA versus control supplementation
(Meyer et al., 2013).
A second limitation is the lack of adequate blinding. 83% of the subjects in the FO and 44% of the subjects in the OO group correctly guessed
their treatment assignment. Unlike in the present study in which no
additional oils or flavors were added to either treatment, previous
studies assessing omega-3 influences on mood, particularly depression,
took measures to enhance treatment blinding such as adding lemon or
orange flavor to both fish oil and placebo capsules (Antypa et al.,
2009; Nemets et al., 2002; Rogers et al., 2008; Silvers et al., 2005) and
adding trace amounts of fish oil to placebo capsules (Freeman et al.,
2008; Lesperance et al., 2011; Lucas et al., 2009). Future trials should
keep study blinding in mind, and either add additional oil or flavors to
better equate the aftertaste or fishy burps.
Third, the sample size and supplementation duration may have
curtailed the findings. The sample size and supplementation duration
exceed the majority of similar studies assessing the influence of n-3
PUFA supplementation on mood and cognition in healthy individuals
(Antypa et al., 2009; Fontani et al., 2005a,b; Karr et al., 2012). However
the mean diet Group effect size was small (i.e., mean between-subjects
η2 = .003), indicating that the current sample size may have been too
low to detect significant effects between the FO and OO groups. Elevations in EPA and DHA in red blood cells have been found after 2 and
4 weeks of fish oil supplementation, respectively (Barcelo-Coblijn
et al., 2008), and red blood cell fatty acid levels may correlate to those
G.E. Giles et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 10–19
in the cerebral cortex (Carver et al., 2001). However longer supplementation periods akin to those employed in numerous randomized
controlled trials, e.g., 8–12 weeks (da Silva et al., 2008; Lucas et al.,
2009; Su et al., 2003, 2008) are warranted to allow sufficient time for
elevations in PUFA composition of the cerebral cortex.
Other limitations do not necessarily pertain to the study design but
rather to how best to determine the extent to which n-3 PUFAs influence mood and cognition, particularly in times of stress. First, young,
healthy students may not benefit from n-3 PUFA supplementation as
much as other populations. Mean n-3 PUFA intake ranged from approximately 8–13 g per week among the foods evaluated in the present sample. The American Heart Association recommends eating two servings
of fatty fish per week, in addition to ALA-rich foods such as flax seed,
walnuts and canola oil. Although fish range in n-3 PUFA content according to type and whether they are farm-raised or wild-caught, on the
upper end of the spectrum, two servings of pink salmon provide approximately 2.18 g/week EPA+DHA (Kris-Etherton et al., 2003), meaning that subjects in the present study consumed well over minimum n-3
PUFA intake requirements.
Second, n-3 PUFA supplementation may counteract impairments
due to chronic stress, rather than acute stress as assessed in our study
by using the Trier Social Stress Test. Despite research showing that
acute stressors such as the Trier Social Stress Test indeed increase proinflammatory cytokines, individuals undergoing more chronic-type
stressors which induce longer-lasting elevations in proinflammatory
cytokines may be more apt to benefit from the anti-inflammatory
actions of n-3 PUFA supplementation (Brydon et al., 2005; Steptoe
et al., 2001; Yamakawa et al., 2009).
Finally, OO is most often used as the control treatment in comparison to n-3 PUFA-rich FO in randomized controlled trials. OO is primarily
comprised of monounsaturated fat (MUFA), which may also have
certain health benefits. For instance, higher MUFA intake was associated
with reduced cognitive decline in older adults (Naqvi et al., 2011;
Okereke et al., 2012; Solfrizzi et al., 2006; Vercambre et al., 2010) and
reduced depressive symptoms in older adults (Kyrozis et al., 2009)
Thus comparing FO to OO supplementation may mask some beneficial
effects of FO on mood and cognition.
5. Conclusion
n-3 PUFA supplementation is thought to mediate stress induced
changes to mood, cognition, and physiological stress response. However
we found that fish oil supplementation did little to negate impairments
in mood, emotion-related cognitive processing, and physiological stress
markers, including cortisol and IL-1β. While some aspects of mood,
including anger and confusion, tended to remain more stable in individuals supplemented with fish oil than those supplemented with olive
oil, these moods effects were insufficient to conclude any anti-stress
benefits of n-3 PUFAs.
There is considerable debate in the literature as to whether n-3 PUFA
intake confers any mood or cognitive benefit. A large body of research
has explored the potential antidepressant effects of n-3 PUFA intake,
and findings from epidemiological studies and randomized controlled
trials are mixed (for a review, see Giles et al., 2013). To date most studies
assessing the cognitive effects of n-3 PUFA supplementation have
focused on infants, children and older adults, and only a handful of studies have evaluated the relationship in young adults. Despite lack of clear
evidence that n-3 PUFA intake improves mood or cognitive processing,
multiple methodological limitations in extant literature may mask beneficial effects. Additionally, young healthy individuals such as those in
the present study may not be an ideal target population in addressing
potential mood and cognitive benefits of n-3 PUFA intake. Individuals
who are older, have a mood disorder such as major depressive disorder,
or have initially low n-3 PUFA levels may comprise populations who
could benefit from n-3 PUFA intake. Future research should address
17
methodological limitations limiting extant research, and specify target
populations for whom n-3 PUFA supplementation may be most helpful.
Acknowledgments
The work of Jackie Hayes, Sarah Schuback, Sarah Tower-Richardi and
Winnie Zhuang in the data collection is greatly appreciated.
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