- Email: [email protected]
Dietary omega-3 polyunsaturated fatty acid supplementation in an animal model of anxiety
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
crossmark
Dietary omega-3 polyunsaturated fatty acid supplementation in an animal model of anxiety ⁎
Brian M. Rossa,b, , Imran Malikb, Slim Babayb a b
Northern Ontario School of Medicine, Canada Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada
A R T I C L E I N F O
A BS T RAC T
Keywords: Anxiety Depression Omega-3 fatty acid Alpha-linolenic acid Eicospentaenoic acid Docosahexaenoic acid
A large body of evidence suggests that dietary supplementation with omega-3 fatty acids may ameliorate depressed mood. The magnitude of the effect varies between studies, however, ranging from none at all to being of clinical significance. Given that substantial comorbidity occurs between mood and anxiety disorders, suggesting that they have one or more pathophysiological mechanisms in common, we hypothesized that omega-3 fatty acids may be acting primarily to reduce anxiety rather than depression per se, a possibility which could underlie their variable effects on mood. To test this hypothesis rats were fed for 8 weeks with diets containing one of three types of omega-3 fatty acids, alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid, as well as a low omega-3 fatty acid control diet. Although brain omega-3 fatty acid concentrations were altered by dietary supplementation with eicospentaenoic acid and docosahexaenoic acid, no significant change in anxiety related behaviors were observed compared to the control group as assessed by the elevated-plus maze test. Our data therefore do not support an anxiolytic effect of omega-3 fatty acids and suggest that any effect of these lipids on mood likely occurs by a mechanism unrelated to reducing anxiety.
1. Introduction Omega-3 polyunsaturated fatty acids have been argued to have psychoactive properties [1,2]. The best evidence for such an effect is their proposed antidepressant action in mood disorders [3]. This includes findings that those with major depressive disorder (a type of mood disorder) have lower concentrations of omega-3 fatty acids in blood cells, and that a variety of clinical trials have shown a beneficial effect of dietary supplementation with these lipids on the mood of depressed patients. Such a mood elevating action is of great potential benefit however not all studies report a positive effect (reviewed in references 3–5), and, even in those that do find benefit, the magnitude of response varies widely. The explanation for such variability is unclear but has been suggested either to be that the true effect size is low and unlikely to be of any clinical relevance [5], or that not all omega-3 fatty acids have the same level of efficacy, with some evidence suggesting that of the most three most biologically abundant examples of the type, eicosapentaenoic acid (EPA), is of greater benefit than either alpha-linoleic acid (ALA) or docosahexaenoic acid (DHA) [3,4,6]. An alternative explanation lies in the fact that mood disorders are highly comorbid with anxiety disorders such as generalized anxiety disorder [7] and are amenable to treatment with the same types of
⁎
pharmacotherapies [8,9]. Mood and anxiety disorders may therefore share some pathophysiological mechanisms with a body evidence indicating that chronic anxiety can lead to a higher risk of a depressive episode occuring [10–13]. We hypothesized that omega-3 fatty acids may be actually reducing anxiety in some patients as their primary mechanims of action, and that it was this that was causing the antidepressant effect. Given that this potential confound was not accounted for in the clinical trials, and that the degree of comorbidity varies depending on the characteristics of the population studied [14,15], a primary effect upon anxiety could explain the variability of response noted in the literature [16,17]. The possibility that omega-3 fatty acids have anxiolytic effects is one which has not been extensively examined although social phobia is associated with reduced cellular omega-3 PUFA concentrations, patients with comorbid anxiety and depression exhibit a greater depletion in cellular omega-3 PUFA concentration than those with depression alone, and omega-3 PUFA supplementation reduced anxiety in both test-takers, and in substance users [18–21]. To investigate the role of omega-3 fatty acids in anxiety we have used an animal model, the elevated plus maze test, a model which is responsive to existing anxiolytic medications [22]. The test makes use of the conflict between the animal's fear of being in open areas versus their desire to explore novel environments. The elevated
Correspondence to: MS3002, Division of Medical Sciences, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada, P7A5E8. E-mail address: [email protected] (B.M. Ross).
http://dx.doi.org/10.1016/j.plefa.2016.09.004 Received 1 June 2016; Accepted 28 September 2016 0952-3278/ © 2016 Published by Elsevier Ltd.
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
chromatograph, with a 60-m DB-23 capillary column (0.32 mm internal diameter), and quantified using flame ionisation. Results are expressed as the relative concentration of each fatty acid species.
plus maze has both enclosed arms which provide security and open arms which have exploratory value. When more anxious animals prefer the enclosed areas, whereas when they are less anxious they will enter the open arms more often. We therefore observed animals tested using the elevated plus maze after being fed with a diet supplemented with either ALA, EPA, or DHA, or an omega-3 fatty acid free control diet.
3. Results 3.1. Brain lipids
2. Materials and Methods
Animal were fed a diet which was either omega-3 fatty acid free or supplemented with one of ALA, EPA or DHA. The results of the fatty acid analysis of brain conducted after the intervention have previously been described in detail [25] and are summarized in Table 2 with liver fatty acids analysis being shown for the purpose of comparison. Omega-3 supplementation resulted in much larger changes in liver fatty acid composition than that in brain. Nevertheless, supplementation with DHA resulted in higher brain DHA, total omega-3 fatty acids concentrations and omega-3/omega-6 fatty acids ration, and lower arachidonic acid and total omega-6 fatty acid concentrations compared to the control group. The brain contained very little ALA and EPA with ALA supplementation having no effect on brain ALA or arachidonic acid concentrations. EPA supplementation did result in small increase in brain EPA concentration, and a somewhat larger increase in total omega-3 fatty acids, omega-3/omega-6 ratio, and a decrease in arachidonic acid and total omega-6 fatty acid concentration compared to the control diet, although the magnitude of response (except for decreased arachidonic acid concentration) was less than that observed
2.1. Dietary intervention Animal experimentation was carried according to an animal utilization protocol approved by the Lakehead University animal care committee. Male Wistar rats were allowed to acclimatize to the lab environment for 2 weeks after arrival. They were randomly assigned into pairs and placed into cages with free access to food and water. Rats were fed for 8 weeks on supplementation diets containing 90% (by weight) rat chow containing all required nutrients except for fats (TD99159 basal mix, Harlan Laboratories) plus 10% added fat. The fat component for the control group was exclusively palm oil (New Directions Aromatics, Canada), containing (as per our own analysis) 1.2% (mole %) 14:0, 42.2% 16:0, 3.5% 18:0, 41.9% 18:1, and 11.2% 18:2, n-6, while other groups received palm oil plus omega-3 fatty acids (“95% purity” preparations from Equatech, UK which contained as per our own analysis over 97% of the described fatty acid) in the ratio 9 parts palm oil to 1 part omega-3 fatty acid as described in Table 1. Food was prepared ahead of time and stored in canisters at −20 °C until use. Body weight was monitored throughout the experiment and did not differ significantly (ANOVA; P > 0.05) between the dietary groups.
Table 1 Composition of test diet. Component
2.2. Elevated plus maze
a
Added fat Caesin DL-Methionine Sucrose Cellulose Calcium monohydrogen phosphate Choline bitartrate Calcium carbonate Potassium dihydrogen phosphate Potassium citrate monohydrate Sodium chloride Potassium sulphate Magnesium oxide Ferric citrate Zinc carbonate Manganese carbonate Copper carbonate Potassium iodate Sodium selenate Ammonium paramolybdate, tetrahydrate Sodium meta-silicate, nonahydrate Chromium potassium sulphate, dodecahydrate Lithiun chloride Boric acid Sodium fluoride Nickel carbonate hydroxide, tetrahydrate Ammonium meta-vanadate Niacin Calcium pantothenate Pyroxidine hydrochloride Thiamin hydrochloride Riboflavin Folic acid Biotin Vitamin B12 (0.1% in mannitol) Vitamin E, DL-alpha tocopheryl acetate (500 IU/g) Vitamin A palmitate (500,000 IU/g) Vitamin D3, cholecalciferol (500,000 IU/g) Vitamin K1, phylloquinone
The maze was purchased from Harvard Apparatus (Massachusetts, USA). It consists of a cross, lifted 660 mm above the ground, with a central portion surrounded by 4 black coloured arms of length 450 mm and width 100 mm. Two arms have 500 mm high opaque grey walls (closed arms) enclosed at one end and open to the central area at the other, while the other two arms have 100 mm clear walls (to prevent falls) and are open at each end. A video camera was suspended above the apparatus to record activity. The movement of animals was analysed using the SMART video analysis software provided by Harvard Apparatus to produce the following measures: relative time spent in the open arms, closed arms and central areas of the maze, and the total number of entries by animals into these three areas. On the day of testing animals were transferred from their housing area to the procedure room and left in the room for 30 min prior to the start of the experiment. Each animal was then transferred from their cage into the central area of the apparatus and the investigator left the room. Rats were recorded for 10 min with the first two minutes being an acclimatisation period followed by data recording for a further 8 min. 2.3. Fatty acid analysis One day after completion of the intervention the rats were euthanized by first anaesthetizing with isoflurane gas followed by decapitation using a small animal guillotine. The brain and liver were rapidly removed and tissue stored in a −80 °C freezer until required. Fatty acids were analysed essentially as described previously [23]. Briefly, the entire organ was ground in liquid nitrogen using a mortar and pestle and approximately 300 mg of tissue powder was homogenized in ice cold water using a Polytron homogenizer. Lipids were then extracted from the samples according to the method of Bligh & Dyer [24] in the presence of the internal standard tritridecanoin. Fatty acid methyl esters were prepared using boron trichloride in methanol, and heating the methylation tubes in a boiling water bath. The resulting fatty acid methyl esters were analysed on a Varian 3400 gas-liquid
Weight per kg 100 g 181.9 g 2.84 g 636.11 g 47.37 g 3.32 g 2.38 g 11.84 g 6.5 g 2.35 g 2.45 g 1.55 g 0.81 g 0.2 g 54.72 mg 20.89 mg 10.28 mg 0.33 mg 0.34 mg 0.27 mg 48.09 mg 9.12 mg 0.58 mg 2.7 mg 2.11 mg 1.05 mg 0.22 mg 28.43 mg 15.16 mg 6.63 mg 5.69 mg 5.69 mg 1.9 mg 0.19 mg 23.69 mg 142.16 mg 7.58 mg 1.9 mg 0.71 mg
a The added fat contained either 100 g palm oil or 90 g palm oil plus 10 g omega-3 fatty acid.
18
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
Table 2 Composition of esterified fatty acids in the brain and liver following dietary intervention. Fatty acid or summary measure
ALA Arachidonic EPA DHA Total omega−3 Total omega−6 omega−3 / omega−6 Total saturated Total monosaturated Total polyunsaturated
Liver
Brain
Ctrl
ALA
EPA
DHA
Ctrl
ALA
EPA
DHA
0.1 ± 0.0 15.2 ± 2.7 0.1 ± 0.1 4.3 ± 0.9 4.8 ± 0.8 23.9 ± 3.1 0.20 ± 0.04 37.6 ± 3.4 33.6 ± 4.1 28.7 ± 2.1
1.3 ± 0.4* 10.1 ± 2.3* 2.1 ± 0.4** 7.1 ± 2.2* 11.8 ± 1.8*** 20.2 ± 3.4 0.59 ± 0.10*** 38.7 ± 3.9 28.9 ± 3.2* 32.0 ± 3.4
0.2 ± 0.1 6.1 ± 0.9** 7.8 ± 2.7*** 10.1 ± 0.8** 18.1 ± 2.4*** 18.6 ± 2.8*** 0.97 ± 0.18*** 38.8 ± 3.1 26.7 ± 3.2*** 34.6 ± 4.0*
0.2 ± 0.3 5.7 ± 0.7** 4.2 ± 1.1** 15.3 ± 2.6*** 20.7 ± 2.6*** 15.5 ± 2.3*** 1.34 ± 0.21*** 40.9 ± 3.2** 23.2 ± 3.0*** 36.2 ± 3.9***
0.0 ± 0.0 9.6 ± 0.6 0.0 ± 0.0 11.9 ± 0.9 12.0 ± 1.2 14.4 ± 0.9 0.83 ± 0.09 43.1 ± 6.0 30.4 ± 2.8 26.4 ± 1.9
0.0 ± 0.0 9.3 ± 0.5 0.0 ± 0.0 12.8 ± 0.5 13.1 ± 0.9 13.5 ± 0.6 0.97 ± 0.10 43.7 ± 3.2 29.4 ± 3.7 26.6 ± 2.2
0.0 ± 0.0 8.2 ± 0.9* 0.2 ± 0.1* 12.6 ± 1.0 13.5 ± 1.0* 12.9 ± 1.2* 1.05 ± 0.12** 44.4 ± 4.6 30.4 ± 2.2 26.6 ± 2.4
0.0 ± 0.0 8.2 ± 0.5* 0.1 ± 0.1* 13.7 ± 0.6* 14.2 ± 0.8*** 12.0 ± 1.0*** 1.18 ± 0.14*** 43.0 ± 5.2 30.1 ± 3.3 26.2 ± 1.9
Values shown are mean (n =8) liver fatty acid relative concentrations (%) ± SD after 8 weeks of dietary intervention and are summary data taken from reference 25. *P < 0.05, **P < 0.01, and ***P < 0.005, as compared to palm oil control group (Ctrl) using a post-ANOVA Tukey test. Ctrl = palm oil control, ALA = palm oil plus alpha-linolenic acid; EPA = palm oil plus eicosapentaenoic acid, DHA = palm oil plus docosahexaenoic acid. Table 3 Effect of dietary intervention on time spent and entries into each section of the elevated plus maze. Group
Open arm
Closed arm
Central area
Time (%)
Entries
Time (%)
Entries
Time (%)
Entries
Ctrl ALA EPA DHA
6.7 7.2 7.6 7.1
3.8 3.3 3.5 4.1
80.7 75.3 78.9 78.0
12.5 14.0 10.4 14.4
14.2 20.5 12.8 17.8
13.6 16.0 13.4 15.3
One way ANOVA by group
F3,28 0.84
± ± ± ±
4.4 3.9 2.6 2.5 P value 0.519
± ± ± ±
F3,28 0.33
1.4 2.6 1.3 1.8 P value 0.807
± ± ± ±
F3,28 0.93
8.1 13.5 9.5 21.5 P value 0.441
± ± ± ±
F3,28 1.91
4.3 3.0 3.7 3.7 P value 0.150
± ± ± ±
F3,28 0.75
11.4 10.1 10.8 7.8 P value 0.532
± ± ± ±
F3,28 0.52
4.8 4.1 6.2 4.4 P value 0.670
Animals received one of four diets for 8 weeks (Ctrl – palm oil control; ALA – palm oil plus alpha-linolenic acid; EPA – palm oil plus eicosapentaenoic acid; DHA – palm oil plus docosahexaenoic acid) prior to testing in the elevated plus maze. Data are shown as mean ± SD of the number of entries or percentage time spent into each part of the apparatus. The results of the one way analysis of variance by group are shown as F values and statistical significance (P value). Note that there were no statistically significant differences between the dietary groups for any of the measures.
evidenced by the very large changes in liver lipid concentrations induced by the different diets, making it unlikely that a higher dosage would have resulted in a different finding. A second limitation is that only a single supplementation time period was used. We cannot rule out that a behavioural effect would have been observed using shorter or longer supplementation times. It should be noted however, that the time used (8 weeks) is typical of that used in human clinical trials designed to test omega-3 fatty acids efficacy in various disorders of mental health [3]. Lastly we only have investigated the effect of omega3 fatty acids using the elevated-plus maze; other models are available such as the open field test. Preliminary work in our laboratory using the latter model, however, also suggested that omega-3 fatty acids have no effect on anxiety state in rats (unpuplished observations). Despite these limitations our data are certainly not supportive of an anxiolytic effect of omega-3 fatty acids although further work is required to confirm our findings.
following DHA supplementation. 3.2. Elevated plus maze Animals assigned to each dietary group were tested after the dietary intervention using the elevated plus maze. There was no statistically significant difference (P > 0.05) between animals in each group in terms of the time spent in the open arms, central area, or closed arms of the apparatus, or the number of entries into these same areas (Table 3). 4. Discussion Our major finding was that dietary supplementation with any of the tested omega-3 fatty acids had no effect on the performance of animals in the elevated plus maze, a well-used model of human anxiety disorders. The animal's performance on the test was typical of that found by other investigators strengthening the conclusion that supplementation had no effect (for example see references [26] and [27]). This finding therefore does not support our primary hypothesis that dietary omega-3 fatty acids act to reduce anxiety. Our study does have a number of limitations which should be borne in mind. Firstly, dietary supplementation had only a small effect upon brain omega-3 fatty acids concentrations, as has been described in detail elsewhere and which is in agreement with other studies in the literature [25]. While we cannot rule out that omega-3 fatty acids, in particular EPA, act on the brain indirectly via extra-CNS actions, the relatively small perturbation by the experimental diets on brain lipids may explain why we found no behavioural changes. The diet we utilised had, however, 10% of total lipids being omega-3 fatty acids, a not insubstantial amount as
Funding Source This work was supported by the National Science and Engineering Council of Canada. References [1] M. Peet, C. Stokes, Omega-3 fatty acids in the treatment of psychiatric disorders, Drugs 65 (2005) 1051–1059. [2] B.M. Ross, The emerging role of eicosapentaenoic acid as an important psychoactive natural product: some answers but a lot more questions, Lipid Insights 2 (2008) 89–96. [3] B.M. Ross, J. Seguin, L.E. Sieswerda, Omega-3 fatty acids as treatments for mental illness: which disorder and which fatty acid?, Lipids Health Dis. 6 (2007) 21. [5] K.M. Appleton, H.M. Sallis, R. Perry, A.R. Ness, R. Churchill, ω−3 Fatty acids for
19
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
[4]
[6] [7] [8] [9] [10] [12]
[11]
[13] [14]
[15]
[16]
[17] K.P. Su, Y. Matsuoka, C.U. Pae, Omega-3 polyunsaturated fatty acids in prevention of mood and anxiety disorders, Clin. Psychopharmacol. Neurosci. 13 (2015) 129–137. [18] S. Yehuda, S. Rabinovitz, D.I. Mostofsky, Mixture of essential fatty acids lowers test anxiety, Nutr. Neurosci. 8 (2005) 265–267. [19] O. Green, H. Hermesh, A. Monselise, S. Marom, G. Presburger, A. Weizman, Red cell membrane omega-3 fatty acids are decreased in nondepressed patients with social anxiety disorder, Eur. Neuropsychopharmacol. 16 (2006) 107–113. [20] L. Buydens-Branchey, M. Branchey, J.R. Hibbeln, Associations between increases in plasma n-3 polyunsaturated fatty acids following supplementation and decreases in anger and anxiety in substance abusers, Prog. Neuropsychopharmacol. Biol. Psychiatry 32 (2008) 568–575. [21] J.J. Liu, H.C. Galfalvy, T.B. Cooper, et al., Omega-3 polyunsaturated fatty acid (PUFA) status in major depressive disorder with comorbid anxiety disorders, J. Clin. Psychiatry 74 (2013) 732–738. [22] S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat, J. Neurosci. Methods 14 (1985) 149–167. [23] R. Maclean, P.E. Ward, I. Glen, S.J. Roberts, B.M. Ross, On the relationship between methylnicotinate-induced skin flush and fatty acids levels in acute psychosis, Prog. Neuropsychopharmacol. Biol. Psychiatry 27 (2008) 927–933. [24] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917. [25] B.M. Ross, S. Babay, I. Malik, Brain and liver headspace aldehyde concentration following dietary supplementation with n-3 polyunsaturated fatty acids, Lipids 50 (2015) 1123–1131. [26] R.J. Rodgers, A. Dalvi, Anxiety, defence and the elevated plus-maze, Neurosci. Biobehav Rev. 21 (1997) 801–810. [27] C. Fernandes, S.E. File, The influence of open arm ledges and maze experience in the elevated plus-maze, Pharm. Biochem Behav. 54 (1996) 31–40.
major depressive disorder in adults: an abridged Cochrane review, BMJ Open 6 (2016) e010172. B. Hallahan, T. Ryan, J.R. Hibbeln, I.T. Murray, S. Glynn, C.E. Ramsden, J.P. SanGiovanni, J.M. Davis, Efficacy of omega-3 highly unsaturated fatty acids in the treatment of depression, Brit. J. Pyschiat 209 (2016) 192–201. A.J. Richardson, n-3 Fatty acids and mood: the devil is in the detail, Brit. J. Nutr. 99 (2008) 221–223. J.M. Gorman, Comorbid depression and anxiety spectrum disorders, Depress. Anxiety 4 (1996) 160–168. C. Höschl, J. Švestka, Escitalopram for the treatment of major depression and anxiety disorders, Expert Rev. Neurother. 8 (2008) 537–552. M.A. Katzman, Current considerations in the treatment of generalized anxiety disorder, CNS Drugs 23 (2009) 103–120. H.S. Mayberg, Limbic-cortical dysregulation: a proposed model of depression, J. Neuropsychiatry Clin. Neurosci. 9 (1997) 471–481. H.U. Wittchen, R.C. Kessler, H. Pfister, M. Höfler, R. Lieb, Why do people with anxiety disorders become depressed? A prospective-longitudinal community study, Acta Psychiatr. Scand. Suppl. 102 (2000) 14–23. H.S. Mayberg, M. Liotti, S.K. Brannan, et al., Reciprocal limbic-cortical function and negative mood: converging PET findings in depression and normal sadness, Am. J. Psychiatry 156 (1999) 675–682. E.J. Nestler, M. Barrot, R.J. DiLeone, A.J. Eisch, S.J. Gold, L.M. Monteggia, Neurobiology of depression, Neuron 34 (2002) 13–25. A.T. Beekman, E. de Beurs, A.J. van Balkom, D.J. Deeg, R. van Dyck, R.W. Van Tilburg, Anxiety and depression in later life: co-occurrence and communality of risk factors, Am. J. Psychiatry 157 (2000) 89–95. R.C. Kessler, W.T. Chiu, O. Demler, E.E. Walters, Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication, Arch. Gen. Psychiatry 62 (2005) 617–627. B.M. Ross, Omega-3 polyunsaturated fatty acids and anxiety disorders, Prostaglandins Leukot. Ess. Fat. Acids 81 (2009) 309–312.
20
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
crossmark
Dietary omega-3 polyunsaturated fatty acid supplementation in an animal model of anxiety ⁎
Brian M. Rossa,b, , Imran Malikb, Slim Babayb a b
Northern Ontario School of Medicine, Canada Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada
A R T I C L E I N F O
A BS T RAC T
Keywords: Anxiety Depression Omega-3 fatty acid Alpha-linolenic acid Eicospentaenoic acid Docosahexaenoic acid
A large body of evidence suggests that dietary supplementation with omega-3 fatty acids may ameliorate depressed mood. The magnitude of the effect varies between studies, however, ranging from none at all to being of clinical significance. Given that substantial comorbidity occurs between mood and anxiety disorders, suggesting that they have one or more pathophysiological mechanisms in common, we hypothesized that omega-3 fatty acids may be acting primarily to reduce anxiety rather than depression per se, a possibility which could underlie their variable effects on mood. To test this hypothesis rats were fed for 8 weeks with diets containing one of three types of omega-3 fatty acids, alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid, as well as a low omega-3 fatty acid control diet. Although brain omega-3 fatty acid concentrations were altered by dietary supplementation with eicospentaenoic acid and docosahexaenoic acid, no significant change in anxiety related behaviors were observed compared to the control group as assessed by the elevated-plus maze test. Our data therefore do not support an anxiolytic effect of omega-3 fatty acids and suggest that any effect of these lipids on mood likely occurs by a mechanism unrelated to reducing anxiety.
1. Introduction Omega-3 polyunsaturated fatty acids have been argued to have psychoactive properties [1,2]. The best evidence for such an effect is their proposed antidepressant action in mood disorders [3]. This includes findings that those with major depressive disorder (a type of mood disorder) have lower concentrations of omega-3 fatty acids in blood cells, and that a variety of clinical trials have shown a beneficial effect of dietary supplementation with these lipids on the mood of depressed patients. Such a mood elevating action is of great potential benefit however not all studies report a positive effect (reviewed in references 3–5), and, even in those that do find benefit, the magnitude of response varies widely. The explanation for such variability is unclear but has been suggested either to be that the true effect size is low and unlikely to be of any clinical relevance [5], or that not all omega-3 fatty acids have the same level of efficacy, with some evidence suggesting that of the most three most biologically abundant examples of the type, eicosapentaenoic acid (EPA), is of greater benefit than either alpha-linoleic acid (ALA) or docosahexaenoic acid (DHA) [3,4,6]. An alternative explanation lies in the fact that mood disorders are highly comorbid with anxiety disorders such as generalized anxiety disorder [7] and are amenable to treatment with the same types of
⁎
pharmacotherapies [8,9]. Mood and anxiety disorders may therefore share some pathophysiological mechanisms with a body evidence indicating that chronic anxiety can lead to a higher risk of a depressive episode occuring [10–13]. We hypothesized that omega-3 fatty acids may be actually reducing anxiety in some patients as their primary mechanims of action, and that it was this that was causing the antidepressant effect. Given that this potential confound was not accounted for in the clinical trials, and that the degree of comorbidity varies depending on the characteristics of the population studied [14,15], a primary effect upon anxiety could explain the variability of response noted in the literature [16,17]. The possibility that omega-3 fatty acids have anxiolytic effects is one which has not been extensively examined although social phobia is associated with reduced cellular omega-3 PUFA concentrations, patients with comorbid anxiety and depression exhibit a greater depletion in cellular omega-3 PUFA concentration than those with depression alone, and omega-3 PUFA supplementation reduced anxiety in both test-takers, and in substance users [18–21]. To investigate the role of omega-3 fatty acids in anxiety we have used an animal model, the elevated plus maze test, a model which is responsive to existing anxiolytic medications [22]. The test makes use of the conflict between the animal's fear of being in open areas versus their desire to explore novel environments. The elevated
Correspondence to: MS3002, Division of Medical Sciences, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada, P7A5E8. E-mail address: [email protected] (B.M. Ross).
http://dx.doi.org/10.1016/j.plefa.2016.09.004 Received 1 June 2016; Accepted 28 September 2016 0952-3278/ © 2016 Published by Elsevier Ltd.
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
chromatograph, with a 60-m DB-23 capillary column (0.32 mm internal diameter), and quantified using flame ionisation. Results are expressed as the relative concentration of each fatty acid species.
plus maze has both enclosed arms which provide security and open arms which have exploratory value. When more anxious animals prefer the enclosed areas, whereas when they are less anxious they will enter the open arms more often. We therefore observed animals tested using the elevated plus maze after being fed with a diet supplemented with either ALA, EPA, or DHA, or an omega-3 fatty acid free control diet.
3. Results 3.1. Brain lipids
2. Materials and Methods
Animal were fed a diet which was either omega-3 fatty acid free or supplemented with one of ALA, EPA or DHA. The results of the fatty acid analysis of brain conducted after the intervention have previously been described in detail [25] and are summarized in Table 2 with liver fatty acids analysis being shown for the purpose of comparison. Omega-3 supplementation resulted in much larger changes in liver fatty acid composition than that in brain. Nevertheless, supplementation with DHA resulted in higher brain DHA, total omega-3 fatty acids concentrations and omega-3/omega-6 fatty acids ration, and lower arachidonic acid and total omega-6 fatty acid concentrations compared to the control group. The brain contained very little ALA and EPA with ALA supplementation having no effect on brain ALA or arachidonic acid concentrations. EPA supplementation did result in small increase in brain EPA concentration, and a somewhat larger increase in total omega-3 fatty acids, omega-3/omega-6 ratio, and a decrease in arachidonic acid and total omega-6 fatty acid concentration compared to the control diet, although the magnitude of response (except for decreased arachidonic acid concentration) was less than that observed
2.1. Dietary intervention Animal experimentation was carried according to an animal utilization protocol approved by the Lakehead University animal care committee. Male Wistar rats were allowed to acclimatize to the lab environment for 2 weeks after arrival. They were randomly assigned into pairs and placed into cages with free access to food and water. Rats were fed for 8 weeks on supplementation diets containing 90% (by weight) rat chow containing all required nutrients except for fats (TD99159 basal mix, Harlan Laboratories) plus 10% added fat. The fat component for the control group was exclusively palm oil (New Directions Aromatics, Canada), containing (as per our own analysis) 1.2% (mole %) 14:0, 42.2% 16:0, 3.5% 18:0, 41.9% 18:1, and 11.2% 18:2, n-6, while other groups received palm oil plus omega-3 fatty acids (“95% purity” preparations from Equatech, UK which contained as per our own analysis over 97% of the described fatty acid) in the ratio 9 parts palm oil to 1 part omega-3 fatty acid as described in Table 1. Food was prepared ahead of time and stored in canisters at −20 °C until use. Body weight was monitored throughout the experiment and did not differ significantly (ANOVA; P > 0.05) between the dietary groups.
Table 1 Composition of test diet. Component
2.2. Elevated plus maze
a
Added fat Caesin DL-Methionine Sucrose Cellulose Calcium monohydrogen phosphate Choline bitartrate Calcium carbonate Potassium dihydrogen phosphate Potassium citrate monohydrate Sodium chloride Potassium sulphate Magnesium oxide Ferric citrate Zinc carbonate Manganese carbonate Copper carbonate Potassium iodate Sodium selenate Ammonium paramolybdate, tetrahydrate Sodium meta-silicate, nonahydrate Chromium potassium sulphate, dodecahydrate Lithiun chloride Boric acid Sodium fluoride Nickel carbonate hydroxide, tetrahydrate Ammonium meta-vanadate Niacin Calcium pantothenate Pyroxidine hydrochloride Thiamin hydrochloride Riboflavin Folic acid Biotin Vitamin B12 (0.1% in mannitol) Vitamin E, DL-alpha tocopheryl acetate (500 IU/g) Vitamin A palmitate (500,000 IU/g) Vitamin D3, cholecalciferol (500,000 IU/g) Vitamin K1, phylloquinone
The maze was purchased from Harvard Apparatus (Massachusetts, USA). It consists of a cross, lifted 660 mm above the ground, with a central portion surrounded by 4 black coloured arms of length 450 mm and width 100 mm. Two arms have 500 mm high opaque grey walls (closed arms) enclosed at one end and open to the central area at the other, while the other two arms have 100 mm clear walls (to prevent falls) and are open at each end. A video camera was suspended above the apparatus to record activity. The movement of animals was analysed using the SMART video analysis software provided by Harvard Apparatus to produce the following measures: relative time spent in the open arms, closed arms and central areas of the maze, and the total number of entries by animals into these three areas. On the day of testing animals were transferred from their housing area to the procedure room and left in the room for 30 min prior to the start of the experiment. Each animal was then transferred from their cage into the central area of the apparatus and the investigator left the room. Rats were recorded for 10 min with the first two minutes being an acclimatisation period followed by data recording for a further 8 min. 2.3. Fatty acid analysis One day after completion of the intervention the rats were euthanized by first anaesthetizing with isoflurane gas followed by decapitation using a small animal guillotine. The brain and liver were rapidly removed and tissue stored in a −80 °C freezer until required. Fatty acids were analysed essentially as described previously [23]. Briefly, the entire organ was ground in liquid nitrogen using a mortar and pestle and approximately 300 mg of tissue powder was homogenized in ice cold water using a Polytron homogenizer. Lipids were then extracted from the samples according to the method of Bligh & Dyer [24] in the presence of the internal standard tritridecanoin. Fatty acid methyl esters were prepared using boron trichloride in methanol, and heating the methylation tubes in a boiling water bath. The resulting fatty acid methyl esters were analysed on a Varian 3400 gas-liquid
Weight per kg 100 g 181.9 g 2.84 g 636.11 g 47.37 g 3.32 g 2.38 g 11.84 g 6.5 g 2.35 g 2.45 g 1.55 g 0.81 g 0.2 g 54.72 mg 20.89 mg 10.28 mg 0.33 mg 0.34 mg 0.27 mg 48.09 mg 9.12 mg 0.58 mg 2.7 mg 2.11 mg 1.05 mg 0.22 mg 28.43 mg 15.16 mg 6.63 mg 5.69 mg 5.69 mg 1.9 mg 0.19 mg 23.69 mg 142.16 mg 7.58 mg 1.9 mg 0.71 mg
a The added fat contained either 100 g palm oil or 90 g palm oil plus 10 g omega-3 fatty acid.
18
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
Table 2 Composition of esterified fatty acids in the brain and liver following dietary intervention. Fatty acid or summary measure
ALA Arachidonic EPA DHA Total omega−3 Total omega−6 omega−3 / omega−6 Total saturated Total monosaturated Total polyunsaturated
Liver
Brain
Ctrl
ALA
EPA
DHA
Ctrl
ALA
EPA
DHA
0.1 ± 0.0 15.2 ± 2.7 0.1 ± 0.1 4.3 ± 0.9 4.8 ± 0.8 23.9 ± 3.1 0.20 ± 0.04 37.6 ± 3.4 33.6 ± 4.1 28.7 ± 2.1
1.3 ± 0.4* 10.1 ± 2.3* 2.1 ± 0.4** 7.1 ± 2.2* 11.8 ± 1.8*** 20.2 ± 3.4 0.59 ± 0.10*** 38.7 ± 3.9 28.9 ± 3.2* 32.0 ± 3.4
0.2 ± 0.1 6.1 ± 0.9** 7.8 ± 2.7*** 10.1 ± 0.8** 18.1 ± 2.4*** 18.6 ± 2.8*** 0.97 ± 0.18*** 38.8 ± 3.1 26.7 ± 3.2*** 34.6 ± 4.0*
0.2 ± 0.3 5.7 ± 0.7** 4.2 ± 1.1** 15.3 ± 2.6*** 20.7 ± 2.6*** 15.5 ± 2.3*** 1.34 ± 0.21*** 40.9 ± 3.2** 23.2 ± 3.0*** 36.2 ± 3.9***
0.0 ± 0.0 9.6 ± 0.6 0.0 ± 0.0 11.9 ± 0.9 12.0 ± 1.2 14.4 ± 0.9 0.83 ± 0.09 43.1 ± 6.0 30.4 ± 2.8 26.4 ± 1.9
0.0 ± 0.0 9.3 ± 0.5 0.0 ± 0.0 12.8 ± 0.5 13.1 ± 0.9 13.5 ± 0.6 0.97 ± 0.10 43.7 ± 3.2 29.4 ± 3.7 26.6 ± 2.2
0.0 ± 0.0 8.2 ± 0.9* 0.2 ± 0.1* 12.6 ± 1.0 13.5 ± 1.0* 12.9 ± 1.2* 1.05 ± 0.12** 44.4 ± 4.6 30.4 ± 2.2 26.6 ± 2.4
0.0 ± 0.0 8.2 ± 0.5* 0.1 ± 0.1* 13.7 ± 0.6* 14.2 ± 0.8*** 12.0 ± 1.0*** 1.18 ± 0.14*** 43.0 ± 5.2 30.1 ± 3.3 26.2 ± 1.9
Values shown are mean (n =8) liver fatty acid relative concentrations (%) ± SD after 8 weeks of dietary intervention and are summary data taken from reference 25. *P < 0.05, **P < 0.01, and ***P < 0.005, as compared to palm oil control group (Ctrl) using a post-ANOVA Tukey test. Ctrl = palm oil control, ALA = palm oil plus alpha-linolenic acid; EPA = palm oil plus eicosapentaenoic acid, DHA = palm oil plus docosahexaenoic acid. Table 3 Effect of dietary intervention on time spent and entries into each section of the elevated plus maze. Group
Open arm
Closed arm
Central area
Time (%)
Entries
Time (%)
Entries
Time (%)
Entries
Ctrl ALA EPA DHA
6.7 7.2 7.6 7.1
3.8 3.3 3.5 4.1
80.7 75.3 78.9 78.0
12.5 14.0 10.4 14.4
14.2 20.5 12.8 17.8
13.6 16.0 13.4 15.3
One way ANOVA by group
F3,28 0.84
± ± ± ±
4.4 3.9 2.6 2.5 P value 0.519
± ± ± ±
F3,28 0.33
1.4 2.6 1.3 1.8 P value 0.807
± ± ± ±
F3,28 0.93
8.1 13.5 9.5 21.5 P value 0.441
± ± ± ±
F3,28 1.91
4.3 3.0 3.7 3.7 P value 0.150
± ± ± ±
F3,28 0.75
11.4 10.1 10.8 7.8 P value 0.532
± ± ± ±
F3,28 0.52
4.8 4.1 6.2 4.4 P value 0.670
Animals received one of four diets for 8 weeks (Ctrl – palm oil control; ALA – palm oil plus alpha-linolenic acid; EPA – palm oil plus eicosapentaenoic acid; DHA – palm oil plus docosahexaenoic acid) prior to testing in the elevated plus maze. Data are shown as mean ± SD of the number of entries or percentage time spent into each part of the apparatus. The results of the one way analysis of variance by group are shown as F values and statistical significance (P value). Note that there were no statistically significant differences between the dietary groups for any of the measures.
evidenced by the very large changes in liver lipid concentrations induced by the different diets, making it unlikely that a higher dosage would have resulted in a different finding. A second limitation is that only a single supplementation time period was used. We cannot rule out that a behavioural effect would have been observed using shorter or longer supplementation times. It should be noted however, that the time used (8 weeks) is typical of that used in human clinical trials designed to test omega-3 fatty acids efficacy in various disorders of mental health [3]. Lastly we only have investigated the effect of omega3 fatty acids using the elevated-plus maze; other models are available such as the open field test. Preliminary work in our laboratory using the latter model, however, also suggested that omega-3 fatty acids have no effect on anxiety state in rats (unpuplished observations). Despite these limitations our data are certainly not supportive of an anxiolytic effect of omega-3 fatty acids although further work is required to confirm our findings.
following DHA supplementation. 3.2. Elevated plus maze Animals assigned to each dietary group were tested after the dietary intervention using the elevated plus maze. There was no statistically significant difference (P > 0.05) between animals in each group in terms of the time spent in the open arms, central area, or closed arms of the apparatus, or the number of entries into these same areas (Table 3). 4. Discussion Our major finding was that dietary supplementation with any of the tested omega-3 fatty acids had no effect on the performance of animals in the elevated plus maze, a well-used model of human anxiety disorders. The animal's performance on the test was typical of that found by other investigators strengthening the conclusion that supplementation had no effect (for example see references [26] and [27]). This finding therefore does not support our primary hypothesis that dietary omega-3 fatty acids act to reduce anxiety. Our study does have a number of limitations which should be borne in mind. Firstly, dietary supplementation had only a small effect upon brain omega-3 fatty acids concentrations, as has been described in detail elsewhere and which is in agreement with other studies in the literature [25]. While we cannot rule out that omega-3 fatty acids, in particular EPA, act on the brain indirectly via extra-CNS actions, the relatively small perturbation by the experimental diets on brain lipids may explain why we found no behavioural changes. The diet we utilised had, however, 10% of total lipids being omega-3 fatty acids, a not insubstantial amount as
Funding Source This work was supported by the National Science and Engineering Council of Canada. References [1] M. Peet, C. Stokes, Omega-3 fatty acids in the treatment of psychiatric disorders, Drugs 65 (2005) 1051–1059. [2] B.M. Ross, The emerging role of eicosapentaenoic acid as an important psychoactive natural product: some answers but a lot more questions, Lipid Insights 2 (2008) 89–96. [3] B.M. Ross, J. Seguin, L.E. Sieswerda, Omega-3 fatty acids as treatments for mental illness: which disorder and which fatty acid?, Lipids Health Dis. 6 (2007) 21. [5] K.M. Appleton, H.M. Sallis, R. Perry, A.R. Ness, R. Churchill, ω−3 Fatty acids for
19
Prostaglandins, Leukotrienes and Essential Fatty Acids 114 (2016) 17–20
B.M. Ross et al.
[4]
[6] [7] [8] [9] [10] [12]
[11]
[13] [14]
[15]
[16]
[17] K.P. Su, Y. Matsuoka, C.U. Pae, Omega-3 polyunsaturated fatty acids in prevention of mood and anxiety disorders, Clin. Psychopharmacol. Neurosci. 13 (2015) 129–137. [18] S. Yehuda, S. Rabinovitz, D.I. Mostofsky, Mixture of essential fatty acids lowers test anxiety, Nutr. Neurosci. 8 (2005) 265–267. [19] O. Green, H. Hermesh, A. Monselise, S. Marom, G. Presburger, A. Weizman, Red cell membrane omega-3 fatty acids are decreased in nondepressed patients with social anxiety disorder, Eur. Neuropsychopharmacol. 16 (2006) 107–113. [20] L. Buydens-Branchey, M. Branchey, J.R. Hibbeln, Associations between increases in plasma n-3 polyunsaturated fatty acids following supplementation and decreases in anger and anxiety in substance abusers, Prog. Neuropsychopharmacol. Biol. Psychiatry 32 (2008) 568–575. [21] J.J. Liu, H.C. Galfalvy, T.B. Cooper, et al., Omega-3 polyunsaturated fatty acid (PUFA) status in major depressive disorder with comorbid anxiety disorders, J. Clin. Psychiatry 74 (2013) 732–738. [22] S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat, J. Neurosci. Methods 14 (1985) 149–167. [23] R. Maclean, P.E. Ward, I. Glen, S.J. Roberts, B.M. Ross, On the relationship between methylnicotinate-induced skin flush and fatty acids levels in acute psychosis, Prog. Neuropsychopharmacol. Biol. Psychiatry 27 (2008) 927–933. [24] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917. [25] B.M. Ross, S. Babay, I. Malik, Brain and liver headspace aldehyde concentration following dietary supplementation with n-3 polyunsaturated fatty acids, Lipids 50 (2015) 1123–1131. [26] R.J. Rodgers, A. Dalvi, Anxiety, defence and the elevated plus-maze, Neurosci. Biobehav Rev. 21 (1997) 801–810. [27] C. Fernandes, S.E. File, The influence of open arm ledges and maze experience in the elevated plus-maze, Pharm. Biochem Behav. 54 (1996) 31–40.
major depressive disorder in adults: an abridged Cochrane review, BMJ Open 6 (2016) e010172. B. Hallahan, T. Ryan, J.R. Hibbeln, I.T. Murray, S. Glynn, C.E. Ramsden, J.P. SanGiovanni, J.M. Davis, Efficacy of omega-3 highly unsaturated fatty acids in the treatment of depression, Brit. J. Pyschiat 209 (2016) 192–201. A.J. Richardson, n-3 Fatty acids and mood: the devil is in the detail, Brit. J. Nutr. 99 (2008) 221–223. J.M. Gorman, Comorbid depression and anxiety spectrum disorders, Depress. Anxiety 4 (1996) 160–168. C. Höschl, J. Švestka, Escitalopram for the treatment of major depression and anxiety disorders, Expert Rev. Neurother. 8 (2008) 537–552. M.A. Katzman, Current considerations in the treatment of generalized anxiety disorder, CNS Drugs 23 (2009) 103–120. H.S. Mayberg, Limbic-cortical dysregulation: a proposed model of depression, J. Neuropsychiatry Clin. Neurosci. 9 (1997) 471–481. H.U. Wittchen, R.C. Kessler, H. Pfister, M. Höfler, R. Lieb, Why do people with anxiety disorders become depressed? A prospective-longitudinal community study, Acta Psychiatr. Scand. Suppl. 102 (2000) 14–23. H.S. Mayberg, M. Liotti, S.K. Brannan, et al., Reciprocal limbic-cortical function and negative mood: converging PET findings in depression and normal sadness, Am. J. Psychiatry 156 (1999) 675–682. E.J. Nestler, M. Barrot, R.J. DiLeone, A.J. Eisch, S.J. Gold, L.M. Monteggia, Neurobiology of depression, Neuron 34 (2002) 13–25. A.T. Beekman, E. de Beurs, A.J. van Balkom, D.J. Deeg, R. van Dyck, R.W. Van Tilburg, Anxiety and depression in later life: co-occurrence and communality of risk factors, Am. J. Psychiatry 157 (2000) 89–95. R.C. Kessler, W.T. Chiu, O. Demler, E.E. Walters, Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication, Arch. Gen. Psychiatry 62 (2005) 617–627. B.M. Ross, Omega-3 polyunsaturated fatty acids and anxiety disorders, Prostaglandins Leukot. Ess. Fat. Acids 81 (2009) 309–312.
20