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Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial
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Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial Andrew Pipingas a,*, Andrew Sinclair b, Kevin D. Croft c, Andrzej S. Januszewski d,e, Alicia J. Jenkins d,e, Trevor A. Mori c, Robyn Cockerell a, Natalie A. Grima f, Con Stough a, Andrew Scholey a, Stephen P. Myers g, Avni Sali h, Matthew P. Pase a a
Centre for Human Psychopharmacology, Swinburne University of Technology, Hawthorn, Australia School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia c School of Medicine and Pharmacology, University of Western Australia, Perth, Australia d Department of Medicine (St. Vincent’s), The University of Melbourne, Parkville, Australia e NHMRC Clinical Trials Centre, University of Sydney, Camperdown, Sydney, Australia f Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Australia g NatMed-Research Unit, Division of Research, Southern Cross University, Lismore, NSW 2480, Australia h National Institute of Integrative Medicine, Hawthorn 3122, Australia b
A R T I C L E
I N F O
A B S T R A C T
Article history:
The effects of daily fish oil supplementation, with and without multivitamins, on biochemi-
Received 8 September 2014
cal markers of health were examined. Healthy adults (N = 160) were randomised to receive
Received in revised form 19 October
3 g of salmon oil with a multivitamin, 6 g of salmon oil with a multivitamin, 6 g of salmon
2014
oil in isolation or placebo in a double-blind fashion on a daily basis for 16 weeks. Relative
Accepted 28 October 2014
to placebo, both 6 g salmon oil groups displayed significantly lower F2-isoprostane levels at
Available online
endpoint. Increases in red blood cell polyunsaturated fatty acids correlated with reductions in F2-isoprostanes. Treatment had no effect on inflammatory cytokines, C-reactive protein,
Keywords: Fish oil
fibrinogen, cholesterol or triacylglycerol. © 2014 Elsevier Ltd. All rights reserved.
PUFA Multivitamins Inflammation Oxidative stress F2-isoprostanes
* Corresponding author. Centre for Human Psychopharmacology, Swinburne University of Technology, ATC Building. PO Box 218, Hawthorn, 3122, Australia. Tel.: +61 3 9214 5215. E-mail address: [email protected] (A. Pipingas). Abbreviations: ANZCTR, Australia and New Zealand Clinical Trials Registry; AA, arachidonic acid; CVD, cardiovascular disease; CRP, C reactive protein; DHA, docosahexaenoic acid; EDTA, ethylenediaminetetraacetic acid; EPA, eicosapentaenoic acid; HDL, high density lipoprotein; LC n-3 PUFA, long chain omega-3 polyunsaturated fatty acids; LDL, low density lipoprotein; n-3, omega-3; n-6, omega-6; RBC, red blood cells; RCT, randomised controlled trial; ROS, reactive oxygen species http://dx.doi.org/10.1016/j.jff.2014.10.028 1756-4646/© 2014 Elsevier Ltd. All rights reserved. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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1.
Introduction
Fish oil supplements containing the long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are one of the most popular dietary supplements in the western world (Morgan et al., 2012). Evidence suggests that higher intake of LC n-3 PUFA is related to better cardiovascular (Kris-Etherton, Harris, & Appel, 2002) and perhaps even neurological outcomes (Fotuhi, Mohassel, & Yaffe, 2009; Tan et al., 2012). Fish oil supplements have been reported to improve the lipid profile, predominantly by lowering triacylglycerol levels (Harris, 1989), exerting vasodilatory effects (Xin, Wei, & Li, 2012) and by lowering blood pressure (Liu, Conklin, Manuck, Yao, & Muldoon, 2014). LC n-3 PUFAs also play an important role in regulating inflammation (Calder, 2006b). The omega-6 (n-6) arachidonic acid (AA) has pro-inflammatory effects through the production of eicosanoids. Both AA and LC n-3 PUFA compete for space inside cell membrane phospholipids (Simopoulos, 2002). Increasing cellular levels of LC n-3 PUFA decreases the amount of AA in cell membranes thus limiting AA derived eicosanoids (Simopoulos, 2002). The ratio of LC n-3 PUFA to omega-6 fatty acid has thus been put forward as a measure of inflammation (Kalogeropoulos et al., 2010). Observational studies have reported that higher circulating levels of LC n-3 PUFA are associated with lower levels of pro-inflammatory cytokines and lower levels of C-reactive protein (CRP) (Farzaneh-Far, Harris, Garg, Na, & Whooley, 2009; Ferrucci et al., 2006; Kalogeropoulos et al., 2010), which are known to be predictive of cardiovascular disease (CVD) (Ridker, Hennekens, Buring, & Rifai, 2000). LC n-3 PUFAs may also have antioxidant effects. A study by Mori et al. (1999) reported that a daily fish meal for eight weeks reduced urinary F2-isoprostane levels in sedentary subjects with non-insulin dependent diabetes. F2-isoprostanes are lipid peroxidation products derived from the non-enzymatic free radical oxidation of AA in membrane lipids and are considered the most reliable biomarkers of in vivo lipid peroxidative damage (Milne, Yin, Hardy, Davies, & Roberts, 2011). Subsequent placebo controlled studies using purified EPA or DHA showed both fatty acids reduced urinary (Mori et al., 2000, 2003) and plasma (Mas et al., 2010) F2-isoprostanes in type 2 diabetic patients, and in overweight mildly dyslipidaemic men, respectively. A recent double-blind, 4 month randomised, controlled trial (RCT) reported that LC n-3 PUFA supplementation reduced plasma F2-isoprostane levels by approximately 15%, relative to placebo (Kiecolt-Glaser et al., 2013). Multivitamin supplements, containing both vitamins and minerals, are commonly used among the general population (Radimer et al., 2004). Evidence suggests that the intake of a range of vitamins and minerals may interact with fatty acid metabolism influencing the overall levels of LC n-3 PUFA measured in vivo (Bertrandt, Klos, & Debski, 2004; Durand, Prost, & Blache, 1996; Pipingas et al., 2014; Stangl & Kirchgessner, 1998). There is thus a need to examine the combined effects of multivitamin supplements and LC n-3 PUFAs on all aspects of human health. The present 16 week RCT was designed to investigate the effect of salmon oil supplementation, with and without the
addition of a multivitamin supplement, on cardiovascular and cognitive health in healthy middle-aged and elderly subjects. To the best of our knowledge, this is the first RCT to investigate the combined effects of fish oils and multivitamins on human health. The results of supplementation on the cognitive, cardiovascular and omega-3 uptake into red bloods cells arising from this RCT have been published (Pase et al., 2015; Pipingas et al., 2014). This report examines the secondary outcomes from this study including the effects of supplementation on measures of inflammation, oxidative stress, serum lipids and liver function, in the same subjects. It was hypothesised that treatment with salmon oil would reduce markers of inflammation and oxidative stress as well as triacylglycerol levels. The study also had the novel aim of exploring whether combining salmon oil with a multivitamin would provide added benefits to human health.
2.
Materials and methods
2.1.
Participants
One hundred and sixty healthy, non-smoking, male and female volunteers aged between 50 and 70 years were recruited from the community using newspaper advertisements, posters and community radio announcements. Participants were not currently taking any medication, such as anti-coagulants, anticholinergic, anti-depressant medications and acetylcholinesterase inhibitors, or vitamin/herbal supplements. Subjects were excluded if they fulfilled any of the following; diagnosis of dementia, diabetes, neurological and psychiatric disorders, overt CVD or past or present drug or alcohol abuse. Long term multivitamin and/or omega-3 supplement users were also excluded. The participant flow diagram is shown in Fig. 1.
2.2.
Setting
The study was conducted at the Centre for Human Psychopharmacology Laboratory at Swinburne University of Technology, Hawthorn, Australia. The study was approved by the Swinburne University Human Research Ethics Committee and all procedures were conducted in accordance with the guidelines of the Australian National Health and Medical Research Council and the Declaration of Helsinki (2008). This trial was registered with the Australian and New Zealand Clinical Trial Registry (ANZCTR no. 12611000094976). Participants gave informed written consent to participate in the study.
2.3.
Interventions, randomisation and blinding
This study was a 16 week randomised, double-blind, placebocontrolled trial comparing the effects of salmon oil supplements and multivitamins on blood biomarkers. Participants were randomly assigned to one of four treatment groups: multivitamin and salmon oil (3 g); multivitamin and salmon oil (6 g); placebo multivitamin and salmon oil (6g); or placebo multivitamin and placebo salmon oil (Sunola oil). Participants were instructed to take their assigned treatment daily for 16 weeks. The study medications were provided
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Fig. 1 – Participant flow diagram.
by Swisse (Melbourne, Australia). The active salmon oil was Swisse Ultiboost Wild Salmon Oil and the active multivitamin was Swisse Ultivite 50 + (Men’s and Women’s formulations). All participants took one multivitamin, or matching placebo, daily and immediately following breakfast. To aid masking, the placebo multivitamin consisted of carrot powder with a small amount of riboflavin to produce a multivitamin like colouration and smell of the urine. The Swisse Ultiboost Wild Salmon Oil capsules contained 1 g of omega-3 (80 mg EPA and 80 mg DHA) each, whilst the matching placebo consisted of Sunola oil combined with 50 IU of vitamin E. All salmon oil containers (including those containing the placebo) included a sachet with a few drops of salmon oil so as to give a distinctive fish scent. Sunola oil, a monounsaturated (omega-9) sunflower oil, was used as a placebo because it is virtually trans-fat free and unlike olive oil there is little research showing health benefits. Both the active and placebo salmon oils were administered in identically appearing soft gelatine capsules. All participants took six active (1 g each) salmon oil or placebo salmon oil capsules daily. Those allocated to 3 g of daily salmon oil received three active salmon oil and three placebo salmon oil capsules daily. A random permuted block procedure with a block size of 4 was used to assign participants to one of the four experimental groups. Randomisation was conducted by the trial sponsor with the treatment packaging labelled according to the randomisation schedule. The research staff were blinded to this allocation. Placebo and treatments were packaged in
identical blister packs for multivitamins and identical sealed plastic containers for salmon oil capsules. Participants received separate sealed plastic containers and participants were instructed to consume three capsules from one container in the morning and three capsules from the other container in the evening. Depending on treatment allocation, this allowed participants to receive either 6 g, 3 g or 0 g of daily salmon oil. Treatments could only be differentiated by the presence of a participant number. Participants were allocated the next sequential number upon enrolment in the study. The masking key was held in a secure password protected electronic file and was only revealed at the end of the study and once analysis of the main study outcomes had been completed. Investigators performing the biochemical analyses were masked to subject treatment and sample order.
2.4.
Outcomes measures
Blood samples were collected via venepuncture from the antecubital vein, using the BD-vacutainer system, following a 12hour overnight fasting period. Biochemistry included electrolyte levels, liver function and kidney function tests. High sensitivity CRP, fibrinogen and lipids were analysed by Healthscope Functional Pathology according to standard procedures. CRP was measured in serum by a latex-enhanced immunoturbidimetric assay. Fibrinogen was quantified by the addition of thrombin to plasma, and measuring the clotting time of the diluted plasma compared to a standardised fibrinogen
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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preparation. Total cholesterol, HDL-cholesterol and LDLcholesterol were measured using the ADVIA system based on the enzymatic method. The method of analysis for triacylglycerol is based on Fossati three-step enzymatic reaction with a Trinder endpoint. Analysis was performed on an ADVIA 1650 analyser (Siemens, USA) by Healthscope Pathology. Blood for plasma F 2 -isoprostanes was collected into ethylenediaminetetraacetic acid (EDTA) and reduced glutathione, and centrifuged at 3000 × g for 20 min at 4 °C. To prevent oxidation of the samples, butylated hydroxytoluene (40 µg per mL of plasma) was added to plasma before samples were stored at −80 °C until analysis. F2-isoprostanes were quantified by negative ion chemical ionisation–gas chromatograph mass spectroscopy as described by Mori et al. (Mori, Croft, Puddey, & Beilin, 1999; Mori et al., 1999). The within and between assay reproducibility is 8.0 and 5.6%, respectively. Levels of cytokines (IL-6, IL-1β and TNFα) were measured using ELISA (R&D, Minneapolis, USA) on a BEP 2000 advance (Siemens, Erlangen, Germany) automated analyser with intra- and inter-assay CVs of <4, <7.5 and <3.6%, respectively. Measurement of F2-isoprostanes and cytokines were included in the protocol after commencement of the study. Once added to the study protocol, all newly recruited subjects provided blood for F2-isoprostanes and cytokines. Consequently 101 subjects had blood samples taken for measurement of F2isoprostanes at baseline (with 91 measurements completed at endpoint) and cytokines were measured on 86 subjects at baseline (with 80 measurements completed at endpoint; see Fig. 1). Fatty acids samples were analysed by Healthscope Functional Pathology according to standard procedures described elsewhere (Pipingas et al., 2014).
2.5.
Procedure
Participants attended three testing sessions at our laboratory including baseline, 6 week and endpoint assessment following 16 weeks of treatment. Blood biomarkers were only measured at baseline and endpoint. Participants attended at the same time of day across all testing sessions.
2.6. Sample size calculations and assessment of compliance
effects of treatment. ANCOVAs were then repeated controlling for gender, to examine if the effects of supplementation were specific to one gender, as has been previously reported (Pipingas et al., 2014). In addition to the ANCOVAs, correlations examined whether absolute changes in blood omega-3 fatty acid levels were associated with changes in inflammatory, oxidative stress and cardiometabolic biomarkers, over the 16-week supplementation period. F2-isoprostanes, CRP and the inflammatory cytokine measures were significantly skewed. Skewness of the F2-isoprostanes and triacylglycerol were corrected by applying natural log transformations. Skewness of the IL-6, IL-Iβ and TNFα variables was corrected by applying negative square root transformations. The distribution of high sensitivity CRP could not be made normal through transformation. High sensitivity CRP was thus analysed using the nonparametric Kruskal–Wallis one way ANOVA. This test examined whether the change from baseline scores in CRP differed according to treatment allocation. All results were considered significant at the level of p < 0.05.
3.
Results
The trial commenced in 2010 and was ceased in 2012 due to attainment of the required sample size. Across the whole sample, 16 participants were lost to follow-up between baseline and endpoint (6 in the 3 g salmon oil multivitamin group, 2 in the 6 g salmon oil multivitamin group, 3 in the 6 g salmon oil group and 5 in the placebo. There were no serious adverse events reported during the study. Characteristics of the participants at baseline are shown in Table 1. Blood levels of omega-3 fatty acids tended to be low (Harris et al., 2013). The effects of treatment on omega-3 fatty acid incorporation into erythrocytes are reported elsewhere (Pipingas et al., 2014). In brief, EPA increased by 96% in the group receiving the combination of the high dose salmon oil and multivitamin. The omega-3 index increased in this group by 40%. EPA increased by 57% in the group receiving the high dose salmon oil only. There were no significant changes in DHA or DPA. The AA/EPA ratio decreased across all treatment groups relative to placebo, indicating adequate compliance across the sample.
3.1.
Effect of the intervention on F2-isoprostane levels
Sample size calculations were based on the primary outcomes of cognitive and cardiovascular function (Pase et al., 2015). Based on the available sample size for cytokines and F2isoprostanes, this study had a 98% change of detecting a small effect size of 0.25 with alpha set to 0.05, for these measures. Power was even greater for the lipid measures given the larger sample size available. Compliance to treatment was assessed by examining changes in the AA/EPA ratio over the 16 week study period (Pipingas et al., 2014).
There was a significant effect of treatment on F2-isoprostane levels at week 16, relative to baseline (F (3, 86) = 11.08, p < 0.001) (Table 2). As compared to placebo, both 6 g salmon oil groups displayed significantly lower F2-isoprostane levels at week 16 (Fig. 2). The two 6 g salmon oil groups, with and without a multivitamin, did not differ significantly to each other (p = 0.40) and the low dose salmon oil group did not differ to placebo (p = 0.22).
2.7.
3.2.
Statistical analyses
Univariate ANCOVAs were used to examine the effects of treatment on blood biomarkers at week 16, adjusting for values at baseline. Pairwise comparisons, applying Bonferroni corrections to adjust for multiple comparisons across the four treatment groups, were used to further examine significant
Effect of the intervention on inflammatory markers
Treatment allocation had no effect on fibrinogen (F (3, 132) = 0.39, p = 0.76), IL-1β (F (3, 75) = 0.07, p = 0.98), IL-6 (F (3, 75) = 0.60, p = 0.62) or TNF-α (F (3, 75) = 0.16, p = 0.92). Changes in CRP over the 16 week study period did not differ according to treatment allocation (p = 0.85).
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Table 1 – Baseline sample means and standard deviations by treatment allocation. Variable Demographics N Age, y Male, % Education, y MMSE BMI Clinical SBP, mmHg DBP, mmHg Heart rate, bpm Fatty acids EPA, % DHA, % DPA, % N3 index, %
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
Placebo
Overall
42 59.5 48 16 28 25.5
40 58.9 48 16 28 24.4
41 59.5 46 16 28 25.3
37 59.2 46 16 28 24.2
(3) (2) (2.8)
160 59.3 (5.7) 47 16 (4) 28 (2) 24.9 (3.4)
(5.6) (3) (2) (3.6)
(5.6) (4) (2) (3.1)
(5.9) (4) (2) (4.0)
(6.0)
126 (21) 77 (13) 66 (11)
123 (17) 75 (11) 67 (10)
126 (17) 78 (10) 65 (12)
121 (21) 75 (12) 66 (9)
124 (19) 76 (12) 66 (11)
0.99 2.66 1.69 5.34
1.01 2.74 1.88 5.63
1.06 2.92 1.94 5.92
1.00 2.82 1.83 5.65
0.10 2.78 1.84 5.63
(0.46) (1.26) (0.75) (2.31)
(0.30) (0.96) (0.50) (1.59)
(0.41) (1.10) (0.65) (2.04)
(0.43) (1.11) (0.61) (2.00)
(0.40) (1.11) (0.64) (1.99)
Note: MMSE = Mini Mental State Examination; WASI = Wechsler Abbreviated Scale of Intelligence; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; N3 = omega-3; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.
Table 2 – Inflammation and oxidative stress measured over the course of supplementation, stratified by treatment allocation. Variable
Oxidative stress F2s, pmol/La Baseline Week 16 %change Inflammation HS-CRPb Baseline Week 16 %change Fibrinogen Baseline Week 16 %change IL-βa Baseline Week 16 %change IL-6a Baseline Week 16 %change TNF-αa Baseline Week 16 %change
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
1659.8 (312.7) 1477.0 (292.6) −11.01%
1651.9 (366.6) 1329.7 (328.2)*** −19.50
1646.8 (336.3) 1383.8 (306.6)** −15.97
Placebo
ANCOVA F
p
11.08
<0.001
−
−
1644.4 (490.8) 1557.0 (337.9) −5.32
1.9 (3.1) 1.2 (1.6) −37.6
2.3 (5.7) 1.1 (1.5) −54.1
1.3 (2.6) 1.2 (2.2) −5.4
1.3 (1.6) 1.00 (1.4) −21.4
2.8 (0.4) 2.9 (0.5) 5.1
2.8 (0.6) 3.0 (0.5) 5.7
2.7 (0.5) 3.0 (0.6) 8.4
2.9 (0.4) 3.0 (0.4) 4.5
0.9 (0.2) 0.9 (0.2) −2.2
0.8 (0.2) 0.8 (0.2) 5.0
0.9 (0.3) 0.9 (0.2) −3.2
1.0 (0.6) 0.9 (0.2) −12.9
1.7 (1.0) 1.5 (1.1) −11.0
2.1 (2.6) 2.1 (2.2) −1.4
1.4 (1.0) 1.7 (1.4) 17.7
1.3 (0.5) 1.4 (0.7) 11.1
1.8 (0.7) 1.7 (0.5) −8.7
1.7 (0.5) 1.6 (0.5) −2.4
1.7 (0.6) 1.6 (0.5) −5.3
1.5 (0.5) 1.5 (0.4) −5.2
0.39
0.76
0.07
0.98
0.60
0.62
0.16
0.92
HS-CRP = high sensitivity C reactive protein; IL-6 = interleukin-6 [pg/mL]; IL-1β = interleukin-1β [pg/mL]; TNFα = tumor necrosis factor α [pg/ mL]; HS-CRP = high sensitivity C reactive protein [mg/L]; F2s = F2-isoprostanes [pmol/L]; ANCOVA = analysis of covariance. Note that ANCOVA statistics refer to the main effect of treatment at week 16. a Statistics performed on transformed values. b Investigated using non-parametric test. *p < 0.01, **p < 0.01, ***p < 0.001. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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significantly from placebo. At week 16, there was no effect of treatment group on HDL-cholesterol (F (3, 134) = 1.23, p = 0.30), LDL-cholesterol (F (3, 134) = 0.54, p = 0.66) or total cholesterol (F (3, 137) = 0.27, p = 0.85).
3.4.
Fig. 2 – Changes in F2-isoprostanes over the 16 week study period stratified according to treatment allocation. SO = salmon oil, MV = multivitamin, *** denotes significant difference from placebo at p < 0.001, ** denotes significant difference from placebo at p < 0.01. Values of F2isoprostanes are presented in original units (without log transformation).
3.3.
Effect of the intervention on blood lipids
There was a significant main effect of treatment for triacylglycerol levels at week 16 (F (3, 137) = 3.43, p < 0.05) (Table 3). Pairwise comparisons showed that the group taking 3 g of salmon oil combined with the multivitamin had lower triacylglycerol levels at week 16 as compared to the group receiving 6 g of salmon oil combined with the multivitamin (p = 0.04). No other significant group differences were found meaning that the treatment groups did not differ
Safety of supplementation
The analysis of the electrolyte, liver function and kidney function tests showed differences from baseline after 16 weeks in only alanine aminotransferase (F (3, 136) = 3.41, p < 0.05), aspartate aminotransferase (F (3, 136) = 6.44, p < 0.001) and estimated glomerular filtration rate (F (3, 80) = 3.15, p < 0.05). Pairwise comparisons revealed that aspartate aminotransferase was higher in both multivitamin groups, relative to placebo, at study-end. For estimated glomerular filtration rate and alanine aminotransferase, none of the treatment groups differed according to placebo. Means, standard deviations and percentage changes for all the safety parameters are shown in Supplementary Table S1 online.
3.5.
Effects of supplementation in men and in women
Controlling for gender did not significantly alter any of the aforementioned results pertaining to the effects of treatment on cardiometabolic risk factors, oxidative stress or inflammation (data for this analysis are not shown).
3.6. Changes in long-chain omega-3 fatty acid levels versus changes in F2-isoprostanes, cardiometabolic risk and inflammation Two-tailed correlations examined absolute changes in red blood cell omega-3 fatty acids with absolute changes in the cardiometabolic, inflammatory and oxidative stress measures over the 16 week study period. Correlations were computed using
Table 3 – Metabolic cardiovascular risk factors measured over the course of supplementation, stratified by treatment allocation. Variable
HDL cholesterol, mmol/L Baseline Week 16 %change LDL cholesterol, mmol/L Baseline Week 16 %change Total cholesterol, mmol/L Baseline Week 16 %change Triacylglycerola, mmol/L Baseline Week 16 %change
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
Placebo
1.5 (0.4) 1.6 (0.5) 5.3
1.6 (0.4) 1.7 (0.4) 3.1
1.6 (0.4) 1.6 (0.4) 1.3
1.6 (0.4) 1.6 (0.3) −1.3
3.3 (0.7) 3.4 (0.7) 2.1
3.5 (0.7) 3.5 (0.9) 0.6
3.4 (0.8) 3.5 (1.1) 4.8
3.3 (0.7) 3.3 (0.8) 0.0
5.4 (0.9) 5.5 (1.0) 2.2
5.7 (0.8) 5.8 (1.0) 2.8
5.5 (1.0) 5.5 (1.1) 1.5
5.5 (0.9) 5.4 (0.7) −1.5
1.2 (0.5) 1.1 (0.6) −6.6
1.2 (0.7) 1.3 (0.8) 10.1
1.2 (0.6) 1.0 (0.4) −16.4
1.3 (0.7) 1.2 (0.7) −7.2
ANCOVA F
p
1.23
0.30
0.54
0.66
0.27
0.85
3.43
<0.05
ANCOVA = analysis of covariance. Note that ANCOVA statistics refer to the main effect of treatment at week 16. a Statistics performed on transformed values. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Fig. 3 – Correlations between change in F2-isoprostanes over the 16 week study period and changes in (A) the n3/n6 fatty acid ratio, (B) total EPA and (C) changes in the AA/EPA fatty acid ratio. Values of F2-isoprostanes have been log transformed.
the entire sample irrespective of treatment allocation. With respect to oxidative stress (Fig. 3), incorporation of both EPA (r = −0.30, p < 0.01) and the ratio of total omega-3 to omega-6 fatty acids (r = −0.30, p < 0.01) were negatively associated with F2-isoprostanes.The AA/EPA ratio was positively associated with F2-isoprostanes (r = 0.35, p < 0.01). There were no other significant correlations between changes in red blood cell omega-3 fatty acids and measures of inflammation or blood lipids.
4.
Discussion
To the best of our knowledge, this is the first study to investigate the effects of salmon oil supplementation, with and without the addition of a multivitamin, on multiple blood biomarkers in healthy older men and women. The principle finding was that 6 g of salmon oil daily for 16 weeks, with or without a daily multivitamin, reduced plasma F2-isoprostane levels, a marker of oxidative stress that has been associated with and predictive of CVD (Zhang, 2013). Relative to placebo, both the 6 g salmon oil and the 6 g salmon oil multivitamin groups reduced plasma F2-isoprostane levels at study endpoint. This suggests that intake of LC n-3 PUFA can reduce oxidative stress, regardless of concomitant vitamin and mineral intake. Interestingly, 3 g of daily salmon oil combined with a multivitamin had no effect on F 2 isoprostane levels suggesting that salmon oil dosage is an important factor in initiating an antioxidant effect. Chemical models show that LC n-3 PUFAs are highly susceptible to oxidation based on their high degree of unsaturation (Holman & Elmer, 1947).This led to the idea of combining vitamin E with fish oils to prevent the oxidation of LC n-3 PUFAs (Armstrong & Koffman, 2000). However, LC n-3 PUFAs do not appear to be highly susceptible to oxidation in the body. As discussed by Richard, Kefi, Barbe, Bausero, and Visioli (2008), the relationship between chemical structure and susceptibility to oxidation in biological systems is complex. Administering LC n-3 PUFA to human aortic endothelial cells reduces the formation of reactive oxygen species (ROS), as compared to the administration of saturated fat, monounsaturated fat or n-6
PUFA (Richard et al., 2008). Other pre-clinical findings have also shown that feeding fish reduces oxidative damage (Wasim Khan et al., 2013). In humans, one previous study showed that the consumption of daily fish meals for 8 weeks reduced F 2 isoprostanes in sedentary, dyslipidaemic non-insulin-dependent diabetic patients (Mori et al., 1999). Further studies have shown that fish oil supplementation decreases plasma F2-isoprostanes in sedentary overweight adults (Kiecolt-Glaser et al., 2013), those with hypertension and type 2 diabetes (Mas et al., 2010) as well as overweight dyslipidaemic men (Mas et al., 2010). Urinary F2isoprostanes have also been shown to reduce in hypertensive diabetic subjects following fish oil supplementation (Mori et al., 2003). Our results extend on these studies by showing that combined EPA + DHA supplementation reduces F2-isopronstanes in a community dwelling sample without diabetes, dyslipidaemia or CVD. We did not demonstrate any changes in inflammatory cytokines or high sensitivity CRP with supplementation in our study population. Consistent with our findings, a 1 year study into the effects of fish oil supplementation on inflammation in healthy monks (mean age of 56 years) reported that fish oil had no effect on ex vivo cytokine production (Blok et al., 1997). In contrast, a RCT in healthy males aged 18–39 years showed fish oil supplementation for 12 weeks reduced IL-6 production but not TNF-α, IL-Iβ, IL-2 IL-4 or IL-10 (Wallace, Miles, & Calder, 2003). As reviewed by Calder (2006a), EPA and DHA can reduce inflammatory cytokines after stimulating cells ex vivo. The ability of fish oil to decrease cytokine production may be dependent on specific genetic polymorphisms related to inflammation (Grimble et al., 2002) and this may explain why inconsistent findings relating to fish oils and cytokines have been reported in the literature (Calder, 2006a). Moreover, across the current sample, levels of high sensitivity CRP were normal (<3 mg/L), perhaps meaning that there was relatively little scope to reduce inflammation in the current sample. Our study showed that, relative to placebo, treatment had no significant effects on cholesterol or triacylglycerol. Fish oils have been shown to decrease plasma triacylglycerol levels whereas decreases in LDL-cholesterol and HDL-cholesterol tend to only be observed when fish oil is compared to a saturated
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fat diet rather than placebo (Harris, 1989). Consistent with this, a recent study in dyslipidaemic adults reported no effect of fish oil supplementation (2 g/d), relative to placebo, on cholesterol levels after 4 weeks (Khandelwal et al., 2013). Although salmon oil treatment failed to lower triacylglycerol levels in the present study, this may be due to the fact that our participants were in good health displaying normal triacylglycerol levels at baseline. Triacylglycerol levels also reduced in the placebo group, potentially masking the reduction in triacylglycerol seen in the high dose salmon oil no multivitamin group. It is also possible that the current salmon oil dose was not large enough to produce an effect on triacylglycerol (Skulas-Ray et al., 2011). A previous study reported that, after 8 weeks of supplementation, triacylglycerol was lowered by 3.4 g/d of EPA + DHA but not 0.85 g/d of EPA+ DHA, relative to placebo (Skulas-Ray et al., 2011). Analysis of blood safety parameters revealed that aspartate aminotransferase, a liver function test, was increased by the multivitamin treatments, relative to placebo. This is unlikely to be clinically meaningful given that the mean scores for all treatments groups was well below 41 U/L at study end, the upper limit of the normal reference range for this test. Moreover, day to day variability in aspartate aminotransferase has been reported to be 5–10% (The National Academy of Clinical Biochemistry, 2000), similar to the magnitude of change reported here. Study strengths include the recruitment of subjects from the community, the prospective nature of the study, the masking of the supplements and placebo tablets, and the detailed biomarker characterisation, with all assays being measured in a masked manner and with all study visits for any subjects being within the same assay run. A limitation of the current study was that cytokines and F2-isoprostanes were not assessed across the whole sample (n = 86 and 101 respectively) due to funding limitations. Although we did not record participants’ changes in dietary habits over the duration of the study, participants were asked to consume their usual diet and our biochemical measure of EPA and DHA would include any dietary contributions. A further limitation is that the optimal dosages of salmon oil needed to reduce inflammation and oxidative stress are unknown, though the present study provides some insight. Although we administered two dosages, larger dosages may produce different effects to those observed in the current investigation. Individuals with disease states, such as diabetes, CVD or dyslipidaemia – which are usually associated with increased oxidative stress and inflammation – may require different doses of supplements.
4.1.
Conclusions
Consumption of 6 g of daily salmon oil for 4 months decreased oxidative stress, as reflected by F2-isoprostance levels, whether taken alone or in combination with a multivitamin. The same effect was not seen for 3 g of daily salmon oil. Relative to placebo, treatment had no effect on inflammation, cholesterol or triacylglycerol levels in this healthy sample. This is perhaps the first study to show that supplementation with salmon oil can reduce oxidative stress in healthy subjects.
Conflicts of interest The study was sponsored by Swisse Wellness Pty Ltd (formerly Swisse Vitamins Pty Ltd) under contract to Swinburne University of Technology and performed independently by the Centre for Human Psychopharmacology. The National Institute of Integrative Medicine, of which Professor Avni Sali is currently director, receives financial support from Swisse Wellness Pty Ltd. Andrew Pipingas and Avni Sali are currently members of the Scientific Advisory Panel for Swisse Wellness Pty Ltd. Aside from input into the supplements utilised and the broad aims of the study as well as the provision of supplements, Swisse Wellness Pty Ltd were not involved in any other aspect of the conduct of the trial including analysis, or interpretation of the trial findings.
Acknowledgements We are grateful to the study participants. During the study, MPP was funded by a Menzies Foundation Scholarship in Allied Health Science.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2014.10.028.
REFERENCES
Armstrong, E. G., & Koffman, R. G. (2000). Oxidation of plasma proteins is not increased after supplementation with eicosapentaenoic and docosahexaenoic acids. American Journal of Clinical Nutrition, 72, 731–737. Bertrandt, J., Klos, A., & Debski, B. (2004). Content of polyunsaturated fatty acids (PUFAs) in serum and liver of rats fed restricted diets supplemented with vitamins B2, B6 and folic acid. Biofactors (Oxford, England), 22, 189–192. Blok, W. L., Deslypere, J. P., Demacker, P. N. M., Van Der VenJongekrijg, J., Hectors, M. P. C., Van Der Meer, J. W. M., & Katan, M. B. (1997). Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year. European Journal of Clinical Investigation, 27, 1003–1008. Calder, P. C. (2006a). n-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases. American Journal of Clinical Nutrition, 83, 1505S–1519S. Calder, P. C. (2006b). Polyunsaturated fatty acids and inflammation. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 75, 197–202. Durand, P., Prost, M., & Blache, D. (1996). Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids. Atherosclerosis, 121, 231–243. Farzaneh-Far, R., Harris, W. S., Garg, S., Na, B., & Whooley, M. A. (2009). Inverse association of erythrocyte n-3 fatty acid levels with inflammatory biomarkers in patients with stable coronary artery disease: The Heart and Soul Study. Atherosclerosis, 205, 538–543.
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
ARTICLE IN PRESS journal of functional foods ■■ (2014) ■■–■■
Ferrucci, L., Cherubini, A., Bandinelli, S., Bartali, B., Corsi, A., Lauretani, F., Martin, A., Andres-Lacueva, C., Senin, U., & Guralnik, J. M. (2006). Relationship of plasma polyunsaturated fatty acids to circulating inflammatory markers. Journal of Clinical Endocrinology and Metabolism, 91, 439–446. Fotuhi, M., Mohassel, P., & Yaffe, K. (2009). Fish consumption, long-chain omega-3 fatty acids and risk of cognitive decline or Alzheimer disease: A complex association. Nature Clinical Practice. Neurology, 5, 140–152. Grimble, R. F., Howell, W. M., O’Reilly, G., Turner, S. J., Markovic, O., Hirrell, S., East, J. M., & Calder, P. C. (2002). The ability of fish oil to suppress tumor necrosis factor α production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor α production. American Journal of Clinical Nutrition, 76, 454–459. Harris, W. S. (1989). Fish oils and plasma lipid and lipoprotein metabolism in humans: A critical review. Journal of Lipid Research, 30, 785–807. Harris, W. S., Pottala, J. V., Varvel, S. A., Borowski, J. J., Ward, J. N., & McConnell, J. P. (2013). Erythrocyte omega-3 fatty acids increase and linoleic acid decreases with age: Observations from 160,000 patients. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 88, 257–263. Holman, R., & Elmer, O. (1947). The rates of oxidation of unsaturated fatty acids and esters. Journal of the American Oil Chemists Society, 24, 127–129. Kalogeropoulos, N., Panagiotakos, D. B., Pitsavos, C., Chrysohoou, C., Rousinou, G., Toutouza, M., & Stefanadis, C. (2010). Unsaturated fatty acids are inversely associated and n-6/n-3 ratios are positively related to inflammation and coagulation markers in plasma of apparently healthy adults. Clinica Chimica Acta, 411, 584–591. Khandelwal, S., Shidhaye, R., Demonty, I., Lakshmy, R., Gupta, R., Prabhakaran, D., & Reddy, S. (2013). Impact of omega-3 fatty acids and/or plant sterol supplementation on non-HDL cholesterol levels of dyslipidemic Indian adults. Journal of Functional Foods, 5, 36–43. Kiecolt-Glaser, J. K., Epel, E. S., Belury, M. A., Andridge, R., Lin, J., Glaser, R., Malarkey, W. B., Hwang, B. S., & Blackburn, E. (2013). Omega-3 fatty acids, oxidative stress, and leukocyte telomere length: A randomized controlled trial. Brain, Behavior, and Immunity, 28, 16–24. Kris-Etherton, P. M., Harris, W. S., & Appel, L. J. (2002). Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation, 106, 2747–2757. Liu, J. C., Conklin, S. M., Manuck, S. B., Yao, J. K., & Muldoon, M. F. (2014). Long-chain omega-3 fatty acids and blood pressure. American Journal of Hypertension, 24, 1121–1126. Mas, E., Woodman, R. J., Burke, V., Puddey, I. B., Beilin, L. J., Durand, T., & Mori, T. A. (2010). The omega-3 fatty acids EPA and DHA decrease plasma F2- isoprostanes: Results from two placebo-controlled interventions. Free Radical Research, 44, 983–990. Milne, G. L., Yin, H., Hardy, K. D., Davies, S. S., & Roberts, L. J. (2011). Isoprostane generation and function. Chemical Reviews, 111, 5973–5996. Morgan, T. K., Williamson, M., Pirotta, M., Stewart, K., Myers, S. P., & Barnes, J. (2012). A national census of medicines use: A 24hour snapshot of Australians aged 50 years and older. Medical Journal of Australia, 196, 50–53. Mori, T. A., Croft, K. D., Puddey, I. B., & Beilin, L. J. (1999). An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry. Analytical Biochemistry, 268, 117–125. Mori, T. A., Dunstan, D. W., Burke, V., Croft, K. D., Rivera, J. H., Beilin, L. J., & Puddey, I. B. (1999). Effect of dietary fish and exercise training on urinary F2-isoprostane excretion in non-
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insulin-dependent diabetic patients. Metabolism: Clinical and Experimental, 48, 1402–1408. Mori, T. A., Puddey, I. B., Burke, V., Croft, K. D., Dunstan, D. W., Rivera, J. H., & Beilin, L. J. (2000). Effect of ω3 fatty acids on oxidative stress in humans: GC-MS measurement of urinary F2-isoprostane excretion. Redox Report, 5, 45–46. Mori, T. A., Woodman, R. J., Burke, V., Puddey, I. B., Croft, K. D., & Beilin, L. J. (2003). Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects. Free Radical Biology and Medicine, 35, 772–781. Pase, M. P., Grima, N. A., Cockerell, R., Stough, C., Scholey, A., Sali, A., & Pipingas, A. (2015). The effects of omega-3 fish oils and multivitamins on cognitive and cardiovascular function. Journal of the American College of Nutrition, In press. Pipingas, A., Cockerell, R., Grima, N., Sinclair, A., Stough, C., Scholey, A., Myers, S., Croft, K., Sali, A., & Pase, M. P. (2014). Randomized controlled trial examining the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells. Nutrients, 6, 1956– 1970. Radimer, K., Bindewald, B., Hughes, J., Ervin, B., Swanson, C., & Picciano, M. F. (2004). Dietary supplement use by US adults: Data from the National Health and Nutrition Examination Survey, 1999–2000. American Journal of Epidemiology, 160, 339– 349. Richard, D., Kefi, K., Barbe, U., Bausero, P., & Visioli, F. (2008). Polyunsaturated fatty acids as antioxidants. Pharmacological Research, 57, 451–455. Ridker, P. M., Hennekens, C. H., Buring, J. E., & Rifai, N. (2000). C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. New England Journal of Medicine, 342, 836–843. Simopoulos, A. P. (2002). The importance of the ratio of omega-6/ omega-3 essential fatty acids. Biomedicine and Pharmacotherapy, 56, 365–379. Skulas-Ray, A. C., Kris-Etherton, P. M., Harris, W. S., Vanden Heuvel, J. P., Wagner, P. R., & West, S. G. (2011). Dose-response effects of omega-3 fatty acids on triglycerides, inflammation, and endothelial function in healthy persons with moderate hypertriglyceridemia. American Journal of Clinical Nutrition, 93, 243–252. Stangl, G. I., & Kirchgessner, M. (1998). Different degrees of moderate iron deficiency modulate lipid metabolism of rats. Lipids, 33, 889–895. Tan, Z. S., Harris, W. S., Beiser, A. S., Au, R., Himali, J. J., Debette, S., Pikula, A., DeCarli, C., Wolf, P. A., Vasan, R. S., Robins, S. J., & Seshadri, S. (2012). Red blood cell omega-3 fatty acid levels and markers of accelerated brain aging. Neurology, 78, 658– 664. The National Academy of Clinical Biochemistry (2000). Laboratory medicine practice guidelines: laboratory guidelines for screening, diagnosis and monitoring of hepatic injury. Washington, DC. Wallace, F. A., Miles, E. A., & Calder, P. C. (2003). Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. British Journal of Nutrition, 89, 679–689. Wasim Khan, M., Arivarasu, N. A., Priyamvada, S., Khan, S. A., Khan, S., & Yusufi, A. N. K. (2013). Protective effect of ω-3 polyunsaturated fatty acids (PUFA) on sodium nitrite induced nephrotoxicity and oxidative damage in rat kidney. Journal of Functional Foods, 5, 956–967. Xin, W., Wei, W., & Li, X. (2012). Effect of fish oil supplementation on fasting vascular endothelial function in humans: A metaanalysis of randomized controlled trials. PLoS ONE, 7. Zhang, Z. J. (2013). Systematic review on the association between F2-isoprostanes and cardiovascular disease. Annals of Clinical Biochemistry, 50, 108–114.
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Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial Andrew Pipingas a,*, Andrew Sinclair b, Kevin D. Croft c, Andrzej S. Januszewski d,e, Alicia J. Jenkins d,e, Trevor A. Mori c, Robyn Cockerell a, Natalie A. Grima f, Con Stough a, Andrew Scholey a, Stephen P. Myers g, Avni Sali h, Matthew P. Pase a a
Centre for Human Psychopharmacology, Swinburne University of Technology, Hawthorn, Australia School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia c School of Medicine and Pharmacology, University of Western Australia, Perth, Australia d Department of Medicine (St. Vincent’s), The University of Melbourne, Parkville, Australia e NHMRC Clinical Trials Centre, University of Sydney, Camperdown, Sydney, Australia f Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Australia g NatMed-Research Unit, Division of Research, Southern Cross University, Lismore, NSW 2480, Australia h National Institute of Integrative Medicine, Hawthorn 3122, Australia b
A R T I C L E
I N F O
A B S T R A C T
Article history:
The effects of daily fish oil supplementation, with and without multivitamins, on biochemi-
Received 8 September 2014
cal markers of health were examined. Healthy adults (N = 160) were randomised to receive
Received in revised form 19 October
3 g of salmon oil with a multivitamin, 6 g of salmon oil with a multivitamin, 6 g of salmon
2014
oil in isolation or placebo in a double-blind fashion on a daily basis for 16 weeks. Relative
Accepted 28 October 2014
to placebo, both 6 g salmon oil groups displayed significantly lower F2-isoprostane levels at
Available online
endpoint. Increases in red blood cell polyunsaturated fatty acids correlated with reductions in F2-isoprostanes. Treatment had no effect on inflammatory cytokines, C-reactive protein,
Keywords: Fish oil
fibrinogen, cholesterol or triacylglycerol. © 2014 Elsevier Ltd. All rights reserved.
PUFA Multivitamins Inflammation Oxidative stress F2-isoprostanes
* Corresponding author. Centre for Human Psychopharmacology, Swinburne University of Technology, ATC Building. PO Box 218, Hawthorn, 3122, Australia. Tel.: +61 3 9214 5215. E-mail address: [email protected] (A. Pipingas). Abbreviations: ANZCTR, Australia and New Zealand Clinical Trials Registry; AA, arachidonic acid; CVD, cardiovascular disease; CRP, C reactive protein; DHA, docosahexaenoic acid; EDTA, ethylenediaminetetraacetic acid; EPA, eicosapentaenoic acid; HDL, high density lipoprotein; LC n-3 PUFA, long chain omega-3 polyunsaturated fatty acids; LDL, low density lipoprotein; n-3, omega-3; n-6, omega-6; RBC, red blood cells; RCT, randomised controlled trial; ROS, reactive oxygen species http://dx.doi.org/10.1016/j.jff.2014.10.028 1756-4646/© 2014 Elsevier Ltd. All rights reserved. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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1.
Introduction
Fish oil supplements containing the long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are one of the most popular dietary supplements in the western world (Morgan et al., 2012). Evidence suggests that higher intake of LC n-3 PUFA is related to better cardiovascular (Kris-Etherton, Harris, & Appel, 2002) and perhaps even neurological outcomes (Fotuhi, Mohassel, & Yaffe, 2009; Tan et al., 2012). Fish oil supplements have been reported to improve the lipid profile, predominantly by lowering triacylglycerol levels (Harris, 1989), exerting vasodilatory effects (Xin, Wei, & Li, 2012) and by lowering blood pressure (Liu, Conklin, Manuck, Yao, & Muldoon, 2014). LC n-3 PUFAs also play an important role in regulating inflammation (Calder, 2006b). The omega-6 (n-6) arachidonic acid (AA) has pro-inflammatory effects through the production of eicosanoids. Both AA and LC n-3 PUFA compete for space inside cell membrane phospholipids (Simopoulos, 2002). Increasing cellular levels of LC n-3 PUFA decreases the amount of AA in cell membranes thus limiting AA derived eicosanoids (Simopoulos, 2002). The ratio of LC n-3 PUFA to omega-6 fatty acid has thus been put forward as a measure of inflammation (Kalogeropoulos et al., 2010). Observational studies have reported that higher circulating levels of LC n-3 PUFA are associated with lower levels of pro-inflammatory cytokines and lower levels of C-reactive protein (CRP) (Farzaneh-Far, Harris, Garg, Na, & Whooley, 2009; Ferrucci et al., 2006; Kalogeropoulos et al., 2010), which are known to be predictive of cardiovascular disease (CVD) (Ridker, Hennekens, Buring, & Rifai, 2000). LC n-3 PUFAs may also have antioxidant effects. A study by Mori et al. (1999) reported that a daily fish meal for eight weeks reduced urinary F2-isoprostane levels in sedentary subjects with non-insulin dependent diabetes. F2-isoprostanes are lipid peroxidation products derived from the non-enzymatic free radical oxidation of AA in membrane lipids and are considered the most reliable biomarkers of in vivo lipid peroxidative damage (Milne, Yin, Hardy, Davies, & Roberts, 2011). Subsequent placebo controlled studies using purified EPA or DHA showed both fatty acids reduced urinary (Mori et al., 2000, 2003) and plasma (Mas et al., 2010) F2-isoprostanes in type 2 diabetic patients, and in overweight mildly dyslipidaemic men, respectively. A recent double-blind, 4 month randomised, controlled trial (RCT) reported that LC n-3 PUFA supplementation reduced plasma F2-isoprostane levels by approximately 15%, relative to placebo (Kiecolt-Glaser et al., 2013). Multivitamin supplements, containing both vitamins and minerals, are commonly used among the general population (Radimer et al., 2004). Evidence suggests that the intake of a range of vitamins and minerals may interact with fatty acid metabolism influencing the overall levels of LC n-3 PUFA measured in vivo (Bertrandt, Klos, & Debski, 2004; Durand, Prost, & Blache, 1996; Pipingas et al., 2014; Stangl & Kirchgessner, 1998). There is thus a need to examine the combined effects of multivitamin supplements and LC n-3 PUFAs on all aspects of human health. The present 16 week RCT was designed to investigate the effect of salmon oil supplementation, with and without the
addition of a multivitamin supplement, on cardiovascular and cognitive health in healthy middle-aged and elderly subjects. To the best of our knowledge, this is the first RCT to investigate the combined effects of fish oils and multivitamins on human health. The results of supplementation on the cognitive, cardiovascular and omega-3 uptake into red bloods cells arising from this RCT have been published (Pase et al., 2015; Pipingas et al., 2014). This report examines the secondary outcomes from this study including the effects of supplementation on measures of inflammation, oxidative stress, serum lipids and liver function, in the same subjects. It was hypothesised that treatment with salmon oil would reduce markers of inflammation and oxidative stress as well as triacylglycerol levels. The study also had the novel aim of exploring whether combining salmon oil with a multivitamin would provide added benefits to human health.
2.
Materials and methods
2.1.
Participants
One hundred and sixty healthy, non-smoking, male and female volunteers aged between 50 and 70 years were recruited from the community using newspaper advertisements, posters and community radio announcements. Participants were not currently taking any medication, such as anti-coagulants, anticholinergic, anti-depressant medications and acetylcholinesterase inhibitors, or vitamin/herbal supplements. Subjects were excluded if they fulfilled any of the following; diagnosis of dementia, diabetes, neurological and psychiatric disorders, overt CVD or past or present drug or alcohol abuse. Long term multivitamin and/or omega-3 supplement users were also excluded. The participant flow diagram is shown in Fig. 1.
2.2.
Setting
The study was conducted at the Centre for Human Psychopharmacology Laboratory at Swinburne University of Technology, Hawthorn, Australia. The study was approved by the Swinburne University Human Research Ethics Committee and all procedures were conducted in accordance with the guidelines of the Australian National Health and Medical Research Council and the Declaration of Helsinki (2008). This trial was registered with the Australian and New Zealand Clinical Trial Registry (ANZCTR no. 12611000094976). Participants gave informed written consent to participate in the study.
2.3.
Interventions, randomisation and blinding
This study was a 16 week randomised, double-blind, placebocontrolled trial comparing the effects of salmon oil supplements and multivitamins on blood biomarkers. Participants were randomly assigned to one of four treatment groups: multivitamin and salmon oil (3 g); multivitamin and salmon oil (6 g); placebo multivitamin and salmon oil (6g); or placebo multivitamin and placebo salmon oil (Sunola oil). Participants were instructed to take their assigned treatment daily for 16 weeks. The study medications were provided
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Fig. 1 – Participant flow diagram.
by Swisse (Melbourne, Australia). The active salmon oil was Swisse Ultiboost Wild Salmon Oil and the active multivitamin was Swisse Ultivite 50 + (Men’s and Women’s formulations). All participants took one multivitamin, or matching placebo, daily and immediately following breakfast. To aid masking, the placebo multivitamin consisted of carrot powder with a small amount of riboflavin to produce a multivitamin like colouration and smell of the urine. The Swisse Ultiboost Wild Salmon Oil capsules contained 1 g of omega-3 (80 mg EPA and 80 mg DHA) each, whilst the matching placebo consisted of Sunola oil combined with 50 IU of vitamin E. All salmon oil containers (including those containing the placebo) included a sachet with a few drops of salmon oil so as to give a distinctive fish scent. Sunola oil, a monounsaturated (omega-9) sunflower oil, was used as a placebo because it is virtually trans-fat free and unlike olive oil there is little research showing health benefits. Both the active and placebo salmon oils were administered in identically appearing soft gelatine capsules. All participants took six active (1 g each) salmon oil or placebo salmon oil capsules daily. Those allocated to 3 g of daily salmon oil received three active salmon oil and three placebo salmon oil capsules daily. A random permuted block procedure with a block size of 4 was used to assign participants to one of the four experimental groups. Randomisation was conducted by the trial sponsor with the treatment packaging labelled according to the randomisation schedule. The research staff were blinded to this allocation. Placebo and treatments were packaged in
identical blister packs for multivitamins and identical sealed plastic containers for salmon oil capsules. Participants received separate sealed plastic containers and participants were instructed to consume three capsules from one container in the morning and three capsules from the other container in the evening. Depending on treatment allocation, this allowed participants to receive either 6 g, 3 g or 0 g of daily salmon oil. Treatments could only be differentiated by the presence of a participant number. Participants were allocated the next sequential number upon enrolment in the study. The masking key was held in a secure password protected electronic file and was only revealed at the end of the study and once analysis of the main study outcomes had been completed. Investigators performing the biochemical analyses were masked to subject treatment and sample order.
2.4.
Outcomes measures
Blood samples were collected via venepuncture from the antecubital vein, using the BD-vacutainer system, following a 12hour overnight fasting period. Biochemistry included electrolyte levels, liver function and kidney function tests. High sensitivity CRP, fibrinogen and lipids were analysed by Healthscope Functional Pathology according to standard procedures. CRP was measured in serum by a latex-enhanced immunoturbidimetric assay. Fibrinogen was quantified by the addition of thrombin to plasma, and measuring the clotting time of the diluted plasma compared to a standardised fibrinogen
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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preparation. Total cholesterol, HDL-cholesterol and LDLcholesterol were measured using the ADVIA system based on the enzymatic method. The method of analysis for triacylglycerol is based on Fossati three-step enzymatic reaction with a Trinder endpoint. Analysis was performed on an ADVIA 1650 analyser (Siemens, USA) by Healthscope Pathology. Blood for plasma F 2 -isoprostanes was collected into ethylenediaminetetraacetic acid (EDTA) and reduced glutathione, and centrifuged at 3000 × g for 20 min at 4 °C. To prevent oxidation of the samples, butylated hydroxytoluene (40 µg per mL of plasma) was added to plasma before samples were stored at −80 °C until analysis. F2-isoprostanes were quantified by negative ion chemical ionisation–gas chromatograph mass spectroscopy as described by Mori et al. (Mori, Croft, Puddey, & Beilin, 1999; Mori et al., 1999). The within and between assay reproducibility is 8.0 and 5.6%, respectively. Levels of cytokines (IL-6, IL-1β and TNFα) were measured using ELISA (R&D, Minneapolis, USA) on a BEP 2000 advance (Siemens, Erlangen, Germany) automated analyser with intra- and inter-assay CVs of <4, <7.5 and <3.6%, respectively. Measurement of F2-isoprostanes and cytokines were included in the protocol after commencement of the study. Once added to the study protocol, all newly recruited subjects provided blood for F2-isoprostanes and cytokines. Consequently 101 subjects had blood samples taken for measurement of F2isoprostanes at baseline (with 91 measurements completed at endpoint) and cytokines were measured on 86 subjects at baseline (with 80 measurements completed at endpoint; see Fig. 1). Fatty acids samples were analysed by Healthscope Functional Pathology according to standard procedures described elsewhere (Pipingas et al., 2014).
2.5.
Procedure
Participants attended three testing sessions at our laboratory including baseline, 6 week and endpoint assessment following 16 weeks of treatment. Blood biomarkers were only measured at baseline and endpoint. Participants attended at the same time of day across all testing sessions.
2.6. Sample size calculations and assessment of compliance
effects of treatment. ANCOVAs were then repeated controlling for gender, to examine if the effects of supplementation were specific to one gender, as has been previously reported (Pipingas et al., 2014). In addition to the ANCOVAs, correlations examined whether absolute changes in blood omega-3 fatty acid levels were associated with changes in inflammatory, oxidative stress and cardiometabolic biomarkers, over the 16-week supplementation period. F2-isoprostanes, CRP and the inflammatory cytokine measures were significantly skewed. Skewness of the F2-isoprostanes and triacylglycerol were corrected by applying natural log transformations. Skewness of the IL-6, IL-Iβ and TNFα variables was corrected by applying negative square root transformations. The distribution of high sensitivity CRP could not be made normal through transformation. High sensitivity CRP was thus analysed using the nonparametric Kruskal–Wallis one way ANOVA. This test examined whether the change from baseline scores in CRP differed according to treatment allocation. All results were considered significant at the level of p < 0.05.
3.
Results
The trial commenced in 2010 and was ceased in 2012 due to attainment of the required sample size. Across the whole sample, 16 participants were lost to follow-up between baseline and endpoint (6 in the 3 g salmon oil multivitamin group, 2 in the 6 g salmon oil multivitamin group, 3 in the 6 g salmon oil group and 5 in the placebo. There were no serious adverse events reported during the study. Characteristics of the participants at baseline are shown in Table 1. Blood levels of omega-3 fatty acids tended to be low (Harris et al., 2013). The effects of treatment on omega-3 fatty acid incorporation into erythrocytes are reported elsewhere (Pipingas et al., 2014). In brief, EPA increased by 96% in the group receiving the combination of the high dose salmon oil and multivitamin. The omega-3 index increased in this group by 40%. EPA increased by 57% in the group receiving the high dose salmon oil only. There were no significant changes in DHA or DPA. The AA/EPA ratio decreased across all treatment groups relative to placebo, indicating adequate compliance across the sample.
3.1.
Effect of the intervention on F2-isoprostane levels
Sample size calculations were based on the primary outcomes of cognitive and cardiovascular function (Pase et al., 2015). Based on the available sample size for cytokines and F2isoprostanes, this study had a 98% change of detecting a small effect size of 0.25 with alpha set to 0.05, for these measures. Power was even greater for the lipid measures given the larger sample size available. Compliance to treatment was assessed by examining changes in the AA/EPA ratio over the 16 week study period (Pipingas et al., 2014).
There was a significant effect of treatment on F2-isoprostane levels at week 16, relative to baseline (F (3, 86) = 11.08, p < 0.001) (Table 2). As compared to placebo, both 6 g salmon oil groups displayed significantly lower F2-isoprostane levels at week 16 (Fig. 2). The two 6 g salmon oil groups, with and without a multivitamin, did not differ significantly to each other (p = 0.40) and the low dose salmon oil group did not differ to placebo (p = 0.22).
2.7.
3.2.
Statistical analyses
Univariate ANCOVAs were used to examine the effects of treatment on blood biomarkers at week 16, adjusting for values at baseline. Pairwise comparisons, applying Bonferroni corrections to adjust for multiple comparisons across the four treatment groups, were used to further examine significant
Effect of the intervention on inflammatory markers
Treatment allocation had no effect on fibrinogen (F (3, 132) = 0.39, p = 0.76), IL-1β (F (3, 75) = 0.07, p = 0.98), IL-6 (F (3, 75) = 0.60, p = 0.62) or TNF-α (F (3, 75) = 0.16, p = 0.92). Changes in CRP over the 16 week study period did not differ according to treatment allocation (p = 0.85).
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Table 1 – Baseline sample means and standard deviations by treatment allocation. Variable Demographics N Age, y Male, % Education, y MMSE BMI Clinical SBP, mmHg DBP, mmHg Heart rate, bpm Fatty acids EPA, % DHA, % DPA, % N3 index, %
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
Placebo
Overall
42 59.5 48 16 28 25.5
40 58.9 48 16 28 24.4
41 59.5 46 16 28 25.3
37 59.2 46 16 28 24.2
(3) (2) (2.8)
160 59.3 (5.7) 47 16 (4) 28 (2) 24.9 (3.4)
(5.6) (3) (2) (3.6)
(5.6) (4) (2) (3.1)
(5.9) (4) (2) (4.0)
(6.0)
126 (21) 77 (13) 66 (11)
123 (17) 75 (11) 67 (10)
126 (17) 78 (10) 65 (12)
121 (21) 75 (12) 66 (9)
124 (19) 76 (12) 66 (11)
0.99 2.66 1.69 5.34
1.01 2.74 1.88 5.63
1.06 2.92 1.94 5.92
1.00 2.82 1.83 5.65
0.10 2.78 1.84 5.63
(0.46) (1.26) (0.75) (2.31)
(0.30) (0.96) (0.50) (1.59)
(0.41) (1.10) (0.65) (2.04)
(0.43) (1.11) (0.61) (2.00)
(0.40) (1.11) (0.64) (1.99)
Note: MMSE = Mini Mental State Examination; WASI = Wechsler Abbreviated Scale of Intelligence; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; N3 = omega-3; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.
Table 2 – Inflammation and oxidative stress measured over the course of supplementation, stratified by treatment allocation. Variable
Oxidative stress F2s, pmol/La Baseline Week 16 %change Inflammation HS-CRPb Baseline Week 16 %change Fibrinogen Baseline Week 16 %change IL-βa Baseline Week 16 %change IL-6a Baseline Week 16 %change TNF-αa Baseline Week 16 %change
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
1659.8 (312.7) 1477.0 (292.6) −11.01%
1651.9 (366.6) 1329.7 (328.2)*** −19.50
1646.8 (336.3) 1383.8 (306.6)** −15.97
Placebo
ANCOVA F
p
11.08
<0.001
−
−
1644.4 (490.8) 1557.0 (337.9) −5.32
1.9 (3.1) 1.2 (1.6) −37.6
2.3 (5.7) 1.1 (1.5) −54.1
1.3 (2.6) 1.2 (2.2) −5.4
1.3 (1.6) 1.00 (1.4) −21.4
2.8 (0.4) 2.9 (0.5) 5.1
2.8 (0.6) 3.0 (0.5) 5.7
2.7 (0.5) 3.0 (0.6) 8.4
2.9 (0.4) 3.0 (0.4) 4.5
0.9 (0.2) 0.9 (0.2) −2.2
0.8 (0.2) 0.8 (0.2) 5.0
0.9 (0.3) 0.9 (0.2) −3.2
1.0 (0.6) 0.9 (0.2) −12.9
1.7 (1.0) 1.5 (1.1) −11.0
2.1 (2.6) 2.1 (2.2) −1.4
1.4 (1.0) 1.7 (1.4) 17.7
1.3 (0.5) 1.4 (0.7) 11.1
1.8 (0.7) 1.7 (0.5) −8.7
1.7 (0.5) 1.6 (0.5) −2.4
1.7 (0.6) 1.6 (0.5) −5.3
1.5 (0.5) 1.5 (0.4) −5.2
0.39
0.76
0.07
0.98
0.60
0.62
0.16
0.92
HS-CRP = high sensitivity C reactive protein; IL-6 = interleukin-6 [pg/mL]; IL-1β = interleukin-1β [pg/mL]; TNFα = tumor necrosis factor α [pg/ mL]; HS-CRP = high sensitivity C reactive protein [mg/L]; F2s = F2-isoprostanes [pmol/L]; ANCOVA = analysis of covariance. Note that ANCOVA statistics refer to the main effect of treatment at week 16. a Statistics performed on transformed values. b Investigated using non-parametric test. *p < 0.01, **p < 0.01, ***p < 0.001. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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significantly from placebo. At week 16, there was no effect of treatment group on HDL-cholesterol (F (3, 134) = 1.23, p = 0.30), LDL-cholesterol (F (3, 134) = 0.54, p = 0.66) or total cholesterol (F (3, 137) = 0.27, p = 0.85).
3.4.
Fig. 2 – Changes in F2-isoprostanes over the 16 week study period stratified according to treatment allocation. SO = salmon oil, MV = multivitamin, *** denotes significant difference from placebo at p < 0.001, ** denotes significant difference from placebo at p < 0.01. Values of F2isoprostanes are presented in original units (without log transformation).
3.3.
Effect of the intervention on blood lipids
There was a significant main effect of treatment for triacylglycerol levels at week 16 (F (3, 137) = 3.43, p < 0.05) (Table 3). Pairwise comparisons showed that the group taking 3 g of salmon oil combined with the multivitamin had lower triacylglycerol levels at week 16 as compared to the group receiving 6 g of salmon oil combined with the multivitamin (p = 0.04). No other significant group differences were found meaning that the treatment groups did not differ
Safety of supplementation
The analysis of the electrolyte, liver function and kidney function tests showed differences from baseline after 16 weeks in only alanine aminotransferase (F (3, 136) = 3.41, p < 0.05), aspartate aminotransferase (F (3, 136) = 6.44, p < 0.001) and estimated glomerular filtration rate (F (3, 80) = 3.15, p < 0.05). Pairwise comparisons revealed that aspartate aminotransferase was higher in both multivitamin groups, relative to placebo, at study-end. For estimated glomerular filtration rate and alanine aminotransferase, none of the treatment groups differed according to placebo. Means, standard deviations and percentage changes for all the safety parameters are shown in Supplementary Table S1 online.
3.5.
Effects of supplementation in men and in women
Controlling for gender did not significantly alter any of the aforementioned results pertaining to the effects of treatment on cardiometabolic risk factors, oxidative stress or inflammation (data for this analysis are not shown).
3.6. Changes in long-chain omega-3 fatty acid levels versus changes in F2-isoprostanes, cardiometabolic risk and inflammation Two-tailed correlations examined absolute changes in red blood cell omega-3 fatty acids with absolute changes in the cardiometabolic, inflammatory and oxidative stress measures over the 16 week study period. Correlations were computed using
Table 3 – Metabolic cardiovascular risk factors measured over the course of supplementation, stratified by treatment allocation. Variable
HDL cholesterol, mmol/L Baseline Week 16 %change LDL cholesterol, mmol/L Baseline Week 16 %change Total cholesterol, mmol/L Baseline Week 16 %change Triacylglycerola, mmol/L Baseline Week 16 %change
Salmon oil, 3 g + multivitamin
Salmon oil, 6 g + multivitamin
Salmon oil, 6 g
Placebo
1.5 (0.4) 1.6 (0.5) 5.3
1.6 (0.4) 1.7 (0.4) 3.1
1.6 (0.4) 1.6 (0.4) 1.3
1.6 (0.4) 1.6 (0.3) −1.3
3.3 (0.7) 3.4 (0.7) 2.1
3.5 (0.7) 3.5 (0.9) 0.6
3.4 (0.8) 3.5 (1.1) 4.8
3.3 (0.7) 3.3 (0.8) 0.0
5.4 (0.9) 5.5 (1.0) 2.2
5.7 (0.8) 5.8 (1.0) 2.8
5.5 (1.0) 5.5 (1.1) 1.5
5.5 (0.9) 5.4 (0.7) −1.5
1.2 (0.5) 1.1 (0.6) −6.6
1.2 (0.7) 1.3 (0.8) 10.1
1.2 (0.6) 1.0 (0.4) −16.4
1.3 (0.7) 1.2 (0.7) −7.2
ANCOVA F
p
1.23
0.30
0.54
0.66
0.27
0.85
3.43
<0.05
ANCOVA = analysis of covariance. Note that ANCOVA statistics refer to the main effect of treatment at week 16. a Statistics performed on transformed values. Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
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Fig. 3 – Correlations between change in F2-isoprostanes over the 16 week study period and changes in (A) the n3/n6 fatty acid ratio, (B) total EPA and (C) changes in the AA/EPA fatty acid ratio. Values of F2-isoprostanes have been log transformed.
the entire sample irrespective of treatment allocation. With respect to oxidative stress (Fig. 3), incorporation of both EPA (r = −0.30, p < 0.01) and the ratio of total omega-3 to omega-6 fatty acids (r = −0.30, p < 0.01) were negatively associated with F2-isoprostanes.The AA/EPA ratio was positively associated with F2-isoprostanes (r = 0.35, p < 0.01). There were no other significant correlations between changes in red blood cell omega-3 fatty acids and measures of inflammation or blood lipids.
4.
Discussion
To the best of our knowledge, this is the first study to investigate the effects of salmon oil supplementation, with and without the addition of a multivitamin, on multiple blood biomarkers in healthy older men and women. The principle finding was that 6 g of salmon oil daily for 16 weeks, with or without a daily multivitamin, reduced plasma F2-isoprostane levels, a marker of oxidative stress that has been associated with and predictive of CVD (Zhang, 2013). Relative to placebo, both the 6 g salmon oil and the 6 g salmon oil multivitamin groups reduced plasma F2-isoprostane levels at study endpoint. This suggests that intake of LC n-3 PUFA can reduce oxidative stress, regardless of concomitant vitamin and mineral intake. Interestingly, 3 g of daily salmon oil combined with a multivitamin had no effect on F 2 isoprostane levels suggesting that salmon oil dosage is an important factor in initiating an antioxidant effect. Chemical models show that LC n-3 PUFAs are highly susceptible to oxidation based on their high degree of unsaturation (Holman & Elmer, 1947).This led to the idea of combining vitamin E with fish oils to prevent the oxidation of LC n-3 PUFAs (Armstrong & Koffman, 2000). However, LC n-3 PUFAs do not appear to be highly susceptible to oxidation in the body. As discussed by Richard, Kefi, Barbe, Bausero, and Visioli (2008), the relationship between chemical structure and susceptibility to oxidation in biological systems is complex. Administering LC n-3 PUFA to human aortic endothelial cells reduces the formation of reactive oxygen species (ROS), as compared to the administration of saturated fat, monounsaturated fat or n-6
PUFA (Richard et al., 2008). Other pre-clinical findings have also shown that feeding fish reduces oxidative damage (Wasim Khan et al., 2013). In humans, one previous study showed that the consumption of daily fish meals for 8 weeks reduced F 2 isoprostanes in sedentary, dyslipidaemic non-insulin-dependent diabetic patients (Mori et al., 1999). Further studies have shown that fish oil supplementation decreases plasma F2-isoprostanes in sedentary overweight adults (Kiecolt-Glaser et al., 2013), those with hypertension and type 2 diabetes (Mas et al., 2010) as well as overweight dyslipidaemic men (Mas et al., 2010). Urinary F2isoprostanes have also been shown to reduce in hypertensive diabetic subjects following fish oil supplementation (Mori et al., 2003). Our results extend on these studies by showing that combined EPA + DHA supplementation reduces F2-isopronstanes in a community dwelling sample without diabetes, dyslipidaemia or CVD. We did not demonstrate any changes in inflammatory cytokines or high sensitivity CRP with supplementation in our study population. Consistent with our findings, a 1 year study into the effects of fish oil supplementation on inflammation in healthy monks (mean age of 56 years) reported that fish oil had no effect on ex vivo cytokine production (Blok et al., 1997). In contrast, a RCT in healthy males aged 18–39 years showed fish oil supplementation for 12 weeks reduced IL-6 production but not TNF-α, IL-Iβ, IL-2 IL-4 or IL-10 (Wallace, Miles, & Calder, 2003). As reviewed by Calder (2006a), EPA and DHA can reduce inflammatory cytokines after stimulating cells ex vivo. The ability of fish oil to decrease cytokine production may be dependent on specific genetic polymorphisms related to inflammation (Grimble et al., 2002) and this may explain why inconsistent findings relating to fish oils and cytokines have been reported in the literature (Calder, 2006a). Moreover, across the current sample, levels of high sensitivity CRP were normal (<3 mg/L), perhaps meaning that there was relatively little scope to reduce inflammation in the current sample. Our study showed that, relative to placebo, treatment had no significant effects on cholesterol or triacylglycerol. Fish oils have been shown to decrease plasma triacylglycerol levels whereas decreases in LDL-cholesterol and HDL-cholesterol tend to only be observed when fish oil is compared to a saturated
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fat diet rather than placebo (Harris, 1989). Consistent with this, a recent study in dyslipidaemic adults reported no effect of fish oil supplementation (2 g/d), relative to placebo, on cholesterol levels after 4 weeks (Khandelwal et al., 2013). Although salmon oil treatment failed to lower triacylglycerol levels in the present study, this may be due to the fact that our participants were in good health displaying normal triacylglycerol levels at baseline. Triacylglycerol levels also reduced in the placebo group, potentially masking the reduction in triacylglycerol seen in the high dose salmon oil no multivitamin group. It is also possible that the current salmon oil dose was not large enough to produce an effect on triacylglycerol (Skulas-Ray et al., 2011). A previous study reported that, after 8 weeks of supplementation, triacylglycerol was lowered by 3.4 g/d of EPA + DHA but not 0.85 g/d of EPA+ DHA, relative to placebo (Skulas-Ray et al., 2011). Analysis of blood safety parameters revealed that aspartate aminotransferase, a liver function test, was increased by the multivitamin treatments, relative to placebo. This is unlikely to be clinically meaningful given that the mean scores for all treatments groups was well below 41 U/L at study end, the upper limit of the normal reference range for this test. Moreover, day to day variability in aspartate aminotransferase has been reported to be 5–10% (The National Academy of Clinical Biochemistry, 2000), similar to the magnitude of change reported here. Study strengths include the recruitment of subjects from the community, the prospective nature of the study, the masking of the supplements and placebo tablets, and the detailed biomarker characterisation, with all assays being measured in a masked manner and with all study visits for any subjects being within the same assay run. A limitation of the current study was that cytokines and F2-isoprostanes were not assessed across the whole sample (n = 86 and 101 respectively) due to funding limitations. Although we did not record participants’ changes in dietary habits over the duration of the study, participants were asked to consume their usual diet and our biochemical measure of EPA and DHA would include any dietary contributions. A further limitation is that the optimal dosages of salmon oil needed to reduce inflammation and oxidative stress are unknown, though the present study provides some insight. Although we administered two dosages, larger dosages may produce different effects to those observed in the current investigation. Individuals with disease states, such as diabetes, CVD or dyslipidaemia – which are usually associated with increased oxidative stress and inflammation – may require different doses of supplements.
4.1.
Conclusions
Consumption of 6 g of daily salmon oil for 4 months decreased oxidative stress, as reflected by F2-isoprostance levels, whether taken alone or in combination with a multivitamin. The same effect was not seen for 3 g of daily salmon oil. Relative to placebo, treatment had no effect on inflammation, cholesterol or triacylglycerol levels in this healthy sample. This is perhaps the first study to show that supplementation with salmon oil can reduce oxidative stress in healthy subjects.
Conflicts of interest The study was sponsored by Swisse Wellness Pty Ltd (formerly Swisse Vitamins Pty Ltd) under contract to Swinburne University of Technology and performed independently by the Centre for Human Psychopharmacology. The National Institute of Integrative Medicine, of which Professor Avni Sali is currently director, receives financial support from Swisse Wellness Pty Ltd. Andrew Pipingas and Avni Sali are currently members of the Scientific Advisory Panel for Swisse Wellness Pty Ltd. Aside from input into the supplements utilised and the broad aims of the study as well as the provision of supplements, Swisse Wellness Pty Ltd were not involved in any other aspect of the conduct of the trial including analysis, or interpretation of the trial findings.
Acknowledgements We are grateful to the study participants. During the study, MPP was funded by a Menzies Foundation Scholarship in Allied Health Science.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2014.10.028.
REFERENCES
Armstrong, E. G., & Koffman, R. G. (2000). Oxidation of plasma proteins is not increased after supplementation with eicosapentaenoic and docosahexaenoic acids. American Journal of Clinical Nutrition, 72, 731–737. Bertrandt, J., Klos, A., & Debski, B. (2004). Content of polyunsaturated fatty acids (PUFAs) in serum and liver of rats fed restricted diets supplemented with vitamins B2, B6 and folic acid. Biofactors (Oxford, England), 22, 189–192. Blok, W. L., Deslypere, J. P., Demacker, P. N. M., Van Der VenJongekrijg, J., Hectors, M. P. C., Van Der Meer, J. W. M., & Katan, M. B. (1997). Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year. European Journal of Clinical Investigation, 27, 1003–1008. Calder, P. C. (2006a). n-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases. American Journal of Clinical Nutrition, 83, 1505S–1519S. Calder, P. C. (2006b). Polyunsaturated fatty acids and inflammation. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 75, 197–202. Durand, P., Prost, M., & Blache, D. (1996). Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids. Atherosclerosis, 121, 231–243. Farzaneh-Far, R., Harris, W. S., Garg, S., Na, B., & Whooley, M. A. (2009). Inverse association of erythrocyte n-3 fatty acid levels with inflammatory biomarkers in patients with stable coronary artery disease: The Heart and Soul Study. Atherosclerosis, 205, 538–543.
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028
ARTICLE IN PRESS journal of functional foods ■■ (2014) ■■–■■
Ferrucci, L., Cherubini, A., Bandinelli, S., Bartali, B., Corsi, A., Lauretani, F., Martin, A., Andres-Lacueva, C., Senin, U., & Guralnik, J. M. (2006). Relationship of plasma polyunsaturated fatty acids to circulating inflammatory markers. Journal of Clinical Endocrinology and Metabolism, 91, 439–446. Fotuhi, M., Mohassel, P., & Yaffe, K. (2009). Fish consumption, long-chain omega-3 fatty acids and risk of cognitive decline or Alzheimer disease: A complex association. Nature Clinical Practice. Neurology, 5, 140–152. Grimble, R. F., Howell, W. M., O’Reilly, G., Turner, S. J., Markovic, O., Hirrell, S., East, J. M., & Calder, P. C. (2002). The ability of fish oil to suppress tumor necrosis factor α production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor α production. American Journal of Clinical Nutrition, 76, 454–459. Harris, W. S. (1989). Fish oils and plasma lipid and lipoprotein metabolism in humans: A critical review. Journal of Lipid Research, 30, 785–807. Harris, W. S., Pottala, J. V., Varvel, S. A., Borowski, J. J., Ward, J. N., & McConnell, J. P. (2013). Erythrocyte omega-3 fatty acids increase and linoleic acid decreases with age: Observations from 160,000 patients. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 88, 257–263. Holman, R., & Elmer, O. (1947). The rates of oxidation of unsaturated fatty acids and esters. Journal of the American Oil Chemists Society, 24, 127–129. Kalogeropoulos, N., Panagiotakos, D. B., Pitsavos, C., Chrysohoou, C., Rousinou, G., Toutouza, M., & Stefanadis, C. (2010). Unsaturated fatty acids are inversely associated and n-6/n-3 ratios are positively related to inflammation and coagulation markers in plasma of apparently healthy adults. Clinica Chimica Acta, 411, 584–591. Khandelwal, S., Shidhaye, R., Demonty, I., Lakshmy, R., Gupta, R., Prabhakaran, D., & Reddy, S. (2013). Impact of omega-3 fatty acids and/or plant sterol supplementation on non-HDL cholesterol levels of dyslipidemic Indian adults. Journal of Functional Foods, 5, 36–43. Kiecolt-Glaser, J. K., Epel, E. S., Belury, M. A., Andridge, R., Lin, J., Glaser, R., Malarkey, W. B., Hwang, B. S., & Blackburn, E. (2013). Omega-3 fatty acids, oxidative stress, and leukocyte telomere length: A randomized controlled trial. Brain, Behavior, and Immunity, 28, 16–24. Kris-Etherton, P. M., Harris, W. S., & Appel, L. J. (2002). Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation, 106, 2747–2757. Liu, J. C., Conklin, S. M., Manuck, S. B., Yao, J. K., & Muldoon, M. F. (2014). Long-chain omega-3 fatty acids and blood pressure. American Journal of Hypertension, 24, 1121–1126. Mas, E., Woodman, R. J., Burke, V., Puddey, I. B., Beilin, L. J., Durand, T., & Mori, T. A. (2010). The omega-3 fatty acids EPA and DHA decrease plasma F2- isoprostanes: Results from two placebo-controlled interventions. Free Radical Research, 44, 983–990. Milne, G. L., Yin, H., Hardy, K. D., Davies, S. S., & Roberts, L. J. (2011). Isoprostane generation and function. Chemical Reviews, 111, 5973–5996. Morgan, T. K., Williamson, M., Pirotta, M., Stewart, K., Myers, S. P., & Barnes, J. (2012). A national census of medicines use: A 24hour snapshot of Australians aged 50 years and older. Medical Journal of Australia, 196, 50–53. Mori, T. A., Croft, K. D., Puddey, I. B., & Beilin, L. J. (1999). An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry. Analytical Biochemistry, 268, 117–125. Mori, T. A., Dunstan, D. W., Burke, V., Croft, K. D., Rivera, J. H., Beilin, L. J., & Puddey, I. B. (1999). Effect of dietary fish and exercise training on urinary F2-isoprostane excretion in non-
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insulin-dependent diabetic patients. Metabolism: Clinical and Experimental, 48, 1402–1408. Mori, T. A., Puddey, I. B., Burke, V., Croft, K. D., Dunstan, D. W., Rivera, J. H., & Beilin, L. J. (2000). Effect of ω3 fatty acids on oxidative stress in humans: GC-MS measurement of urinary F2-isoprostane excretion. Redox Report, 5, 45–46. Mori, T. A., Woodman, R. J., Burke, V., Puddey, I. B., Croft, K. D., & Beilin, L. J. (2003). Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects. Free Radical Biology and Medicine, 35, 772–781. Pase, M. P., Grima, N. A., Cockerell, R., Stough, C., Scholey, A., Sali, A., & Pipingas, A. (2015). The effects of omega-3 fish oils and multivitamins on cognitive and cardiovascular function. Journal of the American College of Nutrition, In press. Pipingas, A., Cockerell, R., Grima, N., Sinclair, A., Stough, C., Scholey, A., Myers, S., Croft, K., Sali, A., & Pase, M. P. (2014). Randomized controlled trial examining the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells. Nutrients, 6, 1956– 1970. Radimer, K., Bindewald, B., Hughes, J., Ervin, B., Swanson, C., & Picciano, M. F. (2004). Dietary supplement use by US adults: Data from the National Health and Nutrition Examination Survey, 1999–2000. American Journal of Epidemiology, 160, 339– 349. Richard, D., Kefi, K., Barbe, U., Bausero, P., & Visioli, F. (2008). Polyunsaturated fatty acids as antioxidants. Pharmacological Research, 57, 451–455. Ridker, P. M., Hennekens, C. H., Buring, J. E., & Rifai, N. (2000). C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. New England Journal of Medicine, 342, 836–843. Simopoulos, A. P. (2002). The importance of the ratio of omega-6/ omega-3 essential fatty acids. Biomedicine and Pharmacotherapy, 56, 365–379. Skulas-Ray, A. C., Kris-Etherton, P. M., Harris, W. S., Vanden Heuvel, J. P., Wagner, P. R., & West, S. G. (2011). Dose-response effects of omega-3 fatty acids on triglycerides, inflammation, and endothelial function in healthy persons with moderate hypertriglyceridemia. American Journal of Clinical Nutrition, 93, 243–252. Stangl, G. I., & Kirchgessner, M. (1998). Different degrees of moderate iron deficiency modulate lipid metabolism of rats. Lipids, 33, 889–895. Tan, Z. S., Harris, W. S., Beiser, A. S., Au, R., Himali, J. J., Debette, S., Pikula, A., DeCarli, C., Wolf, P. A., Vasan, R. S., Robins, S. J., & Seshadri, S. (2012). Red blood cell omega-3 fatty acid levels and markers of accelerated brain aging. Neurology, 78, 658– 664. The National Academy of Clinical Biochemistry (2000). Laboratory medicine practice guidelines: laboratory guidelines for screening, diagnosis and monitoring of hepatic injury. Washington, DC. Wallace, F. A., Miles, E. A., & Calder, P. C. (2003). Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. British Journal of Nutrition, 89, 679–689. Wasim Khan, M., Arivarasu, N. A., Priyamvada, S., Khan, S. A., Khan, S., & Yusufi, A. N. K. (2013). Protective effect of ω-3 polyunsaturated fatty acids (PUFA) on sodium nitrite induced nephrotoxicity and oxidative damage in rat kidney. Journal of Functional Foods, 5, 956–967. Xin, W., Wei, W., & Li, X. (2012). Effect of fish oil supplementation on fasting vascular endothelial function in humans: A metaanalysis of randomized controlled trials. PLoS ONE, 7. Zhang, Z. J. (2013). Systematic review on the association between F2-isoprostanes and cardiovascular disease. Annals of Clinical Biochemistry, 50, 108–114.
Please cite this article in press as: Andrew Pipingas, et al., Fish oil and multivitamin supplementation reduces oxidative stress but not inflammation in healthy older adults: A randomised controlled trial, Journal of Functional Foods (2014), doi: 10.1016/j.jff.2014.10.028