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ARTICLE Effects of vitamin E supplementation on exercise-induced oxidative stress: a meta-analysis1 Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CORNELL UNIV on 02/27/18 For personal use only.

Vahan Stepanyan, Melissa Crowe, Nagaraja Haleagrahara, and Bruce Bowden

Abstract: Tocopherols (commonly referred to as “vitamin E”) are frequently studied antioxidants in exercise research. However, the studies are highly heterogeneous, which has resulted in contradicting opinions. The aim of this review is to identify similar studies investigating the effects of tocopherol supplementation on exercise performance and oxidative stress and to perform minimally biased qualitative comparisons and meta-analysis. The literature search and study selection were performed according to Cochrane guidelines. A 2-dimensional study execution process was developed to enable selection of similar and comparable studies. Twenty relevant studies were identified. The high variability of study designs resulted in final selection of 6 maximally relevant studies. Markers of lipid peroxidation (malondialdehyde) and muscle damage (creatine kinase) were the 2 most frequently and similarly measured variables. Meta comparison showed that tocopherol supplementation did not result in significant protection against either exercise-induced lipid peroxidation or muscle damage. The complex antioxidant nature of tocopherols and low accumulation rates in muscle tissues could underlie an absence of protective effects. Key words: redox imbalance, tocopherols, lipid peroxidation, muscle damage, sport supplements, antioxidants. Résumé : Les tocophérols (habituellement nommés « vitamine E » sont des antioxydants fréquemment sujets de recherche sur l’exercice physique. Néanmoins, ces études très hétérogènes présentent des avis contradictoires. Cette analyse documentaire se propose d’identifier des études similaires traitant des effets de la supplémentation en tocophérol sur la performance physique et le stress oxydatif et d’effectuer des comparaisons qualitatives présentant le moins de biais ainsi qu’une méta-analyse. On a fait la recherche documentaire et sélectionné les études conformément aux directives de Cochrane. On a élaboré un processus bidimensionnel afin de sélectionner des études similaires et comparables. On a identifié vingt études pertinentes. La grande variabilité des devis expérimentaux aboutit a` la sélection de six études hautement pertinentes. Les marqueurs de la peroxydation lipidique (malondialdéhyde) et des lésions musculaires (créatine kinase) sont les variables les plus fréquentes et soumises a` des mesures similaires. D’après la métacomparaison, la supplémentation en tocophérol ne procure pas de protection significative contre la peroxydation lipidique et les lésions musculaires suscitées par l’effort. La nature antioxydante complexe des tocophérols et le faible taux d’accumulation dans les tissus musculaires pourraient expliquer l’absence d’effets protecteurs. Mots-clés : déséquilibre redox, tocophérols, peroxydation lipidique, lésion musculaire, suppléments pour sportif, antioxydants.

Introduction Antioxidant supplements are marketed to decrease oxidative stress and enhance exercise performance. Around 85% of elite track and field athletes (Maughan et al. 2007) and most Olympic athletes (Huang et al. 2006) use dietary supplements, which include antioxidants. The complex nature of antioxidant and exercise research has resulted in highly variable study designs. Type of exercise, biochemical markers assessed, marker assessment timeframe, and supplementation dose and duration are amongst the most variable parameters. Some researchers assessed exercise performance in combination with biochemical markers (McBride et al. 1998; Gaeini et al. 2006). Others assessed biochemical markers only (Niess et al. 2002; Sacheck et al. 2003). Furthermore, the type of biochemical markers (lipid peroxidation or protein oxidation, total antioxidant, or antioxidant enzyme status) and the timing of marker assessment vary widely between studies. Such variability could be explained by an absence of generally recognised and validated markers. As a result, researchers most probably choose

markers based on previous research and possibility to perform on site. Antioxidant supplementation dose and duration also vary. Some researchers utilised 1–2 weeks of supplementation (McBride et al. 1998; Niess et al. 2002; Viitala et al. 2004), while others utilised 2 or more months (Sacheck et al. 2003; Nieman et al. 2004; Gaeini et al. 2006). Broad variation in the abovementioned factors could be a reason for contrasting findings. Some researchers observed positive effects (Itoh et al. 2000; Bryant et al. 2003; Tsakiris et al. 2006) while others observed an absence of effects (Sacheck et al. 2000; Akova et al. 2001; Avery et al. 2003) or negative effects (Nieman et al. 2004) of antioxidant supplementation. Because of the complex interaction between antioxidants and exercise and the increasing interest in this research, Powers et al. (2010) developed guidelines providing directions for high-quality antioxidant research. The guidelines stressed importance of applying holistic approach in antioxidants and exercise area by measuring both exercise performance and various sides of oxidative stress: effects on proteins, lipids, and cell membranes. The guidelines also advised future researchers to pay more attention to the

Received 13 December 2013. Accepted 5 May 2014. V. Stepanyan and M. Crowe. Institute of Sport and Exercise Science, School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, 4811, Australia. N. Haleagrahara. School of Veterinary and Biomedical Sciences, James Cook University, 4811, Australia. B. Bowden. Discipline of Chemistry, School of Pharmacy and Molecular Sciences, James Cook University, 4811, Australia. Corresponding author: Vahan Stepanyan (e-mail: [email protected]). 1This paper is a part of a Special Issue entitled Nutritional Triggers to Adaptation and Performance. Appl. Physiol. Nutr. Metab. 39: 1029–1037 (2014) dx.doi.org/10.1139/apnm-2013-0566

Published at www.nrcresearchpress.com/apnm on 26 May 2014.

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exercise background of participants, type of exercise protocol, and nutritional status. If researchers follow these guidelines it would enable a deeper understanding of the processes happening during exercise-induced oxidative stress (EIOS). However, limited resource availability may restrict the ability of some researchers to implement these guidelines. Similar recommendations were also independently proposed by Nikolaidis et al. (2012). Tocopherols (vitamin E) are believed to be the most important and effective antioxidant throughout the cell lipid phases (Packer 1991; Fisher-Wellman and Bloomer 2009). For this reason, tocopherols have been regarded as having a potentially protective role against EIOS. (Ji 1999; Takanami et al. 2000). The latest reviews (Sacheck and Blumberg 2001; Viitala and Newhouse 2004; Nikolaidis et al. 2012; Lins Draeger et al. 2014), however, have not demonstrated benefit from tocopherol supplementation in protection against EIOS. The high heterogeneity of tocopherol studies and utilization of various types of tocopherols have also been noted in these reviews as possible reasons for the controversial results. Physiological actions of various types of tocopherols can vary widely. For example, d-␣-tocopherol exhibited negative (Tasinato et al. 1995) while dl-␣-tocopherol demonstrated positive (Huang et al. 1988) effects on cells proliferation. Tocopherols also act synergistically with ascorbic acid (Chan 1993). Our review explored the effects of tocopherol supplementation only within maximally similar studies, which could be viewed as a limitation as studies that investigated the synergistic effects with ascorbic acid have been excluded. Although EIOS is currently regarded as a necessary condition for exercise adaptation (Westerblad and Allen 2011), our review considered studies aimed to detect the effect of tocopherols on EIOS reduction. Systematic review and meta-analysis have been suggested as powerful tools in summarising results of randomised controlled trials (Gough et al. 2012). A combination of systematic review and meta-analysis with the current antioxidant research recommendations (Powers et al. 2010) could be a powerful tool in deducing the efficacy of tocopherol supplementation. The aim of this review was to develop a systematic review method and perform meta-analysis with the most appropriate studies and to minimise the confusing methodological and experimental bias. To our knowledge this is the first attempt to perform meta-analysis on tocopherols and exercise studies.

Materials and methods The Cochrane handbook guidelines (Lefebvre et al. 2011) were used to develop the literature search protocol. The main aim of the systematic search was to identify all relevant studies of interest and to establish a comprehensive and unbiased analysis (Gough et al. 2012). To assure inclusion of all relevant published studies, the literature search was performed using the following databases: Medline, PubMed, Scopus, BioMed, CAB Direct, CINAHL, Informit, Cochrane, and Sport Discuss, and by the Google Scholar search engine. The search covered studies published from January 1997 to June 2013 to cover most recent studies. Further articles were also sourced from review articles. The main key words, “exercise-induced oxidative stress” and “tocopherols”, were scanned in the MeSH database for synonyms (Nelson 2012). The list of key words was finalised and applied in the Boolean search (Table 1). No language restrictions were applied during the search. Only studies published in peer-reviewed journals were considered. The studies selection/exclusion process was presented according to PRISMA recommendations (Moher et al. 2010). The study search and selection were performed by 2 authors independently. Identified studies underwent 2 dimensional relevance execution (Gough et al. 2012), allowing on the first dimension selection of specific studies that investigated the effects of tocopherols on

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

Table 1. Search words utilised during systematic search. Search words OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR AND OR OR OR OR OR OR OR NOT OR OR OR OR OR

Aerobic capacity Aerobic power Aerobic endurance Anaerobic power Exercise Physical Endurance Aerobic exercise Anaerobic exercise Running Jogging Swimming Walking Stress test Bicycle ergometry Ergometry Treadmill Jump All-out cycling Tocopherol* Vitamin E Alpha tocopherol Beta tocopherol Gamma tocopherol Tocopherol acetate Tocotrienol* Cancer Obesity Metabolic syndrome Elderly Children

Note: Asterisk (*) used in search engines to indicate that after main word, there could be any types of word endings.

exercise performance and oxidative stress, and on the second dimension, to pool studies with similar and comparable outcomes. First dimension execution was performed based on selection criteria (Table 2) aimed to identify all randomised control studies. Studies of interest were those that assessed exercise performance (anaerobic power, aerobic capacity, aerobic endurance, muscular power, time trials, etc.), oxidative stress markers (malondialdehyde (MDA), protein carbonyls (PC), 8-hydroxy-2deoxyguanosine (8-OHdG), etc.), markers of muscle damage (creatine kinase (CK), rating of soreness, etc.), and body antioxidant status markers (glutathione peroxidase (GLP), total radical scavenging capacity (TRAP), etc.) in combination or alone. Second-dimension execution was performed by scoring the studies according to the following parameters: validity of the exercise protocol and possibility of comparison of results to other studies (Table 3). A valid exercise protocol was defined as a protocol that caused significantly detectable oxidative stress postexercise. To compare outcomes, similar biochemical markers were required to have been measured within similar timeframes. This process minimised comparison errors as noted by Viitala et al. (2004b). Studies gaining scores 5 or 4 were selected for analysis. To obtain these scores a study needed to have the highest scores in any of the 2 parameters and 1 lower score in not more than 1 parameter. Studies gaining a total of 3 or less were excluded from further analysis (Table 4). Numerical data for meta-analysis were collected from published results. The authors were contacted when more data and (or) clarification were required. Transformation of graphical data Published by NRC Research Press

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Table 2. Studies’ selection criteria. No.

Selection criteria

1 2 3 4 5

Human-based randomised control studies One-way or cross-over designs Single- or double-blind More than 1 week supplementation duration No restriction in number, sex, and exercise training level of participants Exclusion of studies conducted on children (60 years old)

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Table 3. Studies’ scoring system. Exercise protocol validity

Score Comparable outcomes

Valid protocol

2

Conditionally valid 1 protocol Not valid protocol 0

Score

Comparable with more than 3 3 studies Comparable with 2 studies 2 Comparable with 1 study Not comparable with any study

1 0 (exclusion)

to numerical was performed when original numerical data were not available from authors. Studies having similar markers measured at similar frequencies in more than 3 cases were selected for further qualitative and meta-analyses. Meta-analysis was performed on the Cochrane Collaboration software, Revman 5. The random effect analysis model was applied because of high variation in experimental factors; type, dose, and duration of tocopherol, and exercise protocols. Mean differences were calculated as the difference in markers pre- and postexercise in placebo and supplemented groups. Effect sizes (Z) were drawn from mean differences (Conger et al. 2011) and were interpreted based on the following reference points: Z values of 0.2, 0.5, and 0.8 were considered small, moderate, and large, respectively (Cohen 1988). Degree of result inconsistency within studies, also referred to as heterogeneity (I2), was interpreted based on the following reference points: 25%, 50%, and 75%, representing low, moderate, and high heterogeneity, respectively (Higgins et al. 2003). Values entered into the software were mean differences and average standard deviations between pre- and postexercise measures. Since markers were measured twice postexercise (e.g., CK was measured 24 and 48 h postexercise), both measurement times were analysed in separate subgroups. Effect size and heterogeneity were presented for each subgroup as well as a combination of both subgroups. Statistical significance was accepted at the level p ≤ 0.05. Regression analysis of the effects of supplementation dose and duration on selected biochemical markers (CK and TBARS) was performed by comparison of dose and duration of supplementation with weighted effect sizes (Hedge’s g and pooled SD as described by Durlak (2009)). Quality appraisal of the final selected studies was performed to assess the research design, its independence, and presence of possible bias (Gough et al. 2012). The assessment parameters (Table 5) were developed based on Powers et al. (2010) recommendations, which stressed the importance of assessing more than 2 markers of oxidative stress and antioxidant status, a marker of muscle damage and bioavailability together in a study. They also strongly recommended employment of reliable and valid exercise protocols and careful selection of antioxidant supplementation dose and duration. Studies that assessed the abovementioned parameters received a low risk of bias score in each individual parameter.

Two more assessment parameters were added in addition to those recommended by Powers et al. (2010): presence of an exercise scientist in the research team and study funding source. Studies having exercise scientists in their team (as a core researcher or a consultant) and an independent funding source received a low risk of bias score in each of these parameters. The overall risk of bias was developed based on the balance of scores received for each parameter. Although it is not yet possible to predict the way in which the results of poorly designed studies will vary from that of better designed ones (Britton et al. 1998; Kunz and Oxman 1998; MacLehose et al. 2000), a number of reviews have found that lower quality studies tend to overestimate the effect of interventions (Gough et al. 2012).

Results Study identification and selection The search was performed in June 2013 and covered all studies published in the previous 16 years. The number of articles initially identified, further screened, and finally selected is shown in Fig. 1. The search resulted in 1660 studies that were screened using the selection criteria (Table 2). Twenty studies that employed tocopherol supplementation were identified. Second dimension relevance analysis identified 14 studies (Table 4), which had more sensitive exercise protocols and comparable markers. Most frequently measured markers in the studies were serum MDA and CK. Other markers such as PC, 8-OHdG, GLP, TRAP, etc., were used in not more than 2 studies disabling further metaanalysis of these markers. Further analysis of the selected studies showed that the frequency of MDA and CK measurements were inconsistent. Studies with similar measurement frequencies were selected to avoid misinterpretation of results. If a marker was measured in a similar timeframe in 3 or more studies, those studies were chosen for final meta-analysis. Six studies were finally identified (Table 6). All 6 studies assessed the MDA marker. Five of these studies (McBride et al. 1998; Itoh et al. 2000; Niess et al. 2002; Avery et al. 2003; Sacheck et al. 2003) assessed CK as well. These studies provided pre- and postexercise measures after supplementation. Most of the studies did not perform baseline, presupplement exercise tests, and biochemical analyses. Presupplement levels of MDA and CK were measured in 2 studies only (Itoh et al. 2000; Sacheck et al. 2003), resulting in exclusion of this measurement stage in the metaanalysis. Studies that used the MDA marker to assess oxidative stress measured its levels after the supplementation at pre-exercise, directly postexercise, and 24-h postexercise periods. Studies that used the CK marker to assess muscle damage measured its levels after the supplementation at pre-exercise, directly postexercise, and 24-h and 48-h postexercise periods. Depending on the exercise protocol, concentration of MDA and CK markers in plasma reaches its maximum at 24, 48, and 72 h postexercise (McBride et al. 1998; Sacheck et al. 2003). Qualitative analysis of the studies Most of the selected studies showed no effect of tocopherol supplementation on inhibition of lipid peroxidation (Table 6). Only 1 study (Itoh et al. 2000) showed peroxidation inhibition in the tocopherol supplemented group. Three out of the 5 studies that measured CK indicated positive effects of supplementation on protection against muscle damage (Table 6). Two others (Niess et al. 2002; Avery et al. 2003) observed no effect of supplementation on CK concentrations. All 6 studies utilised various exercise protocols, such as resistance tests, treadmill, or outdoor running (Table 7). There was no relationship observed between type of tocopherol, its dose, and duration and effects on CK (Table 8). Published by NRC Research Press

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Table 4. Second dimension execution. No.

Study

Exercise protocol validity

Comparable outcomes

Total score

Results/notes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

McBride et al. 1998 Itoh et al. 2000 Sacheck et al. 2000 Akova et al. 2001 Beaton et al. 2002 Niess et al. 2002 Avery et al. 2003 Bryant et al. 2003 Sacheck et al. 2003 Nieman et al. 2004 Viitala et al. 2004 Gaeini et al. 2006 Tsakiris et al. 2006 Hadley et al. 2009 Patil et al. 2009 Vucinic et al. 2010 Chatterjee et al. 2010 Silva et al. 2010 Cobley and Marrin 2012 Garelnabi et al. 2012

2 2 2 1 2 2 2 na 2 2 2 0 2 na 2 2 1 2 2 0

3 3 3 2 3 3 3 na 3 2 3 2 3 na 3 3 3 3 0 2

5 5 5 3 5 5 5 0 5 4 5 2 5 0 5 5 4 5 2 2

Included Included Included Excluded Included Included Included Excluded Included Included Included Excluded Included Excluded Included Included Included Included Excluded Excluded

Note: na, not applicable.

Table 5. Study quality appraisal system. Risk of bias score (Risk level) Parameters assessed

High

Medium

Low

Exercise performance Muscle damage markers Oxidative status Anti-oxidant status Bioavailability Research team Study funding

Not assessed Not assessed Not assessed Not assessed Not assessed No exercise scientist in the team Industry funded

Not applicable Not applicable One marker assessed One marker assessed Not applicable Not applicable Source not mentioned

Assessed Assessed Two or more markers assessed Two or more markers assessed Assessed Exercise scientist in the team Institution/grant funded

Meta-analysis The search criteria identified similar studies so that the outcomes could be compared via meta-analysis. The low number of comparable studies was the main limitation for a strong metaanalysis. Tocopherol supplementation left a large but not statistically significant pro-oxidative effect on lipid peroxidation (Table 9) directly after exercise (Z = 1.84, p = 0.07). Heterogeneity within studies in this subgroup was low (I2 = 28%). Supplementation left a large but not significant protective effect on lipid peroxidation 24 h postexercise (Z = 0.93, p = 0.35). Heterogeneity within studies in this subgroup was moderate (I2 = 56%). Both subgroup synthesis resulted in a small and not significant pro-oxidative effect of tocopherol supplementation on lipid peroxidation (Z = 0.22, p = 0.83). Heterogeneity within studies in both subgroups was moderate (I2 = 57%). Tocopherol supplementation left a large but not significant protective effect against muscle damage (Table 10) with lower CK concentrations 24 h after exercise (Z = 0.92, p = 0.36) although heterogeneity within studies in this subgroup was high (I2 = 91%). Supplementation left a small but not significant muscle protective effect 48 h postexercise (Z = 0.30, p = 0.76), again heterogeneity within studies in this subgroup was high (I2 = 94%). Both subgroups synthesis showed medium but not significant protective effects against muscle damage (Z = 0.51, p = 0.61). Heterogeneity within studies in both subgroups was high (I2 = 91%). Although, in some cases, meta-synthesis resulted in large or medium effect sizes, degree of significance was taken as a final decision indicator.

Regression analysis showed no correlation between supplementation dose and duration and CK and TBARS effect sizes (Table 11). Quality appraisal of the studies Quality of the selected studies is presented in Table 12. No study met requirements of all parameters. The majority of studies received an overall medium risk of bias score, which could be explained by equal presence of both high and low scores in individual parameters.

Discussion Tocopherols are believed to be the most important and effective nutritional antioxidants (Packer 1991; Fisher-Wellman and Bloomer 2009) and are suggested as being possible enhancers of exercise performance (Shephard 1983). Neutralization of exerciseinduced reactive oxygen species by tocopherols could provide protection of muscle cells from deterioration and improve exercise performance and (or) postexercise recovery. A recent study (Atkinson et al. 2010) hypothesised that ␣-tocopherol preferentially incorporates into poly-unsaturated fatty acid domains of cell walls and thus optimises the protection of cell membranes from deleterious oxidation and eventual destruction. Other reasons tocopherols may enhance exercise performance include their ability to increase oxygen pressure tolerance, and to improve myocardial efficiency and peripheral capillary dilation (Shephard 1983). The systematic search and exclusion process applied in our study resulted in only 14 studies for analysis. Uniform measurement frequencies occurred in only 6 studies and on postsupplement MDA and CK markers only. Unfortunately, other more Published by NRC Research Press

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Idenficaon

Studies idenfied through databases search (n= 1,660)

Screening

Studies screened (n= 1,660)

Eligibility

Relevant studies based on first dimension execuon (n= 20)

Relevance

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Fig. 1. Studies selection PRISMA diagram.

Relevant studies based on second dimension execuon (n= 15)

Studies excluded (n= 1,640) Reasons: * Basic science papers * Various disease condions studies * Not exercise trials * Mixture of anoxidants used for supplementaon * Repeon of publicaon in other search engines * Similar arcle in different journals * Nonspecific markers studies (for example effect of anoxidant supplementaon on protecon of Red Blood Cells from exercise induced oxidaon)

Studies excluded (n= 5) Excluded based on received scores

Comparability

Studies excluded (n= 9) Incomparable markers and measurement frequencies Comparable studies by similar markers and frequencies of measurements (n= 6)

Table 6. Final studies and their outcomes summary. Effect on MDA No.

Study

1 2 3 4 5 6

McBride et al. 1998 Itoh et al. 2000 Niess et al. 2002 Avery et al. 2003 Sacheck et al. 2003 Viitala et al. 2004

Positive

Neutral

Effect on CK Negative

X

Positive

Neutral

Negative

X X

X

X X

X X X

X

Note: “X” marks left effect. CK, creatine kinase; MDA, malondialdehyde.

Table 7. Exercise protocols applied in selected studies. No.

Study

Type of exercise applied

1 2 3 4 5 6

McBride et al. 1998 Itoh et al. 2000 Niess et al. 2002 Avery et al. 2003 Sacheck et al. 2003 Viitala et al. 2004

Resistance tests Running training, workload increase Treadmill run, incremental to exhaustion Resistance tests Running, outdoor Resistance tests

sensitive and less variable oxidative stress markers were measured in not more than 2 studies, inhibiting performance of further meta-analysis. The systematic search identified a lack of studies into tocopherol supplementation on other oxidative stress markers such as PC, GLP, TRAP, etc. Inconsistency in marker types and measurement frequencies was also observed by Viitala and Newhouse (2004a) in their review of vitamin E supplementation on exercise and lipid peroxidation. Unfortunately, such inconsistency has continued in studies published after 2004, despite these recommendations. Further recommendations developed by Powers et al. (2010) and Nikolaidis et al. (2012), to foster quality of research into antioxidants and exercise Published by NRC Research Press

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Table 8. Effects of type, daily dose, and duration of supplementation on muscle damage.

No.

Study

Type of supplement

1 2 3 4 5

McBride et al. 1998 Itoh et al. 2000 Niess et al. 2002 Avery et al. 2003 Sacheck et al. 2003

RRR-d-␣-Tocopherol succinate ␣-Tocopherol RRR-␣-Tocopherol RRR-d-␣-Tocopherol succinate RRR-␣-Tocopherol

Daily dose, mg

Duration of supplementation, days

Total supplemented amount, mg

Effect on CK in supplemented group

992 992 413 992 827

14 34 8 31 84

13 888 33 728 3304 30 752 69 468

Decreased CK leakage Decreased CK leakage No effect No effect Decreased CK leakage

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Note: CK, creatine kinase.

Table 9. Effects of tocopherols supplementation on lipid peroxidation. Supplemented Study or subgroup

Mean

SD

Placebo Total

Mean

SD

After 24 h postexercise Gaeini et al. 2006 −2.2 0.4 10 −3.2 1.45 McBride et al. 1988 2.86 1.15 6 1.08 0.918 Viitala et al. 2004a −0.08 0.15 6 −0.12 0.16 Viitala et al. 2004b −0.05 0.265 7 −0.04 0.19 Subtotal 29 Heterogeneity: Tau2 = 0.13; Chi2 = 4.19; df = 3 (p = 0.24); I2 = 28% Test for overall effect: Z = 1.84 (p = 0.07)

Total 10 6 6 7 29

After 24 h postexercise Avery et al. 2003 0.1 0.93 9 −0.09 0.86 9 Itoh et al. 2000 −0.06 0.025 7 −0.27 0.51 7 McBride et al. 1988 0.24 0.645 6 3.84 1.77 6 Sacheck et al. 2003 −0.07 0.0645 8 −0.02 0.061 8 Viitala et al. 2004a 0.14 0.13 6 0.22 0.23 6 Viitala et al. 2004b 0.19 0.375 7 0.16 0.33 7 Subtotal 43 43 Heterogeneity: Tau2 = 0.40; Chi2 = 11.37; df = 5 (p = 0.04); I2 = 56% Test for overall effect: Z = 0.93 (p = 0.35) Total 72 72 Heterogeneity: Tau2 = 0.41; Chi2 = 20.89; df = 9 (p = 0.01); I2 = 57% Test for overall effect: Z = 0.22 (p = 0.83) Test for subgroup differences: Chi2 = 3.77; df = 1 (p = 0.05); I2 = 73.5%

Weight

Standard mean difference IV, [random, 95% CI]

11.6% 8.2% 9.9% 10.6% 40.3%

0.90 [−0.03, 1.83] 1.58 [0.21, 2.95] 0.24 [−0.90, 1.38] −0.04 [−1.09, 1.01] 0.61 [−0.04, 1.26]

11.6% 10.4% 6.5% 10.8% 9.8% 10.6% 59.7%

0.20 [−0.72, 1.13] 0.54 [−0.53, 1.62] −2.49 [−4.16, −0.83] −0.75 [−1.78, 0.27] −0.40 [−1.54, 0.75] 0.08 [−0.97, 1.13] −0.32 [−1.01, 0.36]

100.0%

Standard mean difference IV, [random, 95% CI]

0.06 [−0.47, −0.59]

Note: CI, confidence interval.

should be taken into account as a starting point for researchers to improve reliability and validity of future research. None of the identified studies investigated in this meta-analysis applied similar exercise tests, although all of them initiated muscle damage in terms of changes in CK concentration. Comparison of results from different exercise protocols and durations could potentially increase bias in interpreting results, although Pelletier et al. (2013) found it technically reasonable to combine results of studies with different exercise protocols. Meta-analysis of selected studies showed that tocopherol supplementation did not have significant protective effects against exercise-induced lipid peroxidation (p = 0.83) or muscle cell damage (p = 0.61). High variations of Z values in subgroups were observed (in some cases, effect sizes of supplementation were quite large, but not statistically significant). High heterogeneity (I2) within studies could be a reason for high variations of effect sizes. In some cases (directly after exercise for lipid damage, and 48 h after exercise for muscle cells damage), tocopherol supplementation resulted in altered tissue oxidation, which could be caused by excess tocopherol accumulation. An absence of tocopherol effects on exercise-induced lipid peroxidation was also concluded by Viitala and Newhouse (2004a) in their review. In 1 study (Itoh et al. 2000) tocopherol supplementation showed protective effects against lipid oxidation. In contrast to others, continuous supplementation during short-term (6 days) aerobic training was applied in this study. Continuous supply of tocopherols during the training period could have had suppressive ef-

fects on exercise-induced lipid damage in the Itoh et al. (2000) study. Most of the studies to date utilised acute exercise protocols after a period of supplementation. There is a lack of research into the effects of continuous supplementation combined with continuous training. Tocopherol supplementation had equivocal, although statistically not significant, effects on protection from exercise-induced muscle damage (CK levels). In their review, Peake et al. (2007) similarly concluded that tocopherol supplementation showed controversial results on muscle damage. In contrast, other scientists (McGinley et al. 2009) concluded that tocopherol supplementation does not protect against exercise-induced muscle damage in the majority of cases. The absence of protective effects of tocopherol supplementation on exercise-induced lipid peroxidation (in blood) and muscle damage could be explained by 2 possible reasons. First, oral supplementation dose/duration in selected studies may have been insufficient to cause accumulation of tocopherols in muscle tissues able to neutralise excess reactive oxygen species. In their study, Burton et al. (1998) showed that RRR-␣-tocopheryl acetate accumulation in muscle tissues was approximately 4 times lower than that of in plasma after 41 days of supplementation (150 mg/day). These authors found that accumulation rates in liver and gallbladder tissues were similar to that in plasma. This explains Brady et al.’s (1979) findings that tocopherol supplementation was protective against EIOS in rat liver but not in muscle tissues, suggesting a role not related to exercise. Liver possibly acts as a Published by NRC Research Press

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Table 10. Effects of tocopherols supplementation on muscle damage. Supplemented

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Study or subgroup

Mean

SD

Placebo Total

Mean

SD

Total

After 24 h postexercise Avery et al. 2003 330.3 33.2 9 180.2 30 9 Itoh et al. 2000 423.6 169.4 7 789 222.4 7 McBride et al. 1988 42.2 17.7 6 156.2 31.4 6 Niess et al. 2002 30 18.8 9 92 45.3 9 Sacheck et al. 2003 47.7 46.1 8 254.4 125.6 8 Subtotal 39 39 Heterogeneity: Tau2 = 5.53; Chi2 = 43.75; df = 4 (p < 0.00001); i2 = 91% Test for overall effect: Z = 0.92 (p = 0.36) After 48 h postexercise Avery et al. 2003 287.43 35.39 9 98.67 21.45 9 McBride et al. 1988 3.42 14.25 6 57 27.23 6 Niess et al. 2002 11 15.75 9 54 32.75 9 Subtotal 24 24 Heterogeneity: Tau2 = 11.58; Chi2 = 36.14; df = 2 (p < 0.00001); I2 = 94% Test for overall effect: Z = 0.30 (p = 0.76) Total 63 63 Heterogeneity: Tau2 = 5.87; Chi2 = 80.51; df = 7 (p < 0.00001); I2 = 91% Test for overall effect: Z = 0.51 (p = 0.61) Test for subgroup differences: Chi2 = 0.50; df = 1 (p = 0.48); I2 = 0%

Weight

Standard mean difference IV, [random, 95% CI]

12.1% 13.1% 11.4% 13.3% 13.1% 62.9%

4.52 [2.61, 6.43] −1.73 [−3.02, −0.44] −4.13 [−6.44, −1.82] −1.70 [−2.82, −0.58] −2.07 [−3.35, −0.79] −1.03 [−3.21, 1.16]

11.1% 12.6% 13.3% 37.1%

6.14 [3.69, 8.59] −2.28 [−3.86, −0.69] −1.59 [−2.69, −0.50] 0.61 [−3.38, 4.59]

100.0%

Standard mean difference IV, [random, 95% CI]

−0.46 [−2.25, 1.32]

Note: CI, confidence interval.

Table 11. Correlation between daily supplementation dose, duration, and effects on TBARS and creatine kinase (CK) markers. Correlation between weighted effect size and supplementation daily dose, R2

Correlation between weighted effect size and supplementation duration, R2

14 34 8 31 84

0.170

0.0001

992 413 1200

14 8 31

0.355

0.850

2.65 0.66 0.37 1.58

992 885 885 450

14 14 14 56

0.161

0.339

1.91 0.37 2.70 0.18 0.95 0.35

992 992 992 827 885 885

14 34 31 84 14 14

0.020

0.040

Author (y)

Weighted effect size (Hedge’s g)

Pooled SD

Supplementation daily dose, mg

Supplementation duration, d

CK − 24 h postexercise McBride et al. 1998 Itoh et al. 2000 Niess et al. 2002 Avery et al. 2003 Sacheck et al. 2003

−2.12 −0.71 −0.90 1.40 −1.21

45.35 443.83 61.94 96.40 150.90

992 992 413 1200 827

CK − 48 h postexercise McBride et al. 1998 Niess et al. 2002 Avery et al. 2003

−1.21 −0.77 1.67

37.23 50.21 101.24

TBARS − 24 h postexercise McBride et al. 1998 Viitala et al. 2004 untrained Viitala et al. 2004 trained Gaeini et al. 2006

0.57 −0.01 0.09 0.58

TBARS − 48 h postexercise McBride et al. 1998 Itoh et al. 2000 Avery et al. 2003 Sacheck et al. 2003 Viitala et al. 2004 untrained Viitala et al. 2004 trained

−1.59 0.50 0.06 −0.25 0.03 −0.20

Table 12. Quality assessment of final studies.

No.

Study

Exercise performance assessment

1 2 3 4 5 6

McBride et al. 1998 Itoh et al. 2000 Niess et al. 2002 Avery et al. 2003 Sacheck et al. 2003 Viitala et al. 2004

Low Low High Low High High

Muscle damage markers assessment

Oxidative stress assessment

Anti-oxidant status assessment

Bioavailability assessment

Study funding assessment

Overall ranking of individual studies

Low Low Low Low Low High

Medium Medium High Medium Low Medium

High Medium Low High Medium High

High Low Low High Low Low

Low Low Low Medium High Medium

Medium Low Low Medium Medium Medium

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barrier for further distribution of orally ingested tocopherols into other tissues. Burton et al. (1998) showed that tocopherol accumulation rates in other tissues (skin, nerve, and vein) were similar to that of muscle. Regression analysis in the current study showed no correlation between supplementation dose and duration and CK or TBARS effect sizes (Table 11). Therefore, it can be concluded that applied doses and durations (Table 8) were not sufficient to cause an interaction with EIOS. None of the identified studies measured the extent of tocopherol accumulation in muscle tissues after supplementation, leaving this question open for future research. Second, the inconsistency of tocopherol effects could be related to their complex function and chemical behaviour being able to act as antioxidants, pro-oxidants, or neutral agents in vivo (Rietjens et al. 2002). Moreover, tocopherols could act selectively in neutralizing exercise-induced reactive oxygen species as noted by McGinley et al. (2009). Our review compared studies that utilised various types of tocopherols and found no effect on EIOS. The absence of effect could merely be due to different effects of the various types of tocopherols. More research is needed to determine the effects of specific tocopherols in different tissues during exercise-induced oxidation.

Conclusion and perspectives Different types of tocopherols, daily doses, and durations of supplementation were utilised in the selected studies (Table 8). This inconsistency added extra heterogeneity in results and we do not yet know the impact of these variables on study outcomes. The majority of studies did not have presupplementation measurements, which also added a possible level of error in interpreting results. Damage caused by oxidative stress might occur in other tissues, such as liver, lungs, brain, and blood vessels and not be reflected in the blood (McGinley et al. 2009). Most of the exercise studies, including the ones selected for our qualitative review and meta-analysis, measured oxidative stress via blood markers. Application of 2-dimensional study selection and quality appraisal allowed identification of similar studies from a highly heterogeneous research field. Comparison of the studies showed that tocopherol supplementation did not have protective effects against exercise-induced lipid peroxidation and muscle damage. We speculate that an absence of protective effects in general and exhibition of some pro-oxidative effects in some particular cases could be explained by the complex behaviour of tocopherols and (or) possibly insufficient accumulation in muscle tissues after supplementation. The review identified the following directions for future research in the area: (i) effects of supplementation on other markers of oxidative stress, such as proteins and DNA oxidation markers in various tissues; (ii) effects of continuous supplementation on protection against lipid oxidation and muscle damage; and (iii) extent of tocopherol accumulation in muscle tissues after oral supplementation. Conflict of interest statement The authors wish to declare no conflict of interest.

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