By Dr. Gary Franke

Introduction

Almost everyone has heard about antioxidants. These nutrients, often vitamins or minerals, are found in many types of foods and are widely considered to be beneficial. Fruits and vegetables are healthy partly because of the antioxidants they contain. The aisles of grocery and drug stores and vitamin shops are filled with supplements of all kinds, many containing antioxidants. Antioxidants mop up the damaging reactive compounds (oxidants) in a cell, preventing damage to the cell and its contents. If antioxidants are a good thing, many people reason, more must be better. People take a vast range of vitamins and supplements in an effort to avoid getting sick, improve mental and physical performance and perhaps live longer. Many athletes, for example, take antioxidant supplements, believing they will prevent muscle damage and improve muscle and athletic performance. However, the effect of antioxidant supplements on athletic performance is controversial. Numerous studies have been done on this topic, yet with conflicting results. In order to make sense of this issue, it is helpful to know about the compounds antioxidants neutralize (oxidants). What are oxidants, where do they come from and how do they affect muscle cells? What kind of damage to oxidants cause to muscle cells? Do oxidants benefit muscle cells in any way? Finally, what does the research tell us about the effects of antioxidant supplements on muscle and athletic performance?

What are oxidants?

Life depends on a complex series of chemical reactions. The behavior of electrons is the key to chemical reactions, during which electrons are rearranged in a molecule or moved between molecules. The key property of oxidants is that they attract and thus gain electrons from other molecules5. As their name suggests, oxidants typically contain oxygen, as oxygen strongly attracts electrons5, 6.

Common Oxidants in Cells

Oxygen Superoxide radical Hydrogen peroxide Hydroxyl radical

(O2) (O2-) (H2O2) (OH-)

Redox reactions (Reduction + Oxidation)

Since oxidants gain electrons, there must also be molecules that give up these electrons to them. Such molecules are called reductants5. The balance of reductants (which give up electrons) and oxidants (which gain electrons) determines the redox state of a cell. This is important because each cell has an optimal redox state it must maintain to function properly5, 7, 8.

Redox reactions are chemical reactions that involve the transfer of one or more electrons between molecules (a reduction and oxidation). In these reactions, there is always an oxidant (that gains electron(s)) and a reductant (that loses electron(s))5, 6. In cells, reductants often function as antioxidants, since they can stabilize oxidants by transferring electron(s) to them7.

• Redox Reactions
• Reductant Oxidant

How do oxidants form?

Oxygen allows aerobic organisms to extract energy from food more efficiently, but this comes at a cost. Since oxygen (O2) is inherently reactive, the cost is oxidative damage to the crucial molecules that make up our cells. Chemically reactive and toxic, oxidants form as a result of aerobic metabolism5, 6. Humans, like other complex organisms, require a constant supply of oxygen (O2) to generate enough energy to function. This is because our cells generate most of their energy using a process called oxidative phosphorylation, which requires oxygen. This process involves the flow of electrons through four structures in the mitochondria that form the Electron Transfer Chain (ETC)6. As a very strong oxidant, oxygen pulls electrons through the ETC. As electrons flow through the ETC, most combine with oxygen (as well as protons) at the end of this chain to form water. Occasionally, however, electrons leak from the ETC and combine with oxygen to form the highly reactive oxidant superoxide (O2-)5, 6, 7. As a result, the ETC (and thus mitochondria) are a major source of oxidants in our cells. However, oxidants are also formed in other parts of the cell, such as peroxisomes, by certain oxidase enzymes (that can transfer electrons to oxygen), and from the outside environment (drugs and pollutants, for example)7,8.

As electrons are added to oxygen (O2), various oxidants are formed. When an electron combines with oxygen (O2), it forms superoxide (O2-), which contains an unpaired electron. Molecules with an unpaired electron are very reactive and are called radicals. These molecules are also often called Reactive Oxygen Species (ROS) in cells. If another electron is added to superoxide, it will become hydrogen peroxide (H2O2), which is an oxidant, but not a radical. Finally, if an electron is added to hydrogen peroxide, it will be transformed into hydroxyl radical (OH-), which is a highly reactive and damaging oxidant6.

Why are oxidants harmful?

Oxidants, particularly radicals (ROS) are harmful to our cells because they damage the molecules that make them up. They are especially damaging to the large, complex macromolecules of cells – lipids, proteins and DNA – that are essential for life5, 6, 7. The function of these molecules depends on their precise structure. A tiny change in their structure can completely change their function, especially for proteins and DNA. Since oxidants strip electrons from other molecules, they can alter the structure and function of crucial molecules such as protein, DNA and lipids5, 6.

Damage to the molecules of cells, by oxidants, accumulates over time and is widely thought to contribute to aging. This idea is called the oxidative, or free-radical, theory of aging7. Lipids, proteins, and DNA can all be damaged by ROS5, 6, 7. Examples of such damage include the oxidation of proteins, resulting in the fragmentation and aggregation of proteins within cells. Clumps of damaged proteins (aggregates) accumulate during aging and are the main factor in numerous diseases, including Alzheimer’s7. Oxidants also damage both mitochondrial and nuclear DNA. If this damage is not corrected, the result could be a permanent mutation – potentially harming cell function7.

What are antioxidants?

As aerobic organisms, we require a constant supply of oxygen to generate sufficient energy. Oxygen inevitably results in the formation of oxidants that damage our cells. In order for them to survive and function, there must be mechanisms of adapting to oxidative damage. Cells make two types of antioxidants to prevent damage from oxidants that occurs in all cells. In response to oxidative stress, cells increase the amount and activity of special enzymes that remove oxidants by converting them to a less or non-toxic form5, 8.

The most important antioxidant enzymes include the enzymes Superoxide Dismutase (SOD) and catalase. SOD converts two molecules of superoxide radical (O2-) to one molecule of less toxic hydrogen peroxide (H2O2). Catalase then converts hydrogen peroxide to water6, 8. A special type of antioxidant made by cells is thioredoxin, which is a crucial antioxidant for proteins, maintaining them in a reduced state.7,8

Alternatively, some antioxidants are called scavengers, which convert oxidants to a more stable (and thus less toxic) form, by transferring electrons to them. This type of antioxidant includes several compounds produced by cells, including glutathione, α-lipoic acid, uric acid, bilirubin, and coenzyme Q5, 8. Glutathione is important, for example, because it recycles vitamins C and E by converting them back to their reduced (functional) form. Scavenger antioxidants also includes dietary vitamins and minerals. Vitamins C and E as well as carotenoids are examples of important antioxidant vitamins8.

Why are large amounts of oxidants (ROS) produced during exercise?

Exercise results in the build-up of potentially harmful ROS in skeletal muscle cells5. Why does this happen, and where does the ROS come from? Intense exercise can consume enormous amounts of energy. To meet these energy demands, lots of oxygen must be consumed (to pull electrons through the ETC)5. Some of this oxygen is inevitably converted to ROS because electrons sometimes leak from the ETC. The mitochondrial ETC has therefore been thought to be a major source of ROS in skeletal muscle cells during exercise. More specifically, Complex 1 and complex III of the ETC have been proposed as major sources of ROS6,7. However, recent data suggests that the mitochondria may not be the dominant source of ROS during exercise8.

Besides the mitochondrial ETC, other sources of ROS also exist. Muscle contraction generates heat, which is known to increase levels of ROS. Other possible sources of ROS include the enzymes xanthine oxidase and NADPH oxidase, which can transfer electrons from NADPH to oxygen, forming the radical superoxide6, 7, 8. More research is needed to determine the precise sources of ROS during exercise. Whatever their source, high quantities of ROS are a stress that exercising muscle cells must cope with.

How do oxidants affect muscle cells?

During strenuous exercise, muscle cells use up tremendous amounts of oxygen. A small proportion of this oxygen is converted to oxidants (ROS) that can potentially damage muscle cells. What effect do these ROS have on skeletal muscle cells, and how do they cope with this stress?

The answer depends largely on the intensity and regularity of the exercise that generates the ROS. Regular, moderate exercise is good while infrequent, intense exercise is harmful for skeletal muscle7,8. The reason for this has to do with a concept called hormesis, which means that the effect of a stress depends on its intensity. While a high level of stress is harmful, a low level can be beneficial. This applies to oxidative stress and skeletal muscle cells because it turns out that optimal muscle function depends on a low level of oxidants (ROS) – which regular exercise provides7,8. In other words, there exists an optimal redox (oxidative) state for skeletal muscle function. At either higher or lower levels of ROS, muscle function is reduced.

Regular, moderate exercise is beneficial because it results in this low to moderate level of ROS that is optimal for muscle function (the exact amount is hard to determine because ROS are very difficult to directly measure). Infrequent, intense exercise, by contrast, can be harmful for skeletal muscle cells because the resulting high levels of ROS causes damage to the molecules of the muscle cell – especially oxidation of lipids, proteins, and DNA7.

ROS have both direct and indirect effects on skeletal muscle cells. ROS directly alters the structure and function of muscle fibers (myofilaments). Specifically, ROS oxidize the myofilament proteins myosin heavy chain and troponin C. The result is that a low level of ROS increases the force of muscle contraction, while high levels of ROS have the opposite effect8. The indirect effects of ROS involve cell signaling pathways that sense ROS and trigger events that help the muscle cells adapt to oxidative stress.

ROS, Cell signaling, and Skeletal Muscle Cells

How does this process work? Oxidative stress must first be sensed, and then the muscle cell must respond effectively. In general, a sensor protein first detects oxidative stress. Then, the sensor can trigger events in the cell that culminate in the activation of genes that help protect against oxidative stress.

The sirtuin proteins (sensors of oxidative stress and energy level) and PGC1α (a regulator of skeletal muscle function) illustrate how this process works. Sirtuins sense the energy and redox state of the cell by binding NAD+, which indicates oxidative stress and low energy. When oxidative stress (NAD+) is sensed, sirtuin proteins are activated and alter other proteins by removing acetyl groups from them, which subtly changes their structures7. For example, when SIRT1 (a sirtuin) senses an oxidized state (high levels of NAD+) it removes acetyl groups from key proteins, including PGC1α, an important regulator of skeletal muscle function7. PGC1α then turns on genes that adjust skeletal muscle cells to oxidants by increasing the amount of mitochondria in the cell, increasing blood vessel formation, and making antioxidant enzymes2, 3. These changes help skeletal muscle cells adapt to oxidative stress.

Besides SIRT1, many other signaling proteins also regulate PGC1α. These include the energy sensing protein kinase AMPK, calcium activated protein kinases, and the protein kinase p38 MAPK3. Together, these proteins sense the energy and redox state of the cell and turn on appropriate genes, largely through PGC1α. Like SIRT1, these proteins add or remove small molecules to PGC1α, slightly changing its structure. In this way, skeletal muscle cells first sense and then adjust to changes in their environment – such as oxidative stress due to physical exercise3.

What effects do taking antioxidant supplements have on skeletal muscle cell damage and muscle performance?

Previously, I have described the effects of oxidants on skeletal muscle cells – both positive and negative. A low to moderate level of ROS is essential for optimal muscle function. .Given this, it is reasonable to think that consuming very high levels of antioxidants might harm muscle function by interfering with processes that depend on ROS, especially during exercise. To find out whether or not antioxidants prevent the benefits of exercise, controlled experiments must be done to determine how taking antioxidant supplements actually effects skeletal muscle and athletic performance.

Exactly how taking antioxidant supplements influences skeletal muscle during exercise is a complicated issue. Large numbers of studies have been done but with varying and often conflicting results. Most studies show that taking antioxidant supplements has no effect on actual athletic performance , but other studies show either a benefit  or negative effect. Most studies show that antioxidant supplements reduce oxidative stress in skeletal muscle cells9, 10, 11, 12, but some report an increase in oxidative stress13, 14, 15. What might explain these different conclusions? Part of the explanation is likely the different ways in which these studies are designed and carried out. Studies vary in design, characteristics of subjects, exercise protocols, types of supplements, dosage of supplements, length of time supplements are given, and analysis of data. It is also important to note that ROS are rarely measured directly, because doing so is technically difficult. Instead, various markers of oxidative damage are used to estimate oxidative damage. The most commonly used markers of oxidative damage are products of lipid peroxidation. Oxidized proteins and DNA are also commonly used. However, methods of measuring oxidative damage differ between studies.

Antioxidant supplements and Adaptation of Skeletal Muscle to ROS

Exercising results in the accumulation of ROS in skeletal muscle cells. This ROS then turns on cell signaling pathways that help muscle cells adapt by turning on specific genes. Do antioxidant supplements prevent skeletal muscle from adapting to exercise by interfering with these signaling pathways? An interesting study published in the journal PNAS in 2009 (Ristow et al. 2009) addresses this question1. Physical exercise was already known to improve glucose metabolism and help prevent diabetes in at-risk individuals. The authors asked the question: are these benefits dependent on ROS? The authors performed experiment to find out. These experiments used 40 subjects, 20 trained and 20 untrained. For both trained and untrained subjects, half received a supplement of vitamins C (1g per day total) and E (400 IU per day) while half received a placebo (sugar pill). All subjects participated in a four week exercise training program. As usual, oxidative stress was measured indirectly using an oxidative marker called TBARS. Insulin sensitivity was measured using Glucose Infusion Rate (GIR)1.

Subjects not taking antioxidant supplements (placebo) showed increased insulin sensitivity (GIR) following exercise. By contrast, subjects taking the antioxidant supplements did not have increased insulin sensitivity. Consistently, insulin levels decreased following exercise in the placebo group, indicating increased sensitivity to glucose. In subjects taking supplements, by contrast, this decrease did not occur. These results show that ROS are required for the improved glucose metabolism following exercise.

This study also explored the mechanism for this improvement by measuring changes in gene expression following exercise. In the absence of antioxidant supplements, it was found that three major regulators (transcription factors) are activated by exercise: PGC1α, PGC1β and PPARy. Antioxidant enzymes are a key defense against oxidative damage, and three crucial antioxidant enzymes were found to be activated by exercise: SOD1, SOD2, and GPx1. These enzymes and transcription factors were only activated in the absence of antioxidant supplements1. These results explain why ROS are necessary for the improved glucose metabolism resulting from exercise training.

These results also led to a model showing how ROS signaling triggers the benefits of physical exercise. In this model, ROS activates key cell regulators, such as PGC1α, which then turn on genes for making antioxidant enzymes, as well as more mitochondria and blood vessels. These changes defend the cell from ROS and reduce disease risk1. This is an example of hormesis because a low level of stress (ROS) results in protection from this same stress. More specifically, it is termed mitohormesis because the response involves both ROS generated from mitochondria and the generation of more mitochondria.

Several other studies also show that antioxidant supplements can interfere with the adaptation of skeletal muscle cells to ROS in various ways. For example, antioxidant supplements were found to prevent the activation of heat shock proteins, to increase lipid peroxidation, reactive iron and markers of muscle cell damage22, 23, and to reduce muscle force. Based on these studies, antioxidant supplements can interfere with the benefits of physical exercise in various ways.

Antioxidant supplements and athletic performance

Based on the studies described above, there is reason to believe that antioxidant supplements may actually reduce athletic performance by interfering with critical cell signaling and gene expression in skeletal muscle cells. However, the evidence is mixed and inconclusive. A recent review examined the effects of vitamin C, E, and β-carotene supplementation on physical performance. Of the 12 recent studies (2006-2013) identified, most (seven) reported no significant effect of the antioxidant supplement. Two studies reported a negative effect of supplementation and only two studies reported a benefit. Even in these two studies, the improvement from taking the supplement was very modest. These results are consistent with early reports that vitamin E has no significant effect on swimming performance16, 17. Therefore, there is little evidence that taking antioxidant supplements improves athletic performance while a majority of studies show no significant effect.

An example of the inconsistent results from research on antioxidant supplements is the research on the antioxidant coenzyme Q, which is also sometimes called ubiquinol. Coenzyme Q has a dual role in the cell as part of the mitochondrial ETC and as an antioxidant. Overall, in 12 studies, five reported at least some benefit27, 28, 32, 35, six reported no significant effect30, 32, 33, 34, 36, 37, and one reported reduced performance from coenzyme Q supplementation29. One pattern was that coenzyme Q supplementation tended to reduce fatigue 27, 28 and increase power28, 31, 35 during anaerobic exercise. However, the amount of improvement is often modest. For example, in one study, maximum power output increased by 11% in supplemented group compared to 8.5% in the placebo group. Despite a high dosage of 300 mg per day, the supplemented group improved only 2.5% more than the placebo group35. Therefore, while coenzyme Q supplementation may be beneficial in specific circumstances, there is no consistent evidence that it broadly improves athletic performance.

Conclusion

Based on the current evidence, it is difficult to make any specific recommendations on taking antioxidant supplements to improve athletic performance. Research in redox biology indicates that ROS play many crucial roles in cell signaling and gene expression and taking antioxidant supplements could possibly interfere with these processes in skeletal muscle cells. Most studies, however, show that antioxidant supplements have no actual effect on athletic performance. A smaller number of studies show either a benefit or harm to muscle and athletic performance. Overall, there is a lack of strong evidence that supplements significantly improve athletic performance. However, for specific individuals, it is possible that certain supplements are beneficial for a particular activity. Because of widely varying methods and inconsistent results, more research needs to be done on this issue.

Sources

1. Antioxidants prevent health-promoting effects of physical exercise in humans. PNAS vol 106 no. 21; 8665-70. M. Ristow et al. 2009.

2. Exercise-induced hormesis may help healthy aging. Dose-Response 8 (1). L. L. Ji et al. 2010.

3. PGC-1α regulation by exercise training and its influences on muscle function and insulin sensitivity. Am. J. Physiol. Endocrinol. Metab. 299 E145-61. V. A. Lira et al. 2010.

4. Critical role for free radicals on sprint exercise-induced CaMKII and AMPKα phosphorylation in human skeletal muscle. Journal of Applied Physiology vol. 114 no. 5; 566-77. D.M. Alamo et al. 2013.

5. Antioxidant supplementation during exercise training: Beneficial or detrimental? Sports Med 41 (12); 1043-69. T.T. Peternelj and J.S. Coombes 2011.

6. Textbook of Biochemistry with Clinical Correlations: 6^th Edition. Thomas M. Devlin 2006.

7. Oxygen consumption and usage during physical exercise: The balance between oxidative stress and ROS-dependent adaptive signaling. Antioxidants and Redox Signaling vol. 18 no. 10; 1209-31. Z. Radak et al. 2013.

8. Exercise-induced oxidative stress: Cellular mechanisms and impact on muscle force production. Physiol. Rev. 88; 1243-76. S.K. Powers and M.J. Jackson 2008.

9. Effects of exercise, vitamin E and ozone on pulmonary function and lipid peroxidation. J. Appl. Physiol. 45 (6); 927-32. C.J. Dillard et al. 1978.

10. The effects of endurance exercise and vitamin E on oxidative stress in the elderly. Biological Research for Nursing 5 (1); 47-55. J.V. Jessup et al. 2003.

11. Exercise-induced oxidative stress before and after vitamin C supplementation. Int. J. Sports Nutr. 7 (1); 1-9. H.M. Alessio et al. 1997.

12. Protective effects of vitamin E on exercise-induced oxidative damage in young and older adults. American Journal of Physiology, Regulatory, Integrative and Comparative Physiology 264 (5); R992-98.

13. Effects of vitamin E and C supplementation either alone or in combination on exercise-induced lipid peroxidation in trained cyclists. J. Strength Cond. Res. 17 (4); 792-800. R.J. Bryant et al. 2003.

14. Effect of alpha-tocopherol supplementation on plasma homocysteine and oxidative stress in highly trained athletes before and after exhaustive exercise. J. Nutr. Biochem. 16 (9); 530-37. S.R. McAnulty et al. 2005.

15. Increased lipid peroxidation in trained men after 2 weeks of antioxidant supplementation. Int. J. Sports Nutr. Exerc. Metab. 19 (4); 383-99. M. Lamprecht et al. 2007.

16. The effects of vitamin E and training on physiological function and athletic performance in adolescent swimmers. Br. J. Nutr. 26 (2); 265-76. I.M. Sharman et al. 1971.

17. The effects of alpha-tocopherol acetate on the swimming endurance of trained swimmers. Am. J. Clin. Nutr. 28 (3); 205-08. J. Lawrence et al. 1975.

18. Role of alpha-tocopherol in cardiopulmonary fitness in endurance athlete cyclists. Ind. J. Physiol. Pharmacol. 53 (4); 375-79. S.M. Patil et al. 2009.

19. Exercise performance, red blood cell deformability, and lipid peroxidation effects of fish oil and vitamin E. J. Appl. Physiol. 83 (3); 746-52. G.S. Oostenbrug et al. 1997.

20. Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process. Br. J. Nutr. 95 (5); 976-81. G.L. Close et al. 2006.

21. Vitamin E isoform specific inhibition of the exercise-induced heat shock protein 72 expression in humans. J. Appl. Physiol. 100 (5); 1679-87. C.P. Fischer et al. 2006.

22. Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise. Free Radical Biol. Med. 31 (6); 745-53. A. Childs et al. 2001.

23. Effects of vitamin E supplementation on recovery from repeated bouts of resistance exercise. J. Strength Cond. Res. 17 (4); 801-09. N.G. Avery et al. 2003.

24. Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties. J. Appl. Physiol. 90 (4); 1424-30. J.S. Coombes et al. 2001.

25. Controversies of antioxidant vitamins supplementation in exercise: Ergogenic or ergolytic effects in humans? Journal of the Int. Society of Sports Nutrition 11: 1; 4. C.L. Draeger et al. 2014.

26. Vitamin and mineral supplementation and neuromuscular recovery after a running race. Med. Sci. Sports Exerc. 38; 2110-17. E. Gauche et al. 2006.

27. Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition 24 (4); 293-99. K. Mizuno et al. 2008.

28. The effects of coenzyme Q10 supplementation on performance during repeated bouts of supramaximal exercise in sedentary men. J. Strength Cond. Res. 24 (1); 97-102. H. Gokbel et al. 2010.

29. Effects of ubiquinone-10 supplementation and high intensity training on physical performance in humans. Acta Physiol. Scand. 161 (3); 379-84. C. Malm et al. 1997.

30. Coenzyme Q10 supplementation and exercise-induced oxidative stress in humans. Nutrition 28 (4); 403-17. B. Ostman et al. 2012.

31. Impact of coenzyme Q-10 on parameters of cardiorespiratory fitness and muscle performance in older athletes taking statins. The Physician and Sports Medicine 40 (4); 88-95. R.E. Deichmann et al. 2012.

32. Effects of acute and 14-day coenzyme Q10 supplementation on exercise performance in both trained and untrained individuals. Journal of the International Society of Sports Nutrition 5 (1); 1-14. M. Cooke et al. 2008.

33. Effects of coenzyme Q10 supplementation on exercise performance, VO2 max, and lipid peroxidation in trained cyclists. International Journal of Sports Nutrition 1; 353-65. B. Braun et al. 1991.

34. Impact of oral ubiquinol on blood oxidative stress and exercise performance. Oxidative Medicine and Cellular Longevity vol. 2012 I.D. 465020. R.J. Bloomer et al. 2012.

35. Ubiquinol supplementation enhances peak power production in trained athletes: a double-blind placebo controlled study. Journal of the Int. Society of Sports Nutrition 10 (1): 24. A. Dietmar et al. 2013.

36. Does exogenous coenzyme Q10 affect aerobic capacity in endurance athletes? Sports Nutrition and Exercise Metabolism vol 7 no. 3; 197-206. S.B. Weston et al. 1997.

37. Muscle and plasma coenzyme Q10 concentration, aerobic power and exercise economy of healthy men in response to four weeks of supplementation. Journal of Sports Medicine and Physical Fitness 45 (3); 337-46. S. Zhou et al. 2005.