Update on Antioxidants & Exercise - Neither Vitamin C Nor E Have ANY Effect on the Response to Intense Exercise.

Image 1: If you add some reactive oxygen species to this mitochondrium, this will trigger beneficial, (mito-)hormetic adaptations, that could be blunted by too many antioxidants.

As a diligent reader of the SuppVersity, you have probably been following my posts on antioxidants and their potentially negative effect on the adaptive (hormetic) response to the exercise induced formation of reactive oxygen specimen. Although, I still believe that the theory may have its merit - especially in metabolically deranged people, where the exercise induced ROS formation would initially have to overcome the low-grade chronic "background" stress - it appears that for healthy people, and "moderately trained young men" on an intense exercise protocol, in particular, the ingestion of reasonable amounts (<1g of vitamin C and <400IU of vitamin E) does not pose a problem. At least this is what the results of two relatively recent studies by scientists from Washington School of Medicine (Higashida. 2011) and researchers from universities in Denmark and France (Yfanti. 2011) would suggest.

The hormetic benefits of inflammation

According to the mitohormesis hypothesis, the beneficial effects of exercise on health, in general, and glucose metabolism, in particular, are at least partly mediated by an increase in reactive oxygen species, which triggers downstream "hormetic" adaptation processes which result in increased oxidative capacity and insulin sensitivity, as well as a reduction in total inflammation (cf. previous posts on the work of S. Schmeisser and M. Ristow from the Department of Human Nutrition at the University of Leipzig. If this theory held true, or let's be more specific, if this theory which is largely based on observations in metabollically derranged, i.e. obese and/or type II diabetic subjects, was applicable to healthy people and athletes, as well, this could mean that the multi-vitamin, the vitamin C pills, the alpha-tocopherol (vitamin E) and all the other little helpers you have been taking religiously to increase your exercise performance would actually have hampered, not promoted your muscle gains, fat loss and whatever else you may have had in mind, when you hit the gym, the road, the field, the court or the green ;-)

Figure 1: Neither the high-dose supplementation protocol in rodents (HED: Human Equivalent Dose for 80kg), nor the moderate dose protocol in humans did block any of the measured beneficial adaptations to exercise in the studies by Higashida (2011) and Yfanti. (2011), respectively.

Let me say this right away: As long as you have not been following the recommendations of dubious nutritional gurus and self-proclaimed fitness "experts" to take 10g+ of vitamin C and vitamin E supplements in the 3000IU+ range, the little vitamin pills and powders are probably not the reason that your biceps is not growing and your belly is just as fat as it was, when you began training.

Figure 2: Serum vitamin C and vitamin E levels (µmol/L) in 21 subjects before, at the beginning and after 12 weeks of 5x a week strenuous cycling exercise with and without supplemental vitamin C & E (adapted from Yfanti. 2011)

As you can see in figure 2, supplementation with 500mg of vitamin C and 400 IU of vitamin E (more on the protocols used in the studies in figure 1) before and during 12 weeks of strenuous bicycle exercise training with a frequency of 5 sessions per week (HIIT, HIT and stead state, cf. figure 6) did increase the concentration of antioxidants in the blood of the 21 healthy, physically active subjects (age 18-40years) of the Yfanti study, who had not participated in physical exercise more than twice a week before the experiment. Despite higher vitamin C and E plasma levels, and contrary to the research hypothesis of the scientists, who had expected that the anti-oxidant supplementation would blunt the adaptive response to the exercise protocol,

[...] the present study showed that combined supplementation with vitamins C and E before and during 12 weeks of supervised, strenuous bicycle exercise training of a frequency of 5 days/week had no effect on maximal oxygen consumption, maximal power output, workload at lactate threshold, glycogen content, and CS and β-HAD activity in muscle.

In other words, supplementing with "reasonable" amounts of vitamin C and vitamin E had absolutely NO EFFECT (!) on the exercise induced metabolic adaptations or performance increases - that does yet also imply that taking anti-oxidants is of little benefit as long as the minimal dietary requirements are met... and if you still insist to poor money down the literally rat hole, you may be interested to hear that (assuming that the results from Higashida's rat study translate to humans), even 10g of vitamin C and 3000IU of vitamin E a day probably would not really make a difference - as long as you train heavy enough.

Figure 4: Exercise induced changes in GLUT-4 expression (arbitrary units) and 2DG transport (µmol/ml/20min) in rats subjected to 8 weeks of high dose antioxidant supplementation and 6days/week swimming exercise in the last 3 weeks (data adapted fro Higashida. 2011)

Of particular interest in this context is the effect of "mega-dosing" anti-oxidants on the exercise induced increase in insulin sensitivity, which has been reported to be impaired in previous studies (Ristow. 2009). As the data in figure 3 shows, the increase in glucose transporter (GLUT4) expression is slightly greater in the non-supplemented rats, BUT neither this difference nor the difference in measured 2-Deoxy-D-glucose (2DG) transport reach statistical significance.

Figure 5: Exercise induced changes in MDA, SOD and PGC-1α in rats subjected to 8 weeks of high dose antioxidant supplementation and 6days/week swimming exercise in the last 3 weeks (data adapted fro Higashida. 2011)

Moreover, Higashida et al. found no statistically significant differences in the increases of malondialdehyde (MDA), superoxide dismutase (SOD1 & SOD2) or PGC-1α, a marker for the mitochondrial fatty acid oxidation, between the rats in the two groups (cf. figure 5).

Now, I a confused and don't know what to believe

So what does all that tell us? Well, we can now be relatively certain that supplementing with vitamin C and vitamin E is a waste of time and money for most of us. What we still cannot say for sure, though is why the Ristow study from 2009, which even made it to mainstream media news, found detrimental effects of supplementing with 1g of vitamin C and 400IU of vitamin E on the adaptive response to 4 weeks of 5days/week 20min steady state aerobic training + 45 minute circuit training + 20 min warm up + cool down (Ristow. 2009), while the Yfanti study, with 500mg of vitamin C and 400IU of vitamin E did not find any effects of supplementation...

Figure 6: Exercise protocol that was used in the Yfanti study.

...the only reasonable explanation I have is that the protocol in the Ristow study may not have been intense enough. Unfortunately there is no detailed information on what the subjects did in the course of the "circuit training", but if that was your usual sissy type walk from one machine to the next, chances are that the level of ROS that was induced by this "exercise" protocol was so low that it was completely blocked by the supplemental anti-oxidants. The "cycling protocol" in the Yfanti study, on the other hand, is pretty intense. If you look at the schedule in figure 6 you will concede that this is almost the way athletes (and maybe you) train.

All that being said, it appears that all is coming back to what I have been writing (and also saying on SHR) several times before: Controlled oxidation is likely to be beneficial. It's like the fire in the oven that keeps you warm - the one you carefully take care of, in order to prevent your whole house to catch fire... if you are a marathon runner, the latter can happen pretty quickly and you will need (tons of ;-) antioxidants and even that will probably not suffice. If you are the housewife on the treadmill, who walks at 5km/h for 20min two times a week, on the other hand, even a few milligrams of vitamin C and a few units of vitamin E will blunt the little oxidative "damage" that you do and your "efforts" to increase your insulin sensitivity or whatever your intentions may be will be sabotaged by your vitamin supplements.

Mitohormesis - Suffocated Mitochondria Live Longer: Scientists Probe Longevity-Effect of Low-Level Stressors.

Image 1: Walter Breuning died in April 2011at the biblical age of 114! And you bet that a man who has seen two world wars has had his share of mitohormetic stress in his life.

As a diligent reader of the SuppVersity you will be familiar with the work of S. Schmeisser and M. Ristow from the Department of Human Nutrition at the University of Jena, here in good old Germany (where not everyone eats Sauerkraut und Weisswurst, even now that the Oktoberfest is in full swing). In previous publications, the scientists have (at least in my mind conclusively) argued against the publicly accepted free-radical hypothesis of aging, which implies that the presence of free radicals is one of the fundamental mechanisms of aging. Now, a few month after the publication of their last review back in May 2011, they are presenting the latest results from their own lab in a paper that is going to be published in the October issue of Hormone and Metabolic Research (Schmeisser. 2011).

Want to live longer? Then you better put another log on the fire

Schmeisser, Zarse, and Ristow used lonidamine (LND), a indazole-3-carboxylic acid derivate, to inhibit cellular respiration in the infamous round-worm (Caenorhabditis elegans) model for aging processes (for a review on the pharmacology, biochemistry and toxicology of lonidamine see Silvestrini. 2008). In essence, they thusly made it more difficult for the cells to "breath", which as you may probably imagine, is a major stressor, which will inevitably increase the formation of purportedly dangerous free radicals (ROS) and should thus increase the aging process, if... yes, if there was any truth to the nonsensical idea that you better sit there, don't eat, don't drink, don't move - in essence - don't live to avoid any potential ROS formation, if you want to extend your lifespan... I guess, you as a self-educated SuppVersity reader won't be surprised that the roundworms did not only survive the "torture" (of life), but - after an initial mitohormetic response, i.e. an adaptational response to the the scientists' effort to suffocate their mitochondria (the initial reduction in oxygen consumption was -37 %!) - thrived on the purportedly life-shortening inhibitor of mitochondrial respiration!

Figure 1: Lifespan of C. elegans treated with 5µM lonidamine, n-acetyl-L-cysteine (NAC) or both (data calculated based on (Schmeisser. 2011)

As you can see in figure 1, the "pro-oxidant" treatment with lonidamine, of which Schmeisser's, Zarse's and Ristow's data shows that it increased respiration and thus mitochodrial ROS formation, increased both median as well as maximal life-expectancy of the nematodes (roundworms) by ~8% - an increase with statistical significance, as the p-value of p<0.001 (= chances that the increased lifespan observed in the study is just coincidence are <0.1%). The latter cannot be said of either the slight increase in maximal lifespan nor the slight decrease in median lifespan in the group of nematodes that was treaded with n-acetyl-L-cysteine (p=0.17; non-significant) or a combination of the anti-oxidant sulfur-amino acid and lonidamine (p=0.95; absolutely non-significant; cf. figure 1).

These observations may be considered further experimental "evidence" for Schmeisser's and Ristow's previously formulated mitohormesis theory ("evidence" in the sense that the results do not falsify their hypothesis - they do yet falsify the ROS hypothesis of aging). A theory that refutes the idea that the aging process is driven by reactive oxygen species and emphasizes (mitochondrial) adaptation processes to (external) stressors that strengthen, not weaken the organism in the long-run - or as the scientists phrase it:

[...] the induction of endogenous defense mechanisms as a secondary response to a stressful condition is assumed to contribute to longevity [...] a lifetime low dose oxidative stress with a subsequent secondary induction of defense mechanisms could delay the aging process

It is thus the interplay of manageable stress and metabolic adaptation which extends life and not an overall reduction of reactive oxygen species, as the vendors of some "super-potent" anti-oxidants would have you believe. What is still missing though, is a tool which would help us to identify the critical point, where the endogenous adaptation processes cannot keep pace with ever-increasing (mainly) exogenous stressors... in case any scientist finds an answer, I guess you will soon be able to download the respective app on your shiny new iPhone - I just hope that this app will account for the significantly (!) decreased glucose metabolism the iPhone itself will induce in the temporoparietal junction and anterior temporal lobe of the right hemisphere of your brain within less than 30 minutes (Kwon. 2011), as well.

N-Acetylcysteine Hampers Adaptive Response To Exercise. 50% Reduction in JNK Phosphorylation Entail Reduced Expression of Genes Involved in Cell Proliferation, Apoptosis, Inflammation and DNA Repair.

Image 1: The "beneficial" bad guys under
the microscope: Reactive oxygen species
(green-yellow) within endosomes
of human smooth muscle cells
(Circulation Research. 09/2007)

If you listened to my dissertation on the sulfur-amino acids on Carl Lenore's Super Human Radio (cf. shownotes), you will be aware that I was and still am quite skeptical as far as the touted beneficial effects of n-acetylcysteine (NAC) supplementation on exercise performance are concerned. A very recent study that has been conducted by a team of Australian scientists from Deakin and Victoria University in Melbourne appears to warrant this skepticism.

In a 2006 study (McKennah. 2006) the same group had found that N-acetylcysteine can attenuate the decline in muscle Na+,K+-pump activity and thus delay fatigue during prolonged exercise in humans. But even then, the data on real-world and long-term benefits of n-acetylcysteine supplementation was conflictive and the authors' conclusion that NAC exerted it's effect mainly via the suppression of ROS (reactive oxygen species) generation, prompted questions on whether the suppression of exercise-induced ROS-generation would have any downstream effects on the hormetic (=positive adaptation / strengthening reaction after an insult) response scientists suspect to be the major driving force of the beneficial effects of exercise on perfmormance, as well as general and metablic health.

Illustration 1: Hypothetical dose-response curve of the hormetic response to reactive oxygen species inducing exercise (x-axis, arbitrary units); positive units on the y-axis indicate beneficial, negative units negative effects.

Indeed, a 2009 study by a group of scientists from the University of Jena (Ristow. 2009) was able to show that administration of an anti-oxidant supplement that contained 1,000mg vitamin C and 400 IU of vitamin E prevented the health-promoting effects of exercise on in trained, as well as untrained subjects.

Consistent with the concept of mitohormesis, exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. Supplementation with antioxidants may preclude these health-promoting effects of exercise in humans.

In view of the latter results, the major news here is neither that an N-acetylcysteine infusion before a 45min. cylcing trial at 71% of the individual VO2max that was followed by a bout of all out sprinting to fatique partially blocked the release of reactive oxygen species in the eight male subjects (age, 27.1±5.6 years; height, 180.3±5.4 cm; body mass, 76.7±10.9 kg), nor the related prolongation in time to fatigue the scientists observed. What is new, however, is the data Petersen et al. obtained from sophisticated analyses of the activation of signaling pathways and genes, which have been implicated in exercise adaptation in human skeletal muscle (Petersen. 2011).

[...] NAC infusion blocked the exercise-induced increase in JNK phosphorylation, but not ERK1/2, or p38 MAPK.  Nuclear factor-κB p65 phosphorylation was unaffected by exercise; however it was reduced in NAC at fatigue by 14% (P&amp;amp;amp;amp;lt;0.05) compared to pre-infusion.

This is an important finding, in so far, as it goes to show that the induction of JNK phosphorylation by exercise is ROS-dependent. Now, a -49% reduction in phosphorylation of JNK, a protein that has been shown to be activated as a consequence of strenuous aerobic and/or strength training, would not be a bad thing, if its activation would not play a significant role in the regulation of genes "involved in cell proliferation, apoptosis, inflammation and DNA repair (Karin and Gallagher. 2005) and thus [...] exercise adaptation."

It is difficult to say how the results of this short term study with intravenous n-acetylcysteine will translate into athletic practice. The (over-)consumption of large doses >>1-2g of oral NAC to facilitate exercise recovery, as it has been implicated by some of the advocates of the acetylated version of the sulfur-amino-acid cysteine, by all means, seems to be counter-indicated, as another dreaded foe, exercise-induced reactive oxygen specimen (ROS), eventually exhibits its complementary, hormetic face. The real challenge is thus not extinguish the fire, but to keep it burning at an optimal rate, or, metaphorically speaking, to generate and/or suppress ROS in a way which facilitates a precision landing on the maximum of the graph in illustration 1 ;-)

Are You Stressed Enough for a Longer Life? Reactive Oxygen Specimen and Oxidative Stress May Contribute to Longevity

In June 2010 my fellow countrymen Michael Ristow from the Department of Human Nutrition at the University of Leipzig formed an interesting hypothesis (Ristow. 2010) stating that the dreaded reactive oxygen species [ROS], most scientists associate with accelerated cell-aging and degenerative diseases were in fact "essential signaling molecules which are required to promote health and longevity". In a more recent paper he picks this hypothesis up and, in cooperation with Sebastian Schmeisser from the Department of Clinical Nutrition at the German Institute of Human Nutrition in Nuthetal, Germany, reviews the current literature on selected longevity-promoting intervention.

Figure 1: Health and longevity as a function of mitochondrial reactive oxygen species (ROS) formation.
Both too much and too little ROS are detrimental, due to either insufficient stimulus for or overtaxing of hormetic processes (this graph is a mere illustration and is not based on any existing experimental data)

Ristow and Schmeisser conclude that, as different as they may at first appear, calorie restriction, the reduction of specific macronutrients (esp. low-carb diets), life-extension related to insulin and IGF-1 modulation and physical exercise, have one common denominator, which is

enhanced mitochondrial activity and subsequently increased ROS formation that ultimately induce an adaptive response (increased defense mechanisms and improved stress resistance) which culminates in metabolic health and extended longevity.

For someone, who has been told for years, to avoid the formation of radical oxygen species at all cost, and, for that purpose, to supplement with as much anti-oxidants as possible, this proposition may sound absurd. It also goes against the free radical hypothesis of aging (FRTA) and the hitherto accepted hypothesis that calorie restriction, the one and only relatively established means of life-extension in several animal models (I just want to remind you that the results still await direct confirmation in human beings), would work, because it would reduce the metabolic rate of the fasting critter and thus reduce the amount of potentially harmful reactive oxygen specimen (ROS) which are produced by the down-regulated mitochondria. The authors' response to this hypothesis is that

[...] more recent findings regarding the question whether CR [calorie restriction] actually decreases metabolic rate are, at least in part, inconsistent with FRTA [the free radical theory of aging]. Hence, it has been reported that CR increases metabolic rate (quantified by both oxygen consumption and heat production) in the nematode and well-established model organisms for aging research,  Caenorhabditis elegans. Furthermore, a positive correlation between low metabolic rate and enhanced lifespan could also not be observed in the fruitfly Drosophila melanogaster.

As a diligent student of the SuppVersity you will also be familiar with the lack of effect of antioxidant supplementation in human intervention studies to increase lifespan, promote health or prevent cancer, of which Ristow and Schmeisser write:

[...] in contrast to some of the above-mentioned work in lower organisms, several prospective clinical intervention studies were unable to found a positive association between the supplementation of antioxidants and health beneficial effects. Whereas most studies found a lack of effect in regards to health promotion in humans, other reports even suggest that antioxidants may promote cancer growth. Moreover, supplementation with antioxidants has been linked to increased incidence of a number of diseases to adverse effects of human longevity. 

These observations stand in line with the previously reported findings of Ristow et al. (Ristow. 2009) on the detrimental effect of antioxidant supplementation on exercise induced health promotion, which caused quite a stir in the world of supplement addicted exercise junkies. In all these cases, the German researchers suspect that the inhibition of the formation of an adequate amount of reactive oxygen species (ROS) blocks the initiation of hormetic physiological responses - just like using the 0.5 pound dumbbells for biceps curls to avoid injury would, at the same time, forestall muscular adaptations in form of muscle growth.

Ristow and Schmeisser also point out that, just as it is the case for resistance training, where using too heavy weights may easily lead to injury, the same "so-called adaptive response processes [which] may explain how increased ROS formation culminates in promotion of health and lifespan" may eventually turn against you, if the rate of ROS formation exceeds certain limits:

Interestingly, low doses of ROS seem to exert such effects [promotion of health and lifespan], while higher doses are unquestionably detrimental. Such biphasic responses to a potentially harmful compound are commonly named hormesis, a concept which was initially postulated in 1943 by Southam and Ehrlich and which was shown to have significant impact on aging with a variety of stressors described.

It is this concept of "mitochondrial hormesis or mitohormesis" you should thus keep in mind, when you read about the latest and greatest antioxidant supplement that just hit the market. As an athlete on a two-a-day intensive training regimen, you may well find yourself deep within the red zone on the right of the "optimal health" window in my little graph in figure 1 and thus need additional antioxidants to reduce the amount of ROS and return into the 'hormetic window' of optimal health and longevity. As a couch potato lounging in the red zone to the left of the hormetic window, you better get your metabolism going in order to produce some reactive oxygen specimen and to reap the health and longevity benefits of ROS-triggered hormetic adaptations.

Antioxidant Gear: Anabolic Steroid Stanozolol Decreases Mitochondrial ROS Generation and Oxidative Stress Induced by Acute Exercise in Rat Skeletal Muscle

Figure 1: Molecular structure of Stanozolol
(from Wikipedia)

Oswaldosalcedo, a member of the Mind&Muscle forum came up with a recent study (Saborido. 2011) that found some surprisingly healthy "side-effects" of Stanozolol, a synthetic anabolic orally available steroid derived from testosterone, also known as Winstrol.

The scientists tested the effect of Stanozolol administration on markers of mitochondrial oxidative stress in rats after an acute bout of exhaustive exercise and found: 

Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats.

Those of you who frequent the Mind & Muscle Forum, may already have read my comment on the changes in plasma fatty acid composition of the cell membranes in the respective thread: These changes vaguely remind me of a recent study on the effects of fish oil supplementation in elite athletes (Omega-3 Fatty Acids PRO(!)-Inflammatory in Athletes) where an increase in DHA (docosahexaenoic acid) also increased oxidative stress in the participants. The finding that Winstrol (Stanozolol) protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties by inhibiting the increase in DHA in the cell membrane may thus also be of relevance in view of the benefits / pitfalls of fish oil supplementation. Since (manageable) structural damage and super-compensatory repairs are also a prerequisite at the heart of training adaptation and muscle growth, the decrease in cellular integrity induced by increases in long-chain PUFAs in the cell membranes could also explain the "anabolic" effect of fish oil that has been observed in a handful of studies.