Ascorbic Acid Supplementation Does Not Alter Oxidative Stress Markers in Healthy Volunteers Engaged in a Supervised Exercise Program.

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1 Ascorbic Acid Supplementation Does Not Alter Oxidative Stress Markers in Healthy Volunteers Engaged in a Supervised Exercise Program. Journal: Applied Physiology, Nutrition, and Metabolism Manuscript ID apnm r2 Manuscript Type: Article Date Submitted by the Author: 04-Oct-2015 Complete List of Authors: Bunpo, Piyawan; Chiang Mai University, Medical Technology Anthony, Tracy; Rutgers University, Nutritional Sciences Keyword: stress < exercise, vitamin C, free radicals, supplementation, antioxidants

2 Page 1 of 34 Applied Physiology, Nutrition, and Metabolism Ascorbic Acid Supplementation Does Not Alter Oxidative Stress Markers in Healthy Volunteers Engaged in a Supervised Exercise Program Piyawan Bunpo 1,*, Tracy G. Anthony 2 1 Division of Clinical Chemistry, Department of Medical Technology, Faculty of Associated 6 7 Medical Sciences, Chiang Mai University, 50200, Thailand. 2 Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ *To whom correspondence should be addressed: Piyawan Bunpo, Ph.D., Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand, Tel: (+66)-(0) : ; pbunpo@gmail.com 12

3 Page 2 of Abstract The purpose of this study was to investigate the impact of ascorbic acid (AA) consumption on the oxidative stress status of untrained volunteers participating in a supervised exercise program. The study included 46 young adults (average age 23.5±0.59 years; 37 females, 9 males) who remained sedentary (sedentary 0AA, n=16) or participated in 30 min of outdoor aerobic running (n=30) at intensity corresponding to 65-75% of maximum heart rate three times per week for 12 weeks. Exercised subjects were randomly assigned to exercise group without ascorbic acid supplementation (control 0AA, n=10) or received either 250 mg (250AA, n=10) or 500 mg (500AA, n=10) previous to each exercise session. Blood samples were taken on day 0 and day 84 to evaluate metabolic profiles and antioxidant status. Sedentary subjects underwent in a single bout aerobic running to determine total antioxidant status (TAS) and malondiadehyde (MDA) at pre- and post-exercise with or without AA supplementation. No significant change in TAS was observed. Plasma MDA significantly increased at post-exercise (P<0.05), and AA supplementation decreased MDA level significantly (P<0.05). After 3 months of exercise, there was no significant change in blood glucose, lipid profile, MDA, TAS, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities amongst

4 Page 3 of 34 Applied Physiology, Nutrition, and Metabolism groups. Supplementation of AA was associated with minor and inconsistent reductions in SOD, GPx and catalase activities (P<0.05). These findings indicate that pre-exercise supplementation of ascorbic acid does not alter oxidative stress markers in the plasma and erythrocytes of young adults engaged in a supervised exercise program. Keywords: exercise; vitamin C; oxidative stress markers; free radicals; supplementation; antioxidants

5 Page 4 of Introduction Regular exercise or physical activity promotes overall health and wellness by helping many of the body s systems function better. Conversely, a sedentary lifestyle increase the chances of becoming overweight and developing a variety of diseases associated with oxidative stress and chronic inflammation (Dunstan et al. 2010; Patel et al. 2010; Warren et al. 2010). Enhanced oxidative stress results from an imbalance between free-radical generation and antioxidant defense mechanisms. However, free radicals are not always deleterious (Powers and Jackson 2008). Indeed, while high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can cause cellular damage and promote aging, low-tomoderate levels of oxidants play multiple regulatory roles in cells such as modulation of gene expression, regulation of cell signaling pathways, and control of skeletal muscle force production as the body adapts to exercise (Droge 2002; Reid 2001a, b; Smith and Reid 2006). The delicate balance between beneficial and harmful effects of free radicals is a very important aspect of living organisms and is achieved by utilizing antioxidant enzymes and non-enzymatic antioxidant processes to maintain redox balance (Droge 2002).

6 Page 5 of 34 Applied Physiology, Nutrition, and Metabolism Enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) attenuate the generation of reactive oxygen species by removing potential oxidants or by transferring them into relatively stable compounds. SOD forms the first line of defense against superoxide radicals. It catalyzes the transformation of the superoxide radical into hydrogen peroxide, which can then be transformed by enzyme catalase into water and molecular oxygen (Culotta et al. 2006). Elimination of superoxide radical can attenuate the formation of hydroxyl radical. Glutathione peroxidase (GPx) catalyze the reduction of H 2 O 2 or organic hydroperoxide 60 (ROOH) to water (H 2 O) and alcohol (ROH), respectively (Bjornstedt et al. 1994). Vitamin C or ascorbic acid is a water-soluble vitamin that is required as a co-factor for at least eight enzymes, including prolyl hydroxylase, important in collagen formation (Peterkofsky and Udenfriend 1965). Humans are among the few animals that lack the ability to synthesize the compound from glucose. The recommended daily intake for women is 75 mg and for men is 90 mg. Supplementing vitamin C in the diet to higher levels is suggested to provide antioxidant protection against oxidative stress. Population studies show that individuals with high intakes of vitamin C have lower risk of a number of chronic diseases, including heart disease, cancer, eye diseases, and neurodegenerative conditions (Jacob and Sotoudeh 2002). Antioxidant

7 Page 6 of supplements have also been used to attenuate the injury resulting from strenuous resistance exercise (McHugh 2003). However, recently there is a growing evidence of the negative effects of antioxidant supplementation on exercise training. The efficacy of vitamin C supplementation on exercise-induced oxidative stress remains unclear, some studies demonstrating no effect (Gomez-Cabrera et al. 2008; Ristow et al. 2009; Roberts et al. 2011), and others reporting a reduction in oxidative stress markers following exercise (Higashida et al. 2011; Ryan et al. 2010). Therefore, this study was aimed to evaluate whether ascorbic acid supplementation has 76 any surplus benefits during exercise training intervention in untrained healthy adults

8 Page 7 of 34 Applied Physiology, Nutrition, and Metabolism Materials and methods Subjects and Design Forty-six healthy untrained adult volunteers participated in this study. Table 1 shows the main characteristics of participants in the study. All the participants were informed of the purpose and demands of the study before giving their written consent to participate. The protocol was approved by the Ethics Research Committee from Faculty of Associated Medical Sciences, Chiang Mai University. Volunteers were screened by interview. Exclusion criteria were cigarette 86 smoking and regular use of any medications. Study participants were asked to abstain from alcohol and caffeine and were instructed to maintain their normal physical activity and dietary habits throughout the study. Blood samples were collected at the beginning of the study (day 0) and at study completion (day 84). The subjects were randomly assigned to sedentary control group without ascorbic acid supplementation (n=16), outdoor running group (n=30). Study subjects in the outdoor running groups participated in a supervised aerobic running program for 12 weeks consisting of 30 min/session of outdoor running at intensity corresponding to 65-75% of maximum heart rate three times per week for 12 weeks on 3 nonconsecutive days per week. Exercise subjects were randomly assigned to one of three groups (n=10 per group). Group 1

9 Page 8 of received no supplementary ascorbic acid throughout the study (0AA,control group). Group 2 received 250 mg of ascorbic acid before each supervised exercise session (250AA). Group 3 received 500 mg of ascorbic acid before each supervised exercise session (500AA). On a separate occasion, sixteen subjects who remained sedentary were instructed not to perform any exercise for 2 days preceding single bout of outdoor running at intensity corresponding to 65-75% of maximum heart rate and were overnight fasted. The single bout exercise session was conducted with or without ascorbic acid supplementation at a dose of 500 mg on a different 102 day. Blood samples were collected pre- and 15 minutes post-exercise to measure total antioxidant status (TAS) and malondialdehyde (MDA) levels in plasma. Ascorbic acid (The Government Pharmaceutical Organization, Bangkok, Thailand) was consumed in pill form with water Blood sampling and measurements Blood samples were collected in the morning after an overnight fast of at least 12 hours at the same time before (day 0) and after the 12-week of exercise program. Fasting blood samples were collected into BD VacutainerTM test-tubes (Becton Dickinson Immunocytometry

10 Page 9 of 34 Applied Physiology, Nutrition, and Metabolism Systems-BDIS, USA). Plasma was obtained from the ethylenediaminetetraacetic acid-treated blood samples within 30 min after blood collection by centrifugation (15 min, 1,000 g, 4 C). Serum was obtained by allowing the whole blood to clot for 30 min, followed by chilled centrifugation (15 min, 1,000 g, 4 C). Plasma and erythrocytes were separated and erythrocytes were washed three times with 0.9% NaCl solution, and then hemolyzed with four volumes of cold distilled water. Prepared samples were maintained or stored (at -80 C) before 117 further analyses Determination of blood lipids and glucose Total cholesterol, HDL-cholesterol, triglyceride and glucose levels in whole blood were measured by an enzymatic spectrophotometric technique with an auto-analyser (Dimension EXL 200 Integrated Chemistry System, SIEMENS, Germany). These values were then used to calculate the LDL-cholesterol with the Friedewald equation (Friedewald et al. 1972): LDLcholesterol = (total cholesterol - HDL-cholesterol) - triglyceride/ Determination of plasma TBARS

11 Page 10 of MDA level was determined by thiobarbituric acid reactive substances (TBARS) in plasma (Ohkawa et al. 1979). In the TBARS assay, one molecule of malondialdehyde reacts with two molecules of thiobarbituric acid (TBA) and thereby produces a pink pigment with absorption peak at 532 nm. Amplification of peroxidation during the assay is prevented by the addition of the chain-breaking antioxidant, butyryl hydroxy toluene (BHT). The reaction mixture consisted of 0.4 ml serum, 1.1 ml of 20 mg/dl trichloroacetic acid and mixture was allowed to stand for 10 minutes at room temperature. This was further centrifuged at 3,000 rpm for 10 minutes and the 134 supernatant was discarded. About 1.5 ml of thiobarbituric acid reagent was added to the pellet and was heated in boiling water bath for 10 minutes. The tubes were placed on ice to stop the reaction, 1.5 ml of n-butyl alcohol was added, then mixed and centrifuged at 3,000 rpm for 20 minutes. Color intensity of the chromogen in organic phase was measured spectrophotometrically at 532 nm. Results were compared using standard solution of 1,1,3,3-tetramethoxypropane and expressed as μmol per liter Determination of Total Antioxidant Status

12 Page 11 of 34 Applied Physiology, Nutrition, and Metabolism Antioxidant quantification was done using 2,2' -azino-bis (3ethylbenzthiazoline-6-sulfonic acid) (ABTS + ) radical formation kinetics (Randox Laboratories, Ltd., Crumlin, UK). Manufacturer's instructions were followed to determine the activity. In this method, ABTS was incubated with peroxidase (metmyoglobin) and H 2 O 2 to produce the radical cation ABTS +. This product has a relatively stable blue-green color, which is measured at 600 nm. The antioxidants present in plasma suppressed the bluish-green staining of the ABTS + cation, which was proportional to the antioxidant concentration level. Millimole per liter (mmol/l) unit was using for expression of 149 results. This assay was measured on Olympus AU400 auto analyzer after running control and 150 calibration Determination of red blood cell superoxide dismutase The SOD activity was evaluated in the erythrocyte samples (cell lysates) by using the commercially available RANSOD Kit (Randox Laboratories, Ltd., Crumlin, UK) according to the manufacturer instructions. This method employs xanthine and xanthine oxidase (XOD) to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-

13 Page 12 of phenyltetrazolium chloride (INT) to form a red formazan dye. The SOD activity is then measured by the degree of inhibition of this reaction. One unit of SOD is that which causes 50% inhibition of the rate of reduction of INT under the conditions of the assay. SOD levels were recorded at 505 nm and through a standard curve and expressed unit per gram of hemoglobin (U/g Hb). The SOD assay was measured on Olympus AU400 auto analyzer after running control and calibration Determination of red blood cell glutathione peroxidase The activity of GPx of erythrocytes lysate was evaluated with GPx detection RANSEL kit (Randox Laboratories, Ltd., Crumlin, UK) according to the manufacturer instructions, by using the appropriate control (calibrator). GPx catalyze the oxidation of glutathione (GSH) by cumene hydroperoxide. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP +. The decrease in absorbance at 340 nm against blank was measured spectrophotometrically. One unit (U) of GPx activity was defined as the amount of enzyme that

14 Page 13 of 34 Applied Physiology, Nutrition, and Metabolism converts 1 μmol of NADPH to NADP + per minute. The GPx activity was expressed as unit per gram of hemoglobin (U/g Hb) Determination of erythrocyte catalase The catalase activity in erythrocytes was assayed spectrophotometrically by monitoring the decomposition of H 2 O 2 using the procedure of Aebi (Aebi 1984). Briefly, 0.5 ml of 30 mm/l H 2 O 2 solution in 0.95 ml of 50 mm/l phosphate buffer (ph=7.0), 0.05 ml of 1:50 diluted 179 erythrocyte lysates was added and the consumption of H 2 O 2 was followed spectrophotometrically at 240 nm for 30 and 90 seconds at 25 C. The molar extinction coefficient was 43.6 M 1 cm 1 for H 2 O 2. Catalase activity was expressed as the unit that is defined as μm H 2 O 2 consumed/min/g hemoglobin Determination of hemoglobin Concentration of hemoglobin was determined by cyanmethemoglobin method following the method of Dacie & Lewis (Dacie and Lewis 1975). Hemoglobin is converted into cyanmethemoglobin by the addition of potassium cyanide and ferricyanide. The colour of

15 Page 14 of cyanmethemoglobin is read in a photoelectric colorimeter at 540 nm against a standard solution. Since cyanide has the maximum affinity for hemoglobin, this method estimates the total hemoglobin Determination of plasma ascorbic acid Plasma ascorbic acid was stabilized immediately with equal amounts of 10% (w:v) meta- phosphoric acid. The formed precipitate was spun down by centrifugation at 10,000 rpm for min (4 C) and the protein-free supernatant was collected and stored at 80 C before being analyzed. The samples were diluted 1:1 with HPLC water and filtered through a 0.22 µm-nylon syringe filter prior analyzed on an automated HPLC system. A Knauer HPLC system (Berlin, Germany), all controlled by a Eurochrome 2000 operating and data-acquisition software (Knauer, Berlin, Germany), was used. A reversed phase Agilent ZORBAX Eclipse Plus C18 column ( mm, 5 μm particle size) was used for separation. The mobile phase comprised of a (70:30, v/v) mixture of acetrobitrile-100 mm ammonium acetate was filtered through a 0.45 µm-filter under vacuum and degased before use. The flow rate was 1 ml/min, the injection volume was 20 μl, and the temperature was set at 25±0.5 C.

16 Page 15 of 34 Applied Physiology, Nutrition, and Metabolism Determination of plasma total protein Total protein concentration was measured by biuret method with bovine serum protein (Sigma- Aldrich) as standard. Briefly, the peptide bonds of protein react with the copper II ions in alkaline solution to form a blue-violet complex with an absorption maximum at 545 nm. The color formed is proportional to the protein concentration Statistical Analysis Results were expressed as mean ± SEM. Data were processed by standard statistical software SPSS 17.0 (SPSS Software, Thailand). Two-way repeated measures analysis of variance (group vs. time) were used, differences among groups and times were subsequently identified using a Bonferroni post-hoc analysis with statistical significance of p<

17 Page 16 of Results 3.1. Subject characteristics and metabolic parameters Average body weights of study subjects are shown in Table 1. Average body weights did not change significantly across time. Baseline values of body weights and lipid profiles in sedentary control group were significantly different from exercise groups. The exercise program did not significantly alter glucose and lipid profile levels. Also, no effect of vitamin C supplementation was observed. However, there was a slight reduction in plasma triglycerides in control 0AA group but this effect was blunted in vitamin C supplementation Changes in levels of MDA, TAS, SOD, GPx and catalase The acute responses in MDA levels to a single bout of exercise are presented in Table. 2. Plasma MDA levels in subjects not supplemented with ascorbic acid were significantly (p<0.05) elevated 15 minutes post-exercise, whereas no significant (p>0.05) differences were found between the ascorbic acid-supplemented subjects. No significant changes were observed in TAS. After 3 months of exercise program, volunteers in exercise groups who were administered 0, 250 and 500 mg of vitamin C, respectively, showed no change in MDA levels. Similarly, TAS

18 Page 17 of 34 Applied Physiology, Nutrition, and Metabolism did not exhibit a significant difference. The impact of ascorbic acid and exercise on the antioxidant enzymes studied is presented in Table 3. Baseline values of SOD levels in sedentary control group were significantly different from control 0AA group. SOD activity was reduced in ascorbic acid treated group at a dose of 500 mg. On the other hand, the activity of GPx and catalase enzymes were significantly decreased by ascorbic acid at a dose of 250 mg Ascorbic acid concentrations 240 Plasma ascorbic acid concentrations of volunteers at different time points are shown in Table Volunteers treated with 500 mg of ascorbic acid demonstrated 7.2% higher plasma ascorbic acid at day 84 as compared with their own baseline values at day 0. Nevertheless, this difference was not statistically significant (p>0.05). No other statistical differences were observed among the three experimental groups

19 Page 18 of Discussion Reactive oxygen species (ROS) are unavoidable products during normal intracellular metabolism. They play essential roles in cell differentiation, proliferation, and host defense response (Powers et al. 2010; Sroka and Madeja 2009). However, overproduction of free radicals can cause tissue damage. Various cell components are believed to be damaged by oxygen-derived free radicals, of which lipid peroxidation (Wolters and Konings 1984), DNA damage (Kryston et al. 2011), and protein oxidation (Gieseg et al. 2000) are probably the most critical. The excessive free radicals generated during exercise training may cause the accumulation of reactive oxygen species (Fisher-Wellman and Bloomer 2009; Jackson et al. 2007). To cope with the damaging actions of ROS, organisms have evolved a ROS defense system, consisting endogenous antioxidants, such as SODs, CAT, GPxs, and various thio-, peroxi-, and glutaredoxins glutathione and exogenous antioxidants, such as ascorbic acid, tocopherol. Antioxidant supplements are widely used by athletes to avoid elevated oxidative stress. To date, the efficacy of vitamin C supplementation on exercise-induced oxidative stress remains unclear. The equivocal results between studies, some demonstrating no effect (Gomez- Cabrera et al. 2008; Ristow et al. 2009; Roberts et al. 2011), and others reporting a reduction in

20 Page 19 of 34 Applied Physiology, Nutrition, and Metabolism oxidative stress markers following exercise (Higashida et al. 2011; Ryan et al. 2010), make it difficult to determine if vitamin C provide protection against exercise-induced oxidative damage. In the present study, a single bout of exercise was shown to contribute to lipid peroxidation as reflected by increased levels of plasma MDA. Lipid peroxidation following a single bout exercise was inhibited by vitamin C supplementation. Polidori et al. (Polidori et al. 2004) also reported that vitamin C can prevent lipid peroxidation by directly intercepting ROS. However, after 3 months of exercise program, the plasma MDA levels of exercise control group did not change 270 significantly. This appeared to be due in part to adaptations to exercise mediated by ROS Azizbeigi et al. (Azizbeigi et al. 2014) reported that endurance, resistance and concurrent training, consisting of 3 times/week on nonconsecutive days for 8 weeks, significantly decreased resting MDA levels by 32.7, 32 and 29.1 percent respectively. This indicated that regular exercise training induces an adaptation within the body, which relates to an up regulation of antioxidant protection mechanisms. Furthermore, MDA levels in response to regular exercise did not differ in vitamin C supplementation. The enzyme activities of SOD, GPx and catalase values were slightly reduced in exercised subjects supplemented with vitamin C. However, no dose response were observed in either SOD, GPx or catalase. In a human study,

21 Page 20 of the negative effects of ascorbic acid supplementation on the adaptive responses of endogenous antioxidant enzymes such as SOD and catalase were demonstrated (Khassaf et al. 2003). It has been reported that vitamin C prevented the exercise-induced messenger RNA (mrna) expression of cytochrome C and of the antioxidant enzymes SOD and GPx (Gomez- Cabrera et al. 2008). ROS generated in response to exercise and other physiological or pathological stimuli might be important signaling molecules rather than acts as toxic agents. Many studies have documented a role of ROS as second messengers responsible for 286 adaptations to exercise (Gomez-Cabrera et al. 2005; Ji 2007; Kang et al. 2009). In the current study, a slight increase in plasma ascorbic acid level was seen in 500 mg of vitamin C treated group. The pharmacokinetics of vitamin C is quite complex and involves several different transport mechanisms, as well as localization within specific tissues (Herrmann and Obeid 2011). Following the intestinal epithelium absorption, vitamin C is released into the bloodstream as ascorbic acid (>95%) (Dhariwal et al. 1991; Liang et al. 2001), less than 5% are oxidized into dehydroascorbic acid (DHA) and rapidly taken up through GLUT1 transporters on the erythrocytes (Lykkesfeldt et al. 2003). However, an increase in total antioxidant status was not seen with vitamin C supplementation. Results of total antioxidant status were variable, perhaps

22 Page 21 of 34 Applied Physiology, Nutrition, and Metabolism due to differences in diet consumption containing other antioxidants of each individual. Significant improvements in HDL-cholesterol levels were reported in volunteers who exercised at 75% of maximal heart rate for 12 weeks, but no changes were observed in those who exercised at less than 75% of maximal heart rate (Escalante et al. 2012). In our study, there was no improvement of total cholesterol, HDL-cholesterol and LDL-cholesterol levels in any group and that vitamin C did not have significant effects on lipid profiles. However, aerobic exercise (60 min, 3 times/week, less than 75% maximum heart rate) improve the LDL-C and triglyceride 302 concentrations (Escalante et al. 2012). As indicated in our study, exercise group without vitamin C supplementation showed a slight reduction of triglyceride, on the other hand supplementation with vitamin C mitigated this effect. According to various reviewers, vitamin C supplementation with 0.2 to 1 g daily will reduce oxidative stress (Dekkers et al. 1996; United States. Department of Health and Human Services. et al. 2010). Our results suggested that vitamin C at doses between 250 to 500 mg do not offer additional antioxidant benefits in young, healthy adults engaged in a supervised exercise program. Vitamin C appears to reduce training-induced adaptations by reducing antioxidant enzyme activity including SOD, GPx and catalase. A small dose of vitamin C less than 200 mg may be sufficient to reduce oxidative stress but will not

23 Page 22 of impair optimal training adaptations. Further research is required to clarify a dose-response and nutrient timing protocols on vitamin C. There were some limitations to this study that would make it difficult to extrapolate these results to the general populations. One limitation of this study is its small sample size. The limited sample size could have affected the capacity to identify differences in ascorbic acid supplementation that might be significant in a larger study. A second limitation concerns the dietary habits of subjects, which could influence the antioxidant status of subjects. Subjects 318 were free-living with no dietary restrictions, no dietary recall collected and supplement not taken every day. The intensity of the exercise used in the present study was perhaps not strenuous enough to provoke significant changes in antioxidant enzyme levels. In the present study, the exercise protocol was conducted below anaerobic threshold. That is, exercise-induced oxidative stress may not exceed a certain minimal threshold. During low-intensity and duration protocols, antioxidant defenses appear sufficient to meet the free radical production. If oxidative stress does occur, it is possible that oxidative stress may have occurred prior to or after blood collection or in tissue other than blood. Therefore, from these finding suggested that future investigations employ sufficiently strenuous exercise protocols, and utilize a wide array of

24 Page 23 of 34 Applied Physiology, Nutrition, and Metabolism oxidative stress markers and take multiple samples post exercise (through several hours of recovery) in an attempt to provide valid findings. In addition, the physiological adaptive response using VO2 max or maximal oxygen uptake is one factor that can determine physiological adaptation of each volunteer at the beginning of exercise protocol as well as at the end. The major limitation of this study is that it is done in healthy subjects. The results could not be generalized to all individuals because the older subjects or less healthy individuals were not included in the study. Nevertheless, the results of this study provide valuable insight into this 334 subject populations In summary, the present study reports that vitamin C supplementation before exercise does not alter oxidative stress markers in the plasma and erythrocytes of young adults engaged in a supervised exercise program. Future studies should examine the effects of vitamin C supplementation in older adults Acknowledgements This work was supported by the National Research Project Management (NRPM). 342

25 Page 24 of Conflict of Interest The authors declare no conflict of interest.

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33 Page 32 of Table 1. Biochemical characteristics of subjects before and after 3 months of vitamin C supplement in combination with exercise. Parameter Sedentary (n=16) 0AA (n = 10) 250AA (n = 10) 500AA (n = 10) Baseline 3 months Baseline 3 months Baseline 3 months Baseline 3 months Age, years 22±0.5 24±1.6 24±1.2 25±1.7 Weight, kg 51±2.1 52±2.1 60±2.7 a 60±2.7 58±2.7 a 58±2.6 62±2.1 a 62±2.1 (+2.0%) (0%) (0%) (0%) Glucose, mg/dl 93±2.4 94±2.2 92±2.5 93±2.3 89±2.5 89±2.3 92±2.0 91±2.3 (+1.1%) (+1.1%) (0%) (-1.1%) Cholesterol, 238± ± ±10.2 a 189± ±8.9 a 176± ±13.0 a 194±13.4 mg/dl (-1.3%) (+4.4%) (-3.8%) (+8.4%) Triglycerides, 45±4.6 46±4.6 93±17.1 a 73±9.1 82±12.0 a 79± ±6.5 a 70±10.4 mg/dl (+2.2%) (-21.5%) (-1.3%) (+9.4%) HDL-C, mg/dl 68±3.7 65±3.6 55±5.0 60±5.1 52±4.3 51±3.8 57±2.7 60±3.3 (-4.4%) (+9.1%) (-3.8%) (+5.3%) LDL-C, mg/dl 161± ± ±9.6 a 115± ±8.3 a 109± ±11.1 a 121±12.1 (-0.0%) (+7.5%) (-5.2%) (+10.0%) Values are means ± SE; * p < 0.05, significant different from baseline values (within groups, baseline vs. 3 months); a p < 0.05, significant different from baseline values of sedentary control group. 0AA (exercise control), 250AA (exercise plus 250 mg of vitamin C), 500AA (exercise plus 500 mg of vitamin C), dl = deciliter.

34 Page 33 of 34 Applied Physiology, Nutrition, and Metabolism 33 Table 2. Total antioxidant status and malondialdehyde in single bout exercise. Total antioxidant status and malondialdehyde was measured pre- and post-exercise with or without vitamin C supplementation. No supplement Vitamin C supplement (500 mg) Test Pre-exercise Post-exercise Pre-exercise Post-exercise Total antioxidant status 1.99± ± ± ±0.05 (TAS), mm (n=16) Plasma TBARS, µmol/g protein (n=16) 0.031± ± * 0.029± ± a Values are means ± SE; * p<0.05, significant different from pre-exercise value (within group); a p<0.05, significant different from post-exercise value (between group).

35 Page 34 of Table 3. Antioxidant status of study subjects before and after 3 months of vitamin C supplement in combination with exercise. Test Group Baseline 3 months Ascorbic acid, mg/l Plasma TBARS, µmol/g protein Total antioxidant status (TAS), mm Superoxide dismutase (SOD), U/g Hb Glutathione peroxidase (GPx), U/g Hb Catalase, U/g Hb 0AA 250AA 500AA Sedentary control 0AA 250AA 500AA Sedentary control 0AA 250AA 500AA Sedentary control 0AA 250AA 500AA Sedentary control 0AA 250AA 500AA Sedentary control 0AA 250AA 500AA 30.7± ± ± ± ± ± ± ± ± ± ±0.03 2,325±50 2,140±32 a 2,193±133 2,279±100 83±2.0 81±4.2 86±3.0 84±3.5 87±1.5 79±2.6 80±4.0 82± ± ± ± ± ± ± ± ± ± ± ±0.02 2,329±41 2,118±52 1,999±88 2,020±67 * 84±1.9 77±4.4 71±2.5 * 75±2.9 87±2.5 75±1.5 64±2.6 * 72±1.9 Values are means ± SE; * p<0.05, significant different from baseline values (within groups, baseline vs. 3 months); a p<0.05, significant different from baseline values of sedentary control group. 0AA (exercise control), 250AA (exercise plus 250 mg of vitamin C), 500AA (exercise plus 500 mg of vitamin C).