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1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Title page Effects of six weeks of β-alanine administration on VO2max, time to exhaustion and lactate concentrations in male physical education students Reza Ghiasvand 1, 2* Gholamreza Askari 1, 2 Janmohamad Malekzadeh 3 Maryam Hajishafiee 1, 2 Pooya Daneshvar4 Fahimeh Akbari1, 2 Maryam Bahreynian 1, 2 * Corresponding Author: Reza Ghiasvand, address: Department of Nutrition, School of Health, Isfahan University of Medical Sciences, Hezar Jerib Street, Isfahan, Iran. Tel: 98-311-7922742, Fax: 98-3116682509, email: [email protected] 1. Department of Community Nutrition, School of Nutrition and Food Sciences, Isfahan University of Medical Sciences, Isfahan, Iran 2. Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran 3. Department of Nutrition, School of Health, Yasouj University of Medical Sciences, Yasouj, Iran 4. Esfahan Sport Medicine Association, Isfahan, Iran 2 37 Abstract 38 Objectives 39 Supplementation with β-alanine has been proposed to improve performance in some exercises 40 such as cycling and running. Also, it has been demonstrated that great deals of proton ions are 41 produced in the skeletal muscles during exercise that result in acidosis, whereas β-alanine may 42 reduce this effect. Therefore, the aim of this study was to assess the effects of alanine 43 supplementation on VO2max, time to exhaustion and lactate concentrations in male physical 44 education students 45 Methods 46 Thirty nine male students volunteered for this study. Participants were supplemented orally for 6 47 wk with either β-alanine or placebo (dextrose), randomly. VO2max, Time To Exhaustion (TTE) 48 and lactate and glucose concentrations were measured before and after supplementation. 49 Results 50 Supplementation with β-alanine showed a significant increase in VO2max (p < 0.05) but a 51 significant decrease in TTE and lactate concentrations (p < 0.05). A significant elevation in 52 lactate concentrations, but a non significant increase in TTE was shown in placebo group. 53 Plasma glucose concentrations didn’t change significantly in two groups after intervention. 54 Conclusion 55 It can be concluded that β-alanine supplementation can reduce lactate concentrations during 56 exercise and thus can improve exercise performance in endurance athletes. 57 58 Keywords 59 β-alanine, supplementation, performance 60 61 Running Title 62 β-alanine and performance 3 63 64 Introduction 65 Ingestion of some amino acids have the presumably roles in performance improvement in 66 athletes (Ivy et al. 2003; Koopman et al. 2004; Niles et al. 2001). Among them, β-alanine 67 supplementation has been suggested to improve performance in high intensity exercises (Harris 68 et al., 2003; Hill et al., 2005). On the other hand, it has been shown that large amounts of H+ are 69 produced in the muscles during high-intensity exercise and result in pH reduction (Hultman and 70 Sahlin 1980). There are many cellular pH buffers defend against exercise-induced acidosis 71 (Parkhouse et al., 1985) include phosphocreatine, inorganic phosphates and histidine- 72 containing dipeptides. Carnosine (β-alanyl-L-histidine) is the main histidine-containing dipeptide 73 in humans (Quinn et al. 1992). Additionally, Hill et al. (2005) and Harris et al. (2005) showed 74 that 28 days of β-alanine supplementation increased intramuscular levels of carnosine by nearly 75 60%. Antioxidant function (Boldyrev et al., 1987), muscle contractility regulation (Batrukova and 76 Rubstov, 1997) and pH buffering (Begum et al., 2005; Harris et al., 1990) are the possible 77 physiological roles of carnosine in skeletal muscle. Thus, preventing of skeletal muscle acidity 78 could be a possible role of carnosine in improving exercise performance (Suzuki et al., 2002; 79 Harris et al., 2003). 80 Doing exercise results in oxidative stress and muscle fatigue (Powers and Jackson 2008) that 81 may be prevented by carnosine, due to its antioxidative properties (Kohen et al. 1988). On the 82 other hand, β-alanine administration could increase carnosine content of skeletal muscles by 83 40–80% (Harris et al. 2006; Hill et al. 2007; Derave et al. 2007). Increase of Carnosine 84 concentrations in muscle, results in altered buffering capacity (Harris et al., 2006; Hill et al., 85 2007) and thus performance. Furthermore, some studies have shown that carnosine act as a 86 Ca++ sensitizer for the sarcomeres in muscles, too (Dutka and Lamb 2004; Lamont and Miller 87 1992) and thus could prevent of fatigue (Rubtsov 2001). However, synthesis of carnosine in 4 88 muscle is limited by the availability rate of β-alanine that can be influenced by β-alanine 89 supplementation (Bakardjiev and Bauer 1994; Dunnett and Harris 1999; Hill et al., 2007). 90 While it has been estimated that carnosine is responsible for near ten percent of the total 91 buffering capacity in human muscle (Hill et al., 2007), the importance of acidosis control in 92 exercise performance is still controversial. There are few studies on β-alanine supplementation 93 and its possible effects on endurance exercise; therefore, the purpose of this study was to 94 assess the effects of β-alanine administration on VO2max, time to exhaustion and lactate 95 concentrations in male physical education students. 96 Materials and methods 97 Thirty nine male physical education students volunteered for this investigation. These students 98 were fit and active, but not involved in professional sports. Characteristics of the participants 99 include age, weight and height were 21.1 ± 0.7 years, 71.8 ± 8.8 kg and 178 ± 7 cm for β- 100 alanine (n = 20) and 21.9 ± 1.5 years, 74.9 ± 8.3 kg and 180 ± 5 cm for placebo group (n = 19), 101 respectively (NS). Before initiating the study, all participants were informed of all procedures of 102 the study and signed an informed consent. None of the participants had ingested β-alanine, or 103 any other nutritional supplements, for a minimum of 3 months prior the initiation of the study. 104 Participants were asked to abstain from exercise 24 h before trial initiation and to maintain their 105 current physical activity and dietary patterns. Participants were asked to fill a “food record” for 106 the 2 days before intervention. After pre-testing, the participants were randomly assigned to one 107 of two groups: a) β-alanine (2 g/day), b) placebo (2 g dextrose per day). The supplements were 108 same in appearance, and ingested four times per day for 42 consecutive days before post- 109 testing. All participants completed all experiments, and there were no complaints of side-effects 110 of the supplements. 5 111 This study was a placebo-controlled, double-blind clinical trial. Participants were supplemented 112 orally for 6 wk with either β-alanine (Ajinomoto, USA, Inc) or placebo (dextrose). The study was 113 approved by the Ethics Committee (Esfahan Sport Medicine Association, Iran). Supplements 114 were provided in capsules of 400 mg and were administered each day as five divided single 115 doses, with at least 2 h in between ingestions. Thus, daily doses consisted of 2 g/day during the 116 study. Venous blood samples were obtained from all participants between 5:00 and 6:00 p.m., 117 after intensive endurance exercising, at the baseline and after intervention. All measurements 118 were done before the start of the supplementation (Pre) and after the intervention (Post). 119 Prior to and following the supplementation protocol, participants performed a continuous graded 120 exercise test (GXT) on an electronically braked cycle ergometer (Lode, The Netherlands) to 121 determine VO2max and time to exhaustion (TTE). 122 For each GXT, the primary power output was set at 30 watts and elevated 30 watts every two 123 minutes until the participant couldn’t maintain the required power output at a pedaling rate of 70 124 rpm due to fatigue. 125 Plasma samples were obtained for the determination of plasma lactate and glucose 126 concentrations immediately prior to each GXT and two minutes post-exercise. 127 Glucose and lactate were analyzed using YSI auto-analyzer (Yellow Springs, OH). 128 Dietary analyses were performed using Nutritionist IV software. 129 Statistical analyses were conducted using the Statistical Program for the Social Sciences 130 (SPSS version 13, Inc., Chicago, IL) computer software package. Data are presented as 131 means ± standard deviation. Independent t test was used to analyze the differences in 132 performance between the trials. Paired t test was used to analyze before and after test data for 133 each group differences. An alpha level of p < 0.05 was considered statistically significant. 134 6 135 Results 136 Table 1 shows the mean±SD values of exercise performance indices for the pre- and post- 137 supplementation. Supplementation with β-alanine demonstrated a significant increase in 138 VO2max (p < 0.05) .On the other hand, TTE and lactate concentrations decreased after 6 weeks 139 of supplementation with β-alanine (p < 0.05). The placebo group showed a significant increase 140 in lactate concentrations (p < 0.05), but a non significant increase in TTE. No significant 141 changes in plasma glucose concentrations were detected after exercise in two groups. 142 The post-exercise concentrations of plasma lactate were significantly higher (p < 0.05) than 143 baseline in two groups. However, the post-exercise concentrations of lactate were significantly 144 lower (p < 0.05) during the alanine supplementation compared to the placebo group. Dietary 145 intake before each trial was similar for energy and macronutrients (Table 2). 146 147 Discussion 148 Findings of our study suggest that supplementation with β-alanine may improve the endurance 149 exercise performance as measured by the VO2max, TTE and plasma lactate concentrations. 150 Several studies support our findings (Zoeller et al., 2007, Klein et al., 2009, Baguet et al., 2010, 151 Derave et al. 2007). 152 Also, Harris et al. (2006) showed that supplementation with creatine+β-alanine, resulted in 153 significant increases (P<0.01) in muscle carnosine content. So that, the increased muscle 154 carnosine content was accompanied with an improvement in VO2max and TTE in response to a 155 maximal graded exercise test performed on a cycle ergometer. Their results were agreed with 156 ours. 157 Another study demonstrated that supplementation with both β-alanine and creatine improved 158 cycling performance (TTE), too (Hill et al. (2005)). 7 159 However, β-alanine administration alone improved performance just in the first minute of 160 exercise (Harris et al., 2003). They concluded that this was due to H+ buffering by carnosine 161 during this transitional period. 162 Our data demonstrate that the significant improvements in the performance indices with β- 163 alanine supplementation were due to pH reduction, too. 164 The improvement in TTE seen in the placebo group participants appears to be related to their 165 encouragement by our staff or due to psychological status. 166 Also, the findings of the present study may have been influenced by the fluctuations in the 167 skeletal muscle response to oral supplementation with β-alanine. 168 Our data suggest that supplementation with β-alanine especially may delay the onset of fatigue 169 and thus improve performance during incremental cycle exercise in men. 170 The glucose concentrations didn’t change significantly in our study, maybe due to different 171 individual response and insufficient dose or duration. The participants in this study, however, 172 were male physical education students rather than untrained participants. 173 Although we did not measure the muscle carnosine content, but according to the hypothesis, β- 174 alanine supplementation would prevent the drop in intracellular pH during high-intensity 175 contractions and results in less circulating acidosis finally due to elevation of myocellular 176 carnosine content. It has long been suggested that the acidosis limits muscle contractility 177 (Dennig et al., 1931). Furthermore, several studies have shown the pH regulation importance on 178 performance during endurance exercise, by a pre-exercise alkalosis intervention (Messonnier et 179 al., 2007). 180 This suggests that the difference between groups is related to the presumable enhancement of 181 muscle carnosine content (Harris et al. 2006; Baguet et al. 2009). 182 Future studies should examine muscle carnosine levels along with VO2max and plasma lactate 183 concentration during strenuous exercise with variable quantities of β-alanine supplementation. 8 184 Also, further investigations are necessary to determine the effects of β-alanine supplementation 185 during more prolonged and submaximal exercise. 186 187 Conclusion 188 Overall, it can be concluded from this study that β-alanine administration can reduce acidosis 189 during high-intensity exercise and thus can improve exercise performance in endurance athletes 190 and that 6 weeks of supplementation with β-alanine at the mentioned prescribed dose did not 191 result in significant changes in glucose concentrations. 192 Acknowledgments 193 This study was financially supported by grants from the “Esfahan Sport Medicine Association”. 194 There is not any conflict of interest for authors. The contribution of Mr. Mohammad 195 Khoshnevisan, Mr. Ehsan Bayat and Mrs. Fatemeh Shahryarzadeh is greatly acknowledged. 196 197 198 References Baguet A., Koppo K., Pottier A., Derave W. 2010. Beta-alanine supplementation reduces acidosis but not oxygen uptake response during high-intensity cycling exercise. Eur. J. Appl. Physiol. 108: 495-503 Baguet A., Reyngoudt H., Pottier A., Everaert I., Callens S., Achten E., Derave W. 2009. Carnosine loading and washout in human skeletal muscles. J. Appl. Physiol. 106:837–842 Bakardjiev A., Bauer K. 1994. Transport of beta-alanine and biosynthesis of carnosine by skeletal muscle cells in primary culture. Eur. J. Biochem. 225: 617–623 9 Batrukova M.A., Rubstov A.M. 1997. Histidine-containing dipeptides as endogenous regulators of the activity of sarcoplasmic reticulum Ca-release channels. Biochim. Biophys. Acta. 1324: 142–150 Begum G., Cunliffe A., Leveritt M. 2005. Physiological role of carnosine in contracting muscle. Int. J. Sport. Nutr. 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Comparison of exercise performance indices, pre- and post-supplementation (mean ±SD) β-alanine variables VO2max Placebo Pre Post Pre Post 2.62 ± 0.82 2.79 ± 0.73a, b 2.85 ± 0.67 2.81 ± 0.79 (L.min-1) TTE (sec) 923.6 ±237.5 992.4 ± 225.5 a, b 917.5 ± 243.8 926.5 ± 240.5 a Lactate (mg/dl) 15.5 ± 6.2 Glucose (mg/dl) 79 ± 14 202 203 204 205 206 207 208 a b 210 14.8 ± 7.1 36.0 ± 12.6 a, b 83 ± 11 84 ± 8 80 ± 14 Shows significant differences after exercise between two groups Shows significant differences after exercise within a group Table 2. Dietary intake of subjects for 2 days before intervention (Mean ± SD) Trial 209 27.9 ± 14.4a,b Energy (kcal) Protein (g) Carbohydrates (g) Fat (g) β-alanine 2,625 ± 820 105 ± 32 326 ± 185 105 ± 82 Placebo 99 ± 27 312 ± 154 102 ± 93 2,577 ± 625