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1 113 Journal of Exercise Physiologyonline August 2014 Volume 17 Number 4 Editor-in-Chief Official Research Journal of Tommy the American Boone, PhD, Society MBA of Review Exercise Board Physiologists Todd Astorino, PhD Julien Baker, ISSN PhD Steve Brock, PhD Lance Dalleck, PhD Eric Goulet, PhD Robert Gotshall, PhD Alexander Hutchison, PhD M. Knight-Maloney, PhD Len Kravitz, PhD James Laskin, PhD Yit Aun Lim, PhD Lonnie Lowery, PhD Derek Marks, PhD Cristine Mermier, PhD Robert Robergs, PhD Chantal Vella, PhD Dale Wagner, PhD Frank Wyatt, PhD Ben Zhou, PhD Official Research Journal of the American Society of Exercise Physiologists ISSN JEPonline Vitamin B6 and Maltodextrin Sport Drink Modify Glucose Levels of Elite Mountain Biking Athletes Irene Bronkhorst 1 ; Luiz Silva 1 ; Leandro Freitas 1, Marcos Martins 2, Hilana Martins 2, Carlos Malfatti 1, 1 Laboratory of Biomedical Sciences/Department of Physical Education/ Midwest State University, Guarapuava, Brazil, 2 Physical Education Post-Graduation Program/UFPR, Curitiba, Brazil ABSTRACT Bronkhorst I, Silva L, Freitas L, Martins M, Martins H, Malfatti C. Vitamin B6 and Maltodextrin Sport Drink Modify Glucose Levels of Elite Mountain Biking Athletes. JEPonline 2014;17(4): The aim of this study was to analyze the effects of vitamin B6 and/or maltodextrin in pre- and post-exercise conditions and changes in performance and blood glucose (BG) levels in maximal test performed in laboratory on elite mountain biking (MTB) athletes. Eight male bikers [age, 28.4 ± 10.6 yrs; body fat, 9.46 ± 3.76%; VO 2 max, ± 8.4 ml kg -1 min -1 ] received supplementation drinks composed of carbohydrate (CHO) maltodextrin (1 g kg -1 ) and/or vitamin B6 (30 mg kg -1 ) or a placebo (juice light with 3.5 g of maltodextrin) in 500 ml of water. The subjects performed a maximal cycling test on a cycle ergometer at 60 rev min -1, starting at 50 W that was increased by 25 W every 3 min until exhaustion. Blood glucose was measured at rest, post-supplementation, and post-exercise testing. The comparisons between variables were made using analysis of variance (ANOVA) with post hoc Bonferroni test. A P<0.05 was considered significant. The laboratory test indicated that the maltodextrin associated with vitamin B6 increased the blood glucose pre-exercise (CHO+B6: ± 20.6 vs. CHO: ± 23.9; vitamin B6: 88.3 ± 7.16, and placebo: ± 6.29 mg kg -1 ; P<0.01). CHO+vitamin B6 increased blood glucose during the cycle ergometer test, thus indicating that maltodextrin and vitamin B6 supplementation is a good strategy to increase availability of glucose fuel during exercise. Key Words: Cyclist, Performance, Blood Glucose, Carbohydrate

2 114 INTRODUCTION Vitamin B6 is a collective term for a group of three related compounds, pyridoxine, pyridoxal, pyridoxamine, and their phosphorylated derivatives (30). Pyridoxal 5 -phosphate (PLP) is an essential cofactor in all living systems and participates in catalysis by a wide diversity of enzymes including oxidoreductases, transferases, hydrolases, lyases, isomerases. decarboxylases, and cleavage enzymes used in transformations of amino acids (19,23). Pyridoxal 5 -phosphate is the catalytically active form of vitamin B6. It is well established in numerous reports (6,13,16,18,21,22) that plasma PLP concentrations increase during submaximal exercise. Pyridoxal 5 -phosphate is needed during exercise for gluconeogenesis and for glycogenolysis in which it serves as a cofactor for glycogen phosphorylase (21). In regards to skeletal muscle, the concentration of PLP can change during exercise due to need of carbohydrate consumption. Leklem and Hollenbeck (19) demonstrated that exercise of relatively long duration results in a significant increase in blood levels of PLP. They suggested that exercise is a form of acute energy deficit that initiates the release of vitamin B6 from muscle. Under this hypothesis the coenzyme would be needed in the liver for aminotransferase reactions, providing amino acid carbon skeletons for gluconeogenesis (17). Carbohydrate (CHO) supplementation pre-exercise, during exercise, and post-exercise has been shown to increase the amount of work that can be performed (4,8,32) as well as increase the duration of aerobic exercise (8,33). The increase in blood glucose (BG) associated with CHO supplementation is suggested to improve aerobic performance through reduction of muscle glycogen use (5,15,28,33) or through the use of BG as a predominant fuel source as glycogen becomes depleted (5,12,25). The type of CHO supplementation is very important. It has been suggested that because of the lower osmolalities, glucose polymer solutions (such as maltodextrin) are preferable to isocaloric glucose solutions as a source of ingested CHO before and during exercise (16). Indeed, several studies have shown that the rates of gastric emptying for glucose polymer solutions are faster than those of isocaloric glucose solutions (9,24). In addition, it has been suggested that glucose feedings (75 g of glucose solutions) 30 to 45 min before exercise in cyclists might impair exercise performance by causing a sudden drop in BG and an accompanying acceleration of muscle glycogen oxidation. Relatively little has been published concerning the effects of exercise on vitamin B6 metabolism and maltodextrin supplementation. The purpose of this study was to investigate the effect of consuming vitamin B6 and/or maltodextrin in pre-exercise and post-exercise conditions on exercise performance and blood glucose levels in athletes. METHODS Subjects Eight elite athletic mountain bikers participated in double-blind study. Resting anthropological and physiological characteristics of the subjects are presented in Table 1. An informed consent was obtained from each subject in accordance with Resolution 196/96 of the National Council of Health in Brazil, which was approved by local Ethics Committee.

3 115 Table 1. Resting Anthropological and Physiological Measures of the Subjects. Characteristics N = 8 Age (yrs) 28.4 ± 10.6 Body Fat (%) 9.46 ± 3.76 SBP (mmhg) ± 11 DBP (mmhg) 76.7 ± 10.3 HR (beats min -1 ) 75.3 ± 8 Fasting Glucose (mg dl -1 ) 82.6 ± 7 Maximal Power Output (W) ± 12.9 WVT (W) ± 8.7 Data are means ± SE. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; WVT, power output at ventilatory threshold; HR, heart rate Procedures Prior to the prescription of exercise and treatments (1 wk before), the subjects was evaluated in the laboratory to obtain resting physiological and anthropometrical measures. Body mass and height were measured using anthropometrical devices (Welmy Corporation, EUA). The subjects body fat was measured by means of the skinfold technique (14,29), using a skinfold caliper (Cescorf Corporation, EUA). Blood pressure was measured with a pressure device (Protec ). The subjects had previously fasted for 6 hrs before the measurements were taken. At 30 min prior to each test, the subjects received supplementation drinks composed of maltodextrin (1 g kg -1 ) and/or vitamin B6 (30 mg kg -1 ) or a placebo (juice light with 3.5 g of CHO) in 500 ml of water. The subjects were not to exercise 24 hrs prior to the laboratory tests. They were informed that the test consisted of a maximal cycling effort on a cycle ergometer at 60 rev min -1 at 50 W. The workload would increase 25 W every 3 min until exhaustion with each supplementation (cycle ergometer Biotec 1800-CEFISE ) to determined the influence, if any, on maximal heart rate (HR max), maximal power output (W max), maximal oxygen uptake (VO 2 max), and ventilatory threshold (VT). Ventilation threshold (VT) was determined as a plot of ventilation (V E ) versus oxygen consumption (VO 2 ), as described previously (27). Two linear regression lines were fit to the lower and upper portions of the V E versus VO 2 curve before and after the break points, respectively. The intersection of these two lines was defined as VT. Open circuit spirometry was used to analyze the gas exchange data using the Parvo-Medics TrueOne 2400 Metabolic Measurement System (Sandy, Utah, USA). Oxygen and carbon dioxide were analyzed through a sampling line after the gases passed through a heated pneumotach and mixing chamber. The data were averaged over 15-sec intervals. The highest average VO 2 value during the test was recorded as the VO 2 max if it coincided with at least two of the following criteria: (a) a plateau

4 in HR or HR values within 10% of the age-predicted HR max; (b) a plateau in VO 2 (defined by an increase of no more than 150 ml min -1 ); and/or (c) an respiratory exchange ratio of 1.15 or greater. Heart rate (HR) was monitored every 5 sec during the test (Polar Team System ). Exhaustion was defined as the point when the subject was no longer capable of maintaining the pedaling rate of 60 rev min -1. The highest VO 2 value obtained from the last minute of exercise was considered the VO 2 max (7). Capillary blood samples were used to determine the glucose concentrations using a digital glucosimeter (ACCU CHEK Performa, Roche ), and ACCU-CHEK Multiclix lancetador, with grade 5 on the distal phalange of the right hand 3rd finger at rest (basal), 20 min after maltodextrin, vitamin B6 or placebo use and immediately at the end of maximal cycle test. Statistical Analyses The data are presented as means ± SE. Statistical analysis included the use of a one-way analysis of variance (ANOVA) with statistical significance set at P<0.05 probability. Post-hoc analysis through the Bonferroni test was carried out when appropriate. 116 RESULTS Maltodextrin supplementation resulted in a significant increase in BG in the pre-test compared to control group (CHO: 110 ± 21.2 vs. B6: 88.3 ± 7.16 vs. Placebo: ± 6.29 mg dl -1, P<0.01), and the vitamin B6+maltodextrin supplementation resulted in a significant increase in BG in the pre-test compared to the maltodextrin group (CHO+B6: ± 20.6 vs. CHO: 110 ± 21.2, P<0.05). After the maximal test on the cycle ergometer (post-exercise), BG levels were significantly different between groups that received either the B6+maltodextrin supplement or maltodextrin compared to the vitamin B6 and placebo alone (CHO+B6: ± 26 vs. CHO: ± 20 vs. B6: 86.3 ± 8 vs. Placebo: 86.6 ± 6 mg dl -1, P<0.01) (Figure 1). There were no differences between vitamin B6 group alone versus the placebo group and the maltodextrin group alone versus the vitamin B6+maltodextrin group. Figure 1. Plasma Glucose Values during Testing, Before Supplementation (Rest), 20 Min after Supplementation (Pre-Test), and After a Maximal Test on a Cycle Ergometer (Post-Test) Using Maltodextrin (CHO) and/or Vitamin B6 or Placebo (data are mean ± SE; N = 8). Different letters (a, b, c) indicate statistical differences between the groups (P<0.05, one-way ANOVA with post hoc Bonferroni test).

5 During the cycle ergometer test, VT was significantly lower in the vitamin B6 group versus the CHO group and even more so in the vitamin B6 + CHO group versus the CHO and placebo groups (P<0.05). There were no significant differences in W max, HR at VT, and VO 2 across the groups (Table 2). 117 Table 2. Physiological Responses during Cycle Ergometer Test. Vitamin B6 (N = 8) CHO (N = 8) Vitamin B6 + CHO (N = 8) Placebo (N = 8) Maximal Power Output (W max) Ventilatory Threshold (W) Heart Rate at VT (beats min -1 ) VO 2 max (ml kg -1 min -1 ) 273 ± 30 a 280 ± 26 a 284 ± 15 a 270 ± 37 a 242 ± 37 a ± 36 b ± 18 a ± 26 b 91.9 ± 5 a 93.7 ± 2 a 94.5 ± 3 a 93.8 ± 2 a 59.5 ± 7 a ± 3 a 60.1 ± 6 a 58.3 ± 7 a Data are means ± SE. Different letters (a, b) indicate statistical differences between the groups. (P<0.05, one-way ANOVA with post hoc Bonferroni test). DISCUSSION Relatively little has been published regarding the effects of exercise on the metabolism of vitamin B6. Various studies (6,13,22) have reported that the plasma concentration of vitamin B6 increases during acute submaximal exercise. In addition, the increase in vitamin B6 can be seen in the urine excreted in trained adults with supplemental B6 following a 20-min cycle ergometer test (22). The present study indicates that there was a significant difference in blood glucose after 20 min of supplementation with maltodextrin and vitamin B6 in relation with the placebo group. The average variation in glucose levels with supplementation was 40 mg dl -1 (52%). While the change in vitamin B6 metabolism and the increase concentrations during exercise have not been thoroughly explained in relation to acute exercise, Leklem and Shultz (18) indicate that the change occurs as a function of the increased need for fuel during exercise (13). The increase is essential to meet the metabolic costs of exercise, and the high glucose concentration aids the synthesis of muscle glycogen. Thus, by avoiding a decrease in glycogen concentrations, fatigue during competition may be delayed (7). Black et al. (3) reported that vitamin B6 activates the enzyme glycogen synthase in skeletal muscle. Leklem and Shultz (18) indicate that exercise stimulates the release and activation of the enzyme glycogen phosphorylase pyridoxal phosphate. Thus, the increase in plasma pyridoxal phosphate seen with exercise may be due to this stimulation of muscle reserves. The release of pyridoxal phosphate during exercise is probably related to gluconeogenesis in the liver (1).

6 During exercise, the body depends on the production of glucose from the liver to maintain normal glucose levels in the plasma (20). Therefore, supplementation seems to contribute to more vitamin B6 storage in the muscle and its subsequent release during exercise (22). Note in Figure 1, the plasma glucose levels are higher for maltodextrin and maltodextrin supplementation associated with vitamin B6 compared to the placebo and vitamin B6 alone (21 mg dl -1 ; 24%; P<0.05). Okada et al. (26) showed a significant decrease in plasma glucose in rats with a deficiency of vitamin B6 when compared to rats with normal concentrations of the vitamin. The findings indicate that following the vitamin B6 supplementation, there was a significant increase in BG without exercise to the ingestion of maltodextrin more vitamin B6 in relation to maltodextrin alone group. As expected, supplementation produced higher concentrations of BG post maltodextrin associated with vitamin B6 supplementation in this study. The results demonstrate that may occurred an increase in glycolysis and glycogenolysis in liver by supplementation with vitamin B 6 together with maltodextrin, better than maltodextrin alone. In Manore et al. (22), the young trained individuals excreted more pyridoxic acid than the two untrained groups, for both the no supplemented and supplemented diets with the vitamin. The VT is related to the production of lactate during exercise. The higher the VT the more fatigue the athlete is likely to experience. The nonlinear increase in expired ventilation (V E ) during exercise, in general, is associated with an increase in plasma concentration of carbon dioxide (CO 2 ), which is due to the buffering of lactic acid (10). The findings in the present study support the association between lactic acid and CO 2, where VT was significantly lower for the groups that ingested vitamin B6, compared to other groups (refer to Table 2). Hence, the ergogenic aid of PLP and its role in increasing energy intake during exercise may be an effective way to combat fatigue stress. Although severe vitamin B6 deficiency impairs gluconeogenesis, possibly via decreased flux through aminotransferases (2), there is no evidence that aminotnansferase activity is impaired during exercise in vitamin B6 deficiency subjects. Oxidation of amino acids during exercise requires initial PLPdependent aminotransferase activity. However, this does not mean that exercise induces deficiency of PLP in muscle that must be offset by transfer of PLP into muscle from some other site in the body (6,11). 118 CONCLUSIONS Maltodextrin and association with vitamin B6 enhanced levels of BG during the cycle ergometer test. This shows that maltodextrin and vitamin B6 supplementation are likely to be a good combination and strategy to increase the availability of glucose as a fuel substrate during exercise. While the results are encouraging, it is important to carry out more studies and, in particular, with different doses of maltodextrin and vitamin B6 to further increase our understanding of the relationship between the two and glycogen levels related to athletic performance. Address for correspondence: Malfatti CRM, Dr, Department of Physical Education, Midwest State University, Guarapuava, Parana, Brazil, , Phone: +55(42) ; . crmalfatti@gmail.com

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