Betalain-Rich Concentrate Supplementation Improves Exercise Performance in Competitive Runners

This study aimed to determine the effects of a betalain-rich concentrate (BRC) of red beets, containing antioxidant and anti-in ammatory properties, on performance and exercise-related muscle damage. Thirteen (25.3 5.4 years) competitive male runners completed two double-blind, cross-over, randomized trials (BRC and control) separated by seven days. Each trial was preceded by six days of supplementation with 100 mg of BRC or control. On the seventh day, exercise trials commenced 150 min after supplementation with 50 mg BRC or control and consisted of 30 min of treadmill running (77 4% VO2max) followed by a 5-km time trial (TT). During exercise at the same intensity, BRC resulted in a 3% lower heart rate, a 15% lower rate of perceived exertion (RPE) and a 14% lower blood lactate concentration compared to the control (p= 0.05). Five-kilometer TT duration (23.0 4.2 versus 23.6 4.0 min) was faster in 10 of the 13 subjects, and RPE was lower (p< 0.05) with the BRC treatment compared to the control. Lactate dehydrogenase, a marker of muscle damage, increased less from baseline to immediately and 30 min after the 5-km TT with the BRC treatment, despite no differences in subjective measures of muscle soreness and fatigue. In summary, BRC supplementation improved 5-km performance time in male competitive runners. Keywords:beetroot; time trial; muscle damage markers; rate of perceived exertion 1. Introduction Beetroot has received much attention from competitive athletes, coaches and scientists as a natural food supplement for improving exercise performance [1,2].

with the BRC treatment, despite no differences in subjective measures of muscle soreness and fatigue. In summary, BRC supplementation improved 5-km performance time in male competitive runners. Keywords:beetroot; time trial; muscle damage markers; rate of perceived exertion 1. Introduction Beetroot has received much attention from competitive athletes, coaches and scientists as a natural food supplement for improving exercise performance [1,2]. Several studies have found a correlation between nitrate-rich beetroot juice (5–9 mmol of nitrate or about 85 g of beets) and 5–16.1-km time-trial performances in competitive runners and cyclists [3–6]. The nitrate in the beetroot juice was thought to increase the production of nitric oxide through the nitrate-nitrite-nitric oxide reduction pathway induced by bacterial nitrate reductases in the oral cavity [1,2]. Higher nitric oxide could improve muscle blood ow and oxygenation and, thus, exercise time trial performance. Beetroot also contains a high concentration of betalains, highly bioactive phytochemicals [7] lauded for their free radical scavenging capacity [8] and protecting cell membranes from lipid peroxidation and heme decomposition [9]. This property is likely due to the phenol and cyclic amine groups in betalains, which are good electron and proton donors [9]. The ability of betalains to neutralize superoxide radicals may also lead to an increase of nitric oxide availability in the blood and subsequently increase blood ow and oxygen delivery. Betalains have also been shown Sports2016,4, 40; doi:10.3390/sports4030040

Sports2016,4, 40 2 of 9 to express potent anti-in ammatory properties by reducing the pro-in ammatory cytokines tumor necrosis factor alpha (TNF- ) and interleukin-6 in patients with osteoarthritis [10]. Since intense exercise has been shown to increase muscle damage, in ammation and oxidative stress [11], betalain supplementation may be able to reduce that oxidative stress and in ammation and result in improved exercise performance. Although data supports beetroot juice intake and improved exercise performance, research investigating the health and exercise performance bene ts of betalains is limited. To our knowledge, no study has investigated the effects of a nitrate- and sugar-depleted, betalain-rich concentrate (BRC) on exercise performance. Therefore, the primary aim of this study was to examine the effects of BRC on 5-km time trial time in competitive, male runners, while secondary outcomes included assessing BRC effects on muscle damage, muscle soreness and overall fatigue. We hypothesized that BRC supplementation would improve exercise performance by decreasing muscle damage and subjective measures of muscle soreness and fatigue compared to the control. 2. Materials and Methods 2.1. Subjects We recruited 15 recreationally-competitive male runners (25.3 5.4 years; 173.5 4.9 cm; 70.6 7.9 kg ) from the University of California at Davis campus and local venues to participate in the study. Twelve subjects were needed based on a power analysis [12] (power = 0.8, signi cance p= 0.05, mean difference (MD) = 1.0 min for performance time of supplement versus water and SD of the MD = 1.1 min) [13]. Two subjects were excluded due to noncompliance, leaving 13 subjects for data analysis. Participants had to be healthy, nonsmokers and run more than 8 miles per week. Written informed consent was obtained as approved by the Institutional Review Board of the University of California at Davis. 2.2. Screening and Baseline Measures Day 0 consisted of medical-clearance and measurements of height in cm using a stadiometer, body mass in kg using a scale and body composition via 7-site skinfolds using a Harpenden caliper [14]. Subjects completed a maximal treadmill test (Stairmaster Clubtrack at 1% slope) to determine work intensities for the experimental trials.

of California at Davis. 2.2. Screening and Baseline Measures Day 0 consisted of medical-clearance and measurements of height in cm using a stadiometer, body mass in kg using a scale and body composition via 7-site skinfolds using a Harpenden caliper [14]. Subjects completed a maximal treadmill test (Stairmaster Clubtrack at 1% slope) to determine work intensities for the experimental trials. Every 2 min of the test, oxygen consumption (VO2max; TrueOne 2400, ParvoMedics, Sandy, UT, USA), heart rate (HR) in beats per minute (Model 5410, Polar, Woodbury, NY, USA) and rate of perceived exertion (RPE) (0–10-point scale) [15] were measured. Exercise began at 8–11 kph and increased by 0.8 kph every 2 min to exhaustion. The metabolic cart was calibrated prior to each trial at various ow rates (50–400 L/min) and with a standard gas mixture of 16% O2and 4% CO2. 2.3. Experimental Trials Through a double-blind, randomized and counterbalanced method, subjects were assigned BRC (Racerunner ® , FutureCeuticals, Momence, IL: serving size: 1 capsule (50 mg beetroot concentrate), 5 kcal, 0.1 mg protein, 1 mg carbohydrate, 0 mg fat, 0.3 mg ber and 12.5 mg betalains) or control (serving size: 1 capsule, oat ß-glucans, Nutrim ® , FutureCeuticals, Momence, IL: 19 kcal, 1 mg protein, 3 mg carbohydrate, 0.4 mg fat and 0.9 mg ber). Oat ß-glucans were used as a control to match as close as possible all contents other than the betalains in the experimental treatment. Treatments were supplied inside a blue and white pill capsule and were identical in appearance, taste and smell. Six subjects started with the betalain treatment, and seven started with the control treatment. Prior to each trial, subjects supplemented with 50 mg of treatment, twice per day (30 min before breakfast and 30 min before dinner) for 6 days, recorded 7 days of training (training log; type, duration, intensity and miles of training) and logged 3 days of their diet (Day 5–7) (MyFitnessPal, Inc., San Francisco, CA, USA). We chose 50 mg, as that was shown to be the minimal effective dose in a

min before dinner) for 6 days, recorded 7 days of training (training log; type, duration, intensity and miles of training) and logged 3 days of their diet (Day 5–7) (MyFitnessPal, Inc., San Francisco, CA, USA). We chose 50 mg, as that was shown to be the minimal effective dose in a

Sports2016,4, 40 3 of 9 study byPietrzkowski et al., 2010 [10] . Diet and exercise were followed exactly prior to the second trial. Subjects reported to the lab between 8:30 and 10:00 a.m. in a fasted state. Baseline muscle soreness and fatigue were recorded with 100-mm visual analogue scales (VAS) from “no pain” to “extreme pain” and from “not tired” to “utterly exhausted” [13]. Baseline blood was sampled via a 22-gauge forearm vein catheter, and VO2, the respiratory exchange ratio (RER) and HR were measured. Following baseline measurements, 50 mg of BRC or control were given with 7 mL/kg of water. Subjects rested for 140 min before commencing exercise to allow BRC to reach peak concentrations in the blood [7]. Thirty minutes post-supplementation, subjects consumed a snack (Smuckers' Uncrustables ® , Strawberry, 210 kcal: 56% CHO, 22% fat, and 22% protein; The T.M. Smucker Company, Orrville, OH, USA) with 3 mL/kg of water to prevent hypoglycemia and to simulate pre-training/competition behavior. At 140 min, subjects completed a 10-min warm up, voided their bladder and had body mass measured. 2.4. Submaximal Exercise Treadmill exercise commenced 150 min post-supplementation. Speed was adjusted to elicit 75% of VO2max during the rst 15 min. After 15 min, subjects straddled the treadmill while blood was drawn; 3 mL/kg of water were consumed, and they were connected to the metabolic cart. HR and RPE were averaged over the last 10 min of exercise when the steady state had been reached. A blood sample was collected immediately after the 30-min submaximal exercise period, and 3 mL/kg of water were provided. Blood values were reported as an average of the 15- and 30-min time points. The same workload and rest periods were used for the second trial. 2.5. Time Trial Following the submaximal exercise bout, subjects completed a 5-km time trial (TT), where the subjects controlled their speed and had access to their distance, but were blinded to their actual speed and HR. Elapsed time, RPE and HR were recorded every 1.67 km and then reported as an average over the entire 5-km time period. Post TT, blood

Time Trial Following the submaximal exercise bout, subjects completed a 5-km time trial (TT), where the subjects controlled their speed and had access to their distance, but were blinded to their actual speed and HR. Elapsed time, RPE and HR were recorded every 1.67 km and then reported as an average over the entire 5-km time period. Post TT, blood was immediately collected, and 3 mL/kg of water were ingested while subjects completed 5 min of active recovery at 4.8 kph. 2.6. Post-Exercise Lastly, 30 min after the 5-km TT, VO2, RER and HR and body mass were recorded; a blood sample was collected, and VAS scales were completed. Before leaving, subjects ingested 50 mg of supplement with 3 mL/kg of water to maximize BRC's effects on recovery [7]. Twenty four hours post-exercise, VAS scales were recorded followed by a 24-h blood draw. 2.7. Blood Analysis Standard serum biochemistry analyses for lactate dehydrogenase (LDH), creatine kinase (CK) and glucose were performed on collected blood utilizing a Piccolo Xpress Chemistry Analyzer (Abaxis, Union City, CA, USA). Blood lactate was determined with the Lactate Plus analyzer (Nova Biomedical, Waltham, MA, USA), and hematocrit was measured using microhematocrit tubes (Statspin, Norwood, MA, USA). Serum samples were stored at 80 C prior to analysis. 2.8. Statistical Analysis Data are presented as the means standard deviation (SD). The normality of the distribution for each variable was tested using the Shapiro–Wilk test. All variables except time to complete the 5-km TT were normally distributed (p> 0.05). Pairedt-tests were used for baseline, submaximal exercise and time trial comparisons of HR, VO2, RER, blood lactate, serum glucose, serum LDH and CK, as well as baseline measures of whole body muscle soreness and fatigue. Paired tests were also used for change values for LDH (Figure) and CK. The time trial data were not normally distributed, and a nonparametric analysis was used with the Wilcoxon signed rank test (StatView software, Version 5.0.1, SAS Institute Inc., Cary, NC, USA). Signi cance was accepted atp¤0.05.

for change values for LDH (Figure) and CK. The time trial data were not normally distributed, and a nonparametric analysis was used with the Wilcoxon signed rank test (StatView software, Version 5.0.1, SAS Institute Inc., Cary, NC, USA). Signi cance was accepted atp¤0.05.

Sports2016,4, 40 4 of 9 3. Results 3.1. Subjects Physical characteristics of the subjects are presented in Table. The average daily amount of calories consumed and macronutrient proportions from the 3-day diet records were 2388 617 kcal, 45 8% carbohydrate, 21 5% fat and 34 7% protein for BRC and 2361 608 kcal, 44 7% carbohydrate, 21 5% fat and 35 6% protein for control. Weekly training volumes were identical between treatments at 24.8 10.2 miles, 6.2 3.4 h and at an average RPE of 5.4 1.6. Table 1.Subject physical characteristics;n= 13 men; VO 2, oxygen consumption. Variable mean SD Age, y 25.3 5.4 Height, cm 173.5 4.9 Weight, kg 70.6 7.9 Body fat, % 9.6 2.3 Fat-free mass, kg 63.7 6.6 Fat mass, kg 6.8 2.1 VO 2max, mL kg 1 min 1 55.6 3.7 Training hours per week 6.0 3.3 Running km per week 39.8 3.3 3.2. Baseline Measures Baseline HR, VO2and RER were similar between treatments. There were no treatment differences in baseline blood lactate, serum glucose serum CK and whole body muscle soreness and fatigue. Baseline LDH was higher with the BRC treatment (144.6 26.7 versus 136.0 16.8 U/L;p= 0.04). 3.3. Physiological Responses to Submaximal Exercise Submaximal exercise values are reported in Table. Speed and % VO 2max were similar between treatments. We found no signi cant differences in VO2or RER; however, HR was 2.7 0.8%, and RPE was 14.8 1.3% lower with BRC. Six subjects had a lower average HR with Trial 1 (two control and four BRC), and seven had a lower average HR for Trial 2 (two control and ve BRC). Five subjects had a lower average RPE with Trial 1 (two control and three BRC), and eight had a lower average RPE for Trial 2 (two control and six BRC). Table 2. Physiological responses with the 30-min submaximal exercise bout;n= 13 men; VO 2, oxygen consumption; LDH, lactate dehydrogenase. * Signi cantly different from the control. BRC, betalain-rich concentrate. Variable BRC Control p-Value Average speed, kph 12.2 0.7 12.2 0.7 1.0 Heart rate, bpm 165.0 11.2 * 169.5 10.7 0.04 VO 2,

for Trial 2 (two control and six BRC). Table 2. Physiological responses with the 30-min submaximal exercise bout;n= 13 men; VO 2, oxygen consumption; LDH, lactate dehydrogenase. * Signi cantly different from the control. BRC, betalain-rich concentrate. Variable BRC Control p-Value Average speed, kph 12.2 0.7 12.2 0.7 1.0 Heart rate, bpm 165.0 11.2 * 169.5 10.7 0.04 VO 2, L min 1 3.01 0.39 3.01 0.33 0.99 % VO 2max 76.9 4.4 77.0 3.7 0.88 Respiratory exchange ratio 0.92 0.04 0.93 0.04 0.09 % energy from carbohydrate 71.1 15.3 76.6 13.6 0.09 % energy from fat 28.9 15.3 23.4 13.6 0.09 Rate of perceived exertion 3.79 1.47 * 4.35 1.28 0.04 Blood lactate, mmol L 1 2.9 1.6 * 3.3 1.4 0.05 Serum glucose, mmol L 1 5.0 0.4 5.0 0.5 0.70 Serum creatine kinase, U L 1 379.9 242.4 369.3 225.7 0.75 Serum LDH, U L 1 173.6 23.5 169.2 27.2 0.49

Sports2016,4, 40 5 of 9 There were no differences in serum glucose, lactate dehydrogenase or creatine kinase between treatments, but blood lactate was 13.8 1.6% lower with BRC treatment compared to the control with the submaximal exercise bout (Table). Four subjects had a lower average lactate with Trial 1 (one control and three BRC), and nine had lower average lactate for Trial 2 (three control and six BRC). 3.4. Time Trial Data from the 5-km TT are presented in Table. BRC supplementation was associated with a 3.0 1.9% increase in 5-km speed and a 6.8 1.0% decrease in RPE. There was also a 36-s reduction in 5-km TT time with BRC supplementation. Ten of the 13 subjects had improved TT times with BRC compared to the control. Eight subjects had a faster 5-km TT for their second trial (three with the control and ve with BRC), and ve subjects had a faster TT for Trial 1 (one control and four BRC). Table 3. Physiological responses with the 5-km time trial (TT);n= 13 men. LDH, lactate dehydrogenase; * signi cantly different from the control. BRC, betalain-rich concentrate. Variable BRC Control p-Value Average speed, kph 13.3 1.9 * 12.9 1.8 0.04 Time to complete the TT, min 23.0 4.2 * 23.6 4.0 0.04 Average heart rate, bpm 176.0 14.5 178.3 13.3 0.31 Rate of perceived exertion 5.9 1.1 * 6.3 1.0 0.03 Blood lactate, mmol L 1 6.7 4.1 6.4 3.2 0.36 Serum glucose, mmol L 1 5.6 1.6 5.5 1.1 0.59 Serum creatine kinase, U L 1 427.3 267.3 424.7 250.7 0.94 Serum LDH, U L 1 187.2 30.4 189.2 32.5 0.67 The changes in LDH from baseline to after the 5-km TT were 25.1 1.2% (p= 0.001) lower with BRC treatment (Figure). There was no difference in the change in serum CK, whole body muscle soreness and whole body fatigue from baseline to after the 5-km TT. Hematocrit increased more from baseline to immediately post the 5-km TT with BRC treatment (2.9 1.3%) compared to the control (1.3 2.1% ) (p= 0.004). However, subjects were well hydrated during the trials,

treatment (Figure). There was no difference in the change in serum CK, whole body muscle soreness and whole body fatigue from baseline to after the 5-km TT. Hematocrit increased more from baseline to immediately post the 5-km TT with BRC treatment (2.9 1.3%) compared to the control (1.3 2.1% ) (p= 0.004). However, subjects were well hydrated during the trials, as body weight changes were minimal and not different between treatments ( 0.76 0.3 kg and 0.73 0.3 kg for BRC and the control respectively;p= 0.75).Sports 2016, 4, 40 5 of 9 There were no differences in serum glucose, lactate dehydrogenase or creatine kinase between treatments, but blood lactate was 13.8 ± 1.6% lower with BRC treatment compared to the control with the submaximal exercise bout (Table 2). Four subjects had a lower average lactate with Trial 1 (one control and three BRC), and nine had lower average lactate for Trial 2 (three control and six BRC). 3.4. Time Trial Data from the 5-km TT are presented in Table 3. BRC supplementation was associated with a 3.0 ± 1.9% increase in 5-km speed and a 6.8 ± 1.0% decrease in RPE. There was also a 36-s reduction in 5-km TT time with BRC supplementation. Ten of the 13 subjects had improved TT times with BRC compared to the control. Eight subjects had a faster 5-km TT for their second trial (three with the control and five with BRC), and five subjects had a faster TT for Trial 1 (one control and four BRC). Table 3. Physiological responses with the 5-km time trial (TT); n = 13 men. LDH, lactate dehydrogenase; * significantly different from the control. BRC, betalain-rich concentrate. Variable BRC Control p-Value Average speed, kph 13.3 ± 1.9 * 12.9 ± 1.8 0.04 Time to complete the TT, min 23.0 ± 4.2 * 23.6 ± 4.0 0.04 Average heart rate, bpm 176.0 ± 14.5 178.3 ± 13.3 0.31 Rate of perceived exertion 5.9 ± 1.1 * 6.3 ± 1.0 0.03 Blood lactate, mmol·L −1 6.7 ± 4.1 6.4 ± 3.2 0.36 Serum glucose, mmol·L −1 5.6 ± 1.6 5.5

BRC supplementation improved 5-km performance time in male competitive runners.