Influence of High vs. Low Carbohydrate Ingestion on Substrate Oxidation Patterns of Males and Females During Running Bouts at the Individual Anaerobic Threshold

Background: To date, it remains unclear how pre-exercise CHO availability modulates the oxidative regulation of substrates when exercise is conducted at the intensity (V IAT) where the Individual Anaerobic Threshold (IAT) is located. This study aimed in assessing the impact of High CHO (HC) vs. Low CHO (LC) diets (where on the LC day a combination of low CHO diet and a glycogen depleting exercise was implemented) on the oxidative regulation of CHOs and lipids while exercise is conducted at V IAT. Methods: 16 recreational runners (m=8; f=8; 28 ± 3 y; 1.76 ± 0.09 m; 72 ± 13 kg; 23 ± 2 kg/m 2 ) performed 3 different running protocols, each allocated on a different day. At day 1, a maximal stepwise incremental test was implemented to assess the IAT and V IAT. During days 2 and 3, participants ran a constant-pace bout (30 min) at V IAT that was combined with randomly assigned HC (7g/kg/d) or LC (3g/kg/d) diets for the 24 h before testing. Breath-by-breath gas exchange data was measured continuously and used to determine substrate oxidation. Dietary data and differences in substrate oxidation were analyzed with a paired t-test. A two-way ANOVA tested the diet X gender interaction (α = 0.05). Results: Overall, the IAT and V IAT were 2.74 ± 0.39 mmol/l and 11.1 ± 1.4 km/h, respectively. CHO oxidation was 3.45 ± 0.08 and 2.90 ± 0.07 g/min during HC and LC bouts respectively (P < 0.05). Likewise, lipid oxidation was 0.13 ± 0.03 and 0.36 ± 0.03 g/min

ANOVA tested the diet X gender interaction (α = 0.05). Results: Overall, the IAT and V IAT were 2.74 ± 0.39 mmol/l and 11.1 ± 1.4 km/h, respectively. CHO oxidation was 3.45 ± 0.08 and 2.90 ± 0.07 g/min during HC and LC bouts respectively (P < 0.05). Likewise, lipid oxidation was 0.13 ± 0.03 and 0.36 ± 0.03 g/min (P < 0.05). Females had 14% (P < 0.05) and 12% (P > 0.05) greater lipid oxidation compared to males during HC and LC bouts, respectively. Conclusion: Twenty-four hours of high CHO consumption results in concurrent higher CHO oxidation rates and overall utilization, whereas maintaining a low systemic CHO availability significantly increases the contribution of lipids to the overall energy metabolism. The observed gender differences underline the necessity of individualized dietary planning before exerting at intensities associated with performance exercise. Ultimately, it remains to be established how these findings can be extrapolated to training and competitive situations and with that provide trainers and nutritionists with improved data to derive training prescriptions. *Corresponding author: Raul De Souza Silveira, University of Potsdam Outpatient Clinic, Center of Sports Medicine, Potsdam University, Am Neuen Palais 10, Haus 12, 14469 Potsdam, Germany, Tel: +49 03319771439; E-mail: desouzas@uni-potsdam.de; raul.desouzasilveira@ph-gmuend.de Citation: Silveira RDS, Kopinski S, Mayer F, Carlsohn A (2016) Influence of High vs. Low Carbo- hydrate Ingestion on Substrate Oxidation Patterns of Males and Females During Running Bouts at the Individual Anaerobic Threshold. FoodNutrJ1:102.DOI:10.29011/2575-7091.100002 Received: 30 March, 2016; Accepted: 20 May, 2016; Published: 3 June, 2016

Volume 1; Issue 01 2 Citation: Silveira RDS, Kopinski S, Mayer F, Carlsohn A (2016) Influence of High vs. Low Carbohydrate Ingestion on Substrate Oxidation Patterns of Males and Females During Running Bouts at the Individual Anaerobic Threshold. FoodNutrJ1:102.DOI:10.29011/2575-7091.100002 Keywords Carbohydrate intake; Individual anaerobic threshold; Running; Substrate oxidation Abbreviations CHO : Carbohydrate VO2max : Maximal oxygen uptake IAT : Individual Anaerobic Threshold VIAT : Individual Anaerobic Threshold’s intensity HC : High Carbohydrate LC : Low Carbohydrate BMI : Body Max Index %BF : Percentage Body Fat VO2peak : Peak Oxygen uptake HRmax : Maximal Heart Rate VO2 : Oxygen uptake VCO2 : Carbon dioxide output RER : Respiratory Exchange Ratio Med : Medical check Glyc depl : Glycogen depleting bout Introduction Carbohydrate (CHO) and lipids are the main substrates fueling exercise, each having its oxidation patterns regulated by several factors such as intensity and duration of the activity, dietary intake pattern, gender and training status [1-5]. When described as a sole function of exercise intensity, the oxidative metabolism of these two substrates has a clear pattern. At low and moderate intensities, lipid (intramyocellular lipids and plasma free fatty-acids) is the main substrate being oxidized while CHO metabolism (blood glucose and stored muscle glycogen) increases parallel to exercise intensity and predominates at times of high physical exertion [6-8]. Steady-state exercise on the other hand (i.e., an exercise level that can be maintained for a prolonged period of time), normally favors lipid oxidation [8]. Based upon these regulatory mechanisms and depending on individual goals, professional and recreational athletes may be advised to vary their training regimen around different intensities (using and conditioning both aerobic and anaerobic energetic pathways), while aiming to expand endurance capacity, power and performance [9]. Likewise, nutrition has the potential to alter the metabolic regulation of substrates with the intake of CHOs in particular, being not only crucial to fuel exercise at intensities above 65% of maximal oxygen uptake (VO2max), but also directly assisting in the post-exercise recovery phase [3,10]. For instance, CHO-loading strategies (7-10 g/kg/d) may increase not only glycogen storage (up to 42% post-prandial) but also its

potential to alter the metabolic regulation of substrates with the intake of CHOs in particular, being not only crucial to fuel exercise at intensities above 65% of maximal oxygen uptake (VO2max), but also directly assisting in the post-exercise recovery phase [3,10]. For instance, CHO-loading strategies (7-10 g/kg/d) may increase not only glycogen storage (up to 42% post-prandial) but also its overall usage, which in turn delays fatigue allowing exercise to be prolonged and endurance performance to be improved [3,11-14]. Still, this latter mechanism is somewhat restricted to the male athletic population as females are well known for having a greater reliance on lipid metabolism compared to males [15]. In addition, female athletes have had mixed results when it comes to increasing muscle glycogen storage capacity and/or enhancing endurance exercise performance (i.e., despite CHO-loading equivalent to ~75% of the energy intake during 4-6 days) [7,13,16,17]. Yet, it remains unclear how pre-exercise CHO intake modulates the oxidative regulation of CHOs and lipids, when exercise is conducted at the intensity where the Individual Anaerobic Threshold (IAT) is located (VIAT). Namely, a metabolic marker delineating the upper levels of endurance capacity in which a shift in the oxidative regulation of substrates is expected favoring a CHO driven metabolism [18-20]. The IAT represents the upper border where constant load endurance exercise can be sustained, being commonly used to guide athletic training (e.g., when aiming to improve endurance capacity) or in performance diagnostics [19-22]. Exertion at VIAT can be generally sustained for up to 60 minutes, though the average speed of a marathon is only slightly under it [18,19]. Consequently, in order to assist coaches, trainers and nutritionists in their pre-exercise nutritional plans, it is necessary to investigate and understand how pre-exercise nutrition (especially CHO intake) affects the metabolic regulation of substrates as individuals exercise in accordance to such specific biomarkers of performance and exercise capacity [23,24]. Thus, the aim of the present investigation was to assess the impact of High CHO (HC) vs. Low CHO (LC) diets on the oxidative regulation of CHOs and lipids while moderately endurance-trained males and females run at VIAT. Methods

affects the metabolic regulation of substrates as individuals exercise in accordance to such specific biomarkers of performance and exercise capacity [23,24]. Thus, the aim of the present investigation was to assess the impact of High CHO (HC) vs. Low CHO (LC) diets on the oxidative regulation of CHOs and lipids while moderately endurance-trained males and females run at VIAT. Methods Subjects Sixteen healthy recreational runners (8 males/8 females) voluntarily took part in this investigational study. The ethics committee of the University of Potsdam approved the study and participants gave their written informed consent after receiving detailed information on the investigational protocol and study aims. To increase the cohort’s homogeneity in regards to physical conditioning, subjects, were only included if weekly training was ≥3 hours. Anthropometric characteris- tics are provided in table 1. General design All examinations were conducted at the Outpatient Clinic from Potsdam University. A full medical check (anamnesis, Overall (n=16)Males (n=8)Females (n=8)P values Age (yrs.) 28 ± 3 30 ± 3 26 ± 2 0.005 Height (m) 1.76 ± 0.091.83 ± 0.081.70 ± 0.03 0.001 Weight (kg) 72 ± 13 83 ± 8 61 ± 5 0.000 BMI (kg/m 2 ) 23 ± 2 24.9 ± 1.1 21.2 ± 1.3 0.000 %BF 14.7 ± 3.3 14.1 ± 3.5 15.3 ± 3.0 0.510 Table 1: Anthropometric data of subjects. BMI - Body mass index; %BF - Percentage Body Fat (determined from skin folds); All values are mean±SD; P values reflect gender comparisons only

3 Volume 1; Issue 01 Citation: Silveira RDS, Kopinski S, Mayer F, Carlsohn A (2016) Influence of High vs. Low Carbohydrate Ingestion on Substrate Oxidation Patterns of Males and Females During Running Bouts at the Individual Anaerobic Threshold. FoodNutrJ1:102.DOI:10.29011/2575-7091.100002 anthropometrics, physical examination, resting ECG) was carried out preceding the first exercise appointment as recommended by the German Federation for Cardiovascu- lar Prevention and Rehabilitation [25]. At day 1, participants performed a baseline running test in which the IAT [26], VIAT, peak oxygen uptake (VO2peak) and maximal Heart Rate (HRmax) (RS 400, ©Polar Electro, Finland) were determined. On days 2 and 3, a submaximal running test at VIAT was carried out on the same treadmill ergometer (H/P/Cosmos Pulsar Graphics 2005®, Germany). A breath-by-breath Metamax 3B system (Cortex Biophysik GmbH. Leipzig, Germany) was used to monitor respiratory data and to determine CHO and lipid oxidation rates via indirect calorimetry (detailed below). For the 2 submaximal runs, HC (7g/kg/d) and LC (3g/kg/d) dietary protocols were prescribed for the 24 hours preceding each test (detailed below). As part of the LC protocol, a glycogen-de- pleting running bout was additionally performed (60 min at 75% HRmax in the evening, 12 h prior to the actual submaximal running bout; figure 1 depicts a flowchart of the investigational design). Participants were additionally advised to refrain from any other exercise practices during the 48 hours preceding each submaximal bout. Experimental design Baseline test: Subjects performed a stepwise incremental test until volitional exhaustion. The initial stage (6 km/h), stage increment (2 km/h) and stage duration (3 min) were defined to exhaust subjects in not less than 4 stages [27]. Lactate concen- trations were measured in between stages from capillary blood samples taken from the hyperemized earlobe (Biosen S line, EKF diagnostic GmbH, Magdeburg, Germany). Submaximal runs: Forty-eight hours after the baseline test, subjects performed the first submaximal run. This bout was composed of a 30 minutes, constant-pace endurance run at VIAT. The second submaximal bout was then carried out 7 days later at the same time for each participant (07:15, 8:00 or 8:45 am). Before commencing the tests, a

EKF diagnostic GmbH, Magdeburg, Germany). Submaximal runs: Forty-eight hours after the baseline test, subjects performed the first submaximal run. This bout was composed of a 30 minutes, constant-pace endurance run at VIAT. The second submaximal bout was then carried out 7 days later at the same time for each participant (07:15, 8:00 or 8:45 am). Before commencing the tests, a 3 min run at 80% VIAT served as a warm up not only so subjects could adapt to the forthcoming brisk exercise pace, but also to stabilize cardio- pulmonary parameters and reduce possible breathing artifacts that may arise at the beginning of exercise testing [28]. Nutritional intervention & managing CHO availability: The HC and LC dietary protocols were randomly assigned for the 24 hours preceding each submaximal run. This one day nutritional intervention has its caloric content calculated for each individual based on the basal metabolic rate and the World Health Organization’s PAL-Score [29,30]. Dietary protocols were only prescribed with no food being supplied throughout the investigation. Therefore for compliance control, food intake was documented in a standardized diet record form [31] and analyzed later on. Nutrient and energetic values, including possible deviations from the prescribed protocols were computed based on the German Nutrition database (PRODI 5.7, Nutri-Science GmbH, Hausach, Germany). The dietary plan was designed for breakfast, lunch and dinner (plus in between snacks), and consisted of foods typically eaten in Germany. The plan was standardized with no caffeine (with the exception of a standardized morning coffee) alcohol or supplements included, and individually adapted to body mass to achieve CHO aims. As part of the LC protocol, an exercise bout with duration and intensity proven to deplete glycogen stores was implemented [32,33]. This bout combined to the LC diet (which subsequently avoids glycogen recovery or super compensation) [34,35], would then create a metabolic state where low CHO availability can be assumed. The amounts of CHO intake (i.e., 7 vs. 3 g/kg/d) were chosen, as these are common thresholds used in both clinical and scientific settings. Gas exchange data analysis & calculations: Values from respiratory volume and gas concentrations were

(which subsequently avoids glycogen recovery or super compensation) [34,35], would then create a metabolic state where low CHO availability can be assumed. The amounts of CHO intake (i.e., 7 vs. 3 g/kg/d) were chosen, as these are common thresholds used in both clinical and scientific settings. Gas exchange data analysis & calculations: Values from respiratory volume and gas concentrations were transmitted directly to the analysis software (Metasoft 3, version 3.9). All tests had the investigated gas exchange parameters viewed with an average time interval of 10 seconds. VO2peak was defined as the highest Oxygen uptake (VO2) recorded during the baseline test within a period of 30 seconds. For the two submaximal runs, calculations of CHO and lipid oxidation rates were performed using stoichiometric equations in accordance to the non-protein respiratory quotient technique [36]. Lipid oxidation rate (mg/min -1 )=–1.7012 VCO2 + 1.6946 VO2 CHO oxidation rate (mg/min -1 )=4.585 VCO2 - 3.2255 VO2 This technique provides calculations for substrate oxidation under the assumption that urinary nitrogen excretion is negli- gible. Markers were set every 5 minutes during the possible 30 minutes of each submaximal exercise bout. Respiratory data as well as CHO and lipid oxidation values were averaged from the last 30 seconds preceding every marker. Statistics: All of the analyzed parameters are descriptively reported as mean and Standard Deviation (±SD). Statistical analysis was performed using a commercial software package SPSS, version 20, IBM, USA and Microsoft Excel 2011. Samples were checked for normality using the Shapiro-Wilk test. Gender differences in anthropometry, baseline parameters and within nutritional protocols were tested with an unpaired t-test. Differences in dietary data, cardiopulmonary parameters as well as differences in substrate oxidation between the trials with different nutritional protocols (including gender compari- sons) were computed with a paired t-test. The interaction of the gas-exchange variables between diet and gender was analyzed with a two-way ANOVA for repeated measures (diet x gender). Significance was set at an alpha level of 0.05. Figure 1: Flowchart depicting investigational design of the randonmly assigned protocols. Med - Medical check; Glyc depl - Glycogen depleting bout

with a paired t-test. The interaction of the gas-exchange variables between diet and gender was analyzed with a two-way ANOVA for repeated measures (diet x gender). Significance was set at an alpha level of 0.05. Figure 1: Flowchart depicting investigational design of the randonmly assigned protocols. Med - Medical check; Glyc depl - Glycogen depleting bout

Volume 1; Issue 01 4 Citation: Silveira RDS, Kopinski S, Mayer F, Carlsohn A (2016) Influence of High vs. Low Carbohydrate Ingestion on Substrate Oxidation Patterns of Males and Females During Running Bouts at the Individual Anaerobic Threshold. FoodNutrJ1:102.DOI:10.29011/2575-7091.100002 Results Baseline characteristics: As presented in table 2, the overall values for IAT, VIAT and HRmax were 2.74 ± 0.39 mmol/l, 11.1 ± 1.4 km/h and 194 ± 10 beats/min respectively, with no significant gender differences. VO2peak differed significantly between genders with males achieving 50 ± 0 ml/min/kg and females 44 ± 5 ml/min/kg. Dietary intake: Overall CHO intake differed significantly (P = 0.005) between HC (7.1 ± 1.5 g/kg/d) and LC (3.4 ± 0.8 g/ kg/d) protocols (Figure 2). Accordingly, the mean deviations from the targeted CHO intake were 2 ± 21% (P = 0.566) and 15 ± 28% (P = 0.001) for HC and LC protocols respectively. As shown in figure 3, during the HC days, intake was 7.0 ±1.4 g/kg/d for males and 7.2 ± 1.7 g/kg/d for females (P = 0.661), resulting in a deviation of 0.3 ± 19.5% (P = 0.902) and 3.3 ± 24.2% (P = 0.871) respectively. Accordingly, the intake during the LC days amounted to 3.1 ± 0.7 and 3.8 ± 0.9 g/kg/d (P = 0.078), which reflects a target deviation of 3 ± 24% (P = 0.520) and 26 ± 29% (P = 0.003) respectively. Cardiopulmonary parameters during HC and LC bouts: As shown in figure 4a, with no significant differences between bouts at any of the measured points (P = 0.756 at rest; P = 0.768 at 5 min; P = 0.145 at 10 min; P = 0.067 at 15 min; P = 0.069 at 20 min; P = 0.089 at 25 min; P = 0.079 at 30 min), the overall HR ranged from 161 ± 11 (at 5 min) to 176 ± 13 beats/min (at 30 min; P = 0.001) during the HC bout, and from 165 ± 12 to 178 ± 11 beats/min (P = 0.007) during the LC bout respectively. Mean VO 2 was 38 ± 5 and

25 min; P = 0.079 at 30 min), the overall HR ranged from 161 ± 11 (at 5 min) to 176 ± 13 beats/min (at 30 min; P = 0.001) during the HC bout, and from 165 ± 12 to 178 ± 11 beats/min (P = 0.007) during the LC bout respectively. Mean VO 2 was 38 ± 5 and 39 ± 5 ml/min/kg during HC and LC bouts respectively (P = 0.086 at 5 min; P = 0.060 at 10 min; P = 0.189 at 15 min; P = 0.518 at 20 min; P = 0.059 at 25 min; P = 0.132 at 30 min; figure 4b). The Respiratory Exchange Ratio (RER) was significantly higher during the HC bout at all measure points (P = 0.006 at 5 min; P = 0.000 at 10 min; P = 0.003 at 15 min; P = 0.001 at 20 min; P = 0.007 at 25 min) but the last (P = 0.059 at 30 min; figure 4c). CHO and lipid oxidation: Relative (%) and absolute (g/min) values for overall CHO and lipid oxidation recorded during the two submaximal runs are presented in figure 5a-d. Substrate oxidation differed significantly between HC and LC bouts at Overall Males Females P values VO2peak (ml/min/kg) 47 ± 5 50 ± 0 44 ± 5 0.003 HRmax (beats/min)194 ± 10 193 ± 12 195 ± 5 0.808 VIAT (km/h) 11.1 ± 1.411.4 ± 0.810.7 ± 1.80.366 IAT (mmol/l) 2.74 ± 0.392.71 ± 0.432.77 ± 0.400.750 Table 2: Performance at baseline test. All values are mean±SD; P values reflect gender comparisons only Figure 2: Reported CHO intake during HC and LC days. Dotted lines mark the targeted amounts for CHO intake. Figure 3: Gender comparisons of CHO intake within dietary protocols. Values are expressed as median, quartiles and extremes; * - P < 0.05 Figure 4: Cardiopulmonary parameters during HC and LC runs. a) Displays the average HR measured during submaximal runs at rest and each of the 5 min marker points; b) Displays the average VO 2 measured during submaximal runs at each of the 5 min

within dietary protocols. Values are expressed as median, quartiles and extremes; * - P < 0.05 Figure 4: Cardiopulmonary parameters during HC and LC runs. a) Displays the average HR measured during submaximal runs at rest and each of the 5 min marker points; b) Displays the average VO 2 measured during submaximal runs at each of the 5 min maker points; and c) Displays the average RER (i.e., the ratio between Oxygen uptake (VO2) and Carbon dioxide output (VCO2)) measured during the submaximal runs at each of the 5 min marker points; All values are mean±SD; * - P < 0.05

The study investigates how carbohydrate intake affects substrate oxidation in runners.