Point: Carbohydrate loading does improve endurance performance. Counterpoint: Carbohydrate loading does not improve endurance performance.

Transcription

1 Rey Arcega, Danielle Contreras, Jason Orey BPK 312: P-CP debates April 04, 2017 Carbohydrate Loading Does Improve Endurance Performance Point Hypothesis Hypotheses Point: Carbohydrate loading does improve endurance performance. Counterpoint: Carbohydrate loading does not improve endurance performance. Non-clinical and clinical settings Clinically, carbohydrate loading is used to improve recovery of gut function following colorectal surgery. Evidence from a 2006 study at the Freeman Hospital showed carbohydrate loading led to bowel movements faster than control groups (5). In a non-clinical setting, endurance athletes participating in events longer than 90 minutes were encouraged to increase their carbohydrate intake to decrease muscular fatigue, and therefore, shorten their finishing time (1). Mechanism of action Carbohydrate loading increases the process of glycogenesis, which increases the amount of stored muscle glycogen (5). A classic carbohydrate loading method involves depleting muscle glycogen stores with prolonged, exhaustive exercise and 2-3 days of low-carbohydrate dieting. This is followed by a high-carbohydrate diet, while performing light exercise to replenish glycogen stores (3). This glycogen can then be used by the muscle as a source of ATP production through glycogenolysis, and glycolysis to make pyruvate. Pyruvate then gets converted to Acetyl-CoA and Oxaloacetate (in the presence of oxygen), which enters the Krebs Cycle to 1

2 produce ATP for use by the muscle (5). The more glycogen that is stored within the muscle, the more available energy. Maintaining blood glucose concentration delays the onset of fatigue, allowing exercise intensity to be sustained, and result in improved performance (1). Recommended Daily Allowance (RDA) and safe levels of consumption The USDA recommends that an individual should get between 45-65% of their daily caloric intake from carbohydrates. Excessive carbohydrate intake is associated with weight gain, and decreased insulin sensitivity which may lead to diabetes (10). For endurance athletes it is recommended that their carbohydrate intake increases to around 75-85% of their daily caloric intake (8). Research outcomes supporting the ergogenic effects A study done by Williams et al. examined the influence of a high carbohydrate diet on running performance in 18 runners during a 30-km treadmill time trial. Notable findings include the carbohydrate group running faster during the last 5km, as well as the 6 men in the carbohydrate loading group finished faster (P<0.05). No such improvement in time of the men in the control group was found (9). Only the control group had a decrease in blood glucose concentrations during trial 2. These results confirm that dietary carbohydrate loading improves endurance performance during prolonged running (9). In a study conducted by Andrews et al., significant differences were found in substrate utilization and in blood glucose, lactate, and glycerol responses. This significant treatment effect observed 2

3 more carbohydrate utilization during Supplemented (S) and Loaded+Supplemented (L+S) trials compared with Placebo (P) trials (1). The mean performance time of the S and L+S trials were about 4 and 2 minutes faster than the P trial (1). Evidence to refute the purported ergogenic effects An experiment done by Tarnopolsky and their colleagues looked at the effects of carbohydrate loading on subjects performing a cycle ergometer test to exhaustion. They found no overall increase in time to exhaustion in the carbohydrate loaded group when compared to a low carbohydrate group (7). When gender is controlled for within the study there was a notable increase in both time to exhaustion and muscle glycogen levels in men, but not in women. A few reasons there may not have been an improvement in women is the phase of menstrual cycle was controlled for, and women had a low gross intake of carbohydrates when compared to the men who saw improvements. Previous research has shown that the effect of carbohydrate loading may better relate to the gross amount of carbohydrate intake rather than percentages (8). Another study done by Burke et al. studied the effectiveness of carbohydrate loading on 100 km time trials (TT) in trained cyclists. This study found no effect of carbohydrate loading on reducing the amount of time taken to complete the 100 km TT. While the experimenters reported no difference in the time to completion, on average the carbohydrate loaded group improved by 1.5 minutes which, in a high level competitive environment, can be the difference between 1st 3

4 and finishing outside the top 10. The other issue with the study is they provided monetary rewards (no value given) for finishing fastest in one of the sprints, or finishing fastest overall (2). Conclusion In light of all the research and supporting evidence presented, we conclude that carbohydrate loading does improve endurance performance. 4

5 References 1. Andrews, J. L., Sedlock, D. A., Flynn, M. G., Navalta J.W., & Hongguang Ji. Carbohydrate Loading and Supplementation in Endurance-trained Women Runners. J. Appl. Physiol. 95; Burke, L. M., Hawley, J. A., Schabort, E. J., St Clair Gibson, A., Mujika, I., & Noakes, T. D. Carbohydrate loading failed to improve 100-km cycling performance in a placebo-controlled trial. J. Appl. Physiol. 88 (4); Fogelholm, G. M., Tikkanen, H. O., Naveri, H. K., Naveri, L. S., & Harkonen, M. H. Carbohydrate loading in practice: high muscle glycogen concentration is not certain. Br. J. Sports Med. 25(1); Korla, K., Chanchal, K. M. Modelling the Krebs cycle and oxidative phosphorylation. J. Biomo. Struct. Dyn. 32(2) Noblett, S. E., Watson, D. S., Huong, H., Davison, B., Hainsworth, P. J., & Horgan, A. F. Pre-operative oral carbohydrate loading in colorectal surgery: a randomized controlled trial. Colorectal Disease. 8(7); Pfeiffer, B., Stellingwerff, T., Hodgson, A. B., Randell, R., Pottgen, K., Res, P., Jeukendrup, A.E. Nutritional Intake and Gastrointestinal Problems During Competitive Endurance Events. Med. Sci. Sports Exerc. 44(2); Tarnopolosky, M. A., Atkinson, S. A., Phillips, S. M., & MacDougall, J. D. Carbohydrate loading and metabolism during exercise in men and women. J. Appl. Physiol. 78(4);

6 8. Piacentini, M. F., Parisi, A., Verticchio, N., Comotto, S., Meeusen, R., & Capranica, L. No changes in time trial performance of master endurance athletes after 4 weeks on a low carbohydrate diet. Sport Sci. Health. 8(1); Williams, C., Brewer, J., & Walker, M. The effect of a high carbohydrate diet on running performance during a 30-km treadmill time trial. Eur. J. Appl. Physiol. 65; Yunsheng, M., Youfu, L., Chiriboga, D. E., Olendzki, B. C., Hebert, J. R., Wenjun, L., Leung K., Hafner, A. R., & Ira S. Ockene, I. S. Association between Carbohydrate Intake and Serum Lipids, J. Am. Coll. Nutr. 25(2);

7 J Appl Physiol 95: , First published April 25, 2003; /japplphysiol Carbohydrate loading and supplementation in endurance-trained women runners Jessica L. Andrews, 1 Darlene A. Sedlock, 1 Michael G. Flynn, 1 James W. Navalta, 1 and Hongguang Ji 2 1 Wastl Human Performance Laboratory, Department of Health and Kinesiology, Purdue University, West Lafayette, Indiana 47907; and 2 Department of Nautical Hygiene, Faculty of Naval Medicine, Second Military Medical University, Shanghai , People s Republic of China Submitted 19 September 2002; accepted in final form 17 April 2003 Andrews, Jessica L., Darlene A. Sedlock, Michael G. Flynn, James W. Navalta, and Hongguang Ji. Carbohydrate loading and supplementation in endurance-trained women runners. J Appl Physiol 95: , First published April 25, 2003; /japplphysiol The purpose of this study was to examine the effect of carbohydrate (CHO) augmentation on endurance performance and substrate utilization in aerobically trained women. Eight endurance-trained women completed a 24.2-km (15 mile) self-paced treadmill performance run under three conditions: CHO supplementation (S), CHO loading and supplementation (L S), and placebo (P). Dietary CHO was 75% of energy intake for L S and 50% for both S and P. A 6% CHO-electrolyte solution (S and L S) or placebo (P) was ingested preexercise (6 ml/kg) and every 20 min during exercise (3 ml/kg). Blood glucose was significantly higher at 40, 60, and 100 min during L S, and at 60, 80, and 100 min during S compared with P (P 0.05). Blood lactate was significantly higher (P 0.05) during L S than S and P. Blood glycerol was significantly lower (P 0.05) at 20, 80, and 100 min during L S, and at 80 and 100 min during S than P. The proportion of CHO (%) utilized during exercise was significantly higher (P 0.05) during L S ( %) and S ( %) than P ( %). Performance times (P 0.05) were min (S), min (L S), and min (P). In conclusion, it appears that when CHO availability in women is increased through CHO loading and/or CHO supplementation, there is a concomitant increase in CHO utilization. However, this may not necessarily result in significantly improved performance. endurance performance; substrate utilization; glucose; glycerol; lactate CARBOHYDRATE (CHO) loading is known to produce an increase in stored muscle glycogen, often allowing exercise to be prolonged and/or performance to be improved (6, 9, 20, 34). Whereas the performance-enhancing effect of CHO augmentation has been demonstrated in male athletes, CHO loading has not been shown to be equally effective in female athletes. For example, Tarnopolsky et al. (29) found that women neither increased muscle glycogen concentration nor improved cycling performance after 4 days of ingesting a high-cho diet (75% of energy intake). In contrast, Walker et al. (32) reported a 13% increase in muscle glycogen and a significant increase in cycling time to fatigue in women after 6 days on a high-cho diet (78% of energy intake). However, the magnitude of these changes was smaller than those previously observed in male athletes. CHO supplementation during prolonged endurance exercise is thought to prevent a decline in blood glucose concentration, thus facilitating a high rate of CHO oxidation during the latter stages of exercise (4, 12, 16, 31, 35). Maintaining blood glucose concentration may also delay the onset of fatigue, allow exercise intensity to be sustained, and result in improved performance. The relationship among CHO ingestion, blood glucose, and performance enhancement has been extensively investigated (9, 12, 15, 22, 24, 31), particularly with respect to male athletes. Although there is an increased interest in understanding the interaction of CHO intake and exercise performance in women, investigators have primarily examined CHO loading (29, 32) with less attention being given to CHO supplementation (18). Thus the response of women to CHO feedings during endurance exercise, alone or in combination with CHO loading, requires more research. It is important to understand how women respond to CHO augmentation during endurance running for two reasons. First, female endurance runners believe that CHO intake can be beneficial despite the fact that there is a paucity of scientific evidence in support of this notion. Second, during prolonged endurance exercise, men tend to utilize CHO to a greater extent than women, whereas women tend to preferentially utilize more lipid (13, 17, 18, 28). Possible reasons include differences between men and women in the distribution and/or activation of - and -adrenergic receptors (5), aerobic capacity and/or fitness level (13), exercise intensity (13, 17), and the lack of a sufficient amount of CHO ingested by women (compared with men) during and before exercise (5, 13, 17, 30). However, the gen- Downloaded from by on February 6, 2017 Address for reprint requests and other correspondence: D. A. Sedlock, Dept. of Health and Kinesiology, 800 W. Stadium Ave., Purdue Univ., W. Lafayette, IN ( The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /03 $5.00 Copyright 2003 the American Physiological Society

8 CARBOHYDRATE SUPPLEMENTATION IN WOMEN 585 eral consensus among researchers is that it is likely mediated by endocrine differences. Regardless of the mechanisms involved, it could be hypothesized that CHO feedings would not enhance performance in female athletes to the extent previously seen in similarly trained male athletes because of their relatively greater reliance on lipid. Cycling has been selected most often as the mode of exercise used to investigate the effect of CHO feedings on performance (18, 22, 24, 36). This may be due to the fact that athletes tend to have more difficulty ingesting fluid and experience a greater level of discomfort from fluid ingestion during running than cycling (9, 10). Additionally, it is logistically easier for researchers to obtain physiological and hematologic measures during cycling compared with running. To our knowledge, there is no information regarding the effect of CHO supplementation or the combination of CHO loading and supplementation on run performance in women. Thus the purpose of this study was to examine the combined effects of CHO loading and supplementation, as well as the effects of CHO supplementation alone, on metabolic, hematologic, and performance variables in endurance-trained female athletes during prolonged exercise. We hypothesize that augmenting CHO intake in women, either by supplementation alone or supplementation combined with loading, will result in greater CHO utilization during exercise but little increase in performance. METHODS Subjects. Eight female athletes, yr of age, volunteered to participate in the study. Criteria for inclusion were a training history of at least 12 mo of endurance-type physical activity at a frequency of four times per week and/or a duration of 6 7 h/wk, weight stable ( 2.5 kg) for at least 1 yr, eumenorrheic with a normal cycle length of day, and on a dietary regimen in which CHO intake was not 65% of the total energy intake. Potential subjects with unstable eating tendencies (e.g., skipping meals on a daily basis, macronutrient deficiencies such as a no-fat or no-protein diet, and so forth) were excluded from participation. All procedures were approved by the Institutional Committee on the Use of Human Subjects in Research before data collection. Additionally, subjects provided written, informed consent before admission into the study. Experimental design. Subjects completed three different 24.2-km (15 miles) treadmill performance runs. With the use of a Latin square (counterbalanced) design, subjects completed each of the following three trials: no CHO loading no CHO supplementation (placebo; P), CHO supplementation only (S), and CHO loading CHO supplementation (L S). Each trial was completed during the midfollicular phase of the menstrual cycle (between days 5 and 10 from the first day of menses) to minimize the effects of gonadotrophic hormones on fuel metabolism. Thus subjects performed one trial per month. Preliminary measurements and procedures. Before experimental testing, subjects completed health, training, and menstrual cycle history questionnaires. They were also asked to complete a 3-day food diary and a 3-wk training log to ensure that they met the dietary and training criteria necessary for inclusion into the study. On meeting the study requirements, participants were given a list of food choices and asked to identify foods they consumed on a regular basis (familiar foods). From these choices, menus were generated for the women to follow for 4 days immediately before each experimental trial (Computer Planned Menus, Nutritional Computing Concepts, Indianapolis, IN). Additionally, subjects were given a dietary exchange book to help them approximate the amount of food they consumed on a daily basis and adhere to the prescribed dietary menus before each trial. Maximal oxygen consumption (V O 2 max) was measured 2 wk before the first experimental trial by using a continuous, incremental treadmill running test to exhaustion. On a separate day, subjects completed a self-paced 60-min treadmill run to familiarize themselves with the procedure for controlling treadmill speed and the drinking patterns and measurements to be used during the experimental trials. Exercise taper. Subjects ran for 60, 40, 40, 20, 20, and 0 min for the 6 days immediately before each trial, respectively. They were also required to log their daily activity during the week preceding each trial to ensure that they were following the prescribed tapering protocol. Diet. Subjects followed a prescribed diet for 4 days before each experimental trial and recorded their dietary intake during this time to ensure they were ingesting a high-cho diet (75% energy intake) before the L S trial and a moderate-cho diet (50% energy intake) before the S and P trials. Total energy intake was unchanged, but dietary composition was altered to either increase or decrease the percentage of dietary CHO. The high-cho regimen required subjects to ingest a greater percentage of energy in the form of complex CHO and commercially available glucose polymers in solution (Gatorlode, Quaker Oats, Chicago, IL). Experimental procedure. Experimental beverages were administered in a double-blind fashion during the performance trials. Subjects received a 6% CHO-electrolyte solution (Gatorade, Quaker Oats) during trials S and L S and a similarly flavored artificially sweetened placebo during the P trial. The placebo solution was similar to the supplement solution in electrolyte and mineral content, coloring, and flavoring, but it did not contain the CHO. Subjects arrived at the laboratory between 0700 and 0800 on the morning of the trial after a 10-h fast. Body weight was measured, a fingertip blood sample was obtained from a prewarmed hand, and 6 ml/kg of either the placebo or the CHO supplement was ingested. Subjects completed a 3-min self-paced warm-up run, rested for 2 min, and then began the 24.2-km time trial. Every 20 min throughout exercise, subjects ingested 3 ml/kg of the prescribed solution. The 24.2-km time trial was self-paced, and subjects were encouraged to complete the exercise in the shortest time possible. Pacing was accomplished through the use of photocells positioned at the front and rear of the treadmill, which allowed treadmill speed to be controlled by the subject simply by moving forward or drifting back on the treadmill to interrupt a light beam to increase or decrease speed, respectively. The treadmill was interfaced with a desktop computer and a software program that allowed subjects to see continuous updates of speed and distance on a nearby monitor. Oxygen consumption (V O 2), heart rate (HR), and ratings of perceived exertion (RPE) were measured every 30 min during the run starting at minute 25. Subjective level of gastrointestinal (GI) discomfort was obtained throughout the run by using a nonvalidated scale (1 comfortable, 2 partially full, 3 full, 4 uncomfortably full, 5 nauseous). A small blood sample (200 l) was collected from a fingertip every 20 min, with the beverage administered immediately after the blood collection. No measurements were taken and no bever- Downloaded from by on February 6, 2017 J Appl Physiol VOL 95 AUGUST

9 586 CARBOHYDRATE SUPPLEMENTATION IN WOMEN ages were administered after the 21-km ( 13 mile) mark; i.e., subjects ran uninterrupted for the remainder of the run. On completion of the run, subjects were weighed and a final blood sample was obtained 5 min postexercise. Measurements. Expired air was collected into Douglas bags. Volume was determined by forcing the contents of the bag through a dry-gas meter (Parkinson-Cowan). Respiratory gases were measured by using O 2 (paramagnetic) and CO 2 (infrared) analyzers (ParvoMedics, Salt Lake City, UT). The analyzers were calibrated before each series of analyses by using gases with known concentrations. V O 2 and carbon dioxide production were determined from expired volume and the percentages of O 2 and CO 2. Nonprotein respiratory exchange ratio (RER) and V O 2 were used to estimate the amount of CHO oxidized during the run. RPE was measured by using the Borg 6 20 category-ratio scale of perceived exertion, and HR was measured telemetrically (Polar Vantage XL, Polar, Stamford, CT). Blood sample preparation, storage, and analysis. Blood collected before, during, and after the run was used for the subsequent determination of glucose, glycerol, and lactate concentration. Approximately 200 l of whole blood (WB) were obtained at each sampling point and dispensed into a prechilled tube. Fifty microliters of WB were deproteinized in 200 l of cold 8% perchloric acid. After centrifugation, an aliquot of the perchloric acid extract was stored at 80 C for subsequent analysis of lactate concentration. Of the remaining acid extract, 120 l were mixed with 50 l of cold potassium hydroxide and then stored at 80 C for subsequent analysis of glycerol. WB was centrifuged and stored at 80 C for future determination of serum glucose concentrations. Glucose was determined spectrophotometrically by using a commercially available glucose kit [Infinity Reagent, procedure no. 18-UV; standards and controls (Accutrol); Sigma- Aldrich, St. Louis, MO]. The perchloric acid extract was assayed for lactate by using an enzymatic technique (23). Twenty microliters of sample, standard, or controls were added to a cuvette with 1 ml of a reagent cocktail containing excess NAD ( 12 mmol/l), lactate dehydrogenase (bovine heart, 300 U/ml), and a1mglycine-0.8 M hydrazine buffer (ph 9.2). Glycerol was analyzed fluorometrically by using an enzymatic method (3). Statistical analyses. Values are presented as means SE. A repeated-measures one-way ANOVA was used to determine the effects of the experimental treatments on performance time and total grams of CHO oxidized. Blood lactate, glucose, and glycerol values were analyzed by using separate two-way ANOVA with repeated measures. Tukey s post hoc tests were used where appropriate. Additionally, a Friedman s nonparametric matched-sample statistical test was conducted to test for a possible order or training effect. Statistical significance was accepted at P RESULTS Subject characteristics. Subject characteristics are presented in Table 1. Subjects were endurance trained and had been running an average of 53 km/wk for at least 12 mo before the study. Pretrial conditions. Training diaries collected on the morning of each trial showed that all subjects complied with pretrial training requirements; i.e., each subject followed the prescribed exercise taper. Diet records for the 4 days preceding each trial showed that subjects achieved the CHO intake goals for each experimental Table 1. Descriptive data for the trained female runners Variable Mean SE Range Height, cm Weight, kg V O 2max, ml kg 1 min HR max, beats/min Training history km/wk days/wk Values are for 8 subjects. V O 2max, maximal oxygen consumption; HR max, maximal heart rate. condition. Significantly greater CHO intake (P 0.05) was consumed before the L S trial ( % CHO) than either the P trial ( % CHO) or S trial ( % CHO). This corresponded to g of CHO ( g/kg) for the L S trial vs g CHO ( g/kg) and g CHO ( g/kg) for the P and S trials, respectively. Total energy intake was not significantly different for the 4 days before each trial (P 0.05). Laboratory conditions. Laboratory temperature was , , and C, and relative humidity was , , and % for the L S, S, and P trials, respectively (P 0.05). The women were cooled by an electric fan throughout the run. Performance time. There was no significant difference in performance time among the trials (P 0.05). Mean performance times were min for the S trial, min for the L S trial, and min for the P trial. Six of the eight subjects completed the run faster with CHO augmentation (S and L S trials) compared with ingestion of the placebo. Results of the Friedman s test indicated that there was no order effect for performance times (P 0.05), suggesting that learning and/or training effects did not occur over the course of the investigation. HR. No significant treatment effect or treatment time interaction was found. HR was maintained at , , and beats/min during the L S, P, and S trials, respectively (P 0.05). RPE. The RPE response did not differ among experimental conditions, nor was there a treatment time interaction (P 0.05). There was a significant time effect such that RPE was higher (P 0.05) at 120 min ( ) than at 30 min ( ) across all trials. V O 2. There were no treatment, time, or treatment time effects for V O 2 (P 0.05). Exercise elicited an average V O 2 of , , and ml kg 1 min 1, which corresponded to , , and % of V O 2 max for the S, P, and L S trials, respectively. RER and substrate utilization. There was no significant time effect or treatment time interaction for RER, but there was a significant treatment effect (P 0.05). As shown in Fig. 1, RER values were significantly higher during the L S ( ) and S Downloaded from by on February 6, 2017 J Appl Physiol VOL 95 AUGUST

10 CARBOHYDRATE SUPPLEMENTATION IN WOMEN 587 Fig. 1. Respiratory exchange ratio (RER) of trained female runners during a 24-km treadmill performance run. Values are means SE. L S, carbohydrate loading and supplementation; S, carbohydrate supplementation; P, placebo. *P significantly lower than L S and S, P ( ) trials compared with the P trial ( ). A significant treatment effect was found (P 0.05) for CHO oxidation, such that more CHO (expressed as a percentage of energy expenditure) was utilized during the L S ( %) and S ( %) trials compared with the P trial ( %). No significant time effect or treatment time interaction was noted. Blood glucose. Mean glucose values are presented in Fig. 2. A significant treatment by time interaction was noted (P 0.05). Compared with the P trial, blood glucose levels were significantly higher during the L S trial at 40 min, 60 min, 100 min, and postexercise, and during the S trial at 60 min, 80 min, 100 min, and postexercise. No significant differences were detected between the S and L S trials. Plasma glucose was similar among treatments before each run, averaging , , and mmol/l for the P, L S, and the S trials, respectively (P 0.05). Blood lactate. As shown in Fig. 3, blood lactate concentration was similar among treatments before each run (P 0.05). A significant treatment effect was observed (P 0.05) such that blood lactate during exercise was significantly higher in the L S ( mmol/l) compared with both the P trial ( mmol/l) and the S trial ( mmol/l). There was no significant treatment time interaction (P 0.05). Blood glycerol. Mean glycerol values are displayed in Fig. 4. Glycerol levels were similar among treatments before each run (P 0.05). A significant treatment time interaction was found (P 0.05) such that glycerol levels were significantly higher at 20 min, 80 min, 100 min, and postexercise during the P trial compared with the L S trial. Glycerol levels were also significantly higher at 80 min, 100 min, and postexercise during the P trial compared with the S trial (P 0.05). No significant differences were detected between the S and L S trials. Fluid consumption and weight loss. Total fluid consumption was 1, ml and did not differ among treatments. The initial bolus was ml, and the amount ingested every 20 min throughout the run was ml. Thus the hourly fluid consumption averaged ml. Total CHO intake during the S and L S trials averaged g ( g/kg), which included g CHO (0.37 g/kg) in the initial bolus and an additional g/h ( g kg 1 h 1 ) during the run. Body weight was significantly lower after exercise Downloaded from by on February 6, 2017 Fig. 2. Blood glucose concentration measured in trained female runners before, during, and 5 min after a 24-km treadmill performance run. Values are means SE. Pre, before exercise; Post, after exercise. *P significantly different from S, P P significantly different from L S, P Fig. 3. Blood lactate concentration measured in trained female runners before, during, and 5 min after a 24-km treadmill performance run. Values are means SE. *L S significantly higher than S and P, P Significantly lower than all other time points, P Significantly higher than minute 20, P J Appl Physiol VOL 95 AUGUST

11 588 CARBOHYDRATE SUPPLEMENTATION IN WOMEN Fig. 4. Blood glycerol concentration measured in trained female runners before, during, and 5 min after a 24-km treadmill performance run. Values are means SE. *P significantly higher than L S, P P significantly higher than S and L S, P ( kg) than before ( kg), with no significant difference in weight loss among the trials. GI discomfort. GI discomfort was similar among the three trials, averaging , , and for the L S, P, and S trials, respectively. On average, subjects felt somewhere between full and uncomfortably full throughout the run. They tended to experience increased GI discomfort later in the run during all three trials (P 0.05), suggesting that the discomfort was a result of the volume rather than the type of beverage ingested. DISCUSSION Both CHO loading (31) and CHO supplementation (2) have been shown to increase cycling performance in women. However, to our knowledge, there is no information on the effect of CHO supplementation and/or supplementation combined with CHO loading on metabolism, substrate utilization, and endurance run performance in women. The main finding of this study was no significant difference in performance time among the three experimental trials. Although mean performance time of the S and L S trials were 4 and2min faster than the P trial, respectively, these differences did not reach statistical significance. Additionally, the RPE, HR, and V O 2 responses did not differ among experimental conditions, indicating that subjects were running at a similar level of physical exertion for each experimental condition. A post hoc power analysis indicated that it would have taken 20 female subjects to detect a performance difference in this study with 80% power at Therefore, it is possible that the inclusion of more subjects would have resulted in a different outcome with respect to performance. However, despite the lack of a significant difference in performance across the three exercise trials, significant differences were noted in substrate utilization and in the blood glucose, lactate, and glycerol responses. Blood glucose was maintained at or above resting values throughout exercise in all three trials and was significantly higher in the L S and S trials compared with the P trial. Maintenance of blood glucose concentration during the P trial is not unusual and has been observed in previous run studies (6, 9, 27, 34). Lactate concentration was significantly higher during the L S trial compared with both the P and S trials, indicating that CHO loading in addition to supplementation may significantly increase CHO oxidation, perhaps by utilizing muscle glycogen. It has been shown that the primary substrate for lactate production in muscle is glycosyl units derived from local glycogen stores (14). Previous researchers have reported higher blood lactate levels after CHO loading compared with normal or depressed glycogen levels (1, 21), indicating that glycogen levels influence its utilization during exercise. It has also been shown that when lactate and glucose are infused simultaneously during exercise, lactate turnover exceeds that of glucose, indicating that muscle glycogen provides most of the CHO oxidized during exercise (7). Muscle glycogen concentrations were not measured in the present study, so it is uncertain whether CHO ingestion throughout exercise had any effect on muscle glycogen utilization during exercise. However, the significantly higher RER values observed during the L S and S vs. P trials are reflective of a higher rate of CHO metabolism, indicating that when CHO is made available through CHO augmentation, trained female runners will preferentially utilize CHO. Glycerol concentrations were significantly higher in the P trial compared with the L S and S trials, indicating a greater contribution of fat metabolism during the P condition. Although glycerol concentrations rose steadily during all trials, the increase was significantly smaller during both CHO trials, suggestive of a possible blunting effect as a result of CHO augmentation. Previous researchers have also reported decreased glycerol levels when CHO was consumed before or during an endurance run (6, 10, 31). Few researchers have examined the combined effect of CHO loading and supplementation on endurance performance. Mean performance time of the L S trial in the present study was not significantly different from the S or P trials, indicating that the combination of CHO loading and supplementation was of no added benefit to the women runners. These results are similar to those from a cycling study conducted by Burke et al. (8). However, other researchers found performance to be bettter in subjects when CHO loading and supplementation were combined before and during exercise (19), suggesting that loading and supplementation may have a synergistic effect on performance. In contrast, Widrick et al. (33) found that cycling performance was significantly improved when preexercise glycogen levels were elevated by CHO loading, regardless of whether CHO was ingested throughout exercise. It is unclear from these contradictory findings whether CHO loading provides any additional benefit to CHO ingestion. Downloaded from by on February 6, 2017 J Appl Physiol VOL 95 AUGUST

12 CARBOHYDRATE SUPPLEMENTATION IN WOMEN 589 Women in the present study consumed 335gCHO/ day, or 5.45 g CHO kg 1 day 1, before the L S trial. Because only one subject performed optimally during that trial, it is possible that the amount of CHO consumed was inadequate for improving performance. These findings are similar to results of a previous study by Tarnopolsky et al. (29), where CHO loading failed to increase glycogen stores and also failed to significantly improve cycling performance. The women in their study consumed 6.4gCHO kg 1 day 1 (370 g CHO/day) during the CHO loading regimen. However, in a study by Brewer et al. (6), male and female subjects significantly increased run time to exhaustion after consuming g CHO kg 1 day 1 for 3 days before the run trial. In absolute terms, the subjects were consuming g CHO/day. Because male and female data were pooled, the relative and absolute amount of CHO the women were consuming is unclear. In another study (32), female cyclists had a significant increase in glycogen stores and a significant improvement in performance after a CHO-loading regimen requiring them to consume 8.14 g CHO kg 1 day 1 (464 g CHO/day). Tarnopolsky et al. (30) recently suggested that perhaps women fail to increase their muscle glycogen stores to the same extent as male athletes in response to CHO loading because they do not consume the same absolute amount of CHO as men. It has been suggested that a CHO dosage of g/h is necessary for subjects to benefit from CHO supplementation (12, 15). However, Murray et al. (26) found on two separate occasions that the ingestion of 26 and 78 g CHO/h increased cycle performance to a similar extent, suggesting that 26 g/cho h was adequate to enhance performance. CHO dosage throughout the S and L S trials of the present study was 29 g/h (0.47 g kg 1 h 1 ), with an additional 22.4 g (0.37 g/kg) administered in the preexercise bolus. Relative to body weight, this was comparable to the amount of CHO administered to male runners in previous studies (10, 31). Run time to exhaustion in those men was significantly increased when ingesting CHO at a rate of 0.33 g kg 1 h 1 (39 g/h) in addition to a 0.44 g/kg bolus (31), and 0.30 g kg 1 h 1 (35 g/h) plus an additional 0.55 g/kg in the bolus (10). Women in the present study oxidized significantly more CHO with the ingestion of 29 g CHO/h when compared with a placebo, suggesting that this dosage was adequate to increase CHO utilization. The optimal dose eliciting performance improvements when CHO feedings are administered throughout prolonged exercise has not been established. Hargreaves (15) has suggested there is little added benefit in ingesting CHO solutions more concentrated than 6 8% because consuming higher dosages does not increase the rate of exogenous glucose oxidation. Evidently, there is a plateau in exogenous CHO oxidation during prolonged exercise whereby additional CHO ingestion above a certain amount will have no effect. On the basis of findings from Mitchell et al. (25), ingesting a CHO beverage that is too concentrated (i.e., 18% CHO) can impair gastric emptying and cause discomfort. Similarly, ingesting a less concentrated beverage in a greater volume of fluid also causes discomfort to the subjects. The difficulty is to administer a beverage that will provide an adequate amount of CHO to the subject without causing GI discomfort. Subjects in the present study experienced GI distress late in exercise during all three experimental trials, indicating that fluid volume rather than CHO concentration caused the discomfort. Chryssanthopoulos et al. (11) suggested that one cause of GI distress is that the volume of fluid prescribed and consumed by runners in laboratory studies is greater than the volume consumed during competition. Therefore, it is possible that subjects in the present study were experiencing GI distress because they were consuming more beverage during the experimental trials than they would during training and/or competition. In summary, the aim of the present study was to gain a better understanding and provide more information on the response of women runners to CHO augmentation. On the basis of the findings, it appears that when CHO availability was increased through CHO loading and/or CHO supplementation there was a concomitant increase in CHO utilization. However, this did not translate into significantly improved performance. DISCLOSURES This investigation was supported by a grant from the Gatorade Sport Science Institute. REFERENCES 1. Asmussen BJ, Klausen K, Egelund-Nielson L, Techon OSA, and Tonder PJ. Lactate production and anaerobic work capacity after prolonged exercise. Acta Physiol Scand 90: , Bailey SP, Zacher CM, and Mittleman KD. Effect of menstrual cycle phase on carbohydrate supplementation during prolonged exercise to fatigue. J Appl Physiol 88: , Bergmeyer HV. Methods of Enzymatic Analysis. Weinheim, Germany: Verlag Chemie, 1974, vol. 13, p Bosch AN, Dennis SC, and Noakes TD. Influence of carbohydrate ingestion on fuel substrate turnover and oxidation during prolonged exercise. J Appl Physiol 76: , Braun B and Horton T. Endocrine regulation of exercise substrate utilization in women compared to men. Exerc Sport Sci Rev 29: , Brewer J, Williams C, and Patton A. The influence of high carbohydrate diets on endurance running performance. Eur J Appl Physiol 57: , Brooks GA. Current concepts in lactate exchange. Med Sci Sports Exerc 23: , Burke LM, Hawley JA, Schabort EJ, Gibson AS, Mujika I, and Noakes TD. Carbohydrate loading failed to improve performance in a placebo-controlled trial. J Appl Physiol 88: , Chryssanthopoulos C, Hennessy LM, and Williams C. The influence of preexercise glucose ingestion on endurance running capacity. Br J Sports Med 28: , Chryssanthopoulos C and Williams C. Pre-exercise carbohydrate meal and endurance running capacity when carbohydrates are ingested during exercise. Int J Sports Med 18: , Chryssanthopoulos C, Williams C, Wilson W, Asher L, and Hearne L. Comparison between carbohydrate feedings before and during exercise on running performance during a 30-km treadmill time trial. Int J Sport Nutr 4: , Downloaded from by on February 6, 2017 J Appl Physiol VOL 95 AUGUST

13 590 CARBOHYDRATE SUPPLEMENTATION IN WOMEN 12. Coyle EF. Carbohydrate feeding during exercise. Int J Sports Med 13: , Friedlander AL, Casazza GA, Horning MA, Huie MJ, Piacentini MF, Trimmer JK, and Brooks GA. Training-induced alterations of carbohydrate metabolism in women: women respond differently from men. J Appl Physiol 85: , Gollnick PD, Bayly WM, and Hodgson DR. Exercise intensity, training, diet, and lactate concentration in muscle and blood. Med Sci Sports Exerc 18: , Hargreaves M. Metabolic responses to carbohydrate ingestion: effects on exercise performance. In: Perspectives in Exercise Science, edited by Lamb DR and Murray R. Carmel, IN: Cooper, 1999, p Hargreaves M, Costill DL, Coggan A, Fink WJ, and Nishibata I. Effect of carbohydrate feedings on muscle glycogen utilization and exercise performance. Med Sci Sports Exerc 16: , Horton TJ, Pagliassotti MJ, Hobbs K, and Hill JO. Fuel metabolism in men and women during and after long-duration exercise. J Appl Physiol 85: , Jarvis AT, Felix SD, Sims S, Jones MT, Coughlin MA, and Headley SA. Carbohydrate supplementation fails to improve the sprint performance of female cyclists [Online]. J Exerc Physiol 2: 16 22, april99.htm. 19. Kang J, Robertson RJ, Denys BG, DaSilva SG, Visich P, Suminski RR, Utter AC, Goss FL, and Metz KF. Effect of carbohydrate ingestion subsequent to carbohydrate supercompensation on endurance performance. Int J Sport Nutr 5: , Karlsson J and Saltin B. Diet, muscle glycogen, and endurance performance. J Appl Physiol 31: , Kelman GR, Maughan RJ, and Williams C. The effect of dietary modification on blood lactate during exercise. J Physiol 251: 34P 35P, Langenfeld ME, Seifert JG, Rudge SR, and Bucher RJ. Effect of carbohydrate ingestion on performance of non-fasted cyclists during a simulated 80-mile time trial. J Sports Med Phys Fitness 34: , Lowry OH and Passoneau JV. A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p McConnell G, Kloot K, and Hargreaves M. Effect of timing of carbohydrate ingestion on endurance exercise performance. Med Sci Sports Exerc 28: , Mitchell JB, Costill DL, Houmard JA, Fink WJ, Pascoe DD, and Pearson DR. Influence of CHO dosage on exercise performance and glycogen metabolism. J Appl Physiol 67: , Murray R, Paul GL, Seifert JG, and Eddy DE. Responses to varying rates of carbohydrate ingestion during exercise. Med Sci Sports Exerc 23: , Sullo A, Monda M, Brizzi G, Menino V, Papa A, Lombardi P, and Fabbri B. The effect of a carbohydrate loading on running performance during a 25-km treadmill time trial by level of aerobic capacity in athletes. Eur Rev Med Pharm Sci 2: , Tarnopolsky LJ, MacDougall JD, Atkinson SA, Tarnopolsky MA, and Sutton RJ. Gender differences in substrate for endurance exercise. J Appl Physiol 68: , Tarnopolsky MA, Atkinson SA, Phillips SM, and MacDougall JD. Carbohydrate loading and metabolism during exercise in men and women. J Appl Physiol 78: , Tarnopolsky MA, Zawada C, Richmond LB, Carter S, Shearer J, Graham T, and Phillips SM. Gender differences in carbohydrate loading are related to energy intake. J Appl Physiol 91: , Tsintzas OK, Williams C, Wilson W, and Burrin J. Influence of carbohydrate supplementation early in exercise on endurance running capacity. Med Sci Sports Exerc 28: , Walker JL, Heigenhauser GF, Hultman E, and Spriet LL. Dietary carbohydrate, muscle glycogen content, and endurance performance in well-trained women. J Appl Physiol 88: , Widrick JJ, Costill DL, Fink WJ, Hickey MS, McConnell GK, and Tanaka H. Carbohydrate feedings and exercise performance: effect of initial muscle glycogen concentration. J Appl Physiol 74: , Williams C, Brewer J, and Walker M. The effect of a high carbohydrate diet on running performance during a 30-km treadmill time trial. Eur J Appl Physiol 71: , Wright DA, Sherman WM, and Dernbach AR. Carbohydrate feedings before, during, or in combination improve cycling endurance performance. J Appl Physiol 71: , Yaspelkis BB III, Patterson G, Anderla PA, Ding Z, and Ivy JL. Carbohydrate supplementation spares muscle glycogen during variable-intensity exercise. J Appl Physiol 75: , Downloaded from by on February 6, 2017 J Appl Physiol VOL 95 AUGUST

14 J Appl Physiol 88: , Carbohydrate loading failed to improve 100-km cycling performance in a placebo-controlled trial LOUISE M. BURKE, 1 JOHN A. HAWLEY, 2 ELSKE J. SCHABORT, 3 ALAN ST CLAIR GIBSON, 3 IÑIGO MUJIKA, 4 AND TIMOTHY D. NOAKES 3 1 Department of Sports Nutrition, Australian Institute of Sport, Belconnen, Canberra, Australian Capital Territory 2616; 2 Exercise Metabolism Group, Department of Human Biology and Movement Science, Royal Melbourne Institute of Technology University, Bundoora, Victoria 3083; 3 Medical Research Council/University of Cape Town Bioenergetics of Exercise Research Unit, Department of Physiology, University of Cape Town Medical School, Cape Town 7701, South Africa; and 4 Departmento de Investigación y Desarollo Servicios Médicos, Athletic Club de Bilbao, Basque Country, Spain Burke, Louise M., John A. Hawley, Elske J. Schabort, Alan St Clair Gibson, Iñigo Mujika, and Timothy D. Noakes. Carbohydrate loading failed to improve 100-km cycling performance in a placebo-controlled trial. J Appl Physiol 88: , We evaluated the effect of carbohydrate (CHO) loading on cycling performance that was designed to be similar to the demands of competitive road racing. Seven well-trained cyclists performed two 100-km time trials (TTs) on separate occasions, 3 days after either a CHO-loading (9 g CHO kg body mass 1 day 1 ) or placebocontrolled moderate-cho diet (6 g CHO kg body mass 1 day 1 ). A CHO breakfast (2 g CHO/kg body mass) was consumed 2 h before each TT, and a CHO drink (1 g CHO kg. body mass 1 h 1 ) was consumed during the TTs to optimize CHO availability. The 100-km TT was interspersed with four 4-km and five 1-km sprints. CHO loading significantly increased muscle glycogen concentrations ( vs mmol/kg dry wt for CHO loading and placebo, respectively; P 0.05). Total muscle glycogen utilization did not differ between trials, nor did time to complete the TTs ( and min; P 0.4) or the mean power output during the TTs ( and W, P 0.4). This placebo-controlled study shows that CHO loading did not improve performance of a 100-km cycling TT during which CHO was consumed. By preventing any fall in blood glucose concentration, CHO ingestion during exercise may offset any detrimental effects on performance of lower preexercise muscle and liver glycogen concentrations. Alternatively, part of the reported benefit of CHO loading on subsequent athletic performance could have resulted from a placebo effect. glycogen loading; road cycling; blood glucose DURING PROLONGED ( 90 min) continuous, moderateintensity [70 80% of maximal oxygen uptake (V O2max )] cycling, the onset of fatigue is associated with very low ( 100 mmol/kg dry wt) muscle glycogen concentrations (6, 8, 11) and hypoglycemia (6, 11). Muscle and liver The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. glycogen stores can be increased above normal resting values by combining an exercise taper with the intake of large quantities of dietary carbohydrate (CHO) in the days before an endurance event (29). A recent review (18) concluded that such CHO loading improves endurance by 20% during prolonged continuous exercise at a fixed submaximal work rate. Furthermore, performance of a fixed distance or workload of 90 min is also increased by 2 3% after CHO loading. The common assumptions from these studies are that 1) CHO loading acts exclusively by increasing muscle glycogen stores and 2) these studies establish the value of CHO loading for a wide variety of competitive sporting activities. However, the certainty of these conclusions is influenced by aspects of the experimental designs used in many of these studies. First, to our knowledge, only one CHO-loading study (17) has included a placebo control in the research design. Without placebo control, the influence on subsequent exercise performance of psychological factors, in particular the expectation of an effect by both researchers and subjects, cannot be excluded. Interestingly, that placebo-controlled study failed to show any benefit of CHO loading on performance, albeit in a 1-h cycling time trial (TT) in which any effect would seem unlikely. Second, some studies have tested performance under conditions of an overnight fast and/or intake of water during the endurance test. These conditions are neither typical nor recommended for optimizing performance in an endurance event. Importantly, little is known about the effects of CHO loading on performance when there are continual changes in work rate, an exercise pattern typical of many competitive sports. For example, road cycling races are characterized by periods of sustained steadystate exercise punctuated by bouts of high- and lowintensity work in accordance with the course profile, terrain, environmental conditions, and riding strategies of the cycling group (25). Exercise tasks that include such alternating work bouts frequently elicit physiological responses that are different from those in which work, power, or duration is held constant (26). Thus stochastic exercise might be expected to cause a Downloaded from by on March 31, /00 $5.00 Copyright 2000 the American Physiological Society

15 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING 1285 different pattern of fuel utilization compared with continuous, moderate-intensity work. It is only recently that laboratory studies have attempted to simulate actual race conditions in the laboratory (26, 28). Accordingly, the aims of this study were to examine the reported ergogenic action of CHO loading on cycling performance tested on a reliable laboratory protocol (28) that simulates the demands of competitive road racing and, second, to exclude a possible placebo effect of this CHO loading on exercise performance. The study was also designed to include other dietary practices that are considered optimal for the performance of competitive road cycling. METHODS Table 1. Subject characteristics Mean SD Range Age, yr Mass, kg V O2peak ml kg 1 min l/min Peak power output, W Power/weight, W/kg Values are means SD for 7 subjects. V O2peak, peak oxygen uptake. Subjects. Seven endurance-trained male cyclists and triathletes accustomed to riding for prolonged periods (3 4 h) participated in this study. At the time of the investigation, these subjects were riding between 250 and 500 km/wk. Before commencement of the trial, all subjects were informed that the purpose of the investigation was to test two different sports supplements designed for race preparation. Subjects gave written informed consent in accordance with the guidelines outlined by the American College of Sports Medicine (2). The characteristics of the subjects are shown in Table 1. Preliminary testing. On their first visit to the laboratory, subjects were tested for peak oxygen uptake (V O2peak ) (16) and peak sustained power output on their own bicycles, which were mounted on the Kingcycle ergometer (described below). After a 5- to 10-min warm-up at a self-selected intensity, the test commenced at a workload of 200 W; the load was then increased by 20 W/min until the subject could no longer maintain the required power output. The subject s peak power was taken as the highest average power during any 60-s period of the exercise test. During these incremental tests to exhaustion, subjects were requested to remain in a seated position. Throughout the maximal test, subjects wore a face mask attached to an Oxygen Alpha automated gas analyzer (Jaeger, Wuerzburg, The Netherlands). Before each test the gas analyzer was calibrated by using a Hans Rudolph liter syringe and a 5% CO 2-95% N 2 gas mixture. Analyzer outputs were processed by an IBM computer that calculated minute ventilation, oxygen consumption, and rates of carbon dioxide production by using conventional equations. Each subject s V O2peak was taken as the highest oxygen uptake measured during any 60-s period of the test. After completing the maximal test, subjects performed a familiarization ride on a Kingcycle ergometry system (Kingcycle, High Wycombe, UK). This system allowed cyclists to ride on their own racing bicycles in the laboratory. After the front wheel was removed, the subject s bicycle was attached to the ergometer by the front fork and supported by an adjustable pillar under the bottom bracket. The bottom bracket support was used to position the rolling resistance of the rear wheel correctly on an air-braked flywheel, and the ergometer was calibrated as previously reported in detail (24). The familiarization ride consisted of the first 25 km of the 100-km TT (described in Experimental trials), which was performed to familiarize each subject with the stochastic nature of the trial. Subjects were requested to ride as fast as possible and were not given any feedback other than their elapsed distance. Dietary intervention. Individual food plans were constructed for each subject on the basis of body mass (BM) and food preferences. Each subject was supplied with his food intake for the 72-h period before each trial. Menu plans were provided in written form, and the food assigned to each meal was individually prepared and packaged so that the need for further preparation by subjects was minimized. Subjects were required to record their actual food and fluid intake on dietary logs to account for any portion of meals left unconsumed or for any additional intake. Although some food items differed between subjects, each cyclist received identical breakfast, lunch, and dinner menus for both of his trials, with the CHO intake from these meals designed to provide 6 g CHO kgbm 1 day 1. Snacks were also provided for each day and provided the source of differentiation between trials. On the CHO loading diet, subjects received 1,200 ml of water each day and a number of sports bars (Gijima, Sasko, Paarl, South Africa) calculated to provide an additional3gcho kg BM 1 day 1. Each sports bar had a composition of 27 g CHO, 6.5 g fat, and 2.7 g protein. The sports bars were used to mimic what is used by competitive athletes during training and competitive events. On the placebo trial, daily snacks were provided in the form of 1,200 ml of an artificially sweetened low-calorie drink that was described to subjects as a CHO-loading drink. Experimental trials. Each subject completed a random crossover design of two experimental trials separated by 7 days. Subjects performed their TTs at the same time of day under standard laboratory conditions ( 20 C, 55% relative humidity). Subjects were requested to perform the same type of training for the duration of the trial and to refrain from heavy physical exercise on the day before a TT. Training diaries were kept to assess compliance to this condition. On the morning of an experiment subjects reported to the laboratory between 0700 and 0800, h after an overnight fast. At this time the Kingcycle ergometer was calibrated, and then subjects consumed a standard breakfast providing 2 g CHO/kg BM. After the subjects rested quietly for 105 min, a preexercise muscle biopsy sample was taken from the vastus lateralis of the right leg according to the technique of Bergstrom (5) as modified by Evans et al. (12). After this procedure, the subjects mounted their cycle and began a 5-min self-paced warm-up. Exactly 2 h after they had finished breakfast, subjects commenced the 100-km TT. To mimic the stochastic nature of cycle road races (24), the TT included a series of sprints: five 1-km sprints after 10, 32, 52, 72, and 99 km, as well as four 4-km sprints after 20, 40, 60, and 80 km. Subjects were instructed to complete the total distance in the fastest time possible, taking into consideration the sprints that were included. To ensure subjects participated at maximum capacity, a financial reward was offered to the subject who completed either the entire TT or sprints component in the fastest time for the group. Just before commencement of a sprint, an investigator gave a distance countdown and instructed the cyclist to complete the sprint in the fastest possible time as soon as he reached the specific distance at which the sprint Downloaded from by on March 31, 2017

16 1286 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING Table 2. Dietary intake during 72 h before trial commencement CHO, g CHO, g/kg Energy, kcal Protein, g Fat, g Water, g CHO loading , , Placebo * * 2, * 88 6* 79 7* 3, Values are means SD. CHO, carbohydrate. *CHO intake in CHO-loading diet was significantly greater than in placebo diet; energy, protein, and fat intake were significantly greater in CHO-loading diet than in placebo diet (P 0.05). started. Subjects viewed a diagram of the course profile that graphically illustrated where the 1- and 4-km sprints occurred, before and during each ride. Instantaneous power output was recorded at each 500-m split of both the 1- and 4-km sprints to provide an estimate of the average power output for the sprint. The only feedback given to subjects during the TT was their elapsed distance and heart rate (HR). Subjects were not informed of the elapsed total time or the times for the sprints until completion of the experiment. Throughout each trial, power output, speed, and elapsed time were monitored continuously and stored on computer. HR was recorded by using a SportTester HR monitor (Polar Electro, Kempele, Finland). We have previously reported that the between-test correlation for the time taken by eight well-trained cyclists and triathletes to complete this protocol was 0.93 [95% confidence interval (CI) ], and the within-cyclist coefficient of variation (CV) was only 1.7% (95% CI %) (28). During the 100-km ride, subjects ingested a 7 g/100 ml glucose polymer solution at a rate of 15 ml kg BM 1 h 1. This drinking regimen was intended to replace 80% of sweat loss (23) while providing CHO at 1 g kg BM 1 min 1, the maximum rate at which muscle can oxidize exogenous glucose (15). A fan was positioned to cool the subjects during their TT. The average sweat loss (liters) was estimated as the average fluid intake weight change over the trial of all seven subjects. Immediately on completion of the TT, a second biopsy was taken from the same leg, a distance of 4 cm distal to the first incision. No metabolic measurements were taken during the trials because the TTs were performance trials, and it was believed that interfering with the athletes during the trials would impair their performance by spoiling their concentration. Analytic techniques. Muscle samples were subsequently freeze-dried; dissected free of blood, connective tissue, and fat; and weighed. Muscle glycogen content was determined after acid hydrolysis by a hexokinase method (21). The CV for this assay in our laboratory is 5% for duplicate glycogen assays of a single piece of muscle and 7% for assays of the glycogen content of separate pieces of the same muscle biopsy sample (17). Statistical analyses. Differences in the sprint times and sprint power outputs and in the glycogen concentrations were examined by using repeated-measures ANOVA, whereas glycogen utilization, total 100-km time, and total power outputs were compared by using Student s t-tests. A Sheffé s post hoc test was used to assess differences revealed by the ANOVA. All data are reported as means SD, and all calculations were performed by using Statistica for Windows (version 6, Statsoft, Tulsa, OK). RESULTS Training diaries collected on the morning of each trial showed that all subjects complied with the pretrial training conditions: each subject undertook identical exercise sessions before each of his trials and achieved a training taper over this time. Reported dietary intake for the 72 h before each trial (Table 2) showed that subjects achieved the CHO intake goals predetermined for the study. Significantly greater CHO intake was consumed with the CHO-loading diet than with the placebo diet (9 vs. 5.8 g CHO kg BM 1 day 1 ), with this additional CHO being provided exclusively by the sports bar. Muscle glycogen levels were significantly higher after CHO loading (Table 3). Total energy, protein, and fat intake were also significantly higher with CHO loading (Table 2). This resulted from the high protein and fat content of the energy bar. Despite higher preexercise muscle glycogen concentrations after CHO loading, glycogen utilization during the 100-km TT was not significantly different between trials but tended to be greater after CHO loading. Postexercise glycogen concentrations were not significantly different between treatments. BM changes over the TT were not different between treatments ( vs kg for CHO loading and placebo, respectively), indicating that a mild but similar degree of fluid deficit occurred over the TT. There was no significant difference in overall TT performance between treatments ( vs min, for CHO loading and placebo, respectively; P 0.4). The time difference between the CHO-loaded and placebo trial was 1.1% (95% CI %). The average power output over the total TT was not different between trials ( vs ; P 0.4). There were no differences between groups for HR and rating of perceived exertion data during each trial. There were also no significant differences between groups for either the time to complete (Fig. 1) or the mean power (Fig. 2) of each of the nine 1- and 4-km sprints. Time and power data for the first and final of each of these sprints and for the total 100-km TT are presented in Table 4. In both groups, mean power output declined in both 1- and 4-km sprints over the duration of the 100-km TT, with a corresponding increase in time to complete the sprints. Table 3. Muscle glycogen concentrations after the dietary treatments and 100-km TT Preexercise Postexercise Utilization CHO loading (9 g CHO kg body mass 1 day 1 ) Placebo (6 g CHO kg body mass 1 day 1 ) * Values are means SD given in mmol/kg dry wt. TT, time trial. * Preexercise muscle glycogen content was significantly lower compared with CHO-loaded trial, P Downloaded from by on March 31, 2017

17 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING 1287 Fig. 1. Time taken by carbohydrate (CHO) and placebo groups in 4-km (A) and 1-km (B) sprints during 100-km time trial. Values are means SD. DISCUSSION The major finding of this study is that, when tested against placebo, CHO loading did not improve performance during a prolonged ( 2.5-h) self-paced TT that included high-intensity workbouts and CHO ingestion before and during the trial. This finding contrasts with Fig. 2. Power output by CHO and placebo groups in 4-km (A) and 1-km (B) sprints during 100-km time trial. Values are means SD. Table 4. Time and power data for individual sprints and 100-km TT CHO Loading Placebo Time, min:s km sprint 1:11.7 0:3.5 1:12.8 0: km sprint 1:20.7 0:6.6 1:22.2 0:7.4 1-km sprint 0:8.8 0:8.6 0:9.4 0: km sprint 5:24.1 0:20.6 5:19.2 0: km sprint 5:35.3 0:19.1 5:44.7 0: km sprint 0:11.2 0:14.2 0:25.5 0:17.2 Total 100-km TT 147:30 10:0 149:07 11:0 Power, W km sprint km sprint * km sprint km sprint km sprint km sprint Total 100-km TT Values are means SD., Change. *Power output of final 1-km sprint significantly lower compared with first 1-km sprint in CHOloaded trial, P the majority of studies that show that CHO loading enhances performance during prolonged exercise 90 min under a wide variety of laboratory and field conditions (18). A number of characteristics, unique to this trial, could explain this unexpected finding. First was the use of a double-blind placebo-controlled design. This is an essential characteristic of any intervention study measuring an effect that might be influenced by psychological factors and is an accepted requirement in studies of CHO feeding during exercise. The failure of the majority of CHO-loading studies to include a placebo control group reduces the certainty of the conclusions inferred from their findings. Because endurance athletes are now well educated about the principles of CHO loading (9) and the reported benefits of this popular practice, it is likely that many subjects participating in current studies of CHO loading would expect to perform better after that intervention, thus introducing a psychological bias. To date, only one other study of CHO loading has attempted to include a full placebo control, by providing subjects with their food intake during the pretrial period and disguising the true CHO-enriched menu by matching the other diet with a low-energy placebo supplement (17). That study also failed to find an improvement in performance associated with elevated preexercise glycogen stores, although their exercise protocol involved only 60 min of cycling. Although in this trial a third nonplacebo group was not included, this does not alter the results of our present trial because subjects were blinded to the nature of the CHO or placebo ingestion. Our present study therefore invites the possibility that the subjects knowledge that they were CHO loading could be an important determinant of the measured ergogenic effect of CHO-loading studies that are not placebo controlled. Future studies of CHO loading must be appropriately controlled either to refute or support this unexpected interpretation. Downloaded from by on March 31, 2017

18 1288 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING A second characteristic that may have influenced the outcome of this trial was the exercise protocol used to evaluate cycling performance. The exercise task was designed to mimic the requirements of a 100-km road cycle race (25) rather than to have subjects exercise at fixed submaximal workloads to exhaustion as in previous studies of cycling (1, 6, 8, 20) or running (7, 14, 22). In other studies, CHO loading has been shown to reduce the time taken to complete a prolonged running or cycling task (21, 31, 32). Under these conditions, enhanced performance after CHO loading was achieved by the maintenance of greater average speeds because the rate of decline in power output during the latter stages of the task was reduced. Although these protocols involved self-paced efforts and the possibility of a changing workload, it is unlikely that the typical pacing strategies used in these trials involved the random injection of extremely highintensity efforts, as are characteristic of mass start road cycling events (25). Thus it is possible that elevated pretrial glycogen concentrations might increase performance in those forms of exercise in which the intensity is relatively more constant but not in the stochastic form of exercise evaluated in this trial, in which there are bouts of exercise of very high intensity. Indeed the inclusion of repeated high-intensity sprints in our TT protocol produced postexercise muscle glycogen concentrations as low as have been reported in any previous study (6, 8, 11). Third, although preexercise glycogen concentrations were significantly elevated by 20% at the beginning of the CHO-loaded TT compared with the placebo TT, this increase may not have been sufficient to ensure a physiological benefit to the performance of the repeated high-intensity sprints and therefore the overall time to complete the TT. However, the 20% increase in muscle glycogen concentrations induced by CHO loading is in agreement with values previously reported by other investigators (17, 22, 27) and was sufficient to enhance performance in these studies (22, 27). It should, however, be remembered that the original CHO-loading studies compared the performance of subjects with very large ( 100%) differences in initial muscle (and liver) glycogen concentrations (6, 21) because the comparison was usually between high- and low-cho diets rather than between high- and normal-cho diets as in this study. A fourth feature of our study design was the inclusion of several dietary strategies to optimize CHO availability during performance. CHO was consumed in a preevent meal and during the TT in accordance with current sports nutrition guidelines (3, 4) and the typical practices of competitive cyclists. Several studies have partially or systematically studied the interaction of CHO loading and CHO ingestion during exercise on exercise performance. Flynn and co-workers (13) reported that CHO intake during cycling did not improve performance in CHO-loaded subjects during 2 h of self-paced cycling at 80% V O2max. In contrast, the study of Kang et al. (20) showed that CHO feeding in CHO-loaded subjects enhanced performance during 3 h of exercise at 70% V O2max. The study of Widrick et al. (31) evaluated performance during a self-paced 70-km TT when preexercise glycogen content was manipulated by CHO loading and CHO or placebo was ingested during the ride (31). CHO ingestion during the TT prevented a decline in blood glucose concentrations regardless of the starting muscle glycogen content. Performance was best in subjects who were CHO loaded and who ingested CHO during exercise and was worst in those who neither CHO loaded nor ingested CHO during exercise. However, performance differences were relatively small, especially between the CHO-loaded group who ingested placebo during exercise and the CHO-depleted group who ingested CHO during exercise. This occurred despite the fact that the CHO-depleted group had exercised for 45 min 24 h before the performance trial, whereas they rested for 48 h before the CHO-loaded trial. Thus, whereas CHO ingestion during exercise did not influence performance in CHO-loaded subjects, CHO ingestion enhanced the performance of CHOdepleted subjects, perhaps by preventing the development of hypoglycemia (mean blood glucose concentrations of 3.2 mmol/l) and a synchronous fall in power output in subjects who did not CHO load before exercise. Higher blood glucose concentrations with CHO ingestion than with placebo were also associated with superior exercise performance in the CHO-loading studies of Kang et al. (20). Similarly, superior performance in a 30-km run after CHO loading was associated with higher blood glucose concentrations during exercise than when only water was consumed (33). As in the study of Widrick et al. (31), reductions in running speed fell synchronously with the blood glucose concentrations in both CHO-loaded and control groups. In contrast, performance was not enhanced by CHO ingestion in the trial of Williams et al. (32), even though blood glucose concentrations were higher with ingestion of CHO than placebo during exercise. The opposite result was reported by Tsintzas et al. (30), where performance was enhanced by CHO ingestion during exercise even though blood glucose concentrations were essentially unaffected. Accordingly, performance in this trial may have been the same in CHO-loaded and placebo groups because blood glucose concentrations may have been similar in both trials due to the ingestion of CHO during the ride. Hence the optimization of CHO ingestion before and during exercise in this trial may have negated any additional beneficial ergogenic effect of CHO loading alone. Finally, although CHO loading did not produce a significant ergogenic effect in this trial, the results should be examined in light of what might be meaningful in a competitive sports event. In this study, CHO loading was associated with a 1.1% improvement in TT performance, with true differences in the performance of a similar population likely to range from a 1.6% decrement to a 3.6% improvement. At best, this improvement would not greatly change the outcome of the Downloaded from by on March 31, 2017

19 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING 1289 performances of the back of the pack cyclists; even a 4% improvement would not move these athletes to the front of the field. However, such an improvement, if real, is meaningful to the top cyclists in a race in that it would enhance the likelihood of an improvement in finishing order (19). Hopkins and colleagues (19) have calculated that the smallest intervention likely to enhance the performance of a top-finishing athlete is % of the typical within-athlete CV in performance between events. The typical within-athlete CV for cyclists competing in such road events is currently not known. However, in other sports such as road running and track cycling, this CV is 1 2% for good competitors and often 1% for the best international competitors (W. G. Hopkins, personal communication). Thus a 1.1% improvement in performance is likely to be worthwhile to an elite road cyclist. Should performance in massstart road cycling races be less reliable than for other sports (e.g., CV 4%), then the impact of our effect would need to be recalculated. Power analysis shows that a sample size of 30 subjects would be needed in a similar study to reduce the confidence intervals of the true performance change to approximately 1%, that is 0 2%, and therefore discount the likelihood of a meaningful effect. In summary, this study shows that a CHO-loading protocol that increased preexercise muscle glycogen concentrations resulted in a minimal effect on the performance of a 100-km TT involving high-intensity sprints when CHO was ingested before and during the event according to contemporary sports nutrition guidelines. Although we cannot completely discount the possibility that CHO loading has a worthwhile effect on the performance of competitive cyclists under conditions similar to our trial, our data suggest that this effect is small and, at best, only likely to rearrange the finishing order of the top cyclists in the field. Furthermore, this study raises the possibility that part or all of the ergogenic effect of CHO loading reported in previous studies could result either from a placebo effect or from higher preexercise liver glycogen stores that could delay the onset of hypoglycemia during prolonged exercise. If this is the case, CHO ingestion during exercise, as included in this study, would minimize any ergogenic effect of CHO loading by preventing the development of hypoglycemia in persons beginning exercise with lower liver (and muscle) glycogen concentrations. The research was supported by SASKO Pty, Ltd; the Harry Crossley Research Fund of the University of Cape Town; and the Medical Research Council of South Africa. This study was undertaken by L. M. Burke during her sabbatical study leave at the Medical Research Council/University of Cape Town Bioenergetics of Exercise Unit at the Sports Science Institute of South Africa. Address for reprint requests and other correspondence: T. D. Noakes, MRC/UCT Bioenergetics of Exercise Research Unit, Dept. of Physiology, Univ. of Cape Town Medical School, PO Box 115, Newlands 7700, South Africa ( tdnoakes@sports.uct.ac.za). Received 3 May 1999; accepted in final form 6 December REFERENCES 1. Ahlborg B, Bergstrom J, Brohult J, Ekelund LG, and Maschio G. Human muscle glycogen content and capacity for prolonged exercise after different diets. Forsvarsmedicin 3: 85 99, American College of Sports Medicine. Policy statement regarding the use of human subjects and informed consent Med. Sci. Sports Exerc 20: v, American College of Sports Medicine. Position stand: exercise and fluid replacement. Med Sci Sports Exerc 28: i vii, American Dietetic Association and Canadian Dietetic Association. Position stand: nutrition for physical fitness and athletic performance for adults J Am Diet Assoc 93: , Bergstrom J. Muscle electrolytes in man. Scand J Lab Invest 14, Suppl. 68: 1 47, Bergstrom J, Hermansen L, Hultman E, and Saltin B. Diet, muscle glycogen and physical performance. Acta Physiol Scand 71: , Brewer J, Williams C, and Patton A. The influence of high carbohydrate diets on endurance running performance. Eur J Appl Physiol 57: , Bosch AN, Dennis SC, and Noakes TD. Influence of carbohydrate loading on fuel substrate turnover and oxidation during prolonged exercise. J Appl Physiol 74: , Burke LM and Read RSD. A study of carbohydrate loading techniques used by marathon runners. Can J Sport Sci 12: 6 10, Coggan AR and Coyle EF. Effect of carbohydrate feedings during high-intensity exercise. J Appl Physiol 65: , Coyle EF, Coggan AR, Hemmert MK, and Ivy JL. Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate. J Appl Physiol 61: , Evans WJ, Phinney SD, and Young VR. Suction applied to muscle biopsy maximizes sample size. Med Sci Sports Exerc 14: , Flynn MG, Costill DL, Hawley JA, Fink WJ, Neufer PD, Fielding RA, and Sleeper MD. Influence of selected carbohydrate drinks on cycling performance and glycogen use. Med Sci Sports Exerc 19: 37 40, Galbo H, Holst J, and Christensen NJ. The effect of different diets and of insulin on the hormonal response to prolonged exercise. Acta Physiol Scand 107: 19 32, Hawley JA, Dennis SC, and Noakes TD. Oxidation of carbohydrate ingested during prolonged exercise. Sports Med 14: 27 42, Hawley JA and Noakes TD. Peak sustained power output predicts V O2max and performance time in trained cyclists. Eur J Appl Physiol 65: 79 83, Hawley JA, Palmer GS, and Noakes TD. Effects of 3 days of carbohydrate supplementation on muscle glycogen content and subsequent utilisation during a1hcycletimetrial. Eur J Appl Physiol 75: , Hawley JA, Schabort EJ, Noakes TD, and Dennis SC. Carbohydrate loading and exercise performance: an update. Sports Med 24: 73 81, Hopkins WG, Hawley JA, and Burke LM. Design and analysis of research of sport performance enhancement. Med Sci Sports Exerc 31: , Kang J, Robertson RJ, Denys BG, DaSilva SG, Visich P, Suminski RR, Utter AC, Goss FL, and Metz KF. Effect of carbohydrate ingestion subsequent to carbohydrate supercompensation on endurance performance. Int J Sport Nutr 5: , Karlsson J, Diamant B, and Saltin B. Muscle metabolites during submaximal and maximal exercise in man. Scand J Clin Lab Invest 26: , Lamb DR, Snyder AC, and Baur TS. Muscle glycogen loading with a liquid carbohydrate supplement. Int J Sport Nutr 1: 52 60, Montain SJ and Coyle EF. The influence of graded dehydration on hyperthermia and cardiovascular drift during exercise. J Appl Physiol 73: , Downloaded from by on March 31, 2017

20 1290 NO IMPROVED ENDURANCE PERFORMANCE WITH CHO LOADING 24. Palmer GS, Dennis SC, Noakes TD, and Hawley JA. Assessment of the reproducibility of performance testing on an airbraked cycle ergometer. Int J Sports Med 17: , Palmer GS, Hawley JA, Dennis SC, and Noakes TD. Heart rate responses during a 4-d cycle stage race. Med Sci Sports Exerc 26: , Palmer GS, Hawley JA, and Noakes TD. Effects of steadystate versus stochastic exercise on subsequent cycling performance. Med Sci Sports Exerc 29: , Rauch LHG, Rodger I, Wilson GR, Belonje JD, Dennis SC, Noakes TD, and Hawley JA. The effects of carbohydrate loading on muscle glycogen content and cycling performance. Int J Sport Nutr 5: 25 36, Schabort EJ, Hawley JA, Hopkins WG, Mujika I, and Noakes TD. A new reliable laboratory test of endurance performance for road cyclists. Med Sci Sports Exerc 30: , Sherman WM, Costill DL, Fink WJ, and Miller JM. The effect of exercise and diet manipulation on muscle glycogen and its subsequent use during performance. Int J Sports Med 2: , Tsintzas OK, Williams C, Singh R, Wilson W, and Burrin J. Influence of carbohydrate-electrolyte drinks on marathon running performance. Eur J Appl Physiol 70: , Widrick JJ, Costill DL, Fink WJ, Hickey MS, McConell GK, and Tanaka H. Carbohydrate feedings and exercise performance: effect of initial muscle glycogen concentration. J Appl Physiol 74: , Williams C, Brewer J, and Walker J. The effect of a high carbohydrate diet on running performance during a 30-km treadmill time trial. Eur J Appl Physiol 65: 18 24, Williams C, Nute MG, Broadbank L, and Vinall S. Influence of fluid intake on endurance running performance. Eur J Appl Physiol 60: , Downloaded from by on March 31, 2017

21 Br J Sp Med 1991; 25(1) Carbohydrate loading in practice: high muscle glycogen concentration is not certain G. M. Fogelholm MSc', H. 0. Tikkanen MD2, H. K. Naveri MD, PhD3, L. S. Naveri MD2 and M. H. A. Harkonen MD4 1 Department of Nutrition, University of Helsinki, Finland 2 Department of Clinical Chemistry, University of Helsinki, Finland 3 First Department of Medicine, University of Helsinki, Finland 4 Professor of Clinical Chemistry, University of Helsinki, Finland It is believed that muscle glycogen resynthesis can be stimulated by depleting the glycogen stores by heavy physical exercise and then eating a diet rich in carbohydrates. In this study, we compared muscle glycogen concentrations after two different depletion and loading procedures in six male runners. The depletion runs for the procedures were a half-marathon race and an easier fartlek. The mean muscle glycogen concentrations (±s.e.m.), analysed after the procedures, did not differ significantly between the race and the fartlek being 285 (±25) mmol/kg d.w. (dry weight) versus 315 (±32) mmol/kg d.w. (P > 0.05). Moreover, the subjects' glycogen concentrations were not clearly increased above the predepletion values following either procedure. The results show that higher glycogen levels do not necessarily occur after classical carbohydrate-loading procedures. Keywords: Carbohydrate diet, hormones, marathon Studies in the late 1960s showed a positive correlation between the pre-exercise muscle glycogen concentration and the ability to perform prolonged, severe exercises'. An elevation of muscle glycogen stores can be achieved by a manipulation of the diet2-5. In the classical method2, muscle glycogen stores are first depleted by prolonged, exhaustive exercise, followed by 2-3 days of low-carbohydrate diet, i.e. fat and protein mainly. Then the stores are replenished by eating a high-carbohydrate diet and taking only light exercise. The exhaustive depletion exercise has been criticized, because it interferes with the peaking for an important race6. Sherman and Costill4 and Blom et al.7 have shown that an exhaustive depletion brought about by running might even be totally ineffective in stimulating glycogen synthesis. Despite this, the traditional procedure (exhaustive depletion followed by a carbohydrate-rich diet) is carried out by many endurance athletes as a final preparation for a race. The aim of the present study was to find out whether muscle glycogen concentrations would Address for correspondence: G. M. Fogelholm, University of Helsinki, Department of Nutrition, SF Helsinki, Finland 1991 Butterworth-Heinemann Ltd /91/ differ after two dissimilar carbohydrate-loading procedures, performed in field conditions. The depletion exercises for the procedures involved: a halfmarathon race and an easier fartlek. Both procedures are commonly used by marathon runners. We also investigated the extent of the physical strain caused by the two depletion runs. Changes in serum testosterone, free testosterone and cortisol concentrations are used as indicators of strain8. The rationale for measuring these hormones is that serum testosterone and free testosterone concentrations have been shown to decrease and serum cortisol concentrations to increase as a consequence of heavy physical exercise9 10. Subjects and methods Subjects and experimental design After being informed of the experimental procedures and inherent risks, six well trained, national level, endurance runners (age 29 (±2) years, weight 66 (±2) kg, height 171 (±1) cm and training 552 (±35)km during the previous month - all figures being mean (±s.e.m.)) gave their written consent to participate in the study. Easy training, no more than 1 h per day, was allowed for the 3 days before the depletion run. The subjects performed two carbohydrate-loading procedures, but the depletion runs were different. Twenty-one days separated the two depletion runs, in order to avoid possible carry-over effects from the first procedure. Depletion and loading In the 'race' procedure, the depletion run involved a half-marathon road-race on relatively flat terrain. The subjects ran the distance in 74.2 (±0.5) min and their running speed was 3.5 min/km (4.8 mls), roughly equivalent to 70-80% of their Vo2, max. The race was followed by maximal 200 m spurts until voluntary exhaustion occurred (about 15 spurts). Their total running time was min. In the 'fartlek' procedure the running course differed and it was not totally flat; the athletes' total running time was the same as for the race ( min), but their mean Br J Sp Med 1991; 25(1) 41

22 Carbohydrate loading in practice: G. M. Fogelholm et al. Table 1. Experimental procedures Procedure 1: Race Procedure 2: Fartlek Samples Day-1 Easy running 60 min Easy running 60 min Depletion DayO Half-marathon race 100 min running Bm,M (3.5 min/km) and (4.0 min/km) x 200m until exhaustion Ba Day 1 Running min Running min Fr Loading Day 2 Easy running min Easy running min Bm Day 3 Easy running min Easy running min Fr Day 4 Easy running min Easy running min Fr Day 5 Easy running min Easy running min Fr Day 6 Rest Rest Bm,M Ba, post-run blood samples taken in the afternoon; Bm, fasting blood sample taken in the morning; Fr, food record; M, muscle biopsy taken in the morning running speed was slower (4.0 mi/km or 4.2 m/s), close to their normal training pace. On the first day after the depletion run, an min run was executed. The subjects ate a reduced carbohydrate diet for these 2 days. During the 4-day carbohydrate-loading phase, the subjects executed one easy 45-60min (4.5min/km) run per day. The experimental procedures are shown in Table 1. The athletes were instructed on how to choose foods and liquids that would ensure a reducedcarbohydrate diet during the depletion phase. More detailed instructions were provided for the highcarbohydrate diet. All subjects were instructed how to compose a basic diet1' that would ensure a daily carbohydrate intake of 400 g. They were then able to choose from several different food items, providing additional carbohydrate. This was to make certain that they increased their daily intake of carbohydrate to 9 g/kg body weight. The daily nutrient intake was calculated from the subjects' food diaries by a computer program12. To aid their food-recording process, all participants were given postal scales for weighing portions. During loading, there were no significant differences in the carbohydrate intake between the intense and moderate procedures (Table 2). The mean percentages of carbohydrate in the total energy intake were 70% for the race procedure and 67% for the fartlek. Table 2. Energy and carbohydrate intake of six marathon runners during two carbohydrate-loading procedures. Results are expressed as mean (±s.e.m.) Procedure 1: Procedure 2: Race Fartlek Energy (MJ/day) Depletion (days 0-1) 9.2 (±1.1) 7.9 (±0.8) Loading (days 2-5) 14.3 (±0.8) 15.6 (±1.0)* Carbohydrates (g/day) Depletion (days 0-1) 205 (±68) 145 (±35) Loading (days 2-5) 598 (±44) 626 (±54) * Significant difference (P < 0.05) between the procedures Blood analyses All blood samples were taken after a 15 min rest. The venous blood samples were drawn from the antecubital vein with the subjects in a sitting position. After an overnight fast, the morning samples were taken between 8.00 and hours. Samples were also drawn within 1 h after each depletion run. Only water, in unlimited amounts, was allowed between the run and the time of taking the sample. Radioimmunological methods were used for the measurement of serum cortisol and testosterone9. Serum sex hormone-binding globulin (SHBG) concentrations were determined by an immunoradiometric (IRMA) method (Farmos Diagnostica, Oulunsalo, Finland)13. Serum free-testosterone concentrations were calculated using testosterone and SHBG values'4. All measurements were carried out in duplicate, and in order to avoid interassay variation, all the assays of each subject were run in the same series. The intra-assay coefficients of variation for the analyses were 9.1% for plasma cortisol, 8.3% for plasma testosterone and 8.8% for SHBG9. Muscle analyses After local anaesthesia of the skin with lignocaine, without adrenaline, muscle biopsies were taken from the lateral portion of the quadriceps femoris muscle. Because we had previously taken several consecutive biopsies from that muscle, without the subjects experiencing complications, the vastus lateralis was chosen as the biopsy site'5. The muscle samples were taken before the depletion runs and at the end of the loading phase. The concentration of muscle glycogen was measured from jig of freeze dried cryostat sections after alkaline digestion and ethanol precipitation16. The samples were then analysed for glucose'7. The results are expressed in glycosyl units. Statistical analyses All results are expressed as mean (±s.e.m.). Analysis of variance for repeated measurements (BMDP 2V-package) was used for the statistical analyses. Significant (P < 0.05) time, procedure or time x procedure effects were further identified by Wilcoxon's signed rank test18. Results Normal muscle glycogen concentrations were observed before both procedures: the group mean values were 343 (±10) mmol/kg d.w. before the race procedure, and 345 (±11) mmol/kg d.w. before the fartlek. There were no differences (P > 0.05) in glycogen concentrations resulting from the two carbohydrate-loading procedures (Figure 1): the range of post-loading muscle glycogen concentration was mmol/kg d.w. after the race, and mmol/kg d.w. after the fartlek. The two very low glycogen levels (one after the race, the other after the fartlek) were not found in the same subject. After a total of 12 depletion and loading procedures (two 42 Br J Sp Med 1991; 25(1)

23 Carbohydrate loading in practice: G. M. Fogelholm et al _ E 0- -c ID a5 0 a) cn 2B um E 50 a 0 Predepletion Post-loading Before After After After run run depletion loading -0 0) Figure 2. Serum free-testosterone concentration during two glycogen depletion and loading procedures in six male runners. The procedures differed in the depletion run which was either hard (race) or moderate (fartlek). The results are expressed as mean ±s.e.m. Significant differences (P < 0.05) between the procedures and the changes from pre-run values are denoted with an asterisk. *, Moderate run; 0, hard run E a) CR CY$ -a) b Predepletion Post - loading Figure 1. Individual changes of muscle glycogen concentrations in the lateral portion of the quadriceps muscle before and after two depletion and loading procedures performed by six male runners: a procedure 1, race; b procedure 2, fartlek for each subject) were carried out, only five resulted in increased glycogen levels. A significant (P < 0.05) decrease in the serum free-testosterone concentrations (Figure 2) was observed after both depletion runs, but the decrease was more pronounced (P < 0.05) after the race run. Even 6 days after the half-marathon race, at the end of the loading phase, the free-testosterone concentrations were still significantly lower than the pre-run values. In five out of six subjects, the serum cortisol concentrations were higher after the race than those obtained after the fartlek. This difference did not reach statistical significance. Discussion In the present study, neither the race nor the fartlek procedure resulted in clearly increased glycogen concentrations in the subjects. In addition, no differences in final muscle glycogen levels between the procedures were observed. The failure to demonstrate an increase in glycogen concentration following either experiment was rather surprising, since these kinds of procedures are commonly carried out by marathon runners in Finland. It seems that higher glycogen concentrations, in the vastus lateralis muscle, do not necessarily occur after carbohydrateloading procedures in field conditions. Hence, the results agree well with the laboratory studies carried out by Blom et al.7. We admit that the easy runs, performed daily by all subjects during the loading phase, may be one reason why much higher muscle glycogen concentrations did not occur19. However, we did not want the subjects to rest because, in practice, marathon runners often perform easy exercises during the loading phase. A study to compare glycogen levels after complete rest and after easy running during the loading phase would be interesting. The muscle biopsies in the present study were taken from the vastus lateralis muscle, because we were familiar with the technique. It is possible that glycogen synthesis occurs more readily in the gastrocnemius muscle20. On the other hand, Karlsson and Saltin21 found increased glycogen levels in the vastus lateralis after a carbohydrate-loading regimen. Moreover, in the study of Blom et al.7, the results (normal glycogen concentrations, despite carbohydrate loading) were the same as ours, although they used biopsies taken from the gastrocnemius muscle. They also theorized that muscle fibre damage, sometimes associated with prolonged running22, might interfere with glycogen resynthesis. We doubt that the diets unduly influenced the above findings. We did not use a strict fat and protein diet during the depletion phase, but this should not Br J Sp Med 1991; 25(1) 43

24 Carbohydrate loading in practice: G. M. Fogelholm et al. impede glycogen resynthesis23. The carbohydrate intake, about 600g/day during the fartlek and race procedures, should have been high enough to ensure a maximal rate of glycogen synthesis6. Moreover, 40% of total carbohydrate was derived from wholegrain cereals, which contain a lot of complex carbohydrates. Complex carbohydrates might even stimulate glycogen resynthesis more effectively than mono- or disaccharides3. In other studies, a lower intake of carbohydrate has resulted in clearly increased glycogen concentrations in the vastus lateralis muscle All the subjects felt that the fartlek run was much less exhausting than the race. This subjective feeling of fatigue after the intense run was confirmed by hormonal analyses: serum testosterone and free testosterone concentration decreased after the intense run, which is in agreement with Dessypris et al.24 and Kuoppasalmi et al.9. Increased serum cortisol values, observed in five out of six subjects after the intense run, further indicated heavy physical strain. It can be conjectured that the low concentrations of free testosterone throughout the loading phase after the race impaired glycogen resynthesis. In rats, low serum testosterone concentrations may cause a reduction in glycogen synthesisl0'25. However, it is not known whether this occurs in man as well. In conclusion, we studied a total of 12 glycogen depletion and loading procedures in six athletes in field conditions, and only five out of 12 resulted in higher glycogen concentrations in the vastus lateralis muscle after rather than before the procedure. Different depletion runs did not affect post-loading glycogen levels. Since an exhaustive depletion exercise might lead to physical overstrain, we do not recommend it for marathon runners. Acknowledgements This study was supported financially by the Finnish Olympic Committee. We also thank Ms Sirpa Tamminen and Ms Helena Taskinen for their skillful technical assistance. References 1 Bergstrom J. Hermansen L, Hultman E, Saltin B. Diet, muscle glycogen and physical performance. Acta Physiol Scand 1967; 71: Saltin B, Hermansen L. Glycogen stores and prolonged severe exercise. In: Blix G, ed. Symposia of the Swedish Nutrition Foundation V: Nutrition and Physical Activity. Stockholm: Almqvist & Wiksell, Costill DL, Sherman WM, Fink WJ, Maresh C, Witten M, Miller JM. The role of dietary carbohydrates in muscle glycogen resynthesis after strenuous running. Am J Clin Nutr 1981; 34: Sherman WM, Costill DL, Fink WJ, Hagerman FC, Armstrong LE, Murray TF. Effect of a 42.4-km footrace and subsequent rest or exercise on muscle glycogen and enzymes. I Appl Physiol 1983; 55: Roberts KM, Noble EG, Hayden DB, Taylor AW. Simple and complex carbohydrate-rich diets and muscle glycogen content of marathon runners. Eur J Appl Physiol 1988; 57: Sherman WM, Costill DL. The marathon: dietary manipulation to optimize performance. Am i Sports Med 1984; 12: Blom PCS, Costill DL, Vollestad NK. Exhaustive running: inappropriate as a stimulus of muscle glycogen supercompensation. Med Sci Sports Exerc 1987; 19: Adlercreutz H, Harkonen M, Kuoppasalmi K et al. Effect of training on plasma anabolic and catabolic steroid hormones and their response during physical exercise. Int J Sports Med 1986; 7 Suppl: Kuoppasalmi K, Naveri H, Harkonen M, Adlercreutz H. Plasma cortisol, androstenedione, testosterone and luteinizing hormone in running exercise of different intensities. Scand J Clin Lab Invest 1980; 40: Urhausen A, Kullmer T, Kinderman W. A 7-week follow-up study of the behaviour of testosterone and cortisol during the competition period in rowers. Eur J Appl Physiol 1987; 56: Fogelholm GM, Tikkanen HO, Naveri, HK, Harkonen MHA. High-carbohydrate diet for long distance runners - a practical view-point. Br J Sports Med 1989; 23: Ahlstrbm A, Rasanen L, Kuvaja K. A method of data processing for food consumption surveys. Ann Acad Sci Fenn [Al IV. 1972; 194: Hamalainen E, Tikkanen H, Harkonen M, Naveri H, Adlercreutz H. Serum lipoproteins and sex hormone binding globulin in middle-aged men of different physical fitness and risk of coronary heart disease. Atherosclerosis 1987; 67: Anderson D. The role of sex hormone binding globulin in health and disease In: James VHT, Serio M, Giusti G, eds. The Endocrine Function of the Human Ovary. Proceedings of the Serono Symposia Vol. 7. London: Academic Press, Rehunen S, Harkonen MHA. High-energy phosphate compounds in human slow-twitch and fast-twitch muscle fibres. Scand J Clin Lab Invest 1980; 40: Good CA, Kramer H, Somogyi M. The determination of glycogen. I Biol Chem 1933; 100: Harkonen M, Ndveri H, Rehunen S, Kuoppasalmi K. Determination of metabolites and enzymes in muscle. Ann Clin Res 1982; 14 (Suppl. 34): Langley RL. Practical Statistics. London: Pan Books, Bonen A, Ness GW, Belcastro AN, Kirby RL. Mild exercise impedes glycogen repletion in muscle. J Appl Physiol 1985; 58: Costill DL, Jansson E, Goilnick PD, Saltin B. Glycogen utilization in leg muscles of men during level and uphill running. Acta Physiol Scand 1974; 91: Karlsson J, Saltin B. Diet, muscle glycogen, and endurance performance. J Appl Physiol 1971; 31: Hikida RS, Staron RS, Hagerman FC, Sherman WM, Costill DL. Muscle fiber necrosis associated with human marathon runners. J Neurol Sci 1983; 59: Sherman WM, Costill DL, Fink WJ, Miller JM. Effect of exercise-diet manipulation on muscle glycogen and its subsequent utilization during performance. Int J Sports Med 1981; 2: Dessypris A, Kuoppasalmi K, Adlercreutz H. Plasma cortisol, testosterone, androstenedione and luteinizing hormone (LH) in a non-competitive marathon run. J Steroid Biochem 1976; 7: Guezennec CV, Ferre P. Serrurier DB, Merino D, Pesquies PC. Effects of prolonged physical exercise and fasting upon plasma testosterone level in rats. Eur J Appl Physiol 1982; 49: Br J Sp Med 1991; 25(1)

25 Journal of Biomolecular Structure and Dynamics ISSN: (Print) (Online) Journal homepage: Modelling the Krebs cycle and oxidative phosphorylation Kalyani Korla & Chanchal K. Mitra To cite this article: Kalyani Korla & Chanchal K. Mitra (2014) Modelling the Krebs cycle and oxidative phosphorylation, Journal of Biomolecular Structure and Dynamics, 32:2, , DOI: / To link to this article: Published online: 25 Mar Submit your article to this journal Article views: 384 View related articles View Crossmark data Citing articles: 5 View citing articles Full Terms & Conditions of access and use can be found at Download by: [Simon Fraser University] Date: 02 April 2017, At: 12:17

26 Journal of Biomolecular Structure and Dynamics, 2014 Vol. 32, No. 2, , Modelling the Krebs cycle and oxidative phosphorylation Kalyani Korla and Chanchal K. Mitra* School of Life Sciences, University of Hyderabad, Hyderabad , India Communicated by Ramaswamy H. Sarma (Received 21 September 2012; final version received 19 December 2012; final version received 21 December 2012) The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen setup for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the ph gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance. Keywords: Krebs cycle; electron transport chain; ETC; kinetic simulation; ATP synthesis Introduction Mitochondria are the so-called Powerhouses of the cell ; the major part of ATP under normal conditions is synthesised by oxidative phosphorylation, which essentially takes place in mitochondria (Figure 1). Oxidative phosphorylation is preceded by the Krebs cycle and is tightly coupled with it in terms of both spatial location and function. The Krebs cycle along with the oxidative phosphorylation, together pave the way for ATP synthesis in the mitochondria. The overall process can be divided into three parts: (i) the Krebs cycle, (ii) the electron transport chain (ETC) and (iii) the ATP synthesis. We have attempted to study the overall reaction kinetic model using conventional tools and the reactions in the near steady state. Although the metabolic pathways have been worked out in great detail by experimentalists using various tools and techniques, the overall dynamics of the processes remain poorly understood. Whereas the pathways represent a static picture, the dynamics can accurately predict the roles of the various components under different conditions, in particular under disease or stress conditions. Therefore these results are likely to be useful in various studies, including studies on the effects of drugs, predicting the cell cycle (including the dynamics leading to cell division) and related processes and also in studies involving several diseases originating from metabolic disorders. The Krebs cycle The Krebs cycle is a nearly universal key metabolic pathway which forms the second step of cellular respiration after glycolysis. Acetyl-CoA (Ac-CoA) formed by the oxidation of pyruvate (also from fatty acid and amino acid metabolism) enters the Krebs cycle to combine with oxaloacetate to form citrate in a reaction catalysed by citrate synthase. Pyruvate can also enter the Krebs cycle through a side reaction catalysed by pyruvate carboxylase (Kornberg, 1966) to give oxaloacetate, an intermediate in the cycle (Figure 2). The formation of citrate is followed by seven sequential reactions to regenerate oxaloacetate with the release of two CO 2 molecules. In one complete cycle, one molecule of Ac-CoA is oxidised and the energy is trapped by the reduction of three molecules of NAD + to NADH, one molecule of FAD to FADH 2 and formation of one molecule of nucleoside triphosphate (ATP or GTP). *Corresponding author. addresses: c_mitra@yahoo.com; ckmsl@uohyd.ernet.in Ó 2013 Taylor & Francis

27 Journal of Biomolecular Structure & Dynamics 243 Figure 1. Structure of mitochondrion (Figure taken from wikipedia: Mitochondria in animals, including humans, possess two succinyl-coa synthetases: one that produces GTP from GDP and another that produces ATP from ADP (Johnson, Mehus, Tews, Milavetz, & Lambeth, 1998). NAD + and NADH are the components belonging to the common pool within the mitochondrion and shared by various reactions in the cell, whereas FAD FADH 2 couple is tightly bound to the enzyme, succinate dehydrogenase, and are directly channelled into the ETC (Stryer, Berg, & Tymoczko, 2002), further leading to ATP synthesis. This enzyme (succinate dehydrogenase) is a transmembrane protein and is common to both the Krebs cycle and the ETC. The overall reaction, along with the component reactions, is compiled in Table 1. The intermediates of the Krebs cycle do not appear in the overall reaction which consists of oxidation of Ac-CoA to CO 2. The energy released in the process is used for synthesis of GTP and/ or ATP (via the ETC). The reduction of NAD + to NADH is also accomplished during this process. As these reactions are well known in the literature, we shall limit ourselves to the essential features needed in this study. The individual reactions are summarised in Table 1 and a scrutiny of the overall reaction clearly shows that the complete cycle acts like a giant catalyst, oxidising Ac-CoA to carbon dioxide by NAD and FAD and a concomitant formation of GTP from GDP. The overall rate is therefore dependent on the amount of the catalyst present and this is controlled by injecting oxaloacetate into the cycle through a side reaction. The complete cycle is under tight regulation and is maintained by the rate of conversion of pyruvate to Ac-CoA and by the flux through citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase Figure 2. Overview of the Krebs cycle (taken from wikipedia:

28 244 K. Korla and C.K. Mitra Table 1. Reactions involved in the Krebs cycle. Acetyl CoA þ Oxaloacetate þ H 2 O! Citrate þ CoA SH (1) Citrate! Isocitrate (2) Isocitrate þ NAD þ! a Ketoglutarate þ NADH þ H þ þ CO 2 (3) a Ketoglutarate þ CoA SH þ NAD þ! Succinyl CoA þ NADH þ H þ þ CO 2 (4) Succinyl CoA þ GDP þ Pi! Succinate þ GTP þ CoA SH (5) Succinate þ FAD! Fumarate þ FADH 2 (6) Fumarate þ H 2 O! Malate (7) Malate þ NAD þ! Oxaloacetate þ NADH þ H þ (8) Acetyl CoA þ 2H 2 O þ 3NAD þ þ GDP þ Pi þ FAD!3NADH þ 3H þ þ FADH 2 þ GTP þ CoA SH þ 2CO 2 Note: The last line shows the overall reaction (Pi = inorganic phosphate). (they are the regulatory components in the cycle). These fluxes (enzyme turnover rates) are mainly controlled by the concentration of substrates and products: the end products as NADH, ATP and citrate show an inhibitory effect (Nelson & Cox, 2005) and the substrates as NAD + and ADP are stimulatory (Table 2). Oxidative phosphorylation Oxidative phosphorylation is the next step of cellular respiration preceded by glycolysis and the Krebs cycle. It includes the ETC and ATP synthesis. The first four enzyme complexes in Figure 3 comprise ETC and the fifth complex carries out ATP synthesis. Electron transport chain There are two independent paths for electron transfer i.e. (a) from NADH via complexes I, III and IV and (b) from FADH 2 via complexes II, III and IV. Both processes result in the reduction of quinone to hydroquinone (quinol). The oxidation of NADH by quinone begins with complex I (NADH: ubiquinone oxidoreductase) that facilitates the exergonic transfer of a hydride ion from NADH and a proton from the matrix to ubiquinone. This reaction is tightly coupled with translocation of upto four protons from the mitochondrial matrix to the inter-membrane (IM) space (Wikstrom, 1984). As this reaction is coupled to proton translocation, the rate of the overall oxidation depends on the ph gradient across the membrane (in other words, if the ph gradient is too high, the proton translocation stops and the NADH oxidation also stops). The electrons are next transferred from reduced quinone (hydroquinone) to cytochrome c (cyt c) by complex III (ubiquinone:cyt c oxidoreductase). Cyt c is a heme protein and two molecules of cyt c are needed for the oxidation of one molecule of quinol. It appears that two protons from QH 2 and two protons from the matrix are also translocated to the IM space in this step. In other words, this complex is also regulated by the ph gradient across the membrane. The final transport of electrons is from the reduced cyt c to O 2 reducing it to H 2 O by complex IV (cyt c oxidase). In this step, two protons are translocated from the matrix to the IM space and two protons are consumed to form water. Complete Table 2. The enzymes of the Krebs cycle with their reactants, products, inhibitors, activators and EC number (Brenda database) (Pi = inorganic phosphate) Reaction step Enzyme (EC number) Input Output Inhibitor Activator Citrate formation Isocitrate formation α-ketoglutarate formation Succinyl-CoA formation Succinate formation Fumarate formation Malate formation Oxaloacetate formation citrate synthase (EC ) Oxaloacetate, Acetyl Citrate, CoA-SH CoA, H 2 O Aconitase (EC ) Citrate Isocitrate NADH, succinyl-coa, citrate, ATP 2+ Isocitrate dehydrogenase Isocitrate, NAD + α-ketoglutarate, (EC ) NADH + +H +,CO 2 ATP Ca, ADP α-ketoglutarate A-Ketoglutarate, Succinyl-CoA, NADH + Succinyl-CoA, NADH Ca 2+ dehydrogenase (EC ) CoA-SH, NAD + +H +,CO 2 Succinyl-CoA synthetase Succinyl-CoA, GDP Succinate, GTP, CoA- (EC ) +Pi SH Succinate dehydrogenase Succinate, FAD Fumarate, FADH 2 (EC ) Fumarase (EC ) Fumarate, H 2 O Malate Malate dehydrogenase (EC ) Malate Oxaloacetate, NADH + +H + ADP

29 Journal of Biomolecular Structure & Dynamics 245 Figure 3. Diagrammatic representation of oxidative phosphorylation. Figure taken from show_pathway?map Figure shows the flow of two electrons from left to right, resulting in the formation of one molecule of water. About three molecules of ATP are produced from the oxidation of one molecule of NADH. reduction of one molecule of oxygen takes four electrons and therefore four cytochromes c (reduced form) are involved in the reduction of oxygen molecule in a complex set of reactions (Table 3). The oxidation of FADH 2 starts at the complex II (succinate dehydrogenase), which is also a part of the Krebs cycle. It facilitates the transfer of two electrons and two protons from FADH 2 to quinone (Cecchini, 2003). There is no IM proton translocation in this step. Further transfer of electrons from ubiquinone remains the same for FADH 2 as for NADH via complexes III and IV stated above. Complex II is regulated internally within the Krebs cycle. The oxidations of NADH and FADH 2 are essentially independent and either or both of them can be indifferently used by the ETC system. For one NADH oxidation, translocation of upto 10 protons (for FADH 2 translocation of upto six protons) from mitochondrial matrix to IM space is expected. This translocation of protons from the matrix to the IM space results in the formation of ph gradient (ΔpH). Because of the unequal volume of the two sides of the membrane, translocation of a given number of protons across the membrane will produce unequal changes of ph on two sides. Also, an electrical (or membrane) potential gradient (ΔΨ) is formed due to separation of charges. The flux of other ions (Ca 2+,K +,Na + etc.) across the inner Table 3. The reactions involved in ETC. NADH þ H þ þ Q! NAD þ þ QH 2 (9) FADH 2 þ Q! FAD þ QH 2 (10) QH 2 þ 2cytc ðoþ! Q þ 2cytc ðrþ (11) O 2 þ 4cytc ðrþ þ 4H þ! 4cytc ðoþ þ 2H 2 O (12) mitochondrial membrane also contributes to the electrical potential gradient. This electrochemical gradient, also called the protonmotive force (pmf), drives ATP synthesis via ATP synthase. It has been reported that one component of the electrochemical gradient can drive synthesis even when the other opposes and the rate of synthesis of ATP depends on the algebraic sum of the two (i.e. the pmf) and both contribute to the kinetics for any combination of ΔpH between 0.3 and 2.2 and ΔΨ between 30 and 140 mv (pmf up to 250 mv), irrespective of the individual magnitudes and signs (Soga, Kinosita, Yoshida, & Suzuki, 2012). The accurate measurement of each component of the pmf and the factors involved therein is experimentally difficult. pmf ¼ DW 2:303RT F DpH The change in chemical gradient (ΔpH) is bound to influence the electrical gradient as it also involves a charge separation and therefore individual parameters (ΔΨ and ΔpH) cannot be studied in isolation. Membrane potential is regulated essentially by cation concentration imbalance across the membrane and is controlled by active pumps. The role of such pumps (present elsewhere within the mitochondrion) in the ETC has not been clearly established. The details of the enzymes involved in the ETC are mentioned in Table 4. It has to be noted that there are no activators/ inhibitors in the native system; the ETC is regulated by the influx of its substrates (NADH and FADH 2 ) and the complexes I, III and IV are functionally ph dependent (complex II is internally regulated by the Krebs cycle but is ph independent).

30 246 K. Korla and C.K. Mitra Table 4. The enzymes of the ETC with their reactants and products. Reaction step Enzyme (EC number) [5] Input Output NADH to Ubiquinone NADH:ubiquinone oxidoreductase (EC ) NADH, H +, Q NAD +,QH 2 Succinate to Ubiquinone Succinate dehydrogenase (EC ) FADH 2, Q FAD, QH 2 Ubiquinone to cyt c Ubiquinone: cyt c oxidoreductase (EC ) QH 2, oxidised cyt c, H + Q, reduced cyt c cyt c to O 2 Cytochrome oxidase (EC ) O 2, reduced cyt c, H + H 2 O, oxidised cyt c ATP synthesis The last part of oxidative phosphorylation is ATP synthesis. It can be considered as coupled indirectly to the ETC via IM potential and ph gradient (chemiosmotic coupling). Experimental evidence suggests that P/O ratios of oxidative phosphorylation (the ATP produced per oxygen atom reduced by the respiratory chain) is about 2.5 with NADH-linked substrates (via complexes I, III and IV) and 1.5 with FADH 2 (via complexes II, III and IV) (Hinkle, 2005). Although the two processes, ETC and ATP synthesis, are physically independent and have been shown to function independently in experiments using inhibitors/uncouplers, they remain coupled to each other via the pmf across the membrane (Table 5). In the present work, the three processes explained above are simulated to present a behavioural perspective of the components of the system. For this purpose, we have used GNU Octave ( Table 5. The details of the reactions involved in ATP Synthesis (Weber & Senior, 2003). P þ E þ H þ out! E P þ Hþ in E P þ MgADP þ H þ out! E P MgADP þ Hþ in E P MgADP þ H þ out! E MgATP þ Hþ in E MgATP þ H þ out! E þ MgATP þ Hþ in MgADP þ P þ 4H þ out! MgATP þ 4Hþ in (13) (14) (15) (16) Notes: The last line shows the overall reaction. P stands for phosphate and E is the enzyme. H + out stands for proton in the IM space and H + in stands for protons in the matrix. Materials and methods Octave GNU Octave is a high-level interpreted language, primarily intended for numerical computations. It provides the numerical solution of linear and non-linear problems, and performs other numerical experiments. It also provides extensive graphics capabilities for data visualization and manipulation (GNU Octave). We have used Octave for simulation of the pathways using ODE (ordinary differential equation solver). Rate equations were derived for each component. The three sets of reactions viz. the Krebs cycle, ETC and ATP synthesis were first individually simulated and were then synchronised to form a single programme including all reactions and their components. Each of the components has been given a unique name and the same nomenclature is used every time the particular component was addressed. In this report, we have considered 1:1:1 stoichiometry (the Krebs cycle produces 3NADH + 1FADH 2 : we need three runs of complexes I, III, IV and a single run for complexes II, III and IV; producing effectively 36 proton translocation and producing nine ATP molecules) for the three sets of reactions but this can be very easily modified and the results obtained for different conditions. The rate equations We have used Michaelis Menten approximation as the basic model for single substrate, single intermediate enzyme catalysis. For bisubstrate reactions, we have made a few reasonable assumptions and extended this approach: can be reduced to v ¼ V max½sš K M þ½sš v V max ¼ ½SŠ=K M 1 þ½sš=k M and both v/v max and [S]/K M are now dimensionless quantities and are good candidates in numerical simulations. For multiple substrates, we shall use an extended formula: v V max ¼ ½S 1Š=K M1 ½S 2 Š=K M2 ½S 3 Š=K M3 1 þ½s 1 Š=K M1 1 þ½s 2 Š=K M2 1 þ½s 3 Š=K M3 which can be considered as an approximation, at least to the first order. two types of inhibitions are widely known: competitive inhibition and non-competitive inhibition. In case of the former, the inhibitor binds at the substrate binding site (active site) and increases the apparent K M of the substrate (V max is unaffected). In case of the latter, the inhibitor binds at an unrelated site (regulatory site) and decreases

31 Journal of Biomolecular Structure & Dynamics 247 the V max of the enzyme (K M is unaffected). None of the listed enzymes show allostery. For competitive inhibition, we need to replace K M in the first equation with K M ð1 þ½iš=k I Þ, where [I] is the concentration of the inhibitor and K I is the enzyme inhibitor dissociation constant. Therefore the final equation is: v ¼ and can be simplified to V max ½SŠ K M ð1 þ½iš=k I Þþ½SŠ v ½SŠ=K M ¼ : V max 1 þ½iš=k I þ½sš=k M For non-competitive inhibition: here K M is not affected but V max is reduced by the same factor, i.e. V max! V max =ð1 þ½iš=k I Þ, which gives, using the same notation as before for the inhibitor and the enzyme-inhibitor dissociation constant (i.e. K I ), v ¼ V max½sš=ð1 þ½iš=k I Þ K M þ½sš which simplifies to give the following equation: v ½SŠ=K M ¼ V max ð1 þ½iš=k I Þð1þ½SŠ=K M Þ which is approximately the same as for the competitive inhibition, at least to the first order (i.e. if we ignore terms containing both [I] and [S]). This clearly suggests that the exact nature of inhibition is not too important, as far as the kinetics is concerned. We have generated the numerical solutions for the one substrate and two substrates ordered sequential reactions. The results are shown in Figure 4 along with the models used in the present study (Michaelis Menten and modified Michaelis Menten). In early stages of the reaction, MM equation always predicts a higher turnover. The simulated product concentration graph is tangent to the x-axis in both cases. The enzyme substrate complex (ES, AE and AEB) concentrations show maxima in both cases. For two substrate reactions, the MM equation is symmetric but the actual reaction is not. In all cases, early reaction time refers to time on the left of the maximum of the enzyme substrate complex. We also have used the stoichiometric equations in cases where the exact reaction mechanism is not known. The quasi steady state approximation (QSSA) has been widely used in chemical kinetics but its significance, applicability and accuracy has been subject matter of extensive discussion (Ciliberto, Capuani, & Tyson, 2007; Flach & Schnell, 2006; Pedersen, Bersani, & Bersani 2008; Schnell & Maini, 2003). The QSSA (and its variants) assumes that all intermediates attain constant concentrations rapidly shortly after the reaction initiation. This is however an approximation and as we can see in Figure 4, the concentrations of the intermediates (ES, AE and AEB) are zero initially and also very small at long times. These concentrations therefore go through maxima somewhat early in the reaction processes. During this early phase of the reaction, the QSSA is obviously inapplicable. However, after these maxima of the intermediates the concentrations change rather slowly and the QSSA is applicable as a first approximation (Pedersen, Bersani, & Bersani et al., 2007). The quality of the approximation is very difficult to estimate and the approximations depend on the exact values of the parameters used in simulation. In Figure 4, we can observe that the MM equation is a poor fit to the exact equation in the early stages, but becomes better as the reaction progresses. Moreover, MM equation is well behaved in simulation under various conditions. However, the present simulation reported in this work must be considered as semi-quantitative only. Simulation of the Krebs cycle Table 6 shows all the rate equations used for simulation of the Krebs cycle, this includes all the regulatory feedback components at various stages as given in Table 2. These equations do not include the side injection of oxaloacetate via pyruvate carboxylase (Figure 2) into the cycle. Simulating the ETC The first step towards the modelling of the ETC is deriving the rate equations. We have found that all complexes are very complex in structure and function and the experimental evidences do not suggest Michaelis Menten behaviour. The detailed reaction mechanisms and the rate determining steps are not available for all the complexes in the literature. Complexes I, III and IV are tightly coupled with proton translocation. It has been reported that if protons are not translocated, the electron transfer also ceases. This proton pumping results either in the increase in proton concentration or the decrease in ph of the IM space. The exact change in ph will depend on the number of protons translocated and the total volume of the IM space and the matrix. As the catalytic sites are located on the matrix side, they are more affected by the ph of the matrix and this was taken into account in our simulations. The important task is to couple the proton translocation with the rate equations of oxidation reductions taking place at the enzyme complexes. There are few conditions which have to be satisfied by the final rate

32 248 K. Korla and C.K. Mitra Figure 4. Simulation results for one substrate and two substrate reactions using Octave. The figure on the left shows one substrate reaction using standard MM equation (thin black curve). We have also plotted the exact solutions for the substrate (blue) and enzyme-substrate complex (cyan) and the final product (magenta). The MM curve shows higher concentrations compared to the exact curve. The general behaviour of both the curves is however the same at long times. The figure on the right shows the kinetics for two substrate, one enzyme sequential binding (A + E AE + B AEB P), for both exact solution (magenta) and using the modified MM equation (black thin line). For reference, we have also shown the concentrations of substrates A and B along with the concentrations of AE and AEB. In the modified MM equation, both A and B are symmetrical but the exact equation shows distinct differences. equations. If proton gradient becomes high, it should inhibit further translocation and electron transfer. Rate of translocation has to be coupled with the reactant concentrations. The redox reactions are theoretically and practically reversible, although in this case the reversible reactions will be effectively uncoupled from the proton translocation pump. The protons translocated will not be part of the rate equations (Table 7) but will be incorporated in the rate constant, thus the rate constant would be dependent on the ph and on the enzyme characteristics. The ph gradient would change with the translocation of each proton, as one proton translocated from the matrix to IM would mean one proton less in matrix and one proton more in the IM, thus making a difference of two protons (as far as the ΔpH is concerned). Keeping this in view, the ph has to be varied after each step of the ETC, particularly after the reactions of complexes I, III and IV. Rate constants for these reactions would be varied due to ph by a factor of 1/(1+(ΔpH) 2 ). We assume the ph of the IM to be essentially constant (as the difference in ph is important and not the individual ph values) and ph of the matrix to be variable. The ETC components are embedded in small invaginations of the cristae, thus the local change in ph due to transfer of few protons is expected to be substantial. Increased surface charge density of protons is predicted to change the ph by 0.5 ph units (Strauss, Hofhaus, Schröder, & Kühlbrandt, 2008). It appears that the basic reactions within the ETC are not subject to any feedback regulations but the proton translocation for each complex is determined by the ph gradient and therefore the overall ETC is regulated by the ph of the matrix. It is well known (Figure 3) that the chemically functional part of the enzymes of the ETC is located on the matrix side of the membrane. These enzymes are more susceptible to the ph of the matrix rather than to the ΔpH existing across the membrane. However, this may not apply to ATP synthase, where the ΔpH drives the protons across and ΔΨ determines the electric field and hence the energy of the proton. Computing the ΔpH It is sufficient to show the ph change taking place on the matrix side as computing the ph change on the IM space will follow an identical procedure. Assuming that the ph of the matrix is x, the hydrogen ion concentration is 10 x. We further assume that n moles of protons are translocated into the matrix due to some process and the volume of the matrix space (cristae region; locally accessible volume) is v. The concentration of protons in the matrix phase is therefore increased to ð10 x þ n=vþ; corresponding to a new ph equal to log x þ n ¼ x log v 10 1 þ 10x n : v When the term 10x n v is less than 1, we can linearise the logarithm getting x 2:303 1 n v 10x as the new ph, from which one gets DpH ¼ 0:4343ðn=vÞ10 ph :

33 Journal of Biomolecular Structure & Dynamics 249 Table 6. Rate equations used for simulating the Krebs cycle. d dt ½OxAcŠ ¼ ½OxAcŠ 1 þ½oxacš : ½AcCoAŠ 1 þ½accoaš : 1 1 þ½accoaš : 1 1 þ½citš : 1 1 þ½succoaš þ ½MalŠ 1 þ½malš : ½NADŠ 1 þ½nadš d dt ½AcCoAŠ ¼ ½OxAcŠ 1 þ½oxacš : ½AcCoAŠ 1 þ½accoaš : 1 1 þ½accoaš : 1 1 þ½citš : 1 1 þ½succoaš d dt ½CitŠ ¼ ½OxAcŠ 1 þ½oxacš : ½AcCoAŠ 1 þ½accoaš : 1 1 þ½accoaš : 1 1 þ½citš : 1 ½CitŠ 1 þ½succoaš 1 þ½citš d dt ½IsoCitŠ ¼ ½CitŠ ½Iso CitŠ 1 þ½citš 1 þ½iso CitŠ : ½NADŠ 1 þ½nadš d dt ½KeGluŠ ¼ ½Iso CitŠ 1 þ½iso CitŠ : ½NADŠ ½KeGluŠ 1 þ½nadš 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 1 þ½succoaš d dt ½SucCoAŠ ¼ ½KeGluŠ 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 ½SucCoAŠ 1 þ½succoaš 1 þ½succoaš : ½GDPŠ 1 þ½gdpš d dt ½SucŠ ¼ ½SucCoAŠ 1 þ½succoaš : ½GDPŠ ½SucŠ 1 þ½gdpš 1 þ½sucš : ½FADŠ 1 þ½fadš d dt ½FumŠ ¼ d dt ½MalŠ ¼ d dt ½CoASHŠ ¼ d dt ½SucŠ 1 þ½sucš : ½FADŠ ½FumŠ 1 þ½fadš 1 þ½fumš ½FumŠ ½MalŠ 1 þ½fumš 1 þ½malš : ½NADŠ 1 þ½nadš ½OxAcŠ 1 þ½oxacš : ½AcCoAŠ 1 þ½accoaš : 1 1 þ½accoaš : 1 1 þ½citš : 1 ½KeGluŠ 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 ½NADŠ ¼ ½IsoCitŠ 1 þ½isocitš : ½NADŠ 1 þ½nadš d dt ½NADHŠ ¼ d dt ½CO 2Š¼ d dt 1 þ½succoaš þ ½SucCoAŠ 1 þ½succoaš : ½GDPŠ 1 þ½gdpš 1 þ½succoaš ½KeGluŠ 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 ½MalŠ 1 þ½succoaš 1 þ½malš : ½NADŠ 1 þ½nadš ½IsoCitŠ 1 þ½isocitš : ½NADŠ 1 þ½nadš þ ½KeGluŠ 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 1 þ½succoaš þ ½MalŠ 1 þ½malš : ½NADŠ 1 þ½nadš ½IsoCitŠ 1 þ½isocitš : ½NADŠ 1 þ½nadš þ ½KeGluŠ 1 þ½kegluš : ½CoASHŠ 1 þ½coashš : ½NADŠ 1 þ½nadš : 1 1 þ½succoaš ½GDPŠ ¼ ½SucCoAŠ 1 þ½succoaš : ½GDPŠ 1 þ½gdpš d dt ½GTPŠ ¼ ½SucCoAŠ 1 þ½succoaš : ½GDPŠ 1 þ½gdpš d dt ½FADŠ ¼ ½SucŠ 1 þ½sucš : ½FADŠ 1 þ½fadš d dt ½FADH 2Š¼ ½SucŠ 1 þ½sucš : ½FADŠ 1 þ½fadš Notes: OxAC = oxaloacetate, AcCoA= acetyl CoA, Cit = citrate, SucCoA= succinyl CoA, IsoCit = isocitrate, KeGlu = α-ketoglutarate, Suc = succinate, Fum = fumarate, Mal = malate, NAD = NAD +. where n is usually known exactly from the reaction mechanism but estimation of v is usually approximate. From electron microscopy data, we can assume that the typical linear size for a mitochondrion is 500 nm, corresponding to a volume of l. We assume the total matrix volume to be 50% of the mitochondrion and also note that one hydrogen ion moles. Using the above formula, we note that one proton translocation

34 250 K. Korla and C.K. Mitra Table 7. Rate equations used for simulating the ETC. 1 (17) k ¼ 1 þðdphþ 2 d ½NADHŠ ½NADHŠ ¼ k dt 1 þ½nadhš : ½QŠ (18) 1 þ½qš d dt ½FADH 2Š¼ ½FADH 2Š 1 þ½fadh 2 Š : ½QŠ (19) 1 þ½qš d ½NADHŠ ½QŠ ¼ k dt 1 þ½nadhš : ½QŠ 1 þ½qš ½FADH 2Š 1 þ½fadh 2 Š : ½QŠ 1 þ½qš þ k ½QH 2Š ½Cyt c ðoþ Š 2 (20) : 1 þ QH 2 1 þ½cyt c ðoþ Š 2 d dt ½QH ½NADHŠ 2Š¼k 1 þ½nadhš : ½QŠ 1 þ½qš þ ½FADH 2Š 1 þ½fadh 2 Š : ½QŠ 1 þ½qš k ½QH 2Š ½Cyt c ðoþ Š 2 (21) : 1 þ QH 2 1 þ½cyt c ðoþ Š 2 d dt ½Cyt c ðoþš ¼ k ½QH 2Š ½Cyt c ðoþ Š 2 : 1 þ QH 2 1 þ½cyt c ðoþ Š 2 þ k ½Cyt c ðrþ Š ½O 2 Š 1 þ½cyt c ðrþ Š 4: 1 þ½o 2 Š d dt ½Cyt c ðrþš ¼k ½QH 2Š ½Cyt c ðoþ Š 2 4 : 1 þ QH 2 1 þ½cyt c ðoþ Š 2 k ½Cyt c ðrþ Š ½O 2 Š 1 þ½cyt c ðrþ Š 4: 1 þ½o 2 Š d dt ½O ½Cyt c ðrþ Š 4 ½O 2 Š 2Š¼ k 1 þ½cyt c ðrþ Š 4: 1 þ½o 2 Š Notes: Q = quinone, QH 2 = quinol, cyt c (o) = cytochrome c oxidised form, cyt c (r) = cytochrome c reduced form. 4 (22) (23) (24) can change the matrix ph by 0.04 ph units at ph = 7. However, the matrix usually contains dozens of cristae and ΔpH was somewhat arbitrarily chosen as 0.2 (per proton translocated out of the matrix). Without loss of generality, we have assumed that the ph of the IM is fixed at 6.0 and the changes in the matrix cause the ph gradient. However, we explicitly consider the different number of protons translocated in different reactions (complexes I, III, IV and V). These assumptions can be easily modified in the simulation. ph dependence of the rate constants Although the rate constants of the ETC have no specific feedback regulation, experimental evidences show that the reaction kinetics is strongly ph dependent (Jain & Nath, 2001). This is understandable as complexes I, III and IV are also proton pumps and they are strongly coupled to the electron transfer process. Once a ph gradient has been established, the proton pumps stop and consequently the coupled redox reactions also stop. We note that the functional units of the above complexes are on the matrix side of the membrane and therefore more sensitive to the matrix ph. Using arguments similar to those used by Monod for allostery (Monod, Changeux, & Jacob 1963), we propose to model the kinetic constants as function of ph with the relation: 1 k! k 1 þðph 6Þ ¼ k þðdphþ 2 1 ¼ k 1 þð0:4343ðn=vþ10 ph Þ 2 where ph is the matrix ph and six is the IM space ph. As ph increases, k decreases in a sigmoidal fashion, as evidenced experimentally (Jain & Nath, 2001). For the ATP synthase enzyme, a similar functional dependence is proposed: ðph 6Þ 4 4 k! k 1 þðph 6Þ ¼ k ðdphþ 4 1 þðdphþ 4 Here, we see a strongly sigmoidal increase of the rate of ATP synthesis with the matrix ph. Simulating ATP synthesis ATP synthesis is catalysed by ATP synthase, an enzyme localised on the inner mitochondrial membrane. The enzymatic mechanism of ATP synthesis is widely explained by the binding change mechanism proposed by Boyer (1993). So the rate of ATP synthesis = k [MgADP] [Pi] [H + out] 4. The proton concentration is usually included in the rate constant (k) value and therefore we introduce a new k, which becomes ph dependent new k = k [H + out] 4 indicating the ph dependence of k. Thus, the reaction used for forming the rate equation is as follows: MgADP þ Pi! MgATP Rate constants for these reactions would be varied due to ph by a factor as suggested above. The efflux of protons will be mainly responsible for the change of ph of the matrix, but the actual changes depend on the local (and relative) volumes of the cristae (for the matrix) and the inter-membrane space.

35 Journal of Biomolecular Structure & Dynamics 251 d dt ½ATPŠ ¼k ðdphþ 4 1 þðdphþ 4½ADPŠ½PiŠ This equation is empirical, but broadly corresponds to the experimental results available in the literature (Jain & Nath, 2001). Molar equivalence Simulation of the Krebs cycle, ETC and ATP synthesis together demands for stoichiometric analysis of the substrates and the products. One round of the Krebs cycle produces three molecules of NADH and one molecule of FADH 2. So to oxidise NADH and FADH 2 produced in the Krebs cycle, the ETC chain has to run multiple times: complex I thrice, complex II once, complexes III and IV four times (3NADH + 1FADH 2 ). For three NADH and one FADH 2, the total number of protons translocated would be {3 (4+4+2)+1(4 + 2)} = 36 protons. We have seen that four protons are required for the release of ATP from ATP synthase, therefore the ATP synthase produces 36/4 = 9 ATP. These values have to be incorporated in the rate equations to facilitate the coupling of the system. At this stage, this might appear to be a tight coupling, but in vivo there exists a loose coupling of these pathways. This is because NAD/NADH, ADP/ATP and protons are part of the cellular pool and may be utilised and produced in other pathways too. Assumptions While simulating this system comprising the Krebs cycle, ETC and ATP synthesis, few reasonable assumptions have been made: (1) The rate constant for all the reactions has been assumed equal to one, since different experimental works report different K M values and we have observed that the rate constant varies depending on the experimental conditions and a particular value cannot be considered universal. So, for the sake of studying the behaviour of the reaction and reaction components, all the rate constants have been generally assumed to be 1. In other words, all the concentrations reported here are reduced concentrations, concentrations expressed in terms of the respective K M. (2) The reactions simulated specifically include the reactions of three pathways and therefore all the components are shown to be conserved in the given system, i.e. all the NADH produced in the Krebs cycle is fed into ETC, and all the NAD + from the ETC is fed back to the Krebs cycle. Other cellular processes may use these components but this has not been considered here (this is a trivial modification to the existing rate equations). (3) Components which are not regenerated in the system are supplied from outside at a constant rate to keep the system proceeding. Such components include Ac-CoA, ADP, Pi and oxygen. Other components, like citrate, FAD, NAD etc. were given a fixed initial value. (4) The ph was set to 7.6 and 6.88 for the mitochondrial matrix and the IM space, respectively (Porcelli et al., 2005), for all the calculations, as and when required. Chemiosmotic principle requires a difference in ph values between the two sides and we can arbitrarily suppose that the IM ph is fixed at an initial value. Results and discussion The Krebs cycle The results of the simulations are represented graphically. The results obtained are as expected for the three individual processes. Figure 5 shows the simulation of the Krebs cycle. In this system, a continuous input of acetyl CoA is provided. The initial concentration for oxaloacetate and acetyl CoA is taken equal to 1 and the initial concentration of all the intermediates of cycle is taken equal to 0. It is seen that with input of oxaloacetate and acetyl CoA, the concentration of all the other components reaches a peak and then attains a steady state. Figure 5 also shows the sequence of occurrence of peaks for each component, first citrate, followed by isocitrate, α-ketoglutarate and succinyl CoA, as they come in the cycle. In the absence of any perturbation, the system continues to stay in the steady state. In this system, components as NAD +, FAD and GDP are externally supplied. Figure 5. Simulation curve for selected components of the Krebs cycle. (Ox-Ac = Oxaloacetate, Ac-CoA = Acetyl CoA, Iso- Cit = Isocitrate, Ke-Glu = α-ketoglutarate, Suc-CoA = Succinyl CoA). The graphs after time 50 essentially reflect steady state behaviour.

36 252 K. Korla and C.K. Mitra Figure 6. Simulation curve for selected components of ETC (Q = Quinone, QH 2 = Quinol, cyt c o = cytochrome c oxidised form, cyt c r = cytochrome c reduced form). Electron transport chain The simulation curves of the ETC components are shown in Figure 6. In this system, continuous input of oxygen is given. Also, NADH and FADH 2 are fed to the system in the ratio of 3:1 and the increment given is also in the same ratio, to match the output of one round of the Krebs cycle. The initial concentration of oxygen, cyt c (oxidised form cyt c o) and quinone (Q) is taken equal to 1 and for quinol (QH 2 ) and cyt c (reduced form cyt c r) is taken equal to 0. It is seen that in the presence of constant supply of oxygen, NADH and FADH 2, the system attains a steady state in the due course of time. Further, it is observed that the curves for [Q/QH 2 ] and [cyt c o/cyt c r] Figure 8. The simulation curve of all the three pathways combined together (Ac-CoA = Acetyl CoA, cyt c ox = cytochrome c oxidised form). A constant input of oxygen is continuously fed at units per cycle. couple are complementary as expected, since these are inter-convertible species. ATP synthesis The rate of ATP synthesis depends on the electrochemical gradient across the inner membrane potential. In this work, we have considered only the effect of ph, since it is responsible for chemical gradient and also contributes to the electrical gradient (Ψ). The effect of Ψ is not explicitly involved in the present work. This is reasonable as the membrane potential is mostly determined by Figure 7. The curves show the rate of ATP synthesis at different ph values of the mitochondrial matrix. The lowest curve corresponds to ph 6 and the subsequently following curves correspond to increasing ph values in steps of 0.5 units. The inset image shows the curve for rate constant vs. ph which shows a sigmoidal dependency. Figure 9. Combined simulation with fixed initial input of oxygen. Oxygen concentration decreases 50% and this is sufficient to convert 100% of ADP into ATP. In the ETC, NADH and FADH 2 utilization has been fixed in the script as 1:1 but it can be variable. One round of Krebs cycle produces three molecules of NADH and one molecule of FADH 2 and therefore NADH is underutilised. FADH 2 concentration increases from the Krebs cycle but reduces afterwards. The final steady state behaviour is not attained in this graph.

37 Journal of Biomolecular Structure & Dynamics 253 the concentration imbalance of Na +,K +,Mg ++ and Ca ++ but only a little by the ph. This is understandable as the membrane is rich in phosphates, which act as buffers at or near the membrane surface. The concentrations of the major cations (also Cl ) are not directly controlled by the reactions considered and hence the basic assumption that the membrane potential remains essentially constant appears reasonable. The curves in Figure 7 show the formation of ATP at different ph levels of the mitochondrial matrix ranging from 6 to 9.5. However, the ph across the inner mitochondrial membrane has been reported to be around In the present set-up, we have assumed the ph in the IM space to be constant at six and we have simulated the formation of ATP at different ph in the matrix. The graph shows that the concentration of ATP at ph 6 is zero and there is no conversion of ADP to ATP. As the ph increases in the matrix gradient (ΔpH, ph difference between ph in the matrix and the ph within the IM space (Santo-Domingo & Demaurex, 2012)), the rate of ATP formation also increases. Here, the initial concentration of ADP, phosphate and ATP were 0.5, 0.5 and 0, respectively. At ph 6.5, only a small amount of ATP is formed even after 500 time units, whereas at ph 9.5, more than 95% of ADP has been converted to ATP in just 200 time units. Figure 7 prominently indicates that small changes in ph between 6.5 and 8, significantly affect the rate of ATP formation, while beyond ph 8, the effects of further increase of ph on the rate is not substantial. This result corresponds to the experimental findings related to the mitochondrial ph dependence. The mitochondrial system functions in a narrow ph range, where the maximum effect (production of ATP) can be achieved with minimal ph variations. These curves for ATP formation at different ph resemble the Michaelis Menten curve. Figure 10. Keeping constant all the other parameters, the amount of oxygen given as constant supply to the system was varied (0, , and 0.002) (Ac-CoA= Acetyl CoA, cyt c ox = cytochrome c oxidised form).

38 254 K. Korla and C.K. Mitra Combined simulation The final simulation involves the reactions from all the three processes. In such a system, reactants which are not regenerated in the due course of pathway are given constant stoichiometric input (Ac-CoA, ADP, phosphate, oxygen). NAD + and FAD are fed in the cycle just once at the beginning and are then recycled. Therefore, the Krebs cycle taken together with oxidative phosphorylation constitutes a giant catalytic unit with four inputs and two outputs (ATP and carbon dioxide). Figure 8 shows the kinetics of this giant catalytic unit. It is seen that the concentration of ATP rises as the reactions proceed and is influenced by the concentration of ADP + Pi and ph (Figure 9). The concentration of Ac-CoA, ADP and oxygen falls as the reactions proceed. Such curves are important to study the impact of one component/perturbation on the whole system. For instance, if we change the concentration of oxygen replenished after each cycle, the curves of other components are also affected. Thus, impact of a single component or a particular combination of components can be studied by keeping constant all the other parameters. Effect of O 2 input The concentration of all the components was kept unchanged except oxygen, although the initial concentration of oxygen is the same in all the four cases. Oxygen is replenished in the system after each cycle, so as to keep the system moving. The change in the amount of oxygen fed after every cycle reflects a change in the other system components. These effects are depicted in Figure 10. As the concentration of oxygen pumped increases, there is a decrease in the concentration of NADH and FADH 2 (the decrease in NADH is not prominently Figure 11. Keeping constant all the other parameters, the amount of Ac-CoA given as constant supply to the system was varied (0, , and 0.002) (Ac-CoA= Acetyl CoA, cyt c ox = cytochrome c oxidised form).

39 Journal of Biomolecular Structure & Dynamics 255 visible in Figure 10 but indeed there is a change), i.e. NADH and FADH 2 are oxidised. Further, concentration of oxygen also has a small impact on the consumption of Ac-CoA in the system, with the increase in concentration of oxygen, Ac-CoA concentration decreases. Concentration of oxygen shows its impact on the cyt c oxidation state, too, as expected. As the concentration of oxygen increases, concentration of oxidised form of cyt c also increases. Oxygen is the terminal electron acceptor, when it is depleted in the system, the electron flow ceases and the intermediate components tend to exist in their reduced form as they can no more pass on the electron to the next carrier. Further, with an increase in oxygen input, the intermediate components (as cyt c) tend to exist in their oxidised form as validated by the graphs here. Effect of Ac-CoA input In Figure 11, the concentration of all the components remained unchanged except Ac-CoA. The initial concentration of Ac-CoA is the same in all the four cases. Ac- CoA is oxidised to give two molecules of CO 2 in one cycle of the Krebs cycle, therefore was continuously fed into system. When the concentration of Ac-CoA is increased, rise in the concentration of NADH and FADH 2 is observed. Conclusions The Krebs cycle and oxidative phosphorylation pathway were collectively simulated using GNU-Octave. The products from the former are used as the substrates in the latter to produce ATPs. Both the processes were first simulated individually. The effect of ph on the formation of ATP was studied. Then these simulations were combined and behavioural patterns between various components were studied. Results found in this paper resemble the expected outcome. This approach can also predict unknown associations, which can be confirmed in wet-lab experiments. This approach can be extended to a larger scale to study minor perturbations and their effects on the whole system and its individual components. Acknowledgements One of the authors (KK) acknowledges receipt of Junior Research Fellowship from the University Grants Commission, Government of India. Preliminary results of this study were presented in the 2012 International German/ Russian Summer School on Integrative Biological Pathway Analysis and Simulation, Bielefeld, Germany. The authors wish to thank the anonymous reviewer who has made several constructive and useful comments and suggestions. References Boyer, P. D. (1993). The binding change mechanism for ATP synthase some probabilities and possibilities. Biochimica et Biophysica Acta, 1140, doi: / (93)90063-L Cecchini, G. (2003). Function and structure of complex II of the respiratory chain. Annual Review of Biochemistry, 72, doi: /annurev.biochem Ciliberto, A., Capuani, F., & Tyson, J. J. (2007). Modeling networks of coupled anzymatic reactions using the total quasisteady state approximation. PLoS Computational Biology, 3, doi: /journal.pcbi Flach, E. H., & Schnell, S. (2006). Use and abuse of the quasisteady state approximation. IEE Proceedings Systems Biology, 153, doi: /ip-syb: Hinkle, P. C. (2005). P/O ratios of mitochondrial oxidative phosphorylation. Biochimica et Biophysica Acta, 1706, doi: /j.bbabio Jain, S., & Nath, S. (2001). Catalysis by ATP synthase: Mechanistic, kinetic and thermodynamic characteristics. Thermochimica Acta, 378, doi: /s (01) Johnson, J. D., Mehus, J. G., Tews, K., Milavetz, B. I., & Lambeth, D. O. (1998). Genetic evidence for the expression of ATP and GTP-specific succinyl-coa synthetases in multicellular eucaryotes. Journal of Biological Chemistry, 273, doi: /jbc Kornberg, H. L. (1966). Anaplerotic sequences and their role in metabolism. In P. N. Campbell & R. D. Marshall (Eds.), Essays in biochemistry (pp. 1 31). London: Academic Monod, J., Changeux, J. P., & Jacob, F. (1963). Allosteric proteins and cellular control systems. Journal of Molecular Biology, 6, doi: /s (63) Nelson, D. L., & Cox, M. M. (2005). Lehninger principles of biochemistry (4th ed.). New York, NY: WH Freeman. Pedersen, M. G., Bersani, A. M., & Bersani, E. (2007). The total quasi steady-state approximation for fully competitive enzyme reactions. Bulletin of Mathematical Biology, 69, doi: /s Pedersen, M. G., Bersani, A. M., & Bersani, E. (2008). Quasi steady-state approximations in complex intracellular signal transduction networks a word of caution. Journal of Mathematical Chemistry, 43(4). doi: /s Porcelli, A. M., Ghelli, A., Zanna, C., Pinton, P., Rizzuto, R., & Rugolo, M. (2005). PH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant. Biochemical and Biophysical Research Communications, 326, doi: /j.bbrc Santo-Domingo, J., & Demaurex, N. (2012). The renaissance of mitochondrial ph. Journal of General Physiology, 139, doi: /jgp Schnell, S., & Maini, P. K. (2003). A century of enzyme kinetics. Reliability of the KM and vmax estimates. Comments on Theoretical Biology, 8, doi: / Soga, N., Kinosita, K., Yoshida, M., & Suzuki, T. (2012). Kinetic equivalence of transmembrane ph and electrical potential differences in ATP synthesis. Journal of Biological Chemistry, 287, doi: /jbc.m Strauss, M., Hofhaus, G., Schröder, R. R., & Kühlbrandt, W. (2008). Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. The EMBO Journal, 27, doi: /emboj

40 256 K. Korla and C.K. Mitra Stryer, L., Berg, J., & Tymoczko, J. L. (2002). Biochemistry (5th ed.). San Francisco, CA: W.H. Freeman. Weber, J., & Senior, A. E. (2003). ATP synthesis driven by proton transport in F1 F0 ATP synthase. FEBS Letters, 545, doi: /s (03) Wikstrom, M. (1984). Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone. FEBS Letters, 169, doi: / (84)

41 Original article doi: /j x Pre-operative oral carbohydrate loading in colorectal surgery: a randomized controlled trial S. E. Noblett, D. S. Watson, H. Huong, B. Davison, P. J. Hainsworth and A. F. Horgan Department of Colorectal Surgery, Freeman Hospital, Newcastle-upon-Tyne, UK Received 26 September 2005; accepted 22 November 2005 Abstract Objective Surgery induces a catabolic response with stress hormone release and insulin resistance. The aim of this study was to assess the effect of pre-operative carbohydrate administration on grip strength, gastrointestinal function and hospital stay following elective colorectal surgery. Methods Thirty-six patients undergoing elective colonic resection were randomized into one of three groups. Group 1 were fasted; Group 2 were given pre-operative oral water, Group 3 received equivalent volumes of a Maltodextrin drink. Time to first flatus, first bowel movement and hospital stay were recorded. Muscle strength was measured pre-operatively, and on alternate days thereafter until discharge using a grip strength dynamometer. Results Patients in the carbohydrate group had a median postoperative hospital stay of 7.5 days compared with 13 days in the water group (P > 0.01) and 10 days in the fasted group (P ¼ 0.06). The median time postsurgery to first flatus was 3 days for both the fasted and water groups compared with 1.5 days in the carbohydrate group (P ¼ 0.13). First bowel movement occurred on day 3 in the carbohydrate group, day 4 in the fasting group and day 5 in the water group. The fasted group showed a significant reduction in postoperative grip strength (P <0.05) with a median drop of 10% at discharge. Neither the water nor the carbohydrate groups showed significant reductions in muscle strength. Conclusion We found that pre-operative administration of oral carbohydrate leads to a significantly reduced postoperative hospital stay, and a trend towards earlier return of gut function when compared with fasting or supplementary water. Keywords Colorectal surgery, carbohydrate loading, optimization, fast-track Introduction Correspondence to: Alan Horgan, Freeman Hospital, High Heaton, Newcastleupon-Tyne, Tyne and Wear NE7 7BR, UK alan.horgan@nuth.nhs.uk The strict nil-by-mouth from midnight rule, before elective surgery was introduced to ensure an empty stomach at the time of anaesthesia and reduce the risk of pulmonary aspiration. Over the last decade several studies have questioned the need for such a lengthy pre-operative fast and have shown that not only can more liberal rules be applied without putting the patient at increased risk, but may also be of considerable benefit to the patient [1 7]. Following surgical trauma there is a reduction in the effects of insulin and a compensatory rise in insulin release. This leads to raised blood glucose levels and a metabolic state comparable with untreated Type-1 Diabetes Mellitus [2]. Insulin resistance has been shown to be an independent predictive factor for length of postoperative hospital stay [2]. The postoperative catabolic state is also associated with loss of body fat and protein stores. The fasted state at the time of operation represents an additional metabolic stress to the patient. Indeed, animal studies have shown a significantly reduced capacity to cope with haemorrhagic or endotoxaemic stress when in the fasted state (6 24 h fast) compared to the fed state [3]. More recently patients have been allowed to drink clear fluids up to 90 min before surgery [8], This has been shown to improve subjective well being in terms of thirst, dryness of mouth and anxiety [2,4,5]. These clear fluids however, provide little metabolic benefit. Studies altering the anabolic catabolic milieu using pre-operative intravenous glucose have shown reduced nitrogen losses immediately following surgery [9], whilst the infusion of glucose, potassium and insulin before cardiac surgery has been reported to reduce morbidity and hospital stay [6,7]. Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

42 Pre-operative carbohydrate loading S. E. Noblett et al. The use of intravenous glucose (10 20% to avoid fluid overload) requires concomitant insulin infusion, access to large veins and frequent monitoring of blood glucose levels by trained staff [10]. These disadvantages can be overcome by using the enteral route. To provide a significant carbohydrate load, an oral glucose drink would be significantly hyperosmolar, which would slow gastric emptying and pose an aspiration risk. Workers in Stockholm [11] have used a near isotonic carbohydrate rich beverage (12.5% carbohydrate but only 285mOsm) composed of maltodextrins in water. They demonstrated that 400 mls of this solution was completely emptied from the stomach after 90 minutes in both healthy volunteers and in pre-operative patients. Preliminary studies have suggested several beneficial effects of using this carbohydrate rich beverage preoperatively. Intake of the solution on the night before surgery and 3 h before anaesthesia has been shown to reduce postoperative insulin resistance and lead to a lesser reduction of insulin stimulated glucose disposal in patients undergoing major abdominal surgery and total hip replacement surgery. Patient pre-operative discomfort (as measured on a visual analogue scale) for thirst, hunger and anxiety was reduced [12], whilst postoperative discomfort was improved in a group of colorectal patients [13]. Our aim was to assess the effect of pre-operative oral carbohydrate supplementation on outcome following elective colorectal resection. The primary outcome measurement is length of postoperative hospital stay with secondary outcomes being return of gastrointestinal function and grip strength. prior to surgery. The final group were fasted from midnight the night before surgery. Patients with diabetes mellitus, gastro-oesophageal reflux disease or disorders of gastric emptying were excluded from the study. Muscle strength was measured using a grip strength dynamometer pre-operatively and on alternate days thereafter until discharge; the patient s dominant hand was used for this measurement, and the best out of three consecutive strengths was used. All other aspects of patient care were standardized, with free fluids permitted immediately after surgery, and light diet allowed from day 1, as tolerated by the patient. As social and rehabilitation services often delay actual discharge from hospital in the UK, time until the patient was fit for surgical discharge was noted. Fitness for surgical discharge was defined by the following criteria; passing flatus bowel action, tolerating oral diet, adequate pain control (oral analgesia) and the patient should be able to self-care with minimal presurgery level of assistance. The consultant and junior medical staff in charge of the day-to-day care of patients in the study, were blinded as to which arm of the study individual patients were in. The approval of the hospital research and development committee and the local regional ethics committee were obtained prior to the commencement of the study. Distribution and type of operative procedure The distribution and type of operative procedure is shown in Table 2. Patients and methods Analysis of a historical cohort of colorectal resections showed a median stay of 10 days with a standard deviation of 2.5 days. Subsequent power calculation showed a sample size of 36 patients (12 per group) would be necessary to show a 3 day difference in hospital stay, giving the study a power of 80% and P-value of Following written informed consent, 36 patients undergoing elective colorectal resection were randomised into one of three groups. Sealed envelopes were constructed using random number allocation and opened the night prior to surgery by a researcher independent of the clinical team. Progress of patients through the randomised trial is shown in Fig. 1. The first group received 800 mls of water the night before surgery and 400 mls of water 3 h prior to induction of anaesthesia. The second group received 100 g PrecarbÒ (Vitaflo Limited, Liverpool, UK; Table 1), dissolved in 800 mls of water the night before surgery and 50 g of VitajouleÒ dissolved in 400 ml of water 3 h Statistical analysis Results are presented as median values. Statistical significance was accepted at the probability level of < 0.05 with the use of nonparametric statistical testing using the SPSS version 10.0 for windows. Grip strength was analysed in terms of percentage drop in strength of an individual patient compared with their pre-operative value, intergroup comparisons were made using the Wilcoxon s signed rank test. Comparisons between groups were made using the Mann Whitney U-test. There was no cross over between groups and results were analysed on an intention to treat basis. Results Demographic data The age distribution was similar in all 3 groups; fasting group mean age 55 years (range years), water group mean age 59 years (range years) and 564 Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

43 S. E. Noblett et al. Pre-operative carbohydrate loading Figure 1 Flow Chart showing Progress of Patients through randomised trial. R randomization. Table 1 Constituents of the carbohydrate supplement used (Vitajoule Ò). Typical constitution per 100 g Carbohydrate 96.0 g Energy 1610 kj (kcal 380) Sodium < 1.9 mmol Potassium < 0.2 mmol Osmolarity 285mOsm carbohydrate (CHO) group mean age 58 years (range years). All groups had a median American Society of Anaesthesiologists (ASA) grade of 2. Stoma formation was similar in all groups, as was the distribution of colostomy and ileostomy formation. Exclusions Twelve patients were recruited into the water group. 1 patient was excluded from study due to cancellation of surgery. Twelve patients were recruited into each of the fasting and carbohydrate groups; no patients were excluded in either of these groups. Complications In the 12 patients in the fasting arm of the study one patient suffered from diarrhoea and vomiting on day 8 postsurgery, no other complications were recorded. Postoperative complications occurred in 3 patients in the water group; one perineal wound breakdown, one anastomotic leak and one prolonged ileus. In the carbohydrate group, 1 patient suffered from prolonged nausea in the postoperative period, a further patient Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

44 Pre-operative carbohydrate loading S. E. Noblett et al. Table 2 Distribution of operative procedures between the 3 groups of patients. Operation Fast Water CHO Total Right hemicolectomy Anterior resection APR Panproctocolectomy Proctectomy Sigmoid colectomy Ileocolic resection developed symptomatic atrial fibrillation. This patient was in rate controlled atrial fibrillation pre-operatively but omitted to take her B-blocker on the first and second postoperative days, she subsequently became tachycardic with a rate related ischaemia. This responded to recommencement of B-blockade and a subsequent Troponin level was normal. Length of hospital stay The median time to fitness for surgical discharge for patients in the fasting group was 10 days, the water group 13 days and the carbohydrate group 7.5 days. Statistically significant differences were found between patients who received carbohydrate and those who received water (P ¼ 0.019). Although statistical significance was not reached (P ¼ 0.06), an observed trend to earlier fitness for discharge was seen between patients who received carbohydrate and those who were fasted. There was no significant difference in hospital stay between those patients who received water and those who were fasted (P > 0.25) (Fig. 2). Gastrointestinal function The median time to passage of first flatus was 3 days in patients who were fasted pre-operatively, day 3 in those who received water and day 2 in those patients who received carbohydrate supplement. When comparing carbohydrate with fasting (P ¼ 0.3) or water (P ¼ 0.13) although a trend towards earlier return could be observed no statistically significant difference was found (Fig. 3). The median time to first bowel movement was 3.5 days in the fasted patients, 5 days in the patients who received water and 2 days in the patients who received carbohydrate. There is a trend towards earlier bowel movement in patients who received pre-operative carbohydrate compared with those who were fasted (P ¼ 0.2) and those who received water (P ¼ 0.06)3(Fig. 4). Time to first flatus (days) * Fasting Water Group Carbohydrate Figure 3 Boxplot showing median, range and interquartile range of time until first flatus for each of the 3 patient groups. Time until fit for surgical discharge (days) Fasting Water Group Carbohydrate Time to first bowel movement (days) Fasting Water Group Carbohydrate Figure 2 Boxplot showing median, range and interquartile range of time until fit for surgical discharge for each of the 3 patient groups. Figure 4 Boxplot showing median, range, and interquartile range of time until first bowel movement for each of the 3 patient groups. 566 Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

45 S. E. Noblett et al. Pre-operative carbohydrate loading Grip strength recovery (%) Pre-op Grip strength There was a significant reduction in grip strength on discharge in the fasted patients when compared with their pre-operative values (P <0.05), with a mean drop of 11%. The patients in the water group had a mean drop in grip strength of 8% on discharge and the carbohydrate group a drop of 5%. Neither the water (P ¼ 0.7) nor the carbohydrate (P ¼ 0.6) groups had a significant reduction in their postoperative grip strength on discharge when compared with their pre-operative levels (Fig. 5). Discussion Carbohydrate Water Fasting Days postop. Figure 5 Graph showing percentage recovery of hand grip strength compared with pre-operative value. Pre-operative carbohydrate loading with a maltodextrin drink before elective colorectal resection leads to a reduced postoperative hospital stay when compared with fasting or supplementary water. This finding supports other studies on patients having major abdominal or orthopaedic procedures, which although individually did not reach statistical significance, when analysed retrospectively as part of a meta-analysis found carbohydrate loading significantly reduced hospitalization times by 20% [10]. Whilst we appreciate our study involved only small numbers of patients, we feel the findings suggest the need for further research into the use of carbohydrate supplements before elective colorectal surgery. The maltodextrin drink was not associated with any increase in patient morbidity in our study group, and the anaesthetic team reported no problems at induction. Patients were not informed whether they were receiving water or water plus carbohydrate, however, complete blinding could not be achieved with regards to study group as although the carbohydrate drink is not specifically flavoured it can be distinguished from plain water. No negative feedback with regards to the palatability of the beverage or of the consumption volume was received from the patients. Nasogastric tubes were not inserted in any of the study patients in either the peri-operative period or postoperatively. Residual gastric volumes were therefore not recorded. Studies assessing the safety of pre-operative carbohydrate beverages have demonstrated gastric emptying to safe residual volumes of 400 mls of supplement occurs within 90 min despite the presence of anxiety [11,12]. The supplement used consists of glucose polymers and therefore is iso-osmolar allowing gastric emptying times comparable to water [11,12]. This allows the supplement to be used safely in the preanaesthetic period without increasing aspiration risk. It should be noted however, that not all nutritional supplements are safe in the pre-operative period, supplements containing similar calorific values but composed of fats, proteins or monomers of carbohydrate can delay gastric emptying and pose an aspiration risk [11]. Although statistical significance has not been reached there is a trend towards earlier return of gut function as measured by time to first passage of flatus and first bowel movement in the group of patients given pre-operative carbohydrate. This earlier return of bowel function may be a contributary factor for shorter hospital stay. Short periods of fasting such as is seen in the preoperative period alter the metabolic state of the patient and thereby affect their ability to mount a response to physical stress. The carbohydrate load alters the bodies metabolic state from an overnight fast, with decreased glycogen reserves and low basal insulin levels, to that of glycogen loading and insulin release. This, as shown in animal models is a better way of preparing the body for stress [3]. It has been shown that fasted animals have increased stress hormone release and impaired fluid homeostasis when subjected to haemorrhagic stress compared with their nonfasted counterparts [14,15]. Researchers have studied the biochemical and metabolic effects of glucose administration (oral intravenous) in the pre-operative period, and based on this preexisting evidence in the literature we did not repeat these measurements but chose to concentrate on the clinical outcomes. The supplement used was an isoosmoler solution composed of glucose polymers, which is designed to provide the carbohydrate equivalent of a standard hot meal in the quantity administered. This supplement therefore provided a normal, rather than Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

46 Pre-operative carbohydrate loading S. E. Noblett et al. supra-normal carbohydrate load and was used in nondiabetic patients before the period of surgical stress commenced. Glucose metabolism would be expected to have been normal in our study group as the supplement simply reversed the nutritional effect of the overnight fast Indeed, previous studies have not reported abnormal glucose handling related to oral carbohydrate supplementation to normal levels [12,16,17]. Ljungqvist et al. [14] demonstrated that the major mechanism of insulin resistance following surgical trauma was inhibition of nonoxidative glucose disposal. They showed that pre-operative oral carbohydrate administration better preserved insulin sensitivity by increasing glucose oxidation rates, leading to lesser reductions in peripheral tissue glucose disposal. Other workers have concentrated on the protein sparing effects of glucose [9], demonstrating reduced urea and 3-methylhistidine excretion implying reduced protein breakdown in patients treated with glucose infusion, they further demonstrated that pre-operative administration compared with postoperative infusion leads to an increase in protein synthesis in the early postoperative period. In the fasted state there is demand on amino-acids for gluconeogenesis rather than tissue repair, in addition this study demonstrated decreased protein synthesis compared with protein breakdown when in the fasted state. Altering these changes in protein metabolism by glucose administration may play a role in improving tissue repair following surgery. Our study compared carbohydrate loading with both fasting and water groups. We were interested to assess whether any changes seen were likely due to the preoperative calories received or simply due to hydration and maintenance of gut peristalsis by oral fluid. With regards to length of hospital stay and postoperative gastrointestinal function, receiving pre-operative oral water in our study incurred no benefit over the traditional overnight fast. Studies have shown however, that allowing patients to drink water prior to surgery improves psychological well-being [4,5]. Hand grip strength has been shown to be an indicator of nutritional status and predictor of postoperative complications [18,19]. It is thought that loss of body protein may be important in the development of these postoperative complications; and it has been demonstrated that grip strength is a sensitive measurement of the degree of protein loss [20]. A Danish study in which 48 patients undergoing elective colorectal surgery were randomised into 3 groups (fasting carbohydrate or carbohydrate peptide mix) showed a trend towards improved muscle strength following pre-operative carbohydrate both in the immediate postoperative period and at 1 month follow-up [21]. There was less reduction in dominant hand, grip strength when compared with pre-operative strength at time of discharge in the carbohydrate group. This is supported by the Danish study [21]. The mechanisms underlying this improvement are unclear but may be related to a reduction in protein loss. Interestingly, the patients in the water arm of our study also had less grip strength reduction when pre-operative and discharge values were compared. As the anabolic catabolic milieu was not attenuated in this group the reason for this maintenance in muscle strength is unclear. Fluctuations in recorded grip strength in this group combined with small numbers of patients and longer hospital stays may be responsible. In the UK there are approximately new cases of colorectal malignancy diagnosed per annum [22]. Based on a conservative estimate of 80% of these patients being suitable for surgery, approximately patients will undergo colorectal resection in the U.K. each year. The average cost of a 24-h stay on a surgical ward in our unit is 217 per patient exclusive of specific medical or surgical interventions, and the cost of the carbohydrate supplement is per patient. Based on a 3-day reduction in hospitalization time this gives a potential saving to the national health service of million per year. Pre-operative carbohydrate loading may represent a cost-effective intervention to reduce inpatient hospital stay, which is of benefit to the patient and may help current pressures on availability of acute surgical beds. In conclusion, we have found the maltodextrin drink, a safe and effective way to administer pre-operative carbohydrate to colorectal surgical patients, which may have a positive effect on outcome and be of benefit as part of an integrated fast-track surgical regimen. References 1 Ljungqvist O, Soreide E. Preoperative fasting. Br J Surg 2003; 90: Thorell A, Nygren J, Ljungqvist O. Insulin resistance: a marker of surgical stress. Curr Opin Clin Nutr Metab Care 1999; 2: Ljungqvist O, Nygren J, Hausel J, Thorell A. Preoperative nutrition therapy novel developments. Scand J Nutr 2000; 44: Agarwal A, Chari P, Singh H. Fluid deprivation before operation. The effect of a small drink. Anaesthesia 1989; 44: Maltby JR, Sutherland AD, Sale JP, Shaffer EA. Preoperative oral fluids: is a five-hour fast justified prior to elective surgery? Anaesth Analg 1986; 65: Quinones-Galvan A, Ferrannini E. Metabolic effects of glucose-insulin-potassium infusions: myocardium and whole body. Curr Opin Nutr Metab Care 2001; 4: Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

47 S. E. Noblett et al. Pre-operative carbohydrate loading 7 Lazar HL, Phillppides G, Fitzgerald C et al. Glucose-insulinpotassium solutions enhance recovery after urgent coronary artery bypass grafting. J Thorac Cardiovasc Surg 2001; 113: Report by the American Society of Anesthesiologists (Chair: MA Warner). Practice Guidelines for Pre-Operative Fasting and the Use of Pharmacologic Agents to Reduce the Risk of Pulmonary Aspiration. URL (accessed June 2006). 9 Crowe PJ, Dennison A, Royle GT. The effect of preoperative glucose loading on postoperative nitrogen metabolism. Br J Surg 1984; 71: Nygren J, Thorell A, Ljungqvist O. Preoperative oral carbohydrate nutrition: an update. Current Opinion Clin Metabolic Care 2001; 4: Nygren J, Thorell A, Jacobson H, et al. Preoperative gastric emptying: Effects of anxiety and oral carbohydrate administration. Annals of Surgery 1995; 222: Nygren J, Thorell A, Lagercranser M et al. Safety and patient well being after pre-operative oral intake of a carbohydrate rich beverage. Clin Nutr 1996; 16 (Suppl. 1): Hausel J, Nygren J, Almstrom C et al. Preoperative oral carbohydrates improve well-being after elective colorectal surgery. Clin Nutr 1999; 18 (Suppl. 1): Ljungqvist O, Efendic S, Eneroth P, Hamberger B, Nylander G, Ware J. Nutritional status and endocrine response to hemorrhage. Can J Physiol Pharmacol 1986; 64: Ljungqvist O, Jansson E, Ware J. Effects of food deprivation on survival after hemorrhage in the rat. Circ Shock 1987; 22: Nygren J, Soop M, Thorell A et al. Preoperative oral carbohydrate administration reduces postoperative insulin resistance. Clin Nutrition 1998; 17: Soop M, Nygren J, Myrenfors P et al. Preoperative oral carbohydrate treatment attenuates immediate postoperative insulin resistance. Am J Physiol Endocrinol Metab 2001; 280: E576 E Hunt DR, Rowlands BJ, Johnston D. Hand grip strength a simple prognostic indicator in surgical patients. J Parenter Enteral Nutr 1985; 9: Griffith CD, Whyman M, Bassey EJ, Hopkinson BR, Makin GS. Delayed recovery of hand grip strength predicts postoperative morbidity following major vascular surgery. Br J Surg 1989; 76: Windsor JA, Hill GL. Grip strength: a measure of the proportion of protein loss in surgical patients. Br J Surg 1988; 75: Henrikson M, Hansen H, Dela F et al. Preoperative feeding might improve postoperative voluntary muscle function. Clin Nutr, 1999; 18 (Suppl. 1): Cancer Research UK. URL org (date accessed June 2005). Ó 2006 Blackwell Publishing Ltd. Colorectal Disease, 8,

48 Nutritional Intake and Gastrointestinal Problems during Competitive Endurance Events BEATE PFEIFFER 1, TRENT STELLINGWERFF 2, ADRIAN B. HODGSON 1, REBECCA RANDELL 1, KLAUS PÖTTGEN 3, PETER RES 4, and ASKER E. JEUKENDRUP 1 1 School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, UNITED KINGDOM; 2 Nestlé Research Center, Lausanne, SWITZERLAND; 3 B.A.D. Gesundheitsvorsorge und Sicherheitstechnik GmbH, Darmstadt, GERMANY; and 4 Department of Human Movement Sciences, Maastricht University, Maastricht, THE NETHERLANDS ABSTRACT PFEIFFER, B., T. STELLINGWERFF, A. B. HODGSON, R. RANDELL, K. PÖTTGEN, P. RES, and A. E. JEUKENDRUP. Nutritional Intake and Gastrointestinal Problems during Competitive Endurance Events. Med. Sci. Sports Exerc., Vol. 44, No. 2, pp , There is little information about the actual nutrition and fluid intake habits and gastrointestinal (GI) symptoms of athletes during endurance events. Purpose: This study aimed to quantify and characterize energy, nutrient, and fluid intakes during endurance competitions and investigate associations with GI symptoms. Method: A total of 221 endurance athletes (male and female) were recruited from two Ironman triathlons (IM Hawaii and IM GER), a half-ironman (IM 70.3), a MARATHON, a 100/150-km CYCLE race. Professional cyclists (PRO) were investigated during stage racing. A standardized postrace questionnaire quantified nutrient intake and assessed 12 GI symptoms on a scale from 0 (no problem) to 9 (worst it has ever been) in each competition. Results: Mean CHO intake rates were not significantly different between IM Hawaii, IM GER, and IM 70.3 (62 T 26, 71 T 25, and 65 T 25 gih j1, respectively), but lower mean CHO intake rates were reported during CYCLE (53 T 22 gih j1, P = 0.044) and MARATHON (35 T 26 gih j1, P G 0.01). Prevalence of serious GI symptoms was highest during the IM races (È31%, P = 0.001) compared with IM 70.3 (14%), CYCLE (4%), MARATHON (4%), and PRO (7%) and correlated to a history of GI problems. In all data sets, scores for upper and lower GI symptoms correlated with a reported history of GI distress (r = 0.37 and r = 0.51, respectively, P G 0.001). Total CHO intake rates were positively correlated with nausea and flatulence but were negatively correlated with finishing time during both IM (r = j0.55 and r = j0.48, P G 0.001). Conclusions: The present study demonstrates that CHO intake rates vary greatly between events and individual athletes (6 136 gih j1 ). High CHO intake during exercise was related not only to increased scores for nausea and flatulence but also to better performance during IM races. Key Words: CHO INGESTION, GASTROINTESTINAL DISTRESS, RUNNING, CYCLING, TRIATHLON, FIELD STUDY APPLIED SCIENCES The popularity of mass participation in endurance and ultraendurance events is ever increasing. Athletes participating in these events are required to sustain relatively high work rates for a prolonged period, which results in high sweat rates and energy expenditure. Fatigue during endurance events is generally not caused by a single factor but is the result of a multifaceted phenomenon that often coincides with dehydration, hyperthermia, CHO depletion, central fatigue, and hypoglycemia (1,22). To delay the onset Address for correspondence: Asker E. Jeukendrup, Ph.D., School of Sport and Exercise Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom; A.E.Jeukendrup@bham.ac.uk. Submitted for publication November Accepted for publication July /12/ /0 MEDICINE & SCIENCE IN SPORTS & EXERCISE Ò Copyright Ó 2012 by the American College of Sports Medicine DOI: /MSS.0b013e31822dc809 of fatigue and optimize prolonged endurance performance, it is recommended to compensate fluid and electrolyte losses as well as to fuel the body with energy from CHOs (for review, see position stand [2]). Because CHO intake has been shown to improve endurance capacity and performance (for review, see Jeukendrup [21]), the current position stand of the American College of Sports Medicine (ACSM) and the American Dietetics Association (ADA) advises athletes to consume CHO at rates of 0.7 gikg j1 body weight per hour (30 60 gih j1 ) during endurance events (2). An alternative contemporary recommendation (21) suggests higher CHO intake rates of up to 90 gih j1 for athletes competing in intense (ultra)endurance events longer than 2 h. The rationale to recommend higher CHO intake rates is based on recent research that revealed higher exogenous CHO oxidation rates (19,23) and superior performance (7) with the ingestion of much glucose + fructose blends compared with isoenergetic amounts of glucose. However, whether athletes actually manage to meet these recommendations remains to be established. 344 Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

49 There are only limited and partly contradicting data available about nutritional intake strategies of athletes during endurance events. Several studies have investigated CHO and fluid intake of athletes during events longer than 2 h (Table 1). However, excluding retrospective studies, to the best of our knowledge, all previous studies have focused on a single event, and most studies were conducted with fewer than 20 subjects. For example, reported CHO intake rates in professional cyclists range from as little as 25 gih j1 (9) up to 94 gih j1 (37). The difference among studies could be because of difficulties measuring nutrient intake during competitions, variation in the methodology, environmental conditions, or changing nutritional practices during the years between the studies. Furthermore, a large interindividual difference in nutritional intake habits has been reported in numerous studies investigating athletes during various events (8,9,13,17,26,37), and it is therefore difficult to draw conclusions from small sets of data. It may also be inappropriate to extrapolate findings from one sporting event to another. A retrospective questionnaire based study by Peters et al. (27) compared food and fluid intakes among different endurance sports in 422 athletes. Higher liquid and food intakes were reported by triathletes compared with runners. Hence, although current recommendations do not differentiate between runners, cyclists, and triathletes (2), it is possible that the nutritional intake strategies used are very different. Furthermore, a limitation of nutritional recommendations during exercise is the limited consideration of the negative effect that gastrointestinal (GI) distress might have on exercise performance. The intake of several nutrients, such as fat, fiber, and protein, has previously been linked to GI distress during exercise (29). In several studies, ingestion of CHO, and in particular hypertonic drinks, has been related to GI distress (27,29,34). In contrast, a recent series of studies has suggested that the intake of high rates of CHO (È1.4 gimin j1 ) in the form of gels is well tolerated from most athletes during simulated 10-mile (16-km) running races under mild environmental conditions (30). However, it has to be kept in mind that the incidence of GI problems increases with exercise time (28) and might be increased under hotter and more humid weather and competition conditions. Therefore, the effect of nutrient intake on GI symptoms during more extreme events with longer duration remains unclear. The purposes of the present study were 1) to quantify and characterize the food and fluid intake of athletes during marathon running, road cycling, and long-distance triathlon using a large subject pool with the same GI questionnaire based methodology and 2) to investigate whether nutrient (especially CHO) intake, fluid consumption, training status, and race distance is correlated with the incidence of GI distress and/or performance outcomes. According to previous research, we speculated that fat and fiber intake and hot and humid weather conditions would be positively correlated with GI distress. We also hypothesized that high CHO TABLE 1. Reported nutritional intake of (ultra-)endurance athletes during events in previous studies (mean T SD). Ambient Temperature (-C) CHO Intake Fluid Intake Exercise Time (h:min) Reference Sport Event/Distance Survey Method Subjects Saris et al. (37) Cycling Tour de France (3 wk) a Food diary questionnaires 5 M 5:14 NA 94 gih j1 È1250 mlih j1 Garcia-Roves et al. (9) Cycling Tour de France Food diary questionnaires 10 M NA NA 25 gih j1 (10 43 gih j1 ) 6700 T 200 mlid j1 (three stages) a Havemann and Goedecke (17) Cycling Road race (210 km) Food diary questionnaires 45 M 7:18 T 1: T 23 gih j1 ( gih j1 ) 600 T 178 mlih j1 ( mlih j1 ) Kimber et al. (26) Triathlon Ironman Seven interviews during race 18 (10 M and 8 F) M: 12:00 T 0:36 21 M: 82 gih j1b M: È763 mlih j1b F: 12:36 T 0:54 NA 60 gih j1 (36 90 gih j1 ) c F: 62 gih j1b F: È628 mlih j1b 12 18:36 (17: mlih j1 ( mlih j1 ) c 19:48) c 19 (18 M and 1 F) 24:18 NA 50 gih j1 765 mlih j1 Colombani et al. (4a) Multisport Gigathlon (244 km) Questionnaires before/after; interview at transition/finish 26 (21 M and 5 F) 26:12 T 3:36 up to gih j1d 740 mlih j1d 7 M 10: T 16 gih j1 540 T 210 mlih j1 Glace et al. (13) Running Ultramarathon (160 km) Interview during race (every 13 km) Glace et al. (14) Running Ultramarathon (160 km) Interview during race (every 13 km) Fallon et al. (8) Running Ultramarathon (100 km) Dietary record by investigators during event 10 M 67:00 NA È36 gih j1 NA Dietary record by investigators during event Zimberg et al. (40a) Adventure race Laboratory simulation (477 km) When no SD or raw data were published, only means are reported. a PRO athletes. b Calculated from relative fluid intake per kilogram of body weight. c Values are median (range). d Calculated for 16 finishers. M, male participants; F, female participants. APPLIED SCIENCES HABITUAL NUTRIENT INTAKE AND ENDURANCE EVENTS Medicine & Science in Sports & Exercise d 345 Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

50 intake rates during prolonged exercise would be correlated with increased performance. METHODS Subjects. For this study, different levels of athletes were recruited: amateur and pro triathletes, amateur cyclists, professional cyclists from two different pro cycling teams (both in the top 10 of the International Cycling Union ranking), and amateur runners. Athletes were recruited via or were personally approached on the event exhibition. All participants of the study were informed about the purpose of the study, the practical details, and the risks associated with the procedure before giving their written consent. The study was approved by the School of Sport and Exercise Sciences ethics subcommittee, University of Birmingham, Birmingham, United Kingdom. Events. Table 2 highlights the different events, including subject characteristics and race conditions. All competitions occurred between June and October Triathlon. Three different triathlons were investigated, featuring two different race distances: two full-distance Ironman (IM) races, each covering a 3.8-km swim, 180-km bike, and a 42.2-km run, and an Ironman 70.3 (IM 70.3) race, which covers half of the IM distances (1.9-km swim, 90-km bike, and 21.1-km run). Data were collected during the IM European Championships in Frankfurt, Germany (IM GER), the IM World Championships in Hawaii (IM Hawaii), and during the IM Germany 70.3 in Wiesbaden, Germany (IM 70.3). Road cycling. Professional cyclists (PRO) from two different teams were investigated. One professional cycling team was investigated during two flat stages (228 and 182 km) of the Dauphine Liberé, France. The other professional cycling team was studied during the Vuelta a España (Tour of Spain). Two mountain stages (204.7 and km) and a flat stage (171.2 km) were investigated. Amateur cyclists were investigated at the Vattenfall Cyclassics cycling race, Hamburg, Germany (CYCLE). Half of the subjects participated in a shorter 100-km event, whereas the other half completed 155 km on a slightly hilly course. Running. A city marathon (42.2 km) with a relatively flat course profile was investigated in Munich, Germany (MARATHON). Experimental design. Subjects were recruited via or at the event exhibition and had at least one personal contact with the investigators before the event where they were carefully instructed and briefed about the importance of the accuracy in their responses. During the briefing, athletes were also given strategies to remember food and fluid consumption during the race, such as for participants with a nutrition plan to remember any deviations from the plan. The participants then filled in one questionnaire before the event to assess training history, nutritional habits, and history of GI discomfort and one after the event to accurately quantify their fluid and food intake and rate their GI discomfort during the event. To ensure the accuracy of data, replies were followed up via or in personal communications whenever possible. Professional cyclists were individually interviewed immediately after the race days rather than asked to fill in the questionnaires themselves. Prerace questionnaire. One or two days before the events, subjects were asked to complete a first questionnaire to assess personal characteristics, training history, nutritional habits, and history of GI problems. Race-day questionnaire. In the evening after the races, all participants received an with the second questionnaire, reminding them to fill it in as soon as possible, but no longer than 2 d after the race. Race environmental conditions were collected from local weather stations, and these are expressed as heat index. The heat index takes increased humidity into account, which can lead to increased heat stress and is described elsewhere (15). The questionnaire after the race asked the participants to accurately write down what they ingested in the morning, before the race, and during the entire race. The food and fluid intake was assessed by mentioning the available food and fluid options from the event organizer and giving examples on precision of amounts (e.g., water in milliliters or cups or bottles). For the triathlon races, all food and fluid intake at the start (up to 30 min before the race) was counted into the swim section of the event, the first and second transition were counted into the cycle and APPLIED SCIENCES TABLE 2. Subjects characteristics and ambient conditions for all endurance events (mean T SD). Heat Index (-C) Participants (Male/ Female) Age (yr) Height (m) Body Weight (kg) Finish Time (h:min) Endurance Training Experience (yr) Running Training (hiwk j1 ) Cycling Training (hiwk j1 ) Total Training (hiwk j1 ) IM Hawaii a 29 (26 36) 53 (34/19) 41 T T T 16 11:40 T 2:05 13 T 9 6 T 3 11 T 4 19 T 8 IM GER b 25 (15 33) 54 (45/9) 38 T T T 11 11:09 T 1:23 12 T 8 5 T 1 12 T 1 17 T 6 IM 70.3 c 24 (15 34) 43 (36/7) 38 T T T 13 6:05 T 0:44 11 T 10 4 T 2 7 T 3 13 T 5 MARATHON 12 (7 14) 28 (22/6) 45 T T T 13 3:46 T 0:34 13 T 9 6 T 3 NA NA CYCLE 20 (15 24) 28 (28/0) 41 T T 8 81 T 13 3:32 T 0:42 14 T 11 NA 7 T 4 NA PRO team Dauphine 17 (10 23) 7 (7/0) 31 T T 5 70 T 5 5:04 T 0:32 17 T 5 NA 23 T 2 NA Liberé PRO team VUELTA 25 (19 31) 8 (8/0) 29 T T 5 71 T 7 5:22 T 1:01 16 T 5 NA 23 T 2 NA a Amateurs and two PRO athletes. b Amateur and three PRO athletes. c Amateurs and one PRO athlete. NA question was not assessed. 346 Official Journal of the American College of Sports Medicine Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

51 run section, respectively. Afterward, data on food and fluid intake were evaluated by a trained nutritionist using NutritionistProi (Axxya Systems, Stafford, TX), following up estimated energy, nutrient, and fluid intakes in personal or conversations if there were doubts. Furthermore, participants were asked to answer questions on GI problems, adapted from a previous research (30). The questions about GI symptoms were similar in the prerace questionnaire, which assessed race occurrence and history of GI symptoms. The questionnaires were organized in three sections, and each section included between four and seven questions. Section 1 addressed upper abdominal problems (reflux/ heartburn, belching, bloating, stomach cramps/pain, nausea, vomiting); section 2 addressed lower abdominal problems (intestinal/lower abdominal cramps, flatulence, urge to defecate, side ache/stitch, loose stool, diarrhea, intestinal bleeding); and section 3 addressed systemic problems (dizziness, headache, muscle cramp, urge to urinate). Each question was assessed on a 10-point scale, ranging from 0 or no problem at all to 9 or the worst it has ever been. Statistical analysis. Nutrient intake data, training details, and performance data were normally distributed and evaluated with a parametric statistical approach. Mean values from different events were compared using one-way ANOVA. A Tukey post hoc test was applied where a significant F-ratio was detected. For comparison of mean values between modes of exercise within one triathlon, a repeated-measures one-way ANOVA was used and followed up with a Tukey post hoc test if a significant F-ratio. Possible correlations between race performance (finish time) and nutrient intake during the races were analyzed using the Pearson correlation coefficient. To evaluate data on GI symptoms, a nonparametric statistical approach was chosen, as scores on GI symptoms were mainly recorded on the low end of the scale and not normally distributed. Mean values were compared with the use of Mann Whitney tests. Factors that have previously been linked to GI distress, such as environmental conditions and history of GI distress, were analyzed using the Spearman rank correlation coefficient. Because the overall 12 GI symptoms were answered after 6 different events, the falsepositive rate of 5% could be inflated owing to the multiple tests. To reduce multiplicity, analysis was restricted to data from triathlon events, which revealed highest frequency for GI symptoms and took place under similar conditions. First, analyses were performed on averages over a section of symptoms (upper and lower abdominal problems). Second, correlations were performed for individual questions during each triathlon. The P values of those tests were not adjusted for multiple tests. Therefore, the P values serve as a flag to indicate interesting results. Furthermore, GI symptoms that were scored 9 4 were classified as serious. For all tests, P values G 0.05 were considered significant. All data are reported as means T SD. In addition, minimum and maximum scores are reported for nutrient intake data. Statistics were performed using SPSS version 15 for Windows (SPSS, Inc., Chicago, IL). RESULTS Race Conditions and Participants Characteristics Race conditions (ambient temperature, expressed as heat index), participant characteristics, including finishing times of the events, and details about training experience are shown in Table 2. Nutrient Intake (CHO, Fluid, Sodium, and Caffeine) An overview of energy, nutrient, and fluid intakes during the different races is shown in Table 3. CHO intake rates. Mean CHO intake rates were not significantly different between IM Hawaii, IM GER, and IM 70.3 (62 T 26, 71 T 25, and 65 T 25 gih j1, respectively, F 2,145 = 1.6, P = 0.2). Comparison of mean CHO intake rates between triathlons, marathon, and cycling races showed a significant effect of the event on CHO intake rates (F 3,216 = 13.9, P G 0.001). In contrast to the triathlons, the average CHO intake rate during CYCLE was significantly lower (53 T 22 gih j1, P = 0.044). The lowest mean CHO intake rates were reported during MARATHON (35 T 26 gih j1 ), which were significantly lower than intake rates during CYCLE (P = 0.034) and all triathlon events (P G 0.001). Regardless of the event, individual CHO intakes among athletes varied greatly (range = gih j1 ). Within all triathlon events, CHO intake rates depended significantly on the mode of exercise, with significantly higher intakes during the cycling section compared with the run (P G 0.05; Fig. 1). Form of CHO intake. Carbohydrate was ingested in solid, liquid, and gel form during all events. CHO-containing drinks supplied between 29% (MARATHON) and 49% (IM Hawaii) of the total CHO intake. CHO gels accounted for 28% (CYCLE) to 45% (MARATHON) of the race s CHO intake. Only 15% of CHO was ingested in solid form during IM Hawaii, whereas solid CHO intake made up 37% of the CHO intake during the PRO races. TABLE 3. Nutrient intakes during endurance events (mean T SD). Fluid (mlih j1 ) kcalih j1 CHO (gih j1 ) Protein (gih j1 ) Fat (gih j1 ) Fiber (gih j1 ) Sodium (mgih j1 ) Caffeine (mgih j1 ) IM Hawaii 794 T 309 ( ) 258 T 96 ( ) 62 T 26 (22 124) 2 T 2 (0 6) 1 T 1 (0 7) 1 T 1 (0 3) 422 T 213 ( ) 26 T 22 (0 78) IM GER 703 T 238 ( ) 292 T 104 ( ) 71 T 25 (33 126) 2 T 2 (0 10) 1 T 1 (0 4) 1 T 1 (0 2) 444 T 216 (69 975) 33 T 29 (0 100) IM T 254 ( ) 265 T 105 (96 494) 65 T 25 (31 122) 3 T 3 (0 12) 1 T 1 (0 5) 1 T 1 (0 4) 403 T 193 (91 936) 28 T 24 (0 107) MARATHON 354 T 187 (81 918) 146 T 102 (18 527) 35 T 26 (6 136) 3 T 5 (0 16) 1 T 1 (0 13) 2 T 3 (0 12) 118 T 87 (9 321) 23 T 32 (0 125) CYCLE 643 T 599 ( ) 233 T 103 (53 557) 53 T 22 (13 114) 3 T 3 (0 13) 1 T 1 (0 5) 1 T 1 (0 4) 208 T 183 (15 857) 21 T 29 (0 108) PRO 711 T 270 ( ) 284 T 76 ( ) 64 T 20 (29 107) 4 T 2 (0 8) 2 T 2 (0 6) 1 T 1 (0 2) 311 T 156 ( ) 12 T 14 (0 45) APPLIED SCIENCES HABITUAL NUTRIENT INTAKE AND ENDURANCE EVENTS Medicine & Science in Sports & Exercise d 347 Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

52 APPLIED SCIENCES FIGURE 1 CHO intake during IM Hawaii, IM GER, and IM 70.3 split into swim (black), cycle (dark gray), and run (light gray) sections of the event (means T SD). Intake up to 30 min before race start is included into the swim section. *Significantly different from cycle and run section. f Significantly different from run section. P G When analyzing CHO (from solid, gel, and liquid) together with fluid intake rate, an estimate of percent CHO solution consumed was calculated. On average, the percent CHO solution across all six events was 10.6% T 6.2%. The consumed percent CHO solution was significantly more diluted during IM Hawaii (8.8% T 4.4%, P G 0.05) compared with all other events. The ingested CHO expressed as percent solution ranged from 3.5% to 27%. Fluid intake. Fluid intake rates were not significantly different between all triathlon races (F 2,145 = 1.6, P = 0.20; Table 3). However, ANOVAs showed that fluid intake rates were different between triathlons and the other races (F 3,216 = 20.9, P G 0.001). During CYCLE, significantly lower fluid volumes were ingested compared with those ingested during triathlon (643 T 599 mlih j1, P G 0.001). Compared with all other events, the lowest fluid intake rates were reported during MARATHON (354 T 187 mlih j1 ; P G 0.001). Fluid intake was higher during the cycle compared with the run section within all three triathlon events (849 T 339 and 729 T 377 mlih j1, respectively, P G 0.001). Heat index highly correlated with fluid intake rates (r = 0.9, P G 0.01). Within IM Hawaii and IM GER, fluid intake tended to be higher within males compared with females (IM Hawaii: 849 T 280 and 675 T 289 mlih j1, respectively, P = 0.03; IM GER: 729 T 245 and 575 T 216 mlih j1, respectively, P = 0.08). However, corrected for body weight, the differences were not significant. GI Symptoms during the Races Frequency of serious GI problems and ratings of upper and lower abdominal problems during events are displayed in Table 4. Significantly more participants reported serious GI problems (GI scores 9 4) during IM Hawaii and IM GER compared with those during IM 70.3 (Table 4; P = 0.001). Only 4% of the athletes during CYCLE and MARATHON and 7% of all PRO cyclists reported serious GI problems. History of GI symptoms. During all events, scores for upper and lower abdominal symptoms were positively correlated with the reported history of upper and lower abdominal symptoms (r = 0.37 and r = 0.51, respectively, P G 0.001). CHO intake. Mean scores for upper and lower abdominal problems were not correlated with CHO intake rates in any of the triathlon events. When scores for single GI symptoms were evaluated, some significant low to moderate correlations were detected. However, none of these individual GI symptoms were corrected for multiple tests. Nausea and flatulence were correlated with CHO intake rate in two data sets (r = 0.33 and r = 0.34 for nausea and r = 0.34 and r = 0.35 for flatulence during IM Hawaii and IM 70.3, respectively, P G 0.05). When triathletes were divided into subjects experiencing serious GI problems and subjects with mild or without GI problems, CHO intake rates were not significantly different between both groups (65 T 25 and 69 T 27 gih j1, respectively, P = 0.49). Race performance. During both IM events and MARATHON, faster finish times were correlated (r = j0.55, r = j0.45, and r = j0.49, P G 0.01) with high CHO intake rates (Fig. 2). DISCUSSION The present study featured food and fluid intake habits of athletes that were quantified with the use of the same standardized GI questionnaire methodology and a large subject pool (n = 221) during different endurance events. A major finding of the present study was that CHO intake rates varied greatly among running, cycling, and triathlon events but also among individual athletes. Previously recommended high CHO intake rates (up to 90 gih j1 ) (21) were achieved by È50% of the triathletes, 30% of the cyclists, and 15% of the marathon runners. High CHO intake rates were significantly correlated with faster finishing times, and although they were not associated with higher average scores for upper or lower GI symptoms, they did seem to be a risk factor for nausea and flatulence. However, it seems reasonable to advise athletes to aim for a relatively high CHO intake as tolerated by the individual. TABLE 4. GI symptoms during endurance events: any of the 12 GI symptoms rated 9 4 were considered as serious. % Serious Symptoms Upper Abdominal Symptoms Lower Abdominal Symptoms IM Hawaii T T 0.80 IM GER T T 1.02 IM T T 0.54 MARATHON T T 0.27 CYCLE T T 0.27 PRO T T 0.23 Values for upper and lower GI symptoms are means T SD. 348 Official Journal of the American College of Sports Medicine Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

53 FIGURE 2 Correlation between finishing time and CHO intake rate during IM Hawaii (A; r = j0.55, P G 0.001) and IM GER (B; r = j0.45, P G 0.001). CHO and fluid intakes during endurance events. The highest CHO intake rates within the present study have been reported in ultraendurance triathlon events. In agreement with a previous study by Kimber et al. (26), average CHO intake rates during IM and IM 70.3 (67 gih j1 ) exceeded ACSM recommendations (2). During the amateur cycling event (CYCLE), significantly lower average CHO intake rates (53 gih j1 ) were reported. However, the lowest average CHO intake rates (35 gih j1 ) were found during MARATHON. Consequently, 73% of marathon runners failed to meet the comparatively low ACSM recommendations. Altogether, it has to be recognized that the intraindividual differences in intake rates are large (6 136 gih j1 ) and a substantial number of athletes ingested less or more CHO than recommended. The average fluid intakes during the events were between 354 (MARATHON) and 794 mlih j1 (IM Hawaii). Interestingly, the average resulting CHO solution was 10.6%, with individual percent CHO solution ranging from 3.5% to 27%. The lowest average CHO concentration (8.8% during IM Hawaii) still exceeds the general recommendations for the composition of sports drinks of 4% 8% (2,12). Strategies to achieve CHO and fluid intake rates generally consisted of a mixed intake of CHO forms (solid, semisolid, and liquid), and only 1% of athletes ingested CHO solely from solutions. In support of the strategy to ingest different CHO forms, recent studies have shown similar exogenous CHO oxidation rates between fluids, semisolid gels (32), and solid bars (31). Factors influencing CHO and fluid intakes. Several factors varied among the different events that might have influenced food and fluid intakes. For example, ambient conditions were different among events with hot conditions (24-C 29-C), namely, during the triathlon events compared with moderate temperatures during MARATHON (12-C). Previous studies have shown that hot conditions lead to high voluntary fluid intake rates (13), and our data support this. The highest proportion of CHO ingested in the form of liquid (49%) was found during the hottest event (IM Hawaii), whereas the lowest percentage CHO intake (29%) in the form of liquid was reported during the coolest event (MARATHON). In addition, the investigated events varied in average race duration between 3.5 and 11.7 h. Furthermore, in this study and in previous studies, CHO intake rates have been correlated to faster finishing times (26), indicating that a higher CHO intake potentially improved endurance performance. Although there is still debate about a dose response effect with CHO ingestion on performance, recent intervention-based laboratory studies seem to substantiate this effect (38,39). However, it is also possible that faster athletes tend to ingest more CHO compared with slower athletes (17). Alternatively, it could be speculated that faster athletes have a greater ability to ingest and absorb larger quantities of CHO. Furthermore, during this study, it was not possible to assess or control prerace nutrition. Hence, we cannot exclude the possibility that prerace nutrient intake was different between athletes and had an influence on race performance. Previous research has clearly demonstrated varying nutrient intakes during different endurance sports (26,27), which is supported by the current study. For example, a retrospective questionnaire based study by Peters et al. (27) reported a higher intake of both liquids and food by triathletes during competition than runners. In the present study, CHO intake rates during all triathlons were considerably higher compared with MARATHON and CYCLE. Furthermore, CHO and fluid intake rates during MARATHON were substantially lower compared with CYCLE and triathlon. Part of this difference might be explained by the considerably colder weather during MARATHON compared with all other events. However, the difference in CHO and fluid intake rates between running and cycling also persists within the triathlon events (Fig. 1). A similar nutrition pattern has been reported in a study by Kimber et al. (26), and the authors suggested that the lower nutrient intakes during running are due to practical difficulties ingesting large fluid volumes or solid foods. APPLIED SCIENCES HABITUAL NUTRIENT INTAKE AND ENDURANCE EVENTS Medicine & Science in Sports & Exercise d 349 Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

54 APPLIED SCIENCES Prevalence of GI symptoms. A further aspect of our study was to investigate GI distress, which is common during ultraendurance events (25,27,36). In the present study, a high prevalence for serious GI distress (È30%) was reported during the IM races. Significantly lower frequencies of serious complaints were reported during IM 70.3 (14%), MARATHON (4%), and CYCLE (4%). It is known that prevalence for GI distress is augmented with increasing exercise duration (28), possibly caused by increasing dehydration and decreased blood supply to the GI tract (11). Furthermore, hot conditions have previously been linked to a higher prevalence of GI symptoms (4,10), most likely due to increased cutaneous blood flow and associated restricted blood flow to the GI tract (11). In the present study, environmental conditions were more extreme (hot and humid) during all triathlons compared with MARATHON and CYCLE and, accordingly, heat index correlated with scores of upper and lower GI symptoms. Therefore, the combined effects of exercise duration and hot environmental conditions are most likely the causes for significantly increased GI distress during both IM races compared with IM 70.3 and for higher levels of GI distress during IM 70.3 compared with MARATHON and CYCLE. However, the most important factor that influenced GI problems within our study was an individual predisposition and history of GI distress among athletes. Independent of the event, we detected a positive correlation between GI symptoms during the races and reported history of GI distress. This finding is consistent with our previously published data (30) and suggests an individual predisposition for GI distress during exercise. CHO intake and GI symptoms. The ingestion of CHO, especially the excessive consumption of hypertonic drinks, has previously been linked to altered GI distress (34). It has been speculated that hypertonic drinks cause GI distress via water retention to the human intestines (34). Furthermore, much CHO can lead to incomplete absorption (35), and residual CHO in the intestine has been linked to GI problems in studies about rest (5,33). In contrast to previously reported links between CHO intake and GI distress, a recent series of studies has suggested that the intake of high rates of CHO (È1.4 gimin j1 ) in the form of glucose + fructose gels is well tolerated from most athletes during È70 min of endurance running under mild environmental conditions (30). However, it has to be kept in mind that the incidence of GI problems increases with exercise time (28) and might be increased under more extreme weather and race conditions. In the present study, we detected no clear relationship between CHO intake rates and GI distress. Mean upper and lower abdominal symptoms were not associated with CHO intake rates. Furthermore, mean CHO intake rates were not different between athletes with and without serious GI symptoms. However, we detected correlations between scores for nausea and flatulence with high CHO intake rates in more than one data set (IM Hawaii and IM 70.3). This confirms the finding of a previous study where we reported higher scores for nausea with high (90 gih j1 )comparedwithlowerchointakerates(60gih j1 ) during a 16-km outdoor run (30). Similarly, in a study by van Nieuwenhoven et al. (40), flatulence was previously linked to CHO consumption when the ingestion of a CHO sports drink was compared with water intake. Altogether, these data suggest that CHO intake can indeed be a risk factor for nausea and flatulence during exercise. However, those more minor symptoms are less likely to impair performance compared with symptoms such as diarrhea or stomach cramps (24), and it should be kept in mind that high CHO ingestion rates were correlated with faster finishing times. Benefits and limitations of measurements. One aspect that has to be recognized with all dietary measurements is the difficulty to estimate food and fluid intakes, even more so when subjects are exercising. Direct measurement of food and fluid intake on the race course is not possible with the large number of investigated subjects. Hence, the measures rely on the memory of athletes, which is a challenge especially regarding correct estimates of fluid intake during prolonged races. However, all athletes received detailed instructions before the event and were supplied with strategies to remember their race intake such as to recall from where the actual intake deviated relative to a prerace nutrition plan. Furthermore, any answers that caused doubt, such as very low or high fluid intakes, were directly followed up with individual athlete interviews after the race. Consequently, this is the only study using strict subject and dietary control that features more than 200 subjects and six different competitive situations. CONCLUSIONS In summary, the present study showed that CHO intake rates vary greatly not only between events but also between individual athletes (6 136 gih j1 ). The incidence of serious GI distress was quite variable in the present study (4% 32%). High CHO intakes rates were significantly positively correlated with finishing times during IM events but, at the same time, were linked to higher scores of nausea and flatulence. Moreover, a correlation between reported GI symptoms and history of GI distress was reported in this and previous research (32), suggesting an individual predisposition for GI distress. Altogether, the findings of the present study suggest a need for more individualized nutritional advice for endurance athletes, where each athlete finds his/her unique balance between the ergogenic effects of optimal CHO and fluid intake and the potential ergolytic effects of substantial CHO intakes causing GI distress. This study was supported by a grant of Nestec Ltd., Vevey, Switzerland. The authors thank all the athletes who enthusiastically participated in the study. Special thanks go to the Rabobank professional cycling team as well as to Helge Riepenhof, Rolf Aldag, and Team HTC-Highroad for their participation and help with the study. This study would also not have been possible without the support of the 350 Official Journal of the American College of Sports Medicine Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

55 organizers of the investigated endurance events, in particular the incredibly helpful organizational teams of IM GER and IM Hawaii. REFERENCES 1. Allen DG, Lamb GD, Westerblad H. Skeletal muscle fatigue: cellular mechanisms. Physiol Rev. 2008;88(1): American College of Sports Medicine. Position Stand: nutrition and athletic performance. Med Sci Sports Exerc. 2009;41(3): Benini L, Castellani G, Brighenti F, et al. Gastric emptying of a solid meal is accelerated by the removal of dietary fibre naturally present in food. Gut. 1995;36(1995): Brouns F, Beckers E. Is the gut an athletic organ? Digestion, absorption and exercise. Sports Med. 1993;15(4): a. Colombani PC, Mannhart C, Wenk C, Frey WO. Nutritional intake during a 244 km multisport ultraendurance race. Pakistan J Nutr. 2002;1(3): Corazza G, Strocchi A, Rossi R, Sirola D, Gasbarrini G. Sorbitol malabsorption in normal volunteers and in patients with coeliac disease. Gut. 1988;29(1): Cox RS, Snow RJ, Burke LM. Race-day carbohydrate intakes of elite triathletes contesting Olympic-distance triathlon events. Int J Sport Nutr Exerc Metab. 2010;20: Currell K, Jeukendrup A. Superior endurance performance with ingestion of multiple transportable carbohydrates. Med Sci Sports Exerc. 2008;40(2): Fallon KE, Broad E, Thompson MW, Reull PA. Nutritional and fluid intake in a 100-km ultramarathon. Int J Sport Nutr. 1998; 8(1): Garcia-Roves PM, Terrados N, Fernandez SF, Patterson AM. Macronutrients intake of top level cyclists during continuous competition change in the feeding pattern. Int J Sports Med. 1998;19(1): Gil S, Yazaki E, Evans D. Aetiology of running-related gastrointestinal dysfunction. Sports Med. 1998;26(6): Gisolfi CV. Is the GI system built for exercise? News Physiol Sci. 2000;15: Gisolfi CV, Duchman SM. Guidelines for optimal replacement beverages for different athletic events. Med Sci Sports Exerc. 1992;24(6): Glace B, Murphy C, McHugh M. Food and fluid intake and disturbances in gastrointestinal and mental function during an ultramarathon. Int J Sport Nutr Exerc Metab. 2002;12(4): Glace BW, Murphy CA, McHugh MP. Food intake and electrolyte status of ultramarathoners competing in extreme heat. J Am Coll Nutr. 2002;21(6): Gonzalez RR, Pandolf KB, Gagge AP. Heat acclimation and decline in sweating during humidity transients. J Appl Physiol. 1974;36(4): Harris A, Lindeman AK, Martin BJ. Rapid orocecal transit in chronically active persons with high energy intake. J Appl Physiol. 1991;70(4): Havemann L, Goedecke JH. Nutritional practices of male cyclists before and during an ultraendurance event. Int J Sport Nutr Exerc Metab. 2008;18(6): Holt S, Reid J, Taylor T, Tothill P, Heading R. Gastric emptying of solids in man. Gut. 1982;23: Jentjens RL, Moseley L, Waring RH, Harding LK, Jeukendrup AE. Oxidation of combined ingestion of glucose and fructose during exercise. J Appl Physiol. 2004;96(4): Jeukendrup A. Carbohydrate intake during exercise and performance. Nutrition. 2004;20(7 8): Jeukendrup A. Carbohydrate feeding during exercise. Eur J Sport Sci ;8(2): Results from the present study do not constitute endorsement by the American College of Sports Medicine. 22. Jeukendrup AE, Jentjens RL, Moseley L. Nutritional considerations in triathlon. Sports Med. 2005;35(2): Jeukendrup A, Moseley L, Mainwaring G, Samuels S, Perry S, CH M. Exogenous carbohydrate oxidation during ultraendurance exercise. J Appl Physiol. 2006; Jeukendrup A, Vet-Joop K, Sturk A, et al. Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men. Clin Sci. 2000;98: Keeffe E, Lowe D, Goss J, Wayne R. Gastrointestinal symptoms of marathon runners. West J Med. 1984;141(4): Kimber NE, Ross JJ, Mason SL, Speedy DB. Energy balance during an Ironman triathlon in male and female triathletes. Int J Sport Nutr Exerc Metab. 2002;12(1): Peters H, Bos M, Seebregts L, et al. Gastrointestinal symptoms in long-distance runners, cyclists, and triathletes: prevalence, medication, and etiology. Am J Gastroenterol. 1999;94(6): Peters H, Van Schelven F, Verstappen P, et al. Gastrointestinal problems as a function of carbohydrate supplements and mode of exercise. Med Sci Sports Exerc. 1993;25(11): Peters H, Zweers M, Backx F, et al. Gastrointestinal symptoms during long-distance walking. Med Sci Sports Exerc. 1999;31(6): Pfeiffer B, Cotterrill A, Grathwohl D, Stellingwerff T, Jeukendrup A. The effect of carbohydrate gels on gastrointestinal tolerance during a 16-km run. Int J Sport Nutr Exerc Metab. 2009;19(5): Pfeiffer B, Stellingwerff T, Zaltas E, Jeukendrup AE. Oxidation of solid versus liquid CHO sources during exercise. Med Sci Sports Exerc. 2010;42(11): Pfeiffer B, Stellingwerff T, Zaltas E, Jeukendrup AE. CHO oxidation from a CHO gel compared with a drink during exercise. Med Sci Sports Exerc. 2010;42(11): Ravich W, Bayless T, Thomas M. Fructose: incomplete intestinal absorption in humans. Gastroenterology. 1983;84(1): Rehrer N, van Kemenade M, Meester W, Brouns F, Saris W. Gastrointestinal complaints in relation to dietary intake in triathletes. Int J Sport Nutr. 1992;2(1): Rehrer N, Wagenmakers A, Beckers E, et al. Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise. J Appl Physiol. 1992;72: Riddoch C, Trinick T. Gastrointestinal disturbances in marathon runners. Br J Sports Med. 1988;22(2): Saris WH, van Erp-Baart MA, Brouns F, Westerterp KR, ten Hoor F. Study on food intake and energy expenditure during extreme sustained exercise: the Tour de France. Int J Sports Med. 1989;10(1 suppl):s Smith JW, Zachwieja JJ, Horswill CA, et al. Evidence of a carbohydrate dose and prolonged exercise performance relationship. Med Sci Sports Exerc. 2010;42(5): Smith JW, Zachwieja JJ, Peronnet F, et al. Fuel selection and cycling endurance performance with ingestion of [ 13 C]glucose: evidence for a carbohydrate dose response. J Appl Physiol. 2010; 108(6): van Nieuwenhoven M, Brouns F, Kovacs E. The effect of two sports drinks and water on GI complaints and performance during an 18-km run. Int J Sports Med. 2005;26(4): a. Zimberg IZ, Crispim CA, Juzwiak CR, et al. Nutritional intake during a simulated adventure race. Med Sci Sports Exerc. 2008; 18(2): APPLIED SCIENCES HABITUAL NUTRIENT INTAKE AND ENDURANCE EVENTS Medicine & Science in Sports & Exercise d 351 Copyright 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

56 Sport Sci Health (2012) 8:51 58 DOI: /s ORIGINAL ARTICLE No changes in time trial performance of master endurance athletes after 4 weeks on a low carbohydrate diet Maria Francesca Piacentini Attilio Parisi Nicole Verticchio Stefania Comotto Romain Meeusen Laura Capranica Received: 10 January 2012 / Accepted: 17 January 2012 Springer-Verlag 2012 Abstract The purpose of the present study was to evaluate the effects of a 4-week low-carbohydrate (CHO) diet regimen on body weight, exercise performance and hormonal response to running in master athletes. Six endurance master athletes performed three 30-min time trials, before (TT1), after 15 days (TT2) and after 30 days (TT3) on a low CHO diet. Blood samples were collected for hormonal and lactate measurements. After 15 days body weight had decreased (TT ± 2.4 kg, TT ± 2.7 kg; P = 0.006) and then remained stable. No differences were observed in performance (TT1 7,015 ± 273 m, TT2 6,920 ± 286 m, TT3 7,202 ± 315 m) and in the insulin/glucagon ratio. After 2 and 4 weeks, adrenocorticotropic hormone decreased significantly both at rest (baseline: TT ± 7.8 pg. ml 1, TT ± 3.2 pg. ml 1 ) and during exercise (end of exercise: TT1 120 ± 20 pg. ml 1, TT2 80 ± 16 pg. ml 1, TT3 31 ± 2 pg. ml 1 ). Baseline cortisol concentrations had increased significantly after as little as 15 days on the low CHO diet. The results of the present M.F. Piacentini ( ) N. Verticchio S. Comotto L. Capranica Department of Human Movement and Sport Sciences University of Rome Foro Italico, Rome, Italy mariafrancesca.piacentini@uniroma4.it A. Parisi Department of Health Sciences University of Rome Foro Italico, Rome, Italy R. Meeusen Department of Human Physiology and Sportsmedicine, Vrije Universiteit Brussel, Brussels, Belgium study demonstrate no changes in time trial performance in master endurance athletes after 4 weeks on a low CHO diet. However, an effect on the hypothalamic pituitary adrenal axis emerged. Key words Carbohydrate loading Master athletes HPA axis Zone diet Introduction Master endurance athletes are the largest proportion of participants in marathon and half-marathon competitions. Although they regularly train to improve performance, they often have to squeeze work-outs and competitions into very busy daily schedules that include family and work. Previous research in top-level endurance athletes [1] has shown that a correct diet to meet energy requirements should include 60 65% carbohydrate (CHO) and 20 25% fat, and the remaining percentage protein. Not having comparable technical support to elite athletes, master and recreational athletes tend to adopt word-of-mouth nutritional and training programmes. Unfortunately, incorrect or generalized training programmes often lead to nonfunctional overreaching [2] while nutritional word-ofmouth programmes could result in incorrect practices and insufficient quantity and quality of food intake [3]. One of the most publicized nutritional regimens that claims to improve athletic performance, reduce body fat and increase muscle mass is the Zone diet [4]. This regimen is based on the principles that altering the macronutrient composition (40% CHO, 30% protein and 30% fat) and controlling the protein to CHO ratio (range ) makes it possible to avoid the adverse effects of an increase in insulin. Actually, the ideal macronutrient distribution and

57 52 subsequent caloric intake of the Zone diet is determined primarily by the high protein intake that would lower insulin concentrations, thus promoting free fatty acid utilization and sparing muscle glycogen [4]. For this reason, athletes are recommended to increase their protein intake up to g. kg LBM 1 and to consume five meals per day (three main meals and two snacks) precisely organized in blocks with one block comprising 9 g CHO, 7 g protein and 1.5 g fat in a 1:1:1 ratio [4, 5]. According to the Zone s promises, after as little as 7 10 days on this regimen an increase in athletic performance and lean body mass, and a reduction of body fat should be observed [4]. In Italy, the Zone diet is receiving increasing attention from master and recreational athletes despite the lack of direct evidence of better performances and the severe criticism that it has already received concerning its low CHO content in relation to top level endurance performances [6]. According to a preliminary survey conducted in different fitness and sport centres (personal communication), it appears that 38% of those interviewed had tried the Zone diet. In particular, 79% of competitive master longdistance runners had tried the Zone diet with the precise aim of increasing their performance by reducing their body weight. At present, only two studies [7, 8] have evaluated the effects of the Zone diet on performance and training parameters in endurance, recreational and master athletes. In 2002, Jarvis et al. [7] reported decreases in performance, blood lipid levels and body composition in recreational athletes. The authors claimed that the Zone diet calculation resulted in a significant reduction in caloric intake with respect to the previous dietary regimen that resulted in a reduction in body weight and body fat. In 2009, Piacentini et al. [8] evaluated the performance and training parameters in six master endurance runners who underwent a 2-week Zone diet regimen which was kept isocaloric with respect to their energy expenditure. One athlete dropped out of the study after 10 days because an inability to train. Although no difference emerged in their running performance, the training diary and profile of mood state parameters showed a maladaptation towards the diet. In particular, a decrease in vigour (15%) and increases in fatigue (12%), depression (21%) and strain (169%) were evident. Alterations in the macronutrient components of a diet have been shown to alter the responses of the hypothalamic pituitary adrenal (HPA) axis [9]. In particular, increasing the protein content of meals (30 40% total energy) resulted in an increase in cortisol concentrations [9]. At present, no study has addressed the hormonal changes due to the Zone diet, which could be critical for the stress response to exercise and changes in mood status of endurance athletes. There - fore, the aim of the present study was to evaluate the effects of the Zone diet on endurance performance and on the hormonal response to exercise during a 30-min time trial (TT) in master athletes. In particular, it was hypothesized that no effect on performance would be evident. Materials and methods Experimental approach A repeated-measures experimental design in which participants served as their own control was conducted after obtaining approval from the ethics committee. Four experimental sessions separated by 15 days were scheduled. The first session was designed to determine each athlete s height, body mass and maximal oxygen consumption (VO 2max ) on a treadmill (RunRace HC 1200; Technogym, Gambettole, Italy), while the other three experimental sessions were designed to investigate the effects of the Zone diet on running performance and hormonal responses. Participants Six male master endurance athletes (age 48.0 ± 1.5 years, body mass 72.2 ± 2.4 kg, height 171 ± 4 cm, VO 2max 59.0 ± 2.3 ml. kg 1. min 1 ) signed written informed consent to participate in this study. Because different levels of training and changes in nutritional regimens could affect the results, the criteria for inclusion were: (1) runners had to have trained regularly (at least 5 days per week) and to have competed in long-distance races (i.e km) for the past 10 years; (2) athletes had not adopted diets that altered their regular nutritional regimen in the past 3 years. Maximal exercise test Sport Sci Health (2012) 8:51 58 Athletes were requested to perform a graded incremental exercise test to exhaustion on a treadmill. During the test, heart rate (HR) was monitored (Sport Tester; Polar Electro, Kempele, Finland) and oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ) and ventilation were recorded on a breath-by-breath basis (K4b 2 COSMED, Italy). The K4 Cosmed flow meter was calibrated with a 3-l syringe (Hans Rudolph, Dallas), and the gas analyser was calibrated with known gas mixtures (O 2 16% and 20.9%, CO 2 5% and 0.03%).

58 Sport Sci Health (2012) 8: Participants started running at 9 km. h 1 and speed was increased by 1 km. h 1 every minute until voluntary exhaustion [10]. The individual s VO 2max was identified by the occurrence of one of the following criteria: (a) a plateau or an increase of less than 1 ml. kg 1. min 1 in oxygen consumption despite further increases in exercise intensity; or (b) a respiratory gas exchange ratio higher than 1.1. If the test ended before the attainment of the maximal oxygen consumption, peak oxygen consumption (VO 2peak ) was calculated averaging the values from the final 30 s of the exercise test. A 10-μl capillary sample was taken from the ear lobe to measure blood lactate concentration at rest, at the end of exercise, and after 3, 6 and 9 min of recovery using and Accusport Lactate Analyser (Roche, Basel, Switzerland) with a single-trial interclass reliability of [11]. Dietary control Using standard forms, the participants were asked to record the weight of all food items and beverages consumed (other than water) for seven consecutive days and to keep a detailed training log to measure their caloric intake, diet composition and energy expenditure due to exercise. Before recording, the participants were given standardized instructions by a dietician. Brand names, methods of preparation and recipes for consumed mixed dishes were requested. Indeed, a dietary intake record relies heavily on the honesty and accuracy of the participant, so the results were interpreted with some caution, taking into account the under-reporting often found in athletes [12]. The completed records were analysed by a medical doctor nutritionist with at least 15 years experience with athletes and a 20% under-reporting was estimated. Energy intake and food quotient were calculated for each individual and presented as the average over the 7 days [13]. In considering that the ideal macronutrient distribution and subsequent caloric requirement determined on the basis of protein intake of the Zone diet was hypocaloric with respect to the reported caloric intake of the athletes, the dietician meticulously placed the participants in energy balance, keeping their total caloric intake constant and modifying the macronutrient content according to the 40% CHO, 30% fat and 30% proteins principle of the Zone diet (0.75 protein to CHO ratio distributed in five meals a day, maintaining the 1:1:1 block ratio between macronutrients). Each subject received a precise list of ingredients and recipes to correctly prepare all the different Zone diet dishes. Meetings with the nutritionist were scheduled on a weekly basis. Running performance The day before the experimental sessions athletes were asked to refrain from hard physical training, caffeine and alcohol intake. Participants were evaluated in the laboratory immediately before starting the diet (TT1), and after 15 and 30 days on the diet (TT2 and TT3, respectively). Furthermore, the day before TT2 and TT3 they were asked to replicate their meal and training. On the experimental days, athletes reported to the laboratory between 8.30 and am and performed their test at the same time of day. After collection of their baseline data, body mass was determined to an accuracy of 100 g. The athletes were then instructed to drink water ad libitum and to perform as many kilometres as possible on the treadmill in 30 min as if they were engaged in a race, with no feedback from the experimenter. They were allowed to increase or decrease the speed according to their personal feelings. Body mass was measured at the beginning and at the end of the exercise session. At the beginning, at 15 min and at the end of the exercise, running HR, distance covered and the athlete s fatigue state according to Borg s rate of perceived exertion (RPE) scale [14] were recorded. At the beginning, at 15 minute, at the end of exercise and after 10 min of recovery a 10-μl sample was taken from the ear lobe for immediate measurement of lactate concentration (Accusport Lactate Analyser, Roche, Basel, Switzerland) and a blood sample was collected from an antecubital vein by venepuncture for subsequent adrenocorticotropic hormone (ACTH), cortisol, insulin and glucagon measurements. Blood analysis Blood samples were collected in EDTA K3 tubes, immediately centrifuged at 3,000 rpm for 10 min, and frozen at 20 C until ACTH and haematological analysis including haemoglobin, haematocrit and leucocyte counts. A 10-ml blood sample was drawn into plain serum tubes, centrifuged at 3,000 rpm for 10 min and frozen for determination of cortisol, insulin and glucagon concentrations. A 0.5-ml aliquot of whole blood was extracted and used for determination of haematocrit and haemoglobin in order to estimate the percentage changes in plasma volume relative to the resting sample [15]. All samples were tested in the same series to avoid any variation between tests. Glucagon was measured using a DRG diagnostic kit (DRG Instruments, Marburg, Germany) by radioimmunoassay, insulin and cortisol were analysed with the chemiluminescence methodology (Access Method, Beckman

59 54 Sport Sci Health (2012) 8:51 58 Coulter, Fullerton, CA) and ACTH was analysed by radioimmunoassay (Riamat 280 Wizard 1470 Wallac; Fa. Stratec, Hameln, Germany). Statistical analysis Data are presented as means ± standard error (SE), unless otherwise stated. To evaluate differences in hormone concentrations a 3 4 (three TTs, four sampling times) ANOVA for repeated measures design was used. Data were normalized to the experimental session baseline values. A 3 3 (three TTs, three sampling times) ANOVA for repeated measures was performed for metabolic and RPE measurements. Performance was analysed via a repeated measures design. Post-hoc comparisons were performed by means of Fisher s PLSD test. A level of significance of 0.05 was used throughout the study and the Bonferroni alpha level correction was applied to eliminate any inflated type 1 errors in multiple comparisons. To compare differences between the nutritional regimens (normal diet and Zone diet) a paired Student s t-test was performed. Finally, to provide meaningful analysis in comparing small groups, the Cohen s effect size was calculated considering values of <0.2, <0.6, <1.2 and >1.2 as trivial, small, moderate and large, respectively. Results Table 1 shows the quantities of each macronutrient for the athletes 7-day food records and the Zone diet. The 7-day food records showed that the participants had on average a total intake of 2,580 kcal distributed as 58% CHO, 15% proteins and 27% fat. With the isocaloric Zone diet, CHO intake decreased significantly from 359 to 246 g. day 1 Table 1 Composition of the pre-experimental normal diet and the Zone diet. Values are means ± SEM, grams per day Diet CHO Fat Protein Normal 359 ± ± ± 21 Zone 246 ± 28* 84.1 ± ± 22* *P <0.05 vs. normal diet (P = 0.01, SE 0.8), while protein intake increased significantly (from 105 to 193 g. day 1 ; P = 0.001, SE 0.9). Body mass, running performance, RPE, blood lactate concentration and HR are presented in Table 2 for the three experimental sessions. After 2 weeks on the Zone diet, the athletes body mass had decreased significantly (P = 0.006, SE 0.2). As expected, during exercise increases from baseline values in blood lactate concentration (P <0.05), RPE (P <0.01) and HR (P <0.001) were found; however, no difference emerged between the trials. After 4 weeks on the Zone diet no difference in the total distance covered during the 30-min TT was observed (TT1 distance 7,015 ± 273 m, TT2 distance 6,920 ± 286 m, TT3 distance 7,202 ± 315 m). Figure 1 shows the hormonal values recorded before exercise, and after 15 and 30 min of exercise during the three experimental TTs. ACTH plasma concentrations (Fig. 1a) increased (P = 0.02) during exercise in all three trials, with a much lower percentage increase in TT3. In fact, when normalized as percentage increases with respect to baseline values, at the end of exercise increases in ACTH concentration were seen in TT1 (184%), TT2 (79%) and TT3 (41%), and this difference was significant (P <0.05). At baseline, in the post hoc analysis, ACTH concentrations in TT3 (21.6 ± 3.2 pg. ml 1 ) were lower than in TT2 (44.3 ± 9.6 pg. ml 1 ; P = 0.02), while no difference was seen in the other comparisons. After 15 min of exercise, ACTH concentrations were lower in Table 2 Running performance, RPE, blood lactate concentration and HR recorded during the three time trials. Values are means ± SEM Variable Trial TT1 TT2 TT3 0 min 15 min 30 min 0 min 15 min 30 min 0 min 15 min 30 min Body mass 72.3 ± ± 2.7* 69.7 ± 2.9* (kg) Running 7,015 ± 273 6,920 ± 286 7,202 ± 316 distance (m) RPE 7.0 ± ± ± 1.7 # 8.0 ± ± ± 0.9 # 6.0 ± ± ± 1.0 # Blood lactate 1.6 ± ± ± 1.3 # 1.7 ± ± ± ± ± ± 1.4 (mm) HR (bpm) 63 ± ± ± 2.1 # 67 ± ± ± 3.3 # 65 ± ± ± 2.8 * *P <0.05 vs. TT1, P <0.05 vs. 0 min, # P <0.05 vs. 15 min

60 Sport Sci Health (2012) 8: ACTH 150 * # * # pg/ml * * # TT1 TT2 TT3 0 baseline 15 min end recovery Time (min) 35 Cortisol 30 mcg/dl * # TT1 TT2 TT3 5 0 baseline 15 min end recovery Time (min) Insulin 10,0 8,0 mcu/ml 6,0 4,0 2,0 TT1 TT2 TT3 0,0 baseline 15 min end recovery Time (min) 80 Glucagon pg/ml TT1 TT2 TT3 0 baseline 15 min end recovery Time (min) Fig. 1 ACTH (a), cortisol (b), insulin (c) and glucagon (d) concentrations at rest, after 15 and 30 min of exercise and during recovery before starting the Zone diet (TT1, white bars), and after 15 days (TT2, light-grey bars) and 30 days (TT3, dark-grey bars) on the Zone diet. *P <0.05 vs. TT3, # P <0.05 vs. TT2

61 56 TT3 than in TT2 (P = 0.02) with no further differences. At the end of exercise, in the post hoc analysis ACTH concentrations in TT3 (30.4 ± 2.1 pg. ml 1 ) were lower than in TT1 (121 ± 20.5 pg. ml 1 ; P = ) and TT2 (P = 0.02) and lower in TT2 than in TT1 (P = 0.001). During recovery, ACTH concentrations were lower in TT3 (30.4 ± 2.15 pg. ml 1 ) lower than in TT1 (114.8 ± 23.5 pg. ml 1 ; P = 0.02) and TT2 (68.9 ± 10.6 pg. ml 1 ; P = 0.01), and were lower in TT2 than in TT1 (114.8 ± 23.5 pg. ml 1 ; P = ). Cortisol concentrations increased during exercise in TT1 (P = ) and TT2 (P = 0.01) but not in TT3. In fact, when data collected during exercise were normalized as percentages of increases from the relative baseline concentrations, a significantly lower response (P <0.05) was seen only in TT3 in relation to TT1. In TT1 a steady increase from baseline was observed at all exercise time points (36% at 15 min, 83% at 30 min and 125% during recovery), whereas in TT3 a 10% decrease after 15 min of exercise was seen followed by 5% decrease at the end of exercise and a 7% increase during recovery. Baseline cortisol concentrations were higher (P <0.05) in TT2 (17.2 ± 0.8 μg. dl 1 ) and TT3 (17.3 ± 0.66 μg. dl 1 ) than in TT1 (12.1 ± 1.1 μg. dl 1 ). Insulin concentrations increased during exercise in TT1 (P = 0.01), but not in TT2 or TT3. Moreover, no difference between trials was observed (Fig. 1c). Glucagon concentrations (Fig. 1d) did not increase during exercise. No significant differences in glucagon concentrations were observed between trials. Blood lactate concentrations significantly increased during exercise in TT1 (P = 0.01) and TT2 (P = 0.01), but not in TT3. No differences were observed between trials. Discussion Due to the fact that a primary limiting factor in endurance events such as marathon running, triathlon and cycling is the depletion of CHO in the blood, muscle and liver [16 18], the purpose of the present study was to evaluate the effects of a 4-week Zone diet (isocaloric with respect to the previous dietary regimen) on running performance and hormonal response to exercise in master athletes. The main findings were: (1) the Zone diet did not induce significant differences in the performance parameters, although body mass decreased; and (2) the Zone diet did not modify the insulin to glucagon ratio, but elicited other hormonal changes. In particular, increases in cortisol concentrations at rest (after after as little as 15 days on the Zone diet) and decreases in ACTH concentrations were seen. The importance of CHO as a major energy source for endurance events is not a novelty. Thus, the Zone diet for Sport Sci Health (2012) 8:51 58 top-level endurance athletes has been criticized [6, 19, 20]. However, according to Sears [4], high CHO diets habitually consumed by athletes elicit an insulin response that causes hypoglycaemia, already exaggerated during exercise, preventing the athletes from performing at their best. Furthermore, the presence of excess insulin will prevent the athletes from optimally using their fat as a fuel. Instead the 40% CHO, 30% fat and 30% protein Zone diet promises to increase endurance performance through changes in the insulin to glucagon ratio, decreases in body weight and increases in muscle mass [4]. Several studies have examined the effects of high and low CHO diets, with similar percentages to that used in this study. Comparing a 60% and a 40% CHO diet in élite hockey players, Akermark et al. [21] found an increase in performance and muscle glycogen content only with the high CHO diet. Pitsiladis and Maughan [22] reported no effects on performance or blood metabolites in high-intensity short-duration exercise of 7 days on a high (70%) or a low (40%) CHO diet. Subjects performed almost 2 min less (in exercise of approximately 10 min in duration) while on the low CHO diet, although the difference in performance was reported to be nonsignificant. Kavouras et al. [23] evaluated a high and low isocaloric CHO diet (600 or 100 g of CHO) on high-intensity moderate-duration exercise, and found that performance was unaffected even though glycogen concentrations were lower on the low CHO diet, when total energy intake was adequate. Burke et al. [12] found that performance is more affected by the total amount of CHO ingested rather than its percentage of energy intake. In fact, with high total energy intakes (around 4,000 5,000 kcal per day) a CHO percentage of 45 60% may be sufficient to meet the athlete s requirements and to restore glycogen content. Thus, athletes are recommended to eat four or five times per day and habitually consume a diet containing 60 65% CHO (usually 6 10 g. kg 1 per day), 20 25% fat, and the balance protein (between 1.2 and 1.4 g. kg 1 per day) with no further need for supplemental sources of protein [24]. Although the Zone diet has been claimed to be beneficial for performance, no scientific evidence has been provided. The only study that the author cites in support of his diet is an unpublished work performed in swimmers who were divided according to a 43% or an 80% CHO diet. However, the athletes on the 43% CHO diet were also able to perform, most probably because their reported high caloric intake guaranteed 502 g per day of CHO, which is sufficient to maintain proper muscle glycogen concentrations [25]. Unfortunately, no information regarding other crucial factors influencing athletic performance. (e.g. modifications in training or personal life-style) was provided. The Zone diet is particularly popular in Italy, especially among sports and fitness participants.

62 Sport Sci Health (2012) 8: There are so far only two studies that have evaluated the effects of the Zone diet on exercise performance, these concentrated in particular on recreational and/or master athletes who are less well informed and receive less nutritional support than top level athletes. Jarvis et al. [7] found a decreased time to exhaustion in moderately trained athletes performing a trial at 80% of their VO 2max after a 1-week diet regimen during which they also showed a 14% decrease in energy intake. However, a meta-analysis on the effects of high CHO or high-fat diet studies [17] showed that speed of metabolic adaptation to dietary changes is still ambiguous and trials up to 7 days might more likely reflect changes in acute glycogen status than chronic metabolic effects. A second study [8] evaluated the effects of a 2-week isocaloric Zone diet on performance and training parameters in master marathon runners. No difference emerged for running performance, but subjects reported an increase in fatigue and a decrease in vigour while training, and their total strain measured using an online training diary increased during the Zone diet period. Another study [26] found no effects of the Zone diet on lipid profile and performance measured with a graded incremental exercise test to exhaustion in students and university staff members. To provide further information on the effects of a diet on endurance performance, in the present study only long distance runners were recruited. To be confident that the variations in performance, if any, could be attributed to the new diet regimen, a 4-week period was scheduled, and a 30-min TT test was considered more valid to test the runners performance. In fact, the high HR (on average 93% of the individual HR max ) and blood lactate concentration (around 5.6 mm, corresponding to a 2.5-fold increase from baseline) values recorded at the end of the exercise confirmed that a high running intensity was maintained. Although running performance tended to decrease after 2 weeks and an average 2.6% increase was observed at the end of the experimental period, no significant differences emerged. In the present study, the average energy intake reported by the athletes was 2,580 kcal. Thus, they would fall into the category of those individuals who need to increase their CHO intake above 65% in order to meet their CHO intake requirements [16]. On the basis of the 7-day food record they reported prior to the Zone diet, it appears that the amount of CHO in their normal diet was in accordance with the amounts reported in the literature for club-level and recreational marathon runners [16]. To avoid any confounding factor that could possibly have influenced their performance, we decided to maintain the athletes diet isocaloric from their 7-day food record, but a decrease in body weight was observed. Subjects probably underreported their true weekly intake by 20%, as normally estimated [16]. If we had not maintained their diet isocaloric by solely using the Zone diet indications, they should have had an intake of only 1,836 kcal. Thus, the nutritionist prepared a detailed menu for the whole study period that was in accordance with the Zone diet s principles. Since our results strongly depend on the accuracy in food preparation and assumptions, we required the subjects to provide a strict report every 15 days. Although the caloric intake did not vary from the pre-experimental period, most of the athletes reported that the Zone diet did not provide enough satiety and a significant 3% reduction in body mass was observed after 2 weeks of the new regimen during which subjects reported mood disturbances. In our previous study [8] we found that, despite the lack of performance results, in the athletes who followed the Zone diet for 2 weeks all the mood scores increased (fatigue, anger, depression, tension) except vigour that decreased by 15% during the second week on the Zone diet. The control group, in contrast, showed the typical changes due to normal training: increases in positive scores and decreases in fatigue. Another purpose of the present study was to determine the hormonal changes claimed to occur after as little as 7 days on the Zone diet. Up to now, no direct scientific evidence has been provided of the effects of the Zone diet on the insulin/glucagon ratio. Several studies have evaluated different macronutrient proportions on the insulin response. In particular, Spiller et al. [27] feeding subjects with different combinations of liquid/protein/cho food boluses found a different insulin response only in the first 30 min after ingestion while blood glucose levels returned to normal values 1 h after ingestion. Linn et al. [28] demonstrated that consuming a diet with a Zone-recommended protein/cho ratio has no greater potential to promote fat burning, weight loss or any other phenomenon related to insulin/glucagon alterations when compared to diets consistent with conventional nutritional recommendations. Similarly, in the present study, neither the insulin and glucagon responses to exercise nor their baseline concentrations differed between trials. In accordance with certain criticisms already put forward by some authors [6, 19], the present results found no direct evidence that a 0.75 protein/cho diet reduces the insulin response when compared to traditional dietary guideline food intakes. An interesting result of the present study was the depressed ACTH response to exercise after 30 days on the Zone diet (Fig. 1a) and the alterations in cortisol concentrations, which were significantly higher at rest in TT2 and TT3 but showed a dampened response to exercise in TT3. Thus, it can be hypothesized that a central feedback mechanism is disrupted from cortisol to ACTH. Chronically reducing the CHO to protein ratio of diets has also been associated with deterioration of mood.

63 58 Consumption of a diet containing 8.5 g. kg 1 per day CHO (65% of total energy) compared with a diet of 5.5 g. kg 1 per day CHO (40% of total energy) results in better maintenance not only of physical performance but also of mood state in general over the course of a period of intensified training [21]. Although we did not measure mood during the present study, some of the subjects complained about mood changes during the experimental period. The higher pre-exercise cortisol values observed before TT2 and TT3 in the present study can be explained by the fact that the HPA activity can be modified by changes in feeding patterns [9]. Gibson et al. [9] found increases in cortisol salivary concentrations after a protein-rich meal (30 40% protein). The high cortisol concentrations measured in the present study at rest after 15 and 30 days on the diet may be part of a homeostatic mechanism in response to a high influx of amino acids. Cortisol concentrations control the feedback mechanism on the HPA axis because it regulates central serotonergic activity, due to the fact that it is able to enter the brain compartment [29]. Therefore, it seems that the high cortisol concentrations observed in the present study may send central signals to decrease the ACTH concentration. Practical applications Athletes who tend to adopt word-of-mouth nutritional regimens that claim to improve endurance performance should be aware that the Zone diet will not result in an improvement in their performance. Conversely, the significant reduction in body mass observed in the athletes suggests that the Zone diet could be effective in individuals seeking to lose weight. Conflict of interest statement None. References 1. Tarnopolsky MA, Gibala M, Jeukendrup AE, Phillips SM (2005) Nutritional needs of elite endurance athletes. Part II: Dietary protein and the potential role of caffeine and creatine. Eur J Sport Sci 5: Meeusen R, Duclos M, Gleeson M et al (2006) Prevention, diagnosis and the treatment of the overtraining syndrome. Eur J Sport Sci 6(1): Rosenbloom CA, Dunaway A (2007) Nutrition recommendations for master athletes. Clin J Sport Med 26: Sears B (1995) Mastering the Zone. Harper Collins, New York 5. Sears B (2000) The Zone diet and athletic performance correspondence. Sports Med 29(4): Cheuvront SN (1999) The Zone diet and athletic performance. Sports Med 27(4): Jarvis M, McNaughton L, Seddon A, Thompson D (2002) The acute 1-week effects of the Zone diet on body composition, blood Sport Sci Health (2012) 8:51 58 lipid levels and performance in recreational endurance athletes. J Strength Cond Res 16(1): Piacentini MF, Salvatori G, Di Cesare C et al (2009) Effects of the Zone-diet on training parameters in recreational master athletes. In: Reilly T, Atkinson G (eds) Contemporary sport, leisure and ergonomics. Routledge, London, pp Gibson EL, Checkley S, Papadopulos A et al (1999) Increased salivary cortisol reliably induced by a protein rich meal. Psychosom Med 61: Wasserman K, Hansen JE, Sue DY et al (1999) Principles of exercise testing and interpretation, 3rd edn. Lippincott Willians & Wilkins, Philadelphia, pp Bishop D (2001) Evaluation of the Accusport lactate analyser. Int J Sports Med 22(7): Burke LM, Kiens B, Ivy JL (2004) Carbohydrates and fat for training and recovery. J Sport Sci 22: Black AE (1986) The use of recommended daily allowances to assess dietary adequacy. Proc Nutr Soc 45(3): Borg G (1998) Borg s perceived exertion and pain scales. Human Kinetics, Champaign 15. Dill DB, Costill DL (1974) Calculation of percentage changes in volumes of blood, plasma and red cells in dehydration. J Appl Physiol 37: Burke LM, Cox GR, Cummings NK, Desbrow B (2001) Guidelines for daily carbohydrates intake. Do athletes achieve them? Sports Med 31(4): Erlenbusch M, Haub M, Munoz K et al (2005) Effect of high-fat carbohydrate diets on endurance exercise: a meta-analysis. Int J Sport Nutr Exerc Metab 15: Hargreaves M, Hawley JA, Jeukendrup A (2004) Pre-exercise carbohydrate and fat ingestion: effects on metabolism and performance. J Sport Sci 22: Cheuvront SN (2000) The Zone diet and athletic performance: author s reply. Sports Med 29(4): Cheuvront SN (2000) The Zone diet phenomenon: a closer look at the science behind the claims. J Am Coll Nutr 22(1): Akemark C, Jacobs I, Ramusson M, Karlsson J (1996) Diet and muscle glycogen concentration in relation to physical performance in Swedish elite ice hockey players International/J Sports Nutr 6: Pitsiladis YP, Maughan R (1999) The effects of alteration in dietary carbohydrate intake on the performance of high intensity exercise in trained individuals. Eur J Appl Physiol 79: Kavouras SA, Troup JP, Berning JR (2004) The influence of low versus high carbohydrate diet on a 45-min strenuous cycling exercise. Int J Sports Nutr Exerc Metab 14: American College of Sports Medicine; American Dietetic Association; Dieticians of Canada (2000) Joint Position Statement: nutrition and athletic performance. American College of Sports Medicine, American Dietetic Association, and Dieticians of Canada. Med Sci Sports Exerc 32: Lamb DR, Rinehart KF, Bartels RL et al (1990) Dietary carbohydrate and intensity of interval swim training. Am J Clin Nutr 52: Bosse MC, Davis SC, Puhl SM et al (2004) Effects of Zone diet macronutrient proportions on blood lipids, blood glucose, body composition and treadmill exercise performance. Nutr Res 24: Spiller GA, Jensen CD, Pattison TS et al (1987) Effect of protein dose on serum glucose and insulin response to sugars. Am J Clin Nutr 46: Linn T, Santosa D, Aygen N et al (2000) Effect of long term dietary protein intake on glucose metabolism in humans. Diabetologia 43: Chaouloff F (1993) Physiopharmacological interactions between stress hormones and central serotonergic systems. Brain Res Rev 18:1 32

64 kg- kg- min? min. Carbohydrate loading and metabolism during exercise in men and women M. A. TARNOPOLSKY, S. A. ATKINSON, S. M. PHILLIPS, AND J. D. MAcDOUGALL Departments of Pediatrics and Kinesiology, McMaster University Medical Center, Hamilton, Ontario L8N 325, Canada Tarnopolsky, M. A., S. A. Atkinson, S. M. Phillips, and J. D. MacDougall. Carbohydrate loading and metabolism during exercise in men and women. J. AppZ. Physiol. 78(4): , During endurance exercise at -65% maximal O2 consumption, women oxidize more lipids, and therefore decrease carbohydrate and protein oxidation, compared with men (L. J. Tarnopolsky, M. A. Tarnopolsky, S. A. Atkinson, and J. D. MacDougall. J. Appl. Physiol. 68: , 1990; S. M. Phillips, S. A. Atkinson, M. A. Tarnopolsky, and J. D. MacDougall. J. AppZ. PhysioZ. 75: ,1993). The main purpose of this study was to examine the ability of similarly trained male (n = 7) and female (n = 8) endurance athletes to increase muscle glycogen concentrations in response to an increase in dietary carbohydrate from to 75% of energy intake for a period of 4 days (carbohydrate loading). In addition, we sought to examine whether gender differences existed in metabolism during submaximal endurance cycling at 75% peak O2 consumption (VO,~~~~) for 60 min. The men increased muscle glycogen concentration by 41% in response to the dietary manipulation and had a corresponding increase in performance time during an 85% v02 peak trial (45%), whereas the women did not increase glycogen concentration (0%) or performance time (5%). The women oxidized significantly more lipid and less carbohydrate *and protein compared with the men during exercise at 75% Vo2 _ peak. We conclude that women did not increase muscle glycogen in response to the 4-day regimen of carbohydrate loading described. In addition, these data support previous observations of greater lipid and lower carbohydrate and protein oxidation by women vs. men during submaximal endurance exercise. glycogen; nutrition; female athlete; endurance exercise A POSITIVE RELATIONSHIP between dietary carbohydrate content, muscle glycogen concentration (29, 30), and endurance exercise performance has been established (1, 19). Carbohydrate loading procedures have been shown to result in a doubling of muscle glycogen concentration (1, 19, 30) and an improvement in both endurance cycling (1) and running (19) performance. It is generally assumed that these relationships apply equally to both men and women, but no studies have yet explored the potential for gender differences. Previous studies of carbohydrate loading have used predominantly or exclusively male subjects (1, 19, 29, 30). Recent studies in our laboratory (27,31) have demonstrated that women had a lower respiratory exchange ratio (RER) when exercising at 65% maximal O2 consumption (VO, m,> compared with trained-matched men. These observations indicated that women had greater lipid oxidation during endurance exercise, which resulted in a sparing of muscle glycogen (31) and lower protein utilization (27,31). The estradiols (20,21) and differences in,&oxidation/citric acid cycle potential (12) may explain the observed gender differences in glycogen utilization during exercise. The lesser reliance on carbohydrates as a metabolic fuel for endurance exercise observed for women (27, 31) may impair their ability to supracompensate for muscle glycogen in response to a carbohydrate-loading regimen compared with men. Lower concentrations of muscle glycogen are a stimulus for glycogen repletion (35); less depletion of muscle glycogen in women during tapering exercise while on a carbohydrate-loading regimen (31) may result in a reduced stimulus for glycogen repletion compared with men. The purpose of the present study was to examine selected metabolic and performance variables in male and female endurance athletes during submaximal exercise under conditions of moderate (55-60%) and high (75%) carbohydrate intakes. Our a priori hypotheses were that women compared with men would demonstrate a lesser increase in muscle glycogen in response to a carbohydrate-loading protocol and no benefit in time to fatigue during endurance exercise even after a high-carbohydrate (75%) diet. We also hypothesized that women would have a greater exercise tolerance time compared with men because of a sparing of muscle glycogen. MElTHODS Subjects. Two groups of male and female athletes volunteered for the study. Informed written consent was obtained after a description of the study and advisement of the risks and benefits of participation in accordance with and under approval of the McMaster University Research Advisory Committee. The men (n = 7) were selected on the basis of a training history demonstrating at least 6 mo of consistent endurance-type physical activity at a frequency of at least four times per week and a peak O2 consumption (Vo2peak) of at least 60 ml l l The women (n = 8) were matched to the men by training history and having a vo2peak of at least 50 ml l l All female subjects were eumenorrheic for at least 2 yr before the testing, and four were taking oral contraceptives. The subjects taking oral contraceptives were placed on a triphasic oral contraceptive for 1 mo before testing and for the duration of the testing (Triphasil, Wyeth, Toronto, Ontario). The women were tested during the midfollicular phase of their menstrual cycle [low-carbohydrate diet (Low CHO), day 8.6 t 5.6; high-carbohydrate diet (High CHO), day 9.5,t The midfollicular phase was chosen because of a better ability to determine the onset of the follicular phase (day 1 of menses) compared with the luteal phase (ovulation) and for comparison with our previous work (27, 31). VO 2 peak was determined within 2 wk of the start of the experimental protocol by using an electrically braked cycle ergometer and a computerized open-circuit gas collection system as previously described (27, 31). All of the subjects had been involved in at least one prior research project using the same system within the past year. vo2peak was considered to /95 $3.00 Copyright the American Physiological Society Downloaded from by on March 28, 2017

65 kg mine1 workload- ) min- GENDER DIFFERENCES IN CARBOHYDRATE LOADING 1361 TABLE 1. Subject characteristics Characteristic Men Women l l Weight, kg LBM, kg ~5.2 Body fat, % Height, cm Age9 yr VO 2peak, ml*kg- *rnin- 64.6~ peak, ml LBM Training history Time training, yr Frequency, times/wk 4.9t Duration, h/wk Significance (P value)* <O.Ol co.01 <0.05 co.01 NS <O.Ol co.01 NS NS NS Values are means + SD for 7 men and 8 women. %702peak, peak O2 uptake; LBM, lean body mass. * Nonpaired t-test. l l be the highest value recorded during a standard incremental ergometer protocol, with termination of the test either after O2 consumption ('VOW) values reached a plateau (an increase of < or pedal revolutions could not be maintained at >50 revolutions/min. The same system was used for the subsequent testing described below. Lean body mass (LBM) was determined by hydrostatic weighing, with residual lung volume being estimated by helium dilution (Collins, Braintree, MA) on the same day as the TO2 peak test. Percent body fat was estimated from the following equation, as outlined previously (27, 31): [495(density>-l] The group characteristics are given in Table 1. Experimentul protocol. During each 5day experimental period, subjects were randomly (counter balanced) assigned to receive a High CHO diet [75% of energy intake (Ein) from carbohydrate] or a Low CHO diet (55.60% of Ei, from carbohydrate). The Low CHO diet was set at 55-60% carbohydrate to match the habitual carbohydrate intake that we have previously measured in similarly trained male and female athletes (27,31). The amount of energy provided in the diet was matched to each subject s habitual intake as determined from a weighed/measured 4-day food record collected immediately before the study. Diets were analyzed by using a computerbased nutrient analysis program (Nutritionist III, N-Squared Computing, Silverton, OR). All subjects completed both testing sessions, with the exception of one female who did not start the second fatigue cycling test because of transient local pain from the biopsy. Her fatigue data were not included in the final analysis. During each experimental period food was provided in a prepackaged form with each item weighed to to.05 g on a digital scale (model E400D, Ohaus, Florham Park, NJ). Each diet consisted of a combination of three major food categories: 1) defined formula diet (Ensure, Ross Laboratories, Montreal, Quebec) supplying % of the total Ein, 2) supplement of glucose polymers (Polycose, Ross Laboratories, Montreal, Quebec) (O-20% of Ein>, and 3) miscellaneous foods (spaghetti, spaghetti sauce, jam, whole wheat bread, cookies, margarine, apple juice, lettuce, corn flake cereal, 2% milk, peanut butter, and granola bars; 35-65% of Ei,). The High CHO diet differed from the Low CHO diet by the addition of simple sugars (glucose polymers and increased amounts of jam and apple juice) and by decreased amounts of peanut butter, butter, and spaghetti sauce. Simple sugars appear to be as effective as complex carbohydrates in carbohydrate-loading protocols (29). Each subject s individual diet was identical for all 5 days of the testing procedure. Subjects were instructed to adhere strictly to each diet and were permitted to consume water ad libitum and up to 750 ml of coffee and/or 750 ml of diet soda per day (except this was restricted in the 14 h before the final test day). Subjects recorded all foods immediately after consumption on a checklist to maximize compliance. Reported Eh compliance was determined to be >95%. The characteristics of the habitual and test diets are given in Table 2. Subjects cycled for 90,60, and 30 min/day at -65% VO, peak during the first 3 days of each of the testing periods to serve as an exercise taper. No exercise was performed on the day before the exercise trial day. During each diet trial the subjects were given two $-liter urine collection containers pretreated with 5 ml of glacial acetic acid. Urine was collected on the rest and exercise days, as described previously (31). Aliquots were taken and stored at -70 C until subsequent analysis for urea concentration, as described below. Subjects were tested either at 0830 or at (n = 2/ group). Each subject completed both exercise trials at the same time. Subjects did not consume caffeine-containing beverages for at least 14 h before the testing session. Three hours before the start of the test the subjects received a snack equivalent to 20% of their habitual daily energy intake, which was comprised of 62% carbohydrate, 23% fat, and 15% protein. The subjects tested at 1800 were allowed to eat their prepackaged breakfast and lunch but had to finish lunch by On arrival, subjects were weighed and a al-gauge plastic catheter was inserted into the antecubital vein for blood sampling. Patency of the catheter was maintained by a saline drip (250 ml, 0.9% NaCl). Blood samples (10 ml) were taken before exercise (0 min); at 20, 40, and 60 min of exercise; at +20 min of recovery; and after a final fatigue trial (Fatigue) for subsequent determination of lactate, glucose, hematocrit, urea, glycerol, K+, Na+, and free fatty acids (FFA) (see below). After the first blood sample a muscle biopsy was obtained from the vastus lateralis (12-15 cm proximal to the lateral joint line and at a depth of 1 cm below the fascia) by using the Bergstrom needle technique. The samples (>75 mg) were immediately dissected free of any connective tissue and were quenched in liquid nitrogen at 15 s for subsequent analysis of muscle glycogen and lactate content. Pressure was maintained for 10 min, and a pressure dressing was applied before the exercise trial was started. The subjects then exercised on a cycle ergometer at a power output preset to elicit 75% of To2 ped for 1 h (75% trial). Testing was conducted in a climate chamber with the temperature controlled at 15 C and the relative humidity at %. At each of the blood sampling times, subjects provided a rating of perceived exertion (RPE) by using the 10 point Borg scale (5) and expired gas was collected over 3 min for determination of vo2, CO2 production, and RER. In addition to the 250 ml of saline that were slowly infused over the trial, the subjects were also allowed ad libitum consumption of water during their first trial, and this volume was kept constant between dietary trials. There were no differences between the men and women with respect to the total relative fluid intake during the testing trial (men, 14.0 t 3.3 ml/kg; women, 14.6 t 5.8 ml/kg; NS). After each cycling trial was completed, a biopsy was taken from the contralateral vastus lateralis and a +20=min blood sample was taken. This rest period was 21.6 t 2.5 and 21.8 t 2.5 min for Low and High CHO trials, respectively, for the men and 25.3? 8.1 and min for Low and High CHO trials, respectively, for the women (NS between or within groups). Subjects then exercised on the ergometer at a constant workload predetermined to elicit 85% of ftoaped until Fatigue. Subjects were not given visual cues as to the total time that had elapsed during the exhaustion ride. One of the investigators determined the onset of fatigue for each subject while blinded to the actual time, and the other investigator recorded the actual time from outside the climate chamber. Fatigue was defined as the inability to maintain 60 revo- Downloaded from by on March 28, 2017

66 kg- min- ) 1362 GENDER DIFFERENCES IN CARBOHYDRATE LOADING TABLE 2. Diet analysis l l Protein, Diet Energy, kcal g kg-l day- Protein Fat Carbohydrate Ethanol Habitual Men Women High CHO Men Women Low CHO Men Women 3,372? t5 24k , * * ,364t NA 1, * * lot NA 3, tl NA 1, * * 12tl ~2 ~ ~~~~~ NA Values are mean t SD. * Significantly lower than values for men (P < 0.01). High CHO, high-carbohydrate diet; Low CHO, lowcarbohydrate diet; NA, not applicable. lutions/min with verbal encouragement during a 20-s period. A postexercise blood sample was taken within 20 s of fatigue (Fatigue). The catheters were removed, and the subjects were asked not to perform any exercise until the next day. Analyses. Urinary urea nitrogen (UN) was determined by using the urease-phenol method (kit no. 640, Sigma Chemical, St. Louis, MO). The inter- and intra-assay coefficients of variation (CVs) were 7.4 and 4.2%, respectively. Plasma lactate was determined by using the Yellow Springs Instruments micro method (model 23L, Yellow Springs, OH). Plasma UN was determined by using the urease-phenol method (kit no. 640, Sigma). Plasma glucose was determined by using a glucose oxidase calorimetric assay (kit no. NO- DA, Sigma). Inter- and intra-assay CVs for the above methods were all <5%. Plasma FFAs were determined by using an enzymatic method (Wako Pure Chemicals, Osaka, Japan). The inter- and intra-assay CVs for the FFA method were 8.2 and 2.1%, respectively. Plasma glycerol was determined by using glycerol kinase/glucose 3-phosphate dehydrogenaselinked fluorometric method as in previous studies (31). The inter- and intra-assay CVs were 4.5 and 5.7%, respectively. Na and K were determined by using an ion-selective electrode (model KNA2, Radiometer, Copenhagen, Denmark) with inter- and intra-assay CVs of <1.5% for each ion. The change in plasma volume was determined from the hematocrit values (32), and Na and K were also expressed after correction for these changes to estimate the net appearance or disappearance of the ion from the plasma pool. Muscle samples were lyophilized and divided into two separate pieces before analysis. Glycogen concentration was determined without perchloric acid pretreatment by using 1.0 M HCl digestion and subsequent fluorometric glucose determination (16). Glycogen standards were obtained from Sigma, and the results are expressed as millimoles of glycogen per kilogram of dry weight. The second sample was treated with 3.0 M perchloric acid, and the extract was assayed for lactate concentration by using a standard fluorometric assay (22). Estimates of the utilization of lipid and carbohydrate were made from the RER values obtained during exercise and were corrected for the estimated protein contribution on the basis of the difference between rest and exercise day UN excretion values (27, 31). The UN measurement included both the l-h and fatigue trials because it was not considered feasible to attempt to measure urinary UN excretion before and after each exercise period. StatisticaL analyses. The physical characteristics of the subjects were compared by using an independent t-test. The time to fatigue was examined a number of ways. First, the data were analyzed as a two-way analysis of variance (AN- OVA) with gender (female vs. male) as a between factor and diet (Low CHO vs. High CHO) as the other variable. Given that our a priori hypothesis was that gender differences in metabolism exist and that this would confound that interpretation of studies with mixed genders, we also analyzed the data as pooled genders and as separated genders by using a paired t-test to further demonstrate how gender differences can confound data interpretation if not controlled for. In addition, the data were expressed as the absolute change from Low to High CHO diets and were expressed as the 95% confidence interval (CI) (i.e., boundaries within which the same mean difference between the diets would be expected to fall 95% of the time if the study were repeated). Correlations between muscle glycogen and performance times were examined by using simple linear regression. All other data were analyzed by using a between-within split-plot ANOVA with group (female vs. male) as the between-subject variable and diet (Low CHO vs. High CHO) and time as the two withinsubject variables. When a significant within group F-ratio was obtained, the location of pairwise differences was performed with the Student-Newman-Keuls post hoc test (Sigma Stat, Jandel Scientific, San Rafael, CA). P < 0.05 was taken to indicate significance. All data in Tables l-5 and Figs. l- 3 are means t SD. RESULTS Testing and performance variables. As expected, the absolute power output for the men was significantly greater than for the women for both the 75 and 85% vo 2peak trials (P < 0.001). When the power output was expressed per kilogram of LBM, however, there were no gender differences in relative power output during the 75% VOzpeak trial, but the women still had lower relative power outputs during the 85% vozpeak trial (P < 0.05; Table 3, top). There were no gender differences in the percentage of VOz peak attained during either the 75 or 85% trials (Table 3, middze). The rela- l l tive iro, (in ml for the men was greater than for the women during the testing (P < 0.01) and the values during the 85% trial were greater than for the 75% trial (P < O.OOl), particularly for the men (P < 0.001; Fig. la). Within a group, the men had a significant increase in the time to fatigue during the 85% trial on the High vs. Low CHO diet [45%, +5.9 min, 95% CI = 0.48,11.30 min (P < 0.05)], whereas the women did not [4.9%, +0.7 min, 95% CI = -8.62, min (NS)] (Table 3, bottom). When the data from the men and women were pooled together, there was no significant effect of diet Downloaded from by on March 28, 2017

67 GENDER DIFFERENCES IN CARBOHYDRATE LOADING 1363 TABLE 3. Testing variables Significance Variables Men Women (P value)* Power output 75% Trial, W <O.OOl 75% Trial, W/kg LBM NS 85% Trial,? W ~11 <O.OOl 85% Trial, W/kg LBM 4.6~ co.05 Oxygen consumption, % of VClzpeak 75% Trial Low CHO NS High CHO NS 85% Trial? Low CHO NS High CHO NS Time to fatigue (85% trial) Absolute time to fatigue, min Low CHO 13.Ok NS High CHO NS Change $ Values are means + SD for 7 men and 8 women. * Between group comparisons. t Significant increase for 85 vs. 75% trial for both groups (P < 0.001). $ Significantly different from 0 (i.e., 0 = no change in time to exhaustion). on performance time. When the groups were analyzed as between-group comparisons, there were no differences in the time to fatigue during the 85% trial between the men and women on either the High or Low CHO diets, possibly because of the high variance observed for the women (Table 3, bottom). Regression analysis revealed a significant correlation between the change in muscle glycogen and the change in the time to fatigue from the Low to High CHO diet for both groups combined [time to fatigue = O.O917(change in glycogen); r2 = 81.2%; P < For the men the equation was time to fatigue = O.l02(change in glycogen); r2 = 88.2% (P < O.Ol), and for the women it was time to fatigue = O.l92(change in glycogen); r2 = 78.6% (P < 0.05). The other indicator of a potential ergogenic effect of the dietary intervention was the subjective RPE during the l-h 75% VO 2 peak ergometer ride. Both genders rated the High CHO trial as being easier than the Low CHO trial (P < 0.05; Fig. 1B). The men had a greater RPE at 60 vs. 20 min, and the women had a greater RPE at 40 and 60 vs. 20 min (P < 0.001; Fig. 1B) for both diets. Respiratory and urinary metabolic data. The men had a significantly greater RER than did the women at all time points (P < 0.001). The RER was significantly lower at 60 min vs. Fatigue for both groups (P < 0.001; Fig. 10. The men excreted more urinary UN than did the women (P < 0.001) and significantly more urea on the exercise vs. rest day (P < 0.05). In addition, the men increased their urinary UN excretion from the rest to exercise day to a greater extent on the High vs. Low CHO diet (P < 0.05; Table 4, top). There were no gender differences in urinary UN excretion during the rest day, but the men still increased their urinary urea excretion on the exercise vs. rest day, whereas the women did not (P < 0.05; Table 4, bottom). DJ 2 d a os I I I 11 1 B C l It It I I I 1 1) t FATIGUE TIME (min) FIG. 1.. A: relative oq.gen consumption #o,>. * Both groups had greater Voz during 85% Vo2 trial compared with 75% Voz trial (P < 0.001). 7 Men had significantly greater Voz at all time points compared with women (P < 0.01). B: rating of perceived exertion (RPE). * Both groups rated 40 and 60 min higher than 20 min (P < 0.001). f Both groups rated high-carbohydrate (High CHO) trial lower (i.e., easier) than low-carbohydrate (Low CHO) trial (P < 0.05). C: respiratory exchangft ratio (RER). * Both groups had greater RER during fatigue (85% VO,) ride compared with 60 min (P < 0.001). t Men had significantly greater RER at all time points, for both diets, compared with women (P < 0.001). Men: q, Low CHO diet; n, High CHO diet. Women: o, Low CHO diet; l, High CHO diet. Fatime, final fatigue trial. Downloaded from by on March 28, 2017

68 1364 GENDER DIFFERENCES IN CARBOHYDRATE LOADING TABLE 4. Urinary urea nitrogen excretion Men* Women Rest day Exercise dayt Rest day Exercise day Absolute urinary UN excretion, g/day Low CHO lo High CHO 10.3k ?3.2$ Relative urinary UN excretion, g l g dietary protein- l day- Low CHO t kO.16 High CHO kO Values are means t SD. UN, urea nitrogen. * Significantly greater than women (P < 0.001). t Significantly greater than rest day (P < O.OS>. $ Significantly greater than Low CHO diet (P < 0.05). 2- The total energy cost of the l-h exercise bout at 75% vozpeak on the Low and High CHO diets was 1,008 and 1,030 kcal, respectively, for the men and was 663 and 668 kcal, respectively, for the women. Protein contributed significantly more (P < 0.001) to the total energy cost of exercise for the men (Low CHO, 4.0%; High CHO, 8.8%) compared with the women (Low CHO, 2.5%; High CHO, 0.0%). Carbohydrate contributed significantly (P < 0.01) more to the energy cost of the l-h exercise bout for the men (Low CHO, 83.6%; High CHO, 82.5%) than for the women (Low CHO, 68.9%; High CHO, 75.4%). Lipid contributed significantly (P < 0.001) more to the energy cost of the l-h exercise bout for the women (Low CHO, 25.3%; High CHO, 23.0%) than for the men (Low CHO, 9.3%; High CHO, 5.9%). Plasma measurements. Plasma lactate concentrations were greater at all time points compared with the resting level for both groups (P < O.OOl), and the men increased their concentrations to a greater extent after both fatigue trials compared with the women (P < 0.001; Fig. 2A). Plasma glucose concentrations were greater for the men compared with the women at 20 min and Fatigue (P < 0.001; Fig. 2B). Plasma urea concentrations were greater for the men than for the women (P < O.OOl), and the levels were lower on the High vs. Low CHO diets for the men at 60 and +20 min and Fatigue and for the women at +20 min and Fatigue (Fig. 2C). Hematocrit was lower for the women vs. the men (P < O.Ol>, and both groups had a significantly lower hematocrit at +20 min (P < 0.05; data not shown). The men and the women had significant reductions in plasma volume at 20,40, and 60 min and Fatigue vs. 0 min (P < 0.05), and the women had greater reductions in plasma volume compared with the men at all exercise time points (P < 0.01; data not shown). There were no gender differences in plasma FFA concentrations. As expected, the concentrations were significantly lower and increased to a lesser degree during exercise on the High vs. Low CHO diets (P < 0.05; diet x time interaction; Fig. 3A). Interestingly, the women had significantly lower plasma glycerol levels compared with the men (P < O.Ol), and both groups had significantly greater glycerol concentrations at 40 and 60 min and Fatigue vs. 0 min (P < 0.001; Fig. 3B). Plasma K+ was higher at all time points compared with 0 min for both groups (P < 0.01). The men had greater nlasma K+ at 0 min compared with women (P b- B O s- E w' J, I I I I lb J, I I I 1 Ill FATIGUE TIME (min) FIG. 2. A: plasma lactate concentrations. * Concentrations were greater at all time points, for both groups, compared with 0 min (P < 0.001). t Men had greater lactate concentrations compared with women under both dietary conditions at Fatigue (P < 0.001). B: plasma glucose concentrations. * Plasma glucose was greater for men compared with women at 20 min and at Fatigue (P < 0.001). C: plasma urea nitrogen. * Plasma urea was lower at +20 and 60 min and at Fatigue for men and at +20 min and at Fatigue for women on High vs. Low CHO diet (P < 0.05). T At all time points and on both diets plasma urea was lower for women compared with men (P < 0.001). Men: q, Low CHO diet; n, High CHO diet. Women: 0, Low CHO diet; l, High CHO diet. Downloaded from by on March 28, 2017

69 GENDER DIFFERENCES IN CARBOHYDRATE LOADING TIAE (min) 60 +I#) 120 J FATIGUE FATIGUE TIME (min) FIG. 3. A: plasma free fatty acids. * Significant diet x time interaction (P < 0.05; men and women had greater plasma free fatty acids at 40, 60, and +20 min and Fatigue vs. 0 min, which was greater for the Low vs. High CHO diet). B: plasma glycerol. * Greater for all time points vs. 0 min (P < 0.001). t Women had lower glycerol at all time points compared with men (P < 0.01). C: plasma K. * Both groups had significantly greater K+ at all time points relative to 0 min (P < 0.01). f Men had greater K for both diets at 0 min compared with women (P < 0.05). D: plasma Na. * Both groups had greater Na at 20 and 40 min and Fatigue vs. 0 min (P < 0.01). t Men had significantly greater Na compared with women at 20 min on both diets (P < 0.05). Men: q, Low CHO diet; n, High CHO die< Women: 0, Low CHO diet; a, High CHO diet. Downloaded from by on March 28, 2017 < 0.05; Fig. 3C). When expressed relative to plasma volume changes, both the men and women had a net appearance of K+ into the plasma pool at all time points vs. 0 min (P < 0.001; data not shown). The increase in net plasma K+ was less for the men on the High vs. Low CHO diet at all time points (P < 0.05; data not shown). The men and women increased plasma Na at 20 and 40 min and Fatigue vs. 0 min (P < 0.01). At 20 min the men had greater Na than did the women (P < 0.05; Fig. 30). When expressed relative to plasma volume changes, the men had a net appearance of Na at 20 and 40 min and Fatigue vs. 0 min, whereas the women had a net loss of Na at 20,40, and 60 min and Fatigue vs. 0 min (P < for gender difference). Muscle measurements. Glycogen concentrations decreased after 1 h of exercise for both groups with no gender differences (P < 0.001). The men increased preexercise intramuscular glycogen concentrations by 41% on the High CHO diet (P < O.Ol), whereas the women did not (Table 5, top). In addition, the men used more glycogen during exercise on the High vs. Low CHO diet (P < 0.05; Table 5, top). Intramuscular lactates increased for both groups immediately after exercise (P < 0.001) with no diet or gender effects (Table 5, bottom). TABLE 5. Intramuscular glycogen and lactate Diet Pre Men Women Post Pre post* Glycogen, mmol glycogen /kg dry wt Low CHO High CHO 565.4k88.lt $ Lactate, mmol lactate/kg dry wt Low CHO 6.2kl.l k3.7 High CHO 6.5& ~ 10.7 Values are means + SD. * Significant change Pre vs. Post (P < 0.001). t Significantly greater preglycogen for men on High vs. Low CHO diet (P < 0.01). $ Significantly greater glycogen use for men on High CHO diet (P < 0.05).

70 day-l). kg day 1366 GENDER DIFFERENCES IN CARBOHYDRATE LOADING DISCUSSION l The main purpose of this study was to determine whether men and women increase muscle glycogen to the same extent in response to a carbohydrate-loading dietary regimen and, if so, whether this would be reflected in a longer time to fatigue during endurance exercise. In the women muscle glycogen concentrations did not change in response to an increase in dietary CHO intake from 5560% to 75% of &, for a period of 4 days, whereas in the men muscle glycogen increased by 41%. The magnitude of the increase in muscle glycogen by the men is in agreement with previous reports (1, 19, 29,30). The glycogen loading protocol used in this study was very similar to the method described by Sherman et al. (30) and is now widely used by endurance athletes. The mechanisms responsible for this gender-specific response to carbohydrate loading are not clear at this point. First, the rate and magnitude of muscle glycogen recovery appear to be related to the extent of the depletion phase (35). However, in the current study the absolute amount of muscle glycogen measured after the -75%Vo 2 peak performance trial was not different between the genders. The extent of glycogen depletion on the days before the performance trial was not measured in the present study; however, care was taken to ensure that no between-gender differences existed in the duration or intensity of the pretrial exercise during the depletion and repletion phases of this study. The intensi.ty of the training in. th e days before t he performance ride was -65% vo 2 peak compared with the 75% VO2 peak used in the performance trial. We have reported - that men experienced a greater glycogen depletion during endurance * exercise compared with women at 65% of v02peak (31). Therefore, the higher muscle glycogen in response to the dietary carbohydrate loading observed for the men may be partly due to a greater postexercise glycogen depletion during the 4-day pretrial depletion-repletion phase (35). A second important factor that may contribute to the observed gender differences in muscle glycogen response could be that the absolute amount of carbohydrate ingested and not the relative amount is more important in carbohydrate loading. By design, the relative amount of carbohydrates was not different between the genders (High CHO, 74% Ei,; Low CHO, 55-60% Ei,); however, beta.use of differences in E irl val ues, the absolute amount of carbohydrates on the Low and High CHO diets differed (men, 492 and 614 g; women, 274 and 370 g). When the data were expressed relative to LBM (the largest pool of glucose disposal), the men compared with the women still had a greater carbohydrate intake for both the Low and High CHO on the Low CHO trials (men, 7.7 and 9.6 g* kg LBM I; women, 5.9 and 7.9 g* kg LBM 1 l In the studies that have examined the m.etabolic an.d performance effects of glycogen loading, the me an dietary carbohydrate intake has been >500 g/day (1, 15, 19, 29, 30). It may be that either an absolute (i.e., 550 g carbohydrate/ day) or relative (i.e., >8.5 g carbohydrate l LBM 1 l day- ) intake is required to carbohydrate load. How- ever, the attainment of these levels may not be tenable for many women. For example, a female athlete consuming 2,200 kcal/day would have to consume 100% of her dietary energy from carbohydrate to meet a recommendation of 550 g carbohydrate/day. Ahernatively, a female would have to consume 2,900 kcal/day to reach 550 g carbohydrate/day if 75% of dietary energy were from carbohydrate. Given that female endurance athletes have habitual energy intakes of ~2,200 kcal/day (Refs. 9,26; S. M. Phillips, S. A. Atkinson, M. A. Tarnopolsky, and J. D. MacDougall, unpublished observations), the feasibility of a relative recommendation [i.e., 75% of Ei, (14)] in carbohydrate loading for women is limited. From a practical standpoint, we have demonstrated that women who consume their habitual energy intake do not increase muscle glycogen concentrations in response to increasing dietary carbohydrate from 55-60to 75% of Ei,. Although a mechanism to explain the observed gender differences in carbohydrate loading was not determined in this study, it could relate to the uptake of glucose via the insulin-sensitive glucose transporter (GLUT-4), th e p h osphorylation of glucose (hexokinase), and/or the activity of glycogen synthase. To our knowledge there have been no studies that examined gender differences in glucose transporters. Green et al. (12) have found a greater hexokinase activity for men, which could increase glucose 6-phosphate and promote glycogen synthesis during a glycogen-loading phase, particularly if levels of phosphoglucomutase are similarly increased in men compared with women. Exercise-induced depletion of muscle glycogen causes an increase in glycogen synthase nonphosphorylated/ phosphorylated (increased/decreased activity), which facilitates the repletion of muscle glycogen in the presence of a high-carbohydrate intake (35). Unfortunately, this report involved only male subjects (35). One final factor that needs to be considered in the research on glycogen repletion/carbohydrate loading is the phase of the menstrual cycle. A study by Nicklas and colleagues (23) found that the extent of glycogen repletion was greater in the luteal phase compared with the follicular phase. Because the women in our study were tested in the follicular phase of their menstrual cycle (by design), glycogen synthesis may have been limited by their endocrine status. During the luteal phase of the menstrual cycle the levels of progesterone (testosterone-like metabolic effects) are 2 lo- 15 times greater than during the follicular phase, whereas estrogen is only 3 times greater (23). Progesterone is known to reverse the increased utilization of fats and decreased carbohydrate oxidation as demonstrated in oophorectomized rats (13). Future research efforts should examine gender differences in the capacity for glycogen storage during different menstrual phases in humans. Contrary to our hypothesis, women compared with men did not achieve a longer time to fatigue during the 85% VO, peak performance trial. This hypothesis was based partially on our previous finding of muscle glycogen sparing in women who exercised at 65% vo2peak compared with men (31). In addition, several studies in rats have found a sparing of glycogen and a prolonged Downloaded from by on March 28, 2017

71 kg- min) GENDER DIFFERENCES IN CARBOHYDRATE LOADING 1367 l l time to exhaustion in male rats after 17-P-estradiol administration (20) or in female rats that were oophorectomized (21). Because the postexercise muscle glycogen concentrations in this study after the 75% VO, peak trial were not significantly different between the genders, it is not surprising that we failed to demonstrate an increase in the time to fatigue for the women. It is possible that the glycogen-sparing effect for the women occurs at lower exercise intensities (~65% vo2 peak), where lipid becomes a more important metabolic fuel (24). In addition, at the intensity of the performance trial used in this study (85% vo 2 peak), the predominant metabolic fuel is carbohydrate and not lipid (25). This would tend to disadvantage women because they appear to metabolize a relatively greater proportion of energy from lipids during endurance exercise (12, 27, 31). Time to exhaustion on the High CHO vs. Low CHO diet was increased in six of seven men by an average of 45% (P < 0.05), whereas four of seven women increased their time to fatigue by an average of 4.9% (NS). However, when the genders were pooled there was no increase in the time to exhaustion on the High vs. Low CHO diet despite increasing n to 15 (7 + 8 subjects). This calculation illustrates that the failure to control for gender may have an impact on the significance of an outcome measurement, particularly if there is high inherent variance in the measurement (such as for time to fatigue). This is an important observation because the failure to consider gender differences in a metabolic study could result in a conclusion that a given dietary or pharmacological intervention was not efficacious, when in fact it was confounded by gender differences (type II error). We observed a lower RER for the women during endurance exercise at 75% v02peak and a greater calculated lipid utilization (on the basis of the nonprotein RER). In previous studies (27,31), we have also demonstrated a reduction in RER, indicating greater lipid oxidation in women during submaximal endurance exercise. Animal studies have consistently demonstrated that 17-P-estradiol is a potent promoter of increased lipid oxidation during endurance exercise (13, 2l), resulting in a sparing of muscle glycogen (13, 20, 21). Moreover, in humans, several studies have found greater lipid use in women (3, 6, 11, 27, 31), whereas some have not (7,lO). However, it appears that studies with contrary findings were not well matched for important variables or did not report important outcome variables. For example, in one study the male and female subjects both ran km/wk, the men were 12. yr older than the women, and the women had a vo 2mw (ml that was 30% greater than that of the men (7). It is likely that some or all of the women were anovulatory and that the gender matching was not optimal. In another study (IO), there were no reported RER or lipid oxidation calculations. The mechanism behind the enhanced lipid utilization in women compared with men may be related to gender differences in 17-P-estradiol (13, 20), insulin (10, 31), or growth hormone (6, 10) and does not appear to be related to gender differences in glucagon, epinephrine, norepinephrine, or cortisol(lo,31). Previously, we have reported a significantly lower insulin level during exercise at 65% 902 peak (and 15 min of recovery) for women compared with men (31). Similar findings have also. been reported at the termination of exercise at 75% vo 2 peak (10). Unfortunately, no studies have examined gender differences in the postexercise insulin response for longer time periods when glycogen storage may be affected by plasma insulin levels. It has been suggested that the higher level of growth hormone observed during endurance exercise in women may promote lipolysis and a greater lipid oxidation (6); however, this has not been a consistent finding (31). Estrogen is known not only to decrease adipocyte lipolysis/lipoprotein lipase activity in both rats (28,34) and humans (8) but also to increase cardiac and skeletal muscle lipoprotein lipase activity (34). Taken together, these data suggest that women may have a propensity to increase the flux of triglyceride-derived FFA into muscle and away from adipocytes (34). This may explain the finding of increased lipid oxidation in women with no or inconsistent changes in plasma FFA and glycerol (3, 7, 10, 31). Green and colleagues (12) found that women have an increased capacity for,0- oxidation relative to citric acid cycle capacity (increased 3-hydroxyacyl-CoA dehydrogenase-to-succinic dehydrogenase ratio) compared with men, which would favor lipid oxidation, particularly at lower intensities (50-65% vo 2 peak) of endurance exercise. No gender differences in muscle carnitine (18) or in total carnitine palmitoyl transferase activity (7) have been found. We are unaware of any studies to date that have examined gender differences in hormone-sensitive lipase or intramuscular triglycerides. There is a need for such studies along with stable isotopic investigations of FFA and glycerol flux to further understand why women oxidize more fats than do men during endurance exercise. A final observation in this study was that the men oxidized significantly more protein than did the women on the basis of urinary urea excretion from rest to exercise. We have found that women do not increase urinary urea excretion (31) and oxidize less leucine (27) in response to endurance exercise compared with men. The lower plasma urea observed in this study is also consistent with the findings of others (17, 27, 31). The results of the present study suggest that the lack of increase in protein oxidation for women during endurance exercise is not solely related to a carbohydratesparing effect on amino acid oxidation (33) but may relate to other factors such as the gender differences in the activity and/or regulation of branched-chain ketoacid dehydrogenase [rate-limiting step in branchedchain amino acid oxidation in muscle (33)]. Summary. This study has demonstrated that women do not increase muscle glycogen concentrations in response to an elevation in the percentage of dietary carbohydrates from 60 to 75% Ei, by a modified carbohydrate-loading regimen. The women also oxidized less carbohydrate and protein and more lipid during endurance exercise at 75% v02peak compared with trainingmatched men. Knowledge of these gender differences is important because nutritional recommendations on the basis of data collected by using predominantly male subjects may not be valid for women. In addition, this study highlights the need to control for the potential Downloaded from by on March 28, 2017

72 1368 GENDER DIFFERENCES IN CARBOHYDRATE LOADING confounding effect of gender in any physiological study. Given the implications of the gender differences described in this study on nutritional recommendations, it is important that future studies attempt to reproduce these findings and further explore the mechanisms behind such differences. We give special thanks to Anne Borgmann for technical assistance. We gratefully acknowledge the generous donations of Triphasil contraceptive pills by Wyeth (Toronto, Ontario) and of the Ensure and Polycose by Ross Laboratories (Montreal, Quebec). This study was supported by a grant from Sport Canada. Address for reprint requests: M. A. Tarnopolsky, Dept. of Pediatrics, Rm. 3V42, McMaster Univ., 1200 Main St. West, Hamilton, Ontario L8N 325, Canada. Received 11 April 1994; accepted in final form 28 November REFERENCES 1. Bergstrom, J., L. Hermansen, E. Hultman, and B. Saltin. Diet, muscle glycogen and physical performance. Acta PhysioZ. Stand. 71: , Bergstrom, J., E. Hultman, and A. E. Roth-Norlund. Muscle glycogen synthetase in normal subjects. &and. J. CZin. Lab. Invest. 29: , , Blat&ford, F. K., R. G. Knowlton, and D. A. Schneider. Plasma FFA responses to prolonged walking in untrained men and women. Eur. J. Appl. Physiol. Occup. PhysioZ. 53: , Bonen, A., F. W. Haynes, and T. E. Graham. Substrate and hormonal responses to exercise in women using oral contraceptives J. Appl. Physiol. 70: , Borg, G. A. V. Perceived exertion: a note on history and methods. Med. Sci. Sports 5: 90-93, Bunt, J. C., R. A. Boileau, J. M. Bahr, and R. A. Nelson. Sex and training differences in human growth hormone levels during prolonged exercise. J. AppZ. PhysioZ. 61: , Costill, D. L., W. J. Fink, L. H. Getchell, J. L. Ivy, and F. A. Witzmann. Lipid metabolism in skeletal muscle of endurancetrained men and women. J. Appl. Physiol. 47: , Depres, J. P., C. Bouchard, L. Bukowiecki, R, Savard, and J. Lipien. Effects of sex, fatness and training status on human fat cell lipolysis. In: Biochemistry of Exercise, edited by H. G. Knuttgen, J. A. Voget, and J. Poortmans. Champaign, IL: Human Kinetics, 1983, vol. 13, p Duester, P. A., S. B. Kyle, P. B. Moser, R. A. Vigersky, A. Singh, and E. B. Schoomaker. Nutritional survey of highly trained women runners. Am. J. CZin. Nutr. 45: , Friedmann, B., and W. Kindermann. Energy metabolism and regulatory hormones in women and men during endurance exercise. Eur. J. Appl. Physiol. Occup. Physiol. 59: l-9, Froberg, K., and P. K. Pedersen. Sex differences in endurance capacity and metabolic response to prolonged, heavy exercise. Eur. J. Appl. Physiol. Occup. Physiol. 52: , Green, H. J., I. G. Fraser, and D. A. Ranney. Male and female differences in enzyme activities of energy metabolism in vastus lateralis muscle. J. NeuroZ. Sci. 65: , Hatta, H. Y. Atomi, S. Shinohara, Y. Yamamoto, and S. Yamada. The effects of ovarian hormones on glucose and fatty acid oxidation during exercise in female ovarectomized rats. Horm. MetaboZ. Res , H&man, E. Nutritional effects on work performance. Am. J. Clin. Nutr. 49: , Hultman, E., and J. Bergstrom. Muscle glycogen synthesis in relation to diet studied in normal subjects. Acta Med. Stand. 182: , Jansson, E. Acid soluble and insoluble glycogen in human skeletal muscle. Acta Physiol. Stand. 113: , Jansson, G. M. E., Degenaar, P. P. C. A. Menheere, H. M. L. Habets, and P. Geurten. Plasma urea, creatinine, uric acid, albumin, and total protein concentrations before and after 15-, 25-, and 42-km contests. Int. J. Sports Med. Sl32-S138, Jansson, G. M. E., H. R. Scholte, M. H. M. Vaandrager- Verduin, and J. D. Ross. Muscle carnitine level in endurance training and running a marathon. Int. J. Sports Med. 10: Sl53- S155, Karlsson, J., and B. Saltin. Diet, muscle glycogen, and endurance performance. J. Appl. PhysioZ. 31: , Kendrick, Z. V., and G. S. Ellis. Effect of estradiol on tissue glycogen metabolism and lipid availability in exercised male rats. J. Appl. Physiol. 71: , Kendrick, Z. V., C. A. Steffen, W. L. Rumsey, and D. I. Goldberg. Effect of estradiol on tissue glycogen metabolism in exercised oophorectomized rats. J. AppZ. PhysioZ. 63: , Lowry, 0. H., and J. V. Passonneau. A FZexibZe System of Enzymatic AnaZysis. New York: Academic, 1972, p Nicklas, B. J., A. C. Hackney, and R. L. Sharp. The menstrual cycle and exercise: performance, muscle glycogen, and substrate responses. Int. J. Sports Med. 10: , Nygaard, E. Women and exercise-with special reference to muscle morphology and metabolism. In: Biochemistry of Exercise, edited by J. Poortmans and G. Niset. Baltimore, MD: University Park, 1981, vol. IV-B, p O Brien, M. J., C. A. Viguie, R. S. Mazzeo, and G. A. Brooks. Carbohydrate dependence during marathon running. Med. Sci. Sports Exercise 25: , Pate, R. R., R. G. Sargent, C. Baldwin, and M. L. Burgess. Dietary intake of women runners. Int. J. Sports Med. 11: , Phillips, S. M., S. A. Atkinson, M. A. Tarnopolsky, and J. D. MacDougall. Gender differences in leucine kinetics and nitrogen balance in endurance athletes. J. AppZ. Physiol. 75: , Ramirez, I. Estradiol-induced changes in lipoprotein lipase, eating, and body weight in rats. Am. J. Physiol. 240 (Endocrinol. Metab. 3): E533-E538, Roberts, K. M., E. G. Noble, D. B. Hayden, and A. W. Taylor. Simple and complex carbohydrate-rich diets and muscle glycogen content of marathon runners. Eur. J. AppZ. Physiol. Occup. Physiol. 57: 70-74, Sherman, W. M., D. L. Costill, W. J. Fink, and J. M. Miller. The effect of exercise-diet manipulation on muscle glycogen and its subsequent utilization during performance. Int. J. Sports Med. 2: , Tarnopolsky, L. J., M. A. Tarnopolsky, S. A. Atkinson, and J. D. MacDougall. Gender differences in metabolic responses to endurance exercise. J. AppZ. PhysioZ. 68: , Van Beaumont, W. Evaluation of hemoconcentration from hematocrit measurements. J. AppZ. PhysioZ. 32: , Wagenmakers, A. J. M., J. H. Brookes, J. H. Coakley, T. Reilly, and R. H. T. Edwards. Exercise-induced activation of the branched-chain 2-0~0 acid dehydrogenase in human muscle. Eur. J. Appl. Physiol. Occup. Physiol. 59: , Wilson, D. E., C. M. Flowers, S. I. Carlile, and K. S. Udall. Estrogen treatment and gonadal function in the regulation of lipoprotein lipase. Atherosclerosis 24: , Zachwieja, J. J., D. L. Costill, D. D. Pascoe, D. D. Pascoe, R. A. Robergs, and W. J. Fink. Influence of muscle glycogen depletion on the rate of resynthesis. Med. Sci. Sports Exercise 23: 44-48, Downloaded from by on March 28, 2017

73 Eur J Appl Physiol (1992) 65 : European ou.o, Applied Physiology and Occupational Physiology Springer-Verlag 1992 The effect of a high carbohydrate diet on running performance during a 30-km treadmill time trial Clyde Williams, John Brewer, and Moya Walker Department of Physical Education, Sports Science and Recreation Management, Loughborough University, Loughborough, LEll 3TU, UK Accepted January 29, 1992 Summary. The purpose of the present study was to examine the influence of a high carbohydrate diet on running performances during a 30-km treadmill time trial. Eighteen runners (12 men and 6 women) took part in this study and completed a 30-kin time trial on a level treadmill without modifying their food intake (trial 1). The runners were then randomly assigned to a control or a carbohydrate (CHO) group. The CHO group supplemented their normal diets with additional carbohydrate in the form of confectionery products during the 7 days before trial 2; the control group matched the increased energy intake of the CHO group by consuming additional fat and protein. The mean (SEM) carbohydrate intake of both groups was 334 (22) g before trial 1, after which the CHO group consumed 566 (29) g. day-1 for the first 3 days and 452 (26) g. day- 1 for the remaining 4 days of recovery. Although there was no overall difference between the performance times for the two groups during trial 2, the CHO group ran faster during the last 5 km of trial 2 than during trial 1 [3.64 (0.24) m's -1 vs 3.44 (0.26) m's- 1; p< 0.05]. Furthermore, the 6 men in the CHO group ran the 30 km faster after carbohydrate loading [131.0 (5.4) min vs (4.9) rain; P<0.05], whereas there was no such improvement in times of the men in the control group. Blood glucose concentrations of both groups decreased below pre-exercise values during trial 1 (P<0.001), but only the control group had a decrease in blood glucose concentrations during trial 2 (P< 0.001). There were no differences between the concentrations of plasma catecholamines of the control group during the two trials. However, the adrenaline concentrations of the CHO group were lower (P< 0.05) during trial 2 than during trial 1, even though they ran faster during trial 2. These results confirm that dietary carbohydrate loading improves endurance performance during prolonged running and that confectionery can be used as'an effective means of supplementing the normal carbohydrate intake in preparation for endurance races. Offprint requests to: C. Williams Key words: Diet - Endurance - Carbohydrate metabolism - Catecholamines - Running Introduction The benefits of increasing the carbohydrate stores of skeletal muscles and liver before prolonged exercise were reported over two decades ago (for review, see Hultman 1967). The focus of these studies was the influence of dietary carbohydrate loading on endurance capacity rather than on endurance performance. Endurance capacity is defined as the time to fatigue at a fixed exercise intensity whereas, endurance performance is the time to complete a prescribed distance or work load. The one early study on the influence of carbohydrate loading on endurance performance showed that running times, during a 30-km cross-country race, improved after carbohydrate loading (Karlsson and Saltin 1971). In contrast, a more recent study reported no improvements in the running times of well-trained runners competing over a flat 20.9-km indoor course after carbohydrate loading (Sherman et al. 1981). Their method of carbohydrate loading differed from the traditional approach in that it did not include 2-3 days on a low carbohydrate diet. Instead, their subjects tapered their training and increased their carbohydrate intake during the 4 days before competition. The muscle glycogen concentrations achieved were the same as the values obtained after the traditional method of carbohydrate loading (Astrand 1967). The question about what types of carbohydrates are most effective in carbohydrate loading diets is one which is often raised. Costill and colleagues suggested that simple carbohydrates are just as effective in replacing muscle glycogen stores during the first few days of recovery, but that complex carbohydrates may be more effective thereafter (Costill et al. 1981). On the other hand, Roberts et al. (1987) suggest that simple carbohydrates are as effective as complex carbohydrates in restoring muscle glycogen concentrations after prolonged exercise. Of course, the timing of carbohydrate intake as

74 19 well as the influence of different carbohydrates on blood glucose concentration (glycaemic index) play an important role in dictating the rate of glycogen resynthesis (Coyle 1991). In a previous study we showed that endurance capacity during treadmill running can be improved by supplementing normal mixed diets with either complex or simple carbohydrates (Brewer et al. 1988). Simple carbohydrates, in the form of confectionery, were used because on many occasions sportsmen and sportswomen do not have the opportunity to increase the carbohydrate content of their meals. Confectionery products, therefore, provide a convenient way of increasing carbohydrate intake on these occasions. The purpose of the present study was to examine the effects of increasing the carbohydrate content of normal mixed diets with confectionery products on endurance performance during a simulated race over 30 km on a level treadmill. This distance is sufficiently long to detect fatigue, but short enough for the subjects to maintain their motivation to achieve personal best times. A 7-day recovery period was chosen because this is often the maximum time an athlete has between races during the height of the road racing season. Methods Subjects. The 18 subjects (12 men and 6 women) who volunteered to take part in this study were experienced endurance runners and familiar with racing over marathon and half-marathon distances. The subjects were fully informed about the nature of the experiments and what was required of them before they volunteered to take part in this study. The experimental procedures employed in this study were approved by the Ethical Advisory Committee of Loughborough University. Experimental design. The subjects were required to complete two treadmill runs (trial 1 and trial 2) over a distance of 30 km (18.64 miles), separated by a period of 7 days. During these trials, the subjects were encouraged to complete the 30-km runs as fast as possible. They controlled the treadmill speed with a small handheld switch. After completing trial 1, the subjects were randomly assigned to one of two dietary groups (6 men and 3 women in each group). For the 7 days after the first run, one group (CHO group) was prescribed a diet high in simple carbohydrates which was designed to increase their carbohydrate consumption by 70 7o during the first 3 days and by 35% during the remaining 4 days. This was achieved by supplementing their normal diets with simple carbohydrates in the form of confectionery. The subjects in the control group maintained their normal carbohydrate intake during the 7- day period between the two trials but they consumed additional protein and fat in order to achieve energy intakes that were isocaloric with the diets of the CHO group. Procedures. The subjects were fully familiarized with laboratory procedures and completed two preliminary tests before undertaking the two 30-km time trials. Each individual's maximum oxygen uptake value (1702max) (Table 1) was measured on a motorized treadmill, using methods previously described (Williams et al. 1990). The second test assessed the oxygen cost of running over a range of submaximl speeds, and consisted of 16 min continuous running on a level treadmill. The running speed was increased every 4 min, and for the last minute of each 4-min period, expired air was collected through a low resistance respiratory valve and lightweight, wide bore (40 mm) tubing into plastic Douglas Table 1. The physiological characteristics of the 18 subjects assigned to the control and carbohydrate (CHO) groups; values are means (SEM) Group Age Mass J/'O2 max ~re... Maximum (years) (kg) (ml. kg - 1 (1. min - 1) heart rate rain - 1) (beats min -1) Control (1.2) (1.9) (1.6) (4.9) (2) CHO (1.9) (0.6) (1.6) (4.7) (3) ~re... Maximum expiratory volume; l?oz... consumption maximum oxygen bags. The results from these two preliminary tests were used to calculate the running speeds equivalent to 70 7o of each individual's I20~... Following the 3 days on their "normal" diets, the subjects arrived at the laboratory in the morning after an overnight fast. They were weighed and had chest electrodes attached, for monitoring heart rates. Resting samples of expired air were collected for a period of 5 min, followed by a venous blood sample from an ante-cubital vein and capillary blood samples from the thumb of a pre-warmed hand. The subjects completed a 5-min "warm up" on the treadmill at running speeds equivalent to 60O/o I202m~. A 60-S expired air collection was obtained at the end of the warm-up and then the treadmill speed was increased to 70 7o for the start of the 30-kin run. This speed was an appropriate "guide-line" for each subject and it was maintained for the first 5 km. Thereafter, the runners could change the treadmill speed at any time during the time trials. They were, however, encouraged to complete the distance in the shortest possible time. The chosen speeds, distances and time elapsed were displayed on a computer screen in front of the treadmill and in view of the subjects (Williams et al. 1990). Throughout the run, expired air samples were collected at 5- km intervals, along with capillary blood samples from the thumb. Immediately after completion of the 30-kin run, a venous blood sample was taken. Throughout the run, heart rates were monitored and recorded every 30 s. Stride rate was also measured every 5 km by recording the small changes in treadmill speed which occurred each time the feet of the subjects made contact with the treadmill belt. This caused a slight fluctuation in the voltage passing through the treadmill speed indicator, which was amplified and linked to a flat bed chart recorder (model CR503: Lloyd). Stride length was calculated from stride frequency and running speed. Water, placed adjacent to the treadmill, was allowed ad libitum throughout the run, and a moist sponge was also available for cooling purposes. After completing the run the masses of the subjects were recorded before they were allowed to ingest further amounts of fluid or consume any food. Mean (SEM) laboratory temperatures for trial 1 were 16.3 (0.4) C and 15.0 (0.4) C for the control and CHO groups, respectively. During trial 2 the corresponding values were 16.3 (0.5) C and 15.9 (0.6) C. Relative humidity values during trial 1 and trial 2 were 52 (2) O7o and 54 (2) 7o for the control group; the corresponding values for the CHO group trials were 66 (4) 7o and 59 (3) %, respectively. There were no significant differences between these values for the two groups. Nutritional status. Two to 3 weeks before trial 1 the subjects completed 7-day weighed food intake diaries They were analysed to provide a quantitative description of each subject's "normal" daily food intake (Paul and Southgate 1978). Using this information, the subjects were prescribed their normal diets during the 3 days before trial 1 to ensure that they did not change their CHO intake. In order to achieve this degree of nutritional control the subjects

75 20 Table 2. Daily energy and macro-nutrient intakes of the two groups during the 3 days before trial 1 (pre-t1), during the consecutive 3 (lst-3 days) and 4 days (2nd-4 days) before trial 2; values represent means (SEM) Observation Energy Fat CHO Protein period (MJ) (g) (g) (g) Control Pre-T (0.9) (11) (22) (6) lst-3 days 18.2" 248* " (1.3) (19) (23) (15) 2nd-4 days 15.0' 187" " (1.0) (15) (20) (9) CHO Pre-T (0.7) (8) (18) (8) 1st-3 days 16.9" 158" 566* 120" (0.9) (9) (29) (8) 2nd-4 days 13.9" 133" 452* 105 (0.8) (8) (26) (7) * Denotes significant increases (P< 0.001) compared to pre-trial 1 values also completed weighed food intake diaries during the 3 days before trial 1, and during the 7 days between trial 1 and trial 2. The dietary patterns for the two groups during the study are shown in Table 2. During the 3-day period immediately after trial 1, the CHO group increased their carbohydrate intake by 70 (3) % (P<0.001). The consumption of this additional food increased overall energy intake by 50 (3)% (P< 0.001). Over this same period, there was no change in the carbohydrate intake of the control group, but they increased their energy intake by the same amount as the CHO group, which was largely the result of a 99 (6.3)% increase in fat consumption (P<0.001). Nevertheless, there were no significant differences between the energy intakes of 18.2 (1.3) MJ and 16.9 (0.9) MJ of the control and CHO groups respectively, over this 3-day period. During the following 4 days which preceded trial 2, the CHO group reduced their carbohydrate intake to 452 (26) g-day -1. This change in their carbohydrate and energy intakes was still 35 (2)% and 24 (1)% (P<0.001) respectively, above their normal values. Over the same 4-day period, the control group also reduced their energy intake to 25 (3)% (P< 0.001) above their normal intake, by reducing their intake of fat, but without changing their carbohydrate intake. There was no change in the body masses of the control group during the 7-day period between the two trials whereas, the CHO group gained 0.83 (0.2) kg (P< 0.001). Analyses. The percentages of oxygen and carbon dioxide in the expired air samples were determined using a paramagnetic oxygen analyser (model 570A; Sybron Taylor) and an infra-red carbon dioxide analyser (model 303; Mines Safety Appliances). Prior to, and during each series of analyses, both of the analysers were calibrated with a known calibration gas, room air and nitrogen. The volume of each expired air sample was determined by evacuating the contents of each Douglas bag through a dry gas meter (Parkinson Cowan), which had been previously calibrated with a Tissot spirometer (Collins, USA). The venous blood samples were collected in lithium heparin tubes and samples analysed for haemoglobin concentrations by the cyanmethemoglobin method (Boehringer, Mannheim, FRG), and packed cell volumes using a microcentrifuge (Hawksley). Plasma samples were obtained by centrifugation of the venous blood at 2 C, and analysed for free fatty acids (FFA) and glycerol by methods previously reported (Williams et al. 1990), as well as for plasma catecholamines (Brooks et al. 1988). Capillary blood samples (25 ~tl) were depro- teinised in 0.4 mol.l-i perchlorid acid, frozen at -20 C and later analysed for lactate and glucose (Maughan 1982). Changes in plasma volume were calculated from the pre- and postexercise haemoglobin and haematocrit values, according to the method described by Dill and Costill (1974). Statistical analyses. The analyses of the results were based on standard statistical techniques. Tests of homogeneity were carried out on the results. Parametric t-tests were used to examine differences between results which were distributed normally, whilst the non-parametric Wilcoxon matched pairs test was used to examine differences between sets which were not distributed normally. Pages L trend analyses was used to examine trends in data over a period of time (Cohen and Holliday 1982). In all analyses, the 95% level of confidence was taken to be indicative of statistical significance. Throughout the text, tables and figures, values are reported as means (SEM). Results The performance times for the control group during trials 1 and 2 were (4.5) min and (4.7) min respectively (NS). The CHO group covered the 30-km run in (5.5) min during trial 1 and in (5.5) min during trial 2, representing an improvement of 2.6 rain (1.9%). In the CHO group, 8 of the 9 subjects ran the 30-km run faster during trial 2 than during trial 1. The running speeds over each successive 5 km of trial 1 and trial 2 are shown for the two groups in Fig. 1. There were no differences for the control group during the two trials. However, the running speed of the CHO group over the last 5 km during trial 2 was faster than the speed they achieved over the last 5 km during trial 1 A g o == O Control Group 5 ' ','o 15 ' 2 'o 2 '6 30 Distance (km) CHO Group ' 20 ' 2f5 ' 30 Distance (km) Fig. 1. Mean (SEM) running speeds during trial 1 (--[]--; T1) and trial 2 (--I~--; T2) for the control group and the carbohydrate group (CHO) during the two 30-km treadmill time trials. ** P< 0.001

76 [3.64 (0.24) m.s -1 vs 3.44 (0.26) m.s-i; P<0.001]. g 0.9, Trend analysis revealed significant decreases in running ~ 0.92 speed for the control and CHO groups during both trials : and 2. A closer examination of the overall perform- ~. ance times of the men and the women in this study $ o.88 showed that the men in the CHO group had faster running times during trial 2 than trial 1 [127.4 (4.9) min vs _ ~ o., (5.4) min; P<0.05], but there were no such differ- ~ o.8, ences between trials for the men in the control group [129.6 (4.1) min vs (3.6) min; NS]. The small number of women in this study prevented a similar analysis of their performance times, o.96 The oxygen uptakes of the control group during both trials were equivalent to 70.6% P-Oamax [41.5 (2.1) ~ o.94 ml'kg-l'min -1] during the first 5 km. By the end of ~ 092 the time trial running speed had fallen to the equivalent ~ 0.9o of ~ro2max (P< 0.005). There were no significant ~" 0.= differences between the oxygen uptake values for the CHO group over the first 5 km during the two trials [trial 1:41.3 (0.4) vs trial 2:41.7 (0.1) ml.kg -~.min-1]..84 Heart rates ranged between 169 and 180 beats.min -1 for both groups, during the two trials. No differences were found in the heart rate responses of either group on trial 2 when compared to trial 1. During trial 1, both groups decreased their average stride lengths (P< 0.01); the CHO group decreased from 1.33 (0.06) m to 1.24 (0.08) m, whilst the control group decreased from 1.39 (0.05) m to 1.15 (0.08) m. Only the control group de- 5.0 creased their stride lengths (P< 0.001) during trial 2. An = examination of the relationships between performance times, stride length, and ~ro2max values for the whole g 5.0 group (n = 18) during trial 1 showed a stronger correla- o~ 4.5 tion between performance times and average stride 4.0 lengths (r= -0.94; P<O.O1) than between performance times and DrO2raax values (r= -0.75; P<0.01). ~ ~.9 The respiratory exchange ratios (R) of the control 5.0 and CHO groups decreased (P<0.001) during both trials (Fig. 2). There were no differences between the R values on trial 1 and trial 2 for the control group. However, there was a trend towards higher R values in the CHO group during trial 2 compared with trial 1, and this trend became significant at 5km and 20km (P< 0.05). There were no differences in blood glucose concentrations of the control group during the two trials. They did, however, decrease towards the end of the 30-kin run on both occasions (P<0.001; Fig. 3). In contrast, the blood glucose concentrations of the CHO group decreased during trial 1 (P< 0.001), but not during trial 2. Compared with trial 1, the blood glucose concentrations of the CHO group during trial 2 were higher at 20 km, 25 km and at 30 km (P< 0.05). Blood lactate concentrations for the two groups during the two trials are shown in Fig. 4. Trend analysis revealed a decrease (P< 0.05) in the values of the control group during trial 1. The CHO group had higher blood lactate concentrations after 30 km during trial 2, compared to the values obtained after the same distance during trial 1 (P<0.05). Both groups increased their plasma FFA and glycerol concentrations during trial 1 and trial 2 (P<0.001) (Figs. 5, 6). go Control Group ; ','0,;'='0'2'5 3'0 Distance (kin) CHO Group ';,'0,'9 ;o '2'5 3' Distance (km) Fig. 2. Mean (SEM) respiratory exchange ratios (R) during trial 1 (--El--; T1) and trial 2 (--~--; T2) for the control group and the carbohydrate group (CHO) during the two 30-kin treadmill time trials. * P<0.05 A i 5.0 o 4.51 O 4,0 _~ 3.5 Control Group f r T I i 1 I Distance (km) CHO Group 3.05T ~ 10T 151, 2[0, Distance (km) Fig. 3. Mean (SEM) blood glucose concentrations during trial 1 (--El--; T1) and trial 2 (--~--; T2) for the control group and the carbohydrate group (CHO) during the two 30-kin treadmill time trials. * P<0.05 The plasma catecholamine concentrations of the two groups before and immediately after trial 1 and trial 2 are shown in Table 3. Plasma noradrenaline concentrations of the control group increased during both trials by similar amounts (P< 0.001). The plasma noradrenaline concentrations of the CHO group also increased in both trials by similar amounts, but the values achieved were not identical to those obtained for the control 21

77 22 Control Group 0.8 Control Group 0.7 E g O 0.6 E 0.5 o f 210 ' Distance (kin) O 0.4 ej O 0.3 o t~ 0.2 [ Trial 1 Trial 2 [] Pre [] Po= 5 4 g 3 w..l CHO Group 0.8 ~ 0.7 E o.6 g o o~ 0.4 O 0.3 CHO Group 0 I, I ~ I, 2tO, 2f5 ~ Dlsiance (km) Fig. 4. Mean (SEM) blood lactate concentrations during trial 1 (--E]--; T1) and trial 2 (--,--; T2) for the control group and the carbohydrate group (CHO) during the two 30-km treadmill time trials. * P< O. 0.1 ~ [] Pre [] POSt Trial 1 Trial 2 Fig. 6. Mean (SEM) plasma glycerol concentrations before and after trial 1 and trial 2 for the control group and the carbohydrate group (CHO) during the two 30-km treadmill time trials 1.2 A "m 1.0 E 0.8 g u a i 0.8 v < 0.6 i.4 [ 0.2 Control Group Trial 1 Trial 2 CHO Group Table 3. Plasma catecholamine concentrations for the control and the CHO group before and after the two 30-km time trials; values are means (SEM) Noradrenaline (nmol.1-1) Adrenaline (nmol.1-1) Group Trial 1 Trial 2 Trial 1 Trial 2 Control CHO Pre (0.22) (0.29) (0.08) (0.05) Post (1.34) (0.92) (0.68) (0.61) Pre (0.12) (0.13) (0.04) (0.04) Post ' (1.79) (0.90) (0.04) (0.19) * Denotes significantly lower (P< 0.05) than trial 1 value 0.0 Trial 1 Trial2 Fig. 5. Mean (SEM) plasma free fatty acid (FFA) concentrations before and after trial 1 and trial 2 for the control group and the carbohydrate group (CHO) during the two 30-km treadmill time trials group. The plasma adrenaline concentrations of the control group increased during trials 1 and 2 (P< 0.01) and there were no differences between trials. However, the increase in plasma adrenaline concentration of the CHO group during trial 2 was only 63 7o of the change recorded for trial 1 (P<0.05). Body masses of the runners in the CHO group decreased by 1.99 (0.09) kg during trial 1 and by 2.07 (0.11) kg during trial 2. The runners in the control group had a decrease in body mass of 1.92 (0.1) kg during trial 1 and 1.94 (0.08) kg during trial 2. The fluid intake for the control and CHO groups during trial 1 was 353 (151) ml and 349 (63) ml, respectively and during trial 2 the values were 298 (70) ml and 370 (84) ml, respectively. There were no significant differences between trials or between groups. The mean plasma volume changes for the CHO group were -7.0 (1.2) 70 during trial 1, and -5.9 (1.1)o70 during trial 2. For the control group, the mean plasma volume changes were +0.8 (2.0) 7o and -4.7 (2.4) 7o on trial 1 and trial 2 respectively. These changes in plasma volumes were not significantly different.

78 23 Discussion The main finding of this study was that the subjects ran faster during the last 5 km of the 30-km time trial after 7 days on a high carbohydrate diet than they did after consuming their normal mixed diets. The control group showed no such improvement in performance, even though their energy intake was the same as the CHO group. Although there was no significant improvement in the performance time for the CHO group as a whole, it is worth noting that 8 of the 9 subjects completed the 30-kin run in a shorter time during the CHO trial. Furthermore, an analysis of the results of the male runners showed that the 6 men in the CHO group had significantly better overall performance times during trial 2. They improved their times by 3.6 min (2.8%) after carbohydrate loading, whereas the men in the control group did not improve their performance times during trial 2. These improvements were not as great, however, as those reported by Karlsson and Saltin (1971) in their study on the influence of carbohydrate loading on running performance during cross-country races over 30 km. Their subjects improved their performance times by 5.4% after carbohydrate loading. This improvement is twice as great as that achieved by the men in the present study. These differences may be explained in two ways. The first is that Karlsson and Saltin (1971) had two groups of runners in their study who were clearly of different ability. One group was made up of experienced runners with high ~ro2max values and the other consisted of active physical education students who had only modest lko2max values. The reductions in running times for the 30-km run were 5 min (3.2%) and 12 min (7.6%) for the experienced and less experienced runners respectively. The second and more important explanation for the differences in performances reported by the two studies is that the subjects in the earlier study ran over a cross-country course, whereas the subjects in the present study completed the 30-km run on a level treadmill. The demanding nature of the undulating cross-country course is reflected by the performance times of the runners. The ~ro2max values of their subjects were 67.7ml'kg-l'min -1, but their average performance time was rain. In contrast the CHO group, in the present study, with an average lko2max value of only 58.7ml'kg-l'min -~, covered the 30-kin distance in 137.6min. Uphill running uses more muscle glycogen than running at the same speeds on the level (Costill et al. 1974). Therefore, there was probably a greater reduction in the muscle glycogen stores of the runners who completed the 30-km cross country races than occurred in the runners who completed the 30-km treadmill time trials. Carbohydrate loading would, therefore, have been more important in preparation for the 30-km races over the cross-country course than in the present study. The overall rate of carbohydrate oxidation by the CHO group during trial 1 was approximately 2.2 g.min -~ and 2.5 g.min -1 during trial 2. The total amounts of carbohydrate oxidized during trial 1 and during trial 2 were 297 g and 330 g, respectively. The carbohydrate intake of the CHO group during the 7-day recovery period was approximately 3476g, which is 1138 g more than they would have consumed had they not undertaken carbohydrate loading (Table 2). After replacing the 297 g carbohydrate used during trial 1, the excess carbohydrate intake amounted to about 840 g. This approaches the predicted saturation level for carbohydrate storage beyond which additional carbohydrate intake is converted into fat (Acheson et al. 1988). The additional 33 g carbohydrate used in trial 2 represents, therefore, only a small fraction of the carbohydrate consumed over the 7-day recovery period. A similar result was obtained in a recent study using the same exercise procedures to assess the influence of consuming carbohydrate/electrolyte solutions on endurance performance during 30-km treadmill time trials (Williams et al. 1990). We found that when our highly trained runners drank water during these simulated endurance races they slowed down during the last 10 km. However, when they drank the carbohydrate/electrolyte solutions, throughout the time trials, they were able to sustain their optimum running speeds for the whole 30 km. They consumed 1 1 of the 5% carbohydrate/electrolyte solutions and oxidized approximately 30g carbohydrate more than during the water trial. Carbohydrate loading does improve endurance performance but the changes in running times are relatively modest compared with improvements in endurance capacity. For example, the 5.4% improvement in running times for a 30-kin cross country race (Karlsson and Saltin 1971) contrasts quite markedly with an improvement in running time to exhaustion of (Brewer et al. 1988). There is not a strong relationship between initial muscle glycogen concentration and endurance performance as there appears to be with endurance capacity (Bergstrom et al. 1967). There are factors other than the carbohydrate stores of the competitor which influence endurance performance. To sustain high running speeds, an athlete requires a high J~rO2max and to be sufficiently well trained to utilize a large % 1202max for a long time. An adequate supply of muscle glycogen is, nevertheless, an essential prerequisite for endurance competitions. An additional important finding in this study was the lower plasma adrenaline concentrations of the runners in the CHO group during trial 2. They ran the last 5 km of the 30-km run faster after carbohydrate loading. Therefore, it might have been expected that they would have had higher, rather than lower, adrenaline concentrations because of the greater physiological stress of running at faster speeds. The explanation for this change is probably associated with the consequences of carbohydrate loading, in general, and blood glucose concentrations in particular. Previous studies have shown that a reduction in blood glucose concentrations, to hypoglycaemic levels, increases the concentration of plasma adrenaline. Conversely, restoring low blood glucose concentrations to normal resting levels, by glucose infusion, reduces plasma adrenaline concentration (Galbo et al. 1979). During trial 1, the blood glucose concentrations of both the control and the CHO groups decreased below pre-exercise values towards the end of the

79 24 30 km. This was also the response of the control group during trial 2. Thus their blood glucose concentrations and their plasma adrenaline concentrations were similar in both trials. In contrast, the CHO group maintained their normal blood glucose concentrations for the whole of trial 2 and had lower adrenaline concentrations than in trial 1. These results are consistent with the proposed relationship between plasma adrenaline and blood glucose concentrations (Galbo et al. 1979). There were no differences in the plasma noradrenaline concentrations between trials for both groups. However, the mean noradrenaline values for the CHO group after both trials appeared to be higher, though not significantly so, than those of the control group. The only explanation we can offer is that during trial 1 the CHO group ran for a slightly longer time than the control group. During trial 2 they ran the last 5 km faster than in trial 1 and so the contribution of the neuro-transmitter, noradrenaline, to cardiovascular control was probably somewhat greater. Even though carbohydrate loading improved the endurance performance of the runners, trend analysis showed a significant decrease in their speeds as they approached 30 kin. This raises the question as to what other factors contributed to the onset of fatigue in this group. Savard and colleagues (1987) suggested that myocardial fatigue may contribute to fatigue, during prolonged exercise, as a result of the competing demands on the cardiovascular system for oxygen transport and thermoregulation. The contribution of inadequate thermoregulation to the onset of fatigue cannot be assessed in the present study because we did not include measurements of body temperature. Nevertheless, it is worth noting that the subjects drank very little, even though water was freely available throughout the time trials. Their average water intake was between 300 and 370 ml and some runners did not drink at all during either trial. The runners lost approximately 3 7o of their body mass during each of the trials which was almost entirely the result of sweating. The low fluid intake may have contributed to dehydration and possibly the accumulation of an intolerable heat load towards the end of the time trials (Sawka et al. 1985). Although dehydration may not have a detrimental influence on muscle metabolism (Nielsen et al. 1990), it will, nevertheless, lead to either a decrease in skin blood flow or be a threat to blood pressure. The consequence of either of these events occurring during prolonged exercise would contribute to fatigue, irrespective of the status of the body's glycogen stores. In summary, the results of this study confirm the benefits of carbohydrate loading as a means of improving endurance-running performance. Furthermore, they show that supplementing a normal diet with confectionery products is an effective way of increasing carbohydrate intake in preparation for endurance competitions. Acknowledgements. The authors wish to acknowledge their gratitude to their colleague Steve Brooks for the catecholamine analyses and to Dr. Maureen Edmondson, of Mars Confectionery UK, for her support throughout this study. References Acheson K J, Schutz Y, Bessard T, Anantharaman K, Flatt J-P, Jequier E (1988) Glycogen storage capacity and de novo lipogenesis during massive carbohydrate overfeeding in man. Am J Clin Nutr 48 : Astrand PO (1967) Diet and athletic performance. Fed Proc 26: Bergstrom JB, Hermansen L, Hultman E, Saltin B (1967) Diet, muscle glycogen and physical performance. Acta Physiol Scand 71 : Brewer J, Williams C, Patton A (1988) The influence of high carbohydrate diets on endurance running performance. Eur J Appl Physiol 57 : Brooks S, Burrin J, Cheetham ME, Hall GM, Yeo T, Williams C (1988) The responses of the catecholamines and B-endorphin to maximal exercise in man. Eur J Appl Physiol 57: Cohen L, Holliday M (1982) Statistics for Social Sciences. Harper and Row, London Costill DL, Jansson E, Gollnick PD, Saltin B (1974) Glycogen utilization in leg muscles of men during level and uphill running. Acta Physiol Scand 91: Costill DL, Sherman WM, Fink W J, Maresh C, Witten M, Miller JM (1981) The role of dietary carbohydrates in muscle glycogen resynthesis after strenuous running. Am J Clin Nutr 34: Coyle EF (1991) Timing and method of increased carbohydrate intake to cope with heavy training, competition and recovery. J Sports Sci 9 [Suppl] : Dill DB, Costill DL (1974) Calculation of percentage change in volumes of blood, plasma and red cells in dehydration. J Appl Physiol 37 : Galbo H, Hoist J J, Christensen NJ (1979) The effects of different diets and of insulin on the hormonal response to prolonged exercise. Acta Physiol Scand 107:19-32 Hultman E (1967) Studies on muscle metabolism of glycogen and active phosphate in man with special reference to diet. Scand J Clin Lab Invest [Suppl] 94:19 Karlsson J, Saltin B (1971) Diet, muscle glycogen and endurance. J Appl Physiol 31 : Maughan RJ (1982) A simple rapid method for the determination of glucose, lactate, pyruvate, alanine, 3-hydroxybutyrate and acetoacetate on a single 20 ~tl blood sample. Clin Chim Acta 122: Nielsen B, Savard G, Richter EA, Hargreaves M, Saltin B (1990) Muscle blood flow and muscle metabolism during exercise and heat stress. J Appl Physiol 69: Paul AA, Southgate DAT (1978) The composition of foods. HMSO, London Roberts KM, Noble EG, Hayden DB, Taylor AW (1987) Simple and complex carbohydrate-rich diets and muscle glycogen content of marathon runners. Eur J Appl Physiol 57 : Savard G, Kiens B, Saltin B (1987) Central cardiovascular factors as limits to endurance; with a note on the distinction between maximal oxygen uptake and endurance fitness. In: Macleod D, Maughan R, Nimmo M, Reilly T, Williams C (eds) Exercise, benefits, limits and adaptations. Spon, London, pp Sawka MN, Young A J, Francesconi RP, Muza SR, Pandolf KB (1985) Thermoregulatory and blood responses during exercise at graded hypohydration levels. J Appl Physiol 59: Sherman WM, Costill DL, Fink WJ, Miller J (1981) Effect of exercise-diet manipulation on muscle glycogen and its subsequent utilization during performance. Int J Sports Med 2: Williams C, Nute MG, Broadbank L, Vinall S (1990) Influence of fluid intake on endurance running performance: a comparison between water, glucose and fructose solutions. Eur J Appl Physiol 60 :

80 Journal of the American College of Nutrition ISSN: (Print) (Online) Journal homepage: Association between Carbohydrate Intake and Serum Lipids Yunsheng Ma MD, PhD, Youfu Li MD, MPH, David E. Chiriboga MD, MPH, Barbara C. Olendzki RD, MPH, James R. Hebert MSPH, ScD, Wenjun Li PhD, Katherine Leung MPH, Andrea R. Hafner BS & Ira S. Ockene MD To cite this article: Yunsheng Ma MD, PhD, Youfu Li MD, MPH, David E. Chiriboga MD, MPH, Barbara C. Olendzki RD, MPH, James R. Hebert MSPH, ScD, Wenjun Li PhD, Katherine Leung MPH, Andrea R. Hafner BS & Ira S. Ockene MD (2006) Association between Carbohydrate Intake and Serum Lipids, Journal of the American College of Nutrition, 25:2, , DOI: / To link to this article: Published online: 14 Jun Submit your article to this journal Article views: 59 View related articles Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at Download by: [Simon Fraser University] Date: 07 February 2017, At: 22:33