FORWARD: CREATABOLIN C10 WAS USED IN ALL THE TESTS BY MIKE PREVOST. MR. PREVOST CONTACTED US TO TEST THE EFFECTS OF CREATABOLIN C10 AND WHY C10 IS VERY EFFECTIVE. THE EFFECTS OF CREATINE SUPPLEMENTATION(CREATABOLIN C10) ON TOTAL WORK OUTPUT AND METABOLISM DURING HIGH INTENSITY INTERMITTENT EXERCISE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements of the degree of Doctor of Philosophy in The Department of Kinesiology by Michael Cory Prevost B.A., University of Southwestern Louisiana, 1990 December 1995 TABLE OF CONTENTS List of Figures .........................................................................................................iv List of Tables ..........................................................................................................vii Abstract ...................................................................................................................viii Chapter 1. Introduction ..............................................................................................1 1.1 Project Rationale .......................................................................5 1.2 Hypothesis ....................................................................................6 1.3 Objectives ......................................................................................6 2. Review of Literature ...............................................................................8 2.1 Intermittent Exercise ...............................................................8 2.2 Creatine and Control of the Creatine Pool ......................17 2.3 Creatine Supplementation Studies ....................................20 3. Materials and Methods .........................................................................24 3.1 Basic Design..................................................................................24 3.2 Subjects..........................................................................................26 3.3 Supplementation........................................................................27 3.4 VO2 Peak.......................................................................................28 3.5 Standardized Exercise Bouts.................................................28 3.6 Blood Draws and Analysis.....................................................29 3.7 Oxygen Consumption................................................................31 3.8 Urinary Creatinine ...................................................................31 3.9 Statistical Analysis....................................................................31 4. Results.........................................................................................................33 4.1 Time to Exhaustion...................................................................33 4.2 Oxygen Consumption...............................................................35 4.3 Plasma Lactic Acid....................................................................43 4.4 Urinary Creatinine....................................................................52 4.5 Regression of Body Weight and Fitness on Improvement in Total Work Output.................................53 5. Discussion...................................................................................................54 6. Summary and Conclusions.................................................................70 Bibliography................................................................................................................73 Appendix A: Consent Form..................................................................................78 Appendix B: Physical Activity Readiness Questionnaire.......................80 Appendix C: Raw Data...........................................................................................81 Vita...............................................................................................................................112 LIST OF FIGURES Figure 3.1 Study Flowchart................................................................................26 Figure 4.1 Time to Exhaustion - Phase x Bout, Creatine Group..........34 Figure 4.2 Time to Exhaustion - Phase x Bout, Placebo Group............34 Figure 4.3 VO2 For Phase 1 and 2, Bout D, Creatine Group..................34 Figure 4.4 VO2 - Time x Phase, Bout D, Creatine Group........................36 Figure 4.5 VO2 For Bout C Phase 1 and 2, Creatine Group...................36 Figure 4.6 VO2 - Phase x Time, Bout C, Creatine Group.........................37 Figure 4.7 VO2 For Bout D Phase 1 and 2, Placebo Group....................37 Figure 4.8 VO2 - Phase x Time, Bout D, Placebo Group..........................38 Figure 4.9 VO2 For Bout C Phase 1 and 2, Placebo Group.....................38 Figure 4.10 VO2 - Phase x Time, Bout C, Placebo Group.........................39 Figure 4.11 VO2 For Bout B Phase1 And 2, Creatine Group..................39 Figure 4.12 VO2 - Phase x Time, Bout B, Creatine Group.......................40 Figure 4.13 VO2 For Bout B Phase 1 and 2, Placebo Group...................40 Figure 4.14 VO2 - Phase x Time, Bout B, Placebo Group........................41 Figure 4.15 VO2 For Bout A Phase 1 and 2, Creatine Group.................41 Figure 4.16 VO2 - Phase x Time, Bout A, Creatine Group......................42 Figure 4.17 VO2 For Bout A Phase 1 and 2, Placebo Group..................42 Figure 4.18 VO2 - Phase x Time, Bout A, Placebo Group........................43 Figure 4.19 Lactate For Phase 1 and 2, Bout D, Creatine Group.................................................................................44 Figure 4.20 Lactate - Phase x Time, Bout D, Creatine Group...................................................................................................44 Figure 4.21 Lactate For Phase 1 and 2, Bout D Placebo Group..................................................................................45 Figure 4.22 Lactate - Phase x Time, Bout D, Placebo Group...................................................................................................45 Figure 4.23 Lactate For Phase 1 and 2, Bout C Creatine Group.................................................................................46 Figure 4.24 Lactate - Phase x Time, Bout C, Creatine Group.................46 Figure 4.25 Lactate For Phase 1 and 2, Bout C Placebo Group..................................................................................47 Figure 4.26 Lactate - Phase x Time, Bout C, Placebo Group..................47 Figure 4.27 Lactate For Phase 1 and 2, Bout B Creatine Group.................................................................................48 Figure 4.28 Lactate - Phase x Time, Bout B, Creatine Group.................48 Figure 4.29 Lactate For Phase 1 and 2, Bout B Placebo Group..................................................................................49 Figure 4.30 Lactate - Phase x Time, Bout B, Placebo Group..................49 Figure 4.31 Lactate For Phase 1 and 2, Bout A Creatine Group.................................................................................50 Figure 4.32 Lactate - Phase x Time, Bout A, Creatine Group................50 Figure 4.33 Lactate For Phase 1 and 2, Bout A Placebo Group..................................................................................51 Figure 4.34 Lactate - Phase x Time , Bout A, Placebo Group................51 Figure 4.35 Urinary Creatinine For the Creatine Supplementation Group...................................................................................................52 Figure 4.36 Urinary Creatinine For the Placebo Supplementation Group...................................................................................................53 LIST OF TABLES Table 3.1. Subject Characteristics....................................................................27 Table C.1. Time to Exhaustion Data.................................................................81 Table C.2. VO2 Data Bout A................................................................................84 Table C.3. VO2 Data Bout B.................................................................................86 Table C.4. VO2 Data Bout C.................................................................................89 Table C.5. VO2 Data Bout D.................................................................................93 Table C.6. Lactate Data Bout A.......................................................................102 Table C.7. Lactate Data Bout B........................................................................104 Table C.8. Lactate Data Bout C........................................................................106 Table C.9. Lactate Data Bout D........................................................................108 Table C.10. Urinary Creatinine.........................................................................110 ABSTRACT The effects of creatine supplementation on endurance and metabolism during high intensity intermittent exercise were examined using 18 males and females (age 19-26). The subjects were randomly divided into 2 groups (creatine and placebo) and the testing proceeded in two phases. During phase 1 both groups received a placebo. During phase 2 the placebo group again received a placebo, while the creatine group received creatine (3.75g - 5 times daily for 5 days). Testing consisted of high intensity intermittent cycling protocols on a stationary cycle ergometer. Both groups performed all test bouts (in random order) in phase 1 and again in phase 2. Testing consisted of: Continuous (Bout A) - Continuous pedaling at 150% VO2 peak until exhaustion. 30/60 (Bout B) - 30 seconds of pedaling at 150% VO2 peak followed by 60 seconds rest, repeated until exhaustion. 20/40 (Bout C) - 20 seconds pedaling at 150% VO2 peak followed by 40 seconds rest, repeated until exhaustion. 10/20 (Bout D) - 10 seconds pedaling at 150% VO2 peak followed by 20 seconds rest, repeated until exhaustion. The placebo group showed no significant change in time to exhaustion from phase 1 to phase 2 for any of the bouts tested. The creatine group, however, showed a significant increase in time to exhaustion on all bouts. Bout D was impacted significantly more than the other bouts with a more than twofold increase in time to exhaustion. (note that bout D was truncated before subjects reached exhaustion because even at twice the performance time of phase 1 subjects reported feeling very little fatigue and the ability to continue indefinitely). Oxygen consumption did not change from phase 1 to phase 2 for the placebo group, while the creatine group showed a significantly lower rate of oxygen consumption on bouts D and C. Blood lactic acid values were also lower on bouts C and D for the creatine group only. Therefore creatine supplementation significantly impacted performance of high intensity intermittent exercise. The Effects of Creatine Supplementation (C10) on Endurance and Metabolism During High Intensity Intermittent Exercise Mike C. Prevost (Ph.D.)* * Department of Kinesiology, Louisiana State University Methods- The effects of creatine supplementation on endurance and metabolism during high intensity intermittent exercise was examined using 18 males and females (age 19-26). The subjects were randomly divided into 2 groups (creatine and placebo) and the testing proceeded in two phases. During phase 1 both groups received a placebo. During phase 2 the placebo group again received a placebo, while the creatine group received creatine (C10). Testing consisted of a several high intensity intermittent cycling protocols on a stationary cycle ergometer. Both groups performed all test bouts (in random order) in phase 1 and again in phase 2.Testing consisted of : Continuous (Bout A) - Continuos pedaling at 150% VO2 peak until exhaustion. 30/60 (Bout B) - 30 seconds of pedaling at 150% VO2 peak followed by 60 seconds rest, repeated until exhaustion. 20/40 (Bout C) - 20 seconds pedaling at 150% VO2 peak followed by 40 seconds rest, repeated until exhaustion. 10/20 (Bout D) - 10 seconds pedaling at 150% VO2 peak followed by 20 seconds rest, repeated until exhaustion. Oxygen consumption was measured during all bouts using a Quinton Q-Plex I. Blood samples were also taken periodically during each bout for blood lactate analysis. Results- The placebo group showed no significant change in time to exhaustion from phase 1 to phase 2 for any of the bouts tested. The creatine group, however, showed a significant increase in time to exhaustion on all bouts (see figure 1). Bout D was impacted significantly more than the other bouts with a more than twofold increase in time to exhaustion (note that bout D was truncated before subjects actually reached exhaustion because even at twice the performance time of phase 1 subjects reported feeling very little fatigue and the ability to continue indefinitely). Oxygen consumption showed no change from phase 1 to phase 2 for the placebo group, while the creatine group showed a significantly lower rate of oxygen consumption on bouts D and C (see figure 2 for Bout D VO2). Blood lactic acid values were also lower on bouts C and D for the creatine group only (see fig 3 & 4 for bout D lactate values). Discussion- It has recently been demonstrated that supplementation with creatine can lead to an increase in skeletal muscle creatine levels of up to 50% (Harris et. al. 1992). Once creatine enters the muscle it is phosphorylated to creatine phosphate. Creatine phosphate is a major provider of energy during high intensity short term exercise. It is not surprising then that creatine supplementation can enhance the performance of this type of exercise. From a practical standpoint (based on the results of the present study) any sports that involve short bursts of activity followed by rest periods or periods of reduced activity might benefit tremendously from creatine supplementation (ex. football, basketball, soccer, track events, weightlifting etc...). For example, a football player that normally begins to slow down during the third quarter because of fatigue might (with creatine supplementation) be able to maintain his speed for the entire game. Since creatine also lowers lactic acid levels and the feeling of fatigue, this might allow for more intense or longer training sessions. The present study also showed that oxygen consumption was reduced by creatine supplementation. Since oxygen consumption is related to energy expenditure we might conclude that exercise efficiency (work/cost) was also improved. This might also allow for more intense training. Therefore creatine supplementation appears to be a valuable aid for the enhancement of training and performance of many athletic events. Harris, R.C., Soderlund, K., and Hultman, E., (1992) Elevation of creatine in resting and exercised muscle of normal subjects by creatine supplementation, Clinical Science, 83, 367-374. Figure 1 Figure 2 Figure 3 Figure 4. The Effects of Creatine Supplementation (C10) on Endurance and Metabolism During High Intensity Intermittent Exercise Mike C. Prevost (Ph.D.)* * Department of Kinesiology, Louisiana State University Methods- The effects of creatine supplementation on endurance and metabolism during high intensity intermittent exercise was examined using 18 males and females (age 19-26). The subjects were randomly divided into 2 groups (creatine and placebo) and the testing proceeded in two phases. During phase 1 both groups received a placebo. During phase 2 the placebo group again received a placebo, while the creatine group received creatine (C10). Testing consisted of a several high intensity intermittent cycling protocols on a stationary cycle ergometer. Both groups performed all test bouts (in random order) in phase 1 and again in phase 2.Testing consisted of : Continuous (Bout A) - Continuos pedaling at 150% VO2 peak until exhaustion. 30/60 (Bout B) - 30 seconds of pedaling at 150% VO2 peak followed by 60 seconds rest, repeated until exhaustion. 20/40 (Bout C) - 20 seconds pedaling at 150% VO2 peak followed by 40 seconds rest, repeated until exhaustion. 10/20 (Bout D) - 10 seconds pedaling at 150% VO2 peak followed by 20 seconds rest, repeated until exhaustion. Oxygen consumption was measured during all bouts using a Quinton Q-Plex I. Blood samples were also taken periodically during each bout for blood lactate analysis. Results- The placebo group showed no significant change in time to exhaustion from phase 1 to phase 2 for any of the bouts tested. The creatine group, however, showed a significant increase in time to exhaustion on all bouts (see figure 1). Bout D was impacted significantly more than the other bouts with a more than twofold increase in time to exhaustion (note that bout D was truncated before subjects actually reached exhaustion because even at twice the performance time of phase 1 subjects reported feeling very little fatigue and the ability to continue indefinitely). Oxygen consumption showed no change from phase 1 to phase 2 for the placebo group, while the creatine group showed a significantly lower rate of oxygen consumption on bouts D and C (see figure 2 for Bout D VO2). Blood lactic acid values were also lower on bouts C and D for the creatine group only (see fig 3 & 4 for bout D lactate values). Discussion- It has recently been demonstrated that supplementation with creatine can lead to an increase in skeletal muscle creatine levels of up to 50% (Harris et. al. 1992). Once creatine enters the muscle it is phosphorylated to creatine phosphate. Creatine phosphate is a major provider of energy during high intensity short term exercise. It is not surprising then that creatine supplementation can enhance the performance of this type of exercise. From a practical standpoint (based on the results of the present study) any sports that involve short bursts of activity followed by rest periods or periods of reduced activity might benefit tremendously from creatine supplementation (ex. football, basketball, soccer, track events, weightlifting etc...). For example, a football player that normally begins to slow down during the third quarter because of fatigue might (with creatine supplementation) be able to maintain his speed for the entire game. Since creatine also lowers lactic acid levels and the feeling of fatigue, this might allow for more intense or longer training sessions. The present study also showed that oxygen consumption was reduced by creatine supplementation. Since oxygen consumption is related to energy expenditure we might conclude that exercise efficiency (work/cost) was also improved. This might also allow for more intense training. Therefore creatine supplementation appears to be a valuable aid for the enhancement of training and performance of many athletic events. Harris, R.C., Soderlund, K., and Hultman, E., (1992) Elevation of creatine in resting and exercised muscle of normal subjects by creatine supplementation, Clinical Science, 83, 367-374. Figure 1 Figure 2 Figure 3 Figure 4. CHAPTER 1 - INTRODUCTION Short duration (5-20 second) maximal intensity exercise relies mainly on the phosphagen system to meet energy demands and anaerobic glycolysis to replenish adenosine triphosphate (ATP) and phosphocreatine (PCr) stores. This type of high intensity exercise can be maintained for only brief periods unless it is performed in intermittent fashion. strand et al. (1960) showed that when a subject exercised at a very high intensity (412 watts), exercise could be maintained for only 3 minutes. If the subject then exercised at the same intensity for 1 minute and then rested for 1 minute, exercise could be maintained for 24 minutes before the subject became completely exhausted. However, since rest periods were included, the total work done per unit of time was reduced. strand et al. (1960) found that if both the total work done in 30 minutes and the work to rest ratio is held constant (1:2) that the most important factor in determining the onset of fatigue is the length of the exercise period. It was found that blood lactate levels increased with work to rest ratios of 60:120 seconds and 30:60 seconds, but not a ratio of 10:20 seconds. Therefore, high intensity exercise of a 10 second duration could yield the same amount of work in 30 minutes as the longer exercise periods with no blood lactate accumulation and no feeling of fatigue. strand et al. (1960) suggested that during the shorter work periods ATP demand was met by oxidative metabolism (since lactate levels did not increase) of myoglobin oxygen stores. Later research (Saltin et al. 1976 and Essen 1978) showed that during intense exercise lasting 5 to 20 seconds that ATP and PCr levels were decreased. These ATP and PCr stores were replenished during rest periods. More recent work by Gaitanos et al. (1993) suggests that work bouts of only 6 seconds can proceed with a significant contribution from anaerobic glycolysis. It was demonstrated that 6 second maximal work bouts resulted in increased lactate levels indicating a significant contribution from anaerobic glycolysis. This is possibly because the ATP and PCr stores were depleted more rapidly at this higher intensity level. These results suggest that high intensity intermittent exercise with brief work periods (10s) may rely mainly on the phosphagen system to meet energy demands with anaerobic glycolysis contributing significantly only during longer work periods (>30s) or during shorter work periods if the intensity is high. It has also been shown by Gaitanos et al. (1993) that during the final bout (bout 10) of 6 second maximal cycle sprints that the contribution of glycolysis to ATP resynthesis was decreased and that the contribution from PCr to ATP production was increased from 49.6% (bout 1) to 80% (bout 10). Also, Bogdanis et al. (1993) found that the recovery of power output during 30 second cycle ergometer sprints is correlated with muscle PCr levels and not lactate levels. These findings suggest that PCr plays a dominant role in the maintenance and recovery of power during high intensity intermittent exercise. PCr levels can be increased in skeletal muscle through oral creatine supplementation. Harris et al. (1992) found that supplementation with 5g of creatine monohydrate four to six times a day for a total of 20-30g for two or more days resulted in a significant increase in total creatine content of the quadriceps femoris muscle. Creatine uptake during the first two days accounted for 32% of the administered dose with 20% to 40% of the sequestered creatine present as PCr. Total creatine content in skeletal muscle was increased by up to 50%. Further work by Greenhaff et al. (1993) showed that oral creatine supplementation accelerated PCr resynthesis following intense isometric contractions. Creatine supplementation has been shown to enhance performance during high intensity exercise. Balsom et al. (1993) found a smaller decline in work output during high intensity intermittent cycling following creatine supplementation. There was also a decrease in lactate and hypoxanthine accumulation. In addition, Greenhaff et al. (1993) showed that plasma ammonia accumulation was lower during and after exercise during the final 10 contractions of five bouts of 30 maximum voluntary isokinetic contractions. These decreases in plasma ammonia and hypoxanthine accumulation (indicating a reduced adenine nucleotide degradation) indicate an increased reliance on creatine phosphate rather than the myokinase reaction to remove ADP. The increased PCr levels in skeletal muscle following creatine supplementation may have enhanced performance of intense exercise by increasing the rate of ATP resynthesis during exercise (Greenhaff et al., 1993A) and by increasing the rate of PCr resynthesis during recovery from intense exercise (Greenhaff et al. 1993B). 1.1 - Project Rationale In normal daily activities muscular work is seldom performed for an extended period of time and so steady state is seldom achieved. Most muscular work involves short bursts of activity followed by periods of rest (intermittent exercise). Also many popular sports involve short bursts of activity followed by a rest period or a period of reduced activity (football, basketball, baseball, tennis) and so performance in these sports may be related to the ability to maintain high performance levels in intermittent exercise rather than maximum aerobic power or endurance. Since many of these activities involve extended performance of intermittent exercise, any treatment that could prolong the onset of fatigue during these activities would be of considerable benefit. Although creatine has led to a greater amount of total work during repeated Wingate tests (Earnest et al., 1993) and a smaller decline in work output during high intensity cycling (Balsom et al., 1993) it has not yet been demonstrated that creatine supplementation can prolong the performance of high intensity intermittent exercise. 1.2 - Hypothesis 1. Creatine will increase total work output during all of the exercise bouts. 2. Plasma lactic acid accumulation will be lower during exercise on creatine versus placebo. 3. Exercise will proceed with a lower rate of oxygen consumption during the creatine supplementation period. 4. Response to creatine supplementation will be inversely related to fitness level and body weight. 5. The 10/20 bout will be impacted by creatine supplementation to a greater extent than the other bouts. 6. Urinary creatinine will be significantly increased by creatine supplementation. 1.3 - Objectives 1. Determine if creatine supplementation increases total work output during high intensity intermittent exercise by examining time to exhaustion during several intermittent exercise bouts on creatine versus placebo. 2. Determine if creatine supplementation leads to a lower accumulation of lactic acid during high intensity intermittent exercise. 4. Determine if oxygen consumption during intermittent exercise is affected by creatine supplementation. 5. Determine which bout (continuous, 10/20, 20/40,30/60) benefits the most from creatine supplementation. 6. Determine if body weight or fitness level is related to magnitude of response to creatine supplementation. 7. Determine if urinary creatinine concentration is increased following creatine supplementation. CHAPTER 2 - LITERATURE REVIEW 2.1 - Intermittent exercise Intermittent exercise can be defined as exercise consisting of repeated bouts of work interspersed with recovery periods. Intermittent exercise has an advantage over continuous exercise in that it can be maintained for a longer period of time before fatigue ensues. strand (1970) showed that a subject whose VO2 max. was 4.6 liters.min-1 could exercise at 350 watts for 8 minutes. If the same subject exercised at 350 watts in an intermittent fashion, 3 minutes of exercise alternated with 3 minute rest periods, the subject could continue exercise for one hour. However, since rest periods were included there was less work done per unit of time than continuous exercise at the same intensity. In a second series of experiments strand (1970) exercised the same subject (same subject used in the previous study) at 412 watts for 30 minutes in several bouts; continuous, 60 sec. work /120 sec. rest, 30 sec. work / 60 sec. rest, 10 sec. work / 20 sec. rest. By holding the work to rest ratio constant (1/2) the same amount of work would be done per unit of time during all of the bouts. This allowed strand to investigate the effect of adjusting the length of the work period, while maintaining the same work rate, on the performance of intermittent exercise. It was found that even though the same amount of work was produced in 30 minutes (247 KJ) in all of the intermittent bouts that during the intermittent bout with the shortest work period (10/20) there was no feeling of fatigue and lactate levels did not exceed 2 mM, while during the 60/120 bout lactate levels rose to 15.7 mM and the subject could continue for only 24 minutes. Intermediate results were found with the 30/60 intermittent bout. Thus the critical factor in determining the onset of fatigue was the length of the work bout. strand et al. (1970) suggested that during the 10/20 intermittent bout that ATP demand was met by oxidative metabolism of myoglobin oxygen stores which were replaced during the rest periods. In the longer bouts myoglobin oxygen stores were not adequate to supply all of the needed ATP and so anaerobic glycolysis was initiated to generate the necessary ATP. There was a greater reliance on anaerobic glycolysis to replenish ATP supplies as the length of the bout increased and this (especially in type II fibers) lead to a greater production and accumulation of lactate and a more rapid onset of fatigue. It is probable that during the 10/20 bout mentioned in strands study (1970) that the majority of the ATP demand was met by the stored phosphagens which were replaced by aerobic metabolism during the rest periods. Although it is probable that the intensity of the workload (412 watts) was sufficient to recruit the high threshold type IIb motor units, blood lactate levels did not increase, possibly because ATP depletion was not great enough to call for a large contribution from glycolysis during the relatively short 10 second work periods. During the longer bouts, however, glycolysis was engaged to a significant degree to buffer the decrease in ATP concentration with a subsequent decrease in muscle pH and an earlier onset of fatigue. Essen (1978) has shown that the overall metabolic response to intermittent exercise (in terms of substrate utilization and lactate accumulation) is more similar to that of continuous exercise at about half of the load. There are several reasons for this finding. The most obvious is that metabolic demand is greatly reduced during the rest periods. Also, myoglobin oxygen stores are reloaded allowing for a greater contribution of oxidative metabolism (strand et al. 1960). Essen (1978) has shown that glycogen depletion patterns are similar for intermittent or continuous exercise of the same intensity, ruling out differences in fiber recruitment as the factor controlling the divergent metabolic response. Essen (1978) also mentions that the increase in the key metabolic regulators, ATP, CP and citrate during the rest periods act to synergistically affect metabolism, retarding glycolysis and allowing for a greater utilization of lipids and a lower utilization of carbohydrates. Upon the initiation of exercise there is a considerable utilization of ATP by the myosin ATPases. However, ATP concentration is not decreased until PCr levels are depleted significantly. PCr donates a high energy phosphate to ADP to reform ATP via the reaction catalyzed by creatine phosphokinase (CPK). Bessman and Geiger (1981) have presented evidence that PCr is locally concentrated near the MM-isozyme of creatine phosphokinase (CPK) where it would be positioned optimally for the replenishment of ATP during muscle contraction. The skeletal muscle PCr stores provide enough energy for about 10 seconds of maximal intensity exercise (Miller, 1992). In addition to the CPK reaction the myokinase reaction can buffer the increase in ADP by the following reaction: ADP + ADP --myokinase--> ATP + AMP. However, during low intensity exercise the high affinity cytosolic CPK competes more favorably than myokinase for the available ADP and thus AMP levels are not significantly increased. During high intensity exercise, however, ADP levels rise significantly, providing ample substrate for myokinase (Hochachka and Somero, 1984). The AMP formed is converted to IMP and NH4 via the AMP deaminase reaction. This process is further enhanced by a low pH (the pH optima for AMP deaminase is 6.1-6.5)(Dobson et al., 1987). Therefore during high intensity exercise IMP serves as an adenine nucleotide sink with the net effect being a reduction in the total adenine nucleotide pool. It has been shown that during high intensity intermittent exercise ATP concentration decreases at the beginning of each successive bout and that the drop in ATP is stoichiometrically matched with the rise in IMP (Dobson et al., 1987). A reduction in ATP concentration has been linked to fatigue during high intensity exercise (Nagesser et al., 1992). Also increased IMP concentration has been correlated with a reduction in force production (Nagesser et al., 1992 and Westra et al. 1986). Berden et al. (1986) have shown that there is a binding site for IMP on the actin-myosin complex and have suggested that IMP might have a direct influence on the contractile apparatus. Therefore, during high intensity intermittent exercise the decrease in ATP on successive bouts and the concomitant increase in IMP may contribute to the fatigue response. The magnitude of high energy phosphate store (ATP and PCr) restoration during recovery periods has also been linked to performance during intermittent exercise (Bogdanis et al. , 1993). It was shown that the recovery of PCr correlated with the recovery of power output (r=.76, p<0.05) during repeated bouts of 30 second cycle ergometer sprints with 90 second recovery periods. During high intensity exercise it is the depletion of ATP and the consequent accumulation of ADP and AMP that begins to activate glycolysis by stimulating phosphofructokinase (PFK) (Passonneau and Lowry, 1962). Also, glycogenolysis is activated almost immediately due to the calcium calmodulin stimulation of phosphorylase b (Miller, 1992). Although anaerobic glycolysis can contribute significantly to ATP production, the maximum rate of ATP production is at best about half that of the phosphagen system. It has been estimated by McGilvery (1975) that the maximum power output for skeletal muscle utilizing ATP, ADP and PCr (phosphagen system only) is 96-360 moles ATP gm wet weight -1 min -1. For muscle using glycogen fermentation or glycogen oxidation the values are 60.0 and 30.0 moles ATP gm wet weight -1 min -1 respectively. Therefore if exercise intensity is maximal the depletion of the phosphagen systems high energy phosphate stores is accompanied by a decrease in performance as ATP utilization exceeds demand. During very high intensity intermittent exercise, as previously mentioned, the length of the work bout becomes a primary factor in determining the onset of fatigue (strand, 1960). As the length of the work bout is increased the contribution of glycolysis to ATP production increases. Gaitanos et al. (1993) have shown that maximal intensity intermittent exercise leads to a reduction in the ATP production rate of glycolysis on each subsequent bout. This led to a lower ATP production rate overall (from 14.9 mM/Kg dry wt. To 5.3 mM/Kg dry wt per minute) and thus a drop in performance. Also, glycogen degradation was decreased by 10 fold. It was proposed that the large increase in lactate and the concomitant decrease in muscle pH was responsible for the reduction in glycolytic and glycogenolytic rates. Consequently, as the length of the work bout is increased, there is an increasing reliance on glycolysis to maintain ATP levels. However, the capacity of glycolysis to generate ATP may be reduced during each subsequent bout due to the reduction in muscle pH. Golnick et al. (1974), have shown that if exercise intensity exceeds maximal aerobic power there is an increasing reliance on fast twitch, glycolytic fibers and a concomitant increase in muscle lactate concentration. During high intensity exercise (above VO2 max.) this would inevitably lead to fatigue because of the inability to maintain ATP levels due to an ever decreasing rate of ATP production. It can be concluded then that there are at least 4 important metabolic considerations that affect the performance of high intensity intermittent exercise: 1. The maintenance of high energy phosphates during the exercise bout (or according to Atkinson, the maintenance of the adenylate energy charge): This is accomplished by first utilizing the stored phosphagens. Then as the phosphagens become significantly depleted anaerobic glycolysis is activated to replenish the high energy phosphates. (Recall that the maximum rate of ATP production by anaerobic glycolysis is 60 moles ATP gm wet weight -1 min -1 . If the rate of ATP utilization exceeds this rate then the ability to maintain performance would be determined entirely by the quantity of stored phosphagens.) 2. The recovery of high energy phosphates during the rest periods: (As previously mentioned the recovery of power output during cycle ergometer sprints was correlated with the recovery of PCr stores. ) 3. The restoration of the ability to generate ATP during the exercise bouts: Recall that Gaitanos et al. (1993) found that repeated bouts led to a fall in the rate of ATP production overall (14.9 mM/Kg dry wt. To 5.3 mM/Kg dry wt) and thus a drop in performance. PCr recovery was not a limiting factor (since the rate of ATP production from PCr was not reduced). This would suggest that the recovery of glycolysis (the ability to generate ATP at the desired rate) between bouts would also be an important consideration (provided that the exercise bout is of sufficient intensity or duration to require a significant contribution from glycolysis.) 4. The management of adenine nucleotides: Recall that ATP concentrations are decreased at the beginning of each bout during intermittent exercise and the decrease in the concentration of ATP is stoichiometrically matched with the rise in IMP (Dobson et al., 1987). 2.2 - Creatine and Control of the Creatine Pool Creatine exists in skeletal muscle in a concentration of approximately 125 mmol/kg dry weight and is present in phosphorylated and free forms (Greenhaff, 1994). The two forms of creatine are in a reversible equilibrium. Approximately 60% of the muscle creatine in skeletal muscle at rest is in the form of creatine phosphate (Greenhaff, 1994). Creatine phosphate (PCr) is the primary high energy phosphate store in vertebrate skeletal muscle. PCr has a G of -10.3 kcal/mole allowing it to donate a high energy phosphate to ADP for ATP resynthesis in the following reaction catalyzed by creatine phosphokinase: PCr + ADP <--- --> ATP + Creatine. PCr levels are highest in fast twitch skeletal muscle with typical values approaching 30 moles/gm wet weight. Bessman and Geiger (1981) have presented evidence that PCr is locally concentrated near the MM- isozyme of creatine phosphokinase (CPK) where it would be positioned optimally for the replenishment of ATP during muscle contraction. Skeletal muscle contains 95% of the bodys approximately 120g of creatine (Greenhaff, 1994). The control of this creatine pool is achieved primarily by the regulation of the synthesis of creatine by the transamidinase enzyme. Although skeletal muscle contains the primary creatine pool, creatine is not synthesized in skeletal muscle. Exogenous synthesis of creatine occurs in the kidney, liver and pancreas. Creatine synthesis is controlled by negative feedback inhibition of the transamidinase enzyme by creatine (Walker, 1973). Since the location of the bulk of the creatine pool (skeletal muscle) and the sites of synthesis of creatine (liver, kidney and pancreas) are physically separated, the control of creatine synthesis must be regulated by the small amount of circulating creatine. Additionally, it has been shown by Crim et al. (1976) that feeding of the creatine precursors glycine and arginine stimulated creatine synthesis in excess of the amount required to maintain steady state. It was proposed that (a) substrate limitation was controlling creatine synthesis or (b) transamidinase enzyme inhibition by creatine was reversed by the precursors or (c) synthesis of the transamidinase enzyme was increased by precursor feeding or (d) that insulin concentrations were elevated in the plasma due to precursor feeding (insulin has been shown to enhance creatine uptake into skeletal muscle by Koszalka et al. 1972). Since creatine is not synthesized in skeletal muscle and does not readily cross the cell membrane there must be a second level of control of the size of the creatine pool in skeletal muscle, the transport of creatine into skeletal muscle. The Na+ dependent creatine transporter in skeletal muscle has been identified and its cDNA has recently been cloned and sequenced (Guimbal and Kilimann, 1993). The transporter specifically interacts with the amidine group of creatine (Fitch et al., 1968). Fitch and Shields (1966) have shown that creatine uptake in rat skeletal muscle is an energy requiring saturable process that is capable of replacing about 4% of the total skeletal muscle creatine each day. It has been shown by Loike et al. (1988) that creatine transport is down regulated in the presence of extracellular creatine concentrations of 45 M. It was also shown that there was a downregulation of creatine transporters by extracellular creatine. Although creatine can be synthesized de novo from the precursors glycine and alanine, the skeletal muscle creatine pool has been shown to respond to creatine supplementation. In a recent study Harris et al. (1992) have shown that supplementation with 5g of creatine monohydrate four to six times a day for 2 or more days resulted in a significant increase in the total creatine content of the quadriceps femoris muscle in man. The increase in the total creatine pool was in excess of 20% with 20% or more of the increase being present as creatine phosphate. Uptake was greatest during the first two days of supplementation accounting for 32% of the administered dose. It was also shown that exercise apparently increased the uptake of creatine. Creatine uptake was greater in subjects with low initial levels of creatine. 2.3 - Creatine Supplementation Studies Since Harris et al. (1992) demonstrated that skeletal muscle creatine can be increased by creatine supplementation, several studies have demonstrated the beneficial effects of creatine supplementation. Using a high intensity intermittent cycling protocol (880 watts) Balsom et al. (1993) showed that oral creatine supplementation (5 6-g doses daily for 6 days) led to a smaller decline in work output from baseline than a placebo group. The creatine group also showed a decrease in lactate accumulation (from 7.0 to 5.1 mmol l-1 ) and a lower oxygen consumption (2.84 to 2.78 l min-1). Plasma hypoxanthine accumulation was also lowered (21 to 16.7 (mol l-1) indicating a reduced adenine nucleotide degradation. The increased availability of PCr may have led to a preferential use of creatine phosphokinase to rephosphorylate ADP to ATP (ADP + PCr --creatine phosphokinase--> ATP + Cr) rather than myokinase (ADP + ADP --myokinase-->ATP + AMP) with a reduced production of NH4 and IMP (AMP --AMP deaminase--> NH4 + IMP). These findings have been supported by Greenhaff et al. (1993) who showed that during bouts of maximal isokinetic contractions that there was a lower accumulation of ammonia following creatine supplementation. In the same study it was shown that during the final 10 contractions of each bout that torque was better preserved on creatine vs. Controls. Since it had been proposed that creatine may be enhancing performance by increasing the rate of high energy phosphate resynthesis during recovery (Balsom et al. 1993) this was investigated by Greenhaff et al. (1994). Muscle biopsy samples were taken during recovery from intense electrically evoked isometric contractions. Creatine increased muscular creatine levels by 15-32% and substantially increased PCr resynthesis during recovery (6%). It has also been shown that creatine supplementation can lead to a decrease in ATP degradation during exercise (Greenhaff et al. 1994). Cr supplementation had no effect on ATP degradation during the first bout (30 s bouts of maximal isokinetic contractions) but reduced ATP loss by 50% versus pre creatine values during the second bout even though more work was done. Creatine supplementation has not always been shown to enhance performance, however. Cooke et al. (1995) showed that during high-intensity short-duration cycle sprints creatine supplementation (the same supplementation regime shown by Harris et al. 1992 to increase muscle creatine levels) did not lead to enhanced performance. However this study used 15 second maximal sprints (as opposed to the 6 second sprints used by Balsom et al., 1993). In maximal sprints of this duration the large contribution of glycolysis may overshadow any improvements in the phosphagen system due to creatine supplementation. Recall that Gaitanos et al. (1993) have shown that during 6 second maximal bouts that there is a significant contribution of glycolysis. This would be expected to increase as the duration of the bout is increased. Also, Balsom et al. (1993) have shown that creatine supplementation does not enhance endurance exercise. There was no improvement in run times during a treadmill run to exhaustion at supramaximal intensities (>VO2 max.) and creatine supplementation actually led to an increase in run times for a 6 km terrain run. It was proposed that this increase in run times might be attributed to a 2.3 kg weight gain for the creatine group. Therefore creatine supplementation appears to enhance exercise performance during very brief (<10 seconds) high intensity exercise that stresses primarily the phosphagen system. Creatine can also lead to a lower lactate accumulation (because energy provision during the exercise bout can be met by a larger contribution of the phosphagen system with decreased reliance on glycolysis) and a lower accumulation of NH4 and hypoxanthine (due to a decreased reliance on myokinase to replenish ATP and thus less substrate available to AMP deaminase). CHAPTER 3 - MATERIALS AND METHODS 3.1 - Basic Design Eighteen subjects were divided into two groups, a placebo-control and an experimental. The experimental group was given a placebo before and during the first phase of testing and was given creatine supplementation before and during the second phase of testing. The control group received a placebo before and during the first and second phases of testing. Both groups underwent identical testing protocols. The testing then proceeded in two phases (see figure 3.1) as follows: Phase 1(placebo) Day 1 - Begin placebo administration - VO2 peak test Day 5 - Exercise bout at 150% VO2 peak* Day 7 - Exercise bout at 150% VO2 peak* Day 9 - Exercise bout at 150% VO2 peak* Day 11 - Exercise bout at 150% VO2 peak* - end placebo supplementation regimen Phase 2 (creatine-placebo) Day 20 - begin creatine or placebo supplementation regimen Day 25 - (two weeks following last test) -(after 5 days on creatine supplement or placebo) - Exercise bout at 150% VO2 peak* Day 27 - Exercise bout at 150% VO2 peak* Day 29 - Exercise bout at 150% VO2 peak* Day 31 - Exercise bout at 150% VO2 peak* - end supplementation regimen *Exercise bouts consisted of four different protocols. One protocol was performed per testing session and the protocols were assigned in random order during each phase. The protocols consisted of: Protocol A-Continuous Protocol B-30 seconds work:60 seconds rest Protocol C-20 seconds work:40 seconds rest Protocol D-10 seconds work:20 seconds rest. Also, finger prick blood draws and VO2 measurements were taken during each test. Details on blood draws and analysis and VO2 measures will be given in a later section. 3.2 - Subjects Subjects were apparently healthy (apparently healthy = satisfactory answers on PARQ - see appendix B) male and female volunteers recruited from Kinesiology classes at LSU. Both groups consisted of 5 males and 4 females (see table 3.1. for subject characteristics). The Subjects gave written consent to act as a subject in a research experiment and the experiment proceeded following the approval of the LSU Institutional Review Board. Table 3.1. Subject Characteristics 3.3 - Supplementation Creatine supplementation consisted of 3.75g creatine monohydrate 5 times daily (5 tablets 5 times daily for a total of 18.75 g/day) for 5 days followed by 2.25g creatine once daily (3 tablets once daily) for 6 days. Creatine supplementation was given to the creatine group only, before phase 2 testing only. Placebo supplementation consisted of 1.0g of placebo (calcium) 5 times daily (5 tablets 5 times daily) for 5 days followed by .6g placebo once daily (3 tablets once daily) for 6 days. The placebo was indistinguishable in appearance from the creatine supplement. Placebo regimen administration began 5 days prior to phase 1 (both groups) and phase 2 testing (placebo group) and ended following the last testing session during each phase. 3.4 - VO2 peak Subjects were instructed to refrain from intense physical exercise the day before testing and fasted for at least 4 hours prior to the test session (Note - VO2 peak measures were used to standardize the proceeding intermittent work bouts and have no other experimental significance). The seat height was adjusted to subject satisfaction and recorded to standardize its position for each test. Subjects first engaged in a standardized warm up procedure consisting of 5 minutes of pedaling at a low tension level. Subjects began the test by pedaling at 100 rpm against no load for 1 minute. The load was then be increased by 50 watts each minute until the subject was no longer able to maintain the required pedaling rate of 100 rpm. The work rate associated with the last completed stage (last 1 minute stage) was considered the VO2 peak. Expired respiratory data (VO2 and VCO2) was collected continuously using the Quinton Q-Plex I. 3.5 - Standardized Exercise Bouts Protocol A - Subjects repeated the standardized warm up procedure administered before the VO2 peak test. Subjects then pedaled at 100 rpm at 150% of their VO2 peak work rate until exhaustion (the subject could no longer maintain the required pedaling rate). Protocol B - Subjects will repeat the standardized warm up procedure. Subjects will then begin intermittent exercise consisting of repeated 100 rpm cycling bouts at 150% VO2 peak for 30 seconds followed by 60 seconds rest. Protocol C - Standardized warm up followed by 100 rpm cycling (150% VO2 peak) for 20 seconds followed by 40 seconds rest. Protocol D - 100 rpm (150% VO2 peak) cycling for 10 seconds followed by 20 seconds rest. All intermittent protocols were repeated until exhaustion (the subject could no longer maintain the required pedaling rate). 3.6 - Blood draws and analysis One hundred microliters of blood was collected in heparinized microcapillary tubes by the finger prick method. New lancelets were used for each prick and sanitary conditions were maintained throughout. Blood samples were taken on the following schedule: Continuous bout - Before exercise, immediately after exercise, and 3 minutes post exercise. 10/20 intermittent bout - Immediately prior to exercise, immediately after the fourth 10 second pedaling period (2 minutes), immediately after the eighth 10 second pedaling period(4 minutes) and immediately post exercise. 20/40 intermittent bout - Immediately prior to exercise, after the second 20 second pedaling period (2 minutes), after the fourth 20 second pedaling period (4 minutes) and immediately post exercise. 30/60 intermittent bout - Immediately prior to exercise, immediately after the first 30 second pedaling period (30 seconds), immediately after the second 30 second pedaling period (2 minutes) and immediately post exercise. Blood samples were placed in an ice water bath and were centrifuged immediately following the test and stored at -70 C for further analysis. Blood lactate concentration was determined using the Analox GM7 micro-stat analyzer (Analox Instruments Ltd. , 8 Godkhawk Industrial Estate, Brackenbury Road, Hammersmith, London W6 OBQ, England). The micro-stat analyzer functions by measuring the oxygen change when oxyreductase enzymes react with their substrates under controlled semi-anaerobic conditions. For lactate analysis the reaction proceeds as follows: L-lactate + O2 ----Lactate oxyreductase--> pyruvate +H2O2 3.7 - Oxygen Consumption Expired respiratory data (VO2 and VCO2) was collected continuously using the Quinton Q-Plex I during all exercise tests. 3.8 - Urinary Creatinine Urine samples were collected from both groups during phase 2. The first sample was collected before the phase 2 supplementation period begins. Samples two and three were collected on day 2 and 3 of supplementation respectively. Urinary creatinine was determined by the use of Sigma kit #555-A . 3.9 - Statistical Analysis Time to exhaustion was analyzed using ANOVA with repeated measures (group x phase x bout). Post-ANOVA analysis involved, where appropriate, the use of Tukeys range test. The experimentwise error rate was set at 0.05 and was maintained throughout all post-ANOVA tests. Each bout was analyzed independently for differences in lactate and respiratory data at specific time intervals using ANOVA with repeated measures (group x phase x time). Differences within groups in urinary creatinine concentration were determined by using ANOVA with repeated measures. Post ANOVA analysis involved the use of Tukeys range test. The relationship of fitness level and body weight with increases in total work output was examined utilizing multiple linear regression. Prior to drawing conclusions concerning the relationship of fitness levels and body weight with total work output, the regression model was examined for departure from the linear model (i.e., lack of error variance linearity/constancy, presence of outliers, lack of error term normality, etc.) using established diagnostics. CHAPTER 4 - RESULTS 4.1 Time to Exhaustion Creatine supplementation had a significant impact on time to exhaustion (and thus total work output). The creatine supplementation group showed a greater than 100% increase in time to exhaustion on bout D, phase 2 (p<0.01) (see fig.4.1), while the placebo group showed no significant change (see fig. 4.2). Bout D was impacted significantly more than the other bouts with a greater than twofold increase in time to exhaustion. (Note that for all subjects on creatine, phase 2 bout D was truncated at twice the performance time of phase 1. At this time point all subjects on creatine reported feeling very little fatigue and the ability to continue indefinitely. The bout was truncated due to factors related to subject compliance and because a twofold increase in time to exhaustion was sufficient to show a significant impact of creatine supplementation). Bout C was also significantly impacted by creatine supplementation with a 61.9% increase in time to exhaustion (P<0.01), while the placebo group showed no significant change from phase 1 to phase 2 (see fig.4.1 and 4.2). The creatine group showed a 61.0% increase in time to exhaustion (p<0.01) on bout B, Phase 2, while the placebo group, again, showed no significant change. Finally, bout A was also significantly impacted by creatine supplementation (p<0.01) with a 23.5% increase in time to exhaustion, while again, there was no significant change for the placebo group (see fig.4.1 and 4.2). Figure 4.1. Time to Exhaustion - Phase x Bout, Creatine Group. (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.2. Time to Exhaustion - Phase x Bout, Placebo Group. (Data are presented as means + standard deviations) 4.2 Oxygen Consumption Oxygen consumption increased with time, as expected for all exercise bouts. The creatine supplementation group, however, showed a significantly lower oxygen consumption rate (p<0.01) during bouts D (see fig. 4.3 and 4.4) and C (see fig. 4.5 and 4.6) for phase 2. The placebo group showed no significant change in oxygen consumption rate during these bouts (see fig. 4.7,4.8,4.9 and 4.10). Both groups showed a significantly lower rate of oxygen consumption (p<0.01) during bout B, phase 2 (see fig. 4.11, 4.12,4.13 and 4.14). For bout A neither group showed a significantly lower rate of oxygen consumption during phase 2. (See fig. 4.15,4.16,4.17 and 4.18). Figure 4.3. VO2 For Bout D Phase 1 and 2, Creatine Group (Data are presented as means + standard deviations. * Significantly different from phase 1, p<0.01) Figure 4.4. VO2 - Time x Phase, Bout D, Creatine Group (Data are presented as means + standard deviations.) Figure 4.5. VO2 For Bout C Phase 1 and 2, Creatine Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.6. VO2 - Phase x Time, Bout C, Creatine Group (Data are presented as means + standard deviations) Figure 4.7. VO2 For Bout D Phase 1 and 2, Placebo Group (Data are presented as means + standard deviations) Figure 4.8. VO2 - Phase x Time, Bout D, Placebo Group (Data are presented as means + standard deviations) Figure 4.9. VO2 For Bout C Phase 1 and 2, Placebo Group (Data are presented as means + standard deviations) Figure 4.10. VO2 - Phase x Time, Bout C, Placebo Group (Data are presented as means + standard deviations) Figure 4.11. VO2 For Bout B Phase 1 and 2, Creatine Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.12. VO2 - Phase x Time, Bout B, Creatine Group (Data are presented as means + standard deviations) Figure 4.13. VO2 For Bout B Phase 1 and 2, Placebo Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.14. VO2 - Phase x Time, Bout B, Placebo Group (Data are presented as means + standard deviations) Figure 4.15. VO2 For Bout A Phase 1 and 2, Creatine Group (Data are presented as means + standard deviations) Figure 4.16. VO2 - Phase x Time, Bout A, Creatine Group (Data are presented as means + standard deviations) Figure 4.17. VO2 For Bout A Phase 1 and 2, Placebo Group (Data are presented as means + standard deviations) Figure 4.18. VO2 - Phase x Time, Bout A, Placebo Group (Data are presented as means + standard deviations) 4.3 Plasma Lactic Acid Plasma lactic acid concentration increased with exercise as expected. Lactate concentration was significantly lower (P<0.01) for the creatine group on bout D, phase 2 (see fig. 4.19 and 4.20) while the placebo group showed no significant change from phase 1 to 2 (see fig. 4.21 and 4.22). Bout C also showed a significant decrease (p<0.01) from phase 1 to phase 2 for the creatine group only (see fig. 4.23 and 4.24), while the placebo group showed no significant change (see fig. 4.25 and 4.26). Also for bout B there was a significant decrease in lactate concentration from phase 1 to phase 2 (p<0.01) for the creatine group (see fig. 4.27 and 4.28), while the placebo group showed no change (see fig.4.29 and 4.30). There was no change by either group for bout A from phase 1 to 2 (see fig. 4.31, 4.32, 4.33 and 4.34). Figure 4.19. Lactate Values For Phase 1 and 2, Bout D, Creatine Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.20. Lactate - Phase x Time, Bout D, Creatine Group (Data are presented as means + standard deviations) Figure 4.21. Lactate For Phase 1 and 2 Bout D, Placebo Group (Data are presented as means + standard deviations) Figure 4.22. Lactate - Phase x Time, Bout D, Placebo Group (Data are presented as means + standard deviations) Figure 4.23. Lactate For Phase 1 and 2, Bout C, Creatine Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.24. Lactate - Phase x Time, Bout C, Creatine Group (Data are presented as means + standard deviations) Figure 4.25. Lactate For Phase 1 and 2, Bout C, Placebo Group (Data are presented as means + standard deviations) Figure 4.26. Lactate - Phase x Time, Bout C, Placebo Group (Data are presented as means + standard deviations) Figure 4.27. Lactate For Phase 1 and 2, Bout B, Creatine Group (Data are presented as means + standard deviations. *Significantly different from phase 1, p<0.01) Figure 4.28. Lactate - Phase x Time, Bout B, Creatine Group (Data are presented as means + standard deviations) Figure 4.29. Lactate For Phase 1 and 2, Bout B, Placebo Group (Data are presented as means + standard deviations) Figure 4.30. Lactate - Phase x Time, Bout B, Placebo Group (Data are presented as means + standard deviations) Figure 4.31. Lactate For Phase 1 and 2, Bout A, Creatine Group (Data are presented as means + standard deviations) Figure 4.32. Lactate - Phase x Time, Bout A, Creatine Group (Data are presented as means + standard deviations) Figure 4.33. Lactate For Phase 1 and 2, Bout A, Placebo Group (Data are presented as means + standard deviations) Figure 4.34. Lactate - Phase x Time, Bout A, Placebo Group (Data are presented as means + standard deviations) 4.4 Urinary Creatinine Urinary creatinine concentration was significantly (p<0.01) elevated on day 2 and 3 of supplementation for the creatine group (see fig. 4.35), while the placebo supplementation group showed no change (see fig. 4.36). Figure 4.35. Urinary Creatinine For the Creatine Supplementation Group (Data are presented as means + standard deviations. *Significantly different from pre-supplementation values, p<0.01) Figure 4.36. Urinary Creatinine For the Placebo Supplementation Group (Data are presented as means + standard deviations) 4.5 Regression of Body Weight and Fitness on Improvement in Total Work Output Neither body weight (r=0.074) nor fitness level (maximum VO2 during the VO2 peak test)(r=0.038) or the combination (r=0.076) were a good predictor of the magnitude of improvement in total work output due to creatine supplementation on bouts C, B and A (note that bout D was not included because this bout was truncated at twice the performance time of phase 1. CHAPTER 5 - DISCUSSION Creatine supplementation (5g of creatine monohydrate four to six times a day for 2 or more days) has been shown to significantly increase the total creatine content of the quadriceps femoris muscle in man (Harris et al. 1992). The increase in the total creatine pool was in excess of 20%, with 20% or more of the increase being present as creatine phosphate. Greenhaff et al. (1994) have also found a 25% increase in total creatine content of the vastus lateralis after creatine supplementation. Although skeletal muscle contains 95% of the bodys creatine pool (Greenhaff, 1994), creatine is not synthesized in muscle. Therefore the size of the creatine pool in skeletal muscle is regulated in part by blood creatine concentrations (Crim et al., 1976). In the present study, all subjects on creatine showed a significantly elevated urinary creatinine concentration on day 2 and 3 of the supplementation period. Subjects in the placebo group showed no significant change. Assuming a daily urine output of .6 to 2.5 liters/day, creatine turnover averaged 1.2 - 5.0 grams/day for the placebo group, which agrees with Fitch and Shields (1966) estimate of 2.0 grams/day under normal conditions. Creatine turnover for the creatine group on supplementation day 2 and 3, based on these estimates, would be 6 to 10 times this value. This increase in urinary creatinine indicates that creatine entered the bloodstream in significant quantities and was subsequently eliminated by excretion in the urine. It is therefore likely that the supplementation regime used in the present study provided an increased availability of circulating creatine and thus successfully elevated skeletal muscle creatine content. An increase in total creatine content and the concomitant increase in PCr due to creatine supplementation has been shown to: 1. Reduce the decrease in ATP concentration following high intensity exercise (Greenhaff, 1994). 2. Increase the rate of PCr recovery following high intensity exercise (Greenhaff, 1994B). 3. Reduce the decline in power output following repeated bouts of maximal isokinetic cycling (Birch et al., 1994) . 4. Lower plasma lactic acid and hypoxanthine accumulation during high intensity intermittent exercise (Balsom et al., 1993). As such, creatine supplementation may be able to blunt the metabolic consequences of high intensity exercise and therefore be of ergogenic benefit. Surprisingly, it has not yet been demonstrated whether creatine supplementation can enhance total work output during high intensity intermittent exercise. Therefore the present study was undertaken to investigate the effects of creatine supplementation on the performance of 4 different high intensity intermittent work bouts. The most significant finding of the present study is that creatine supplementation increased total work output (as measured by time to exhaustion) during all exercise bouts (A, B, C, and D), with the greatest response occurring during bout D. As previously mentioned, bout D was truncated at twice the performance time of phase 1. The decision to truncate the bout was based on the request by several subjects to terminate the bout prior to exhaustion as they felt as though they could continue indefinitely. For example, subject #7 (the first subject on creatine to perform phase 2, bout D) continued for 10 minutes to exhaustion on phase 1. On phase 2, at 29 minutes (nearly a threefold increase) the subject reported feeling very little fatigue and felt capable of continuing indefinitely. At the request of the subject the bout was terminated. Based on this experience and due to the request by several subjects to truncate the bout prior to exhaustion, all of the subsequent phase 2, bout D tests for the creatine group were truncated at twice the performance time of phase 1. Supporting this decision was the fact that at this time point all subjects experienced little fatigue and felt capable of continuing indefinitely. It is generally accepted that the energy requirements (ATP) of brief (<20 seconds), high intensity exercise are met by the available phosphagen stores (ATP, PCr), the resynthesis of these phosphagens and the anaerobic degradation of glucose. A reduction in performance occurs in this type of physical activity when ATP and PCr stores become depleted and lactate, the end product of anaerobic glycolysis, begins to accumulate. It could be anticipated that any mechanism which delays the occurrence of either of these events should extend the time an individual can work at this intensity level i.e. be of ergogenic benefit. Data from the current study demonstrates that creatine supplementation substantially delays the onset of fatigue. This ergogenic benefit may have been achieved by better meeting the energy needs of the exercising muscles through one or more of the following mechanisms: 1. Increasing the concentration of stored phosphagens. 2. Increasing the rate of PCr resynthesis during the recovery periods. 3. Decreasing the reliance on myokinase to buffer the drop in ATP levels, thus lowering the production of IMP and the concomitant loss in adenine nucleotides. 4. Decreasing the reliance on glycolysis to provide ATP during the work bouts, hence reducing lactate accumulation and its ergolytic effect of reduced pH. Employing a dose similar to the present study (20g/day versus 18.75g/day in the present study), Harris et al. (1992) and Greenhaff et al. (1994) demonstrated a significant increase in muscle creatine concentration (20% and 25% respectively) due to supplementation. Therefore, it is most probable that the supplementation regime used in the present study resulted in an increase in muscle creatine and PCr concentrations. An increased concentration of stored phosphagens would have allowed more work to be done before high energy phosphates were depleted significantly, or a reduced depletion of high energy phosphates for same amount of work. A reduced depletion of high energy phosphates during the work periods would result in a reduced activation of ATP generating metabolic pathways. Note that this is supported by the fact that both aerobic (reduced VO2) and anaerobic (reduced lactate) metabolism during the exercise bouts were reduced following creatine supplementation. Therefore the increased quantity of stored phosphagens due to creatine supplementation may have reduced the metabolic consequences of high intensity exercise (reduced ATP and PCr concentrations and increased lactate) and consequently delayed the onset of fatigue. In addition, creatine supplementation may have extended time to exhaustion by increasing the rate of PCr resynthesis during the recovery periods of the intermittent bouts. Greenhaff et al. (1993) have shown that creatine supplementation accelerates the rate of phosphocreatine resynthesis following intense muscle contractions. This increase in PCr resynthesis resulted in a greater concentration of PCr at the beginning of each subsequent exercise period during intermittent exercise. Furthermore, the recovery of PCr following exercise is highly correlated with the recovery of power output during repeated cycle sprints (Bogdanis et al., 1993). It was found that a higher concentration of PCr prior to each subsequent work bout led to a greater power output and thus more work done during the bouts. Therefore, in the present study, creatine may have impacted performance by accelerating the rate of PCr resynthesis during recovery, thus increasing the concentration of PCr at the beginning of each subsequent exercise period. With regards to increasing the rate of PCr resynthesis, Greenhaff et al. (1993) suggested that creatine feeding may have accelerated the rate of PCr resynthesis from mitochondrial ATP. It is argued that the increase in muscle creatine due to supplementation may have increased the rate of flux through the CPK reaction at the mitochondrial membrane. The availability of creatine may possibly be a limiting factor in PCr resynthesis following intense exercise because: A) The Km of CPK for creatine (19mmol/l) is very close to the concentration of creatine in skeletal muscle at rest and after maximal exercise (Bergmeyer, 1965). B) The affinity of CPK for creatine is much lower than for ATP. As a result, even after high intensity exercise when creatine levels are highest, CPK would not be operating near Vmax because of a limiting supply of creatine (creatine concentration at this time would still not be significantly higher than the Km of CPK for creatine). Therefore the sequestering of creatine by CPK might be rate limiting. Greenhaff et al. (1993) argue that after supplementation muscle Cr. levels may have reached 44 mmol/l, significantly higher than the km for creatine (19 mmol/l). It is proposed that this near saturation of CPK is the mechanism whereby creatine supplementation accelerates the rate of PCr recovery following high intensity exercise. This increased rate of PCr resynthesis could have enhanced performance during the present study by providing a greater high energy phosphate pool for each succeeding work bout. Again, this increase in the high energy phosphate pool would reduce the metabolic impact of the exercise bouts. Increased PCr levels caused by creatine supplementation might also affect the management of adenine nucleotide concentrations via the myokinase reaction. As exercise is initiated ATP is degraded rapidly. PCr buffers this drop in ATP concentration via the reaction catalyzed by CPK (ADP + PCr--CPK-- >ATP + Cr.). Therefore ATP concentration is not significantly reduced until PCr levels are depleted. A second reaction, catalyzed by myokinase (ADP + ADP--myokinase--> ATP + AMP), competes with CPK for the available ADP. During low intensity exercise the higher affinity CPK competes more successfully than myokinase for the available ADP. During high intensity exercise, however, ADP levels rise significantly, providing ample substrate for myokinase which converts two molecules of ADP to ATP and AMP (Hochachka and Somero, 1984). The AMP formed is in turn converted to IMP and NH4 via the AMP deaminase reaction. Therefore, during high intensity exercise there is a shunting of adenine nucleotides from ATP to IMP with the result being a net loss of adenine nucleotides from the muscle cell. This reaction shunts adenine nucleotides away from the resynthesis of ATP. Evidence is available which demonstrates that during high intensity intermittent exercise, ATP concentration continues to decreases with each successive exercise period and that the drop in ATP levels is stoichiometrically matched with a rise in IMP (Dobson et al., 1987). Some of the adenine nucleotides lost in the production of IMP are restored during the recovery periods via the purine nucleotide cycle. However, there is likely to be a progressive loss of adenine nucleotides during each successive bout of high intensity exercise. Therefore there would be a shunting of adenine nucleotides away from the pools available for ATP synthesis, leading to a reduction in ATP levels during each subsequent exercise period. A reduction in ATP concentration during exercise has been linked to a decrease in force production (Nagesser et al., 1992). In addition to this metabolically driven decrease in force production (due to a decreased ATP concentration) an increased IMP concentration may physically interfere with force production of the actin-myosin complex (Nagesser et al., 1992 and Westra et al. 1986). Berden et al. (1986) have shown that there is a binding site for IMP on the actin-myosin complex and have suggested that IMP might have a direct influence on the contractile apparatus due to an, as yet undetermined mechanism. Therefore, during high intensity intermittent exercise there is a progressive decrease in ATP levels on successive bouts due to the loss of adenine nucleotides to the production of IMP. This decrease in ATP levels at the beginning of each successive exercise period may contribute to the fatigue response. Also, the increased IMP concentration may physically interfere with force production of the actin-myosin complex by an unknown mechanism. Due to the high intensity nature of the exercise bouts, this cause of fatigue is likely to have occurred during the placebo supplementation regimen. The aforementioned cause of fatigue (decrease in ATP and an increase in IMP) might have been attenuated by creatine supplementation due to a decreased reliance on myokinase to buffer the rise in ADP (by providing more PCr substrate for the competing CPK reaction). This would result in a lower production of IMP and a lower total adenine nucleotide loss. This reduced adenine nucleotide loss would have resulted in better maintenance of ATP levels during intermittent exercise by providing more adenine nucleotides for ATP resynthesis rather than shunting the adenine nucleotides to the synthesis of IMP. In support of this it has been shown that creatine supplementation can reduce hypoxanthine (Balsom et al., 1993) and ammonia (Greenhaff et al., 1993) accumulation during high intensity intermittent exercise. Plasma ammonia and hypoxanthine are established markers of adenine nucleotide loss during high intensity exercise (Harris et al., 1991). This reduced adenine nucleotide loss due to creatine supplementation is associated with a reduction in the decrease in ATP concentration following high intensity exercise (Greenhaff, 1994). This also would result in better maintenance of the high energy phosphate pool and thus greater phosphagen stores proceeding each successive bout of exercise. Finally, creatine supplementation may have reduced the reliance on glycolysis to replace ATP during the bouts. During high intensity intermittent exercise the ability to generate ATP rapidly via glycolysis may limit performance. As high intensity exercise is initiated there is a rapid degradation of high energy phosphates. Glycolysis is then engaged to buffer the drop in ATP. It has been shown by Gaitanos et al. (1993) that after a 6 second cycle sprint PCr levels were decreased by 57%. Bogdanis et al. (1993) have found that after a 30 second cycle sprint both PCr and ATP contents were reduced to 17.6 and 71% of their initial values respectively. This decrease in PCr and ATP increases flux through glycolysis almost immediately by the removal of the inhibitory effect of these metabolites on PFK and phosphorylase (Morgan and Parmeggiani, 1964, Mansour, 1963). Gaitanos et al. (1993) have found that glycolysis can provide up to 50% of the ATP required during maximal work bouts as short as 6 seconds. As the length of the work period is increased the contribution from glycolysis would be expected to increase as well. This reliance on glycolysis to maintain ATP levels may be a primary factor in determining the onset of fatigue (strand, 1960). Although glycolysis is required to replace the rapidly diminishing ATP stores, maximal intensity intermittent exercise leads to a reduction in the ATP production rate of glycolysis for each subsequent bout. In a study by Gaitanos et al. (1993) this reduction in the ability of glycolysis to generate ATP during intermittent exercise led to a lower ATP production rate overall (from 14.9 mM/Kg dry wt. to 5.3 mM/Kg dry wt per minute) after 10 six second sprints and thus a drop in performance. Also, glycogen degradation was decreased by 10 fold. It was proposed that the large increase in lactate and the concomitant decrease in muscle pH was responsible for the reduction in glycolytic and glycogenolytic rates. Danforth (1965) has shown that a lowering of pH slows glycolysis by inhibiting PFK and phosphorylase. Consequently, as the volume of work is increased, there is an increasing reliance on glycolysis to maintain ATP levels. However, the capacity of glycolysis to generate ATP may be reduced during each subsequent bout due to the reduction in pH. This would inevitably lead to a reduction in performance because of an inability to maintain the necessary ATP production rate. In the present study plasma lactate accumulation was reduced following creatine supplementation. Assuming that lactate efflux and clearance were similar, this would suggest a reduced reliance on anaerobic glycolysis due to creatine supplementation. As previously mentioned creatine supplementation has been shown to: A) Reduce the decline in ATP concentration following high intensity exercise (Greenhaff, 1994). B) Increase the rate of PCr recovery following high intensity exercise (Greenhaff, 1994B) C) Lower plasma lactic acid and hypoxanthine accumulation (indicating a reduction in adenine nucleotide loss) during high intensity intermittent exercise (Balsom et al., 1993). All of these factors would result in greater high energy phosphate stores at the beginning of each subsequent exercise bout. This might allow more work to be done prior to ATP levels being decreased significantly. This would result in a delay in the accumulation of the PFK stimulators ADP and AMP. Also creatine supplementation has been shown to reduce ammonia production, another stimulator of PFK. Thus, the overall effect is the reduced stimulation of anaerobic glycolysis following creatine supplementation. In the present study this could have resulted in a lower reliance on glycolysis during each exercise bout (This is supported by the observed reduction in plasma lactate concentrations.). A decreasing reliance on glycolysis would result in a lower production of lactate and thus a smaller reduction in pH. A reduction in pH has been linked to a reduced ability of glycolysis to generate ATP (Gaitanos et al., 1993). Therefore a reduced decline in pH might extend time to exhaustion by maintaining the ATP production rate of glycolysis. A reduced reliance on glycolysis might also explain the greater impact of creatine supplementation on bout D. Due to the relatively short work periods during bout D (10 seconds), it is likely that glycolysis contributed less to ATP production during this bout than during bouts C, B, and A (note that lactate accumulation was lower during bout D than during the other bouts). Therefore even a small reduction in the stimulation of glycolysis would represent a relatively large reduction in the contribution of glycolysis during bout D. Consequently, during longer work bouts, a similarly small reduction in the already large contribution from glycolysis would be expected to have less effect. This is best exemplified by comparing bout D to Bout A. Note that lactate accumulation was significantly lower during bout D following creatine supplementation. Conversely, bout A showed no significant change. In summary, creatine supplementation may have increased PCr levels resulting in an increased phosphagen concentration, a more rapid resynthesis of PCr during the recovery periods and a reduced adenine nucleotide loss. A reduced adenine nucleotide loss would provide more adenine nucleotides for the resynthesis of ATP rather than the production of IMP. All of these factors would result in a better maintenance of high energy phosphate concentrations during high intensity intermittent exercise. Also, because of the high intensity nature of the work bouts, anaerobic glycolysis was engaged to buffer the depletion of ATP. However, the ability to generate ATP via glycolysis is reduced following repeated bouts of exercise due to a reduction in muscle pH. Creatine supplementation reduced lactate accumulation (indicating a reduced reliance on glycolysis to produce ATP) and thus may have better preserved the ability of glycolysis to generate ATP. The reduction in lactate accumulation was greatest during bout D. This may explain the relatively large impact of creatine supplementation on bout D. Therefore because of a higher concentration of stored phosphagens prior to each subsequent exercise period during the bouts, the metabolic impact (decreased ATP and PCr concentrations and increased lactate) of the exercise bouts was reduced, thus delaying the onset of fatigue. In addition to reducing lactate accumulation and increasing total work output, oxygen consumption was also impacted by creatine supplementation. The rate of oxygen consumption was decreased during bout D and C following creatine supplementation while the placebo group showed no change during these bouts. Since oxygen consumption was measured at the same time points on creatine versus placebo, the same amount of work was done at a lower net oxygen cost. This suggests that exercise efficiency (cost/work) was affected by creatine supplementation. Creatine may have delayed the increase in oxygen consumption upon the initiation of exercise. A decrease in ATP and an increase in ADP upon the initiation of exercise serves as a stimulator of mitochondrial respiration (Scott, 1995). An increase in PCr levels due to creatine supplementation might have delayed the decrease in the ATP/ADP ratio that is responsible for stimulating mitochondrial respiration. In support of this argument it has been found that competition between aerobic and anaerobic systems for ADP can inhibit mitochondrial respiration (Gatt, S., and Racker, E., 1959). CPK would be poised to have such an influence on mitochondrial respiration. An increase in PCr might allow for a more rapid rephosphorylation of ADP by CPK and thus reduce the ADP concentration that might stimulate respiration. Alternatively, a reduction in fatigue due to creatine supplementation may have delayed changes in neuromuscular coordination that may have occurred due to fatigue. It was noted (although not actually measured) that subjects showed an increased body movement as they approached exhaustion (i.e. swaying side to side and leaning forward). This may have represented a shift in muscle recruitment (due to fatigue) from the primary muscles used during cycling to secondary muscles that are in a less favorable position to produce force (i.e. gluteus maximus, hamstrings). Utilizing these secondary muscles would result in a need to generate greater force to produce the same amount of external work, since these muscles are in a less favorable position to produce force. As a result oxygen consumption would be increased. If creatine supplementation delayed the fatigue of the primary muscles involved in cycling there would be a delay in this shift in whole muscle recruitment patterns and thus a lower rate of oxygen consumption. Note that although muscle recruitment patterns were not measured, changes in recruitment patterns cannot be ruled out as a plausible explanation for the decrease in oxygen consumption and may present a plausible explanation since both aerobic (decreased VO2) and anaerobic (decreased lactate) metabolism were reduced following supplementation. It was proposed that fitness levels might affect response to creatine supplementation since Harris et al. (1992) have shown that creatine uptake due to creatine feeding was greatest in subjects that had a lower initial level of creatine. Because exercise training has been shown to increase PCr stores (Macdougall et al., 1977) the relationship between fitness level (highest VO2 during VO2 peak test) and magnitude of response to creatine supplementation was investigated. No significant relationship between these two variables was found. Perhaps the index of fitness chosen has little relationship to muscle creatine levels. Alternatively, creatine may have impacted performance regardless of initial creatine levels. Also, the relationship of body weight to magnitude of response to creatine supplementation was investigated. The subjects encompassed a broad range in body weights from 115 to 290 pounds. Therefore the lightest subject in the study would have received nearly three times the relative dose (dose/body wt.) of the heaviest subject. However, no relationship between body weight and response to creatine supplementation was observed. This might suggest that the dose administered to the lighter subjects might have been in excess of what was necessary to produce a response since nearly 1/3 of that relative dose led to a similar response in the largest subject. CHAPTER 6 - SUMMARY AND CONCLUSIONS The purpose of the present study was to determine if creatine supplementation could increase total work output during high intensity continuous and intermittent exercise. The results indicate that creatine does enhance the performance of high intensity intermittent exercise with a lower accumulation of lactate and a lower rate of oxygen consumption. Creatine supplementation may have enhanced performance by affecting the management of adenine nucleotides and high energy phosphates during the bouts. The effects of creatine supplementation on intermittent exercise are greatest when the length of the work bout is short. This may be due to a greater relative reduction in the contribution from glycolysis during the shorter work periods. Creatine supplementation might be of considerable benefit for athletes participating in sports involving repeated, short bursts of activity followed by rest or periods of reduced activity (i.e. football, soccer, tennis, hockey etc...) Creatine supplementation has been associated with no known side effects as creatine is rapidly converted to creatinine in the blood stream in a nonenzymatic-nonsaturable process. The resulting creatinine is easily excreted by the kidney. Also, since creatine is a naturally occurring component of a meat containing diet it is unlikely to become a banned substance. In light of these findings, creatine is likely to find use in many sport and recreational activities. Three important, yet unanswered questions with regard to creatine supplementation are: What is the dose-response relationship? What is the washout period (How long after the cessation of supplementation does it take for muscle creatine levels to return to normal baseline levels)? What are the long-term effects of creatine supplementation? Creatine supplementation studies generally employ a dose of 20g a day during the loading phase, apparently because this dose was shown by Harris et al. (1992) to increase skeletal muscle creatine concentration. However, this dose may be more than is necessary. Due to the relatively high cost of creatine supplementation, especially during the loading phase, it would be beneficial to investigate further the dose-response relationship of oral creatine supplementation. Also, Greenhaff (1994) has mentioned that skeletal muscle creatine concentration does not return to baseline levels for weeks after the cessation of supplementation. Therefore the washout period for creatine supplementation may be from weeks to months. With regards to research, this may rule out implementing cross over designs and reversal of treatment designs. Finally, the long term effects of supplementing with creatine while training are unknown. It is interesting to note that creatine depletion upregulates glucose transporter and oxidative enzyme expression in skeletal muscle (Shields et al. (1975). It would be interesting to determine if long term creatine supplementation affects any of these parameters. 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(1987) Extracellular creatine regulates creatine transport in rat and human muscle cells, Proc. Natl. Acad. Sci. USA, 85: 807-811. MacDougall, J.D, Ward, G.R, Sale, D.G., and Sutton, J.R. (1977) Biochemical adaptation of human skeletal muscle to heavy resistance training and immobilization, J. Appl. Physiol., 43(4), 700-703. McGilvery, R.W. (1975) The use of fuels for muscular work. In Metabolic Adaptation to Prolonged Physical Exercise, ed. H. Howald and J.R. Poortmans, 12-30. Basel: Birkhauser Verlag. Miller, W.G. (1992) Regulation of energy production during exercise, In Biochemistry of Exercise and Metabolic Adaption, Brown and Benchmark, Duburke, IA. Mansour, T.E., (1963) Studies on heart phosphofructokinase: Purification, inhibition, and activation. J. Biol. Chem., 238, 2285-2292. Morgan, H.E. and Parmeggiani, A., (1964) Regulation of glycogenolysis in muscle. III. Control of glycogen phosphorylase activity, J. Biol. Chem., 239, 2440-2445. Nagesser, A.S., Van Der Laarse and Elzinga, W. J. (1992) Metabolic changes with fatigue in different types of single muscle fibers of Xeopus laevis. Journal of Physiology, 448, 511-523. Passonneau, J.V. and Lowry, O.H. (1963) Phosphofructokinase and the control of the citric acid cycle, Biochem. Biophys. Res. Commun., 13, 372-379. Saltin, B., Essen, B., and Pendersen, P.K., (1976) Intermittent exercise:its physiology and some practical applications, in E. Jokl, R.L. Anand, and H. Stoboy (eds.), Advances in Exercise Physiology, p. 23, S. Karger, Basel. Scott, C.B. (1995) Anaerobic metabolic influences on oxygen uptake behavior, Journal of Strength and Conditioning Research, 9(1), 59-62. Shields, R.P., Whitehair, C.K., Carrow, R.E, Heusner, W.W., and Van Huss, W.D. (1975) Skeletal muscle function and structure after depletion of creatine, Labratory Investigation, 33(2), 151-158. Soderlund, K., Greenhaff, P.L., and Hultman, E., (1992) energy metabolism in type I and type II human muscle fibers during short term electrical stimulation at different frequencies, Acta Physio Scand., 144, 15- 22. Walker, J.B. (1973) Metabolic control of creatine biosynthesis, II. Restoration of transamidinase activity following creatine repression. J. Biol. Chem. 236, 493-498. Westra, H.G. De Haan, A., Van Doorn, J.E. and De Haan, E.J. (1986) IMP production and energy metabolism during exercise in rats in relation to age. Biochemical Journal 239, 751-755. APPENDIX A: CONSENT FORM The Effects of Creatine Supplementation on Total Work Output and Metabolism During High Intensity Intermittent Exercise. I_________________________________________, Hereby agree to participate as a subject in the research project entitled: The Effects of Creatine Supplementation on Total Work Output and Metabolism During High Intensity Intermittent Exercise. I understand that I shall perform a series of high intensity cycle ergometer tests to exhaustion. I understand that since the tests are of a high intensity nature I will experience fatigue and the associated symptoms (shortness of breath, tiredness, muscle ache). I also understand that during the test data collection will include: finger prick blood samples - finger prick blood samples will be taken with sterile lancets in a sanitary manner. Oxygen consumption - Oxygen consumption will be determined by the use of a Quinton gas analyzer. Urinary creatinine - Urinary creatinine will be measured and will be performed on the 5 urine samples that I will provide. I understand that the risks of this study are minimal and no greater than that I would experience during intense physical exertion (for example, fatique, localized muscle ache, shortness of breath, soreness). I am aware that I may choose to discontinue my participation in the study at any time without any penalty or consequences. I understand that I will be given creatine monohydrate (20g per day for 5 days) or a calcium placebo (6g per day for 5 days). Both are nutritional supplements that are available over the counter and do not exceed the manufacturers suggested maximum dosage. I understand that the results of this study may be published, but upon publication or presentation of the collected data, in any form, my name will not be used. Also, if I wish, at the conclusion of the study I will be informed as to the results. I have completely read this form. I understand the purpose of the study and the proceedures and risks involved. I understand that if I have any questions I may contact the researchers at 388-2036 or 766-6328. Signed _________________ Date ____________________ SS# _________________ Date ____________________ _________________________________________________ Micheel C. Prevost, Principal Investigator. _________________________________________________ Arnold Nelson, Ph.D., Co-Investigator APPENDIX B: PHYSICAL ACTIVITY READINESS QUESTIONNAIRE PHYSICAL ACTIVITY READINESS QUESTIONNAIRE For most people, physical activity should not pose any problem or hazard. PAR-Q has been designed to identify the small number of adults for whom physical activity might be inappropriate or those who should have medical advice concerning the type of activity most suitable. 1. Has your doctor ever said you have heart trouble? 2. Do you frequently suffer from pains in yuour chest? 3. Do you often feel faint or have spells of severe dizziness? 4. Has a doctor ever said yhour blood pressure was too high? 5. Has a doctor ever told yo that you have a bone or joint problem shch as arthritis that has been aggravated by exercise, or might be made worse with exercise? 6. Is there a good physical reason not mentioned here why yuou should not follow an activity program even if yo wanted to? 7. Are you over the age of 65 and not accustomed to vigorous exercise? Can you answer yes to any of the above questions?__________ If so which one?________ Signature _________________________ Date__________________. APPENDIX C: RAW DATA VITA Michael Cory Prevost was born on August 7, 1967 in Opelousas, Louisiana. He graduated from Opelousas Senior High School in 1985. He then enrolled at the University of Southwestern Louisiana to pursue a B.S. in psychology. During his years at USL he vigorously pursued a bodybuilding program, adding 60 pounds of muscle to his physique. He began working as an instructor in local health clubs and also trained a local handicapped powerlifting team. He graduated with a B.A. in general studies in 1990. Due to his newfound interest in exercise and fitness he entered the graduate program in exercise physiology at Louisiana State University in 1991. While at LSU he received training in exercise physiology, biochemistry, molecular biology and radiotracer methodology. He has plans to be married immediately after completing degree requirements and then enter the Navy as an aerospace physiologist.