Effect of oral creatine supplementation on skeletal muscle phosphocreatine resynthesis

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1 Effect of oral creatine supplementation on skeletal muscle phosphocreatine resynthesis P L GREENHAFF, K BODIN, K SODERLUND, AND E HULTMAN Queens Medical Center, Department of Physiology and Pharmacology, University Medical School, NG7 2UH Nottingham, United Kingdom; and Department of Clinical Chemistry, Karolinska Institute, Huddinge University Hospital, S Huddinge, Sweden Greenhaff, P L, K Bodin, K Soderlund, and E Hultman Effect of oral creatine supplementation on skeletal muscle phosphocreatine resynthesis Am J Physiol 266 (Endocrinol Metab 29): E725-E730, 1994-Biopsy samples were obtained from the vastus lateralis muscle of eight subjects after 0, 20, 60, and 120 s of recovery from intense electrically evoked isometric contraction Later (10 days), the same procedures were performed using the other leg, but subjects ingested 20 g creatine (W/day for the preceding 5 days Muscle ATP, phosphocreatine (PCr), free Cr, and lactate concentrations were measured, and total Cr was calculated as the sum of PCr and free Cr concentrations In five of the eight subjects, Cr ingestion substantially increased muscle total Cr concentration (mean 29 t 3 mmol/kg dry matter, 25 t 3%; range mmol/kg dry matter, 15-32%) and PCr resynthesis during recovery (mean 19 t 4 mmol/kg dry matter, 35 t 6%; range mmol/kg dry matter, 23-53%) In the remaining three subjects, Cr ingestion had little effect on muscle total Cr concentration, producing increases of 8-9 mmol/kg dry matter (5-7%), and did not increase PCr resynthesis The data suggest that a dietary-induced increase in muscle total Cr concentration can increase PCr resynthesis during the 2nd min of recovery from intense contraction energy metabolism; recovery CREATINE (Cr) is a naturally occurring compound found principally in skeletal muscle, and in-its free and phosphorylated forms it plays a pivotal role in the regulation and homeostasis of skeletal muscle energy metabolism (5, 6, 24, 31) It is now generally accepted that the maintenance of phosphocreatine (PCr) availability is important to the-continuation of muscle force production (19,23) Endogenous synthesis of Cr occurs in liver, kidney, and pancreas However, it has been known for some time that oral ingestion of Cr in the form of meat and fish or supplements will add to the whole body Cr pool (7,8,18) It has recently been shown that ingestion of g Cr/day for several days can lead to a > 20% increase in human skeletal muscle total Cr content, of which N 20-30% is in the form of PCr (16) It also appears that muscle Cr uptake is augmented if submaximal exercise is performed during the period of supplementation (16) Further recent evidence of our own (11, 17) and others (1) demonstrates that Cr ingestion can significantly increase the amount of work that can be performed during repeated bouts of maximal exercise It was postulated in these studies that the ergogenic effect of Cr ingestion may be attributable to an increased muscle Cr content, accelerating PCr resynthesis between exercise bouts As a result, the required rate of ADP rephosphorylation would have been sustained longer during contraction This suggestion was sup- ported by the lower accumulation of plasma ammonia and hypoxanthine, which was observed during exercise after Cr ingestion The aim of the present experiment was therefore to investigate the effect of oral Cr ingestion on muscle PCr resynthesis after a contraction-induced depletion of muscle PCr stores METHODS Eight male subjects volunteered to take part in the present experiment All undertook some form of recreational exercise, but none was highly trained Their physical characteristics were as follows (mean * SE): age 291 t 16 yr; weight 800 t 47 kg, and height 184 t 3 cm Before the commencement of the study, all subjects gave voluntary consent to take part in the experiment; all were informed of the experimental procedures to be undertaken and were aware that they were free to withdraw from the study at any point The study was approved by the Ethics Committee of the Karolinska Institute, Huddinge, Sweden Each subject reported to the laboratory in a normal fed state on the morning of the study, having abstained from strenuous physical exercise the previous day, and had their nude body weight recorded The experiment began with each subject lying in a semi-supine position on a bed with both legs flexed over one end at an angle of 90 The leg to be investigated on this visit was chosen at random and was attached via an ankle strap to a strain gauge built into the frame of the bed The subject was then asked to perform three maximal voluntary contractions to determine the maximal voluntary isometric force of the knee extensors The isometric force produced was measured with a strain gauge (AB Bofors, Karlskoga, Sweden) and, after amplification (direct current amp Medelec AD6; Medelac, Surrey, UK), was displayed on an oscilloscope and recorded on ultraviolet paper (Medelec) The leg was then prepared for electrical stimulation, as described previously (20) Briefly, the muscles of the anterolateral portion of the thigh were stimulated to contract with square wave impulses of 05 ms duration, at a frequency of 50 Hz, and at a voltage sufficient to elicit maximal contraction Approximately 35% of the musculature that extends the knee is activated in this way (20) Stimulation was intermittent with 20 trains of 16 s stimulation being separated by rest periods of 16 s The total contraction time was therefore 32 s Before initiation of stimulation (30 s), a cuff surrounding the proximal portion of the thigh was inflated (250 mmhg) to occlude limb blood flow and remained inflated until a muscle biopsy had been obtained from the vastus lateralis immediately after the final contraction (4) This stimulation protocol was chosen because it has been previously shown to result in almost total degradation of muscle PCr stores (12) The cuff was then deflated, and further muscle biopsy samples were obtained after 20,60, and 120 s During this time, subjects remained resting in a semi-supine position on the bed Subjects reported back to the laboratory 10 days later for the second part of the study However, for the 5 days preceding Downloaded from by on March 30, /94 $300 Copyright o 1994 the American Physiological Society E725

2 E726 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION their second visit, each ingested 4 x 5 g (total 20 g) creatine monohydrate (Chemi, Linz, Austria) Subjects were instructed to ingest a single 5-g dose in the early morning, at noon, in the late afternoon, and in the evening They were also instructed to totally dissolve each 5-g dose in warm-hot tea or coffee before ingestion Upon reporting back to the laboratory, subjects underwent exactly the same experimental procedures that they had performed 10 days previously, but on this occasion their other leg was electrically stimulated and biopsied Upon removal from the muscle, all biopsy samples were immediately frozen by plunging the biopsy needle into liquid nitrogen The time delay between the insertion of the biopsy needle and freezing of the sample ranged from 3 to 5 s All samples were freeze dried and stored at -80 C until analyzed at a later date After fat extraction with petroleum ether, a portion of each freeze-dried muscle sample was dissected free of all visible blood and connective tissue and was pulverized The powdered muscle was used for the spectrophotometric determination of ATP, PCr, free Cr, and lactate concentrations (15) Muscle total Cr concentration was calculated by summing PCr and free Cr concentrations Unless stated otherwise, values presented in the text, Tables 1 and 2, and Figs 1 and 2 refer to means t SE Comparison of metabolite levels during recovery before and after Cr ingestion was performed by using repeated-measures two-way analysis of variance with respect to treatment (with/ without Cr) and time (O-120 s) When a significant F value was achieved (P < 005), Fisher s test for pairwise comparisons was used to locate the difference between paired means across treatments On all occasions, statistical difference was declared at P < 005 RESULTS Mean body weight before Cr ingestion was 800 t 47 kg After Cr ingestion, body weight had increased to 816 t 48 kg (P < 005) A body weight increase was observed in seven of the eight subjects Table 1 shows mean muscle ATP, PCr, free Cr, total Cr (sum of PCr + free Cr), and lactate concentrations for all eight subjects during recovery from contraction, Table 1 Muscle ATP, PCr, free Cr, total Cr, and lactate concentrations in muscle biopsy samples Seconds Before Cr Ingestion ATP _+ 04 PCr Free Cr Total Cr Lactate f 56 After Cr ingestion ATP k 06 PCr Free Cr , t Total Cr 1429 t Lactate t Values are means * SE; n = 8 subjects Muscle ATP, phosphocreatine (PCr), free creatine (Cr), total Cr, and lactate concentrations (mmol/kg dry matter) in muscle biopsy samples obtained at 0, 20, 60, and 120 s after intense electrically evoked isometric contraction, both before and after oral Cr ingestion E W 60 A 1 MO- MO- g E 5 p G lzo- llo- 100 i d B Pre-Ingestion 1 0 Post-Ingestion Fig 1 A individual values for muscle total creatine (TCr) concentration before (CI) and after (N) creatine (Cr) ingestion Subjects have been numbered 1-8, based on their initial muscle TCr content, which was calculated to be equal to sum of PCr and free Cr dm, dry matter B: individual increases in muscle TCr after Cr ingestion for same subjects in A, plotted against change in PCr resynthesis during recovery after Cr ingestion Values on y-axis were calculated by subtracting PCr resynthesis during 2 min of recovery before Cr ingestion from corresponding values after Cr ingestion l '0-3'0 4'0 Increase in TCr after Cr ingestion (mmol/kg dm) before and after Cr ingestion As expected, electrical stimulation resulted in a decline in muscle ATP, almost total degradation of muscle PCr, and marked increases in muscle free Cr and lactate All variables returned toward resting values during recovery On average, Cr ingestion resulted in an - 15% increase in muscle total Cr concentration PCr resynthesis was similar during the 1st min of recovery when comparing subjects before and after Cr ingestion However, during the 2nd min, the mean rate of PCr resynthesis was increased by -42% after Cr feeding Despite this trend, the mean Downloaded from by on March 30, 2017

3 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION E727 Table 2 Muscle ATP, PCr, free Cr, total Cr, and lactate concentrations (mmollkg dry matter) in muscle biopsy samples obtained from responders and nonresponders Before Cr Supplementation, s After Cr Supplementation, s ATP PCr Free Cr Total Cr Lactate ATP PCr Free Cr Total Cr Lactate k ?z f * * _ f t t f t f f Responders ii k f $ * Nonresponders PCr concentration was not significantly different at the end of recovery when comparing treatments Figure la shows the change in muscle total Cr concentration with Cr feeding for each subject Subjects have been numbered one to eight, based on their initial muscle total Cr concentration As can be seen, five subjects experienced a marked 29 t 3 mmol/kg dry matter (25 t 3%) increase in total Cr concentration (subjects l-4 and 7), which ranged from 19 to 35 mmol/kg dry matter or 15 to 32% of the initial total Cr content In particular, the four subjects with the lowest initial total Cr concentration (120 mmol/kg dry matter; subjects l-4) experienced the most dramatic increase in total Cr (25-35 mmol/kg dry matter), which was equivalent to 20-32% of their initial total Cr concentration The remaining three subjects (subjects 5, 6, and S), each had an initial total Cr concentration of > 125 mmol/kg dry matter, and each experienced a relatively small increase in total Cr concentration with Cr ingestion (7-9 mmol/kg dry matter), which was equivalent to - 5% of their initial total Cr concentration Figure 1B shows, for each subject, the increase in muscle total Cr after Cr ingestion plotted against the change in PCr resynthesis during the 2 min of recovery after Cr ingestion (the latter being PCr resynthesis before Cr ingestion - PCr resynthesis after Cr ingestion) As can be seen, the same five subjects who experienced a substantial increase in muscle total Cr concentration with Cr ingestion also showed an increased rate of PCr resynthesis during recovery after Cr ingestion (mean 19 t 4 mmol/kg dry matter, 35 t 6%; range mmol/kg dry matter, 23-53%) Conversely, the three subjects who had a < 10 mmol/kg dry matter increase in muscle total Cr concentration with feeding showed very little change or even a lower rate of PCr resynthesis during recovery after Cr feeding As in Table 1, Table 2 shows mean muscle metabolite concentrations during recovery: but on this occasion subjects have been categorized into the following two groups: those who demonstrated a marked increase in 231 f IL t f 42-f * F $ t ~fr f 44f * * $ 854 AI _ f f 44 f k 47* * $ _ t t 43t muscle total Cr after oral Cr supplementation (responders, n = 5) and those who showed only a small change (nonresponders, n = 3) For the sake of clarity, mean muscle PCr and free Cr concentrations during recovery in the group of responders have been plotted in Fig 2 Figure 2 shows data obtained before (open symbols) and after (closed symbols) Cr feeding, and metabolite levels are represented as millimoles per kilogram dry matter and millimoles per liter intracellular water Figure 2 illustrates that muscle free Cr concentration declined during recovery on both occasions but that the free Cr concentration was higher throughout recovery after Cr ingestion As expected, PCr resynthesis began soon after exercise, and concentrations were almost identical during the first 40 s of recovery when comparing treatments However, during the remainder of recovery, the rate of PCr resynthesis was greater after Cr ingestion, resulting in the mean muscle concentration being 30% higher at the end of recovery (P < 005) Table 2 also demonstrates that muscle lactate concentration was significantly higher immediately after contraction in the group of responders With the exception of the reported small increase in muscle total Cr concentration, dietary Cr supplementation had no influence on muscle metabolite concentrations in the nonresponders DISCUSSION The major finding of the present experiment is that oral Cr ingestion markedly influences muscle Cr uptake in those individuals who have a total muscle Cr concentration of close to or < 120 mmol/kg dry matter before ingestion (Fig la), and these same individuals demonstrate an accelerated rate of PCr resynthesis after 1 min of recovery from intense muscular contraction (Fig 1B) Cr ingestion has for some time been known to result in an increase in the body Cr pool in humans (7, 8, 18) Only recently has it been shown that the ingestion of a dose similar to that used in the present experiment can Downloaded from by on March 30, 2017

4 E728 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION Recovery Fig 2 Phosphocreatine (PCr; O,O) and free creatine (Cr; q I,~) concentrations measured in muscle biopsy samples obtained after 0, 20,60, and 120 s of recovery from intense contraction before (0,~) and after (~,m) Cr ingestion Values (means IT SE) are represented as mmol/l intracellular water and mmol/kg dry matter (dm) Differences between treatments are indicated as follows: **P < 001, *P < 005 result in a substantial increase in muscle total Cr concentration, of which -2O-30% was in the form of PCr (16) In agreement with the present experiment, it was found that the greatest increases were in those individuals with the lowest prefeeding levels and that muscle total Cr concentration did not increase above mmol/kg dry matter with ingestion Although not commented on at the time, the largest increases were also found in those individuals with a total muscle Cr concentration close to or < 120 mmol/kg dry matter The total Cr concentration of human skeletal muscle has been shown to be 1244 t 112 mmol/kg dry matter (mean + - SD of 81 biopsy samples; see Ref 15) and to follow a normal distribution ranging from - 90 to 170 mmol/kg dry matter (13) A muscle total Cr concentration of mmol/kg dry matter should therefore not be viewed as appreciably low The present results demon- (s) strate that Cr loading is achievable in normal healthy individuals However, they also suggest that increases may be most dramatic in vegetarians, who obviously will have a low dietary Cr intake and have been shown to have a reduced total body Cr pool (9), and in patients with diseased or atrophied skeletal and heart muscle, where low total Cr concentrations have been observed (10, 22) We are unclear about the factors that will dictate muscle Cr content under normal dietary conditions A comparison of the subjects who participated in the present study showed no obvious differences in lifestyle or eating habits As far as we are aware, the present experiment is the first to show that an increase in muscle Cr concentration, resulting from dietary Cr supplementation, can accelerate the rate of muscle PCr resynthesis during recovery from exercise We have recently demonstrated that a regimen of Cr ingestion similar to that used in the present experiment can significantly increase performance during repeated bouts of 400 and 1,000 m running (17) and can reduce the decline in muscle torque production seen during repeated bouts of maximal isokinetic contraction in humans (11) More recently, these findings relating to exercise performance have been confirmed by others (1) The authors showed that Cr ingestion significantly improved work capacity during the 4th-6th s of ten 6-s sprints when each sprint was interspersed with 30 s of rest The depletion of muscle PCr stores is generally considered to be one of the limitations to muscle force production during maximal exercise (19, 23) In the above study (ll), it was suggested that the increase in work capacity observed after Cr ingestion may have been attributable to the rate of PCr resynthesis being accelerated during recovery between exercise bouts, possibly as a result of Cr availability displacing the equilibrium reaction catalyzed by creatine kinase (CK) Thus it was postulated that, after Cr ingestion, subjects began each bout of exercise with PCr levels at a relatively higher level and thereby may have delayed the depletion of muscle PCr stores during exercise In general, PCr resynthesis follows an exponential curve after intense muscle contraction (14, 25, 26) and the half-time for resynthesis in mixed-fibred human skeletal muscle is s (14) It is generally accepted that the resynthesis of PCr during recovery is mediated by mitochondrial membranebound CK, thus linking oxidative ATP production to cytoplasmic PCr resynthesis (5, 24, 31) Factors that will undoubtedly influence the rate of resynthesis will include free ATP, ADP, H+, and Cr concentrations, due to their role in the CK equilibrium reaction The in vitro Michaelis constant (K,) values of CK for ATP and ADP are relatively low, being -06 and 1 mmol/l, respectively (3) Conversely, the K, of CK for Cr is comparatively high, being close to 19 mmol/l It is known that muscle free Cr can range in concentration from - 13 mmol/l intracellular water at rest (16) to - 40 mmolll intracellular water after maximal exercise (29) Thus, during the initial stages of recovery from maximal exercise, when the rate of mitochondrial ADP rephosphorylation to ATP will be at its highest, it is unlikely Downloaded from by on March 30, 2017

5 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION E729 that the rate of PCr formation by mitochondrial CK will be dependent on the availability of free Cr, as its concentration will be far in excess of the K, value However, as PCr resynthesis continues and as the muscle free Cr concentration declines toward 19 mmol/l, it is suggested that free Cr concentration may then begin to be a determinant of the rate of PCr resynthesis The results of the present experiment support this suggestion Figure 2 demonstrates that, in those subjects who responded to Cr feeding during the initial 20 s of recovery, the rates of PCr resynthesis were almost identical when comparing values obtained before and after Cr ingestion This, based on the above explanation, might be expected, as the free Cr concentration was at all times in excess of 27 mmol/l intracellular water However, as recovery proceeded beyond 60 s, it can be seen that Cr feeding was associated with a higher rate of PCr resynthesis, producing a 30% higher PCr concentration at the end of recovery (P < 005) This, we suggest, was a result of Cr ingestion maintaining the muscle free Cr concentration throughout the second half of recovery higher than the K, of CK for Cr (19 mmol/l) and thereby sustaining a high flux rate through the CK reaction in favor of PCr resynthesis and ADP formation The latter, in turn, will have provided mitochondria with substrate to maintain a high rate of ATP formation The role of Cr as an acceptor of mitochondrial ATP has been discussed in a series of previously published papers (2,5,6,24,31) The rate of muscle PCr resynthesis during recovery from intense contraction has been suggested to be at least partly limited by intracellular ph (14, 27) However, it is unlikely that a difference in intracellular ph between treatments could explain the faster PCr resynthesis observed after Cr ingestion in the present study, as lactate accumulation was greater after Cr ingestion (Table Z), ind ica t ing that, if anything, muscle ph would have been lower during recovery after Cr ingestion As previously stated, data from this laboratory have shown that Cr ingestion can significantly aid performance during repeated bouts of maximal isokinetic exercise in humans, when each bout of exercise is separated by 60 s of recovery Figure 2 demonstrates that, in those subjects who responded to Cr feeding in the present study, muscle PCr concentrations were not statistically different after 60 s of recovery when comparing pre- and postingestion values It could be argued therefore that the increase in exercise performance that we have previously observed could not be attributable to an accelerated rate of PCr resynthesis during the 60 s of recovery between exercise bouts However, it perhaps should be made clear that, after 60 s of recovery, the mean muscle PCr concentration was 8 mmol/kg dry matter greater after Cr ingestion (P = 009, Fig 2) and that this increase was similar to that observed in resting skeletal muscle after Cr feeding (16) Furthermore, it is unlikely that the exercise protocol previously used would have depleted PCr stores to the same extent as the model used in the present experiment Tesch et al (30) demonstrated that, after 30 maximal voluntary isokinetic contractions at 314 radians/s, 36 mmol/kg dry matter of PCr were still present in the muscle Thus the pattern of PCr resynthesis in our previous study (11) would have probably been more similar to that recorded between 60 and 120 s in the present experiment On the basis of the lactate data shown in Table 2, another explanation for the increase in exercise performance could be that anaerobic glycolysis made a greater contribution to ATP production during exercise after Cr ingestion ( - 10 mmol/kg dry matter) However, the similarity in blood lactate concentrations in our previous study (11) and the lower lactate accumulation observed after Cr ingestion by Balsom et al (1) is not in agreement with this suggestion In the present experiment, muscle force production was not measured with sufficient precision to enable it to be accurately related to lactate accumulation In conclusion, the results demonstrate that oral Cr ingestion markedly increases the muscle total Cr concentration of those individuals who have a concentration of close to or < 120 mmol/kg dry matter before ingestion Furthermore, these same individuals show an accelerated rate of PCr resynthesis in the 2nd min of recovery from intense muscular contraction, which has depleted muscle PCr stores Present addresses: K Bodin, Dept of Clinical Physiology, Karolinska Institute, Huddinge University Hospital, S Huddinge, Sweden; K Soderlund, Department of Physiology III, Karolinska Institute, Box 5626, Stockholm, Sweden Address for reprint requests: P L Greenhaff, Queens Medical Center, Dept of Physiology & Pharmacology, University Medical School, Nottingham NG7 2UH, UK Received 22 October 1993; accepted in final form 22 December 1993 REFERENCES Balsom, P D, B Ekblom, K Soderlund, B Sjodin, and E Hultman Creatine supplementation and dynamic highintensity intermittent exercise Stand J Med Sci Sports 3: ,1993 Belitzer, V A La regulation de la respiration par le transformation du phosphagene Enzymalogia 6: 1-5, 1939 Bergmeyer, H U Methods of Enzymatic Analysis (2nd ed) London: Academic, 1965 Bergstrom, J Muscle electrolytes in man Determination by neutron activation analysis on needle biopsy specimens A study on normal subjects, kidney patients and patients with cronic diarrhoea Stand J Clin Lab Invest Suppl 68: l-110, 1962 Bessman, S P, and A Fonyo The possible role of mitochondrial bound creatine kinase in regulation of mitochondrial respiration Biochem Biophys Res Commun 22: , 1966 Bessman, S P, and P J Geiger Transport of energy in muscle The phosphorylcreatine shuttle Science Wash DC 211: ,198l Bloch, K, and R Schoenheimer The metabolic relation of creatine and creatinine studied with isotopic nitrogen J BioZ Chem 131: ill-118,1939 Chanutin, A The fate of creatine when administered to man J Biol Chem 67: 29-34, 1926 Delanghe, J, J-P DeSlypere, M Debuyzere, J Robbrecht, R Wieme, and A Vermeulen Normal reference values for creatine, creatinine and carnitine are lower in vegetarians CZin Chem 35: ,1989 Fitch, C D Significance of abnormalities of creatine metabolism In: Pathogenesis of Human Muscular Dystrophies, edited by L P Rowland Amsterdam: Excerpta Medica, 1977, p Greenhaff, P L, A Casey, A H Short, R C Harris, K Soderlund, and E Hultman Influence of oral creatine supplementation on muscle torque during repeated bouts of Downloaded from by on March 30, 2017

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