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1Neuromuscular Function Laboratory, Department of Occupational Therapy, Temple University, Philadelphia, Pennsylvania 19140; 2United States Army Research Institute of Environmental Medicine, Natick 01760; 3Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston 02115; and 4Boston University, Sargent College of Health and Rehabilitation Sciences, Boston, Massachusetts 02215
Submitted 23 September 2003 ; accepted in final form 3 February 2004
ABSTRACT
Recent human isolated muscle fiber studies suggest that phosphocreatine (PCr) and creatine (Cr) concentrations play a role in the regulation of mitochondrial respiration rate. To determine whether similar regulatory mechanisms are present in vivo, this study examined the relationship between skeletal muscle mitochondrial respiration rate and end-exercise PCr, Cr, PCr-to-Cr ratio (PCr/Cr), ADP, and pH by using 31P-magnetic resonance spectroscopy in 16 men and women (36.9 ± 4.6 yr). The initial PCr resynthesis rate and time constant (Tc) were used as indicators of mitochondrial respiration after brief (10–12 s) and exhaustive (1–4 min) dynamic knee extension exercise performed in placebo and creatine-supplemented conditions. The results show that the initial PCr resynthesis rate has a strong relationship with end-exercise PCr, Cr, and PCr/Cr (r > 0.80, P < 0.001), a moderate relationship with end-exercise ADP (r = 0.77, P < 0.001), and no relationship with end-exercise pH (r = -0.14, P = 0.34). The PCr Tc was not as strongly related to PCr, Cr, PCr/Cr, and ADP (r < 0.77, P < 0.001–0.18) and was significantly influenced by end-exercise pH (r = -0.43, P < 0.01). These findings suggest that end-exercise PCr and Cr should be taken into consideration when PCr recovery kinetics is used as an indicator of mitochondrial respiration and that the initial PCr resynthesis rate is a more reliable indicator of mitochondrial respiration compared with the PCr Tc.
skeletal muscle; creatine kinase; mitochondria; phosphocreatine; phosphorous-31 magnetic resonance spectroscopy
Recently, PCr and Cr concentrations were shown to affect mitochondrial respiration rate in human isolated muscle fiber preparations (19, 22). Walsh et al. (22) found that mitochondrial respiration rate increased as the PCr-to-Cr ratio (PCr/Cr) declined and suggest that ADP production from mitochondrial creatine kinase serves as a regulator of mitochondrial oxidative respiration and that PCr/Cr modulates mitochondrial sensitivity to ADP. Similar results have been shown in animal isolated muscle fiber studies (2, 6, 14).
To determine whether similar relationships exist in vivo, we combined data from two studies (15, 16) and retrospectively examined the relationship between human vastus lateralis muscle mitochondrial respiration rate and end-exercise PCr, Cr, PCr/Cr, and ADP by using 31P-MRS. To obtain a range of end-exercise PCr and Cr concentrations for comparison, mitochondrial respiration was determined after exercise of variable duration in both placebo and creatine-supplemented conditions. In addition, the initial PCr resynthesis rate (mmol·kg-1· min-1) and the PCr recovery time constant (Tc; s) have been used independently as indicators of the mitochondrial respiration rate. Therefore, we present PCr Tc and initial resynthesis rate results and compare both with end-exercise PCr, Cr, PCr/Cr, ADP, and pH to elucidate which measure may be a more reliable indicator of maximal mitochondrial respiration. The results herein provide plausible explanations for the inconsistent findings on the effects of creatine ingestion on muscle metabolism and provide further support for a regulatory link between mitochondrial creatine kinase activity and oxidative phosphorylation.
METHODS
Exercise. Eight men (41.6 ± 4.4 yr) and 8 women (32.1 ± 4.7 yr) gave their written, informed consent and performed single-leg dynamic knee-extension exercise while lying supine inside a whole body 1.5-T MR system (General Electric Signa, Milwaukee, WI). Knee extensions were performed at 35 contractions/min through a range of motion of 
110–145°. A 10- to 12-s brief exercise bout (n = 9) and a 1- to 4-min exhaustive exercise bout (n = 16) were performed during each trial. Nine subjects performed both brief and exhaustive exercise bouts, and seven subjects performed only exhaustive bouts. Exhaustion was defined as the time when the subject could not maintain the rate and/or range of motion. For each subject, exercise resistance was constant across placebo and creatine trials. This study was approved by the affiliated Institutional Review Boards and was conducted in accordance with the Declaration of Helsinki and US Fedral Regulations governing the protection of human subjects.
Creatine supplementation. Two single-blind exercise trials were performed, a placebo trial followed by a creatine trial 7–14 days later. The trials were not randomized, because creatine levels may remain elevated in skeletal muscle for 3–4 mo after supplementation. Five days before each trial, subjects began consuming 0.3 g·kg-1·day-1 of either a placebo (granulated sugar) or creatine monohydrate combined with a flavored powder drink mix. The mixture was dissolved in water and consumed four times per day.
31P-MRS. 31P spectra were collected every 10 s during exercise and recovery through a 1H/31P dual radio frequency transmit-receive 11-cm surface coil placed over the vastus lateralis muscle. For details on spectral parameters and processing see Smith et al. (15, 16). Relative concentrations of PCr, inorganic phosphate (Pi), and 
-ATP were determined from spectral peak areas. Resting PCr/ATP values were converted to millimoles per kilogram of wet weight by assuming the area of -ATP at rest = 5.5 mmol/kg wet weight. End-exercise free Cr was calculated by adding the change in PCr during exercise to 15% of the resting PCr value (3). ADP (µmol) was calculated by using the equation ADP = [Cr][ATP]/[PCr][H+](Keq), with Keq (creatine kinase equilibrium constant) =1.66 x 109 and H+ (hydrogen ion concentration) = 10-pH with brackets indicating concentration (1). Resting and end-exercise pH was calculated by using the chemical shift between the Pi and PCr frequencies (17). The PCr resynthesis rate (mmol·kg-1·min-1) and Tc (s) were determined from a monoexponential curve fit to the PCr recovery data: y = a[1 - exp(-bx)] + c, where y represents the PCr value at any given time x, a is the change in PCr during recovery, b is the rate constant, and c is the initial PCr value at the onset of recovery. PCr recovery starts at c, rises to a + c with a Tc of 1/b. The initial PCr resynthesis rate was determined by calculating the amount of PCr resynthesized in the first second of recovery by using the exponential equation and multiplying that quantity by 60 to obtain initial PCr resynthesis rate in millimoles per kilogram per minute (15, 16). This was accomplished by setting x = 1 s, c = 0 mmol/kg, a = change in PCr (mmol/kg), and b = rate constant and by multipling the y resultant by 60. To check the reliability of this method, the initial PCr resynthesis rate results calculated from the exponential equation were compared with resynthesis rates calculated using a linear fit across the first 30 s of PCr recovery and the change in PCr during the first 20 s of recovery (3, 9, 11).
Analysis. Regression analyses were used to determine the relationship of the PCr Tc and the initial PCr resynthesis rate to end-exercise PCr, Cr, PCr/Cr, ADP, and pH. The regression equation that resulted in the best fit for each comparison was selected. Given the possible interactions of PCr/Cr availability, pH, and ADP on mitochondrial ATP production and PCr recovery kinetics, it is likely that basic Michaelis-Menton kinetics may not best represent relationships between PCr recovery rate and end-exercise metabolites. Additional regression analyses were performed on PCr Tc data corrected for pH when end-exercise pH was <6.95 by using an arithmetic correction (5, 20). A partial correlation between PCr Tc and end-exercise metabolites statistically correcting for pH was also performed. Paired t-tests were used to determine differences in variables between placebo and creatine conditions. The significance level was P < 0.05 for all tests, and results are means ± SE.
RESULTS
Resting vastus lateralis muscle PCr was 38.6 ± 1.2 mmol/kg in the placebo and 45.8 ± 5.4 mmol/kg in the creatine conditions, an increase of 19% (n = 16, P < 0.01). Resting muscle pH was not different between placebo (7.12 ± 0.01) and creatine (7.12 ± 0.008) conditions (P = 0.87). Data for the brief exercise bouts were from three men and six women (29.9 ± 1.4 yr). During both the brief and exhaustive exercise bouts, the mean power output was the same for the placebo (18.4 ± 0.6 W/knee extension) and creatine (18.6 ± 0.7 W/knee extension) conditions (P = 0.83). The brief and exhaustive end-exercise pH was not different between the placebo and creatine conditions with pH 7.19 ± 0.02 vs. 7.18 ± 0.01 (P = 0.42) for brief and 6.54 ± 0.06 vs. 6.57 ± 0.06 (P = 0.56) for exhaustive exercise, respectively. Time to exhaustion was increased from 197 ± 17 to 225 ± 18 s from the placebo to creatine condition (P < 0.01).
PCr recovery kinetics after brief and exhaustive exercise are shown in Fig. 1, A and B, for the placebo and creatine conditions. The end-brief exercise PCr was greater after creatine ingestion, and the change in PCr during brief exercise tended to be greater after creatine ingestion (Fig. 1A). The end-brief exercise Cr was 12.3 ± 0.9 and 14.2 ± 0.7 mmol/kg for the placebo and creatine conditions (P = 0.02), and PCr/Cr ratio 2.8 ± 0.1 and 2.8 ± 0.2, respectively (P = 0.74). Initial PCr resynthesis rate tended to be greater in the placebo condition (P = 0.16), and the PCr Tc was less during the placebo condition (Fig. 1A). In addition, initial PCr resynthesis rates calculated by using a linear fit across the first 30 s of PCr recovery and from the change in PCr during the first 20 s of recovery provided similar resynthesis rate results compared with those calculated by using the exponential equation (r > 0.92, P < 0.001).
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End-exhaustive exercise PCr is not different after creatine ingestion, and the change in PCr during exercise was greater (Fig. 1B). The end-exhaustive exercise Cr was 30.7 ± 2.1 and 38.7 ± 2.1 mmol/kg for the placebo creatine conditions (P < 0.01), and PCr/Cr was 0.51 ± 0.11 and 0.41 ± 0.09, respectively (P = 0.03). Initial PCr resynthesis rate was greater in the creatine condition, and there was no difference in PCr Tc between the placebo and creatine conditions (Fig. 1B).
Regression analyses found that initial PCr resynthesis rate and end-exercise PCr, Cr, PCr/Cr, and ADP were all significantly related (Fig. 2, A–D). PCr, Cr, and PCr/Cr (r > 0.8) were more strongly related to initial PCr resynthesis rate than ADP (r = 0.77). Equivalent regression results were obtained when initial PCr resynthesis rates were calculated from a linear fit across the first 30 s of recovery and from the change in PCr in the first 20 s of recovery. We present the initial PCr rate calculated from the exponential equation to illustrate that reliable resynthesis rate data can be derived from the same equation used to determine the PCr Tc. The regression coefficients for raw PCr Tc and PCr Tc corrected for pH by using statistical and arithmetic methods (5, 20) compared with end-exercise metabolites and pH are presented in Table 1. The arithmetic pH correction eliminated the significant relationship between PCr Tc and pH. The raw PCr Tc was significantly related to end-exercise pH (r = -0.43, P < 0.01), whereas the initial PCr resynthesis rate was not (r = -0.14, P = 0.34). For the regression analyses, the best fit for PCr Tc and PCr initial resynthesis rate was obtained with a double-exponential decline for end-exercise PCr and PCr/Cr, a single-exponential rise for ADP and a linear regression for Cr and pH.
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DISCUSSION
The results of this study show that the initial PCr resynthesis rate in vastus lateralis muscle was greater after an exhaustive exercise bout compared with a brief exercise bout (Fig. 1), which has also been shown by others (11, 13). Creatine ingestion tended to reduce initial PCr resynthesis rate after brief exercise and increase initial PCr resynthesis rate after exhaustive exercise, with the differences appearing to coincide with the magnitude of muscle PCr utilization and free Cr accumulation at the end of exercise (Fig. 1). Regression analyses (Fig. 2) in fact show that there is a strong relationship between initial PCr resynthesis rate and end-exercise PCr, Cr, and PCr/Cr and a moderate relationship with ADP. Given that PCr resynthesis rate is dependent on mitochondrial ATP production, these results indicate that PCr and Cr concentrations influence mitochondrial oxidative respiration rate. A similar relationship is clearly shown in human (19, 22) and animal (2, 6, 14) skinned isolated muscle fiber studies. These studies and the in vivo results herein strongly suggest that muscle PCr and Cr modulate mitochondrial respiration rate, possibly by affecting mitochondrial sensitivity to ADP activation (22) and/or through other mechanisms such as improving ATP transport from the mitochondria to metabolically active sites by enhancing creatine kinase kinetics (10).
The PCr Tc, which is also used as an indicator of mitochondrial respiration, was not as strongly related to end-exercise PCr, Cr, PCr/Cr, and ADP as the initial PCr resynthesis rate. In addition, the raw PCr Tc was significantly related to changes in end-exercise pH, whereas PCr resynthesis rate was not as previously shown (1, 13). Correction of PCr Tc for pH with the use of statistical or arithmetic (5, 20) methods did not improve the relationship between PCr Tc and end-exercise PCr, Cr, PCr/Cr, and ADP as shown in Table 1. Unlike the initial PCr resynthesis rate, the Tc depends heavily on PCr kinetics throughout recovery and is more likely to be influenced by factors that impact the latter stages of PCr recovery, such as muscle pH and changes in hemodynamics (9). It has been shown that the initial PCr resynthesis rate is not affected by end-exercise pH (23) and that the initial PCr resynthesis rate is more strongly related to maximum whole body oxygen consumption and maximum citrate synthase activity (mitochondrial marker of oxidative capacity) than the PCr Tc and other measures of PCr resynthesis rate that incorporate latter stages of recovery (9). The Tc is analogous to half time of recovery and alone does not indicate the initial quantity of PCr resynthesized per unit time. This is particularly apparent when comparing groups or conditions that differ in preexercise PCr availability and subsequent hydrolysis as demonstrated in Fig. 1B. Creatine ingestion produced a marked increase in the initial rate of PCr recovery, presumably supported by an increase in mitochondrial ATP production driven by a significantly lower PCr/Cr compared with the placebo condition. However, the PCr Tc was the same for both placebo and creatine conditions. These findings suggest that the initial PCr resynthesis rate may better predict maximal mitochondrial oxidative capacity compared with PCr Tc and other rate measures that rely on PCr kinetics throughout recovery.
In addition to PCr Tc vs. initial resynthesis rate considerations, the influence of PCr and Cr on initial PCr resynthesis rate (Fig. 2) further demonstrates that end-exercise PCr and Cr concentrations should be accounted for when determining mitochondrial respiration rate from PCr recovery kinetics. Otherwise, differences in respiration rate between groups or conditions resulting from a difference in end-exercise PCr/Cr-mediated mitochondrial activation may be mistaken for differences in intrinsic mitochondrial oxidative capacity. Alternatively, actual differences in mitochondrial oxidative capacity may be missed if the effects of PCr and Cr are not considered.
The use of the PCr Tc or related recovery measures vs. the initial PCr resynthesis rate may in part explain the inconsistent findings reported in human 31P-MRS studies of the effects of creatine ingestion on muscle metabolism. The potential for discrepancies between the PCr Tc and the initial rate is illustrated in Fig. 1. After brief exercise (Fig. 1A), the initial PCr resynthesis rate tended to be less and the Tc was greater after creatine ingestion, showing agreement between the measures (a slower Tc indicates a faster resynthesis rate). However, after exhaustive exercise (Fig. 1B), the initial PCr rate was greater and the Tc was unchanged after creatine ingestion, which may be interpreted as either an increase in mitochondrial respiration or no change depending on the measure chosen. Results from Kreis et al. (7) show similar inconsistencies between PCr rate and Tc. They report slower PCr recovery after creatine ingestion by using the PCr rate constant (reciprocal of the Tc); however, in a model of PCr recovery after complete PCr depletion, they show that more PCr is resynthesized per unit time after creatine ingestion.
Differences in end-exercise PCr and Cr between 31P-MRS studies have probably also contributed to the inconsistent effects of creatine supplementation on mitochondrial respiration. As shown in Fig. 1, the initial PCr resynthesis rate after creatine ingestion tends to be reduced after brief exercise where PCr/Cr is >1. After exhaustive exercise, where PCr/Cr is <1, the initial PCr resynthesis rate is increased after creatine ingestion. Figure 2 further illustrates the relationship between end-exercise metabolite concentrations and the initial PCr resynthesis rate. These results agree with isolated muscle fiber studies where the mitochondrial respiration rate has been measured over a range PCr and Cr concentrations (2, 6, 14, 19, 22).
In summary, our results show that there is a significant relationship in vivo between the initial PCr resynthesis rate and end-exercise PCr, Cr, and PCr/Cr in human skeletal muscle, suggesting that PCr and Cr may modulate mitochondrial ATP production. Given the potential influence of PCr and Cr on mitochondrial respiration, it is recommended that end-exercise PCr and Cr be accounted for when postexercise PCr recovery kinetics are used to determine mitochondrial respiration rate. Not correcting for the influence of PCr and Cr may lead to inaccurate conclusions regarding maximal mitochondrial respiration capacity. These considerations may be applicable in future studies as well as the comparison of data from previous studies. In addition, the initial PCr resynthesis rate is more strongly related to end-exercise PCr, Cr, PCr/Cr, and ADP compared with the PCr Tc, and the initial PCr rate is not significantly influenced by end-exercise pH. Therefore, the initial PCr resynthesis rate appears to be a more reliable indicator of maximal end-exercise mitochondrial respiration compared with the PCr Tc. These findings are particularly important to consider when determining the effects of oral creatine ingestion on muscle metabolism and when comparing groups or conditions that may differ in initial muscle PCr and Cr availability.
ACKNOWLEDGMENTS
The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision.
FOOTNOTES
Address for reprint requests and other correspondence: S. A. Smith, Temple Univ., Neuromuscular Function Laboratory, Dept. of Occupational Therapy, 3307 North Broad St., Philadelphia, PA 19140 (E-mail: sinclair.smith{at}temple.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
O2 and phosphorylcreatine level. Implications for the control of respiration. J Gen Physiol 86: 135-165, 1985.This article has been cited by other articles:
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  A. M. Jones, D. P. Wilkerson, and J. Fulford Influence of dietary creatine supplementation on muscle phosphocreatine kinetics during knee-extensor exercise in humans Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1078 - R1087. [Abstract] [Full Text] [PDF]
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 G. Kemp, S. A. Smith, and S. J. Montain Mitochondrial respiration in creatine-loaded muscle: is there 31P-MRS evidence of direct effects of phosphocreatine and creatine in vivo? J Appl Physiol, April 1, 2006; 100(4): 1428 - 1430. [Abstract] [Full Text] [PDF]
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PCr; mmol/kg), end-exercise PCr (End ex; mmol/kg), PCr resynthesis time constant (Tc; s), and initial PCr resynthesis rate (ratei; mmol · kg-1 · min-1) for brief and exhaustive exercise. Values are means ± SE.
and 
) and exhaustive (1–4 min) dynamic exercise (
and 
) are shown.