INTRODUCTION

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THE TOTAL CREATINE (TCr) pool in skeletal muscle is the sum of creatine (Cr) and phosphocreatine (PCr), hence TCr = PCr concentration ([PCr]) + Cr concentration ([Cr]). The fraction of the Cr pool in the phosphorylated state [ratio of PCr to TCr (PCr/TCr)] was termed the PCr energy charge by Connett (9). In resting skeletal muscle, PCr/TCr normally ranges from 0.6 to 0.8 (9). Dietary Cr supplementation has been shown to be an effective intervention to expand the TCr pool in human skeletal muscle, typically by ~20% (4, 7, 13, 16, 17, 19, 21, 28). However, most reports in the literature indicate that increased TCr after dietary supplementation is attended by a fall in PCr/TCr, i.e., the additional Cr taken into the TCr pool is not phosphorylated at the same PCr/TCr as that obtained in the pretreatment condition (4, 7, 16, 17, 19, 21, 28). These results are surprising when one considers the bioenergetic determinants of PCr/TCr.

In resting skeletal muscle, the Cr kinase reaction (see Eq. 1) is generally considered to be maintained near equilibrium, with an apparent equilibrium constant, K'= 1.66 ×10⁹ (23)

(1)

At a constant pH = 7.0, the equilibrium expression can be rearranged as

(2)

where K' now equals 166, [ATP] is the ATP concentration, and [ADP]_f is the free, or thermodynamically active, ADP concentration. As Eq. 2 indicates, the ratio of PCr to Cr will remain proportional to the ratio of ATP to free ADP (ADP_f; ATP/ADP_f) at constant pH. Green et al. (16) used Eqs. 1 and 2, along with their reported finding of a decreased PCr/TCr in resting muscle after Cr supplementation, to conclude that supplementation significantly increased (by up to 24%) the [ADP]_f.

In resting cells, the ATP hydrolysis reaction, which is shown as the second arrow in Eq. 3, is held far away from equilibrium, at a free energy of ATP hydrolysis (G'_ATP) of roughly 14 to 16 kcal/mol (22)

(3)

This very high potential energy in ATP (G'_ATP) is sustained at rest by the combined effects of inactivated enzymes of ATP hydrolysis "downstream" and the high-mitochondrial driving forces of oxidative phosphorylation (ox phos) applied from the reducing power of fuel "upstream" (the first arrow in Eq. 3). The resulting G'_ATP is given by

(4)

where G_ATP^o' is the standard free energy. R is the gas constant, and T is the temperature in degrees absolute. Equation 4 reveals that, at a constant P_i concentration ([P_i]), the ratio of ATP to ADP reflects G'_ATP, which, in turn, is established by the mitochondrial driving forces. Thus, if Cr supplementation does indeed decrease PCr/TCr of resting muscle, then Cr loading also decreases [P_i], pH, and/or mitochondrial driving forces in the resting condition.

The purpose of this study was to evaluate the effect of dietary Cr supplementation on the energy phosphate status of resting soleus and gastrocnemius muscles in the rat. These muscles were chosen for their fiber-type composition, predominantly type I in the soleus and type II in the gastrocnemius (1). Based on the bioenergetic relationship of PCr/TCr to G'_ATP, and the prediction of stable cellular [P_i] and pH, we hypothesized that dietary Cr supplementation would not alter PCr/TCr in resting type I or type II skeletal muscle. We found that dietary Cr supplementation simply increased skeletal muscle TCr in the gastrocnemius and had no effect on any other energy phosphate variable.

	METHODS

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Animal care. All aspects of the experimental protocol were approved by the Animal Care and Use Committee at Arizona State University. Twenty male Sprague-Dawley rats (200-250 g) were housed in the University Animal Research Center at Arizona State University at 21°C with a 12:12-h light-dark cycle. The animals were randomly assigned to either a dietary control group (n = 11) or a dietary Cr-supplemented group (Cr-S; n = 9). Animals were housed two or three to a cage. Control rats were fed a standard rodent chow for 2 wk, whereas Cr-S rats were fed the same diet with 2% (wt/wt) Cr monohydrate (Sigma Chemical, St. Louis, MO) incorporated into the chow (Harlan TekLad, Madison, WI). Water was supplied ad libitum to all rats. Body mass was measured at days 1, 3, 5, 8, 10, 12, 13, and 15.

Blood and tissue collection. After 10 days on the diet, tail blood samples were taken from control (n = 5) and Cr-S (n = 7) animals to measure blood [Cr]. Because the animals tended to begin eating shortly after the lights were turned off (6:00 PM) and [Cr] in the blood peaks ~1 h after intake (19), blood was sampled at 10:00 PM. After 2 wk on the dietary regimen, the animals were taken from their cages, weighed, and then anesthetized via an intramuscular injection of an anesthetic cocktail containing xylazine (0.86 mg/100 g body mass) plus ketamine (4.3 mg/100 g body mass) plus acepromazine (0.14 mg/100 g body mass). From the posterior aspect of the hindlimbs, the entire musculature was exposed through a vertical incision beginning at the heel and continuing past the knee joint. With the use of blunt dissection, the gastrocnemius was gently separated, freeze clamped with tongs previously cooled in liquid nitrogen, and removed. The soleus was then removed by using the same procedure. Up to the moment that the freeze clamp was applied, the blood supply to the muscles remained intact, and no contractile activity was visible. Immediately after removal, muscle was immersed in liquid nitrogen and stored at 80°C. The gastrocnemius and soleus were removed from both legs of each animal. Once all muscles were removed, the animal was killed via exsanguination.

Tissue preparation. Frozen muscles were broken into smaller pieces under liquid nitrogen in a mortar and pestle and were weighed, and metabolites were extracted in 0.5 M perchloric acid plus 1 mM EDTA. Acidified muscle was homogenized with a TekMar Tissumizer (Cincinnati, OH), and the extract was centrifuged for 2 min at 10,000 rpm. The supernatants were then neutralized by adding 2.1 M KHCO₃ containing 0.3 M MOPS. Finally, the neutralized extracts were recentrifuged for 3 min at 10,000 rpm, and the supernatants were stored at 80°C until analysis.

Blood preparation. Immediately after sampling, 100 µl of blood were added to 400 µl of 0.5 M perchloric acid plus 1 mM EDTA, centrifuged, and neutralized as described for the tissue samples. Analysis for [Cr] was performed the following day with the same methods used for tissue measurement.

Biochemical analysis. Cr and TCr were measured by using the nonenzymatic technique of De Saedeleer and Marechal (11). PCr was calculated by subtracting the [Cr] from TCr concentration ([TCr]) for each sample. ATP and P_i were determined enzymatically by using the techniques of Trautschold et al. (27) and Gawehn (14), respectively. All assays were carried out at least in duplicate.

Calculations. The G_ATP^o' was taken to be 7,566 cal/mol (15). The [ADP]_f was calculated using a value of 166 for the K' of the Cr kinase reaction (23). In both estimations, resting muscle temperature, pH, and free Mg²⁺ were assumed to be 310 K, 7.0, and 1.0 mM, respectively.

Statistics. Data are reported as means ± SE. Significant differences between two mean values were determined by using Student's t-tests for independent samples. Significance was accepted at P < 0.05.

	RESULTS

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Body masses before and after the 2-wk dietary intervention were, respectively, 223.3 ± 4.5 and 324.8 ± 7.3 g for control animals and 214.9 ± 6.3 and 335.6 ± 7.1 g for Cr-S rats. Body masses of the two groups were similar at every time point (data not shown).

Blood Cr levels. The [Cr] of blood taken at 10:00 PM on day 10 of the feeding protocol was 260 ± 15 µM in control rats and 2,420 ± 350 µM in Cr-S rats. Thus, during the period of daily feeding, dietary Cr supplementation elevated blood [Cr] by roughly 10-fold (P < 0.005).

Energy phosphates. In Fig. 1, the [Cr], [PCr], and [TCr] are reported for the soleus in 1A and the gastrocnemius in 1B. Dietary Cr supplementation did not influence [Cr], [PCr], or [TCr] in the soleus muscle. The mean soleus TCr of Cr-S rats was 6% higher than that for control, but statistical significance was not achieved.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Creatine (Cr), phosphocreatine (PCr), and total creatine (TCr) in control and creatine-supplemented (Cr-S) skeletal muscle. A: data for the soleus (n = 11 control, n = 9 Cr-S). B: data for the gastrocnemius (n = 10 control, n = 9 Cr-S). Values are means ± SE reported in µmol/g wet muscle. * Significant difference, Cr-S vs. control, P < 0.05.

View this table: [in this window] [in a new window]   Table 1.   Concentrations of Pi, ADPf, and ATP and the calculated values of ATP/ADPf, G'ATP, and PCr/TCr

	DISCUSSION

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Several studies in the literature have reported roughly 20% increases in the TCr content of skeletal muscle after dietary Cr supplementation in humans (4, 7, 13, 16, 17, 19, 21, 28) and in the rat (6). The present study showed 10-fold higher blood [Cr] in rats consuming the Cr-supplemented diet. A numerical increase of 6% in the TCr of soleus and a significant increase of 20% in gastrocnemius TCr followed 14 days of such dietary Cr supplementation in rats. The major finding of the present study was that the increase in TCr was achieved with no other apparent changes in the cellular energy phosphate status. There were no changes in the measured [ATP] and [P_i] of treated muscles, nor were there any changes in the calculated ADP_f, ATP/ADP_f, or G'_ATP effected by the elevated TCr of supplemented muscles. Thus the extra Cr taken up by the muscle of Cr-S rats was phosphorylated at the same PCr/TCr as was present before the dietary intervention.

The [Cr] and [PCr] data reported in most previous studies indicate that muscle PCr/TCr falls after TCr augmentation (4, 7, 16, 17, 19, 21, 26, 28). This finding is curious when we consider that the equation for the G'_ATP can be rewritten to take into account the equilibrium at the Cr kinase reaction (K_ck^') (Eq. 5). Assuming constant pH

(5)

Equation 5 shows that the ratio PCr/(Cr × P_i) can act as the concentration term of G'_ATP, when the K'_ck is taken into account. Thus, if dietary Cr supplementation results in decreased muscle PCr/TCr, then it may be concluded that Cr supplementation either decreases the [P_i], weakens the mitochondrial driving forces responsible for establishing the value of G'_ATP, or some combination of the two. In the present study, dietary Cr supplementation did not affect [P_i], nor did it affect the value of G'_ATP, calculated on the basis of the measured energy phosphates. Our data, and recent data from Febbraio et al. (13), support the contention that dietary Cr supplementation simply increases the TCr pool size in skeletal muscle without affecting PCr/TCr or mitochondrial energy transduction. We are not surprised by this outcome, because we can think of no compelling reasons to suspect that Cr supplementation would compromise the forces or flows generated by mitochondria in resting muscle. Because the resting cellular [P_i] in striated muscle is also energy linked (24), we would also not predict changes in the [P_i] of muscle adapted to augmented TCr.

It might be proposed that the 3- to 5-day dietary interventions used in several previous studies (4, 7, 16, 19, 26) may have been too brief for equilibration across the sarcolemma to occur. According to this view, depressed PCr/TCr in muscle after Cr supplementation would be a transient phenomenon, occurring because of inadequate time for myocytes to establish a stable relationship with the high plasma [Cr] and to reestablish PCr/TCr. However, the data of Hultman et al. (21) would seem to argue against this proposition, because they reported that PCr/TCr fell and tracked, as the mirror image, the rise in TCr observed over a period of at least 1 mo. Moreover, Hochachka and Mossey (20) recently reported the results of tracer experiments, suggesting that recently taken up Cr actually has greater access to equilibration in the Cr kinase reaction than does the preexisting intracellular pool. Specifically, they showed that, when isotopically labeled Cr is provided for 2 h to fish white muscle, acid extraction of the muscle reveals three- to fivefold lower specific activity in the cellular Cr compared with PCr, suggesting that a cellular compartment of unlabeled ("cold") Cr is released on acid extraction. This very large compartment of Cr would, therefore, presumably not have access to the Cr kinase reaction, whereas recently taken up Cr, according to the data of Hochachka and Mossey, apparently does. Thus any temporary change in PCr/TCr induced by acutely stimulated Cr uptake would, according to this interpretation, be predicted to raise, not depress, the chemically measured PCr/TCr.

The soleus and gastrocnemius of the rat were chosen for this study on the basis of their fiber-type characteristics. The rat soleus contains predominantly type I muscle: specifically, 13% fast-oxidative glycolytic (type IIa) and 87% slow oxidative (type I) fibers. The mixed gastrocnemius contains predominantly type II muscle: 28% type IIa, 65% fast glycolytic (type IIb), and 7% type I (1). Homogeneous type II muscles have larger [PCr] and [ATP] and smaller [P_i] and [ADP]_f compared with type I muscles (22). Some of these differences are apparent in Table 1, despite the noteworthy, albeit small, fiber-type heterogeneity of the rat soleus and gastrocnemius. In this study, gastrocnemius muscles responded to Cr supplementation with a 20% increase in TCr, whereas the soleus failed to achieve a significant increase in TCr. Brannon et al. (6) recently reported that 24 days of Cr supplementation resulted in a significant rise in TCr in the fast-twitch plantaris but no significant increase in rat soleus TCr, although there were increases observed at earlier time points in the supplementation regimen. Thus our results, and those of Brannon et al., indicate that type II muscle becomes even better suited for burst-type activity. Higher resting TCr, concomitant with a stable PCr/TCr, i.e., a higher [PCr], would provide augmented metabolic capacitance.

Humans have shown improved exercise performance in high-intensity, short-duration, and repetitive-type exercise after dietary Cr supplementation (2, 5, 7, 12, 18, 19, 28). These results are consistent with the view that the increased metabolic capacitance, provided by elevated [PCr], better defends energetic homeostasis during burst activity, when phosphagen bond splitting occurs without replacement (9, 25).

Type I skeletal muscle contains a smaller TCr pool than does type II (9, 22), and this difference was amplified when supplemental Cr was made available in the present study. The lack of a response of the type I soleus muscle to greater Cr availability in this study may reflect the primary physiological mission of the type I fiber, i.e., to support aerobic work. Specifically, a disadvantage of a greater PCr pool during steady-state aerobic exercise is the necessity to discharge a greater capacitance, thus producing a larger quantity of P_i to fall to a given cytosolic G'_ATP (9). Because muscle oxygen consumption rate rises as a roughly linear function of falling G'_ATP (25), greater TCr means that more P_i must accumulate to elicit a given muscle oxygen consumption. In turn, the higher steady-state [P_i] may result in a metabolic environment less conducive to endurance performance, e.g., greater inhibition of muscle force production (10) and greater stimulation of carbohydrate mobilization (8). In fact, the limited data presently available suggest that dietary Cr supplementation may impair muscle aerobic endurance capacity (3).

In conclusion, this study showed that Cr supplementation increased TCr in rat fast-twitch skeletal muscle. Because no change in PCr/TCr occurred, Cr supplementation simply provided an increased TCr content and metabolic capacitance in skeletal muscle. Finally, the additional Cr taken up during dietary Cr supplementation was phosphorylated at the same fraction as the original TCr pool, apparently because mitochondrial driving forces and the cellular [P_i] remain unaffected by the expansion of the TCr pool.