The relationship between creatine kinase kinetics and exercise intensity in human forearm is unchanged by age

¹ Gerontology Research Center, ² Nuclear Magnetic Resonance Unit, and ³ Laboratory of Cardiovascular Sciences, National Institutes of Health, National Institute on Aging, Baltimore, Maryland 21224

	ABSTRACT

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Using ³¹P magnetic resonance spectroscopy, creatine kinase (CK) reaction kinetics was assessed in the forearm flexor digitorum profundus muscle of healthy young (n = 11, age 34.7 ± 5 yr) and older (n = 20, age 73.5 ± 8 yr) subjects at rest, intermittent exercise at 20% maximum voluntary contraction (MVC), and 40% MVC. Exercise resulted in a significant increase in the average ratio of inorganic phosphate (P_i) to phosphocreatine (PCr) from resting values of 0.073 ± 0.031 (young) and 0.082 ± 0.037 (older) to 0.268 ± 0.140 (young, P < 0.01) and 0.452 ± 0.387 (older, P < 0.01) at 40% MVC. At 40% MVC, intracellular pH decreased significantly, from resting values of 7.08 ± 0.08 (young) and 7.08 ± 0.11 (older) to 6.84 ± 0.19 (young, P < 0.05) and to 6.75 ± 0.25 (older, P < 0.05). Average values of the pseudo-first-order reaction rate k_(PCrATP) at rest were 0.07 ± 0.04 s¹ in the young and 0.07 ± 0.03 s¹ in the older group. At both exercise levels, the reaction rate constant increased compared with the resting value, but only the difference between the resting value and the 20% MVC value, which showed an 86% higher reaction rate constant in both groups, reached statistical significance (P < 0.05). No difference in the reaction rate constant between the young and older groups was observed at either exercise level. As with k_(PCrATP), the average phosphorus flux through the CK reaction increased during exercise at 20% MVC (P < 0.05 in the older group) but decreased toward resting values at 40% MVC in both groups. The data in our study suggest that normal aging does not significantly affect the metabolic processes associated with the CK reaction.

skeletal muscle; ³¹P magnetic resonance spectroscopy; reaction rate; magnetization transfer

	INTRODUCTION

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ALTHOUGH THE DECLINE IN PHYSIOLOGICAL and biochemical performance of tissue and organs with age is associated with impairment of cellular bioenergetics (27), ³¹P magnetic resonance spectroscopy (MRS) data on bioenergetic correlates of age in human skeletal muscle are inconclusive. Three groups, including our own (4, 20, 37, 39), found no correlation between age and steady-state high-energy phosphate concentrations with acute exercise or recovery, whereas such correlations were reported in other studies (5, 28, 33, 38). However, the kinetics of the creatine kinase (CK) reaction
that catalyzes the transphosphorylation between PCr and ADP and is central to regulation of muscle bioenergetics (40) is not directly observed in concentration measurements. Compared with such measurements, analysis of the CK reaction rate, reflecting the rate of ATP turnover (34), provides a more sensitive test for monitoring muscle high-energy phosphate metabolism. Accordingly, we hypothesized that, in skeletal muscle, the phosphorus flux through the CK reaction, acting as a shuttle for high energy phosphate transport (29), would be decreased in older subjects compared with young subjects during physiological stress.

Traditional methods of MRS for measurement of the CK rate constant and flux in vivo, based on magnetization transfer techniques (10, 11), are time consuming and subject to several sources of significant error (36). These considerations may account for the fact that few studies of CK reaction kinetics in human skeletal muscle in vivo have been reported to date (13, 18, 32). To measure the CK reaction rate in vivo more accurately and rapidly than is possible with classical magnetization transfer techniques, we have recently developed alternative methods for measuring reaction rates (16- 18). In the present study, we used these methods to measure CK reaction kinetics in the human flexor digitorum profundus (FDP) muscle at rest, at 20% maximum voluntary contraction (MVC), and at 40% MVC.

	METHODS

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Subject population. Two groups of healthy male and female volunteers from the Baltimore Longitudinal Study of Aging were studied: 11 young subjects (6 male, 5 female, age 34.7 ± 5 yr, range 27.4-41.6 yr) and 20 older subjects (11 male, 9 female, age 73.5 ± 8 yr, range 60.8-87.5 yr). All subjects were free of cardiac or peripheral vascular disease, untreated hypertension, significant arthritis of the upper limbs, and neuromuscular disease. The experimental protocol was approved by the Johns Hopkins Bayview Medical Center Institutional Review Board for Human Subjects Research, and informed consent was obtained from each individual before the study.

³¹P MRS. ³¹P MR spectra were obtained with a 1.9-T, 31-cm superconducting magnet (Oxford Instruments, Oxford, UK) interfaced to a Bruker Biospec ABX console (Bruker Meolizindechnik, Erlangen, Germany). A homebuilt double tuned three-turn elliptical surface coil (2.9 × 3.8 cm) was positioned over the FDP muscle of the nondominant arm. The muscle was located by palpation while the subject was squeezing the ring and little fingers (9, 21). A half-passage sin/cos adiabatic observe pulse of 2 ms duration was applied to achieve uniform spin excitation over the sensitive volume of the coil (1). The field homogeneity was adjusted by shimming on water to proton linewidths of <45 Hz. A spectral width of 2,000 Hz with 2,048 data points was used in all experiments.

Spin-lattice relaxation time (T₁) measurements were performed using a progressive saturation experiment modified to account for chemical exchange (16, 17, 35). Data were acquired at repetition times (TR) of 0.6 s with 320 acquisitions after 16 dummy scans (spectra in trace d, Fig. 1, A-C), and at 25 s with 12 acquisitions (spectra in trace a, Fig. 1, A-C), in the absence of any saturation. A 1-ms homospoil pulse was applied after the data acquisition at the short TR to dephase transverse magnetization (17).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   31P magnetic resonance spectra of the nondominant forearm in a 69-yr-old male subject at rest (A), during exercise at 20% maximum voluntary contraction (MVC) (B), and during exercise at 40% MVC (C). Signals of inorganic phosphate (Pi), phosphocreatine (PCr), and ATP were detected. The steady-state saturation transfer experiment was performed with the -ATP resonance saturated (spectrum b) and with saturation contralateral to PCr (spectrum c). The arrow denotes the frequency of selective irradiation. Spectra a-c were acquired with a repetition time of 25 s and 12 acquisitions; spectrum d was acquired with a repetition time of 0.6 s and 320 acquisitions.

The steady-state saturation transfer experiment was performed both at rest and during exercise. Saturation of the -ATP resonance was achieved with a 6-s low-power square pulse on the observe channel (16, 17; spectra in trace b, Fig. 1, A-C). Control spectra were obtained by irradiating at an offset contralateral to the PCr resonance (spectra in trace c, Fig. 1, A-C). The spectra with -ATP saturation and contralateral irradiation were obtained with TR = 25 s and 12 acquisitions. Spectra traces a-c were acquired sequentially in one block, with 12 acquisitions. The pseudo-first-order rate constant k_(PCrATP) was calculated according to the formula (36)

where T₁(PCr) is the spin-lattice relaxation time of PCr, M_con(PCr) is the steady-state magnetization of PCr in the contralateral saturation experiment, M_sat(PCr) is the steady-state magnetization of PCr in the -ATP saturation experiment, M₀(PCr) and M₀(-ATP) are the equilibrium magnetizations, and M_res(-ATP) is the residual magnetization in the -ATP saturation experiment. The contralateral saturation resulted in an average attenuation of the PCr resonance by 14%. The product [PCr] k_(PCrATP), which is the phosphorus flux through the CK reaction, was calculated assuming muscle ATP concentration of 8.2 mM (3). This concentration was taken to be unaffected by exercise and to be equal in the young and older subjects (30).

All data were processed with NMR1 software (Tripos, St. Louis, MO). The free induction decays were corrected for DC offset, and a line broadening of 8 Hz was applied. After Fourier transformation and phasing of the spectra, manual baseline correction was performed by use of a polynomial fit. The resulting resonance lines were fit to a Lorentzian shape and integrated.

The intracellular pH was calculated from the relative chemical shift of the P_i signal with the formula pH = 6.75 + log [(  3.27)/(5.69  )], where  is the chemical shift difference (in ppm) between the P_i and the PCr signals (32).

Free ADP concentration ([ADP]) was calculated from the CK equilibrium expression
The apparent equilibrium constant K'_CK for 0.25 mol l¹ ionic strength, 1 mM [Mg²⁺], and temperature of 38°C was adjusted for pH (12). The total creatine concentration was assumed to be 42.5 mM (13).

Relative ATP concentration ATP/P_total was calculated as a ratio of -ATP signal intensity and the total phosphorus signal intensity in the fully relaxed spectra.

Exercise protocol. Experiments were conducted on two consecutive days and were performed at rest and during two levels of exercise. On the first day, experiments at rest and with intermittent exercise at 20% MVC were performed; the experiment at 40% MVC was performed on the next day. The MVC was determined before the study, with the subject's arm extended in the magnet bore, and the highest value of three trials was taken. During exercise, subjects squeezed a hand dynamometer with their ring and little fingers every 3 s for 1 s with prompting by an acoustic signal. The subjects were also provided a display indicating the actual intensity of exercise to assist them in maintaining constant exercise intensity throughout the experiment. In the experiments performed during exercise, data acquisition was started 5 min after the initiation of the exercise to allow for establishment of steady-state metabolite levels. The total experimental time, including adjustments for field homogeneity and determination of power of the saturation pulse, did not exceed 30 min.

Statistical analysis. Data are presented as means ± SD. The data were analyzed by repeated-measures analysis using the mixed-effects modeling procedure (PROC MIXED; SAS, Cary, NC). The variables {T₁(PCr), PCr/ATP, P_i/PCr, k_(PCrATP), free [ADP], pH, and forward phosphorus flux through the CK reaction} were expressed as a function of age group, exercise level (rest, 20% exercise, and 40% exercise), gender, and interaction terms (exercise level × gender, age group × gender, age group × exercise level, and age group × gender × exercise level). The Fisher method (31) was applied for post hoc analysis. The correlations of k_(PCrATP), forward phosphorus flux through the CK reaction, free [ADP], PCr/ATP, P_i/PCr, and pH were examined with linear regression analysis. Statistical significance in all tests was taken as P < 0.05.

	RESULTS

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Representative ³¹P MR spectra of the FDP at rest, 20% MVC, and 40% MVC are shown in Fig. 1, A-C. The data summary is presented in Table 1.

View this table: [in this window] [in a new window]   Table 1.   Summary of data

Metabolite concentrations and pH. No significant effects of age or gender on P_i/PCr, PCr/ATP, ATP/P_total, free [ADP], or pH were detected.

We found a significant effect of exercise on PCr/ATP, P_i/PCr, and pH (all P < 0.01; mixed effects model). Data at 20% MVC exercise were obtained in 9 young and 12 older subjects. All subjects maintained a constant level of force within 10%. Post hoc analysis revealed a significant increase in P_i/PCr (P < 0.01 and P < 0.05 in the young and older groups, respectively) at 20% MVC exercise compared with values measured at rest. We also observed a small decrease in the average PCr/ATP and intracellular pH in both groups, but the difference between the values obtained at rest and during 20% MVC exercise did not reach statistical significance. Data at 40% MVC exercise were obtained in 8 young and 11 older subjects. Two subjects in the young group and five subjects in the older group experienced significant fatigue during the exercise; their relative force levels decreased below 30% MVC. However, because no differences in average measured variables {PCr/ATP, P_i/PCr, ATP/P_total, free [ADP], pH, k_(PCrATP), forward CK phosphorus flux} were found between the subjects experiencing fatigue and those not experiencing fatigue in both young and older groups, the data on all subjects in both groups were averaged together. In both groups, exercise at 40% MVC resulted in a significant decrease in intracellular pH, compared with the values obtained at rest (P < 0.05) and at 20% MVC (P < 0.05) (Table 1). P_i/PCr was significantly higher in both groups compared with resting values (P < 0.01 in both groups). The difference in P_i/PCr between 40% MVC exercise and 20% MVC exercise also reached statistical significance in the older group (P < 0.05). A further nonsignificant decline in the average value of PCr/ATP compared with the exercise at 20% MVC was found in both age groups.

No significant effect of exercise level on free [ADP] was found. Similarly, ATP/P_total did not change between rest and exercise (Table 1).

CK reaction rates and phosphorus fluxes through the CK reaction. Statistical analysis of k_(PCrATP) and the phosphorus flux revealed a significant interaction between age and gender (both P < 0.01), indicating that the gender effect on k_(PCrATP) and the phosphorus flux was different in the young and older groups. Post hoc analyses showed that young men had a lower overall average [calculated from all data measured at rest and exercise; k_(PCrATP) = 0.07 ± 0.04 s¹; phosphorus flux = 1.85 ± 0.95 mM s¹] than older men [k_(PCrATP) = 0.11 ± 0.06 s¹; phosphorus flux = 2.86 ± 1.62 mM s¹; both P < 0.05]. No differences between young and older female subjects were detected.

Exercise had a significant effect on both reaction rate k_(PCrATP) and phosphorus flux through the CK reaction (both P < 0.01; mixed effects model). At 20% MVC, the reaction rate k_(PCrATP) increased in both age groups compared with the average value at rest (P < 0.05). This increase in k_(PCrATP) resulted in a 50% higher average forward phosphorus flux in the young group (P < 0.10) and a 79.8% higher average forward phosphorus flux in the older group (P < 0.05). At 40% MVC exercise, the average k_(PCrATP) was unchanged in the older group and somewhat lower in the young group [not significant (NS)] compared with exercise at 20% MVC. We also observed a decrease in the average forward CK phosphorus flux at 40% MVC compared with 20% MVC (NS).

The linear regression analysis showed a significant dependence of k_(PCrATP) on PCr/ATP (slope 0.026 s¹; P < 0.01), P_i/PCr (slope 0.178 s¹; P < 0.001), and pH (slope 0.13 s¹; P < 0.01). We also found a linear relationship between phosphorus flux and free [ADP] (slope 20.4 s¹; P < 0.01).

We also note that large standard deviations in metabolite concentrations, reaction rates, and CK phosphorus fluxes found in our study (Table 1) may be due to an interindividual variability in measures of intracellular bioenergetics (23).

T₁(PCr). Throughout the experiment, we did not observe any significant effect of age and exercise on T₁(PCr). The statistical analysis of T₁(PCr) revealed a significant effect of gender (P < 0.05). The overall average (calculated from all data measured at rest and exercise) in men (T₁ = 6.50 ± 1.11 s) was higher than in women (T₁ = 5.81 ± 1.52 s; P < 0.05).

	DISCUSSION

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Effect of age on muscle metabolism as detected by ³¹P MR spectroscopy. The decline in muscular strength and performance with age has been well documented (8, 26). In vivo ³¹P MR spectroscopy, which permits the noninvasive detection of high-energy phosphates and intracellular pH, has been used extensively to examine muscle bioenergetics in many settings (7). However, the results of ³¹P MRS studies on aged human muscle, performed both at rest and at exercise, have been contradictory. One early study comparing the metabolism of forearm muscle at rest and during exercise and recovery found no difference between elderly and young subjects (37), in agreement with our previous results (20, 39). However, subsequent investigations in which measurements were made on leg musculature found that older subjects had a lower resting PCr/P_i (28, 33) and PCr/ATP (38) than did younger subjects, indicating a lower phosphorylation potential. After exercise, a lower PCr recovery rate in the elderly was detected in one investigation in which the workload was not individually adjusted for each subject (28). A later study reported no differences in PCr recovery between young and older subjects when the workload was individually adjusted for lean body mass (38). Similarly, no age-related effect on PCr recovery after progressive exercise was found in a recent study on PCr kinetics in young and older subjects (4). In related work, a lower intracellular pH threshold relative to peak work rate was found in older subjects; however, the study did not fully account for differences in muscle strength among the subjects (5). Differences in fiber type composition between young and older subjects might account for some of the observed differences among the published studies. Direct fiber measurements have demonstrated no significant difference in fiber type distribution in the gastrocnemius muscle with age (6, 19). In contrast, lower mitochondrial enzyme activities in aged muscle found in the same studies (6, 19) suggest a decrease with age in oxidative metabolic capacity. Because the fiber type composition of the forearm flexors and the gastrocnemius muscle is similar (22), the discrepancy between our results (present study, 20, 39) and other work (28, 33, 38) is likely to be due to other age-related changes, such as deconditioning, which may be of greater significance in the lower than in the upper extremity.

The current study differs from previous work in many methodological respects. Rather than performing the same absolute amount of work, all subjects, carefully screened healthy volunteers, performed at the same workload relative to their individual maximum. Thus we attempted to obtain results that reflect intrinsic metabolism rather than extrinsic variables such as muscle mass and absolute strength. Furthermore, in the current study, we measured CK reaction kinetics rather than steady-state metabolite concentrations. Measurement of the CK reaction rate provides a more sensitive test for monitoring muscle high-energy phosphate metabolism than simple measurement of relative metabolite concentrations, as typically performed with MRS.

CK reaction rate and phosphorus flux at rest and exercise. In both the young and older groups, the average value of the pseudo-first-order reaction rate k_(PCrATP) measured at rest was 0.07 s¹. Using the same methodology, we previously reported k_(PCrATP) = 0.2 s¹ in rat skeletal muscle at rest (17), in agreement with literature values (15, 34). The lower value of the CK reaction rate in human skeletal muscle found in our study is consistent with the nearly twofold smaller CK catalytic activity compared with rat skeletal muscle (25). The comparable but somewhat larger k_(PCrATP) value of 0.2 s¹ found in previous studies of CK reaction kinetics in young healthy volunteers may be attributable in part to differences in study populations.

Aside from these potential physiological differences, discrepancies between this work and previous studies may result from the different experimental methodology employed. In contrast to an earlier study of the forearm (32), we carefully accounted for incomplete saturation in saturation transfer experiments. As previously shown (36), even apparently small degrees of incomplete saturation may result in large errors in reaction rate measurements. In addition, we determined T₁(PCr) using a rapid and reliable method that does not require selective saturation of a resonance. This allowed us to determine T₁(PCr), and thus the CK reaction rate, in each individual, both at rest and at each of the two exercise levels, without making the assumption of equal T₁(PCr) among individuals and between rest and exercise (32). Although one study on human calf (13) attempted to use progressive saturation to determine T₁(PCr), the effect of chemical exchange between PCr and -ATP was not accounted for. We have previously shown that this can lead to a large underestimate of T₁(PCr) (16). Indeed, T₁(PCr) was found to be 50% lower in the resting muscle in this earlier work (13) compared with our current results, in which the effect of chemical exchange between PCr and -ATP has been accounted for. An underestimate of T₁(PCr) results directly in an overestimate of the CK reaction rate, consistent with the differences found between previous work (13) and the present study. The differences in measured T₁(PCr) may be accounted for by different magnetic fields used, although magnetic field differences do not result in changes in reaction rates.

Exercise at 20% MVC resulted in an increase in the k_(PCrATP) in both subject groups (P < 0.05) and an increase in the forward phosphorus flux (P < 0.05 in the older group, P < 0.10 in the young group). In comparison with exercise at 20% MVC, we found a decrease (NS) in the forward CK flux at the 40% MVC exercise level in both groups. A similar decrease in forward CK flux was also observed in previous studies of human forearm and gastrocnemius muscles (13, 32). In contrast to previous studies (13, 32), in our current study the pH at the highest exercise level was significantly lower in both groups compared with the resting state, reflecting the high intensity of the exercise protocol that we used. The present results of a decreased phosphorus flux through the CK reaction at the highest workload are also consistent with those in stimulated rat muscle (24, 34). In all cases, the results argue against functional increase in CK flux in response to increasing workload, as has been reported in perfused hearts (2, 14). There are a number of possible explanations, as outlined previously (13). Modeling studies demonstrate that a decrease in [ADP], consistent with the trend we observed in the older group with increasing exercise load, may result in decreased CK flux (13). Indeed, we found a statistically significant decrease in flux with increasing [ADP]. Local alterations in [ADP] may result directly from intracellular heterogeneity; however, this possibility cannot be directly assessed by our measurements. Direct loss of enzymatic activity could also result from membrane damage as a result of the strenuous exercise protocol undertaken; this would be reflected in the decreased CK flux at high workload. A further possibility is the accumulation of inhibiting metabolites, such as P_i and protons (41). In addition, the observed flux pattern may reflect the effect of anion complexes. For further discussion, see the study by Goudemant et al. (13).

In conclusion, CK reaction flux did not vary monotonically with workload in the forearm muscle of healthy young and older subjects. No differences in this variation were found between young and older subjects, despite the application of physiological stress sufficient to reduce intracellular pH and PCr/ATP and thus probe metabolic reserve. Our results thus suggest that aging does not significantly affect the metabolic processes associated with the CK reaction.

	ACKNOWLEDGEMENTS

We would like to thank Denis Muller for help with the statistical analyses.

	FOOTNOTES

Address for reprint requests and other correspondence: A. Horská, Johns Hopkins University, Dept. of Radiology, 217 Traylor Bldg., 720 Rutland Ave., Baltimore, MD 21205 (E-mail: ahorska{at}mri.jhu.edu), or R. G. S. Spencer, National Institutes of Health/National Institute on Aging, GRC 4D-08, 5600 Nathan Shock Drive, Baltimore, MD 21224 (E-mail: spencer{at}helix.nih.gov).

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. §1734 solely to indicate this fact.

Received 12 October 1999; accepted in final form 8 March 2000.

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