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
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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 (Pi)
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 s
1 in the young and 0.07 ± 0.03 s
1 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(PCr
ATP), 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
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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
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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.
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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).
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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, Pi/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(PCr
ATP), forward
phosphorus flux through the CK reaction, free [ADP], PCr/ATP,
Pi/PCr, and pH were examined with linear regression
analysis. Statistical significance in all tests was taken as
P < 0.05.
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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.
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Metabolite concentrations and pH. No significant effects of age or gender on Pi/PCr, PCr/ATP, ATP/Ptotal, free [ADP], or pH were detected.
We found a significant effect of exercise on PCr/ATP, Pi/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 Pi/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, Pi/PCr, ATP/Ptotal, free [ADP], pH, k(PCrCK 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(PCr
ATP) 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(PCr
ATP) = 0.07 ± 0.04 s
1; phosphorus flux = 1.85 ± 0.95 mM s
1] than older men
[k(PCr
ATP) = 0.11 ± 0.06 s
1; phosphorus flux = 2.86 ± 1.62 mM
s
1; both P < 0.05]. No differences
between young and older female subjects were detected.
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).
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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/Pi (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 s
1. Using the same
methodology, we previously reported
k(PCr
ATP) = 0.2 s
1 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(PCr
ATP) value of 0.2 s
1 found
in previous studies of CK reaction kinetics in young healthy volunteers
may be attributable in part to differences in study populations.
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ACKNOWLEDGEMENTS |
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We would like to thank Denis Muller for help with the statistical analyses.
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FOOTNOTES |
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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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