1 Nutrition, Exercise Physiology, and Sarcopenia Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston 02111; and 2 Department of Physical Medicine and Rehabilitation, Harvard Medical School and Spaulding Rehabilitation Hospital, Boston, Massachusetts 02114
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ABSTRACT |
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Whole muscle strength and cross-sectional area (WMCSA), and contractile properties of chemically skinned segments from single fibers of the quadriceps were studied in 7 young men (YM, 36.5 ± 3.0 yr), 12 older men (OM, 74.4 ± 5.9 yr), and 12 older women (OW, 72.1 ± 4.3 yr). WMCSA was smaller in OM compared with YM (56.1 ± 10.1 vs. 79.7 ± 13.1 cm2; P = 0.031) and in OW (44.9 ± 7.5; P < 0.003) compared with OM. Age-related, but not sex-related, differences in strength were eliminated after adjusting for WMCSA. Maximal force was measured in 552 type I and 230 type IIA fibers. Fibers from YM (type I = 725 ± 221; type IIA = 792 ± 271 µN) were stronger (P < 0.001) than fibers from OM (I = 505 ± 179; IIA = 577 ± 262 µN) even after correcting for size. Type IIA fibers were stronger (P < 0.005) than type I fibers in YM and OM but not in OW (I = 472 ± 154; IIA = 422 ± 97 µN). Sex-related differences in type I and IIA fibers were dependent on fiber size. In conclusion, differences in WMCSA explain age-related differences in strength. An intrinsic defect in contractile proteins could explain weakness in single fibers from OM. Sex-related differences exist at the whole muscle and single fiber levels.
sarcopenia; chemically skinned segments of single fibers; aging; specific force
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INTRODUCTION |
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THE CAPACITY OF SKELETAL MUSCLE to generate force declines with age (17). A loss of muscle mass can explain part of this decline, but it is possible that alterations in muscle quality also occur (26). The extent and nature of qualitative changes in muscle and their contribution to sarcopenia and muscle dysfunction in old age are poorly understood. Since impairment of skeletal muscle function leads to disability and loss of independence (36), it is important to understand the basic cellular mechanism underlying muscle dysfunction in the elderly. This knowledge is essential to optimize rehabilitation and preventive strategies for this population.
Whole muscle quality has been estimated by normalizing strength to muscle cross-sectional area (specific force, SF). In humans, some reports show a significant reduction in SF with age, whereas other reports show no change (17, 26; for review see Ref. 5). Significant methodological limitations in the estimates of muscle mass or size in humans may explain these conflicting results. For example, imaging techniques produce measurements of whole muscle cross-sectional areas (WMCSA), but in pennate muscles (like m. quadriceps femoris), where muscle fibers attach to tendons at an angle, WMCSA underestimates the amount of muscle generating force; i.e., the physiological cross-sectional area (PCSA). Even improved measurements of PCSA suggest that factors other than WMCSA account for part of the force produced by a muscle (12).
The ability of skeletal muscle to generate force and produce movement may be compromised by an incomplete activation of motor units (central motor drive), peripheral nerve dysfunction, loss of hormonal influences, changes in the excitation-contraction coupling mechanisms, or by alterations in the contractile elements of muscle cells. Since elderly men and women can fully activate their muscles (34, 38) and show minimal changes in nerve conduction velocity (7), it appears likely that one site of possible qualitative alterations is in the contractile elements of the muscle cell. The skinned muscle fiber preparation allows the direct investigation of the function of muscle proteins in segments of a cell with an intact myofilament lattice, but without the confounding effects of neural influences, the variations in fiber architecture and the spatial orientation of fibers, the effects of intercellular connective tissue, or of the protein heterogeneity between cells found in multicellular preparations (22, 46). Finally, it has been suggested that changes in the levels of hormones such as estrogen and its effects on the number and function of cross-bridges could result in different rates of loss of strength between the sexes (35, 44). Direct measurements of muscle fiber quality could be used to test this hypothesis.
The purpose of the present investigation was to compare the SF of m. vastus lateralis in vivo and the SF of segments of single muscle fibers from young and old men and old women. Our hypotheses were: 1) there is a difference in muscle strength and WMCSA between young and old men but no difference in SF at the whole muscle level; 2) compared with fiber segments from younger subjects expressing the same myosin heavy chain (MyHC) isoform, type I and IIA fibers from older subjects show lower SF; and 3) there are no sex-related differences in SF in whole muscle, type I, or IIA single fiber segments.
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MATERIALS AND METHODS |
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Subjects.
A total of 31 healthy subjects not involved in strength training
volunteered for the study. The general characteristics of the subjects
are presented in Table 1. The volunteers
received a complete explanation of the purposes and procedures and gave their written consent. A comprehensive medical evaluation including a
medical history, physical examination, and routine blood and urine
tests was performed prior to their participation in the study. A
resting electrocardiogram was also done in older volunteers. The number
of self-reported diagnoses was 1.3 ± 1.2 in women (2 reported no
diagnoses) and 1.8 ± 1.0 in men (2 reported no diagnoses). The
number of medications taken by the subjects was 0.9 ± 1.4 in
women (6 were not taking medications) and 1.5 ± 1.0 in men (2 were not taking medications). Subjects with conditions that could
interfere with neuromuscular function were excluded.
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Muscle strength measurements. An isokinetic dynamometer (Cybex II, Medway, MA) was used to measure strength (Newton-meters) of the left knee extensors at 1.04 rad/s as previously reported (11). The subjects performed five maximal voluntary contractions, and the highest or peak torque was recorded.
Computerized tomography. A computerized tomography (CT) scan of the thigh was performed between 38 and 62% (mean ± SD = 50 ± 4%) of the length of the femur measured from the distal end of the bone. The scan was obtained using a GE Highspeed Advantage CT scanner (Milwaukee, WI) operating at 100 kV and 170 mA. A slice width of 10 mm and a scanning time of 1 s were used. From the CT image, the WMCSA occupied by m. quadriceps femoris was measured. The image was analyzed according to optical density on a computer (Macintosh Power PC 8500; Apple Computer, Cupertino, CA) by a single investigator in a blinded fashion with IMAGE software (developed at the National Institutes of Health and available on the Internet) modified by G. Solares of our group for quantification of cross-sectional areas of fat, muscle, and bone to the nearest 0.1 cm2 (32). The coefficient of variation (CV) for repeated measurements of muscle area in a single scan by the same observer was <0.25%. The CV for measurements done 3 wk apart in two subjects was <0.8%.
Muscle biopsies and permeabilization of fibers.
Biopsy specimens were taken from m. vastus lateralis under local
anesthesia using biopsy needles (4) and suction
(8). The specimens were placed in relaxing solution (see
below) at 4°C. Bundles of ~30 fiber segments were dissected free
from the samples and then tied with surgical silk to glass capillary
tubes at slightly stretched lengths. The fiber segments were chemically skinned for 24 h in relaxing solution containing 50% (vol/vol) glycerol at 4°C and were subsequently stored at 20°C for up to 4 wk before use.
Experimental procedure. A detailed explanation of the general methods used in this study has been published by others (22, 30). Briefly, on the day of an experiment, fiber segments were placed for 30 min in relaxing solution (see below) containing 0.5% Brij-58 (polyoxyethylene 20 cetyl ether; Sigma Chemical) prior to mounting in an experimental apparatus, similar to the one described previously by Moss (30). A fiber segment length of 1 to 2 mm was left exposed to the solution between connectors leading to a force transducer (model 400A, Cambridge Technology) and a lever arm system (model 308B, Cambridge Technology). The apparatus was mounted on the stage of an inverted microscope (model IX70; Olympus, Tokyo, Japan). While the fiber segments were in relaxing solution, sarcomere length (SL) was set to 2.75-2.85 µm by adjusting the overall segment length. The segments were observed through the microscope at a magnification of ×320.
The sarcomere length, the segment diameter, and the length of segment between the connectors were measured with an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). Fiber depth was measured by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fiber. The focusing control of the microscope was used as a micrometer. Fiber cross-sectional area (FCSA) was calculated from the diameter and depth, assuming an elliptical circumference, and was corrected for the 20% swelling that is known to occur during skinning (14, 30). Maximum force (Po) was adjusted for FCSA. Relaxing and activating solutions contained (in mM) 4 Mg-ATP, 1 free Mg2+, 20 imidazole, 7 EGTA, 14.5 creatine phosphate, and KCl to adjust the ionic strength to 180 mM. The pH was adjusted to 7.0. The concentrations of free Ca2+ were 10MyHC composition.
After mechanical measurements, each fiber was placed in SDS sample
buffer in a plastic microcentrifuge tube and stored at 20°C for up
to 1 wk or at
80°C if the gels were to be run later. The MyHC
composition of single fibers was determined by SDS-PAGE (21). The acrylamide concentration was 4% (wt/vol) in the
stacking gel and 6% in the running gel, and the gel matrix included
30% glycerol. Sample loads were kept small (equivalent to ~0.05 mm of fiber segment) to improve the resolution of the MyHC bands (types I,
IIA, and IIB). Proteins were identified using a combination of human
myosins from vastus lateralis muscles and from reports in the
literature (see Ref. 22).
Statistical analysis. Multiple regression analysis was used to test all three hypotheses. For the first hypothesis, a regression using only males was used to test for age differences, whereas a regression using only older subjects, but including both sexes, was used to test for differences between men and women.
The second and third hypotheses were tested using a regression with standard errors corrected for the lack of independence (clustering effect) caused by using multiple fibers from the same person. In Tables 4 and 5 the corrected standard errors are referred to as "robust standard errors." This approach allowed both the inclusion of standard covariates [age, gender, fiber type (I and IIA), fiber size] and the inclusion of the interaction terms to correctly model a ratio (SF). The use of ratios in correlation and regression analysis in medicine and physiology has been discussed by Tanner in 1949 (41) and Kronmal in 1993 (20). The residuals from all regressions were examined for possible violations of underlying assumptions. The CV for three measurements done by the same observer in four fibers was 0.5% for diameter and 3.7% for depth. We thought that the error in the depth measurement could increase the variability in CSA. Therefore, a sensitivity analysis was used to determine whether the amount of error in the measure might affect any results. For each of the two regressions, four sets of 10,000 new sets of depth measurements and CSA were simulated; for each Monte Carlo simulation, the regression was re-estimated (thus 80,000 regressions were estimated). The new depth measures were simulated from a uniform distribution (first, ±10% error; and second, ±15% error), from a normal distribution, and from a log-normal distribution. The results of all simulations showed that the error in the measurement of depth did not alter the results. The relationship between the SF at the whole muscle and single fiber levels was examined using a simplified multilevel analysis. We calculated the mean SF across individual fibers for each subject in the study and correlated this with the whole muscle SF. Weighted correlations for number of fibers per person and for SD of the SF among fibers for each person were tried with the same results. Due to the nonlinear nature of the association, we also calculated Kendall's tau rank correlation coefficient. Sample size was determined using the PinT software for two level (number of subjects and fibers) analysis (40). At a power of about 0.80, a sample size of 24 older persons and 20 fibers per person were needed to detect a significant difference. Analyses were performed using Stata, version 5 (Stata, College Station, TX), and Systat Mac version 5.2.1. ![]() |
RESULTS |
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In vivo measurements of muscle function and size.
The results of the multiple regression analysis of isokinetic peak
torque and WMCSA are presented in Table
2. There was no interaction between age
or sex and WMCSA. Older men (56.1 ± 10.1 cm2) showed
smaller WMCSA compared with younger men (79.7 ± 13.1 cm2) and larger WMCSA compared with older women (44.9 ± 7.5 cm2). Significant differences in strength between
young (209 ± 50 Nm) and older (130 ± 32 Nm) men were
eliminated after adjusting for WMCSA. On the other hand, differences in
strength between older men and women (86 ± 14 Nm) persisted even
after adjusting for WMCSA.
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Contractile properties of single fibers.
The results for the maximal force (Po) and fiber diameter, depth, CSA,
and SF of type I and IIA single muscle fibers are presented in Table
3. A total of 552 type I and 230 type IIA
fibers were studied (an average per subject of 17 and 8 type I and IIA
fibers, respectively).
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Age comparisons.
The results of the multiple regression analysis comparing single fiber
maximal force in young and old males are presented in Table
4. Fibers (I and IIA) from older men had
lower maximal force compared with fibers from younger men. The
differences in force between young and older men were significant
even after adjusting for differences in fiber size (diameter, depth).
There was no interaction between age and fiber type. Therefore,
in both age groups, type IIA fibers were significantly stronger than
type I fibers.
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Sex comparisons.
The results of the multiple regression analysis comparing single fiber
maximal force in older men and women are presented in Table
5. The relationship between FCSA (in
percentiles) and maximal force for both fiber types in older men and
women is presented in Fig. 2.
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Whole muscle and single fiber relation.
The association between the whole muscle and single fiber SF
for all subjects is shown in Fig. 3. The
unweighted correlation between the two variables was significant
(r = 0.37; P = 0.042). Kendall's tau
rank correlation coefficient (used because of the nonlinear
nature of the association) was 0.25 (P = 0.054).
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DISCUSSION |
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To our knowledge, this is the first study to investigate the relationship between strength and size at the whole muscle and single fiber levels in the same cohort of young men and older men and women. Our main results were: 1) strength and WMCSA of the knee extensors were significantly higher in younger men compared with older subjects and in older men compared with older women; 2) the age-related, but not the sex-related, differences in whole muscle strength were eliminated after adjusting for WMCSA; 3) fibers from younger men were stronger than fibers from older men expressing the same MyHC isoform even after adjusting for size; 4) in general, type I and IIA fibers from older men were stronger than similar fibers from older women even after adjusting for size; and 5) in men, but not in women, type IIA fibers were stronger than type I fibers.
Whole muscle. Many studies have evaluated the association between age and changes in whole muscle SF or quality (force/size) with variable results (see Refs. 5 and 27). In the present study, adjusting for WMCSA eliminated differences in strength between young and older men. This is similar to our previous report (11), in which differences in isokinetic strength of the knee extensors tested at 1.04 rad/s among men and women (age range = 45-78 yr) were significantly reduced or eliminated after adjusting for whole body muscle mass estimated using urinary creatinine. Häkkinen et al. (15) and Kent-Braun and Ng (19) also reported no age-related differences in isometric strength of the knee extensors and ankle dorsiflexors, respectively, after adjusting for muscle CSA.
Other studies have reported significantly higher isokinetic (26, 33) and isometric (48) strength-to-WMCSA ratios in younger men compared with older men. Lynch et al. (26) recently reported a reduction in muscle quality with age in both men and women (range 19-93 yr). However, they estimated muscle mass from appendicular fat-free mass determined by dual-energy X-ray absorptiometry and measured isokinetic strength at an angular velocity of 0.52 rads/s. Metter et al. (27) estimated muscle quality in cross-sectional and longitudinal analyses of men and women (range 18-93 yr) and concluded that the relationship between muscle quality and age is dependent, among other factors, on how muscle mass is estimated. For example, they reported no significant age effect in strength expressed per kilogram of creatinine but a linear decline when expressed per CSA. In the present study we also report sex-related differences in isokinetic muscle strength that persisted after adjusting for size. Our data are similar to that reported by Häkkinen et al. (15) who tested isometric strength in 70-yr-old men and women. However, Miller et al. (29) found no differences in the isometric strength-to-WMCSA ratio between younger men and women (mean age = 23-25 yr). Discrepancies among studies could be explained by differences in: 1) the architecture of the muscle groups tested; 2) techniques used to estimate muscle CSA, not all of which are precise in vivo; 3) measurements of strength (static vs. dynamic) (33); and 4) statistical techniques (ratio vs. allometric) to assess the relationship between muscle strength and size (31). Several experimental interventions have been shown to alter whole muscle quality, including compensatory hypertrophy after tenotomy (18), bed rest (23), hind limb suspension (13), and strength training (10). These studies suggest that the relationship between strength and size is not constant. The fact that in several of the experimental models the measurements are made after isolating the muscle from neural influences suggests that the intrinsic properties of muscle fibers may be affected. To test this hypothesis, we studied permeabilized single muscle fibers.Age comparisons at the single fiber level. Our single fiber SF values (Table 3) for younger men of 20.8 (type I) and 23.8 N/cm2 (type IIA) are in the same range as those published by others. For type I fibers, published values for SF measured under similar conditions are 19 (24), 21 (22), and 20.5 N/cm2 (16). For type IIA fibers, published values are 25 (24), 20 (22), and 22.3 N/cm2 (16).
In the present study we found significantly lower maximal force in both type I and type IIA fibers from older men compared with similar fibers from younger men. These differences were significant even after adjusting for fiber size and confirm previous observations by Larsson et al. (24). That study, however, was limited by a small sample size (4 young and 4 older men) and the absence of WMCSA comparisons. The results of the present study suggest that the intrinsic ability (quality or SF) of muscle fibers to generate force is lower in older subjects. Conceptually, the events leading to the generation of force could be classified into three domains: 1) excitation-contraction coupling, 2) fuel metabolism and energy supply, and 3) cross-bridge formation and myofilament sliding. It is possible that with advanced adult age, one or more of these domains is altered, resulting in muscle weakness and disability. The elderly maintain the capacity to fully activate all motor units (34), and mean motor unit firing rates at given force levels are similar in young and older men (38). However, the effectiveness of the neural impulse in generating force may be limited by the excitation-contraction events in the muscle cell. This hypothesis is supported by the work of Delbono et al. (6) showing that aging interferes with the linking of the dihydropyridine receptors in the transverse tubules and the ryanodine receptors in the sarcoplasmic reticulum, resulting in uncoupling of calcium release and reduced availability of calcium to initiate contraction. In the present study it is unlikely that the above alterations could explain the differences between young and older men, since the permeabilization of fibers and the use of Brij solution removes the sarcoplasmic reticulum. Furthermore, only concentrations of calcium known to produce maximal activation were used. Another important difference between our study and that of Delbono et al. (6) is that we found lower SF values in both type I and IIA fibers, whereas they reported changes that were limited to fast-twitch fibers. It appears that under the conditions of the present study, weakness resulted from alterations beyond the excitation-contraction coupling steps. Energy supply could potentially limit force generation. In this regard, Taylor et al. (42) used nuclear magnetic resonance to study muscle energetics in young and old volunteers. Under resting conditions, no differences were seen in intracellular pH or levels of ATP and phosphocreatine (PCr). Similar reductions in pH and PCr were seen during hand-grip exercise (7.5 min of fatiguing exercise), and resynthesis of ATP during recovery (12 min) was similar among age groups. The authors concluded that the aging process does not alter the energetics of human skeletal muscle. Furthermore, since energy supply is carefully controlled in the present experiments and the composition of the solutions standardized, alterations in this domain cannot explain the observed differences in force production. Abnormalities in the quantity and/or function of the contractile or regulatory proteins (including troponin and tropomyosin) could explain the reduction in the intrinsic capacity of muscle cells to generate force. In younger subjects (mean age = 28.3 yr), a 6-wk bed rest period resulted in a 40% reduction in single fiber SF with a decline in myofibrillar protein content (23). The mean fractional rate of myofibrillar protein synthesis is slower in older muscles, probably due to a slower elongation of the peptide chain (45). Also, an age-related decline of MyHC synthesis rate (but not of sarcoplasmic protein) has been observed (2). Since, in that study, the MyHC synthesis rate correlated with in vivo measurements of muscle strength, the reported decline could contribute to weakness in the elderly. Although there is no direct evidence that with age newly synthesized proteins are abnormal, a slow synthesis rate could delay the replacement of dysfunctional proteins. Thompson and Brown (43) reported in single fibers from rat soleus muscle a dissociation between the loss of FCSA and force with age and attributed this to changes in the molecular dynamics of myosin. Brooks and Faulkner (5) have concluded that in mammals the deficit in SF may result from a reduction in the number of cross-bridges or in the average force per cross-bridge. Alterations in the internal architecture of the cell could potentially limit force generation. For example, the amount of force generated by each cross-bridge may be reduced if the space between myofilaments increases. Evidence to support this hypothesis has been reported in single fibers from rat and frog muscle (1, 14, 28). Studies in single fibers from older humans testing this hypothesis are needed. Another possible explanation is a loss of thin myofilaments as reported in human soleus after bed rest (37). However, in the latter study, SF was only slightly decreased. The reported reduction in force with age after adjusting for size suggest that atrophy of muscle fibers in itself offers only a partial explanation for the muscle weakness of old age. Atrophy can result in the loss of muscle mass without appreciable changes in the physiological properties of the cells (39). Degeneration and cell death, on the other hand, involve changes in both structure and function. It remains to be seen whether the strength training that reverses muscle atrophy in the elderly (10) can also reverse the age-related alterations in the intrinsic quality of muscle fibers.Sex comparisons at the single fiber level. To our knowledge, this is the first report to compare single human muscle fibers from both sexes. Previous studies have not included women (24) or have combined the results from both sexes (22). Our results at the single fiber level demonstrated significant sex-related differences in maximal force that were dependant on fiber type and not explained by differences in fiber size.
Other authors have noted the influence of sex on the age-related changes in skeletal muscle. Young et al. reported that the age-related reduction in strength was proportional to the reduction in WMCSA in women (47) but not in men (48). These differences in younger subjects have been attributed to greater fiber area, greater fiber number, and/or higher voluntary motor unit activation in men (3). Since our experimental preparation isolated single fibers from the influence of the nervous system and we adjusted for the differences in size, those hypotheses cannot explain our observations and suggest that differences may exist in the contractile elements of the muscle cell. The fact that the sex-related differences were fiber type dependent supports this hypothesis. The reduction in estrogen levels after menopause could explain the different rates of loss of strength between sexes (35). This hypothesis is supported by the observation that 50-yr-old men and women have similar strength-to-WMCSA ratio but 70-yr-old women show lower values than 70-yr-old men (15). Balagopal and colleagues (2) observed that the correlation between muscle strength and MyHC synthesis rate is stronger in women and suggested that protein synthesis in sarcopenia could be regulated by different parameters in men and women. The mechanism by which estrogen may exert this influence remains to be determined, but it may be due to an influence on the number of cross-bridges or the force generated per cross-bridge (44) or the sensitivity of the cross-bridges to metabolites, such as hydrogen ions or inorganic phosphate, that reduce force (35). It is also possible that immunologic, neurological, or genetic influences play a role. Experiments with whole muscle and with single fibers represent different levels of study. Extrapolation of observations made in experiments with single fiber segments should be made with caution. The whole muscle condition represents a summation of various influences and intermediate steps eliminated in the single fiber preparation. It would be of scientific interest and practical significance to understand the nature of the association between the single fiber level and determinations of whole muscle function. Figure 3 illustrates the complex and nonlinear nature of the relationship between the two levels. We have initiated a more in-depth analysis, including random effects modeling. In conclusion, the present study demonstrates an age-related reduction in the intrinsic ability of single muscle fibers expressing type I and IIA MyHC isoforms to generate force. It is likely that alterations at the level of cross-bridge formation and interaction play a significant role. More research is needed to understand the basic mechanisms underlying muscle dysfunction in old age. ![]() |
ACKNOWLEDGEMENTS |
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This material is based upon work supported by the US Department of Agriculture, under agreement 58-1950-9-001 and contract 53-K06-1.
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FOOTNOTES |
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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture.
Address for reprint requests and other correspondence: W. R. Frontera, Dept. of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital, 125 Nashua St., Boston, MA 02114 (E-mail: frontera.walter{at}mgh.harvard.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. §1734 solely to indicate this fact.
Received 6 October 1999; accepted in final form 5 April 2000.
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