Context Sarcopenia is associated with loss of strength and function, eventually
leading to loss of independence. Some studies suggest that basal muscle protein
turnover is reduced with aging, but other studies do not confirm this finding.
Objective To determine if aging per se affects basal muscle protein turnover in
men.
Design and Setting Cross-sectional study conducted from June 1997 to July 2000 in a general
US community.
Participants Twenty-six young (mean [SE] age, 28 [2] years) and 22 older (mean [SE]
age, 70 [1] years) men, who were healthy and independent based on activities
of daily living, physical examinations, and screening tests. Subjects were
excluded if they had cardiac, pulmonary, liver, or kidney disease; any impairment
in activities of daily living; or steroid use.
Main Outcome Measures We measured basal muscle protein and amino acid kinetics, based on stable
isotope techniques with femoral arteriovenous catheterization and muscle biopsies.
Three models (arteriovenous balance, three-pool, and fractional synthesis
rate) were used to estimate the metabolic parameters.
Results Mean (SE) total leg volume was 9.60 (0.32) L in older men vs 10.83 (0.43)
L in younger men, which suggests muscle loss in the older men. Net muscle
protein balance was similar in both groups (older men, − 19 [2] nmol/min
per 100 mL of leg volume vs younger men, − 21 [2] nmol/min per 100 mL
of leg volume; P = .51). Small differences were found
in mean (SE) muscle protein synthesis in comparisons of older vs younger men:
arteriovenous balance, 48 (5) nmol/min per 100 mL of leg volume vs 32 (3)
nmol/min per 100 mL of leg volume; P = .004; three-pool,
58 (5) nmol/min per 100 mL of leg volume vs 43 (4) nmol/min per 100 mL of
leg volume; P = .04; and fractional synthesis rate,
0.0601 (0.0046) %/h vs 0.0578 (0.0047) %/h; P = .73.
Small differences were also found in mean (SE) muscle protein breakdown: arteriovenous
balance, 66 (5) nmol/min per 100 mL of leg volume in older vs 53 (4) nmol/min
per 100 mL of leg volume in younger men, P = .045;
and three-pool, 76 (6) nmol/min per 100 mL of leg volume vs 64 (5) nmol/min
per 100 mL of leg volume, P = .14.
Conclusion Differences in basal muscle protein turnover between older and younger
men do not appear to explain muscle loss that occurs with age.
Sarcopenia, the involuntary decrease in muscle mass with aging, is associated
with loss of strength and function, eventually leading to loss of independence.1-4 The reduction
in lean body mass also may contribute to the development of metabolic alterations,
such as diabetes mellitus and osteoporosis.1,5
The mechanisms leading to sarcopenia are still unclear. Numerous hypotheses
have been suggested, including DNA damage,6
reduced protein synthesis,7 fiber type changes,2 inactivity,8 inadequate
nutrition,9,10 and hormonal changes.11-14 A
combination of several factors likely is responsible for the age-related changes
in muscle mass and function.5 Overall, any
of the primary factors responsible for sarcopenia should, at some point, affect
muscle protein turnover by creating an imbalance between muscle protein synthesis
and breakdown, thus inducing skeletal muscle loss. Therefore, a better understanding
of the metabolic alterations characterizing sarcopenia may help to clarify
the etiological characteristics of this condition and to determine the efficacy
of potential treatments.
Initial studies on muscle protein metabolism have suggested that the
metabolic alteration responsible for sarcopenia is a reduction in basal muscle
protein synthesis rate in older people as compared with younger controls.15-19
However, more recent data did not confirm those results.20-22
A careful review of these studies allows the exclusion of methodological problems
as a reason for these discrepancies, since the methods used were similar and
already validated in a variety of conditions.
Thus, 3 other possible explanations need to be considered. First, protein
synthesis alone does not provide enough information about muscle loss or gain
because protein breakdown influences net muscle protein balance as well. Muscle
protein breakdown was directly measured only in the 2 studies from our group,20,22 whereas the other studies did not
measure breakdown16 or used indirect measures
of breakdown (3-methylhistidine or creatinine urinary excretion),15,17-19,21
which are less sensitive and less specific than the methods used to measure
protein synthesis, and they do not allow the estimation of muscle net protein
balance. Second, the older population is more heterogeneous than the younger
population, because the distinction between health and disease, activity and
inactivity, or appropriate and inappropriate nutrition becomes less clear,
thus increasing variability. Therefore, different investigators may have obtained
contrasting results because they studied slightly different segments of the
older population. Third, differences in the study design and insufficient
power may have contributed to the discrepancies between different studies.
To assess if age is associated with a reduction in basal net muscle
protein synthesis, we measured basal muscle protein turnover and amino acid
kinetics in healthy young and older men, and we applied 3 different models
to estimate muscle protein turnover to reduce a possible methodological bias.
The volunteers were carefully selected to exclude any detectable confounders
(eg, diseases, medications, exercise training) that might have affected measures
of muscle protein turnover, and they were encouraged to maintain their usual
lifestyle prior to the study to avoid the potential bias introduced by acute
changes in diet, physical activity level, or both.
Forty-eight young and older men were recruited from June 1997 to July
2000 through the Sealy Center on Aging of the University of Texas Medical
Branch (UTMB), Galveston, Tex (Table 1).
There were 26 men in the young group with a mean (SE) age of 28 (2) years,
and 22 men in the older group with a mean age of 70 (1) years. Women were
not included because of difficulties in recruiting an adequate number representative
of the population makeup ( ˜ 50% per sex). After explaining the purpose
and risks of the study, which was approved by the institutional review board
of UTMB, written consent was obtained from each volunteer. Four young and
4 older subjects had participated in a previous study.22
Volunteers were considered to be eligible if they were found to be healthy
on the basis of clinical history, including a standard questionnaire on the
activities of daily living (ADLs) in use at UTMB, physical examination, and
screening tests, including complete blood count, blood chemistry workup, hepatitis
panel, human immunodeficiency virus test, urinalysis, blood pressure, oral
glucose tolerance test if necessary,23 and
electrocardiogram.
Exclusion criteria were the following: cardiac, pulmonary, liver, or
kidney disease; peripheral vascular disease; cerbrovascular disease; recurrent
deep venous thrombosis; acute, subacute, or chronic infection; active cancer;
autoimmune disease; metabolic disease; diabetes mellitus and glucose intolerance
as defined by the American Diabetes Association23;
other endocrinopathies; anemia; hypertension treated with calcium channel
blockers, angiotensin-converting enzyme inhibitors, β-blockers, or angiotensin
receptor blockers; drug or alcohol abuse; coagulation defects; strength or
aerobic training; impairment in the ADLs; history of falls; and anabolic steroid,
corticosteroid, or antiandrogen therapy. Subjects with a history of anabolic
steroid use or long-term corticosteroid therapy were excluded. Mild osteoarthritis
was not an exclusion criterion, provided that it did not impair the ADLs.
However, volunteers who could not discontinue analgesic or anti-inflammatory
therapy for the time necessary for a complete washout (eg, 5 days for aspirin)
were excluded. A careful clinical assessment of the amount of daily physical
activity was performed to exclude any subjects engaged in regular aerobic
or resistance exercise training, subjects whose jobs included daily heavy
weight lifting or long walks, subjects with a history of falls (≥2/y),
or subjects with any impairment in ADLs. We did not use a standardized questionnaire
to measure the level of physical activity because several studies have shown
that the individual variability is high, and the validity of these questionnaires
for the estimation of the daily total energy expenditure is limited.24,25
The volunteers performed their regular activities and maintained their
usual diet during the week preceding the study. This routine was preferred
to an early admission to the General Clinical Research Center (GCRC) and to
the administration of a standardized diet for the 3 to 5 days preceding the
study, as previously reported,15-19,21
because these manipulations might influence basal muscle protein turnover.
The night before the study each subject was admitted to the GCRC. After
10 PM the subject was allowed only water ad libitum. At 6 AM on the morning
of the study, a catheter was inserted in a forearm vein for phenylalanine
tracer infusion and another catheter was inserted in the opposite forearm
for blood sampling (Figure 1). Catheters
were inserted in the femoral artery and femoral vein of one leg for blood
sampling. The arterial catheter also was used for indocyanine green (ICG)
(IC-Green, Akorn Inc, Buffalo Grove, Ill) infusion. Leg volume was measured
using an anthropometric method.26 After the
insertion of the femoral arteriovenous (AV) catheters and the collection of
a blood sample for background phenylalanine enrichment and ICG concentration,
a primed-continuous infusion of L-[ring-2H5]-phenylalanine
(priming dose: 2 µmol/kg; infusion rate: 0.05 µmol · kg−1· min−1) was started at 6:30 AM (0 minutes)
and continued for 5 hours. The background sample also was used to measure
total testosterone concentration in 19 young and 21 older men.
At 120 minutes, a first muscle biopsy sample was taken from the lateral
aspect of the vastus lateralis of the leg with the femoral catheters, using
a 5-mm Bergström needle. The muscle tissue sample (50-150 mg) was quickly
blotted and frozen in liquid nitrogen and kept at –80°C until analysis.
At 230 minutes, the continuous infusion of ICG was started in the femoral
artery (0.5 mg/min) and carried out until 270 minutes. To measure ICG concentration,
4 blood samples were taken every 10 minutes from the femoral vein and the
forearm vein during the infusion. After stopping the ICG infusion, to measure
phenylalanine concentration and enrichment, 4 blood samples were taken every
10 minutes from the femoral artery and vein until 300 minutes. At 300 minutes,
before stopping the tracer infusion, a second muscle biopsy sample was taken
as described above.
Approximately 1 week after the study, leg muscle volume was measured
using magnetic resonance imaging (MRI) (GE Signa 1.5 T whole body imager,
General Electric, Milwaukee, Wis)20 in the
first 10 older and 7 young subjects participating in the study. The remaining
subjects could not undergo the procedure because the instrumentation was unavailable.
Muscle volume was calculated from the MRI scans as previously described.20 Indocyanine green concentration in infusate and serum
samples was measured spectrophotometrically at λ = 805 nm. Total testosterone
was measured using a commercial radioimmunoassay (Diagnostic Products Corporation,
Los Angeles, Calif). Phenylalanine concentrations and enrichments in blood
samples were measured using gas chromatography mass spectrometry (GCMS; Hewlett
Packard, Palo Alto, Calif).27
Muscle tissue from biopsy samples were processed as previously described.27 Free phenylalanine enrichment and concentration were
measured after extraction from the muscle tissue samples using GCMS.27 Protein-bound phenylalanine enrichment was analyzed
with GCMS after protein hydrolysis and amino acid extraction,27
using the external standard curve approach for very low enrichments.28,29
Muscle phenylalanine kinetic parameters were calculated using 2 different
approaches: the AV balance method30 and the
three-pool model recently described and validated.31
The AV balance method relies on the measurement of phenylalanine enrichments
and concentrations in the femoral artery and vein to estimate muscle protein
synthesis, breakdown, and net balance. These parameters are based on the extraction
of labeled phenylalanine (tracer) from the femoral artery, the rate of appearance
(Ra) of unlabeled phenylalanine (tracee) from the muscle in the femoral vein,
and the net AV difference in phenylalanine concentrations, respectively.30 The three-pool model is an expansion of the AV method
and relies not only on the measurement of phenylalanine enrichments and concentrations
in the femoral artery and vein but also on the direct measurement of free
phenylalanine enrichment in the tissue water. This method allows for the direct
calculation of phenylalanine intracellular utilization for protein synthesis
and release from protein breakdown as well as the calculation of the rates
of phenylalanine transport from the artery into the tissue and from the tissue
into the venous blood.31
Phenylalanine is used with both methods because it is an essential amino
acid, and it is not oxidized in the muscle tissue. Therefore, its utilization
in the muscle is an index of muscle protein synthesis, and its release from
the muscle is a measure of muscle protein breakdown.
The following 3 parameters are common to both the AV balance and the
three-pool model:
A
V
AV
CA and CV indicate plasma phenylalanine concentrations
in the femoral artery and vein, respectively; and BF, leg blood flow. Data
are presented per 100 mL of leg volume.30
The other kinetic parameters of the AV method were calculated as follows:
AAV
AAV
AAVV
EA and EV indicate phenylalanine enrichments (tracer/tracee
ratio) in the femoral artery and vein, respectively. Data are presented per
100 mL of leg volume.30 In these calculations
it is assumed that the phenylalanine enrichment in the vein is the closest
to that of the intracellular pool.
From the AV balance data, it is also possible to calculate the fractional
uptake of labeled phenylalanine (%), which is another indirect index of muscle
protein synthesis:
AAVVAA
The parameters specific to the three-pool model for labeled phenylalanine
were calculated as follows:
M,AMVAMVA
V,MMVAMVV
V,AM,A
M,0M,AAM
0,MM,0
EM indicates the phenylalanine enrichment (tracer/tracee
ratio) in the muscle. Data are presented per 100 mL of leg volume.31
Intracellular amino acid availability is given by the sum of transport
into the muscle (FM,A) and the muscle protein breakdown rate (FM,0). Thus, it is possible to calculate protein synthesis efficiency
as follows:
0,MM,AM,0
Leg plasma flow was calculated at dye dilution steady state, as previously
described.32,33 Leg blood flow
was calculated by correcting the plasma flow by the hematocrit.
We also determined the fractional synthesis rate (FSR) of mixed muscle
proteins by measuring the incorporation rate of L-[ring-2H5]-phenylalanine into the proteins and using the precursor-product model34 to calculate the synthesis rate as follows:
PM(1)M(2)
where ΔEP is the increment of protein-bound phenylalanine
enrichment between 2 sequential biopsy samples, t is the time interval between
the 2 sequential biopsy samples, and EM(1) and EM(2)
are the phenylalanine enrichments (tracer/tracee ratio) in the free muscle
pool in the 2 subsequent biopsy samples. The results are presented as percentage
per hour.
We also calculated whole body phenylalanine Ra, an index of whole body
protein breakdown, using the single-pool model35:
A
where i is the infusion rate of L-[ring-2H5]-phenylalanine.
Differences between young and older men were analyzed for each variable
using the 2-tailed 2-sample t test. Since the prior
studies reported contrasting results with regards to muscle protein synthesis,
some observing a reduction with age15-19
and others finding no age-related differences,20-22
we could not calculate a priori the least significant number of subjects to
be studied to reach a set significance with a set power. Thus, we opted for
a large-scale study, relative to the technology used. On the basis of the
results obtained, we calculated for each measured parameter the power and
the least significant number of observations, which we defined as the total
number of observations that would produce an α = .05 with a β =
.80, with δ and σ equal to those of the data, to provide additional
information regarding sample size for future investigations (JMP statistical
software, version 4.0.2; SAS Institute Inc, Cary, NC). The relationships between
total leg volume and leg muscle volume and between FSR and total testosterone
concentration were assessed with the Pearson correlation coefficient. Significance
was set at P<.05.
Subjects' Physical Features
Height, weight, and body mass index were similar in the older and in
the young men (Table 1). However,
the total leg volume was significantly lower in the older than in the young
men, suggesting a reduced leg muscle mass (Table 1). Mean (SE) leg muscle volume measured by MRI in 10 older
and 7 young men was lower in the older than in the young men (young men, 6.16
[0.23] L and older men, 4.64 [0.26] L; P = .001).
There was a significant correlation between total leg volume and leg muscle
volume (r2 = 0.77; P<.001) in the 17 subjects who underwent MRI measurement of leg
muscle volume (Figure 2). Leg blood
flow was not different between groups (Table 1).
Basal total testosterone concentration was measured in 19 young and
21 older subjects (Table 1). Total
testosterone was slightly lower in the older than in the young men.
Phenylalanine Concentrations and Enrichments
Phenylalanine concentrations in the femoral artery and vein were significantly
higher in the older than in the young men (P<.001)
(Table 2). However, no difference
in muscle free phenylalanine concentration was found between groups.
Phenylalanine enrichments were slightly but significantly higher in
the femoral artery (P = .04) and in the muscle tissue
water in the older men (P = .03). Phenylalanine enrichment
in the femoral vein was not different in the 2 groups. The enrichments were
at steady state during the sampling period (data available from authors).
Phenylalanine delivery to the leg, release from the leg, and NB: across
the leg were similar in older and young men (Table 3). Using the AV balance method, phenylalanine Ra was not
different in both groups. Muscle proteolysis was slightly but significantly
higher in the older men (P = .045) (an N = 91 would
have been needed to achieve a power of β = .80). Phenylalanine Rd, an
index of protein synthesis, was significantly higher in the older than in
the young men (P = .004).
Using the three-pool model, phenylalanine transport into the muscle
(FM,A) was slightly but not significantly higher in the older men
(P = .054). Power analysis indicated that an additional
51 subjects should have been studied to achieve an α = .05 and β
= .80. Phenylalanine AV shunting (FV,A) was slightly but significantly
higher in the older men (P = .03), whereas the transport
from the muscle (FV,M), and the release from proteolysis (FM,O) did not differ in the 2 groups. Phenylalanine utilization for protein
synthesis was significantly higher in the older group (P = .04), confirming the AV model data. Also, the fractional uptake
of phenylalanine, another index of protein synthesis, was significantly higher
in the older group (P<.001). Protein synthesis
efficiency was similar in young and older men.
Mean (SE) mixed muscle FSR was not different in the older and in the
young groups with a power of β = .06 (Table 3 and Figure 3).
The FSR was 3.5% higher in the older group. We estimated that we should have
studied at least 3208 subjects to achieve an α = .05, with β =
.80 and σ equal to that of the present data. We calculated that 8
subjects per group would be required to observe a 30% difference in FSR between
young and older men. No relationship was found between total testosterone
concentration and FSR (Figure 4).
Whole Body Phenylalanine Ra
Mean (SE) whole body phenylalanine Ra, an index of whole body proteolysis,
was not different (P = .07) in the older group (0.64
[0.02] µmol·kg−1·min−1)
and in the young group (0.70 [0.02] mol·kg−1·min−1), with a power of β = .47. To achieve an α = .05
and a β = .80, 105 subjects should have been studied.
Contrary to the general notion that muscle protein synthesis is reduced
with age, we found that the kinetic indicators of muscle protein synthesis
(phenylalanine Rd, fractional uptake, utilization for protein synthesis, and
mixed protein FSR) all tended toward the opposite direction: a higher protein
synthesis rate in older men. Nevertheless, phenylalanine net balance across
the leg was similar in both age groups, due to a slightly higher breakdown
rate in the older subjects, indicating that the degree of net protein catabolism
was similar in the basal state in both the young and older men. In addition,
the power analysis performed in this study provides evidence that when the
study design is not longitudinal very large numbers of subjects are needed
to observe significant age-related differences in basal muscle protein turnover
with sufficient power.
The contrast between the present data and previous reports15-19
that showed a difference in muscle myofibrillar protein synthesis, mixed protein
synthesis, or both may be explained by a combination of factors. First, this
investigation is the first large study to date to report not only basal muscle
protein synthesis values but also direct measures of muscle protein breakdown
and net muscle protein balance in older men. In the absence of quantification
of breakdown, it is not possible to draw conclusions on the mechanisms leading
to muscle loss. A lower protein synthesis rate would not induce muscle loss
if the breakdown was reduced as well. The results of direct (present study
and earlier studies20,22) and
indirect15,17-19,21
measurement of muscle protein breakdown have consistently indicated that breakdown
does not change with age. If true, the 30% to 40% reduction in basal myofibrillar
and mixed muscle protein synthesis with aging found in previous studies15-19
would have resulted in a net loss of approximately 60% of the muscle mass
within 1 year, assuming that an individual is in the basal state for approximately
16 h/d and that the response to feeding and other stimuli is preserved with
age. This reduction is inconsistent with the natural history of sarcopenia
that develops over decades and suggests that the measurement of that large
of a decrease in basal myofibrillar and mixed muscle protein synthesis may
reflect an acute response to the experimental design.
We purposely avoided any dietary manipulations prior to the study, whereas
in some of the previous reports volunteers were given a standardized diet
for several days prior to the study.15-19,21
Recent data suggest that the recommended protein dietary allowance for people
older than 55 years might be higher than that reported for adults younger
than 55 years,36 which is the cohort in which
dietary allowance was assessed. Thus, standardized diets may not be adequate
for healthy older men who may be compensating in their everyday life with
increased ingestion of protein. Also, we cannot exclude that the older muscle
is more sensitive to inactivity, so that an early admission to the GCRC (3
days before the study) as described in previous studies15-18
might have contributed to a reduction in physical activity and, consequently,
muscle protein turnover. This hypothesis is indirectly confirmed by the fact
that 1 group, who found a slower muscle protein synthesis in older subjects
after a 3-day admission to the GCRC,18 did
not confirm those findings when studying the subjects the day after admission.21
To assess the influence of age alone on muscle protein kinetics, we
intentionally selected healthy and active, although not participating in exercise
training, older subjects and compared them with healthy younger men. In addition,
the older volunteers of our study had total testosterone concentrations within
the mid- to-normal range, and we found no difference in total testosterone
concentrations between the young and older men. Since other investigators
have found age-related differences in testosterone concentrations,11 the lack of a difference in testosterone concentration
between the older and young men in our study might be explained by a high
variability of total testosterone concentration in the older age group and
by the fact that free testosterone was not measured. If our selection criteria
allowed us to assess the effect of age per se on muscle protein turnover,
our older volunteers may have not been representative of the general older
population. Furthermore, it is possible that the subjects of other studies
were not as healthy as the subjects in our study, although they were not clinically
ill.
One question that our study cannot answer is whether age affects the
turnover rates and the net balance of specific muscle proteins. Although the
synthesis rate of isolated muscle proteins, such as myofibrillar proteins
or, more specifically, myosin heavy chain, can be measured,15-17,19
at present no methods are available to determine their individual breakdown
rates. However, myofibrillar proteins and myosin heavy chain represent the
bulk of muscle proteins, and previous studies in which both mixed muscle proteins
and muscle myosin heavy chain synthesis rates were measured together reported
similar qualitative results.16,19
Thus, we believe that our data closely reflect contractile muscle protein
turnover.
In conclusion, since differences in the basal rate of muscle protein
turnover are not apparently responsible for the loss of muscle with aging,
it follows that research on sarcopenia should focus on the response of muscle
to specific stimuli, such as feeding and physical activity. Recent data indicate
that a blunted response to feeding may be in part responsible for the slow
loss of muscle with aging22 and that inactivity
may be responsible as well, since exercise training results in increased muscle
mass and muscle protein synthesis rate in both healthy and frail older individuals.18,19,21,37 Furthermore,
age-related changes in the hormonal pattern and the possible pharmacological
correction of these imbalances should be considered. Specifically, the reduction
in sex hormones and growth hormone/insulinlike growth factor I in some subjects
are appealing targets for a replacement therapy, which could positively impact
sarcopenia.11,12,14
From a clinical perspective, it is encouraging that age-related reduction
of basal muscle protein synthesis does not appear to explain sarcopenia, as
it would be difficult to target such a fundamental response with an appropriate
therapeutic intervention. On the other hand, it may be more feasible to treat
alterations stemming from inactivity, altered response to nutrients, or hormonal
imbalances.
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