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McGill Nutrition and Food Science Centre and
*
Division of Geriatric Medicine, Royal Victoria Hospital, Montreal, Canada;
School of Health and Physical Education, Queen’s University, Kingston, Ontario, Canada;
Research Institute, Hospital for Sick Children, Departments of Paediatrics and Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada; and
§
School of Dietetics and Human Nutrition, Mac Donald Campus, McGill University, Montreal, Canada
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-methylhistine excretion and lean tissue volumes
defined by whole-body magnetic resonance imaging, from eight
healthy elderly subjects (5 females and 3 males, mean age 71.5 y)
were compared with those of seven young persons (3 females and 4 males,
mean age 28 y). There were no significant age or gender effects on
rates of protein kinetics per L total lean tissue. There was a lower
(P < 0.004) rate of muscle protein catabolism in
the elderly (1.8 ± 0.2 vs. 2.6 ± 0.1 g ·
L-1 · d-1) and a trend
(P = 0.08) for lower muscle volume (19.7 ± 1.5 vs. 25.0 ± 2.4 L). This contrasted with intraabdominal lean
tissue, where the rate of protein catabolism (13.8 ± 0.6 vs. 13.2
± 0.9 g · L-1 · d-1) and
volume (7.5 ± 0.3 vs 8.0 ± 0.5 L) did not differ between
age groups. Thus, the decrease in the contribution by muscle to
whole-body protein metabolism with age is associated with an
increase from 62 to 74% (P < 0.001) in the
contribution by nonmuscle lean tissues. These findings have potential
implications for the nutrition of both normal and sick elderly
persons.
KEY WORDS: • protein turnover • aging • N-methylhistidine • magnetic resonance imaging • elderly humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Roberts et al. 1995
), resulting in
decreased total energy requirement to maintain equilibrium. This fall
in RMR is attributable to a loss of lean tissue with aging since there
is little or no change when RMR is expressed relative to lean body mass
(Visser et al. 1995
). Body composition studies suggest
that the decrease in lean tissues is primarily related to loss of
muscle mass with age (Cohn et al. 1980
, Forbes and Reina 1970
). The skeletal muscle mass plays an active role
in whole-body amino acid metabolism and is especially important
during restricted energy and protein intakes (Young 1990
). Although rates of whole-body protein turnover per kg
of body weight (BW) have been reported to decrease with age
(Golden and Waterlow 1977
, Young et al. 1975
), most studies (Fukagawa et al. 1989
,
Robert et al. 1984
, Uauy et al. 1978
),
including our own (Morais et al. 1997
) found no
differences when rates of protein kinetics were expressed per kg lean
tissue.
The finding that whole-body protein kinetics are unchanged per unit
of lean body mass with aging does not preclude the possibility of
changes in the turnover within individual lean tissues and thereby
their relative contributions to whole-body turnover. Indeed, we and
others have shown that the contribution of muscle protein breakdown to
whole-body protein catabolism is decreased, with a proportional
increase in the visceral organ protein catabolism (Morais et al. 1997
, Uauy et al. 1978
). Furthermore, muscle
fractional protein synthesis has also been found to be decreased with
age (Welle et al. 1993
, Yarasheski et al. 1993
). On the other hand, the effect of aging on visceral
protein turnover has not been studied directly in humans because it
requires invasive techniques that are difficult to perform in older
individuals. We therefore tested the hypothesis that nonmuscle lean
tissue mass and its rate of protein catabolism remain constant with
aging in the presence of unchanged whole-body protein turnover per
unit of lean tissue and therefore, a reduced contribution and turnover
of muscle protein mass. The aim of this study was thus to determine the
effects of aging on rates of protein turnover of the whole-body and
of muscle and nonmuscle lean tissues in a noninvasive fashion. To
achieve this, we concurrently applied the stable isotope technique
using the oral 60-h [15N]glycine method to
study whole-body protein metabolism, measurements of urinary
N-methylhistidine excretion to estimate total
body myofibrillar protein catabolism, and state-of-the-art
whole-body magnetic resonance imaging (MRI) analysis to define
muscle, nonmuscle lean tissue and adipose tissue volumes. We compared
results in healthy elderly to those of young adult persons.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eight healthy, moderately active elderly persons (5 women and 3 men),
classified as demonstrating characteristics of "usual aging"
(Rowe and Khan 1987
) and seven lean young persons (3
women and 4 men) were recruited through advertisements in local
newspapers. They were screened by history, physical examination and
cognitive status using the Mini Mental State Examination
(Folstein et al. 1975
) (for older persons only), a
laboratory investigation which included fasting blood sampling for
complete blood count, serum electrolytes, plasma glucose, renal and
liver function tests, total proteins and albumin, lipid profile,
thyroid hormones, a urine analysis, a chest X-ray and an
electrocardiogram (except for the young persons). Two elderly persons
were on thyroid hormone replacement therapy, and two others were on
treatment for mild arterial hypertension with drugs not known to
interfere with the metabolic measurements. Subjects were admitted to
the Royal Victoria Hospital Clinical Investigation Unit (CIU) and gave
written informed consent. The elderly group was studied first.
Following the in-patient stay, each group underwent total body MRI.
The protocol was approved by the Human Ethics Review Committee of the
Hospital. Subject characteristics are shown in Table 1
.
|
MR images were obtained with a GE Signa Advantage, 1.5 Tesla scanner using software version 5.4.2. (Madison, WI). A T1-weighted, spin-echo pulse sequence with a 210 ms repetition time, 17 ms echo time and a rectangular field of view (48 cm x 36 cm) was used to acquire the MRI data. The MR images obtained in the abdomen and thorax regions were acquired using a 1/2 Fourier transformation-pulse sequence (1/2 NEX), meaning that a set of 7 images could be obtained in 26 s. During this 26 s the subjects were asked to take a normal inspiration and hold their breath. For the appendicular regions, the same pulse sequence was used with the exception that a single NEX was employed, resulting in a 43-s acquisition time for a set of 7 images. The total time required to obtain all MRI data (41 images) for each subject was ~25 min. During this time, the subjects lay in the magnet in a prone position. All image data were transferred onto an Indigo 2 computer (Silicon Graphics, Mountain View, CA) for analysis using specialized computer software (Tomovision, Montreal, QC).
Calculation of lean and adipose tissue area and volume.
The segmentation method used to determine tissue areas is described in
detail elsewhere (Ross et al. 1996
). Briefly, the method
is based on image morphology and employs a combination of edge
detection filters and Watershed techniques. Initially a filter is used
to distinguish between different gray level regions on the image. Once
the edges are determined, lines are drawn on the image using a
Watershed algorithm. If the regions (i.e., a group of voxels) are too
small, they can be merged using statistical parameters inherent to the
image. Once the regions representing the various tissues (i.e.,
skeletal muscle and adipose tissue) are identified, the observer uses a
mouse pointer to identify each tissue using color codes. Each image is
then reviewed using an interactive slice editor program which allows
for verification and, where necessary, correction of the segmentation
result (Ross et al. 1996
). This operation is facilitated
by superimposing the original gray level image on the binary segmented
image using a transparency mode (Ross et al. 1996
). To
calculate tissue area (cm2), the respective
tissue regions in each slice are computed automatically by summing the
given tissues’ pixels and multiplying by the pixel surface area. The
tissue volume (cm3) for each slice is calculated
by multiplying the tissue area (cm2) by slice
thickness (10 mm). Adipose tissue (AT) and lean tissue (LT) volumes
were calculated by adding the volumes of truncated pyramids defined by
pairs of consecutive slices (Ross et al. 1996
).
Whole-body tissue volumes were calculated using all 41 slices.
Intraabdominal LT volume was derived using four abdominal images
extending from L4-L5 to three images above.
Validation and reliability of MRI.
We have recently reported that the arm and leg AT-free skeletal
muscle cross-sectional areas in cm2
(n = 119) using MRI were not different from cadaveric
estimates (38.9 ± 22.3 vs. 39.5 ± 23.0
cm2; P < 0.001). Similar good
results were observed between MRI-measured and cadaver-measured
interstitial and subcutaneous adipose tissue (Mitsiopoulos et al. 1998
).
For AT-free skeletal muscle cross-sectional areas the
intraobserver correlation for duplicate (same day) measurements in vivo
was 0.99 and the standard error of estimate 2.9% (Mitsiopoulos et al. 1998
). For MRI-LT measurements, we have previously
reported the results obtained when duplicate (same day) MR images
obtained on 19 female subjects at the L4-L5 level were compared
(Ross et al. 1992
). The correlation coefficient obtained
between the two measurements was 0.94 (P < 0.001). The
difference between the two mean values was 1.0 ± 5.1%
(P > 0.10). The repeatability of whole-body LT
volume measurements (in liters) was assessed from repeated measurements
on two obese men. For each subject, a complete data set was acquired
(41 images) on two separate occasions during the same day. The mean
difference between test 1 and 2 for MRI-LT (L) was < 2%. The
MRI-LT calculations were determined by a single individual and thus
represent the intraindividual error associated with repeated
LT-volume calculations. In previous studies we have reported that
for total AT volume (L), the mean difference between tests 1 and 2 was
2.6% with a range of 0.9 to 4.3% (Ross et al. 1992
).
Anthropometric measurements.
Daily morning body weights of each subject were measured in the fasted state after voiding, and wearing the same light clothing, to the nearest 100 g on a Scale-Tronix digital scale (Ingram & Bell-Meditron, Le Groupe, Don Mills, ON). Body height without shoes was measured to the nearest 0.1 cm with a wall-mounted stadiometer. Body mass index (BMI) was calculated as weight in kg/height in m2, using the average daily weight from the d 2 onward.
Diets.
The elderly subjects received an individualized diet based on a 6-d
food diary record analyzed by the CBORD Diet Analyzer V 3.0.3 (the CBORD Group, Natural Software Limited, 1988, Ithaca,NY). On the basis
of this assessment, the average protein intake was 1.20 g/(kg BW · d)
and the average energy intake was 130 kJ/(kg BW · d) (31 kcal/(kg BW
· d), which represented 148 to 182% of the RMR, as measured by
indirect calorimetry. Each young subject received 1.27 g/(kg BW · d)
of protein with energy intake based on measured RMR multiplied by an
activity factor of 1.7 (Table 3
). The diet was divided into
six small meals given every 3 h from 0800 to 2300. For breakfast,
subjects received 30 g of whole-bran cereal (All Bran Cereal;
Kellogg Canada, Etobicoke, ON) with 200 mL milk (2% fat). The rest of
protein and energy intake was provided as a meal replacement product
(BoostR, Mead Johnson, Montreal, QC) with
additional energy sources as a combination of two-thirds glucose
polymer (Polycose, Ross Laboratories, Montreal, QC) and one-third
corn oil. The energy distribution was 15% as protein, 65% as
carbohydrate and 20% as fat for the elderly and 13, 62 and 25%,
respectively, for the young people. The elderly group received the diet
for 9 d and the young group for 7 d, all at the CIU except
for the first 2 d of the protocol for young subjects, who consumed
it at home.
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Following a training session and before admission, RMR was measured in
the elderly and young subjects by continuous indirect calorimetry with
a Deltatrac ventilated hood metabolic monitor (Sensor Medics, Yorba
Linda, CA). All subjects were fasting for at least 10 h, arrived
by car or by public transportation and were allowed to rest in a supine
position in a thermally neutral and quiet room for a minimum of 30 min
before oxygen consumption and carbon dioxide production measurements
were begun. Subjects breathed under the plastic canopy for 20 min, and
the average of the last 15 min was used for calculation of the 24-h RMR
based on the de Weir equation (de Weir 1949
).
Urinary nitrogen measurements.
Daily 24-h urine collections were made. Total urinary nitrogen (N) was analyzed by chemiluminescence (Anteck Pyro-Chemiluminescent Nitrogen System, Houston, TX) according to methods described by Ward et al. 1980. Standards were prepared from analytical grade urea (ICN Biomedicals, Aurora, OH) ranging from 1.0 to 10.0 g N/L. Standards and urine samples were diluted 1:100 or 1:200 with water and delivered in 5 µL aliquots in duplicate into the pyrolysis chamber by a quartz boat filled with quartz wool in a water-jacketed pyrolysis tube (Antek Syringe Driver 735, Houston, TX).
Protein turnover.
Protein kinetic studies were done using the 60-h oral
[15N]glycine method during the last 3 d of
the CIU stay. Details of the procedures are described by Gougeon et al.
1994. The method requires that within 60 h the enrichment of
15N in urinary urea has reached a plateau. This
determination, based on the isotopic enrichment curves, is defined as
the first plateau that extends for at least four points (12 h). Mean CV
of plateau values was 4.2 and 3.5% for the elderly and young persons,
respectively. The rate of entry of N (Q) into the metabolic N pool can
be calculated from the mean plateau value, assuming that the fraction
of the administered isotope that is excreted as urinary
15N equals the fraction of total amino N entering
the metabolic pool excreted as urinary urea N (Stefee et al. 1976
, Waterlow et al. 1978
). Since N intake is
known and total urinary N was measured, protein synthesis and breakdown
can be calculated from the Picou and Taylor-Roberts equation: Q
= I + B = S + E, (Picou and Taylor-Roberts 1969
) where Q is nitrogen flux, I is nitrogen intake, B is
protein breakdown, S is protein synthesis, and E is total urinary
nitrogen. 15N enrichment of urea N was measured
with a dual-inlet, triple collector isotope-ratio mass
spectrometer (Vacuum Generators, Micromass 903D, Winsford, Cheshire,
UK) after correction for background values determined on a urine sample
taken immediately before the test.
Urinary N-methylhistidine and calculations of
individual protein turnover.
Skeletal muscle protein breakdown was calculated from measurements of
urinary N-methylhistidine (3MH).
Methylation of histidine occurs after its incorporation into the
peptide chains of actin and myosin. After degradation of these
proteins, the 3MH liberated is not recycled but quantitatively excreted
in urine. 3MH is thus an index of myofibrillar proteolysis (Ward and Buttery 1978
). The rate of muscle protein breakdown was
estimated using a value of 4.2 µmol 3MH per g of mixed protein
catabolized in skeletal muscle. Results are then expressed as a
percentage of whole-body protein breakdown (Young and Munro 1978
). We estimated the nonmuscle component by subtracting the
muscle protein breakdown calculated in this manner from the
whole-body breakdown from the stable isotope measurements
(Morais et al. 1997
). Protein breakdown rates per unit
of muscle and nonmuscle lean tissues were estimated by factoring the
values for their individual volumes in L assessed by MRI. Urinary 3MH
excretion was measured by reverse-phase HPLC (Hewlett-Packard 1090,
Mississauga, ON) after derivatization with orthophthalaldehyde and
mercaptopionic acid (Garrel et al. 1995
). The
sensitivity of the assay was 0.1 µmol/L. Interassay and intraassay
variation was <5% (Garrel et al. 1995
).
Other analytical measurements.
Venous blood samples were drawn with minimal stasis in the
overnight-fasted state on d 1 and 3 of the
[15N]glycine study. The samples were drawn into serum
separation tubes and into heparinized tubes containing one tenth of the
volume of blood as aprotinin (Trasylol, 10,000 Kallikrein inhibitor
ku/L, FBA, Pointe Claire, QC). The latter were cooled, centrifuged at
2000 x g at 4°C for 15 min and stored in multiple
aliquots at -20°C. Plasma was assayed for insulin by
single-antibody charcoal precipitation radioimmunoassay with human
standards and labeled hormone from Linco Research (St. Louis, MO) by
methods described previously (Marliss et al. 1978
).
Immunoreactive glucagon was measured by double antibody RIA (Linco).
Fatty acids were assayed in duplicate Dole extracts of plasma by the
radiochemical microtechnique (Ho 1970
). Plasma glucose
was measured by the glucose oxidase method on a Beckman II glucose
analyzer (Beckman Instruments, Fullerton, CA). Plasma values were
corrected for dilution by aprotinin, based on a concurrently measured
hematocrit. Serum human growth hormone (hGH) was assayed using National
Institutes of Health Standards (NIDDK-hGH-RP-1) and antibody
(NIDDK-anti-hGH-2) and hGH obtained from NIH (product
AFP-11019BNIDDK-NIDDK-hGH-I-3) that was labeled with
125I in the Royal Victoria Hospital’s
polypeptide laboratory. Serum cortisol was measured at the Royal
Victoria Hospital endocrinology laboratory by an automated
immunoluminescence technique (CIBA Corning ACS 180, Cooperstown, NY).
After thorough mixing, aliquots of the 24-h urine collections were analyzed daily for urea nitrogen, creatinine, and electrolytes (Na, K, Cl) and frozen at -20°C until assayed by the methods listed above. Daily urinary urea N and creatinine were obtained by the autoanalyzer method in the Clinical Biochemistry laboratory of the Hospital; the creatinine served as a means of assuring completeness of collections.
Statistical methods.
Data are presented as means ± SEM Outcomes were
analyzed with two-factor ANOVA models for age group and gender
using the generalized linear model procedure. The main effects are to
be interpreted as the overall effect of age adjusted for gender and the
overall effect of gender adjusted for age. An interaction term for age
group and gender was included in all models. Since no interaction term
was found (P > 0.05), the models include only the main
effects. Residual plots were used to examine model assumptions.
Normality of the residuals was further tested using the
Shapiro-Wilk statistic. When model assumptions were violated,
analyses were redone after transforming the dependent variable;
reported P-values are from the transformed model. Analyses
were performed with the SAS (SAS Institute, Cary, NC) software
programs. On the basis of a standard deviation of 15% of the protein
turnover results in our laboratory using the
[15N]glycine method (Gougeon et al. 1994
), and applying a power calculation formula for dietary
studies (Hall 1983
), we estimated that seven subjects
per group would need to be studied for a 15% change in protein
turnover to be detectable with a power of 90% (one-tailed 
= 0.05, ß = 0.10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Groups were distinct with regard to age, as required by the design
(Table 1
). Elderly persons had higher (P < 0.04) BMI, and lower creatinine and 3MH excretions, compared with the
young people. A gender effect independent of age for lower
(P < 0.03) weight, height, creatinine and 3MH
excretions was observed for females. The ratios of urine 3MH to
creatinine were not significantly different between age groups.
MRI measurements.
There were no significant differences between age groups for volumes of
whole-body LT, muscle, or intraabdominal LT (Table 2
). However, there was a trend for lower muscle volumes in
elderly persons (19.7 vs. 25.0 L; P = 0.08). There
was a overall gender effect for females to have lower (P
< 0.03) volumes of LT. AT volumes as a whole, and at each site
measured, were greater (P < 0.02) in the elderly
compared with young people. Percentage intraabdominal AT, and
percentage abdominal subcutaneous AT (vs. total AT) and the ratio of
intraabdominal/abdominal subcutaneous AT were also higher
(P < 0.04) in elderly compared with young people.
This is in contrast with the percentage subcutaneous AT that was lower
(P < 0.02) in the aged group. As an overall gender
effect, females also showed increased (P = 0.03)
volumes of intraabdominal AT. Their percentage subcutaneous and
percentage intraabdominal AT (vs. total AT) were also higher than in
males, as was their ratio of intraabdominal/abdominal subcutaneous AT
(P < 0.004).
|
Elderly persons had lower (P < 0.03) energy intake and
RMR per day and per kg BW than young persons (Table 3
). However, once these results were corrected for volumes of LT, no
differences remained between groups. Protein intake was not
significantly different between age groups irrespective of the
denominator. As an overall gender effect, females had lower
(P < 0.02) protein intakes per day but not per kg BW
or per L of LT.
Kinetics of protein metabolism.
When rates were expressed in g N/(kg BW · d), the elderly group had a
16% lower mean Q that did not reach significance (P = 0.057) and 20% lower (P < 0.04) S and B (Fig. 1
). However, when expressed per L of LT/d, differences were no longer
significant (Fig. 2
). No gender effect was observed for protein kinetics. Results of net
endogenous protein balance (S-B; in mg N) showed no significant
differences whether expressed per kg BW-1 ·
d-1 or per L of LT/d.
|
|
Rates of muscle protein breakdown (mB) in g, estimated from 3MH
excretion were lower (P < 0.004) in the elderly,
whether expressed per day, per kg BW-1 ·
d-1 or per L of LT/d (Fig. 3
). The estimation of the rate of myofibrillar protein breakdown,
presented as the ratio of mB to muscle volume was also lower
(P < 0.004) in the elderly. There was a gender effect
independent of age responsible for lower (P < 0.02)
rates of all components of muscle protein breakdown in females. Rates
of nonmuscle body protein breakdown (B-mB) were not significantly
different between age groups whether expressed per day, per kg
BW-1 · d-1 or per L of LT/d
(Fig. 4
), nor was the estimation of the rate of visceral protein breakdown,
obtained by factoring (B-mB)/d for nonmuscle LT volume. No gender
effect was observed for any of the components of (B-mB).
|
|
). The reciprocal values and corresponding significant differences were
observed for the percentage contribution made by nonmuscle LT
[(B-mB)/B], namely higher values for older individuals and females.
|
Postabsorptive concentrations of energy substrates and of principal
hormones implicated in the regulation of protein metabolism are
presented in Table 4
. Fasting plasma glucose was higher (P = 0.049) values
in the elderly. There was a gender effect with lower (P
< 0.03) fasting plasma insulin values in females. No significant
age or gender effects were observed for the other measurements.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Morais et al. 1997
, Robert et al. 1984
,
Uauy et al. 1978
). Since these results represent the
overall rate of individual lean tissues turning over at different
rates, it could very well be that a decrease in one of them, e.g.,
muscle with slower protein turnover is concealed by another with a
faster turnover, i.e., visceral tissue. As previously reported
(Morais et al. 1997
, Uauy et al. 1978
),
we found that the contribution of muscle to whole-body protein
catabolism is significantly reduced in elderly persons (25.8 vs.
38.4%; P < 0.001). This could be explained by a
change in the amount of tissue participating, alterations in the
intrinsic rates of turnover within tissues, i.e., their individual
rates of protein turnover, or both. It is well known that muscle mass
decreases with aging (Cohn et al. 1980
, Forbes and Reina 1970
). In our subjects, the muscle volume of elderly
subjects was not significantly different from that of the young group,
perhaps due to the number of subjects studied, as a trend for
lower values was present. However, our findings indicate that there is
a significant age-related decrease in protein breakdown within
muscle of about 30%, reducing further the contribution of muscle to
whole-body protein catabolism.
In support of our results of a lower rate of muscle protein breakdown
per unit of tissue using 3MH and muscle volumes assessed by MRI are
published data using [13C]leucine incorporation into
muscle in the postabsorptive state, showing that older subjects had
lower fractional muscle protein synthesis than young subjects. These
results suggest an age-related decline of muscle protein turnover
(Welle et al. 1993
, Yarasheski et al. 1993
) in addition to the loss of total mass. Because muscle
mass is relatively stable over long periods even in older persons, we
can infer that the rates of skeletal myofibrillar protein breakdown
would also reflect the decremental effect of aging. No reduction in
rates of myofibrillar protein breakdown with aging was inferred by Uauy
et al., using the ratio of 3MH to creatinine excretion (Uauy et al. 1978
). Our 3MH/creatinine data also showed no significant
differences, though both 3MH and creatinine excretion were
substantially lower in the elderly subjects. The most plausible
explanation for this lack of age-related change using 3MH to
creatinine is that creatinine is not as good a surrogate for muscle
mass as imaging.
The measurement of urinary 3MH is a reproducible and reliable method to
quantify the turnover of skeletal myofibrillar protein (Young and Munro 1978
). However, there are skin and endogenous gut
sources contributing to the total urinary 3MH excretion (Afting et al. 1981
, Harris 1981
). Since aging affects
primarily muscle mass (Cohn et al. 1980
, Forbes and Reina 1970
), elderly individuals are expected to have a
higher contribution of nonskeletal muscle urinary 3MH to the total
output than young subjects. Although there is uncertainty as to the
precise amount of 3MH produced by the skeletal muscle, these
considerations serve to further strengthen the argument for our finding
of lower skeletal muscle protein turnover with aging.
The finding of lower relative rates of myofibrillar protein catabolism
in women than men is of interest. This gender effect was observed
independent of age. In all but one study (Welle et al. 1995
) comparing old with young individuals, the statistical
analyses performed were not appropriate to demonstrate gender
differences. Welle and coauthors did not report gender differences, but
they may not have been sought in the analyses. In any case, this
question needs to be confirmed by more direct measurements of muscle
protein metabolism.
A novel finding of the present study is that the rate of nonmuscle
protein breakdown tissue mass appears to remain unchanged with aging
regardless of the denominator used. Our data indicate that volumes of
nonmuscle LT and of intraabdominal LT were not affected by age, which
is in accordance with other studies of body composition suggesting
maintenance of nonmuscle LT with aging (Cohn et al. 1980
). The finding of unchanged rates of visceral protein
turnover is not surprising as visceral organs tend to maintain their
functions, in contrast to skeletal muscle that decreases, because of
the decrease in physical activity with aging. Rates of nonmuscle LT
protein breakdown were 6.8 ± 0.4 vs. 6.5 ± 0.4 g/(L · d)
(NS) for elderly and young persons, respectively. For young persons,
these nonmuscle tissue rates are 2.5 times higher than those of muscle
which is in agreement with the nearly three-fold higher splanchnic
than leg muscle leucine uptake in one human study using
[14C]leucine (Gelfand et al. 1988
). As a consequence of the unchanged volumes and
rates of protein breakdown of visceral tissue, the percentage
contribution of nonmuscle LT to whole-body protein breakdown
significantly increased in older persons (74 vs. 62%) making up for
the decreased contribution of muscle.
To our knowledge, the present study is the first to have assessed the
effects of aging on rates of protein turnover of visceral lean tissues
in humans. However, two other publications in human subjects support
the notion that no decrease in visceral protein metabolism is observed
with aging (Boirie et al. 1997
, Gersovitz et al. 1980
). The rates of albumin synthesis using the continuous oral
[15N]glycine method (and estimating the enrichment of the
free arginine guanidine-N in the liver from the plateau
15N in urinary urea) were found to be unchanged in older
compared with young males (Gersovitz et al. 1980
).
Recently, Boirie et al. 1997, using a combination of intravenous
[13C]leucine and oral
[2H3]leucine tracers, have found that there
is a higher leucine extraction by the gut and liver in older males.
Interestingly, they also found that the ratio of -ketoisocaproic
acid to leucine tracer enrichment, an indirect way of estimating
splanchnic leucine transamination, was higher in old than young males.
These results suggest that both liver and gut maintain their activity
of protein metabolism with aging in humans. In vivo animal data looking
at the effects of aging on different visceral tissues have given
conflicting results. Some have shown a decline with age in liver
protein synthesis rates in rats (Ward and Richardson 1991
), whereas others that have found unchanged rates of
fractional liver protein synthesis and breakdown (Goldspink and Kelly 1984
, Mosoni et al. 1993
). Increased heart
and lung protein turnover rates have also been found (Mays et al. 1991
).
MRI was able to discriminate among the different lean tissues, giving
volumes of muscle and nonmuscle LT, and defining volumes of AT and its
distribution. We are thereby able to adduce direct evidence of the
preservation of visceral organ mass with aging in a cross-sectional
design. Previous studies have concluded this by interpreting the faster
decline of potassium to nitrogen using 40K and the prompt
gamma neutron-activation technique (Cohn et al. 1980
). Although this was not primarily a study on body
composition measurement, and conclusions must be circumspect due to the
small number of subjects, we showed a trend toward a reduced muscle
mass with aging, as well as preservation of visceral lean tissues and
increased adipose tissue in the elderly. Similar conclusions can be
derived from reports that used a diversity of techniques (Cohn et al. 1980
, Durnin and Wormersley 1974
,
Flynn et al. 1989
). However, MRI enabled us to quantify
the tissues by region, including the amount and distribution of AT. The
AT is increased not only in total but also at all the sites evaluated:
intraabdominal, subcutaneous and abdominal subcutaneous. The notion
that there is a central distribution of fat with aging (Durnin and Wormersley 1974
) has also been confirmed since there was an
aging effect responsible for the increase in the percentage
intraabdominal AT to total AT and in the ratio of intraabdominal AT to
abdominal subcutaneous AT, with an opposite effect on the percentage
subcutaneous AT. Female sex contributed to increased intraabdominal AT
but contrary to aging, it contributed also to increase the percentage
subcutaneous AT/whole-body AT. However, because no increase in the
abdominal subcutaneous AT was found for females compared with males,
the resulting effect is an "internalization" of fat in females with
aging, in the abdominal region (T9-T10 to L4-L5). Although this
distribution of fat in females is not a universal finding and could be
due to a selection bias, the higher total fat in females compared with
males is a well-recognized fact (Flynn et al. 1989
).
Our panel of energy substrate measurements showed a significantly
elevated fasting plasma glucose with age (4.8 ± 0.1 vs. 4.5
± 0.1 mmol/L), although its value was still within the normal
range. The magnitude of this difference is compatible with other
studies comparing healthy elderly with young subjects (Meneilly et al. 1997
, Robert et al. 1984
) and is
considered a manifestation of insulin resistance of aging
(Jackson 1989
). Although hyperglycemia due to diabetes
mellitus is known to accelerate protein turnover (Gougeon et al. 1994
and 1997
), this magnitude of glucose
elevation per se is unlikely to be associated with altered protein
turnover rates in the elderly. Fasting plasma insulin levels showed a
gender effect, with lower values in females. This is of interest
because of the increased intraabdominal AT which has been linked with
insulin resistance and elevated plasma levels (Després et al. 1989
). Other factors are known to influence plasma insulin
levels, including the current levels of energy intake (Ivy et al. 1991
) and physical activity (Kohrt et al. 1992
, Rosenthal et al. 1983
), which could
possibly account for this discrepancy. Of note is also the lack of
significant aging effect on growth hormone levels in our subjects.
Growth hormone deficiency affects approximately half of the elderly
population (Rudman 1985
) and thus is not a universal
accompaniment of aging. This result and the absence of any significant
aging effect on the level of plasma insulin support the statement that
our aged group was composed of healthy elderly persons.
In summary, we found that despite unchanged rates of protein turnover
per liter of lean tissue with aging, there is a significant reduction
in the contribution by muscle to whole-body catabolism due to loss
of muscle mass and to a slower myofibrillar protein catabolism. This is
in contrast with nonmuscle lean tissue whose mass and rates of protein
catabolism remain constant with aging, thus increasing its percentage
contribution to the whole body. The implications of a lesser
contribution of muscle to whole-body protein breakdown are not
fully understood, but it is postulated (Young 1990
) that
this could diminish the capacity of the elderly to respond successfully
to restricted dietary intakes or to stressful conditions that require
mobilization of amino acids from the myocyte for protein synthesis in
vital organs, including cells of the immune system. Furthermore, the
significance of the changing pattern of whole-body protein
metabolism with aging on protein requirements remains to be defined.
Debate prevails on whether the present FAO/WHO/UNU protein
recommendations should remain the same (Millward et al. 1997
) or be increased (Campbell et al. 1994
) in
older persons. Since the elderly are more prone to a variety of
illnesses that can affect nutrient intake, efficiency of nutrient
assimilation and metabolism and/or increased demand for nutrients, they
are further at risk. Studies are needed in frail elderly subjects to
ascertain what their body composition and protein turnover are, as well
as alterations in nutrient requirements such illnesses impose, as they
are expected to differ from those of younger persons.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: AT, adipose tissue; B-mB, nonmuscle protein breakdown; BMI, body mass index; BW, body weight; CIU, Clinical Investigation Unit; LT, lean muscle; 3MH,
N-methylhistidine; MRI, magnetic resonance imaging; mB, muscle protein breakdown; RMR, resting metabolic rate.
Manuscript received July 8, 1999. Initial review completed August 17, 1999. Revision accepted December 21, 1999.
|
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