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in PresS. Am J Physiol Endocrinol Metab (May 29, 2007). doi:10.1152/ajpendo.00185.2007
Manuscript E-00185-2007 (FINAL ACCEPTED VERSION)
Measurement of human mixed muscle protein fractional synthesis rate depends on the
choice of amino acid tracer
Gordon I. Smith, Dennis T. Villareal, and Bettina Mittendorfer
Washington University, School of Medicine, St. Louis, MO, USA
Running head
Tracer dependence of fractional muscle protein synthesis rate
Corresponding author
Bettina Mittendorfer, PhD
Division of Geriatrics and Nutritional Science
Washington University School of Medicine
660 South Euclid Avenue; Campus Box 8031
Saint Louis, MO 63110
Phone: (314) 362 8450
Fax: (314) 362 8230
E-mail: [email protected]
1
Copyright © 2007 by the American Physiological Society.
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Abstract
The goal of this study was to discover whether using different tracers affects the measured rate of
muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein
fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions
and mixed meal feeding (70 mg protein·kg fat-free mass-1·h-1 × 2.5 h) by simultaneous intravenous
infusions of [5,5,5-2H3]leucine and either [ring-13C6]phenylalanine or [ring-2H5]phenylalanine and
analysis of muscle tissue samples by gas-chromatography mass-spectrometry. Both the basal FSR and
the FSR during feeding were ~20% greater (P < 0.001) when calculated from the leucine labeling in
muscle tissue fluid and proteins (fasted: 0.063 ± 0.005 %·h-1; fed: 0.080 ± 0.007 %·h-1) than when
calculated from the phenylalanine enrichment data (0.051 ± 0.004 and 0.066 ± 0.005 %·h-1,
respectively). The feeding induced increase in the FSR (~20%; P = 0.011) was not different with
leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine and
phenylalanine derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We
conclude that when using stable isotope labeled tracers and the classic precursor product model to
measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly
dependent on the tracer amino acid used; however, the anabolic response to feeding is independent of
the tracer used. Thus, different precursor amino acid tracers cannot be used interchangeably for the
evaluation of muscle protein synthesis and data from studies using different tracer amino acids can be
compared qualitatively but not quantitatively.
Key words
tracer, amino acid, muscle protein turnover
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Introduction
Muscle mass is maintained by a balance between the rate of muscle protein synthesis and breakdown
(29), which can be measured in vivo by using a variety of tracer methods (4, 27, 28, 32, 40, 42). The
most commonly used approach to measure the rate of muscle protein synthesis is based upon the
incorporation of a stable isotope labeled amino acid (usually phenylalanine or leucine) during
continuous intravenous infusion of the tracer to determine the muscle protein fractional synthesis rate
(FSR) (5, 22, 31, 35, 37, 41). The reported rates of mixed muscle protein synthesis from these studies
varies substantially even during standard overnight fasted conditions in young, healthy individuals
(from ~0.04 to 0.08 %·h-1) (11-13, 16, 23, 24, 36, 37, 41). The reason(s) for this variability in results
is not clear.
One potential explanation for the disparity in results may be the use of different tracers and/or
surrogate precursor pools for the calculation of the FSR. Although the FSR is known to be influenced,
to some degree, by the precursor pool used for calculating the FSR (6-8, 18, 38), theoretically the FSR
should be the same regardless of which labeled amino acid is infused (39). However, substantial
differences in the measured muscle protein synthesis rate have been observed when using different
radioactively labeled amino acid tracers ([14C]leucine vs [3H]phenylalanine) in rats (20). This casts
doubt on this important assumption but could be due to errors in deriving the exact amino acid
composition of muscle proteins which is necessary when using radioactive amino acid tracers to
measure muscle protein turnover. Studies in which the flooding dose (21) or limb AV-balance (3, 33)
techniques were used to examine this issue in human muscle also suggest that there may be an amino
acid tracer dependence of the measured muscle protein synthesis rate. However, the results are
difficult to interpret due to the small number of subjects included in the flooding dose study (21) and
potential errors in measuring limb leucine oxidation or the leucine and phenylalanine content in
muscle proteins when using the AV-balance technique (3, 33). Whether the measured rate of human
muscle protein synthesis is tracer dependent when using a continuous intravenous infusion of stable
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isotope labeled tracers with simultaneous measurement of tracer incorporation into muscle proteins
has never been investigated.
The purpose of the present study, therefore, was to evaluate how the choice of stable isotope labeled
tracer affects the calculated rate of human skeletal muscle protein synthesis. To do so, we measured
the muscle protein FSR in the quadriceps of older men and women by simultaneous continuous
intravenous infusion of [5,5,5-2H3]leucine and either [ring-2H5]phenylalanine or [ring13
C6]phenylalanine during basal, post-absorptive conditions and feeding.
Methods
Subjects
Fifteen older adults (6 men and 9 women; age: 70 ± 1 years; BMI: 38.0 ± 1.6 kg/ m2; means ± SEM),
who underwent measurements of their muscle protein FSR as part of a larger, currently ongoing,
clinical trial participated in this study. All subjects were considered to be in good health after
completing a comprehensive medical evaluation. Written informed consent was obtained from all
subjects before their participation in the study, which was approved by the Human Studies Committee
and the General Clinical Research Center (GCRC) Advisory Committee at Washington University
School of Medicine in St. Louis, MO.
Experimental Protocol
Five of the fifteen subjects underwent one, the other ten subjects underwent two separate studies
(approximately 3 months apart) to determine the FSR of muscle proteins by simultaneous intravenous
infusion of stable isotope labeled leucine and phenylalanine tracers and analysis of blood and muscle
tissue samples by mass spectrometry. The repeat studies in ten of the subjects were performed after
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various types of "intervention" (e.g., exercise training, weight loss) and are therefore not
reproducibility studies - hence data from these studies were treated as independent observations.
Subjects were admitted to the GCRC the evening before the tracer infusion study. At 2000 h, they
consumed a standard meal, containing 12 kcal per kg body weight (55% of total energy from
carbohydrates, 30% from fat, and 15% from protein) and then rested in bed until completion of the
study the next day. At 0600 h on the following morning, a cannula was inserted into an antecubital
vein for infusion of the stable isotope labeled amino acids; another one was inserted into a vein of the
contralateral forearm for blood sampling. At 0800 h, a baseline blood sample was obtained to
determine the background enrichments of leucine and phenylalanine in plasma and a muscle biopsy
was taken from the quadriceps femoris to determine the background leucine and phenylalanine
enrichments in muscle protein and muscle tissue fluid. Immediately afterwards, primed constant
infusions of [5,5,5-2H3]leucine (priming dose: 4.8 µmol/kg body wt, infusion rate: 0.08 µmol·kg body
wt-1·min-1) and either [ring-13C6]phenylalanine or [ring-2H5]phenylalanine (priming dose: 5.4 µmol/kg
body wt, infusion rate: 0.05 µmol·kg body wt-1·min-1) were started and maintained until completion of
the study some 6 h later. [Ring-13C6]phenylalanine was used in 14, and [ring-2H5]phenylalanine was
used in 11 of the 25 studies. 210 min after the start of the tracer infusion, an additional muscle biopsy
was obtained to determine the basal rate of muscle protein synthesis (as incorporation of labeled
amino acids into proteins; see Calculations). Immediately following the biopsy procedure, a liquid
meal (Ensure®, Abbott Laboratories, Abbott Park, IL, USA), containing 15% of total energy as
protein, 55% as carbohydrate, and 30% as fat, was given in small boluses every 10 minutes for 150
min so that every person received a total of 70 mg protein per kg fat-free mass (FFM) per hour plus a
priming dose of 23 mg protein/kg FFM during the 2.5 h feeding period. At the onset of the feeding
regimen, the infusion rates of labeled leucine and phenylalanine were increased to 0.12 µmol·kg body
wt-1·min-1 and 0.08 µmol·kg body wt-1·min-1, respectively to adjust for the increased amino acid
availability. A muscle biopsy was obtained again 150 min after ingesting the first aliquot of the meal
to determine the rate of muscle protein synthesis during mixed meal feeding. The second and third
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biopsies were obtained through the same incision on the leg, with the forceps directed in distal and
proximal direction, respectively so that the two biopsies were collected from sites that were
approximately 10-20 cm apart; the first biopsy was taken from the opposite leg. Additional blood
samples were obtained every 30 min during the entire experiment to determine plasma leucine and
phenylalanine enrichment. The tracer infusions were stopped and cannulae were removed after the
last (third) biopsy and the final blood draw were completed.
Sample Collection and Storage
Blood samples ( 3 ml) were collected in pre-chilled tubes containing EDTA; plasma was separated by
centrifugation within 30 min of collection and then stored at -80°C until final analyses were
performed. Muscle tissue (~100 mg) was obtained under local anaesthesia (lidocaine, 2%) by using a
Tilley-Henkel forceps. The tissue was immediately frozen in liquid nitrogen and then stored at -80°C
for determination of leucine and phenylalanine enrichments in muscle protein and tissue fluid.
Sample Processing and Analyses
To determine plasma leucine and phenylalanine enrichments (tracer-to-tracee ratios, TTR), proteins
were precipitated and the supernatant, containing free amino acids, was collected to prepare their tbutyldimethylsilyl (t-BDMS) derivatives for analysis by gas-chromatography/mass-spectrometry (GCMS; MSD 5973 System, Hewlett-Packard) via electron-ionization and selective ion monitoring (26).
To determine leucine and phenylalanine enrichments in muscle proteins and muscle tissue fluid,
muscle samples (~20 mg) were homogenized in 1 ml trichloroacetic acid solution (3% w/v), proteins
were precipitated by centrifugation, and the supernatant, containing free amino acids, was collected.
The pellet containing muscle proteins was washed and then hydrolyzed in 6 N HCl at 110°C for 24 h.
Amino acids in the protein hydrolysate and supernatant samples were then purified on cationexchange columns (Dowex 50W-X8-200, Bio-Rad Laboratories, Richmond, CA), and leucine and
phenylalanine were converted to their t-BDMS derivatives to determine their respective TTRs by GC-
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MS (MSD 5973 System, Hewlett-Packard) analysis with [5,5,5-2H3]leucine, [ring-2H5]phenylalanine,
and [ring-13C6]phenylalanine enrichment standards and selective ion monitoring after electronionization (26). The following mass-to-charge ratios were monitored in the muscle protein extracts:
202 (m+2) and 203 (m+3) for [5,5,5-2H3]leucine; 237 (m+3) and 239 (m+5) for [ring2
H5]phenylalanine; and 238 (m+4) and 240 (m+6) for [ring-13C6]phenylalanine.
Calculations
The FSR of mixed muscle protein was calculated based on the incorporation rate of [5,5,5-2H3]leucine,
[ring-2H5]phenylalanine, or [ring-13C6]phenylalanine into muscle proteins by using a standard
precursor-product model as follows: FSR = Ep/Eic × 1/t × 100, where Ep is the change in enrichment
(TTR) of protein-bound leucine or phenylalanine in two subsequent biopsies, Eic is the mean
enrichment over time of the precursor for protein synthesis and t is the time between biopsies. The
free leucine or phenylalanine enrichment in muscle tissue fluid was chosen to represent the immediate
precursor for muscle protein synthesis (i.e., aminoacyl-t-RNA) (38). Values for FSR are expressed as
%·h-1.
Statistical Analysis
All data sets were tested for normality and the differences in FSR derived from the leucine and
phenylalanine tracer enrichments were evaluated by using either repeated measures analysis of
variance with tracer and study condition (fasted vs. fed) as the factors or paired Student's t-test as
appropriate. A P-value 0.05 was considered statistically significant.
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Results
Leucine and phenylalanine concentrations in plasma and enrichments in plasma, muscle tissue fluid,
and muscle proteins
Fasting plasma free leucine concentration was 121±5 µM and fasting plasma phenylalanine
concentration was 71±3 µM; both increased by ~15% during feeding to 134 ± 6 µM and 81±3 µM,
respectively. Plasma free leucine and phenylalanine enrichments were constant during both basal,
post-absorptive conditions and feeding (data not shown), but the enrichment was ~35% greater during
feeding than post-absorptively (Table 1). Muscle free leucine and phenylalanine enrichments were
~45% greater during feeding compared with post-absorptive conditions (Table 1). Muscle proteins
were significantly enriched above baseline levels with the leucine and phenylalanine tracers
(~0.011%) at the end of the basal, post-absorptive period; the enrichment increased further (to
~0.028%) during feeding until the end of the study, 2.5 h later (Table 1).
Muscle protein synthesis
The FSR calculated from the leucine labeling in muscle tissue fluid and proteins was ~20% greater (P
< 0.001), both during basal, post-absorptive conditions and feeding, than the respective FSRs
calculated from the phenylalanine enrichment data (Figure 1). However, the feeding induced increase
in the FSR (P = 0.011) was not different with the two tracers (Figure 2). The discrepancy between the
leucine and phenylalanine derived FSRs was independent of the phenylalanine isotopomer used; the
differences between the leucine derived FSR and the FSR derived from either the [ring2
H5]phenylalanine or the [ring-13C6]phenylalanine enrichment data were 0.013 ± 0.008 %·h-1 (FSRleu -
FSR[2H5]phe) and 0.012 ± 0.005 %·h-1 (FSRleu - FSR[13C6]phe) during basal, postabsorptive conditions (P =
0.92) and 0.013 ± 0.005 %·h-1 (FSRleu - FSR[2H5]phe) and 0.015 ± 0.007 %·h-1 (FSRleu - FSR[13C6]phe)
during feeding (P = 0.75).
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Discussion
It was our goal to discover whether or not primed, constant intravenous infusions of different stable
isotope labeled amino acid tracers with GC-MS analysis of muscle tissue samples provide similar
values for the human muscle protein synthesis rate during basal, postabsorptive conditions and during
feeding. We therefore measured the mixed muscle protein FSR simultaneously with a [2H3]leucine
and either a [2H5]phenylalanine or [13C6]phenylalanine tracer. We found that the FSR derived from
leucine labeling in muscle tissue fluid and proteins was approximately 20% greater than the FSR
derived from phenylalanine labeling in muscle tissue fluid and proteins, both during fasted and fed
conditions. The difference in results was independent of the phenylalanine tracer labeling (i.e., 2H5 vs
13
C6). Furthermore, the anabolic response to feeding was also independent of the tracer used. Thus,
qualitative comparison of the results from studies in which different tracer amino acids were used is
valid but quantitative comparison is not.
Both the phenylalanine and leucine tracer infusions in conjunction with muscle tissue analysis by
GCMS provided equally robust measurements of the muscle protein FSR. The population variance in
our study was ~35-40% independent of the tracer used. This is generally comparable to reports in the
literature (11, 16, 17, 24, 31, 34), in particular one in which the same techniques were used in a similar
subject population (37). Although, we (22) and others (10, 13, 25) have previously obtained much
tighter values in healthy, young men when using GC-combustion isotope ratio mass spectrometry
(GC-C-IRMS) for the analysis of muscle protein samples, probably because of the greater sensitivity
of GC-C-IRMS compared with GC-MS analysis (39).
The absolute mixed muscle protein FSR values obtained in our present study are also in good
agreement with the values reported in the literature, particularly those in a very similar study
population (37). We found that the basal rate of mixed muscle protein FSR was 0.051 ± 0.004 %·h-1
with the phenylalanine tracer and Volpi et al (37) report a value of 0.058 ± 0.005 %·h-1 for healthy
elderly men and women. Nonetheless, there is a wide range of values for the basal rate of mixed
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muscle protein FSR in the human quadriceps in the literature - the source of this variability cannot
readily be explained. Our data suggest that some of this variability may be due to the choice of tracer;
however, this can only account for a small portion of the wide range in values reported. We
determined that the difference in FSR that stems from the choice of tracer amino acid is ~20% whereas
literature values for mixed muscle protein FSR (obtained by using stable isotope labeled leucine or
phenylalanine tracers) vary by two-fold (from ~0.04 - 0.08%·h-1) even when restricted to reports that
studied healthy young individuals during basal post-absorptive conditions (11-13, 16, 23, 24, 36, 37,
41). The reliance of different precursor pools for the calculation of FSR may further add to the
variability in results from different studies but is not expected to account for more than an additional
20-40% (2, 7, 8, 13, 38). Thus, factors that are unrelated to the tracer and analyses employed must be
responsible for the inconsistency in values found in the literature.
Feeding, as expected, stimulated muscle protein synthesis in our study and the response (relative
increase in FSR) was independent of the tracer used to measure the FSR. Although, the feedinginduced increase in FSR in our study was small because of the age of our subjects and the small
amount of food, in particular protein, provided. Amino acids stimulate muscle protein synthesis in a
dose dependent-manner (5), and muscle of older adults is resistant to the anabolic effects of amino
acids (10, 12, 35). Thus, while feeding or hyperaminoacidemia has often been found to cause a
doubling or even greater increase in muscle protein FSR (5, 22, 35), we only found a ~20% increase in
muscle protein FSR when providing ~10 g of protein; this compares well with a ~doubling of muscle
protein FSR in older adults in response to oral administration of a mixed amino acid solution,
containing a total of 40 g of an amino acids given in small aliquots during 3 h (36).
The reasons for the discrepancy in the measured muscle protein synthesis rate with leucine and
phenylalanine tracers in our study are not clear. Since the difference in FSR obtained with the leucine
and phenylalanine tracers was independent of the phenylalanine isotopomer infused we can rule out
differences due to potential errors in the preparation of enrichment standards and speculate that the
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discrepancy must be attributed to the amino acid itself. In fact, Iresjö et al. (15) recently described a
phenomenon that would fit this notion in cell culture where phenylalanine stimulated the incorporation
of a phenylalanine but not a tyrosine tracer into protein and vice versa. This was observed when large
amounts of these amino acids (405 µM) were in the medium and could therefore be related to amino
acid competition for transporters. In our study, we can rule this mechanism out because plasma amino
acid availability is far below the concentrations which cause saturation of transmembrane leucine and
phenylalanine transport (Km 20 mM) (14); furthermore, the phenylalanine and leucine
transmembrane enrichment gradients in muscle were very similar, which would not be expected if
there were selective inhibition of leucine or phenylalanine uptake into muscle. It is possible that the
enrichment of phenylalanine and leucine in muscle tissue fluid, which we used as the precursor pool
for calculating the FSR, do not provide equally good estimates of the respective aminoacyl-tRNA
enrichment. It has been suggested that leucine exhibits functional compartmentalization within the
muscle, with a pool of leucine directed towards oxidation and a separate pool directed towards protein
synthesis (30). In contrast, phenylalanine is not oxidized within skeletal muscle (39); thus, the
phenylalanine enrichment in muscle tissue fluid probably reflects its tRNA charged pool better than
the leucine enrichment in muscle tissue fluid, which represents a mixture of its different functional
compartments. We did not directly measure the aminoacyl-tRNA enrichment because the amino acid
enrichment in muscle fluid has been shown to be a suitable surrogate (2, 18, 38). Furthermore, it
requires a large amount of tissue (~1 g) to analyze the aminoacyl-tRNA (38) and it is technically very
challenging to obtain accurate and robust results (38, 39). In fact, differences between amino acid
labeling in muscle tissue fluid and amnioacyl-tRNA that could account for the differences in FSR
obtained with leucine and phenylalanine tracers in our study are probably too small to be measured
accurately. Most important of all, it is (for those reasons) usually not done. Furthermore, the
conclusions from our study are the same even if we assume, as suggested by Lobley et al (19), that the
amino acid precursor enrichment in the extracellular space (blood) provides a closer approximation of
the true precursor enrichment for muscle protein synthesis than an intracellular source. When we
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calculated the FSR with plasma leucine and phenylalanine enrichments as the precursors, FSRPhe was
also ~20% less than FSRLeu (during fasting conditions FSRLeu was 0.044 ± 0.004 %/h and FSRPhe was
0.035 ± 0.003 %/h, P<0.01; during feeding, FSRLeu was 0.059 ± 0.005 %/h and FSRPhe was 0.049 ±
0.004 %/h, P<0.02; ;means ± SEM).
Interestingly, very similar differences to the ones in our study have been found by comparing the basal
rate of muscle protein synthesis by using the flooding dose technique (21) and when the basal rate of
muscle protein synthesis was calculated from non-oxidative leg leucine and phenylalanine uptake with
the classic 2-pool arterio-venous balance model (3). McNurlan et al. (21) found that when using the
flooding dose technique, the FSR determined with a leucine tracer is approximately 15% greater than
the FSR determined with a phenylalanine tracer; the difference failed to reach statistical significance
in some of the comparisons, most likely due to lack of statistical power. The arterio-venous balance
approach used by Bennet et al (3) is based on entirely different assumptions and independent of the
precursor pool issue discussed, which suggests that other factors also contribute to the amino acid
tracer dependent rate of muscle protein synthesis. However, unlike in our study, the relative increase
in muscle protein synthesis in response to hyperaminoacidemia was also more or less marked
depending on the tracer used for evaluation of muscle protein metabolism with leucine and
phenylalanine tracers in a limb arterio-venous-balance model (i.e., greater with phenylalanine than
leucine in the study by Bennet et al (3) and greater with leucine than phenylalanine by a study by
Tessari et at (33)). This, however, could largely be due to errors in measuring limb leucine oxidation.
Preferential incorporation of leucine into fast turning over muscle proteins (e.g. sarcoplasmic and
mitochondrial proteins (1, 22)), or vice versa, preferential incorporation of phenylalanine into slower
turning over proteins (e.g., myofibrillar proteins (1, 22)) could cause the apparent discrepancy in FSR.
However, the relative contents of leucine and phenylalanine in the major muscle protein fractions
(myofibrillar, sarcoplasmic and insoluble proteins) are very similar (9). It is therefore unlikely that
such a phenomenon contributed significantly to the differences in FSR obtained by using a leucine vs
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a phenylalanine tracer. Thus, although our study leaves some questions concerning the potential
source of discrepancy unanswered, it provides meaningful and important novel information
concerning the use of leucine and phenylalanine tracers for the investigation of muscle protein
metabolism.
In summary, we demonstrate that there is a considerable and statistically highly significant difference
in the rate of muscle protein synthesis obtained via constant infusion of leucine and phenylalanine
tracers. However, the anabolic response to feeding is independent of the tracer used. Therefore,
different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle
protein synthesis. Furthermore, data from studies that applied different amino acid tracers to measure
muscle protein FSR can only be compared quantitatively with respect to the relative changes in FSR
due to anabolic stimuli such as hyperaminoacidemia or feeding whereas comparison of absolute rates
of protein synthesis is not valid.
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Acknowledgements
The authors wish to thank Nicole Wright for subject recruitment and technical assistance, and the
study subjects for their participation.
The study was supported by US National Institutes of Health grants AR 49869, AG 025501, RR 00036
(General Clinical Research Center), RR 00954 (Biomedical Mass Spectrometry Resource), and DK
56341 (Clinical Nutrition Research Unit).
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Figure Legends
Figure 1. Skeletal muscle protein fractional synthesis rate (FSR) during basal, post-absorptive
conditions (fasted) and feeding measured by using stable isotope labeled leucine and phenylalanine
tracers. Values are means ± SEM. ANOVA revealed significant effects for tracer amino acid used (P
< 0.001) and feeding (P = 0.011), but no interaction (P = 0.80).
Figure 2. Feeding-induced increase in skeletal muscle protein fractional synthesis rate (FSR) when
using stable isotope labeled leucine and phenylalanine tracers. Values are means ± SEM and are
statistically not significantly different (P = 0.69).
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Table 1. Leucine and phenylalanine enrichments in plasma, muscle tissue fluid, and muscle proteins
during basal, post-absorptive conditions (fasted) and feeding.
Plasmaa
Muscle tissue fluidb
Muscle proteinsb
Fasted
0.0708 ± 0.0027
0.0472 ± 0.0020
0.00011 ± 0.00001
Fed
0.0943 ± 0.0030
0.0681 ± 0.0023
0.00026 ± 0.00002
Fasted
0.0969 ± 0.0071
0.0687 ± 0.0043
0.00011 ± 0.00002
Fed
0.1290 ± 0.0069
0.1006 ± 0.0037
0.00028 ± 0.00002
Fasted
0.0994 ± 0.0067
0.0626 ± 0.0049
0.00012 ± 0.00001
Fed
0.1359 ± 0.0085
0.0951 ± 0.0073
0.00029 ± 0.00003
[5,5,5-2H3]leucine
[ring-2H5]phenylalanine
[ring-13C6]phenylalanine
Values (tracer to tracee ratios corrected for the background enrichment) are means ± SEM. [5,5,52
H3]leucine was used in all 25 studies; [ring-2H5]phenylalanine was used in 11 studies; [ring-
13
C6]phenylalanine was used in 14 studies.
a
Average values during basal, post-absorptive and fed conditions.
b
Values obtained at the end of the basal period (i.e., 210 min after the start of the tracer infusion) and
the end of the feeding period (i.e., 360 min after the start of the tracer infusion).
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Mixed muscle protein FSR (%·h-1)
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Phenylalanine
Fasted
Leucine
Phenylalanine
Fed
Leucine
Percent change in FSR from fasted condition
Page 23 of 23
40
30
20
10
0
Phenylalanine
Leucine