Essential amino acids and muscle protein recovery from
resistance exercise
Elisabet
Børsheim,
Kevin
D.
Tipton,
Steven E.
Wolf, and
Robert R.
Wolfe
Metabolism Unit, Department of Surgery, Shriners Hospital
for Children/Galveston, University of Texas Medical Branch,
Galveston, Texas 77550
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ABSTRACT |
This study tests the
hypothesis that a dose of 6 g of orally administered essential
amino acids (EAAs) stimulates net muscle protein balance in healthy
volunteers when consumed 1 and 2 h after resistance exercise.
Subjects received a primed constant infusion of
L-[2H5]phenylalanine and
L-[1-13C]leucine. Samples from femoral
artery and vein and biopsies from vastus lateralis were obtained.
Arterial EAA concentrations increased severalfold after drinks. Net
muscle protein balance (NB) increased proportionally more than arterial
AA concentrations in response to drinks, and it returned rapidly to
basal values when AA concentrations decreased. Area under the curve for
net phenylalanine uptake above basal value was similar for the first
hour after each drink (67 ± 17 vs. 77 ± 20 mg/leg,
respectively). Because the NB response was double the response to two
doses of a mixture of 3 g of EAA + 3 g of nonessential
AA (NEAA) (14), we conclude that NEAA are not necessary
for stimulation of NB and that there is a dose-dependent effect of EAA
ingestion on muscle protein synthesis.
muscle protein metabolism; essential amino acids; stable isotopes
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INTRODUCTION |
MUSCLE PROTEIN
SYNTHESIS is stimulated in the recovery period after resistance
exercise (4, 8). However, the rate of muscle protein
breakdown is also increased, thereby blunting the change in the net
balance between synthesis and breakdown. Although net muscle protein
balance is generally improved after resistance exercise, it remains
negative. Therefore, nutrient intake is necessary to achieve positive
net muscle protein balance.
The optimal composition and amount of nutrient ingestion to maximally
stimulate muscle protein synthesis after resistance exercise are not
known. It is clear that amino acids or protein should be a component,
as we have previously shown that either the infusion (5)
or ingestion of a large amount (17) (30-40 g) of
amino acids after exercise stimulates muscle protein synthesis. Furthermore, muscle protein synthesis was increased 3.5-fold when only
a small amount (6 g) of a mixture of essential amino acids (EAAs) was
given along with 35 g of carbohydrate after resistance exercise
(16). The latter results (16) suggest that a
relatively small amount (i.e., 6 g) of EAAs can effectively
stimulate muscle protein synthesis, but the independent effects of
amino acids and carbohydrate were not assessed. Thus a principal goal
of this study was to determine the independent effect of ingestion of a
bolus of 6 g of EAAs on net muscle protein synthesis after
resistance exercise.
The proportional contributions of individual amino acids to a
mixture ingested after resistance exercise can potentially affect the
response. In an earlier study (17), we concluded that the ingestion of only EAAs was necessary for stimulation of muscle protein
synthesis, because the effect on net muscle protein synthesis was
similar when subjects were given either 40 g of a balanced mixture
[21.4 g EAAs and 18.6 g nonessential amino acids (NEAAs), roughly
in proportion to their relative contributions to muscle protein], or
40 g of only EAAs. However, if the NEAAs are not necessary for
stimulation of synthesis, it is unclear why ingestion of the EAAs alone
did not stimulate muscle protein synthesis to a greater extent than did
the balanced mixture. It could be that, in fact, the NEAAs served no
function, but the amount of EAAs in the balanced mixture (21 g)
exceeded the maximal effective dose. In this case, intake of more than
21 g of EAAs (i.e., 40 g) would have no further effect than
that already elicited by 21 g. If true, then comparison of less
than the maximally effective dose of EAAs with a comparable amount of a
balanced mixture of EAAs + NEAAs should reveal a significantly
greater effect of the EAAs. Thus a secondary goal of this project was
to compare the response to 6 g of an EAA mixture with the response
to 6 g of a balanced mixture of amino acids, which included ~3 g
of EAAs and 3 g of NEAAs. We speculated that 6 g of EAA would
be less than the maximally effective dose of EAA, and thus there would be a greater response to the EAAs than to the mixture of EAAs and
NEAAs. The results of the response to 6 g of the balanced mixture
have been published previously (14).
The composition of the mixture of EAAs we have tested in this and
previous studies (16) was originally based on the
composition of muscle protein, with the idea being to increase the
availability of each EAA in proportion to its requirement for the
synthesis of muscle protein. However, different clearance rates of
individual amino acids could result in rates of uptake that do not
directly correspond to the composition of the ingested mixture. This
would be reflected by disproportionate changes in concentrations of blood amino acids compared with the composition of the ingested mixture. Therefore, another goal of the current study was to measure blood and intramuscular concentrations of all amino acids before and
after ingestion of the mixture to determine whether the mixture achieved the goal of causing proportional increases in all EAAs, and if
any NEAA dropped sufficiently to become potentially rate limiting for
protein synthesis. This information could help to formulate a new
mixture that might be more effective than the EAA mixture we have used.
Not only is the composition of nutrient ingestion after exercise
potentially important, but also the pattern of ingestion may affect the
response of muscle protein synthesis. We have previously shown that
amino acid concentration, per se, is not a direct determinant of muscle
protein synthesis. Thus, when blood amino acid concentrations were
elevated to a steady-state level about twice the basal values for
6 h, muscle protein synthesis was stimulated over the first 2 h but thereafter returned to the resting level despite persistent elevations in blood amino acid concentrations (6).
Similarly, in our earlier study after exercise (16), the
rate of net muscle protein synthesis returned to the basal level 60 min
after the ingestion of a bolus of EAAs + carbohydrate, even though
the blood amino acid concentrations were still approximately double the basal level. These observations are best explained by the muscle becoming refractory to a persistent elevation in amino acid
concentrations. If true, it follows that the synthetic response to
ingestion of a bolus of amino acids would be refractory to a second
dose until the concentrations from the first dose returned to the basal
level. It was therefore a further goal of this study to determine
whether the response of net muscle protein synthesis to a second dose of amino acids would be affected by the ingestion of an initial dose
1 h earlier.
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MATERIALS AND METHODS |
Subjects.
Six healthy subjects (3 men and 3 women), 19-25 yr of age,
participated in the study (Table 1).
Subjects were recreationally active but were not involved in a
consistent resistance or endurance-training program. They were fully
informed about the purpose and procedures of the study before written
consent was obtained. Before participation in the experiments, each
subject had a complete medical screening, including vital signs, blood
tests, urine tests, and a 12-lead electrocardiogram, for determination
of health status at the General Clinical Research Center (GCRC) of the
University of Texas Medical Branch (UTMB) at Galveston, TX. The
protocol was approved by the Institutional Review Board of the UTMB.
Preexperimental procedures.
At least 1 wk before an experiment, subjects were familiarized with the
exercise protocol, and their one repetition maximum (1RM, the maximum
weight that can be lifted for one repetition) was determined by the
procedure described by Mayhew et al. (13) (Table 1). The
leg volume of each subject was estimated from anthropometric measures
of leg circumference and height at multiple points down the length of
the leg (Table 1).
Experimental protocol.
Each subject was studied once. The subjects were instructed not to
exercise for 2 days before an experiment, not to make any changes in
their dietary habits, and not to use tobacco or alcohol during the last
24 h before an experiment. The subjects reported to the GCRC in
the evening before an experiment for an overnight stay and were fasted
from 10:00 PM.
The experimental protocol is shown schematically in Fig.
1. At ~6:00 AM, an 18-gauge
polyethylene catheter (Cook, Bloomington, IN) was inserted into an
antecubital arm vein for the primed continuous infusion of stable
isotopes of amino acids. After obtaining a blood sample for measurement
of background amino acid enrichment, a primed, constant infusion of
[15N2]urea was started at
180 min (~6:30
AM). At
120 min (~7:30 AM), a primed, constant infusion of
L-[ring-2H5]phenylalanine
and L-[1-13C]leucine was started. The
following infusion rates (IR) and priming doses (PD) were used
Isotopes were purchased from Cambridge Isotopes (Andover, MA).
They were dissolved in 0.9% saline and were filtered through a 2-µm
filter before infusion. The infusion protocol was designed to allow the
quantification of the effect of the drink on muscle protein synthesis
and breakdown. The net balance between protein synthesis and breakdown
(net muscle protein synthesis) was considered to be the primary end
point of the study, and urea production was measured isotopically to
assess short-term changes in total amino acid oxidation.
At ~7:30 AM, 3-Fr 8-cm polyethylene catheters (Cook) were inserted
into the femoral vein and the femoral artery with the subject under
local anesthesia. Both femoral catheters were used for blood sampling,
and the femoral arterial catheter was also used for indocyanine green
dye (ICG) infusion for determination of leg blood flow
(3). A constant infusion of ICG (0.5 mg/min) was given at
intervals during the experiment (Fig. 1). The infusion ran for
10 min
before peripheral and femoral venous blood samples were drawn for
measurement of blood flow. The peripheral venous blood samples were
drawn from an 18-gauge polyethylene catheter inserted into an
antecubital vein of the arm opposite that into which the amino acids
were infused. Patency of catheters was maintained by saline infusion.
Subjects rested in bed until the exercise started at
45 min (8:45
AM). Subjects performed 10 sets of 10 repetitions of leg presses and 8 sets of 8 repetitions of leg extensions at 80% of the 1RM. Each set
was completed in ~30 s with a 2-min rest between sets, and the entire
bout was completed in ~40 min. This exercise bout was difficult for
all subjects to complete. The exercise ended 3 h after the start
of the urea infusion and 2 h after the start of the amino acid
infusion. Subjects then returned to bed, and the first samples were
taken 30 min after the end of exercise.
At 1 and 2 h postexercise, the subjects were given an oral
supplement of 0.087 g of essential amino acids (EAA)/kg body weight. The nutritional composition was designed to increase intramuscular availability of EAA in proportion to the composition of muscle protein
(Table 2). Each supplement solution was
composed of 425 ml of double-distilled water, the appropriate mixture
of EAA, and an artificial sweetener.
L-[ring-2H5]phenylalanine
and L-[13C]leucine were added to the drink in
amounts to equal 8% enrichment, to allow maintenance of isotopic
equilibrium during ingestion of the AA drinks.
To measure the isotopic enrichment of free amino acid tracers in the
muscle, muscle biopsies were sampled at 30, 90, 150, and 240 min after
exercise (Fig. 1). With subjects under local anesthesia, the biopsies
were taken from the lateral portion of the vastus lateralis
~10-15 cm above the knee. A 5-mm Bergstrom biopsy needle (Depuy,
Warsaw, IN) was used to sample ~30-50 mg of mixed muscle tissue.
The sample was quickly rinsed, blotted, immediately frozen in liquid
nitrogen, and stored at
80°C for later analysis.
Blood samples were drawn for determination of net muscle protein
balance from the femoral artery and venous catheters at 30 min after
exercise, at 70, 80, 90, 105, 130, 140, 150, and 165 min after exercise
(corresponding to 10, 20, 30, and 45 min after each drink), and at 180, 210, 220, and 240 min after exercise (Fig. 1). The samples were
analyzed for phenylalanine and leucine enrichments and concentrations.
To allow sampling from the femoral artery, the dye infusion was stopped
for <10 s and then quickly resumed.
Sample analyses.
Blood samples for determination of amino acid enrichment and
concentrations were immediately precipitated in preweighed tubes containing 15% sulfosalicylic acid (SSA), and a weighed amount of an
appropriate internal standard consisting of amino acids labeled
differently from the infused amino acids was added (3, 4,
15). The supernatant was passed over a cation exchange column
(Dovex AG 50W-8X, 100-200 mesh H+ form; Bio-Rad
Laboratories, Richmond, CA) and dried under vacuum with a Speed Vac
(Savant Instruments, Farmingdale, NY). Enrichments of intracellular
free amino acids were then determined on the tertiary-butyldimethylsilyl (t-BDMS) derivatives
using GC-MS (Hewlett-Packard 5973, Palo Alto, CA) and selected ion
monitoring (21). Enrichments were expressed as
tracer-to-tracee ratios. Appropriate corrections were made for
overlapping spectra (21).
To determine muscle intracellular enrichment of infused tracers, muscle
tissue was weighed and the protein precipitated with perchloroacetic
acid. The tissue was then homogenized and centrifuged, and the
supernatant was collected. The procedure was then repeated, and the
pooled supernatant was processed in the same way as the supernatant
from the blood samples.
Urea production was calculated from enrichment and tracer infusion
rates, as described previously (20). ICG concentration in
serum for the determination of leg blood flow was measured spectrophotometrically at
= 805 nm (10, 19).
Plasma samples and muscle intracellular fluid were also analyzed for
amino acid concentrations by high-performance liquid chromatography
(Waters Alliance HPLC System 2690, Milford, MA). Plasma glucose
concentration was determined enzymatically by an automated system (YSI
1500, Yellow Spring Instruments, Yellow Springs, OH). Plasma insulin concentration was determined by a radioimmunoassay method (Diagnostic Products, Los Angeles, CA).
Calculations.
Net muscle phenylalanine balance, which was considered as the
primary end point, was calculated as follows: (phenylalanine arterial
concentration
venous concentration) × blood flow. Because phenylalanine is neither produced nor metabolized in muscle, net phenylalanine balance reflects net muscle protein synthesis, provided there are no significant changes in the free intracellular pool of
phenylalanine. Area under the curve (AUC) of net phenylalanine uptake
was determined for each individual hour after drink ingestion, with net
uptake at t = 30 min postexercise used as the zero
point for each hour of the recovery period. This approach assumes a constant net balance after the basal sample if amino acids are not
given. The basis for this assumption is the relatively constant net
balance for 3 h after exercise in the absence of nutrient intake
that we have previously observed when using the same exercise protocol
(16).
Leg amino acid kinetics were calculated according to a three-pool
compartment model previously presented (2, 3). Hourly averages for blood flow and blood and muscle amino acid concentrations and enrichments were used in the calculation of leg amino acid kinetics. Kinetic parameters calculated for both amino acid tracers included intracellular de novo appearance, irreversible disappearance from the intracellular compartment, and the rate of transport from
blood into muscle. In the case of phenylalanine, irreversible loss from
the intracellular pool can only be to protein synthesis, because it is
not oxidized in the muscle. Because leucine can be oxidized in muscle,
the irreversible loss is due to synthesis + oxidation. Neither
leucine nor phenylalanine can be synthesized in muscle, so de novo
appearance of both amino acids is due entirely to breakdown. We also
calculated the rate of release of phenylalanine and leucine from
protein breakdown into blood (Ra) and incorporation of
phenylalanine from blood into muscle protein (Rd).
Calculation of Ra and Rd, as well as the
three-pool kinetic factors, requires an isotopic, but not
physiological, steady state. By adding an appropriate amount of tracer
to the ingested amino acids, we were able to maintain a relatively
stable isotopic steady state, despite changing concentrations of plasma
amino acids (see below). Thus the difference between total protein
synthesis and Rd is the amount of recycling of
phenylalanine that was released from breakdown and directly
incorporated into protein without entering the blood. Similarly, the
difference between total protein breakdown and Ra is the
amount of phenylalanine from breakdown that was directly reincorporated
into protein, rather than being released into blood.
Statistical methods.
Overall significance of differences in response with time was tested by
repeated-measures analysis of variance followed by Dunnett's test
(SigmaStat 2.03, SPSS, Chicago, IL). The response to the second drink
was compared with the response to the first drink by Tukey's test.
Results were considered significant if P < 0.05. The
results are presented as means ± SE unless otherwise noted.
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RESULTS |
Phenylalanine concentration and balance.
At 30 min after exercise, the arterial blood phenylalanine
concentration was 59 ± 4 nmol/ml. The concentration rose
significantly within 10 min of ingestion of the EAA drink (Fig.
2). Phenylalanine concentration declined
before the second drink but stayed significantly above predrink values
until 240 min after exercise. The response to the second drink was
comparable to that of the first.

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Fig. 2.
Changes in arterial phenylalanine concentration and net
phenylalanine balance during the recovery period after a resistance
exercise bout (means ± SE; n = 6). Essential
amino acid (EAA) drink was consumed at 60 and 120 min after exercise.
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Phenylalanine net balance followed the same time pattern as the blood
concentration changes, with rapid changes in response to arterial blood
phenylalanine changes (Fig. 2). However, the early increase of net
uptake was proportionately greater than the change in arterial
concentration, and the rate of net balance returned to the basal value
40 min after ingestion of the first drink, despite persistent elevation
of arterial phenylalanine concentration. Net balance also increased
significantly in response to the second drink (Fig. 2) and returned to
the basal value during the 3rd h. The AUC for net uptake of
phenylalanine above the basal value was similar for the 1st h after the
first drink (67 ± 17 mg/leg) and the 1st h after the second drink
(77 ± 20 mg/leg). However, the AUC decreased significantly by the
3rd h (
5 ± 20 mg/leg), despite the fact that the arterial
concentration was significantly elevated until 240 min after the first drink.
Muscle intracellular phenylalanine concentration was 57 ± 3 nmol/ml before intake of the drinks. After intake of drinks, the concentration increased, and the value at 150 min (115 ± 24 nmol/ml) was significantly higher than the baseline value. At 240 min, the concentration (88 ± 14 nmol/ml) was not statistically
different from the baseline value.
Phenylalanine kinetics.
Enrichment of phenylalanine in blood was relatively constant throughout
the experiment despite the large changes in concentration (Fig.
3). No statistical changes in enrichment
were observed during the different calculation periods. This was
accomplished by adding tracer to the ingested amino acids.

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Fig. 3.
Arterial
L-[2H5]phenylalanine and
L-[1-13C]leucine enrichments during the
recovery period after a resistance exercise bout (means ± SE;
n = 6). EAA drink was consumed at 60 and 120 min after
exercise.
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The rate of appearance of phenylalanine into the blood from the muscle
(Ra) did not change during the first 2 h after the drink but increased significantly during the 3rd h (Fig.
4). The average rate of disappearance
(Rd) of phenylalanine from the blood into the muscle (i.e.,
protein synthesis from plasma phenylalanine) increased significantly
from the basal value during the first 2 h after intake of drink
but was not different from the basal value during the 3rd h.

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Fig. 4.
Phenylalanine kinetics across leg before intake of drink
and during the 1st, 2nd, and 3rd h after the first drink (means ± SE; n = 6). Ra, rate of appearance of
phenylalanine into the blood; Rd, rate of disappearance of
phenylalanine out of the blood. *P < 0.05, value vs.
predrink value.
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Ingestion of the EAA drink caused inward transport of phenylalanine
from the artery to the muscle to increase (Table
3, P = 0.055). At 30 min
after exercise, the total rate of intracellular phenylalanine release
from protein breakdown was 43 ± 9 nmol · min
1 · 100 ml leg
1
(Table 3). There was no change in Ra during the first
2 h after the first drink, but it increased significantly during
the 3rd h to 75 ± 13 nmol · min
1 · 100 ml leg
1.
The rate of utilization of phenylalanine for protein synthesis was
30 ± 9 nmol · min
1 · 100 ml
leg
1 at 30 min after exercise, and this value
increased significantly during the 1st and 2nd h after drink to
121 ± 16 and 139 ± 21 nmol · min
1 · 100 ml leg
1,
respectively (Table 3). During the 3rd h after drink, the value returned to the basal level, despite the fact that the total
intracellular appearance of phenylalanine (inward transport + breakdown) was elevated approximately threefold above the basal value.
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Table 3.
Calculated kinetics for phenylalanine before drink and during 1st, 2nd,
and 3rd h after intake of first drink
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Leucine concentration and kinetics.
The pattern of response of blood concentration and kinetics of leucine
was similar to that of phenylalanine. As was the case for
phenylalanine, leucine tracer was added to the ingested amino acids.
Therefore, enrichment of leucine in blood was essentially constant
throughout the experiment despite changes in concentrations (Fig. 3).
Leucine concentration increased significantly within 10 min of
ingestion of the first EAA drink, from a value of 118 ± 4 nmol/ml at 30 min after exercise to a peak of 407 ± 24 nmol/ml at 20 min after ingestion of the drink. The concentration declined before the
second drink, but not to the basal level. Leucine concentration increased again after ingestion of the second drink, and although the
value again began falling 30 min after ingestion of the second drink,
it was still elevated above basal at 210 min after ingestion of the
first drink. Muscle intracellular leucine concentration increased
significantly from the basal value of 144 ± 7 to 307 ± 52 nmol/ml at 150 min. At 240 min, the concentration was not different
from basal value at 30 min after exercise.
Leucine net uptake increased as a result of drink. No significant
difference was found between the AUC value for net uptake during the
1st h after the first drink (159 ± 36 mg/leg) and that during the
1st h after the second drink (165 ± 32 mg/leg), but the area was
significantly smaller by the 3rd h (20 ± 22 mg/leg).
The average Ra of leucine into the blood over each of the
hours after intake of drink did not change, whereas the average rate of
muscle protein synthesis and oxidation from plasma leucine increased
significantly from basal values during the first 2 h after intake
of drink but was not different from basal values during the 3rd h (Fig.
5). As a result, leucine net balance
increased from a slightly negative basal value to ~250-260
nmol · min
1 · 100 ml leg
1
during the 2 h after the first drink (not significant between hours; Fig. 5). During the 3rd h, net balance decreased again.

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Fig. 5.
Leucine kinetics across leg before intake of drink and
during the 1st, 2nd, and 3rd h after the first drink (means ± SE;
n = 6). *P < 0.05, value vs. predrink
value.
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Table 4 shows leucine kinetics. The
delivery of leucine to the leg muscle increased as a result of intake
of the drink. Also, the movement of leucine into the muscle increased
and stayed elevated above the predrink value throughout the following
3 h. The rate of intracellular Ra of leucine increased
slightly after the drink, and during the 1st h the value was
significantly different from the predrink value. The rate of
utilization of intracellular leucine, which includes utilization for
both protein synthesis and oxidation, increased significantly during
the 1st and 2nd h after drink, whereas it returned to basal value
during the 3rd h.
The rate of incorporation of leucine into protein can be calculated
from the rate of protein synthesis as calculated with the phenylalanine
tracer and the ratio between leucine and phenylalanine in mixed muscle
protein. The ratio between leucine and phenylalanine in mixed muscle
protein was calculated from the ratio between the rates of leucine and
phenylalanine release from muscle protein breakdown at the predrink
time point (30 min). The mean ratio for the group was 2.6 ± 0.2. When calculated accordingly, the rate of leucine incorporated into
protein increased significantly during the 1st h after each drink, but
during the 3rd h it decreased to a value not different from the
predrink rate (Table 4). The rate of leucine oxidation was calculated
from the difference between the rate of utilization of intracellular
leucine and the rate at which leucine was used for protein synthesis.
This calculation showed that leucine oxidation increased significantly
during the 1st h after the first drink but that, during the 2nd and 3rd
h, the values were not different from the predrink value.
Plasma and muscle intracellular amino acid concentrations.
Intake of drinks increased the arterial plasma concentrations of total
EAAs significantly. At 150 min after exercise (90 min after the first
drink and 30 min after the second drink), the concentrations for most
of the EAAs given in the drinks were increased by ~75-150%,
except for isoleucine and leucine, which showed greater responses
(317 ± 69 and 212 ± 29% increase, respectively; Fig. 6A). Arterial plasma
concentrations of the amino acids not given in the drink generally
showed smaller changes (Fig. 6B). Tyrosine increased
significantly, whereas glycine, alanine, and tryptophan decreased
during the study.

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Fig. 6.
Percentage changes in arterial plasma concentrations of EAAs
(A), arterial plasma concentrations of nonessential
amino acids (NEAAs) and tryptophan (B), intracellular
concentrations of EAAs (C), and intracellular
concentrations of NEAAs and tryptophan (D) from 30 min to
150 min after exercise (means ± SE; n = 6). EAA
drink was consumed at 60 and 120 min after exercise.
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Whereas there was no change in intracellular concentration of total
EAAs or in total NEAAs during the recovery period, some individual
changes were observed. Threonine was not different between first and
second biopsies but increased thereafter to a concentration at 150 min
significantly higher than predrink concentration (741 ± 99 vs.
561 ± 59 nmol/ml H2O). Similarly, valine, isoleucine,
and leucine concentrations all increased after the first drink. The
concentrations increased further at 150 min but decreased again at the
end of the study at 240 min. For valine, the values at 150 and 240 min
were significantly higher than the predrink value (228 ± 17 and
212 ± 16 vs. 168 ± 9 nmol/ml, respectively). For
isoleucine, 90- and 150-min values were higher than the predrink value
(86 ± 5 and 98 ± 6 vs. 61 ± 2 nmol/ml, respectively).
For leucine, 90-, 150-, and 240-min values were higher than the
predrink value (249 ± 5, 297 ± 22, and 240 ± 15 vs.
186 ± 7 nmol/ml, respectively). For the amino acids not given in
the drink, a significant fall during the day was found for asparagine,
serine, arginine, and alanine. The relative changes in intracellular
concentrations of EAAs were small compared with the changes in arterial
plasma (Fig. 6).
Urea production.
Plasma urea enrichment was stable at ~4% throughout the study. Thus
no change in urea production was seen over time. The value was stable
at 330 µmol · kg
1 · h
1.
Plasma glucose and insulin concentrations.
Arterial glucose concentration decreased slightly during the experiment
from a value of 91 ± 3 mg/dl at 30 min after exercise to 87 ± 3 mg/dl at the end of the study (P < 0.05). No
change was found in net uptake of glucose during the study. Similarly, insulin concentrations did not change over time.
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DISCUSSION |
The principal finding of this study was that ingestion of a
relatively small amount (6 g) of EAA effectively stimulated net muscle
protein balance after resistance exercise. The response of net muscle
protein balance could be explained largely by a change in synthesis, as
the rate of breakdown was not significantly affected. The response of
net balance was about twice the previous published response to 6 g
of mixed amino acids (14), leading to the conclusions that
there is a dose response to the amount of EAAs given and that ingestion
of NEAAs is not necessary to stimulate net protein synthesis. The
latter conclusion is supported by changes in NEAA concentrations after
ingestion of the EAA drink. Although plasma concentrations of some
individual NEAAs fell after ingestion of EAAs, intracellular
concentrations of NEAAs were generally maintained, indicating that
availability of NEAAs did not limit the response of muscle protein
synthesis. Finally, the response of net muscle protein synthesis to the
drink ingested 2 h after exercise was comparable to that of the
drink ingested 1 h after exercise.
Independent effect of EAA on net protein balance.
In previous studies, both intravenous infusion (5) and
oral intake of amino acids (17) after resistance exercise
have been shown to stimulate muscle protein synthesis. However, optimal proportions and amounts of individual amino acids to stimulate muscle
protein synthesis are not known. Intake of only a small amount (6 g) of
a mixture of EAA given along with 35 g of carbohydrate after
resistance exercise transiently increased muscle protein synthesis
3.5-fold (14), but the independent effects of amino acids
and carbohydrate were not assessed. The results of the present study
show that ingestion of 6 g of EAA alone without addition of
carbohydrate effectively stimulated muscle protein synthesis after
resistance exercise (Figs. 4 and 5, Tables 3 and 4).
In a recent study, Miller et al. (14) compared the
independent and combined effects of a balanced mixture of amino acids (i.e., EAAs + NEAAs) and carbohydrate on muscle protein synthesis after resistance exercise. Addition of 35 g of carbohydrate to 6 g of mixed AA did not cause a greater stimulation of net muscle protein synthesis than the AAs alone. The effect of adding carbohydrate to 6 g of EAA can be seen in Fig. 7,
which compares the AUC for net phenylalanine uptake for the 1st h after
intake of drink (i.e., 60-120 min) in the present study with the
previously published response to 6 g of EAAs plus 35 g of
carbohydrate (16). The additional carbohydrate provided no
advantage to EAAs alone. From these results, it is clear that the
stimulation of protein synthesis by EAAs is not a caloric effect,
because ingestion of an additional 3 g of EAA (difference in EAA
content between mixed AA and EAA groups) caused a much larger effect
than addition of 35 g of carbohydrate to the amino acid mixture
(Fig. 7), and 35 g of carbohydrate alone had a minimal effect
(14). Although direct comparison with historical data may
be problematic, the cited studies (14, 16) were performed in the same laboratory, approximately contemporaneously, and by use of
the same general experimental protocol and techniques.

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Fig. 7.
Area under curve for net uptake of phenylalanine over
1 h after ingestion of 6 g of different amino acid drinks.
MAA, mixed amino acids (n = 7; values from Ref.
14); MAA + CHO, MAA + 35 g carbohydrate
(n = 7; values from Ref. 14); EAA, present
study; EAA + CHO, EAA + 35 g carbohydrate
(n = 6; values from Ref. 16).
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The results of the current study indicate that ~3.5 ± 1.1 g of muscle protein were synthesized during the 3 h after the
first drink in one leg, or 7.0 ± 2.0 g in both legs, on the
basis of irreversible loss of phenylalanine and the composition of
muscle protein. This represents ~27% of ingested EAAs, because each
gram of muscle protein synthesized includes both EAAs and NEAAs. When account of the water content of muscle (~73%) is taken, this would represent a net gain of ~26 g of muscle tissue synthesized in response to the drinks. This magnitude of gain in muscle mass would
therefore require many weeks of comparable response to become detectable by available means to quantify changes in muscle mass, during which time all variables such as activity and other nutritional intake would have to be absolutely controlled. Thus, although the
stimulation in net muscle synthesis resulting from the total of 12 g of EAA reported in this study would eventually be expected to enhance
the rate of muscle gain during a resistance training program, an
outcome study based on measured differences in muscle mass would have
to be carefully designed and executed to demonstrate an effect of an
EAA supplement.
EAA vs. NEAA.
Because ingestion of 6 g of EAAs alone stimulates muscle protein
synthesis after resistance exercise, NEAAs are apparently not required
to stimulate protein synthesis. In a previous study, Tipton et al.
(17) found no difference in net muscle protein balance
response to the ingestion of 40 g of mixed AAs (roughly in
proportion to their relative contributions to muscle protein) or
40 g of EAAs. These results could also be interpreted to indicate that NEAAs are not needed for stimulation of muscle protein synthesis. However, if the NEAAs were not necessary for stimulation of synthesis, it was unclear why ingestion of the EAAs alone did not stimulate muscle
protein synthesis to a greater extent than did the balanced mixture.
The response in the present study was about twice that when 6 g of
mixed AAs were given after a similar exercise bout (Fig. 7)
(14). Thus, it seems likely that there is a dose response of muscle protein synthesis to the intake of EAAs and that the amount
of EAAs in the balanced mixture (21 g) in the study by Tipton et al.
was equal to or exceeded the maximal effective dose. However, it should
be noted that, in this study by Tipton et al., the amino acids were
ingested as small boluses over 3 h, and the different pattern of
intake may also have contributed to the lack of difference between the
two mixtures of amino acids.
Pattern of ingestion.
No differences were found in the protein synthesis response between the
first and the second dose (Tables 3 and 4, Figs. 4 and 5), and the AUC
for net phenylalanine uptake was similar after the first and the second
drinks. Net balance increased rapidly after intake of the drink, and
the relative increase in net balance was greater than the change in
arterial phenylalanine concentrations (Fig. 2). However, when arterial
AA concentrations started to drop, net balance rapidly decreased to the
basal level, even though the arterial AA concentrations were still
elevated (Fig. 2). In fact, at 60 min after ingestion of the first
drink, the phenylalanine concentration was still higher than the
maximal concentration previously observed to coincide with stimulation
of protein synthesis when 6 g of mixed amino acids were given
(14). Thus muscle protein synthesis is apparently
stimulated when there is an increase in arterial, and presumably
interstitial, AA concentrations, rather than by the absolute AA
concentration. This observation is consistent with our previous
findings (6) that, when blood amino acid concentrations
were elevated to a steady-state level about twice the basal values for
6 h, muscle protein synthesis was stimulated over the first 2 h but thereafter returned to the resting level despite the persistent
elevation in blood amino acid concentrations.
The similarity of response to both boluses suggests that there is
little effect of the exact time of ingestion after exercise. This is
consistent with our previous work, in which we observed similar
responses to single doses of amino acids and glucose given either
immediately, 1 h, or 3 h after resistance exercise (16, 18). In contrast, Esmarck et al. (9) recently
reported that a protein-carbohydrate-fat supplement was effective in
stimulating muscle protein gain over a period of resistance training in
elderly men only when ingested immediately after, as opposed to 2 h after, exercise. Differences between that study and our studies
include age of subjects, ingestion of protein rather than free amino
acids, and end point. Whereas we measured the acute response of muscle protein, they measured net muscle gain, which includes the response to
all food intake over a period of time. Thus it is possible that
ingestion of a protein-carbohydrate-fat supplement 2 h after exercise might have interfered with either the amount eaten at the next
meal or the response to the next ingested meal. In another study that
addressed the timing of intake on response, Levenhagen et al.
(11) found a greater stimulation of net muscle protein synthesis when a protein-carbohydrate-fat supplement was given immediately after aerobic exercise than when it was given 2 h later. There likely is a difference between timing after aerobic and
timing after resistance exercise, as performed in the current study.
The stimulation of muscle protein synthesis is modest after aerobic
exercise (7). Rather, the interaction effect of exercise and supplement ingestion may be due to increased muscle blood flow, and
thus substrate delivery, immediately after exercise. In contrast,
fractional synthetic rate remains elevated for
48 h after
resistance exercise (15), so an effect on timing of supplement ingestion after resistance exercise is less likely.
Composition of drink.
The composition of the mixture of EAA we have tested in this and
previous studies (16, 18) was originally based on the composition of muscle protein, with the notion of increasing the availability of each EAA in proportion to its requirement for synthesis
of muscle protein. The results of the current study show that
isoleucine and leucine increased more than the others in the blood
(Fig. 6), meaning that the goal of causing proportional increases in
all EAA was not fully achieved. Different changes in concentrations of
blood amino acids after ingestion of the drink may be caused by
different clearance rates of individual amino acids, thereby resulting
in rates of uptake that do not directly correspond to the composition
of the ingested mixture. Furthermore, it is possible that the effect is
largely, or even entirely, mediated by leucine alone (1).
Therefore, it is possible that adjustments in the composition of the
drink could further improve the response of net muscle balance.
Whereas NEAAs can be synthesized within the body at a rate generally
sufficient to meet requirements, certain amino acids may be limited in
the rapidity with which changes in production can occur. Thus glycine
is known to be slowly transaminated (12), and this likely
explains the fall in plasma glycine concentration as its utilization is
increased because of the stimulated rate of protein synthesis after
EAA, not only in muscle but also throughout the body. The fact that the
EAA mixture alone was twice as effective as the same amount of the
balanced mixture of AAs (14) in stimulating muscle protein
synthesis indicates that glycine was probably not rate limiting,
despite the decrease in plasma concentration, because the muscle
concentration of free glycine was maintained. Similarly, the fall in
alanine concentration in plasma probably reflected increased uptake
elsewhere than in muscle for incorporation into protein, as muscle is
normally highly efficient in producing alanine, and its rate of
production is increased during exercise (22).
Methodological considerations.
Drinks normally are ingested as boluses. However, quantifying the
response to a bolus ingestion of unlabeled amino acids introduces potential methodological problems because of rapid dilution of the
tracer. The resulting isotopic nonsteady state violates a fundamental
assumption of the three-pool compartment model (2, 3). We
therefore added tracer to the ingested amino acids so that a relatively
constant enrichment in the blood was maintained, even during absorption
of the bolus. Thus the calculated values for synthesis and breakdown
should theoretically be accurate. Nonetheless, we used net protein
balance, which is not dependent on the measurement of isotopic
enrichment, as our primary end point, and the other variables were
considered as secondary end points. This is not only because of
methodological issues but also because, in terms of gain or loss of
muscle protein, the net balance (i.e., synthesis minus breakdown) is
the most relevant parameter. Furthermore, it can be determined in
non-steady-state conditions. In relation to net balance,
the pertinent kinetic parameters are the rate of synthesis from plasma
amino acids (phenylalanine and leucine, respectively) and the rate of
appearance of amino acids into plasma from protein breakdown. This is
because, although the synthesis of protein from amino acids derived
from protein breakdown (i.e., intracellular recycling of amino acids)
is important from the standpoint of understanding the regulation of the
process of synthesis, it does not represent any net gain or loss of
protein. The protein synthesis from plasma amino acids and appearance
in plasma from protein breakdown are calculated by the two-pool model. We also have calculated rates of synthesis and breakdown by use of the
three-pool model that we have described previously, which includes
synthesis from all sources and the total rate of breakdown. However,
even though we avoided changes in enrichment by adding tracer to the
exogenous amino acids, the calculated values of synthesis and breakdown
were variable. This is because calculation of the parameters from the
three-pool model requires a gradient in enrichment from blood to the
intracellular phenylalanine pools, and the rapid influx of amino acids
from plasma caused the gradient in enrichment between compartments to
narrow to the point where accurate measurement is difficult.
The primary potential problem in the interpretation of net balance
results in the nonsteady state is the possibility that amino acids
entered the intracellular pool from the plasma that eventually
reentered the blood at some time after the final blood sample was
drawn. To the extent that this occurred in this experiment, we would
have overestimated net uptake. However, the free intracellular phenylalanine concentration was not significantly elevated at the time
we drew our last sample, so the magnitude of error due to this
potential problem was likely not large.
Because previous studies have shown that net muscle protein balance
remains slightly negative for several hours after resistance exercise
in the absence of nutrient intake (4, 8), a control group
was not included in this study. Furthermore, in a previous study from
our laboratory, Rasmussen et al. (16) gave a placebo drink at 60 min after a similar resistance exercise bout. They found no
significant change in net balance over the first 3 h after
exercise. Hence, the significant positive net balance observed after drinks in the present study can be ascribed to the intake of the
amino acids, rather than to changes that would have occurred anyway.
 |
ACKNOWLEDGEMENTS |
We thank the nurses and the staff at the General Clinical
Research Center (GCRC) at the University of Texas Medical Branch (UTMB)
in Galveston, TX. We thank Julie M. Vargas for skillful technical
assistance. We also thank the volunteers who participated in the study.
 |
FOOTNOTES |
This work was supported by Shriners Hospital for Children Grant 8490 and the National Institutes of Health (NIH) Grants DK-38010 and
AG-98-006. Studies were conducted at the GCRC at the UTMB in
Galveston, funded by Grant M01 RR-00073 from the National Center for
Research Resources, NIH.
Address for reprint requests and other correspondence:
R. R. Wolfe, 815 Market St., Galveston, TX 77550 (E-mail:
rwolfe{at}utmb.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00466.2001
Received 15 October 2001; accepted in final form 26 May 2002.
 |
REFERENCES |
1.
Anthony, JC,
Anthony TG,
Kimball SR,
Vary TC,
and
Jefferson LS.
Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation.
J Nutr
130:
139-145,
2000[Abstract/Free Full Text].
2.
Biolo, G,
Chinkes D,
Zhang XJ,
and
Wolfe RR.
A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle.
J Parenter Enteral Nutr
16:
305-315,
1992[Abstract].
3.
Biolo, G,
Fleming RY,
Maggi SP,
and
Wolfe RR.
Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle.
Am J Physiol Endocrinol Metab
268:
E75-E84,
1995[Abstract/Free Full Text].
4.
Biolo, G,
Maggi SP,
Williams BD,
Tipton KD,
and
Wolfe RR.
Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans.
Am J Physiol Endocrinol Metab
268:
E514-E520,
1995[Abstract/Free Full Text].
5.
Biolo, G,
Tipton KD,
Klein S,
and
Wolfe RR.
An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.
Am J Physiol Endocrinol Metab
273:
E122-E129,
1997[Abstract/Free Full Text].
6.
Bohe, J,
Low JFA,
Wolfe RR,
and
Rennie MJ.
Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids.
J Physiol
532:
575-579,
2001[Abstract/Free Full Text].
7.
Carraro, F,
Stuart CA,
Hartl WH,
Rosenblatt J,
and
Wolfe RR.
Effect of exercise and recovery on muscle protein synthesis in human subjects.
Am J Physiol Endocrinol Metab
259:
E470-E476,
1990[Abstract/Free Full Text].
8.
Chesley, A,
MacDougall JD,
Tarnopolsky MA,
Atkinson SA,
and
Smith K.
Changes in human muscle protein synthesis after resistance exercise.
J Appl Physiol
73:
1383-1388,
1992[Abstract/Free Full Text].
9.
Esmarck, B,
Andersen JL,
Olsen S,
Richter EA,
Mizuno M,
and
Kjær M.
Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans.
J Physiol
535:
301-311,
2001[Abstract/Free Full Text].
10.
Jorfeldt, L,
and
Wahren J.
Leg blood flow during exercise in man.
Clin Sci (Colch)
41:
459-473,
1971.
11.
Levenhagen, DK,
Gresham JD,
Carlson MG,
Maron DJ,
Borel MJ,
and
Flakoll PJ.
Postexercise nutrient intake timing in humans is critical to recovery of leg glucose and protein homeostasis.
Am J Physiol Endocrinol Metab
280:
E982-E993,
2001[Abstract/Free Full Text].
12.
Matthews, DE,
Conway JM,
Young VR,
and
Bier DM.
Glycine nitrogen metabolism in man.
Metabolism
30:
886-893,
1981[ISI][Medline].
13.
Mayhew, JL,
Ball TE,
Arnold TE,
and
Bowen JC.
Relative muscular endurance as a predictor of bench press strength in college men and women.
J Appl Sport Sci Res
6:
200-206,
1992.
14.
Miller SL, Tipton KD, Wolf SE, and Wolfe RR. Independent and
combined effects of amino acids and glucose ingestion on muscle protein
metabolism following resistance exercise. In press.
15.
Phillips, SM,
Tipton KD,
Aarsland A,
Wolf SE,
and
Wolfe RR.
Mixed muscle protein synthesis and breakdown after resistance exercise in humans.
Am J Physiol Endocrinol Metab
273:
E99-E107,
1997[Abstract/Free Full Text].
16.
Rasmussen, BB,
Tipton KD,
Miller SL,
Wolf SE,
and
Wolfe RR.
An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise.
J Appl Physiol
88:
386-392,
2000[Abstract/Free Full Text].
17.
Tipton, KD,
Ferrando AA,
Phillips SM,
Doyle D, Jr,
and
Wolfe RR.
Postexercise net protein synthesis in human muscle from orally administered amino acids.
Am J Physiol Endocrinol Metab
276:
E628-E634,
1999[Abstract/Free Full Text].
18.
Tipton, KD,
Rasmussen BB,
Miller SL,
Wolf SE,
Owens-Stovall SK,
Petrini BE,
and
Wolfe RR.
Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise.
Am J Physiol Endocrinol Metab
281:
E197-E206,
2001[Abstract/Free Full Text].
19.
Wahren, J,
and
Jorfeldt L.
Determination of leg blood flow during exercise in man: an indicator-dilution technique based on femoral venous dye infusion.
Clin Sci Mol Med Suppl
42:
135-146,
1973[Medline].
20.
Wolfe, RR.
Measurement of urea kinetics in vivo by means of a constant tracer infusion of di-15N-urea.
Am J Physiol Endocrinol Metab
240:
E428-E434,
1981[Abstract/Free Full Text].
21.
Wolfe, RR.
Radioactive and Stable Isotope Tracers in Biomedicine. New York: Wiley-Liss, 1992.
22.
Wolfe, RR,
Wolfe MH,
Nadel ER,
and
Shaw JH.
Isotopic determination of amino acid-urea interactions in exercise in humans.
J Appl Physiol
56:
221-229,
1984[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 283(4):E648-E657
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