Essential amino acids and muscle protein recovery from resistance exercise

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-[(2)H(5)]phenylalanine and L-[1-(13)C]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.

Full text

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
Received 15 October 2001; accepted in final form 26 May 2002
Børsheim, Elisabet, Kevin D. Tipton, Steven E. Wolf,
and Robert R. Wolfe. Essential amino acids and muscle
protein recovery from resistance exercise. Am J Physiol En-
docrinol Metab 283: E648–E657, 2002; 10.1152/ajpendo.
00466.2001.—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-[
2
H
5
]phenylalanine
and
L-[1-
13
C]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 up-
take above basal value was similar for the first hour after
each drink (67 ⫾ 17 vs. 77 ⫾ 20 mg/leg, respectively). Be-
cause the NB response was double the response to two doses
of a mixture of3gofEAA⫹ 3 g of nonessential AA (NEAA)
(14), we conclude that NEAA are not necessary for stimula-
tion 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
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 be-
tween synthesis and breakdown. Although net muscle
protein balance is generally improved after resistance
exercise, it remains negative. Therefore, nutrient in-
take is necessary to achieve positive net muscle protein
balance.
The optimal composition and amount of nutrient
ingestion to maximally stimulate muscle protein syn-
thesis after resistance exercise are not known. It is
clear that amino acids or protein should be a compo-
nent, 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 pro-
tein 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 exer-
cise (16). The latter results (16) suggest that a rela-
tively small amount (i.e., 6 g) of EAAs can effectively
stimulate muscle protein synthesis, but the indepen-
dent 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
of6gofEAAs on net muscle protein synthesis after
resistance exercise.
The proportional contributions of individual amino ac-
ids 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 neces-
sary 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 mix-
ture [21.4 g EAAs and 18.6 g nonessential amino acids
(NEAAs), roughly in proportion to their relative contri-
butions to muscle protein], or 40 g of only EAAs. How-
ever, 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 max-
imal 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 com-
parable 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 to6gofanEAAmixture with the response to
6 g of a balanced mixture of amino acids, which included
⬃3 g of EAAs and3gofNEAAs. 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 to6gofthebalanced 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
Address for reprint requests and other correspondence: R. R. Wolfe,
815 Market St., Galveston, TX 77550 (E-mail: rwolfe@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.
Am J Physiol Endocrinol Metab 283: E648–E657, 2002;
10.1152/ajpendo.00466.2001.
0193-1849/02 $5.00 Copyright
©
2002 the American Physiological Society http://www.ajpendo.orgE648

idea being to increase the availability of each EAA in
proportion to its requirement for the synthesis of mus-
cle protein. However, different clearance rates of indi-
vidual 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 dispropor-
tionate changes in concentrations of blood amino acids
compared with the composition of the ingested mix-
ture. Therefore, another goal of the current study was
to measure blood and intramuscular concentrations of
all amino acids before and after ingestion of the mix-
ture 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 pat-
tern of ingestion may affect the response of muscle
protein synthesis. We have previously shown that
amino acid concentration, per se, is not a direct deter-
minant 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 persis-
tent 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 ele-
vation 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 pro-
tein synthesis to a second dose of amino acids would be
affected by the ingestion of an initial dose 1 h earlier.
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 screen-
ing, 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 Uni-
versity 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 exper-
iment, 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 anthro-
pometric measures of leg circumference and height at multi-
ple 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 infu-
sion of [
15
N
2
]urea was started at ⫺180 min (⬃6:30 AM). At
⫺120 min (⬃7:30 AM), a primed, constant infusion of L-[ring-
2
H
5
]phenylalanine and L-[1-
13
C]leucine was started. The fol
-
lowing infusion rates (IR) and priming doses (PD) were used
L-[
2
H
5
]phenylalanine: IR⫽0.10 ␮mol䡠 kg
⫺1
䡠 min
⫺1
,
PD⫽2 ␮mol/kg
L-[1-
13
C]leucine: IR⫽0.12 ␮mol䡠 kg
⫺1
䡠 min
⫺1
,
PD⫽4.8 ␮mol/kg
关
15
N
2
]urea: IR⫽0.2267 ␮mol䡠 kg
⫺1
䡠 min
⫺1
,
PD⫽88 ␮mol/kg
Isotopes were purchased from Cambridge Isotopes (An-
dover, MA). They were dissolved in 0.9% saline and were
filtered through a 2-␮m 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
Table 1. Physical characteristics and exercise data for all subjects
Subject No. Gender, F/M Age, yr Height, m Weight, kg Leg Volume, l 1RM Leg Press, kg 1RM Leg Extension, kg
1 F 25 1.72 57.2 7.48 130.5 113.5
2 F 24 1.76 71.4 11.87 85.0 79.5
3 M 24 1.90 90.7 11.65 146.5 130.5
4 M 25 1.83 82.0 10.51 141.5 113.5
5 F 23 1.63 56.0 7.54 79.5 60.0
6 M 19 1.63 56.8 6.91 136.0 136.0
Means ⫾ SD 23⫾ 2 1.74⫾ 0.11 69.0⫾ 14.9 9.33⫾2.27 119.8⫾ 29.6 105.5⫾ 29.7
1RM, one repetition maximum.
E649ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

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 cathe-
ters 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 ⱖ10 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 com-
plete. 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 com-
position of muscle protein (Table 2). Each supplement solu-
tion was composed of 425 ml of double-distilled water, the
appropriate mixture of EAA, and an artificial sweetener.
L-[ring-
2
H
5
]phenylalanine and L-[
13
C]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 phenylala-
nine 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% sulfosali-
cylic acid (SSA), and a weighed amount of an appropriate
internal standard consisting of amino acids labeled differ-
ently 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 Labo
-
ratories, Richmond, CA) and dried under vacuum with a
Speed Vac (Savant Instruments, Farmingdale, NY). Enrich-
ments of intracellular free amino acids were then determined
on the tertiary-butyldimethylsilyl (t-BDMS) derivatives us-
ing GC-MS (Hewlett-Packard 5973, Palo Alto, CA) and se-
lected 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 precipi-
tated with perchloroacetic acid. The tissue was then homog-
enized and centrifuged, and the supernatant was collected.
The procedure was then repeated, and the pooled superna-
tant 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 con-
centration 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 nei-
ther produced nor metabolized in muscle, net phenylala-
nine balance reflects net muscle protein synthesis, pro-
vided 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
Fig. 1. Infusion protocol. ICG, indocyanine green; D, drink; *, blood
sample; B, biopsy.
Table 2. Amino acid composition of drink
Amino Acid % of Total AAs Grams in EAA Drink
Histidine 10.9 0.6540
Isoleucine 10.1 0.6060
Leucine 18.6 1.1160
Lysine 15.5 0.9300
Methionine 3.1 0.1860
Phenylalanine 15.5 0.9300
Threonine 14.7 0.8820
Valine 11.5 0.6900
Total 99.9 5.994
AA, amino acid; EAA, essential amino acids. Composition is based
on a 70-kg person. Drink was given at 1 and 2 h after completion of
exercise.
E650 ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

constant net balance after the basal sample if amino acids
are not given. The basis for this assumption is the rela-
tively constant net balance for 3 h after exercise in the
absence of nutrient intake that we have previously ob-
served 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 intracellu-
lar 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 break-
down. We also calculated the rate of release of phenylala-
nine and leucine from protein breakdown into blood (R
a
)
and incorporation of phenylalanine from blood into muscle
protein (R
d
). Calculation of R
a
and R
d
, 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, de-
spite changing concentrations of plasma amino acids (see
below). Thus the difference between total protein synthesis
and R
d
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 R
a
is the
amount of phenylalanine from breakdown that was di-
rectly reincorporated into protein, rather than being re-
leased into blood.
Statistical methods. Overall significance of differences in
response with time was tested by repeated-measures analy-
sis 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.
RESULTS
Phenylalanine concentration and balance. At 30 min
after exercise, the arterial blood phenylalanine concen-
tration 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 re-
sponse to the second drink was comparable to that of
the first.
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 in-
creased 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 con-
centration (88 ⫾ 14 nmol/ml) was not statistically
different from the baseline value.
Phenylalanine kinetics. Enrichment of phenylala-
nine 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.
The rate of appearance of phenylalanine into the
blood from the muscle (R
a
) did not change during the
first 2 h after the drink but increased significantly
during the 3rd h (Fig. 4). The average rate of disap-
pearance (R
d
) of phenylalanine from the blood into the
muscle (i.e., protein synthesis from plasma phenylala-
nine) increased significantly from the basal value dur-
ing the first 2 h after intake of drink but was not
different from the basal value during the 3rd h.
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 ⫾ 9nmol䡠 min
⫺1
䡠 100
ml leg
⫺1
(Table 3). There was no change in R
a
during
the first 2 h after the first drink, but it increased signif-
icantly during the 3rd h to 75 ⫾ 13 nmol䡠 min
⫺1
䡠 100 ml
leg
⫺1
. The rate of utilization of phenylalanine for protein
synthesis was 30 ⫾ 9nmol䡠 min
⫺1
䡠 100 ml leg
⫺1
at 30 min
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.
E651ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

after exercise, and this value increased significantly dur-
ing the 1st and 2nd h after drink to 121 ⫾ 16 and 139 ⫾
21 nmol䡠 min
⫺1
䡠 100 ml leg
⫺1
, respectively (Table 3). Dur
-
ing 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.
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 ex-
periment 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 in-
creased significantly from the basal value of 144 ⫾ 7to
307 ⫾ 52 nmol/ml at 150 min. At 240 min, the concen-
tration 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 R
a
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 oxi-
dation 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䡠 min
⫺1
䡠 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.
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 R
a
of leucine increased slightly after
the drink, and during the 1st h the value was signifi-
cantly different from the predrink value. The rate of
utilization of intracellular leucine, which includes uti-
lization for both protein synthesis and oxidation, in-
creased 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 dur-
ing 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 calcu-
lated 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
Fig. 3. Arterial L-[
2
H
5
]phenylalanine and L-[1-
13
C]leucine enrich
-
ments during the recovery period after a resistance exercise bout
(means ⫾ SE; n ⫽ 6). EAA drink was consumed at 60 and 120 min
after exercise.
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). R
a
, rate of appearance of phenylalanine into the blood; R
d
, rate of
disappearance of phenylalanine out of the blood. *P ⬍ 0.05, value vs.
predrink value.
E652 ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

the 2nd and 3rd h, the values were not different from
the predrink value.
Plasma and muscle intracellular amino acid concen-
trations. 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.
Whereas there was no change in intracellular con-
centration of total EAAs or in total NEAAs during the
recovery period, some individual changes were ob-
served. Threonine was not different between first and
second biopsies but increased thereafter to a concen-
tration at 150 min significantly higher than predrink
concentration (741 ⫾ 99 vs. 561 ⫾ 59 nmol/ml H
2
O).
Similarly, valine, isoleucine, and leucine concentra-
tions all increased after the first drink. The concentra-
tions 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 concen-
trations of EAAs were small compared with the
changes in arterial plasma (Fig. 6).
Urea production. Plasma urea enrichment was sta-
ble at ⬃4% throughout the study. Thus no change in
urea production was seen over time. The value was
stable at 330 ␮mol䡠 kg
⫺1
䡠 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.
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 pre-
vious published response to6gofmixed amino acids
(14), leading to the conclusions that there is a dose
response to the amount of EAAs given and that inges-
tion 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, intra-
cellular concentrations of NEAAs were generally main-
tained, 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 synthe-
sis. However, optimal proportions and amounts of in-
dividual amino acids to stimulate muscle protein syn-
thesis are not known. Intake of only a small amount (6
g) of a mixture of EAA given along with 35 g of carbo-
hydrate after resistance exercise transiently increased
muscle protein synthesis 3.5-fold (14), but the indepen-
dent effects of amino acids and carbohydrate were not
assessed. The results of the present study show that
ingestion of6gofEAAalone without addition of
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.
Table 3. Calculated kinetics for phenylalanine before
drink and during 1st, 2nd, and 3rd h after intake of
first drink
Predrink 1st h 2nd h 3rd h
Inflow 219⫾ 27 514⫾ 64* 598⫾87* 547⫾92*
Inward transport 201⫾ 31 590⫾ 74 659⫾ 193 454⫾ 105
Protein synthesis 30⫾ 9 121⫾ 16* 139 ⫾21* 75 ⫾31
Protein breakdown 43⫾ 940⫾655⫾575⫾ 13
Inflow, rate at which phenylalanine enters leg via artery; inward
transport, rate of net phenylalanine movement from artery to mus-
cle; protein synthesis, rate of phenylalanine incorporation into pro-
tein; protein breakdown, rate of intracellular release of phenylala-
nine from protein breakdown. Values are nmol䡠 min
⫺1
䡠 100 ml leg
⫺1
(means ⫾ SE; n ⫽ 6). Value vs. predrink value: *P ⬍ 0.05.
E653ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

carbohydrate effectively stimulated muscle protein
synthesis after resistance exercise (Figs. 4 and 5, Ta-
bles 3 and 4).
In a recent study, Miller et al. (14) compared the
independent and combined effects of a balanced mix-
ture of amino acids (i.e., EAAs ⫹ NEAAs) and carbo-
hydrate on muscle protein synthesis after resistance
exercise. Addition of 35 g of carbohydrate to6gof
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 phenylala-
nine uptake for the 1st h after intake of drink (i.e.,
60–120 min) in the present study with the previously
published response to6gofEAAs plus 35 g of carbo-
hydrate (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, ap-
proximately contemporaneously, and by use of the
same general experimental protocol and techniques.
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 ⬃26gof
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
Fig. 6. Percentage changes in arterial
plasma concentrations of EAAs (A),
arterial plasma concentrations of non-
essential amino acids (NEAAs) and
tryptophan (B), intracellular concentra-
tions of EAAs (C), and intracellular
concentrations of NEAAs and trypto-
phan (D) from 30 min to 150 min after
exercise (means ⫾ SE; n ⫽ 6). EAA
drink was consumed at 60 and 120 min
after exercise.
Table 4. Calculated kinetics for leucine before drink and during 1st, 2nd, and 3rd h after intake of first drink
Predrink 1st h 2nd h 3rd h
Inflow 433⫾ 42 1228⫾ 128* 1366⫾ 143* 1318⫾ 167*
Inward transport 302⫾ 64 1008⫾ 210* 990 ⫾104* 718⫾ 91*
Utilization 100⫾ 16 458 ⫾58* 416⫾ 54* 176⫾43
Protein synthesis 73⫾ 17 306 ⫾25* 360⫾ 60* 158⫾41
Oxidation 27⫾ 7 151⫾45* 56⫾ 34† 18 ⫾10
Protein breakdown 106⫾14 195 ⫾30* 164⫾26 147⫾ 30
Utilization, rate of utilization of intracellular leucine (i.e., protein synthesis and oxidation); oxidation, rate of leucine oxidation. Values are
nmol䡠 min
⫺1
䡠 100 ml leg
⫺1
(means ⫾ SE; n ⫽ 6). *P ⬍ 0.05, value vs. predrink value. † P ⬍ 0.05, 2nd h vs. 1st h value.
E654 ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

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 resis-
tance exercise, NEAAs are apparently not required to
stimulate protein synthesis. In a previous study, Tip-
ton 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 stimu-
lation 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
when6gofmixed 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). How-
ever, when arterial AA concentrations started to drop,
net balance rapidly decreased to the basal level, even
though the arterial AA concentrations were still ele-
vated (Fig. 2). In fact, at 60 min after ingestion of the
first drink, the phenylalanine concentration was still
higher than the maximal concentration previously ob-
served to coincide with stimulation of protein synthesis
when6gofmixed 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 ab-
solute 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 concentra-
tions.
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 immedi-
ately, 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 resis-
tance training in elderly men only when ingested im-
mediately after, as opposed to 2 h after, exercise. Dif-
ferences 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 resis-
tance 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 in-
creased muscle blood flow, and thus substrate delivery,
immediately after exercise. In contrast, fractional syn-
thetic rate remains elevated for ⱖ48 h after resistance
exercise (15), so an effect on timing of supplement
ingestion after resistance exercise is less likely.
Fig. 7. Area under curve for net uptake of phenylalanine over 1 h
after ingestion of6gofdifferent 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).
E655ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

Composition of drink. The composition of the mix-
ture 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 avail-
ability 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 com-
position of the drink could further improve the re-
sponse 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 mix-
ture 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 con-
centration, 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 pro-
ducing alanine, and its rate of production is increased
during exercise (22).
Methodological considerations. Drinks normally are
ingested as boluses. However, quantifying the re-
sponse 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 main-
tained, 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 mea-
surement of isotopic enrichment, as our primary end
point, and the other variables were considered as sec-
ondary end points. This is not only because of method-
ological 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. Further-
more, it can be determined in non-steady-state condi-
tions. 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 un-
derstanding 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 calcu-
lated 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 exoge-
nous amino acids, the calculated values of synthesis
and breakdown were variable. This is because calcula-
tion of the parameters from the three-pool model re-
quires 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 exper-
iment, we would have overestimated net uptake. How-
ever, the free intracellular phenylalanine concentra-
tion 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 mus-
cle protein balance remains slightly negative for sev-
eral 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.
We thank the nurses and the staff at the General Clinical Re-
search 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.
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.
E656 ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org

REFERENCES
1. Anthony JC, Anthony TG, Kimball SR, Vary TC, and Jef-
ferson LS. Orally administered leucine stimulates protein syn-
thesis in skeletal muscle of postabsorptive rats in association
with increased eIF4F formation. J Nutr 130: 139–145, 2000.
2. Biolo G, Chinkes D, Zhang XJ, and Wolfe RR. A new model
to determine in vivo the relationship between amino acid trans-
membrane transport and protein kinetics in muscle. J Parenter
Enteral Nutr 16: 305–315, 1992.
3. Biolo G, Fleming RY, Maggi SP, and Wolfe RR. Transmem-
brane transport and intracellular kinetics of amino acids in
human skeletal muscle. Am J Physiol Endocrinol Metab 268:
E75–E84, 1995.
4. Biolo G, Maggi SP, Williams BD, Tipton KD, and Wolfe RR.
Increased rates of muscle protein turnover and amino acid trans-
port after resistance exercise in humans. Am J Physiol Endocri-
nol Metab 268: E514–E520, 1995.
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.
6. Bohe J, Low JFA, Wolfe RR, and Rennie MJ. Latency and
duration of stimulation of human muscle protein synthesis dur-
ing continuous infusion of amino acids. J Physiol 532: 575–579,
2001.
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.
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.
9. Esmarck B, Andersen JL, Olsen S, Richter EA, Mizuno M,
and KjærM.Timing of postexercise protein intake is important
for muscle hypertrophy with resistance training in elderly hu-
mans. J Physiol 535: 301–311, 2001.
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.
12. Matthews DE, Conway JM, Young VR, and Bier DM. Gly-
cine nitrogen metabolism in man. Metabolism 30: 886–893,
1981.
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 resis-
tance exercise in humans. Am J Physiol Endocrinol Metab 273:
E99–E107, 1997.
16. Rasmussen BB, Tipton KD, Miller SL, Wolf SE, and Wolfe
RR. An oral essential amino acid-carbohydrate supplement en-
hances muscle protein anabolism after resistance exercise.
J Appl Physiol 88: 386–392, 2000.
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.
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.
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.
20. Wolfe RR. Measurement of urea kinetics in vivo by means of a
constant tracer infusion of di-
15
N-urea. Am J Physiol Endocrinol
Metab 240: E428–E434, 1981.
21. Wolfe RR. Radioactive and Stable Isotope Tracers in Biomedi-
cine. 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.
E657ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab • VOL 283 • OCTOBER 2002 • www.ajpendo.org