Kevin D. Tipton, Elisabet Borsheim, Steven E. Wolf, Arthur P.

Sanford and
Robert R. Wolfe
Am J Physiol Endocrinol Metab 284:76-89, 2003. First published Sep 11, 2002;
doi:10.1152/ajpendo.00234.2002

You might find this additional information useful...

This article cites 27 articles, 22 of which you can access free at:
http://ajpendo.physiology.org/cgi/content/full/284/1/E76#BIBL

This article has been cited by 22 other HighWire hosted articles, the first 5 are:
Fasting and recovery from exercise
L. Burke
Br. J. Sports Med., June 1, 2010; 44 (7): 502-508.
[Abstract] [Full Text] [PDF]

Translational signaling responses preceding resistance training-mediated myofiber

hypertrophy in young and old humans
D. L. Mayhew, J.-s. Kim, J. M. Cross, A. A. Ferrando and M. M. Bamman
J Appl Physiol, November 1, 2009; 107 (5): 1655-1662.

Downloaded from ajpendo.physiology.org on September 20, 2010

[Abstract] [Full Text] [PDF]

The Role of Milk- and Soy-Based Protein in Support of Muscle Protein Synthesis and
Muscle Protein Accretion in Young and Elderly Persons
S. M. Phillips, J. E. Tang and D. R. Moore
J. Am. Coll. Nutr., August 1, 2009; 28 (4): 343-354.
[Abstract] [Full Text] [PDF]

Human muscle protein synthesis and breakdown during and after exercise
V. Kumar, P. Atherton, K. Smith and M. J. Rennie
J Appl Physiol, June 1, 2009; 106 (6): 2026-2039.
[Abstract] [Full Text] [PDF]

Nutritional and contractile regulation of human skeletal muscle protein synthesis and
mTORC1 signaling
M. J. Drummond, H. C. Dreyer, C. S. Fry, E. L. Glynn and B. B. Rasmussen
J Appl Physiol, April 1, 2009; 106 (4): 1374-1384.
[Abstract] [Full Text] [PDF]

Updated information and services including high-resolution figures, can be found at:
http://ajpendo.physiology.org/cgi/content/full/284/1/E76

Additional material and information about AJP - Endocrinology and Metabolism can be found at:
http://www.the-aps.org/publications/ajpendo

This information is current as of September 20, 2010 .

AJP - Endocrinology and Metabolism publishes results of original studies about endocrine and metabolic systems on any level of
organization. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD
20814-3991. Copyright © 2003 by the American Physiological Society. ISSN: 0193-1849, ESSN: 1522-1555. Visit our website at
http://www.the-aps.org/.
Am J Physiol Endocrinol Metab 284: E76–E89, 2003.
First published September 11, 2002; 10.1152/ajpendo.00234.2002.
Acute response of net muscle protein balance reflects
24-h balance after exercise and amino acid ingestion
KEVIN D. TIPTON, ELISABET BORSHEIM, STEVEN E. WOLF,
ARTHUR P. SANFORD, AND ROBERT R. WOLFE
Metabolism Unit, Shriners Hospitals for Children, Galveston 77550; and Department
of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555
Submitted 29 May 2002; accepted in final form 26 August 2002
Tipton, Kevin D., Elisabet Borsheim, Steven E.
Wolf, Arthur P. Sanford, and Robert R. Wolfe. Acute
response of net muscle protein balance reflects 24-h bal-
ance after exercise and amino acid ingestion. Am J Physiol
Endocrinol Metab 284: E76–E89, 2003. First published
September 11, 2002; 10.1152/ajpendo.00234.2002.—The
purpose of this study was to determine if the acute ana-
bolic muscle response to resistance exercise and essential
amino acids (EAA) reflects the response over 24 h. Seven
subjects participated in the following two 24-h studies: 1)
resting (REST) and 2) rest plus resistance exercise and
consumption of EAA (ES). Net balance (NB) across the leg
was determined for four amino acids. [13C6]phenylalanine
was infused to determine mixed muscle fractional syn-
thetic rate (FSR). Twenty-four-hour FSR was significantly
greater for ES than for REST (P ⫽ 0.003). Exchange of
phenylalanine across the leg was ⫺194 ⫾ 74 (SE) mg for
ES and ⫺371 ⫾ 88 mg for REST (P ⫽ 0.07) over 24 h and
229 ⫾ 42 mg (ES) and 28 ⫾ 15 mg (REST; P ⬍ 0.01) over
3 h corresponding to exercise and EAA consumption for
ES. The difference in phenylalanine exchange between
REST and ES was not different for measurements over 24
and 3 h. Increases in NB during ES were primarily the
result of increases in protein synthesis. Results for other
amino acids were similar. The acute anabolic response of
muscle to EAA intake and exercise is additive to the
response at rest and thus reflects the 24-h response.
muscle protein synthesis; muscle protein breakdown; stable
isotopic tracers
IT IS CLEAR THAT the combination of exogenous amino
acids, administered either intravenously (7) or orally
(21, 25, 26), and resistance exercise is a potent stimu-
lator of acute net muscle protein synthesis (PS). Stim-
ulation of net muscle PS by amino acids and exercise is
of large magnitude and is primarily the result of an
increase in muscle PS rather than a decrease in break-
down. Interpretation of the results of these studies is
dependent on the assumption that the response of net
muscle PS is additive to the balance that would occur
during a normal day in the absence of the exercise and
amino acid ingestion. Although the magnitude of the
response to oral ingestion of amino acids after exercise
is large, it is also transient (21, 25, 26). Thus there is
Address for reprint requests and other correspondence: K. D.
Tipton, Metabolism Unit, Shriners Hospitals for Children, 815 Mar-
ket St., Galveston, TX 77550 (E-mail: ktipton@utmb.edu).
E76 0193-1849/03 $5.00 Copyright © 2003 the American Physiological Society
uncertainty as to the impact of these transient re-
sponses on chronic changes in muscle metabolism and
muscle mass.
It has been proposed that protein metabolism exhib-
its a homeostasis whereby dietary-induced stimulation
Downloaded from ajpendo.physiology.org on September 20, 2010
of protein balance during the day would be countered
by a subsequent nadir in the middle of the night and
the net change would be zero for the full day (16, 17,
20). Furthermore, in our previous studies, the large
acute changes were measured in the fasting state. A
recent study showed that the response of muscle PS
may become refractive to hyperaminoacidemia in cer-
tain situations (8). Thus it is possible that changes in
net muscle protein balance resulting from hyperami-
noacidemia after exercise would be diminished by a
meal before the exercise or may diminish the response
to a subsequent meal. Either scenario would affect the
total response of muscle over a full 24-h day, possibly
diminishing the total response.
Therefore, the purpose of the present study was to
determine if the response of net muscle protein metab-
olism to resistance exercise and amino acid ingestion
measured acutely was reflective of that measured over
a full 24-h day. We measured net muscle protein bal-
ance of healthy volunteers for 24 h on two separate
occasions, once while they rested comfortably and once
when they also performed a resistance exercise bout
and consumed 30 g of essential amino acids (EAA).
METHODS
Subjects
Seven healthy volunteers (4 females, 3 males) participated
in each of two trials. The study design, purpose, and possible
risks were explained to each subject before written consent
was obtained. The Institutional Review Board and the Gen-
eral Clinical Research Center (GCRC) of the University of
Texas Medical Branch at Galveston approved the study pro-
tocol. All subjects were healthy, nondiabetic, and normoten-
sive and had normal cardiac rhythm with no abnormalities,
as judged by medical history, physical examination, resting
electrocardiogram, and laboratory blood and urine tests. All
volunteers were recreationally active, but none had partici-
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.
http://www.ajpendo.org
 24-H MUSCLE PROTEIN BALANCE
pated in resistance training for at least 1 yr. Subjects were
instructed to refrain from physical exercise for 48 h before
each study. Mean age was 26.7 ⫾ 1.0 (SE) yr, weight was
73.0 ⫾ 5.0 kg, height was 1.75 ⫾ 0.15 m, and body mass index
was 23.7 ⫾ 0.6 kg/m2. At least 1 wk before the initial infusion
protocol, each subject was familiarized with the leg extension
machine, and his or her one-repetition maximum (1 RM; the
maximum weight that can be lifted for one repetition) was
determined. Mean 1 RM for the leg extension was 116.7 ⫾
16.1 kg.
Experimental Protocol
A schematic diagram of the study protocol is presented in
Fig. 1. The protocol was designed to determine if the acute
response of net muscle protein balance to the interaction of
resistance exercise and EAA ingestion was reflective of that
over 24 h. Each subject participated in two 24-h infusion
protocols, a resting protocol (REST) and an exercise-supple-
mentation protocol (ES), in random order. Net muscle protein
balance was calculated for the entire 24-h period and for a
3-h period from 1100, i.e., the beginning of the resistance
exercise protocol for ES, including ingestion of 15 g of EAA
immediately before the exercise and a second EAA ingestion
1 h after completion of the exercise bout during ES. During
REST, 3-h net muscle protein balance was calculated for the
corresponding time period.
The possible influence of dietary fluctuations on net mus-
cle protein balance during each infusion study was mini-
mized by standardization of the diet for each subject for 4
days before each study. Before the first infusion protocol,
subjects completed a 3-day diet record. The GCRC dietitian
analyzed the diet utilizing Nutritionist IV software (N2 Com-
Table 1. Habitual diet and diet consumed for 4 days of diet standardization and the day
of the infusion study (day 5)
Habitual Day 1 Day 2
Energy, MJ 8.49 ⫾ 1.24 8.49 ⫾ 1.23 8.44 ⫾ 1.23
Protein, g 90 ⫾ 12 91 ⫾ 12 90 ⫾ 12
Carbohydrates, g 223 ⫾ 32 240 ⫾ 36 239 ⫾ 36
Lipids, g 80 ⫾ 21 80 ⫾ 20 79 ⫾ 20
Values are means ⫾ SE. Habitual is the diet calculated from 3-day diet records. Days 1–4, consumption on the 1st through 4th days of the
diet standardization period; study day 5, day of the 24-h study period; total, total amount consumed on the study day; meal 1, amount
consumed during the 1st meal on the study day; meal 2, amount consumed during the 2nd (liquid) meal during the study day.
AJP-Endocrinol Metab • VOL
E77
Fig. 1. Study protocol. Rest (REST) and exercise-sup-
plementation (ES) trials are identical with the excep-
tion of the addition of the ingestion of 2 ⫻ 15 g essential
amino acids (D) and a resistance exercise bout indi-
cated below the timeline. Net muscle protein balance
and fractional synthesis rate (FSR) were determined
for the entire 24-h period of both trials and for 3 h
corresponding to the acute period of drink ingestion and
exercise (EX) from 1100 to 1400. M, ingestion of meals;
BF, breakfast period, 0900–1100; PE, postexercise pe-
riod, 1400–1700; ML, 2nd meal period, 1700–2000; NT,
middle of the night, 0000–0300; AM, morning fasting
period, 0500–0800; CON, control; B, muscle biopsy.
puting, Silverton, OR) and designed a 5-day diet for each
Downloaded from ajpendo.physiology.org on September 20, 2010
subject that matched the subject’s habitual diet in energy,
protein, carbohydrate, and fat intake. Habitual dietary in-
take and standardized diet are summarized in Table 1. For 4
days before each protocol, the GCRC kitchen prepared all
meals consumed by each subject, and subjects were in-
structed not to consume anything other than water outside of
the prepared diet. Energy intake was 100% of habitual on the
first 2 days of the diet period, 110% on the second 2 days, and
80% of habitual on the day of the study (day 5). This pattern
was used to ensure that subjects were in energy balance over
the entire 5-day diet period while consuming only two meals
on the day of the study. Subjects consumed only two meals so
that the response to the EAA ingestion and resistance exer-
cise could be clearly delineated by measuring net muscle
protein balance over a sufficiently long time period after
exercise. Protein intake was 100% of habitual for all 5 days of
the diet standardization period, including the study day.
The REST trial consisted of a 26-h isotope infusion and
24-h sampling period while the subjects rested comfortably in
a bed at the GCRC. Movement was not prohibited, and
subjects were free to stand up and move around, but the
nature of the study, i.e., the catheters and infusion pumps,
made it uncomfortable for the subject to do more than mini-
mal movement. Thus subjects spent the majority of their
time lying in bed. Two meals were consumed during the
protocol. The first meal was consumed in the morning, im-
mediately after the first biopsy and arteriovenous sample.
The second meal was divided into eight small doses of a
liquid supplement (condensed milk or Boost; Mead Johnson,
Evansville, IN) consumed at 15-min intervals over a 2-h
period in an attempt to maintain isotopic steady-state during
Study Day 5
Day 3 Day 4 Total Meal 1 Meal 2
9.32 ⫾ 1.36 9.30 ⫾ 1.35 6.83 ⫾ 1.00 2.90 ⫾ 0.46 3.93 ⫾ 0.57
91 ⫾ 12 92 ⫾ 12 88 ⫾ 12 44 ⫾ 7 44 ⫾ 7
289 ⫾ 40 290 ⫾ 41 154 ⫾ 31 50 ⫾ 12 104 ⫾ 20
80 ⫾ 20 80 ⫾ 20 70 ⫾ 19 33 ⫾ 7 48 ⫾ 15
284 • JANUARY 2003 • www.ajpendo.org
E78 24-H MUSCLE PROTEIN BALANCE

this time (for enrichment values, see Tables 2 and 3). Ap-
proximately 40% of the energy and 50% of the protein were
consumed during the first meal of the study day, and 60 and
50% of the energy and protein, respectively, were consumed
during the second meal (Table 1).
Subjects were admitted to the GCRC on the evening before
each study day. The next morning, an 18-gauge, polyethylene
catheter was inserted in a large peripheral vein of each arm.
One catheter was for infusion of isotopic tracers, and the
second, in the contralateral arm, was used for blood sampling
for blood flow measurement. Catheters were inserted in po-
sitions to prevent occlusion by bending of the arms. After
background blood sampling, a primed-constant infusion of
L-[ring-13C6]phenylalanine was started at 0600. The priming
dose and infusion rate were 2 ␮mol/kg and 0.05 ␮mol 䡠
min⫺1 䡠 g⫺1. The infusion continued throughout the 24-h sam-
pling protocol (i.e., until the final muscle biopsy at 0800 the
next morning). Catheters were then placed in the femoral
artery and vein for arteriovenous sampling across the leg.
The femoral arterial catheter was also used for infusion of
indocyanine green dye (ICG) for blood flow measurement
concentration in each. Blood was placed in serum separator
tubes for later spectrophotometric measurement of the dye in
the samples.
At ⬃0800, after 2 h of tracer infusion for establishment of
isotopic steady state, the first arteriovenous blood samples
were taken from the femoral vessels, and the first of five
muscle biopsies was taken from the vastus lateralis. The first
meal was then served and completely consumed by no later
than 0900. Arteriovenous blood samples were taken on the
hour throughout the next 24 h (until 0800 the next morning)
and more frequently at times when amino acid concentra-
tions were changing, e.g., after meal and amino acid inges-
tion and at six selected periods when blood flow was mea-
sured. All blood samples for measurement of arterial and
venous concentrations and isotopic enrichments were imme-
diately placed in preweighed tubes containing 1 ml sulfosal-
icylic acid/ml blood.
ICG infusion was initiated 10 min before blood flow sam-
pling. Blood samples were taken from the femoral vein and a
peripheral vein two times during each blood flow-sampling

Downloaded from ajpendo.physiology.org on September 20, 2010

using the dye-dilution technique, as previously described (4, period. Blood flow sampling was performed from 1200 to
6). Briefly, ICG (0.5 mg/ml) was infused (60 ml/h) in the 1220, 1600 to 1620, 1800 to 1820, 0230 to 0250, and 0730 to
femoral artery. Blood samples were taken simultaneously 0750. One subject had no blood flow sampling during the
from the femoral vein and peripheral vein to measure ICG 1200–1220 time period for the REST trial. The second meal

Table 2. Phenylalanine enrichment in arterial samples during 24 h of sampling at rest

Subject No.

Time, h 1 2 3 4 5 6 7

0 0.0855 0.0702 0.0686 0.0815 0.0578 0.0650 0.0622

1 0.0465 0.0617 0.0617 0.0515 0.0420 0.0618 0.0544
2 0.0626 0.0599 0.0618 0.0449 0.0501 0.0495 0.0483
3 0.0570 0.0528 0.0740 0.0464 0.0445 0.0481 0.0494
3.5 0.0707 0.0220 0.0441 0.0459 0.0507
4 0.0610 0.0567 0.0791 0.0171 0.0491 0.0456 0.0509
4.5 0.0805 0.0537 0.0519 0.0489 0.0404
5 0.0607 0.0744 0.0765 0.0561 0.0497 0.0471 0.0482
5.3 0.0566 0.0665 0.0780 0.0676 0.0621 0.0480 0.0495
5.5 0.0632 0.0661 0.0814 0.0690 0.0476 0.0500 0.0440
6 0.0658 0.0721 0.0861 0.0707 0.0771 0.0470 0.0518
7 0.0653 0.0591 0.0810 0.0860 0.0698 0.0626 0.0642
8 0.0880 0.0666 0.0844 0.0928 0.0992 0.0743 0.0601
8.5 0.0833 0.0808 0.0752 0.0851 0.0840 0.0666 0.0623
9 0.0879 0.0765 0.0783 0.0826 0.0659 0.0700
9.5 0.0613 0.0428 0.0662 0.0969 0.0471 0.0591 0.0569
10 0.0638 0.0449 0.0568 0.0607 0.0502 0.0553 0.0547
10.5 0.0587 0.0402 0.0489 0.0674 0.0567 0.0578 0.0541
11 0.0628 0.0349 0.0533 0.0342 0.0450 0.0584 0.0550
11.5 0.0549 0.0404 0.0509 0.0229 0.0534 0.0628 0.0554
12 0.0553 0.0445 0.0591 0.0696 0.0521 0.0578 0.0586
12.5 0.0473 0.0456 0.0546 0.0725 0.0574 0.0673 0.0649
13 0.0460 0.0540 0.0643 0.0703 0.0532 0.0722 0.0481
13.5 0.0445 0.0481 0.0704 0.0866 0.0560 0.0642 0.0650
14 0.0492 0.0438 0.0836 0.0694 0.0675 0.0652 0.0558
15 0.0667 0.0669 0.0773 0.0636 0.0597 0.0719 0.0725
16 0.0779 0.0766 0.0803 0.0685 0.0812 0.0781 0.0500
17 0.0834 0.0669 0.0815 0.0773 0.0935 0.0689 0.0632
18 0.0909 0.0766 0.0771 0.0831 0.0757 0.0690 0.0576
18.5 0.0800 0.0702 0.0874 0.0843 0.0842 0.0816 0.0684
19 0.0892 0.0617 0.0826 0.0857 0.0631 0.0769 0.0646
20 0.0911 0.0524 0.0850 0.0858 0.0735 0.0795 0.0703
21 0.0860 0.0703 0.0857 0.0890 0.0722 0.0827 0.0660
22 0.0899 0.0660 0.0913 0.0802 0.0683 0.0735 0.0688
23 0.0939 0.0745 0.0829 0.0845 0.0631 0.0838 0.0627
23.5 0.0949 0.0812 0.0782 0.0795 0.0833 0.0839 0.0812
24 0.0958 0.0856 0.0860 0.0813 0.0776 0.0758 0.0685
Units are tracer-to-tracee ratios (t/T).

AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 • www.ajpendo.org

 24-H MUSCLE PROTEIN BALANCE E79

Table 3. Phenylalanine enrichment in arterial samples during 24 h of sampling with resistance exercise
and ingestion of eccentric amino acid solutions
Subject No.

Time, h 1 2 3 4 5 6 7

0 0.0727 0.0747 0.0665 0.0706 0.0578 0.0688 0.0627

1 0.0621 0.0686 0.0615 0.0526 0.0459 0.0630 0.0525
2 0.0669 0.0575 0.0522 0.0475 0.0370 0.0422 0.0469
3 0.0528 0.0585 0.0762 0.0476 0.0438 0.0471 0.0440
3.5 0.0539 0.0506 0.0631 0.0493 0.0565 0.0448 0.0481
4 0.0532 0.0612 0.0569 0.0578 0.0770 0.0523 0.0481
4.5 0.0528 0.0564 0.0586 0.0528 0.0505 0.0508 0.0503
5 0.0521 0.0577 0.0626 0.0533 0.0524 0.0513 0.0509
5.3 0.0531 0.0573 0.0570 0.0571 0.0524 0.0537 0.0513
5.5 0.0539 0.0605 0.0516 0.0535 0.0541 0.0561 0.0611
6 0.0481 0.0620 0.0581 0.0567 0.0520 0.0598 0.0583
7 0.0420 0.0640 0.0664 0.0630 0.0536 0.0571 0.0571
8 0.0365 0.0689 0.0703 0.0667 0.0606 0.0624 0.0528
8.5 0.0373 0.0788 0.0759 0.0660 0.0619 0.0540 0.0557
9 0.0514 0.0839 0.0748 0.0654 0.0465 0.0574 0.0598
9.5 0.0527 0.0569 0.0637 0.0621 0.0753 0.0567 0.0899

Downloaded from ajpendo.physiology.org on September 20, 2010

10 0.0627 0.0416 0.0570 0.0554 0.0482 0.0552 0.0562
10.5 0.0644 0.0448 0.0580 0.0387 0.0446 0.0583 0.0550
11 0.0730 0.0388 0.0510 0.0429 0.0490 0.0528 0.0522
11.5 0.0645 0.0385 0.0511 0.0494 0.0342 0.0514 0.0528
12 0.0612 0.0392 0.0621 0.0503 0.0447 0.0452 0.0576
12.5 0.0622 0.0432 0.0507 0.0582 0.0443 0.0514 0.0527
13 0.0528 0.0470 0.0783 0.0527 0.0498 0.0494 0.0640
13.5 0.0640 0.0449 0.0565 0.0567 0.0483 0.0592 0.0571
14 0.0663 0.0491 0.0760 0.0576 0.0646 0.0622
15 0.0652 0.0682 0.0899 0.0611 0.0655 0.0849 0.0514
16 0.0836 0.0799 0.0864 0.0651 0.0647 0.0709 0.0558
17 0.0803 0.0799 0.0866 0.0738 0.0557 0.0648 0.0571
18 0.1066 0.0822 0.0811 0.0756 0.1076 0.0705 0.0579
18.5 0.1027 0.0819 0.0676 0.0802 0.0761 0.0736 0.0707
19 0.0943 0.0786 0.0818 0.0739 0.0817 0.0640 0.0508
20 0.0906 0.0777 0.0820 0.0726 0.0708 0.0748 0.0576
21 0.0946 0.0874 0.0897 0.0739 0.0702 0.0776 0.0629
22 0.1009 0.0871 0.1071 0.0728 0.0748 0.0671 0.0608
23 0.0787 0.0836 0.0794 0.0689 0.0795 0.0681 0.0722
23.5 0.0976 0.0788 0.0777 0.0734 0.0798 0.0608 0.0727
24 0.0948 0.0790 0.0802 0.0637 0.0785 0.0653 0.0339
Units are t/T.

was begun at 1700, and further doses were ingested every 15
min until 1845.
Five percutaneous muscle biopsies were taken from the
lateral portion of the vastus lateralis using sterile technique
over the 24-h period. Biopsies were taken at 0800, 1630,
1830, 0300, and 0800 (next morning). For each biopsy, skin
and subcutaneous tissue were anesthetized with 1% lido-
caine, and an ⬃6-mm incision was made in the skin and
muscle fascia. No more than three biopsies were taken from
any single incision; thus, two incisions were necessary for a
total of five biopsies. Each individual biopsy was separated
from the previous biopsy by at least 1 cm in an attempt to
minimize the influence of local inflammatory responses. A
5-mm Bergström biopsy needle (Depuy, Warsaw, IN) was
advanced through the skin and fascia deep into the muscle
with the cutting needle closed. With suction applied, the
cutting cylinder was opened and then closed two to three
times. A sample of ⬃50 mg of mixed muscle tissue was
obtained with each biopsy, rinsed of excess blood with ice-
cold saline, blotted dry, and quickly (within 1 min) frozen in
liquid N2. Blood samples for insulin measurements were
taken at 0800, 0900, 1330, 1830, 2000, 0215, and 0700 from
the femoral artery and placed in serum separator tubes.
The ES protocol was identical to REST with the addition of
a resistance exercise bout and two amino acid drinks. During
ES, subjects were transported by gurney to the Exercise
Metabolism Laboratory in the Shriners Hospitals for Chil-
dren at ⬃1100 for the resistance exercise bout. During ES,
there was an additional blood flow measurement period from
1115 to 1135 during the exercise bout. Infusion of ICG in the
femoral artery for measurement of leg blood flow was initi-
ated at 1114 followed immediately by commencement of the
resistance exercise bout. Resistance exercise consisted of
eight sets of eight repetitions of knee extension exercise at
80% of 1 RM. There were 2 min of rest between sets. Blood
samples were taken from the femoral vein and the peripheral
vein contralateral to the infusion catheter for measurement
of leg blood flow after the fourth and the eighth (final) set of
knee extensions. Arteriovenous blood samples were taken
after the sixth set of leg extensions (i.e., ⬃15 min after
ingestion of the first drink). Subsequent to completion of the
exercise bout, the subject was returned to the GCRC for the
remainder of the 24-h protocol.
EAA Solution
During ES, each subject consumed 2 ⫻ 15 g of an EAA
solution in 350 ml of ddH2O. The drinks were ingested
immediately before the resistance exercise bout (1115) and
1 h after completion of the bout (1235). This pattern of timing

AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 • www.ajpendo.org

E80 24-H MUSCLE PROTEIN BALANCE
and amount of EAA ingestion was utilized to maximize the
response of net muscle protein balance (21, 26). Each solution
contained 15 g EAA in amounts designed to increase muscle
free intracellular amino acid levels in proportion to their
respective requirements for PS. Thus subjects consumed 30 g
of amino acids and ⬃502 kJ more energy during ES than
REST. Amounts of EAA in 350 ml solution were (g and mmol,
respectively) 1.64 and 10.54 for histidine, 1.52 and 11.56 for
isoleucine, 2.79 and 21.27 for leucine, 2.33 and for 15.96
lysine, 0.47 and for 3.12 methionine, 2.31 and 13.99 for
phenylalanine, 2.21 and 18.52 for threonine, and 1.73 and
14.73 for valine. The amount of phenylalanine includes
L-[ring-13C6]phenylalanine (0.2619 g), which was added to
minimize the disruption of isotopic steady state during bolus
ingestion of the solution. A small amount of artificial sweet-
ener, containing aspartame, was added to the solution to
improve palatability. The artificial sweetener contained
⬍200 mg amino acids, and this amount is factored into the
totals.
Analysis of Samples
Blood. Amino acid enrichment and concentration of phe-
nylalanine in whole blood were measured by gas chromatog-
raphy-mass spectrometry (GC-MS; Hewlett Packard 5973,
Palo Alto, CA) and expressed as tracer-to-tracee ratios (t/T)
(5, 6, 13). Upon thawing, 500 ␮l of the sulfosalicylic extract
was passed over a cation exchange column (Dowex AG 50W-
8X, 100–200 mesh H⫹ form; Bio-Rad Laboratories, Rich-
mond, CA) and dried under vacuum using a Speed Vac
(Savant Instruments, Farmingdale, NY). To determine the
enrichment of infused amino acids in whole blood, the ter-
tiary-butyldimethylsilyl (t-BDMS) derivative of each amino
acid was made according to previously described procedures
(12, 13, 18). Concentrations of free amino acids were deter-
mined using an internal standard solution, as previously
described (4–6, 13, 18). The internal standards used were
[U-13C9-15N]phenylalanine (50 ␮mol/l), L-[13C6]leucine (115
␮mol/l), [2H8]valine (207 ␮mol/l), and [15N]glycine (198
␮mol/l) added in a ratio of ⬃100 ␮l/ml blood. Because the
tube weight and the amount of blood were known, the blood
amino acid concentration could also be determined from the
internal standard enrichments measured by GC-MS based
on the amount of blood and internal standard added (4, 18).
Appropriate corrections were made for overlapping spectra
that contributed to the t/T (22). Leg blood flow was deter-
mined by spectrophotometrically measuring the ICG con-
centration in serum from the femoral vein and the periph-
eral vein, as described previously (3, 4, 6, 18). Leg plasma
flow was calculated from steady-state values of dye con-
centration and converted to blood flow using the hematocrit
(4). Plasma insulin levels were determined by RIA (Diagnos-
tic Products, Los Angeles, CA). Intra-assay coefficient of
variation was 1.45%.
Muscle. Muscle biopsy tissue samples were analyzed for
mixed protein-bound and free intracellular amino acid en-
richment and concentration, as previously described (3, 4, 6,
12, 13, 18). Tissue biopsies (⬃50 mg) of the vastus lateralis
were immediately blotted and frozen in liquid nitrogen. Sam-
ples were then stored at ⫺80°C until processed. Upon thaw-
ing, ⬃20–25 mg tissue were weighed, and protein was pre-
cipitated with 0.5 ml of 10% perchloroacetic acid. The tissue
was then homogenized and centrifuged, and the supernatant
was collected. This procedure was repeated two more times,
and the pooled supernatant (⬃1.3 ml) was processed as were
the blood samples described in Blood. To determine intracel-
lular enrichment of infused tracers, the t-BDMS derivative
AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 •
was prepared as previously described (4, 9, 18) and analyzed
by GC-MS. Intracellular enrichment was determined by cor-
rection for extracellular fluid based on values determined
from the chloride method (2, 4). Muscle free amino acid
concentration was measured with the internal standard
method, with corrections for the contribution of extracellular
fluid and for overlapping spectra, as described in Blood and
previously (4, 6, 9, 18).
The remaining pellet of muscle tissue was further washed
two times with ddH2O and two times with absolute ethanol.
It was then placed in an oven and dried at 50°C overnight.
The dried pellet was then hydrolyzed at 110°C for 24 h with
6 N HCl. The protein hydrolysate was then passed over a
cation exchange column, dried by a Speed Vac, and derivat-
ized with t-BDMS, as described for Blood. Enrichment of
protein-bound L-[ring-13C6]phenylalanine was determined by
GC-MS (model 5973; Hewlett-Packard) with a splitless injec-
tion and positive electron-impact ionization, as described
previously (18, 25). Mass-to-charge ratios 237 and 240 were
monitored. These ions are the m ⫹ 3 and m ⫹ 6 enrichments,
respectively, where m ⫹ 0 is the lowest molecular weight of
Downloaded from ajpendo.physiology.org on September 20, 2010
the ion. The ratio of m ⫹ 6 to m ⫹ 3 was used because it is
more sensitive than the traditional m ⫹ 6/m ⫹ 0 (used for
blood samples). Enrichment from the protein-bound samples
was determined with a linear standard curve of known m ⫹
6-to-m ⫹ 3 ratios and corrected back to the absolute change
in m ⫹ 6 enrichment over the incorporation period.
Calculations
Chemical net amino acid balance (NB) across the leg was
calculated from the difference between the femoral arterial
and venous concentrations multiplied by the blood flow. Thus
NB ⫽ (C a ⫺ C v) ⫻ BF
where Ca is the arterial amino acid concentration, Cv is the
venous amino acid concentration, and BF is leg blood flow.
The primary endpoint of the study is the comparison of the
amino acid (phenylalanine) uptake or release (i.e., exchange)
over 24 h and that for an acute period including the exercise
and EAA ingestion. Area under the curve was used to calcu-
late total amino acid exchange (mg), i.e., uptake (⫹) or
release (⫺), across the leg for the entire 24-h period and for
3 h from 1100 to 1400. The 3-h time period represents the
acute measurement period around the resistance exercise
bout and ingestion of EAA during ES. For the 24-h balance
across the leg, the baseline was zero. For the acute 3-h
period, the immediately preceding resting value (1100 sam-
ple) was used as baseline so that all values reflected the
uptake resulting from the ingestion of EAA combined with
exercise. The difference in milligrams between the 24-h value
for ES and for REST, and the difference between the 3-h
value for ES and for REST, is also calculated for each amino
acid. Additionally, amino acid balance was calculated for
leucine, valine, and glycine to assess the responses of other
amino acids.
Because phenylalanine is not metabolized in muscle, mus-
cle PS from blood-borne amino acids and the appearance of
amino acids in the blood from muscle protein breakdown (PB)
can be estimated using the NB across the leg and the arterial
and venous enrichments of L-[ring-13C6]phenylalanine (24,
28)
PB ⫽ (E a / E v ⫺ 1) ⫻ C a ⫻ BF
where Ea is the arterial enrichment of L-[ring-13C6]-
phenylalanine and Ev is venous enrichment and
www.ajpendo.org
 24-H MUSCLE PROTEIN BALANCE
PS ⫽ NB ⫹ PB
PB, PS, and NB were calculated for six time periods by
combining the individual measurements within each period
and using the mean values in the calculations (see below).
These time periods were chosen to delineate the temporal
changes during the 24-h periods with and without exercise
and amino acid ingestion.
The fractional rate of mixed muscle PS (FSR) was deter-
mined for the entire 24-h period (0800–0800) and for periods
of the 24-h labeled day (0800–1630), meal (1630–1830), and
night (1830–0800). FSR (%/h) was calculated as outlined
previously (13, 18). Briefly, mixed muscle protein FSR was
determined using the free intracellular phenylalanine en-
richment as the precursor pool, which appears to be the
superior surrogate for the true precursor, phenylalanine
tRNA enrichment, over blood phenylalanine enrichment (1,
15). The actual mixed-muscle FSR was calculated as the
mean of the incorporation of L-[ring-13C6]phenylalanine in
mixed muscle protein over time divided by the precursor
enrichment.
Data Presentation and Statistical Analysis
Data are presented as means ⫾ SE. Phenylalanine,
leucine, valine, and glycine arterial and venous concentra-
tions and NB across the leg are presented across time. For
valine and glycine, data are available only from five subjects
for REST and four for ES. Because data are available from
only four to five subjects for valine and glycine, concentration
and NB over time were statistically evaluated for phenylal-
anine and leucine only. Differences between REST and ES at
each time point were evaluated with a paired t-test. Although
a more complex model, such as ANOVA or parametric func-
tional curve fitting yielding confidence bands for a mean
response, could be used to analyze the differences across
time, the lack of uniform variability exhibited by our data
make it unlikely that one of these models would yield mean-
ingful results. Bonferroni corrections were used to reduce the
possibility that even one test will be false at P ⬍ 0.05.
Paired Student’s t-test was used to detect the differences
between REST and ES for total amino acid uptake or release,
FSR and the area under the insulin curve, as well as the
differences between 24 and 3 h for the difference between
REST and ES exchange (i.e., the net effect of the EAA
ingestion and resistance exercise bout). Significance was set
at P ⬍ 0.05. Additionally, total amino acid uptake or release
for each amino acid was compared against zero using the
paired t-test.
Leg blood flow, amino acid delivery to the muscle, PS from
blood-borne amino acids, amino acid release in blood from
PB, and net muscle protein balance calculated from L-[ring-
13
C6]phenylalanine infusion are presented for six separate
periods during the study day. The periods were chosen a
priori to illustrate the changes resulting from meals, amino
acid ingestion, and exercise over the study day. Time periods
are as follows: 0900–1100, period after ingestion of the first
meal; 1100–1400, period during ingestion of EAA drinks and
resistance exercise; 1400–1700, period from influence of ex-
ercise and drinks until beginning of ingestion of second meal;
1700–2000, period from beginning of ingestion of second
meal to 1 h after ingestion of the last dose of the second meal;
0000–0300, period in the middle of the night; and 0500–
0800, fasting period on the second morning of study. For each
period, the measured values used to calculate each parame-
ter were averaged over the time period for each subject, and
the parameters were calculated from the mean values. Pa-
AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 •
E81
rameters for the first period during ingestion of the first meal
were calculated from the mean of three values, the second
period, i.e., during exercise and supplementation from the
mean of six values, the postexercise night and early morning
fasted periods from the mean of five values, and the period
during the second meal ingestion from the mean of four
values. Only the last four values for the period during the
ingestion of the second meal were used for calculations so
that an isotopic steady state was obtained. Muscle intracel-
lular phenylalanine, leucine, valine, and glycine concentra-
tions are presented for each of five muscle biopsies taken
throughout the 24-h period (see Fig. 1).
Two-way ANOVA with repeated measures on both fac-
tors was performed to evaluate differences between means
for leg blood flow, amino acid delivery to the muscle, PS
from blood-borne amino acids, amino acid release in blood
from PB and net muscle protein balance, and muscle
intracellular concentration of phenylalanine and leucine.
A subject ⫻ trial (REST and ES) ⫻ time period (or biopsy
in the case of the muscle intracellular concentration) fac-
torial design was used. If a significant trial ⫻ time period
Downloaded from ajpendo.physiology.org on September 20, 2010
interaction was detected, a subsequent one-way, repeated-
measure ANOVA was performed for each trial (REST and
ES) to detect differences across time. For differences be-
tween trials at each time point, Bonferroni posttests were
used for pairwise comparisons.
RESULTS
Insulin, Leg Blood Flow, and Amino Acid Delivery
to the Leg
Area under the curve of arterial insulin above the
basal value was not significantly different between ES
(263 ⫾ 78 ␮IU/ml) and REST (432 ⫾ 147 ␮IU/ml). Leg
blood flow and phenylalanine delivery to the leg are
summarized for five time periods of the 24-h study in
Table 4. Leg blood flow was similar for all measure-
ment periods during REST (Table 4). During ES, leg
blood flow was similar to REST values for all time
periods except the supplementation and exercise pe-
riod. Blood flow was ⬃1.5–2 times greater during the
supplementation and exercise period of the ES trial
than all other time periods (P ⬍ 0.05). Overall, 24-h
mean blood flow was greater for ES than for REST
(6.20 ⫾ 1.23 vs. 3.42 ⫾ 0.33 ml 䡠 min⫺1 䡠 kg⫺1; P ⬍ 0.05).
Phenylalanine delivery during REST was greater for
the second meal period than for the postexercise peri-
ods in the middle of the night and the early morning
(P ⬍ 0.05). During ES, phenylalanine delivery was
increased by approximately six times during the sup-
plementation and exercise period, decreased signifi-
cantly during the postexercise period (P ⬍ 0.05), but
remained approximately two to three times greater
than during other periods (P ⬍ 0.05).
Blood Amino Acid Concentrations
The pattern of concentration changes for all four
amino acids was similar in the artery and the vein.
Thus, for clarity, only the arterial concentrations are
presented in Fig. 2. EAA (phenylalanine, leucine, and
valine, which were included in the EAA solution) ex-
hibited similar patterns of blood concentrations. Amino
acid concentrations were slightly increased during
www.ajpendo.org
E82 24-H MUSCLE PROTEIN BALANCE

Table 4. Mean blood flow and delivery (blood flow ⫻ arterial concentration) of phenylalanine to and from the
leg for rest and ES trials
BF EX PE ML NT AM

Blood flow, ml 䡠 min⫺1 䡠 100 ml leg vol⫺1

Rest 3.6 ⫾ 0.4 3.5 ⫾ 0.4a 3.6 ⫾ 0.3 3.4 ⫾ 0.3 3.2 ⫾ 0.3 3.5 ⫾ 0.5
ES 4.2 ⫾ 0.6 7.6 ⫾ 0.7b 4.7 ⫾ 0.7c 4.2 ⫾ 0.5c 4.2 ⫾ 0.4c 4.1 ⫾ 0.5c
Phe delivery, nmol 䡠 min⫺1 䡠 100 ml leg vol⫺1
Rest 274 ⫾ 22 272 ⫾ 27a 222 ⫾ 22d 320 ⫾ 36 216 ⫾ 20d 235 ⫾ 32d
ES 375 ⫾ 65 2,004 ⫾ 144b 802 ⫾ 89b,e 498 ⫾ 69c,e 298 ⫾ 29c,e 288 ⫾ 44c,e
Values are means ⫾ SE. Rest, value when subjects rested quietly for entire 24-h period; ES, value when subjects consumed 2 ⫻ 15 g of
essential amino acid mixture immediately before and 1 h after resistance exercise; BF, mean value for samples taken 2 h after ingestion of
first meal (0900–1100); EX, mean value for samples taken 3 h from first drink ingestion and beginning of exercise (1100–1400); PE, mean
value for 4 h from the exercise period to the beginning of the 2nd meal (1400–1700); ML, mean value for samples taken during ingestion of
the 2nd meal (1830–2000); NT, mean value for 3 h during the middle of the night (0000–0300); AM, mean value for samples taken on the
2nd morning (0500–0800). P ⬍ 0.05, significantly different between REST and ES in a given time period (a), BF within a trial (REST or ES;
b), EX within a trial (REST or ES; c, PE within a trial (REST or ES; e), from ML within a trial (REST or ES; d).

both meals in REST and ES and declined after each well as leucine, valine, and glycine, for both REST and

Downloaded from ajpendo.physiology.org on September 20, 2010

meal. During ES, amino acid concentrations were in-
creased ⬃3.5–4 times when the EAA solutions were
ingested and remained elevated for ⬃4 h. Phenylala-
nine concentration was significantly greater for ES
than for REST from 270 to 420 min, i.e., 1230–1500.
Leucine concentration was significantly greater for ES
than REST from 270 to 480 min, i.e., 1230–1600.
Concentration of the nonessential amino acid, glycine,
did not seem to change dramatically during the entire
24-h study.
Muscle Intracellular Amino Acid Concentrations
Figure 3 summarizes the intracellular concentra-
tions of phenylalanine, leucine, valine, and glycine in
five muscle biopsy samples taken during the 24-h study
period for both REST and ES. Muscle amino acid con-
centrations did not change significantly during REST.
During ES, intracellular phenylalanine concentrations
were significantly greater (P ⬍ 0.05) for the second
biopsy (1630) than all others. Phenylalanine concen-
tration from the third biopsy (taken during the liquid
meal feeding at 1830) was also greater (P ⬍ 0.05) than
from the first, fourth, and fifth biopsies. Phenylalanine
concentration was significantly greater (P ⬍ 0.05) dur-
ing ES than REST for only the second biopsy. Mean
phenylalanine value of the five biopsies was higher
(P ⫽ 0.02) for ES (86 ⫾ 3 nmol/ml of intracellular
water) than for REST (72 ⫾ 4 nmol/ml of intracellular
water). A similar pattern was exhibited by the concen-
tration of leucine, but the two-way ANOVA did not
reveal a significant interaction nor was there a signif-
icant difference between ES and REST for the mean
leucine concentration across all biopsies. Valine con-
centration seemed to exhibit a similar pattern. There
was little change in glycine concentration.
Amino Acid Balance Across the Leg
Phenylalanine exchange across the leg, i.e., the area
under the curve for NB, is the primary endpoint of the
study. Exchange was calculated for phenylalanine, as
AJP-Endocrinol Metab • VOL
ES over the entire 24-h study period and a 3-h period
corresponding to the ingestion of the first EAA drink
and resistance exercise during ES, i.e., 1100–1400
(Table 5). There was a net release of phenylalanine for
the 24-h study period of both REST and ES. Net re-
lease of phenylalanine was greater during REST than
ES, but this difference did not reach statistical signif-
icance (P ⬍ 0.07). For the 3 h corresponding to the time
of amino acid ingestion and exercise during ES, phe-
nylalanine exchange was no different from zero for
REST, but a total of 229 ⫾ 42 mg was taken up during
ES (P ⫽ 0.003). The difference in phenylalanine ex-
change between ES and REST was almost identical for
the 24-h time period and the 3-h time period corre-
sponding to EAA ingestion and exercise during ES
(Fig. 4).
Leucine exchange was not different from zero during
REST for either the 24- or 3-h time period but was
positive (net uptake) during ES for both time periods.
Net leucine uptake was greater for ES than REST for
both 24 h (P ⫽ 0.002) and 3 h (P ⫽ 0.001). The
difference in net leucine uptake between ES and REST
was significantly greater (P ⫽ 0.03) for the 24-h period
(1,456 ⫾ 313 mg) than the 3-h period (727 ⫾ 144 mg).
Valine exchange for the five subjects from whom
data were available was similar to leucine. During
REST, valine exchange was not significantly different
from zero for 24 h but was negative during the 3-h
period. For ES, there was net uptake of valine for both
the 24- and 3-h periods. Valine uptake was greater
during ES than REST for both 24-h (P ⫽ 0.03) and 3-h
periods (P ⫽ 0.003). The difference in valine exchange
between ES and REST was not statistically different
for 24-h and 3-h periods (1,094 ⫾ 406 and 766 ⫾ 182
mg, respectively).
Glycine exchange was significantly different from
zero only for the 24-h REST period, but glycine tended
to be released during 24 h ES (P ⫽ 0.057). Glycine
exchange for REST was not different from ES for the
24- or 3-h period. The difference between ES and REST
284 • JANUARY 2003 • www.ajpendo.org
 24-H MUSCLE PROTEIN BALANCE E83

Downloaded from ajpendo.physiology.org on September 20, 2010

Fig. 2. Twenty-four-hour arterial concentrations (nmol/ml) for phenylalanine (A), leucine (B), valine (C), and
glycine (D) during REST and ES. AA, indicates ingestion of 15 g of essential amino acids during the ES trial only.
M, meal ingestion.

was not statistically different for 24 h (⫺1,133 ⫾ 519
mg) and 3 h (418 ⫾ 576 mg).
Net amino acid balances across the leg over time for
the entire 24-h period are presented in Fig. 5. During
REST, phenylalanine, leucine, and valine balance in-
creased slightly after the first meal, declined, and then
increased again when the second meal was consumed.
After the meals, the values changed from negative to
positive, indicating a change from net release to net
uptake. Values returned to negative at night and in the
morning. During ES, there was a slight increase of
phenylalanine, leucine, and valine in response to the
first meal but a very large increase from negative to
positive levels in response to ingestion of EAA imme-
diately before exercise and 1 h after exercise. Values
decreased rapidly back to negative levels and then
returned to positive in response to the second meal.
Paired t-tests with Bonferroni corrections indicated
significant differences for both phenylalanine and
leucine NB across the leg only at 300 min despite the
AJP-Endocrinol Metab • VOL
large differences in means apparent from Fig. 5. Dur-
ing the night and second morning, values of phenylal-
anine, valine, and leucine declined back to negative
levels similar to those during REST. Glycine NB was
below zero for most of the 24-h period for both REST
and ES and exhibited a large degree of variability.
PS from Blood-Borne Amino Acids and Amino Acid
Appearance in Blood from PB
Figure 6 summarizes muscle PS from blood-borne
amino acids (PS), amino acid appearance from protein
breakdown (PB), and net muscle protein balance (NB)
calculated from infusion of [13C6]phenylalanine. There
was no change in PB during REST. PB was signifi-
cantly lower during the postexercise period and the
period during the second meal than during the supple-
mentation and exercise period for the ES trial. PB was
significantly greater (P ⬍ 0.05) for ES than REST
during the first and second periods, i.e., during inges-
284 • JANUARY 2003 • www.ajpendo.org
E84 24-H MUSCLE PROTEIN BALANCE

Fig. 3. Muscle intracellular concentra-

tions (nmol/ml intracellular water)
from 5 muscle biopsies (see Fig. 1) for
phenylalanine (A), leucine (B), valine
(C), and glycine (D) during REST and
ES. * Significant difference between
REST and ES, P ⬍ 0.05. Significantly
different from m1 (a), m2 (b), and m3 (c)
within a trial (REST or ES), P ⬍ 0.05.

Downloaded from ajpendo.physiology.org on September 20, 2010

tion of the first meal and the period of supplementation
and exercise. Overall, PB was greater (P ⫽ 0.003)
during ES than REST. PS was significantly reduced for
the night and early morning fasted periods compared
with the first early morning period (breakfast) and the
supplementation and exercise period during REST.
During the ES trial, PS was approximately two to six
times greater during the supplementation and exercise
period than all other time periods (P ⬍ 0.05). PS
declined to ⫺3 nmol 䡠 min⫺1 䡠 100 ml leg vol⫺1 during
the postexercise period, and this value was signifi-
cantly lower than PS during the supplementation and
exercise period and first early morning period (break-
fast; P ⬍ 0.05). PS was greater for ES than REST
during the early morning when breakfast was con-
sumed and during the exercise period. Overall, PS was
greater during ES than during REST. During REST,
NB was significantly greater for the first early morning
period (breakfast) than for the postexercise period. The
first early morning period (breakfast) NB was greater
than NB for the postexercise, night, and early morning
fasted periods, and the supplementation and exercise
period was greater than night and early morning
fasted periods during REST. During the ES trial, the
supplementation and exercise period NB was signifi-
cantly greater than for all other time periods. NB
during the first early morning meal period and the
second meal period was greater than during the post-
exercise period for the ES trial. NB was greater during
ES than REST during the supplementation and exer-
cise period, whereas it was significantly lower for ES
than REST during the postexercise period. NB
changed over the 24-h period for both REST and ES.
During the first early morning meal period and the
second meal period for both study days and the sup-
plementation and exercise period for REST, NB was no
different from zero. NB was negative during the post-
exercise period for REST and during the middle of the
night hours and at the end of the study on the second
morning for both ES and REST.

Table 5. Amino acid exchange across the leg (area under the curve for net balance) over the entire 24-h period
and over 3 h of exercise and amino acid supplementation period for REST and ES treatments
Phenylalanine Leucine Valine Glycine

24 h
Rest ⫺371 ⫾ 88† 245 ⫾ 174 534 ⫾ 326 ⫺2,163 ⫾ 1,015†
ES ⫺194 ⫾ 74† 1,702 ⫾ 306*† 1,789 ⫾ 458*† ⫺2,748 ⫾ 1,245
3h
Rest 28 ⫾ 15 111 ⫾ 37 ⫺161 ⫾ 69† ⫺361 ⫾ 237
ES 229 ⫾ 42*† 788 ⫾ 140*† 594 ⫾ 96*† 88 ⫾ 272
Values are means ⫾ SE in mg; n ⫽ 7 experiments for phenylalanine and leucine; n ⫽ 5 for valine and glycine REST; and n ⫽ 4 for valine
and glycine ES. 24 h, Area under curve for net amino acid balance over the entire 24-h study period; 3 h, area under the curve for net amino
acid balance over 3 h from ingestion of first drink and beginning of exercise (1100–1400). P ⬍ 0.05, significantly different from REST (*) and
0 (†).

AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 • www.ajpendo.org

 24-H MUSCLE PROTEIN BALANCE E85

FSR of Mixed Muscle PS

Fig. 4. Difference in phenylalanine exchange (area under the curve
for net muscle protein balance in mg) between REST and ES over the
entire 24 h and over the 3 h, in both trials, corresponding to the time
of amino acid ingestion and exercise in the ES trial.
During ES, mean 24-h FSR was 41% greater (P ⫽
0.003) than during REST. During the day, i.e., from the
0800 biopsy to the 1630 biopsy, FSR was not signifi-
cantly different between ES (0.0586 ⫾ 0.0062%/h) and
REST (0.0476 ⫾ 0.0115%/h). For the 2 h during inges-
tion of the second meal, FSR was significantly in-
creased (145%; P ⫽ 0.045) during ES (0.1882 ⫾
0.0607%/h) than REST (0.0762 ⫾ 0.0089%/h). During
the night, i.e., from 1830 to 0800, mean ES FSR was
29% greater than REST FSR (0.0709 ⫾ 0.0096 vs.
0.0552 ⫾ 0.0059%/h, respectively), but the difference
failed to reach statistical significance (P ⫽ 0.07).
DISCUSSION

This study was designed to determine if the response

of net muscle protein balance to resistance exercise and

Downloaded from ajpendo.physiology.org on September 20, 2010

amino acid ingestion previously noted on an acute

Fig. 5. Twenty-four-hour net muscle protein balance (mg/min) for phenylalanine (A), leucine (B), valine (C), and
glycine (D) during REST and ES.

AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 • www.ajpendo.org

E86 24-H MUSCLE PROTEIN BALANCE
Fig. 6. Muscle protein synthesis, breakdown, and net muscle protein
balance determined for 6 discrete time periods (see Fig. 1) over the
entire 24-h study for both REST and ES trials. EX, exercise period,
1100–1400. * Significantly different between REST and ES in a given
time period, P ⬍ 0.05. Significantly different from BF (a), EX (b), PE
(c), and ML (d) within a trial (REST or ES), P ⬍ 0.05.
basis (21, 25, 26) reflects the response of net muscle
protein balance over an entire 24-h period. To our
knowledge, these are the first data to describe the
response of muscle protein balance to exercise and
amino acids over a 24-h period. The results support the
notion that measured changes in muscle NB over a
short time period, e.g., 3 h, are representative of
changes in NB for a full 24-h period. Exchange of
amino acids, i.e., area under the curve for net muscle
protein balance, was measured over the 3-h period
corresponding to the period of amino acid ingestion and
AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 •
exercise and over the entire 24-h period. Ingestion of
the EAA solution immediately before and 1 h after
exercise improved amino acid exchange when mea-
sured both on a 24- and 3-h basis (Table 5). More
importantly, if the response of net muscle protein bal-
ance to the amino acids and exercise is overestimated
by acute measurement over 3 h, then the difference
between the response during ES and REST periods,
i.e., anabolic response to the exercise bout and the
ingestion of EAA, should be greater when measured
over 3 h than over the full 24 h. However, the differ-
ence of net phenylalanine balance between ES and
REST, i.e., the response of muscle to the exercise and
EAA ingestion, was similar when measured over 3 h,
encompassing the exercise and amino acid ingestion
vs. the full 24 h (Fig. 4). Furthermore, the responses of
leucine, valine, and glycine balance were also similar
for 24 and 3 h. These amino acids include three EAA
that were included in the ingested EAA solution, as
Downloaded from ajpendo.physiology.org on September 20, 2010
well as a nonessential amino acid that was not in-
gested. Consistent with our previous observations (e.g.,
21 and 26), we found that NB was elevated in the time
surrounding and immediately subsequent to resistance
exercise and the ingestion of EAA (Fig. 5). During most
of the rest of the 24-h period, NB across the leg was
similar for REST and ES. Thus the acute stimulation of
muscle protein by exercise and EAA ingestion is addi-
tive to the balance that normally occurs in resting
muscle.
An examination of NB across time during ES (Fig. 5)
suggests that there is a nadir in the response of NB
after the initial large increase. If these data are sepa-
rated into discrete time periods (Fig. 6C), it is apparent
that there is, indeed, a decline in net muscle balance
that dips below the level seen during rest. This tran-
sient nadir is likely because of an efflux of some of the
amino acids taken up from the blood when the zenith of
concentration favored inward movement that was sub-
sequently not incorporated into protein. Transport of
amino acids into the muscle cell results in both in-
creased muscle PS and expansion of the muscle intra-
cellular pool. Thus not all of the amino acids taken up
by the muscle while these measurements were being
made were incorporated into muscle protein; therefore,
elevated PS during the supplementation and exercise
period probably overestimates muscle PS. Subse-
quently, outward efflux from transport of the amino
acids would contribute to an underestimation of the
calculated PS value and, thus, the negative values
noted for the postexercise period. Indeed, the magni-
tude of the outward efflux of phenylalanine during the
postexercise period in relation to the delivery to the
muscle (Table 5) is consistent with the notion that
there was a large amount of phenylalanine remaining
in the intracellular space that leaves the muscle during
the postexercise period. Hence, although the actual
rate of PS is clearly reduced during this period, it is
almost certainly underestimated because the negative
values reported here are reflections of the large efflux
of amino acids from the cell.
www.ajpendo.org
 24-H MUSCLE PROTEIN BALANCE
Although there is likely an overestimation of PS
during the supplementation and exercise period and an
underestimation during the postexercise period, it is
still clear that PS was dramatically elevated by the
resistance exercise and amino acid ingestion. If we
assume that the basal level of PS for ES is equal to that
measured during the same period for REST, we can
estimate PS for the supplementation and exercise pe-
riod of ES as 223 ⫾ 36 nmol 䡠 min⫺1 䡠 100 ml leg⫺1,
indicating that PS was originally overestimated for the
supplementation and exercise period during ES by
⬃17%. Even using the more conservative, corrected
value, PS for ES is 350% greater (P ⬍ 0.001) than
REST. Hence, despite a likely overestimation of PS
during the supplementation and exercise period and an
underestimation during the postexercise period, it is
clear that the performance of resistance exercise and
ingestion of EAA stimulates muscle PS, resulting in an
improvement in net muscle protein balance over rest-
ing levels. Thus the total NB is greater during ES than
REST, primarily because of differences in the period
immediately surrounding the exercise and EAA inges-
tion.
Although the main endpoint of the study is the
response of net amino acid balance, the response of
mixed muscle FSR supports the conclusion that the
acute response of muscle anabolism to exercise and
amino acids reflects that of a full 24-h period. Mixed-
muscle FSR measured over the entire 24 h was ⬃40%
greater when subjects performed resistance exercise
and ingested EAA than when subjects rested. Although
we did not measure FSR for the 3 h after exercise in
this study, we previously demonstrated that the com-
bination of exogenous amino acids and resistance ex-
ercise acutely stimulates FSR over resting levels (7).
Taken together, these data support the notion that
exogenous amino acids combined with resistance exer-
cise improve net muscle protein balance primarily by
increasing PS (7, 25, 26).
Temporal patterns of muscle PS and breakdown
were examined by calculating PS, i.e., PS from plasma
bound phenylalanine, and PB, i.e., phenylalanine re-
leased from muscle PB that appeared in plasma using
mean values from discrete time periods during ES and
REST (Fig. 6). There were no dramatic changes in PS
or PB throughout the 24-h period when subjects rested
quietly during REST. However, as expected (7, 25, 26),
resistance exercise and EAA ingestion resulted in dra-
matic changes in PS. Furthermore, the mean value of
PS for the full 24-h day, calculated from the data
presented in Fig. 6, is significantly greater (P ⫽ 0.0005)
for ES than for REST (79 ⫾ 11 and 36 ⫾ 5
nmol 䡠 min⫺1 䡠 100 ml leg⫺1, respectively). It is interest-
ing to note the increase in PB that accompanies in-
creased PS. If the changes in net muscle protein bal-
ance were influenced by an inhibition of PB, then we
would expect PB to be reduced during ES compared
with REST. In fact, the opposite is true. PB is signifi-
cantly greater for ES (67 ⫾ 11 nmol 䡠 min⫺1 䡠 100 ml
leg⫺1) than for REST (49 ⫾ 7 nmol 䡠 min⫺1 䡠 100 ml
leg⫺1). Thus these data support the concept that in-
AJP-Endocrinol Metab • VOL
E87
creased muscle PS is primarily responsible for the
increase in net muscle protein balance resulting from
resistance exercise and ingestion of exogenous amino
acids. Previously, we reported that muscle PS and
muscle PB are correlated (6, 18, 19). Similarly, when
all of the PS data are compared with the respective PB
in the present study, a significant (P ⬍ 0.001) correla-
tion is noted (r ⫽ 0.71).
The subjects were in overall negative net phenylal-
anine balance during the resting study. This observa-
tion is a bit puzzling, since normal, healthy, adult
humans would be expected to be in muscle protein
balance rather than negative muscle protein balance.
It is possible that inactivity for this period of time is
sufficient to induce negative muscle protein balance
and the beginning of muscle loss. Negative muscle
protein balance resulting from only 24–36 h of inactiv-
ity would be a novel finding. Decreased lean body mass
has been reported in as little as 7 days of strict bed rest
Downloaded from ajpendo.physiology.org on September 20, 2010
(10, 23); however, the precision of methods to measure
muscle loss are probably not sufficient to reliably mea-
sure muscle loss for shorter time periods. No measure-
ment of activity was made on subjects in the present
study, and they were not instructed to remain immo-
bile. However, subjects typically rested comfortably in
bed with only minimal movement during REST. In-
cluding sleeping the night before the infusion study,
activity was limited for ⬃30–36 h. These data suggest
that a minimum amount of activity may be necessary
to maintain net muscle protein balance and, ulti-
mately, muscle mass. Further research specifically fo-
cused on short-duration inactivity, and perhaps the
minimum amount of activity necessary to maintain net
muscle protein balance, appears necessary. On the
other hand, negative muscle protein balance may have
been an artifact of the study design. It is possible that
limiting the subjects to only 80% of their normal en-
ergy intake for the 24-h study day was enough to
initiate muscle loss. Although acute effects of energy
imbalance have not been examined previously, Todd et
al. (27) demonstrated that chronic energy balance was
critical for the maintenance of nitrogen balance, espe-
cially in the absence of exercise. We attempted to
normalize energy intake for 5 days, including the study
day, but we cannot exclude the possibility that even an
energy deficit over as short a period as 24 h may be
enough to initiate net muscle protein degradation. Fi-
nally, it is also possible that the acute stress resulting
from catheterization and muscle biopsies may be
enough to contribute to net negative muscle protein
balance. Furthermore, inactivity, at least for as much
as 2 wk, seems to interact with cortisol to increase
muscle PB and net muscle loss (11). These results raise
the possibility that even shorter durations of inactivity
may contribute to initiation of muscle loss, especially if
combined with stress.
Our previous results (7, 25) demonstrate that there
is an elevation of net muscle protein balance in asso-
ciation with hyperaminoacidemia and resistance exer-
cise. It has been suggested that there may be a physi-
ological diurnal homeostasis of the body protein pool
284 • JANUARY 2003 • www.ajpendo.org
E88 24-H MUSCLE PROTEIN BALANCE
such that an elevation during feeding may be coun-
tered by a nadir in PS during fasting situations (16, 17,
20). If this were true for muscle, we would expect to see
a large decrease in net muscle protein balance during
the night when our subjects were fasted. This decrease
would be expected to be much greater during ES than
REST, since there was a larger increase with EAA
intake and exercise. In this study, there was no evi-
dence that stimulation of muscle PS and elevation of
net muscle protein balance during the day in ES re-
sulted in a subsequent nadir. Both PS and NB were
similar for ES and REST in the nighttime period.
Furthermore, there was no change in PS or NB from
the middle of the night to early morning. Thus there
seems to be no evidence of a physiological homeostasis
that compensates for excessive levels of muscle PS
during the day.
During the morning meal period, both PS and PB
were elevated during ES compared with REST, raising
the possibility that a difference during the first period
may have influenced the net 24-h response during
ES and therefore our conclusions. These results are
somewhat puzzling, since this period was the initial
measurement period, and there should have been no
difference between the treatments. The most parsimo-
nious explanation would be for an inadvertent proce-
dural difference between the two trials. The order of
the trials was randomized so that four subjects did the
REST trial first while three did the ES trial first. There
was no significant difference between PS values from a
subject’s first trial vs. second trial, so no order effect
was apparent. There were slight differences in the time
of meal ingestion on any given trial day because of
differences in timing of catheter placement and muscle
biopsy. However, there was no correlation of the PS to
the time of meal ingestion nor was there any difference
in the time from meal ingestion to the first arterio-
venous sample between REST and ES. Accordingly,
there is no obvious procedural explanation for the
observed difference between ES and REST for phenyl-
alanine concentration, PS, or PB. More importantly,
NB is not significantly different in the first early morn-
ing meal period of REST compared with ES, and this is
the primary endpoint of the study.
The question remains as to whether this difference
in the first early morning meal period PS and PB
affected the results during the remainder of the day. If
anything, one might a priori expect a greater stimula-
tion in one time period to result in a refractory re-
sponse rather than further stimulation. Bohe et al. (8)
demonstrated that elevated levels of amino acids ini-
tially stimulated muscle PS, but subsequently muscle
PS returned to basal levels despite continued hy-
peraminoacidemia. Therefore, the elevated levels of PS
during the first early morning meal period for ES
might be expected to result in a blunting of the re-
sponse to resistance exercise and EAA. Although there
was not a statistically significant difference in NB
between REST and ES for the first early morning meal
time period, ES was numerically higher, and it is
conceivable that this difference may contribute to the
AJP-Endocrinol Metab • VOL
overall difference between ES and REST. To address
this concern, we calculated the phenylalanine ex-
change, i.e., area under the curve for NB, for both
REST and ES without the breakfast time period in-
cluded. The difference in phenylalanine exchange be-
tween ES and REST without the first early morning
meal periods (21 h) was not significantly lower than
that for the whole 24 h (132 ⫾ 88 and 177 ⫾ 104 mg,
respectively). Furthermore, as with the full 24-h pe-
riod, there was no significant difference between the
21 h without the breakfast period and the 3-h period
for the difference between ES and REST phenylalanine
exchange, i.e., the response to EAA ingestion and re-
sistance exercise. Thus it appears that the unantici-
pated increase in phenylalanine turnover during the
first time period of ES compared with REST does not
affect the conclusion that resistance exercise plus
amino acids stimulate net muscle PS through an in-
crease in muscle PS and that acute measurements
Downloaded from ajpendo.physiology.org on September 20, 2010
reflect those over 24 h.
Net muscle protein balance has been demonstrated
to increase dramatically in response to exercise and
amino acid ingestion when measured acutely (7, 25).
However, the question remained as to whether this
acute response reflected the response of muscle over a
longer time period. In this study, we confirmed that
muscle protein balance is increased, primarily because
of an increase in muscle PS, when measured acutely
and found that this response is additive to the basal
response over a full 24-h period. Thus measurement of
the acute metabolic response of muscle metabolism in
future studies will provide information that may be
interpreted to reflect the likelihood of changes to mus-
cle mass when the intervention is carried out over
longer time periods.
We thank the nurses and staff of the General Clinical Research
Center at the University of Texas Medical Branch-Galveston. We
also thank Dr. Judah Rosenblatt for statistical assistance and the
volunteers who participated in the studies for their time and effort.
This work was supported, in part, by National Institutes of Health
(NIH) Grants RO1-AR-45382 and RO1-DK-38010 and by grants
8940 and 15489 from the Shriners Hospitals for Children. Studies
conducted at the General Clinical Research Center at the University
of Texas Medical Branch at Galveston were funded by NIH Grant
M01 RR-00073.
REFERENCES
1. Baumann PQ, Stirewalt WS, O’Rourke BD, Howard D, and
Nair KS. Precursor pools of protein synthesis: a stable isotope
study in a swine model. Am J Physiol Endocrinol Metab 267:
E203–E209, 1994.
2. Bergstrom J, Furst P, Noree LO, and Vinnars E. Intracel-
lular free amino acid concentration in human muscle tissue.
J Appl Physiol 36: 693–697, 1974.
3. Biolo G, Declan Fleming RY, and Wolfe RR. Physiologic
hyperinsulinemia stimulates protein synthesis and enhances
transport of selected amino acids in human skeletal muscle.
J Clin Invest 95: 811–819, 1995.
4. 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.
5. Biolo G, Gastaldelli A, Zhang XJ, and Wolfe RR. Protein
synthesis and breakdown in skin and muscle: a leg model of
284 • JANUARY 2003 • www.ajpendo.org
 24-H MUSCLE PROTEIN BALANCE
amino acid kinetics. Am J Physiol Endocrinol Metab 267: E467–
E474, 1994.
6. 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.
7. 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.
8. Bohe J, Low JF, 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.
9. Ferrando AA, Lane HW, Stuart CA, Davis-Street J, and
Wolfe RR. Prolonged bed rest decreases skeletal muscle and
whole body protein synthesis. Am J Physiol Endocrinol Metab
270: E627–E633, 1996.
10. Ferrando AA, Stuart CA, Brunder DG, and Hillman GR.
Magnetic resonance imaging quantitation of changes in muscle
volume during 7 days of strict bed rest. Aviat Space Environ Med
66: 976–981, 1995.
11. Ferrando AA, Stuart CA, Sheffield-Moore M, and Wolfe
RR. Inactivity amplifies the catabolic response of skeletal mus-
cle to cortisol. J Clin Endocrinol Metab 84: 3515–3521, 1999.
12. Ferrando AA, Tipton KD, Bamman MM, and Wolfe RR.
Resistance exercise maintains skeletal muscle protein synthesis
during bed rest. J Appl Physiol 82: 807–810, 1997.
13. Ferrando AA, Tipton KD, Doyle D, Phillips SM, Cortiella
J, and Wolfe RR. Testosterone injection stimulates net protein
synthesis but not tissue amino acid transport. Am J Physiol
Endocrinol Metab 275: E864–E871, 1998.
14. Forslund AH, Hambraeus L, Olsson RM, El Khoury AE, Yu
YM, and Young VR. The 24-h whole body leucine and urea
kinetics at normal and high protein intakes with exercise in
healthy adults. Am J Physiol Endocrinol Metab 275: E310–
E320, 1998.
15. Ljungqvist OH, Persson M, Ford GC, and Nair KS. Func-
tional heterogeneity of leucine pools in human skeletal muscle.
Am J Physiol Endocrinol Metab 273: E564–E570, 1997.
16. Millward DJ and Rivers JP. The nutritional role of indispens-
able amino acids and the metabolic basis for their requirements.
Eur J Clin Nutr 42: 367–393, 1988.
17. Pacy PJ, Price GM, Halliday D, Quevedo MR, and Mill-
ward DJ. Nitrogen homeostasis in man: the diurnal responses
of protein synthesis and degradation and amino acid oxidation to
AJP-Endocrinol Metab • VOL 284 • JANUARY 2003 •
E89
diets with increasing protein intakes. Clin Sci (Colch) 86: 103–
116, 1994.
18. 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.
19. Phillips SM, Tipton KD, Ferrando AA, and Wolfe RR.
Resistance training reduces the acute exercise-induced increase
in muscle protein turnover. Am J Physiol Endocrinol Metab 276:
E118–E124, 1999.
20. Price GM, Halliday D, Pacy PJ, Quevedo MR, and Mill-
ward DJ. Nitrogen homeostasis in man: influence of protein
intake on the amplitude of diurnal cycling of body nitrogen. Clin
Sci (Colch) 86: 91–102, 1994.
21. 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.
22. Rosenblatt J, Chinkes D, Wolfe M, and Wolfe RR. Stable
isotope tracer analysis by GC-MS, including quantification of
isotopomer effects. Am J Physiol Endocrinol Metab 263: E584–
E596, 1992.
23. Shangraw RE, Stuart CA, Prince MJ, Peters EJ, and Wolfe
Downloaded from ajpendo.physiology.org on September 20, 2010
RR. Insulin responsiveness of protein metabolism in vivo follow-
ing bedrest in humans. Am J Physiol Endocrinol Metab 255:
E548–E558, 1988.
24. Thompson GN, Pacy PJ, Merritt H, Ford GC, Read MA,
Cheng KN, and Halliday D. Rapid measurement of whole body
and forearm protein turnover using a [2H5]phenylalanine model.
Am J Physiol Endocrinol Metab 256: E631–E639, 1989.
25. 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.
26. 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.
27. Todd KS, Butterfield GE, and Calloway DH. Nitrogen bal-
ance in men with adequate and deficient energy intake at three
levels of work. J Nutr 114: 2107–2118, 1984.
28. Wolfe RR. Radioactive and Stable Isotope Tracers in Biomedi-
cine: Principles and Practice of Kinetic Analysis. New York:
Wiley-Liss, 1992.
www.ajpendo.org