1. Exogenous amino acids stimulate human muscle anabolism
without interfering with the response to mixed meal ingestion
Douglas Paddon-Jones,1,3
Melinda Sheffield-Moore,1,3
Asle Aarsland, 1,2,3
Robert R. Wolfe,1,2,3
and Arny A. Ferrando1,3
Departments of 1
Surgery and 2
Anesthesiology, The University of Texas
Medical Branch, and 3
Shriners Hospitals for Children, Galveston, Texas
Submitted 2 July 2004; accepted in final form 29 November 2004
Paddon-Jones, Douglas, Melinda Sheffield-Moore, Asle Aars-
land, Robert R. Wolfe, and Arny A. Ferrando. Exogenous amino
acids stimulate human muscle anabolism without interfering with the
response to mixed meal ingestion. Am J Physiol Endocrinol Metab
288: E761–E767, 2005. First published November 30, 2004; doi:
10.1152/ajpendo.00291.2004.—We sought to determine whether in-
gestion of a between-meal supplement containing 30 g of carbohy-
drate and 15 g of essential amino acids (CAA) altered the metabolic
response to a nutritionally mixed meal in healthy, recreationally active
male volunteers. A control group (CON; n ϭ 6, 38 Ϯ 8 yr, 86 Ϯ 10
kg, 179 Ϯ 3 cm) received a liquid mixed meal [protein, 23.4 Ϯ 1.0 g
(essential amino acids, 14.7 Ϯ 0.7 g); carbohydrate, 126.6 Ϯ 4.0 g;
fat, 30.3 Ϯ 2.8 g] every 5 h (0830, 1330, 1830). The experimental
group (SUP; n ϭ 7, 36 Ϯ 10 yr, 87 Ϯ 12 kg, 180 Ϯ 3 cm) consumed
the same meals but, in addition, were given CAA supplements (1100,
1600, 2100). Net phenylalanine balance (NB) and fractional synthetic
rate (FSR) were calculated during a 16-h primed constant infusion of
L-[ring-2
H5]phenylalanine. Ingestion of a combination of CAA sup-
plements and meals resulted in a greater mixed muscle FSR than
ingestion of the meals alone (SUP, 0.099 Ϯ 0.008; CON, 0.076 Ϯ
0.005%/h; P Ͻ 0.05). Both groups experienced an improvement in
NB after the morning (SUP, Ϫ2.2 Ϯ 3.3; CON, Ϫ1.5 Ϯ 3.5
nmol⅐minϪ1
⅐100 ml leg volumeϪ1
) and evening meals (SUP, Ϫ9.7 Ϯ
4.3; CON, Ϫ6.7 Ϯ 4.1 nmol⅐minϪ1
⅐100 ml leg volumeϪ1
). NB after
CAA ingestion was significantly greater than after the meals, with
values of 40.2 Ϯ 8.5 nmol⅐minϪ1
⅐100 ml leg volumeϪ1
. These data
indicate that CAA supplementation produces a greater anabolic effect
than ingestion of intact protein but does not interfere with the normal
metabolic response to a meal.
protein metabolism; diet; supplement; skeletal muscle
THE RATIONALE FOR THE USE of amino acid supplements is based
on the assumption that they will improve net muscle protein
synthesis above and beyond that afforded by regular food
ingestion alone. To be effective, a dietary amino acid supple-
ment should fulfill several requirements. Among these, a sup-
plement should provide an energetically efficient anabolic
response greater than that achieved by ingestion of regular
meals. In addition to this direct anabolic effect, the additional
energy and nutrient content provided by a supplement should
not interfere with or blunt the normal anabolic response to
protein consumed as part of normal daily meals. Specifically, a
supplement should not be so energetically dense as to blunt an
individual’s appetite or desire to ingest regular meals. This
appetite suppression has been demonstrated in elderly individ-
uals, where the provision of a nutritionally mixed 360-kcal
supplement resulted in a compensatory caloric redistribution,
with the supplement serving as a caloric replacement rather
than a true supplement per se (21).
We have previously demonstrated that the bolus oral inges-
tion of essential amino acids produces a rapid severalfold
increase in plasma amino acid concentrations and can stimulate
skeletal muscle protein synthesis to a greater extent than
nonessential amino acids (13, 34). Whereas fat has no direct
anabolic effect, carbohydrate ingestion increases insulin secre-
tion in healthy individuals and may also inhibit proteolysis (12)
while stimulating amino acid uptake and muscle protein syn-
thesis (8, 42). Furthermore, in young individuals, the combined
effect of essential amino acid and carbohydrate supplementa-
tion on muscle protein synthesis is greater than the sum of their
independent effects (36).
Protein balance over a 24-h period is governed by periods of
net protein degradation (postabsorptive) and periods of net
protein synthesis (postprandial). In most healthy ambulatory
individuals, muscle protein synthesis and breakdown are
closely matched, resulting in no readily discernable change in
muscle mass. However, in many situations, this balance is
skewed. For example, the sarcopenia of aging is characterized
by a progressive loss of contractile tissue that is facilitated by
a combination of factors including the adoption of a less-than-
optimal diet (17, 18, 32). Similarly, after debilitating injury, the
normal anabolic stimulus to feeding is disrupted, and many
severely injured individuals fail to maintain lean body mass
despite elevated caloric intakes (22, 35). The goal of amino
acid supplementation in such circumstances is to provide an
anabolic stimulus capable of reducing or ameliorating the
catabolic process. This goal would be compromised should the
stimulus afforded by a supplement be offset by a concomitant
reduction in the response to regular meal ingestion.
We have previously demonstrated that, after ingestion of
15 g of essential amino acids, plasma amino acid concentra-
tions remain elevated for upwards of 3 h, whereas net phenyl-
alanine balance returns to postabsorptive levels after ϳ60 min
(28). It remains uncertain, however, whether the protein met-
abolic effect of a meal ingested during a period of elevated
plasma amino acid concentrations (i.e., 2–3 h after an essential
amino acid supplement) would be diminished or perhaps ben-
efit from the prior nutrient ingestion.
The purpose of this study was twofold. Our first goal was to
determine whether ingestion of three carbohydrate and essen-
tial amino acid supplements and three nutritionally mixed
meals (SUP) over a 16-h period would produce a greater
Address for reprint requests and other correspondence: D. Paddon-Jones, Dept.
of Surgery, 815 Market St., Galveston, TX 77550 (E-mail: djpaddon@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 288: E761–E767, 2005.
First published November 30, 2004; doi:10.1152/ajpendo.00291.2004.
0193-1849/05 $8.00 Copyright © 2005 the American Physiological Societyhttp://www.ajpendo.org E761

2. protein synthetic response than ingestion of the meals alone
(CON). Our second goal was to determine whether prior
ingestion of carbohydrate and essential amino acid supple-
ments altered the normal skeletal muscle metabolic response to
a subsequent meal.
METHODS
Thirteen healthy, recreationally active male volunteers aged be-
tween 28 and 48 yr participated in this project. Subjects were ran-
domly assigned to a supplement [SUP: n ϭ 7, 36 Ϯ 10 (SD) yr, 87 Ϯ
12 kg, 180 Ϯ 3 cm, 18.2 Ϯ 2.0% body fat] or placebo group [CON:
n ϭ 6, 38 Ϯ 8 (SD) yr, 86 Ϯ 10 kg, 179 Ϯ 3 cm, 19.9 Ϯ 1.9% body
fat]. Body mass index (BMI) was 26.5 Ϯ 1.2 kg/m2
(SUP) and 26.9 Ϯ
1.1 kg/m2
(CON), respectively. All subjects gave informed, written
consent according to the guidelines established by the Institutional
Review Board at the University of Texas Medical Branch (Galveston,
TX). Subject eligibility was assessed by a battery of medical screening
tests, including medical history, physical examination, electrocardio-
gram, blood count, plasma electrolytes, blood glucose concentration,
and liver and renal function tests. Exclusion criteria included recent
injury, the presence of a metabolically unstable medical condition,
low hematocrit or hemoglobin, vascular disease, hypertension, or
cardiac abnormality.
The experimental protocol was similar to several previous studies
performed in this laboratory (13, 20, 26, 37) (Fig. 1). Volunteers were
instructed to maintain their normal diet and refrain from strenuous
activity during the weeks after medical screening and preceding
admission. Subjects completed 5 days of dietary stabilization before
the stable isotope infusion study. During this period, subjects were
housed in the General Clinical Research Center at the University of
Texas Medical Branch. Subjects were sedentary but remained ambu-
latory. Consistent with previous studies from our laboratory (19), the
Harris-Benedict equation with an activity factor (AF) of 1.6 was used
to estimate daily energy requirements during diet stabilization, ac-
cording to the following formula:
daily energy requirement (kcal)
ϭ ͓66 ϩ ͑13.7 ⅐ kg͒ ϩ ͑5 ⅐ cm͒ Ϫ ͑6.8 ⅐ yr͔͒ ⅐ AF
The AF was lowered to 1.3 during the stable isotope infusion study to
better reflect energy requirements during a period of inactivity. During
the diet stabilization period and stable isotope infusion study, daily
nutrient intake was evenly distributed between three meals (0830,
1300, 1830), with carbohydrate, fat, and protein representing 59, 27,
and 14%, respectively (19) (Table 1). Subjects consumed water ad
libitum.
At approximately 0600 on the morning of the stable isotope
infusion study, an 18-gauge polyethylene catheter (Insyte-W; Becton-
Dickinson, Sandy, UT) was inserted into an antecubital vein. Baseline
blood samples were drawn for the analysis of background amino acid
concentration and enrichment and insulin and glucose concentrations.
A second 18-gauge polyethylene catheter was placed in the contralat-
eral wrist for blood sampling for the spectrophotometric determina-
tion of leg blood flow (23). A primed (2 ␮mol/kg) continuous infusion
(0.05 ␮mol⅐kgϪ1
⅐minϪ1
) of [ring-2
H5]phenylalanine was initiated
and maintained for the duration of the study. At approximately 0700,
3-Fr 8-cm polyethylene Cook catheters (Bloomington, IN) were
inserted into the femoral artery and vein of one leg under local
anesthesia. Femoral arterial and venous blood samples were obtained
at 15- to 30-min intervals from 0800 to 2400. Samples were analyzed
to determine phenylalanine kinetics and plasma concentrations of
glucose and insulin as previously described (28). Briefly, femoral
artery and vein blood samples were immediately mixed and precipi-
tated in preweighed tubes containing a 15% sulfosalicylic acid solu-
tion and an internal standard. The internal standard (100 ml/l blood)
contained 49.3 ␮mol/l L-[ring-13
C6]phenylalanine. Samples were re-
weighed and centrifuged, and the supernatant was removed and frozen
(Ϫ80°C) until analysis. On thawing, blood amino acids were extracted
from 500 ␮l of supernatant by cation exchange chromatography
(Dowex AG 50W-8X, 100–200 mesh Hϩ form; Bio-Rad Laborato-
ries, Richmond, CA) and dried under vacuum (Savant Instruments,
Farmingdale, NY). Phenylalanine enrichments and concentrations
were determined on the tert-butyldimethylsilyl derivative by GC-MS
(HP model no. 5989; Hewlett-Packard, Palo Alto, CA) with electron
impact ionization. Ions 336, 341, and 342 were monitored (29, 43).
Plasma insulin concentrations were determined by radioimmuno-
assay (Coat-A-Count; Diagnostic Products, Los Angeles, CA).
Muscle biopsy samples were immediately rinsed, blotted, and
frozen in liquid nitrogen until analysis. On thawing, samples were
weighed, and the protein was precipitated with 800 ␮l of 14%
perchloroacetic acid. To measure intracellular phenylalanine con-
centration, an internal standard (2 ␮l/mg wet wt) containing 3
␮mol/l L-[ring-13
C6]phenylalanine was added. Approximately 1.5
ml of supernatant was collected after tissue homogenization and
centrifugation and processed in the same manner as the supernatant
from blood samples. Intracellular phenylalanine enrichment and
concentrations were determined with the tert-butyldimethylsilyl
derivative (6, 40). The remaining muscle pellet was washed and
dried, and the proteins were hydrolyzed in 6 N HCl at 50°C for
24 h. The protein-bound L-[ring-2
H5]phenylalanine enrichment
was determined using GC-MS (HP model no. 5989, Hewlett-
Packard) with electron impact ionization (14).
To measure leg blood flow, indocyanine green (ICG) was infused
into the femoral artery for ϳ20 min on two occasions (1000 and
2000). Three 2-ml blood samples were drawn simultaneously from the
femoral and wrist vein during the final 10 min of each ICG infusion
period, as previously described (7, 23). This technique does not
account for acute or transient changes in blood flow after meal or
supplement ingestion. Rather, it is a representation of resting blood
flow over the study period.
Fifty-milligram muscle biopsy samples were taken from the lateral
portion of the vastus lateralis ϳ10–15 cm above the knee, using a
5-mm Bergstrom biopsy needle as previously described (5). Samples
were obtained at 0800 and 2400 and used to calculate mixed muscle
Fig. 1. Study design and timeline. Control group
(CON; n ϭ 6) received a liquid, mixed meal (MEAL)
containing protein (23.4 Ϯ 1.0 g), carbohydrate
(126.6 Ϯ 4.0 g), and fat (30.3 Ϯ 2.8 g). Experimental
group (SUP; n ϭ 7) consumed the same meals but, in
addition, were given a supplement (CAA) containing
15 g of essential amino acids (EAAs) and 30 g of
sucrose. Muscle protein kinetics were determined
during a 16-h primed constant infusion of L-[ring-
2
H5]phenylalanine with femoral arterial and venous
(A-V) blood samples.
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3. fractional synthetic rate (FSR), as described previously (13, 28, 39).
Femoral arterial and venous blood samples taken between 0800 and
0830 were used to calculate postabsorptive phenylalanine kinetics.
During the stable isotope infusion study, all subjects received three
500-ml nutritionally mixed liquid meals (Boost Plus, Polycose, and
Microlipid). The meals contained the same nutrient distribution pro-
vided during the diet stabilization period (59% carbohydrate, 27% fat,
and 14% protein). The caloric content was based on the Harris-
Benedict equation (AF 1.3; Table 1). The meals were consumed over
a 5-min period at 0830, 1300, and 1830 and were chosen to optimize
nutrient delivery by controlling nutrient/protein content and reducing
variability associated with digestion and gastric emptying of whole foods.
Although the diet stabilization period was identical for all volun-
teers, during the stable isotope infusion study, subjects in the SUP
group also received three supplements (1100, 1600, 2100), each
containing 30 g of sucrose and 15 g of essential amino acids (EAAs).
The proportion of EAAs in the supplement was based on the distri-
bution required to increase the intracellular concentration of EAAs in
proportion to their respective contribution to the synthesis of skeletal
muscle protein (Table 2) (26). The amino acids and sucrose were
dissolved in 250 ml of a noncaloric, noncaffeinated soft drink.
Subjects in the CON group received only the diet soft drink.
We have previously determined that a constant infusion of [ring-
2
H5]phenylalanine (0.05 ␮mol⅐kgϪ1
⅐minϪ1
) results in an isotopic
enrichment (tracer-to-tracee ratio) in the femoral artery of ϳ8% (26).
To maintain an isotopic steady state after ingestion of unlabeled
phenylalanine, an additional 0.186 g of [ring-2
H5]phenylalanine was
added to each carbohydrate-EAA (CAA) drink, and 0.133 g of
[ring-2
H5]phenylalanine was added to the Boost meals (27).
Calculations. Phenylalanine was selected to trace muscle protein
kinetics, because it is neither produced nor metabolized in skeletal
muscle. Net phenylalanine balance (NB) was calculated as
NB ϭ ͑Ca Ϫ Cv͒ ⅐ BF
where Ca and Cv represent the phenylalanine concentrations in the
femoral artery and vein, respectively (40). BF represents leg blood
flow, as determined by the ICG dye dilution method (23). Total
phenylalanine uptake was determined by calculating net balance area
under the curve.
The FSR of mixed muscle protein was calculated by measuring the
direct incorporation of L-[ring-2
H5]phenylalanine into protein, using
the precursor-product model
FSR ϭ ͓͑EP2 Ϫ EP1͒/͑Em ⅐ t͔͒ ⅐ 60 ⅐ 100
where EP1 and EP2 are the enrichments of bound L-[ring-
2
H5]phenylalanine in the first and second muscle biopsies, respec-
tively; t is the time interval between biopsies (i.e., ϳ16 h); and Em is
the mean L-[ring-2
H5]phenylalanine precursor enrichment in the mus-
cle intracellular pool from the first and second muscle biopsies (3).
Statistical analysis. Within- and between-group comparisons for
each period (postabsorptive, meal, supplement/placebo) were per-
formed with two-way ANOVA. Two-tailed t-tests were used to
compare FSR and blood flow variables. A Bonferroni correction was
applied to account for the multiple comparisons. Data are presented as
means Ϯ SE. Differences were considered significant at P Ͻ 0.05.
RESULTS
Comparison of meal vs. supplement. The nutrient distribu-
tion of the meals was similar in SUP and CON groups (Table
1). The EAA content of the meal and supplement was also
similar (Table 2).
Arterial and venous phenylalanine enrichments followed a
similar pattern and were maintained throughout the study.
Arterial phenylalanine enrichments after meal and CAA/pla-
cebo ingestion are presented in Fig. 2. Over the 16-h study
period, ingestion of the CAA supplement and meals resulted in
an anabolic stimulus greater than that effected by the meals
alone. Mixed muscle FSR (0800–2400) in the SUP group was
ϳ25% greater than in the CON group (P Ͻ 0.05; Fig. 3).
Similarly, 16-h net phenylalanine uptake (Fig. 4) and net
phenylalanine balance (Fig. 5) were significantly greater in the
SUP group compared with the CON group (P Ͻ 0.05).
Muscle intracellular phenylalanine concentrations (MIC)
from the two muscle biopsies (0800 and 2400) were similar in
the CON group (1st MIC 77.9 Ϯ 9.6 nmol/ml; 2nd MIC 70.5 Ϯ
7.2 nmol/ml; P Ͼ 0.05). There was, however, a residual
expansion of the intracellular phenylalanine pool at the time of
the final biopsy in the SUP group (1st MIC 80.4 Ϯ 7.8 nmol/ml;
2nd MIC 113.8 Ϯ 7.6 nmol/ml; P Ͻ 0.05). This is equivalent to
ϳ48 mg phenylalanine/leg remaining in the MIC pool at the
completion of the study period. It is likely that a portion of this
intracellular phenylalanine would eventually be incorporated
Fig. 2. Arterial L-[ring-2
H5]phenylalanine enrichment after meal and CAA
supplement/placebo ingestion. Values are means Ϯ SE.
Table 1. Energy and nutrient distribution
Energy, kcal Protein, g Carbohydrate, g Fat, g
Meal
SUP 856.3Ϯ39.9 23.1Ϯ1.1 127.2Ϯ4.3 32.6Ϯ2.6
CON 859.1Ϯ34.1 23.4Ϯ1.0 126.6Ϯ4.0 30.3Ϯ2.8
CAA
SUP 180 15 30
Meal values are means Ϯ SE. Energy and nutrient distribution of a single
meal (0830, 1330, 1830) and carbohydrate-essential amino acid (CAA) sup-
plement (1100, 1600, 2100), consumed by the supplement (SUP; n ϭ 7) and
control (CON; n ϭ 6) groups.
Table 2. Essential amino acid profile
Amino Acid Supplement, g Meal, g
Histidine 1.64 0.95Ϯ0.04
Isoleucine 1.56 1.69Ϯ0.08
Leucine 2.79 3.31Ϯ0.02
Lysine 2.33 2.71Ϯ0.01
Methionine 0.46 0.88Ϯ0.04
Phenylalanine 2.33 1.67Ϯ0.07
Threonine 2.20 1.40Ϯ0.06
Valine 1.73 2.09Ϯ0.09
Total 15 14.7Ϯ0.7
Meal values are means Ϯ SE. Essential amino acid profile of the CAA
supplement and meals, consumed by the SUP (n ϭ 7) and CON (n ϭ 6)
groups.
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4. into bound protein; however, even if all the residual intracel-
lular phenylalanine were ultimately released back into the
circulation without being incorporated into skeletal muscle
protein, it represents a relatively small proportion of the total
phenylalanine taken up in the SUP group (Fig. 4).
Peak femoral artery phenylalanine concentrations occurred
15–45 min after ingestion of both the meal and the supplement.
The increase in arterial phenylalanine concentration was sig-
nificantly greater after CAA ingestion (SUP 145.9 Ϯ 6.7; CON
70.8 Ϯ 2.0 ␮mol/l) compared with the morning or evening
meal (Table 3P Ͻ 0.05). Furthermore, the duration of the
increase in arterial phenylalanine concentration was signifi-
cantly greater after CAA ingestion compared with the meal
(Fig. 6).
Effect of prior supplement ingestion on meals. Compared
with postabsorptive values, insulin, glucose, and phenylalanine
concentrations in the SUP and CON groups were significantly
greater after ingestion of both the morning and evening meals
(Table 3). Plasma phenylalanine and insulin concentrations in
the SUP group were higher after the evening meal than at other
sampling times (P Ͻ 0.05). This value was also higher (P Ͻ
0.05) than the corresponding CON group value, suggesting a
cumulative effect of previous meal/supplement ingestion. The
timing of the meal (morning vs. evening) did not affect plasma
phenylalanine and insulin concentrations in the CON group.
However, CON group blood glucose concentrations were
lower after the evening meal (P Ͻ 0.05; Table 3).
Net phenylalanine balance values during the 2.5 h after meal
ingestion are presented in Fig. 7. Compared with postabsorp-
tive values (SUP Ϫ18.6 Ϯ 3.3 and CON Ϫ23.7 Ϯ 2.3
nmol⅐minϪ1
⅐100 ml leg volumeϪ1
), ingestion of the morning
(SUP Ϫ2.2 Ϯ 3.3 and CON Ϫ1.5 Ϯ 3.5 nmol⅐minϪ1
⅐100 ml
leg volumeϪ1
) and evening (SUP Ϫ9.7 Ϯ 4.3 and CON
Ϫ6.7 Ϯ 4.1 nmol⅐minϪ1
⅐100 ml leg volumeϪ1
) meals im-
proved net phenylalanine balance in both groups (P Ͻ 0.05).
No differences attributable to the timing of the meal (i.e.,
morning vs. evening) were identified (P Ͼ 0.05). Furthermore,
the previous ingestion of CAA in the SUP group did not affect
net phenylalanine balance during the 2.5-h period after the
evening meal (P Ͼ 0.05).
Leg blood flow. Representative leg blood flow values were
similar in both the SUP and CON groups during the morning
(1000) and evening (2000) measurement periods. Mean leg
blood flow values (morning and evening) in the SUP group
were 3.4 Ϯ 0.4 and 3.6 Ϯ 0.5 ml⅐minϪ1
⅐100 ml leg volumeϪ1
(P Ͼ 0.05). CON group values were 3.4 Ϯ 0.4 and 3.7 Ϯ 0.4
ml⅐minϪ1
⅐100 ml leg volumeϪ1
(P Ͼ 0.05).
Fig. 5. Net phenylalanine balance across the leg during the 2.5-h period after
ingestion of 3 meals (0830, 1330, 1830) and 3 supplements (1100, 1400, 2100)
in healthy young males. Values are means Ϯ SE. a, Ingestion of the CAA
supplement (SUP group; n ϭ 7); b, ingestion of a placebo (CON group; n ϭ
6). *Different from meal and placebo ingestion (P Ͻ 0.05). Net phenylalanine
balance after meal and placebo ingestion was not different from zero (P Ͼ
0.05).
Table 3. Mean postabsorptive and postprandial insulin,
glucose, and phenylalanine concentrations
Plasma Postabsorptive Meal (AM) Meal (PM)
Insulin, pmol/l
SUP 47.2Ϯ7.6 336.1Ϯ38.9† 434.7Ϯ50.0†‡
CON 50.7Ϯ5.6 395.2Ϯ41.7† 405.6Ϯ58.3†
Glucose, mmol/l
SUP 5.1Ϯ0.1 8.3Ϯ0.8† 8.9Ϯ0.4†
CON 5.0Ϯ0.3 9.3Ϯ0.5† 8.2Ϯ0.2†‡
Arterial phenylalanine,
nmol Phe/ml
SUP 64.3Ϯ5.0 76.3Ϯ4.4† 106.9Ϯ6.5*†‡
CON 68.0Ϯ3.8 81.4Ϯ3.5† 86.5Ϯ5.0†
Values are means Ϯ SE. Mean postabsorptive and postprandial insulin,
glucose, and phenylalanine (Phe) concentrations in the SUP (n ϭ 7) and CON
(n ϭ 6) groups during the 2.5-h period after ingestion of the morning and
evening meals. *Different from CON (P Ͻ 0.05). †Different from the post-
absorptive period (P Ͻ 0.05). ‡Different from AM meal (P Ͻ 0.05).
Fig. 3. Sixteen-hour mixed muscle fractional synthetic rate (FSR) after meal
(CON; n ϭ 6) or meal and CAA supplement (SUP; n ϭ 7) ingestion in healthy
young men. Values are means Ϯ SE. *Different from CON (P Ͻ 0.05).
Postabsorptive data are presented for reference with the permission of Volpi et
al. (38).
Fig. 4. Sixteen-hour net phenylalanine uptake by the leg after meal (CON; n ϭ
6) or meal and CAA supplement (SUP; n ϭ 7) ingestion in healthy young men.
Values are means Ϯ SE. *Different from CON (P Ͻ 0.05). Both groups were
significantly different from zero (P Ͻ 0.05).
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5. DISCUSSION
Amino acid supplementation is an effective means of stim-
ulating muscle protein synthesis (26, 30, 37). However, the
effect of an amino acid supplement on the normal response to
a subsequent nutritionally mixed meal has not been previously
investigated. This study demonstrated that supplementation
with 30 g of carbohydrate and 15 g of EAAs (CAA) produces
a greater anabolic effect than ingestion of nutritionally mixed
meals alone. This effect was evident despite the fact that the
supplement and meal contained a similar total amount of EAA.
Furthermore, although the increase in net phenylalanine bal-
ance afforded by a CAA supplement was significantly greater
than the response to a meal, ingestion of a CAA supplement
does not significantly affect the normal modest improvement in
net balance after a regular mixed meal.
The anabolic potential of an amino acid supplement can be
evaluated in several ways. Perhaps the most common method
is to examine specific outcome variables such as strength, lean
muscle mass, and nitrogen balance after a prolonged period
(Ͼ2 mo) of supplementation (15, 41). However, with stable
isotope methodology, we can accurately quantify acute
changes in muscle protein kinetics after ingestion of a single
nutrient-controlled meal or supplement without inherent vari-
ation due to daily activities. Although there is some uncertainty
that increases in muscle protein synthesis occurring during the
3- to 4-h period after ingestion (10, 13, 30, 37) translate to a
measurable change in muscle mass over a period of weeks or
months, recent data from our laboratory suggest that there is
indeed a correlation between acute stimulation of muscle
protein synthesis and chronic changes in muscle mass (27, 33).
In a recent study in which subjects were provided with an EAA
supplement three times a day for 28 days during bed rest, the
repeated acute stimulation of muscle protein synthesis pro-
vided by the CAA supplement on day 1 of bed rest resulted in
a predicted net gain of ϳ7.5 g of muscle over a 24-h period
(27). When extrapolated over the entire 28-day study, the
predicted change in muscle mass corresponded to the actual
change in muscle mass (ϳ210 g) measured by dual-energy
X-ray absorptiometry. Although it may be tempting to specu-
late that such a supplement regimen would enable sedentary
individuals to increase muscle mass without any accompanying
physical activity, it must be noted that the estimated change in
muscle mass was small (ϳ7.5 g/day) and may not represent a
linear and/or continuous increase in muscle mass for extended
periods greater than a month.
Compared with the SUP group, the CON group remained in
negative net balance for the duration of the 16-h study, despite
the protein/energy content of the meals. We (27) have previ-
ously demonstrated that the energy content of the meals was
sufficient to produce an increase in body fat over 28 days of
bed rest. Consequently, while it is possible that the total protein
content of the meals was insufficient to maintain net balance in
the CON group, it is likely that this was also influenced by the
minimal amount of physical activity performed during the
overnight period before the study and the 16-h data collection
period.
In nonathletic groups in particular, it is often proposed that
dietary protein/amino acid supplementation is not warranted
and similar benefits can be obtained by ingestion of additional
dietary protein (2, 16). This theory would be plausible if the
anabolic effect provided by normal dietary protein was similar
in magnitude to the response to EAA ingestion. However, in
the current study, the increase in net phenylalanine balance
after supplement ingestion far exceeded the response to the
meals. The fact that the meal and the supplement both con-
tained ϳ15 g of EAA (Table 2) indicates that some aspect
related to the mode of delivery of the amino acids likely plays
a substantial role in the ability to stimulate muscle protein
anabolism. Furthermore, the fact that the mixed meal contained
a total of 23 g of protein supports our previous contention that
ingestion of nonessential amino acids does not play a major
role in the stimulation of muscle protein synthesis (13, 34).
The smaller improvement in net balance produced by inges-
tion of the meals, compared with the CAA supplement, sug-
gests that some intrinsic component of the meal may have
blunted the muscle protein synthetic stimulus of the EAAs
contained in the meal. There are a number of possibilities that
may have contributed to this response. It is possible that the
formulation of an EAA supplement influences its anabolic
effect. The relative proportion of EAAs in the meal and CAA
supplement was similar but not exactly the same. It is unlikely,
however, that the specific profile of EAAs in the meal and the
supplement was responsible for the differences in responses, as
Fig. 6. Femoral artery phenylalanine concentrations after ingestion of 3 meals
and 3 placebo drinks in the CON group (n ϭ 6) and 3 meals and 3 CAA
supplements in the SUP group (n ϭ 7) in healthy young males. Values are
means Ϯ SE. *Different from meal and placebo response (P Ͻ 0.05).
†Different from placebo response (P Ͻ 0.05).
Fig. 7. Net phenylalanine balance during the postabsorptive period and during
the 2.5-h period after the morning and evening meals in healthy young males.
The CON (n ϭ 6) and SUP (n ϭ 7) groups ingested meals at 0830, 1330, and
1830. The SUP group also ingested CAA supplements at 1100, 1600, and
2100. Values are means Ϯ SE. †Different from morning and evening meals
(P Ͻ 0.05). Net phenylalanine balance after the morning and evening meals
was not different from zero (P Ͼ 0.05).
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6. there were only minor differences (Table 2). Furthermore, the
proportion of leucine, an EAA strongly linked to muscle
protein synthesis (1, 24, 31), was higher in the meal than the
supplement.
Perhaps the most likely difference between the CAA sup-
plement and the meal was the speed of digestion and subse-
quent effect on splanchnic uptake. It has been well established
that meals containing a mixture of nutrients (fat, carbohydrate,
and protein) are more slowly digested and released from the
gut than meals containing glucose and free amino acids.
Similarly, it has been demonstrated that the milk protein
casein, a major constituent of the Boost Plus meal, is a
nonsoluble protein that coagulates in the stomach and is there-
fore digested more slowly than whey protein (10, 25) and
presumably also free-form amino acids. The tissues of the
splanchnic bed are responsible for the initial uptake of amino
acids from the gut and their subsequent release into the circu-
lation (37). We propose that a slightly slower release of amino
acids from the gut after ingestion of a mixed meal enables a
more efficient uptake by the splanchnic bed and therefore
reduces the magnitude of the acute increase in the concentra-
tion of amino acids in the peripheral circulation that are
available for skeletal muscle protein synthesis. In contrast, a
more rapid clearance of the CAA supplement from the gut
would likely result in a comparatively lower first-pass splanch-
nic uptake of EAAs. This greater increase in peripheral arterial
plasma amino acid concentrations and extracellular availability
may act as a signal for the stimulation of muscle protein
synthesis (9).
In terms of speed of digestion influencing protein kinetics,
studies investigating whole body protein kinetics after protein
ingestion have demonstrated that slowly digested proteins such
as casein produce a greater whole body net protein deposition
than more rapidly digested proteins such as whey (4, 10, 11).
However, as a function of the methodology, these studies
cannot distinguish splanchnic and skeletal muscle amino acid
uptake and therefore are not able necessarily to directly quan-
tify skeletal muscle protein anabolism.
In conclusion, ingestion of a CAA supplement produces a
greater anabolic effect than ingestion of a nutritionally mixed
meal, despite similar EAA content. Furthermore, ingestion of
the CAA supplement does not result in a subsequent compen-
satory nadir in net phenylalanine balance and does not effect
the normal anabolic response to ingestion of a nutritionally
mixed meal.
ACKNOWLEDGMENTS
We gratefully acknowledge Melissa Bailey, Stephaine Blase´, David
Chinkes, Christopher Danesi, Dessa Gemar, Guy Jones, and Anthony Obuseh
for invaluable assistance with data processing.
GRANTS
This project was supported by National Space Biomedical Research Insti-
tute Grant NPFR00205, National Aeronautics and Space Administration Grant
NAG9-1155, and Shriners Hospital Grant 8490. Studies were conducted at the
General Clinical Research Center at The University of Texas Medical Branch
at Galveston and supported in part by National Institutes of Health Grant
M01-RR-00073 from the National Center for Research Resources.
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