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Applied nutritional investigation
Pharmacokinetics, safety, and effects on exercise performance of
L-arginine
␣
-ketoglutarate in trained adult men
Bill Campbell, M.S., Mike Roberts, B.S., Chad Kerksick, M.S., Colin Wilborn, M.S.,
Brandon Marcello, M.S., Lem Taylor, M.S., Erika Nassar, M.S., Brian Leutholtz, Ph.D.,
Rodney Bowden, Ph.D., Chris Rasmussen, M.S., Mike Greenwood, Ph.D., and
Richard Kreider, Ph.D.*
Exercise and Sport Nutrition Laboratory at the Center for Exercise, Nutrition, and Preventative Health Research, Department of Health, Human
Performance, and Recreation, Baylor University, Waco, Texas, USA
Manuscript received August 29, 2005; accepted June 5, 2006.
Abstract Objective: We evaluated the pharmacokinetics, safety, and efficacy of L-arginine
␣
-ketoglutarate
(AAKG) in trained adult men.
Methods: Subjects participated in two studies that employed a randomized, double-blind, con-
trolled design. In study 1, 10 healthy men (30 –50 y old) fasted for 8 h and then ingested4gof
time-released or non–timed-released AAKG. Blood samples were taken for 8 h after AAKG
ingestion to assess the pharmacokinetic profile of L-arginine. After 1 wk the alternative supplement
was ingested. In study 2, which was placebo controlled, 35 resistance-trained adult men (30 –50 y
old) were randomly assigned to ingest4gofAAKG (three times a day, i.e., 12 g daily, n⫽20) or
placebo (n⫽15). Participants performed4dofperiodized resistance training per week for 8 wk.
At 0, 4, and 8 wk of supplementation the following tests were performed: clinical blood markers,
one repetition maximum bench press, isokinetic quadriceps muscle endurance, anaerobic power,
aerobic capacity, total body water, body composition, and psychometric parameters tests. Data were
analyzed by repeated measures analysis of variance.
Results: In study 1, significant differences were observed in plasma arginine levels in subjects
taking non–timed-release and timed-release AAKG. In study 2, significant differences were ob-
served in the AAKG group (P⬍0.05) for 1RM bench press, Wingate peak power, blood glucose,
and plasma arginine. No significant differences were observed between groups in body composition,
total body water, isokinetic quadriceps muscle endurance, or aerobic capacity.
Conclusion: AAKG supplementation appeared to be safe and well tolerated, and positively
influenced 1RM bench press and Wingate peak power performance. AAKG did not influence body
composition or aerobic capacity. © 2006 Elsevier Inc. All rights reserved.
Keywords: Sports nutrition; Ergogenic aid; Resistance training; Supplementation; Exercise
Introduction
L-arginine is a “semi-essential” amino acid used by all
cells [1]. It plays a critical role in cytoplasmic and nuclear
protein syntheses, the biosynthesis of other amino acids,
creatine synthesis, and the urea cycle. In this essential bio-
chemical pathway, urea is synthesized from arginine to
enable the body to remove excess ammonia, which is toxic
to cells. L-arginine is classified as a glucogenic amino acid
This study was funded by a research grant from the Medical Re-
search Institute (San Francisco, CA) to Baylor University. Researchers
at Baylor University independently collected, analyzed, and interpreted
the results from this study and have no financial interests in the results
of this study.
Some of the results reported were presented in preliminary fashion at
the First Annual International Society of Sports Nutrition; Lake Las Vegas,
Nevada; June 18 –19, 2004.
* Corresponding author: Tel.: ⫹254-710-3526; fax: ⫹425-928-9371.
E-mail address: richard_kreider@baylor.edu (R. Kreider).
Nutrition 22 (2006) 872– 881
www.elsevier.com/locate/nut
0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nut.2006.06.003
because it can be metabolized into
␣
-ketoglutarate (AKG)
and enter the citric acid cycle. In addition, L-arginine has
been reported to improve the immune response and increase
the release of growth hormone and insulin [2].
In one of its most important roles, L-arginine serves as a
precursor for the biosynthesis of nitric oxide (NO), an en-
dogenously produced, cellular signaling molecule that is
involved in a variety of endothelium-mediated effects in the
vasculature [3,4]. NO serves as a second messenger to
trigger blood vessel dilation and increase blood flow. Nisoli
et al. [5] recently reported that NO stimulates the synthesis
of mitochondria. Although NO was first identified in endo-
thelial cells [6], the generation of NO from L-arginine oc-
curs in a variety of other cell types including skeletal muscle
[3,7,8].
Nitric oxide is produced endogenously from L-arginine
in a complex reaction that is catalyzed by the enzyme NO
synthase [9]. The other product that is formed in this
reaction is citrulline. L-arginine is the only endogenous
nitrogen-containing substrate of NO synthase and thus
governs the production of NO. In consequence, under
certain conditions, the plasma concentration of L-arginine
may be rate limiting for NO production. Research has
indicated that exogenously administered L-arginine
promotes NO-mediated biological effects [10]. Studies in
animals have reported that acute and long-term adminis-
tration of L-arginine improves blood flow and vascular
health [11]. In humans, studies have reported beneficial
effects after oral L-arginine supplementation including
improved blood flow, reductions in blood pressure, and
improved immune function [11,12]. Theoretically,
L-arginine supplementation may support general health,
enhance blood flow to tissues, and/or enhance protein
synthesis [13,14]. These outcomes would potentially be
beneficial for athletes undergoing heavy resistance train-
ing. However, the available literature on the effects of
L-arginine on exercise and/or training adaptations is
limited and conflicting [15].
␣
-Ketoglutarate is a five-carbon dicarboxylic acid pro-
duced in the citric acid cycle from the oxidative decar-
boxylation of isocitrate. In a subsequent decarboxylation
reaction, AKG is converted to succinyl coenzyme A, a
reaction catalyzed by the AKG dehydrogenase complex.
Hammarqvist et al. [16] reported that AKG supplemen-
tation after surgery limited postoperative decreases in the
concentration of glutamine and other amino acids. Orni-
thine AKG has been reported to modulate protein tissue
metabolism in rats with burn injury and muscle-wasting
conditions [17,18]. In addition, Czernichow et al. [19]
reported that intergastrically administered ornithine AKG
improved the early adaptive hypertrophic response to
resection in rats. Theoretically, if oral AKG or ornithine
AKG supplementation influences protein metabolism or
catabolism, then it may help athletes undergoing intense
training increase muscle mass and/or promote positive
training adaptations. However, it should be noted that
some studies have involved enteral administration of
AKG or ornithine AKG and less is known regarding the
effects of oral arginine, AKG, or ornithine AKG supple-
mentation on protein metabolism. Wiren et al. [20] re-
ported that oral supplementation of AKG after surgery
had no significant effects on protein metabolism or ca-
tabolism in patients undergoing elective abdominal sur-
gery. In consequence, some have doubted the potential
value of orally supplemented AKG as a means of mod-
ulating protein synthesis [21].
The reported physiological and biochemical effects of
L-arginine and AKG have apparently served as the rationale
behind the development and marketing of a number of NO
potentiating dietary supplements to resistance-trained ath-
letes. These supplements are purported to stimulate NO
production, improve blood flow to muscle during resistance
training, increase protein synthesis, and/or reduce catabo-
lism, leading to greater training adaptations. Although there
is some supportive theoretical rationale, it is unclear
whether oral dietary supplementation of L-arginine and
AKG influence training adaptations in resistance-trained
athletes. Therefore, this study evaluated the pharmacokinet-
ics, safety, tolerability, and effects on exercise performance
of L-arginine/
␣
-ketoglutarate (AAKG) in trained adult men.
Materials and methods
Two separate studies were conducted to assess the phar-
macokinetic profile of ingesting two forms of AAKG in the
blood (study 1) and the effects of dietary supplementation of
AAKG on training adaptations in resistance-trained men
(study 2). The following paragraphs describe the methods
employed in each study.
Study 1: pharmacokinetic profile
Ten male subjects volunteered to participate in this phase
of the study (37 ⫾5 y old, 180 ⫾5 cm, 85 ⫾17 kg). The
study was conducted using a double-blind, randomized, and
crossover design separated by 1 wk. All testing sessions
were started at the same time of day for each testing session.
All subjects were asked to replicate their diets for the 24-h
period before each testing session. Participants came after
fasting for 8 h and ingested 12 oz of non–sugar-fortified
orange juice 2 h before donating their baseline blood sam-
ple. In a randomized and double-blind manner, participants
ingested four caplets of non–time-released (NTR) AAKG
(i.e., form of AAKG that is released immediately into the
gut) or time-released (TR) AAKG (i.e., form of AAKG that
is released from the delivery system matrix in a planned,
predictable, and slower-than-normal manner). The total
weight of each caplet was 1500 mg and consisted of 1000
mg of L-arginine/AKG and 500 mg of excipients including
the time-released matrix material. These excipients included
dicalcium phosphate, cellulose ethers and composites, sea-
873B. Campbell et al. / Nutrition 22 (2006) 872– 881
weed extract, corn extract, pectin, and magnesium stearate.
The formulation did not contain yeast, wheat, soy, sugar or
other sweeteners, artificial flavors, colors, or preservatives.
Ingestion of four caplets of AAKG provided approximately
2gofL-arginine and2gofAKG.
After ingestion of the supplements, subjects donated
blood samples at 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h. Four hours
after ingestion of the supplementation, each participant was
provided with an additional 12 oz of orange juice. Because
the fasting and data collection protocol lasted 18 h, orange
juice was provided to subjects as described above to prevent
hypoglycemia and/or undue hunger pangs. Because both
groups followed the same fasting protocol and ingested
identical amounts of orange juice at the same time, provi-
sion of orange juice to the subjects would not have been a
limiting factor in the ability to determine whether the dif-
ferent forms of AAKG had different time courses in the
appearance of arginine in the blood.
The AAKG used for the pharmacokinetic study (study 1)
and for the safety and efficacy study (study 2) was pur-
chased from Rexim (Degussa-SAS, Paris, France). This
material is of the highest purity commercially available
(⬎99.0%) and produced in accordance with current good
manufacturing practices. AAKG caplets were formulated
and manufactured for the Medical Research Institute (MRI;
San Francisco, CA, USA) in the United States in accordance
with current good manufacturing practices. AAKG is cur-
rently manufactured and distributed in the United States by
MRI under the trade name of NO2. The proprietary nature
of this product is protected under U.S. patent number
6,905,707 (B2), and additional patents are pending. The
quality and integrity of the active ingredient and finished
product were assessed and certified by an independent,
third-party analytical laboratory (Eurofins Scientific Inc.,
Petaluma, CA, USA). In addition, every finished lot of
AAKG (NO2) is tested for the presence of illegal or other-
wise unwanted substances under the terms and conditions of
an agreement with Banned Substances Control Group (Los
Angeles, CA, USA), an independent analytical laboratory
certified and accredited by the International Olympic Com-
mittee (IOC).
Study 2: training study
Subjects
Thirty-five male, experienced, resistance-trained males
volunteered to participate in the study. Subjects were in-
formed of the risks and benefits and signed informed con-
sent statements in adherence with the institutional review
board of Baylor University. To qualify for participation,
subjects had to be 30 to 50 y of age, been involved in a
systematic resistance-training program for ⱖ1 y before the
start of the study, and not have used any ergogenic aids
within the previous 6 mo. Subjects who volunteered to
participate in this study underwent two familiarization/prac-
tice exercise sessions to become accustomed to the exercise
testing to be employed in the study. Demographic informa-
tion of the subjects were 38.9 ⫾5.8 y of age, 86 ⫾13.7 kg
of body weight, 178 ⫾8.4 cm in height, and 18.8 ⫾5%
body fat. Subjects reported resistance training 4 ⫾1 d/wk
for 6 ⫾3 h/wk.
Experimental design
Subjects reported to the laboratory to sign informed
consent statements, complete medical and training history
questionnaires, and become familiarized with the testing
protocol. The subjects were then scheduled to participate in
two practice testing trials to establish reliability in perform-
ing the exercise protocol. The testing protocol had the sub-
jects perform a bench press, isokinetic leg extension test,
and a Wingate anaerobic test. The procedures for these tests
are described in detail below. After the practice trials were
completed, the subjects were scheduled for baseline testing.
Subjects were asked to not change their dietary habits in
any way throughout the 8-wk investigative period. This was
monitored by having each subject document dietary intake
for4d(3weekdays and 1 weekend day) before each testing
session. In addition, each subject was instructed to fast for
12 h and not to perform any physical activity for the 2 d
preceding each testing session. Upon entering the laboratory
for baseline testing, each subject completed the 36-item
Short-Form Health Survey (SF-36) quality-of-life inventory
[22], the Profile of Mood States (POMS) psychological
inventory [23], the Beck Depression Inventory [24], the
Occupational Strain Questionnaire [25], and a Likert scale
libido/energy questionnaire. After this, the following tests
were conducted in the order listed: (1) body mass; (2) the
donation of approximately 30 mL of fasting venous blood
from an antecubital vein; (3) total body water; (4) body
composition; (5) resting heart rate; and (6) resting blood
pressure.
After these assessments, the subjects then began the
performance tests. These tests were conducted in the fol-
lowing order, with the rest periods between tests listed in
parentheses: one repetition maximum (1RM) bench press (5
min), isokinetic right quadriceps muscle endurance test (10
min), Wingate anaerobic capacity test (20 min), and a max-
imal cardiopulmonary aerobic capacity test. Rest periods
were based on the estimated time for the energy system
utilized to recover adequately [26]. Follow-up testing was
conducted at the midway point of the study (4 wk) and at the
end of the investigative period (8 wk). The 4-wk and 8-wk
testing sessions were identical and compared with baseline
testing. On a weekly basis, each participant was asked to
complete a weekly follow-up assessment chart that ad-
dressed possible symptoms or side effects related to the
supplementation and training.
Supplementation protocol
Subjects were assigned in a double-blind and random-
ized manner to ingest a dextrose placebo (PLA, n⫽15) or
AAKG (n⫽20). All supplements were supplied to the
874 B. Campbell et al. / Nutrition 22 (2006) 872– 881
investigators in a double-blinded fashion by MRI. Subjects
were instructed to ingest four caplets of the supplements
three times per day (12 caplets daily) for the duration of the
8-wk investigation. Each 1.5-g caplet provided 0.5 g of
L-arginine and 0.5 g of AKG in a 1:1 ratio. The remaining
0.5 g in each caplet contained the TR and other excipients
required to produce a caplet form of the supplement. There-
fore, ingestion of 12 caplets/d provided approximately 6 g
of L-arginine and6gofAKG. Supplements were tested for
purity and the presence of unwanted and/or banned sub-
stances by an IOC-certified laboratory (Banned Substances
Control Group). Subjects were instructed to ingest their
supplements 30 min before breakfast, 1 h before lunch, and
late afternoon (⬃1500 h). Subjects were also informed to
take their supplements on an empty stomach and not to
ingest them with food. This precaution was taken to elimi-
nate the competitive inhibition of amino acids in their trans-
port across the blood-brain barrier [27].
Training protocol
All subjects were required to follow the same workout
routine. Participants were required to participate in a 4-d/wk
resistance-training program split into two upper and two
lower body workouts per week for a total of 8 wk. This
8-wk training protocol was periodized in 4-wk increments
consisting of selected exercises for the following muscle
groups: chest (two exercises for a total of six sets), back
(two exercises for a total of six sets), shoulders (one exer-
cise for a total of three sets), biceps (one exercise for a total
of three sets), triceps (one exercise for a total of three sets),
abdominals and quadriceps (two exercises for a total of six
sets), hamstrings (two exercises for a total of six sets),
calves (one exercise for a total of three sets), and lower back
(one exercise for a total of three sets). Each exercise con-
sisted of three sets of 10 repetitions (weeks 1– 4) or 8
repetitions (weeks 4 – 8) performed with as much weight as
the participant could perform per set (typically 70 – 85%
1RM). Rest periods between exercises lasted no longer than
3 min and the rest periods between sets lasted no longer than
2 min [26]. The resistance-training workout was approxi-
mately1hinduration. In addition to the resistance-training
program, each participant was instructed to perform some
type of aerobic activity three times per week for 30 min in
duration. Subjects were informed that the intensity of effort
must result in a heart rate of 70% of maximal heart rate for
the 30-min duration.
Procedures
Subjects recorded the amount of weight lifted and the
number of repetitions performed on training cards. Training
sessions were monitored by a training partner or fitness
instructor who signed off that the session was completed.
Total training volume was calculated by multiplying the
number of repetitions by the amount of weight lifted for
each exercise. Total daily lifting volume was calculated for
each subject per exercise session and for the entire training
program. Subjects recorded all caloric intake from food and
fluids for 4 d (including 1 weekend day) before each testing
session. Dietary records were interpreted by a registered
dietitian and analyzed with ESHA Food Processor 8.3
(ESHA Research Inc., Salem, OR, USA). Subjects were
instructed to maintain their normal diet throughout the sup-
plementation and training periods. Resting heart rate was
determined by palpation of the radial artery using standard
procedures and resting blood pressure was also assessed
using standard procedures [28]. Total body mass was mea-
sured on a calibrated digital scale with a precision of ⫾0.02
kg (Health-O-Meter, Bridgeview, IL, USA). Total body
water was estimated with a Xitron Multi-frequency analyzer
(Xitron Technologies Inc., San Diego, CA, USA). Bioelec-
trical impedance analysis has been determined to be a valid
measurement for total body water [29]. Whole-body com-
position measurements (excluding cranium) were deter-
mined with a Hologic 4500W Dual-Energy X-ray Absorp-
tiometer (DEXA; Hologic, Bedford, MA, USA) by using
procedures previously described [30,31]. This test evaluates
body composition and body density by scanning the entire
body with a low dose of radiation and takes approximately
6 min. The DEXA scans regions of the body (right arm, left
arm, trunk, right leg, and left leg) to determine bone mass,
fat mass, and lean mass within each region. The scanned
bone, fat, and lean masses for each region are then subto-
taled to determine whole-body values (excluding cranium).
Percentage of body fat was determined by dividing the
amount of fat mass by the total scanned mass (bone mass,
fat mass, and lean mass). Test-retest reliability studies per-
formed on male athletes with this DEXA machine yielded
mean deviation for total bone mineral content and total fat
free/soft tissue mass of 0.31%, with a mean intraclass cor-
relation of 0.985.
Subjects abstained from exercise for 48 h before testing
and provided fasting blood samples (8 h) via venipuncture
from an antecubital vein in the forearm according to stan-
dard phlebotomy procedures. Serum and plasma blood sam-
ples were collected into two 10-mL serum separation tubes
and two 5-mL anticoagulant tubes containing K
3
(ethylene-
diaminetetra-acetic acid). One serum separation tube was
inverted several times and quickly centrifuged (1318g) for
20 min on a bench top centrifuge. The other serum separa-
tion tube was allowed to sit at room temperature until the
sample clotted and was then centrifuged. Plasma and serum
samples were transferred into microcentrifuge tubes and
frozen at ⫺80°C for subsequent analyses. A serum sample
and whole blood from the tube containing ethylene-
diaminetetra-acetic acid were refrigerated and shipped in
cold containers to Quest Diagnostics (Dallas, TX, USA) for
clinical analysis. A complete clinical chemistry panel was
run on serum samples using the Olympus AU5400 (Dia-
mond Diagnostics, Holliston, MA, USA) according to stan-
dard clinical procedures. Cell blood counts with percent
differentials were run on whole-blood samples using a
Coulter GEN S (Diamond Diagnostics) analyzer according
875B. Campbell et al. / Nutrition 22 (2006) 872– 881
to standard procedures. Test-test reliability (within and be-
tween) of performing these assays ranged from 2% to 6%
for individual assays, with an average variation of ⫾3%.
Samples were run in duplicate to verify results if the ob-
served values were outside control values and/or clinical
norms according to standard procedures.
Plasma samples were shipped frozen to the Nichols
Institute (San Diego, CA, USA) for plasma L-arginine
analysis according to blood collection and handling pro-
cedures outlined by the Nichols Institute. The specific
methods for the assay of L-arginine are proprietary in
nature but basically involve being analyzed by a Waters
2690 Alliance high performance liquid chromatographic
(HPLC) system (Waters, Milford, MA, USA). When us-
ing the Waters Pico-Tag method, amino acids are pre-
column derivatized with phenylisothiocyanate and sepa-
rated by reversed-phase chromatography. The interassay
precision (coefficient of variation) of L-arginine analysis
by this method is typically 5–9%.
Plasma agmatine was analyzed by a high performance
liquid chromatography-mass spectrometry/mass spec-
trometry (HPLC-MS/MS) method recently developed by
Song et al. [32]. Pretreatment of plasma samples were as
follows: 50
L of trichloroacetic acid solution (30% w/v)
was added to 150
L of plasma sample and vortexed
thoroughly. This mixture was placed on ice for approx-
imately 30 min and centrifuged (20 937g) at 9000 rpm for
10 min. Twenty microliters of borate buffer (100 nM at
pH 9.0) and 60
L of 7-flouro-4-nitrobenzoxadiazole
solution (10 mM in acetonitrile) were added to 10
Lof
supernatant. This mixture was vortexed and heated at
65°C for 20 min in a dry heating block. After heating, the
derivative solutions were cooled in running tap water and
kept at 5°C until analysis. Portions (5
L) of the solution
were injected for HPLC-MS/MS analysis with an Auto-
injector. This method has an intraday precision of 3.6%
and interday precision of 5.7%.
Upper body strength tests were performed on the Nebula
(Versailles, OH, USA) flat bench. Participants were in-
structed to conduct two warm-up sets with approximately
50% of their estimated 1RM. After the two warm-up sets,
resistance was continually added until subjects obtained
their 1RM. Three minutes of rest was allowed between each
attempt. Subjects then performed successive 1RM lifts start-
ing at about 70% of anticipated 1RM and increasing by
5–10 kg until participants reached their 1RM. Test-test
reliability of performing these strength tests in our labora-
tory on resistance-trained subjects yielded low mean coef-
ficients of variation and high reliability for the bench press
(1.9%, intraclass r⫽0.94).
Before the isokinetic leg extension was conducted, sub-
jects warmed up on a stationary bicycle for 2–3 min at a
self-selected pace. Subjects then performed five warm-up
repetitions on the Biodex (Biodex Medical Systems,
Shirley, NY, USA) isokinetic leg extension machine. After
these warm-up repetitions, subjects performed unilateral leg
extensions (right leg) for one set of 50 repetitions [33] at
180 degrees/s. Test-test reliability has been shown to be
0.78 to 0.82 [34]. Subjects then warmed up for 2 min at a
self-selected pace on a stationary bicycle ergometer before
performing the Wingate test. This warm-up was continued
into the start of the sprinting portion of the Wingate test,
which allows for a flying start. The Wingate anaerobic
capacity test was performed on the LODE (Amsterdam,
Netherlands) with a resistance of 0.7 Nm/kg. Test-test vari-
ability in performing repeated Wingate tests in our labora-
tory yielded correlation coefficients of r⫽0.98 ⫾15% for
mean power. The maximal cardiopulmonary test used the
Bruce protocol. Metabolic gases were obtained with the
Parvo Medics 2400 TrueMax metabolic measurement sys-
tem (Sandy, UT, USA) on a Trackmaster TMX425C tread-
mill (Newton, KS, USA). The mean coefficient of variation
(assessing maximum oxygen consumption) for this protocol
was 6.5% (range, 2–14%) [35].
The SF-36 was designed for use in clinical practice and
research and includes one multi-item scale that assesses
health-related quality of life via eight health concepts
[22,36]. The POMS questionnaire is a standardized and
validated scale that allows subjects to self-rate on six mood
states [23]. The Occupational Roles Questionnaire measures
stressful work roles [25]. The Beck Depression Inventory is
a 21-item self-report rating inventory that measures charac-
teristic attitudes and symptoms of depression [24,37]. The
libido/activity questionnaire was composed of seven ques-
tions with Likert-scale rankings from 0 (very poor) to 10
(very good). The seven questions contained in this assess-
ment included such items as positive attitude about training,
ability to recover from training sessions, body satisfaction,
sexual desire, erectile function, quality of sleep, and feeling
of energy when waking up.
Statistical analysis
In study 1, data were analyzed by a 2 (groups) ⫻10
(samples) repeated measures analysis of variance
(ANOVA) to determine whether there were any differ-
ences in appearance of arginine in the blood between the
NTR and TR versions of AAKG. Area under the curve
analyses from the pharmacokinetic study was determined
with PK Solutions 2.0 (Summit Research Services, Mon-
trose, CO, USA). In study 2, data were analyzed by a 2 ⫻
3 repeated measures ANOVA. Delta scores (post minus
pre values) were calculated on selected variables and
analyzed using repeated measures ANOVA. SPSS 11.5
for Windows (SPSS, Inc., Chicago, IL, USA) was used to
analyze data in both phases of the study. Post hoc pro-
cedures were conducted when necessary by using least-
significant difference post hoc procedures. Statistical sig-
nificance was accepted at P⬍0.05. Data are presented as
means ⫾standard deviations.
876 B. Campbell et al. / Nutrition 22 (2006) 872– 881
Results
Study 1: pharmokinetic profile
Figure 1 shows that a significant interaction was ob-
served across groups in arginine levels over the 8-h obser-
vation. However, no significant differences were observed
across groups in mean changes in arginine levels (NTR 26.6
⫾11 versus TR 23.9 ⫾12
mol/L, P⫽0.62) or accumu-
lated area under the curve (NTR 788 ⫾63 versus TR 741 ⫾
79
mol·h
⫺1
·L
⫺1
,P⫽0.17) during the 8-h observation
period. These findings indicate that the pattern of appear-
ance of arginine differed in the blood between the TR and
NTR forms of AAKG but that similar amounts of arginine
appeared in the blood over time.
Study 2: training study
Medical monitoring
No significant clinical side effects, related or unrelated to
the study, were reported to the research nurse by any subject
throughout the entire course of the study. All subjects tol-
erated the training and supplementation protocols without
any problems.
Training and diet
No statistically significant differences were observed
across groups in total lifting volume during the training
period (AAKG 350 336 ⫾67 867 versus PLA 386 990 ⫾
83 087 kg, P⫽0.51). There were also no statistically
significant interactions observed across groups in absolute
caloric intake (P⫽0.52), relative caloric intake (P⫽0.90),
carbohydrate intake (P⫽0.26), protein intake (P⫽0.71),
or fat intake (P⫽0.10).
Blood analysis
No significant differences were observed between groups
in lipid profiles, liver enzymes, renal function (creatinine,
serum urea nitrogen), electrolytes, markers of cytolysis (cre-
atine kinase, lactate dehydrogenase), calcium, total protein,
albumin, globulin, total bilirubin, alkaline phosphatase, ni-
trate/nitrite, agmatine, red blood cells, hematocrit, or white
blood cells. Table 1 presents data for glucose, hemoglobin,
and plasma arginine. Fasting plasma arginine levels were
significantly increased from baseline (108 ⫾17 to 126 ⫾21
mol/L) and were significantly higher than PLA values
(106 ⫾14
mol/L) after 8 wk of supplementation. Fasting
glucose levels were significantly increased from 90 ⫾10 to
97 ⫾8 mg/dL during the course of the study. However,
glucose values remained within normal ranges and were not
significantly different from PLA values (96 ⫾7 mg/dL)
after 8 wk of supplementation. A significant interaction was
observed among hemoglobin values as a result of values in
the PLA group decreasing (14.9 ⫾0.9 to 14.5 ⫾0.9 g/dL),
whereas values in the AAKG group were maintained (15.0
⫾0.7 to 15.2 ⫾1.0 g/dL). All hematologic values remained
within normal clinical ranges.
Muscular strength and muscular endurance
The analysis of 1RM bench press (Fig. 2)over8wk
showed a significant difference between groups (AAKG
8.82 ⫾7.33 versus PLA 2.67 ⫾9.11 kg, P⫽0.03). No
Fig. 1. Plasma arginine levels across all time points in subjects taking
non–time-released (circles) and time-released (squares) forms of L-arginine
␣
-ketoglutarate.
Table 1
Selected blood markers
AAKG Placebo P(group ⫻
time)
Glucose (mmol/L)
T1 5.0 ⫾0.56 5.2 ⫾0.33
T2 5.1 ⫾0.44 5.4 ⫾0.5
T3 5.4 ⫾0.44* 5.3 ⫾0.39 0.03
Hemoglobin (g/dL)
T1 15.0 ⫾0.7 14.9 ⫾0.9
T2 15.2 ⫾0.9 14.9 ⫾1.0
T3 15.2 ⫾1.0* 14.5 ⫾0.9 0.04
Plasma arginine (
mol/L)
T1 108 ⫾17 105 ⫾18
T2 122 ⫾30 112 ⫾22
T3 128 ⫾21* 106 ⫾14 0.01
AAKG, L-arginine/
␣
-ketoglutarate; T1 to T3, time 1 to time 3
*P⬍0.005, different from baseline or from placebo.
Fig. 2. Changes in 1RM strength for bench press in subjects taking
L-arginine
␣
-ketoglutarate (circles) and placebo (squares).
877B. Campbell et al. / Nutrition 22 (2006) 872– 881
significant difference was observed between groups in these
isokinetic right quadriceps muscle endurance variables:
peak torque, time to peak torque, total work, and work
fatigue.
Anaerobic power and aerobic capacity
Table 2 presents anaerobic power data for the AAKG
and PLA groups. Significant differences were observed be-
tween groups in peak power and rate to fatigue. A trend was
observed between groups in time to peak power and fatigue
index. No significant differences were observed between
groups in mean power, minimum power, or total work.
Table 3 presents selected aerobic capacity variables. No
significant differences were observed between groups in
time to exhaustion, oxygen consumption (milliliters per
kilogram per minute), minute ventilation, metabolic equiv-
alents, respiratory exchange ratio, systolic or diastolic blood
pressure, or maximal heart rate.
Body composition
Table 4 presents body composition results. No significant
differences were observed between groups in changes in
total body mass (AAKG 0.6 ⫾0.3 versus PLA 0.2 ⫾0.1 kg,
P⫽0.53), fat mass (AAKG 0.7 ⫾0.4 versus PLA ⫺0.6 ⫾
0.3 kg, P⫽0.25), lean mass (AAKG 0.8 ⫾0.1 versus PLA
0.9 ⫾0.4 kg, P⫽0.90), or percentage of body fat (AAKG
⫺0.5 ⫾0.2% versus PLA ⫺1.0 ⫾0.2%, P⫽0.31).
Psychometric analysis
No significant differences were observed for the SF-36
health-related quality-of-life domains. Likewise, no signif-
icant differences were observed between groups for the six
mood states of the POMS questionnaire. For the Occupa-
tional Roles Questionnaire, no significant differences were
observed between groups for five of the scales, but there
was a significant interaction between groups for the role
overload scale (AAKG ⫺1.5 ⫾5 versus PLA 3.4 ⫾8, P⫽
0.04). This shows an improvement by decreasing the feel-
ings that workload is increasing, unreasonable, and unsup-
ported by needed resources. In relation to the Beck Depres-
sion Inventory, no significant differences were observed
between groups (AAKG ⫺2.3 ⫾5 versus PLA ⫺0.2 ⫾4,
P⫽0.20). No significant differences were observed be-
tween groups in the libido questionnaire.
Table 2
Wingate anaerobic power indices.
AAKG Placebo P(group ⫻
time)
Peak power (W)
T1 1251 ⫾236 1271 ⫾257
T2 1291 ⫾254 1282 ⫾219
T3 1331 ⫾242* 1202 ⫾241 0.005
Time to peak power (s)
T1 3.77 ⫾.55 3.83 ⫾1.02
T2 3.80 ⫾.80 4.12 ⫾0.84
T3 3.88 ⫾.48* 3.32 ⫾1.25 0.050
Rate to fatigue (W/s)
T1 34.9 ⫾8.9 35.6 ⫾8.6
T2 36.4 ⫾10.0 35.6 ⫾9.1
T3 37.6 ⫾8.8* 31.9 ⫾9.5 0.005
AAKG, L-arginine/
␣
-ketoglutarate; T1 to T3, time 1 to time 3
* AAKG greater than placebo at T3 (P⬍0.05).
Table 3
Aerobic capacity variables.
AAKG Placebo P(group ⫻
time)
Time to exhaustion (min)
T1 11.0 ⫾1.2 10.5 ⫾1.1
T2 11.3 ⫾1.1 11.0 ⫾1.3
T3 11.6 ⫾1.4 11.0 ⫾1.5 0.76
Oxygen consumption
(mL·kg
⫺1
· min
⫺1
)
T1 41.0 ⫾6.1 40.4 ⫾5.1
T2 42.4 ⫾7.0 41.8 ⫾5.9
T3 43.8 ⫾6.1 42.2 ⫾6.0 0.39
Minute ventilation
T1 94.7 ⫾14.8 93.4 ⫾13.9
T2 98.1 ⫾14.8 96.0 ⫾12.4
T3 95.1 ⫾24.4 97.3 ⫾15.3 0.62
METS
T1 11.7 ⫾1.7 11.5 ⫾1.5
T2 12.1 ⫾2.0 11.9 ⫾1.7
T3 12.5 ⫾1.8 12.0 ⫾1.7 0.41
Respiratory exchange ratio
T1 1.19 ⫾0.07 1.14 ⫾0.07
T2 1.20 ⫾0.06 1.17 ⫾0.05
T3 1.20 ⫾0.07 1.15 ⫾0.07 0.86
AAKG, L-arginine/
␣
-ketoglutarate; METS, metabolic equivalents; T1 to
T3, time 1 to time 3
Table 4
Body composition variables
AAKG Placebo P(group ⫻
time)
Lean mass (kg)
T1 59.8 ⫾8.0 63.6 ⫾9.4
T2 60.0 ⫾8.0 64.3 ⫾9.3
T3 60.6 ⫾7.9 64.5 ⫾8.9 0.90
Fat mass (kg)
T1 13.9 ⫾6.6 15.7 ⫾4.7
T2 14.8 ⫾6.2 15.4 ⫾4.9
T3 14.6 ⫾6.3 15.1 ⫾4.9 0.25
Total body mass (kg)
T1 77.0 ⫾12.7 81.9 ⫾12.7
T2 77.1 ⫾12.6 82.2 ⫾13.0
T3 77.6 ⫾13.0 82.1 ⫾12.5 0.53
Body fat (%)
T1 18.7 ⫾5.8 19.1 ⫾3.8
T2 18.7 ⫾5.6 18.6 ⫾3.8
T3 18.2 ⫾5.5 18.1 ⫾3.9 0.31
AAKG, L-arginine/
␣
-ketoglutarate; T1 to T3, time 1 to time 3
878 B. Campbell et al. / Nutrition 22 (2006) 872– 881
Discussion
This study examined the hypothesis that L-arginine in
combination with AKG might serve as an effective ergo-
genic aid for resistance-trained athletes. The theoretical
rationale was based on research indicating that L-arginine
may increase NO production and/or protein synthesis and
that AKG may increase protein synthesis and/or reduce
catabolism during training [14,15]. Although numerous
NO-potentiating supplements containing L-arginine and/or
AKG are marketed to resistance-trained athletes, no study
has evaluated the potential ergogenic value of AAKG sup-
plementation. In consequence, this is the first report of the
development, use, and clinical assessment of a TR formu-
lation of AAKG as a potential ergogenic aid in this popu-
lation.
To examine the potential ergogenic value, we conducted
two studies to examine the pharmacokinetics, safety, toler-
ability, and effects of AAKG on training adaptations in
trained adult men. The first study was conducted to examine
the pharmacokinetic profiles of ingesting NTR and TR
forms of AAKG. Results showed that plasma arginine levels
peaked to a greater degree after ingesting the NTR version
of AAKG but that the total area under the curve for plasma
arginine was similar between groups. These findings indi-
cated that that NTR and TR forms of AAKG have different
pharmacokinetic patterns that may affect arginine release,
uptake, and/or physiologic effect over time. However, it
should be noted that orange juice, which was provided to the
subjects during the course of the observation period in an
effort to prevent hypoglycemia during the 18-h fasting pe-
riod, contained macro- and/or micronutrients that may have
influenced arginine levels to some degree because observed
peak values were not increased in an overly impressive
manner. Although this does not detract from the finding that
plasma arginine profiles differed between the TR and NTR
forms of arginine supplements, the absolute values observed
may have been influenced to some degree. Additional re-
search should examine the physiologic effect of more rapid
or delayed appearance of arginine in the plasma in response
to NTR and TR forms of AAKG.
The present study is the first to examine the safety and
efficacy of AAKG supplementation during resistance train-
ing in well-trained men. Results indicated that AAKG sup-
plementation (12 g/d for 8 wk) was well tolerated and
produced no significant changes in liver enzymes, liver or
kidney function, or hematologic profiles. Moreover, no se-
rious side effects were observed. These results are in good
agreement with the history of safety and tolerability of
L-arginine, whether administered by intravenous infusion or
orally [4].
In terms of potential ergogenic value during training, the
major finding of this study was that AAKG treatment re-
sulted in significantly greater gains in 1 RM bench press and
anaerobic power in resistance-trained men. These findings
may be of interest to athletes interested in gaining strength
and/or anaerobic power during training and warrant addi-
tional research to examine the possible mechanisms of ac-
tion and potential ergogenic value of AAKG supplementa-
tion on additional anaerobic strength and power indices.
However, AAKG supplementation did not promote statisti-
cally significant changes in fat-free mass, muscular endur-
ance, or aerobic capacity. These findings indicate that, al-
though there was evidence of greater gains in strength and
anaerobic power, the improved performance capacity did
not promote muscle hypertrophy or improve body compo-
sition results during training. These findings do not support
contentions that AAKG promotes lean tissue accretion dur-
ing resistance training. However, it is possible that the
sample and/or use of well-trained resistance-trained subjects
who are more resistant to gains in muscle mass during
training as opposed to untrained subjects may have influ-
enced the results.
We are aware of one other study that used resistance-
trained men as subjects that investigated the effects of
arginine on body composition and muscle function.
Walberg-Rankin et al. [38] gave male weight trainers
who were consuming a hypocaloric diet approximately
8 g of arginine daily for 10 d. The investigators con-
cluded that arginine supplementation had no influence on
fat or lean tissue loss, muscle function (as determined by
biceps and quadriceps isokinetic testing), or overall
growth hormone status. Results of the present study sup-
port these findings because 8 wk of AAKG supplemen-
tation did not significantly affect body composition,
lower body muscle function as measured by quadriceps
isokinetic testing, or anabolic hormonal profiles. Con-
versely, Santos et al. [39] examined the effects of short-
term arginine supplementation (3 g/d for 15 d) on muscle
function in healthy but untrained men. The researchers
reported that arginine supplementation promoted an 8.5%
improvement in isokinetic knee extension fatigue index.
They concluded that arginine supplementation improved
muscular resistance capacity to fatigue.
The significant effects observed from AAKG supple-
mentation on anaerobic sprint capacity in the present study
are interesting. To date, there has only been one study [40]
that has evaluated the effects of an arginine-containing
compound on anaerobic sprint performance. Buford et al.
[40] examined the effects of ingesting 11.2 g of glycine-
arginine-[
␣
]-ketoisocaproic acid (GAKIC) 45 min before
exercise on repetitive cycling sprint performance (5 ⫻10-s
sprints with 1-min rest recovery between sprints). The re-
searchers reported that GAKIC supplementation before ex-
ercise significantly improved retention of mean power dur-
ing the first two 10-s sprints. They concluded that GAKIC
appears to attenuate the decline in mean power during
repeated bouts of supramaximal exercise. In the present
study, subjects performed a 30-s Wingate anaerobic capac-
ity test. This test assesses peak power and the ability to
maintain power output during a prolonged sprint and
thereby assesses phosphagen and glycolytic capacity. Re-
879B. Campbell et al. / Nutrition 22 (2006) 872– 881
sults showed that AAKG supplementation increased peak
power and thereby influenced rate of fatigue. The reason
that AAKG increased peak power is unclear. Because argi-
nine is one of three amino acids used in the synthesis of
creatine [41] and low-dose creatine supplementation (e.g., 3
g/d for 4 wk) has been shown to increase muscle phospho-
creatine concentrations [42], 6 g/d of L-arginine supplemen-
tation may have influenced phosphocreatine availability and
thus anaerobic power indices [43,44]. Additional research
should be conducted to examine the effects of AAKG sup-
plementation on muscle phosphagen stores and anaerobic
capacity.
Arginine supplementation has also been reported to in-
crease aerobic capacity in patients with congestive heart
failure, stable angina, and pulmonary hypertension. For
example, Bronislaw et al. [45] studied the effects of arginine
supplementation on exercise duration in patients who had
congestive heart failure and concluded that arginine supple-
mentation prolonged exercise duration. In another study,
Ceremuzynski et al. [46] investigated the effects of oral
supplementation with arginine on exercise capacity in pa-
tients with stable angina after myocardial infarction. Argi-
nine supplementation resulted in an increase in mean exer-
cise time to maximal ST-segment depression and an
increase in the maximum workload (metabolic equivalents).
Mizuno et al. [47] reported that infusion of arginine coun-
teracted myocardial ischemia. Nagaya et al. [48] studied the
effects of oral supplementation of arginine on exercise ca-
pacity in patients with pulmonary hypertension. A signifi-
cant increase in peak oxygen consumption was observed,
leading the investigators to conclude that arginine improves
exercise capacity in patients with pulmonary hypertension.
Results of the present study in healthy resistance-trained
men do not support these findings.
Analysis of blood markers showed that AAKG supple-
mentation had no influence on nearly all parameters studied
and that all values remained within normal clinical ranges.
These findings indicate that AAKG supplementation is rel-
atively benign in terms of markers of clinical safety. A
statistically significant increase was observed in plasma
arginine and fasting glucose in the AAKG group. Increases
in plasma arginine would be expected from the ingestion of
12 g of AAKG daily for 8 wk. The observed values of
plasma arginine for the AAKG-supplemented group were in
the upper end of the normal reference range for plasma
arginine. Although the AAKG-supplemented group showed
a significant increase in blood glucose as compared with the
PLA group (7.7% versus 2.1%), the blood glucose values
were within normal blood glucose reference ranges, within
the observed standard deviation range of the baseline assays
(8 –10%), and not significantly different from PLA values
after 8 wk of supplementation (i.e., AAKG 97 ⫾10 versus
PLA 96 ⫾7 mg/dL). Hence, it is unclear whether the
differences observed were directly related to AAKG sup-
plementation, a result of natural changes in blood glucose
levels, assay variability, and/or of any clinical significance.
Interestingly, Siani et al. [49] reported that fasting glucose
levels decreased by 4% (P⫽0.10) in subjects consuming an
arginine-rich diet (10 g/d) and by 9% (P⫽0.008) in
subjects supplementing their diet with 10 g/d of L-arginine
for 1 wk. This reported reduction in fasting glucose in
response to L-arginine supplementation was a primary rea-
son that we provided orange juice to our subjects during the
18-h fasting and experimental pharmacokinetic trial. Obvi-
ously, further research is needed to examine the effects of
AAKG supplementation on fasting glucose levels before
any conclusions can be drawn.
In conclusion, AAKG supplementation appeared to be
safe and well tolerated and positively influenced 1RM
bench press and Wingate peak power performance. AAKG
did not influence body composition or aerobic capacity.
Further research is needed to examine the role of AAKG
supplementation during training in untrained and trained
populations before definitive conclusions can be drawn re-
garding its potential ergogenic value.
Acknowledgments
The authors thank the subjects who participated in this
study and the laboratory assistants in the Exercise & Sport
Nutrition Laboratory (ESNL) who assisted with data col-
lection and analysis. Researchers in the ESNL indepen-
dently collected, analyzed and interpreted the results from
this study and have no financial interests in the results of
this study. Presentation of results in this study does not
constitute endorsement by Baylor University or its authors
of the supplements investigated.
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