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Effects of 7 Days of Arginine-Alpha-Ketoglutarate Supplementation on Blood Flow, Plasma L-Arginine, Nitric Oxide Metabolites, and Asymmetric Dimethyl Arginine After Resistance Exercise


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Arginine-alpha-ketoglutarate (AAKG) supplements are alleged to increase nitric oxide production, thereby resulting in vasodilation during resistance exercise. This study sought to determine the effects of AAKG supplementation on hemodynamics and brachial-artery blood flow and the circulating levels of L-arginine, nitric oxide metabolites (NOx; nitrate/nitrite), asymmetric dimethyl arginine (ADMA), and L-arginine:ADMA ratio after resistance exercise. Twenty-four physically active men underwent 7 days of AAKG supplementation with 12 g/day of either NO(2) Platinum or placebo (PLC). Before and after supplementation, a resistance-exercise session involving the elbow flexors was performed involving 3 sets of 15 repetitions with 70-75% of 1-repetition maximum. Data were collected immediately before, immediately after (PST), and 30 min after (30PST) each exercise session. Data were analyzed with factorial ANOVA (p < .05). Heart rate, blood pressure, and blood flow were increased in both groups at PST (p = .001) but not different between groups. Plasma L-arginine was increased in the NO(2) group (p = .001). NOx was shown to increase in both groups at PST (p = .001) and at 30PST (p = .001) but was not different between groups. ADMA was not affected between tests (p = .26) or time points (p = .31); however, the L-arginine:ADMA ratio was increased in the NO(2) group (p = .03). NO(2) Platinum increased plasma L-arginine levels; however, the effects observed in hemodynamics, brachial-artery blood flow, and NOx can only be attributed to the resistance exercise.
Content may be subject to copyright.
International Journal of Sport Nutrition and Exercise Metabolism, 2011, 21, 291-299
© 2011 Human Kinetics, Inc.
The authors are with the Dept. of Health, Human Performance,
and Recreation, Baylor University, Waco, TX.
Effects of 7 Days of Arginine-Alpha-Ketoglutarate
Supplementation on Blood Flow, Plasma L-Arginine,
Nitric Oxide Metabolites, and Asymmetric
Dimethyl Arginine After Resistance Exercise
Darryn S. Willoughby, Tony Boucher, Jeremy Reid, Garson Skelton, and Mandy Clark
Background: Arginine-alpha-ketoglutarate (AAKG) supplements are alleged to increase nitric oxide produc-
tion, thereby resulting in vasodilation during resistance exercise. This study sought to determine the effects
of AAKG supplementation on hemodynamics and brachial-artery blood ow and the circulating levels of
L-arginine, nitric oxide metabolites (NOx; nitrate/nitrite), asymmetric dimethyl arginine (ADMA), and
L-arginine:ADMA ratio after resistance exercise. Methods: Twenty-four physically active men underwent
7 days of AAKG supplementation with 12 g/day of either NO2 Platinum or placebo (PLC). Before and after
supplementation, a resistance-exercise session involving the elbow exors was performed involving 3 sets of
15 repetitions with 70–75% of 1-repetition maximum. Data were collected immediately before, immediately
after (PST), and 30 min after (30PST) each exercise session. Data were analyzed with factorial ANOVA (p <
.05). Results: Heart rate, blood pressure, and blood ow were increased in both groups at PST (p = .001) but
not different between groups. Plasma L-arginine was increased in the NO2 group (p = .001). NOx was shown
to increase in both groups at PST (p = .001) and at 30PST (p = .001) but was not different between groups.
ADMA was not affected between tests (p = .26) or time points (p = .31); however, the L-arginine:ADMA ratio
was increased in the NO2 group (p = .03). Conclusion: NO2 Platinum increased plasma L-arginine levels;
however, the effects observed in hemodynamics, brachial-artery blood ow, and NOx can only be attributed
to the resistance exercise.
Keywords: vasodilation, hemodynamics, amino acid, skeletal muscle
Nitric oxide is a gaseous signaling molecule known
to contribute to the control of vascular tone (Thomas,
Shaul, Yuhanna, Froehner, & Adams, 2003) and is con-
sidered to play a role in the vasodilation of muscle resis-
tance vessels during exercise (Tschakovsky & Joyner,
2008). Nitric oxide is biosynthesized endogenously in the
endothelium from L-arginine by endothelial nitric oxide
synthase (eNOS; Böger & Bode-Böger, 2001); however,
asymmetric dimethyl arginine (ADMA) can interact with
eNOS and inhibit its activity (Leiper & Vallance, 1999).
L-arginine (2-amino-5-guanidino-pentanoic acid), a
conditionally essential, proteinogenic amino acid that
is a natural constituent of dietary proteins (McConnell,
2007), has been used clinically to improve vasodilatory
capacity and blood ow (McConnell, 2007), based on
the premise that L-arginine supplementation has been
shown to affect the release of nitric oxide (Hishikawa
et al., 1992).
In recent years, various nutritional supplements have
been developed containing L-arginine and other com-
pounds (mainly arginine-alpha-ketoglutarate; AAKG)
and are being marketed as ergogenic aids because of their
function as “vasodilators” as a result of up-regulation
of the endothelial L-arginine-nitric-oxide pathway. The
AAKG-enhanced vasodilation and blood ow to working
muscles during resistance exercise is alleged to provide
an even greater impetus for increasing muscle strength
and hypertrophy than exercise alone. This is based on
the premise that as nitric oxide is elevated in circula-
tion, blood ow increases to active muscles. However,
it has been shown that L-arginine supplementation does
not increase muscle blood ow after resistance exercise
(Tang, Lysecki, Manolakos, Tarnopolsky, & Phillips,
2011). Furthermore, 8 weeks of resistance training com-
bined with AAKG supplementation at a daily dose of 12 g
appeared to be safe and well tolerated but had only caused
modest improvements in muscle strength and power and
had no effects on body composition or aerobic capacity
(Campbell et al., 2006).
Little research has been published to substantiate
manufacturers’ claims of these allegedly vasodilating,
292 Willoughby et al.
ergogenic nutritional supplements. In a study examin-
ing the effects of acute L-arginine supplementation and
resistance exercise on arterial function in young men,
there was no signicant change in blood ow and hemo-
dynamic and vascular responses when 7 g of L-arginine
was given immediately before resistance exercise (Fahs,
Heffernan, & Fernhall, 2009). It has been shown that
single doses of alleged nitric-oxide-inducing supplements
were ineffective at increasing circulating nitric oxide
levels and blood ow in response to resistance exercise
(Bloomer et al., 2010). Bode-Böger, Böger, Galland,
Tsikas, and Frölich (1998) found that 6 g of L-arginine
delivered either intravenously or orally did not result in
any signicant changes in blood pressure, heart rate, or
cardiac output. Similarly, L-arginine provided orally at 6
g/day for 3 days was shown to have no effect on plasma
nitric oxide levels and muscle power generated during
an anaerobic cycle-ergometer test in well-trained male
athletes (Liu et al., 2009).
In light of unsubstantiated product claims by supple-
ment manufacturers supporting the notion that AAKG
supplements increase blood ow, and because of the
paucity of data involving AAKG supplementation and
the conicting results of available research, the purpose
of this study was to determine the effects of 7 days of
AAKG supplementation, using the nutritional supplement
NO2 Platinum, on resting and resistance-exercise-induced
hemodynamics (heart rate, blood pressure, and mean
arterial blood pressure [MAP]), arterial blood ow, cir-
culating levels of nitric oxide metabolites (NOx [nitrate/
nitrite]) and ADMA, and the L-arginine:ADMA ratio.
Twenty-four apparently healthy, resistance-trained (regu-
lar, consistent resistance training—i.e., thrice weekly—
for at least 1 year before the onset of the study) men age
18–25 with body-mass index of 18.5–30 kg/m2 volun-
teered to participate in the double-blind study. Enrollment
was open to men of all ethnicities. Only participants
considered at low risk for cardiovascular disease, with no
contraindications to exercise as outlined by the American
College of Sports Medicine, who had not consumed any
nutritional supplements (excluding multivitamins) 1
month before the study were allowed to participate. All
participants provided written informed consent and were
cleared for participation by passing a mandatory medical
screening. With the exception of the exercise involved
in the study, participants were instructed not to engage
in any other resistance exercise during the course of the
study. All eligible subjects signed university-approved
informed-consent documents, and approval was granted
by the Institutional Review Board for Human Subjects of
Baylor University. All experimental procedures involved
in the study conformed to the ethical considerations of
the Helsinki Code. A diagram of the experimental design
can be seen in Figure 1.
Baseline Muscle-Strength Testing
A familiarization/baseline muscle-strength testing ses-
sion was scheduled approximately 72 hr before the rst
Figure 1 An illustration of the experimental design highlighting the protocol used at both testing sessions (T1 and T2). It should
be noted that the procedures involved for T1 and T2 were identical. Baseline muscle-strength testing was performed 72 hr before
T1. T1 was performed between 2 and 4 p.m. T2 occurred 7 days later and at the same time of day as T1 for each participant.
AAKG Supplementation and Blood Flow 293
resistance-exercise session. To determine maximum
strength of the elbow exors, participants performed
a one-repetition maximum (1-RM) test on the same
“preacher curl” machine to be used in the two resistance-
exercise sessions. They warmed up by completing 5–10
repetitions at approximately 50% of their estimated
1-RM. Participants rested for 1 min and then completed
3–5 repetitions at approximately 70% of their estimated
1-RM. The weight was then increased conservatively, and
the participants attempted to lift it for one repetition. If the
lift was successful, the participant rested for 2 min before
attempting the next weight increment. This procedure was
continued until the participant failed to complete the lift.
The 1-RM was recorded as the maximum weight that the
participant was able to lift for one repetition.
Venous Blood Sampling
Venous blood samples were collected from the antecubital
vein into 10-ml serum- and plasma-collection tubes using
a standard Vacutainer apparatus. Blood samples were
allowed to stand at room temperature for 10 min and then
centrifuged. The serum and plasma were removed and
frozen at –80 °C for later analysis. Three blood samples
per participant were obtained at Testing Session 1 (T1)
and 7 days later at Testing Session 2 (T2) for a total of six
blood samples per participant. The rst blood samples at
T1 and T2 were obtained immediately before resistance
exercise (PRE) and after an 8-hr fast, the second sample
immediately after resistance exercise (PST), and the third
sample 30 min after exercise (30PST).
Supplementation Protocol
In a randomized, double-blind, placebo-controlled design
participants were assigned either an alleged nitric-oxide-
inducing supplement containing AAKG (NO2 Platinum,
Medical Research Institute, San Francisco, CA) or a pla-
cebo supplement (PLC; apple pectin, General Nutrition
Corp., Pittsburgh, PA). The Medical Research Institute
recommends that individuals weighing 160–200 lb
(73–90 kg) take 8 tablets/day and those weighing over
200 lb take 10 tablets/day (http://www.mri-performance.
com/no2.php). However, a previous study (Campbell et
al., 2006) using NO2 Platinum provided 12 tablets/day;
therefore, we chose to adopt this dosing protocol. In the
current study, both supplements contained 1 g/tablet, and
12 tablets were ingested daily for 7 days. On Days 1–6,
four tablets were ingested in the morning on an empty
stomach 30 min before breakfast, four tablets 30 min
before lunch, and four tablets on an empty stomach 30
min before dinner (Campbell et al., 2006). On Day 7,
however, six tablets were ingested in the morning on an
empty stomach 30 min before breakfast, and six tablets,
30 min before lunch. Supplementation compliance was
monitored by participants’ returning empty containers
of their supplement on Day 7 and also by documenting
their daily supplement ingestion. With the exception of
ingesting the supplement, participants were instructed not
to change their normal dietary intake during the course
of the study.
Resistance-Exercise Protocol
For T1, testing was performed between 12 and 2 p.m. To
standardize the time of day for the two testing sessions,
each participant performed T2 at the same time of day
as T1. Each resistance-exercise session involved the
“preacher curl” bicep-exion exercise on a Selectorized
weight machine (Body Master, Rayne, LA). Participants
performed three sets of 15 repetitions with as much
weight as they could lift per set (typically 70–75% of
1RM). Rest periods between sets were timed and lasted
10 s.
Assessment of Plasma L-Arginine Levels
Plasma L-arginine was assessed using high-performance
liquid chromatography/mass spectrometry (Huang,
Guo, Liang, Yang, & Cheng, 2004). Because we were
primarily concerned about whether NO2 Platinum was
effective at increasing circulating L-arginine, only the
PRE blood samples obtained at T1 and T2 were evalu-
ated. Whole blood was collected in heparinized tubes,
and plasma was obtained by centrifugation for 10 min.
Twenty milligrams of 5-SSA were added to 1 ml of
plasma, and the mixture was incubated on ice for 10 min.
The precipitated protein was removed by centrifugation
for 10 min. Separations were performed on a Shimadzu
model system that consisted of an LC-10Advp solvent
delivery pump, a FCV-10ALvp low-pressure gradi-
ent unit, a DGU-14A degasser, a CTO-10Avp column
oven, and a SPD-M10Avp photo-diode array detector.
The column used for separation was a 2.0 × 150-mm
Shimadzu VP-ODS column with a particle size of 5 μm.
The analytical column was protected by a C18 Guard-Pak
cartridge (2.0 × 10 mm, 5 μm, Waters, Milford, MA). The
mobile phase consisted of water/acetonitrile (90/10, v/v)
containing 0.5% TFA, which was degassed ultrasonically
before use. Each component of the mobile phase was
ltered through a 0.22-μm membrane. All separations
were at ambient temperature and a ow rate of 0.2 ml/
min. The wavelength of the photo-diode array detector
was 200–300 nm. The amount of injection was 5 μl.
Mass-spectrometry experiments were performed
using a LCMS-2010 quadrupole mass spectrometer (Shi-
madzu Kyoto, Japan) interfaced with the Shimadzu model
system coupling with an atmospheric-pressure chemical-
ionization interface. The mass spectrum of L-arginine
was obtained by positively scanning between m/z 100
and 400/s. Selective ion-monitoring mode involved the
use of the positively protonated molecular ion [M+H]+
at m/z 175 (IS ion). The L-arginine standard and IS were
injected into the LCMS system, and sensitivity optimiza-
tion was performed by injection of an L-arginine standard
(20 μmol/L). Mass-spectrometric detection conditions for
294 Willoughby et al.
both scan and selective ion monitoring were as follows:
atmospheric-pressure chemical ionization temperature
380 °C, curved desolvation line temperature 240 °C,
block temperature 200 °C, probe voltage 4.0 kV, detec-
tor voltage 1.6 kV, curved desolvation line voltage –30
V, Q-array Bios voltage 22 V, and nebulizing gas ow
3.0 L/min.
Assessment of Serum NOx and ADMA
From the six blood samples obtained at T1 and T2,
serum NOx (nitrate/nitrite) was determined using a
commercially available colorimetric assay kit (Cayman
Chemical, Ann Arbor, MI) according to the procedures
provided by the manufacturer. This assay determines the
measurement for total nitrate and nitrite concentrations
involving the conversion of nitrate to nitrite using nitrate
reductase. The absorbance was then detected photo-
metrically at 540 nm. Quantication was performed with
calibration curves using nitrate and nitrite standards of
known concentrations. The sensitivity of the assay is 2.0
μM. The manufacturer has demonstrated the interassay
coefcient of variation on ve samples to be 3.4%. We
have demonstrated the interassay coefcient of variation
for this assay in our laboratory on approximately 500
samples to be 6.4%.
The serum level of ADMA (Alexis Biochemicals,
San Diego, CA) was determined using a commercially
available ELISA kit, based on manufacturer’s guidelines.
The ADMA assay is a competitive ELISA involving
polyclonal capture and secondary antibodies specic for
human ADMA. Samples were assessed at a wavelength of
450 nm using a standard curve generated from a known
concentration of ADMA with a Wallac 1420 Multilabel
Counter (Wallac, Turku, Finland). Data analysis was
performed using MicroWin microplate data-reduction
software (Mikrotek Laborsysteme, Germany). The
sensitivity of the assay is 0.05 μmol/L. The interassay
coefcient of variation on six samples established by
the manufacturer is 4.5%. In our laboratory, we have
demonstrated the interassay coefcient of variation on
approximately 200 samples to be 5.6%.
Assessment of Brachial-Artery
Blood Flow
Blood ow (peak velocity) of the brachial artery was
assessed before each blood draw and determined imme-
diately before and after (within 2–3 min) and 30 min after
exercise by way of high-resolution, real-time pulsed-wave
Doppler ultrasound (SonoSite M-Turbo, SonoSite, Inc.,
Bothell, WA), employing an electronic 13-6-MHz multi-
frequency and 25-mm linear array with a maximum depth
of 6 cm. The brachial artery was located by palpation
while the participants lay supine with the elbow in full
extension. Using the Doppler probe, maximal arterial
blood ow (cm/s) was determined by using the average
value from a 30-s sampling period at each time point.
Doppler ultrasonography has been well studied and is
widely used in medical practice as the gold standard for
vascular examination. Multiple studies have concluded
that Doppler ultrasound is a reliable and valid objec-
tive measure of blood ow (Billinger & Kluding, 2009;
Hotoleanu, Fodor, & Suciu, 2010; Matthiessen, Zeitz,
Richard, & Klemm, 2004; Thomson, Thomson, Woods,
Lannos, & Sage, 2001).
Assessment of Heart Rate,
Blood Pressure, and MAP
At T1 and T2, heart rate and blood pressure were assessed
and MAP calculated at PRE, PST, and 30PST. Heart rate
and blood pressure were assessed in the supine position
with an automated blood pressure monitor (Arial BP
2400, Medquip, Bluffton, SC) using standard procedures.
MAP was determined using the equation MAP = DBP
+ [0.333(SBP DBP)], where DBP = diastolic blood
pressure and SBP = systolic blood pressure.
Reported Side Effects From Supplements
At T2, after 7 days of supplementation, participants
reported by questionnaire whether they had tolerated the
supplement and supplementation protocol and reported
any medical problems or symptoms they may have
Statistical Analysis
For plasma L-arginine and L-arginine:ADMA, separate
two-way (Group × Testing Session) factorial ANOVAs
were used to determine differences between groups and
testing sessions and interactions (p .05). For all other
data, separate three-way (Group × Testing Session ×
Time Point) factorial ANOVAs were used to determine
differences between groups, testing sessions, and time
points and interactions (p .05). Tukey’s post hoc tests
were used to locate signicant differences among time
points (p .05). Effect sizes are presented as partial eta-
squared (η2) values.
Participant Demographics, Supplement
Compliance, and Reported Side Effects
Of the 24 participants who began the study, all nished
successfully. For the PLC group, participants had a mean
height, body mass, and age of 179.62 ± 6.19 cm, 83.82
± 12.51 kg, and 21.75 ± 2.17 years, respectively. The
respective height, body mass, and age for the NO2 group
were 175.66 ± 9.98 cm, 84.17 ± 12.51 kg, and 22.58 ±
AAKG Supplementation and Blood Flow 295
3.31. Results showed no signicant differences between
groups for any of these variables, indicating homogeneity
between groups
Overall participant compliance with supplement
ingestion was 95%; 1 participant in the NO2 group
accidentally only ingested 9 tablets/day rather than 12;
however, a review of his data showed no difference from
the rest of the group, so they were retained. In addition,
none of the participants reported any negative side effects
associated with ingesting either of the supplements.
Heart rate (p = .001, η2 = 0.17), systolic (p = .001, η2 =
0.44) and diastolic blood pressure (p = .001, η2 = 0.07),
and MAP (p = .001, η2 = 0.27) were shown to signi-
cantly increase at PST but were not different between
groups (p > .05) or testing sessions (p > .05). Resistance
exercise signicantly increased brachial-artery blood
ow in both groups (p = .001, η2 = 0.42) at PST, but it
was not different between groups (p = .14, η2 = 0.02) or
testing sessions (p = .47, η2 = 0.04; Table 1).
Plasma L-Arginine
From the preexercise blood samples at T1 and T2, a
signicant Group × Time interaction was observed (p =
.003, η2 = 0.18). Results demonstrated that L-arginine
signicantly increased in the NO2 group compared with
T1 and PLC values (Table 2).
Table 1 Hemodynamic Variables in Response to 7 Days of Arginine-Alpha-Ketoglutarate
Supplementation and Resistance Exercise, M ± SD
Variable PLC T1 PLC T2 NO
< .05
Heart rate (beats/min) Time: p = .001
PRE 60.66 ± 8.74 62.91 ± 11.42 61.58 ± 10.47 63.91 ± 12.59
PST 72.08 ± 18.89 79.33 ± 18.76 83.58 ± 25.96 80.00 ± 28.43 PST > PRE
30PST 66.16 ± 11.99 66.75 ± 10.26 65.75 ± 12.22 65.75 ± 12.22
Systolic blood pressure (mmHg) Time: p = .001
PRE 119.08 ± 11.30 119.50 ± 10.24 120.01 ± 13.91 122.17 ± 11.07
PST 142.58 ± 16.45 140.75 ± 9.10 150.50 ± 16.47 143.75 ± 12.57 PST > PRE
30PST 124.33 ± 13.93 122.08 ± 10.65 124.33 ± 9.79 126.75 ± 9.56
Diastolic blood pressure (mmHg) Time: p = .001
PRE 71.83 ± 7.22 75.83 ± 10.32 73.83 ± 6.97 72.25 ± 6.51
PST 74.50 ± 8.82 74.33 ± 8.46 78.75 ± 6.92 76.75 ± 9.55 PST > PRE
30PST 69.33 ± 8.91 69.50 ± 13.63 72.25 ± 7.31 70.97 ± 6.14
Mean arterial pressure (mmHg) Time: p = .001
PRE 87.56 ± 7.95 90.37 ± 9.15 89.26 ± 8.12 89.03 ± 6.56
PST 97.17 ± 9.39 96.45 ± 7.31 102.64 ± 9.04 99.06 ± 8.08 PST > PRE
30PST 88.64 ± 7.95 87.01 ± 10.34. 89.59 ± 7.06 89.51 ± 5.04
Brachial artery blood ow (cm/s) Time: p = .001
PRE 85.69 ± 20.34 90.55 ± 18.20 85.85 ± 17.76 82.15 ± 15.
PST 116.91 ± 8.09 119.88 ± 10.21 115.51 ± 11.18 113.31 ± 11.18 PST > PRE
30PST 90.74 ± 20.34 94.50 ± 21.43 85.01 ± 10.74 91.52 ± 24.56
Note. PLC = placebo; T1 = Testing Session 1; T2 = Testing Session 2; NO2 = NO2 Platinum; PRE = preexercise; PST = immediately postexercise;
30PST = 30 min PST.
296 Willoughby et al.
Serum NOx, eNOS, ADMA,
and L-Arginine:ADMA Ratio
Resistance exercise was shown to signicantly increase
NOx in both groups at PST (p = .001, η2 = 0.10) but
was not different between groups (p = .73, η2 = 0.01) or
testing sessions (p = .44, η2 = 0.04). For ADMA, NO2
was signicantly less than for PLC (p = .04, η2 = 0.15)
at PRE for T2; however, there were no signicant differ-
ences between testing sessions (p = .26, η2 = 0.01) or time
points (p = .31, η2 = 0.05). In regard to L-arginine:ADMA
ratio, NO2 was signicantly greater than PLC at T2 (p =
.03, η2 = 0.10), but there were no signicant differences
between testing sessions (p = .419, η2 = 0.02; Table 2).
This study examined the effects of 7 days of AAKG
supplementation using the nutritional supplement NO2
Platinum on arterial blood ow after a single bout of
resistance exercise. Despite a signicant increase in
plasma L-arginine, the primary nding of this study was
that 7 days of AAKG supplementation at 12 g/day had no
signicant impact on hemodynamic function, brachial-
artery blood ow, NOx, or ADMA in response to a single
bout of resistance exercise.
The endothelium produces numerous paracrine
substances, including nitric oxide, that help regulate
vasomotor function. Nitric oxide is a labile, lipid-soluble
gas synthesized in endothelial cells from the amino acid
L-arginine through the action of eNOS (Palmer, Rees,
Ashton, & Moncada, 1988). It is released both basally
and in response to pharmacological stimulation (Vallance,
Collier, & Moncada, 1989) and shear stress (Cabral,
Hong, & Garvin, 2010). Because shear stress is a con-
sequence of blood ow and viscosity, the likely physi-
ological stimulus to endothelial nitric oxide production
has been identied as increased ow through the vessel
lumen (Pohl, Holtz, Busse, & Bassenge, 1986), with acute
nitric-oxide-mediated vasodilation tending to normalize
shear stress (Dimmeler & Zeiher, 2003). This raises the
possibility that nitric oxide may contribute to exercise
hyperemia, because exercise is associated with increased
pulse pressure and pulsatility that results in concomitant
increases in shear stress. However, nitric oxide may also
affect exercise hyperemia by mechanisms associated
with ow mediation (Maiorana, O’Driscoll, Taylor, &
Green, 2003), mechanical vessel distortion (Clifford,
Kluess, Hamann, Buckwalter, & Jasperse, 2006), muscle
activation (Van Teeffelen & Segal, 2006), and red blood
cell oxyhemoglobin desaturation (Ellsworth, 2004). In
addition, indirect evidence for a role of nitric oxide in
skeletal-muscle exercise hyperemia is provided by the
increased levels of plasma and urinary nitrite in response
to prolonged aerobic exercise but does not directly reect
endothelium-derived nitric oxide (Bode-Böger, Böger,
Scröder, & Frölich, 1994). Furthermore, it has been
shown that there is a signicant correlation between the
changes in forearm blood ow and serum nitrite concen-
tration, suggesting that serum nitrite reects changes in
endothelial nitric oxide formation in human forearm cir-
culation (Kelm, Preik-Steinhoff, Preik, & Strauer, 1999).
Table 2 Plasma and Serum Variables in Response to 7 Days of Arginine-Alpha-Ketoglutarate
Supplementation and Resistance Exercise, M ± SD
Variable PLC T1 PLC T2 NO
< .05
L-arginine (μmol/L) Group × Time: p = .001
PRE 92.83 ± 13.12 91.01 ± 16.27 104.17 ± 30.50 179.33 ± 75.79 NO2 > PLC
NOx (μmol/L) Time: p = .001
PRE 9.20 ± 5.29 8.28 ± 7.64 11.22 ± 5.26 15.27 ± 6.38
PST 17.63 ± 1.96 15.77 ± 8.98 27.79 ± 9.03 22 .79 ± 12.66 PST > PRE
30PST 22.21 ± 13.96 18.28 ± 7.62 11.61 ± 10.50 16.45 ± 10.73
ADMA (μmol/L) Group: p = .04
PRE 1.53 ± 0.76 1.31 ± 0.72 1.38 ± 0.75 1.10 ± 0.59 NO2 < PLC
PST 1.38 ± 0.75 1.31 ± 0.86 1.01 ± 0.65 1.19 ± 0.62
30PST 1.34 ± 0.76 1.42 ± 0.84 0.97 ± 0.75 1.12 ± 0.65
L-arginine:ADMA Group: p = .03
PRE 83.84 ± 58.69 95.78 ± 54.18 165.37 ± 261.10 234.80 ± 211.24 NO2 > PLC
Note. PLC = placebo; T1 = Testing Session 1; T2 = Testing Session 2; NO2 = NO2 Platinum; PRE = preexercise; NOx = nitric oxide metabolites;
PST = immediately postexercise; 30PST = 30 min PST; ADMA = asymmetric dimethyl arginine.
AAKG Supplementation and Blood Flow 297
The infusion of 30 g of L-arginine has been shown
to affect heart rate, blood pressure, and blood ow at rest,
but they were not affected by 6 g of either intravenous or
oral L-arginine (Bode-Böger et al., 1998). In the current
study, we observed 7 days of AAKG supplementation at
12 g/day to have no effect on the resting levels of heart
rate, blood pressure, MAP, blood ow, and NOx (Tables
1 and 2). This is also in line with a study that involved
providing alleged nitric-oxide-inducing supplements
before resistance exercise and found no change in the
baseline heart rate and NOx values after the supplementa-
tion period (Bloomer et al., 2010). With respect to acute
exercise, 6 mg/kg L-arginine has been shown to have little
effect on hemodynamics in healthy humans in response to
a 12-min exercise test (Haram, Kemi, & Wisloff, 2008).
Similarly, no changes in heart rate or NOx levels were
noted after single bouts of anaerobic or resistance exercise
after 7 days of supplementation with alleged nitric-oxide-
inducing supplements (Bloomer et al., 2010). However,
in the current study we observed increases in heart rate,
blood pressure, MAP, blood ow, and NOx immediately
after single bouts of resistance exercise (Tables 1 and 2).
In comparison with Bloomer et al.’s study, the results of
the current study may differ because we used a smaller
muscle mass with the arm-curl exercise compared with
the bench press. In addition, Bloomer et al. estimated
arterial blood ow by way of muscle-tissue oxygen
saturation using near-infrared spectroscopy, whereas the
current study measured arterial blood ow using Doppler
ADMA is derived from the proteolysis of methyl-
ated arginine residues on various proteins. The meth-
ylation is carried out by a group of enzymes referred
to as protein-arginine-methyltransferases (Leiper &
Vallance, 1999). On proteolysis of methylated proteins,
free methylarginines are released and can function as
competitive inhibitors of nitric oxide activity. Many fac-
tors such as inammatory cytokines (which have been
shown to increase in response to resistance exercise;
Izquierdo et al., 2009) have been shown to up-regulate
ADMA accumulation, thereby inhibiting nitric oxide
synthesis (Stuhlinger et al., 2001). Increased circulating
ADMA levels and reduced L-arginine:ADMA ratio are
correlated with a decreased endothelial-dependent ow-
mediated vasodilation (Sydow et al., 2003). As such, an
inhibition of eNOS activity can potentially be overcome
with increases in the extracellular L-arginine:ADMA
ratio through excess L-arginine substrate (i.e., AAKG
Because ADMA competes with L-arginine for bind-
ing to eNOS, it is considered an endogenous inhibitor of
nitric oxide synthesis. Because L-arginine is a substrate
for eNOS activity, we were curious to see if any changes
in the levels of NOx because of 7 days of AAKG supple-
mentation might provide an exacerbated increase in blood
ow in response to resistance exercise because of eNOS
inhibition. However, based on our inability to directly
assess endothelial eNOS activity in our experimental
model, we chose to indirectly assess it by evaluating
the levels of ADMA and the L-arginine:ADMA ratio.
After 7 days of supplementation, we observed decreases
in ADMA of ~14% and ~25% at PRE in the PLC and
NO2 groups, respectively, with the decrease in the NO2
group being signicantly different (p = .035; Table 2).
In addition, at the same time points we also observed
increases in the L-arginine:ADMA ratio of ~15% and
~42%, respectively, with NO2 being greater than PLC (p =
.032; Table 2). Based on our data, we observed increases
in circulating L-arginine with concomitant decreases in
ADMA with AAKG supplementation. Therefore, because
we observed decreases in ADMA and the increase in the
L-arginine:ADMA ratio in NO2 as a result of the increase
in L-arginine substrate, it is unlikely that ADMA had any
inhibitory effect on eNOS-induced nitric oxide synthesis.
In the current study, the fact that the AAKG supple-
ment had no effect on blood ow could be a result of
the amount of L-arginine ingested. Even so, the lack of
effect on blood ow from the AAKG supplement may
be a result of the bioavailability of L-arginine from the
daily ingested dose. A single dose as high as 30 g of
L-arginine administered intravenously during a 30-min
period has been shown to induce vasodilation in humans
(Bode-Böger et al., 1998). L-arginine-induced vasodila-
tion was associated with increased release of nitric oxide
metabolites—nitrite and nitrate—into urine, suggesting
that nitric oxide release induced by such high doses of
L-arginine contributed to the vasodilation effect. In a
study examining the effects of 7 g of acute L-arginine
supplementation and resistance exercise on arterial
function in young men, there was no signicant change
in blood ow and hemodynamic and vascular responses
when L-arginine was given immediately before resistance
exercise (Fahs et al., 2009). Similarly, in the current study,
we observed that 12 g/day of AAKG supplementation
for 7 days had no preferential effects on the resting and
exercise values for blood ow, hemodynamics, NOx, and
ADMA. However, we must caution that a limitation of
our results is that we measured nitric oxide metabolites
and not actual levels of circulating nitric oxide.
There is no empirical evidence to date demonstrat-
ing any acute vasodilating effects of oral L-arginine
with doses below 15 g/day. Apparently, an acute vaso-
dilator effect has been shown only in studies in which
L-arginine was administered parenterally, particularly
at a dose greater than 15 g (Böger, 2007). Any acute
hemodynamic effects of L-arginine at higher parenteral
doses are most likely related to endocrine secretagogue
vasodilator actions, which are absent with lower doses.
Although intracellular L-arginine levels have been
demonstrated to be considerably higher than L-arginine
levels in the extracellular uid or in plasma (Böger et
al., 2000), extracellular L-arginine can be taken up rap-
idly by endothelial cells, thereby contributing to nitric
oxide production (Schmidt et al., 1988). Moreover, the
298 Willoughby et al.
issue of L-arginine’s bioavailability must be taken into
consideration. It is absorbed in the small intestine and
transported to the liver, where the majority is taken up
and used in the hepatic urea cycle; however, a small part
of dietary L-arginine passes through the liver and is used
as a substrate for nitric oxide production (Böger, 2004).
This can be further illustrated from the standpoint that
the bioavailability of 6 g of orally ingested L-arginine has
been shown to be only ~68% (Bode-Böger et al., 1998).
As a result, it is conceivable that lower doses of L-arginine
(either alone or within AAKG supplements) may lack the
bioavailability needed to stimulate eNOS. Therefore, it is
plausible that the lack of effect on blood ow we observed
in the current study from AAKG supplementation resulted
from an inadequate amount of bioavailable L-arginine,
and it is possible that with a dosage greater than 12 g/
day we may have observed a more positive response to
the AAKG supplement.
At the dosage used, we have presented data herein
that appear to refute the alleged supposition and manu-
facturers’ claims that “vasodilating supplements” con-
taining L-arginine are effective at causing vasodilation,
thereby resulting in increased blood ow to active skeletal
muscle during resistance exercise. We have specically
demonstrated that a single bout of resistance exercise
increases vasomotor function, arterial blood ow, and
circulating NOx levels but that the AAKG supplement
provided no additive, preferential response compared
with placebo. Therefore, based on our collective data,
we conclude that 7 days of AAKG supplementation at a
dose of 12 g/day with NO2 Platinum effectively increased
plasma L-arginine levels; however, the effects observed in
brachial-artery blood ow and serum NOx were attributed
to resistance exercise rather than the AAKG supplement.
The authors would like to thank all the participants for their
involvement in the study. This study was supported by fund-
ing from the Exercise and Biochemical Nutrition Laboratory
at Baylor University.
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... Venous blood was collected from the antecubital vein into a serum separator tube using a Vacutainer apparatus and needle (Becton, Dickinson and Company, Franklin lakes, NJ, USA). Blood samples were allowed to clot at room temperature for 10 min [14] and the samples were then centrifuged (1000 g) for 15 min and serum was aliquoted into 1.5 mL microtubes. The aliquoted samples were frozen at −80 • C for future analysis. ...
... Before filtration, the filters were pre-rinsed with ultrapure water. Total serum nitric oxide metabolites (nitrate/nitrite) were determined [14] using a plate-based colorimetric assay at 540 nm (SpectraMax 384 Plus, SoftMax Pro Software, Molecular Devices, San Jose, CA, USA) Sports 2020, 8,22 4 of 10 against a standard curve according to manufacturer specifications (Cayman Chemical; Ann Arbor, MI, USA; Item # 780001). In brief, for total nitrate/nitrite measurement, samples and standards were added to a microplate followed by the addition of enzyme cofactor mixture (Cayman Chemical, Ann Arbor, MI, USA; Item # 780012) and nitrate reductase mixture (Cayman Chemical, Ann Arbor, MI, USA; Item # 780010). ...
... Interestingly, even though large increases in brachial artery diameter were observed, serum nitric oxide metabolite concentration remained unchanged. A previous study with a similar resistance exercise design reported an increase in nitric oxide metabolites at similar time points as used in this study [14]. In contrast, in the current study, three sets of barbell curls with minimal rest was not sufficient to increase nitric oxide metabolites in venous blood. ...
Full-text available
(-)-Epicatechin is a polyphenol previously shown to enhance vascular health. The purposes of the current studies were to determine the effect of acute (-)-epicatechin supplementation on local vasodilation in conjunction with resistance exercise (study 1) and on high-intensity exercise performance (study 2). For study 1, 11 men participated in two resistance exercise sessions, where they performed three sets of barbell curls while consuming 200 mg of 98% pure (-)-epicatechin or placebo. Measurements of total serum nitrate/nitrite and brachial artery diameter were acquired at baseline (pre-supplement), 90 min after supplement consumption (post-supplement), immediately post-exercise (post-exercise), and 30 min post-exercise (30 min post-exercise). For serum nitric oxide metabolites, no significant interaction between supplement and time nor significant main effect of time was observed (p = 0.38 and p = 0.20; respectively). For brachial artery diameter, no significant interaction between supplement and time was observed (p = 0.24). A significant main effect of time was observed for brachial artery diameter (p < 0.01) with post-exercise brachial artery diameter significantly greater diameter than all other time points (all p < 0.01). For study 2, six women and five men completed the 15.5 CrossFit® Open Workout three times. A familiarization session was performed first where the workout was performed without the consumption of a supplement. In a randomized, balanced fashion, 100 mg of 98% pure (-)-epicatechin or cellulose (placebo) was consumed two times per day for two days before testing sessions two and three. On the day of testing sessions two and three, 60 to 90 min before completing the workout, 200 mg of the assigned supplement was ingested with water. No significant difference was observed for time to complete the workout between testing sessions (p = 0.49). In conclusion, under the conditions of the current studies, acute (-)-epicatechin supplementation did not augment vasodilation in combination with resistance exercise, nor did it increase exercise performance in humans.
... Moderateintensity resistance exercise may induce to a laminar pattern of shear stress, stimulating the influx of endothelial calcium (Ca 2+ ) (Olesen et al., 1988;Cooke et al., 1991), increasing the activity of endothelial nitric oxide synthase (eNOS) and consequently elevating NO bioavailability (Bussell, 2002). In agreement with our results, Willoughby et al. (2011) demonstrated elevation in NOx levels after exercise in similar conditions (Willoughby et al., 2011). As a matter of fact, elevated levels of NOx after exercise may represent a greater bioactivity of NO, because 85% of NOx plasma levels seem to be related to NO formation (Lundberg et al., 2009). ...
... Moderateintensity resistance exercise may induce to a laminar pattern of shear stress, stimulating the influx of endothelial calcium (Ca 2+ ) (Olesen et al., 1988;Cooke et al., 1991), increasing the activity of endothelial nitric oxide synthase (eNOS) and consequently elevating NO bioavailability (Bussell, 2002). In agreement with our results, Willoughby et al. (2011) demonstrated elevation in NOx levels after exercise in similar conditions (Willoughby et al., 2011). As a matter of fact, elevated levels of NOx after exercise may represent a greater bioactivity of NO, because 85% of NOx plasma levels seem to be related to NO formation (Lundberg et al., 2009). ...
Full-text available
Regular resistance exercise is associated with metabolic, neuromuscular and cardiovascular adaptations which improve quality of life and health. However, sedentary subjects have shown acute impairments in endothelial function after high-intensity resistance exercise. The aim of this study was to evaluate endothelial function in sedentary middle-aged men after a single session of resistance exercise at different intensities. Eleven sedentary middle-aged men (40.1 ± 3.9 years; 27.3 ± 1.4 kg/m2) underwent three different conditions of assessment: (1) single knee extension exercise at moderate intensity (MI) [4 sets of 12 repetitions at 50% of one repetition maximum (1RM) for each leg], (2) single knee extension exercise at high intensity (HI) (4 sets of 8 repetitions at 80% of 1RM for each leg), (3) resting for the control condition (CON). Flow-mediated dilation (FMD) was assessed before, 30 and 60 min after exercise. Plasma concentrations of endothelin-1 (ET-1), nitrites and nitrates (NOx) and thiobarbituric acid reactive substances (TBARS) were measured before, immediately after and 60 min after exercise. Blood pressure (BP) was measured prior to the experimental protocols, and in the following times: immediately following, and 2, 5, 10, 15, 30, and 60 min after exertion. There was a significant improvement in FMD 30 min after MI condition (12.5 ± 4.10 vs. 17.2 ± 3.9%; p = 0.016). NOx levels were significantly higher immediately after MI (6.8 ± 3.3 vs. 12.6 ± 4.2 μM; p = 0.007) and there was a significant increase in the concentration of ET-1 immediately after HI (20.02 ± 2.2 vs. 25.4 ± 2.1 pg/mL; p = 0.004). However, there was no significant difference for BP (MI vs. HI) and TBARS among the experimental conditions. Resistance exercise performed at moderate intensity improved vasodilatation via increases on NOx levels and FMD in sedentary middle-aged men.
... 20 Moreover, there is a hypothesis that L-arginine can enhance endogenous NO production which is the only endogenous nitrogen-containing substrate of NOS that may increase muscle glucose uptake via NO and insulin release. 22,23 Our results are close to this hypothesis on insulin levels. Besides, there is no significant change in glucose uptake in the research which may return to accurate regulating of glucose during and after exercise by liver and related biochemical factors. ...
... In a study including 24 male athletes, the experimental group was given 12 g arginine per day for seven days. Based on the results, after the arginine supplementation, there was a decrease in the heart rate measured immediately after the exercise in the experimental group, compared to the pre-supplementation heart rate measurements while there was an increase in the placebo group (52). In another study, where NG-nitro-L-arginine methyl ester (L-NAME) supplementation was given to healthy male volunteers, although blood lactate level of the L-NAME group at the beginning of exercise was found to be higher than the control group, no significant difference was found between the groups after the exercise. ...
Full-text available
Background: The beneficial effect of popular supplements and use of nitric oxide (NO) precursor nutrients in athletes which aim at increase of sports performance. Objective: The aim of this study is to investigate the effect of nitric oxide precursor nutrients and some nutrients with the ability to increase NO levels in the body on increased NO production in metabolism and on the recovery after acute exercise. Materials and Methods: 8 volunteers, male athletes, in shape and trained participated into this study. Athletes were subject to research protocol every other day, a total of three times. Heart rate and lactate levels were determined at resting and recovery. Results: Statistically significant difference was detected between control and cocoa/caffeine (CC) groups in the 1 minute lactate levels. Lactate levels significantly decreased in the CC group compared to the control group in the 1 minute lactate levels (p<0.05). Statistical significance difference was found between the nitric oxide supplement (NOX) and CC groups in the 1 and 10 minutes heart rate levels. Heart rate levels significantly decreased in the CC group compared to the NOX group (p<0.05). Heart rate levels were significantly decreased in the CC group compared to the control and NOX groups in the 15 minute measurement (p<0.05). Conclusions: Nitric oxide consumed as a supplement in line with nutrition and recovery strategies in athletes improved the recovery by accelerating lactate excretion from the body after the exercise. All of these metabolic responses in the present study suggest that NO will have a positive effect on exercise performance and recovery.
... Besides that, L-arg supplementation impaired the gain of muscle strength in Nx RT + L-arg group; there is no experimental studies demonstrating an interference of L-arg supplementation on muscle strength of rodents submitted to RT, furthermore, studies with RT in humans had failed to show muscle strength additional improvements after oral L-arg supplementation [36][37][38][39], but none of them demonstrated impairments of RT effects after L-arg supplementation. ...
A decade-old meta-analytic work indicated that L-arginine supplementation might have a blood pressure (BP) lowering effect in different populations. However, several relevant investigations have emerged in the last 10 years, and an up-to-date systematic review and meta-analysis on this topic is currently lacking. Therefore, we aimed to examine the impact of L-arginine supplementation on BP by conducting a systematic review and dose-response meta-analysis of randomized placebo-controlled clinical trials (RCTs). We searched online databases using relevant keywords up to April 2021 to identify RCTs using oral L-arginine on systolic (SBP) and diastolic BP (DBP) in adults. Inclusion criteria were adult participants and an intervention duration ≥4 days. Exclusion criteria were the use of L-arginine infusion and acute interventions. A random-effects model was used to estimate the weighted mean difference (WMD) and 95% confidence (CI). Twenty-two RCTs with 30 effect sizes were included in this meta-analytic work. The pooled analysis demonstrated significant decreases in SBP (WMD = –6.40 mmHg; 95%CI: –8.74, –4.05; P<0.001) and DBP (WMD = –2.64 mmHg; 95%CI: –3.94, –1.40; P<0.001) after L-arginine supplementation. Sub-group analysis showed significant reductions in SBP and DBP regardless of baseline BP category (normotensive, hypertensive), study duration (≤24 days, >24 days), sex (female, male), health status (healthy, unhealthy), and body mass index (normal, overweight, obese). No significant changes were observed with dosages >9 g/day, trial duration >24 days, or in obese individuals. L-arginine supplementation also appears to decrease DBP more effectively in females than in males. Moreover, meta-regression analysis for DBP demonstrated a significant relationship between the dose of L-arginine intake and changes in DBP (P = 0.020). In the nonlinear dose-response analysis, the effective dosage of L-arginine supplementation was detected to be ≥4 g/day for SBP (P = 0.034) independent of trial duration. Overall, L-arginine supplementation may be effective for decreasing BP.
Full-text available
Abstract This study evaluated the ability of resistance training (RT) of moderate intensity to promote vascular changes in insulin-induced vasodilation in healthy animals. Wistar rats were divided into two groups: control (CON) and trained (eight weeks of training, performing 3 sets with 10 repetitions at 60% of maximum intensity). Forty-eight hours after the last session of the RT, the animals were sacrificed and vascular reactivity to insulin in the absence and presence of LY294002 (phosphatidylinositol 3-kinase inhibitors (PI3K), L-NAME (nitric oxide synthase (NOS) inhibitors) and BQ123 (endothelin A antagonist (ET-A) receptor). In addition, phenylephrine (Phe)-induced vasoconstriction in the absence and presence of L-NAME was also evaluated. The RT group showed greater vasodilation in maximal response compared to the CON group. After PI3K inhibition, vasodilation was reduced in both groups. However, when the NOS participation was evaluated, the RT group showed contraction in relation to the CON group, which was abolished by BQ123. In addition, the RT group had an increase in nitrite levels compared to the CON group. When the Phe response was evaluated, there was a reduction in tension in the RT group compared to the CON group. The results suggest that RT improves vascular reactivity.
In this study, we examined the effects of acute intravenous administration of l‐arginine on circulating levels of metabolites in the portal‐drained viscera (PDV) of 12 barrows surgically fitted with chronic catheters in the portal vein. At day 14 post‐surgery, the pigs were fasted for 12 hr and then randomly allocated to one of three groups to receive administration of normal saline, l‐alanine [103 mg/kg body weight (BW), isonitrogenous control] or l‐arginine‐HCl (61 mg/kg BW), via the portal vein. Blood samples were obtained from the carotid artery before and at 30‐min intervals for 5 hr after the administration of saline or amino acid in order to determine metabolic profiles. The results showed that, compared with the saline treatment, arginine infusion increased plasma concentrations of insulin‐like growth factor‐I, arginine and cystine in the portal vein plasma, whereas plasma concentrations of threonine, serine, leucine and methionine were reduced. These findings indicate that increasing arginine concentrations in the portal vein alters the metabolic profile in swine, an established animal model for studying human nutrition and metabolism.
Azizi M, Behpour N, Zari F, Nikseresht A. Metabolic Responses and Muscular Performance during 7-Days of Arginine Supplementation in Young Resistance-Trained Women. JEPonline 2018;21(6):82-91. The purpose of this study was to evaluate a dietary supplementation containing arginine, which is one of the latest ergogenic aids for enhancing strength, power, and muscle recovery combined with resistance training. Sixteen young resistance-trained women (age, 22.7 ± 0.6 yrs, BMI, 22.0 ± 2.09 kg·d-1) were randomly assigned to the Supplementation Group (N = 8, 7 days, 0.1 g·kg-1 ·BW-1) or the Placebo Group (N = 8, Rice flour with capsules of same size and shape). The subjects performed a similar resistance exercise (bench press and leg press exercises, 3 sets × 10-repetition maximum) in both groups before and after 7 days. The findings indicate that the concentration of lactate after 7 days of arginine supplementation was lower than the concentration of lactate in the Placebo Group (P = 0.01). The decrease in lactate production after resistance exercise is the most important finding of this study. However, while increasing the production 83 of lactate in high intensity resistance exercise is a reasonable finding, the profound effect of arginine may be the result of NO-mediated glycolysis inhibition, especially in the female subjects. On the other hand, the inconsistency of other metabolites with lactate may have been related to the time of blood sampling, because it seems there were more latent responses to blood rather than lactate.
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Unlabelled: Although the assessment of deep venous thrombosis (DVT) is based on clinical examination, the complementary tests and especially Doppler ultrasound plays the most important role in DVT diagnosis. Objectives: We aimed to establish the correlations between the clinical probability of DVT based on Wells score and the results of Doppler ultrasound exam. Method: We included 382 patients with clinical supposition of DVT divided into 3 groups based upon the probability of DVT (Wells score): low, moderate and high, respectively. All the patients were examined by Doppler ultrasonography. Results: We noticed that DVT was confirmed by Doppler ultrasonography in more than half of the cases; the highest percent of confirmed cases were in the patients with a high probability of DVT (70.58%) whereas the lowest percent was associated with the low clinical probability (14.63%). Discussions: These findings show the importance of the correct management of cases, starting with clinical data and including a complete anamnesis to identify the risk factors for DVT. Conclusions: There is a significant correlation between the Wells score reflected in the probability of DVT and the Doppler ultrasonography findings.
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The aim of this study was to investigate the ergogenic potential of arginine on NO synthesis, muscle blood flow, and skeletal muscle protein synthesis (MPS). Eight healthy young men (22.1 ± 2.6 y, 1.79 ± 0.06 m, 76.6 ± 6.2 kg; mean ± SD) participated in 2 trials where they performed a bout of unilateral leg resistance exercise and ingested a drink containing either 10 g essential amino acids with 10 g l-arginine (ARG) or an isonitrogenous control (CON). Femoral artery blood flow of both the nonexercised and exercised leg was measured continuously using pulsed-wave Doppler ultrasound, while rates of mixed and myofibrillar MPS were determined using a primed continuous infusion of L-[ring-(13)C(6)] or L-[ring-(2)H(5)]phenylalanine. The plasma arginine concentration increased 300% during the ARG trial but not during the CON trial (P < 0.001). Plasma nitrate, nitrite, and endothelin-1, all markers of NO synthesis, did not change during either the ARG or CON trial. Plasma growth hormone increased to a greater degree after exercise in the ARG trial than CON trial (P < 0.05). Femoral artery blood flow increased 270% above basal in the exercised leg (P < 0.001) but not in the nonexercised leg, with no differences between the ARG and CON trials. Mixed and myofibrillar MPS were both greater in the exercised leg compared with the nonexercised leg (P < 0.001), but did not differ between the ARG and CON treatments. We conclude that an oral bolus (10 g) of arginine does not increase NO synthesis or muscle blood flow. Furthermore, arginine does not enhance mixed or myofibrillar MPS either at rest or after resistance exercise beyond that achieved by feeding alone.
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We compared Glycine Propionyl-L-Carnitine (GlycoCarn(R)) and three different pre-workout nutritional supplements on measures of skeletal muscle oxygen saturation (StO2), blood nitrate/nitrite (NOx), lactate (HLa), malondialdehyde (MDA), and exercise performance in men. Using a randomized, double-blind, cross-over design, 19 resistance trained men performed tests of muscular power (bench press throws) and endurance (10 sets of bench press to muscular failure). A placebo, GlycoCarn(R), or one of three dietary supplements (SUPP1, SUPP2, SUPP3) was consumed prior to exercise, with one week separating conditions. Blood was collected before receiving the condition and immediately after exercise. StO2 was measured during the endurance test using Near Infrared Spectroscopy. Heart rate (HR) and rating of perceived exertion (RPE) were determined at the end of each set. A condition effect was noted for StO2 at the start of exercise (p = 0.02), with GlycoCarn(R) higher than SUPP2. A condition effect was also noted for StO2 at the end of exercise (p = 0.003), with SUPP1 lower than all other conditions. No statistically significant interaction, condition, or time effects were noted for NOx or MDA (p > 0.05); however, MDA decreased 13.7% with GlycoCarn(R) and increased in all other conditions. Only a time effect was noted for HLa (p < 0.0001), with values increasing from pre- to post-exercise. No effects were noted for HR, RPE, or for any exercise performance variables (p > 0.05); however, GlycoCarn(R) resulted in a statistically insignificant greater total volume load compared to the placebo (3.3%), SUPP1 (4.2%), SUPP2 (2.5%), and SUPP3 (4.6%). None of the products tested resulted in favorable changes in our chosen outcome measures, with the exception of GlycoCarn(R) in terms of higher StO2 at the start of exercise. GlycoCarn(R) resulted in a 13.7% decrease in MDA from pre- to post-exercise and yielded a non-significant but greater total volume load compared to all other conditions. These data indicate that 1) a single ingredient (GlycoCarn(R)) can provide similar practical benefit than finished products containing multiple ingredients, and 2) while we do not have data in relation to post-exercise recovery parameters, the tested products are ineffective in terms of increasing blood flow and improving acute upper body exercise performance.
Hyperhomocysteinemia is a putative risk factor for cardiovascular disease, which also impairs endothelium-dependent vasodilatation. A number of other risk factors for cardiovascular disease may exert their adverse vascular effects in part by elevating plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Accordingly, we determined if homocysteine could increase ADMA levels. When endothelial or nonvascular cells were exposed to DL-homocysteine or to its precursor L-methionine, ADMA concentration in the cell culture medium increased in a dose- and time-dependent fashion. This effect was associated with the reduced activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Furthermore, homocysteine-induced accumulation of ADMA was associated with reduced nitric oxide synthesis by endothelial cells and segments of pig aorta. The antioxidant pyrrollidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation. Moreover, homocysteine dose-dependently reduced the activity of recombinant human DDAH in a cell free system, an effect that was due to a direct interaction between homocysteine and DDAH. Homocysteine post-translationally inhibits DDAH enzyme activity, causing ADMA to accumulate and inhibit nitric oxide synthesis. This may explain the known effect of homocysteine to impair endothelium-mediated nitric oxide-dependent vasodilatation.
Objectives: Hyperhomocyst(e)inemia is a risk factor for atherosclerotic vascular disease, and it is associated with endothelial dysfunction. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia may involve impaired bioavailability of NO, possibly secondary to accumulation of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) and increased oxidative stress. We investigated whether oral treatment with B vitamins or l-arginine normalizes endothelium-dependent, flow-dependent vasodilation (FDD) in patients with peripheral arterial occlusive disease (PAOD) and hyperhomocyst(e)inemia. Methods: 27 patients with PAOD and hyperhomocyst(e)inemia were assigned to oral treatment with combined B vitamins (folate, 10 mg; vitamin B-12, 200 μg; vitamin B-6, 20 mg/day), l-arginine (24 g/day) or placebo, for 8 weeks in a double-blind fashion. FDD was determined by high-resolution ultrasound in the radial artery. Results: Vitamin B supplementation significantly lowered plasma homocyst(e)ine concentration from 15.8±1.8 to 8.7±1.1 μmol/l (P<0.01). However, B vitamins had no significant effect on FDD (baseline, 7.8±0.7%, B vitamins, 8.3±0.9%, placebo 8.9±0.7%; P = n.s.). In contrast, l-arginine treatment did not affect homocyst(e)ine levels, but significantly improved FDD (10.2±0.2%), probably by antagonizing the impact of elevated ADMA concentration (3.8±0.3 μmol/l) and reducing the oxidative stress by lowering urinary 8-iso-prostaglandin F2α (baseline, 76.3±7.1 vs. 62.7±8.3 pmol/mmol creatinine after 8 weeks). Conclusions: Oral supplementation with combined B vitamins during 8 weeks does not improve endothelium-dependent vasodilation in PAOD patients with hyperhomocyst(e)inemia, whereas l-arginine significantly improved endothelial function in these patients. Thus, accumulation of ADMA and increased oxidative stress may underlie endothelial dysfunction under hyperhomocyst(e)inemic conditions. These findings may have importance for evaluation of homocyst(e)ine-lowering therapy.
Objective: A reduced bioactivity of endothelial nitric oxide (NO) has been implicated in the pathogenesis of atherosclerosis. In humans, the endothelial l-arginine–NO pathway has been indirectly assessed via the flow response to endothelium-dependent vasodilators locally administered into the coronary, pulmonary or forearm circulation. However, biochemical quantification of endothelial NO formation in these organ circulations has been hampered so far because of the rapid metabolism of NO. Therefore, we aimed to work out a reliable biochemical index to assess endothelial NO formation in human circulation. Methods: In 33 healthy volunteers, forearm blood flow (FBF) was measured by standard techniques of venous occlusion plethysmography at rest, after local application of the endothelium-dependent vasodilator acetylcholine (ACH), the endothelium-independent vasodilator papaverine (PAP), the stereospecific inhibitor of endothelial NO synthase (eNOS) L-NMMA, and l-arginine (ARG), the natural substrate of eNOS. In parallel, nitrite and nitrate concentrations in blood samples taken from the antecubital vein were measured by HPLC using anion-exchange chromatography in combination with electrochemical and ultraviolet detection following a specific sample preparation method. Results: ACH dose-dependently increased resting FBF (from 3.0±0.3 to 10.4±0.9 ml/min per 100 ml tissue) and serum nitrite concentration (from 402±59 to 977±82 nmol/l, both p<0.05, n=12). A significant correlation was observed between the changes in FBF and the serum nitrite concentration (r=0.61, p<0.0001). L-NMMA reduced resting FBF and endothelium-dependent vasodilation by 30% and this was paralleled by a significant reduction in serum nitrite concentration at the highest dose of ACH (n=9, p<0.001). PAP increased FBF more than fourfold, but did not affect serum nitrite concentration (n=11), whereas ARG significantly increased both FBF and nitrite. Basal serum nitrate amounted to 25±4 μmol/l and remained constant during the application of ACH, PAP and L-NMMA. Conclusions: The concentration of serum nitrite sensitively reflects changes in endothelial NO formation in human forearm circulation. This biochemical measure may help to characterize the l-arginine–NO pathway in disease states associated with endothelial dysfunction and to further elucidate its pathophysiological significance for the development of atherosclerosis in humans.
Administration of L-arginine by intravenous infusion or via oral absorption has been shown to induce peripheral vasodilation in humans, and to improve endothelium-dependent vasodilation. We investigated the pharmacokinetics and pharmacokinetic-pharmacodynamic relationship of L-arginine after a single intravenous infusion of 30 g or 6 g, or after a single oral application of 6 g, as compared with the respective placebo, in eight healthy male human subjects. L-arginine levels were determined by h.p.l.c. The vasodilator effects of L-arginine were assessed non-invasively by blood pressure monitoring and impedance cardiography. Urinary nitrate and cyclic GMP excretion rates were measured as non-invasive indicators of endogenous NO production. Plasma L-arginine levels increased to (mean +/- s.e.mean) 6223+/-407 (range, 5100-7680) and 822+/-59 (527-955) micromol l(-1) after intravenous infusion of 30 g and 6 g L-arginine, respectively, and to 310+/-152 (118-1219) micromol l(-1) after oral ingestion of 6 g L-arginine. Oral bioavailability of L-arginine was 68+/-9 (51-87)%. Clearance was 544+/-24 (440-620), 894+/-164 (470-1190), and 1018+/-230 (710-2130) ml min(-1), and elimination half-life was calculated as 41.6+/-2.3 (34-55), 59.6+/-9.1 (24-98), and 79.5+/-9.3 (50-121) min, respectively, for 30 g i.v., 6 g i.v., and 6 g p.o. of L-arginine. Blood pressure and total peripheral resistance were significantly decreased after intravenous infusion of 30 g L-arginine by 4.4+/-1.4% and 10.4+/-3.6%, respectively, but were not significantly changed after oral or intravenous administration of 6 g L-arginine. L-arginine (30 g) also significantly increased urinary nitrate and cyclic GMP excretion rates by 97+/-28 and 66+/-20%, respectively. After infusion of 6 g L-arginine, urinary nitrate excretion also significantly increased, (nitrate by 47+/-12% [P<0.05], cyclic GMP by 67+/-47% [P= ns]), although to a lesser and more variable extent than after 30 g of L-arginine. The onset and the duration of the vasodilator effect of L-arginine and its effects on endogenous NO production closely corresponded to the plasma concentration half-life of L-arginine, as indicated by an equilibration half-life of 6+/-2 (3.7-8.4) min between plasma concentration and effect in pharmacokinetic-pharmacodynamic analysis, and the lack of hysteresis in the plasma concentration-versus-effect plot. The vascular effects of L-arginine are closely correlated with its plasma concentrations. These data may provide a basis for the utilization of L-arginine in cardiovascular diseases.
We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min (P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min (P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min (P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O(2)(-) scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm(2) produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively (P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.