Content uploaded by Lee Wylie
Author content
All content in this area was uploaded by Lee Wylie on Apr 28, 2016
Content may be subject to copyright.
Content uploaded by Asker Jeukendrup
Author content
All content in this area was uploaded by Asker Jeukendrup on Feb 02, 2016
Content may be subject to copyright.
doi: 10.1152/japplphysiol.00372.2013
115:325-336, 2013. First published 2 May 2013;J Appl Physiol
Winyard, Asker E. Jeukendrup, Anni Vanhatalo and Andrew M. Jones
Lee J. Wylie, James Kelly, Stephen J. Bailey, Jamie R. Blackwell, Philip F. Skiba, Paul G.
dose-response relationships
Beetroot juice and exercise: pharmacodynamic and
You might find this additional info useful...
41 articles, 13 of which you can access for free at: This article cites
http://jap.physiology.org/content/115/3/325.full#ref-list-1
including high resolution figures, can be found at: Updated information and services
http://jap.physiology.org/content/115/3/325.full
can be found at: Journal of Applied Physiology about Additional material and information
http://www.the-aps.org/publications/jappl
This information is current as of August 5, 2013.
http://www.the-aps.org/.
Copyright © 2013 the American Physiological Society. ESSN: 1522-1601. Visit our website at
year (twice monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991.
physiology, especially those papers emphasizing adaptive and integrative mechanisms. It is published 24 times a
publishes original papers that deal with diverse area of research in appliedJournal of Applied Physiology
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
Beetroot juice and exercise: pharmacodynamic and dose-response
relationships
Lee J. Wylie,
1
James Kelly,
1
Stephen J. Bailey,
1
Jamie R. Blackwell,
1
Philip F. Skiba,
1
Paul G. Winyard,
2
Asker E. Jeukendrup,
3
Anni Vanhatalo,
1
and Andrew M. Jones
1
1
Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, St. Luke’s Campus, Exeter,
United Kingdom;
2
University of Exeter Medical School, St. Luke’s Campus, Exeter, United Kingdom; and
3
Gatorade Sports
Science Institute, Barrington, Illinois
Submitted 25 March 2013; accepted in final form 25 April 2013
Wylie LJ, Kelly J, Bailey SJ, Blackwell JR, Skiba PF,
Winyard PG, Jeukendrup AE, Vanhatalo A, Jones AM. Beet-
root juice and exercise: pharmacodynamic and dose-response re-
lationships. J Appl Physiol 115: 325–336, 2013. First published
May 2, 2013; doi:10.1152/japplphysiol.00372.2013.—Dietary sup-
plementation with beetroot juice (BR), containing approximately 5– 8
mmol inorganic nitrate (NO
3
⫺
), increases plasma nitrite concentration
([NO
2
⫺
]), reduces blood pressure, and may positively influence the
physiological responses to exercise. However, the dose-response re-
lationship between the volume of BR ingested and the physiological
effects invoked has not been investigated. In a balanced crossover
design, 10 healthy men ingested 70, 140, or 280 ml concentrated BR
(containing 4.2, 8.4, and 16.8 mmol NO
3
⫺
, respectively) or no sup-
plement to establish the effects of BR on resting plasma [NO
3
⫺
] and
[NO
2
⫺
] over 24 h. Subsequently, on six separate occasions, 10 subjects
completed moderate-intensity and severe-intensity cycle exercise
tests, 2.5 h postingestion of 70, 140, and 280 ml BR or NO
3
⫺
-depleted
BR as placebo (PL). Following acute BR ingestion, plasma [NO
2
⫺
]
increased in a dose-dependent manner, with the peak changes occur-
ring at approximately 2–3 h. Compared with PL, 70 ml BR did not
alter the physiological responses to exercise. However, 140 and 280
ml BR reduced the steady-state oxygen (O
2
) uptake during moderate-
intensity exercise by 1.7% (P ⫽ 0.06) and 3.0% (P ⬍ 0.05), whereas
time-to-task failure was extended by 14% and 12% (both P ⬍ 0.05),
respectively, compared with PL. The results indicate that whereas
plasma [NO
2
⫺
] and the O
2
cost of moderate-intensity exercise are
altered dose dependently with NO
3
⫺
-rich BR, there is no additional
improvement in exercise tolerance after ingesting BR containing 16.8
compared with 8.4 mmol NO
3
⫺
. These findings have important impli-
cations for the use of BR to enhance cardiovascular health and
exercise performance in young adults.
nitrate; nitrite; nitric oxide; blood pressure; exercise economy; O
2
uptake; exercise tolerance
NITRIC OXIDE (NO) IS A GASEOUS signaling molecule that modu-
lates human physiological function via its role in, for example,
the regulation of blood flow, neurotransmission, immune func-
tion, glucose and calcium homeostasis, muscle contractility,
and mitochondrial respiration (9, 36).
1
NO is generated
through the oxidation of the amino acid
L-arginine in a reaction
catalyzed by NO synthase (NOS), with nitrite (NO
2
⫺
) and
nitrate (NO
3
⫺
) being oxidation products of NO (30). It is now
appreciated that under appropriate physiological conditions,
NO can also be produced via the reduction of NO
2
⫺
, a process
that may be particularly important in situations where oxygen
(O
2
) availability is low, and/or NOS function is impaired (12).
Interestingly, administration of dietary inorganic NO
3
⫺
has
been shown to increase plasma NO
2
⫺
concentration ([NO
2
⫺
])
and to produce NO-like bioactivity (19, 23, 39). Up to 25% of
ingested NO
3
⫺
enters the enterosalivary circulation and is
concentrated in the saliva, whereupon facultative, anaerobic
bacteria in the oral cavity reduce the NO
3
⫺
to NO
2
⫺
(30). When
swallowed into the acidic environment of the stomach, some of
the NO
2
⫺
is converted further into NO, whereas the remainder
is absorbed to increase circulating plasma [NO
2
⫺
]. This NO
2
⫺
may be reduced further to NO and other reactive nitrogen
intermediates, particularly in tissues that may be relatively
hypoxic, such as contracting skeletal muscle (30).
We (3, 37) and others (19, 23, 39) have demonstrated that
NO
3
⫺
ingestion, either in the form of NO
3
⫺
salts or via the
consumption of high NO
3
⫺
vegetable products, such as beetroot
juice (BR), reduces resting blood pressure (BP) profoundly and
consistently. Consequently, dietary NO
3
⫺
supplementation has
emerged as a potential nutritional agent for the prevention and
treatment of hypertension and cardiovascular disease (30).
Webb et al. (39) assessed the effects of acute BR consumption
(⬃23 mmol NO
3
⫺
) on plasma [NO
2
⫺
] and BP over 24 h. Plasma
[NO
2
⫺
] peaked 3 h postingestion, remained close to peak values
until 5 h postingestion, and returned to baseline after 24 h (39).
The systolic and diastolic BP and the mean arterial pressure
(MAP) were reduced significantly, by ⬃10, ⬃8, and ⬃8
mmHg, respectively, at 2.5–3 h after BR intake. The same
research group later reported a dose-dependent increase in
plasma [NO
3
⫺
] and [NO
2
⫺
] and reduction in BP following
ingestion of potassium NO
3
⫺
(KNO
3
) (19). In this study,
plasma [NO
2
⫺
] rose by ⬃1.3-, approximately two-, and approx-
imately fourfold following consumption of 4, 12, and 24 mmol
KNO
3
, respectively. The peak rise in plasma [NO
2
⫺
] was
accompanied by significant reductions in both systolic BP (of
⬃2, ⬃6, and ⬃9 mmHg, respectively) and diastolic BP (of ⬃4,
⬃4, and ⬃6 mmHg, respectively). However, since BR contains
polyphenols and antioxidants, which can facilitate the synthe-
sis of NO from NO
2
⫺
in the stomach (30), it is unclear whether
BP is similarly impacted when different doses of BR are
ingested compared with equivalent doses of NO
3
⫺
salts. Given
the growing interest in dietary NO
3
⫺
supplementation in the
form of BR amongst athletes and the general population, it is
important to determine the pharmacokinetic-pharmacodynamic
relationship between different volumes of BR consumption and
changes in plasma [NO
2
⫺
] and BP to establish an optimal dose
for beneficial effects.
Recent investigations suggest that dietary NO
3
⫺
supplemen-
tation has the potential to influence human physiology beyond
1
This article is the topic of an Invited Editorial by L. Burke (5a).
Address for reprint requests and other correspondence: A. M. Jones, College
of Life and Environmental Sciences, Univ. of Exeter, St. Luke’s Campus,
Exeter EX1 2LU, UK (e-mail: a.m.jones@exeter.ac.uk).
J Appl Physiol 115: 325–336, 2013.
First published May 2, 2013; doi:10.1152/japplphysiol.00372.2013.
8750-7587/13 Copyright
©
2013 the American Physiological Societyhttp://www.jappl.org 325
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
the above hemodynamic effects (3, 26). Specifically, we (2, 3,
22) and others (6, 24 –26) have demonstrated that 3– 6 days of
dietary NO
3
⫺
supplementation reduces the O
2
cost of moderate-
intensity exercise and may enhance exercise tolerance in
healthy, young adults. It appears that these effects are related to
NO
2
⫺
or NO-mediated enhancements of muscle contractile
function (2, 17) and/or mitochondrial efficiency (24) and/or
enhanced muscle blood flow, especially to type II fibers (14).
Importantly, a reduction of the O
2
cost of exercise (25, 37) and
improved exercise performance (21) has also been reported as
early as 2.5 h following a single dose of dietary NO
3
⫺
, which is
consistent with the time required for the peak plasma [NO
2
⫺
]to
be attained (39). However, since all exercise-performance
studies completed to date with BR have administered approx-
imately 5– 8 mmol NO
3
⫺
, it is unclear whether a dose-response
relationship exists between acute NO
3
⫺
intake and the physio
-
logical responses to exercise. The establishment of the dose-
response relationship between NO
3
⫺
intake and the physiolog
-
ical responses to exercise and the ascertainment of the optimal
NO
3
⫺
dose for enhancing exercise performance are important,
given the increasing popularity of BR supplementation in both
basic research and applied exercise settings.
Therefore, the purpose of the present study was twofold:
firstly, to characterize the plasma [NO
3
⫺
] and [NO
2
⫺
] pharma
-
cokinetics and the changes in BP after ingestion of three
different quantities of NO
3
⫺
-rich BR; and secondly, to investi
-
gate the dose-response relationship between BR/NO
3
⫺
intake
and the physiological responses to exercise. In two separate
experiments, we administered a BR concentrate that enabled a
substantial NO
3
⫺
load to be ingested quickly and easily. We
investigated: 1) the influence of acute NO
3
⫺
doses of 4.2, 8.4,
and 16.8 mmol consumed in 70, 140, and 280 ml concentrated
BR on plasma [NO
3
⫺
] and [NO
2
⫺
] and BP over a 24-h period;
and 2) the physiological responses to step transitions to mod-
erate- and severe-intensity exercise, 2.5 h postingestion of the
same NO
3
⫺
doses. We hypothesized that the effects of dietary
inorganic NO
3
⫺
on plasma [NO
3
⫺
] and [NO
2
⫺
], BP, the O
2
cost
of moderate-intensity exercise, and exercise tolerance (as-
sessed as the time-to-task failure) during severe-intensity ex-
ercise would be dose dependent.
METHODS
The study was conducted in two phases [study 1 (S
1
), pharmaco
-
kinetics; and S
2
, dose response], with the results generated in S
1
used
to inform the experimental design in S
2
. There was distinct subject
recruitment for each experiment. Ten healthy, recreationally active
men volunteered for each experiment [mean ⫾ SD: S
1
, age 23 ⫾ 5 yr,
height 1.79 ⫾ 0.07 m, body mass (BM) 79 ⫾ 9 kg; S
2
, age 22 ⫾ 5 yr,
height 1.77 ⫾ 0.05 m, BM 74 ⫾ 8 kg]. None of the subjects in S
1
and
S
2
was a tobacco smoker or user of dietary supplements. All subjects
recruited for S
2
were fully familiar with laboratory exercise-testing
procedures, having participated previously in studies using cycle
ergometry in our laboratory. The procedures used in S
1
and S
2
were
granted full ethics approval by the Institutional Research Ethics
Committee. All subjects gave their written, informed consent to
participate after the experimental procedures, associated risks, and
potential benefits of participation had been explained in detail.
All subjects in S
1
and S
2
were instructed to keep a food and
physical-activity diary in the 24 h preceding their first laboratory visit
and to replicate food consumption and physical activity in the 24 h
preceding subsequent visits. The subjects were required to arrive at
the laboratory in a rested and fully hydrated state, following an
overnight fast, and to avoid strenuous activity in the 24 h preceding
each testing session. Subjects were instructed to refrain from caffeine
and alcohol-containing drinks for 6 and 24 h before each laboratory
visit, respectively, and to abstain from using antibacterial mouthwash
and chewing gum throughout the study, because these are known to
eradicate the oral bacteria that are necessary for the conversion of
NO
3
⫺
to NO
2
⫺
(16).
S
1
: Pharmacokinetics and Pharmacodynamics
Procedures. All subjects reported to the laboratory on four separate
occasions over a period of 3 wk. Upon arrival to the laboratory,
resting BP was measured, and a venous blood sample was obtained for
the measurement of plasma [NO
2
⫺
] and [NO
3
⫺
]. Subjects then con
-
sumed an acute dose of 70, 140, or 280 ml NO
3
⫺
-rich BR (organic BR
containing ⬃4.2, ⬃8.4, or ⬃16.8 mmol NO
3
⫺
, respectively; Beet It;
James White Drinks, Ipswich, UK) or 140 ml water [control (CON)],
in addition to a standardized breakfast (72 g porridge oats with 180 ml
semiskimmed milk). BP was measured, and a venous blood sample
was obtained, 1, 2, 4, 8, 12, and 24 h postingestion. For each 24-h
period of data collection, subjects were provided with a standardized,
low NO
3
⫺
diet. The quantity and timing of food and drink intake were
recorded on visit 1 and replicated in subsequent visits. A washout
period of at least 3 days separated the laboratory visits.
Measurements. The BP of the brachial artery was measured using
an automated sphygmomanometer (Dinamap Pro; GE Medical Sys-
tems, Tampa, FL), with the subjects in a seated position. After arrival
at the laboratory and following 10 min of rest in an isolated room, four
measurements were recorded, and the mean of the final three mea-
surements was used for data analysis.
Venous blood samples were drawn into lithium-heparin tubes (7.5
ml Monovette lithium heparin; Sarstedt, Leicester, UK). Samples
were centrifuged at 4,000 rpm and 4°C for 7 min, within 1 min of
collection. Plasma was subsequently extracted and immediately fro-
zen at ⫺80°C for later analysis of [NO
2
⫺
] and [NO
3
⫺
].
All glassware, utensils, and surfaces were rinsed with deionized
water to remove residual [NO
2
⫺
] and [NO
3
⫺
] before blood analyses.
The [NO
2
⫺
] of the undiluted (nondeproteinized) plasma was deter
-
mined by its reduction to NO in the presence of glacial acetic acid and
4% (w/v) aqueous sodium iodide. The spectral emission of electron-
ically excited nitrogen dioxide product, from the NO reaction with
ozone, was detected by a thermoelectrically cooled, red-sensitive
photomultiplier tube, housed in a Sievers gas-phase chemilumines-
cence NO analyzer (NOA; Sievers NOA 280i; Analytix, Durham,
UK). The [NO
2
⫺
] was determined by plotting signal (mV) area against
a calibration plot of 100 nM–1 M sodium NO
2
⫺
. Before determina
-
tion of [NO
3
⫺
], samples were deproteinized using zinc sulfate
(ZnSO
4
)/sodium hydroxide (NaOH) precipitation. Aqueous ZnSO
4
[400 l 10% (w/v)] and 400 l 0.5 M NaOH were added to 200 lof
sample and vortexed for 30 s before being left to stand at room
temperature for 15 min. Thereafter, samples were centrifuged at 4,000
rpm for 5 min, and the supernatant was removed for subsequent
analysis. The [NO
3
⫺
] of the deproteinized plasma sample was deter
-
mined by its reduction to NO in the presence of 0.8% (w/v) vanadium
trichloride in 1 M HCl. The production of NO was detected using the
chemiluminescence NOA, as described above.
To determine more precisely the time-to-peak plasma [NO
2
⫺
] fol
-
lowing NO
3
⫺
ingestion, a one-compartment model with first-order
absorption and elimination kinetics was used, as described in the
following equation
Y ⫽
共
exp(⫺ Ke ⫻ X ⁄ (Ke ⁄ Ka)
兲
⫺ exp
共
⫺ Ke ⫻ X)
兲
⁄(Ke⁄Ka⫺ 1)
where Y represents fraction absorbed; X represents time; and, Ka and
Ke represent the first-order absorption and elimination rate constants,
respectively.
Statistical analysis. Two-way repeated-measures ANOVA was
used to assess the difference across conditions (4.2, 8.4, and 16.8
326 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
mmol NO
3
⫺
and CON) and across time (0, 1, 2, 4, 8, 12, and 24 h) for
plasma [NO
2
⫺
] and [NO
3
⫺
] and BP. Significant main or interaction
effects were analyzed further using simple contrasts. One-way repeated-
measures ANOVA was used to assess the differences in time-to-peak
plasma [NO
2
⫺
]. Relationships between plasma [NO
2
⫺
] and BP were
analyzed using Pearson product moment correlation coefficients. Sta-
tistical significance was accepted at P ⬍ 0.05. Results are presented as
mean ⫾ SD unless stated otherwise.
S
2
: Dose Response
Protocol. Subjects were required to report to the laboratory on
seven separate occasions, over a 4- to 5-wk period. During the first
visit to the laboratory, subjects completed a ramp incremental exercise
test for determination of peak O
2
uptake (V
˙
O
2 peak
) and gas-exchange
threshold (GET). All tests were performed on an electronically braked
cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands).
Initially, each subject completed 3 min of “unloaded” baseline cy-
cling; then, the work rate was increased by 30 W/min until the subject
was unable to continue. The subjects cycled at a self-selected pedal
rate (70 –90 rpm), and this pedal rate along with the saddle and
handlebar height and configuration were recorded and reproduced in
subsequent tests. The breath-by-breath pulmonary gas-exchange data
were collected continuously during the incremental tests and averaged
over consecutive 10-s periods. V
˙
O
2 peak
was taken as the highest 30-s
mean value attained before the subject’s volitional exhaustion. The
GET was determined as described previously (3, 37). The work rates
that would require 80% of the GET (moderate-intensity exercise) and
75% of the difference between the power output at GET and V
˙
O
2 peak
plus the power output at GET, i.e., severe-intensity exercise (⌬) were
subsequently calculated.
On test days, subjects arrived at the laboratory at ⬃8 AM. A
venous blood sample was drawn for measurement of plasma [NO
2
⫺
]
and NO
3
⫺
. Subjects then ingested 70, 140, or 280 ml NO
3
⫺
-rich BR
(containing 4.2, 8.4, or 16.8 mmol NO
3
⫺
, respectively; Beet It) or 70,
140, or 280 ml NO
3
⫺
-depleted BR as a placebo (PL70, PL140, or
PL280; containing ⬃0.04, ⬃0.08, or ⬃0.12 mmol NO
3
⫺
; Beet It). All
BR and PL doses were administered using a randomized, double-blind
crossover design. Subjects were asked to consume the beverage within
a 5-min period and, after doing so, were served a standardized
breakfast (72 g porridge with 180 ml semiskimmed milk). A washout
period of at least 72 h separated each visit.
After ingestion of the beverage, subjects were given a period of 2.5
h, during which they were allowed to leave the laboratory but were
asked to refrain from strenuous physical activity. Subjects were also
asked to fast during this time, although water was permitted ad
libitum. Following this 2.5-h period, a second venous blood sample
was drawn for measurement of plasma [NO
2
⫺
] and [NO
3
⫺
]. Subjects
then completed “step” exercise tests, from a 20-W baseline to mod-
erate-intensity (93 ⫾ 11 W) and severe-intensity (258 ⫾ 23 W) work
rates for the determination of pulmonary V
˙
O
2
dynamics. On each visit,
subjects completed two, 5-min bouts of moderate-intensity exercise
and one bout of severe-intensity exercise that was continued until task
failure as a measure of exercise tolerance. All bouts of exercise on
each day were separated by 5 min of passive rest. The time-to-task
failure was recorded when the pedal rate fell by ⬎10 rpm below the
self-selected pedal rate. In the severe-intensity bouts, the subjects
were verbally encouraged to continue for as long as possible.
Measurements. During all exercise tests, pulmonary gas exchange
and ventilation were measured breath by breath, with subjects wearing
a nose clip and breathing through a low dead-space (90 ml), low-
resistance (0.75 mmHg1
⫺1
·s
⫺1
at 15 l/s) mouthpiece and impeller
turbine assembly (Jaeger Triple-V; Jaeger GmbH, Hoechberg, Ger-
many). The inspired and expired gas volume and gas concentration
signals were sampled continuously at 100 Hz—the latter using para-
magnetic (O
2
) and infrared [carbon dioxide (CO
2
)] analyzers (Oxycon
Pro; Jaeger GmbH) via a capillary line connected to the mouthpiece.
These analyzers were calibrated before each test with gases of known
concentration, and the turbine volume transducer was calibrated using
a 3-liter syringe (Hans Rudolph, Kansas City, MO). The volume and
concentration signals were time aligned by accounting for the delay in
capillary gas transit and analyzer rise time relative to volume signal.
O
2
uptake, CO
2
output, and minute ventilation were calculated using
a standard formula and displayed breath by breath. Heart rate (HR)
was measured using short-range radiotelemetry (model RS400; Polar
Electro Oy, Kempele, Finland).
Capillary blood samples were collected from the fingertip into a
capillary tube during the baseline, preceding each step transition in
work rate; during the final 30 s of each moderate-intensity exercise
bout; and following exhaustion in the severe-intensity exercise bout.
These samples were analyzed immediately to determine blood lactate
concentration ([lactate]; model YSI 1500; Yellow Springs Instrument,
Yellow Springs, OH). Venous blood samples were treated and ana-
lyzed as described in S
1
.
The breath-by-breath data from each exercise test were linearly
interpolated to provide second-by-second values, and the two identi-
cal, moderate-intensity repetitions performed on each visit were time
aligned to the start of exercise and ensemble averaged. Baseline V
˙
O
2
(V
˙
O
2baseline
), expired CO
2
at baseline (V
˙
CO
2baseline
), and respiratory
exchange ratio (RER) at baseline were defined as the mean values
measured over the final 90 s of baseline pedaling. The end-exercise
V
˙
O
2
,V
˙
CO
2
, and RER were defined as the mean values measured over
the final 30 s of exercise. The amplitude of the V
˙
O
2
response was
calculated by subtracting V
˙
O
2baseline
from V
˙
O
2
at the end of exercise.
Subsequently, the functional gain of the entire response was calcu-
lated by dividing the V
˙
O
2
amplitude by the change (⌬) in work rate.
The amplitude of the V
˙
O
2
slow component during the severe-intensity
exercise bout was estimated by subtracting the mean V
˙
O
2
at 2 min
from the mean V
˙
O
2
at 6 min.
Statistical analysis. Two-way repeated-measures ANOVA was
used to assess the difference in pulmonary gas-exchange variables,
blood [lactate], and HR across dose (70, 140, and 280 ml) and
treatment (PL and BR). Differences in pre- and postplasma [NO
2
⫺
]
and [NO
3
⫺
] were assessed separately in PL and BR, across dose and
time (pre and post) using two-way repeated-measures ANOVAs.
Significant main and interaction effects were analyzed further using
simple contrasts. Statistical significance was accepted at P ⬍ 0.05.
Results are presented as mean ⫾ SD unless stated otherwise.
RESULTS
Ingestion of BR was tolerated well by all subjects in S
1
and
S
2
. Subjects did, however, report beeturia (red urine) and red
stools, consistent with previous studies (3, 39). The absolute
NO
3
⫺
doses used in S
1
and S
2
(4.2, 8.4, and 16.8 mmol) were
equivalent to ⬃0.05 ⫾ 0.01 (range: 0.05– 0.07), ⬃0.11 ⫾ 0.01
(range: 0.09 – 0.13), and ⬃0.22 ⫾ 0.03 mmol (range: 0.19 –
0.26) NO
3
⫺
/kg BM, respectively.
S
1
: Pharmacokinetics and Pharmacodynamics
The effects of different volumes of BR (and therefore,
different amounts of ingested NO
3
⫺
) on plasma [NO
3
⫺
] and
[NO
2
⫺
] are presented in Fig. 1.
There were significant main
effects by dose and time and an interaction effect for both
plasma [NO
3
⫺
] (Fig. 1A; all P ⬍ 0.01) and plasma [NO
2
⫺
] (Fig.
1B; all P ⬍ 0.01).
At resting baseline, before the ingestion of any beverage,
plasma [NO
3
⫺
] was not significantly different between doses
(Fig. 1A; all P ⬎ 0.05). ANOVA analyses revealed significant
dose-dependent increases in plasma [NO
3
⫺
] following BR sup
-
plementation (P ⬍ 0.05). The peak elevation above baseline in
plasma [NO
3
⫺
] occurred 1 h postadministration of 4.2 (160 ⫾
327Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
43 M) and 8.4 mmol NO
3
⫺
(269 ⫾ 92 M) and 2 h postad
-
ministration of 16.8 mmol NO
3
⫺
(581 ⫾ 209 M; Fig. 1A; all
P ⬍ 0.05). Plasma [NO
3
⫺
] remained elevated above baseline
and CON at all time points after administration of 4.2, 8.4, and
16.8 mmol NO
3
⫺
(P ⬍ 0.05).
At baseline, before ingestion of any beverage, plasma
[NO
2
⫺
] was not significantly different between doses (Fig. 1B;
P ⬎ 0.05). ANOVA analyses revealed significant dose-depen-
dent increases in plasma [NO
2
⫺
] following BR supplementation
(P ⬍ 0.05). The peak elevation above baseline in plasma
[NO
2
⫺
] occurred 2 h postadministration of 4.2 (220 ⫾ 104 nM)
and 8.4 mmol NO
3
⫺
(374 ⫾ 173 nM) and 4 h postadministra
-
tion of 16.8 mmol NO
3
⫺
(653 ⫾ 356 nM; Fig. 1B; all P ⬍ 0.05).
Kinetic analyses revealed that plasma [NO
2
⫺
] peaked signifi
-
cantly later (198 ⫾ 64 min; range: 130 –367 min) following
ingestion of 16.8 mmol relative to both 8.4 mmol (146 ⫾ 38
min; range: 77 ⫾ 213 min; P ⬍ 0.05) and 4.2 mmol BR (106 ⫾ 39
min; range: 63–192 min; P ⬍ 0.05). Peak plasma [NO
2
⫺
],
following ingestion of 8.4 mmol, tended to occur later com-
pared with 4.2 mmol (P ⫽ 0.06). Plasma [NO
2
⫺
] remained
elevated above baseline and CON at 1, 2, 4, and 8 h after
administration of 4.2, 8.4, and 16.8 mmol NO
3
⫺
(all P ⬍ 0.05).
At 12 h, plasma [NO
2
⫺
] remained elevated above baseline and
4.2 mmol BR following ingestion of 8.4 and 16.8 mmol NO
3
⫺
(all P ⬍ 0.05). In addition, plasma [NO
2
⫺
] remained elevated at
24 h following administration of 16.8 mmol NO
3
⫺
compared
with all other doses (P ⬍ 0.05).
The effects of different volumes of BR (and therefore,
different amounts of ingested NO
3
⫺
) on systolic and diastolic
BP and MAP are presented in Fig. 2. The changes in systolic
BP across all conditions are presented in Fig. 2A. There were
significant main effects by dose and time and an interaction
effect on systolic BP (all P ⬍ 0.05). Systolic BP at baseline,
before administration of any beverage, was lower (P ⬍ 0.05) in
the 16.8-mmol NO
3
⫺
condition (118 ⫾ 5 mmHg) relative to CON
(121 ⫾ 5 mmHg) but not relative to 4.2 (119 ⫾ 6 mmHg) and 8.4
mmol NO
3
⫺
(120 ⫾ 6 mmHg). Compared with baseline, sys
-
tolic BP was lowered significantly following ingestion of 4.2,
8.4, and 16.8 mmol NO
3
⫺
(all P ⬍ 0.05). The peak reduction in
systolic BP occurred 4 h postadministration of 4.2 (5 ⫾ 5
mmHg), 8.4 (10 ⫾ 5 mmHg), and 16.8 mmol NO
3
⫺
(9 ⫾ 4
mmHg), respectively, relative to baseline (all P ⬍ 0.05).
Systolic BP was reduced relative to baseline, CON, and 4.2
mmol NO
3
⫺
, at 2, 4, and 8 h postadministration of 8.4 mmol
and 16.8 mmol NO
3
⫺
(all P ⬍ 0.05). There were no differences
in systolic BP between 8.4 and 16.8 mmol NO
3
⫺
at any time
point (P ⬎ 0.05). At 24 h, systolic BP remained significantly
lower (by 5 ⫾ 5 mmHg) than baseline, following consumption
of 16.8 mmol NO
3
⫺
(P ⬍ 0.05). In contrast, systolic BP was not
significantly different than CON or baseline at 24 h postad-
100
200
300
400
500
600
700
100
200
300
400
500
600
700
800
900
0
2
4
6
8
10
12
24
0
2
4
6
8
10
12
24
Time (h)
Plasma [NO
2
-
](nM)
Plasma [NO
3
-
](μM)
a b c
*
a b c
*
a b c
a b c
a b c
*
*
*
*
*
*
*
*
*
*
*
*
*
a b
a b
a b
a b
a b
a
a
a
a
a
a b c
*
a b
*
*
a
a b c
*
a b c
*
a b c
*
a b
*
*
*
a b
a b
a b
*
a b
*
a b c
*
a b
*
*
a
a
*
a
*
a
*
*
a
A
B
Fig. 1. Plasma nitrate concentration ([NO
3
⫺
]; A) and
nitrite concentration ([NO
2
⫺
]; B) following consump
-
tion of water (control;
) and 4.2 (Œ), 8.4 (), and
16.8 (}) mmol NO
3
⫺
(group mean ⫾ SE). Plasma
[NO
3
⫺
] and [NO
2
⫺
] rose significantly in a dose-depen
-
dent manner. See text for further details. *Significant
difference from presupplemention baseline (P ⬍
0.05);
a
significant difference from control (P ⬍ 0.05);
b
significant difference from 4.2 mmol NO
3
⫺
(P ⬍
0.05);
c
significant difference from 8.4 mmol NO
3
⫺
(P ⬍ 0.05).
328 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
ministration of 4.2 and 8.4 mmol NO
3
⫺
(P ⬎ 0.05). Overall, the
mean systolic BP across 24 h, relative to CON, was lowered
dose dependently by ⬃3, ⬃4, and ⬃6 mmHg after adminis-
tration of 4.2, 8.4, and 16.8 mmol NO
3
⫺
, respectively (all P ⬍
0.05). The change in systolic BP was correlated with the
change in plasma [NO
3
⫺
](r ⫽⫺0.27; P ⬍ 0.05) and the
change in plasma [NO
2
⫺
](r ⫽⫺0.37; P ⬍ 0.05). The peak
reduction in systolic BP was not correlated with the baseline
systolic BP.
The changes in diastolic BP following the ingestion of
different doses of NO
3
⫺
-rich BR are presented in Fig. 2B. There
was a significant interaction effect (dose ⫻ time) on diastolic
BP (P ⬍ 0.05). Diastolic BP at baseline was not significantly
different among conditions (CON: 67 ⫾ 5; 4.2 mmol: 68 ⫾ 4;
8.4 mmol: 68 ⫾ 6; 16.8 mmol: 67 ⫾ 6 mmHg; P ⬎ 0.05).
Follow-up tests revealed that ingestion of 8.4 and 16.8 but not
4.2 mmol NO
3
⫺
reduced diastolic BP significantly, relative to
baseline and CON (all P ⬍ 0.05). The peak reduction in
diastolic BP from baseline occurred at 4 h postadministration
of 8.4 mmol NO
3
⫺
(3 ⫾ 3 mmHg) and 2 h postadministration
of 16.8 mmol NO
3
⫺
(4 ⫾ 4 mmHg; both P ⬍ 0.05) relative to
baseline (both P ⬎ 0.05) and returned to near-baseline values
by 24 h (P ⬎ 0.05). There were no differences in diastolic BP
between 8.4 and 16.8 mmol NO
3
⫺
at any time point (P ⬎ 0.05).
The change in diastolic BP was correlated with the change in
plasma [NO
3
⫺
](r ⫽⫺0.35; P ⬍ 0.05) and the change in
plasma [NO
2
⫺
](r ⫽⫺0.39; P ⬍ 0.05). Moreover, the peak
change in diastolic BP was correlated with the baseline dia-
stolic BP (r ⫽⫺0.49; P ⬍ 0.05).
The changes in MAP following the ingestion of different
doses of NO
3
⫺
-rich BR are presented in Fig. 2C. There were
significant main effects by dose and time and an interaction
effect on MAP (all P ⬍ 0.05). At baseline, before the ingestion
of any beverage, MAP was not significantly different among
conditions (CON: 85 ⫾ 4; 4.2 mmol: 85 ⫾ 4; 8.4 mmol: 85 ⫾
5; 16.8 mmol: 84 ⫾ 5 mmHg; P ⬎ 0.05). MAP was signifi-
cantly lower following ingestion of 4.2, 8.4, and 16.8 mmol
NO
3
⫺
relative to baseline and CON (all P ⬍ 0.05). Following
ingestion of 4.2 mmol NO
3
⫺
, the peak reduction (2 ⫾ 2 mmHg)
in MAP occurred at 1 h, and MAP remained reduced by ⬃2
mmHg at 2 h relative to baseline (P ⬍ 0.05). In contrast, the
peak reduction in MAP (5 ⫾ 3 mmHg) occurred 4 h postad-
ministration of 8.4 and 16.8 mmol NO
3
⫺
relative to baseline
(P ⬍ 0.05). MAP was not different between 8.4 and 16.8 mmol
NO
3
⫺
at any time point (P ⬎ 0.05). Overall, the mean MAP
across 24 h, relative to CON, was reduced dose dependently by
⬃1, ⬃2, and ⬃4 mmHg after administration of 4.2, 8.4, and
16.8 mmol NO
3
⫺
, respectively (all P ⬍ 0.05). The change in
MAP was correlated significantly with the change in plasma
[NO
3
⫺
](r ⫽⫺0.35; P ⬍ 0.05) and the change in plasma [NO
2
⫺
]
(r ⫽⫺0.41; P ⬍ 0.05).
S
2
: Dose Response
Plasma [NO
3
⫺
] and [NO
2
⫺
]. The group mean plasma [NO
3
⫺
]
and [NO
2
⫺
] responses in the BR and PL conditions are illus
-
trated in Fig. 3, A and B, respectively. Presupplementation
plasma [NO
3
⫺
] was not significantly different between condi
-
tions (P ⬎ 0.05), and no significant change in plasma [NO
3
⫺
]
was observed following PL supplementation (P ⬎ 0.05).
ANOVA analyses revealed a significant dose-dependent in-
crease in plasma [NO
3
⫺
] at 2.5 h following BR supplementation
(P ⬍ 0.05). An elevation in plasma [NO
3
⫺
] above baseline was
apparent following 4.2 (130 ⫾ 17 M; P ⬍ 0.05), 8.4 (282 ⫾
54 M; P ⬍ 0.05), and 16.8 mmol NO
3
⫺
(580 ⫾ 89 M; P ⬍
0.05). Presupplementation plasma [NO
2
⫺
] was not significantly
different among conditions (P ⬎ 0.05), and no significant
change in plasma [NO
2
⫺
] was observed following PL supple
-
mentation (P ⬎ 0.05). ANOVA analyses revealed a significant
dose-dependent increase in plasma [NO
2
⫺
] at 2.5 h following
BR supplementation (P ⬍ 0.05). Following administration of
4.2, 8.4, and 16.8 mmol NO
3
⫺
, plasma [NO
2
⫺
] was elevated
above baseline by 150 ⫾ 73 nM, 291 ⫾ 145 nM, and 425 ⫾
225 nM, respectively (all P ⬍ 0.05). Plasma [NO
2
⫺
] was
significantly greater after ingestion of 16.8 mmol compared
with 4.2 mmol NO
3
⫺
(P ⬍ 0.05) and tended to be greater
compared with 8.4 mmol NO
3
⫺
(P ⫽ 0.06). Plasma [NO
2
⫺
] was
02468101224
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
024681012 24
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
02468101224
-12
-10
-8
-6
-4
-2
0
2
4
B
A
C
Δ MAP (mmHg)
Δ Systolic BP (mmHg)
Δ Diastolic BP (mmHg)
Time (h)
*
*
*
*
*
*
*
*
a b
a b
a b
a
a
*
*
a b
*
*
a
a
a
a
a
*
*
*
*
*
a b
a b
a
a
a
*
*
a
a
*
Fig. 2. Change (⌬) relative to presupplementation baseline in systolic blood
pressure (BP; A), diastolic BP (B), and mean arterial pressure (MAP; C)
following consumption of water (control; ) and 4.2 (Œ), 8.4 (), and 16.8 (})
mmol NO
3
⫺
(group mean ⫾ SE). *Significant difference from presupplemen
-
tion baseline (P ⬍ 0.05);
a
significant difference from control (P ⬍ 0.05);
b
significant difference from 4.2 mmol NO
3
⫺
(P ⬍ 0.05).
329Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
significantly greater following ingestion of 8.4 mmol NO
3
⫺
compared with 4.2 mmol NO
3
⫺
(P ⬍ 0.05).
Moderate-intensity exercise. The pulmonary gas exchange
and ventilatory responses to moderate-intensity exercise across
all doses and conditions are summarized in Table 1. The V
˙
O
2
measured during the period of baseline cycling at 20 W was not
affected by dose or condition (P ⬎ 0.05). However, the
absolute end-exercise V
˙
O
2
, measured over the final 30 s of
moderate-intensity exercise, was altered significantly by BR
ingestion (P ⬍ 0.05; Fig. 4A). Follow-up tests indicated that
end-exercise V
˙
O
2
was lowered significantly by ⬃3% following
administration of 16.8 mmol NO
3
⫺
relative to the respective PL
(PL280: 1.65 ⫾ 0.19 vs. BR280, 1.60 ⫾ 0.23 l/min; P ⬍ 0.05).
In addition, there was a trend toward a significant reduction
(⬃2%) in end-exercise V
˙
O
2
following administration of 8.4
mmol NO
3
⫺
relative to the respective PL (PL140: 1.67 ⫾ 0.21
vs. BR140, 1.64 ⫾ 0.23 l/min; P ⫽ 0.06). The change in
plasma [NO
2
⫺
] from baseline to postingestion of 4.2, 8.4, and
16.8 mmol NO
3
⫺
was correlated with the change in end-
exercise V
˙
O
2
(r ⫽⫺0.47; P ⬍ 0.05). There was no significant
difference in end-exercise V
˙
O
2
following ingestion of 4.6
mmol NO
3
⫺
(BR70) compared with PL70 (P ⬎ 0.05).
The amplitude of the V
˙
O
2
response (end-exercise ⫺
V
˙
O
2baseline
; Table 1) was affected by dose (P ⬍ 0.05) and
tended to be affected by condition (P ⫽ 0.07). Follow-up tests
revealed that there was a trend toward a significant reduction in
the V
˙
O
2
amplitude (by ⬃6%) after administration of 16.8 mmol
NO
3
⫺
compared with 8.4 mmol NO
3
⫺
(BR140: 0.70 ⫾ 0.16 vs.
BR280: 0.66 ⫾ 0.16 l/min; P ⫽ 0.06). The change in plasma
[NO
2
⫺
] from baseline to postingestion of 4.2, 8.4, and 16.8
mmol NO
3
⫺
was correlated with the change in V
˙
O
2
amplitude
(r ⫽⫺0.38; P ⬍ 0.05). There was no significant difference in
V
˙
O
2
amplitude between PL and BR at any dose (P ⬎ 0.05).
The V
˙
CO
2baseline
, measured over the last 90 s of 20 W
pedaling, and the end-exercise V
˙
CO
2
, measured over the last 30
s of exercise, were affected by dose (P ⬍ 0.05 for both) but not
condition (P ⬎ 0.05 for both; Table 1). Follow-up tests
revealed that V
˙
CO
2baseline
was increased significantly, as the
volume of supplement ingested increased (P ⬍ 0.05), irrespec-
tive of the condition (i.e., PL or BR). Specifically, V
˙
CO
2baseline
was increased by ⬃7% and ⬃5% following consumption of
280 ml of supplement relative to 70 and 140 ml, respectively
(P ⬍ 0.05 for both). There were no significant differences in
V
˙
CO
2
between the ingestion of 70 and 140 ml of supplement
(P ⬎ 0.05). Furthermore, post hoc analysis revealed that the
end-exercise V
˙
CO
2
was significantly higher following inges
-
tion of both 140 and 280 ml of supplement relative to 70 ml
(P ⬍ 0.01 for both). There was, however, no significant
difference in end-exercise V
˙
CO
2
between ingestion of 140
and 280 ml of supplement (P ⬎ 0.05).
Baseline and end-exercise RER were affected by dose
(P ⬍ 0.05 for both) but not condition (P ⬎ 0.05). The
follow-up tests indicated that RER increased as the volume
of supplement ingested increased (P ⬍ 0.05; Table 1).
Specifically, RER at baseline was increased by ⬃5% and
⬃4%, following consumption of 280 ml of supplement
relative to 70 and 140 ml, respectively (P ⬍ 0.05 for both).
Although there was no significant interaction effect or main
effect by condition, baseline RER tended to be higher (by
⬃3%) following administration of 16.8 mmol NO
3
⫺
com
-
pared with the respective PL (P ⫽ 0.08). End-exercise RER
was increased significantly by ⬃4% and ⬃3%, following
consumption of 280 ml compared with 70 and 140 ml of
supplement, respectively (P ⬍ 0.05 for both). In addition,
the ingestion of 140 ml increased end-exercise RER com-
pared with ingestion of 70 ml of supplement (P ⬍ 0.05). The
baseline, end-exercise, and change in blood [lactate] and HR
were not altered significantly by dose or condition (Table 2;
P ⬎ 0.05).
Severe-intensity exercise. The pulmonary gas exchange and
ventilatory responses to severe-intensity exercise across all
doses and conditions are summarized in Table 1. In contrast to
the effects observed for moderate-intensity exercise, the V
˙
O
2
and V
˙
CO
2
measured at baseline and at task failure were not
altered by dose or treatment (all P ⬎ 0.05). Moreover, neither
the dose nor the treatment altered the V
˙
O
2
slow component
amplitude (P ⬎ 0.05 for both). There was a trend toward
significant main effects by dose (P ⫽ 0.09) and treatment (P ⫽
0.08) but no interaction effect on RER at baseline (P ⬎ 0.05).
Follow-up tests revealed that there was a trend toward signif-
icant increases in RER at baseline by ⬃4% and ⬃3% following
consumption of 280 ml of supplement compared with the
consumption of 70 (P ⫽ 0.06) or 140 ml (P ⫽ 0.08) of
supplement, respectively. RER, at task failure, was not altered
by dose or treatment (P ⬎ 0.05). The baseline, end-exercise,
and change in blood [lactate] and HR were not altered signif-
icantly by dose or condition (Table 2; P ⬎ 0.05).
There was a significant main effect by condition (P ⬍ 0.05)
but not dose (P ⬎ 0.05) on time-to-task failure (Table 1 and
200
400
600
800
200
400
600
800
70 ml
140 ml
280 ml
70 ml
140 ml
280 ml
Nitrate
Placebo
70 ml
140 ml
280 ml
70 ml
140 ml
280 ml
Placebo
Nitrate
Plasma [NO
3
-
](μM)
Plasma [NO
2
-
](nM)
B
A
*
a b
*
a
*
*
*
*
a
a
Fig. 3. Mean ⫾ SE plasma [NO
3
⫺
](A) and [NO
2
⫺
](B) preingestion (black bars)
and 2.5-h postingestion (gray bars) of 70, 140, and 280 ml NO
3
⫺
-rich beetroot
juice (BR) (NO
3
⫺
)orNO
3
⫺
-depleted BR [placebo (PL)]. See text for further
details. *Significant difference from baseline (P ⬍ 0.05);
a
significant differ
-
ence postconsumption of 70 ml NO
3
⫺
-rich BR (P ⬍ 0.05);
b
significant
difference from postconsumption of 140 ml NO
3
⫺
-rich BR (P ⬍ 0.05).
330 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
Fig. 4B). Follow-up tests revealed that consumption of 8.4
mmol NO
3
⫺
(BR140) and 16.8 mmol NO
3
⫺
(BR280) resulted in
a significant increase in time-to-task failure by 71 ⫾ 77 s and
59 ⫾ 61 s, respectively, relative to PL140 and PL280 (P ⬍
0.05; Fig. 4B). There was no difference in time-to-task failure
between BR70 and PL70 (P ⬎ 0.05). The change in plasma
[NO
2
⫺
] from baseline to postingestion of 4.2, 8.4, and 16.8
mmol NO
3
⫺
was correlated significantly with the change in
time-to-task failure (r ⫽ 0.55; P ⬍ 0.05). There was no
significant difference in time-to-task failure among 4.2, 8.4,
and 16.8 mmol BR (all P ⬎ 0.05) or among PL70, PL140, and
PL280 (P ⬎ 0.05).
In terms of positive changes in time-to-task failure, there
were three “nonresponders” in the 4.2-mmol condition, two in
the 8.4-mmol condition, and one in the 16.8-mmol condition.
Individual subjects who did not respond at lower doses did
respond at higher doses. The increase in plasma [NO
2
⫺
] from
baseline to pre-exercise for the nonresponders was similar to
the other subjects who did respond. For example, the three
nonresponders at the lowest NO
3
⫺
dose had an increase in
plasma [NO
2
⫺
] of 140, 208, and 161 nM compared with a group
mean increase of 150 nM. In addition, the nonresponders did
not have high baseline values of plasma [NO
2
⫺
] (70 –121 nM)
compared with the group mean.
DISCUSSION
This study is the first to characterize the pharmacokinetic-
pharmacodynamic effects of NO
3
⫺
-rich BR ingestion and to
investigate the dose-response relationship between BR inges-
tion and the physiological responses to exercise. Specifically,
we studied how acute ingestion of three different BR volumes
(and thus three different NO
3
⫺
doses) impacted on plasma
[NO
3
⫺
] and [NO
2
⫺
], resting BP, the pulmonary gas-exchange
responses to moderate- and severe-intensity exercise, and ex-
ercise tolerance. Our principal findings were that plasma
[NO
3
⫺
] and [NO
2
⫺
] increased dose dependently up to 16.8
mmol NO
3
⫺
with there being a dose-dependent peak reduction
in BP up to 8.4 mmol NO
3
⫺
.ANO
3
⫺
dose of 16.8 mmol was
required to elicit a significant reduction in the O
2
cost of
moderate-intensity cycle exercise, although there was a
trend (P ⫽ 0.06) for a reduction with 8.4 mmol. A signifi-
cant improvement in time-to-task failure during severe-
intensity exercise was evident after ingestion of 8.4 mmol
NO
3
⫺
, with no further benefits observed following the inges
-
tion of 16.8 mmol NO
3
⫺
.
S
1
: BR Pharmacokinetics and Pharmacodynamics—Effects
on Plasma [NO
3
⫺
], [NO
2
⫺
], and BP
The results of S
1
demonstrated that concentrated BR con
-
sumption causes dose-dependent increases in plasma [NO
3
⫺
]
and [NO
2
⫺
]. Plasma [NO
3
⫺
] increased by approximately five-
and eightfold, 1 h after the ingestion of 4.2 and 8.4 mmol NO
3
⫺
,
and by ⬃18-fold, 2 h after the ingestion of 16.8 mmol NO
3
⫺
.In
contrast, the increase in plasma [NO
2
⫺
] occurred later, peaking
at approximately 2–2.5 h postadministration of 4.2 and 8.4
mmol NO
3
⫺
and ⬃3 h postadministration of 16.8 mmol NO
3
⫺
.
Table 1. Pulmonary gas-exchange variables during moderate- and severe-intensity exercise following supplementation with
3 different volumes of beetroot juice and placebo
70 ml 140 ml 280 ml
Placebo Nitrate, 4.2 mmol Placebo Nitrate, 8.4 mmol Placebo Nitrate, 16.8 mmol
Moderate-intensity exercise
V
˙
O
2
Baseline, l/min 0.94 ⫾ 0.10 0.93 ⫾ 0.09 0.92 ⫾ 0.12 0.94 ⫾ 0.13 0.95 ⫾ 0.12 0.94 ⫾ 0.08
End-exercise, l/min 1.64 ⫾ 0.21 1.61 ⫾ 0.21 1.67 ⫾ 0.21
a
1.64 ⫾ 0.23 1.65 ⫾ 0.19 1.60 ⫾ 0.18
b
Primary amplitude, l/min 0.70 ⫾ 0.16 0.68 ⫾ 0.16 0.74 ⫾ 0.16 0.70 ⫾ 0.16 0.70 ⫾ 0.14 0.66 ⫾ 0.16
Primary gain, ml · min
⫺1
·W
⫺1
9.5 ⫾ 1.0 9.2 ⫾ 1.1 10.1 ⫾ 0.9 9.5 ⫾ 0.9 9.6 ⫾ 0.6 9.0 ⫾ 1.1
c
V
˙
CO
2
Baseline, l/min 0.82 ⫾ 0.07 0.81 ⫾ 0.05 0.82 ⫾ 0.09 0.83 ⫾ 0.12 0.86 ⫾ 0.07
a
0.89 ⫾ 0.07
a,d
End-exercise, l/min 1.48 ⫾ 0.17 1.45 ⫾ 0.17 1.51 ⫾ 0.17 1.50 ⫾ 0.17 1.52 ⫾ 0.14
a
1.52 ⫾ 0.17
d
V
˙
E
Baseline, l/min 23 ⫾ 322⫾ 223⫾ 323⫾ 424⫾ 323⫾ 2
End-exercise, l/min 37 ⫾ 536⫾ 537⫾ 537⫾ 538⫾ 537⫾ 4
RER
Baseline 0.88 ⫾ 0.05 0.88 ⫾ 0.04 0.89 ⫾ 0.04 0.89 ⫾ 0.04 0.91 ⫾ 0.05 0.94 ⫾ 0.04
c,d
End-exercise 0.91 ⫾ 0.04 0.90 ⫾ 0.04 0.91 ⫾ 0.03 0.92 ⫾ 0.05 0.93 ⫾ 0.04 0.95 ⫾ 0.04
c,d
Severe-intensity exercise
V
˙
O
2
Baseline, l/min 1.00 ⫾ 0.10 0.99 ⫾ 0.11 0.99 ⫾ 0.13 0.99 ⫾ 0.11 0.99 ⫾ 0.11 0.97 ⫾ 0.11
End-exercise, l/min 3.89 ⫾ 0.40 3.97 ⫾ 0.34 3.96 ⫾ 0.38 3.99 ⫾ 0.40 3.98 ⫾ 0.35 3.94 ⫾ 0.28
Overall gain, ml · min
⫺1
·W
⫺1
12.1 ⫾ 0.8 12.5 ⫾ 1.0 12.5 ⫾ 0.9 12.6 ⫾ 1.2 12.6 ⫾ 0.9 12.5 ⫾ 0.8
Slow-phase amplitude, 6–2 min; l/min 0.66 ⫾ 0.14 0.65 ⫾ 0.15 0.67 ⫾ 0.17 0.62 ⫾ 0.17 0.75 ⫾ 0.09 0.69 ⫾ 0.11
V
˙
CO
2
Baseline, l/min 0.91 ⫾ 0.06 0.90 ⫾ 0.08 0.89 ⫾ 0.08 0.91 ⫾ 0.09 0.92 ⫾ 0.08 0.93 ⫾ 0.11
End-exercise, l/min 4.16 ⫾ 0.36 4.17 ⫾ 0.27 4.21 ⫾ 0.38 4.18 ⫾ 0.32 4.20 ⫾ 0.25 4.20 ⫾ 0.31
RER
Baseline 0.91 ⫾ 0.05 0.91 ⫾ 0.06 0.91 ⫾ 0.06 0.92 ⫾ 0.06 0.94 ⫾ 0.05 0.96 ⫾ 0.05
End-exercise 1.07 ⫾ 0.06 1.05 ⫾ 0.05 1.06 ⫾ 0.05 1.05 ⫾ 0.05 1.06 ⫾ 0.05 1.07 ⫾ 0.05
Time-to-task failure(s) 470 ⫾ 81 508 ⫾ 102 498 ⫾ 113 570 ⫾ 153
e
493 ⫾ 114 552 ⫾ 117
b
Values are means ⫾ SD.V
˙
O
2
, oxygen uptake; V
˙
CO
2
, expired carbon dioxide; V
˙
E
, ventilation; RER, respiratory exchange ratio.
a
Significantly different from
placebo (PL)70 (P ⬍ 0.05);
b
significantly different from PL280 (P ⬍ 0.05);
c
Significantly different from beetroot juice (BR)140 (P ⬍ 0.05);
d
significantly
different from BR70 (P ⬍ 0.05);
e
significantly different from PL140 (P ⬍ 0.05).
331Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
As expected, the rise in plasma [NO
2
⫺
] was smaller compared
with plasma [NO
3
⫺
], with peak increases of ⬃2.5-fold, approx
-
imately fourfold, and approximately eightfold, respectively.
The delayed peak increases in plasma [NO
2
⫺
] compared with
plasma [NO
3
⫺
] reflect the importance of the enterosalivary
circulation and subsequent reduction of NO
3
⫺
to NO
2
⫺
by
lingual bacteria (16, 39). These pharmacokinetic responses to
BR supplementation are consistent with those reported previ-
ously following acute ingestion of KNO
3
(19). Together, these
data suggest that the pharmacokinetics of plasma [NO
3
⫺
] and
[NO
2
⫺
] are dose dependent when NO
3
⫺
is administered, either
as NO
3
⫺
salt or in the form of a natural vegetable supplement.
Ingestion of concentrated BR dose dependently lowered
systolic BP and MAP up to an intake of 8.4 mmol NO
3
⫺
. More
specifically, acute ingestion of 4.2, 8.4, and 16.8 mmol inor-
ganic NO
3
⫺
, administered in the form of BR, resulted in peak
reductions of systolic BP of ⬃5, ⬃10, and ⬃9 mmHg and peak
reductions of MAP of ⬃2, ⬃5, and ⬃5 mmHg, respectively.
Moreover, BR ingestion resulted in a similar “threshold” effect
on diastolic BP, with peak reductions of ⬃3 and ⬃4 mmHg
following administration of 8.4 and 16.8 mmol NO
3
⫺
; however,
ingestion of 4.2 mmol NO
3
⫺
did not reduce diastolic BP
significantly. These reductions in BP are similar to those
reported by Kapil et al. (19) following acute administration of
KNO
3
, except that Kapil et al. (19) reported a dose-dependent
reduction in BP up to 24 mmol KNO
3
. The reason for this
discrepancy between studies is unclear. Interestingly, com-
pared with Kapil et al. (19), who reported 6 mmHg and 9
mmHg reductions in systolic BP following the consumption of
12 mmol and 24 mmol KNO
3
, respectively, we observed larger
reductions in BP following the consumption of BR (e.g., a peak
reduction of 10 mmHg in systolic BP with 8.4 mmol NO
3
⫺
contained in 140 ml BR). It is possible that this apparent
greater potency of BR compared with NO
3
⫺
salt in reducing BP
is related to the polyphenols and other antioxidants present in
BR, which may facilitate a more efficient conversion of NO
3
⫺
to NO
2
⫺
(30). Interestingly, although the peak reduction in BP
was not significantly different between 8.4 and 16.8 mmol
NO
3
⫺
, the mean reduction in BP over 24 h was dose dependent,
with MAP, for example, reduced by 1, 2, and 4 mmHg
following administration of 4.2, 8.4, and 16.8 mmol NO
3
⫺
,
respectively.
The results of the present study suggest that BR (and
presumably other NO
3
⫺
-rich vegetable) consumption can pro
-
vide a natural approach to maintaining or improving BP and
Time to Task Failure (s)
0
450
500
550
600
650
End-exercise o
2
(L.min
-
1
)
1.75
1.70
1.65
1.60
1.55
0.00
PL
BR
70 ml
PL
BR
140 ml
PL
BR
280 ml
*
B
A
.
*
*
Fig. 4. Mean ⫾ SE steady-state oxygen consumption (V
˙
O
2
) during moderate-
intensity exercise (A) and time-to-task failure during severe-intensity exercise
(B), following consumption of 70, 140, and 280 ml NO
3
⫺
-rich BR (gray bars)
or NO
3
⫺
-depleted BR (PL; black bars). End-exercise V
˙
O
2
during moderate-
intensity exercise was reduced significantly following the ingestion of 280 ml
BR. Time-to-task failure during severe-intensity exercise was extended after
consumption of 140 ml BR with no further increase following 280 ml BR.
*Significant difference from PL (P ⬍ 0.05).
Table 2. Heart rate and blood lactate responses to moderate- and severe-intensity exercise following supplementation with 3
different volumes of beetroot juice and placebo
70 ml 140 ml 280 ml
Placebo Nitrate, 4.2 mmol Placebo Nitrate, 8.4 mmol Placebo Nitrate, 16.8 mmol
Moderate-intensity exercise
Heart rate, beats/min
Baseline 89 ⫾ 989⫾ 888⫾ 888⫾ 889⫾ 889⫾ 6
End-exercise 116 ⫾ 11 116 ⫾ 12 115 ⫾ 10 116 ⫾ 8 115 ⫾ 9 115 ⫾ 10
Blood [lactate], mM
Baseline 1.1 ⫾ 0.3 1.1 ⫾ 0.5 1.1 ⫾ 0.4 1.0 ⫾ 0.4 1.0 ⫾ 0.3 1.1 ⫾ 0.3
End-exercise 1.2 ⫾ 0.2 1.2 ⫾ 0.5 1.2 ⫾ 0.5 1.1 ⫾ 0.4 1.1 ⫾ 0.4 1.2 ⫾ 0.5
⌬ 0.1 ⫾ 0.2 0.1 ⫾ 0.4 0.1 ⫾ 0.3 0.1 ⫾ 0.2 0.1 ⫾ 0.3 0.1 ⫾ 0.2
Severe-intensity exercise
Heart rate, beats/min
Baseline 99 ⫾ 9 100 ⫾ 899⫾ 9 100 ⫾ 10 100 ⫾ 10 99 ⫾ 8
End-exercise 186 ⫾ 11 186 ⫾ 12 185 ⫾ 12 187 ⫾ 10 186 ⫾ 11 185 ⫾ 10
Blood [lactate], mM
Baseline 0.9 ⫾ 0.4 0.9 ⫾ 0.4 0.9 ⫾ 0.4 0.9 ⫾ 0.6 1.0 ⫾ 0.3 0.9 ⫾ 0.2
Task failure 9.7 ⫾ 1.4 9.4 ⫾ 1.6 9.4 ⫾ 1.6 9.6 ⫾ 1.8 9.5 ⫾ 1.5 9.5 ⫾ 1.1
⌬ 8.7 ⫾ 1.2 8.5 ⫾ 1.7 8.5 ⫾ 1.7 8.7 ⫾ 1.3 8.5 ⫾ 1.4 8.6 ⫾ 1.2
Values are means ⫾
SD. [lactate], lactate concentration; ⌬, change.
332 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
vascular health in young adults. The reductions in BP, evident
in the present study, are noteworthy. For example, it has been
suggested that lowering systolic BP by 10 mmHg may reduce
the risk of ischemic heart disease by ⬃25% and the risk of
stroke by ⬃35% (27–29, 31). The beneficial hemodynamic
effects of NO
3
⫺
supplementation are thought to be due to the
reduction of NO
3
⫺
to NO
2
⫺
and then to NO within the blood
vessel (13), resulting in arterial dilatation and a reduced pe-
ripheral resistance (39). However, it is possible that NO
2
⫺
itself
may also exert a direct effect on the vascular system, indepen-
dent of NO formation (1). There are several advantages to
using inorganic rather than organic NO
3
⫺
for the prevention or
treatment of hypertension (33). These include a slow and
controlled increase in plasma [NO
2
⫺
] following inorganic NO
3
⫺
intake (due to NO
3
⫺
uptake into the enterosalivary circulation)
compared with the more abrupt changes in plasma [NO
2
⫺
]
(perhaps to toxic levels) and BP, which can occur with
organic NO
3
⫺
administration (33). Moreover, unlike the
chronic administration of organic NO
3
⫺
, inorganic NO
3
⫺
does
not appear to lead to the development of tolerance (37) and
endothelial dysfunction (33).
S
2
: Dose Response
The results of S
2
confirm that concentrated BR consumption
causes a dose-dependent increase in plasma [NO
3
⫺
] by 334%,
778%, and 1,556% and plasma [NO
2
⫺
] by 121%, 218%, and
338%, 2.5 h postingestion of 4.2, 8.4, and 16.8 mmol NO
3
⫺
,
respectively. The magnitude of the increase in plasma [NO
2
⫺
]
following consumption of 8.4 and 16.8 mmol NO
3
⫺
in the
present study was much larger than the approximate 15–150%
rise in plasma [NO
2
⫺
], reported previously, following acute
(approximately 4 – 6 mmol) (5, 21, 25, 37) and chronic (ap-
proximately 5– 6 mmol/day) (2, 3, 22, 26, 37) dietary NO
3
⫺
supplementation. This finding is likely a consequence of the
relatively higher NO
3
⫺
doses (8.4 and 16.8 mmol NO
3
⫺
) admin
-
istered in the present study. Interestingly, the group mean
plasma [NO
3
⫺
] and [NO
2
⫺
] reported in S
2
are somewhat lower
than those reported at 2– 4 h postingestion of BR in S
1
. Given
that there was distinct subject recruitment for S
1
and S
2
,itis
likely that this discrepancy is due to individual variations in the
pharmacokinetic response to BR consumption. For example,
when the individual plasma [NO
2
⫺
] responses to the ingestion
of 16.8 mmol NO
3
⫺
in S
1
are considered, peak concentrations
ranged from 493 to 1,523 nM, and the time-to-peak concen-
tration ranged from 130 to 367 min. The cause of this wide
interindividual variability in the response of plasma [NO
2
⫺
]to
NO
3
⫺
ingestion is unclear, although it may depend, in part, on
salivary flow rate; also, it is known that the reduction of NO
3
⫺
to NO
2
⫺
is highly dependent on the activity of oral bacteria (16,
39). Another consideration is that the absolute NO
3
⫺
doses
administered in the present study (4.2, 8.4, and 16.8 mmol in 1,
2, and 4 BR shots, respectively) resulted in somewhat different
NO
3
⫺
doses when expressed relative to BM (0.05– 0.07, 0.09–
0.13, and 0.19 –0.25 mmol NO
3
⫺
/kg BM, respectively).
Dose Response: Moderate-Intensity Exercise
This is the first study to assess the acute dose-dependent
physiological responses to exercise following dietary NO
3
⫺
supplementation in humans. We assessed the acute response to
three different doses of BR at 2.5 h postingestion, based on the
significant dose-dependent elevation in plasma [NO
2
⫺
]ob
-
served at 2–3 h postingestion in S
1
(Fig. 1B). The steady-state
V
˙
O
2
measured over the final 30 s of moderate-intensity cycle
exercise was unaffected by 4.2 mmol NO
3
⫺
, tended to be lower
(⬃30 ml/min) following administration of 8.4 mmol NO
3
⫺
, and
was reduced significantly (by ⬃50 ml/min) following admin-
istration of 16.8 mmol NO
3
⫺
.
The reduction in steady-state V
˙
O
2
(⬃3%), observed follow
-
ing acute ingestion of 16.8 mmol NO
3
⫺
(⬃0.23 mmol/kg BM),
is similar to that reported 2.5 h postingestion of 5.2 mmol NO
3
⫺
(⬃0.07 mmol/kg BM) in the form of nonconcentrated BR (37)
but is smaller than the 6% reduction reported 1 h postingestion
of 0.033 mmol/kg BM sodium nitrate (25). In contrast to acute
ingestion, longer-term BR supplementation (3– 6 days at ap-
proximately 5–7 mmol NO
3
⫺
/day) resulted in an approximate
5–7% reduction in steady-state V
˙
O
2
during moderate-intensity
cycling (3, 26) and running (22).
Previous studies have indicated that the lowering of sub-
maximal exercise V
˙
O
2
, following dietary NO
3
⫺
supplementa
-
tion, may result from improved mitochondrial efficiency (25)
and/or a reduction in the ATP cost of muscle force production
(4). Alterations in protein expression have been proposed as
the mechanistic basis for these effects (17, 24); however, it is
unlikely that these alterations occur quickly enough to explain
the effects observed so soon (1–2.5 h) after NO
3
⫺
ingestion (25,
37). Alternatively, NO may acutely and reversibly impact
protein function through post-translational protein modifica-
tions. For instance, S-nitrosation of adenine nucleotide trans-
locase or other mitochondrial or calcium-handling proteins
(35) may contribute to the acute reduction in O
2
cost of
exercise following BR ingestion. The mechanistic basis for the
acute changes in the O
2
cost of exercise following BR inges
-
tion warrants further investigation.
An interesting observation was the dose-dependent increase
in baseline and end-exercise V
˙
CO
2
, irrespective of condition
(i.e., PL or BR). This small but significant rise in V
˙
CO
2
led to
a dose-dependent increase in RER that was more pronounced
during baseline cycling compared with the exercising steady-
state. An elevation in RER is indicative of a shift in substrate
use toward a relatively greater reliance on carbohydrate and is
likely due to the sugar content of the concentrated BR and PL
beverages (⬃16 g/70 ml).
Dose Response: Severe-Intensity Exercise
A novel finding of the present study was that 8.4 and 16.8
mmol NO
3
⫺
, but not 4.2 mmol NO
3
⫺
, administered acutely in
the form of concentrated BR, significantly improved the time-
to-task failure by 14% and 12%, respectively, during severe-
intensity exercise. These findings are similar to the 14 –16%
improvement in exercise tolerance reported previously follow-
ing 5– 6 days of BR supplementation at a lower dose (5– 6
mmol NO
3
⫺
) (3, 22). Although the mechanism(s) responsible
for the ergogenic potential of NO
3
⫺
supplementation remain
uncertain, they are believed to be mediated via a biochem-
ical reduction of ingested NO
3
⫺
to biologically active NO
2
⫺
and NO (4).
NO has been linked to the efficiency of aerobic respiration
(9) and the regulation of muscle contraction (35). Indeed, both
more efficient mitochondrial oxidative phosphorylation, via a
reduced proton leak across the inner mitochondrial membrane
333Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
(24) and a reduced ATP and phosphocreatine cost of muscle
force production (2, 15), has been reported following dietary
NO
3
⫺
supplementation. In addition, recent evidence suggests
that BR supplementation results in a marked increase in muscle
blood flow during exercise in rats, with the blood flow prefer-
entially distributed to muscle groups that principally contain
type II fibers, which are recruited during severe-intensity ex-
ercise (14). Furthermore, NO
3
⫺
supplementation has been
shown to increase muscle force production in mice via modu-
lation of intracellular calcium ion (Ca
2⫹
) handling in fast-
twitch fibers (17). It is possible that these mechanisms operate
simultaneously and/or synergistically, resulting in enhanced
exercise tolerance. It is, however, important to note that the
studies that demonstrated effects of NO
3
⫺
supplementation on
muscle metabolic and vascular control mechanisms (2, 14, 17,
24) used chronic, rather than acute, NO
3
⫺
supplementation
protocols. On the other hand, Cosby et al. (10) reported acutely
increased blood flow to exercising forearm muscle following
infusion of NO
2
⫺
into the brachial artery. It is possible that the
improved time-to-task failure that we observed with 8.4 and
16.8 mmol NO
3
⫺
was related to improved blood flow to muscle
or to a NO-mediated enhancement of local matching of O
2
delivery to metabolic rate. This would be consistent with
reports that BR supplementation results in a preferential dis-
tribution of blood flow to type II fibers (14) and improves
oxidative function in hypoxic muscle (38). The lack of a
further improvement in time-to-task failure with 16.8 mmol
compared to 8.4 mmol NO
3
⫺
mirrors the lack of an additional
effect of consuming the higher NO
3
⫺
dose on the peak
reduction in BP that we observed in S
1
, suggesting that the
acute effects of BR ingestion on exercise tolerance may be
related, at least in part, to effects on the vasculature. Further
studies are needed to establish which mechanisms may be
responsible for the ergogenic potential of NO
3
⫺
, at least at
high doses, as early as 2.5 h after ingestion of BR.
The results of the present study indicate a dose-dependent
effect of BR supplementation on exercise tolerance up to 8.4
mmol, with no further benefit (indeed a small reduction in
exercise tolerance compared with 8.4 mmol) following inges-
tion of 16.8 mmol NO
3
⫺
. A possible explanation for this
threshold might be a NO-dependent reduction in skeletal mus-
cle force via modulation of excitation-contraction coupling. It
has been reported that the opening of the Ca
2⫹
release channels
of the sarcoplasmic reticulum (SR) is inhibited by NO (32, 35)
and highly related to NO availability (35). In addition, Ca
2⫹
transport (35), SR Ca
2⫹
-ATPase activity (18), and cytochrome
c-oxidase inhibition (9) may be influenced by NO and contrib-
ute to a dose-dependent modulation of excitation-contraction
coupling. Therefore, whereas an increase in NO bioavailability
may result in a more efficient mitochondrial function (24) and
changes to type II fiber contractility (17) and blood flow (14),
it is possible that these positive effects may be offset by
impairments of mitochondrial or contractile function at higher
NO levels that might promote nitrative stress. These sugges-
tions are naturally speculative and await further investigation.
The improvements in time-to-task failure during severe-
intensity exercise, following ingestion of 8.4 and 16.8 mmol
NO
3
⫺
in the present study, were evident without any significant
changes in the V
˙
O
2
response to exercise. Neither the amplitude
of the V
˙
O
2
slow component nor the end-exercise V
˙
O
2
was
influenced by acute ingestion of up to 16.8 mmol NO
3
⫺
. This
finding is consistent with some (20) but not all previous reports
(3, 22). For example, Bailey et al. (3) reported that 3 days of
BR supplementation reduced the V
˙
O
2
slow-component ampli
-
tude by 23% and improved exercise tolerance by ⬃16%. In
contrast, Kelly et al. (20) reported that 3 days of BR supple-
mentation improved exercise tolerance at three different severe
intensities by 12–17%, without any accompanying changes in
the V
˙
O
2
response. We found no difference in end-exercise V
˙
O
2
between BR and PL at any dose. In the severe exercise-
intensity domain, the V
˙
O
2
at the point of volitional exhaustion
would be expected to equal the maximum V
˙
O
2
(V
˙
O
2 max
) (11).
Our results are therefore consistent with some (3, 37) but not
all (5, 25) previous studies that indicate that NO
3
⫺
supplemen
-
tation does not reduce V
˙
O
2 max
. Interestingly, there was a
disconnect between the effects of BR on steady-state V
˙
O
2
during moderate-intensity exercise (where the greatest reduc-
tion occurred at the highest dose of NO
3
⫺
) and the effects of BR
on exercise tolerance (where the increased time-to-task failure
was similar with 8.4 and 16.8 mmol NO
3
⫺
). Collectively, these
results appear to indicate that the effects of BR on severe-
intensity exercise performance may be independent from the
effects of BR on the O
2
cost of submaximal exercise.
It should be noted that while an approximate 12–14%
extension of time-to-task failure during severe-intensity, con-
stant work-rate exercise, following acute BR ingestion, may
appear impressive, this is likely to translate into no more than
a 1–2% reduction in the time to complete a given distance, for
example, during a short endurance time-trial (TT) event (34).
This is similar to the magnitude of improvement in perfor-
mance reported previously for 4 km and 16.1 km TT after acute
BR ingestion (21) and for 10 km TT following 6 days of BR
supplementation (6). A 1% improvement in performance is
highly meaningful in elite sport. For example, it could improve
1,500-m running performance by ⬃2 s or 3,000-m running
performance by approximately 4 –5 s in international standard
athletes. It remains unclear, however, whether elite athletes
may confer a performance benefit from NO
3
⫺
supplementation.
Several studies now indicate that at least when NO
3
⫺
is ingested
acutely, TT performance is not enhanced in highly trained
endurance athletes (7, 8, 40). This may be related to factors
such as greater NOS activity, better muscle oxygenation and
mitochondrial efficiency, and a lower fraction of type II fibers
in the muscles of highly endurance trained compared with
moderately trained subjects (40). It is possible that the dose-
response relationship between NO
3
⫺
ingestion and changes in
exercise performance are different in elite compared with
sub-elite subjects, such that larger NO
3
⫺
doses and/or longer
supplementation periods may be required to elicit improved
exercise performance. The significant correlation between the
change in plasma [NO
2
⫺
] and the change in time-to-task failure
indicates that the dietary NO
3
⫺
intervention must be sufficient
to increase plasma [NO
2
⫺
] if performance is to be improved. In
this regard, an important consideration may be the timing of
supplementation relative to the start of exercise. The present
study shows that on average, plasma [NO
2
⫺
] takes longer to
peak when larger doses of NO
3
⫺
are imbibed. However, there
are appreciable interindividual differences in the speed with
which ingested NO
3
⫺
is reduced to NO
2
⫺
, which may preclude
any more specific advice other than to consume NO
3
⫺
some 2–3
h before the start of exercise.
334 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
It has been suggested previously that there may be “respond-
ers” and nonresponders to dietary NO
3
⫺
supplementation (40),
and there was evidence of this in the present study. Interest-
ingly, the number of nonresponders (in terms of exercise
capacity) decreased as the dose ingested increased. For exam-
ple, there were three nonresponders in the 4.2-mmol condition,
two in the 8.4-mmol condition, and one in the 16.8-mmol
condition. Two of the subjects who did not respond at the
lowest dose did respond to the larger doses, and one subject
who did not respond following administration of 4.2 or 8.4
mmol did respond to the 16.8-mmol dose. This suggests that
some individuals will require a larger acute dose than others to
elicit any positive effects on exercise capacity from dietary
NO
3
⫺
ingestion. Unlike in our previous study (40), the increase
in plasma [NO
2
⫺
] from baseline to pre-exercise for the nonre
-
sponders was not smaller than that measured in other subjects
who did respond, and the nonresponders did not have partic-
ularly high baseline plasma [NO
2
⫺
]. In a recent study, we found
that the subjects who demonstrated improvement in high-
intensity, intermittent exercise performance following dietary
NO
3
⫺
supplementation were those whose plasma [NO
2
⫺
] fell
significantly during exercise (41). We did not measure plasma
[NO
2
⫺
] postexercise in the present study. The explanation for
the existence of responders and nonresponders to dietary NO
3
⫺
supplementation is presently obscure.
In conclusion, dietary supplementation with NO
3
⫺
-rich BR
dose dependently increased plasma [NO
3
⫺
] and [NO
2
⫺
]upto
16.8 mmol NO
3
⫺
and caused peak reductions in systolic BP and
MAP dose dependently, up to 8.4 mmol NO
3
⫺
. These results
suggest that the consumption of high NO
3
⫺
foodstuffs may be
an effective strategy for maintaining and perhaps enhancing
vascular health in young adults. The present study also dem-
onstrated that the O
2
cost of moderate-intensity exercise is
reduced dose dependently, up to 16.8 mmol NO
3
⫺
. Supplemen
-
tation with 4.2 mmol NO
3
⫺
did not enhance time-to-task failure
relative to PL; however, supplementation with 8.4 mmol NO
3
⫺
significantly improved time-to-task failure relative to PL, with
no further improvement evident following supplementation
with 16.8 mmol NO
3
⫺
. Although the mechanistic bases for the
reduction in the O
2
cost of submaximal exercise and enhance
-
ments in exercise tolerance following acute dietary BR remain
unclear, these results provide important, practical information
that may underpin the potential use of BR/NO
3
⫺
supplementa
-
tion for improving cardiovascular health in the general popu-
lation and for enhancing exercise performance in athletes.
ACKNOWLEDGMENTS
The authors thank Beet It for providing the beverages used in this study,
gratis.
GRANTS
This study was funded in part by a research grant of GSSI, a division of
PepsiCo, Inc. The views expressed in this manuscript are those of the authors
and do not necessarily reflect the position or policy of PepsiCo, Inc.
DISCLOSURES
The views expressed in this article are those of the authors and do not
necessarily reflect the position or policy of PepsiCo.
AUTHOR CONTRIBUTIONS
Author contributions: L.J.W., S.J.B., P.G.W., A.E.J., A.V., and A.M.J.
conception and design of research; L.J.W., J.K., and J.R.B. performed exper-
iments; L.J.W., J.K., P.F.S., and A.V. analyzed data; L.J.W., S.J.B., P.F.S.,
A.V., and A.M.J. interpreted results of experiments; L.J.W. and P.F.S. pre-
pared figures; L.J.W. and A.M.J. drafted manuscript; L.J.W., J.K., S.J.B.,
P.G.W., A.E.J., A.V., and A.M.J. edited and revised manuscript; L.J.W., J.K.,
S.J.B., J.R.B., P.F.S., P.G.W., A.E.J., A.V., and A.M.J. approved final version
of manuscript.
REFERENCES
1. Alzawahra WF, Talukder MA, Liu X, Samouilov A, Zwuier JL. Heme
proteins mediate the conversion of nitrite to nitric oxide in the vascular
wall. Am J Physiol Heart Circ Physiol 295: H499 –H508, 2008.
2. Bailey SJ, Fulford J, Vanhatalo A, Winyard PG, Blackwell JR,
DiMenna FJ, Wilkerson DP, Benjamin N, Jones AM. Dietary nitrate
supplementation enhances muscle contractile efficiency during knee-ex-
tensor exercise in humans. J Appl Physiol 109: 135–148, 2010.
3. Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ,
Wilkerson DP, Tarr J, Benjamin N, Jones AM. Dietary nitrate supple-
mentation reduces the O
2
cost of low-intensity exercise and enhances
tolerance to high-intensity exercise in humans. J Appl Physiol 107:
1144 –1155, 2009.
4. Benjamin N, O’Driscoll F, Dougall H, Duncan C, Smith L, Golden M,
McKenzie H. Stomach NO synthesis. Nature 368: 502–503, 1994.
5. Bescos R, Rodriguez FA, Iglesias X, Ferrer MD, Iborra E, Pons A.
Acute administration of inorganic nitrate reduces V
˙
O
2 peak
in endurance
athletes. Med Sci Sports Exerc 43: 1979 –1986, 2011.
5a.Burke LM. To beet or not to beet. J Appl Physiol. doi:10.1152/jappl-
physiol.00612.2013.
6. Cermak NM, Gibala MJ, van Loon LJ. Nitrate supplementation’s
improvement of 10-km time-trial performance in trained cyclists. Int J
Sport Nutr Exerc Metab 22: 64 –71, 2012.
7. Cermak NM, Res P, Stinkens R, Lundberg JO, Gibala MJ, van Loon
LJ. No improvement in endurance performance following a single dose of
beetroot juice. Int J Sport Nutr Exerc Metab 22: 470 –478, 2012.
8. Christensen PM, Nyberg M, Bangsbo J. Influence of nitrate supplemen-
tation on VO
2
kinetics and endurance of elite cyclists. Scand J Med Sci
Sports 23: e21–e31, 2013.
9. Clerc P, Rigoulet M, Leverve X, Fontaine E. Nitric oxide increases
oxidative phosphorylation efficiency. J Bioenerg Biomembr 39: 158 –166,
2007.
10. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S,
Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H,
Kim-Shapiro DB, Schechter AN, Cannon III RO, Gladwin MT. Nitrite
reduction to nitric oxide by deoxyhemoglobin vasodilates the human
circulation. Nat Med 9: 1498 –1505, 2003.
11. Day JR, Rossiter HB, Coats EM, Skasick A, Whipp BJ. The maximally
attainable V
˙
O
2
during exercise in humans: the peak vs. maximum issue. J
Appl Physiol 95: 1901–1907, 2003.
12. Dejam A, Hunter CJ, Schechter AN, Gladwin MT. Emerging role of
nitrite in human biology. Blood Cells Mol Dis 32: 423–429, 2004.
13. Feelisch M, Fernandez BO, Bryan NS, Garcia-Saura MF, Bauer S,
Whitlock DR, Ford PC, Janero DR, Rodriguez J, Ashrafian H. Tissue
processing of nitrite in hypoxia: an intricate interplay of nitric oxide-
generating and -scavenging systems. J Biol Chem 283: 33927–33934,
2008.
14. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones
AM, Musch TI, Poole DC. Impact of dietary nitrate supplementation via
beetroot juice on exercising muscle vascular control in rats. J Physiol 591:
547–557, 2013.
15. Fulford J, Winyard PG, Vanhatalo A, Bailey SJ, Blackwell JR, Jones
AM. Influence of dietary nitrate supplementation on human skeletal
muscle metabolism and force production during maximum voluntary
contractions. Pflugers Arch 465: 517–528, 2013.
16. Govoni M, Jansson EÅ, Weitzberg E, Lundberg JO. The increase in
plasma nitrite after a dietary nitrate load is markedly attenuated by an
antibacterial mouthwash. Nitric Oxide 19: 333–337, 2008.
17. Hernández A, Schiffer TA, Ivarsson N, Cheng AJ, Bruton JD, Lun-
dberg JO, Weitzberg E, Westerblad H. Dietary nitrate increases tetanic
[Ca
2⫹
]i and contractile force in mouse fast-twitch muscle. J Physiol 590:
3575–3583, 2012.
18. Ishii T, Sunami O, Saitoh N, Nishio H, Takeuchi T, Hata F. Inhibition
of skeletal muscle sarcoplasmic reticulum [Ca
2⫹
]-ATPase by nitric oxide.
FEBS Lett 440: 218 –222, 1998.
19. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F,
Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S,
335Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from
Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A. Inorganic nitrate
supplementation lowers blood pressure in humans: role for nitrite-derived
NO. Hypertension 56: 274 –281, 2010.
20. Kelly J, Vanhatalo A, Blackwell JR, Wilkerson DP, Wylie LJ, Jones
AM. Effects of nitrate on the power-duration relationship for severe-
intensity exercise. Med Sci Sports Exerc. In press.
21. Lansley KE, Winyard PG, Bailey SJ, Vanhatalo A, Wilkerson DP,
Blackwell JR, Gilchrist M, Benjamin N, Jones AM. Acute dietary
nitrate supplementation improves cycling time trial performance. Med Sci
Sports Exerc 43: 1125–1131, 2011.
22. Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Black-
well JR, DiMenna FJ, Gilchrist M, Benjamin N, Jones AM. Dietary
nitrate supplementation reduces the O
2
cost of walking and running: a
placebo-controlled study. J Appl Physiol 110: 591–600, 2011.
23. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of
dietary nitrate on blood pressure in healthy volunteers. N Engl J Med 355:
2792–2793, 2006.
24. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg
JO, Weitzberg E. Dietary inorganic nitrate improves mitochondrial effi-
ciency in humans. Cell Metab 13: 149 –159, 2011.
25. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Dietary nitrate
reduces maximal oxygen consumption while maintaining work perfor-
mance in maximal exercise. Free Radic Biol Med 48: 342–347, 2010.
26. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate
on oxygen cost during exercise. Acta Physiol (Oxf) 191: 59 –66, 2007.
27. Law MR, Wald NJ, Morris JK, Jordan RE. Value of low dose
combination treatment with blood pressure lowering drugs: analysis of 354
randomised trials. BMJ 326: 1427–1434, 2003.
28. Lawes CM, Rodgers A, Bennett DA, Parag V, Suh I, Ueshima H,
MacMahon S, Asia Pacific Cohort Studies Collaboration. Blood pres-
sure and cardiovascular disease in the Asia Pacific region. J Hyperten 21:
707–717, 2003.
29. Lewington S, Clarke R, Qizibash N, Peto R, Collins R. Prospective
Studies Collaboration. Age-specific relevance of usual blood pressure to
vascular mortality: a meta-analysis of individual data for one million
adults in 61 prospective studies. Lancet 360: 1903–1913, 2002.
30. Lundberg JO, Carlström M, Larsen FJ, Weitzberg E. Roles of dietary
inorganic nitrate in cardiovascular health and disease. Cardiovasc Res 89:
525–532, 2011.
31. MacMahon S, Peto R, Cutler J, Collins R, Sorlie P, Neaton J, Abbott
R, Godwin J, Dyer A, Stamler J. Blood pressure, stroke and coronary
heart disease: part 1, prolonged differences in blood pressure: prospective
observational studies corrected for the regression dilution bias. Lancet
335: 765–774, 1990.
32. Mészáros L, Minarovic I, Zahradnikova A. Inhibition of the skeletal
muscle ryanodine receptor calcium release channel by nitric oxide. FEBS
Lett 380: 49 –52, 1996.
33. Omar SA, Artime E, Webb AJ. A comparison of organic and inorganic
nitrates/nitrites. Nitric Oxide 26: 229 –240, 2012.
34. Paton CD, Hopkins WG. Variation in performance of elite cyclists from
race to race. Eur J Sports Sci 6: 25–31, 2006.
35. Reid MB. Role of nitric oxide in skeletal muscle: synthesis, distribution
and functional importance. Acta Physiol Scand 162: 401–409, 1998.
36. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle.
Physiol Rev 81: 209 –237, 2001.
37. Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Pavey TG,
Wilkerson DP, Benjamin N, Winyard PG, Jones AM. Acute and
chronic effects of dietary nitrate supplementation on blood pressure and
the physiological responses to moderate-intensity and incremental exer-
cise. Am J Physiol Regul Integr Comp Physiol 299: R1121–R1131,
2010.
38. Vanhatalo A, Fulford J, Bailey SJ, Blackwell JR, Winyard PG, Jones
AM. Dietary nitrate reduces muscle metabolic perturbation and improves
exercise tolerance in hypoxia. J Physiol 589: 5517–5528, 2011.
39. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S,
Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ,
Ahluwalia A. Acute blood pressure lowering, vasoprotective, and anti-
platelet properties of dietary nitrate via bioconversion to nitrite. Hyper-
tension 51: 784 –790, 2008.
40. Wilkerson DP, Hayward GM, Bailey SJ, Vanhatalo A, Blackwell JR,
Jones AM. Influence of acute dietary nitrate supplementation on 50 mile
time trial performance in well-trained cyclists. Eur J Appl Physiol 112:
4127–4134, 2012.
41. Wylie LJ, Mohr M, Krustrup P, Jackman SR, Ermdis G, Kelly J,
Black MI, Bailey SJ, Vanhatalo A, Jones AM. Dietary nitrate supple-
mentation improves team sport-specific intense intermittent exercise per-
formance. Eur J Appl Physiol 113: 1673–1684, 2013.
336 Nitrate and Exercise Dose Response • Wylie LJ et al.
J Appl Physiol • doi:10.1152/japplphysiol.00372.2013 • www.jappl.org
at Exeter Univ Library on August 5, 2013http://jap.physiology.org/Downloaded from