Embed Size (px)
Citation preview
7/21/2015
1
John Ivy, PhD
Department of Kinesiology and Health Education
University of Texas at Austin
Sponsored by
EAS
Columbus, OH
Dietary Nitrate: Effect on Exercise Performance and Training Adaptation
Disclosure
• John Ivy is:
• The chief science officer for Neogenis Sports, a company that makes products to boost nitric oxide production
• Chairman of the EAS Scientific Advisory Board
• Teresa Lozano Long Chair Emeritus of the University of Texas
Learning Objectives
As a result of this lecture, participants will be able to:• Describe the two major pathways nitric oxide is produced
in the body and the limitations of each system• Discuss the role nitric oxide plays in controlling blood flow
and muscle metabolism during exercise• Describe the ergogenic effects of dietary nitrates on
exercise performance and their limitations • Propose ways of boosting nitric oxide production during
exercise via dietary considerations • Evaluate dietary supplements that purport to booster nitric
oxide levels and improve exercise performance • Describe the chronic effects of a diet high in dietary nitrate
What is Nitric Oxide?
• N-O is a gaseous, inorganic, molecule and a free radical with one unpaired electron in its external orbital.
• It is highly lipophilic and diffusible, so N-O can pass through multiple cell membranes to reach its final target at some distance from the sites of N-O synthesis.
• Considering its very short half-life, less than 1 second, N-O cannot be stored in free form and is generally synthesized on demand with specific biological effects.
• It is one of the most important signaling molecules in the body, and one of the few gaseous signaling molecules known.
Nitric Oxide Plays a Key Role in the Regulation of
Numerous Vital Biological Functions
N-O
Cardiovascular System
Respiratory Tract
Immunology
Cell Proliferation
Central Nervous System
Peripheral Nervous System
Gastrointestinal/Urogenital Tract Vasorelaxation
Blood Cell RegulationMyocardial ContractilityMicrovascular Permeability
Bronchial DilationAsthma, ARDS
NANC nerve-mediatedRelaxation
Learning and MemoryPain SensitizationEpilepsyNeurodegenerationCentral BP Control
ApoptosisAngiogenesisTumor Cell Growth
Unspecific ImmunityInhibition of Viral ReplicationTransplant Rejection
Penile ErectionPre-term Labour
RegenerationMobilization of resident stem cellsTargeted differentiation
Skeletal Muscle
Mitochondrial FunctionMuscle Contractile Efficiency
Production of N-O via Nitric Oxide Synthase (NOS)
7/21/2015
2
Nitric Oxide Synthase Isoforms
NOS-1
NOS-2
NOS-3
Neuronal
Inducible
Endothelial
Förstermann U and Sessa WC Eur Heart J 33:829-837, 2012
Nitric Oxide Production
Limitations
• Depends on a very complex enzyme that requires many co-factors that can be deficient in the diet
• Enzyme requires adequate oxygen and is not very functional when oxygen tension declines to low levels such during exercise or at altitude.
• Enzyme does not function well at low pH
• There is loss of enzyme function with age, i.e. reduced expression and uncoupling
• *L-arginine Km for NOS is 5µM and plasma has 100µM. Usually L-arginine is not a limitation and supplementing with L-arginine has little effect.
Nitric Oxide Producers
Arginine and Glycerol with Caffeine
Arginine a-Ketoglutarate (AKG), Arginine HCl, Ornithine a-Ketoglutarate (AKG)
Why would so important a process as generating a key ubiquitous signaling molecule like N-O be left up to such a
fragile and complex reaction?
Nitric Oxide Production
7/21/2015
3
N-O Produced FromNitrate and Nitrite
The reduction of nitrite to nitric oxide by reduced hemoglobin and reductase enzymes is most effective under low pH and hypoxic conditions making this NO pathway most effective during high intensity exercise and at altitude.
Weitzberg E and Lundberg JO Annu Rev Nutr 33:129-159, 2013
Dietary Sources of Nitrate and Nitrite
Eur Food Saf Auth EFSA J 689:1-79, 2008
Classification of vegetables according to nitrate content
Nitrate content (mg/100g fresh weight) Vegetables
Very low, < 20 Artichoke, asparagus, broad bean, eggplant
garlic, onion, green bean, mushroom, pea
pepper, potato, summer squash, sweet potato, tomato, watermelon
Low, 20 to < 50 Broccoli, carrot, cauliflower, cucumber,
pumpkin, chicoryMiddle, 50 to < 100 Cabbage, dill, turnip, savoy cabbageHigh, 100 to < 250 Celeriac, Chinese cabbage, endive,
fennel, kohlrabi, leek, parsleyVery high, > 250 Celery, cress, chervil, lettuce, red beetroot,
redbeet leaves, spinach, rocket (rucola), arugula, kale, rhubarb, basil,
Indicators Nitric Oxide Influences Physical Performance
Nitric oxide synthase-derived plasma
nitrite predicts exercise capacity
Rassaf T, et al Br J Sports Med 41:669–673, 2007
Post
-exe
rcis
e p
lasm
a n
itri
te (
Δ%
)
250
200
150
100
50
-50
0
0 5 10FMD (%)
FMD – flow-mediated dilation(r = 0.36; p = 0.01)
N = 55 (22 men, 33 females)Mean age 40
Post
-exe
rcis
e p
lasm
a n
itri
te (
nM
)
Maximal stress (Watt)
300
250
200
150
100
0 50 100 150 200 250 300
r = 0.38 p < 0.007N = 55 (22 men, 33 females)Mean age 40
Rassaf et al. Br J Sports Med 41:669-673, 2007
Relationship Between N-O Production And Exercise Performance
Baseline Levels of N-O are related to the lactate threshold in highly
trained athletes
7/21/2015
4
Experimental Design
• N = 11 highly trained athletes
• Tested on two separate occasions and the mean of each parameter determined
• Exercise was on bicycle ergometer
• Exercise protocol: cycled at 100 W for 5 min and then the workload was increased by 50 W every 5 min until fatigue.
• Pre-blood samples were taken from an arm vein for nitrate and nitrite determination and from the ear lobe for lactate determination
Totzeck et al. Nitric Oxide 27:75-81, 2012 Totzeck et al. Nitric Oxide 27:75-81, 2012
r = 0.71, p = 0.0002
14
12
10
8
6
4
2
.04 .06 .08 .10 .12 .14 .16 .18 .20
Flo
w M
edia
ted
Dila
tio
n (
%)
Nitrite [µM]
Totzeck et al. Nitric Oxide 27:75-81, 2012
r = 0.65, p = 0.001
220
200
180
160
140
120
100
.04 .06 .08 .10 .12 .14 .16 .18 .20
Nitrite [µM]
Lact
ate
An
aero
bic
Th
resh
old
(b
pm
)
Subject Mode of Administration
Exercise Work Time (min)Placebo Nitrate
Δ P value
Young healthy Beet Juice, 6 D Cycling 9:43 11:15 15.8% ≤0.05
Young healthy Beet Juice, 6 D Knee extension 9:46 12:14 25.0% ≤0.01
Young healthy Beet Juice, 6 D Running 7:36 8:42 15.0% ≤0.01
Young rowers Beet Juice, 6 D Cycling (TT) 16:05 15:53 1.0% ≤0.05
Young healthy Beet Juice, 2 D Arm/leg cycling 8:44 9:23 7.5% 0.03
Young athlete Na Nitrate, 1 dose Cycling 6:49 6:56 1.7% NS
Young cyclist Beet Juice, 1 dose Cycling (TT) 27:42 26:54 2.7% ≤0.01
Old PAD Beet Juice, 1 dose Walking 7:47 8:53 17.0% ≤0.05
Young kayakers Beet Juice, 1 dose Cycling (TT) 4:35 4:34 0% NS
Young active Beet lozenge, 1 dose Cycling (TT) 41:15 40:09 2.1% ≤0.01
Effect of dietary nitrate on exercise performance
Dietary Nitrate and Exercise Performance
Influence of Dietary Nitrate Supplementation on Exercise Performance in the Highly Trained/Elite
Mean (0.5%)
Hoon et al. (2013)
Hoon et al. (2013)
Muggeridge et al. (2013)
Wilkerson et al. (2012)
Peacock et al. (2012)
Cermak et al. (2012b)
Cermak et al. (2012a)
Peeling et al.( 2014)
-1.0 -0.5 0 0.5 1 1.5 2.0
Performance Change (%)
Bescos et al. 2011
Acute Effects of Nitric Oxide on Exercise Performance and
Mechanisms of Action
7/21/2015
5
1. Effect of Dietary Nitrate on Blood Flow During Exercise
Experimental Design
• N = 19 young adult rats (3-4 months of age)• Placebo or dietary nitrate treatment group
(randomly assigned)• Dietary nitrate from concentrate beetroot juice
was provided daily for 5 days (1 mmol kg/d in 100 ml tap water
• Exercise consisted of treadmill running at 20 m/min on a 5% grade.
• Blood flow was determined by microsphere injection and muscle of the hindlimbs evaluated
Ferguson SK, et al. J Physiol 591:547-557, 2013
Ferguson SK, et al. J Physiol 591:547-557, 2013
150
Pla
sma
NO
3-
( µ
M)
Pla
sma
NO
2-
( n
M)
180
120
90
60
30
0
800
600
400
200
0
Control BR
Control BR
*
*
Plasma Nitrate (NO3-) and Nitrite (NO2
-) Levels
Ferguson SK, et al. J Physiol 591:547-557, 2013
Effects of 5 days of BR supplementation on HR, MAP and blood [lactate] at restand during exercise
HR (beats/min) MAP (mmHg) Blood [lactate] (mM)
Control BR Control BR Control BR
Rest 405±8 409±13 138±3 132±7 0.0±0.1 0.7±0.1
Exercise 525±9† 521±6† 137±3 127±4* 2.6±0.3† 1.9±0.28*†
Data are means ± SEM. *P < 0.05 vs. control, †P < 0.01 vs. rest.
Ferguson SK, et al. J Physiol 591:547-557, 2013
0
BF
(ml/
min
/100
g)V
C (
ml/
min
/mm
Hg)
160
150140
130120
110
100
1.50
1.25
1.00
0.75
0.50
0.25
0Control BR
*
*
Blood Flow and Vascular Compliance
Ferguson SK, et al. J Physiol 591:547-557, 2013
100
80
60
40
20
0
0 20 40 60 80 100
ΔB
F (%
)
% Type IIb + d/x
r = 0.74P < 0.01
ΔV
C (
%)
0 20 40 60 80 100
140
100
60
20
0
% Type IIb + dx
r = 0.71P < 0.01
Change in Blood Flow And Vascular ComplianceAccording to muscle fiber
type
7/21/2015
6
Ferguson SK, et al. J Physiol 591:547-557, 2013
Gast red
200
180
160
140
120
100
ΔB
F %
Control BR
Semi wh*
RF wh*
BF ant*Gast wh*
VL white
100% Type IIb + d/x
≤ 20% Type IIb + d/x
SolAdd longVI
Change in Muscle Blood Flow
The body converts dietary nitrate and nitrite to nitric oxide
(NO), which supports the dilatation of blood vessels,
increasing blood flow to the active muscles.
The effect during exercise is seen primarily in fast twitch –
glycolytic fibers.
This helps the body boost delivery of oxygen and
nutrient to less oxidative muscle fibers, fueling
mitochondrial ATP production, which supports more
intense workouts and increased stamina without
excessive fatigue.
Increased muscle blood flow also helps the body support a
faster rate of recovery by increasing removal of
metabolic waste products such as lactic acid and
carbon dioxide and replenishing depleted fuel stores.
Nitric Oxide and Vasodilation
2. Increases O2 Efficiency
Experimental Design
• Randomized, double-blind, crossover experimental design
• N = 9 club competitive male cyclists• Investigated the effects of beetroot juice on cycling
time trial performance for a 4.0 and a 16.1 km time trials.
• 500 ml of beetroot juice with or without nitrate provided 2.5 h before exercise
• Measure time to complete time trial, power and VO2
Lansley et al. MSSE 43: 1125-1131, 2011.
4 km Cycling Time Trial
Lansley et al. Med Sci Sports & Exerc 43:1125-1131, 2011.
8
7
6
5
0
Tim
e(m
in)
Pow
er o
utp
ut:
VO
2ra
tio
(W
/L/m
in)
2.8%#
PL BR
0 1 2 3 4Distance (km)
** *
* *
100
90
80
70
60
50
BRPL
0
16.1 km Cycling Time Trial
Lansley et al. Med Sci Sports & Exerc 43:1125-1131, 2011.
30
28
26
24
0
PL BR
2.7%*
Tim
e(m
in)
80
70
Pow
er o
utp
ut:
VO
2ra
tio
(W/L
/min
)
60
00 2 4 6 8 10 12 14 16
Distance (km)
BR
PL
7/21/2015
7
N-O Increases Oxygen Efficiency
This means aerobic energy production in the
mitochondria of the muscle is more efficient, i.e. the ATP
production per unit of oxygen converted to H2O is
reduced.
Reducing the O2 cost of exercise is associated with
greater endurance
3. Nitric Oxide Improves Contractile Efficiency
Experimental Design
Subjects ingested 500 ml of beet juice (5.1 mmol NO3/d) for 6 days or placebo.
Subjects completed a series of low-intensity and high-intensity step exercise tests over the last 3 days
Muscle metabolism determined by 31P-MRS
Pulmonary VO2 determined by open circuit spirometry
Bailey SJ, et al. J Appl Physiol 109:135-148, 2010. Bailey SJ, et al. J Appl Physiol 109:135-148, 2010.
[PC
r](m
mo
l)
40
35
30
25
20
15
0-60 0 120 240 360 500 700 900
*
Time (s)
High Intensity ExerciseResistance Exercise
PL
BR
160
120
80
40
0
[AD
P]
(µM
)
*
0 120 240 360 500 700 900-60
Time (s)
PL
BR
High Energy Phosphate Turnover During Standardized Workload
ATP Turnover Rate
Bailey SJ, et al. J Appl Physiol 109:135-148, 2010.
ATP Total ATP Ox ATP PCr ATP Gly
ATP
Tu
rno
ver
(µM
/s)
700
600
500
400
300
200
100
0
*
*
*
Placebo
Beet Juice
Experimental Design
• N = 24 subjects
• Double blind, repeated measure, crossover design
• Subjects received 0.5 L beetroot juice with (10.2 mM) or without nitrate (0.17 mM; placebo) 2.5 hours before exercise
• Exercise was 50 maximal isometric contractions lasting 6.6 sec each
Fulford, J. Pflugers Arch – Eur J Physiol 465:517-528, 2013
7/21/2015
8
Nitrate
Placebo
Fulford, J. et al. Pflugers Arch – Eur J Physiol 465:517-528, 2013
Mea
n f
orc
e o
utp
ut
per
co
ntr
acti
on
(N)
400
300
200
1000 5 10 15 20 25 30 35 40 45 50
Contraction Number
Muscle Fatigue Curves During 50 Isometric Contractions
Fulford, J. et al. Pflugers Arch – Eur J Physiol 465:517-528, 2013
Nitrate
Placebo
25
20
15
10
5
00 100
PC
rco
st (m
M)
200 300 400 500 600 700 800 900 1000
TIME (Sec)
Rate of Creatine Phosphate Breakdown During Contractions
N-O AND MUSCLE CONTRACTION
Improvement in exercise performance isrelated to the ability of N-O to reduce the amount of ATP utilized per force generated by the muscles during exercise
This effect is seen at all exercise intensities and is the reason dietary nitrates have been found to improve exercise performance in all different types of sports such as cycling, running, rowing, swimming team sports, resistance exercise, etc.
N-O improves contractile efficiency
Acute Physiological Effects of N-O
• Increases vasodilation, thereby facilitating blood flow to the working muscles. This increases O2 and nutrient delivery to the muscles and the rapid removal of metabolic waste products.
• Increases mitochondrial efficiency decreasing the O2 cost of exercise.
• Increases muscle contractile efficiency, i.e., less ATP per unit of force generated.
• Also causes bronchiole dilation making breathing easier during high intensity exercise
Pharmacodynamics and dose-response to dietary nitrate
Experimental Design
• N = 10 healthy men• Ingested placebo, 4.2 (~260 mg), 8.4 and 16.8
mmol nitrate from a beet concentrate 2.5 hours to determine NO2 and NO3 dose response over 24 hours.
• On 6 separate occasions the subjects received beetroot concentrate containing either no nitrate or 4.2, 8.4 and 16.8 mmol nitrate 2.5 hours before exercise
• Exercise consisted cycling at moderate-intensity and severe-intensity
Wylie et al. J Appl Physiol. 115:325-336, 2013.
7/21/2015
9
Wylie et al. J Appl Physiol. 115:325-336, 2013.
14% 12%
*
*
650
600
550
500
450
0PL PL PLBR BR BR
4.2 mmol 8.4 mmol 16.8 mmol
Tim
e to
Tas
k Fa
ilure
(s)
Exercise Time
BR
PL
4.2 mmol is ~ 250 mg of nitrate
Chronic Effects of Nitrate on Training Adaptation
Nitric Oxide & Mitochondrial Biogenesis
Brown, GC. Frontiers Biosci 12:1024-1033,2007.
Exercise , Cold, Calorie Restriction
N-O
cGMP cAMPAMP
PGC-1α
NRF-1 mtTFA
Nuclear mtGenes mtDNA Transcription &Replication
Increase Mitochondria
Dietary Nitrate
Structural Changes in Mitochondria Experimental Design
• Randomized, double-blind, and crossover study, N = 14 healthy subjects (VO2peak 56 ± 3 ml kg1 min1).
• Sodium nitrate (0.1 mmol/kg/day, divided in three doses) or an equimolar amount of sodium chloride (placebo) for 3 days prior to experiments, with a washout period of at least 6 days between tests.
• Muscle biopsies were taken from the vastus lateralis
• Exercise was performed by cycling
Larsen, FJ. Cell Metabolism 13:149-159, 2011
Larsen, FJ. Cell Metabolism 13:149-159, 2011
1500
1700
1900
2100
VO
2(L
/min
)
2300 80
70
60
50
Whole body VO2
P = 0.02
Watt/VO2
P = 0.01
placebo nitrate placebo nitrate
Oxygen Consumption and Work at Steady State Exercise
Larsen, FJ. Cell Metabolism 13:149-159, 2011
P/O ratio Maximal ATP production
2.0
1.5
1.0
0.5
0
P = 0.02 P = 0.05
ATP
(%
of
pla
ceb
o)
150
100
50
0placebo placebonitrate nitrate
Muscle ATP Production Relative to O2 Consumption and Maximal ATP Production
Reduction in translocases in mitochondria, which reduces proton leakage and increase efficiency of ATP production
7/21/2015
10
2. Nitric Oxide Increases Contractile Force in Mouse Fast Twitch Fibers
Experimental Design
• Mice were fed 1 mM NaNO3 for 7 days in drinking water or provided just water (control)
• EDL and soleus muscles were isolated and mounted at optimal length.
• Maximal tetanic force was determined for both muscles
• Flexor Digitorum Brevis was isolated and mounted to determine Ca++ kinetics during tetanic stimulation by a fluorescence Ca++
indicator.
Hernandez A. et al. J Physiol 590:3575-3583, 2012
EDL
Soleus
Hernandez A. et al. J Physiol 590:3575-3583, 2012
Forc
e (k
Pa)
Forc
e (k
Pa)
Frequency (Hz)
EDL
Soleus
Nitrate
Control
3. Nitric Oxide Stimulates Muscle Hypertrophy and Angiogenesis
in Old Mice
Experimental Design
• Female mice, 18 months of age (old mice)
• Treatment groups: control, exercise (E), Isosorbide dinitrate (I.66 mg/kg in canola oil), exercise plus isosorbide dinitrate (E + I) for 6 weeks (Isosorbide dinitrate is a N-O donor)
• Exercise was voluntary wheel running
• Muscles examined were the quadriceps and tibialis anterior
Leiter, JRS., et al. Am J Physiol Cell Physiol 3012:C1306-C1315, 2012
Muscle mass of the quadriceps and tibialis anterior after training
Treatment Quadriceps Tibialis Anterior
Control 145±9 37±2
Ex 169±7 36±1
I 172±5 38±1
I + Ex 182±5* 39±1
I + Ex had a 25% increase in muscle mass in the Quadriceps over 6 weeks
Leiter, JRS., et al. Am J Physiol Cell Physiol 3012:C1306-C1315, 2012
7/21/2015
11
Leiter, JRS., et al. Am J Physiol Cell Physiol 3012:C1306-C1315, 2012
Vascular Density
120
100
80
60
40
20
0
C Ex I I+Ex
c
b
ab a
# V
ess
els
pe
r Fi
eld
Results of Leiter et al. Study
• Exercise plus a nitric oxide donor increased hypertrophy and possibly hyperplasia in muscle from old mice.
• Vascular density was also increased
• This suggests that age-related muscle refractoriness to exercise can be overcome with a nitric oxide booster.
Leiter, JRS., et al. Am J Physiol Cell Physiol 3012:C1306-C1315, 2012
Safety of Dietary Nitrate
• Dietary nitrate/nitrite reacts under conditions of high heat (e.g., frying bacon) or high acidity (e.g., stomach) to produce nitrosamines
• Nitrosamines fed at supraphysiological levels are carcinogenic in lab animals
• Some studies have linked nitrosamines from red processed meats to gastrointestinal cancer in humans
These studies did not account for obesity, physical inactivity, low vegetable intake, etc. – which are also associated with increased risk of cancerVegetables are the primary source of dietary nitrate/nitrite, yet vegetable intake is highly correlated with reduced risk of gastrointestinal cancerAscorbic acid in vegetables or added to nitrate/nitrite rich products inhibits the formation of nitrosamines
• Based on epidemiological studies, the European Food Safety Authority in 2008 concluded that nitrate intake from diet or drinking water is not associated with increased risk for cancer
Acceptable Nitrate and Nitrite Intake Levels?
Joint FAO/WHO Expert Panel on Food Additives established Acceptable Daily Intakes (ADI) for
Nitrate and Nitrite
Nitrate (0 to 3.7 mg/kg body wt/d)
Nitrite (0 to 0.07 mg/kg body wt/d)
Translates to 222 mg and nitrite 4.2 mg for 60 kg person
DASH Diet with high nitrate food choices (4 to 5 servings each of vegetables and fruits)
Intake would be nearly 6 times ADI for nitrate and exceeds the ADI for nitrite
People at Risk for Low Conversion of
Nitrate to N-O
Possible Trouble Reducing Nitrate:• Use of Proton Pump Inhibitors
– GERD• Chronic use of Antibiotics• Altered Salivary Bacteria –
Enterosalivary System (25% population)
• Use of Antibacterial Mouthwash• Gastric Bypass Surgery• Athletes should not take nitrite
compounds (NaNO2)
SUMMARY
• Nitric oxide can be produced by conversion of L-arginine to L-citrulline via NOS, and the conversion of nitrate to nitric oxide
• Most studies show that consumption of L-arginine and L-citrulline have little effect on blood N-O production and exercise performance
• Acutely, dietary nitrates provided before exercise increase blood flow to fast twitch-glycolytic muscle, increase O2
efficiency, decrease the energy cost of exercise and improve exercise performance
• Consumption of approximately 7-8 mmol (450-550 mg) of nitrate about 2.5 h before exercise are required to see an enhancement in exercise performance
• The effect is most prominent in less fit and older individuals
7/21/2015
12
Summary concluded
• It is recommended that highly trained athlete’s (elite) consumption 7-8 mmol of dietary nitrate for several days prior to and the day of competition
• Green leafy vegetables have a high concentration of nitrate and should be part of an athletes diet (200 to 300 g/d)
• Chronic consumption of nitrate can, (1) increase muscle mitochondrial density and alter its structure to increase energy efficiency, (2) modify Ca++ kinetics that favor a faster and more forceful muscle contraction, and (3) increase the muscle hypertrophy response to exercise in old muscle and improve its vascularization.
• Daily consumption of high levels of dietary nitrates have many health benefits including improved control of blood pressure, blood glucose, cognitive function, immune cell function, etc.
Thank You!
If you have questions
or for more information,
contact SCAN by email at:
SCAN values your feedback.
Please take a few minutes to complete a short 8-
question survey on this webinar by following the link
bellow:
https://www.surveymonkey.com/r/DietaryNO
