Case 1: Nitrate

Where can nitrate be found? (beetroot, spinach…)

Jones & Furgeson

Consuming food-stuffs such as green leafy vegetables (spinach, lettuce, rocket/ arugula) and
beetroot, which are rich in inorganic NO3-, substantially increases the circulating plasma nitrate
[NO3-] and nitrite [NO2-], thereby increasing the potential for O2-independent NO synthesis.

NO is produced in the canonical pathway in which semi-essential amino acid L-arginine is oxidized in a
reaction catalyzed by the NO synthase (NOS) family of enzymes. The products of this reaction are
nitrite (NO2-) and nitrate (NO3-).
Nitrate (NO3-) is converted to (NO2-) by facultative bacteria residing in the mouth.

Affourtit & Baily

Humans do not only get NO3- from their diet as it is also generated endogenously by oxidation of
nitric oxide (NO) formed via the L-arginine/NO synthase pathway.
Importantly, inorganic NO3- can be reduced to nitrite (NO2-) and then NO, which offers an additional
path of mammalian NO production thatis independent of oxygen (O2).

Salivary NO2- is rapidly protonated in the acidic environment of the stomach resulting in the
formation of NO and other reactive nitrogen species (RNS) including nitrogen dioxide (NO2), nitrous
acid (HNO2), and dinitrogen trioxide (N2O3).

NO2- increases after NO3- ingestion through catalyzation by xanthine oxidase and/or
deoxyhaemoglobin.

- Xanthine oxidase catalysation of NO3- to NO2- is competitively inhibited by O2.

NO2- is reduced to NO under conditions of low oxygen tension (low PO2) and low Ph. NO2- reductase
activity include cytochrome c and mitochondrial respiratory complexes III and IV. However this
process is a very inefficient way to produce NO, partly due to the low Ph environment in happens in.

Oxygenated haem effectively scavenges free NO

NO may be converted to N2O3 that in turn may react with free thiols to generate S-nitrosothiols (via
an S-nitrosation reaction).
Course opening and lecture 01
Found in water and processed meat (to keep it fresh), also found in some medication  to treat
certain heart diseases

Larsen & Schiffer

To ensure NO production throughout the body, complex NO synthases (NOSs) generate NO by

oxidation of the amino acid L-arginine.

What is the optimal dose of nitrate and when should you take it?
Course opening and lecture 01

Currently there is inadequate evidence to formulate specific recommendations for individual sports.
Studies have reported benefits with a nitrate dose of ~5-6 mmol (or ~300 mg), which is equivalent to
250-300 g nitrate-rich vegetables (such as rocket, lettuce, spinach, bok choy, broccoli, radish and
beetroot)

Jones & Furgeson

24h of NO3 supplementation (9.3 mmol NO3-) reduced the extent of muscle metabolic perturbation
during high-intensity knee-extension exercise and enhanced exercise tolerance in moderate hypoxia.

Voedingscentrum: https://www.voedingscentrum.nl/encyclopedie/nitraat.aspx#:~:text=De
%20aanvaardbare%20dagelijkse%20inname%20van%20nitraat%20is%20gesteld,EFSA%29%20een
%20ADI%20van%200%2C07%20mg%2Fkg%20lichaamsgewicht%20vastgesteld.
De aanvaardbare dagelijkse inname van nitraat is gesteld op 3,7 milligram per kilogram
lichaamsgewicht.

Affourtit & Baily

Acute (2.5 h post-ingestion) lowering of V˙O2 during low-intensity exercise is progressively greater at
4.2, 8.4, and 16.8 mmol NO3-, whereas high-intensity exercise tolerance is unaffected by 4.2 mmol
NO3-, but acutely improved to a similar extent by 8.4 and 16.8 mmol.
Therefore, short-term supplementation (≥3 days) with at least 5 mmol NO3- ⋅ day−1, or acute
ingestion of at least 8.4 mmol NO3-, might represent an effective dietary intervention to improve the
economy and performance of human locomotion, at least in healthy, moderately fit adults.

In humans, the ingestion of 10 mg NaNO3·kg−1 has been shown to increase mean plasma NO3-
within 90 min from 30 to 432 μM and mean plasma NO2- from 123 to 392 nM.
Similarly, 500 mL beetroot juice containing 45 mM NO3- on average raises mean plasma NO3- to 380
μM and NO2- to 600 nM within 30 min and 3 h of ingestion, respectively.

Plasma NO3- reaches peak concentrations 1–2 h post-ingestion and NO3- gradually returns to its base
level after about 24 h. The half-life of NO3- in blood is 5–8 h. About 60% of ingested NO3- is excreted
by the kidneys.

Plasma NO2- reaches peak concentrations, respectively, and 2–3 h post-ingestion, and NO3- gradually
returns to its base level after about 24 h. The half-life of NO2- in whole human blood is only about
110 sec as it is rapidly oxidized to NO−3.

300 – 560 mg of nitrate  250 - 500 gram root vegetables

Nitrate should be taken acutely (2-3 hours before exercise) or chronically (Nitrate has to be taken 24
hours before sport).

Who should take nitrate and for which sport/activity should you take nitrate?

Are there any side effects to taking nitrate?

Affourtit & Baily

Inorganic nitrate (NO3-) has long been considered an undesirable food component and pollutant of
drinking water as nitrosation of secondary amines may produce carcinogenic N-nitrosamines (Magee
and Barnes, 1956). People also believed that nitrate could cause cancer. Today no concrete evidence
for that has been found. Beneficial health effects of NO3- are widely accepted.

Course opening and lecture 01

The beneficial effects of eating vegetables and fruit outweigh the potential risk to human health from
exposure to nitrate through vegetables. This is based on the consumption of up to 400g mixed fruit
and vegetabels per day, which does not exceed the recommended daily intake for nitrate.

Larsen & Schiffer

Nitrate, which is also abundant in green leafy vegetables, has previously been considered merely as
an inert end product of NO metabolism (Moncada and Higgs, 1993) or as a potentially toxic
constituent in our diet (Tannenbaum and Correa, 1985). However, during the last decade its
importance in biological processes has been increasingly appreciated.
Peroxinitrate production

High nitrate containing medicine can cause headaches

What are beneficial effects of taking nitrate? i.e. cardiovascular system

Jones & Furgeson

Nitric oxide (NO) is essential to life. It is a gaseous signaling molecule that is produced continuously to
enable or facilitate a wide range of physiological processes including:

- Neurotransmission
- Vasodilatation
- Angiogenesis
- cellular respiration
- mitochondrial biogenesis
- muscular contraction

A failure to produce or use sufficient NO is a characteristic of many cardiovascular and metabolic

diseases.

It has been shown repeatedly that dietary NO3- intake results in a significant reduction of resting
blood pressure for several hours, consistent with an NO-mediated peripheral vasodilatation.

NO3- ingestion also has been reported to reduce the O2 cost of submaximal exercise, at least in
nonelite athletes. This may be a consequence of a reduced adenosine triphosphate (ATP) cost of
muscle force production or to enhanced mitochondrial efficiency.

Nitrate supplementation  Improved muscle efficiency and/or exercise economy  enhanced

exercise performance

Many recreational and elite athletes consume NO3--rich products, usually beetroot juice,
precompetition and/or to support training. However it seems that elite endurance athletes do not
benefit to the same extent as nonelite endurance athletes. This may be caused by:

- dose and duration of NO3- supplementation

- genetic or training effects on NOS activity
- Differences in muscle oxygenation
- Differences in mitochondrial function
- Differences in muscle fiber type composition between these populations

In vivo evidence suggests that the O2 demand of exercising muscle may be reduced during normoxic
exercise after dietary NO3- supplementation. Possibly due to:

- an increased phosphate/oxygen ratio of mitochondrial respiration

- and/or a reduced ATP cost of force production

Most recent studies report improvements in exercise economy and/or fatigue resistance in
moderately trained participants (VO2peak, <55 mL.kg-1.min-1 ) after NO3- supplementation. This
effect is less apparent in well-trained endurance athletes.
It is plausible that elite athletes, in whom NOS activity, plasma [NO2-], and muscle oxygenation will all
be enhanced by intensive training, might benefit from greater and/or more protracted NO3-
supplementation. However, high-level endurance athletes primarily have more developed type I
fibers and not so much type II fibers. NO3-treatment seems effective mainly for type II fibers so it may
not be efficacious on highly trained endurance athletes.

Affourtit & Baily

NO is widely believed to mediate the benefits of NO3- including protection against cardiovascular
disease and the metabolic syndrome.

Larsen and colleagues reported that a 3-day supplementation with 0.1 mmol·kg−1 NaNO3· day−1
lowered pulmonary O2 uptake (V˙O2) by ~3–5% in humans completing sub-maximal cycling exercise.

Bailey and co-workers subsequently observed a 5% lower V˙O2 during low-intensity cycling exercise
and a 16% improvement in the tolerable duration of high-intensity exercise over days 3–6 of a 6-day
supplementation period with 5.6 mmol NO3-⋅ day-1, administered as 500 mL NO3- -rich beetroot
juice· day−1.

NO3--induced improvements have been observed in humans completing walking, running, cycling,
rowing and kayaking exercise, and positive responses arise both acutely, i.e., 1–3 h after NO3-
ingestion, and after prolonged NO3- supplementation over 3–15 days.

Larsen & Schiffer

Several lines of research indicate that the nitrate-nitrite-NO pathway is involved in:

- regulation of blood flow

- blood pressure
- cell signalling
- glucose homeostasis
- tissue responses to hypoxia

Nitrate has an oxygen sparing effect that occurs without changing circulating lactate concentrations
and with maintained or increased work performance. Indicating more efficient aerobic metabolism.

What happens in the muscles when you supplement nitrate?

Jones & Furgeson

O2 tension in muscle fibers (PO2): PO2 fluctuates from approximately 40 mm Hg (5% O2) at rest to
as low as approximately 2 mm Hg (~0.4% O2) during strenuous exertion.
tissue O2 delivery (QO2):
Restriction in QO2 to skeletal muscle reduces maximal oxidative metabolic rate (Qmax), which is
reflected in a slowing of muscle phosphocreatine (PCr) recovery kinetics after exercise.

Collectively, the actions of NO2- and/or NO on O2 consumption and vasodilatation might enable a
more precise local matching of blood flow to metabolic rate in active and non-active muscle tissue.

NO production via NOS enzymes requires O2. NO has an important role in regulating blood flow and
cellular respiration. A back up system is needed to guarantee NO bioavailability under low-PO2 in
skeletal muscle conditions during exercise.

- NO can be produced alternatively via the NOS-independent, single-electron reduction of

NO2- (which is obtained through dietary NO2- and NO3- and oxidation of endogenous NO).
This NO2- reduction is stimulated by low PO2 and low pH.  mainly occurs in type II fibers,
since they operate at a lower PO2.
 - It has been shown that Bolus infusion of NO2- offsets acute pharmacological NOS inhibition
and restores cardiovascular function to control values.

NO-mediated signalling pathways that regulate skeletal muscle tissue O2 delivery (QO2) are also O2
dependent. A low PO2 in active muscle stimulates erythrocyte-derived NO-mediated vascular
relaxation which increases blood flow and provides increased tissue O2 delivery to the active muscle
tissue.
A high PO2 would indicate inactive muscle tissue, with reduced metabolic needs compared active
muscle tissue. A high PO2 therfore causes vasoconstriction as increased blood flow and increased
oxygen delivery is not necessary.

NO regulates myocyte calcium flux via S-nitrosylation of thiols contained in ryanodine receptor (RyR1)
Ca2+ channels. NO only influences this at low PO2 (~10 mm Hg). So that contractility in active muscle
is increased.

NO may also regulate mitochondrial respiration by reversible inhibition of cytochrome c oxidase in

competition with O2, such that greater inhibition by NO would occur at low physiological PO2.

It is important to note here that although it is likely that the effects of dietary NO3- largely are
mediated by NO, it is possible that NO2- and/or other bioactive NO3- derived molecules also play a
role in the physiological effects observed after NO3- supplementation

24h of NO3 supplementation (9.3 mmol NO3-) reduced the extent of muscle metabolic perturbation
during high-intensity knee-extension exercise and enhanced exercise tolerance in moderate hypoxia.
Dietary NO3- supplementation also abolishes the reduction in the rate of pH-independent PCr
recovery, which typically is observed in hypoxia, and essentially restores muscle Qmax to values
similar to those observed in normoxia.  The faster Cr recovery is linked to improved muscle
oxygenation during immediate post exercise period in hypoxia but not in normoxia  NO3-
supplementation may facilitate NO production and enable greater muscle oxygenation, offsetting
deleterious effects of hypoxia.
The ability to drive O2 from the capillary into the myocyte and, thus, to the mitochondria is
dependent on the driving pressure (i.e., microvascular PO2, PmvO2) and the resultant pressure
gradient. PmvO2 = QO2/VO2

Muscles predominantly composed of Type II fibers regulate their PmvO2 very differently from their
slow-twitch (Type I) counterparts.
Specifically, significantly lower blood flow (relative to V˙ O2) results in a much lower contracting
PmvO2 (i.e., ~10 vs 20 mm Hg; Fig. 1), ultimately forcing greater reliance on fractional O2 extraction
in Type II compared with Type I muscle

Affourtit & Baily

Dietary NO−3 benefits on the O2 cost of exercise likely arise from:

- increased efficiency of ATP synthesis: NO3- increases the rate of human skeletal muscle PCr
recovery after exercise in hypoxia suggesting an augmented maximum rate of oxidative ATP
synthesis.
- and/or of skeletal muscle work: NO3- lowers the ATP cost of contractile force production.
In vitro experiments (C2C12 myocytes) show that beetroot juice provokes mitochondrial biogenesis
and modestly increases basal cellular respiration without affecting respiratory capacity and proton
leak.  indicate improved mitochondrial coupling efficiency as beetroot juice has increased the
proportion of total O2 consumption coupled to ATP synthesis.

- Larsen et al. (2011) show that skeletal muscle mitochondria isolated from NO3- -
supplemented subjects exhibit higher respiratory control and P/O ratios than mitochondria
from non-supplemented controls
- Increases in P/O ratio correlate with NO3- -induced decreases in whole-body O2 uptake
during exercise.

NO2- improves efficiency of oxidative ATP synthesis without apparent effect on proton leak, which
agrees with the beetroot juice effects on C2C12 respiration

- NO2- activates AMPK in rat aortic smooth muscle cells thus stimulating mitochondrial
biogenesis, and increasing coupling efficiency and cellular respiratory control.
- NO2- activates PKA in cultured cardiomyocytes, stimulating mitochondrial fusion and again
increasing cellular respiratory control.
- NO2- also activates PKA in cultured adipocytes, increasing mitochondrial fusion, and
stimulating glucose uptake
- NO2- increases proliferation of muscle and epithelial cells.

a. What happens for the type I muscle fibers?

b. What happens for the type II muscle fibers?

Jones & Furgeson

Type II (fast-twitch) fibers differ in terms of myofibrillar protein content and calcium handling,
typically have lower mitochondrial and capillary density, and, therefore, rely relatively more on
nonoxidative compared with oxidative pathways of ATP resynthesis. This results in greater fatigability
compared with Type I muscle.
Type II muscles and muscle fibers operate at a lower PO2 than do Type I muscles and muscle fibers. It,
therefore, is conceivable that the physiological effects of NO2- are more manifest in Type II than Type
I muscle.

Ferguson et al. (12) tested the hypothesis that dietary NO3- preferentially would increase blood flow
and thus PmvO2 in exercising muscles composed of Type II fibers. In rats running at approximately
70% VO2max, beetroot juice ingestion (1 mmolINO3 jj1 Ikgj1 for 5 d) elevated blood flow to muscles
and muscle portions composed of 66% or more Type II muscle fibers. This raises PmvO2 in Type II, but
not Type I, muscles during contractions.

Primarily in type II fibers supplementation with NO3- enriched water resulted in an increased level of
calsequestrin 1 and the dihydropyridine receptor, which ultimately increased the rate of muscle force
development.

Type II fibers experience ADP, K+ and H+ build up during hypoxia resulting in fatigue

How does nitrate improve exercise economy (different intensities: low, mid, high)?
(figure 2)
Jones & Furgeson
During whole body exercise in hypoxia, NO3- supplementation has been shown to reduce the O2 cost
of submaximal exercise by approximately 4% to 7%.

Muscle oxygenation was elevated by 4% to 5% at rest and during both submaximal and maximal
intensity exercise after NO3- supplementation in hypoxia (11% O2).

Dietary NO3- ingestion also attenuated the hypoxia-induced reduction in exercise tolerance during
both severe-intensity constant work rate exercise (by 9%) and exhaustive incremental exercise (by
5%)

NO3- supplementation seems more effective in states of hypoxia then normoxia.

How does it translate into practice for an individual?