Free Radical Research, January 2014; 48(1): 3042

2013 Informa UK, Ltd.

ISSN 1071-5762 print/ISSN 1029-2470 online
DOI: 10.3109/10715762.2013.825043

REVIEW ARTICLE

Free radicals and sprint exercise in humans

D. Morales-Alamo & J. A. L. Calbet
Department of Physical Education, University of Las Palmas de Gran Canaria, Campus Universitario de Tara s/n,
Las Palmas de Gran Canaria, Canary Island, Spain
Abstract
Sprint exercise ability has been critical for survival. The remarkably high-power output levels attained during sprint exercise are achieved
through strong activation of anaerobic, and to a lesser extent, aerobic energy supplying metabolic reactions, which generate reactive oxygen
and nitrogen species (RONS). Sprint exercise may cause oxidative stress leading to muscle damage, particularly when performed in severe
acute hypoxia. However, with training oxidative stress is reduced. Paradoxically, total plasma antioxidant capacity increases during the
subsequent 2 h after a short sprint due to the increase in plasma urate concentration. The RONS produced during and immediately after
sprint exercise play a capital role in signaling the adaptive response to sprint. Antioxidant supplementation blunts the normal AMPK and
CaMKII phosphorylation in response to sprint exercise. However, under conditions of increased glycolytic energy turnover and muscle
acidication, as during sprint exercise in severe acute hypoxia, AMPK phosphorylation is also blunted. This indicates that an optimal level
of RONS-mediated stimulation is required for the normal signaling response to sprint exercise. Although RONS are implicated in fatigue,
most studies convey that antioxidants do not enhance sprint performance in humans. Although currently controversial, it has been reported
that antioxidant ingestion during training may jeopardize some of the benecial adaptations to sprint training.
Keywords: exercise, antioxidant, redox signaling, reactive oxygen species, xanthine dehydrogenase/xanthine oxidase

Introduction
A free radical is a term dening an atom (or molecule)
that contains one or more unpaired electrons that is capable of independent existence. Reactive oxygen species
(ROS) are compounds (or atoms) which may be free radicals containing oxygen or nonradical but have reactive
derivatives of oxygen, like hydrogen peroxide and others.
Reactive nitrogen species (RNS) are nitrogen radicals and
nonradical nitrogen reactive molecules with a nitrogen
reactive center [1]. Collectively, ROS and RNS are termed
reactive oxygen and nitrogen species (RONS). Since
RONS may cause damage to cellular structures and
biomolecules which is limited or prevented by complex
enzymatic and nonenzymatic systems, RONS has been
considered as harmful and emphasis was put to counteract
its eects [2]. Nevertheless, RONS have critical roles as
signaling molecules [3], contribute to the sensing mechanisms of oxygenation [4], and are necessary for normal
force development in skeletal muscle [5] and the adaptive
response to regular exercise [3].
Free radicals, ROS and RNS, may be generated from
multiple reactions undertaken in skeletal muscle during
and after exercise [1]. The primary free radicals generated
in skeletal muscle are superoxide (O2) and nitric oxide
(NO). These two free radicals react with other compounds
and form additional ROS and RNS, such us hydrogen
peroxide (H2O2), hydroxyl radicals (OH), singlet oxygen

(an electronically excited form of oxygen, highly reactive

but is not a radical), nitric oxide (NO, a weak reducing
agent), peroxynitrite (ONOO, a strong oxidizing agent
product of the reaction between superoxide and NO), and
hypochlorite (ClO, predominantly formed in neutrophils).
During sprint exercise, free radicals are mostly produced in chemical reactions coupled to the energy turnover. Therefore in this review, we will rst focus on human
data on the energy pathways and potential sources of
RONS during and following sprint exercise. Subsequently,
the contribution of RONS to oxidative damage, and the
putative antioxidant eects of sprint exercise will be
examined. To follow, recent studies on the inuence of
RONS-mediated signaling in response to sprint exercise
in normoxia and hypoxia will be addressed. To nalize,
the potential ergogenic eects of antioxidants on sprint
performance will be examined.
Energy metabolism during sprint exercise in man
Sprint exercise ability has been critical for survival of
many species either to hunt or to avoid being hunted.
Actually humans can achieve peak power output values
above 3000 W for a few seconds [67] and can sustain
mean power output values above 1000 W during a 30-s
sprint [8]. This requires remarkable amounts of energy in
a short time and the skeletal muscle is designed to provide
this energy using readily available energy sources as free

Correspondence: Jos A. L. Calbet, Departamento de Educacin Fsica, Campus Universitario de Tara, 35017 Las Palmas de Gran Canaria,
Canary Island, Spain. Tel: 0034 928 458 896. Fax: 0034 928 458 867. E-mail:lopezcalbet@gmail.com
(Received date: 29 May 2013; Accepted date: 10 July 2013; Published online: 7 August 2013)

Free radicals and sprint exercise

adenosine triphosphate (ATP) and phosphocreatine (PCr),

which can provide about 50% of the energy required for a
6-s long sprint, while the rest is mainly supplied by a high
glycolytic rate [9]. Glycolysis reaches maximum rates
within the rst 6 s of the sprint [10]. Thus, during 5- to
10-s sprints most of the ATP required is mainly provided
by anaerobic energy sources, however, as the sprint is prolonged the aerobic energy production contributes increasingly to the overall energy turnover [8,10] (Figure 1).
During a 30-s sprint, 7080% of the energy consumed is
provided by anaerobic pathways while the rest is supplied
through muscle glycogen oxidation [8,1011]. During the
rst 510 s of the sprint a rapid reduction of PCr stores
leads to an increase in free adenosine diphosphate (ADP)
[10], which stimulates the adenylate kinase reaction
(ATP AMP 2ADP). The latter prevents excessive accumulation of ADP and subsequent inhibition of myosin
ATPase, which may cause the termination of exercise [12]
(Figure 2). The adenylate kinase reaction produces adenosine monophosphate (AMP) [13], which is subsequently
deaminated to inosine monophosphate (IMP) and accumulates in the muscle [1416] through a process stimulated
by the drop in intramuscular pH [15,17]. A fraction of the
IMP produced during the sprint is converted into inosine
by the cytoplasmic 5nucleotidase and the latter to hypoxanthine by the purine nucleoside phosphorylase which is
then converted into xanthine by the xanthine oxidase
(Figure 2). The formation and release of hypoxanthine and
xanthine is a slow process and most of it occurs during
the rst 90 min of the recovery period [15] due to the low
activities of cytoplasmic 5nucleotidase [16] and purine
nucleoside phosphorylase [18] (Figure 3).
This reliance on anaerobic energy sources explains
why peak and mean power output during a 30-s sprint is
barely aected by severe hypoxia, despite a marked reduction in muscle oxygen supply [19]. This is possible
because the reduction in oxygen supply is compensated
for by increasing the energy produced through glycolysis
at the expense of greater muscle acidication [19].

PCr hydrolysis

15

Glycolysis
Oxidative phosphorylation

ATP turnover rate

(mmol.kg.dw1.s1)

12
9
6
3
0
0

15
Time (s)

30

Figure 1. ATP turnover rates from PCr hydrolysis, glycolysis, and

oxidative phosphorylation during a 30 s sprint [10].

31

Pi
ATP

ATPase

Pi
ATP

ATPase

ATP

ADP

AK
ADP

AMP
deaminase

IMP

AMP

NH3

Cytoplasmic
5-nucleotidase

Pi
Inosine

Pi

Purine nucleoside
phosphorylase

Ribose-1 phosphate

Hypoxanthine
Xanthine
oxidase

Xanthine
Xanthine
oxidase

Uric acid
Figure 2. Nucleotide metabolism during high-intensity exercise
leads to the production of uric acid, mostly in the liver, with net loss
of purines from the skeletal muscles.

Despite the short duration of sprint exercise high

oxidative rates can be achieved. For example, during a
Wingate test (the most common sprint used in research
performed on a cycle ergometer and lasting 30 s) subjects are able to reach 80% of their maximal oxygen
uptake in 25 s [8]. If the sprint is prolonged beyond 60 s,
more than 50% of the energy required is obtained from
aerobic processes [20]. If several sprints are repeated
with short recovery periods in between, the aerobic contribution to the total energy expended in each sprint
increases [21].
Potential sources of free radicals during sprint
exercise in man
Little is unknown about the main sources of RONS
during aerobic exercise [1] and much less is known
regarding sprint exercise. Most RONS are produced in
reactions coupled to energy metabolism, and during aerobic exercise, RONS production is increased as fatigue
ensues, in many instances preceded by an increased
involvement of the anaerobic energy sources. A particularity of sprint exercise is the large reliance on anaerobic
energy metabolism throughout the exercise accompanied
by early appearance of fatigue. Thus, it is likely that
RONS originate from similar sources during aerobic and
sprint exercise, but the relative contribution of each
source is likely to be dierent between both exercise
modalities.

32 D. Morales-Alamo & J. A. L. Calbet

35

Hypoxanthine (M)

30
25
20
15
10
5
0

Inosine (M)

5
4
3
2
1
0

PRE 0

10

15 20

25 30

35 40

45

Time (min) post-exercise

Figure 3. Plasma hypoxanthine and inosine concentration before
(PRE) and after high-intensity exercise at 120130% of VO2max
until exhaustion [16].

During sprint exercise, superoxide may be generated in

the mitochondria. Mitochondrial superoxide is produced
at a rate below 0.15% of the oxygen consumption (VO2)
[22]. However, even during aerobic exercise, when most
of the energy is provided by mitochondrial oxidation, the
contribution of superoxide to the overall RONS produced
is relatively low since skeletal muscle mitochondria are
predominantly in state 3 (ADP-stimulated [23]). Therefore, mitochondrial superoxide production may be even
lesser during sprint exercise than at rest, since in vitro
experiments have shown reduced H2O2 production in state
3 than in state 4 with either succinate or pyruvate/malate
as substrates [2425].
Superoxide may be also generated by the sarcoplasmic
reticulum, transverse tubules, or plasma membrane
NAD(P)H oxidase enzymes [1]. There is some evidence
indicating that contracting muscles can transfer electrons
from cytosolic NAD(P)H to the plasma membranes
through either NADH-cytochrome b5 oxidoreductase or
NAD(P)H quinone oxidoreductase (NQO1) [26]. This can
lead to the formation of superoxide at the cell surface.
Xanthine oxidase, present in low amounts in human skeletal muscle and more abundant in endothelial cells [27],

may also contribute to superoxide generation [28]. Since

hypoxanthine, the substrate for xanthine oxidase is increased
during and, particularly following, sprint exercise, this
reaction may be an important source for superoxide during
and after sprint exercise.
The increase of intracellular calcium during muscle
contraction has been shown in in vitro hemidiaphragm
preparations, which are relatively hypoxic [29], to activate
the calcium-dependent phospholipase A2 (PLA2) isoform
(cPLA2), which stimulates intracellular ROS production
[30]. Nevertheless, cyclooxygenase inhibitors do not blunt
superoxide release from contracting muscle [31], and it
remains unknown what role, if any, cPLA2 plays in the
RONS production during sprint exercise.
Nitric oxide is produced via the conversion of L-arginine to L-citrulline by three isoenzymes called nitric oxide
synthases (NOS), all present in skeletal muscle. Neuronal
(nNOS or NOS-1) and endothelial (eNOS or NOS-3) are
expressed in skeletal muscles, and regulated by the interaction of Ca2 with calmodulin. Inducible NOS (iNOS,
NOS-2) is expressed during inammatory responses [32].
Ca2/calmodulin-dependent protein kinase II (CaMKII) is
phosphorylated in response to sprint exercise and may
upregulate nNOS transcription [33]. nNOs possesses a
calmodulin domain, where Ca2/calmodulin binds during
exercise activating nNOS. The physiological relevance of
this system is emphasized by the increased expression of
nNOS [34] and eNOS in response to high-intensity training [35]. The activity of nNOS is regulated by phosphorylation [36]. Phosphorylation at Ser847 and Ser1412 reduce
and increase nNOS activity, respectively [33]. Although it
has been shown in humans that sprint exercise elicits
CaMKII phosphorylation (activation), it remains unknown
what the eect of CaMKII is on nNOS during and the
following minutes after sprint exercise. CaMKII could
phosphorylate nNOS at Ser847 and inhibit the enzyme by
impeding the binding of calmodulin [33]. However,
AMPK is phosphorylated (activated) during sprint exercise [3738] and AMPK has been reported to phosphorylate nNOS at Ser1412 in human skeletal muscle
immediately after sprint exercise [39]. Additional kinases
and phosphatases, like protein kinase A (PKA), CaMKI,
protein kinase C (PKC), and phosphatase 1, are known to
regulate nNOS phosphorylation [33], however, their role
in sprint exercise has not been dened yet.
Another potential source of RONS during sprint
exercise is through the metabolism of an increased catecholamine production [40]. RONS may be generated due
to autoxidation of catecholamines [4142] and the formation of highly reactive deaminated catecholaldehyde
metabolites by monoamine oxidase [4243].
Is sprint exercise causing oxidative stress in humans?
RONS are produced and released into the circulation during submaximal muscle contractions in humans [44].
Under some circumstances, the rate of RONS production
exceeds the capacity of the antioxidant systems leading to

Free radicals and sprint exercise

oxidative stress and potentially oxidative damage to cellular structures [45]. A major potential contributor to oxidative stress during sprint exercise is acidosis [4647].
During sprint exercise, glycolysis is maximally stimulated
causing high production of pyruvate and lactate [10,19,48].
Lactate may act as a good antioxidant at physiological pH,
due to its scavenging activity toward both superoxide and
OH, which is concentration dependent [49] (Figure 4).
Lactate addition to hepatocytes treated with a ferric nitrilotriacetate solution (Fe-NTA) reduces the amount of malondialdehyde (MDA, a marker of lipid peroxidation)
produced [49]. Likewise, lactate attenuates the oxidant
eects of O2 in ischemic reperfused hearts [50]. However,
lactate is produced equimolarly with 1 H, and H accelerates the rate of dismutation of O2 to H2O2, which can
react with Fe2 and generate OH. Acidosis promotes the
conversion of O2 to the more reactive and more lipid
soluble hydroperoxyl radical HO2 and facilitates the dissociation of protein bound iron [47], a strong oxidant.
Pyruvate may act as an antioxidant during sprint exercise. Muscle pyruvate production is markedly increased
during sprint exercise, leading to intramuscular accumulation, although the concentration reached is much lower
than that of lactate [10,19]. In pyruvate-perfused hearts,
glutathione peroxidase activity is even increased [50].
Moreover, pyruvate is able to act as a hydrogen peroxide
scavenger preventing OH generation by the iron-catalyzed
Fenton reaction [51]. Pyruvate has been observed to
enhance recovery of rat hearts after ischemia and reperfusion by preventing free radical generation [52].
Few studies have reported increased oxidative stress in
response to sprint exercise in humans. In most of these
studies the assessment is indirect, based on measurements
of oxidants, antioxidants, oxidation products, and the antioxidant/pro-oxidant balance in blood. Only in two studies
biomarkers of oxidative stress were assessed in skeletal
muscle biopsies [19,38]. We did not see signicant
changes in skeletal muscle protein carbonylation measured

immediately after and during the rst 2 h after a Wingate

test performed in normoxia [19]. However, plasma protein
carbonylation was increased after a 30 s sprint (Wingate
test) performed in hypoxia [19]. This was likely facilitated
by the greater muscle acidication observed immediately
after the sprint exercise in hypoxia compared to that of
normoxia [19].
Increased lipid peroxidation (lipid hydroperoxides and
MDA) and reduction of blood nonenzymatic antioxidants
(uric acid and -tocopherol) have been reported in blood
(obtained from and arm vein) after a Wingate test in 18
physically active young men [53]. However, no change in
thiobarbituric reactive species (TBARS) has been observed
after one or after four repeated Wingate tests interspaced
by 60-min recovery periods [54]. Reduced levels of tocopherol and -carotene have been reported 5 and
10 min after a Wingate test in seven physically active men,
while ascorbic acid and uric acid concentrations increased
during the recovery period [55]. Twenty minute after the
Wingate test, lipid radicals measured by electron spin
resonance (ESR) spectroscopy are also elevated [5556]
(Figure 5). Likewise, erythrocyte superoxide dismutase
(SOD) activity was reduced immediately after the Wingate
test [56]. Although erythrocyte glutathione peroxidase
(GPx) did not change signicantly, erythrocyte gluthatione
(GSH) was lowered [56]. This was conrmed by Cuevas
et al. [54] who observed reduced blood GSH and increased
gluthatione difulde/gluthatione (GSSG/GSH) values
immediately after a single Wingate test and during the
following 2 h. Interestingly, Cuevas et al. also observed
increased nuclear factor B (NFB) activation in Peripheral blood mononuclear cells (PBMC) immediately after
a single Wingate test and the following 15, 60, and 120 min
of recovery [54], which could indicate activation by free
radicals reaching the PBMC during the Wingate test or
the recovery period [54].
Plasma hydroperoxide concentration (a biomarker of
oxidative stress due to dehydrogenation and peroxidation
(B)

100

80

*
*
*

60
40

20

DMPO-OH adduct (%)

DMPO-OOH adduct (%)

(A)

33

120
100

80

60
40
20

0
0

10 15
30
Lactate (mM)

60

10 15 30
Lactate (mM)

60

Figure 4. Scavenging of superoxide anion (O2) (A) and hydroxyl radical (OH) (B) by lactate. Eect of lactate concentrations on the
electron paramagnetic resonance (EPR) signal corresponding to the O2 and OH adducts with 5,5-dimethylpyrroline-N-oxide (DMPO).
OH was generated by decomposition of H O by ferrous ion in the presence or absence of lactate dissolved in plasma and O was
2 2
2
generated by a xanthinexanthine oxidase system at dierent concentrations of lactate. The 100% reference value corresponds to the level
of DMPO adducts produced in the corresponding control sample without lactate. (Means SE), *P 0.05 compared to the control without
lactate [49].

34 D. Morales-Alamo & J. A. L. Calbet

400

increased oxidative stress [68], which may be related to

the marked acidosis caused by this type of eort [69].

350

Is sprint exercise an antioxidant?

Lipid radical level (%)

300

250

200

150

100
0
R

End
of sprint

10

20

40

Time (min)

Figure 5. Lipid radical levels measured by electron spin resonance

spectroscopy at rest (R), after the Wingate test and during the
recovery period. Results are expressed as means SEM where
100% lipid radical level corresponds to the lipid radical level at rest.
*P 0.05 compared to rest [55].

of several cellular components) was increased after a single Wingate test in young men [5758]. In swimmers,
erythrocyte catalase and GPx activities are increased, and
GSH reduced immediately after a 100 m race [59].
Just 24 h after a single Wingate test, 8-hydroxy2-deoxyguanosine (8-OHdG, a marker of DNA damage)
was increased in PBMC [54]. This has been recently conrmed using a dierent exercise model (100 maximal
continuous knee extensions), in which DNA damage was
observed (as determined by 8-OHdG in PBMC and
muscle tissue) accompanied by increased plasma
lipid hydroperoxides, protein carbonyls, and hydrogen
peroxide [60].
In some studies using repeated sprints, increased biomarkers of oxidative stress have been reported. In sprint
athletes, conjugated dienes are increased 6 h after 6 150
m [61]. Others have reported no signicant changes in
plasma total antioxidant capacity (TAC) a measure of the
antioxidant capacity of the aqueous compartment (i.e.,
excludes the erythrocytes), which is accounted for by urate
(3565%), plasma proteins (1050%), ascorbate (024%),
and vitamin E (510%) [62] or the ratio of GSSG/GSH
immediately after repeated sprints (10 15 s sprints separated by 45 s of low-intensity exercise) [63]. However,
no indication of oxidative stress was observed in trained
men after repeated 60 s or 15 s sprints [64], suggesting
lower production of free radicals [65] or increased antioxidant capacity after training [6667], or the combination
of both. However, high-intensity exercise (maximal exercise that can be performed in 67 min) in well-trained
rowers during a 2000 m competition simulation elicited

Interestingly, TAC has been reported to be increased

immediately after sprint exercise [57,70] or during 1090
min after the sprint [63]. This may due to the increase in
vitamin C [71] and urate after high-intensity exercise
[7274]. During high-intensity exercise and the following
4575 min the muscle releases inosine and hypoxanthine
[7577]. During the recovery period the liver takes up
inosine and hypoxanthine, and oxidizes most of the hypoxanthine to uric acid, which is released into the circulation
[75]. Thus, most of the production of uric acid after intense
exercise occurs in the liver [75]. After high-intensity exercise the exercised (but not the rested) muscles uptake uric
acid, which may be oxidized to allantoin (and other products) [72,76]. Uric acid oxidation scavenges hydroxyl
radicals, lipid hydroperoxide radicals, singlet molecular
oxygen [78], and oxo-haem oxidants [79], while inhibiting
lipid peroxidation [80]. Uric acid also modulates vascular
endothelial function through the downregulation of nitric
oxide production [81]. In addition, formation of urateFe3 complexes dramatically inhibits Fe3-catalyzed
ascorbate oxidation, as well as lipid peroxidation [80]. At
physiological concentrations urate increases the stability
of ascorbate in human serum approximately vefold [82]
and urate may also protect against H-induced ascorbate
oxidation [82].
The increased TAC observed after a single sprint may
be the reason why GSH is reduced after a single sprint but
it is not further reduced when the sprint is repeated 60 min
after the rst, that is, when plasma urate concentration is
elevated, despite a similar power output in both sprints
[54]. This putative antioxidant action of urate accumulation in blood after a rst sprint on subsequent sprints is
supported by the antioxidant eects of urate administration prior to aerobic exercise in humans [83]. Moreover,
Hellsten et al. demonstrated that muscle urate concentration decreases during intense exercise due to conversion
into its oxidation product allantoin [66].
RONS-mediated signaling in sprint exercise
The muscle response to sprint training is initiated from a
single sprint by activating several signaling cascades regulating functional and structural adjustments that permit
adaptation of the muscle to reduce the alteration of homeostasis caused by the sprints. These adaptations allow
for a greater functional capacity, lower perturbation of
homeostasis, and faster recovery. The main functional
adaptations induced by sprint (or high-intensity) training
include improved: maximal oxygen uptake (VO2max)
[8485], glycolytic capacity (and power) [8586], pH
and electrolyte regulation [8788], muscle mass (hypertrophy) [89], rate of force development [90], fat oxidation
capacity [8687,91], and antioxidant capacity [66]. These

Free radicals and sprint exercise

functional changes depend mainly on structural and

functional adaptations mediated by a series of signaling
pathways regulating mitochondrial biogenesis, expression
of enzymes of aerobic and anaerobic metabolism, H,
lactate and electrolyte transporting proteins and pumps,
intracellular calcium homeostasis, antioxidant systems,
vascularization, myosin ATPase activity, protein synthesis
and degradation, glycogen storage, etc. [65,8587,9298]
Several pathways of signaling transduction involved in
these adaptations to exercise in skeletal muscle are regulated by RONS, including SIRT1/LKB1/AMPK/PGC-1
[28,99], mitogen-activated protein kinase (MAP kinase,
ERK1/2, p38) [100101], NF-kB [100,102], NOS,
calmodulin-dependent protein kinases CaMKs [103] and
JNK [101]. However, the potential role of free radicals on
skeletal muscle signaling pathways in response to sprint
exercise in human skeletal muscle has been scarcely
studied [19,38]. We have examined the role played by
RONS on the skeletal muscle signaling response to a
Wingate test by comparing the signaling responses after
the ingestion of either placebo or an antioxidant cocktail
containing -lipoic acid, vitamin C, and vitamin E, ingested
2 h prior to a single sprint. Performance and muscle metabolism were not altered by the antioxidants. In both conditions the NAD/NADH.H ratio was similarly reduced
(84%), and the AMP/ATP ratio similarly increased ( 21
fold) immediately after the sprints. The ingestion of antioxidants prior to the sprint prevented Thr172-AMPK
phosphorylation by two potential mechanisms. First, antioxidant ingestion blunted Thr286-CaMKII phosphorylation,
a potential upstream kinase for AMPK in skeletal muscle
[104105]. Secondly, antioxidants increased Ser485AMPK1/Ser491-AMPK2 phosphorylation immediately
after the sprint [38]. AMPK phosphorylation in Ser485 of
the 1 and Ser491 of the 2 subunits mitigates or completely
inhibits AMPK phosphorylation at Thr172 [106109].
RONS may activate CaMKII through modication of
the Met281/282 pair within the regulatory domain, blocking
reassociation with the catalytic domain and preserving
kinase activity via a similar but parallel mechanism to
Thr286 autophosphorylation [110]. Thus, reducing RONS
by antioxidant administration may lower CaMKII activity
by a mechanism unrelated to sarcoplasmic Ca2. Since
both AMPK and CaMKII stimulate glucose transporter
4 (GLUT4) expression, abrogation of the exercise-induced
upregulation of GLUT4 with antioxidants may blunt the
exercise training-induced increase of insulin sensitivity. In
fact, it has been reported that antioxidants (a combination
1000 mg/day of vitamin C with 400 IU/day of vitamin E)
prevent the increase in insulin sensitivity observed after
training (85 min of exercise, 5 consecutive days of the
week for 4 weeks) as measured by glucose infusion rates
(GIR) during a hyperinsulinemic, euglycemic clamp [111].
Sprint exercise in severe acute hypoxia (equivalent to
5300 m above sea level) elicits higher glycolytic rate,
greater reductions of the NAD/NADH.H ratio, lower
muscle pH and increased protein carbonylation in skeletal
muscle and plasma than a similar sprint in normoxia, suggesting increased RONS production in hypoxia [19]. The

35

increased acidication combined with the greater reduction of the NAD/NADH.H ratio and the associated protein carbonylation indicates that during the sprint in
hypoxia RONS production is greater than in normoxia and
may be eliciting some oxidative stress. This conclusion is
also supported by the changes observed in muscle signaling after the sprint. Despite this increased RONS production, Thr172-AMPK phosphorylation was completely
blunted after sprint exercise in hypoxia [19]. Since the free
AMP/ATP ratio, the main mechanism eliciting Thr172AMPK was increased to the same extent after the sprint
in hypoxia and normoxia, other mechanisms must intervene to explain how Thr172-AMPK phosphorylation after
sprint exercise in severe hypoxia is abrogated. It was found
that SIRT1 protein was reduced after sprint exercise in
hypoxia [19]. SIRT1 is an NAD-dependent deacetylase
that activates (deacetylates) LKB1 [112]. Both the reduction in SIRT1 protein and the lower NAD after the sprint
in hypoxia may result in lower SIRT1 activity [113], and
hence lower activation of LKB1 blunting the expected
Thr172-AMPK phosphorylation.
These experiments support a critical role of RONSmediated signaling for Thr172-AMPK and Thr286CaMKII phosphorylation in response to sprint exercise.
Since both kinases play a role in mitochondrial biogenesis,
fat oxidation, and glucose uptake in skeletal muscle, the
ingestion of antioxidants immediately before a sprint exercise may reduce some of the expected health-related benets of sprint exercise training or blunt the improvement
in performance [114].
Sprint exercise in severe hypoxia induces oxidative stress
and alters signaling
Sprint exercise, particularly in severe hypoxia shares some
similarity with ischemia reperfusion (IR) phenomena.
During skeletal muscle ischemia, the supply of oxygen
and substrates for oxidative phosphorylation is stopped.
The lack of oxygen arrests oxidative phosphorylation and
the cells have to rely on limited anaerobic energy sources.
After some time in ischemia, anaerobic ATP production
is insucient to match the ATP demand, leading to
reduced sodiumpotassium and calcium pump activities,
resulting in the increase of intracellular sodium and calcium. Concomitantly, orthophosphate accumulates; there
is loss of adenine nucleotides and intracellular accumulation of fatty acids, lactic acid, and H. Upon reperfusion,
the mitochondrial respiratory chain produces a burst of
ROS, mostly superoxide from respiratory complexes I and
III [115116]. Superoxide damages the iron sulfur center
in aconitase, releasing ferrous iron [117], and is dismutated to hydrogen peroxide, which reacts with ferrous iron
to form OH, while the reaction of superoxide with NO
generates peroxynitrite [118]. The burst of OH and
peroxynitrite damages most mitochondrial components,
particularly phospholipids [119], altering respiratory complexes I, II, III, and IV [115,120], and increases
membrane permeability [121]. This causes mitochondrial
permeability transition pore opening leading to immediate

36 D. Morales-Alamo & J. A. L. Calbet

depolarization of membrane potential, matrix swelling,
outer mitochondrial membrane rupture, and release of
proapoptotic molecules such as cytochrome c into the cytosolic compartment, where it activates cell apoptosis [122].
An important source of superoxide during IR is the
xanthine oxidase reaction [123]. This enzyme normally
exists as an oxidized nicotinamide-adenine dinucleotide
(NAD)-dependent dehydrogenase (XDH) in nonischemic cells and is converted to its oxidant form xanthine
oxidase with ischemia due to the calcium-mediated activation of a protease that irreversibly converts XDH into xanthine oxidase (Figure 6). Hypoxanthine production is
increased during ischemia, but to be converted into xanthine by xanthine oxidase requires O2 that is absent during
ischemia. On reperfusion O2 is provided and superoxide
is produced by the xanthine oxidase reaction [123].
Endothelial NOS can produce considerable amounts of
superoxide in the absence of sucient tetrahydrobiopterin
[124]. Constitutive NOS monomers consist of a avincontaining reductase domain, a heme containing oxygenase domain, and a regulatory calmodulin-binding
sequence [125]. Calcium-calmodulin binding stimulates
the transfer of electron from NAD(P)H into the avin centers and leading to the activation of the NOS reductase
[126]. Then the electrons are transferred to the heme
[127]. Calmodulin-triggered electron transfer to heme
causes rapid enzymatic oxidation of NAD(P)H in the presence of O2 [126]. In the absence of L-arginine, the Ca2promoted CaM binding triggers an even greater rate of
ATP

Ischemia

AMP

Adenosine

Inosine

Xanthine
dehydrogenase
Protease

Xanthine oxidase

Xanthine+O2

Hypoxanthine

SOD

Catalase
H2O2
H2O

O2

Reperfusion

OH+OH+O2
O2
NOS

Ischemia

Ca2+-CaM
Cytoplasmatic Ca2+ + CaM

Ischemia

Figure 6. Mechanisms of RONS generation during ischemia and

reperfusion.

NAD(P)H oxidation and heme-catalyzed reduction of O2

to superoxide [124,127]. During episodes of prolonged
Ca2 inux, as it might occur during ischemia, NOS might
be capable of depleting L-arginine, further facilitating the
generation of superoxide. RONS production peaks during
the rst 5 min of reperfusion [122].
Despite the metabolic similarity between sprint exercise and the initial events observed during IR episodes,
there are great dierences between the two. During sprint
exercise, even in severe hypoxia, the muscle O2 stores are
not completely depleted, oxidative phosphorylation is not
inhibited, ATP is not depleted, and there is no sign of
disruption of mitochondrial respiration [128]. This is
likely due to the fact that during sprint exercise muscle
blood ow is intermittent but is not completely interrupted. Even after repeated bursts of high-intensity exercise, the capacity to achieve VO2max is preserved [21]. In
contrast, IR is associated with mitochondrial uncoupling
and reduced skeletal muscle mitochondrial VO2max [129].
Upon cessation of exercise reactive hyperemia may facilitate the production of superoxide by xanthine oxidase,
particularly if the sprint exercise is performed in severe
hypoxia and the recovery after exercise takes place in normoxia. However, the physiological relevance of IR phenomena in RONS production during sprint exercise has
not been established.
In agreement with increased oxidative stress during
sprint exercise in hypoxia is the increased levels of protein
carbonylation in plasma and skeletal muscle, combined
with the observed reduction of SIRT1 protein after the
sprint in hypoxia. In cell cultures H2O2 induces JNK1
phosphorylation which then phosphorylates SIRT1 [130].
SIRT1 phosphorylation causes its translocation to the
nucleus and an increase of its deacetylase activity while
committing SIRT1 to degradation at proteasome in few
minutes [113] .
In both conditions, normoxia with antioxidants and
hypoxia the abrogation of the normal Thr172-AMPK
phosphorylation occurred with a concomitant increase
of Ser485-AMPK1/Ser491-AMPK2 phosphorylation,
indicating that an excessively high or low level of RONS
during sprint exercise may elicit Ser485-AMPK1/Ser491AMPK2 phosphorylation and subsequent blunting of the
expected Thr172-AMPK phosphorylation.
phosphorylation
Ser485-AMPK1/Ser491-AMPK2
may be elicited by protein kinase B (Akt) [108] and
cAMP-dependent protein kinase (PKA) [106]. However,
the role played by these two kinases on Ser485-AMPK1/
Ser491-AMPK2 phosphorylation remains to be elucidated. One potential mechanism by which antioxidants
could facilitate an increased Ser485-AMPK1/Ser491AMPK2 phosphorylation is by promoting Thr308-Akt
phosphorylation. Interestingly, resveratrol (a SIRT1 activator) decreases phosphoinositide 3-kinase (PI3K) activation in cultured muscle cell lines [131]. Since PI3K is an
upstream kinase for Thr308-Akt phosphorylation [132], the
reduced SIRT1 protein levels after the sprint in hypoxia
could account, at least in part, for the increased Thr308-Akt
phosphorylation after the sprint in hypoxia (Figure 7).

Free radicals and sprint exercise

37

(A)

(B)

Figure 7. Inuence of RONS in the regulation of AMPK phosphorylation in response to sprint exercise. The expected Thr172-AMPK
phosphorylation in response to sprint exercise is blunted by changes in RONS levels. A. In severe acute hypoxia (FiO2:0.10) there is increased
RONS production resulting in blunted AMPK phosphorylation by at least three potential mechanisms. First, the increase in RONS is
accompanied by a greater reduction of the NAD/NADH.H and, hence LKB1 activation. Secondly, the total amount of SIRT1 protein is
reduced. Thirdly, the sprint in hypoxia elicits an increase in Thr308-Akt phosphorylation which increases Ser485-AMPK1/Ser491-AMPK2
blunting Thr172-AMPK phosphorylation. B. The ingestion of antioxidants before sprint exercise abrogates the expected Thr172-AMPK
phosphorylation by decreasing ROS-mediated activation (autophosphorylation) of CaMKII, leading to reduced Thr172-AMPK phosphorylation
by an unknown mechanism. The reduction of ROS-mediated signaling is accompanied by increased Ser485-AMPK1/Ser491-AMPK2
phosphorylation.

Are antioxidants ergogenic during sprint exercise?

An ergogenic agent is a substance that if administered
before or during exercise increases performance by
enhancing power generation capacity or reducing fatigue.
Most studies on the ergogenic eects of antioxidant

supplementation in humans involved aerobic exercise or

training, and very few have focused on anaerobic exercise
or training (for review see [1,133]).
Maximal force production is achieved at optimal levels
of RONS [5]. Reducing ROS with antioxidants decreases
force production in unfatigued skeletal muscles, while a

38 D. Morales-Alamo & J. A. L. Calbet

slight increase in ROS enhances but an excessive elevation
of RONS decreases force production (for review see [1]).
In vitro and in situ animal studies have shown that ROS
scavenging may delay fatigue during submaximal prolonged exercise but fail at high contraction intensities. In
contrast, the majority of studies in humans show no ergogenic eect of antioxidant vitamins during prolonged
exercise [1,133] and the same applies to sprint [38] or
high-intensity exercise [134]. Moreover, allopurinol ingestion (300 mg/d 5 days) has no eect on sprint exercise
performance [77]. Increasing skeletal muscle carnosine
levels (carnosine may act as antioxidant in skeletal muscle
due to its H-buering properties) through beta-alanine
supplementation does not improve sprint performance in
humans [135136].
It has been reported that the ingestion of ubiquinone-10
(coenzyme Q10) during the training period may reduce the
improvement in sprint performance in humans [114,137].
More recent studies have reported no eect of coenzyme
Q10 supplementation on performance [138].
The ingestion of 250 ml of fresh red-orange juice
(Citrus Sinensis, Sanguinello cultivar) thrice a day, 1 h
before each meal during 4 weeks had no impact on
VO2max or exercise performance during incremental exercise to exhaustion despite improvement in oxidative stress
biomarkers [139].
Conclusions
During sprint exercise a high energy demand is satised
through a maximal or near maximal activation of the
anaerobic energy pathways with a minor contribution of
aerobic metabolism. These chemical reactions are thought
to be the main sources of RONS, particularly superoxide
and NO and derivatives during and immediately after
sprint exercise. During sprint exercise superoxide may be
generated in the mitochondria. However, mitochondrial
superoxide production may be even lower during sprint
exercise than at rest, due to lower ROS generation in state
3 than in state 4 respiration. NAD(P)H oxidase enzymes
from the sarcoplasmic reticulum, transverse tubules or
plasma membrane, and xanthine oxidase could also produce superoxide during sprint exercise or the rst minutes
of recovery. Nitric oxide production is likely increased
during sprint exercise as indirectly indicated by the
increase of NOS enzymes, in response to high-intensity
training.
When the rate of RONS production exceeds the capacity of the antioxidant systems oxidative stress and potentially oxidative damage to cellular structures may occur,
and this is most likely if high levels of muscular acidosis
are reached, as observed during sprint exercise in severe
hypoxia. However, few studies have reported increased
oxidative stress in response to sprint exercise in humans
based indirectly on measurements of oxidants, antioxidants, oxidation products, and the antioxidant/prooxidant balance in blood. Only in two studies biomarkers
of oxidative stress were assessed in skeletal muscle

biopsies, and only is severe hypoxia was the sprint associated with increased muscle protein carbonylation, as a
marker of oxidative stress. Paradoxically, total plasma
antioxidant capacity has been reported to be increased
during the 2 h following sprint exercise due to increased
liver production of urate.
RONS play a critical role as signaling molecules in
response to sprint exercise and antioxidant ingestion may
blunt part of the signaling response and hamper the adaptations to sprint training. Although the ingestion of antioxidants has no eects on exercise performance it blunts
AMPK and CaMKII phosphorylation, while facilitates
inhibitory phosphorylations in serine residues of AMPK.
Despite increased oxidative stress, AMPK phosphorylation is also blunted after sprint exercise in severe hypoxia,
by a mechanism that may implicate a reduced SIRT1
activity and hence lower activation of LKB1, an upstream
kinase for AMPK. More studies examining the inuence
of RONS on the sprint exercise-mediated signaling
responses in human skeletal muscle are necessary.
Declaration of interest
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of this
paper.
The authors are supported by grants from the
Ministerio de Educacin y Ciencia (DEP2009-11638;
DEP2010-21866, and FEDER).
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