in recent decades , intensive research in the field of oxidative damage indicates that exercise exacerbates the generation of reactive oxygen ( ros ) and reactive nitrogen species ( rns ) , some of which are free - radicals     [ 15 ] . a free - radical
ros / rns refer to oxygen or nitrogen containing free - radicals and their non - free - radical derivatives .
aerobic organisms produce free - radicals as a consequence of the oxygen metabolism , and , obviously , exercise causes an increase in oxygen utilization in mitochondria , resulting in elevated radical generation .
an estimated 2% to 5% of the oxygen consumed ( vo2 ) during normal mitochondrial metabolism in aerobic organisms may be converted to radicals and their products . during exercise ,
the vo2 uptake is higher than at rest   because of the increasing energy demand in many tissues , mainly in muscle .
the increase in oxygen uptake is related to substantial rise in the production of free - radicals generated during mitochondrial respiration .
several potential alternative sources of free - radicals , such as oxidase systems associated with membranes , nitric oxide production , and phagocytic processes , as well as an increase in lactate formation , as happens in exhaustive exercise , have been proposed to contribute significantly to the overproduction of free - radicals .   the elevation of metabolism by exercise results in a greater production of superoxide radicals     , which are dismutated to hydrogen peroxide ( h2o2 ) by superoxide dismutase ( sod ) .
h2o2 , a molecule that readily crosses cell membranes , can be detoxified to water and oxygen by other enzymes , that is , catalase ( cat ) and glutathione peroxidase ( gpx ) .
but when these transformations do not take place , h2o2 is , via the fenton reaction , converted to the hydroxyl radical , the most toxic ros because of its high reactivity     [ 6 , 12 ] .
numerous studies have shown that muscle cells also release superoxide into the extracellular space     [ 11 , 13 ] , so free - radicals readily reach the blood and act on other cells .
there is a general consensus that free - radical generation during exercise occurs predominantly in skeletal and heart muscles ; however , the invasive nature of obtaining biopsies from exercising humans limits their access .
therefore , some reports have claimed that the concentration of several oxidative stress markers increase immediately in the plasma after an acute exercise     [ 14 , 15 ] .  
antioxidants are defined as any substance that , when present at low concentration compared to those of an oxidizable substrate , significantly delays or prevents the oxidation of that substrate .
the first are low molecular weight proteins , which minimize oxidative damage by catalyzing chemical reactions to detoxify free - radicals in cells and tissues and can be synthesised due to the redox - activation of specific genes , primarily by affecting the binding of transcription factors to dna . during exercise , free - radicals generated can activate different redox - sensitive transcription factors , including nf-b , which leads to an elevation in the expression of some antioxidant enzymes     .
nonenzymatic antioxidants are generally small molecules that directly scavenge ros , preventing them from disfiguring lipids , proteins , or nucleic acids .
glutathione , an important intracellular antioxidant , is converted to its oxidized form ( gssg ) by gpx when it is used as a scavenger ; it is converted back to its reduced form by glutathione reductase ( gr ) . moreover ,
both animals and humans subjected to a long - term heavy exercise became more resistant to oxidative damage     [ 18 , 19 ] .
fisher et al . studied the activity of sod , cat , and gpx after high - intensity interval training , and they found that all of these enzymes activities were increased .
none of these studies have compared different types of exercise and observed changes in activities of four antioxidant enzymes and in the total antioxidant status in plasma .
herein , we evaluated the antioxidant status after three protocols of acute exercise in untrained healthy men .
plasma samples were collected for the measurement of cat , gr , gpx , and sod activities and total antioxidant status ( tas ) as a general marker of antioxidant defences .
thirty - four healthy male volunteers , aged 1929 ( 23.0  0.41 ) , were involved in this study . only males were included to avoid any distortion in the hormonal response to physical exercise caused by sex differences .
none of the subjects participated regularly in sport competitions , and they did not engage in any form of vigorous exercise or take medications for 24 hours before the study was performed .
all subjects underwent an extensive previous medical evaluation that included a history and physical examination , electrocardiogram , and biochemical profile to discard possible pathologies .
subjects gave their informed written consent to participate in the study , designed according to the principles of the declaration of helsinki ( 1964 ) and approved by the ethical committee of clinical investigation of aragon with the statement code c02/2010 .
exercise was performed on an electronically regulated cycloergometer between 08:00 and 10:30  a.m. after an overnight fast .
three ergometric tests were performed on each subject in random order at intervals of at least 1 week .
firstly , the maximal oxygen consumption ( vo2max ) and the maximal working capacity ( mwc ) were determined using a continuous progressive exercise test , where the work load was increased by 10 watts every minute .
the initial load was 100 watts less than the mwc determined in the first test .
finally , the subjects pedaled for 30 minutes at a submaximal workload chosen at 70% of the expected maximum for each individual     . peripheral venous blood samples obtained before exercise ( a ) , and immediately after a continuous progressive exercise test ( b ) , a strenuous test performed until exhaustion ( c ) , and a submaximal exercise ( 70% of the expected maximum workload ) for 30 minutes ( d ) were drawn by antecubital venepuncture and collected into lithium heparin - containing tubes . 10  ml of blood were collected at each sampling time .
blood samples were immediately centrifuged at 1.000 g for 10 minutes in a beckman allegra 64r refrigerated centrifuge ( fullerton , usa ) .
plasma was stored in 250  l aliquots at 30c until tas and enzyme activities were determined .
the total antioxidant status method relies on the ability of plasma antioxidant substances to inhibit the oxidation of 2,2-azino - di-[3-ethylbenzthiazioline sulphonate ] ( abts ) to the radical cation abts by metmyoglobin .
this molecule formed a relative stable green substance which was measured spectrophotometrically at 600  nm .
the capacity of the antioxidants in the sample to prevent abts oxidation is compared with that of trolox , a water - soluble tocopherol analogue , and is quantified as millimolar trolox equivalents .
when h2o2 was added at low concentration ( 0.2  m ) to a sample with cat , this enzyme catalyzed the transformation of this substrate to oxygen and water . to check the activity , a kinetic curve had to be measured during 30 seconds at  = 240  nm using a molar extinction coefficient 43.6  cm  m   to know the amount of h2o2 eliminated .
catalytic activity of gr was measured following the decrease in the absorbance at  = 340  nm for 3 minutes due to the oxidation of nadph to nadp , in presence of gssg , as described by goldberg and spooner .
the molar extinction coefficient is  = 6.2210  cm  m. to determine gpx activity , the continuous decrease in nadph concentration was measured , while gsh levels were maintained , following the flohe and gunzler method .
this method is based on the rise of the absorbance , during 3 minutes at  = 340  nm , because of the oxidation of nadph in presence of gsh , t - butyl hydroperoxide , gr , and the sample . the molar extinction coefficient used for the calculations is  = 6.2210  cm  m. one cat , gr , or gpx enzymatic unit ( units ) was defined as that amount of the enzyme that catalyzes the conversion of 1 micromole of substrate per minute .
sod activity was measured as the inhibition of the rate of reduction of cytochrome c by the superoxide radical ( o2 ) , observed at 550  nm .
o2 was first produced by the reaction of xanthine and oxygen catalyzed by xanthine oxidase     .
the definition of one unit of sod is the amount of protein that inhibits the rate of cytochrome c reduction by 50% .
data were analysed using repeated - measures anovas .   where significant main effects were found , pairwise comparisons were conducted using bonferroni adjustments for multiple comparisons .
total antioxidant status after all the three acute exercise protocols tested in this study : continuous progressive ( 1.62  0.07 ) , strenuous ( 1.66  0.08 ) and submaximal exercises ( 1.61  0.08 ) was elevated when compared with basal status ( 1.53  0.07 ) as illustrated in figure 1 .
the strenuous protocol produced the highest increase in cat activity ( 42.29  2.84 ) although the continuous progressive ( 24.85  2.35 ) and submaximal exercises ( 29.19  3.89 ) also significantly elevated cat activity relative to that at rest ( 18.54  1.97 ) ( figure 2 ) . both gr and gpx were elevated after submaximal exercises and even higher activity values of these enzymes were obtained after progressive and strenuous exercises ( figures 3 and 4 ) .
gr increases from a basal level of 0.021  0.002 to b : 0.031  0.003 , c : 0.031  0.003 , and d : 0.026  0.003 .
gpx level was also higher after exercises b : 1.03  0.3 , c : 1.02  0.03 , and d : 1.02  0.03 from a basal level of 0.94  0.02 .
the sod activity rose significantly after the strenuous exercise ( 0.028  0.007 ) , but it is after the nonexhaustive test ( 0.033  0.007 ) when the highest increase took place versus the basal value ( 0.018  0.004 ) ( figure 5 ) .
oxidative stress is an imbalance between ros / rns generation and antioxidants levels in favour of the former .
although there is a general consensus that free - radical overproduction due to exercise occurs mainly in the active skeletal muscles     [ 3234 ] , it is also well documented that exercise causes systemic oxidative damage .
.   found an augmentation of malondialdehyde , a marker of lipid peroxidation due to oxidative stress , in lymphocytes after a single bout of intense exercise .
in addition , ajmani et al .      noted an increase in membrane rigidity in erythrocytes after a strenuous exercise because of oxidative stress .
the main finding of our work was the increase of the tas in human blood after a single bout of exercise , either maximal or submaximal .
plasma tas is a combination of various antioxidant defences , including enzymatic and nonenzymatic systems . as shown herein , child et al
.      found higher tas levels in the plasma after an exhaustive exercise , that is , equivalent to a half marathon , and they concluded that uric acid , an important component of the antioxidant system , is responsible for one third of the tas increase . although uric acid may contribute to the antioxidant defences , another possible explanation for the elevation of tas is changes in other antioxidants , for example , gsh , melatonin , or the antioxidant enzymes .
we also demonstrate that cat , gpx , gr , and sod activities were higher after exercise than at rest .
previous reports have shown increases of gpx , gr , and cat activities in the skeletal muscle during exercise .
[ 39 , 40 ] observed in two consecutive studies that an acute bout of exercise increased antioxidant enzymatic activities in rat muscle . in the first report
the second study was designed to compare both maximal and submaximal protocols of exercise , and , as we found , gpx , gr , and cat activities were enhanced by exercise .
sod activity also increased after a single period of exhaustive exercise and during the recovery , following a nondamaging aerobic exercise in a cycloergometer . in this report
, muscle samples were taken from the vastus lateralis muscle of the exercised leg in humans by biopsy .
the effect of acute exercise on the antioxidant enzyme activities appears to be systemic , involving many organs .   following a single exhaustive swimming test
, terblanche   reported that cat activity was higher compared to the rest in several tissues : liver , heart , kidney , or lung in male and in female rats ; thereby , these authors proposed that increasing cat activity during acute exercise may be a defence mechanism in protecting the tissues against hydrogen peroxide generation .
also , cat and sod activities in rat lung tissue was increased significantly after the effort in young rats , a response that was significantly depressed in old rats .
the invasive nature of obtaining muscular biopsies from exercising humans limits their access ; thus , a number of studies have directed their efforts to the blood .
thus , aguil et al .      have recently found an elevation in cat and gr activities in the erythrocytes , while cases et al .     
found cat , gpx , and sod rises in lymphocytes after a single bicycle ride or swimming .
these authors proposed that oxidative stress and the necessity of protection against oxidative damage may be responsible , at least partially , for the elevation in the activity of these enzymes induced by exercise .
however , cat and gpx activities in human neutrophils decreased after a single bout of exercise     , perhaps because these cells released the enzymes into the plasma .
also reported that the activity of extracellular sod and gpx rose from basal status after an acute bout of aerobic .
although one hour later , during recovery , the antioxidant enzyme activities fell back to basal levels .
however , enzyme activities were increased again 24 hours later because a possible genetic upregulation of these enzymes     .
shin et al .   studied the effect of 6 months aerobic endurance training on the response to the acute exercise and reported that antioxidant enzyme activities were much higher than after pretraining test .
a possible explanation for the reinforcement of the antioxidant system due to training is that exercise stimulates the expression of the genes involved in the regulation of the antioxidant enzymes in a redox sensitive signal transduction pathways , mainly nf-b .
it is reported the importance of the nuclear factor in the expression of sod and inducible nitric oxide synthase ( inos ) in rat skeletal muscle .
ji et al .   observed that exercising rats injected with allopurinol , a competitive inhibitor of xanthine oxidase , attenuated ros production compared to untreated group , and also nf-b binding was dramatically enhanced by exercise but inhibited by allopurinol treatment .
it is also reported that physical exercise ( 80% vo2max for 1 hour ) resulted in nf-b activation in peripheral blood cells of physically fit young men . in the other hand ,
another study shows how an eccentric exercise for 45 minutes did not increase the expression of this transcription factor     .  
so , the influence of different exercise protocols of training in the redox status in many cells or tissues remains unclear     [ 48 , 5457 ] .
in conclusion , the data reported herein provide evidence that a single bout of maximal and submaximal acute exercise enhances plasma tas in healthy human subjects . this increase may be a consequence of an improvement in the plasma activities of cat , gpx , gr , and sod . based on our observations and previous studies which reported
that exercise causes a significant augmentation of the concentrations of several antioxidant scavengers , presumably due to its interactions with the free - radical overproduction     [ 58 , 59 ] , it seems reasonable to propose that exercise may play a beneficial role because of its ability to increase the antioxidant defense mechanisms against oxidative stress .
 antioxidant defences are essential for cellular redox regulation . since free - radical production may be enhanced by physical activity , herein , we evaluated the effect of acute exercise on total antioxidant status ( tas ) and the plasma activities of catalase , glutathione reductase , glutathione peroxidase , and superoxide dismutase and its possible relation to oxidative stress resulting from exercise . 
 healthy untrained male subjects ( n = 34 ) performed three cycloergometric tests , including maximal and submaximal episodes . 
 venous blood samples were collected before and immediately after each different exercise . 
 tas and enzyme activities were assessed by spectrophotometry . 
 an increase of the antioxidant enzyme activities in plasma was detected after both maximal and submaximal exercise periods . moreover , under our experimental conditions , exercise also led to an augmentation of tas levels . 
 these findings are consistent with the idea that acute exercise may play a beneficial role because of its ability to increase antioxidant defense mechanisms through a redox sensitive pathway . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions