Since superoxide dismutase (SOD), the first line defense enzyme in red blood cells (RBCs), was the most commonly found to be the one that changes under the influence of both acute and chronic exercise (Miyazaki et al., 2001; Groussard et al., 2003; Ookawara et al., 2003) and the one that differentiates between well trained subjects and controls (Evelson et al., 2002; Metin et al., 2003; Brites et al, 1999), we hypothesized that the level of its pre-exercise (basal) activity may determine the extent of oxidative stress induced by acute exercise. So, one of our recent papers dealt with the differences in response to acute exercise in subjects with different basal level of SOD activity (Djordjevic et al., 2010a). 24 young handball players were subjected to maximal graded exercise test and taken blood samples immediately before and after exercise. Maximal progressive exercise test induced significant changes in five out of six investigated parameters of redox state (O2**•**- was not changed significantly while H2O2, •NO, TBARS, SOD, CAT were) which suggests that either this kind of exercise is a potent oxidative stress inducer, or antioxidative defense system of our subjects was not efficient enough to resist the generated prooxidants. Interestingly, when analyzing the changes of these biochemical parameters in groups of athletes with different basal SOD activity (8 athletes with the lowest, 8 athletes with average and 8 athletes with the highest levels of basal SOD activity), H2O2, •NO, SOD and CAT were changed after exercise only in group of athletes with the lowest basal SOD activity. In other two investigated groups of athletes only TBARS changed significantly. So, statistical significance of the changes seen in the whole group of investigated athletes had its roots in the group of athletes with the lowest basal SOD activity which points out to the role of pre-exercise SOD activity in maintaining desirable redox state, both in exercise-related and exercise-nonrelated conditions.

Exercise and Oxidative Stress 53

state of 33 young handball players (age 16 - 19 years) with redox state of 14 age-matched adolescents who do not have regular physical activity (Djordjevic et al., 2011). The results showed that athletes and non-athletes did not differ significantly in levels of investigated prooxidants (O2•-, H2O2, •NO, TBARS), but athletes had significantly higher levels of SOD and lower level of CAT activity. Higher SOD activity in athletes was also found in other studies, including studies involving karate athletes (Naghizadeh et al, 2009) soccer players (Briites et al., 1999; Cazzola et al., 2003), rugby players (Evelson et al., 2002;), jump-trained subjects (Ortenblad et al., 1997) and students of Physical Education and Sports Sciences (Balakrishnan & Anuradh, 1998). Regarding CAT activity, there is a number of previous studies that also found that it's acitivity is decreased in athletes compared with controls, but there are also a number of studies that reported no change in CAT activity as a consequence of exercise training (Miyazaki et al., 2001; Lekhi et al., 2007; Metin et al., 2003; Ortenblad et al., 1997; Balakrishnan & Anuradh, 1998). According to numerous results from previous papers, it can be concluded that SOD behaviour as a consequence of chronic exercise training is clear (its' activity increases), while CAT has variant tendencies. The second objective of that work was to compare redox state of athletes with different duration of training experience. The results showed that there was no significant correlation between duration of sports engagement and redox state of athletes. The final objective of this work related to the correlation between morphofunctional characteristics and redox state of subjects. Athletes were divided into 3 groups based on ther aerobic capacity expressed through maximal oxygen consumption ((1) athletes with poor cardiorespiratory fitness (VO2max < 38.3 ml/kg/min), (2) athletes with average cardiorespiratory fitness (VO2max = 38.4– 45.1 ml/kg/min), and (3) athletes with good cardiorespiratory fitness (VO2max > 45.2 ml/kg/min)). Interestingly, athletes with low aerobic capacity had higher levels of H2O2 than athletes with average or high aerobic capacity, but lower levels of TBARS. Positive correlation was found between muscle percentage and TBARS. We hypothesize that the highest levels of TBARS in athletes with the highest aerobic power may be a consequence of the higher working capacity and consequently increased oxidative stress in working

**5.2 Systemic adaptation to oxidative stress induced by regular long term exercise 5.2.1 Morphofunctional effects of redox disturbance homeostasis induced by long**

The overall positive impact of exercise on growth and development of children and youth is one of the generally accepted facts. From the aspect of physiology, adaptation to muscle activity is presented through a systematic response in order to provide the best possible performance with the lowest energy deficit. Adaptation is an universal, common characteristic of all living beings, responsible for the survival of the organism under

Football, as a team game of simple rules without significant financial investment in equipment and space to play, is one of the most popular sports today, played by all nations, both sexes and various ages, regardless of skill level (Stolen, 2005). According to the intensity of football loads it can be classified as a highly intensive intermittent team sport (Bangsbo, 1994). For these reasons, it is evident that players are expected to have a high level of aerobic capacity and aerobic endurance. There is an evidently progressive trend of

musculature of these athletes.

**term exercise**

different conditions.

#### **5. The chronic effects of exercise on redox status of athletes**

#### **5.1 Assessment of the redox state of athletes and non-athletes**

During sports training athletes are continously exposed to various kind of stress. Adaptations to stress occur on numerous levels: from adaptations on subcellular, cellular and tissue level, to adaptations of organs and the whole organism of an athlete. Adaptations to stressors, i.e., structural and functional changes, enable improvement to occur in an athlete's sports performance. Since energy demands and oxygen consumption increase several-fold during exercise, it is thought that production of reactive oxygen and nitrogen species (RONS) also increases. Although much of early research viewed exercise-induced RONS production as a potential detriment to physiological function, more recent work investigates an alternative role for RONS production in regards to favorable exerciseinduced adaptations (Fisher-Wellman & Bloomer, 2009). It is thought that the basic principle of exercise, stress-adaptation, also takes place in events related to exercise-induced oxidative stress. The exercise-induced increase in free radical production can be seen as no different from other responses to exercise: a certain load disturbs homeostasis, resulting in adaptations in the body to be able to cope with a similar load in the future (Vollaard et al., 2005). The oxidative challenge-related adaptive process of exercise is systemic and includes increased antioxidant/damage repair enzyme activity, lower oxidative damage, and increased resistance to oxidative stress (Radak et al., 2008a). The upregulation of antioxidant system provides adaptive protection from RONS during subsequent training sessions, as well as during non-exercise related conditions.

Previously published papers on exercise-induced changes in redox state of athletes mainly explored redox state of athletes engaged in dominantly aerobic or anaerobic training protocols, while mixed (aerobic-anaerobic) sports were not so explored. According to some studies, anaerobically trained subjects have a better antioxidant enzyme activity in blood, in tissues and especially in working muscle (Evelson et al., 2002; Marzatiko et al., 1997; (Radak et al., 2008b). It was also shown that a controlled protocol of endurance training is followed by an increase in antioxidant enzyme activity in plasma and other tissues (Tanskanen et al., 2010; Selamoglu et al., 2000; Lekhi et al., 2007). Regarding mixed (aerobic-anaerobic) training, there are only a few studies suggesting that trained football and rugby players show lower oxidative stress at rest than sedentary subjects, and that there is a correlation between redox status and fitness level (Chang et al., 2002; Metin et al., 2003; Cazzola et al., 2003). Currently, there is little information available regarding exercise-induced adaptations of the antioxidant defense system in adolescent and child athletes. The absence of studies investigating the effect of exercise on young population is surprising considering the numerous metabolic and physiologic differences between children, adolescents and adults (Cooper et al., 2004; Armstrong et al., 2008; Boisseau et al., 2000). The lack of information on young individuals' biochemical responses to exercise is mainly attributable to ethical concerns and methodological constraints that limit invasive research in children/adolescents. Thus, our team performed a set of investigations on exercise induced changes in redox state of young, adolescent handball players. Effects of acute exercise on their redox state were described earlier in this chapter. Here we'll focus on relationship between duration of training experience, aerobic capacities and biochemical parameters that represent factors of redox homeostasis. The first objective of our investigation was to explore the relationship between sports engagment and redox state so we compared redox

During sports training athletes are continously exposed to various kind of stress. Adaptations to stress occur on numerous levels: from adaptations on subcellular, cellular and tissue level, to adaptations of organs and the whole organism of an athlete. Adaptations to stressors, i.e., structural and functional changes, enable improvement to occur in an athlete's sports performance. Since energy demands and oxygen consumption increase several-fold during exercise, it is thought that production of reactive oxygen and nitrogen species (RONS) also increases. Although much of early research viewed exercise-induced RONS production as a potential detriment to physiological function, more recent work investigates an alternative role for RONS production in regards to favorable exerciseinduced adaptations (Fisher-Wellman & Bloomer, 2009). It is thought that the basic principle of exercise, stress-adaptation, also takes place in events related to exercise-induced oxidative stress. The exercise-induced increase in free radical production can be seen as no different from other responses to exercise: a certain load disturbs homeostasis, resulting in adaptations in the body to be able to cope with a similar load in the future (Vollaard et al., 2005). The oxidative challenge-related adaptive process of exercise is systemic and includes increased antioxidant/damage repair enzyme activity, lower oxidative damage, and increased resistance to oxidative stress (Radak et al., 2008a). The upregulation of antioxidant system provides adaptive protection from RONS during subsequent training sessions, as

Previously published papers on exercise-induced changes in redox state of athletes mainly explored redox state of athletes engaged in dominantly aerobic or anaerobic training protocols, while mixed (aerobic-anaerobic) sports were not so explored. According to some studies, anaerobically trained subjects have a better antioxidant enzyme activity in blood, in tissues and especially in working muscle (Evelson et al., 2002; Marzatiko et al., 1997; (Radak et al., 2008b). It was also shown that a controlled protocol of endurance training is followed by an increase in antioxidant enzyme activity in plasma and other tissues (Tanskanen et al., 2010; Selamoglu et al., 2000; Lekhi et al., 2007). Regarding mixed (aerobic-anaerobic) training, there are only a few studies suggesting that trained football and rugby players show lower oxidative stress at rest than sedentary subjects, and that there is a correlation between redox status and fitness level (Chang et al., 2002; Metin et al., 2003; Cazzola et al., 2003). Currently, there is little information available regarding exercise-induced adaptations of the antioxidant defense system in adolescent and child athletes. The absence of studies investigating the effect of exercise on young population is surprising considering the numerous metabolic and physiologic differences between children, adolescents and adults (Cooper et al., 2004; Armstrong et al., 2008; Boisseau et al., 2000). The lack of information on young individuals' biochemical responses to exercise is mainly attributable to ethical concerns and methodological constraints that limit invasive research in children/adolescents. Thus, our team performed a set of investigations on exercise induced changes in redox state of young, adolescent handball players. Effects of acute exercise on their redox state were described earlier in this chapter. Here we'll focus on relationship between duration of training experience, aerobic capacities and biochemical parameters that represent factors of redox homeostasis. The first objective of our investigation was to explore the relationship between sports engagment and redox state so we compared redox

**5. The chronic effects of exercise on redox status of athletes**

**5.1 Assessment of the redox state of athletes and non-athletes**

well as during non-exercise related conditions.

state of 33 young handball players (age 16 - 19 years) with redox state of 14 age-matched adolescents who do not have regular physical activity (Djordjevic et al., 2011). The results showed that athletes and non-athletes did not differ significantly in levels of investigated prooxidants (O2•-, H2O2, •NO, TBARS), but athletes had significantly higher levels of SOD and lower level of CAT activity. Higher SOD activity in athletes was also found in other studies, including studies involving karate athletes (Naghizadeh et al, 2009) soccer players (Briites et al., 1999; Cazzola et al., 2003), rugby players (Evelson et al., 2002;), jump-trained subjects (Ortenblad et al., 1997) and students of Physical Education and Sports Sciences (Balakrishnan & Anuradh, 1998). Regarding CAT activity, there is a number of previous studies that also found that it's acitivity is decreased in athletes compared with controls, but there are also a number of studies that reported no change in CAT activity as a consequence of exercise training (Miyazaki et al., 2001; Lekhi et al., 2007; Metin et al., 2003; Ortenblad et al., 1997; Balakrishnan & Anuradh, 1998). According to numerous results from previous papers, it can be concluded that SOD behaviour as a consequence of chronic exercise training is clear (its' activity increases), while CAT has variant tendencies. The second objective of that work was to compare redox state of athletes with different duration of training experience. The results showed that there was no significant correlation between duration of sports engagement and redox state of athletes. The final objective of this work related to the correlation between morphofunctional characteristics and redox state of subjects. Athletes were divided into 3 groups based on ther aerobic capacity expressed through maximal oxygen consumption ((1) athletes with poor cardiorespiratory fitness (VO2max < 38.3 ml/kg/min), (2) athletes with average cardiorespiratory fitness (VO2max = 38.4– 45.1 ml/kg/min), and (3) athletes with good cardiorespiratory fitness (VO2max > 45.2 ml/kg/min)). Interestingly, athletes with low aerobic capacity had higher levels of H2O2 than athletes with average or high aerobic capacity, but lower levels of TBARS. Positive correlation was found between muscle percentage and TBARS. We hypothesize that the highest levels of TBARS in athletes with the highest aerobic power may be a consequence of the higher working capacity and consequently increased oxidative stress in working musculature of these athletes.

#### **5.2 Systemic adaptation to oxidative stress induced by regular long term exercise**

#### **5.2.1 Morphofunctional effects of redox disturbance homeostasis induced by long term exercise**

The overall positive impact of exercise on growth and development of children and youth is one of the generally accepted facts. From the aspect of physiology, adaptation to muscle activity is presented through a systematic response in order to provide the best possible performance with the lowest energy deficit. Adaptation is an universal, common characteristic of all living beings, responsible for the survival of the organism under different conditions.

Football, as a team game of simple rules without significant financial investment in equipment and space to play, is one of the most popular sports today, played by all nations, both sexes and various ages, regardless of skill level (Stolen, 2005). According to the intensity of football loads it can be classified as a highly intensive intermittent team sport (Bangsbo, 1994). For these reasons, it is evident that players are expected to have a high level of aerobic capacity and aerobic endurance. There is an evidently progressive trend of

Exercise and Oxidative Stress 55

erythrocyte SOD positively correlated with CAT, but there is no correlation between GSH-Px and other antioxidant enzymes (Andersen, 1997). The appearance of a negative correlation between SOD, CAT and GPx in the group of older players in our study resulted from the decrease in SOD activity in the older group, compared to the younger players. Reduction of SOD activity is most likely a result of inhibition of CuZn SOD by hydrogen peroxide. It is known that evidently increased antioxidant activity observed under the influence of the training process is the result of altered gene expression and influence on both the mRNA and at the level of protein synthesis. The results of these studies indicate that changes in markers of oxidative stress and antioxidant defense may occur within the normal variation in training and food intake rich in antioxidants. This means that variations in diet can affect markers of oxidative stress and antioxidant defense (for example if food is richer or poorer in antioxidants). All this indicates that under conditions of intense exercise there are numerous sources of hydrogen peroxide and nitric oxide present in the circulation and the potentiated heart and that some of these reactive species can have a detrimental effect on circulating blood elements by propagating systemic damage. Increased amounts of prooxidants in erythrocytes in the circulation may cause vascular spasm which further

Young athletes in constant competition and training process represent a specific subgroup of healthy people with specific lifestyles, who gained the ability to overcome supramaximal physical efforts (Maron, 2003, Maron & Zipes 2005, Maron et al., 1996). "Athlete's heart" was first described by Henchen in the European literature of the 1899th. Applying percutaneus method, he found an increase in the heart of racers–runners (Henchen, 1899). The growing interest in studying the effects of intensive physical loads on the cardiovascular system, and enormous progress in the field of noninvasive cardiac diagnostics, especially echocardiography in the last 30 years, have led to significant process of observing and noticing of heart remodeling. This fact is the focus of scientists in monitoring and understanding of specific changes in the heart of an athlete, known as "athlete's heart" (Morganroth et al., 1975; Martin et al., 1986; Pelliccia et al., 1991; Pelliccia et al., 1996; Douglas et al., 1997; Sharma et al., 2002; Pelliccia et al., 2002; Fagard, 2003; Pelliccia et al., 2005; Maron & Pelliccia, 2006; Pelliccia et al., 2007). Electrophysiological changes associated with athlete's heart syndrome, reflected by changes in heart rhythm, change in heart conductivity, repolarization, and precordial voltage changes, are consequently manifested by changes on ECG. Previous studies related to the search for the causes of these changes were mainly oriented towards the study of the autonomous regulation of heart rate due to changes in terms of increased vagal tone on the one hand and the suppression of sympathetic nerve regulation on the other. It is evident that intensive training influences the autonomic control and intrinsic cardiac pacemaker function (Huston et al., 1985). For these reasons, there is growing interest in more complete understanding of systemic change in the heart of an athlete, with emphasis on differentiation of changes in terms of age, or the influence of sports training and competition. Another, no less important reason is related to the strict classification of ECG changes in terms of successful differentiation of athlete's heart from cardiovascular disease and the possible development of sudden cardiac death of

emphasizes the production of free radicals.

athletes.

**5.2.2 The cardiac conduction system and redox status**

aerobic capacity in the last 20 years (Casajus, 2001), compared with results from eighties of the twentieth century (Ekblom, 1986; Faina et al., 1988). One of our studies was focused on analysis of the aerobic capacity of young soccer players of different age (14-15 versus 16-17 years old) and length of the sports experience (Cubrilo, 2009). Surveys conducted to date show that, when compared to the seniors, young players show lower values of maximum oxygen consumption, lower than 60 ml/kg/min (Stolen, 2005), which is consistent with the results of our research. In fact, comparative analysis of our research between the categories of young athletes in relation to age and length of the sports experience showed that aerobic capacity is significantly changed in terms of age, while the length of training does not affect the value of this parameter. The values of oxygen consumption of 51.05 ± 2.39 ml/kg/min in a group of older athletes with 10-11 years of sport experience approach the values achieved at the level of elite senior players rank of Serbia (Оstojić, 2000; Ponorac, 2005). These results confirm the fact that during the development of top athletes aerobic capacity reaches its maximum between 17-22 years, then linearly decreases with age (Shephard, 1999), which would practically mean that the beginning of a process of adaptation to level of stroke volume, or end-diastolic volume as a key factor of aerobic capacity (Levine, 2008), takes place only after 16 years of age. The level of aerobic capacity in the older group of athletes positively correlated with the H2O2 concentrations, as opposed to a group of young athletes where the level of aerobic capacity negatively correlated with the level of SOD activity. Due to the higher values of SOD in the group of young athletes, a negative correlation could be interpreted as an adaptive response to significantly lower values of aerobic capacity.

The percentages of fat mass (% FM) and fat free mass (% FFM) were not statistically different in the observed groups of athletes, while body mass index (BMI) was higher in older compared to younger group of athletes. However, correlations of measured parameters were age specific. In group of older athletes, fat mass percentage positively correlated with TBARS and H2O2 levels in older group while BMI positively correlated with SOD levels and negatively with CAT levels and GPx levels. Percentage of fat free mass positively correlated with H2O2 in younger athletes. Since there was no statistically significant differences in H2O2 concentrations between groups of younger and older players, observed positive correlation between the concentration of H2O2 in plasma and valuable asset of maximal oxygen uptake in a group of older athletes is especially interesting. Given that in the older group of athletes only TBARS plasma concentrations correlated with ECG parameters at rest, one might assume that the concentration of hydrogen peroxide in plasma indirectly mediates the induction of morphological changes of the left ventricle, with resultant effects on the regulation of myocardial inotropic properties.

Maintenance of optimal amounts of reactive species formed from molecular oxygen in the homeostatic balance is of great importance for preserving health. High levels of ROS are viewed as a toxic mediator of cell and tissue injury. Erythrocytes are the cells most exposed to possible damage caused by reactive oxygen species (Tappel, 1953). The results of correlation analysis in our work indicated that CAT and GPx significantly positively correlated in the group of young players. SOD significantly negatively correlated with CAT and GPx in older players tested. In erythrocytes, a major role in maintaining homeostatic balance of ROS belongs to enzymatic antioxidant system (AOS). Studies in healthy people have shown the expression of antioxidant enzymes is in a positive correlation with SOD activity, CAT and GR. A study of 220 healthy subjects from Danish population showed that

aerobic capacity in the last 20 years (Casajus, 2001), compared with results from eighties of the twentieth century (Ekblom, 1986; Faina et al., 1988). One of our studies was focused on analysis of the aerobic capacity of young soccer players of different age (14-15 versus 16-17 years old) and length of the sports experience (Cubrilo, 2009). Surveys conducted to date show that, when compared to the seniors, young players show lower values of maximum oxygen consumption, lower than 60 ml/kg/min (Stolen, 2005), which is consistent with the results of our research. In fact, comparative analysis of our research between the categories of young athletes in relation to age and length of the sports experience showed that aerobic capacity is significantly changed in terms of age, while the length of training does not affect the value of this parameter. The values of oxygen consumption of 51.05 ± 2.39 ml/kg/min in a group of older athletes with 10-11 years of sport experience approach the values achieved at the level of elite senior players rank of Serbia (Оstojić, 2000; Ponorac, 2005). These results confirm the fact that during the development of top athletes aerobic capacity reaches its maximum between 17-22 years, then linearly decreases with age (Shephard, 1999), which would practically mean that the beginning of a process of adaptation to level of stroke volume, or end-diastolic volume as a key factor of aerobic capacity (Levine, 2008), takes place only after 16 years of age. The level of aerobic capacity in the older group of athletes positively correlated with the H2O2 concentrations, as opposed to a group of young athletes where the level of aerobic capacity negatively correlated with the level of SOD activity. Due to the higher values of SOD in the group of young athletes, a negative correlation could be

interpreted as an adaptive response to significantly lower values of aerobic capacity.

resultant effects on the regulation of myocardial inotropic properties.

The percentages of fat mass (% FM) and fat free mass (% FFM) were not statistically different in the observed groups of athletes, while body mass index (BMI) was higher in older compared to younger group of athletes. However, correlations of measured parameters were age specific. In group of older athletes, fat mass percentage positively correlated with TBARS and H2O2 levels in older group while BMI positively correlated with SOD levels and negatively with CAT levels and GPx levels. Percentage of fat free mass positively correlated with H2O2 in younger athletes. Since there was no statistically significant differences in H2O2 concentrations between groups of younger and older players, observed positive correlation between the concentration of H2O2 in plasma and valuable asset of maximal oxygen uptake in a group of older athletes is especially interesting. Given that in the older group of athletes only TBARS plasma concentrations correlated with ECG parameters at rest, one might assume that the concentration of hydrogen peroxide in plasma indirectly mediates the induction of morphological changes of the left ventricle, with

Maintenance of optimal amounts of reactive species formed from molecular oxygen in the homeostatic balance is of great importance for preserving health. High levels of ROS are viewed as a toxic mediator of cell and tissue injury. Erythrocytes are the cells most exposed to possible damage caused by reactive oxygen species (Tappel, 1953). The results of correlation analysis in our work indicated that CAT and GPx significantly positively correlated in the group of young players. SOD significantly negatively correlated with CAT and GPx in older players tested. In erythrocytes, a major role in maintaining homeostatic balance of ROS belongs to enzymatic antioxidant system (AOS). Studies in healthy people have shown the expression of antioxidant enzymes is in a positive correlation with SOD activity, CAT and GR. A study of 220 healthy subjects from Danish population showed that erythrocyte SOD positively correlated with CAT, but there is no correlation between GSH-Px and other antioxidant enzymes (Andersen, 1997). The appearance of a negative correlation between SOD, CAT and GPx in the group of older players in our study resulted from the decrease in SOD activity in the older group, compared to the younger players. Reduction of SOD activity is most likely a result of inhibition of CuZn SOD by hydrogen peroxide. It is known that evidently increased antioxidant activity observed under the influence of the training process is the result of altered gene expression and influence on both the mRNA and at the level of protein synthesis. The results of these studies indicate that changes in markers of oxidative stress and antioxidant defense may occur within the normal variation in training and food intake rich in antioxidants. This means that variations in diet can affect markers of oxidative stress and antioxidant defense (for example if food is richer or poorer in antioxidants). All this indicates that under conditions of intense exercise there are numerous sources of hydrogen peroxide and nitric oxide present in the circulation and the potentiated heart and that some of these reactive species can have a detrimental effect on circulating blood elements by propagating systemic damage. Increased amounts of prooxidants in erythrocytes in the circulation may cause vascular spasm which further emphasizes the production of free radicals.

#### **5.2.2 The cardiac conduction system and redox status**

Young athletes in constant competition and training process represent a specific subgroup of healthy people with specific lifestyles, who gained the ability to overcome supramaximal physical efforts (Maron, 2003, Maron & Zipes 2005, Maron et al., 1996). "Athlete's heart" was first described by Henchen in the European literature of the 1899th. Applying percutaneus method, he found an increase in the heart of racers–runners (Henchen, 1899). The growing interest in studying the effects of intensive physical loads on the cardiovascular system, and enormous progress in the field of noninvasive cardiac diagnostics, especially echocardiography in the last 30 years, have led to significant process of observing and noticing of heart remodeling. This fact is the focus of scientists in monitoring and understanding of specific changes in the heart of an athlete, known as "athlete's heart" (Morganroth et al., 1975; Martin et al., 1986; Pelliccia et al., 1991; Pelliccia et al., 1996; Douglas et al., 1997; Sharma et al., 2002; Pelliccia et al., 2002; Fagard, 2003; Pelliccia et al., 2005; Maron & Pelliccia, 2006; Pelliccia et al., 2007). Electrophysiological changes associated with athlete's heart syndrome, reflected by changes in heart rhythm, change in heart conductivity, repolarization, and precordial voltage changes, are consequently manifested by changes on ECG. Previous studies related to the search for the causes of these changes were mainly oriented towards the study of the autonomous regulation of heart rate due to changes in terms of increased vagal tone on the one hand and the suppression of sympathetic nerve regulation on the other. It is evident that intensive training influences the autonomic control and intrinsic cardiac pacemaker function (Huston et al., 1985). For these reasons, there is growing interest in more complete understanding of systemic change in the heart of an athlete, with emphasis on differentiation of changes in terms of age, or the influence of sports training and competition. Another, no less important reason is related to the strict classification of ECG changes in terms of successful differentiation of athlete's heart from cardiovascular disease and the possible development of sudden cardiac death of athletes.

Exercise and Oxidative Stress 57

activity, where the subject matter referred to the response of skeletal muscle, liver and brain (Radak, 2008). The potential connection of ROS as second-messenger in terms of transmission of biological information through the modulation of signaling molecules, enzymes and proteins at the level of the heart muscle and heart conduction system of young athletes, represented through ECG changes, in current literature has not yet been processed. The concept of ROS as normal and necessary components of the cellular milieu is emerging as an important homeostatic mechanism that participates in the control of multiple cellular processes (Finkel, 1998; Lander, 1997). Skeletal muscles produce ROS at a rate that is activity dependent. This, plus evidence of ROS induced cellular damage, lead to idea that ROS may participate in the development of fatigue and/or activity induced injury as well as processes of cardiac contraction and relaxation (intracellular calcium cycle). More recently, various steps of the contractile process have proven to be susceptible to redox modulation. First, the opening probability of isolated sarcoplasmic reticulum (SR) Ca2+ release channels of the ryanodine receptors increases upon oxidation of accessible protein thiols. Moreover, SR Ca2+ reuptake is inhibited by high concentration of H2O2. Finally, oxidants alter myofibrillar Ca2+ sensitivity in a time and concentration dependent fashion. This so-called redox signalling function is especially true for the ROS, H2O2, which is more stable and diffusible than radical species such as O2•-, but also applies to nitric oxide. An important consideration in the experimental use of oxidants such as H2O2 is whether the observed changes are physiological or are the results of overt oxidative stress. This issue has become topical in our research on young football players (children of 14-15 years old) where H2O2 positively correlated with the percentage of muscle, while in the older group of athletes demonstrated (16-17 years old) a positive correlation between H2O2 and the percentage of fat was found. A

positive correlation between H2O2 and the sum of (SV1 + RV5) was also observed.

prolongation of the action potential slow phase.

The observed positive correlation between TBARS concentration and QT interval duration, and a negative correlation between the concentration of •NO and PQ interval duration can be observed in the light of altered Ca2+ homeostasis as an indicator of oxidative stress. Andrade and coworkers showed that continuous exposure to relatively high concentrations of H2O2 for extended periods of time caused resting Ca2+ to increase and slowed the return of Ca2+ to resting levels after stimulation ended (Andrade et al., 1998). Prolonged QT syndrome is a functional abnormality probably coupled with neurological severe impacts that can cause lethal arrhythmias (Schwartz et al., 1975). The acquired forms of prolonged QT interval may be due to idiosyncrasies of medicines (antiarrhythmics and psychotropic drugs), electrolyte abnormalities, hypothermia, toxic substances and injuries of the central nervous system (Bhadari & Scheinman, 1985). Correlations obtained in our study may include oxidative stress as the cause of the prolonged QT interval in young athletes. TBARS as index of lipid peroxidation could play a significant role in the modulation of cardiac systolic phase in which the mechanism of action can be linked to Ca2+ homeostasis and the Ca2+ influx through slow calcium channels. TBARS could act on the Ca2+ dependent slow action potential, which is its negative inotropic effect: reduced Ca2+ current with

Heart conductivity disorders are very common finding in active athletes. First-degree atrioventricular block is represented with 6-33 % (Puffer, 2001) or 35-40 % (Crisafulli et al., 2002) in athletes, which is significantly higher than in the general population (0.65 %). The observed negative correlation between the concentration of •NO and PQ interval duration in young athletes called into question a possible link with the myocardial (atrial) contractile

Understanding the specific adaptations of the sport and its specific mechanisms and regularity, especially in a group of young athletes, requires an interdisciplinary approach, which should answer many questions related to the optimization of training, the athletes' health and morbidity in terms of preventing injury and sudden cardiac death in the field. Regular, intense physical activity is associated with increased dimensions of the heart muscle, where these changes are interpreted as benign (Maron, 1998, Maron & Pelliccia, 2006). In addition, it was shown that cardiac dimensions and functional abilities tend to change over training cycle as an adaptation to the specific requirements of a particular sports season (Crouse et al., 1992). The degree of cardiac adaptations can be determined by various factors including age, sex, type and intensity of exercise (Pelliccia et al., 2000), as well as ethnicity and race (Crouse et al., 2009).

Analysis of electrocardiograms of athletes in our study (Cubrilo, 2009), who were included in regular, intense physical activity, showed that some ECG changes may occur in athletes under the influence of training on the one hand or the regular process of growth and development during the normal training cycle on the other. It is shown that athletes' age significantly affects the dynamics of change in P wave voltage to the lead D3, the amount of ST elevation in V1 lead, and the duration of PQ, QRS and QT intervals. On the other hand, the length of the sports experience showed a statistically significant effect on the mean QRS vector, P wave voltage to the lead D2, as well as the value of the sum of (RD1 + SD3). Also, in older athletes it has been reported higher incidence in percentage of sinus arrhythmia and an incomplete right bundle branch block, regardless of the length of the sports experience. The results of our research in terms of classification of ECG findings, based on generally accepted clinical criteria (Pelliccia et al., 2000), show that athletes 57 % of both age groups can be classified as with normal ECG findings while 41 % is classified as partially normal and 2 % significantly abnormal findings with potential structural changes and clinical significance. The largest number of research on electrocardiographic changes include athletes between 18 and 35 years, while very few studies focused on the monitoring of young athletes with the aim of determining the difference in the adaptation of the heart to the training process in relation to the age. In our study ECG voltage criteria of LVH (RD1 + SD3> 25 mm) (Gubner & Ungerleider, 1943) showed statistically significant difference compared to the length of the sports experience. Consideration of the dynamics of average values of this parameter in the observed groups of athletes showed a decreasing trend with increasing length of the sports experience in both age groups. Average values of P waves in leads D2 and D3 showed statistically significant differences observed among groups of athletes, where the dynamics of P waves in the lead D2 showed a statistically significant difference compared to the length of the sports experience, as opposed to the dynamics of the P wave to the lead D3, where it was observed statistically significant differences by athletes age. By analyzing the dynamics of average values of P wave in D2 lead, voltage reduction is observed with increasing length of the sports experience. The mean QRS vector in our study shows a statistically significant upward trend in this parameter with increasing length of the sport experience in both groups.

Increased oxidative stress is involved in the pathophysiology of diverse diseases such as atherosclerosis, neurodegeneration, renal disease and cancer. Over the past 20 years, significant evidence has suggested a role for increased oxidative stress in the pathophysiology of congestive heart failure (CHF). In previous scientific work focus is mainly put on the analysis of adaptive response to redox disturbance during physical

Understanding the specific adaptations of the sport and its specific mechanisms and regularity, especially in a group of young athletes, requires an interdisciplinary approach, which should answer many questions related to the optimization of training, the athletes' health and morbidity in terms of preventing injury and sudden cardiac death in the field. Regular, intense physical activity is associated with increased dimensions of the heart muscle, where these changes are interpreted as benign (Maron, 1998, Maron & Pelliccia, 2006). In addition, it was shown that cardiac dimensions and functional abilities tend to change over training cycle as an adaptation to the specific requirements of a particular sports season (Crouse et al., 1992). The degree of cardiac adaptations can be determined by various factors including age, sex, type and intensity of exercise (Pelliccia et al., 2000), as

Analysis of electrocardiograms of athletes in our study (Cubrilo, 2009), who were included in regular, intense physical activity, showed that some ECG changes may occur in athletes under the influence of training on the one hand or the regular process of growth and development during the normal training cycle on the other. It is shown that athletes' age significantly affects the dynamics of change in P wave voltage to the lead D3, the amount of ST elevation in V1 lead, and the duration of PQ, QRS and QT intervals. On the other hand, the length of the sports experience showed a statistically significant effect on the mean QRS vector, P wave voltage to the lead D2, as well as the value of the sum of (RD1 + SD3). Also, in older athletes it has been reported higher incidence in percentage of sinus arrhythmia and an incomplete right bundle branch block, regardless of the length of the sports experience. The results of our research in terms of classification of ECG findings, based on generally accepted clinical criteria (Pelliccia et al., 2000), show that athletes 57 % of both age groups can be classified as with normal ECG findings while 41 % is classified as partially normal and 2 % significantly abnormal findings with potential structural changes and clinical significance. The largest number of research on electrocardiographic changes include athletes between 18 and 35 years, while very few studies focused on the monitoring of young athletes with the aim of determining the difference in the adaptation of the heart to the training process in relation to the age. In our study ECG voltage criteria of LVH (RD1 + SD3> 25 mm) (Gubner & Ungerleider, 1943) showed statistically significant difference compared to the length of the sports experience. Consideration of the dynamics of average values of this parameter in the observed groups of athletes showed a decreasing trend with increasing length of the sports experience in both age groups. Average values of P waves in leads D2 and D3 showed statistically significant differences observed among groups of athletes, where the dynamics of P waves in the lead D2 showed a statistically significant difference compared to the length of the sports experience, as opposed to the dynamics of the P wave to the lead D3, where it was observed statistically significant differences by athletes age. By analyzing the dynamics of average values of P wave in D2 lead, voltage reduction is observed with increasing length of the sports experience. The mean QRS vector in our study shows a statistically significant upward trend in this parameter with increasing

Increased oxidative stress is involved in the pathophysiology of diverse diseases such as atherosclerosis, neurodegeneration, renal disease and cancer. Over the past 20 years, significant evidence has suggested a role for increased oxidative stress in the pathophysiology of congestive heart failure (CHF). In previous scientific work focus is mainly put on the analysis of adaptive response to redox disturbance during physical

well as ethnicity and race (Crouse et al., 2009).

length of the sport experience in both groups.

activity, where the subject matter referred to the response of skeletal muscle, liver and brain (Radak, 2008). The potential connection of ROS as second-messenger in terms of transmission of biological information through the modulation of signaling molecules, enzymes and proteins at the level of the heart muscle and heart conduction system of young athletes, represented through ECG changes, in current literature has not yet been processed. The concept of ROS as normal and necessary components of the cellular milieu is emerging as an important homeostatic mechanism that participates in the control of multiple cellular processes (Finkel, 1998; Lander, 1997). Skeletal muscles produce ROS at a rate that is activity dependent. This, plus evidence of ROS induced cellular damage, lead to idea that ROS may participate in the development of fatigue and/or activity induced injury as well as processes of cardiac contraction and relaxation (intracellular calcium cycle). More recently, various steps of the contractile process have proven to be susceptible to redox modulation. First, the opening probability of isolated sarcoplasmic reticulum (SR) Ca2+ release channels of the ryanodine receptors increases upon oxidation of accessible protein thiols. Moreover, SR Ca2+ reuptake is inhibited by high concentration of H2O2. Finally, oxidants alter myofibrillar Ca2+ sensitivity in a time and concentration dependent fashion. This so-called redox signalling function is especially true for the ROS, H2O2, which is more stable and diffusible than radical species such as O2•-, but also applies to nitric oxide. An important consideration in the experimental use of oxidants such as H2O2 is whether the observed changes are physiological or are the results of overt oxidative stress. This issue has become topical in our research on young football players (children of 14-15 years old) where H2O2 positively correlated with the percentage of muscle, while in the older group of athletes demonstrated (16-17 years old) a positive correlation between H2O2 and the percentage of fat was found. A positive correlation between H2O2 and the sum of (SV1 + RV5) was also observed.

The observed positive correlation between TBARS concentration and QT interval duration, and a negative correlation between the concentration of •NO and PQ interval duration can be observed in the light of altered Ca2+ homeostasis as an indicator of oxidative stress. Andrade and coworkers showed that continuous exposure to relatively high concentrations of H2O2 for extended periods of time caused resting Ca2+ to increase and slowed the return of Ca2+ to resting levels after stimulation ended (Andrade et al., 1998). Prolonged QT syndrome is a functional abnormality probably coupled with neurological severe impacts that can cause lethal arrhythmias (Schwartz et al., 1975). The acquired forms of prolonged QT interval may be due to idiosyncrasies of medicines (antiarrhythmics and psychotropic drugs), electrolyte abnormalities, hypothermia, toxic substances and injuries of the central nervous system (Bhadari & Scheinman, 1985). Correlations obtained in our study may include oxidative stress as the cause of the prolonged QT interval in young athletes. TBARS as index of lipid peroxidation could play a significant role in the modulation of cardiac systolic phase in which the mechanism of action can be linked to Ca2+ homeostasis and the Ca2+ influx through slow calcium channels. TBARS could act on the Ca2+ dependent slow action potential, which is its negative inotropic effect: reduced Ca2+ current with prolongation of the action potential slow phase.

Heart conductivity disorders are very common finding in active athletes. First-degree atrioventricular block is represented with 6-33 % (Puffer, 2001) or 35-40 % (Crisafulli et al., 2002) in athletes, which is significantly higher than in the general population (0.65 %). The observed negative correlation between the concentration of •NO and PQ interval duration in young athletes called into question a possible link with the myocardial (atrial) contractile

Exercise and Oxidative Stress 59

homeostasis caused by the influence of adaptation at the level of gene transcription

The following research could provide a clearer picture of the possible diagnostic and therapeutic importance (individual antioxidant supplementation) of these correlations in a group of young athletes of both ages. Mechanical stretch and neurohumoral factors induce changes in intracellular signaling pathways resulting in increase protein synthesis and the activation of specific genes promoting growth and potentially leading to ventricular remodeling. Our results demonstrated that oxidative stress and body (systemic) redox state

**6. Effect of different supplementation strategies on nitric oxide and oxidative**

**6.1 The influence of L-Arginin supplementation on nitric oxide and oxidative stress**

Arginine is one of the 20 amino acids (AA) necessary for protein synthesis and coded by DNA. Plasma arginine concentrations are therefore maintained mostly by protein catabolism (≈ 85 %) (Morris et al., 2006) or by synthesis from other AA. Of the total synthesis, de novo synthesis accounts for < 15 %, while 60 % of arginine is derived from citrulline (Wu et al., 1998). Arginine metabolism may give rise to several other AA and molecules indispensable for life. Exogenous arginine is largely destroyed (≈ 40 %) during absorption by the gut itself. Much attention was given to arginine availability due to the discovery in the 1980's that endothelium-dependent relaxing factor' is a gas, nitric oxide (•NO), produced from arginine by endothelial •NO synthase (eNOS) (Ignarro et al., 1987) in a reaction that gives also rise to citrulline. In endothelial cells, eNOS and the two sequential enzymes argininosuccinate lyase (ASL) and argininosuccinate synthase (ASsynth), necessary for recycling citrulline to arginine, are co-localized in the caveolae (Li et al., 2005), a fraction of cell membranes that provides an efficient environment for maintaining arginine available to eNOS. The cytoplasm of endothelial cells has a concentration of arginine largely saturating eNOS, and yet the acute introduction of exogenous arginine elicits an increase in **•**NO production, a puzzling finding known as "the arginine paradox" (Kurz et al., 1997). Another puzzling finding is the peculiar regulation of arginine transport and its effects on **•**NO. The transport of arginine into cells is mediated by the cationic AA transporter 1 (CAT1). Over-expression of CAT1 enhances arginine uptake sixfold, and **•**NO production twofold. The presence of AA competing with arginine decreases arginine uptake, but not •NO production (Li et al., 2005). Moreover, citrulline succeeds in stimulating NO production even in a medium containing saturating levels of arginine, and extracellular citrulline does not influence intracellular arginine levels. Therefore, •NO production depends mostly on the efficient recycling of arginine-derived citrulline back to arginine, and not so much on exogenous arginine supply. Thus, the presence of a "micro-environment", where arginine metabolism and recycling are independently regulated and only partially in balance with plasma arginine concentrations, is the most

influence heart functionality, but the effects are age specific.

**parameters dynamics during maximal exercise testing**

**stress parameters dynamics during maximal exercise testing**

consistent explanation for the endothelial arginine paradox (Flam et al., 2007).

To understand the clinical paradox of arginine, i.e. the fact that the much needed arginine is ineffective or actually detrimental when supplemented chronically, we have to focus on

**6.1.1 Endothelial vasomotion and the exogenous arginine paradox**

(Vollaard et al., 2005).

•NO effect. Conductivity at the AV (atrioventricular) node is mediated by increased parasympathetic tone and/or reduced sympathetic tone in rest. So far some of the most important controversies surrounding the myocardial contractile effect of •NO were if •NO exert a myocardial contractile effect under baseline conditions or only following adrenergic or cholinergic stimulation on the one hand, or can contractile effects of •NO be labeled as positively or negatively inotropic on the other. One might assume that •NO might play an important role in the contraction of the atria (presystolic phase) and the filling of the ventriculi in the physiological resting conditions of the athlete's heart.

Changes in QRS complexes voltage are very common finding in the sports population. However there is considerable variation in the percentage of literature data which goes up to 8-76 %, thus the problem of standardization of criteria for their registration exists. Intensive, long-practicing activity is associated with morphological changes in heart muscle, including increased volume, as well as increased thickness of the walls of the heart (Huston et al., 1985; Fagard, 2003). The main difference between pathologycal and physiological hypertrophy is the nature of the stimulus for the growth of cells, as well as the duration of a given stimulus. For the development of physiological hypertrophy, the most common stimulus is training process, with effect from time to time, episodic, and largely through the sympathetic neurotransmitter.

Several studies showed high incidence (80 %) sportsmen who met electrocardiographic criteria for left ventricular hypertrophy using the Sokolow and Lyon criteria (S wave in V1 + R wave in V5> 35 mm) (Sharma et al., 1999; Pelliccia et al., 2000). The percentage of the right ventricle was 18-69 %, where the sum is taken as a criterion RV1 and SV5 deflection greater than 10.5 mm. ECG voltage criteria of LVH (Gubner & Ungerleider, 1943), ( RD1 + SD3> 25 mm), in our study showed a statistically significant difference compared to the length of the sports experience. Consideration of the dynamics of average values of this parameter in the observed groups of athletes observed a decreasing trend with increasing length of the sports experience in both age groups.

Electrical heart axis in sport depends on age, chest structure and position of heart in chest cavity. It is manifested by the development of left ventricular hypertrophy. In about 60-70 % of all athletes electrical cardiac axis is between 30-70 degrees, and turn 90 degrees to the right and more encounters with 10-20 % of athletes. Turning electrical axis of 30 degrees to the left and more are found in 15-25 % of athletes. Thus, it was determined that it often turns to the right of athletes involved in endurance sports type, while in athletes involved in power sports-type it often turns to the left (Sharma et al., 2002). The literature has shown that the shaft QRS complex becomes more vertical with increasing levels of well trained, often associated with right bundle branch block incomplete which can be explained by increased myocardial mass at the top of the right ventricle. The mean QRS vector in our study shows a statistically significant upward trend (verticalisation) with increasing length of sport experience of the athlete in both groups. In the group of younger athletes, negative corelation between O2**•**- concentration in plasma and P wave voltage in inferior bipolar leads was observed and/or negative correlation with the mean QRS vector. On the other hand, in the older group of athletes a positive correlation between TBARS in plasma with values voltage criteria for LVH (RD1 + SD3) was observed, and negative correlation with the mean QRS vector. The presence of these correlations confirms the assumptions about the role of ROS as signaling molecules ideal, where the level of their production during and after physical activity could be a crucial link between exercise and consequently disturbed

•NO effect. Conductivity at the AV (atrioventricular) node is mediated by increased parasympathetic tone and/or reduced sympathetic tone in rest. So far some of the most important controversies surrounding the myocardial contractile effect of •NO were if •NO exert a myocardial contractile effect under baseline conditions or only following adrenergic or cholinergic stimulation on the one hand, or can contractile effects of •NO be labeled as positively or negatively inotropic on the other. One might assume that •NO might play an important role in the contraction of the atria (presystolic phase) and the filling of the

Changes in QRS complexes voltage are very common finding in the sports population. However there is considerable variation in the percentage of literature data which goes up to 8-76 %, thus the problem of standardization of criteria for their registration exists. Intensive, long-practicing activity is associated with morphological changes in heart muscle, including increased volume, as well as increased thickness of the walls of the heart (Huston et al., 1985; Fagard, 2003). The main difference between pathologycal and physiological hypertrophy is the nature of the stimulus for the growth of cells, as well as the duration of a given stimulus. For the development of physiological hypertrophy, the most common stimulus is training process, with effect from time to time, episodic, and largely through the

Several studies showed high incidence (80 %) sportsmen who met electrocardiographic criteria for left ventricular hypertrophy using the Sokolow and Lyon criteria (S wave in V1 + R wave in V5> 35 mm) (Sharma et al., 1999; Pelliccia et al., 2000). The percentage of the right ventricle was 18-69 %, where the sum is taken as a criterion RV1 and SV5 deflection greater than 10.5 mm. ECG voltage criteria of LVH (Gubner & Ungerleider, 1943), ( RD1 + SD3> 25 mm), in our study showed a statistically significant difference compared to the length of the sports experience. Consideration of the dynamics of average values of this parameter in the observed groups of athletes observed a decreasing trend with increasing length of the sports

Electrical heart axis in sport depends on age, chest structure and position of heart in chest cavity. It is manifested by the development of left ventricular hypertrophy. In about 60-70 % of all athletes electrical cardiac axis is between 30-70 degrees, and turn 90 degrees to the right and more encounters with 10-20 % of athletes. Turning electrical axis of 30 degrees to the left and more are found in 15-25 % of athletes. Thus, it was determined that it often turns to the right of athletes involved in endurance sports type, while in athletes involved in power sports-type it often turns to the left (Sharma et al., 2002). The literature has shown that the shaft QRS complex becomes more vertical with increasing levels of well trained, often associated with right bundle branch block incomplete which can be explained by increased myocardial mass at the top of the right ventricle. The mean QRS vector in our study shows a statistically significant upward trend (verticalisation) with increasing length of sport experience of the athlete in both groups. In the group of younger athletes, negative corelation between O2**•**- concentration in plasma and P wave voltage in inferior bipolar leads was observed and/or negative correlation with the mean QRS vector. On the other hand, in the older group of athletes a positive correlation between TBARS in plasma with values voltage criteria for LVH (RD1 + SD3) was observed, and negative correlation with the mean QRS vector. The presence of these correlations confirms the assumptions about the role of ROS as signaling molecules ideal, where the level of their production during and after physical activity could be a crucial link between exercise and consequently disturbed

ventriculi in the physiological resting conditions of the athlete's heart.

sympathetic neurotransmitter.

experience in both age groups.

homeostasis caused by the influence of adaptation at the level of gene transcription (Vollaard et al., 2005).

The following research could provide a clearer picture of the possible diagnostic and therapeutic importance (individual antioxidant supplementation) of these correlations in a group of young athletes of both ages. Mechanical stretch and neurohumoral factors induce changes in intracellular signaling pathways resulting in increase protein synthesis and the activation of specific genes promoting growth and potentially leading to ventricular remodeling. Our results demonstrated that oxidative stress and body (systemic) redox state influence heart functionality, but the effects are age specific.

#### **6. Effect of different supplementation strategies on nitric oxide and oxidative stress parameters dynamics during maximal exercise testing**

#### **6.1 The influence of L-Arginin supplementation on nitric oxide and oxidative stress parameters dynamics during maximal exercise testing**

Arginine is one of the 20 amino acids (AA) necessary for protein synthesis and coded by DNA. Plasma arginine concentrations are therefore maintained mostly by protein catabolism (≈ 85 %) (Morris et al., 2006) or by synthesis from other AA. Of the total synthesis, de novo synthesis accounts for < 15 %, while 60 % of arginine is derived from citrulline (Wu et al., 1998). Arginine metabolism may give rise to several other AA and molecules indispensable for life. Exogenous arginine is largely destroyed (≈ 40 %) during absorption by the gut itself. Much attention was given to arginine availability due to the discovery in the 1980's that endothelium-dependent relaxing factor' is a gas, nitric oxide (•NO), produced from arginine by endothelial •NO synthase (eNOS) (Ignarro et al., 1987) in a reaction that gives also rise to citrulline. In endothelial cells, eNOS and the two sequential enzymes argininosuccinate lyase (ASL) and argininosuccinate synthase (ASsynth), necessary for recycling citrulline to arginine, are co-localized in the caveolae (Li et al., 2005), a fraction of cell membranes that provides an efficient environment for maintaining arginine available to eNOS. The cytoplasm of endothelial cells has a concentration of arginine largely saturating eNOS, and yet the acute introduction of exogenous arginine elicits an increase in **•**NO production, a puzzling finding known as "the arginine paradox" (Kurz et al., 1997). Another puzzling finding is the peculiar regulation of arginine transport and its effects on **•**NO. The transport of arginine into cells is mediated by the cationic AA transporter 1 (CAT1). Over-expression of CAT1 enhances arginine uptake sixfold, and **•**NO production twofold. The presence of AA competing with arginine decreases arginine uptake, but not •NO production (Li et al., 2005). Moreover, citrulline succeeds in stimulating NO production even in a medium containing saturating levels of arginine, and extracellular citrulline does not influence intracellular arginine levels. Therefore, •NO production depends mostly on the efficient recycling of arginine-derived citrulline back to arginine, and not so much on exogenous arginine supply. Thus, the presence of a "micro-environment", where arginine metabolism and recycling are independently regulated and only partially in balance with plasma arginine concentrations, is the most consistent explanation for the endothelial arginine paradox (Flam et al., 2007).

#### **6.1.1 Endothelial vasomotion and the exogenous arginine paradox**

To understand the clinical paradox of arginine, i.e. the fact that the much needed arginine is ineffective or actually detrimental when supplemented chronically, we have to focus on

Exercise and Oxidative Stress 61

in healthy men (by blockade of •NO synthase) should lead to increased AIx and reduced PP amplification. Sharman and coworkers (Sharman et al., 2008) compared this hypothesis in young men who performed submaximal cycle exercise after intravenous infusion of L-NMMA with an •NO-independent control vasoconstrictor (noradrenaline [NE]) and placebo (saline). This study supports modulatory role of •NO in large artery stiffness, pressure amplification, and myocardial loading under resting conditions. However, this is the first study to report that •NO does not affect large artery hemodynamics and wave reflection such that ventricular-vascular interaction is substantially altered during low-intensity aerobic exercise. Systemic infusion of L-NAME (an inhibitor of NOS) caused significant increase in MAP, but not in VO2max in healthy volunteers at submaximal exercise. Furthermore, that reduction was significant in maximal exercise (Jones et al., 2004). These finding suggest that muscle blood flow was well preserved during submaximal exercise following NOS inhibition, indicating multifactorial influence on blood flow regulation

On the other hand, one other study on healthy subjects showed that L-NAME decreases lactate production during exercise, which is quite logical, taken into consideration well known fact that •NO stimulates glycolysis. These data indicated that skeletal muscle •NO production represents an important limitation to the acceleration of oxidative metabolism

In conclusion, the study by Taddei and coworkers (Taddei et al., 2000) demonstrated that regular physical training protects the vascular endothelium from aging-related alterations. The beneficial effect of exercise is related to preservation of •NO availability by a mechanism probably linked to the prevention of oxidative stress and the consequent •NO breakdown. This beneficial effect could be important in accounting for the positive impact of regular exercise on cardiovascular risk in the elderly population. Arginine and glutamine are two non-essential amino acids than can become "conditionally essential" because of elevated needs during pathological conditions, and metabolism may not be able to maintain their concentrations at sufficient levels to match metabolic requirements. Chronic exogenous arginine supplementation has not proven to exert positive clinical effects in different trials, and sequential articulation of the knowledge of introduction of arginine-driven transcriptional, translational, and epigenetic adaptations may give us a key for interpreting those controversial results. Study by Lerman and coworkers demonstrated that oral Larginine supplementation for 6 months improves coronary endothelial function in association with improvement in symptoms in patients with non-significant coronary artery stenosis. This study proposes a therapeutic role for L-arginine in patients with chest pain

The role of L-arginine supplementation in cardiac patients remains controversial. Furthermore, it is also unclear if arginine supplementation in the sedentary population can have the same results. Further research will be needed to assess the interaction of these factors and to determine the effects of prolonged administration of arginine and antioxidants on exercise performance. Supporting that, a recent study in trained athletes, who were without any cardiovascular problems, showed beneficial effect of L-arginine in elderly physically active subjects (Chen et al., 2010). An arginine and antioxidant-containing supplement increased the anaerobic threshold and the work at anaerobic threshold at both week one and week three in elderly cyclists. No effect on VO2max was observed. This study

following the onset of supramaximal exercise in humans (Wilkerson et al., 2004).

and coronary endothelial dysfunction (Lerman et al., 1998).

during exercise.

another pathway of the complex arginine metabolism, which is controlled by the ubiquitous enzymes arginases (types, 1 and 2), which compete with NOS for arginine as a substrate. The effect of both arginases is to catalyze the cleavage of urea from arginine, thus forming ornithine. Urea controls osmolarity and water content of plasma and cells. The other product of the reaction, ornithine, may be recycled to citrulline and also synthesized de novo by the liver from glutamine, and then transformed by the gut into citrulline. The kidneys provide further recycling of citrulline to arginine, which is finally released into the plasma (Van de Poll et al., 2007). Arginase 1 (ARG 1) is a cytosolic enzyme, mostly expressed in the liver and red blood cells. Deletion of the ARG 1 gene, as occasionally found in humans, is incompatible with prolonged life, and the accompanying hyperargininemia is associated with a several-fold increase in the activity of the mitochondrial arginase (ARG 2), which accounts for the persistent ureagenesis in those patients (Grody et al., 1989). ARG 2 is widely expressed, mostly in the kidneys, gut and brain. ARG 2 overexpression plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction (Schulman et al., 2006). Arginases and NOS compete for arginine, and – under any conditions – arginase activity exceeds NOS activity at all NOS/arginase molar ratios (Santhanam et al., 2008). Moreover, although the KM of arginases is 100-fold higher than that of NOS, the enzymes compete for arginine because the maximal catalytic rate of arginases is more than 1000 times higher than that of NOS (Wu et al., 1998; Topal et al., 2006). Therefore, increased expression and/or activity of ARG have a deep impact on NOS efficiency. As an example, arginase activity is increased in type 2 diabetic subjects with impaired NOS activity, and such impairment correlates with the degree of hyperglycemia and is reduced by insulin (Kashyap et al., 2008). The problem is more interesting because results of experimental and clinical studies are quite controversial and can not definitely support relevant conclusions. There are only few experimental studies dealing with this problem.

The study of Maxwell and coworkers (Maxwell et al., 2001) was performed to determine whether supplementation with L-arginine would prevent the decline in aerobic capacity observed in hypercholesterolemic mice. In one previous study the same authors determined the exercise capacity of wild-type (E1) and apolipoprotein E-deficient mice (E2) at 8 week of age when the cholesterol levels of both strains are low. The observed decline in VO2max is associated with endothelial vasodilator dysfunction and reduced urinary nitrate excretion. This study was designed with the intention of averting the impairment in aerobic capacity associated with hypercholesterolemia through chronic supplementation of L-arginine. Those results clearly showed that administration of L-arginine restores exercise-induced EDNO synthesis and normalizes aerobic capacity in hypercholesterolemic mice. In normal mice, Larginine enhances exercise-induced EDNO synthesis and aerobic capacity. As we noted above, investigation on humans do not clearly support exact role of L-arginine/NO system in exercise-induced vasodilation in healthy subjects, athletes or patients in some pathophysiological statement. Increased vasodilation and large artery distensibility occur after dynamic aerobic exercise (Kingwell et al., 1997; Naka et al., 2003) probably attributed in part to augment •NO release (Green et al., 2002; Gilligan et al., 1994). Although the contribution of •NO to large artery stiffness, wave reflection (AIx), and pressure amplification during exercise has never been tested, it is an important consideration relevant to cardiac structure and cardiovascular risk (Sharman et al., 2007). If reduced •NO bioavailability underlies abnormal exercise hemodynamics in a fashion similar to that found in men with hypercholesterolemia, we may expect that induction of endothelial dysfunction

another pathway of the complex arginine metabolism, which is controlled by the ubiquitous enzymes arginases (types, 1 and 2), which compete with NOS for arginine as a substrate. The effect of both arginases is to catalyze the cleavage of urea from arginine, thus forming ornithine. Urea controls osmolarity and water content of plasma and cells. The other product of the reaction, ornithine, may be recycled to citrulline and also synthesized de novo by the liver from glutamine, and then transformed by the gut into citrulline. The kidneys provide further recycling of citrulline to arginine, which is finally released into the plasma (Van de Poll et al., 2007). Arginase 1 (ARG 1) is a cytosolic enzyme, mostly expressed in the liver and red blood cells. Deletion of the ARG 1 gene, as occasionally found in humans, is incompatible with prolonged life, and the accompanying hyperargininemia is associated with a several-fold increase in the activity of the mitochondrial arginase (ARG 2), which accounts for the persistent ureagenesis in those patients (Grody et al., 1989). ARG 2 is widely expressed, mostly in the kidneys, gut and brain. ARG 2 overexpression plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction (Schulman et al., 2006). Arginases and NOS compete for arginine, and – under any conditions – arginase activity exceeds NOS activity at all NOS/arginase molar ratios (Santhanam et al., 2008). Moreover, although the KM of arginases is 100-fold higher than that of NOS, the enzymes compete for arginine because the maximal catalytic rate of arginases is more than 1000 times higher than that of NOS (Wu et al., 1998; Topal et al., 2006). Therefore, increased expression and/or activity of ARG have a deep impact on NOS efficiency. As an example, arginase activity is increased in type 2 diabetic subjects with impaired NOS activity, and such impairment correlates with the degree of hyperglycemia and is reduced by insulin (Kashyap et al., 2008). The problem is more interesting because results of experimental and clinical studies are quite controversial and can not definitely support relevant conclusions. There are only few experimental studies dealing with this problem.

The study of Maxwell and coworkers (Maxwell et al., 2001) was performed to determine whether supplementation with L-arginine would prevent the decline in aerobic capacity observed in hypercholesterolemic mice. In one previous study the same authors determined the exercise capacity of wild-type (E1) and apolipoprotein E-deficient mice (E2) at 8 week of age when the cholesterol levels of both strains are low. The observed decline in VO2max is associated with endothelial vasodilator dysfunction and reduced urinary nitrate excretion. This study was designed with the intention of averting the impairment in aerobic capacity associated with hypercholesterolemia through chronic supplementation of L-arginine. Those results clearly showed that administration of L-arginine restores exercise-induced EDNO synthesis and normalizes aerobic capacity in hypercholesterolemic mice. In normal mice, Larginine enhances exercise-induced EDNO synthesis and aerobic capacity. As we noted above, investigation on humans do not clearly support exact role of L-arginine/NO system in exercise-induced vasodilation in healthy subjects, athletes or patients in some pathophysiological statement. Increased vasodilation and large artery distensibility occur after dynamic aerobic exercise (Kingwell et al., 1997; Naka et al., 2003) probably attributed in part to augment •NO release (Green et al., 2002; Gilligan et al., 1994). Although the contribution of •NO to large artery stiffness, wave reflection (AIx), and pressure amplification during exercise has never been tested, it is an important consideration relevant to cardiac structure and cardiovascular risk (Sharman et al., 2007). If reduced •NO bioavailability underlies abnormal exercise hemodynamics in a fashion similar to that found in men with hypercholesterolemia, we may expect that induction of endothelial dysfunction in healthy men (by blockade of •NO synthase) should lead to increased AIx and reduced PP amplification. Sharman and coworkers (Sharman et al., 2008) compared this hypothesis in young men who performed submaximal cycle exercise after intravenous infusion of L-NMMA with an •NO-independent control vasoconstrictor (noradrenaline [NE]) and placebo (saline). This study supports modulatory role of •NO in large artery stiffness, pressure amplification, and myocardial loading under resting conditions. However, this is the first study to report that •NO does not affect large artery hemodynamics and wave reflection such that ventricular-vascular interaction is substantially altered during low-intensity aerobic exercise. Systemic infusion of L-NAME (an inhibitor of NOS) caused significant increase in MAP, but not in VO2max in healthy volunteers at submaximal exercise. Furthermore, that reduction was significant in maximal exercise (Jones et al., 2004). These finding suggest that muscle blood flow was well preserved during submaximal exercise following NOS inhibition, indicating multifactorial influence on blood flow regulation during exercise.

On the other hand, one other study on healthy subjects showed that L-NAME decreases lactate production during exercise, which is quite logical, taken into consideration well known fact that •NO stimulates glycolysis. These data indicated that skeletal muscle •NO production represents an important limitation to the acceleration of oxidative metabolism following the onset of supramaximal exercise in humans (Wilkerson et al., 2004).

In conclusion, the study by Taddei and coworkers (Taddei et al., 2000) demonstrated that regular physical training protects the vascular endothelium from aging-related alterations. The beneficial effect of exercise is related to preservation of •NO availability by a mechanism probably linked to the prevention of oxidative stress and the consequent •NO breakdown. This beneficial effect could be important in accounting for the positive impact of regular exercise on cardiovascular risk in the elderly population. Arginine and glutamine are two non-essential amino acids than can become "conditionally essential" because of elevated needs during pathological conditions, and metabolism may not be able to maintain their concentrations at sufficient levels to match metabolic requirements. Chronic exogenous arginine supplementation has not proven to exert positive clinical effects in different trials, and sequential articulation of the knowledge of introduction of arginine-driven transcriptional, translational, and epigenetic adaptations may give us a key for interpreting those controversial results. Study by Lerman and coworkers demonstrated that oral Larginine supplementation for 6 months improves coronary endothelial function in association with improvement in symptoms in patients with non-significant coronary artery stenosis. This study proposes a therapeutic role for L-arginine in patients with chest pain and coronary endothelial dysfunction (Lerman et al., 1998).

The role of L-arginine supplementation in cardiac patients remains controversial. Furthermore, it is also unclear if arginine supplementation in the sedentary population can have the same results. Further research will be needed to assess the interaction of these factors and to determine the effects of prolonged administration of arginine and antioxidants on exercise performance. Supporting that, a recent study in trained athletes, who were without any cardiovascular problems, showed beneficial effect of L-arginine in elderly physically active subjects (Chen et al., 2010). An arginine and antioxidant-containing supplement increased the anaerobic threshold and the work at anaerobic threshold at both week one and week three in elderly cyclists. No effect on VO2max was observed. This study

Exercise and Oxidative Stress 63

Auclair, C. & Voisin, E. (1985). Nitroblue Tetrazolium Reduction. In: CRC *Handbook of*

Balakrishnan, S.D. & Anuradh, C.V. (1998). Exercise, Depletion of Antioxidants and

Banfi, G.; Malavazos, A.; Iorio, E.; Dolci, A.; Doneda, L.; Verna, R. & Corsi, M.M. (2006).

Bangsbo, J. (1994). The Physiology of Soccer with Special Reference to High-Intensity Intermittent Exercise. *Acta Physiologica Scandinavica Suppl,* Vol. 619*,* pp. 1-155 Beutler, E. (1982). Catalase. In: *Red cell metabolism, a manual of biochemical methods.* E. Beutler

Bhandari, A.K. & Scheinman, M. (1985). The Long Q-T Syndrome. *Modern Concepts of*

Bloomer, R.J. & Fisher-Wellman, K.H. (2008). Blood Oxidative Stress Biomarkers: Influence

Boisseau, N. & Delamarche, P. (2000). Metabolic and Hormonal Responses to Exercise in

Borg, G.A. (1982). Psychophysical Bases of Perceived Exertion. *Medicine and Science in Sports*

Brites, F.D.; Evelson, P.A.; Christiansen, M.G.; Nicol, M.F.; Basílico, M.J.; Wikinski, R.W. &

Caiozzo, V.J.; Davis, J.A.; Ellis, J.F.; Azus, J.L.; Vandagriff, R.; Prietto, C.A. & McMaster,

Casajus, J.A. (2001). Seasonal Variation in Fitness Variables in Professional Soccer Players. *J.*

Cazzola, R.; Russo-Volpe, S.; Cervato, G. & Cestaro, B. (2003). Biochemical Assessments of

Chang, C.K.; Tseng, H.F.; Hsuuw, Y.D.; Chan, W.H. & Shieh L.C. (2002). Higher LDL

Chen, S.S.; Chang, L.S. & Wei, Y.H. (2001). Oxidative Damage to Proteins Anddecrease of

*Investigation,* Vol. 33, No. 10, (October 2003)*,* pp. 924–930

*Nutrition & Metabolism,* Vol. 46, No. 3-4, pp. 103-107

Vol. 30, No. 11, (June 2001)*,* pp. 1328-1334

of Sex, Training Status, and Dietary Intake. *Gender Medicine,* Vol. 5, No. 3,

Children and Adolescents. *Sports Medicine,* Vol. 30, No. 6, (December 2000)*,* pp. 405-

Llesuy, S.F. (1999). Soccer Players under Regular Training Show Oxidative Stress but an Improved Plasma Antioxidant Status. *Clinical Science,* Vol. 96, No. 4, (April

W.C. (1982). A Comparison of Gas Exchange Indices Used to Detect the Anaerobic Threshold. *Journal of Applied Physiology,* Vol. 53, No. 5, (November 1982)*,* pp. 1184-

*The Journal of Sports Medicine and Physical Fitness,* Vol. 41, No. 4, (December 2001)*,*

Oxidative Stress, Erythrocyte Membrane Fluidity and Antioxidant Status in Professional Soccer Players and Sedentary Controls. *European Journal of Clinical*

Oxidation at Rest and after a Rugby Game in Weekend Warriors. *Annals of*

Antioxidant Capacity in Patients with Varicocele. *Free Radical Biology & Medicine,*

Raton, Florida

1998)*,* pp. 269-275

(March 2006)*,* pp. 483-486

(Ed.), 105-115, *Grune and Stratton*, New York

*Cardiovascular Disease,* Vol. 54, pp. 45-50

*and Exerc*ise, Vol. 14, No. 5, pp. 377-381

(September 2008) *,*pp. 218-228

422

1189

pp. 463-469

1999)*,* pp. 381-385

*methods for oxygen radical research,* R.A. Greenwald (Ed.), 123-132, *CRC Press,* Boca

Antioxidant Manipulation. *Cell Biochemistry and Function,* Vol. 16, No. 4, (December

Plasma Oxidative Stress Biomarkers, Nitric Oxide and Heat Shock Protein 70 in Trained Elite Soccer Players. *European Journal of Applied Physiology*, Vol. 96, No. 5,

indicates a potential role of L-arginine and antioxidant supplementation in improving exercise performance in elderly.

Our data about different supplementation in different sports are in accordance with those results on elderly cyclist (Cubrilo, 2006). So, both vitamins and combination of vitamins+amino acids (L-arginine+L-citruline, as potential improvement of L-arginine/NO system) increased basal level of •NO in different sports: cycling, rowing and taekwondo. Furthermore, two months supplementation by vitamins+amino acids increased release of •NO and showed antioxidant properties in acute exercise, what can be start point for further strategies in systemic supplementation in elite athletes (Cubrilo, 2006).

#### **7. Conslusions and perspectives**

Regarding to all notes listed above, general conclusion about role of oxidative stress in exercise suggest that reactive oxygen species play in part role in oxidative damage during exercise. Taken into consideration controversial results of many studies, as well as our own results, we can not clearly conclude which ROS is responsible for such events in strenuous exercise. Furthermore, therapeutic interventions with different antioxidants and other supplements are, also, controversial. Based on our results, SOD might be important marker for acute adaptation on oxidative damage, but this note need further support with serious studies with large number of well randomized participants. Also, based on recent results only measurement of isoprostans, 8-Br-Deoxyguanosine and allantoin in body fluids can support conclusion about oxidative damage. Anyway, we are in a big open field and usage of redox status as potential new therapeutic target needs further big clinical trials.

#### **8. References**

indicates a potential role of L-arginine and antioxidant supplementation in improving

Our data about different supplementation in different sports are in accordance with those results on elderly cyclist (Cubrilo, 2006). So, both vitamins and combination of vitamins+amino acids (L-arginine+L-citruline, as potential improvement of L-arginine/NO system) increased basal level of •NO in different sports: cycling, rowing and taekwondo. Furthermore, two months supplementation by vitamins+amino acids increased release of •NO and showed antioxidant properties in acute exercise, what can be start point for further

Regarding to all notes listed above, general conclusion about role of oxidative stress in exercise suggest that reactive oxygen species play in part role in oxidative damage during exercise. Taken into consideration controversial results of many studies, as well as our own results, we can not clearly conclude which ROS is responsible for such events in strenuous exercise. Furthermore, therapeutic interventions with different antioxidants and other supplements are, also, controversial. Based on our results, SOD might be important marker for acute adaptation on oxidative damage, but this note need further support with serious studies with large number of well randomized participants. Also, based on recent results only measurement of isoprostans, 8-Br-Deoxyguanosine and allantoin in body fluids can support conclusion about oxidative damage. Anyway, we are in a big open field and usage

of redox status as potential new therapeutic target needs further big clinical trials.

Vol. 38, No. 9, (May 2005)*,* pp. 1164-1069

*Oxford University Press*, Oxford, United Kingdom

Allen, J.D.; Cobb, F.R. & Gow, A.J. (2005). Regional and Whole-Body Markers of Nitric

Allen, J.D.; Cobb, F.R.; Kraus, W.E. & Gow, A.J. (2006). Total Nitrogen Oxide following

*Free Radical Biology & Medicine*, Vol. 41, No. 5, (September 2006)*,* pp. 740-747 Allen, J.D.; Miller, E.M.; Schwark, E.; Robbins, J.L.; Duscha, B.D. & Annex, B.H. (2009).

Andersen, HR; Nielsen, JB; Nielsen, F. & Grandjean, P. (1997). Antioxidative Enzyme

Andrade, F.H.; Reid, M.B.; Allen, D.G. & Westerblad, H. (1998). Effect of Hydrogen Peroxide

Performance. *Nitric Oxide*, Vol. 20, No. 4, (June 2009)*,* pp. 231-237

Oxide Production following Hyperemic Stimuli. *Free Radical Biology & Medicine*,

Exercise Testing Reflects Endothelial Function and Discriminates Health Status.

Plasma Nitrite Response and Arterial Reactivity Differentiate Vascular Health and

Activity in Human Erythrocytes. *Clinical Chemistry,* Vol. 43, No. 4, (April 1997)*,* pp.

and Dithiothreitol on Contractile Function of Single Skeletal Muscle Fibres from the Mouse. *The Journal of Physiology* (London), Vol. 509, No. 2, (June 1998)*,* pp. 565-575 Armstrong, N.; McManus, A.M. & Welsman, J.R. (2008). Aerobic Fitness. In: *Paediatric*

*Exercise Science and Medicine,* N. Armstrong & W. Van Mechelen (Eds.), 269-282,

strategies in systemic supplementation in elite athletes (Cubrilo, 2006).

exercise performance in elderly.

**7. Conslusions and perspectives**

**8. References**

562-568

Exercise and Oxidative Stress 65

Djordjevic, D.; Jakovljevic, V.; Cubrilo, D.; Zlatkovic, M.; Zivkovic ,V. & Djuric, D. (2010b).

Djordjevic, D. (2011). The Influence of Training Status on Morphofunctional Characteristics

Douglas, P.S.; O'Toole, M.L.; Katz, S.E.; Ginsburg, G.S.; Hiller, W.D. & Laird, R.H. (1997).

Dröge W. (2002). Free Radicals in the Physiological Control of Cell Function. *Physiological*

Ekblom, B. (1986). Applied Physiology of Soccer. *Sports Medicine,* Vol. 3, No. 1, (January-

Evelson, P.; Gambino, G.; Travacio, M.; Jaita, G.; Verona, J.; Maroncelli, C.; Wikinski, R.;

Ferrer, M.D.; Tauler, P.; Sureda, A.; Tur, J.A. & Pons, A. (2009). Antioxidant Regulatory

Finaud, J.; Lac, G. & Filaire, E. (2006). Oxidative Stress: Relationship with Exercise and

Finkel, T. (1998). Oxygen Radicals and Signaling. *Current Opinion in Cell Biology,* Vol. 10, No.

Fisher-Wellman, K. & Bloomer, R.J. (2009). Acute Exercise and Oxidative Stress: a 30 Year

Flam, B.R.; Eichler, D.C. & Solomonson, L.P. (2007). Endothelial Nitric Oxide Production is

Ghafourifar, P. & Cadenas, E. (2005). Mitochondrial Nitric Oxide Synthase. *Trends in*

Gilligan, D.M.; Panza, J.A.; Kilcoyne, C.M.; Waclawiw, M.A.; Casino, P.R. & Quyyumi, A.A.

Vasodilation. *Circulation,* Vol. 90, No. 6, (December 1994)*,* pp. 2853–2858 Goldfarb, A.H.; Bloomer, R.J. & McKenzie, M.J. (2005). Combined Antioxidant Treatment

Tightly Coupled to the Citrulline-NO Cycle. *Nitric Oxide*, Vol. 17, No. 3-4,

(1994). Contribution of Endothelium-Derived Nitric Oxide to Exercise-Induced

Effects on Blood Oxidative Stress after Eccentric exercise. *Medicine and Science in*

Fagard R. (2003). Athlete's Heart. *Heart*, Vol. 89, No. 12, (December 2003)*,* pp. 1455–1461 Faina, M.; Gallozi, C.; Lupo, S.; Colli, S.; Sassi, R. & Marini, C. (1988). Definition of

(December 2010)*,* pp. 145–153

(December 2010)*,* pp. 100-106

February 1986)*,* pp. 50–60

2, (April 1998)*,* pp. 248-253

University of Kragujevac, Kragujevac, Serbia

*Rev*iew, Vol. 82, No. 1, (January 2002)*,* pp. 47–95

Vol. 32, No. 11, (November 2002)*,* pp. 818-825

158-163, *E&FN Spon,* London, United Kingdom

*Sports Sciences,* Vol. 27, No. 1, (January 2009)*,* pp. 49–58

History. *Dynamic Medicine,* Vol. 8, (January 2009)*,* pp. 1-25

*Pharmacological Sciences,* Vol. 26, No. 4, (April 2005)*,* pp. 190–195

*Sports and Exerc*ise, Vol. 37, No. 2, (February 2005)*,* pp. 234-239

Training. *Sports Medicine,* Vol. 36, No. 4, pp. 327-358

(November-December 2007)*,* pp. 115–121

No. 10, (November 1997)*,* pp. 1384–1388.

to Acute Exercise. *Serbian Journal of Experimental and Clinical Research*, Vol. 11, No. 4,

Coordination between Nitric Oxide and Superoxide Anion Radical during Progressive Exercise in Elite Soccer Players. *The Open Biochemistry Journal,* Vol. 4,

and Redox State of Young Handball Players. *PhD Thesis*. Faculty of Medicine,

Left Ventricular Hypertrophy in Athletes. *American Journal of Cardiology,* Vol. 80,

Llesuy, S. & Brites, F. (2002). Higher Antioxidant Defenses in Plasma and Low Density Lipoproteins from Rugby Players. *European Journal of Clinical Investigation,*

Physiological Profile of the Soccer Players. In: *Science and football.* T. Reilly (Ed.),

Mechanisms in Neutrophils and Lymphocytes after Intense Exercise. *Journal of*

Chen, S.; Kim, W.; Henning, S.M.; Carpenter, C.L. & Li, Z. (2010). Arginine and Antioxidant

Chevion, S.; Moran, D.S.; Heled, Y., Shani, Y.; Regev, G.; Abbou, B.; Berenshtein, E.;

Childs, A.; Jacobs, C.; Kaminski, T.; Halliwell, B. & Leeuwenburgh, C. (2001).

Chiueh, C.C. (1999). Neuroprotective Properties of Nitric Oxide. *Annals of the New York*

Cooper, D.M.; Nemet, D. & Galassetti, P. (2004). Exercise, Stress, and Infammation in the

Crisafulli, A.; Melis, F. & Lai, A.C. (2002). Haemodynamics during a Complete Exercise

Crouse, S.F.; Rohack, J.J. & Jacobsen, D.J. (1992). Cardiac Structure and Function in Women

Crouse, S.F.; Meade, T.; Hansen, E.B.; Green, J.S. & Martin, S.E. (2009). Electrocardiograms of

Cubrilo, D. (2006). The Evaluation of L-Arginine/NO System Function and Oxidative Stress

Cubrilo, D. (2009). Systemic Effects of Redox Disturbance Induced by Intensive Training in

Cubrilo, D.; Djordjevic, D.; Zivkovic, V.; Djuric, D.; Blagojevic, D.; Spasic, M. & Jakovljevic,

DeCoursey, T.E. & Ligeti, E. (2005). Regulation and Termination of NADPH Oxidase

Dillard, C.J.; Litov, R.E. & Savin, W.M. (1978). Effects of Exercise, Vitamin E, and Ozone on

Djordjevic, D.; Cubrilo, D.; Zivkovic, V.; Barudzic, N.; Vuletic, M. & Jakovljevic, V. (2010a).

*Biology & Medicine*, Vol. 31, No. 6, (September 2001)*,* pp. 745-753

*States of America,* Vol. 100, No. 9, pp. 5119-5123

*Academy of Sciences,* Vol. 890, pp. 301–311

Vol. 16, No. 3, (June 2004)*,* pp. 286–292

(February 2002)*,* pp. 69-70

Kragujevac, Kragujevac, Serbia

2, (September 2011)*,* pp. 273-279

No. 6, (December 1978)*,* pp. 927 – 932

Kragujevac, Serbia

2173–2193

pp. 393–401

37-42

13

Supplement on Performance in Elderly Male Cyclists: a Randomized Controlled Trial. *Journal of the International Society of Sports Nutrition,* Vol. 7, (March 2010)*,* pp.

Stadtman, E.R. & Epstein, Y. (2003). Plasma Antioxidant Status and Cell Injury after Severe Physical Exercise. *Proceedings of the National Academy of Sciences of the United*

Supplementation with Vitamin C and N-Acetyl-Cysteine Increases Oxidative Stress in Humans after an Acute Muscle Injury Induced by Eccentric Exercise. *Free Radical*

Growing Child: From the Bench to the Playground. *Current Opinion in Pediatrics,*

Induced Atrioventricular Block. *British Journal of Sports Medicine,* Vol. 36, No. 1,

Basketball Athletes—Seasonal-Variation and Comparisons with Nonathletic Controls. *Research Quarterly for Exercise and Sport,* Vol. 63, No. 4, (December 1992)*,*

Collegiate Football Athletes. *Clinical Cardiology,* Vol. 32, No. 1, (January 2009)*,* pp.

Parameters in Elite Athletes. *Master thesis.* Faculty of Medicine, University of

Young Football Players. *PhD thesis.* Faculty of Medicine, University of Kragujevac,

V. (2011). Oxidative Stress and Nitrite Dynamics under Maximal Load in Elite Athletes: Relation to Sport Type. *Molecular and Celular Biochem*istry, Vol. 355, No. 1-

Activity. *Cellular and Molecular Life Sciences,* Vol. 62, No. 19-20, (October 2005)*,* pp.

Pulmonary Function and Lipid Peroxidation. *Journal of Applied Physiology,* Vol. 45,

Pre-Exercise Superoxide Dismutase Activity Affects the Pro/Antioxidant Response

to Acute Exercise. *Serbian Journal of Experimental and Clinical Research*, Vol. 11, No. 4, (December 2010)*,* pp. 145–153

Exercise and Oxidative Stress 67

Huston, T.P.; Puffer, J.C. & Rodney, W.M.; (1985). The Athletic Heart Syndrome. *The New*

Ignarro, L.J.; Buga, G.M.; Wood, K.S.; Byrns, R.E. & Chaudhuri, G. (1987). Endothelium-

Jackson, M. J.; Pye, D. & Palomero, J. (2007). The Production of Reactive Oxygen and

Jakovljevic, V.Lj.; Zlatkovic, M.; Cubrilo, D.; Pantic, I. & Djuric, D.M. (2011). The Effects of

Ji, L.L. & Fu, R. (1993). Antioxidant Enzyme Response to Exercise and Ageing. *Medicine and Science in Sports and Exercise,* Vol. 25, No. 2, (February 1993)*,* pp. 225-231 Ji, L.L. (1999). Antioxidants and Oxidative Stress in Exercise. *Proceedings of the Society for Experimental Biology and Medicine,* Vol. 222, No. 3, (December 1999)*,* pp. 283-292 Jones, M.A. & Carter H. (2000). The Effect of Endurance Training on Parameters of Aerobic Fitness. *Sports Medicine,* Vol. 29, No. 6, (June 2000)*,* pp. 373-386 Jones, A.M.; Wilkerson, D.P. & Campbell, I.T. (2004). Nitric Oxide Synthase Inhibition with

Jungersten, L.; Ambring, A.; Wall, B. & Wennmalm, A. (1997). Both Physical Fitness and

Kakhlon, O. & Cabantchik, Z.I. (2002). The Labile Iron Pool: Characterization, Measurement,

Kashyap, S.R.; Park, Y.M.; Lara, A.; De Fronzo, R.A. & Zhang, R. (2008). Insulin Reduces

Kayatekin, B.M.; Gonenc, S.; Acikgoz, O.; Uysal, N. & Dayi, A. (2002). Effects of Sprint

Kew, S.; Mesa, M.D; Tricon, S.; Buckley, R.; Minihane, A.M. & Yaqoob, P. (2004). Effects of

Kingwell, B.A. (2000). Nitric Oxide-Mediated Metabolic Regulation during Exercise: Effects

Kingwell, B.A.; Berry, K.L.; Cameron, J.D.; Jennings. G.L. & Dart, A.M. (1997). Arterial

*Applied Physiology*, Vol. 82, No. 3, (March 1997)*,* pp. 760-764

*Physiology*, Vol. 87, No. 2, (June 2002)*,* pp. 141-144

*Nutrition*, Vol. 79, No. 4, (April 2004)*,* pp. 674–681

12, (September 2000)*,* pp. 1685–1696

Derived Relaxing Factor Produced and Released from Artery and Vein is Nitric Oxide. *Proceedings of the National Academy of Sciences of the United States of America,*

Nitrogen Species by Skeletal Muscle. *Journal of Applied Physiology,* Vol. 1025, No. 4,

Progressive Exercise on Cardiovascular Function in Top Level Athletes: Focus on Oxidative Stress. *Acta Physiologica Hungarica,* Vol. 98, No. 1, (March 2011)*,* pp. 50-57

L-NAME Reduces Maximal Oxygen Uptake but not Gas Exchange Threshold during Incremental Cycle Exercise in Man. *The Journal of Physiology*, Vol. 56, No. 1

Acute Exercise Regulate Nitric Oxide Formation in Healthy Humans. *Journal of*

and Participation in Cellular Processes. *Free Radical Biology & Medicine,* Vol. 33, No.

Plasma Arginase Activity in Type 2 Diabetic Patients. *Diabetes Care*, Vol. 31, No. 1,

Exercise on Oxidative Stress in Skeletal Muscle and Liver. *European Journal of*

Oils Rich in Eicosapentaenoic and Docosahexaenoic Acids on Immune Cell Composition and Function in Healthy Humans. *The American Journal of Clinical*

of Training in Health and Cardiovascular Disease. *The FASEB journal: Official Publication of the Federation of American Societies for Experimental Biology,* Vol. 14, No.

Compliance Increases after Moderate-Intensity Cycling. *American Journal of*

*England Journal of Medicine,* Vol. 313, No. 1, (July 1985)*,* pp. 24 –32

Vol. 84, No. 24, (December 1987)*,* pp. 9265–9269

(April 2007)*,* pp. 1664-1670

(October 2004)*,* pp. 329-338

8, (October 2002)*,* pp. 1037–1046

(January 2008)*,* pp. 134–139

Gougoura, S.; Nikolaidis, M.G.; Kostaropoulos, I.A.; Jamurtas, A.Z.; Koukoulis, G. &

Green, L.C.; Wagner, D.A.; Glogowski, J.; Skipper, P.L.; Wishnok, J.S. & Tannenbaum, S.R.

Green. D.; Cheetham, C.; Mavaddat, L.; Watts. K.; Best, M.; Taylor. R. & O'Driscoll, G.

Green, D.J.; Maiorana, A.; O'Driscoll, G. & Taylor, R. (2004). Effect of Exercise Training on

Grody, W.W.; Argyle, C.; Kern, R.M.; Dizikes, D.J.; Spector, E.B.; Strickland, A.D.; Klein, D.

Groussard, C.; Rannou-Bekono, F.; Machefer, G.; Chevanne, M.; Vincent, S.; Sergent, O.;

Gubner, R. & Ungerleider, H. (1943). Electrocardiographic Criteria for Left Ventricular

Halliwell, B. & Gutteridge, J.M.C. (1999). *Free Radicals in Biology and Medicine,* Oxford

Halliwell, B. & Gutteridge, J.M.C. (2007). *Free Radicals in Biology and Medicine,* Oxford

Hambrecht, R.; Fiehn, E.; Weigl, C.; Gielen, S.; Hamann, C.; Kaiser, R.; Yu, J.;

Henschen, S. (1899). Skilanglauf und Skiwettlauf. Eine Medizinische Sport Studie. *Mitt Med*

Herrero A. & Barja, G. (1997). ADP Regulation of Mitochondrial Free Radical Production is

Howley, E.T.; Bassett, D.R.Jr. & Welch, H.G. (1995). Criteria for Maximal Oxygen Uptake:

*Clinical Investigation,* Vol. 83, No. 2, (February 1989)*,* pp. 602–609

*of Applied Physiology,* Vol. 89, No. 1, (March 2003)*,* pp. 14-20

*Biochemistry,* Vol. 126, No. 1, (October 1982)*,* pp. 131-138

283, No. 3, (September 2002)*,* pp. H899–H907

Vol. 561, No. 1, (November 2004)*,* pp. 1–25

Vol. 72, No. 2, (August 1943)*,* pp. 196-209

University Press, New York

University Press, New York

1998)*,* pp. 2709–2715

*Klin Uppsala (Jena),* Vol.2, pp. 15-18

(September 1995)*,* pp. 1292-1301

*Biomembranes,* Vol. 29, No. 3, (June 1997)*,* pp. 241-249

235-239

Kouretas, D. (2007). Increased Oxidative Stress Indices in the Blood of Child Swimmers. *European Journal of Applied Physioogy,* Vol. 100, No. 2, (May 2007)*,* pp.

(1982). Analysis of Nitrate, Nitrite and [15 N] Nitrate in Biological Fluids. *Analytical*

(2002). Effect of Lower Limb Exercise on Forearm Vascular Function: Contribution of Nitric Oxide. *American Journal of Physiology. Heart and Circulatory Physiology,* Vol.

Endothelium-Derived Nitric Oxide Function in Humans. *The Journal of Physiology,*

& Cederbaum, S.D. (1989). Differential Expression of the Two Arginase Genes in Hyperargininemia. Enzymatic, Pathologic, and Molecular Analysis. *The Journal of*

Cillard, J. & Gratas-Delamarche, A. (2003). Changes in Blood Lipid Peroxidation Markers and Antioxidants after a Single Sprint Anaerobic Exercise. *European Journal*

Hypertrophy. Factors Determining the Evolution of the Electrocardiographic Patterns in Hypertrophy and Bundle Branch Block. *Archives of Internal Medicine,*

Adams, V.; Niebauer, J. & Schuler, G. (1998). Regular Physical Exercise Corrects Endothelial Dysfunction and Improves Exercise Capacity in Patients with Chronic Heart Failure. *Circulation,* Vol. 98, No. 24, (December

Different with Complex I or Complex II Linked Substrates: Implications for the Exercise Paradox and Brain Hypermetabolism. *Journal of Bioenergetics and*

Review and Commentary. *Medicine and Science in Sports and Exerc*ise, Vol. 27, No. 9,

Exercise and Oxidative Stress 69

Maron, B.J. (1998). Heart Disease and Other Causes of Sudden Death in Young Athletes. *Current Problems in Cardiology,* Vol. 23, No. 9, (September 2003)*,* pp. 477–529 Maron, B.J. (2003). Sudden Death in Young Athletes*. New England Journal of Medicine*, Vol.

Maron, B.J. & Zipes, D.P. (2005). 36th Bethesda Conference: Eligibility Recommendations for

Maron, J.B. & Pelliccia A. (2006). The Heart of Trained Athletes. Cardiac Remodeling and the

Martin, W.H. 3rd.; Coyle, E.F.; Bloomfield, S.A. & Ehsani, A.A. (1986). Effects of Physical

Marzatiko, F.; Pansarasa, O.; Bertorelli, L.; Somenzini, L. & Della Valle, G. (1997). Blood Free

Mastaloudis, A.; Leonard, S.W. & Traber, M.G. (2001). Oxidative Stress in Athletes during

Maxwell, A.J.; Ho, H.K.V.; Le, C.Q.; Lin, P.S.; Bernstein. D. & Cooke, J.P. (2001). L-Arginine

Metin, G.; Gumustas, M.K.; Uslu, E., Belce, A.; & Kayserilioglu, A. (2003). Effect of Regular

Misra, H.P. & Fridovich, I. (1972). The Role of Superoxide-Anion in the Autooxidation of

Miyazaki, H.; Oh-ishi, S.; Ookawara, T.; Kizaki, T.; Toshinai, K.; Ha, S.; Haga, S.; Ji, L.L. &

*Chemistry,* Vol. 247, No. 10, (May 1972)*,* pp. 3170-3175

No. 1-2, (January-February 2001)*,* pp. 1-6

Vol. 94, No. 4, (August 1996)*,* pp. 850–856

349, No. 11, (September 2003)*,* pp. 1064–1075

2006)*,* pp. 1633-1644

1986)*,* pp. 982–989

(October 2001)*,* pp. 911-922

(November 1969)*,* pp. 6056-6063

235-239

35-39

*College of Cardiology,* Vol. 45, No. 8, pp. 1312–1375

(1996). American Heart Association Statement for Health Professionals: Cardiovascular Preparticipation Screening of Competitive Athletes. *Circulation,*

Competitive Athletes with Cardiovascular Abnormalities. *Journal of the American*

Risks of Sports, Including Sudden Death. *Circulation*, Vol. 114, No. 15, (October

Deconditioning after Intense Endurance Training on Left Ventricular Dimensions and Stroke Volume. *Journal of the American College of Cardiology,* Vol. 7, No. 5, (May

Radical Antioxidant Enzymes and Lipid Peroxides following Long-Distance and Lactacidemic Performances in Highly Trained Aerobic and Sprint Athletes. *The Journal of Sports Medicine and Physical Fitness,* Vol. 37, No. 4, (December 1997)*,* pp.

Extreme Endurance Exercise. *Free Radical Biology & Medicine,* Vol. 31, No. 7,

Enhances Aerobic Exercise Capacity in Association with Augmented Nitric Oxide Production. *Journal of Applied Physiology*, Vol. 90, No. 3, (March 2001)*,* pp. 933-938 McCord, J.M. & Fridovich, I. (1969). The Utility of Superoxide Dismutase in Studying Free

Radical Reactions. I. Radicals Generated by the Interaction of Sulfite, Dimethyl Sulfoxide, and Oxygen. *The Journal of Biological Chemistry,* Vol. 244, No. 22,

Training on Plasma Thiols, Malondialdehyde and Carnitine Concentracions in Young Soccer Players. *Chinese Journal of Physiology,* Vol. 46, No. 1, (March 2001)*,* pp.

Epinephrine and a Simple Assay for Superoxide Dismutase. *The Journal of Biological*

Ohno, H. (2001). Strenuous Endurance Training in Humans Reduces Oxidative Stress following Exhausting Exercise. *European Journal of Applied Physiol*ogy, Vol. 84,

*Physiology. Heart and Circulatory Physiology,* Vol. 273, No. 5 Pt 2, (November 1997)*,* pp. H2186–H2191

Kingwell, B.A.; Sherrard, B.; Jennings, G.L. & Dart, A.M. (1997). Four Weeks of Cycle

*Journal of Physiology,* Vol. 272, No. 3 Pt 2, (March 1997)*,* pp. H1070-H1077 Kurz, S. & Harrison, D.G. (1997). Insulin and the Arginine Paradox. *The Journal of Clinical*

Laaksonen, D.E.; Atalay, M.; Niskanen, L.; Uusitupa, M., Hänninen, O. & Sen, C.K. (1999).

Lagranha, C.J.; Alba-Loureiro, T.C.; Martins, E.F.; Pithon-Curi, T.C.; & Curi, R. (2008).

*Societies for Experimental Biology,* Vol. 11, No. 2, (February 1997)*,* pp. 118-124 Lekhi, C.; Gupta, P.H. & Singh, B. (2007). Influence of Exercise on Oxidant Stress Products in

Lerman, A.; Burnett, J.C.; Higano, S.T.Jr.; McKinley, L.J. & Holmes, D.R.Jr. (1998). Long-

Function in Humans. *Circulation*, Vol. 97, No. 21, (June 1998)*,* pp. 2123-2128 Levine, DB (2008). VO2max: What do We Know, and What do We Still Need to Know? *The*

Li, C.; Huang, W.; Harris, M.B.; Goolsky, J.M. & Venema, R.C. (2005). Interaction of

Lim, J.S.; Hwang, J.S.; Lee, J.A.; Kim, D.H.; Park, K.D.; Jeong, J.S. & Cheon, G.J. (2009). Cross-

*Journal of the Japan Pediatric Society,* Vol. 51, No. 2, (April 2009)*,* pp. 63-68 Liochev, S.I.. & Fridovich, I. (2002). The Haber–Weiss Cycle—70 Years Later: an Alternative

Maeda, S.; Miyauchi, T.; Kakiyama, T.; Sugawara, J.; Iemitsu, M.; Irukayama-Tomobe, Y.;

Maiorana, A.; O'Driscoll, G.; Taylor, R. & Green, D. (2003). Exercise and the Nitric Oxide

Maron, B.J.; Thompson, P.D.; Puffer, J.C.; McGrew, C.A.; Strong, W.B.; Douglas, P.S.; Clark,

Vasodilator System. *Sports Medicine,* Vol. 33, No. 14, pp. 1013-1035

*Journal of Physiology*, Vol. 586, No. 1, (January 2008)*,* pp. 25-34

*Journal*, Vol. 386, No. 3 (March 2005)*,* pp. 567–574

Vol. 69, No. 9, (July 2001)*,* pp. 1005-1016

*Investigation,* Vol. 99, No. 3, (February 1997)*,* pp. 369–370

pp. H2186–H2191

2007)*,* pp. 691-693.

*Research,* Vol. 4, No. 1-2, pp. 53-59

*Physiology. Heart and Circulatory Physiology,* Vol. 273, No. 5 Pt 2, (November 1997)*,*

Training Increases Basal Production of Nitric Oxide from the Forearm. *American*

Blood Glutathione Homeostasis as a Determinant of Resting and Exercise-Induced Oxidative Stress in Young Men. *Redox Report: Communications in Free Radical*

Neutrophil Fatty Acid Composition: Effect of a Single Session of Exercise and Glutamine Supplementation. *Amino Acids*, Vol. 35, No. 1, (June 2008)*,* pp. 243–245 Lander H.M. (1997). An Essential Role of Free Radicals and Derived Species in Signal

Transduction. *The FASEB journal: Official Publication of the Federation of American*

Elite Indian Cyclists. *British Journal of Sports Medicine,* Vol. 41, No. 10, (October

Term L-Arginine Supplementation Improves Small-Vessel Coronary Endothelial

Endothelial Nitric Oxide Synthase with the CAT-1 Arginine Transporter Enhances NO Release by a Mechanism not Involving Arginine Transport. *The Biochemical*

Calibration of Multi-Frequency Bioelectrical Impedance Analysis with Eight-Point Tactile Electrodes and Dual-Energy X-Ray Absorptiometry for Assessment of Body Composition in Healthy Children Aged 6-18 Years. *Pediatrics International: Official*

View. *Redox Report : Communications in Free Radical Research,* Vol. 7, No. 1, pp. 55–57

Murakami, H.; Kumagai, Y.; Kuno, S. & Matsuda M. (2001). Effects of Exercise Training of 8 Weeks and Detraining on Plasma Levels of Endothelium-Derived Factors, Endothelin-1 and Nitric Oxide in Healthy Young Humans. *Life Sciences,*

L.T.; Mitten, M.J.; Crawford, M.H.; Atkins, D.L.; Driscoll, D.J. & Epstein, A.E.

(1996). American Heart Association Statement for Health Professionals: Cardiovascular Preparticipation Screening of Competitive Athletes. *Circulation,* Vol. 94, No. 4, (August 1996)*,* pp. 850–856

Exercise and Oxidative Stress 71

Pelliccia, A.; Maron, B.J.; Culasso, F.; Di Paolo, F.M.; Spataro, A.; Biffi, A.; Caselli, G. &

in Trained Athletes. *Circulation,* Vol. 102, No. 3, (July 2000)*,* pp. 278-84 Pelliccia, A.; Maron, B.J.; de Luca, R.; Di Paolo, F.M.; Spataro, A. & Culasso, F. (2002).

Deconditioning. *Circulation*, Vol. 105, No. 8, (February 2002)*,* pp. 944 –949 Pelliccia, A.; Maron, B.J.; Di Paolo, F.M.; Biffi, A.; Quattrini, F.M.; Pisicchio, C.; Roselli, A.;

Pelliccia, A.; Culasso, F.; Di Paolo, M.F.; Accettura, D.; Cantore, R., Castagna, W.;

*European Heart Journal*. Vol. 28, No. 16, (August 2007)*,* pp. 2006-2010 Pick, E. & Keisari, Y. (1980). A Simple Colorimetric Method for the Measurment of

Vol. 46, No. 4, (August 2005)*,* pp. 690–696

*Medica Medianae*. Vol. 44, No. 4, pp. 17-20

No. 5 Pt 1, (May 1995)*,* pp. L699-L722

(January 2008)*,* pp. 153-159

1, (February 2004)*,* pp. 2-6

Vol. 38, No. 1-2, pp. 161-170

559-565

971

Piovano, P. (2000). Clinical Significance of Abnormal Electrocardiographic Patterns

Remodeling of Left Ventricular Hypertrophy in Elite Athletes after Long-Term

Caselli, S. & Culasso, F. (2005). Prevalence and Clinical Significance of Left Atrial Remodeling in Competitive Athletes. *Journal of the American College of Cardiology,*

Ciacciarelli, A.; Costini, G.; Cuffari, B.; Drago, E.; Federici, V.; Gribaudo, C.G.; Lacovelli, G.; Landolfi, L.; Menichetti, G.; Atzeni, O.U.; Vagnini, A.; Casasco, M. & Di Luigi, L. (2007). Prevalence of Abnormal Electrocardiograms in a Large Unselected Population Undergoing Pre-Participation Cardiovascular Screening.

Hydrogen Peroxide Produced by Cells in Culture. *Journal of Immunological Methods,*

R. & Defraigne, J. (2000). Evaluation of Autoantibodies against Oxidized LDL and Antioxidant Status in Top Soccer and Basketball Players after 4 Months of Competition. *Free Radical Biology & Medicine,* Vol. 28, No. 4, (February 2000)*,* pp.

Uptake (VO2max) as the Indicator of Physical Working Capacity In Sportsmen. *Acta*

Berrazueta, J.R. (1997). Contribution of Nitric Oxide to Exercise-Induced Changes in Healthy Volunteers: Effects of Acute Exercise and Long-Term Physical Training. *European Journal of Clinical Investigation,* Vol. 27, No. 11, (November 1997)*,* pp. 967-

Reaction of Nitric Oxide with Superoxide. *American Journal of Physiology,* Vol. 268,

Induced by Regular Exercise. *Free Radical Biology & Medicine,* Vol. 44, No. 2,

Stress and Hormesis. *Ageing Research Reviews,* Vol. 7, No. 1, (January 2008)*,*pp. 34-42

Submaximal Resistance Exercise in Men. *European Journal of Nutrition,* Vol. 43, No.

C.; Meyer, C. & Kelm, M. (2007). Nitric Oxide Synthase-Derived Plasma Nitrite

Pincemail, J.; Lecomte, J.; Castiau, J. ; Collard, E. ; Vasankari, T. ; Cheramy-Bien, J. ; Limet,

Ponorac, N; Matavulj, A; Grujic. N; Rajkovaca, Z; & Kovacevic, P. (2005). Maximal Oxigen

Poveda, J.J.; Riestra, A.; Salas, E.; Cagigas, M.L.; López-Somoza, C.; Amado, J.A. &

Pryor, W.A. & Squadrito, G.L. (1995). The Chemistry of Peroxynitrite: a Product from the

Radak, Z.; Chung, H.Y. & Goto, S. (2008a). Systemic Adaptation to Oxidative Challenge

Radak, Z.; Chung, H.Y.; Koltai, E.; Taylor, A.W. & Goto, S. (2008b). Exercise, Oxidative

Ramel, A.; Wagner, K.H. & Elmadfa, I. (2004). Plasma Antioxidants and Lipidoxidation after

Rassaf, T.; Lauer, T.; Heiss, C.; Balzer, J.; Mangold, S.; Leyendecker, T.; Rottler, J.; Drexhage,

Morganroth, J.; Maron, B.J.; Henry, W.L. & Epstein, S.E. (1975). Comparative Left

Morris, S.M.Jr. (2006): Arginine: beyond Protein. *The American Journal of Clinical Nutrition,*

Muller, F.L., Liu, Y., & Van Remmen, H. (2004). Complex III Releases Superoxide to Both

Naghizadeh, H.; Afzalpour, M.E. & Zarban A. (2009). The Comparison of Antioxidant Status

Naka, K.K.; Tweddel, A.C.; Parthimos. D.; Henderson. A.; Goodfellow. J. & Frenneaux, M.P.

Ohkawa, H.; Ohishi, N. & Yagi, K. (1979). Assay for Lipid Peroxides in Animal Tissues by

Ookawara, T.; Haga, S.; Ha S.; Oh-Ishi ,S.; Toshinai, K.; Kizaki, T.; Ji, L.L.; Suzuki, K. &.

Ørtenblad, N.; Madsen, K. & Djurhuus, M.S. (1997). Antioxidant Status and Lipid

Оstojić, MS. (2000). Physical and Physiological Characteristics of Elite Serbian Soccer

Palazzetti, S.; Richard, M.J.; Favier, A. &. Margaritis, I. (2003). Overload Training Increases

Pastor, N.; Weinstein, H.; Jamison, E. & Brenowitz, M. (2000). A Detailed Interpretation of

Pelliccia, A.; Maron, B.J.; Spataro, A.; Proschan, M.A. & Spirito, P. (1991). The Upper Limit of

Pelliccia, A.; Maron, B.J.; Culasso, F.; Spataro, A. & Caselli, G. (1996). Athlete's Heart in

*Journal of Medicine*, Vol. 324, No. 5, (January 1991)*,* pp. 295–301

*Physiol*ogy, Vol. 28, No. 4, (August 2003)*,* pp. 588-604

1, (November 2000)*,* pp. 55–68

1996)*,* pp. 211–215

4, (April 1975)*,* pp. 521–524

351-358

713-719

29

R1258-R1263

*Vol.* 83, No. 6, (February 2006), pp. 508S–512S

279, No. 47, (November 2004)*,* pp. 49064–49073

*of Medical Sciences,* Vol. 16, No. 3, pp. 54-61

Vol. 284, No. 3, (March 2003)*,* pp. H970–H978

Ventricular Dimensions in Trained Athletes. *Annals of Internal Medicine,* Vol. 82, No.

Sides of the Inner Mitochondrial Membrane. *The Journal of Biological Chemistry,* Vol.

and Lipid Profile of Karate Athletes with Non-Athletes. *Journal of Birjand University*

(2003). Arterial Distensibility: Acute Changes following Dynamic Exercise in Normal Subjects*. American Journal of Physiology. Heart and Circulatory Physiology,*

Thiobarbituric Acid Reaction. *Analytical Biochemistry,* Vol. 95, No. 2, (June 1979)*,* pp.

Ohno H. (2003). Effects of Endurance Training on Three Superoxide Dismutase Isoenzymes in Human Plasma. *Free Radical Research,* Vol. 37, No. 7, (July 2003)*,* pp.

Peroxidation after Short-Term Maximal Exercise in Trained and Untrained Humans. *American Journal of Physiology,* Vol. 272, No. 4 Pt 2, (April 1997)*,* pp.

Players. *Facta Universitatis Series Physical Education andSports,* Vol. 7, No. 7, pp. 23-

Exercise-Induced Oxidative Stress and Damage. *Canadian Journal of Applied*

OH Radical Footprints in a TBPDNA Complex Reveals the Role of Dynamics in the Mechanism of Sequence-Specic Binding. *Journal of Molecular Biology,* Vol. 304, No.

Physiologic Cardiac Hypertrophy in Highly Trained Elite Athletes. *New England*

Women: Echocardiographic Characterization of Highly Trained Elite Female Athletes. *JAMA: the Journal of the American Medical Association,* Vol. 276, No. 3 (July

Exercise and Oxidative Stress 73

Tanskanen, M.; Atalay, M. & Uusitalo, A. (2010). Altered Oxidative Stress in Overtrained Athletes. *Journal of Sports Sciences,* Vol. 28, No. 3, (February 2010)*,* pp. 309-317 Tappel, A.L. (1953). The Inhibition of Hematin-Catalyzed Oxidations by Alpha-Tocopherol.

Tauler, P.; Ferrer, M.D.; Sureda, A.; Pujol, P.; Drobnic, F.; Tur, J.A. & Pons, A. (2008).

Tinken, T.M.; Thijssen, D.H.J.; Black, M.A.; Cable, N.T. & Green, D.J. (2008). Time Course of

*Experimental Therapeutics,* Vol. 318, No. 3, (September 2006)*,* pp. 1368–1374 Valko, M.; Izakovic; M.; Mazur, M.; Rhodes, C.J. & Telser, J. (2004). Role of Oxygen Radicals

Valko M.; Morris H. & Cronin, M.T.D. (2005). Metals, Toxicity and Oxidative Stress. *Current*

Valko, M.; Rhodes, C.J; Moncol, J.; Izakovic, M. & Mazur, M. (2006). Free Radicals, Metals

Van de Poll, M.C.G.; Siroen, M.P.C.; van Leeuwen, P.A.M.; Soeters, P.B.; Melis, G.C.;

Vorbach, C.; Harrison, R. & Capecchi, M.R. (2003). Xanthine Oxidoreductase is Central to

Wasserman, K.; Whipp, B.J.; Koyal, S.N. & Beaver, W.L. (1973). Anaerobic Threshold and

Wilkerson, D.P.; Campbell, I.T. & Jones, A.M. (2004). Influence of Nitric Oxide Synthase

*Physiology,* Vol. 104, No. 5, (November 2008)*,* pp. 777–785

No. 1-2, (November 2004)*,* pp. 37–56

*Medicinal Chemistry,* Vol. 12, No. 10, pp. 1161–1208

*Interactions,* Vol. 160, No. 1, (March 2006)*,* pp. 1–40

Vol. 24, No. 9, (September 2003)*,* pp. 512–517

No. 2, (August 1973)*,* pp. 236-243

225

1045-1062

1430-1442

*Archives of Biochemistry and Biophysics,* Vol. 47, No. 1, (November 1953)*,* pp. 223-

Supplementation with an Antioxidant Cocktail Containing Coenzyme Q Prevents Plasma Oxidative Damage Induced by Soccer. *European Journal of Applied*

Change in Vasodilator Function and Capacity in Response to Exercise Training in Humans. *The Journal of Physiology.* Vol. 586, No. 20, (October 2008)*,* 5003-5012 Topal, G.; Brunet, A.; Walch, L.; Boucher, J.L. & David-Dufilho, M. (2006). Mitochondrial

Arginase II Modulates Nitric-Oxide Synthesis through non Freely Exchangeable L - Arginine Pools in Human Endothelial Cells. *The Journal of Pharmacology and*

in DNA Damage and Cancer Incidence. *Molecular and Celular Biochemistry,* Vol. 266,

and Antioxidants in Oxidative Stress-Induced Cancer. *Chemico-Biological*

Boelens, P.G.; Deutz, N.E.P. & Dejong, C.H.C. (2007). Interorgan Amino Acid Exchange in Humans: Consequences for Arginine and Citrulline Metabolism. *The American Journal of Clinical Nutrition,* Vol. 85, No. 1, (January 2007)*,* pp. 167–172 Vollaard, N.B.; Shearman, J.P. & Cooper, C.E. (2005). Exercise-Induced Oxidative Stress:

Myths, Realities and Physiological Relevance. *Sports Medicine,* Vol. 35, No. 12, pp.

the Evolution and Function of the Innate Immune System. *Trends in Immunology,*

Respiratory Gas Exchange during Exercise. *Journal of Applied Physiol*ogy, Vol. 35,

Inhibition on Pulmonary O2 Uptake Kinetics during Supra-Maximal Exercise in Humans. *The Journal of Physiology*, Vol. 561, No. 2, (December 2004)*,* pp. 623-635 Wolin, M.S. (2000). Interactions of Oxidants with Vascular Signaling Systems.

*Arteriosclerosis, Thrombosis, and Vascular Biology,* Vol. 20, No. 6, (June 2000)*,* pp.

Predicts Exercise Capacity. *British Journal of Sports Medicine,* Vol. 41, No. 10, (October 2007)*,* pp. 669-673

Ridnour, L.A.; Thomas D.D.; Mancardi D.; Espey, M.G.; Miranda K.M.; Paolocci N.; Feelisch,

Santhanam, L.; Christianson, D.W.; Nyhan, D. & Berkowitz, D.E. (2008). Arginase and

Schulman, S.P.; Becker L.C.; Kass, D.A.; Champion, H.C.; Terrin, M.L.; Forman, S.; Ernst

Schwartz, P.J.; Periti, M. & Malliani, A. (1975). The Long QT Syndrome. *American*

Selamoglu, S.; Turgay, F.; Kayatekin, B.M.; Gönenc, S. & Yslegen, C. (2000). Aerobic and

Sharma, S.; Maron, B.J., Whyte, G.; Firoozi, S.; Elliott, P.M. & McKenna, W.J. (2002).

Sharman, J.E.; McEniery, C.M.; Campbell, R.; Pusalkar, P.; Wilkinson, I.B.; Coombes, J.S. &

Shephard, R.J. (1999). Biology and Medicine of Soccer: an Update. *Journal of Sport Sciences,*

Stolen, T.; Chamari, K. & Castagna, C. (2005). Physiology of Soccer: an Update. *Sports*

Sureda, A.; Ferrer, M.D.; Tauler, P.; Tur, J.A. & Pons, A. (2008). Lymphocyte Antioxidant

Taddei, S.; Galetta, F.; Virdis, A.; Ghiadoni, L.; Salvetti, G.; Franzoni, F.; Giusti, C. & Salvetti,

*of America,* Vol. 102, No. 37, (September 2005)*,* pp. 13147–13152

*Heart Journal,* Vol. 89, No. 3, (March 1975)*,* pp. 378-390

*Physiologica Hungarica* Vol. 87, No. 3, pp. 267-273

(October 2002)*,* pp. 1431–1436

No. 4, (April 2008)*,* pp. 856-861

*Medicine,* Vol. 35, No. 6, pp. 501-536

Vol. 17, No. 10, (October 1999)*,* pp. 757-786

*Radical Research,* Vol. 42, No. 4, (April 2008)*,* pp. 312–319

(October 2007)*,* pp. 669-673

1632–1642

pp. 58–64

2901

Predicts Exercise Capacity. *British Journal of Sports Medicine,* Vol. 41, No. 10,

M.; Fukuto, J. & Wink, D.A. (2004). The Chemistry of Nitrosative Stress Induced by Nitric Oxide and Reactive Nitrogen Oxide Species. Putting Perspective on Stressful Biological Situations. *Biological Chemistry,* Vol.385, No. 1, (January 2004)*,* pp. 1–10 Ridnour, L.A.; Isenberg, J.S.; Espey, M.G.; Thomas, D.D.; Roberts, D.D. & Wink, D.A. (2005).

Nitric Oxide Regulates Angiogenesis Through a Functional Switch Involving Thrombospondin-1. *Proceedings of the National Academy of Sciences of the United States*

Vascular Aging. *Journal of Applied Physiology,* Vol. 105, No. 5, (November 2008)*,* pp.

K.V., Kelemen, M.D.; Townsend, S.N.; Capriotti, A.; Hare, J.M. & Gerstenblith, G. (2006). L-Arginine Therapy in Acute Myocardial Infarction. The Vascular Interaction with Age in Myocardial Infarction (VINTAGE MI) Randomized Trial. *JAMA: the Journal of the American Medical Association,* Vol. 295, No. 1, (January 2006)*,*

Anaerobic Training Effects on the Antioxidant Enzymes in the Blood. *Acta*

Physiologic Limits of Left Ventricular Hypertrophy in Elite Junior Athletes: Relevance to Differential Diagnosis of Athlete's Heart and Hypertrophic Cardiomyopathy. *Journal of the American College of Cardiology*, Vol. 40, No. 8,

Cockcroft, J.R. (2008). Nitric Oxide Does not Significantly Contribute to Changes in Pulse Pressure Amplification during Light Aerobic Exercise. *Hypertension*, Vol. 51,

Response and H2O2 Production after a Swimming Session: Gender Differences. *Free*

A. (2000). Physical Activity Prevents Age-Related Impairment in Nitric Oxide Availability in Elderly Athletes. *Circulation,* Vol.101, No. 25, (June 2000)*,* pp. 2896-

**4**

*Bulgaria*

**Transient Cold Shock Induces**

Nedelina Kostadinova, Ekaterina Krumova,

*The Stephan Angeloff Institute of Microbiology,*

*Bulgarian Academy of Sciences,*

**Oxidative Stress Events in Antarctic Fungi**

Tzvetanka Stefanova, Vladislava Dishliyska and Maria Angelova

The Antarctic biota has evolved under the influence of a suite of geological and climatic factors, including the geographic isolation of the landmass and the continental shelves, extremely low temperatures and intense seasonality (Russo et al., 2010). The isolation and environmental history of Antarctica have led to a unique biota. Many groups of organisms became extinct in Antarctica as a result of the extremely cold conditions.  In addition to its well-known inhabitants, such as penguins and seals, it also has a diverse and unique range of microbial diversity (Nichols et al., 1999; Vincent, 2000). Microorganisms successfully colonise cold habitats and play a major role in the processes of nutrient turnover at low temperatures. In recent years, a growing attention in research has been devoted to cold-adapted microorganisms. This interest in Antarctic microorganisms stems from several reasons. Antarctica's environmental extremes present conditions in which microorganisms have evolved unique characteristics for survival, which are of great scientific interest. Moreover, the availability of novel Antarctic species, which are generally isolated from extreme environments, opens the door for biotechnological exploration. Investigations of psychrotolerant and psychrophilic microorganisms are also important for human health because microorganisms can cause food spoilage and food-borne diseases. Research on cold shock raises a number of questions: which cellular function is affected most upon cold shock, what makes cell growth stop, and are there well-conserved or common cold shock proteins as in the case of heat-shock proteins? These questions are no less important

Cold-adapted microorganisms include both psychrophilic (organisms with an optimal growth temperature at or below 15°C and a maximum growth temperature below 20°C) and psychrotrophic or psychrotolerant (organisms exhibiting the ability to grow at temperatures below 15°C but exhibiting maximum growth rates at temperature optima above 18°C) organisms, and they are often subjected to other extreme environmental parameters (Morgan-Kiss et al., 2006). Antarctic microflora is also represented by mesophilic and thermophilic bacteria that are isolated from geothermal soils and by mesophilic fungi, which

**1. Introduction**

than those in the case of heat shock (Inouye, 1999).