16.1. INTRODUCTION

Free radicals are normally produced during numerous physiological processes and play important roles as regulatory mediators in signalling processes (Strobel et al. 2011). Under physiological conditions, the body has adequate antioxidant defences to cope with the production of free radicals. However, oxidant species can become toxic when generated in excess or in the presence of a deficiency in the naturally occurring antioxidant defences. Specifically, the imbalance between free radical generation and antioxidant defence leads to an oxidative stress state, which may be involved in ageing processes and in many pathological conditions (e.g. cardiovascular and neurodegenerative disease, and cancer) (Valko et al. 2007).

Exercise can have positive or negative effects on redox biology depending on the type (acute or chronic), and on training specificity, load and the basal level of training. Beneficial changes on multiple physiological and laboratory parameters have been generally observed as a result of regular moderate training (Haskell et al. 2007). Conversely, acute and strenuous exercise may paradoxically induce oxidative stress and adverse effects on health (Neubauer et al. 2008, Suzuki et al. 2006). Antioxidant nutrients by diet and exogenous supplementation may be helpful to cope with adverse implications on health and performance (Whayne and Maulik 2012, Braakuis 2012). However, caution should be taken against excess antioxidant supplements.

The complexity of the human antioxidant defence/oxidant system and their delicate balance renders it extremely difficult to estimate the oxidative stress status (Lee et al. 2012). A number of oxidative stress biomarkers (belonging to both antioxidant and oxidant counterparts and also to inflammatory processes) have been identified and measured in biological fluids (Lee et al. 2012). The choice of a specific biomarker and the analytical method used may have a great impact on the results obtained (Lee et al. 2012). Moreover, it is clear that the choice of specific markers depends on the function affected by the exercise and/or by the deficit of the nutrient intake. At the moment, no shared consensus exists on which biomarkers or group of biomarkers must be used to estimate exercise effects as well as bioefficacy of dietary/supplemental antioxidants in sport.

In this chapter, aspects related to the effect of exercise on oxidative stress, diet/antioxidant supplementation and biomarkers in trained subjects will be discussed. Moreover, possible future strategies to answer still open questions and to solve problems in the assessment of these complex issues will also be proposed.

16.2. EFFECT OF EXERCISE ON HEALTH: GOOD AND BAD OF TRAINING

There is a large amount of evidence on the fact that regular and moderate physical exercise improves prognosis and quality of life in patients with cardiovascular disease, hypertension and some types of cancers as well as in those affected by cognitive diseases (Mellett and Bousquet 2013, Briet and Schiffrin 2013, Jones and Alfano 2013, Raichlen and Polk 2013). Performance is related to training and physical adaptation, and correct nutrition in individuals with specific genetic characteristics can facilitate such adaptations. Beneficial effects on multiple physiological laboratory parameters have generally been observed as a result of regular moderate training (Banfi et al. 2012).

However, the effect of sustained and strenuous exercise, as in competitive athletes, is still controversial. In fact, while some studies report that strenuous and sustained exercise induces oxidative stress, inflammatory response and even structural damage of skeletal and cardiac muscle cells, other data show that training is able to confer resistance against oxidative stress and reduce inflammation (Neubauer et al. 2008, Pinto et al. 2012, Nunn et al. 2010).

Increased reactive oxygen species (ROS) generation is considered to be the hallmark of ageing and the major determinant of lifespan (Vina et al. 2013). On an epidemiological basis, elite athletes, in particular those performing endurance aerobic exercise, live longer than the general population and an inverse linear dose–response relationship has been reported between the volume of physical training and all-cause mortality (Corbi et al. 2012, Lee et al. 2001). Thus, an apparent contradiction exists in professional athletes between the evidence of oxidative and inflammatory effects of exercise and the beneficial effect of physical activity on health and survival. This contradiction might be explained by a hypothetical adaptive response of the organism to repeated oxidative stress induced by physical training, leading to the acquired ability to cope with oxidative stress of different origins (Teramoto and Bungum 2010). The adaptive response to repetitive and potentially harmful stressors is commonly called ‘hormesis’ (Radak et al. 2008). According to this hypothesis, regular physical exercise could act as a stimulating stressor, which induces physiological and biochemical systemic adaptation (Radak et al. 2008). Such a repeated exposure to increased ROS production from repeated exercise bursts leads to the up-regulation of antioxidants and the shift towards a more reducing environment (Radak et al. 2008). This adaptation might provide protection from increased oxidative stress during successive exercise sections, ultimately leading to improvement in health and/or physical performance. Actually, several experimental data show that regular exercise is associated with reduced oxidative damage and enhanced resistance to oxidative stress, supporting the requirements of hormesis theory. Accordingly, regular exercise leads to up-regulation of gluthatione levels and reduces the generation of ROS and inflammation in rats (Radak et al. 2008, 1999). We have previously reported lower levels of malondialdehyde in endurance athletes with respect to sedentary controls (Vassalle et al. 2002). Interestingly, in accordance with the ROS-hormesis hypothesis, exercise-induced ROS production may play an important role in cell signalling involved in the antioxidant defence network. This response to ROS stress, called mitochondrial hormesis, is supposed to be responsible for the respective lifespan-extending and health-promoting effects of physical exercise (Radak et al. 2008).

Another interesting issue emerging among aspects related to exercise is the overtraining syndrome (OTS) (Meeusen et al. 2013). This condition frequently occurs in athletes who train longer and harder beyond the body’s ability to recover (Meeusen et al. 2013). Its relevance is evidenced by marked decrements in performance and profound fatigue of athletes (Meeusen et al. 2013). The diagnosis of OTS is differential because the underlying causes are essentially unknown, and remains difficult (Meeusen et al. 2013). Different factors that affect performance and mood status must be excluded, including anaemia, magnesium deficiency, viral infections, muscle damage evidenced by creatine kinase levels, eating and endocrinological disorders (e.g. diabetes, thyroid disorders and adrenal dysfunction), depression, allergies, asthma and cardiovascular disease (Purvis et al. 2010). Interestingly, overtraining drives a marked response of oxidative stress biomarkers, which in some cases appeared proportional to the training load (Margonis et al. 2007). Recent data on proteomic profiling of skeletal muscle in animal models of overtraining confirm the role of proteins of oxidative phosphorylation complexes and antioxidants, together with proteins related to lipid metabolism, and chaperones in response to OTS (Gandra et al. 2012). Thus, oxidative stress biomarkers may be helpful as a tool for OTS diagnosis. Moreover, oxidative stress may represent a potential target of future antioxidant interventions, although at the moment there is a lack of data on the effects of diet/supplemental antioxidants in OTS.

An important question to correctly interpret the response of an oxidative stress system to exercise is the complex interrelationship between this system and inflammation, neuroendocrine and immunitary systems. Recent findings from in vitro studies have shown that the inflammatory cytokine interleukin (IL)-8 is significantly and inversely related to antioxidant levels (Vidyashankar et al. 2013). Moreover, ROS generation and cellular redox status are known to lead to the activation of nuclear factor-κB (NF-kB), which in turn leads to the expression of proinflammatory cytokines. Importantly, NF-kB represents the main transcription factor for the expression of IL-8 in response to oxidative stress (Vidyashankar et al. 2013). Among the various proinflammatory cytokines, IL-8 is identified as the most sensitive indicator responding to intracellular ROS intensity (Vidyashankar et al. 2013, Tsuji et al. 2011, Pei et al. 2002, Park et al. 2001). Recently, we showed a close relationship among variations in oxidative stress, IL-8 and N-terminal pro-brain natriuretic peptide (NT-proBNP) in Ironman athletes early after strenuous exercise consisting of a 3.8 km swim, 180 km bike and 42.2 km run (Pingitore et al. 2011a,b). This result most likely reflects a common pattern regulating the physiological acute response to exercise and the hormetic process.

In this context, also intriguing is the recent hypothesis that elevated mitochondrial ROS can exert beneficial effects by triggering hypoxia-inducible factor 1α (HIF-1α) signalling, which in turn stimulated the immune system to improve protection against infective and neoplastic stimulus, thus reducing molecular damages (Hekimi 2013). However, there are still no data corroborating this hypothesis in the adaptive response to exercise.

16.3. AVAILABLE BIOMARKERS OF OXIDATIVE STRESS

In vivo, oxidative stress is a dynamic condition amplified by a continuing vicious cycle leading to increased free radical generation and reduced defence systems that further exacerbate oxidative damage.

The direct measurement of oxidative stress in complex biological systems is extremely difficult, because free radicals are highly reactive and have a very short half-life. Thus, major advances have been made in the development of indirect assays, and it is generally based on the measurement of different oxidised compounds or antibodies directed against oxidised epitopes. Most commonly used oxidative biomarkers include conjugated dienes, hydroperoxides, malondialdehyde, 4-hydroxynonenal, hydrocarbons such as pentane and ethane (in breath), F2-isoprostanes and oxLDL (Valko et al. 2007, Lee et al. 2012) (Table 16.1).

TABLE 16.1

Main Oxidative Stress Biomarkers

The human antioxidant defence consists of antioxidant enzymes (including catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (such as vitamins E, A, C, and glutathione and uric acid) (Table 16.2) (Pinchuk et al. 2012).

TABLE 16.2

Main Antioxidant Biomarkers

Indeed, there are situations in which knowledge of the individual levels of a single, specific antioxidant may be useful, as when determining the antioxidant contribution of specific dietary components and their relationship with antioxidant composition and activities, or during studies of decreased antioxidant capacity in subjects under oxidative stress in specific patho-physiological states. However, global approaches to measure the total antioxidant activity are generally used to overcome the complexity of this system (Somogyi et al. 2007). These tests are easy to perform and measure in a single step the so-called total antioxidant capacity (TAC), a parameter that represents the cumulative effects of all antioxidants present in a sample (food, blood or tissue). However, in this case, the contribution of the antioxidant enzymes may be very small. Moreover, whether this integrated approach allows to assess the capacity of both known and unknown antioxidants and their synergistic interaction, the assumption that an ex vivo exposure of the sample mimics the complexity of a physiological context, clearly represents the main limitation of such assays.

The principle on which these assays are based is the capacity of antioxidants in the sample to inactivate the oxidants added in excess (Pinchuk et al. 2012, Somogyi et al. 2007). The degree of colour change is proportional to the capacity of all antioxidants present, and the reaction endpoint is reached when colour change stops. In these tests, it is assumed that antioxidant capacity is equal to reducing capacity. However, these assays may differ from each other in terms of substrates, reaction conditions and quantification methods (Pinchuk et al. 2012, Somogyi et al. 2007). This fact may render it difficult to compare the results obtained by different assays.

New biological markers of oxidative stress are emerging. The effect of physical activity on molecular biomarkers associated with chronic degenerative diseases has been recently reviewed (Izzotti 2011). Among molecular markers, the gene polymorphisms of glutathione S-transferase, GSTM1/T1, which is involved in antioxidant defence, appear to be closely related to physical activity and consequently to clinical outcomes (Izzotti 2011). Specifically, this gene may present a homozygous deletion polymorphism that results in a lack of gene activity (‘null’ polymorphism). This condition is correlated to higher oxidative DNA alterations (8-oxo-dG) and the mtDNA 4977 deletion in arterial smooth muscle cells of null polymorphism carriers compared to wild-type carriers for sedentary subjects (Izzotti 2011). However, the effect of this polymorphism appeared to be strongly influenced by physical activity. In fact, no significant difference was observed between the wild-type and double null genotype carriers in terms of the level of these oxidative biomarkers (8-oxo-dG and mtDNA 4977 deletion) in physically active subjects (Izzotti 2011).

Interestingly, available evidences give support to the existence of cardioprotective genotypes for the haeme oxygenase gene, which catalyses the degradation of heme antioxidant, in its adaptive response to exercise. In particular, recent results suggest that after endurance training, subjects carrying a CC genotype presented higher values in different cardiac function biomarkers and better cardiac adaptation than those having CT and TT genotypes (He et al. 2008).

Beneficial effects induced by physical activity at the molecular level are also evidenced by the reduction of oxidative DNA damage to nuclear DNA in terms of 8-oxo-dG and DNA adducts and to mitochondrial DNA in terms of the mtDNA 4977 deletion (Izzotti 2011). Thus, subjects lacking antioxidant defences due to their adverse genetic polymorphism (i.e. the double null carriers) might receive the benefit of physical activity and/or antioxidant supplementation, thereby replacing the lack of endogenous antioxidant defences due to their genetic profile.

Moreover, in view of the close correlation of oxidative stress with inflammatory and other physiological processes, an integrative approach including biomarkers such as cytokines (such as ILs such as IL-8), neuroendocrine biomarkers (such as NT-proBNP) or physiological tests (such as those used to assess endothelial function; e.g. flow-mediated dilation) could be additively conducted to indirectly assess the effects of dietary/antioxidant supplementation in athletes.

16.4. HYPER-HOMOCYSTEINEMIA

Mild hyper-homocysteinemia (HHcy) is an independent marker of cardiovascular diseases. A dose response between level and risk, even within the reference interval, is reported. Homocysteine (Hcy)-induced oxidative damage may contribute to increase the risk of vascular events. Only a few studies have been performed on exercise and homocysteinemia.

Bambaeichi et al.’s study on the influence of aerobic exercise on plasma Hcy levels in young men showed that 8 weeks incremental exercise had no significant effect on reducing Hcy as a risk factor for cardiovascular diseases (Bambaeichi et al. 2010). The relationship between physical exercise and plasma Hcy levels, metabolically related to folate and vitamin B12, was investigated in well-trained male triathletes (Konig et al. 2003). After a 30-day endurance training period, athletes had a significant decrease in Hcy levels and a significant increase in folate but not in vitamin B12 levels. Conversely, intense exercise (sprint triathlon) acutely increased Hcy levels (Konig et al. 2003).

Marked circulating HHcy occur in homocystinuria, a recessively inherited disorder of methionine metabolism, due to cystathionine-β-synthase deficiency, associated with a greatly enhanced cardiovascular risk. The mechanisms mediating Hcy-induced vascular changes are not completely defined; however, subjects, also young, with homocystinuria or with HHcy from other causes (e.g. folate or vitamin B12 deficiency) have impaired endothelial function and, consequently, an oxidative stress condition. In Wilcken et al.’s study, the positive relationship between Hcy and superoxide dismutase, an important component of the endogenous antioxidant defence in vascular tissue, could represent a protective antioxidant response to Hcy-induced oxidative damage and contribute to reducing cardiovascular risk in homocystinuric patients (Wilcken et al. 2000).

16.5. SUPPLEMENTAL ANTIOXIDANTS, HEALTH AND RISK

An active debate still exists on the effect of antioxidant supplementation on exercise-induced oxidative stress. Typical treatment generally includes vitamins A, C and E, at various dosages, administered alone or in combination, chronically or acutely (Braakuis 2012, Askari et al. 2012, D’Adamo et al. 2013, Simar et al. 2012, Bobeuf et al. 2011, Fisher-Wellman and Bloomer 2009, Nikolaidis et al. 2012a,b, Bohlooli et al. 2012). Of these, vitamins C and E have been used more frequently in clinical and experimental studies, mostly because of their safety profile and easy availability (Braakuis 2012, Nikolaidis et al. 2012b). Bohlooli et al.’s study showed a protective role of a moderate dose of vitamin C (500 mg), acutely administered in a non-trained male group compared with the placebo group, on exercise-induced lipid peroxidation (MDA and TAC) and muscle damage (creatine kinase); conversely, vitamin C did not show any effect on inflammatory markers (total leukocytes, CRP, IL-6) (Bohlooli et al. 2012). Other less used antioxidants include quercetin, coenzyme Q10 and N-acetylcysteine (Bloomer et al. 2012, Díaz-Castro et al. 2012, Michailidis et al. 2013). Concerning the endpoints, the antioxidant might be effective in particular conditions in terms of exercise and training, such as one type of sport with respect to another one (e.g. aerobic vs. anaerobic) or specific moment of the training (e.g. before, or after the race, during OTS). Thus, the selection and detailed description of the appropriate training stimulus is needed, and/or the monitoring of the athlete during training phases.

Concerning the biomarker used, some authors did not report any significant changes in levels of markers of lipid peroxidation, DNA damage and glutathione redox status following long duration protocols, whereas others reported reductions in F2-isoprostanes, thiobarbituric acid reactive substances, DNA damage as well as inflammatory biomarkers (Fisher-Wellman et al. 2009, Nikolaidis et al. 2012a,b, Bloomer et al. 2012). Indeed, some studies reported that antioxidant supplementation may induce no effect or even an adverse pro-oxidant response (Rytter et al. 2010, Theodorou et al. 2011, Tomasello et al. 2012). In the light of contrasting results in the literature, there are several arguments in favour of or against antioxidant supplementation. One argument against the use of antioxidants is based on the evidence that ROS production during exercise is fundamental to promote the expression of several skeletal muscle proteins, including antioxidants enzymes, mitochondrial and heat shock proteins that represent the molecular basis of the exercise-induced hormetic response. Interestingly, recent experimental data evidenced that ROS mitochondrial production upregulates HIF-1α that is an important regulator of the immune response and that is elevated in heterozygote Mclk1^+/- mutant mice. Mclk1^+/- encodes a mitochondrial protein necessary for ubiquinone biosynthesis (Hekimi 2013). These mice have increased mitochondrial ROS production but have a reduced development of oxidative biomarkers or ageing, a decrease in the age-associated loss of mitochondrial function and increased lifespan. The use of antioxidants may therefore blunt the ROS-induced adaptive response to exercise. Further data from a recent meta-analysis showed that beta-carotene, vitamins A and E given singly or combined with other antioxidant supplements significantly increase overall mortality, whereas there was no evidence that vitamin C may increase longevity (Bjelakovic et al. 2007). Conversely, selenium given singly or in combination with other supplements significantly decrease mortality (Bjelakovic et al. 2007).

One of the main arguments in favour of antioxidant supplementation strengthens the benefit in terms of reduction of muscular fatigue and improvement of performance (Powers et al. 2011, Bjelakovis et al. 2011). In particular, N-acetylcysteine may delay muscular fatigue (Kelly et al. 2009, Cobley et al. 2011). More recently a new antioxidant agent, pycnogenol, administered 4 h before exercise has been found to improve performance in trained cyclists increasing maximal oxygen consumption (Bentley et al. 2012).

Currently, there is no a general agreement to the use of antioxidants for athletes. This is clearly stated in a recent position statement on the maintenance of immune health in athletes, where the more common antioxidants are not recommended due to the absence of documented benefit when compared with placebo (Walsh et al. 2011). Nonetheless, the benefit of antioxidant intake can be more reasonable in athletes not consuming a balanced diet (Machefer et al. 2007). Polyphenols, such as quercetin, curculin, resveratrol and luteolin, are recommended especially when mixed with other flavonoids and nutrients, for their antioxidative, anti-inflammatory, cardioprotective and anticarcinogenic and mitochondrial stimulatory activities (Walsh et al. 2011). In a recent study, 500 mg quercetin + 250 mg vitamin C supplementation for 8 weeks was effective in reducing oxidative stress and inflammation among subjects with regular exercise, and 3 weeks quercetin (1000 mg/day) lowered the incidence of upper respiratory tract pathologies in athletes (Askari et al. 2012, Nieman et al. 2007).

16.6. ANTIOXIDANT SUPPLEMENTATION IN EXERCISE: LIMITS AND CONSIDERATIONS

Marked divergence and often contradiction among studies addressing the effect of antioxidant supplementation on exercise adaptation may be explained by a variety of factors (Table 16.3).

TABLE 16.3

Antioxidant Supplementation in Exercise: Main Endpoints to Be Considered

At the moment, the use of different exercise protocols, different outcomes, in different physically trained subjects, and the use of a variety of laboratory parameters to evidence such effects still make it difficult to evaluate effects of physical activity on health. Thus, in any case, a detailed description of the type of exercise (e.g. aerobic or anaerobic), subject characteristics, oxidative stress biomarkers used and training endpoints examined is always necessary to allow data interpretation.

Subjects presenting higher levels of oxidative stress may clearly benefit more from the antioxidant treatment. An initial screening of the oxidative stress status is therefore essential. Clearly, individual susceptibility related to the presence of specific genetic variants in key enzymes for ROS detoxification can be another important factor (Izzotti 2011, He et al. 2008). Possible drug interaction may be considered, for example, it being known that antioxidant treatment may blunt the effectiveness of hypolipidemic therapy with statins and niacin. Moreover, evaluation of the hydration status could be helpful (Kenefick and Cheuvront 2012). It would be useful to consider the integrated effect of diet and exogenous antioxidant supplementation.

The use of proper oxidative stress biomarkers, as well as valid and reliable procedure, and techniques and assays remains critical to evaluate results. Evaluation of oxidative stress implied careful attention to pre-analytical aspects, which include procedures for collecting and storing samples. In fact, appropriate procedures adopted during blood collection and sample storage are essential for consistent and accurate results. The best conditions include that samples, unless immediately dosed, must be kept on ice soon after collection and rapidly separated by centrifugation at 4 °C. Then haemolysis-free serum samples should be frozen and preferably maintained at ‒80°C until assayed (Vassalle 2008).

Oxidative stress represents a dynamic situation of balance between oxidants and antioxidants (Vassalle 2008). Thus, estimate of redox status should include an appropriate measure of both components, in most cases (Vassalle 2008). However, this principle is not true in all situations. There may be a difference in the contribution of different oxidant/antioxidant classes in different states, potentially limiting the effectiveness of a given antioxidant under certain conditions. As an example, TAC appears to be increased in patients with chronic renal failure (Jackson et al. 1995). This finding may not be representative of the real status of oxidative stress, because the elevation of this biomarker is principally due to high urate concentrations. Thus, in this specific case, other parameters may be more useful, such as malondialdehyde, which resulted high, and ascorbate, whose levels fell in these patients (Jackson et al. 1995). Moreover, much evidence suggests an increased oxidative stress, but the lack of TAC decrease in obese subjects (Vassalle et al. 2013a, Puchau et al. 2010, Mancini et al. 2008, Vigna et al. 2010). Thus, the measure of TAC in such subjects might be less important or negligible.

The number of different antioxidants present in serum or other body fluids makes it difficult to measure how much each contributes separately. Conversely, the possible interactions between different antioxidants in vivo make the measure of each individual component not representative of the entire antioxidant status.

Clearly, lack of consensus may also be partially explained by variability of redox processes. In this context, and last but not least, it is important to consider the complexity of redox reactions in vivo and that the antioxidant itself may act as a pro-oxidant under certain circumstances (Tomasello et al. 2012).

16.7. VITAMIN D: TO BE SUPPLEMENTED OR NOT TO BE SUPPLEMENTED?

The case of vitamin D [25(OH)D] in exercise is of particular interest. In fact, of the well-known effects in maintaining normal calcium–phosphorus homeostasis, this molecule has been involved in many extra bone conditions—including cancer, diabetes, cardiovascular and autoimmune diseases, and osteoporosis (Pludowski et al. 2013).

Vitamin D retains antioxidant properties and affects inflammatory and immunity processes, synthesis of proteins, cell growth and proliferation, and regulates the expression of over 1000 genes in a variety of tissues (Pludowski et al. 2013). In regard to oxidative status, experimental studies (cellular and animal models) indicated a significant reduction in oxidative stress damage and chromosomal aberrations, as well as prevention of telomere shortening and inhibition of telomerase activity following treatment with vitamin D (Nair-Shalliker et al. 2012). Moreover, vitamin D influences the poly-ADP-ribose polymerase activity, affecting the cell cycle to prevent propagation of damaged DNA, and apoptosis to promote cell death (Nair-Shalliker et al. 2012). Treatment with paricalcitol alone or in combination with enalapril protected against inflammatory and oxidative endothelial damage in mice atherosclerotic aorta (Husain et al. 2010). In an in vitro study, a vitamin D analogue prevented neuronal damage caused by H₂O₂-induced toxicity (Tetich et al. 2004). Other data showed that vitamin D reduced lipid peroxidation and induced SOD activity in a hepatic anti-oxidant system in a rat model (Garcion et al. 1999). Calcitriol increases intracellular glutathione pools and reduces nitrite production that is induced by lipopolysaccharides in astrocytes (Bao et al. 2008). Moreover, the activation of 1α-hydroxylase in macrophages increases the level of calcitriol, which inhibits iNOS expression and reduces nitric oxide (NO) production within lipopolysaccharide-stimulated macrophages, providing protection against the oxidative injuries caused by the NO burst (Chang et al. 2004).

Total 25(OH)D is the recognised laboratory parameter accepted to estimate the status of the overall vitamin D status in the clinical field (Vassalle and Pérez-López 2013b).

It is known that the majority of the population the world over presents 25(OH)D deficiency, whose levels are also highly dependent on seasonality (Shoben et al. 2011, Holick et al. 2011). Thus, emerging evidence indicates that 25(OH)D deficiency exists in some athlete categories, especially in players practising indoor sport, such as basketball. In fact, a high prevalence of 25(OH)D insufficiency has been recently reported in Israelian basketball athletes and dancers, with a very higher rate of 25(OH)D insufficiency among participants who practise indoors, and during the winter (Constantini et al. 2010). A high prevalence of 25(OH)D insufficiency/deficiency has been also observed among elite Irish athletes, suggesting that vitamin D supplementation is an appropriate regime to ensure 25(OH)D sufficiency in athletes during winter and early spring (Magee et al. 2013).

However, other evidence also suggests that outdoor athletes may be at risk of 25(OH)D deficiency during winter, and that supplementation may be advisable to maintain adequate 25(OH)D concentrations (Galan et al. 2012).

Vitamin D effects on athletic performance have been recently reviewed (Cannell et al. 2009). Available data suggest that 25(OH)D are related to the size and number of muscle fibres (Type II fast twitch-muscle fibres) (Cannell et al. 2009). Since the 1950s, it has been reported that vitamin D-producing ultraviolet light may improve athletic performance. Moreover, more recent results suggest the seasonality of physical and athletic performance, enhanced or reduced in parallel with 25(OH)D levels variation (Cannell et al. 2009).

Recent data found a correlation between vitamin D levels and bone mass and muscle strength in a cohort of adolescent girls (Foo et al. 2009). Other results, obtained in adolescent girls, evidenced a significant association between 25(OH)D and muscle power, force, velocity and jump height (Ward et al. 2009).

Additional data evidenced that supplementation with vitamin D increased variables related to power, jumping velocity and height increase and consequently the efficiency of the jump, although it did not increase the maximum muscle force and power (Ward et al. 2010). Thus, authors suggest an effect of vitamin D on lower limb function through improvements in the mechanical efficiency of the muscle (Ward et al. 2010).

Vitamin D is essentially produced by skin exposure to ultraviolet irradiation (Holick et al. 2011). However, vitamin D can also be supplied in part from the diet by the intake of a limited number of aliments (e.g. some fish, egg, mushrooms) (Holick et al. 2011). Supplementation is safe, because intoxication for elevated levels of vitamin D is rare, and it may be considered in categories at risk of deficiency (Holick et al. 2011). Actually, there is no shared consensus on the use of vitamin D in athletes; thus the role of vitamin D in athletic performance still has to be determined. Data are also expected on the relationship between vitamin D and oxidative stress in sport studies, an interesting area of research so far still not investigated. Nonetheless, based on the evidence that many athletic cohorts are vitamin D deficient or insufficient, vitamin D could be monitored in athletes to decide whether they would benefit from vitamin D supplementation.

16.8. DIET: THE IMPORTANCE OF BEING HEALTHY

A well-balanced and appropriate diet is essential for sport performance. Actually, there are more existing position papers on athletic basic nutrition adopted in many countries (Rodriguez et al. 2009). However, this matter is not simple, as there is no such thing as ‘one-size-fits-all plan’. Indeed, nutrition requirement in athletes is reasonably influenced by the number of variables that could influence an individual’s diet, including the type of exercise and individual characteristics (sex, age, hormonal changes in women, etc.) and training status. Generally, the quality of the food composition plan for trace elements, carotenoids and flavonoids is still considered to be poor (Soriguer et al. 2007, Bernardi et al. 2007). Moreover, food habits as well as quantities of nutrients within food may vary among countries and studies are not always comparable.

One of the most intensively emerging aspects in nutrition is the development of the so-called ‘functional food’ (Sirò et al. 2008). Functional foods are fortified, enriched or enhanced foods that give additional health benefits (as health-promotion or disease prevention) exceeding the capacity based on their content of nutrients, when consumed as part of a varied diet on a regular basis. The category of functional foods includes processed food or foods fortified with additives like ‘vitamin-enriched’ products. Several such foods have been demonstrated to improve sport performance at a higher level than the one expected with a well-balanced diet (Deldique et al. 2008). However, there is still the need for additive rigorous scientific evidence to classify these food types on the basis of safety and efficacy (Deldique et al. 2008). The Mediterranean diet is characterised by a high consumption of monounsaturated fatty acids, primarily from olives and oil, and daily consumption of fruits, vegetables, whole-grains and other low-fat dairy products, with a relatively low consumption of red meat.

A recent meta-analysis evidenced that adhering to the Mediterranean diet protected against overall mortality and incidences of various chronic degenerative diseases, including cardiovascular incidence or mortality, cancer incidence or mortality and neurodegenerative diseases (Sofi et al. 2010). The Mediterranean diet is recognised to exert a significant anti-inflammatory, cardioprotective and anticancer action (Benetou et al. 2008).

In particular, a diet low in trans fatty acids and glycaemic load, high in cereal fibre, marine omega-3 fatty acids and folate with a high ratio of polyunsaturated fatty acids, non-smoking and moderate to vigorous exercise (≥30 min/day) significantly lowered the relative risk for coronary events in women (Stampfer et al. 2000).

Adhesion to the Mediterranean diet has been found to enhance the regenerative capacity of the endothelium and fitness in subjects with the metabolic syndrome trained with moderate-to-high-intensity endurance (Fernández et al. 2012). Other data indicate that adherence to a Mediterranean dietary pattern in healthy subjects increases the circulating plasma levels of carotenoids, vitamins A and E, and reduced oxidative stress and inflammation (evaluated as levels of uric acid, SH groups, SOD and GPx activities, FRAP and TRAP, tumour necrosis factor-α and IL-10 cytokines, and malondialdehyde in the erythrocytes as a marker of lipid peroxidation) (Azzini et al. 2011).

However, there is a lack of clear results in the field of Mediterranean diet and athlete well-being and exercise performance. Nonetheless, available results will most likely help to stimulate the interest of researchers to better tailor forthcoming studies in the field of nutrition in exercise, and to make uniform and standardise the score and its components.

16.9. CONCLUSIONS

Available data still do not allow us to define the optimal type and intake of antioxidants, as well as better laboratory biomarkers to evaluate these effects. The alternative possibility that antioxidant supplements modulate selective inhibitors of various enzymatic sources of ROS could also be considered (Olukman et al. 2010).

The specificity of the adaptive response is a function of the specific individual characteristics, such as the training period, the type of physical activity performed, the training level, age and sex of the subjects, environmental conditions and inter-individual differences. It is important to determine the individual antioxidant need of each athlete performing a specific sport or in a specific phase of training. Antioxidant supplementation could help athletes with initially low antioxidant levels to improve their antioxidant status. High-intensity training periods are other critical periods for nutritional requirement. In such cases, the risk of deficit can be reduced by a rational application of recommended training loads and the requirement can be satisfied by well-balanced dietary/supplemental help.

For the biomarkers to be used, one possibility could be the shared agreement on some critical oxidative stress biomarkers, which may allow better standardisation and comparison of data. However, given the complexity of the relationship between oxidative stress, antioxidant diet/supplementation and exercise, the careful choice of a specific panel of biomarkers may be more appropriate in different cases in the research area as well in clinical practice. Moreover, available evidence supports that the development of additive integrative and comparative biomarkers/physiological tests could shed light on the interactions of key redox responses at multiple levels of biology/environment interactions, and assist clinicians in selecting the optimal treatment/monitoring on an individual basis.

REFERENCES

1. Askari G, Ghiasvand R, Feizi A, Ghanadian SM, Karimian J. The effect of quercetin supplementation on selected markers of inflammation and oxidative stress. J Res Med Sci. 2012;17:637–41. [PMC free article: PMC3685779] [PubMed: 23798923]
2. Azzini E, Polito A, Fumagalli A. et al. Mediterranean Diet Effect: An Italian picture. Nutr J. 2011;10(125) doi: 10.1186/1475-2891-10-125. [PMC free article: PMC3252250] [PubMed: 22087545]
3. Bambaeichi E, Najary MA, Barjasteh B. Influence of incremental aerobic exercise on homocysteine level in young males. Br J Sports Med. 2010;44 Suppl 1 i22.
4. Banfi G, Colombini A, Lombardi G, Lubkowska A. Metabolic markers in sports medicine. Adv Clin Chem. 2012;56:1–54. [PubMed: 22397027]
5. Bao BY, Ting HJ, Hsu JW, Lee YF. Protective role of 1α, 25-dihydroxyvitamin D₃ against oxidative stress in nonmalignant human prostate epithelial cells. Int J Cancer. 2008;122:2699–706. [PubMed: 18348143]
6. Benetou V, Trichopoulou A, Orfanos P. et al. Conforming to traditional Mediterranean diet and cancer incidence: The Greek EPIC cohort. Br J Cancer. 2008;99:191–5. [PMC free article: PMC2453039] [PubMed: 18594542]
7. Bentley DJ, Dank S, Coupland R, Midgley A, Spence I. Acute antioxidant supplementation improves endurance performance in trained athletes. Res Sports Med. 2012;20:1–12. [PubMed: 22242733]
8. Bernardi E, Delussu SA, Quattrini FM, Rodio A, Bernardi M. Energy balance and dietary habits of America’s Cup sailors. J Sports Sci. 2007;25:1153–60. [PubMed: 17613739]
9. Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: Systematic review and meta-analysis. JAMA. 2007;297:842–57. [PubMed: 17327526]
10. Bloomer RJ, Canale RE, McCarthy CG, Farney TM. Impact of oral ubiquinol on blood oxidative stress and exercise performance. Oxid Med Cell Longev. 2012 doi: 10.1155/2012/465020. [PMC free article: PMC3432554] [PubMed: 22966414]
11. Bobeuf F, Labonte M, Dionne IJ, Khalil A. Combined effect of antioxidant supplementation and resistance training on oxidative stress markers, muscle and body composition in an elderly population. J Nutr Health Aging. 2011;15:883–9. [PubMed: 22159777]
12. Bohlooli S, Rahmani-Nia F, Babaei P, Nakhostin-Roohi B. Influence of vitamin C moderate dose supplementation on exercise-induced lipid peroxidation, muscle damage and inflammation. Med Sport. 2012;65:187–97.
13. Braakhuis AJ. Effect of vitamin C supplements on physical performance. Curr Sports Med Rep. 2012;11:180–4. [PubMed: 22777327]
14. Briet M, Schiffrin EL. Treatment of arterial remodeling in essential hypertension. Curr Hypertens Rep. 2013;15:3–9. [PubMed: 23250721]
15. Cannell JJ, Hollis BW, Sorenson MB, Taft TN, Anderson JJ. Athletic performance and vitamin D. Med Sci Sports Exerc. 2009;41:1102–10. [PubMed: 19346976]
16. Chang JM, Kuo MC, Kuo HT, Hwang SJ, Tsai JC. 1-α,25-Dihydroxyvitamin D₃ regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW264.7 cells. J Lab Clin Med. 2004;143:14–22. [PubMed: 14749681]
17. Cobley JN, McGlory C, Morton JP, Close GL. N-Acetylcysteine’s attenuation of fatigue after repeated bouts of intermittent exercise: Practical implications for tournament situations. Int J Sport Nutr Exerc Metab. 2011;21:451–61. [PubMed: 22089305]
18. Constantini NW, Arieli R, Chodick G, Dubnov-Raz G. High prevalence of vitamin D insufficiency in athletes and dancers. Clin J Sport Med. 2010;20:368–71. [PubMed: 20818195]
19. Corbi G, Conti V, Russomanno G. et al. Is physical activity able to modify oxidative damage in cardiovascular aging? Oxid Med Cell Longev. 2012 doi:10.1155/2012/728547. [PMC free article: PMC3458405] [PubMed: 23029599]
20. D’Adamo E, Marcovecchio ML, Giannini C. et al. Improved oxidative stress and cardio-metabolic status in obese prepubertal children with liver steatosis treated with lifestyle combined with Vitamin E. Free Radic Res. 2013;47:146–53. [PubMed: 23205728]
21. Deldicque L, Francaux M. Functional food for exercise performance: Fact or foe? Curr Opin Clin Nutr Metab Care. 2008;11:774–81. [PubMed: 18827583]
22. Díaz-Castro J, Guisado R, Kajarabille N. et al. Coenzyme Q(10) supplementation ameliorates inflammatory signaling and oxidative stress associated with strenuous exercise. Eur J Nutr. 2012;51:791–9. [PubMed: 21990004]
23. Fernández JM, Rosado-Álvarez D, Da Silva Grigoletto ME. et al. Moderate-to-high-intensity training and a hypocaloric Mediterranean diet enhance endothelial progenitor cells and fitness in subjects with the metabolic syndrome. Clin Sci (Lond). 2012;123:361–73. [PubMed: 22489903]
24. Fisher-Wellman K, Bloomer RJ. Acute exercise and oxidative stress: A 30 year history. Dyn Med. 2009;8:1. [PMC free article: PMC2642810] [PubMed: 19144121]
25. Foo LH, Zhang Q, Zhu K. et al. Low vitamin D status has an adverse influence on bone mass, bone turnover, and muscle strength in Chinese adolescent girls. J Nutr. 2009;139:1002–7. [PubMed: 19321588]
26. Galan F, Ribas J, Sánchez-Martinez PM, Calero T, Sánchez AB, Muñoz A. Serum 25-hydroxyvitamin D in early autumn to ensure vitamin D sufficiency in mid-winter in professional football players. Clin Nutr. 2012;31:132–6. [PubMed: 21843910]
27. Gandra PG, Valente RH, Perales J, Pacheco AG, Macedo DV. Proteomic profiling of skeletal muscle in an animal model of overtraining. Proteomics. 2012;12:2663–7. [PubMed: 22761172]
28. Garcion E, Sindji L, Leblondel G, Brachet P, Darcy F. 1,25-dihydroxyvitamin D3 regulates the synthesis of γ-glutamyl transpeptidase and glutathione levels in rat primary astrocytes. J Neurochem. 1999;73:859–66. [PubMed: 10428085]
29. Haskell WL, Lee IM, Pate RR. et al. American College of Sports Medicine; American Heart Association. Physical activity and public health: Updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation. 2007;116:1081–93. [PubMed: 17671237]
30. He Z, Hu Y, Feng L. et al. Association between HMOX-1 genotype and cardiac function during exercise. Appl Physiol Nutr Metab. 2008;33:450–60. [PubMed: 18461097]
31. Hekimi S. Enhanced immunity in slowly aging mutant mice with high mitochondrial oxidative stress. Oncoimmunology. 2013:1–2. 2:e23793. [PMC free article: PMC3654596] [PubMed: 23734326]
32. Holick MF, Binkley NC, Bischoff-Ferrari HA. et al. Evaluation, treatment, and prevention of vitamin D deficiency: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911–30. [PubMed: 21646368]
33. Husain K, Suárez E, Isidro A, Ferder L. Effects of paricalcitol and enalapril on atherosclerotic injury in mouse aortas. Am J Nephrol. 2010;32:296–304. [PubMed: 20720404]
34. Izzotti A. Genomic biomarkers and clinical outcomes of physical activity. Ann N Y Acad Sci. 2011;1229:103–14. [PubMed: 21793845]
35. Jackson P, Loughrey CM, Lightbody JH, McNamee PT, Young IS. Effect of hemodialysis on total antioxidant capacity and serum antioxidants in patients with chronic renal failure. Clin Chem. 1995;41:1135–8. [PubMed: 7628087]
36. Jones LW, Alfano CM. Exercise-oncology research: Past, present, and future. Acta Oncol. 2013;52:195–215. [PubMed: 23244677]
37. Kelly MK, Wicker RJ, Barstow TJ, Harms CA. Effects of N-acetylcysteine on respiratory muscle fatigue during heavy exercise. Respir Physiol Neurobiol. 2009;165:67–72. [PubMed: 18992854]
38. Kenefick RW, Cheuvront SN. Hydration for recreational sport and physical activity. Nutr Rev. 2012;70(Suppl 2):S137–42. [PubMed: 23121349]
39. Konig D, Bisse E, Deibert P, Muller HM, Wieland H, Berg A. Influence of training volume and acute physical exercise on the homocysteine levels in endurance-trained men: Interactions with plasma folate and vitamin B12. Ann Nutr Metab. 2003;47:114–8. [PubMed: 12743461]
40. Lee IM, Skerrett PJ. Physical activity and all-cause mortality: What is the dose-response relation. Med Sci Sports Exerc. 2001 33:S459–71. [PubMed: 11427772]
41. Lee R, Margaritis M, Channon KM, Antoniades C. Evaluating oxidative stress in human cardiovascular disease: Methodological aspects and considerations. Curr Med Chem. 2012;19:2504–20. [PMC free article: PMC3412204] [PubMed: 22489713]
42. Machefer G, Groussard C, Zouhal H. et al. Nutritional and plasmatic antioxidant vitamins status of ultra endurance athletes. J Am Coll Nutr. 2007;26:311–6. [PubMed: 17906181]
43. Magee PJ, Pourshahidi LK, Wallace JMW. et al. Vitamin D status and supplementation in elite Irish athletes. Int J Sport Nutr Exerc Metab. 2013;23:441–8. [PubMed: 23535936]
44. Mancini A, Leone E, Festa R. et al. Evaluation of antioxidant systems (coenzyme Q10 and total antioxidant capacity) in morbid obesity before and after biliopancreatic diversion. Metabolism. 2008;57:1384–9. [PubMed: 18803943]
45. Margonis K, Fatouros IG, Jamurtas AZ. et al. Oxidative stress biomarkers responses to physical overtraining: Implications for diagnosis. Free Radic Biol Med. 2007;43:901–10. [PubMed: 17697935]
46. Meeusen R, Duclos M, Foster C. et al. European College of Sport Science; American College of Sports Medicine. Prevention, diagnosis, and treatment of the overtraining syndrome: Joint consensus statement of the European College of Sport Science and the American College of Sports Medicine. Med Sci Sports Exerc. 2013;45:186–205. [PubMed: 23247672]
47. Mellett LH, Bousquet G. Cardiology patient page. Heart-healthy exercise. Circulation. 2013;127:e571–2. [PubMed: 23630089]
48. Michailidis Y, Karagounis LG, Terzis G. et al. Thiol-based antioxidant supplementation alters human skeletal muscle signaling and attenuates its inflammatory response and recovery after intense eccentric exercise. Am J Clin Nutr. 2013;98:233–45. [PubMed: 23719546]
49. Nair-Shalliker V, Armstrong BK, Fenech M. Does vitamin D protect against DNA damage? Mutat Res. 2012;733:50–7. [PubMed: 22366026]
50. Neubauer O, Konig D, Wagner K-H. Recovery after an Ironman Triathlon: Sustained inflammatory responses and muscular stress. Eur J Appl Physiol. 2008;104:417–26. [PubMed: 18548269]
51. Nieman DC, Henson DA, Gross SJ. et al. Quercetin reduces illness but not immune perturbations after intensive exercise. Med Sci Sports Exerc. 2007;39:1561–9. [PubMed: 17805089]
52. Nikolaidis MG, Kerksick CM, Lamprecht M, McAnulty SR. Does vitamin C and E supplementation impair the favorable adaptations of regular exercise? Oxid Med Cell Longev. 2012a doi: 10.1155/2012/707941. [PMC free article: PMC3425865] [PubMed: 22928084]
53. Nikolaidis MG, Kyparos A, Spanou C, Paschalis V, Theodorou AA, Vrabas IS. Redox biology of exercise: An integrative and comparative consideration of some overlooked issues. J Exp Biol. 2012b;215:1615–25. [PubMed: 22539728]
54. Nunn AV, Guy GW, Brodie JS, Bell JD. Nutr Metab. Vol. 7. (Lond): 2010. Inflammatory modulation of exercise salience: Using hormesis to return to a healthy lifestyle; p. 87. doi: 10.1186/1743-7075-7-87. [PMC free article: PMC3009972] [PubMed: 21143891]
55. Olukman M, Orhan CE, Celenk FG, Ulker S. Apocynin restores endothelial dysfunction in streptozotocin diabetic rats through regulation of nitric oxide synthase and NADPH oxidase expressions. J Diabetes Complications. 2010;24:415–423. [PubMed: 20226688]
56. Park GY, Le S, Park KH. et al. Anti-inflammatory effect of adenovirus-mediated IkBα overexpression in respiratory epithelial cells. Eur Respir J. 2001;18:801–9. [PubMed: 11757631]
57. Pei XY, Nakanishi Y, Inoue H, Takayama K, Bai F, Kara H. Polycyclic aromatic hydrocarbons induce IL-8 expression through nuclear factor KB activation in A549 cell line. Cytokine. 2002;19:236–41. [PubMed: 12393170]
58. Pinchuk I, Shoval H, Dotan Y, Lichtenberg D. Evaluation of antioxidants: Scope, limitations and relevance of assays. Chem Phys Lipids. 2012;165:638–47. [PubMed: 22721987]
59. Pingitore A, Battaglia D, Prontera C. et al. Oxidative Stress and antioxidant capacity, inflammatory parameters and biomarkers of myocardial dysfunction and damage after an ironman race. IFCC—WorldLab—EuroMedLab Berlin 2011 – Berlin, 15–19 May 2011. Clin Chem Lab Med. 2011a 49, abstract no. 1303.
60. Pingitore A, Garbella E, Piaggi P. et al. Early subclinical increase in pulmonary water content in athletes performing sustained heavy exercise at sea level: Ultrasound lung comet-tail evidence. Am J Physiol Heart Circ Physiol. 2011b;301:H2161–7. [PubMed: 21873499]
61. Pinto A, Di Raimondo D, Tuttolomondo A, Buttà C, Milio G, Licata G. Effects of physical exercise on inflammatory markers of atherosclerosis. Curr Pharm Des. 2012;18:4326–49. [PubMed: 22390642]
62. Pludowski P, Holick MF, Pilz S. et al. Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality—A review of recent evidence. Autoimmun Rev. 2013;12:976–89. [PubMed: 23542507]
63. Powers S, Nelson WB, Larson-Meyer E. Antioxidant and vitamin D supplements for athletes: Sense or nonsense? J Sports Sci. 2011;29 Suppl 1:S47–55. [PubMed: 21830999]
64. Puchau B, Ochoa MC, Zulet MA, Marti A, Martínez JA, Members G. Dietary total antioxidant capacity and obesity in children and adolescents. Int J Food Sci Nutr. 2010;61:713–21. [PubMed: 20528580]
65. Purvis D, Gonsalves S, Deuster PA. Physiological and psychological fatigue in extreme conditions: Overtraining and elite athletes. PM R. 2010;2:442–50. [PubMed: 20656626]
66. Radak Z, Chung HY, Goto S. Systemic adaptation to oxidative challenge induced by regular exercise. Free Radic Biol Med. 2008;44:153–9. [PubMed: 18191751]
67. Radak Z, Kaneko T, Tahara S. et al. The effect of exercise training on oxidative damage of lipids, proteins, and DNA in rat skeletal muscle: Evidence for beneficial outcomes. Free Radic Biol Med. 1999;27:69–74. [PubMed: 10443921]
68. Raichlen DA, Polk JD. Linking brains and brawn: Exercise and the evolution of human neurobiology. Proc Biol Sci. 2013;280:2012–50. [PMC free article: PMC3574441] [PubMed: 23173208]
69. Rodriguez NR, DiMarco NM, Langley S. American Dietetic Association; Dietitians of Canada; American College of Sports Medicine. Nutrition and Athletic Performance. Position of the American Dietetic Association, Dietitians of Canada, and the American College of Sports Medicine: Nutrition and athletic performance. J Am Diet Assoc. 2009;109:509–27. [PubMed: 19278045]
70. Rytter E, Vessby B, Asgard R. et al. Supplementation with a combination of antioxidants does not affect glycaemic control, oxidative stress or inflammation in type 2 diabetes subjects. Free Radic Res. 2010;44:1445–53. [PubMed: 20942575]
71. Shoben AB, Kestenbaum B, Levin G. et al. Seasonal variation in 25-hydroxyvitamin D concentrations in the cardiovascular health study. Am J Epidemiol. 2011;174:1363–72. [PMC free article: PMC3276302] [PubMed: 22112344]
72. Simar D, Malatesta D, Mas E, Delage M, Caillaud C. Effect of an 8-weeks aerobic training program in elderly on oxidative stress and HSP72 expression in leukocytes during antioxidant supplementation. J Nutr Health Aging. 2012;16:155–61. [PubMed: 22323351]
73. Sirò I, Kápolna E, Kápolna B, Lugasi A. Functional food. Product development, marketing and consumer acceptance—A review. Appetite. 2008;51:456–67. [PubMed: 18582508]
74. Sofi F, Abbate R, Gensini GF, Casini A. Accruing evidence on benefits of adherence to the Mediterranean diet on health: An updated systematic review and meta-analysis. Am J Clin Nutr. 2010;92:1189–96. [PubMed: 20810976]
75. Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G. Antioxidant measurements. Physiol Meas. 2007;28:R41–55. [PubMed: 17395989]
76. Soriguer F, Rojo-Martínez G, de Fonseca FR, García-Escobar E, García Fuentes E, Olveira G. Obesity and the metabolic syndrome in Mediterranean countries: A hypothesis related to olive oil. Mol Nutr Food Res. 2007;51:1260–67. [PubMed: 17912723]
77. Stampfer MJ, Hu FB, Manson JE, Rimm EB, Willett WC. Primary prevention of coronary heart disease in women through diet and lifestyle. N Engl J Med. 2000;343:16–22. [PubMed: 10882764]
78. Strobel NA, Fassett RG, Marsh SA, Coombes JS. Oxidative stress biomarkers as predictors of cardiovascular disease. Int J Cardiol. 2011;147:191–201. [PubMed: 20864194]
79. Suzuki K, Peake J, Nosaka K. et al. Changes in markers of muscle damage, inflammation and HSP70 after an Ironman Triathlon race. Eur J Appl Physiol. 2006;98:525–34. [PubMed: 17031693]
80. Teramoto M, Bungum TJ. Mortality and longevity of elite athletes. J Sci Med Sport. 2010;13:410–6. [PubMed: 19574095]
81. Tetich M, Kutner A, Leskiewicz M, Budziszewska B, Lasonń W. Neuroprotective effects of (24R)-1,24-dihydroxycholecalciferol in human neuroblastoma SH-SY5Y cell line. J Steroid Biochem Mol Biol. 2004;89–90:365–70. [PubMed: 15225802]
82. Theodorou AA, Nikolaidis MG, Paschalis V. et al. No effect of antioxidant supplementation on muscle performance and blood redox status adaptations to eccentric training. Am J Clin Nutr. 2011;93:1373–83. [PubMed: 21508092]
83. Tomasello B, Grasso S, Malfa G, Stella S, Favetta M, Renis M. Double-face activity of resveratrol in voluntary runners: Assessment of DNA damage by comet assay. J Med Food. 2012;15:441–7. [PubMed: 22439873]
84. Tsuji G, Takahara M, Uchi H. et al. An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway. J Dermatol Sci. 2011;62:42–9. [PubMed: 21316925]
85. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:44–84. [PubMed: 16978905]
86. Vassalle C. An easy and reliable automated method to estimate oxidative stress in the clinical setting. Methods Mol Biol. 2008;477:31–9. [PubMed: 19082936]
87. Vassalle C, Lubrano V, L’Abbate A, Clerico A. Determination of nitrite plus nitrate and malondialdehyde in human plasma: Analytical performance and the effect of smoking and exercise. Clin Chem Lab Med. 2002;40:802–9. [PubMed: 12392309]
88. Vassalle C, Pérez-López FR. The importance of some analytical aspects and confounding factors in relation to clinical interpretation of results. In. In: Meer C, Smits H, editors. Vitamin D: Daily Requirements, Dietary Sources and Symptoms of Deficiency. Nova Science Publishers, Inc; Hauppauge, NY: 2013b.
89. Vassalle C, Vigna L, Bianchi S. et al. A biomarker of oxidative stress as a nontraditional risk factor in obese subjects. Biomark Med. 2013a;7:633–9. [PubMed: 23905900]
90. Vidyashankar S, Sandeep Varma R, Patki PS. Quercetin ameliorate insulin resistance and up-regulates cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells. Toxicol In Vitro. 2013;27:945–53. [PubMed: 23348005]
91. Vigna L, Novembrino C, De Giuseppe R. et al. Nutritional and oxidative status in occupational obese subjects. Mediterranean J Nutrition and Metabolism. 2010;4:9–74.
92. Vina J, Borras C, Mohamed K, Garcia-Valles R, Gomez-Cabrera MC. The free radical theory of aging revisited. The cell signaling disruption theory of aging. Antioxid Redox Signal. 2013;19:779–87. [PMC free article: PMC3749699] [PubMed: 23841595]
93. Walsh NP, Gleeson M, Pyne DB. et al. Position statement. Part two: Maintaining immune health. Exerc Immunol Rev. 2011;17:64–103. [PubMed: 21446353]
94. Ward KA, Das G, Berry JL. et al. Vitamin D status and muscle function in post-menarchal adolescent girls. J Clin Endocrinol Metab. 2009;94:559–63. [PubMed: 19033372]
95. Ward KA, Das G, Roberts SA. et al. A randomized, controlled trial of vitamin D supplementation upon musculoskeletal health in postmenarchal females. J Clin Endocrinol Metab. 2010;95:4643–51. [PubMed: 20631020]
96. Whayne TF Jr, Maulik N. Nutrition and the healthy heart with an exercise boost. Can J Physiol Pharmacol. 2012;90:967–76. [PubMed: 22812658]
97. Wilcken DEL, Wang XL, Adachi T. et al. Relationship between homocysteine and superoxide dismutase in homocystinuria. Possible relevance to cardiovascular risk. Arterioscler Thromb Vasc Biol. 2000;20:1199–202. [PubMed: 10807733]