14.1. INTRODUCTION

Redox biology has been one of the most rapidly developed fields of biology and one of the most popular in the mass media. Free radicals (reactive species in the present review) have been linked to many different biological processes, such as cell signalling (Forman et al. 2010), enzyme activity (Stubbe and Van Der Donk 1998), synthesis of antibiotic substances (Lesniak et al. 2005) and pathophysiology of diseases (Valko et al. 2007). From the results of thorough investigations conducted in the past three decades, it is now clear that acute exercise induces oxidative stress, whereas chronic exercise enhances the endogenous antioxidant mechanisms (Camiletti-Moirón et al. 2013; Theodorou et al. 2011). Along with the progress of the exercise redox biology, the in vitro molecular and biochemical properties of many nutrient compounds possessing redox properties (i.e. pro-oxidants and mostly antioxidants) have also been revealed. However, despite the long-standing research efforts, it is still uncertain whether and how the exogenous administered antioxidants affect redox homeostasis in vivo and physical performance (Bell et al. 2013; Braakhuis 2012; Nikolaidis 2012c; Peternelj and Coombes 2011; Powers et al. 2010).

Why did it prove to be difficult to reveal the effects of antioxidant supplementation on oxidative stress and human physiology? We believe that the main reason is the methodological uniqueness of each study, particularly regarding the research strategy that investigators adopt on issues relevant to redox biology. Taking into account that redox biology of exercise is a relatively new field, research is driven more on intuition and less on sound methodological evidence. Thus, it is desirable to develop and achieve some agreement on key influencing factors, which investigators should take into account when designing studies in the area of redox biology. Therefore, the aim of this chapter is to provide a methodological framework and broad directions on setting up appropriate experimental set-ups. In particular, we have introduced and tentatively answered eight questions, which a researcher may come across when designing experiments in the redox biology of exercise. It is emphasised, particularly considering the inherent complexity of redox biochemistry, that the following answers are based on the current knowledge; therefore, they can always be amended or disproved by new evidence and should not be accepted as the final answers.

14.2. QUESTION 1: WHICH REDOX BIOMARKERS TO MEASURE?

‘Oxidative stress’ remains until today a term not clearly defined. In this chapter, when referring to oxidative stress we mean ‘any increase in the level of reactive species and/or oxidant biomarkers’, which is consistent with the interpretation of oxidative stress in most studies (Nikolaidis et al. 2012b). In addition, any shift (increase or decrease) in the level of reactive species, oxidant biomarkers, antioxidants and/or redox-active molecules will be referred to as an ‘alteration in redox homeostasis’ (Nikolaidis et al. 2012b).

In most studies, redox biomarkers are traditionally considered as some end or intermediary products of a chemical reaction between a reactive species and a biomolecule. However, other molecules, for example, hydrogen peroxide (H₂O₂), vitamin C and others, can also be considered as redox biomarkers since they can affect redox homeostasis. Therefore, we broadly categorised redox biomarkers as pro-oxidants, reactive species, oxidation products, antioxidants and redox couples (Figure 14.1). Only a small portion of the available redox biomarkers in each category is presented. The selection was based on three factors: (i) the frequency with which they are currently used in research, (ii) the ability to be measured without requiring specialised equipment and (iii) the reliability and validity of their measurement.

FIGURE 14.1

Classification of redox biomarkers.

14.2.3. Oxidation Products

This is the most extensively investigated category of redox biomarkers. This category is composed of oxidatively modified biomolecules, namely products generated by the reaction of free radicals with biomolecules. The term ‘modified’ is preferred instead of the term ‘damaged’, because recent studies do not support the old point of view, whereby every reaction of free radicals with a molecule was associated with harmful consequences. In this section, we limit our analysis (in most cases) to the two most popular and reliable biomarkers reflecting oxidative modifications to each of the three main molecular targets of free radicals (i.e. lipids, proteins and DNA). Carbohydrates (Benov and Beema 2003) and RNA (Jorgensen et al. 2013) are also oxidisable biomolecules; however, only very limited data are available for these targets (Figure 14.2).

FIGURE 14.2

Free radical targets and two of the most frequently used biomarkers in each category. *No adequate data available.

14.2.3.1. Lipids

Lipid peroxidation, namely the reaction of reactive species with lipids (e.g. fatty acids, triacylglycerols, phospholipids, sterols) leads to a wide range of products through complex processes. For a long time, lipid peroxidation was considered only a harmful process. Nowadays, it is certain that many beneficial effects accompany lipid peroxidation (Greenberg et al. 2008). Additionally, most studies tended to focus on cell membrane lipid peroxidation, despite the existence of other lipids found in cytosol such as triacylglycerols, which could also be oxidised (Wu et al. 1999). Various methods have been developed to quantify the products of lipid peroxidation reactions. Two of the most popular and reliable biomarkers for the detection of lipid peroxidation are presented below.

The old and simple thiobarbituric acid (TBA) assay seems inadequate in modern research. This is because most of the TBA-reactive substances (TBARS) formed in vivo are not related to lipid peroxidation (Halliwell and Whiteman 2004). On the bright side, the measurement of (TBA)₂-malondialdehyde (MDA) adduct in human plasma with the use of high-pressure liquid chromatography (HPLC) (in order to separate this adduct from other chromogens) increases markedly the specificity of the assay (Breusing et al. 2010). However, reaction of MDA with TBA still requires treatment at high temperatures for extended incubation times and in strong acidic conditions, which may result in artefactual peroxidation of sample constituents (Mateos et al. 2005). Alternatively, derivatisation of MDA with 2,4-dinitrophenylhydrazine (DNPH) and conversion into pyrazole and hydrazone derivatives has been found to allow specific estimation of this compound if combined with its separation using HPLC (Mateos et al. 2005).

By today’s standards, isoprostanes is considered to be the best lipid peroxidation biomarker (Halliwell 2009). Isoprostanes are peroxidation products of polyunsaturated fatty acids. The most frequently measured isoprostane class is F₂-isoprostanes (Montuschi et al. 2007; Nourooz-Zadeh 2008) and particularly the abundant stereoisomer 15-F_2t-IsoP (frequently also called 8-iso-prostaglandin F_2a). In human studies, 15-F_2t-IsoP has been mostly measured in the blood and urine and, to a lesser extent, in muscle biopsies (Karamouzis et al. 2004). The most reliable methods used for F₂-isoprostane measurement are gas chromatography-mass spectrometry (GC-MS) and the most recently developed–HPLC-MS (Nikolaidis et al. 2011). The aforementioned methods offer sufficiently accurate results; nevertheless, they require sophisticated equipment. As a result, most of the exercise physiology and nutritional laboratories have no access to this equipment and for this reason the use of commercially available immunoassay kits for F₂-isoprostanes measurement is very common. It is important to consider that most of the studies suggest that GC- or HPLC-MS and immunoassay kits do not measure the same compounds (Nikolaidis et al. 2011). Therefore, caution needs to be exercised when comparing results from GC- or HPLC-MS and immunoassay kits. A very important advantage of isoprostanes as redox biomarkers is that their levels in plasma or urine seem not to be confounded by isoprostanes present in food (Gopaul et al. 2000). On the contrary, an important drawback of isoprostanes is that they are unstable in plasma with a half-life of only 20 min (Basu 1998). On a positive note, some of their metabolites (β-oxidation products like 2,3-dinor-8-iso-prostaglandin F_2a and 2,3-dinor-5,6,-dihydro-8-iso-prostaglandin F_2a) are more stable and found in higher concentrations than F₂-isoprostanes [up to 15-fold and 6-fold higher, respectively (Dorjgochoo et al. 2012; Nourooz-Zadeh et al. 2005; Yan et al. 2007)], and thus it is possible that they may prove to be more reliable lipid peroxidation biomarkers in the future (Figure 14.3).

FIGURE 14.3

Metabolism of the most frequently measured isoprostane 8-iso-prostaglandin F_2a. Owing to their higher accumulation, it is possible that these two metabolites of 8-iso-prostaglandin F_2a will prove to be more valid and reliable biomarkers than the parent (more...)

14.2.3.2. Proteins

Protein modifications are caused by the reaction of proteins with free radicals, lipid peroxidation products (e.g. 4-hydroxynonenal) or by glycosylation. Protein modifications have important physiological repercussions because they directly affect the function of receptors, antibodies or enzymes and may lead to indirect modification of biomolecules (e.g. inactivation of enzymes related to DNA repair) (Halliwell and Gutteridge 2007). Oxidative modifications of proteins can be reversible or irreversible. In the case of irreversible modifications, the oxidised protein should be removed or destroyed because it may result in cell death (Davies 2001). Protein oxidation has been mostly measured through protein carbonyls (Levine et al. 2000) and by quantification of specific amino acid oxidation products (Hawkins et al. 2009).

The protein carbonyl assay is a widely used technique that measures carbonyl groups and provides a general picture of the systemic protein oxidation. This is because protein carbonyls can also be originated by protein glycation or by the binding of aldehydes to proteins, processes that overestimate the actual protein oxidation (Negre-Salvayre et al. 2008). In addition, as it is the case with all biomarkers, what is actually measured is the oxidised protein turnover, namely the balance between production and removal of oxidised proteins (Halliwell and Whiteman 2004). Measurements of protein carbonyls in fluids and tissues can be performed spectrophotometrically (Levine et al. 2000) after the reaction of protein-bound carbonyls with DNPH or by using DNPH antibodies in immunochemical techniques, such as western blotting (Shacter et al. 1994) and enzyme-linked immunosorbent assay (ELISA) (Buss et al. 1997).

Reactive species react individually with all the 22 amino acids in many different ways. Oxidation of amino acids produces molecules such as kynurenines, bityrosines, valine hydroxides and l-DOPA (Halliwell and Whiteman 2004). Bityrosine (or dityrosine) is considered to be one of the most reliable redox biomarkers and it is easily detectable in human plasma and urine using the ELISA technique (Davies et al. 1999; Giulivi and Davies 2001). Since amino acids are also oxidised by reactive nitrogen species and reactive chlorine species (e.g. peroxynitrite and hypochlorous acid), substances like nitro- and chloro-tyrosines have also been considered as redox biomarkers (Buss et al. 2003; Leeuwenburgh et al. 1998). Indeed, some of these oxidation products, for example, 3-nitrotyrosine, are thought to be sensitive and stable biomarkers (Hawkins et al. 2009). For the measurement of all the aforementioned oxidised amino acid products, the tools used are antibody techniques, GC-MS and HPLC. The most sensitive and reliable are the GC-MS and the HPLC techniques (Kaur et al. 1998). However, the required hydrolysis of proteins, in order to separate the amino acids, may lead to artefacts (Halliwell and Whiteman 2004).

14.2.3.3. DNA

Oxidative DNA modifications induced by reactive species and followed by repair occur continuously into cells (Halliwell and Gutteridge 2007). DNA bases can be oxidised by several reactive oxygen (especially by ^•OH) and/or nitrogen (e.g. dinitrogen trioxide) species; thus, many products are generated (Dizdaroglu et al. 2002). However, the repair process of the oxidised DNA bases may be overwhelmed and this imbalance between oxidation and repair may lead to cell death. This is why DNA oxidation more than any other redox biomarker has been associated with diseases (Halliwell 2006, 2007b). DNA oxidation has been mostly investigated in leukocytes (especially in lymphocytes) and in urine by HPLC, GC-MS, HPLC-MS and antibody-based techniques.

The measurement of 8-hydroxy-2′-deoxyguanosine (8OHdG) in urine represents a whole-body DNA oxidation assessment and there is a good agreement among laboratories on the concentration of this biomarker in urine (Halliwell and Gutteridge 2007). It is most commonly measured through ELISA, HPLC and MS-based techniques. The ELISA technique needs caution because it may give falsely elevated levels of DNA oxidation (Halliwell and Whiteman 2004). Specifically regarding the measurement of 8OHdG in urine, it is critical to consider that the procedure requires ‘cleaning’ of the sample from interfering substrates unrelated to oxidation (Lin et al. 2004). Measurements of 8OHdG have also been performed in DNA isolated from tissues. However, numerous artefacts have been reported and the results of the several studies seem inconsistent (Collins et al. 2004; Gedik and Collins 2005). Most artefacts are formed during the isolation and preparation of the samples, due to sample exposure to oxygen and to traces of transition metals (Halliwell 2000).

The ‘Comet assay’ (Duthie et al. 1996; Fairbairn et al. 1995), which is applied directly to cells (mostly in leukocytes) and measures DNA strand breaks, is an approach to bypass the artefacts occurring during the isolation and analysis of DNA. This technique is a single-cell gel electrophoresis, easy to perform and does not require DNA isolation. Additionally, since this method is a cell-assay, it has been used for investigating the potential protection effects of an antioxidant specifically to cells (Mastaloudis et al. 2004). Generally, the comet assay indicated decreased levels of DNA oxidation after antioxidant supplementation in humans (Duthie et al. 1996). However, there is no sufficient information whether these effects appear due to minimised artefactual DNA oxidation (i.e. the high reliability of the assay) or due to underestimation of oxidative damage by the assay (Halliwell and Whiteman 2004). This assay is considered to be one of the most reliable in redox biology, although some limitations exist. For instance, strand breaks can be induced not only by DNA damage, but also by enzymatic repair leading to a ‘false increased’ number of strand breaks (Spencer et al. 1996). Moreover, two strand breaks occurring within a short interval may be perceived incorrectly as one, leading artefactually to minimised DNA oxidation (Collins 2013).

14.2.5. Redox Couples

In recent years, there has been an increasing interest in redox couples and particularly the redox potential that is calculated through the Nernst equation (Dimauro et al. 2012; Go and Jones 2011; Kemp et al. 2008). It has been suggested that a cell as a biological entity responds according to redox potential differences (Schafer and Buettner 2001; Jones 2008). The oxidised/reduced pair of some molecules (better known as ‘redox couples’) are in the core of the ‘redox hypothesis’ (Jones 2008) and nicely reflect the concept of redox homeostasis (Nikolaidis et al. 2012b). Virtually, electron(s) transfer is taking place in redox couples, where the reduced molecule loses electron(s) becoming the oxidised form and vice versa.

Redox potential is calculated by the Nernst equation:

Here, ΔE⁰ is the standard reduction potential, R is the universal gas constant, T is the temperature, n is the number of electrons transferred, F is the Faraday constant and Q is the mass law quotient with the actual concentrations of reaction partners. The most frequently studied redox couple is that composed of glutathione (GSH) and glutathione disulphde (GSSG), mostly because of the GSH high cell content (1–10 mM; Jones 2008). For the GSH/GSSG couple, the reaction quotient is

The redox potential calculated by GSH and GSSG concentration in cells/fluids has produced values ranging from −300 mV to −140 mV and also been correlated with the biological status of the cell (Kemp et al. 2008). In addition, the less the redox potential (i.e. more negative), the higher the reductive capacity of the cell/fluid.

In most (if not all) of the relevant exercise literature, in which GSH and GSSG were measured, the redox potential was not calculated. Instead, it has been calculated as the ratio of GSH/GSSG. When the ratio decreases, this was an indication of ‘oxidative stress’, whereas an increased ratio was indicative of ‘reductive stress’. However, this kind of conclusion, based on the GSH/GSSG ratio, ignores the large concentration differences between GSH and GSSG in the blood (or in any other tissue). Indeed, the concentration of GSH in the blood is about 100-fold greater than that of GSSG. The following example illustrates how misleading the uncritical use of the GSH/GSSG ratio could be. For the sake of simplicity, if it is assumed that at rest blood GSH concentration is 1 mM and GSSG concentration is 0.01 mM, then the GSH/GSSG ratio is 100. On the basis of this value, it is frequently assumed that a GSH/GSSG ratio equal to 100 is the ‘basal redox state’, a ratio less than 100 denotes ‘oxidative stress’ and a ratio higher than 100 denotes ‘reductive stress’. Although the ratio and its interpretation work well in cases where acute exercise is employed, it does not seem to work as well with chronic exercise. Acute exercise induces opposite changes in the concentration of glutathione, by decreasing GSH and increasing GSSG (Nikolaidis et al. 2007; Michailidis et al. 2007). As a result, their ratio (GSH/GSSG) magnifies the effect of exercise. However, in a hypothetical scenario after chronic exercise, if the values stated above were doubled at rest, that is, GSH becomes 2 mM and GSSG becomes 0.02 mM, then the GSH/GSSG ratio will remain unaltered, that is 100. In this case, if we adopt the explanation given for the acute exercise, there is no change in the redox state of glutathione. Nevertheless, we believe that this would be a misleading conclusion, because in fact chronic exercise induces redox adaptations by increasing GSH concentration and its antioxidant function. In the case of chronic exercise and based on the changes in the concentrations of GSH and GSSG, we would even conclude that there is a ‘reductive stress’. Therefore, in that particular case-scenario, calculating the redox potential through the Nernst equation can possibly provide a more realistic representation of what is happening in vivo. This is because in the Nernst equation GSH enters the calculation as GSH squared (GSH²), and consequently the redox potential depends not only on the GSH/GSSG ratio but also on the absolute concentrations of GSH and GSSG. Without question, the increasing use of the Nernst equation is not derived solely from the sudden acceptance of its ability to reflect the reductive capacity of the cell, but also by the struggle to achieve mathematical realism using equations and paper chemistry. Therefore, transforming the easily understood GSH/GSSG ratio into abstract redox potentials is not without disadvantages. The major limitation of using the Nernst equation stems from the fact that assumes reversible systems and equilibrium conditions, which actually occur rarely in vivo (Flohé 2013; Kohen and Nyska 2002).

14.3. QUESTION 2: HOW MANY REDOX BIOMARKERS TO MEASURE?

Aside from the redox biomarkers mentioned above, there are countless others. However, very few of them (including many of the biomarkers presented above) have been subjected to a rigorous test of validity and reliability. Taking into account also the complexity of redox biology, it is not surprising that most researchers measure a battery of biomarkers as a means to provide a more satisfactory view on the general redox status of the human body. However, it should be kept in mind that the results produced by measuring, for example, 10 unreliable biomarkers are equally untrustworthy to those produced by a single unreliable biomarker.

Indeed, even within the same biomarker category (for instance, DNA oxidation or lipid peroxidation), the corresponding biomarkers, which presumably indicate the same type of oxidative modifications, may provide partially or completely different responses to the same oxidant stimulus (Breusing et al. 2010; Watters et al. 2009). This is not surprising, since each biomarker is a product of a specific chemical reaction. Considering also that many different techniques (e.g. HPLC, GC-MS or ELISA) are frequently applied to determine the same biomarker and that many different protocols are available (e.g. the determination of MDA via HPLC can be performed using many different derivatisation protocols; Bird and Draper 1984; Mateos et al. 2005), comparisons among studies become even more difficult (Figure 14.4).

FIGURE 14.4

Different techniques for measuring the same redox biomarkers often produce different results both under non-stressed and stressed conditions. (Adapted from Breusing, N., T. Grune, L. Andrisic et al. 2010. An inter-laboratory validation of methods of lipid (more...)

However, assessment of redox homeostasis through only one or few biomarkers is also risky, because almost always the oxidative pathway(s) of the oxidant stimulus are unknown and the chosen biomarker may not respond to this pathway. Unfortunately, due to the absence of validation studies, a specific number of biomarkers cannot be suggested. For these reasons, and until an ‘ideal’ biomarker is found (if it exists), we believe that the safest approach is for each laboratory to validate its own set of redox biomarkers and to choose which biomarkers to measure based on the hypotheses and experimental set-up of their study.

14.4. QUESTION 3: WHICH BODY FLUID TO COLLECT?

One major issue of the studies looking to assess redox homeostasis is the type of sample for analysis. Given the objective difficulties in accessing muscle or other tissue samples in human studies, the present chapter has been focused on body fluids. A key question is in which body fluid (i.e. blood or urine) could alterations in redox biomarkers be more easily and reliably detected (Veskoukis et al. 2009). Blood has a unique place in biomedical science, since its easy collection renders it the most frequently studied tissue (Nikolaidis et al. 2012b). For a long time, researchers have focused on muscle-derived reactive species production (Nikolaidis et al. 2008), particularly in animal studies, disregarding the role of blood. In particular, blood through the circulatory system interacts with almost all organs, tissues and cells of the human body. Clearly, during this interaction, a considerable exchange of redox biomarkers takes place, and this is probably the main reason why biomarkers measured in blood are considered to have been transferred from other tissues.

In addition, blood itself is a noticeable generator of reactive species (Nikolaidis and Jamurtas 2009). In fact, blood plasma contains metal ions that can lead to reactive species generation (e.g. reaction between H₂O₂ with Fe²⁺) as well as carbohydrates (e.g. glucose), proteins (e.g. albumin) and lipids (e.g. polyunsaturated fatty acids), which are potentially oxidisable components. Moreover, blood cells also contain all the three main types of oxidisable substrates. In particular, erythrocytes have an extensive membrane network, which contains several types of carbohydrates, lipids and proteins (Delaunay et al. 1990; Lemaitre et al. 2008) and a haemoglobin solution inside them, which undergoes autooxidation producing O₂^•− (Cimen 2008). Finally, leukocytes (mostly neutrophils) are also a source of reactive species, particularly after muscle-damaging exercise (Cannon et al. 1990; Ookawara et al. 2003). Since reactive species are generated by both blood and tissues, it is reasonable to assume that there is a bidirectional movement of reactive species from the tissues to the blood and vice versa. Supporting the inverse relationship (i.e. from blood to tissues), blood plasma has been reported to considerably affect redox homeostasis of endothelial cells, and particularly this effect was shown to be dependent on the type of exercise activity (Conti et al. 2012).

Compared with blood, the use of urine samples in redox science is more limited, despite its easy collection and the opportunity for repeated measurements offered (spot samples). An advantage of using urine is that it does not contain many reactive substances or catalysts (e.g. metal ions), which implies that the possibility for ex vivo oxidation reactions is limited. A further advantage of urine samples is that they allow accumulation of oxidative products for a longer time period than blood (Nikolaidis et al. 2012a). However, renal function may affect the quantity of biomarkers excreted.

We cannot provide definite directions about which body fluid to collect, because insufficient data are available about the analytical and the biological variability of the same biomarker in different fluids. There are also no data available comparing the magnitude of biomarker stress response after an exercise or nutritional intervention. Additionally, it is clear that each body fluid provides different information about a redox alteration depending on the time point and the redox biomarker chosen (Table 14.1). For example, free F₂-isoprostane levels are peaked at different time points in blood (4 h) and urine (6 h) after carbon tetrachloride-induced lipid peroxidation (Basu 2003). Therefore, each researcher should validate his/her available biomarkers in the various body fluids in order to draw more reliable and interpretable results.

TABLE 14.1

Choice of Body Fluid (Plasma vs. Urine) and Type of Biomarker (Esterified vs. Free F₂-IsoPs) Can Affect the Time of Body Fluid Collection (More on This Issue under Section 14.6)

14.5. QUESTION 4: WHICH TYPE OF EXERCISE TO APPLY?

14.5.1. Non-Muscle-Damaging Exercise versus Muscle-Damaging Exercise

Exercise is probably the most widely studied physiological stimulus to alter redox homeostasis in redox biology research (e.g. Finkler et al. 2013; Neubauer et al. 2008; Radak et al. 2013). However, an important distinction should be made among the different types of exercise and, specifically, between muscle-damaging exercise (e.g. downhill running, containing a strong component of eccentric actions) and non-muscle-damaging exercise (e.g. horizontal running, containing a strong component of concentric contractions). It should be pointed out that even low-intensity exercise may cause some degree of muscle damage and that the term ‘non-muscle-damaging exercise’ is used only to distinguish between types of exercise that cause limited or extensive muscle damage. It is well established that eccentric exercise causes oxidative stress to a greater extent than common aerobic exercise and this stress is prolonged and lasts up to 4 days (Close et al. 2004; Kerksick et al. 2013; Nikolaidis et al. 2007, 2008; Silva et al. 2011), compared with the few hours-lasting (approximately 4 hours post-exercise) oxidative stress caused by mainly concentric exercise (Bloomer 2008; Fogarty et al. 2013; Michailidis et al. 2007; Nikolaidis et al. 2013). Taking into account that eccentric exercise induces long-lasting and extensive alterations in redox homeostasis, muscle-damaging exercise may be a more suitable model to study the effects of experimental interventions on free radical biology (Figure 14.5).

FIGURE 14.5

Urine F₂-isoprostanes after muscle-damaging exercise (solid line) and after non-muscle-damaging exercise (dotted line). Muscle-damaging exercise causes a biphasic wave during the 96 h of recovery, while non-muscle-damaging exercise induces a monophasic (more...)

14.5.2. Acute versus Chronic Exercise

Despite the fact that almost any type of acute exercise causes oxidative stress (Michailidis et al. 2013; Nikolaidis et al. 2012b; Pittaluga et al. 2013), chronic exercise seems to induce reductive stress (i.e. the opposite of what acute exercise does; Gomez-Cabrera et al. 2008a). For example, plasma levels of F₂-isoprostanes are markedly increased after acute exercise (Nieman et al. 2003), whereas F₂-isoprostanes are slightly decreased (Galassetti et al. 2006) or even unchanged (Watson et al. 2005) after chronic exercise. Specifically, changes after acute exercise can be almost up to three times greater compared with those after chronic exercise (56% increase compared with 17% decrease, respectively; Figure 14.6). Therefore, if the aim of a study necessitates the use of a redox stimulus, the use of acute exercise instead of chronic exercise seems more suitable since it causes greater redox alterations.

FIGURE 14.6

Acute exercise induces greater changes in plasma F₂-isoprostane concentration compared to chronic exercise. (Based on data from Nikolaidis, M.G., A. Kyparos, I.S. Vrabas. 2011. Prog Lipid Res. 50(1):89–103.)

14.6. QUESTION 5: WHEN TO COLLECT BODY FLUIDS AFTER EXERCISE?

Most of the redox modification biomarkers do not remain in their initial form for a long time, but they are either metabolised to other products or participate in further reactions often unrelated to redox status. Therefore, ideally, the half-life of a biomarker should be known and remain large enough to allow sufficient accumulation of the biomarker after exposure to an oxidant stimulus. In addition, it would be useful to know the time point at which the biomarker reaches its peak value. The most common oxidant biomarkers exhibit their maximum (or minimum) values at different time points. For example, after an aerobic non-muscle-damaging exercise session, GSH exhibits its minimum value after 2 hours of recovery, TBARS exhibit highest changes after 1 hour, protein carbonyls after 4 hours and catalase immediately after exercise (Michailidis et al. 2007) (Figure 14.7). It is therefore presumed that only one ‘spot’ sampling, if not misleading, surely cannot provide comprehensive information about the time course of a biomarker and, consequently, the effect of an oxidant stimulus. For example, in order to describe the effects of aerobic exercise in more complete dimensions, multiple measurements should be planned over time, the first one taking place immediately after exercise and the following ones during the next few hours.

FIGURE 14.7

Redox biomarkers exhibit different time-courses after the same exercise stimulus indicating the need for multiple sampling points after exercise. (Based on data from Michailidis, Y., A.Z. Jamurtas, M.G. Nikolaidis et al. 2007. Sampling time is crucial (more...)

14.7. QUESTION 6: HOW TO CHOOSE AN ANTIOXIDANT MOLECULE?

A frequently used approach to find out the most effective antioxidant in vivo is by measuring its total antioxidant capacity (TAC) in vitro. Several assays have been developed over the years (Lopez-Alarcon and Denicola 2013; Magalhães et al. 2008) to estimate the TAC of several nutrients (e.g. polyphenol-rich foods) as well as of body fluids and tissues (Buico et al. 2009; Prior and Cao 1999). The major drawback of these ‘TAC’ assays is that they measure antioxidant capacity against only one reactive species or oxidant stimulus (often not normally found in vivo) in a non-physiological medium. Moreover, when investigating the antioxidant capacity of polyphenol-rich foods or plants extracts, there are some further limitations. In particular, most of these substances show high instability and low bioavailability in the human body (Manach et al. 2004), while it is still uncertain if any of their effects arise from their antioxidant activity or from the many other bioactive components they possess (Fraga 2007; Halliwell 2007a). Hence, the approach of screening the antioxidant capacity of a substance in vitro in order to draw conclusions for in vivo conditions is better avoided (Figure 14.8).

FIGURE 14.8

A controversial reductional approach often performed by researchers in order to decide on the most effective antioxidant in vivo.

A multitude of substances have been used as antioxidant agents in redox biology research. However, the objective of each particular study is the crucial determinant of which antioxidant to choose. In nutritional and clinically oriented studies, nutrients derived from the diet should be preferred. However, the low bioavailability and/or bioefficacy of most of the nutrient antioxidants (Halliwell and Gutteridge 2007) render them not appropriate in mechanistic studies. In fundamental research, the researchers may use better characterised pharmaceutical substances like allopurinol or mitoQ.

14.8. QUESTION 7: WHICH DOSE OF THE ANTIOXIDANT MOLECULE TO ADMINISTER?

In the following analysis, we limit our discussion to vitamins C and E since the number of molecules with acclaimed ‘antioxidant’ properties is vast and consists of substances with completely different characteristics. Vitamins C and E are probably the most extensively studied antioxidants. In addition, vitamins C and E are two of the most commonly used supplements by athletes, and thus their antioxidant effects on exercise-induced oxidative stress have been investigated both after acute (Lamprecht et al. 2009; Nieman et al. 2004; Sureda et al. 2013) and chronic exercise (Gomez-Cabrera et al. 2008b; Ristow et al. 2009; Strobel et al. 2011). They act via a common mechanism (Njus and Kelley 1991) and dependency exists between them, since vitamin C recycles vitamin E through the tocopheroxyl radical (Buettner 1993).

A standard dose-protocol about the most efficient amounts of the various antioxidants (even for the well-studied vitamins C and E) does not exist. In most of the recent studies, a combination of these two vitamins is preferred, administering daily about 500–1000 mg of vitamin C and 200–400 mg of vitamin E. However, since antioxidant doses do not follow a specific protocol, the findings of these studies remain partly incomparable. The most important factor for determining the dose of the antioxidant is the research objective of the study set by the investigator. When investigating the physiological role of an antioxidant, the dose should be similar and close to the amounts normally consumed. However, when investigating the potential pharmacological functions of an antioxidant (e.g. supraphysiological amounts of vitamin C against cancer cells), attention should not be focused on the dose administrated, but on the possible desirable result and the mediated pathway. Hence, in fundamental/pharmacological studies, the dose administered can be much higher than the normal daily administration. Another important factor influencing the dose is when the administered antioxidant reaches its peak value in the tissue of interest. For example, daily administration of 200 mg vitamin C seems to be sufficient for all circulating cells (Levine et al. 2001; Figure 14.9).

FIGURE 14.9

Vitamin C concentration in circulating cells as a function of dose. Daily administration of approximately 200 mg vitamin C seems to be sufficient in order to achieve a concentration plateau in all blood cells. (Adapted from Levine, M., Y. Wang, S.J. Padayatty, (more...)

14.9. QUESTION 8: WHEN TO ADMINISTER THE ANTIOXIDANT MOLECULE?

The administration time point of an antioxidant during the day could greatly affect the results of a study. For example, vitamin C is absorbed in a few hours after administration. Considering that oxidative modifications occur continuously, it is better that vitamin C administration should take place not only once (e.g. in the morning) but every few hours (e.g. every 6 hours) in order to achieve high blood concentration throughout the daytime (Padayatty et al. 2004). Moreover, when investigating the acute ingestion of more than one antioxidant (let us consider again the convenient example of vitamins C and E), their administration should take place at different time points in order to exhibit their peak values at the same time point in the fluid and/or tissue of interest (Figure 14.10). This signifies that the sampling point in studies administering a single dose of antioxidants depends on the antioxidant supplement administered. Additionally, the route of administration (intravenously or orally) seems to play a crucial role for the time point at which plasma peak levels appear (Padayatty et al. 2004). Finally, several antioxidant products used in studies do not exhibit the same level of absorption (Julianto et al. 2000). In any case, researchers are encouraged to perform pilot pharmakokinetic studies to confirm the appropriateness of their administration scheme.

FIGURE 14.10

(a) Concentration of plasma vitamin C and vitamin E peak at different time points after a simultaneous administration. (b) Concentration of plasma vitamin C and vitamin E peak at the same time point after asynchronous administration. In a hypothetical (more...)

14.10. CONCLUSIONS

Certainly, many papers published in exercise redox biology have employed inappropriate methodologies leading to doubtful data. The aim of this chapter was to highlight some of the most important questions a researcher faces when setting up an experiment in this field. The careful reader will notice that we avoided giving a definitive answer to any question that we raised. This was mainly for three reasons: first, because redox biology is a relatively new field and not having established maturity. As a result, it still lacks a solid theoretical framework and valid experimental tools. Second, because we are sceptical about the feasibility of defining firm criteria for physiological studies with a huge variety in animals, tissues/fluids, redox biomarkers, antioxidant molecules and exercise protocols. Third, because we believe that establishing ‘gold standards’ in any field of science is implausible. Experimental standardisation implies definite answers and particularly current redox biology is much far from that. We think that, instead of a dogmatic approach, researchers should establish their own reference methods based on scientific evidence and decide which experimental variables are most critical when dealing with a specific question in the vast field of ‘antioxidant’ nutrition and exercise biology. This ad hoc approach does not facilitate the comparison of results obtained in different studies, though it will potentially provide more satisfactory answers to the questions of each specific study. We hope that the limitations of the many usual adopted procedures presented in this chapter, along with the potential solutions provided, will lead to better designed and more carefully executed studies.

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