Exercise modulation of total antioxidant capacity (TAC): towards a molecular signature of healthy aging

Abstract

The objective of this article was to review the biochemical basis of the total antioxidant capacity (TAC) test and its applications to exercise science and promotion of healthy aging. Measurement of TAC allows evaluation of the antioxidant content of muscle, heart and other exercise-target organs. Acute non-regular physical exercise and body exposition to higher altitude have been associated with a considerable decrease of blood TAC. However, regular practice of physical activity and exercise is associated with improved antioxidant response increasing TAC levels. TAC methodologies did not address lipophylic antioxidants and have some other limitations. However, TAC is useful as a complementary test for the evaluation of antiperoxidation assays and blood, erythrocyte and cytosolic antioxidant enzyme assays (glutathion reductase, glutathion peroxidase, superoxide dismutase and catalase). Exercise induces both molecular antioxidant and hormetic responses which have been suggested to be linked with a healthy aging.

Keywords:

• total antioxidant capacity
• mitochondrial biogenesis
• free radicals
• hormesis
• physical exercise

Introduction

The molecular participation of free radicals from oxygen, nitrogen and chlorine sources in cell pathogenesis has been strongly consolidated. At least 100 diseases are closely associated with oxidative and nitrosative stresses and its biochemical consequences – lipid peroxidation (measured by biomarkers such as malonaldehyde, MDA, and 4-hydroxynonenal, HNE and acrolein), protein peroxidation (measured by protein carbonyls), nucleic acid peroxidation (quantification of oxidized DNA bases), and carbohydrate oxidation products (measured by glycosilation products) (Ferrari 2000; Halliwell 2011; Niki 2011).

This molecular pathogenesis is based on an imbalance in favor of oxygen free radicals (or nitrogen and chlorine radicals) in relation to levels of the antioxidant molecules, resulting in oxidative stress (Halliwell 2011; Niki 2011), which is correlated to cell membrane damage, DNA damage and gene mutation, and triggers cell death by necrosis or apoptosis (Ferrari 2000; Gross et al. 2011).

Over many decades, a great number of research groups have been studying the biomarkers of oxidative stress and antioxidant activity. Antioxidant activity has been estimated by measuring antioxidant cell enzymes including superoxide dismutase (SOD), catalase (CAT), glutathion reductase (GSH), glutathion peroxidase (GPx), ceruloplasmin and metallothioneins (Ferrari 2000; Gross et al. 2011; Halliwell 2011).

Miller et al. (1993) developed the first well-replicated antioxidant capacity test: total antioxidant capacity (TAC). The major advantage of this test is that it can measure the antioxidant capacity of virtually all components of a biological sample (blood, urine, feces), vegetable extracts, medicinal plants, beverages and food.

This article summarizes the antioxidant capacity tests and their applications for exercise sciences and healthy aging.

TAC tests

There are three major TAC tests (Rice-Evans 2000):

	(1) Ferric reducing antioxidant power (FRAP), developed by Benzie and Strain (1996), which has been widely used in food and nutritional studies – the essay is based on the reduction of the ferric to ferrous ion by adding a sample (plasma, urine, vegetable/fruit extract or food) with presumptive reducing activity. The samples are measured by spectrophotometry at 593 nm (Benzie and Strain 1996).
	(2) Oxygen radical absorbance capacity (ORAC) created by Cao and Alessio (1993) – this test is based on the capacity of the plasma constituents in scavenging peroxyl radicals formed by thermal decomposition of azo initiators, such as 2,2′-diazobis[2-diaminepropane hydrochloride (ABAP) and measurement of fluorescence decay 540 nm wavelength (excitation) and 565 nm (emission);
	(3) Trolox-equivalent antioxidant capacity (TEAC), created by Miller et al. (2005), and transformed in diagnostic kit by Randox Laboratories (Crumlin, UK) – the test is based on ABTS+ radical formation [2,2′-azino-di-(3-ethylbenzothiazoline sulfonate) , with a green-bluish color, and its removal by sample compounds, e.g. the serum, pharmaceutical or foods, measured by spectrophotometry.

In the TEAC test, the ABTS is incubated with methemoglobin peroxidase enzyme, producing the ABTS⁺ radical (reactions 1 and 2), presenting a stable green-bluish color with absorbance reading at 600 nm.

where .

If the sample has antioxidants, its addition produces proportional inhibition of the ABTS⁺ radical decreasing absorbance at 600 nm. This absorbance is inversely associated with the antioxidant concentration in the sample which can be compared to the Trolox antioxidant (the standard), a hydrophilic vitamin E analog (Figure 1). Results are expressed in mmol of trolox equivalents (TEq)/l (blood, liquids or fluids) or in μ mol TEq/100 g of sample (food or solid samples).

Figure 1. 6-hydroxy-2,5,7,8-tetrametylcroman-2-carboxylic acid (Trolox).

TAC can also be estimated by chemiluminescence methods. These methods are based on the time of induction of oxidation of a lipid dispersion exposed to the 2,2′-azo-bis (2-amidinepropane) (ABAP) in environmental temperature and the measurement of chemiluminescence intensity after sample incubation with luminol or luciferol (Whitehead et al. 1992; Said et al. 2003; Bastos et al. 2006).

Reference values for TAC

Although Table 1 presents reference values for TAC, a very important recommendation is that each laboratory should maintain their own reference data, as gender, age, lifestyle variables, ethnic differences, genome diversity and other factors can change the antioxidant responses.

Table 1. Total antioxidant capacity of plasma or serum.

Method		Source	Value (mmol TEq/l)
FRAP	Plasma
	Serum
ORAC	Serum
	Serum (apc*)
TEAC	Muscle	Benzie and Strain (1996)
	Original test (plasma)
	Kit (plasma)
	Seminal plasma	Cao and Alessio (1993)
	fertile men		1.44 to 2.29
	infertile men		1.04 to 1.60
	Urine (exercised)	Child et al. (1999)
	Urine (at rest)	Child et al. (1999)
*protein precipitation by perchloric acid.

There are three important limitations to TAC tests: they did not measure the role of the important lipophylic antioxidants (vitamin E, fatty acids, carnosine) nor the activity of antioxidant enzyme systems (SOD, CAT, GSH), and they are very much influenced by uric acid levels. Because of its hydrophilic nature and reducing activity, uric acid is considered an important interfering factor in TAC evaluation (Benzie and Strain 1996; Rice-Evans 2000).

Total antioxidant capacity in exercise physiology studies

It is well established that acute or exhausting exercise induces oxidative and nitrosative stresses and subsequent damage to cell membranes, genome and organeles (Möller et al. 1996). Liu et al. (1999) reported that marathon athletes presented increased susceptibility of low density lipoprotein (LDL) cholesterol to peroxidation. They also observed increased levels of both plasma total antioxidant capacity and uric acid, although a significant decrement in potent antioxidant blood thiols also occurred. It was demonstrated that after running a marathon there was a reduction in TAC as well as increased DNA oxidation from human lymphocytes (Briviba et al. 2005). Another interesting study pointed out improved TAC levels as well as DNA peroxidation in blood of athletes after an ultramarathon competition (Skenderi et al. 2008). In this regard, overtraining hampers tissue and organ adaptations to exercise, resulting in rising of oxidative stress levels and reduction of the TAC, a fact that was confirmed later in study of protocols of military acute physical training (Taskanen et al. 2010).

Beyond acute effects of exercise, chronic physical training has been also associated with TAC. Chronic swimming training induced blood TAC and GSH decay with augmentation of blood SOD levels (Aguilar-Silva et al. 2002). Reduction of TAC and increasing lipid peroxidation were also confirmed in athletes after an acute endurance testing (Caimi 2009). In rowing athletes, including kayaking, there was reduction of TAC concomitant with increasing lipid peroxidation and creatine kinase (Teixeira et al. 2009).

After an intensive chronic 18-day physical training, skiing athletes from French Federation submitted to higher altitudes could not recover their plasma TAC levels after two weeks of resting compared to the basal pre-training levels (Pialoux et al. 2010). Training at higher altitudes also decreased TAC of elite swimmers (Subudhi et al. 2004), confirming previous research which demonstrated increased oxidative stress at higher altitudes after nutritional supplementation with antioxidants (Heinicke et al. 2009; Castell et al. 2010). However, in high-fitness trained military marine men, the total antioxidant capacity remained elevated despite training at higher altitudes during the winter (Ji et al. 2009).

Regular physical exercise: adaptation to oxidative stress and induction of total antioxidant capacity

Regular practice of physical activity and exercise can improve TAC by modulating the synthesis of both enzymatic (SOD, CAT, GPX) and non-enzymatic (uric acid, albumin, ceruloplasmin, metallothioneins) cell antioxidants in skeletal muscles, liver, heart, brain and other organs, and can reduce lipid peroxidation, postprandial oxidative and nitrosative stresses and LDL cholesterol oxidation (Heitkamp et al. 2008; Neubauer et al. 2008; Bloomer et al. 2009; Heinicke et al. 2009; Ji et al. 2009; Castell et al. 2010). DNA damage can be reduced with regular physical exercise by enhancing expression of genome repairing enzymes on striatal skeletal muscles (Radák et al. 2003; Kim et al. 2010). The influence of physical exercise regularity on TAC concentrations was tested. After consecutive days of exercise training a progressive decreasing on oxidative stress was observed, whereas the TAC remained at higher levels (Sahlin et al. 2010). Although ultraendurance exercise had increased ROS production in isolated mitochondria this effect was abolished after 28 h and no mitochondrial protein damage was observed (Shing et al. 2007). Mitochondria of untrained muscles released sustained higher levels of reactive oxygen species, whereas progressive adaptation to exercise lowered the production of free radicals through abrogation of NFkB and activator protein-1 (AP-1) pathway (Brookes et al. 2008). Concurrently with the modulation of the antioxidant factors there is induction of other cytoprotective factors such as chaperones or heat shock proteins (HSPs), as well as the regulation of the nuclear kappa beta (NFkB) proliferating factor, increasing synthesis of the anti-inflammatory cytokine IL-10, and decreasing production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, and further reduction of inflammatory lineages of monocytes (CD14⁺/CD16⁺) (Timmerman et al. 2008; Czarkowska-Paczek et al. 2009; Giraldo et al. 2010; Rahman et al. 2010; Ropelle et al. 2010) in response to physical exertion. In this sense, it was observed that skeletal muscle activation of nitric oxide synthesis is dependent on heat shock protein-90 (HSP90) expression (Harris et al. 2008). In fact, acute exercise increases inflammation, but chronic adaptation to physical exercise also leads to a positive anti-inflammatory response (Cunniffe et al. 2010). Physical training also induces antioxidant protection against radiation. A study pointed out that exercised rats exposed to gamma radiation produced more SOD (41%), Mn-SOD (51%), and the mitochondrial enzymes such as cytochrome c oxidase (38%), and citrate synthase (42%) when compared with sedentary radiation-exposed rats (De Lisio et al. 2011).

The exposure of healthy sedentary subjects to strenuous physical exercise has been associated with inhibition of macrophage migration due to TGF-β activation, and this inhibitory effect on migratory activities of macrophages was associated with increased total antioxidant capacity (Czepluch et al. 2011).

In another study, during 12 initial minutes of treadmill running plasma TAC increased by 36%, and the decayed by 59% after 26 minutes of exertion in comparison to the basal levels (de Souza et al. 2005). Although intense physical exercise increases free radical release, regular sports training can increase plasma total antioxidant capacity, as seen in two independent studies with soccer athletes (Brites et al. 1999; Mukherjee and Chia 2009). Regular practice of physical activities leads to tissue adaptation against free radicals. Even anaerobic exercise promotes adaptation to oxidative stress, increasing brain, heart and muscle TAC (Qiao et al. 2006). Thus, exercise induces enhancement of vascular TAC and decrement of endothelial oxidative stress (Suvorava and Kojda 2007). Regular physical activity and exercise increased by 78% to 83% the TAC of adolescent athletes (Carlsohn et al. 2008). After motocross running, increased lipid peroxidation was reported as well as rising of TAC levels, which were associated with increased uric acid levels (Ascensão et al. 2007). Similar results were obtained after mountain bike competitions in two independent research reports (Tauler et al. 2006; Martarelli et al. 2009). In a study with wrestler combat athletes, their physical training provoked an adaptation characterized by increased oxidative stress, but also increased TAC (Kürkçu et al. 2010).

It should be emphasized that TAC levels do not always change; depending on training intensity, frequency and timing, as well as other endogenous (genomic and phenotypic) and exogenous factors. A study with untrained subjects submitted to an aerobic training over eight weeks did not reporte increased TAC, but observed positive association between TAC and VO₂max (Afzalpour et al. 2008). In the same manner, although there were studies demonstrating positive correlations between TAC and oxidative stress biomarkers, there were also many studies demonstrating inverse associations between these two parameters (Falone et al. 2009).

After the above-mentioned questions, it should be noted there are three important limitations to those TAC tests: they did not measure the role of the important lipophylic antioxidants (vitamin E, fatty acids, carnosine) nor the activity of antioxidant enzyme systems (SOD, CAT, GSH), and they are very much influenced by uric acid levels. Because of its hydrophilic nature and reducing activity, uric acid is considered an important interfering factor in TAC evaluation (Benzie and Strain 1996; Liu et al. 1999; Rice-Evans 2000; Neubauer et al. 2008).

Antioxidant capacity and healthy aging: does exercise matter?

Aging per se not always risk oxidative and nitrosative stresses. In fact, oxidative/nitrosative stresses in aging depend on age, gender, smoking, alcohol drinking, body composition, genome, and other intrinsic and extrinsic factors (Lesgards et al. 2002; Gutteridge and Halliwell 2010). However, since the 1950s a great number of studies have been reported increased oxidative/nitrosative stresses and decreased cell antioxidant defenses in aging animals and humans (Ferrari 2004; Barja 2005; Pamplona et al. 2005; da Silva and Ferrari 2011).

The increased oxidative/nitrosative stresses found in aging animals and humans have been linked to peroxidative damage to lipids, DNA, proteins and carbohydrates as well increased levels of SOD and CAT, and impairment of the total antioxidant capacity (İnal et al. 2001; Lasheras et al. 2002; Mutlu-Türkoǧlu et al. 2003; Rizvi and Maurya 2007; Dayhoff-Brannigan et al. 2008; Alexandrova and Bochev 2009). In fact, excessive production of hydrogen peroxide is very harmful to aging muscles. It has been suggested excessive release of H₂O₂ is cytotoxic to muscle cells and markedly increases mitochondrial disruption (Fan et al. 2010), and hydrogen peroxide overload triggers the expression of both autophagy and atrophy genes (McClung et al. 2010). In addition, until the age of 45 years, GSH redox system levels are maintained which do not occur in later life (Jones et al. 2002).

Further, for many elderly patients the presence of multiple pathologies is a common feature, especially overweight/obesity, hypertension, atherosclerosis, type 2 diabetes, chronic kidney disease, neurodegenerative disorders and the metabolic syndrome (Jones et al. 2002; Ferrari 2007; Qureshi and Parvez 2007; Morais et al. 2008; Tsirpanlis 2008; Thorin and Thorin-Trescases 2009). These diseases usually shorten the life span (Fontaine et al. 2003; Qureshi and Parvez 2007). It is important to note that those chronic non-transmissible diseases (atherosclerosis, metabolic syndrome, type 2 diabetes, Alzheimer's disease and Parkinson's disease) are frequently associated with increased production of oxygen and nitrogen free radicals and its reactive species, also including sarcopenia and physical fitness decay (Leiter and Lewanczuk 2005; Qureshi and Parvez 2007; Dayhoff-Brannigan et al. 2008; Schiffrin 2010; Noirez and Butler-Browne 2006). For example, among adult patients with metabolic syndrome higher levels of systemic inflammation and oxidative stress and lower TAC were demonstrated (On et al. 2005). A practice of regular exercise in the elderly leads to adaptation to oxidative and nitrosative stresses, decreasing mitochondrial release of free radicals and their reactive species, increasing TAC and cellular antioxidants such as GSH and nitric oxide, which in turn improve endothelial health and reduce systemic inflammation contributing to decreased risk of hypertension, type 2 diabetes, atherosclerosis and metabolic syndrome (Fatouros et al. 2004; Maeda et al. 2004; Suvorava and Kojda 2007; Seals et al. 2009; Gando et al. 2010; Newsholme et al. 2010; Teixeira-Lemos et al. 2011). Thus, regular exercise can afford an excellent maintenance of lower levels of oxidative stress with adequate levels of total antioxidants, promoting healthy aging. The molecular pathways of exercise regulation of TAC and its health benefits are discussed below.

TAC in aging and detraining: is the lack of exercise the origin of chronic diseases?

Aging is characterized by molecular and cellular changes in tissues and organs. In this sense, aging is associated with accumulation of lipid peroxidation products, and reduction of TAC into the heart, brain, liver and other target organs (Siqueira et al. 2005; Kaplan et al. 2007; Aydin et al. 2010; Terryn et al. 2011). Although aging has been associated with decreasing antioxidant capacity of the organism, dietary supplementation with higher antioxidant capacity plant foods did not enhance lifespan in Caenorhabditis elegans worms (Pun 2010). Cell aging is characterized by mitochondrial failure with ATP synthesis decay and increment of oxygen free radical production (Passarino et al. 2010; Osiewacz 2011). It is well established that the massive free radical production in aging cells and organisms has been closely associated with higher rates of both DNA and RNA mutation (Robert et al. 2010), confirming previous reports of an inverse association between DNA oxidation rates and longevity (Barja and Herrero et al. 2000). It also has been demonstrated that mtDNA mutation impaired oxidative phosphorylation, without further free radical enhancement, leading to apoptosis of skeletal muscle cells and sarcopenia (Hiona et al. 2010). By the same manner, detraining and sedentary behavior reduce mitochondrial biogenesis. Thus, the aging subject frequently has lower muscle mitochondrial density and many senescent mitochondria which are related to reduced vital capacity, decreased physical fitness and to an increased risk of chronic and metabolic diseases (Ferrari 2008; Figueiredo et al. 2008; Hiona et al. 2010; Jackson et al. 2010). Therein, positive associations have been observed between oxygen free radicals and both systolic and diastolic blood pressure values, whereas the association between ROS and arterial stiffness was only observed among hypertensive men (Kruger et al. 2012).

Beyond sedentary behavior, aging per se induces over-expression of NADPH oxidase (phagocyte oxidase) contributing to overreaching release of free radicals which, in turn, degrades nitric oxide cytosolic pools, causing impaired endothelium-dependent arterial dilation in old rats but not among young animals (Trott et al. 2011).

A very interesting experimental study announced that denervation-induced atrophy was associated with a 30-fold increasing on mitochondrial free radical production (Muller et al. 2007). In animal models of sarcopenia it has been suggested that both defective mitochondria and SOD deficiency in senescent skeletal muscle cells resulted in massive release of superoxide anion which decisively contributes to aging-related sarcopenia (Jackson 2009). In spite of this mitochondrial decay of aging with increased free radical production and decreased total antioxidant capacity, mitochondrial biogenesis is not impaired since exercised elderly subjects produce more skeletal muscle mitochondria than elderly sedentary individuals (Figueiredo et al. 2008).

Although the heat shock response was preserved, elderly people had decreased TAC which was associated with lower heat-shock protein synthesis compared with young subjects (Rao et al. 2003). One year of swimming training improved antioxidant enzymes of heart, liver, and lung of older rats compared to the older control (Günduz et al. 2004).

As discussed above, cells and tissues can react against exercise-induced free radicals by molecular pathways involving triggering NFkB, which in turn stimulates protein kinases that can activate other protein mitogen-activated kinases (MAPK), or the zinc-containing nuclear respiratory transcription factor-2 (Nrf-2) (George et al. 2008) or even activating the forkhead box proteins (FOXO) (Rattan and Ali 2007; Rattan and Demirovic 2010; Yin et al. 2010). In the absence of insulin and insulin-growth factor-1 (IGF-1), FOXO can trigger the kinase protein pathway, akt, which in turn activates the adenosine monophosphate activated protein kinase (AMP-kinase or AMPK) resulting in cell proliferation, survival, suppressing of tumors, and longevity (Greer and Brunet 2005; Greer et al. 2007). Chronic AMPK activation due to exercise improves muscle oxidative metabolism, increasing myogenic responses (Llubicic et al. 2011). The FOXO3A gene which encodes information for the synthesis of FOXO proteins has been considered a longevity gene in very different cell types (Anselmi et al. 2009; Rattan and Demirovic 2010). It should be noted that on contrast to other tissues and organs, in skeletal muscles akt is a survival pathway and FOXO proteins mediate myostatin-induced sarcopenia (Greer and Brunet 2005; Favier et al. 2008). Regular exercise activates akt which in turn downregulates FOXO phosphorylation, causing inhibition of the expression of muscle atrophy genes (Anselmi et al. 2009). Another cell longevity group factor is represented by sirtuins – the NAD+-dependent deacetylases (Blander and Guarente 2004; Yang et al. 2007). In response to oxidative stress insults, sirtuins activate FOXO transcription factors leading to decrease of free radical production (Wang et al. 2007). Endurance exercise has been associated with increased sirtuin levels and sirtuin activation in skeletal muscle cells of young and old mammals (Suwa et al. 2008; Koltai et al. 2010).

These transcription factors (MAPK, Nrf-2, akt, AMPK) stimulate the antioxidant response element (ARE) and other target genes in the cell nucleus, resulting in increased synthesis and release of antioxidants, anti-inflammatory factors, and other cytoprotective responses with massive production of HSPs, DNA-repair enzymes, nitric oxide, anti-inflammatory cytokines, and heme-oxygenase. In fact, MAPK signal transduction regulates expression of Nrf-2, and it has been suggested that aging cells have lower expression of Nfr-2 experiencing higher degree of oxidative stress (Shih and Yen 2007). As discussed above, exercise stimulates Nfr-2 and NFkB, improving antioxidant defenses in decreasing free radicals. It has been suggested aging-related sedentary lifestyle is associated with impaired function of the Nfr-2 signaling pathway and increased oxidative stress (Safdar et al. 2010). In fact, polymorphisms of the Nrf-2 encoding gene have been associated with endurance performance of athletes (He et al. 2007; Eynon et al. 2010). These adaptive responses to exercise and toxic insults are known as hormesis, a concept that increases cytoprotection and promotes longevity (Motta et al. 2004; Rattan 2008a). In this regard, any natural or synthetic compound which activates a stress response resulting in positive effects is known as hormetin (Rattan 2008b). So, exercise could also be considered a hormetin factor (Rattan et al. 2009). These molecular pathways of exercise-induced longevity are represented in Figure 2. Regular practice of exercise or physical activity triggers activation of the antioxidant response element and other cytoprotective pathways (antioxidant, anti-inflammatory and anti-apoptotic pathways), whereas a lack of exercise induces NFkB-induction of FOXO phosphorylation and expression of skeletal muscle atrophy genes (Figure 2).

Figure 2. Hormetic and antioxidant pathways modulated by exercise in promotion of healthy aging.

Other total antioxidant capacity modulators

Many different physiological or pathological states result in oxidative stress response. Psychological stress, arterial hypertension, acute massive physical exercise and aging are intimately related to increased release of free radicals, as well as to decreasing on antioxidant defenses, and in a consequently reduction of the TAC (Liu et al. 1999; Briviba et al. 2005; Rattan 2008c; Czepluch et al. 2011; Gross et al. 2011; Halliwell 2011). Aging and detraining in rats were associated with reduction of muscle strength as well as TAC, which was partially recovered by resveratrol administration (Kashyap et al. 2005). Acute stress causes reduction of TAC but chronic exposure to stress can result in an adaptation characterized by improvement in plasma TAC (Torres et al. 2004). Psychological stress reduced plasma levels of TAC, an effect that was partially reverted by an eight week physical exercise program in rats (Rosety-Rodriguez et al. 2006).

Conclusion

Studies of exercise physiology can incorporate TAC evaluation before, during and after physical exercise; this strategy could be very useful to determine the real needs of antioxidant supplementation for athletes.

It should be emphasized that regular engagement in physical activities enhances TAC, reducing the oxidative stress (Shing et al. 2007; Brookes et al. 2008; Ji et al. 2009), being recommended for promotion of a healthy aging of the skeletal muscle and cardiovascular system (Seals et al. 2009).