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In addition to the antioxidant enzyme defence network, skeletal muscle also contains non-enzymatic systems, which regulate reactive species and protect muscle cells from oxidative injury. These are water soluble and fat soluble (Halliwell and Gutteridge, 2007) and are classified into two categories: i) the endogenously produced and ii) dietary antioxidants which cannot be synthesized or induced and must be taken from the diet.

1.3.2.1 Endogenous non-enzymatic antioxidants

The most important non-enzymatic endogenously produced antioxidants are glutathione, uric acid, bilirubin and coenzyme Q10.

1.3.2.1.1 Glutathione

Reduced glutathione (GSH) is the most abundant endogenous antioxidant in eukaryotic cells and is a major player in the regulation of the cellular redox state (Kim and Vaziri, 2010). GSH a tripeptide and is synthesized in the liver, by GSH synthetase (Radak, 2000). The release of GSH from the liver is stimulated by catecholamines, glycagon and vasopressin and is transported to tissues via the circulation (Lu et al., 1992; Sies and Graf, 1985). GSH is the most abundant non-protein thiol in cells and plays an important role in converting disulfides to thiols as well as maintaining substrate levels for GPX to eliminate H2O2 and hydroperoxides (Meister and Anderson, 1983).

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of GSH (Krejsa et al., 2010). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress (Krejsa et al., 2010). Expression of GCL is regulated by the redox- sensitive transcription factor Nrf2 (Kim and Vaziri, 2010) (see Section 1.7) and experimental evidence undertaken in Nrf2-null mice has shown that regulation of GSH in cardiomyocytes is linked to the Nrf2 regulated pathway (Brewer et al., 2011). By donating a pair of hydrogen ions, GSH is oxidised to glutathione disulfide (GSSG) (Powers and Jackson, 2008). Although GSSG levels in most tissues are very low (de la Asuncion et al., 1996), the reduction of GSSG is catalyzed by glutathione reductase

(GR), a flavin containing enzyme, where NADPH is used as the reducing agent (Halliwell and Gutteridge, 2007; Radak, 2000). In many tissues, NADPH is produced by glucose-6-phosphate dehydrogenase, however studies in skeletal muscle have identified that NADPH is primarily supplied by isocitrate dehydrogenase (Vetrano et al., 2005). GSH can react directly with a variety of radicals by donating a hydrogen atom (Yu, 1994) and has been shown to reduce vitamin C and E radicals derived in chain breaking reactions with alkoxyl or lipid peroxyl radicals (Powers and Jackson, 2008; Radak, 2000). The GSH concentration varies from tissue to tissue and studies in skeletal muscle have shown that type I muscle fibres contain a higher GSH content than type IIb although the ratio GSH/GSSG (an additional indicator of redox status) appears to be consistent across various fibre types (Ji, 1995). Intracellular GSH levels are regulated by GSH utilization and GSH synthesis and due to the large muscle mass of the body, the relatively high concentration of GSH in skeletal muscle can influence plasma GSH levels (Kretzschmar et al., 1992; Sen et al., 1992). High intracellular levels of GSSG have shown to inactivate enzymes and cause damage to the cell (Halliwell and Gutteridge, 2007), however skeletal muscle fibres are capable of exporting GSSG to maintain the GSH/GSSG ratio (Meister and Anderson, 1983).

1.3.2.1.2 Uric acid

Uric acid (UA) may also function to protect muscle fibres against oxidative injury and is a strong reducing agent by acting as an electron donor (Halliwell and Gutteridge, 2007). UA is a by-product of purine metabolism and has shown properties in scavenging peroxyl and hydroxyl radicals as well as singlet oxygen (Davies et al., 1986; Sevanian et al., 1985). In humans, over half of the antioxidant capacity of blood

plasma derives from UA (Maxwell et al., 1997). Contractile activity of high intensity has shown to increase plasma UA levels due to increased release of hypoxanthine and xanthine from the muscle into the circulation and the subsequent convertion of these products to UA by xanthine oxidase, which is present in endothelial cells of blood vessels (Samra et al., 1991). At physiological pH, uric acid exists mainly as urate (Powers and Jackson, 2008), which has also been shown to protect against oxidative damage by acting as an electron donor (Halliwell and Gutteridge, 2007). Urate is also capable of preventing hydroxyl radical formation by hindering the Fenton reaction as it is considered a metal ion chelator (Halliwell and Gutteridge, 2007).

1.3.2.1.3 Bilirubin and Coenzyme Q10

Bilirubin is the final product of hemoprotein catabolism, and similarly to UA it possesses antioxidant properties against peroxyl radicals and lipid peroxidation (Stocker et al., 1987a; Stocker et al., 1987b). Contractile activity has shown to increase blood levels of bilirubin and evidence suggests that is can protect cells from the toxic levels of H2O2 (Baranano et al., 2002). Upon oxidation, bilirubin is converted to

biliverdin and then recycled back to bilirubin, a reaction catalysed by biliverdin reductase (Liu et al., 2006; Baranano et al., 2002). Coenzyme Q10 (CoQ10) also known

as ubiquinone contributes to peroxyl radical scavenging and inhibits lipid peroxidation (Sena et al., 2008). CoQ10 is abundant on the inner mitochondrial membrane and plays

an essential role in mitochondrial electron transport as an electron carrier (Halliwell and Gutteridge, 2007). Although few data are available regarding the role of CoQ10 as an

antioxidant in vivo, evidence has shown that exercise can increase the CoQ10 content in

1.3.2.2 Dietary antioxidants

Nutrition significantly impacts the cellular antioxidant system and research has identified that dietary antioxidants such as vitamin C, vitamin E and carotenoids also exert defensive properties and protect muscle cells from RONS toxicity. Vitamin E is a lipid-soluble antioxidant and possesses strong membrane-stabilizing effects, thus it protects cells from lipid peroxidation (Dillard et al., 1978; Jackson et al., 2007). Intervention studies have shown that vitamin E deficiency can disturb cell membrane fluidity (Dillard et al., 1978), alter GSH/GSSG (Anzueto et al., 1993) and exacerbate mitochondrial dysfunction and lipid peroxidation in skeletal muscle (Davies et al., 1982b). Carotenoids which are also located in the membranes of tissues show significant antioxidant defence properties by protecting myocytes from lipid peroxidation (Krinsky, 1998). Due to their structural arrangement, carotenoids are efficient biological antioxidants and can scavenge superoxide and peroxyl radicals (Krinsky, 1998). In contrast, vitamin C is a hydrophilic vitamin and therefore functions in a aqueous environment, the cytosolic cellular compartment and extracellular fluid (Beyer, 1994). Vitamin C scavenges superoxide, hydroxyl radical, and lipid hydroperoxide radicals (Carr and Frei, 1999) and can additionally play an important role in the recycling of vitamin E (Packer et al., 1979). Most mammalian species synthesize vitamin C. High doses can have a prooxidant effect (Yu, 1994) and induce hydroxyl radical formation due to reaction with transition metal ions (Halliwell and Gutteridge, 2007).

1.4. RONS METABOLISM IN GLYCOLYTIC AND OXIDATIVE SKELETAL