**5.1. Intracellular enzymes activity in type 1 diabetes**

A number of natural antioxidants are present in the body to scavenge oxygen free radicals and prevent oxidative damage to biological membranes. Antioxidant defense mechanisms involve both non-enzymatic and enzymatic strategies. One group of these antioxidants is in‐ tracellular enzymes such as manganese superoxide dismutase, catalase, glutathione peroxi‐ dase, and glutathione-S-transferases. These enzymes represent a protective mechanism against the damage caused by the oxidative stress and most of these enzymes are polymor‐ phic (Fang et al., 2002; Mates et al., 1999).

et al., 1995). The low glutathione peroxidase activity could be directly explained by either low glutathione content or enzyme inactivation under sever oxidative stress (Faure et al., 1995). However, some authors found no differences between glutathione peroxidase activity of type 1 diabetic patients and control subjects (Jain et al., 1994; Murakami et al., 1993;

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Catalase, located in peroxisomes, decomposes hydrogen peroxide to water and oxygen (Win‐ terbourn & Metodiewa, 1994). In addition, glutathione peroxidase in the mitochondria and the lysosomes also catalyses the conversion of hydrogen peroxide to water and oxygen (Yung et al., 2006). A significant increase in the catalase activity in lymphocytes was found in 40 chil‐ dren with type 1 diabetes during all phases (at the beginning of diabetes, in remission period and in the later chronic course) compared with the control group. The highest catalase activi‐ ty occurs in the early course of disease followed by a linear decrease and the lowest activity in chronic course (Zivić, 2008). Conversely Dave and colleagues (2007) reported significant de‐ creased glutathione peroxidase, catalase and glutathione, and significant increase in thiobar‐ bituric acid reactive substances concentration in type 1 diabetic patients with and without

In addition to enzymatic antioxidants, the major natural antioxidants, most of which are de‐ rived from dietary sources are vitamin A, vitamin C or ascorbic acid, vitamin E and carote‐ noids. Water-soluble vitamin C and fat-soluble vitamin E together make up the antioxidant system for mammalian cells (Engler et al., 2003). Vitamins A, C, and E are obtained from the diet and function to directly detoxify free radicals. Vitamin C forms the first line of defense against plasma lipid peroxidation is considered the most important antioxidant in plasma (Frei et al., 1990). Vitamin C under certain conditions may foster toxicity by generating prooxidants, and is also engaged in the recycling processes which involved the generation of reduced forms of the vitamins. In the processes of regeneration, α-tocopherol is reconstitut‐ ed when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is

Vitamin E involves all tocopherol and tocotrienol derivatives that comprise the major lipo‐ philic exogenous antioxidant in tissues (Di Mambro et al., 2003). Vitamin E, a component of the total peroxyl radical-trapping antioxidant system reacts directly with superoxide and peroxyl radicals, and singlet oxygen and in so doing protects membranes from lipid peroxi‐ dation (Weber & Bendich, 1997). In a study by Gupta et al. 2011 that evaluated the oxidative stress in 20 type 1 diabetic children, reduced glutathione and vitamin E levels were de‐ creased and malondialdehyde levels were elevated compared with controls. After supple‐ mentation with vitamin E (600 mg/daily for three months) there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E. The find‐ ings indicate that vitamin E ameliorates oxidative stress in type 1 diabetes mellitus patients and improves antioxidant defense system. In a latter study high-dose vitamin E supplemen‐ tation (1200 mg/day) reduces markers of oxidative stress and improves antioxidant defense

nephropathy compared with normal healthy individuals (Dave et al., 2007).

**5.2. Non-enzymatic antioxidant levels in type 1 diabetes**

formed, is recycled by glutathione (Weber, 1997).

Majchrzak et al., 2001).

Superoxide dismutase is considered a primary enzyme since it is involved in the direct elim‐ ination of reactive oxygen synthase (Halliwell, 1994). Isoforms of superoxide dismutase are Cu/Zn-superoxide dismutase which is found in both the cytoplasm and the nucleus, and Mn-superoxide dismutase that is present in the mitochondria. The latter can be released into extracellular space (Reiter et al., 2000). Cu/Zn-superoxide dismutase over-expression inhib‐ its oxidized low density lipoprotein which is can elevate deoxyribonucleic acid binding ac‐ tivity of activator protein-1 and NF-κB (Yung et al., 2006). Superoxide dismutase catalyzes the conversion of superoxide anion radicals produced in the body to hydrogen peroxide. This decreases the possibility of superoxide anion interacting with nitric oxide to form reac‐ tive peroxynitrite (Reiter et al., 2000). Low Cu/Zn-superoxide dismutase is a potential early marker of susceptibility to diabetic vascular disease. Suys et al. (2007) found that erythrocyte superoxide dismutase activity and Cu/Zn-superoxide dismutase were higher in type 1 dia‐ betic subjects and was positively associated with flow-mediated dilatation. Based on these findings the authors suggest that higher circulating Cu/Zn-superoxide dismutase could pro‐ tect type 1 diabetic children and adolescents against endothelial dysfunction (Suys et al., 2007). Furthermore, Reznick and colleagues analyzed both serum and salivary superoxide dismutase activity in 20 patients with type 1 diabetes mellitus. A significant association was found between the level of glycemic control as indicated by the glycated hemoglobin values and an increase in both salivary and serum superoxide dismutase activity (Reznick et al., 2006). On the contrary, in a study which assessed correlations between increase of oxidative stress and the development of microalbuminuria in 87 type 1 diabetic patients (44 with nor‐ mal urinary protein excretion, and 43 with microalbuminuria), there was a decreased in ac‐ tivity of superoxide dismutase. This was associated with an increased microalbuminuria in type 1 diabetic patients (Artenie et al., 2005).

Selenium-dependent glutathione peroxidase works in conjunction with superoxide dismu‐ tase in protecting cell proteins and membranes against oxidative damage. In the literature, glutathione peroxidase response to diabetes has been conflicting. Diabetics have been re‐ ported to be associated with increased glutathione peroxidase activity in 90 pregnant wom‐ en with type 1 diabetes mellitus (Djordjevic et al., 2004) and in young diabetic patients (Ndahimana et al., 1996). On the other hand, decreased glutathione peroxidase activity was reported in the early stages of type 1 diabetes in children and adolescents (Dominguez et al., 1998) or unchanged in type 1 diabetic patients with early retina degenerative lesions (Faure et al., 1995). The low glutathione peroxidase activity could be directly explained by either low glutathione content or enzyme inactivation under sever oxidative stress (Faure et al., 1995). However, some authors found no differences between glutathione peroxidase activity of type 1 diabetic patients and control subjects (Jain et al., 1994; Murakami et al., 1993; Majchrzak et al., 2001).

Catalase, located in peroxisomes, decomposes hydrogen peroxide to water and oxygen (Win‐ terbourn & Metodiewa, 1994). In addition, glutathione peroxidase in the mitochondria and the lysosomes also catalyses the conversion of hydrogen peroxide to water and oxygen (Yung et al., 2006). A significant increase in the catalase activity in lymphocytes was found in 40 chil‐ dren with type 1 diabetes during all phases (at the beginning of diabetes, in remission period and in the later chronic course) compared with the control group. The highest catalase activi‐ ty occurs in the early course of disease followed by a linear decrease and the lowest activity in chronic course (Zivić, 2008). Conversely Dave and colleagues (2007) reported significant de‐ creased glutathione peroxidase, catalase and glutathione, and significant increase in thiobar‐ bituric acid reactive substances concentration in type 1 diabetic patients with and without nephropathy compared with normal healthy individuals (Dave et al., 2007).

### **5.2. Non-enzymatic antioxidant levels in type 1 diabetes**

**5. Enzymatics and non-enzymatic antioxidants**

A number of natural antioxidants are present in the body to scavenge oxygen free radicals and prevent oxidative damage to biological membranes. Antioxidant defense mechanisms involve both non-enzymatic and enzymatic strategies. One group of these antioxidants is in‐ tracellular enzymes such as manganese superoxide dismutase, catalase, glutathione peroxi‐ dase, and glutathione-S-transferases. These enzymes represent a protective mechanism against the damage caused by the oxidative stress and most of these enzymes are polymor‐

Superoxide dismutase is considered a primary enzyme since it is involved in the direct elim‐ ination of reactive oxygen synthase (Halliwell, 1994). Isoforms of superoxide dismutase are Cu/Zn-superoxide dismutase which is found in both the cytoplasm and the nucleus, and Mn-superoxide dismutase that is present in the mitochondria. The latter can be released into extracellular space (Reiter et al., 2000). Cu/Zn-superoxide dismutase over-expression inhib‐ its oxidized low density lipoprotein which is can elevate deoxyribonucleic acid binding ac‐ tivity of activator protein-1 and NF-κB (Yung et al., 2006). Superoxide dismutase catalyzes the conversion of superoxide anion radicals produced in the body to hydrogen peroxide. This decreases the possibility of superoxide anion interacting with nitric oxide to form reac‐ tive peroxynitrite (Reiter et al., 2000). Low Cu/Zn-superoxide dismutase is a potential early marker of susceptibility to diabetic vascular disease. Suys et al. (2007) found that erythrocyte superoxide dismutase activity and Cu/Zn-superoxide dismutase were higher in type 1 dia‐ betic subjects and was positively associated with flow-mediated dilatation. Based on these findings the authors suggest that higher circulating Cu/Zn-superoxide dismutase could pro‐ tect type 1 diabetic children and adolescents against endothelial dysfunction (Suys et al., 2007). Furthermore, Reznick and colleagues analyzed both serum and salivary superoxide dismutase activity in 20 patients with type 1 diabetes mellitus. A significant association was found between the level of glycemic control as indicated by the glycated hemoglobin values and an increase in both salivary and serum superoxide dismutase activity (Reznick et al., 2006). On the contrary, in a study which assessed correlations between increase of oxidative stress and the development of microalbuminuria in 87 type 1 diabetic patients (44 with nor‐ mal urinary protein excretion, and 43 with microalbuminuria), there was a decreased in ac‐ tivity of superoxide dismutase. This was associated with an increased microalbuminuria in

Selenium-dependent glutathione peroxidase works in conjunction with superoxide dismu‐ tase in protecting cell proteins and membranes against oxidative damage. In the literature, glutathione peroxidase response to diabetes has been conflicting. Diabetics have been re‐ ported to be associated with increased glutathione peroxidase activity in 90 pregnant wom‐ en with type 1 diabetes mellitus (Djordjevic et al., 2004) and in young diabetic patients (Ndahimana et al., 1996). On the other hand, decreased glutathione peroxidase activity was reported in the early stages of type 1 diabetes in children and adolescents (Dominguez et al., 1998) or unchanged in type 1 diabetic patients with early retina degenerative lesions (Faure

**5.1. Intracellular enzymes activity in type 1 diabetes**

phic (Fang et al., 2002; Mates et al., 1999).

230 Type 1 Diabetes

type 1 diabetic patients (Artenie et al., 2005).

In addition to enzymatic antioxidants, the major natural antioxidants, most of which are de‐ rived from dietary sources are vitamin A, vitamin C or ascorbic acid, vitamin E and carote‐ noids. Water-soluble vitamin C and fat-soluble vitamin E together make up the antioxidant system for mammalian cells (Engler et al., 2003). Vitamins A, C, and E are obtained from the diet and function to directly detoxify free radicals. Vitamin C forms the first line of defense against plasma lipid peroxidation is considered the most important antioxidant in plasma (Frei et al., 1990). Vitamin C under certain conditions may foster toxicity by generating prooxidants, and is also engaged in the recycling processes which involved the generation of reduced forms of the vitamins. In the processes of regeneration, α-tocopherol is reconstitut‐ ed when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is formed, is recycled by glutathione (Weber, 1997).

Vitamin E involves all tocopherol and tocotrienol derivatives that comprise the major lipo‐ philic exogenous antioxidant in tissues (Di Mambro et al., 2003). Vitamin E, a component of the total peroxyl radical-trapping antioxidant system reacts directly with superoxide and peroxyl radicals, and singlet oxygen and in so doing protects membranes from lipid peroxi‐ dation (Weber & Bendich, 1997). In a study by Gupta et al. 2011 that evaluated the oxidative stress in 20 type 1 diabetic children, reduced glutathione and vitamin E levels were de‐ creased and malondialdehyde levels were elevated compared with controls. After supple‐ mentation with vitamin E (600 mg/daily for three months) there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E. The find‐ ings indicate that vitamin E ameliorates oxidative stress in type 1 diabetes mellitus patients and improves antioxidant defense system. In a latter study high-dose vitamin E supplemen‐ tation (1200 mg/day) reduces markers of oxidative stress and improves antioxidant defense in young patients with type 1 diabetes mellitus. However vitamin E supplementation did not decreased albumin excretion rate in these patients (Giannini et al., 2007).

tion. At low physiological oxygen pressures, it exhibits effective radical-trapping antioxidant behaviour (Frei, 1994). Coenzyme Q10 has been found to have a very important role in mitochondrial bioenergetics. It is an electron carrier-proton translocator in the respi‐ ratory chain and potent antioxidant which works by directly scavenging radicals or indirect‐ ly by regenerating vitamin E. In a study by Menke and colleagues (2008), plasma concentrations of coenzyme Q10 in 39 children with type 1 diabetes mellitus were higher than in healthy children. The findings suggest that elevated plasma concentration and the intracellular redox capacity of coenzyme Q10 in diabetic children may contribute to the body's self-protection during a state of enhanced oxidative stress (Menke et al., 2008). In an‐ other study, Salardi and colleagues (2004) determine whether serum hydroperoxides as oxi‐ dative markers and vitamin E and coenzyme Q10 as indexes of antioxidant capacity could be related to metabolic control in 75 unselected children, adolescents, and young adults with type 1 diabetes. Vitamin E and coenzyme Q10 were not significantly different from agematched control subjects. However, there were significant positive correlations between coenzyme Q10 and glycated hemoglobin, and vitamin E and glycated hemoglobin. It was al‐ so observed that diabetic patients with poor metabolic control and complications had elevat‐

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Small molecules that have antioxidant capacity such as glutathione and uric acid are synthe‐ sized or produced within the body (Engler et al., 2003). A study by Maxwell et al. (1997) found significantly reduced total serum antioxidant status in 28 patients with type 1 diabe‐ tes mellitus as attributed by lower uric acid and vitamin C levels. Furthermore, multiple re‐ gression analysis showed that uric acid, vitamin E and vitamin C were the main

Oxidative stress and its contribution to low-density lipoprotein oxidation have been impli‐ cated in the pathogenesis of vascular diabetic complications. Diabetes produces disturban‐ ces of lipid profiles, especially an increased susceptibility to lipid peroxidation, which is responsible for increased incidence of atherosclerosis, a major complication of diabetes mel‐ litus (Siu & To, 2002). Polyunsaturated fatty acids with multiple bonds and lipoproteins in the plasma membrane are very susceptible to attack by reactive oxygen species (Esterbauer & Schaur, 1991). The hydroxyl radicals extract a hydrogen atom from one of the carbon atoms in the polyunsaturated fatty acid and lipoproteins, initiating a free radical chain reac‐ tion which leads to lipid peroxidation. This characterized by membrane protein damage through subsequent free radical attacks (Halliwell, 1995). Lipid peroxidation can produce advanced products of oxidation, such as aldehydes, alkanes and isoprostanes (Moore & Roberts, 1998). Elevation of lipid peroxidation negatively affects membrane function causing reduced membrane fluidity and changing the activity of membrane bound enzymes and re‐

ed vitamin E levels and coenzyme Q10 levels (Salardi et al., 2004).

contributors to serum total antioxidant activity.

ceptors (Acworth et al., 1997).

**6. Markers of oxidative stress in type 1 diabetes**

**6.1. Biomarkers of lipid peroxidation in type 1 diabetes**

α-Tocopherol is very effective in lipid peroxidation inhibition and is the primary *in vivo* chain-breaking, lipid-soluble antioxidant in human serum. A reduction in serum α-toco‐ pherol could be attributed to its consumption while scavenging free radicals in lipoproteins or biomembranes (Frei, 1994). In the Pittsburgh Epidemiology of Diabetes Complications Study cohort, a 10-year prospective study of childhood-onset type 1 diabetes, α-tocopherol or γ-tocopherol did not showed protection against incident coronary artery disease overall. However, high α-tocopherol levels among patients with renal disease and in those using vi‐ tamin supplements were associated with lower coronary artery disease risk in type 1 diabe‐ tes (Costacou et al., 2006). All the antioxidants work in a synergistic manner with each other and against different types of free radicals. This is shown in the way in which vitamin E suppresses the propagation of lipid peroxidation, and vitamin C working with vitamin E in‐ hibits hydroperoxide formation (Laight et al., 2000).

Glutathione functions as a direct free-radical scavenger, and as a co-substrate for glutathione peroxidase activity (Meister & Anderson, 1983). Glutathione, a tri-peptide present in milli‐ molar concentrations is the most prevalent low-molecular weight peptide antioxidant in cells. Reduced glutathione normally plays the role of a direct intracellular free-radical scav‐ enger through interaction with free radicals and is the substrate of many xenobiotic elimina‐ tion reactions (Gregus et al., 1996). It is also involved in other cellular functions such as the elimination of hydrogen peroxide, detoxification processes such as protection of the sulf‐ hydryl group of cysteine in proteins, and regeneration of oxidized vitamin E (Lu, 1999). In 30 children with type 1 diabetes at onset, there was a significant reduction in all glutathione forms (total, reduced, oxidized, and protein-bound glutathione). This indicates that there is glutathione depletion upon early onset of type 1 diabetes mellitus (Pastore et al., 2012). In another study, Likidlilid et al. (2007) compared the glutathione level, and glutathione perox‐ idase activity in 20 type 1 diabetic patients (with fasting glucose > 140 mg/dL) and a normal healthy group. They found that the level of red cell reduced glutathione was significantly lower in type 1 diabetic patients but red cell glutathione peroxidase activity was significant‐ ly increased. The decrease of red cell glutathione may be due to its higher rate of consump‐ tion, increasing glutathione peroxidase activity or a reduction of pentose phosphate pathway, stimulated by insulin, resulting in lowered glutathione recycle (Likidlilid et al., 2007). In a recent study, reduced glutathione and vitamin E levels were decreased and ma‐ londialdehyde levels were higher in 20 type 1 diabetic children compared with healthy con‐ trols. After supplementation with vitamin E (600 mg/daily for three months), there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E levels. This shows that vitamin E ameliorates oxidative stress in type 1 diabetic patients and improves antioxidant defense system (Gupta et al., 2011).

Other nonenzymatic antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). β-carotene is a lipid soluble and chainbreaking antioxidant that effectively quenches singlet oxygen and inhibits lipid peroxida‐ tion. At low physiological oxygen pressures, it exhibits effective radical-trapping antioxidant behaviour (Frei, 1994). Coenzyme Q10 has been found to have a very important role in mitochondrial bioenergetics. It is an electron carrier-proton translocator in the respi‐ ratory chain and potent antioxidant which works by directly scavenging radicals or indirect‐ ly by regenerating vitamin E. In a study by Menke and colleagues (2008), plasma concentrations of coenzyme Q10 in 39 children with type 1 diabetes mellitus were higher than in healthy children. The findings suggest that elevated plasma concentration and the intracellular redox capacity of coenzyme Q10 in diabetic children may contribute to the body's self-protection during a state of enhanced oxidative stress (Menke et al., 2008). In an‐ other study, Salardi and colleagues (2004) determine whether serum hydroperoxides as oxi‐ dative markers and vitamin E and coenzyme Q10 as indexes of antioxidant capacity could be related to metabolic control in 75 unselected children, adolescents, and young adults with type 1 diabetes. Vitamin E and coenzyme Q10 were not significantly different from agematched control subjects. However, there were significant positive correlations between coenzyme Q10 and glycated hemoglobin, and vitamin E and glycated hemoglobin. It was al‐ so observed that diabetic patients with poor metabolic control and complications had elevat‐ ed vitamin E levels and coenzyme Q10 levels (Salardi et al., 2004).

Small molecules that have antioxidant capacity such as glutathione and uric acid are synthe‐ sized or produced within the body (Engler et al., 2003). A study by Maxwell et al. (1997) found significantly reduced total serum antioxidant status in 28 patients with type 1 diabe‐ tes mellitus as attributed by lower uric acid and vitamin C levels. Furthermore, multiple re‐ gression analysis showed that uric acid, vitamin E and vitamin C were the main contributors to serum total antioxidant activity.
