**4. Free radicals formed during oxidative stress**

#### **4.1. Reactive oxygen species in type 1 diabetes**

available reduced nicotinamide adenine dinucleotide phosphate is depleted resulting in the reduction of glutathione regeneration and nitric oxide synthase activity (Ramana et al., 2003). The oxidation of sorbitol to fructose with the concomitant production of reduced nico‐ tinamide adenine dinucleotide is catalyzed by sorbitol dehydrogenase. The reduced nicoti‐ namide adenine dinucleotide phosphate may be used by nicotinamide adenine dinucleotide phosphate oxidases to generate superoxide anion (Moore & Roberts, 1998). Vitamin C sup‐ plementation has been found to be effective in reducing sorbitol accumulation in the red blood cells of diabetic patients. In a study conducted by Cunningham et al. (1994) who in‐ vestigated the effect of two different doses of vitamin C supplements (100 and 600 mg) dur‐ ing a 58 day trial on young adults with type 1 diabetes mellitus, vitamin C supplementation

Glucose at high concentrations undergoes non-enzymatic reactions with primary amino groups of proteins to form glycated residues called Amadori products. These early glycation products undergo further complex reactions, such as rearrangement, dehydration, and con‐ densation, to become irreversibly cross-linked, heterogeneous fluorescent derivatives called advanced glycation end products (Thornalley, 2002). The advanced glycation end products binds to a cell surface receptor known as receptor for advanced glycation end product. As a result of interaction of advanced glycation end products, with receptor for advanced end product, there is the induction of the synthesis of reactive oxygen species via a mechanism which involves localization of pro-oxidant molecules at the cell surface (Yan et al., 1994) and the participation of activated nicotinamide adenine dinucleotide phosphate oxidase (Wauti‐ er et a., 2001). The reactive aldehydes methylglyoxal and glyoxal are produced from enzy‐ matic and non-enzymatic degradation of glucose, lipid and protein catabolism, and lipid peroxidation. These aldehydes form advanced glycation end products with proteins that are implicated in diabetic complications. Han et al. (2007) assessed plasma methylglyoxal and glyoxal using a novel liquid chromatography-mass spectrophotometry method in 56 young patients (6 - 22 years) with type 1 diabetes mellitus without complications. They found that mean plasma methylglyoxal and glyoxal levels were higher in the diabetic patients com‐ pared with their non-diabetic counterparts. They suggest that increased plasma methyl‐ glyoxal and glyoxal levels give an indication of future diabetic complications and

It has been shown that through receptor for advanced glycation end products mediated ef‐ fects, advanced glycation end product induces reactive oxygen species production possibly through an nicotinamide adenine dinucleotide phosphate oxidase, and the subsequent ex‐ pression of inflammatory mediators and activation of redox-sensitive transcription factors (Wautier et al., 2001; Schmidt et al., 1996). Furthermore, advanced glycation end products, binding to receptor for advanced glycation end product activate protein kinase C-α-mediat‐ ed activation of nuclear factor-κB (NFκβ) and nicotinamide adenine dinucleotide phosphate oxidase. This may cause the generation of mitochondrial reactive oxygen species and induce the production of various inflammatory cytokines further aggravating oxidative stress

at either dose within 30 days normalized sorbitol levels.

226 Type 1 Diabetes

emphasized the need for aggressive management (Han et al., 2007).

(Simm et al., 1997).

Reactive oxygen species consist of oxygen free radicals such as superoxide anion (O2 •−), hy‐ drogen peroxide (H2O2), hydroxyl radical (•OH), singlet oxygen, nitric oxide, and peroxyni‐ trite (Chong et al., 2005). Most of these free radicals are produced at low concentrations during normal physiological conditions in the body and are scavenged by endogenous en‐ zymatic and non-enzymatic antioxidant systems that include superoxide dismutase, gluta‐ thione peroxidase, catalase, and small molecule substances such as vitamins C and E.

Reactive oxygen species induced tissue injury as well as they are involved in signaling path‐ ways and gene expression (Ha & Lee, 2000). Excess generation of reactive oxygen species such as superoxide anion, hydrogen peroxide, hydroxyl radical and reactive nitrogen spe‐ cies such as nitric oxide oxidize target cellular proteins, nucleic acids, or membrane lipids and damage their cellular structure and function (Brownlee, 2001). There is also evidence that reactive oxygen species also regulate the expression of genes encoding for proteins in‐ volved in immune response, inflammation and cell death (Ho & Bray, 1999).

endothelial nitric oxide synthase contains reductase and oxygenase domains that are con‐ nected by a calmodulin-binding region and requires cofactor groups such as heme, flavin mononucleotide, flavin adenine dinucleotide, tetrahydrobiopterin, and Ca2+-calmodulin for activation (Gorren & Mayer, 2002; Andrew & Mayer, 1999). If there is none or insufficient Larginine, the endothelial nitric oxide synthase produce superoxide instead of nitric oxide and this is referred to as the uncoupled state of nitric oxide synthase (Channon, 2004).

Biochemical Evaluation of Oxidative Stress in Type 1 Diabetes

http://dx.doi.org/10.5772/52836

229

Oxidative stress decreases the bioavailability of endothelium-derived nitric oxide in diabetic patients. In a 3-year longitudinal study involving 37 patients with recent-onset (less than 2 years) type 1 diabetes, oxidative stress was evident by elevated malondialdehyde excretion and serum NOx (nitrate and nitrite) (Hoeldtke et al., 2011). In a latter study, NOx was also higher in 99 female subjects with uncomplicated type 1 diabetes (duration disease <10 years) compared with 44 sex-matched controls (Pitocco et al., 2009). Mylona Karayanni et al. (2006) examined possible correlation between oxidative stress parameters and adhesion molecules derived from endothelial/platelet activation, P-selectin and tetranectin in a group of juve‐ niles with type 1 diabetes mellitus. Significantly elevated NOx and lipid hydroperoxide lev‐ els, elevated tetranectin and P-selectin plasma levels, and lower glutathione peroxidase activity were found in the diabetic children compared with healthy controls. Based on these findings the authors suggested that decreased anti-oxidative protection from overproduc‐ tion of lipid hydroperoxide and NOx overproduction is present in juveniles with type 1 dia‐ betes mellitus. There is also a parallel endothelial/platelet activation which contributes to the

vascular complications of type 1 diabetes mellitus (Mylona-Karayanni et al., 2006).

electron transport, and adenosine triphosphate formation (Pacher & Szabó, 2006).

The formation of peroxynitrite can further lead to the generation of peroxynitrous acid. The spontaneous decomposition of peroxynitrous acid results in the formation of hydroxyl radi‐ cals that can cause endothelial damage (Elliott et al., 1993; Beckman & Koppenol, 1996) thereby reduces the efficacy the endothelium-derived vasodilator system that participates in the general homeostasis of the vasculature (Benz et al., 2002). Overproduction of both nitric oxide and superoxide anion has been reported in response to hyperglycemia (Cosentino et al., 1997; Ceriello et al., 2002), and nitric oxide may work through peroxynitrite to directly alter cellular structure and function (Pfeiffer et al., 2001). Increased nitric oxide levels have been reported in both saliva and plasma of diabetic patients in comparison to healthy sub‐

substrate, nicotinamide adenine dinucleotide (NAD+

jects (Astaneie et al., 2005).

Nitric oxide can react with superoxide to form peroxynitrite which in turn oxidizes tetrahy‐ drobiopterin and causes further uncoupling of nitric oxide formation (Yung et al., 2003). In diabetes mellitus, elevated glucose may cause an increase in the expression of both reduced nicotinamide adenine dinucleotide phosphate and of inducible nitric oxide synthase via the activation of NF-κB, (Spitaler & Graier, 2002). The upregulated inducible nitric oxide syn‐ thase will synthesize the superoxide anion instead of nitric oxide, leading to oxidative and nitrosative stress (Llorens & Nava, 2003). The stable protein adduct, nitrotyrosine, is a mark‐ er of peroxynitrite (Ischiropoulos, 1998) and nitrogen dioxide (Prutz et al., 1985). Moreover, increased oxidative and nitrosative stress activates poly(ADP-ribose) polymerase-1, which

) as well as slows the rate of glycolysis,

Hydroxyl radicals, hydrogen peroxide, and superoxide anion are byproducts of xanthine ox‐ idase. Xanthine oxidase and xanthine dehydrogenase catalyze the conversion of hypoxan‐ thine to xanthine and then to uric acid, with the former reducing oxygen as an electron acceptor while the latter can reduce either oxygen or nicotinamide adenine dinucleotide (NAD+ ) (Fatehi-Hassanabad et al., 2010). Superoxide anion is also produced by nicotinamide adenine dinucleotide phosphate oxidases and cytochrome P450, and is the most commonly occurring oxygen free radical that produces hydrogen peroxide by dismutation. This is ach‐ ieved via the Haber-Weiss reaction in the presence of ferrous iron by copper (Cu)-superox‐ ide dismutase or manganese (Mn)-superoxide dismutase. Mitochondrial superoxide anion is produced from excess reduced nicotinamide adenine dinucleotide produced in the Krebs cy‐ cle (Fubini & Hubbard, 2003). Elevated free or non-esterified fatty acids in type 1 diabetic patients enter the Krebs cycle causing the production of acetyl-CoA to subsequently excess reduced nicotinamide adenine dinucleotide (Steinberg & Baron, 2002). The superoxide anion undergo dismutation to hydrogen peroxide, which if not degraded by catalase or gluta‐ thione peroxidase, and in the presence of transition metals, can lead to production of hy‐ droxyl radical, the most active oxygen free radical. Hydroxyl radical alternatively may be formed through an interaction between superoxide anion and nitric oxide (Fubini & Hub‐ bard, 2003; Wolff, 1993).

Superoxide anion can also react with nitric oxide to form the reactive peroxynitrite radicals (Hogg & Kalyanaraman, 1998). Excess production of superoxide anion by the mitochondrial electron transport chain, induced by hyperglycaemia has been reported to have a role in triggering protein kinase C, hexosamine and polyol pathway fluxes, and advanced glycation end product formation pathways which are involved in the pathogenesis of diabetic compli‐ cations (Nishikawa et al., 2000; Brownlee, 2001). In a study conducted by Hsu et al. (2006), plasma superoxide anion (determined by a chemiluminescent assay) gave photoemission which was considerably higher in 47 type 1 diabetic children than those in controls. The findings confirm the presence of oxidative stress in children with type 1 diabetes mellitus (Hsu et al., 2006).

#### **4.2. Reactive nitrogen species in type 1 diabetes**

Nitric oxide is an important regulator of endothelial function and the impairment of its ac‐ tivity is determinant of the endothelial dysfunction (Ignarro, 2002). It is an important vascu‐ lar target for ROS and is produced by constitutive and inducible nitric oxide synthases. These enzymes oxidize L-arginine to citrulline in the presence of biopterin, reduced nicoti‐ namide adenine dinucleotide phosphate, and oxygen (Alp & Channon, 2004). Constitutive endothelial nitric oxide synthase contains reductase and oxygenase domains that are con‐ nected by a calmodulin-binding region and requires cofactor groups such as heme, flavin mononucleotide, flavin adenine dinucleotide, tetrahydrobiopterin, and Ca2+-calmodulin for activation (Gorren & Mayer, 2002; Andrew & Mayer, 1999). If there is none or insufficient Larginine, the endothelial nitric oxide synthase produce superoxide instead of nitric oxide and this is referred to as the uncoupled state of nitric oxide synthase (Channon, 2004).

Reactive oxygen species induced tissue injury as well as they are involved in signaling path‐ ways and gene expression (Ha & Lee, 2000). Excess generation of reactive oxygen species such as superoxide anion, hydrogen peroxide, hydroxyl radical and reactive nitrogen spe‐ cies such as nitric oxide oxidize target cellular proteins, nucleic acids, or membrane lipids and damage their cellular structure and function (Brownlee, 2001). There is also evidence that reactive oxygen species also regulate the expression of genes encoding for proteins in‐

Hydroxyl radicals, hydrogen peroxide, and superoxide anion are byproducts of xanthine ox‐ idase. Xanthine oxidase and xanthine dehydrogenase catalyze the conversion of hypoxan‐ thine to xanthine and then to uric acid, with the former reducing oxygen as an electron acceptor while the latter can reduce either oxygen or nicotinamide adenine dinucleotide

Superoxide anion can also react with nitric oxide to form the reactive peroxynitrite radicals (Hogg & Kalyanaraman, 1998). Excess production of superoxide anion by the mitochondrial electron transport chain, induced by hyperglycaemia has been reported to have a role in triggering protein kinase C, hexosamine and polyol pathway fluxes, and advanced glycation end product formation pathways which are involved in the pathogenesis of diabetic compli‐ cations (Nishikawa et al., 2000; Brownlee, 2001). In a study conducted by Hsu et al. (2006), plasma superoxide anion (determined by a chemiluminescent assay) gave photoemission which was considerably higher in 47 type 1 diabetic children than those in controls. The findings confirm the presence of oxidative stress in children with type 1 diabetes mellitus

Nitric oxide is an important regulator of endothelial function and the impairment of its ac‐ tivity is determinant of the endothelial dysfunction (Ignarro, 2002). It is an important vascu‐ lar target for ROS and is produced by constitutive and inducible nitric oxide synthases. These enzymes oxidize L-arginine to citrulline in the presence of biopterin, reduced nicoti‐ namide adenine dinucleotide phosphate, and oxygen (Alp & Channon, 2004). Constitutive

) (Fatehi-Hassanabad et al., 2010). Superoxide anion is also produced by nicotinamide adenine dinucleotide phosphate oxidases and cytochrome P450, and is the most commonly occurring oxygen free radical that produces hydrogen peroxide by dismutation. This is ach‐ ieved via the Haber-Weiss reaction in the presence of ferrous iron by copper (Cu)-superox‐ ide dismutase or manganese (Mn)-superoxide dismutase. Mitochondrial superoxide anion is produced from excess reduced nicotinamide adenine dinucleotide produced in the Krebs cy‐ cle (Fubini & Hubbard, 2003). Elevated free or non-esterified fatty acids in type 1 diabetic patients enter the Krebs cycle causing the production of acetyl-CoA to subsequently excess reduced nicotinamide adenine dinucleotide (Steinberg & Baron, 2002). The superoxide anion undergo dismutation to hydrogen peroxide, which if not degraded by catalase or gluta‐ thione peroxidase, and in the presence of transition metals, can lead to production of hy‐ droxyl radical, the most active oxygen free radical. Hydroxyl radical alternatively may be formed through an interaction between superoxide anion and nitric oxide (Fubini & Hub‐

volved in immune response, inflammation and cell death (Ho & Bray, 1999).

(NAD+

228 Type 1 Diabetes

bard, 2003; Wolff, 1993).

(Hsu et al., 2006).

**4.2. Reactive nitrogen species in type 1 diabetes**

Oxidative stress decreases the bioavailability of endothelium-derived nitric oxide in diabetic patients. In a 3-year longitudinal study involving 37 patients with recent-onset (less than 2 years) type 1 diabetes, oxidative stress was evident by elevated malondialdehyde excretion and serum NOx (nitrate and nitrite) (Hoeldtke et al., 2011). In a latter study, NOx was also higher in 99 female subjects with uncomplicated type 1 diabetes (duration disease <10 years) compared with 44 sex-matched controls (Pitocco et al., 2009). Mylona Karayanni et al. (2006) examined possible correlation between oxidative stress parameters and adhesion molecules derived from endothelial/platelet activation, P-selectin and tetranectin in a group of juve‐ niles with type 1 diabetes mellitus. Significantly elevated NOx and lipid hydroperoxide lev‐ els, elevated tetranectin and P-selectin plasma levels, and lower glutathione peroxidase activity were found in the diabetic children compared with healthy controls. Based on these findings the authors suggested that decreased anti-oxidative protection from overproduc‐ tion of lipid hydroperoxide and NOx overproduction is present in juveniles with type 1 dia‐ betes mellitus. There is also a parallel endothelial/platelet activation which contributes to the vascular complications of type 1 diabetes mellitus (Mylona-Karayanni et al., 2006).

Nitric oxide can react with superoxide to form peroxynitrite which in turn oxidizes tetrahy‐ drobiopterin and causes further uncoupling of nitric oxide formation (Yung et al., 2003). In diabetes mellitus, elevated glucose may cause an increase in the expression of both reduced nicotinamide adenine dinucleotide phosphate and of inducible nitric oxide synthase via the activation of NF-κB, (Spitaler & Graier, 2002). The upregulated inducible nitric oxide syn‐ thase will synthesize the superoxide anion instead of nitric oxide, leading to oxidative and nitrosative stress (Llorens & Nava, 2003). The stable protein adduct, nitrotyrosine, is a mark‐ er of peroxynitrite (Ischiropoulos, 1998) and nitrogen dioxide (Prutz et al., 1985). Moreover, increased oxidative and nitrosative stress activates poly(ADP-ribose) polymerase-1, which substrate, nicotinamide adenine dinucleotide (NAD+ ) as well as slows the rate of glycolysis, electron transport, and adenosine triphosphate formation (Pacher & Szabó, 2006).

The formation of peroxynitrite can further lead to the generation of peroxynitrous acid. The spontaneous decomposition of peroxynitrous acid results in the formation of hydroxyl radi‐ cals that can cause endothelial damage (Elliott et al., 1993; Beckman & Koppenol, 1996) thereby reduces the efficacy the endothelium-derived vasodilator system that participates in the general homeostasis of the vasculature (Benz et al., 2002). Overproduction of both nitric oxide and superoxide anion has been reported in response to hyperglycemia (Cosentino et al., 1997; Ceriello et al., 2002), and nitric oxide may work through peroxynitrite to directly alter cellular structure and function (Pfeiffer et al., 2001). Increased nitric oxide levels have been reported in both saliva and plasma of diabetic patients in comparison to healthy sub‐ jects (Astaneie et al., 2005).
