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

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

in young patients with type 1 diabetes mellitus. However vitamin E supplementation did

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‐

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

not decreased albumin excretion rate in these patients (Giannini et al., 2007).

hibits hydroperoxide formation (Laight et al., 2000).

232 Type 1 Diabetes

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‐

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‐ ceptors (Acworth et al., 1997).

In diabetes mellitus, persistence of hyperglycemia was reported to cause increased produc‐ tion of oxidative parameters of lipid peroxidation including malondialdehyde. In a study by Firoozrai and colleagues (2007), malondialdehyde levels were significantly elevated in diabet‐ ic patients. The level of malondialdehyde was positively correlated with duration of diabetes and glycated hemoglobin and negatively with ferric reducing ability of plasma (Firoozrai et al., 2007). In a latter study that investigated the effect of glycemic control on oxidative stress and the lipid profile of pediatric type 1 diabetes mellitus patients, total cholesterol, low densi‐ ty lipoprotein-cholesterol, apolipoprotein A, apolipoprotein B, and malondialdehyde levels were significantly elevated compared with controls. In addition, serum malondialdehyde lev‐ els and malondialdehyde/low density lipoprotein-cholesterol index were significantly elevat‐ ed in metabolically poorly controlled in relation to metabolically well-controlled diabetic patients. Based on these findings the authors suggested that type 1 diabetic children, especial‐ ly those who are metabolically poorly controlled are at high risk of atherosclerosis and vascu‐ lar complications of diabetes mellitus, and that there is a significant relationship between the lipid profile and oxidative stress (Erciyas et al., 2004).

and age-matched healthy subjects was reported by Ferretti et al. (2004). These findings confirm a linkage between paraoxonase-1 activity and lipid peroxidation of lipoproteins and suggest that the ability of high density lipoprotein to protect erythrocyte membranes might be related to the paraoxonase-1 activity (Ferretti et al., 2004). The low paraoxonase-1 aryles‐ terase activity suggests insufficient high density lipoprotein capacity to protect against lipid oxidation in patients with type 1 diabetes (Wegner et al., 2011). It is also hypothesized that the lower high density lipoprotein protective action against membrane peroxidation and de‐ crease paraoxonase-1 activity in diabetic patients could contribute to acceleration of arterio‐ sclerosis in patients with type 1 diabetes mellitus (Ferretti et al., 2004). Furthermore, there are several studies linking diabetes and even postprandial hyperglycemia with increased low density lipoprotein oxidative susceptibility (Ceriello, 2000). Decreased insulin in diabe‐ tes mellitus increases the activity of fatty acyl coenzyme A oxidase, which intiates β-oxida‐

Biochemical Evaluation of Oxidative Stress in Type 1 Diabetes

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

235

**6.2. Biomarkers of protein peroxidation and oxidative damage to DNA in type 1 diabetes**

High plasma glucose concentrations can increase the levels of glycation and oxidative dam‐ age to cellular and plasma proteins in diabetes mellitus. Glycation of proteins is a complex series of reactions where early-stage reactions leads to the formation of the early glycation adduct, fructosyl-lysine and NH2-terminal fructosyl-amino acids, and later-stage reactions form advanced glycation end products (Thornalley, 2002). The oxidation of proteins produ‐ ces nitrotyrosine and protein carbonyl derivatives and nitrotyrosine (Adams et al., 2001). The oxidized or nitrosylated products of free radical attack have reduced biological activity, leading to loss of cell signaling, energy metabolism, transport, and other major cellular func‐ tions. These altered oxidized products also are targeted for proteosome degradation, further reducing cellular function. There is also cell death through necrotic or apoptotic mecha‐

Carbonyl group formation is considered an early and stable marker for protein oxidation in the body. Diabetes mellitus is associated with carbonyl stress where there is an increase of reactive carbonyl compounds caused by their enhanced formation and/or decreased degra‐ dation or excretion (Miyata et al., 1999.) This leads to the formation of advanced glycation end products such as pentosidin and carboxymethyllysine and advanced oxidation protein products, and damage to a number of biologically important compounds (Miayta et al. 1999; Witko-Sarsat et al., 1996). Telci et al. (2000) examined the influence of oxidative stress on oxi‐ dative protein damage in 51 young type 1 diabetic patients clinically free of complications and 48 healthy normolipidaemic age-matched controls. The levels of plasma carbonyl and plasma lipid hydroperoxide were increased in adolescent and young adult type 1 diabetic

Modifications in endothelial cell function are proposed to play an important role in athero‐ genesis. These perturbations include increased permeability to circulating lipoproteins par‐ ticularly low density lipoprotein, increased retention of these lipoproteins, the loss of endothelial cell-directed vasodilatation, and the increased expression of intercellular cell ad‐ hesion molecule-1 and vascular cell adhesion molecule-1 (Ross, 1999). Koitka et al. (2004) re‐

tion of fatty acids, resulting in lipid peroxidation (Horie et al., 2006).

nisms as a result of the accumulation of cellular injury (Rosen et al., 2001).

patients compared with controls.

Isoprostanes are prostaglandin-like compounds formed through peroxidation of arachidonic acid, and have been used extensively as biomarkers of lipid peroxidation as a risk factor for atherosclerosis and other diseases (Roberts & Marrow, 2000). Oxidative stress parameters such as advanced oxidation protein products, total peroxyl radical-trapping antioxidant pa‐ rameter, and F2-isoprostanes (8-epi-prostaglandin-F2: 8-isoPGF2alpha) were not significant‐ ly different in 27 pre-pubertal patients with type 1 diabetes mellitus (with less than 5 years of disease) compared with controls (Gleisner et al., 2006). In another study, Flores and col‐ leagues (2004) evaluated the effect of the normalization of blood glucose levels on urinary F2-isoprostanes at the onset of type 1 diabetes in 14 patients. There was a statistically signifi‐ cant reduction in F2-isoprostanes after insulin therapy (after 16 weeks) which was accompa‐ nied by a significant reduction in glycated hemoglobin (Flores et al., 2004).

Lipid hydroperoxides are potentially atherogenic and are degraded by enzymes such as para‐ oxonase-1 and lipoprotein-associated phospholipase A2 (Van Lenten et al., 2001; Macphee et al., 2005). Paraoxonase-1 is an enzyme associated with high density lipoprotein surface and the antioxidant effect of the latter is partially related to paraoxonase. This enzyme is able to hydrolyze lipid hydroperoxides and to delay or inhibit the initiation of oxidation of lipopro‐ teins induced by metal ions (Watson et al., 1995). It has been suggested that individuals with low paraoxonase-1 activity may have a greater risk of developing diseases such as diabetes mellitus in which oxidative damage and lipid peroxidation are involved, compared with those with high paraoxonase-1 activity (Durrington et al., 2001; Nourooz-Zadehet al., 1995).

Wegner et al. (2011) reported that 80 type 1 diabetic patients had lower paraoxonase-1 ary‐ lesterase activity and higher lipid hydroperoxide levels, and that there was a negative corre‐ lation between paraoxonase-1arylesterase activity and lipid hydroperoxide levels. In a latter study, paraoxonase-1 activity was reduced in patients with type 1 diabetes mellitus with ret‐ inopathy, confirming that oxidative stress could play a role in pathogenesis of diabetic retin‐ opathy (Nowak et al., 2010). A similar finding of lower high density lipoproteinparaoxonase-1 activity in 31 type 1 diabetic patients compared with the same number of sexand age-matched healthy subjects was reported by Ferretti et al. (2004). These findings confirm a linkage between paraoxonase-1 activity and lipid peroxidation of lipoproteins and suggest that the ability of high density lipoprotein to protect erythrocyte membranes might be related to the paraoxonase-1 activity (Ferretti et al., 2004). The low paraoxonase-1 aryles‐ terase activity suggests insufficient high density lipoprotein capacity to protect against lipid oxidation in patients with type 1 diabetes (Wegner et al., 2011). It is also hypothesized that the lower high density lipoprotein protective action against membrane peroxidation and de‐ crease paraoxonase-1 activity in diabetic patients could contribute to acceleration of arterio‐ sclerosis in patients with type 1 diabetes mellitus (Ferretti et al., 2004). Furthermore, there are several studies linking diabetes and even postprandial hyperglycemia with increased low density lipoprotein oxidative susceptibility (Ceriello, 2000). Decreased insulin in diabe‐ tes mellitus increases the activity of fatty acyl coenzyme A oxidase, which intiates β-oxida‐ tion of fatty acids, resulting in lipid peroxidation (Horie et al., 2006).

In diabetes mellitus, persistence of hyperglycemia was reported to cause increased produc‐ tion of oxidative parameters of lipid peroxidation including malondialdehyde. In a study by Firoozrai and colleagues (2007), malondialdehyde levels were significantly elevated in diabet‐ ic patients. The level of malondialdehyde was positively correlated with duration of diabetes and glycated hemoglobin and negatively with ferric reducing ability of plasma (Firoozrai et al., 2007). In a latter study that investigated the effect of glycemic control on oxidative stress and the lipid profile of pediatric type 1 diabetes mellitus patients, total cholesterol, low densi‐ ty lipoprotein-cholesterol, apolipoprotein A, apolipoprotein B, and malondialdehyde levels were significantly elevated compared with controls. In addition, serum malondialdehyde lev‐ els and malondialdehyde/low density lipoprotein-cholesterol index were significantly elevat‐ ed in metabolically poorly controlled in relation to metabolically well-controlled diabetic patients. Based on these findings the authors suggested that type 1 diabetic children, especial‐ ly those who are metabolically poorly controlled are at high risk of atherosclerosis and vascu‐ lar complications of diabetes mellitus, and that there is a significant relationship between the

Isoprostanes are prostaglandin-like compounds formed through peroxidation of arachidonic acid, and have been used extensively as biomarkers of lipid peroxidation as a risk factor for atherosclerosis and other diseases (Roberts & Marrow, 2000). Oxidative stress parameters such as advanced oxidation protein products, total peroxyl radical-trapping antioxidant pa‐ rameter, and F2-isoprostanes (8-epi-prostaglandin-F2: 8-isoPGF2alpha) were not significant‐ ly different in 27 pre-pubertal patients with type 1 diabetes mellitus (with less than 5 years of disease) compared with controls (Gleisner et al., 2006). In another study, Flores and col‐ leagues (2004) evaluated the effect of the normalization of blood glucose levels on urinary F2-isoprostanes at the onset of type 1 diabetes in 14 patients. There was a statistically signifi‐ cant reduction in F2-isoprostanes after insulin therapy (after 16 weeks) which was accompa‐

Lipid hydroperoxides are potentially atherogenic and are degraded by enzymes such as para‐ oxonase-1 and lipoprotein-associated phospholipase A2 (Van Lenten et al., 2001; Macphee et al., 2005). Paraoxonase-1 is an enzyme associated with high density lipoprotein surface and the antioxidant effect of the latter is partially related to paraoxonase. This enzyme is able to hydrolyze lipid hydroperoxides and to delay or inhibit the initiation of oxidation of lipopro‐ teins induced by metal ions (Watson et al., 1995). It has been suggested that individuals with low paraoxonase-1 activity may have a greater risk of developing diseases such as diabetes mellitus in which oxidative damage and lipid peroxidation are involved, compared with those with high paraoxonase-1 activity (Durrington et al., 2001; Nourooz-Zadehet al., 1995). Wegner et al. (2011) reported that 80 type 1 diabetic patients had lower paraoxonase-1 ary‐ lesterase activity and higher lipid hydroperoxide levels, and that there was a negative corre‐ lation between paraoxonase-1arylesterase activity and lipid hydroperoxide levels. In a latter study, paraoxonase-1 activity was reduced in patients with type 1 diabetes mellitus with ret‐ inopathy, confirming that oxidative stress could play a role in pathogenesis of diabetic retin‐ opathy (Nowak et al., 2010). A similar finding of lower high density lipoproteinparaoxonase-1 activity in 31 type 1 diabetic patients compared with the same number of sex-

nied by a significant reduction in glycated hemoglobin (Flores et al., 2004).

lipid profile and oxidative stress (Erciyas et al., 2004).

234 Type 1 Diabetes

#### **6.2. Biomarkers of protein peroxidation and oxidative damage to DNA in type 1 diabetes**

High plasma glucose concentrations can increase the levels of glycation and oxidative dam‐ age to cellular and plasma proteins in diabetes mellitus. Glycation of proteins is a complex series of reactions where early-stage reactions leads to the formation of the early glycation adduct, fructosyl-lysine and NH2-terminal fructosyl-amino acids, and later-stage reactions form advanced glycation end products (Thornalley, 2002). The oxidation of proteins produ‐ ces nitrotyrosine and protein carbonyl derivatives and nitrotyrosine (Adams et al., 2001). The oxidized or nitrosylated products of free radical attack have reduced biological activity, leading to loss of cell signaling, energy metabolism, transport, and other major cellular func‐ tions. These altered oxidized products also are targeted for proteosome degradation, further reducing cellular function. There is also cell death through necrotic or apoptotic mecha‐ nisms as a result of the accumulation of cellular injury (Rosen et al., 2001).

Carbonyl group formation is considered an early and stable marker for protein oxidation in the body. Diabetes mellitus is associated with carbonyl stress where there is an increase of reactive carbonyl compounds caused by their enhanced formation and/or decreased degra‐ dation or excretion (Miyata et al., 1999.) This leads to the formation of advanced glycation end products such as pentosidin and carboxymethyllysine and advanced oxidation protein products, and damage to a number of biologically important compounds (Miayta et al. 1999; Witko-Sarsat et al., 1996). Telci et al. (2000) examined the influence of oxidative stress on oxi‐ dative protein damage in 51 young type 1 diabetic patients clinically free of complications and 48 healthy normolipidaemic age-matched controls. The levels of plasma carbonyl and plasma lipid hydroperoxide were increased in adolescent and young adult type 1 diabetic patients compared with controls.

Modifications in endothelial cell function are proposed to play an important role in athero‐ genesis. These perturbations include increased permeability to circulating lipoproteins par‐ ticularly low density lipoprotein, increased retention of these lipoproteins, the loss of endothelial cell-directed vasodilatation, and the increased expression of intercellular cell ad‐ hesion molecule-1 and vascular cell adhesion molecule-1 (Ross, 1999). Koitka et al. (2004) re‐ ported evidence of endothelial dysfunction in patients with type 1 diabetes. In another study of 45 type 1 diabetic children, there was significantly lower peak brachial artery flowmediated dilation response and increased carotid artery intima-media thickness. This sug‐ gests that altered endothelium function in children with type 1 diabetes may predispose them to the development of early atherosclerosis (Jarvisalo et al., 2004). Furthermore, in a double-blind, placebo-controlled, randomized study of 41 young subjects with type I diabe‐ tes mellitus, vitamin E supplementation (1,000 IU for three months) had a positive effect on the endothelial function as evident by improved endothelial vasodilator function in both the conduit and resistance vessels (Skyrme-Jones, 2000).

**7. Conclusion**

the detection of oxidative stress.

choices for type 1 diabetic patients.

**Author details**

Jamaica

Donovan A. McGrowder1

Mona, Kingston, Jamaica

2 Radiology West, Montego Bay, Jamaica

This review presented convincing experimental and clinical evidence that the aetiology of oxidative stress in diabetes mellitus arises from a number of mechanisms that includes ex‐ cessive reactive oxygen species production from the peroxidation of lipids, auto-oxidation of glucose, glycation of proteins, and glycation of antioxidative enzymes, which limit their ca‐ pacity to detoxify oxygen radicals. There is also evidence that supports the role of hypergly‐ cemia in producing oxidative stress and, eventually, severe endothelial dysfunction in blood vessels of individuals with type 1 diabetes mellitus. The induction of oxidative stress is a key process in the onset and development of diabetic complications, but the precise mecha‐ nisms has not been fully elucidated. A number of biomarkers of oxidative stress have been studied in type 1 diabetic patients such as malondialdehyde, F2-isoprostanes, advanced gly‐ cation end product and nitrotyrosine. The introduction of breath microassays has enhanced

Type 1 diabetic patients have been found to have decreased amounts and efficiency of anti‐ oxidant defenses (both enzymatic and non-enzymatic) due to increased consumption of dis‐ tinct antioxidant components (e.g. intracellular glutathione) or to primarily low levels of antioxidant substances (flavonoids, carotenoids, vitamin E and C). This review also presents small clinical studies that have demonstrated improvements in a variety of oxidative stress biomarkers in type 1 diabetic patients who have received vitamin A, C or E supplements. However, the findings of key prospective randomized controlled antioxidant clinical trials have failed to demonstrate a significant benefit, in the prevention of cardiovascular events. There is a need for continued investigation of the association between reactive oxygen spe‐ cies, type 1 diabetes mellitus and its complications in order to clarify the molecular mecha‐ nisms by which increased oxidative stress accelerates the development of diabetic complications. This will have implication for the prevention and development of therapeutic

, Lennox Anderson-Jackson2

1 Department of Pathology, Faculty of Medical Sciences, The University of the West Indies,

3 Health Policy Department, Independent Health Policy Consultant, Christiana, Manchester,

and Tazhmoye V. Crawford3

Biochemical Evaluation of Oxidative Stress in Type 1 Diabetes

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

237

In addition to lipids and proteins, reactive oxygen species reacts with deoxyribonucleic acid resulting in various products, such as 8-hydroxydeoxyguanosine, that is excrete in urine ow‐ ing to deoxyribonucleic acid repair processes. Urinary 8-hydroxydeoxyguanosine has been proposed as an indicator of oxidative damage to deoxyribonucleic acid. Goodarzi and col‐ leagues (2010) evaluated the relationship between oxidative damage to deoxyribonucleic acid and protein glycation in 32 patients with type 1 diabetes. There were elevated levels of urinary 8-hydroxydeoxyguanosine, glycated hemoglobin, plasma malondialdehyde, and glycated serum protein in 32 patients with type 1 diabetes. There was a significant correla‐ tion between urinary 8-hydroxydeoxyguanosine and glycated hemoglobin. The findings in‐ dicate that that deoxyribonucleic acid is associated to glycemic control level (Goodarzi et al., 2010). In a study which investigated whether advanced glycation end product production and oxidative stress are augmented in young patients with type 1 diabetes at early clinical stages of the disease, advanced glycation end products, pentosidine, and 8-hydroxydeoxy‐ guanosine and acrolein-lysine were significantly higher in the patients with type 1 diabetes compared with healthy control subjects (Tsukahara et al., 2003).

#### **6.3. Biomarkers of oxidative stress present in breath**

Oxidative stress has been implicated in the major complications of diabetes mellitus, includ‐ ing retinopathy, nephropathy, neuropathy and accelerated coronary artery disease (Ceriello & Morocutti, 2000; Androne et al., 2000; Mackness et al., 2002). There is a clinical need for markers of oxidative stress which could potentially identify diabetic patients at increased risk for these complications. The introduction of breath microassays has enhanced the detec‐ tion of oxidative stress because reactive oxygen species oxidize polyunsaturated fatty acids in membranes to alkanes such as ethane and pentane. These are excreted in the breath as volatile organic compounds (Kneepkens & Lepage, 1994). Another marker of oxidative stress is the breath methylated alkane contour, comprising a three-dimensional display of C4 to C20 alkanes and monomethylated alkanes in the breath (Phillips et al., 2004). Phillips et al. (2004) reported significantly increased volatile organic compounds and breath methy‐ lated alkane contour in the breath of type 1 diabetic patients which was independent of gly‐ cemic as they did with blood glucose concentration or with glycation hemoglobin levels.
