Free radicals are very reactive chemical species, can cause oxidation injury to the living beings by attacking the macromolecules like lipids, carbohydrates, proteins and nucleic acids. Under normal physiological conditions, there is a critical balance in the generation of oxygen free radicals and antioxidant defense systems used by organisms to deactivate and protect themselves against free radical toxicity 12. Impairment in the oxidant/antioxidant equilibrium creates a condition known as oxidative stress. Oxidative stress is known to be a component of molecular and cellular tissue damage mechanisms in a wide spectrum of human diseases 345.

Diabetes is associated with a number of metabolic alterations and principal among these is hyperglycemia. The precise mechanism by which hyperglycemia may contribute to the development of coronary heart disease (CHD) is a matter of some controversy. Known sequelae of hyperglycemia such as cellular damage, increased extra cellular matrix production and vascular dysfunction have all been implicated in the pathogenesis of vascular disease in type I and type II diabetes 35678910. Mechanisms involved in the increased oxidative stress in diabetes include not only oxygen free radical generation due to nonenzymatic glycosylation (glycation), autooxidation of glycation products, but also changes in the tissue content and activity of antioxidant defense systems. Increased levels of the products of oxidative damage to lipids have been detected in serum of diabetic patients, and their presence correlates with the development of complications 6101112131415. A variety of natural antioxidants exist to scavenge oxygen free radicals and prevent oxidative damage to biological membranes. One group of these antioxidants is enzymatic (intracellular), which include super oxide dismutase, glutathione peroxidase and catalase. In addition to enzymatic antioxidants, the major natural antioxidants, most of them derived from natural sources by dietary intake are vitamin A, vitamin C and vitamin E and carotenoids. Also, numerous small molecules are synthesized or produced within the body that has antioxidant capacity (e.g. glutathione and uric acid) 361415161718.

There are several studies evaluated the free radical induced lipid peroxidation and the antioxidants in diabetic patients. Many of these studies assessed individual antioxidants that act cooperatively in vivo to provide greater protection to the organism against free radical damage than could be provided by any single antioxidant acting alone. Controversial reports have been reported concerning the antioxidant status in diabetic patients 17192021. Protein oxidation, in contrast to lipid peroxidation, does not have the features of chain reactions. The plasma proteins destructed by peroxidation have a quite long period. Therefore, the evaluation of protein oxidation (PCG) in plasma is a respected marker of free radical intensity. There are only a few reports regarding the protein oxidation in various other pathogenic conditions and no reports are available for that processes along with antioxidants in type I diabetic patients. The aim of the present study was to evaluate the free radical reaction intensity in chronic diabetic IDDM patients on basis of protein carbonyl groups of proteins, lipid peroxidation and the status of antioxidants in plasma.

The biochemical particulars of the study subjects including IDDM and non-diabetic controls are given in table 1. The subjects with diabetes had duration of the disease ranged from 5 – 10 years. They all were being treated with insulin. Despite of their treatment, they had hyperglycemia having blood glucose levels more than 3-fold higher than in healthy subjects. The lipid profile of IDDM is statistically insignificant changes with nondiabetics. However, the TBARS (lipid peroxidation) levels increased by 4 fold in diabetics compared to healthy controls (table 2). No significant differences were found between patients with type 1 diabetes and control subjects in the concentration of total, HDL, and LDL cholesterol and triglycerides in age and in BMI.

The protein oxidation (PCG) in IDDM subjects was increased 3.5 fold with decreasing the plasma levels of total protein, albumin, globulin and their ratio compared to non-diabetic subjects. In native PAGE (not shown), the diabetic children had also decreased plasma levels of ceruloplasmin, ceruloglobulin and retinol binding protein (RBP), which are the carrier proteins for copper and vitamin A, respectively and also observed the decreased levels of proteins – albumin, transferrin, and heptoglobulins and variable GC globulin fractions in IDDM compared to normal healthy controls. Table 3 shows a significant decrease in the activities Cu-Zn super oxide dismutase, glutathione peroxidase and catalase in children with IDDM compared to controls. The glutathione peroxidase in the whole blood was also decreased in diabetic children, but their differences with those of the non-diabetic counterparts were not statistically significant. The antioxidant activity containing vitamins retinol, β-carotene, vitamin C and vitamin E were significantly decreased in plasma by 25 – 50 % respectively in diabetic children.

Diabetes mellitus is a chronic, systemic, metabolic disease defined by hyperglycemia and characterized by alterations in the metabolism of carbohydrate, protein and lipid. Oxidative stress thought to be increased in a system where the rate of free radical production is increased and/or the antioxidant mechanisms are impaired. In recent years, the oxidative stress-induced free radicals have been implicated in the pathology of IDDM 14193031323334.

The present study was examined the changes in both extra and intracellular antioxidants and oxidant status in a children suffering from IDDM. The diabetic children were treated with insulin and yet they were hyperglycemic. Prolonged exposure to hyperglycemia and consequent non-enzymatic posttranslational modification of proteins resulting from chemical reaction between glucose and primary amino groups of proteins – glycation, and also increased oxygen free radicals through auto oxidation of glucose 1533343536. Our results are in accordance with those of previous finding clearly show the increased glucose levels induces diabetes, the overproduction of oxygen free radicals and consequently increases the protein oxidation and lipid oxidation (table 2). Plasma MDA and PCG levels were significantly higher, which would indicate that free radical mediated oxidative damage of lipids and proteins is produced at in diabetics 354242. To our knowledge, there are no reports in the literature concerning plasma PCG levels in IDDM patients in relation with antioxidant status. Carbonyl group formation is considered an early and stable marker for protein oxidation. Oxidized proteins constitute a substantial fraction of the catalytically inactive or less active forms of enzymes, which may have direct metabolic consequences 3738394041. According to Gliesner et al 35 showed no statistically significant differences were found for any of the oxidative stress markers (PCG) assessed between patients with DM1 and controls. In addition, weight, height, and routine metabolic tests, including creatininemia and cholesterol levels, were similar between the groups. The lack of significant differences between healthy controls and patients with DM1 suggested that treatment is able to counteract the increase in free radical production. Ahmed et al 41 observed profound increases in proteolytic products of glycated and oxidised proteins in diabetic patients, concurrent with much lower increases in protein glycation and oxidation adduct residues.

Nitric oxide (NO) is an important vascular target for ROS. Superoxide neutralizes NO, and the peroxynitrite formed is a source of hydroxyl radicals that can cause endothelial damage 43. Increased levels of nitric oxide were observed in Type I DM (table 2). Astaneie et al 44 have shown the elevated levels of NO with total antioxidant power. Existence of increased total antioxidant power in the presence of normal lipid peroxidation in plasma of type I diabetic patients indicates the existence of oxidative stress. Oxidative stress therefore diminishes vessel endothelium-dependent relaxation, which is apparent in some experimental preparations even after acute exposure to hyperglycemia. Defective endothelium-dependent relaxation has been observed in chronic diabetic animals, and also in type 1 and type 2 diabetic subjects 414546 and is an important potential target for antioxidant treatment.

The relationship between hyperglycemia and oxygen free radicals is supported by our results demonstrating an association between blood levels of glucose and enzymatic oxidants such as super oxide dismutase and glutathione peroxidase, only in children with IDDM (table 3). The decreased erythrocyte Cu/Zn-SOD and catalase activity in our young diabetic patients also supports the hypothesis of radical mediated injury in this disease. These results are in agreement with others 474849. Circulating RBC act as a sink for free radicals. Consequently, erythrocytes are subject to a continuous flux of O₂ and H₂O₂. It is, therefore, possible that SOD may have an important physiological role in combating this process, since this enzyme can catalyze the dissimulation of two super oxide radicals into H₂O₂. In the present study, on native PAGE, the plasma concentration of ceruloplasmin in IDDM was significantly lower than those of controls. These results are in agreement with others 50 who reported a significant reduction in the plasma concentration of ceuloplasmin in type I diabetic patients. It has been reported that ceruloplasmin is immunologically altered with decrease in enzymatic oxidants. These results may explain the significant reduction in SOD activity in IDDM patients, since increased H₂O₂ and decreased ceruloplasmin 51. Selenium-dependent glutathione peroxidase (GPx), which works in parallel with SOD, protects cell proteins and cell membranes against oxidative damage. In the present sudy, the GPx activity was decreased significantly compared to that of the controls and a negative correlation coefficient between GPx activity and blood glucose concentration was observed in these children (table 3). However, in the published lieterature, the GPx response to diabetes has been conflicting. Diabetics have been reported to be associated with increased 4852 decreased 53 or unchanged 54. In lieu with GPx, the catalase activities were also decreased in diabetes compared to control subjects (table 3). The low GPx activity could be directly explained by either low GSH content or enzyme inactivation under sever oxidative stress.

In addition to antioxidant enzyme activities, the capacity of the antioxidant system to cope with or trap the free radicals generated under normal or pathological conditions was evaluated by measuring the level of total antioxidant status. It reflects the status of α-tocopherol, vitamin A, β-carotene, ascorbic acid, albumin, uric acid and other antioxidants 424957. These extracellular nonenzymatic antioxidants delay or inhibit the oxidative process. Enhanced lipid peroxidation increases the need for lipid soluble antioxidants, such as α-tocopherol and vitamin A and β-carotene. A significance difference in the mean plasma concentration of total antioxidant status was observed in IDDM patients (table 3). Low levels of vitamin E are associated with increased incidence of diabetes and some research suggests that people with diabetes have decreased levels of antioxidants 324255. People with diabetes may also have greater antioxidant requirements because of increased production of free radicals in hyperglycemia 56. The results of Martin-Gallan et al 42 clearly show systemic peroxidative damage associated with insufficient defense mechanisms against ROS to be already present at clinical onset of type 1 diabetes mellitus in children and adolescents. The present study has also demonstrated a significantly lower plasma ascorbate children compared with their controls. A significant negative correlation coefficient between blood glucose and vitamin C has been observed. Patients with diabetes or the metabolic syndrome have low levels of the antioxidant vitamin C 5859 and also control the diabetes 33. Chen et al 59 shown that high-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance.

The findings of the present study suggest that diabetes in an altered metabolic state of oxidation-reduction and that it is convenient to give therapeutic interventions with antioxidants. The loss of antioxidant capacity is statistically associated with accelerated aging processes in diabetic patients, due to an increase in basal oxidation products of etrythrocytes associated with monosaccharide autooxidative glycation 4111860. Our present work takes into account the hypothesis involving the relation between individual components in the intact clinical model and the complex oxidant-antioxidant plasmatic systemic balance. We clearly identified antioxidant markers that were affected in the presence of IDDM; include SOD, plasma albumin, ascorbic acid and α-tocopherol. Theoretically, one should be able to reverse these effects through dietary supplementation, especially of vitamin C and E.

IDDM – Insulin dependent diabetes mellitus, PAGE – Polyacrylamide gel electrophoresis, MDA – Melondialdehyde, PCG – Protein carbonyl groups, TBARS – Thiobarbutyricacid reactives species, TLC – Thin layer chromatography, GPx – Glutathione peroxidase, NO – Nitric oxide, SOD – Superoxide dismutase.