OXIDATIVE STRESS STATUS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

Selma. M Osman. M. Yousif 1 , Mohammed S. M. Abdalla 1 , E. M. A. Elmahdi and Manal. Mohammed. Ramadan 3 . 1. Sudan Atomic Energy Commission, Khartoum. 2. University of Khartoum, Faculty of Medicine. 3. National Research Center in Cairo, Egypt.  ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................  Manuscript History

Diabetes mellitus is a chronic disease with serious metabolic disturbances in carbohydrate, protein and fat metabolism characterized by hyperglycemia resulting from defects in insulin secretion, resistance to insulin action or both. The primary causative factor of oxidative stress in diabetes mellitus is hyperglycemia. Hyperglycemia is known to cause elevation in plasma free radical concentrations [1,2]. Increased production of free radicals and diminishing antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus such as coronary artery diseases, nephropathy, neuropathy, and foot ulceration [3].
Enzymes like Superoxide Dismutase (SOD) and Catalase(CAT) comprise natural cellular defense mechanism against these free radicals. Superoxide dismutase (SOD) is the antioxidant enzyme, plays important protective roles against cellular and histological damages that are produced by ROS. It facilitates the conversion of superoxide radicals into hydrogen peroxide and in the presence of other enzymes hydrogen peroxide is converted into oxygen and water [4]. CAT is the antioxidant enzyme, plays an important role against oxidative stress-generated complications such as diabetes and cardiovascular diseases [5]. CAT acts as main regulator of a highly reactive small molecule, hydrogen peroxide metabolism. It enzymatically converts hydrogen peroxide into oxygen and water and thus neutralizes it [6]. CAT protects pancreatic-cells from damage by hydrogen peroxide [7]. Increased risk of diabetes has been documented in patients with catalase deficiency.
Malondialdehyde (MDA) has been documented as a primary biomarker of free radical mediated lipid damage and oxidative stress [8]. Increased lipid peroxidation in diabetes induced many secondary chronic complications through peroxidative injury, like atherosclerosis and neural disorders [9,10]. Therefore the aim of this study was to evaluate plasma lipid peroxidation marker, malondialdehyde (an oxidant) and antioxidant enzyme (superoxide dismutase and catalase) in type 2 diabetes mellitus patients with and without complications i.e., neuropathy, nephropathy and cardiovascular diseases compared to normal subjects. And also, to access the association between oxidative stress and diabetes mellitus and development of its complication.

Materials and methods:-Study Design and Population:-
A total of 140 type II diabetic patients were recruited from Jabir Abu Eliz (the public diabetes Center) and People Teaching Hospital of the heart and chest in Khartoum State. Medical records were screened by specialist physicians. The patients of type2 diabetes mellitus were divided into four groups: DWC group consisted of diabetic patients without complications; CAD group consisted of patients with coronary artery disease, diagnosed by clinical symptoms of angina pectoris, electrocardiogram examination Or documented myocardial infarction; DN group consisted of patients with diabetic nephropathy, evaluated by significant renal impairment such as abnormal creatinine or macroalbuminuria; DNe group consisted of patients with diabetic neuropathy(nervous system damage), diagnosed by the clinical examination of the patients. The diabetic patients received oral hypoglycemic agents like sulphonylureas or metformin. No patient was taking insulin in all the groups of diabetic patients.
Healthy control group, C group was selected. Healthy Control subjects and diabetic patients were matched with respect to age, sex, body mass index (BMI) as determined by the weight and height of patients. All individuals were non smokers. None had taken antioxidant vitamin supplements.

Results:-General Characteristics of the Study Participants:-
The general characteristics and biochemical parameters of all the study participants were summarized in Table 1. All Observations were recorded as (Mean± SE or %).
Significant difference was found between diabetic groups (DWC, DN, DNe, CAD) and control group for plasma insulin mean level which was high in all diabetic patients regardless of complications (15.2±1.5, 18.1±2.1, 19.0±1.8, 19.8±2.7). versus (9.0±0.35), (P<0.001, P <0.001, P <0.001, P <0.001) respectively, as shown on Table1. The patients of diabetes with complications have a longer duration of disease than those without complication (p < 0.001), as shown on Table1. .001) respectively. The difference was also found to be statistically significant in CAD, DNe, DN groups when compared with DWC group (P<0.001, P <0.001, P <0.001) respectively as shown on Table1.

Correlation of HbA1c with SOD, CAT, MDA and duration of diabetes:-
The study showed significant inverse correlation of HbA1c with SOD, CAT and significant direct correlation with MDA and duration of diabetes in all diabetic groups as shown on Table2.

Correlation of MDA with SOD, CAT and duration of diabetes:-
The study showed significant inverse correlation of MDA with SOD and CAT and significant direct correlation with duration in all diabetic groups as shown on Table3.

Correlation of insulin with SOD, CAT:-
The study showed significant inverse correlation of insulin with SOD and CAT in all diabetic groups as shown on Table3.

Association of oxidative stress parameters with diabetic complications:-
To find out the effect of oxidative stress parameters independently for the development of diabetic complications (coronary artery disease, nephropathy, neuropathy) we performed the multivariate model with diabetes mellitus as a Control variable. Multivariate analysis revealed that SOD, CAT, MDA, HbA1c, insulin and duration of diabetes were associated with CAD, DN and DNe as shown on Table4.  <0.001 ** Correlation is significant at the 0.01 level (2-tailed). **; Correlation is Significant at the 0.05 level (2-tailed).* ; correlation is nonsignificant, ns.

Discussion:-
Type II diabetes mellitus is characterized by chronic hyperglycemia which induces an increase of oxidative stress leading to an overproduction of reactive oxygen species (ROS) and free radical species associated with an impairment of antioxidant defence systems [15].

ISSN: 2320-5407
Int. J. Adv. Res. 6(6), 455-462 The aldehydic product of lipid peroxidation (MDA) is a biomarker of intensified lipid peroxidation and also indirect evidence of high free radical production in diabetes [16]. In the present study, the MDA level was found to be significantly increased in type II diabetic patients with different complications compared to patients without complications and to healthy subjects. The findings of this study are also in a good agreement with the findings of a previous study which showed that MDA levels in patients with diabetic neuropathy are ∼40% higher than diabetics without neuropathy and almost three times higher than healthy controls [17].
In this study, Superoxide dismutase (SOD) and catalase (CAT) levels were found to be significantly reduced in type II diabetic patients with different complications compared to patients without complications and to healthy subjects.
The present findings were in a good agreement with the observations of Kimura et al [18] who have reported decreased SOD level in type II diabetic patients. Uzel et al [19] and Kedziora-Kornatowska et al [20] have also reported low SOD and catalase activities in type II diabetics compared to controls. In contrast, Aydin and his coworkers [21] have noticed that SOD activity is elevated while catalase activities are normal in erythrocytes of type II diabetics.
In this study, the insulin hormone level was found to be significantly increased in type II diabetic patients compared to healthy subjects. This result agrees with the theory of insulin resistance associated with hyperinsulinemia that promotes higher production of free radicals by NADPH-dependent mechanisms [22].
This study specifically focused on the correlation of HbA1c and insulin with antioxidant enzymes, superoxide dismutase and catalase. The study findings showed a significant inverse correlation of HbA1C and insulin with superoxide dismutase and catalase in diabetic patients and even stronger inverse correlation in diabetic patients with different complications. This was consistent with the Hunt et al's proposition that chronic hyperglycemia and hyperinsulinemia may increase oxidative stress [23].
This study also focused on the correlation of MDA with antioxidant enzymes, superoxide dismutase and catalase. The study showed significant inverse correlations of MDA with antioxidant enzymes, superoxide dismutase (SOD) and catalase in diabetic patients, and even stronger inverse correlations were found in diabetic patients with different complications. This evidently showed that oxidative stress in terms of MDA was present in diabetic patients and was still significantly increased in diabetic patients with different complications; this is consistent with the findings of other studies [24, 25, and 26]. Increase in lipid peroxidation products (as indicated by the level of MDA) due to the decrease of the antioxidant enzymes is in line with previous reports [27,28]. Hence complications of diabetes may be the result of this high level of free radicals (increase in the level of MDA and peroxidation index) and the reduction in antioxidant defences.
The findings of the present study clearly demonstrated that oxidative stress occurred early in diabetes and increased in the course of the disease, leading to diabetic complications. This finding was in line with those of other authors [29].
The present study also focused on the correlation of HbA1c with the MDA. The present study showed significant direct correlation of HbA1c with the MDA in diabetic patients and even stronger significant direct correlation in diabetic patients with different complications.
The direct correlation between glycemic control parameter (HbA1c) and MDA in diabetic patients has been reported by other authors [30,31], and it can be partially explained by the existing correlation between hyperglycemia, and increased lipid peroxidation.
The present study showed significant direct correlation of HbA1C and MDA with the duration of diabetes in diabetic patients and even stronger significant direct correlation in diabetic patients with different complications. This is in accordance with the study of Kesavulu MM [32] and Sundaram RK [33].
The present study have assessed the effect of malonaldehyde (MDA), antioxidant enzymes; superoxide dismutase and catalase and other factors; glycated hemoglobin (HbA1c), insulin and duration of diabetes independently for the development of diabetic complications i.e., neuropathy, nephropathy, and cardiovascular diseases using the multivariate model .In this multivariate model diabetes mellitus was taken as a control variable for comparison. The 460 outcome of this multivariate was that, all the oxidative stress biomarkers were found to be independently significantly associated with diabetic complications. Therefore, the overall effect of the individual biomarkers may aggravate the situation and speed up the development of complications. The present study findings have shown that poor glycemic control and long duration of diabetes were associated with nephropathy, neuropathy and cardiovascular disease. This is similar to that reported in several other studies [