Assessment of Oxidative Stress and Lipid Status in Patients of Type 2 Diabetes Mellitus with and without Complications

Oxidative stress plays an important role in different disease processes. Some studies conducted on diabetic patients also support it. But very few studies have been conducted in the Indian subcontinent so far. Lipid peroxidation refers to the oxidative degradation of lipids. In this process free radicals take electrons from the lipids (generally in cell membranes), resulting in cell damage. Quantification of lipid peroxidation is essential to assess oxidative stress in pathophysiological processes. The end products of lipid peroxidation are reactive aldehydes such as malondialdehyde (MDA) as natural bi-products. Measuring the end products of lipid peroxidation is one of the most widely accepted assays for oxidative damage. This study was designed to assess the levels of oxidative stress in patients suffering from diabetes mellitus (DM) and to compare them with controls. Also, the study attempted to evaluate correlation between oxidative stress marker MDA and lipids as well as lipoproteins in type 2 DM subjects both with and without complications so as to analyse the role of lipid peroxidation in causing secondary pathophysiologic changes in multiple organ systems. The present study was conducted in Department of Clinical Biochemistry S.B.K.S. Medical Institute. & Research Centre, Waghodiya, Gujarat. Sixty diabetic patients were divided into two groups. Group A comprised of 30 (thirty) diabetic patients without complications and Group B comprised of 30 (thirty) diabetic patients with complications. Sixty normal healthy persons were selected for the study to serve as controls. The parameters assessed in diabetic subjects as well as healthy controls were: Serum MDA level which is a product of lipid peroxidation and Serum lipids as well as lipoproteins (Total Cholesterol, Triglycerides, High density lipoprotein cholesterol, Low density lipoprotein cholesterol). Mean values of MDA in type 2 diabetic subjects with complications were significantly higher (P<0.001) than values observed in type 2 diabetic subjects without complications. Values obtained for the lipids and lipoproteins in type 2 diabetic subjects with complications were significantly high (P≤ 0.001) compared to type 2 diabetic subjects without complications. The present study concludes that there is a significant elevation as well as correlation between oxidative stress marker MDA and various lipid parameters in type 2 diabetic subjects with complications compared to diabetic subjects without complications. This indicates increased lipid peroxidation in DM subjects with complications which may play a significant role in the development of DM associated vascular complications. oxidative stress in subjects


INTRODUCTION
Diabetes Mellitus (DM) comprises a group of common metabolic disorders that share the phenotype of hyperglycemia. Several distinct types of DM exist and are caused by a complex interaction of genetic, environmental factors and life-style choices. Depending on the etiology of the DM, factors contributing to hyperglycemia may include reduced insulin secretion, decreased glucose utilization and increased glucose production. The metabolic deregulation associated with DM causes secondary pathophysiologic changes in multiple organ systems that impose a tremendous burden on the individual with diabetes and on the health care system. [1] DM has emerged as a major health care problem in India. According to the Diabetes Atlas published by the International Diabetes Federation (IDF), there were an estimated 40 million persons with DM in India in 2007 and this number is predicted to rise to almost 70 million people by 2025 by which time every fifth diabetic subject in the world would be an Indian [2]. Type 2 DM is a combination of resistance to insulin action and an inadequate compensatory insulin secretory response. This form of DM, accounts for approximately 90-95% of those with DM [3].
In type 2 DM increased hepatic glucose production occurs early in the course of DM, though likely after the onset of insulin secretory abnormalities in adipose tissue and obesity. Free fatty acid (FFA) flux from adipocytes is increased, leading to increased lipid [VLDL and triglyceride (TG)] synthesis in hepatocytes. This is responsible for the dyslipidemia found in type 2 DM [elevated TG, reduced high-density lipoprotein (HDL), and increased small dense low density lipoprotein particles (LDL)] [1].
According to Stanislaw et al. [4] good glycemic control (defined as near normoglycemia) as well as effective treatment of high blood pressure and dyslipidemia delay development and progression of microangiopathy [4].
DM is typically associated with increased generation of free radicals and/or impaired antioxidant defence mechanism, representing a central contribution for reactive oxygen species (ROS) in the onset, progression, and pathological consequences of DM. Increased free radical generation leads to a condition of oxidative stress. Oxidative stress leads to generation of Malondialdehyde (MDA) which is formed by both lipid oxidation and as a byproduct of prostaglandin and thromboxane synthesis. Oxidation of complex lipids in vivo is largely caused by oxygen derived free radicals [5]. The major targets of these damaging species are the long chain polyunsaturated fatty acids (PUFAs) of cellular phospholipids, which are particularly prone to attack because of the arrangement of double and single bonds. The resultant lipid peroxide frequently decomposes to a radical [6]. Which reacts with most biological molecules, including proteins and lipids. Further decomposition of these lipid peroxides produces toxic aldehydes, in particular MDA (mainly from arachidonic acid) [7]. According to DeZwart,; Mahboob et al. [8,9] Increased free radical production is said to mediate tissue injury in a wide range of diseases and DM is no exception. Free radicals are formed disproportionately in DM by glucose degradation, which may play an important role in the development of complications in diabetic patients. The generation of free radicals may lead to lipid peroxidation and cause severe damage in DM patients. Oxidative stress is increased in DM owing to an increase in the production of oxygen free radicals, such as superoxide, hydrogen peroxide and hydroxide radicals and deficiency of antioxidant defense mechanisms. Increased nonenzymatic and autooxidative glycosylation is one of the possible mechanisms that contribute to the formation of free radicals and free radical -induced lipid peroxidation in DM [8,9].
The purpose of the present study was to evaluate the role of oxidative stress and abnormal lipid levels in pathogenesis of various complications in type 2 DM.

Sample Collection and Processing
The study subjects were advised to be in 12-hour strict fasting state, after which venous blood samples were drawn to obtain plasma and serum for MDA and other assays. Blood was allowed to clot for 30 minutes at room temperature and then centrifuged at 3000 rotations per minute (rpm) for

Measurement of MDA
Lipid peroxidation in serum was measured by MDA estimation as described previously. [10] Lipoproteins in serum were precipitated by adding 20% trichloroacetic acid and 8.1% sodium dodecyl sulphate. Thereafter, 0.8% aqueous solution of thiobarbituric acid was added to this precipitate, mixed well, and finally heated at 95 • C for 1 hour for coupling of lipid peroxide with thiobarbituric acid reagent. The resulting chromogen was extracted from the precipitate by adding n-butanol and pyridine mixture (15:1). The organic mixture was separated by centrifugation and the intensity of the organic layer was measured spectrophotometrically (Halo DB-20, Dynamica, Mayrwies, Salzburg, Austria) by using 530nm filter against water blank. The concentration of MDA in serum was determined from linear standard curve established by 1 to 8 nm of 1,1,3,3tetramethoxypropane [10].

Measurement of Fasting and Post Prandial Plasma Glucose (FPG & PPG)
FPG and PPG were measured by Glucose oxidase-peroxidase method, [11] using the kits provided by DiaSys Diagnostic Systems GmbH.

Measurement of Serum Lipids and Lipoproteins
The lipid and lipoprotein parameters were measured by standard methods by using commercially available kits (DiaSys Diagnostic Systems GmbH).Serum total cholesterol was estimated by Cholesterol Oxidase-Peroxidase method, [12] triglycerides by Glycerol Phosphokinase-Peroxidase method, [13] high density lipoprotein-cholesterol Phosphotungstic Acid, [14] end point method, low density lipoprotein cholesterol by Direct Immunoseparation method [15] (very low density lipoprotein cholesterol were calculated by Friedwald's equation) [16].
The analyses of various parameters was performed on ERBA Mannheim's Erba XL 300 an open system clinical chemistry analyzer with throughput of 300 tests per hour.

Statistical Analysis
Differences in the parameters between the groups were analyzed by means of the student's t test. Variables were presented as mean ± standard deviation (S.D). Correlations between variables were tested using the Pearson rho (r: correlation coefficient) correlation test. Chisquare (χ 2 ) analysis was used for comparison of groups. Data were compared in the groups using SPSS for Windows (version 16; SPSS Inc., Chicago, IL, USA). P < 0.05 was considered as a statistically significance level.

RESULTS AND DISCUSSION
In the present study type 2 diabetic subjects with complications (6.90 nmol/ml) and without complications (5.81 nmol/ml) showed significantly higher mean values of MDA (P< 0.001; P= 0.003) compared to values of MDA observed in healthy controls (4.06 nmol/ml) (Tables 3 and 4).
MDA is a late-stage lipid oxidation byproduct that can be formed nonenzymatically or as a byproduct of cyclo-oxygenase activity. MDA is a volatile molecule that reacts, via schiff base formation, with free amine groups of protein, lipid, and DNA. It is estimated that up to 80% of MDA is protein bound. In addition, accumulation of MDA affects membrane organization by increasing phosphatidylserine externalization. Accumulation of MDA and MDA adducts is correlated with many disease states, such as diabetes mellitus [17].
Oxidative stress, a state of lost balance between the oxidative and anti-oxidative systems of the cells and tissues, results in the over production of oxidative free radicals and reactive oxygen species (ROS). Excessive ROS generated could attack the cellular proteins, lipids and nucleic acids leading to cellular dysfunction including loss of energy metabolism, altered cell signaling and cell cycle control, genetic mutations, altered cellular transport mechanisms and overall decreased biological activity, immune activation and inflammation. These changes lead to initiation of pathogenic milieu and development of pathologies like diabetes [17].
It has been reported that oxidative stress is enhanced in response to hyperglycemia in vascular tissues of patients with DM, leading to the peroxidation of cellular membrane lipids as well as the increased oxidative modification of amino acids and DNA [18]. Ozdemir et al. [19] observed a significant increase in MDA in patients with type 2 DM compared with control group. Their study suggested that permanent structural membrane alterations occur in DM, due to increased production of ROS and decreased antioxidants in the circulation [19].
Moreover, significantly higher values of MDA (P = 0.002) were observed in type 2 diabetic subjects with complications compared to type 2 diabetic subjects without complications (Table 3 and 4).Enhanced oxidative stress under hyperglycemic conditions causes an increase in peroxide lipids in the cell membrane, which induces the intracellular expression of specific genes. To date, it has been understood that the activity of two transcriptional factors, NF-_B and AP-1 (activator protein-1), is regulated by intracellular redox states. When activated, these transcriptional factors bind to the specific binding sites in the regions upstream of various genes such as VCAM-1, ICAM-1, as well as cytokines and growth factors including MCP-1 and PDGF and then regulate the expression of those genes. Vascular disorders progress through the expression of these proteins which are involved in cell-cell interactions in the vascular wall [20].
There is much evidence that oxidative stress is involved in the etiology of several diabetic complications. Type 2 DM is associated with insulin resistance which results in failure of insulin stimulated glucose uptake by tissues. This causes glucose concentrations in blood to remain high. Consequently, glucose uptake by insulin independent tissues increases. Increased glucose flux enhances oxidant production [21,22].     (Tables 3  and 4).
DM is a condition where hyperlipidemia is very common. Moreover, a positive correlation was observed between TC and MDA (r=0.36; P=0.009), TG and MDA (r = 0.31; P = 0.02) and also between LDL-C and MDA (r=0.27; P=0.05) in type 2 diabetic subjects with complications (Table 5). However, the type 2 diabetic subjects without complications did not show a significant positive correlation between lipids and MDA ( Table 5). These observations indicate the coexistence of atherogenic risk factors and oxidative stress in DM subjects with complications. Chronic oxidative stress in diabetic subjects may be related to the metabolism of excess substrates available such as glucose and fatty acids present in the hyperglycemic state. DM is a condition where hyperlipidemia is very common. Moreover, lipid peroxidation increases with hyperlipidemia [23]. Peroxidation of apolipoproteins may affects the lipoprotein metabolism. It is suggested that apo-A has an antioxidant effect, but due to the peroxidation the antioxidant property of apo-A is lost [24].
Januszweski et al. [25] proposed a variation on the advanced glycation end products hypothesis i.e. the hyperglycemia exacerbates the chemical modification of proteins by lipids and that lipids and advanced glycation end products, rather than carbohydrates and advanced glycation end products, may be the immediate and major source of chemical modifications leading to tissue damage, pro-inflammatory process and chronic complications in diabetes [25].
Thus, while severe hyperlipidemia may be sufficient to induce lipo-oxidative damage, hyperlipidemia compared with hyperglycemia and possibly an increase in oxidative stress in diabetes appears to exacerbate the chemical modifications of proteins in diabetes.
Gambhir et al. [26] also found a significant positive correlation (r = 0.712) between MDA levels and TG in diabetics with complications. According to Gambhir et al. [26] hypertriglyceridemia may lead to increased production of lipid derived free radicals. The resultant oxidative stress may be implicated as a causative factor for endothelial dysfunction, which may be a primary event in the pathogenesis of atherosclerotic vascular disease.
Values obtained for the lipids that is TC, TG, HDL-C, VLDL-C and LDL-C in type 2 diabetic subjects with complications were significantly high (P <0.001) ( Table 4) compared to type 2 diabetic subjects without complications.   According to the study by Soliman [27] hyperlipidemia is reported as one of the causative factors for increased lipid peroxidation in DM. Kesavulu et al. [27] observed that the levels of LDL-C and TG were increased in diabetics with microvascular complications compared to those without these complications. Even levels of TBARS were much higher in diabetics with microvascular complications. The hyperglycemia in association with hyperlipidemia observed in diabetic patients could be the causative factor for the increased production of oxygen free radicals and lipid peroxides [28]. The TBARS levels positively correlated with HbA 1C and total cholesterol in the study by Kesavulu et al. [28]. Thus, lipid peroxides may play a role in the pathogenesis of microvascular complications of DM.
Chi-square analysis (χ 2 ) was performed (employing 2 X 2 contingency table) to compare the percentage distribution of study subjects in various risk ranges of lipids. Significant values were obtained for various lipids in diabetic subjects with complications within high risk range (as per NCEP ATP III Guidelines) compared to diabetic subjects without complications ( Table 6).
The results for lipids observed in the present study are in accordance to observations by Chapman [29]. He stated that type 2 DM is characterized by atherogenic dyslipidemic profile with mild to marked elevation of triglyceride-rich lipoprotein (VLDL and VLDL remnants) concentrations, an increase in small dense LDL and apoliproprotein B (apo B) and low levels of HDL. According to Chapman [29] dyslipidemia is also characterized by a spectrum of qualitative lipid abnormalities reflecting perturbations in the structure, metabolism and biological activities of both atherogenic lipoproteins containing apo B [VLDL, intermediate density lipoprotein (IDL) and LDL] and anti-atherogenic HDL containing apo A-I and / or apo A-II.
In the present study percentage distribution of subjects within various risk ranges of lipids (as per NCEP III guidelines) was evaluated. It was observed that maximum number of subjects in borderline and high risk ranges of lipids were among type 2 diabetics with complications ( Fig. 1 above).

CONCLUSION
In recent years, much attention has been focused on the role of oxidative stress, and it has been reported that oxidative stress may constitute the key and common event in the pathogenesis of secondary diabetic complications These findings point towards the need for early diagnosis and management of Type 2 DM patients in order to prevent the development of oxidative stress associated diabetic complications. It may also be inferred that even in the early stages diabetic patients are exposed to oxidative stress due to hyperglycemia and oxidative stress is known to be the unifying factor in the development of diabetes complications. Hence supplementation of antioxidants may also be considered in the management of newly diagnosed Type 2 DM.
In conclusion, the estimation of lipid peroxide MDA along with lipid profile in diabetes mellitus would serve as a useful monitor to judge the prognosis of the patient. Prevention of lipid peroxidation may help to delay the development of diabetic complications. The detection of the risk factor in the early stage of the disease helps to improve and reduce the mortality rate. It gives reason to look for dependence between the oxidative stress degree, evolution of the disease and its chronic complications. This dependence could be used as prognostic marker of course evaluation of diabetes. Not the least is the possibility of reducing oxidative stress by means of different antioxidants as a supplement. .

ETHICAL APPROVAL
All experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.