Relationship between hyperglycemia, antioxidant capacity and some enzymatic and non-enzymatic antioxidants in African patients with type 2 diabetes

Diabetes in all their forms are characterised by either a hyperglycemia or a relative or absolute lack of insulin action. Type 2 diabetes mellitus (DM) has similar physio-pathological characteristics with type 1 DM but differs in etiology [1]. As an endocrinological disease diabetes is associated with several metabolic alterations including hyperglycemia and high level of oxidative stress. The later plays an important role in the development of diabetes complications both in the microvascular and cardiovascular systems [1]. Oxygen free radical generated from glycosylation, auto-oxidation of glycation products, changes of the tissue content and/or the activity of the antioxidant defence systems are some mechanisms involved oxidative stress in diabetics patients [2]. Free radicals not trapped by anti-oxidants during the period of oxidative stress are known to disturb endothelial dependent vasorelaxation, stimulate growth factors, induce the expression of adhesion molecules, promote blood coagulation and contribute to the formation of advanced glycosylation end products [3]. Several by-products of lipid peroxidation found in a higher level in the serum of patients with diabetes, have been correlated with the development of complications of diabetes [4, 6]. Reactive oxygen species (ROS) have been involved in the induction oxidative damage of several macromolecules including lipids, nucleic acids and proteins in patients with diabetes [7]. Most of these side effects can be prevented by antioxidants [8, 9]. Several studies have provided evidences of oxidative damage of different macromolecules and their impact on antioxidant/pro-oxidant systems in type 2 diabetic patients. A number of antioxidants exists in the cells either enzymatic (superoxide dismutase, glutathione peroxidase and catalase) or non-enzymatic (as glutathione and uric) as scavengers of ROS, to prevent oxidative damage of biological membranes [7, 10, 11]. Beside these antioxidants found in the cells, natural antioxidants exist from vegetables and most of them including vitamin A, vitamin C, vitamin E and carotenoids. To improve our knowledge on the pathogenesis of some metabolic diseases, several clinical and experimental studies have been undertaken in order to understand the role of the oxidative stress in the pathogenesis of diabetes [3, 5]. These investigations have described that free radicals generated during this oxidative process is involved in genesis of diabetes, plays significant role in its development of diabetes and complications [1, 4, 7, 12–15]. In such a context, it appears important to evaluate the level of oxidative stress parameters in blood as biomarkers that can be used to identify and prevent the risk of promoting diabetic complications. Up till now, the methods used to detect the status of oxidative stress in patients at clinical level are scarce in literature. Although some useful methods are available to investigate the oxidative profile, they are unfortunately not applicable for clinical diagnosis. However, some of these methods can be used to better understand the appearance of complications that occur during the development of some metabolic diseases such as diabetes. In this light, it seems important to associate the blood levels of TAC, GSH and fasting blood glucose in order to understand what happens in patients with type 2 diabetes with complications compared to healthy persons. The measure of antioxidant capacity considers the cumulative action of all the antioxidants present in plasma and body fluids, thus providing an integrated parameter rather than the simple sum of the measurable antioxidants. The capacity of the known and unknown antioxidants and their synergistic interaction is therefore assessed, thus giving an insight into the delicate balance in vivo between oxidants and antioxidants. In this study serum levels of enzymatic and non-enzymatic antioxidant markers were determined in three groups of persons (healthy persons, diabetic patients without complications (DPNC) and diabetic patients with complications (DPWC) and discussed the relationships between these markers.

Diabetes does not only alter the metabolism of carbohydrates, lipids and protein, but also the chemistry of these macromolecules. Poorly controlled diabetes accelerates the chemical modification of proteins and their functions which could lead to the development of diabetic complications. In this context, several hypotheses have been emitted in order to understand the origin of the complications observed in diabetic patients. These hypotheses include mitochondria damage, mitochondrial defect in oxidative phosphorylation, increased formation of advanced glycation end products (AGES), increased activity of the polyol pathway, hypoxia, alteration of lipoprotein metabolism, increased protein kinase C activity, alteration of growth factors and cytokine, activities and increased oxidative and reductive stress [15, 22]. Although oxidative stress appears as one of the metabolic events associated to diabetes and its complications the precise mechanisms by which oxidative stress may accelerate the development these complications are still not well understood [7]. Oxidative stress seems to be increased in a system where the rate of free radicals production is increased and/or the antioxidant mechanisms are impaired [22].

The present study examined the variation in both extra and intracellular antioxidants, oxidant status, fating blood glucose as well as HbA1c in a group of diabetic patients without or with complications. The glycated hemoglobin (HbA1c fraction) which estimates the fasting blood glucose over a period of three months is useful and considered as a reference parameter in monitoring glycemic control in diabetic patients. Our results showed the increase in fasting blood glucose and HbA1c levels in a group of diabetic patients with complications (DPWC) compared to those without complications and healthy persons. Indeed hyperglycemia causes tissue damage through 5 major mechanisms: (1) increased flux of glucose and other sugars through the polyol pathway; (2) increased intracellular formation of AGEs (advanced glycation end products); (3) increased expression of the receptor for AGEs and its activating ligands; (4) activation of protein kinase C (PKC) isoforms and (5) the over activity of the hexosamine pathway [13]. Previous studies have shown that an increase in glucose levels induces diabetes, the overproduction of oxygen free radicals which consequently increases the protein and lipid oxidation. Malondiadehyde (MDA) is highly toxic by-products partly produced by oxidation and derived from lipid. MDA reacts both irreversibly and reversibly with proteins and phospholipids with profound effects. In this study, high statistical significant differences were observed in the levels of MDA in these cases. MDA was higher in the case of DPNC and DPWC groups than in the control group. Similar results were found in previous studies [23, 24]. The observed high levels of plasma MDA in diabetic patients reflect lipid peroxidation which is the consequence of oxidative stress. The increase in the level of MDA correlates with hyperglycemia in these patients because of self-oxidation of glucose and could generate free radicals. A significant correlation exists between the biomarkers of oxidative stress and fasting blood glucose in DPWC and DPNC. Our study demonstrated a significant positive correlation between fasting blood glucose and CAT activity while significant negative association was demonstrated between fasting blood glucose and glutathione. Nitrogen monoxide (NO) is an important vascular target for ROS. Ion Superoxide, NO and the peroxynitrite formed are sources of hydroxyl radicals that could cause endothelial damage [2]. Increased levels of NO observed in DPNC and DPWC compared to healthy patients could be explained by the fact that hyperglycemia in diabetic patients affects the vascular endothelium and the associated vascular tone by affecting NO levels. In the diabetic state, the endothelium is exposed to high concentrations of glucose and this likely leads to some of the vascular abnormalities which result due to glucose toxicity. The endothelial dysfunction associated with diabetes has been attributed to lack of bioavailable NO due to reduced ability to synthesise NO from l-arginine [25]. NO is a candidate for mediating hyper filtration and the increased vascular permeability induced by diabetes. The higher level of NO in these groups correlates with the reduction of total antioxidant capacity. The reduction of the total antioxidant capacity in the presence of higher levels of lipid peroxidation in serum of patients with type 2 diabetes indicates the existence of oxidative stress. The significant correlation between HbA1c and serum with NO content found in the present study confirms that hyperglycemia control may directly influence NO synthesis. It is well known that the expression of NO action largely depends on its relative level and on its interaction with superoxide anions. These results support the findings that vascular dysfunction due to exposure to pathologically high d-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.

The synergistic effect of antioxidants in human plasma is known to provide greater protection against free radical aggression than any single antioxidant alone. In addition to the total antioxidant capacity (TAC), the enzyme antioxidant activities to trap the free radicals generated under normal or pathological conditions were evaluated by measuring SOD, CAT and reduced glutathione level. The high concentration of superoxide dismutase causes impairment of endothelial isoform of nitrogen monoxide synthase (eNOS) by triggering advanced glycation end products and poly (ADP-ribose) polymerase [26].

Reduced glutathione (GSH), a non-enzymatic antioxidant plays an excellent role by protecting cells from oxidative damage keeping up the cellular levels of the active forms of vitamins C and E by neutralizing the free radicals. The increase in the GSH level in DPNC and DPWC may be related to increase activity of glutathione peroxidase which acts by neutralizing free radicals produced during the complication of the disease. This extracellular non-enzymatic antioxidant delays or inhibits the oxidative process through different mechanisms. Antioxidant enzyme levels are particularly sensitive to oxidative stress and both increase or decrease these have been reported in different disease states in which an enhancement of oxygen species is a cause or a consequence of the disease [7]. The significant variation in CAT and SOD activities in the serum of diabetic patients with and without complications was also noted. The increase in CAT activity may be a respond to an induction of the enzyme caused by the high level of its substrate (organoperoxides and H₂O₂) [24]. This observation may results in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxides [27]. Our results confirm that diabetes, alcohol and smoking are associated with an increase of oxidative stress, which results in higher serum concentration MDA and nitrogen monoxide radical or in lower level of SOD activity in DPWC group. To the best of our knowledge very few studies demonstrated these findings. However, it has been reported that excessive use of alcohol can contribute to increase BMI [27, 28]. Our results of SOD and CAT indicate a relationship between these two enzymes which may play an important role in the effective control of oxidative stress without the variation of one or the other being sufficient independently [7]. The ratio CAT/SOD is considered to be a biomarker of the glycemic control while the MDA/FRAP ratio is a biomarker of lipid peroxidation [7, 28]. These two ratios are considered as the risk factors for the development of diabetes complications. Increase in lipid peroxidation products (as indicated by the level of MDA) due to the decrease activity of most of the antioxidant enzymes is in line with previous reports [24, 29]. 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 (SOD and total antioxidant capacity). This study provides evidence of oxidative damage of different molecules and its impact on antioxidant/pro-oxidant system in type 2 diabetic patients with or without complications.

Our studies showed that the levels of fasting blood glucose and HbA1c, MD and NO were higher in a group of diabetic patients compare the control group. Except for SOD, HbA1c and fasting blood glucose, there was no significant difference between the two groups of diabetic patients. The increase of fasting blood glucose affected the levels of reduced glutathione and catalase in the group of diabetic patient. The correlation between different markers can provide additional information on the risk of diabetic complications.