Aim: Enhanced oxidative stress contributes to the pathogenesis of diabetic retinopathy. The aim of this study is to detect oxidative stress parameters in type 2 diabetes mellitus with or without retinopathy and to investigate the relationship between oxidative stress and patients with type 2 diabetic retinopathy.
Methods: Oxidative stress parameters included malondialdehyde (MDA), conjugated diene (CD), advanced oxidation protein products (AOPP), protein carbonyl and 8-hydroxydeoxyguanosin (8-OHdG) in serum measured in 33 patients with diabetes mellitus without complications, 27 diabetes mellitus retinopathy and 32 normal control subjects, respectively.
Results: The concentrations of MDA and CD in type 2 diabetes mellitus (DM) were significantly higher than those of the control subjects (p<0.01). The concentration of MDA and CD in patients with retinopathy was significantly elevated in comparison with patients with diabetes without retinopathy (p<0.05). There was a significant increase in serum 8-OHdG in patients with diabetes compared with normal subjects (p<0.01), and serum 8-OHdG was much higher in diabetes mellitus retinopathy than that in patients with diabetes without retinopathy (p<0.05). This was significantly higher in serum AOPP and protein carbonyl in type 2 DM compared with normal subjects (p<0.01). Moreover, diabetes mellitus retinopathy patients had significantly higher AOPP and protein carbonyl compared with patients with diabetes without retinopathy (p<0.05).
Conclusions: These results indicate that there were severe lipid peroxidation, protein oxidation, and oxidative DNA damage in diabetes. The augmented oxidative stress in diabetes may be speculated to contribute to the pathogenesis of diabetes mellitus. Oxidative stress is an important risk factor in the development of diabetic retinopathy. The levels and the types of serum oxidative stress by-products will be in favour of predicting the amount of retinopathy.
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The prevalence of diabetes mellitus (DM), particularly type 2 DM, has increased rapidly in industrialised and many developing countries. In the past few decades, type 2 DM has increased rapidly worldwide. It is estimated there are about 40 million patients with diabetes in China in 2002. It has been estimated that the number will more than double within 10 years.1
The DM occurs as an important disease at elderly people, to whom the micro- and macrovascular complications represent a major cause of morbidity and mortality. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in patients with diabetes.2 The role of oxidative stress in the causation of chronic tissue damage is being increasingly recognised. Experimental research in recent years proved that the oxidative stress may be the common mechanism that intervenes in the occurrence of the diabetic complications as well as in the ageing process and is responsible for the increased prevalence of chronic complications in elderly diabetics.3–5 Oxidative stress is increased in the retina in diabetes, and it is considered to play an important role in the development of retinopathy.6–8
Diabetes mellitus retinopathy is one of the major chronic complications of type 2 DM, finally leading to blindness.9 The pathogenesis of diabetes mellitus retinopathy is multifactorial, and the precise mechanisms are unclear. Several mechanisms have been proposed, including increased production of advanced glycation end products (AGEs), enhanced polyol pathway, activation of protein kinase C and generation of ROS.10–13 A number of studies in vitro and in vivo suggest that oxidative stress is increased in patients with diabetes and animal models of diabetes.3–5 Specifically, in diabetic retinopathy, both the genesis6 7 and the advanced stage of proliferative diabetic retinopathy have been hypothesised to be a result of increased oxidative species.8 It has been hypothesised that hyperglycaemia may damage the vascular endothelium and retina by inducing the synthesis of oxidant reactive species. However, the detailed molecular mechanism remains uncertain.
The objective of the present work was to investigate the relationship between lipid peroxidation, protein damage, oxidative DNA damage and diabetes and diabetes mellitus retinopathy. We used a spectrophotometric assay to detect the concentration of conjugated dienes, MDA, AOPP and protein carbonyl, and measured the content of serum 8-OHdG with ELISA assay in patients with type 2 diabetes with or without retinopathy and healthy control subjects.
PATIENTS AND METHODS
The studied group consisted of 60 patients with type 2 diabetes mellitus, who were recruited from the First Affiliated Hospital of the Harbin Medical University. The patients with diabetes included 33 patients without diabetic complications and 27 patients with diabetes mellitus retinopathy (DR). All patients were diagnosed according to the World Health Organization diagnostic criteria for type 2 diabetes. DR was diagnosed by independent ophthalmologists using fundus photographs and was classified as non-proliferative retinopathy (NPDR) and proliferative retinopathy (PDR), including 21 NPDR patients and six PDR patients. All patients were on a diabetes diet and were treated with insulin. They were not taking other medications. We excluded any patients who had acute and chronic infections, fever, malignancy, acute and chronic nephritis, cirrhosis and congestive heart failure. Thirty-two healthy, age-matched subjects were also included for the control. None of the control subjects was taking any medication.
Venous blood samples were collected after 12 h overnight fasting from each subject. The samples were placed on ice and centrifuged within an hour at 3500 rpm and 4°C for 15 min. Supernatants were stored at −20°C, and determination of the samples occurred within 3 months.
Measurement of lipid peroxidation products
Quantitative estimation of products of lipid peroxidation included assays for conjugated dienes (CD) and malondialdehyde (MDA). CD was determined according to the method of Ward et al.14 MDA levels were measured spetrophotometrically using thiobarbituric acid-reacting substance (TBARS) production.15
Measurement of DNA damage product
DNA damage was evaluated by measuring 8-hydroxydeoxyguanosin (8-OHdG) content in serum as a biomarker of DNA damage. Serum 8-OhdG was measured using the enzyme-linked immunosorbent assay (ELISA) method.16
Measurement of protein damage products
Products of protein damage included advanced oxidation protein products (AOPP) and protein carbonyl content. AOPP were quantified as described by Witko-Sarsat et al.17 Protein carbonyl concentrations in serum were measured by the spectrophotometric assay described by Reznick and Packer.18
The assay variances of all methods described above were <10%.
Other biochemical parameters
Blood glucose, triglycerides and cholesterol were determined using routine clinical chemical assays.
Data are presented as mean (SD). All experimental data in this study were statistically analysed with SAS 9.13. The statistical significance was evaluated using an unpaired Student t test. Results were considerd significant at p<0.05.
As shown in table 1, the study groups were well matched for age and sex. The patients with diabetes mellitus had significantly elevated levels of fasting blood glucose and triglycerides, and the diabetes duration for diabetic retinopathy was longer than that of diabetes mellitus without complications.
As shown in table 2, there was a significant increase in serum MDA in patients with diabetes (6.72 (2.52) nmol/ml) compared with normal subjects (3.12 (1.27) nmol/ml, p<0.01). Serum conjugated dienes in patients with diabetes has significantly increased (0.486 (0.248) vs 0.321 (0.106), p<0.05). Moreover, patients with diabetes mellitus retinopathy have a higher MDA and conjugated dienes compared with patients with diabetes without complications (7.90 (2.59) vs 5.76 (2.02), p<0.05; 0.566 (0.273) vs 0.420 (0.208), p<0.01, respectively). This was significantly higher in serum 8-OHdG in both type 2 diabetics with and without microvascular complications (24.96 (6.36) ng/ml) compared with normal subjects (5.89 (1.68) ng/ml, p<0.01). Moreover, patients with diabetes mellitus retinopathy also had a significantly higher 8-OHdG compared with patients without vascular complications (26.86 (7.08) ng/ml vs 23.40 (5.31) ng/ml, p<0.05).
As shown in table 3, compared with the control, serum AOPP in patients with diabetes has significantly increased (25.66 (7.53) vs 16.66 (5.65) μmol/l, p<0.01). This was significantly higher in serum protein carbonyl in patients with type 2 diabetes mellitus (4.08 (1.24) nmol/ml) compared with normal subjects (3.12 (0.75) nmol/ml, p<0.01). Moreover, patients with diabetes mellitus retinopathy also had significantly higher AOPP and protein carbonyl compared with patients without vascular complications (27.80 (7.19) μmol/l vs 23.91 (8.17) μmol/l, p<0.05; 4.60 (0.94) nmol/ml vs 3.65 (1.30) nmol/ml, p<0.05, respectively).
Hyperglycaemia plays a critical role in the development and progression of retinopathy, but the mechanism by which hyperglycaemia results in the development of retinopathy is not clear. Oxidative stress is increased in the retina in diabetes. The possible sources of increased oxidative stress might include increased generation of free radicals or an impaired anti-oxidant defence system. Enhanced levels of free radicals have been found in diabetes.19 20 The present study shows that the biomarkers of oxidative stress increased in diabetes, especially in diabetic retinopathy.3–8 In general, oxidative stress, including ROS, can cause oxidative damage to DNA, proteins and lipids, leading to DNA and protein modification and lipid peroxidation, and many clinical conditions are associated with increased indices of oxidant stress.
Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy and nephropathy. Diabetes mellitus may be associated with increased lipid peroxidation and oxidative DNA damage caused by oxidative stress. Increasing evidence in both experimental and clinical studies suggests that there is a close link between hyperglycaemia, oxidative stress and diabetic complications. It had been considered that oxidative stress contributes to the pathological processes of diabetic complications, including nephropathy and retinopathy, and so on.21 However, the detailed mechanism remains to be elucidated. In this study, we confirmed that there lipid peroxidation and oxidative DNA damage in diabetes exist to some extent, especially in patients with diabetes mellitus retinopathy.
Oxidants are highly reactive compounds with a half-life of only seconds. Therefore, their in vivo determination is generally not feasible. In contrast, lipids, proteins, carbohydrates and DNA, after being modified by oxyradicals, have lifetimes ranging from hours to weeks and can be measured with biochemical assays, which makes them ideal markers of oxidative stress. Many biomarkers have been developed to evaluate oxidative stress. These markers include lipid peroxidation products (such as acrolein, MDA, conjugated dienes, 4-hydroxynonenal), protein oxidation products (such as AOPP, protein carbonyl) and DNA damage products (8-OHdG).
Early evidence to support the association of oxidative stress and hyperglycaemia was largely indirect. Serum MDA level is a sensitive marker of lipid peroxidation that is a useful measure of oxidative stress status. For example, the plasma from diabetic subjects contains increased levels of MDA, F2-Isoprostanes, 8-isoprostane and lipid hydroperoxides.22–24 Recent studies in type 2 diabetic animal models report that the progressive reduction of islet cells is associated with excessive oxidative stress.25 Japanese patients with type 2 diabetes show a reduction of cell mass and evidence of increased oxidative stress-related tissue damage.26
Production of ROS and lipid peroxidation are increased in patients with diabetes.2 In this study, we selected two biomarkers, CD and MDA, of lipid peroxidation that are widely used, sensitive and appropriate for use in large studies. CD is the initial formation of a lipid peroxide. MDA is a decomposition product of peroxidised polyunsaturated fatty acids. Increased serum MDA and conjugated dienes levels have been found in patients with diabetes mellitus.22 27 In our study, we found that DM patients had a significantly higher MDA and conjugated dienes compared with control subjects. Therefore, the concentration of MDA and CD in patients with retinopathy was significantly elevated in comparison with patients with diabetes without retinopathy. These data are in agreement with those of other authors.28 29
Oxidative stress may play an important role in the development of complications in diabetes. ROS can cause strand breaks in DNA and base modifications, including the oxidation of guanine residues to 8-hydroxydeoxyguanosine (8-OHdG), an oxidised nucleoside of DNA, and is the most frequently detected and studied DNA lesion marker. It has been regarded as a novel biomarker of oxidative DNA damage in vivo. In humans, higher levels of 8-OHdG were observed in the mononuclear cells from patients with diabetes.30
High blood glucose levels in vitro impair cellular DNA repair and increase DNA cleavage.31 Hyperglycaemia itself contributed to increased generation of ROS, and this increased oxidative stress would lead to oxidative DNA damage. Nishikawa et al32 had shown that blockade of hyperglycaemia-induced ROS production reversed the pathways implicated in diabetic angiopathy in cultured endothelial cells.
Oxidative stress can effect nucleic acids and generate various modified bases in DNA. In nuclear and mitochondrial DNA, 8-OHdG is released into blood and urine after being excised from DNA by the repair enzyme. Previous studies had shown that patients with diabetes had higher levels of 8-OHdG in the mononuclear cells, urine, pancreatic islet and mitochondrial DNA.33–38 Kakimoto et al have also shown that the levels of 8-OHdG are increased in kidney tissues of streptozotocin-induced diabetic rats.39 Moreover, the content of 8-OHdG in the urine and mononuclear cells of patients with type 2 diabetes with either retinopathy or nephropathy were much higher than those in patients without complications.4 In our research, we observed the similar results. Therefore, the contents of 8-OHdG in serum could act as a sensitive biomarker for the diabetes mellitus retinopathy, too.
Biomarkers of protein oxidation are often applied when a battery of markers of oxidative stress status are being studied. Elevated biomarkers of protein oxidation have often been associated with diseases such as Alzheimer’s, diabetes mellitus and cancer.40–44 Protein oxidation may, therefore, represent an important mechanism in the onset of eye complications in patients with diabetes. The concentration of protein carbonyl was stable, yielded quantitative results, and appeared to reflect disease endpoints in a biologically significant way. Increased protein carbonyl has been found in patients with diabetes.45–47 In this study, we found that the level of protein carbonyl in DM was increased significantly. Our finding is in line with Dandona et al.30
Recently, a new marker of protein oxidation, advanced oxidation protein products (AOPP), has begun to attract the attention of various investigators.48 49 Advanced oxidation protein products were described by Witko-Sarsat et al17 for the first time. They are formed during oxidative stress by the action of chlorinated oxidants, mainly hypochlorous acid and chloramines (produced by myeloperoxidase in activated neutrophils). They are elevated in patients with renal insufficiency and diabetes mellitus.42 43 In our study, we have also determined the level of AOPP and protein carbonyl, two products of protein oxidation, which were increased, and demonstrated that there was protein oxidative damage in diabetes. The results show that AOPP is a biomarker available for assessing oxidative stress in diabetes, too.
In conclusion, we found that there were severe lipid peroxidation, oxidative DNA damage and protein damage in the diabetes mellitus. These findings suggested the possibility that increased oxidative stress may be associated with diabetes, especially in patients with diabetes mellitus retinopathy. Therefore, assessment of oxidative stress in patients with diabetes may be important for the therapy and prevention of diabetes mellitus and diabetic complications.
This work was supported by Postgraduate Innovative Foundation of Harbin Medical University (No. HCXB2006008) and the Grant from Health Bureau of Heilongjiang Province (No. 2005-87).
Ethics approval: Ethics approval was obtained.
Patient consent: All subjects gave written informed consent to participate in this study.
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