|Year : 2019 | Volume
| Issue : 2 | Page : 69-75
Oxidant/antioxidant status of Sudanese Type II diabetic patients with multiple complications
Selma Mohammed Osman Mahmoud Yousif1, Mohammed S M. Abdalla1, Elmahdi M A. Elmahdi2
1 Research Institute, Sudan Atomic Energy Commission, Khartoum, Sudan
2 Department of Endocrinology, Faculty of Medicine, University of Khartoum, Khartoum, Sudan
|Date of Web Publication||26-Apr-2019|
Dr. Selma Mohammed Osman Mahmoud Yousif
Sudan Atomic Energy Commission, Khartoum
Source of Support: None, Conflict of Interest: None
Background: Diabetes-associated oxidative stress is induced by both increase in production of plasma-free radical concentrations and a significant reduction in antioxidant defence mechanisms, and it is associated with the pathophysiology of diabetes mellitus and its complications. Objectives: To evaluate the oxidant and antioxidant status in Type II diabetic patients with complications and diabetic patients without complications (DWC) compared to normal subjects and their association with diabetic complications. Materials and Methods: Plasma levels of malondialdehyde (MDA), antioxidant Vitamin C and antioxidant enzyme, superoxide dismutase (Cu-Zn SOD) activity were estimated in patients with diabetic nephropathy (DN), patients with diabetic neuropathy (DNe), diabetic patients with coronary artery disease (CAD), DWC, and controls matched for age, sex, body mass index and study region. Spectrophotometry and enzyme-linked immunosorbent assay techniques have been applied for the determination of MDA, Vitamin C and superoxide dismutase (SOD). Results: Comparing the level of Vitamin C and activity of SOD in all groups, it was observed that the lowest concentrations were found in CAD, DN and DNe groups, higher in DWC, and the highest in the control group. On the contrary, MDA levels (as an indicator of oxidative stress) were found to be increased in CAD, DN and DNe groups as compared to DWC and control groups. SOD and Vitamin C were found inversely correlated with glycosylated haemoglobin and MDA levels in all diabetic groups regardless of their complications. Multivariate model showed that all the variables independently associated with the diabetic complications. Conclusions: There is an imbalance between the oxidants and antioxidants in diabetic patients with complications and patients of DWC as compared to healthy groups.
Keywords: Malondialdehyde, oxidative stress, superoxide dismutase, Type II diabetes, Vitamin C
|How to cite this article:|
Mahmoud Yousif SM, Abdalla MS, Elmahdi EM. Oxidant/antioxidant status of Sudanese Type II diabetic patients with multiple complications. J Diabetol 2019;10:69-75
| Introduction|| |
Diabetes mellitus is a chronic disease characterized by hyperglycaemia resulting from defects in insulin secretion, resistance to insulin action or both. Hyperglycaemia is known to cause elevation in plasma-free radical concentrations., Increased production of free radicals and diminishing antioxidant defence 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. Malondialdehyde (MDA) has been documented as a primary biomarker of free radical-mediated lipid damage and oxidative stress. Increased lipid peroxidation in diabetes induced many secondary chronic complications through per-oxidative injuries, such as atherosclerosis and neural disorders.,
There are several mechanisms to counteract oxidative stress by producing antioxidants, either naturally generated (endogenous) constituents or externally supplied through foods (exogenous). Thus, much interest to use the natural antioxidants as means for the prevention and slowing of diabetic complications has been developed.
Antioxidant defence system involves both antioxidant enzyme systems; superoxide dismutase (SOD), catalase and glutathione peroxidases within the body that scavenge free radicals and micronutrient (vitamin) antioxidants; Vitamin E, beta-carotene, Vitamin C and selenium. The body cannot manufacture these micronutrients, so they must be supplied in the diet.
SOD is an antioxidant enzyme that plays an important protective role against cellular and histological damages that are produced by free radicals. 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. Vitamin C is a water-soluble vitamin that can scavenge radicals, among them the hydroxyl radical. Vitamin C is actively taken up in high concentrations by secretary cells of the Islets of Langerhans More Details where it is believed to play a role in antioxidant defence mechanisms. It has been shown to be an important antioxidant to regenerate Vitamin E through redox cycling.
Therefore, the aim of this study was to evaluate the status of oxidants and antioxidant systems in Sudanese diabetic patients and their association with diabetic complications.
| Materials and Methods|| |
Study design and population
A total of 140 Type II diabetic patients were recruited from the Jabir Abu Eliz (the public diabetes centre) and the People Teaching Hospital of the Heart and Chest in Khartoum State. Medical records were screened by specialist physicians; they examined the patients carefully and recorded the patients' nephropathy, neuropathy, cardiovascular diseases, and diabetic control.
The diabetic Type II patients included in this study were divided into four groups: Group 1 consisted of diabetic patients without complications (DWC); Group II consisted of patients with coronary artery disease (CAD), diagnosed by clinical symptoms of angina pectoris, electrocardiogram examination or documented myocardial infarction; Group III consisted of patients with diabetic nephropathy (DN), evaluated by significant renal impairment such as abnormal creatinine or macro albuminuria; Group IV consisted of patients with diabetic neuropathy (DNe), diagnosed by the clinical examination of the patients. Each group includes 35 patients with only one complication.
Patients with cancer, hepatic or respiratory diseases, gestational diabetes mellitus and Type I diabetes mellitus, alcoholics and smokers and patients on antioxidant vitamin supplements were excluded from the study.
All the diabetics were on diet and on hypoglycaemic agents such as glibenclamide or metformin. In addition, diabetic patients with DN were treated by angiotensin-converting enzyme inhibitors. Diabetic patients with CAD were treated by calcium antagonists.
A group of 35 control subjects was selected. Control subjects and diabetic patients were matched with respect to age, sex and body mass index (BMI) as determined by the weight and height of patients. The characteristics of patients are given in [Table 1].
|Table 1: Demographic and biochemical parameters of diabetic and control subjects|
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This study was conducted after taking ethical clearance from The Ministry of Health Khartoum State-Planning Management and Training Unit. Written informed consents were obtained from the all participants before the study.
Samples collection and preparation
Fasting venous blood samples (5 mL) were collected; whole blood was used for determination of glycosylated haemoglobin (HbA1c) concentration. For plasma preparation, blood samples were collected in heparinized tubes, centrifuged for 20 min at the speed of 2000–3000 rpm, for immediate determination of glucose level. Aliquots of plasma were frozen at −20°C for further determinations of insulin, SOD, Vitamin C and MDA.
Glucose was measured spectrophotometrically by oxidase/peroxidase method using reagents and standards obtained from BioSystems S.A., Spain. HbA1c was measured quantitatively by I-chroma™ HbA1c fluorescence immunoassay, using reagents and standards obtained from Boditech Med Incorporated, Korea. Insulin was measured quantitatively by insulin (125I) IRMA assay system using reagents and standards obtained from IZOTOP, Hungary.
MDA was estimated quantitatively by thiobarbituric acid (TBA) assay, using reagents and standards obtained from Biodiagnostic Co., Cairo, Egypt. TBA reacts with MDA to form TBA reactive intermediate product. Cu-Zn SOD activity was estimated using ELIZA Kit obtained from SunLong Biotech Co., LTD, China. Vitamin C (ascorbic acid) was estimated spectrophotometrically using the method of Roe and Keuther.
All analyses were performed using SPSS software (version 20), IBM, Armonk, NY, United States of America for Windows. Statistical significance between the groups was analysed by independent samples t-test. Moreover, correlation between variables was studied using Pearson's correlation coefficient test. Multivariate model was performed with diabetes mellitus as a control variable; to find out the effect of the different variables independently on the development of diabetic complications. All parameters were shown as mean ± standard error (SE) of the mean. The criterion for significance was considered as P < 0.001.
| Results|| |
General characteristics of the study participants
The general characteristics and biochemical parameters of all the study participants are summarized in [Table 1]. All observations were recorded as mean ± SE or %.
No significant difference were observed between diabetic groups (DWC, DN, DNe and CAD) and the control group for mean age in years which was 54.3 ± 1.1, 57.0 ± 1.3, 56.1 ± 1.3 and 57.4 ± 1.1 versus 54.5 ± 0.9 for the control group (P = 0.913, P = 0.144, P = 0.330 and P = 0.06), respectively. The difference was also found to be statistically not significant in DN, DNe and CAD groups when compared with DWC group (P = 0.144, P = 0.315 and P = 0.064), respectively.
No significant difference was observed between diabetic groups (DWC, DN, DNe and CAD) and the control group for BMI mean which was 26.9 ± 0.9, 26.4 ± 0.9, 26.0 ± 0.9 and 26.6 ± 0.9 versus 25.0 ± 0.5 for the control group (P = 0.054, P = 0.064, P = 0.102 and P = 0.052), respectively. The difference was also found to be statistically not significant in DN, DNe and CAD groups when compared with DWC group (P = 0.909, P = 0.515 and P = 0.766), respectively.
Prolonged duration of diabetes was more profound in the complicated groups (DN, DNe and CAD) as the mean ± SE duration in years was 13.4 ± 0.8, 13.0 ± 0.8 and 12.6 ± 1.0 compared to 7.0 ± 0.5 in the group without complications (DWC) (P < 0.001, P < 0.001, P < 0.001 and P < 0.001), respectively.
Plasma glucose, glycosylated haemoglobin and insulin levels
Significant difference were observed between diabetic groups (DWC, DN, DNe and CAD) and the control group for plasma glucose mean level which was higher in all diabetic patients regardless of complications (107.8 ± 2.7, 171.4 ± 13, 183.0 ± 13 and 191.5 ± 12.2 vs. 88.8 ± 0.9 for the control group; P < 0.001, P < 0.001, P < 0.001, P < 0.001, respectively). A highly significant difference was also found in DN, DNe and CAD groups when compared with DWC group (P < 0.001, P < 0.001 and P < 0.001), respectively.
Similar to glucose, significant difference were found between diabetic groups (DWC, DN, DNe and CAD) and control group for plasma HbA1C mean level which was higher in all diabetic patients regardless of complications (5.8 ± 0.08, 8.9 ± 0.37, 10.0 ± 0.42 and 10.3 ± 0.32 vs. 4.9 ± 0.05; P < 0.001, P < 0.001, P < 0.001 and P < 0.001), respectively. A significant difference was also found in DN, DNe and CAD groups when compared with DWC group (P < 0.001, P < 0.001 and P < 0.001, respectively).
Significant difference were found between diabetic groups (DWC, DN, DNe and CAD) and control group for plasma insulin mean level which was higher in all diabetic patients regardless of complications (15.2 ± 1.5, 18.1 ± 2.1, 19.0 ± 1.8 and 19.8 ± 2.7 vs. 9.0 ± 0.35; P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively).
Plasma superoxide dismutase activity and Vitamin C levels
SOD activity was significantly lower in all groups of Type II diabetes mellitus (DWC, CAD, DNe and DN) when compared with control (254.9 ± 4.9, 174.7 ± 1.3, 150.0 ± 1.4 and 126.8 ± 1.4 vs. 363.9 ± 4.9; P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). The difference was also found to be statistically significant in CAD, DNe and DN groups when compared with DWC group (P < 0.001, P < 0.001 and P < 0.001, respectively).
Vitamin C level was significantly lower in all groups of Type II diabetes mellitus (DWC, CAD, DN and DNe) when compared with control (0.72 ± 0.01, 0.60 ± 0.01, 0.52 ± 0.01, 0.49 ± 0.01 vs. 0.88 ± 0.01; P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). The difference was also found to be statistically significant in CAD, DN and DNe groups when compared with DWC group (P < 0.001, P < 0.001 and P < 0.001, respectively).
Plasma malondialdehyde levels
MDA level was significantly increased in all groups of Type II diabetes (DWC, DN, CAD and DNe) when compared with control (9.2 ± 0.2, 10.5 ± 0.4, 11.4 ± 0.4, 12.4 ± 0.4 vs. 6.7 ± 0.1; P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). The difference was also found to be statistically significant in DN, CAD and DNe groups when compared with DWC group (P = 0.014, P < 0.001, P < 0.001, respectively).
Correlation analysis of glycosylated haemoglobin
The study showed significant direct correlation of HbA1c with FBG, insulin and duration of diabetes, and a significant inverse correlation with SOD and Vitamin C in all diabetic groups as shown in [Table 2].
|Table 2: Correlation analysis of glycated haemoglobin levels in diabetic patients|
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Correlation analysis of malondialdehyde
The study showed significant direct correlation of MDAwith FBG, insulin and duration of diabetes, and a significant inverse correlation with SOD and Vitamin C in all diabetic groups as shown in [Table 3].
|Table 3: Correlation analysis of malondialdehyde levels in diabetic patients|
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Association of biochemical parameters with diabetic complications
To find out the effect of biochemical parameters independently for the development of diabetic complications (CAD, nephropathy and neuropathy), the general linear multivariate model was applied with diabetic complications as dependant variables and SOD, Vitamin C, MDA, HbA1c, insulin and duration of diabetes as an independent variables and adjusting for age, sex, BMI. The results of the multivariate model are given in [Table 4]. From [Table 4], it is observed that the levels of all the parameters were statistically significant factors on diabetic complications.
|Table 4: Multivariate model determining biochemical variables associated with different diabetic complications|
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| Discussion|| |
The present study was conducted with an objective to evaluate the status of oxidants and antioxidant systems and their association with diabetic complications.
Since there are contradictory results on the oxidant and antioxidant status in diabetic patients in the literature, the present study was conducted to fill this gap concerning the Sudanese patients.
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 hyperinsulinaemia that promotes higher production of free radicals by NADPH-dependent mechanisms.
The aldehyde product of lipid peroxidation (MDA) is a biomarker of intensified lipid peroxidation and also indirect evidence of high free radical production in diabetes. 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 DNe are 40% higher than diabetics without neuropathy and almost three times higher than healthy controls.
In the present study, SOD activity was 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 Kedziora-Kornatowska et al., who have reported low SOD activity in Type II diabetic patients compared to controls. The marked reduction of SOD activity observed in this study and other literature reports might be due to the direct inactivation of the enzyme by its product hydrogen peroxide and by superoxide anion itself; it might also be due to deactivation of the enzyme by glycation. In contrast, Aydin et al. have noticed that SOD activity is elevated in Type II diabetes mellitus.
The findings of the present study have shown that Vitamin C level was significantly lower in Type II diabetic patients with different complications compared to patients without complications and to healthy subjects. Similar findings were also reported by Sundaram et al. This marked reduction of Vitamin C level in Type II diabetic patients could reflect its high utilization rate, suggesting that this vitamin was intensively consumed in the process of reduction of oxidative stress in diabetic patients. It is well known that oxidative stress is induced by both the increase in free radicals concentration and disturbance of the free radical scavenging system in diabetes mellitus., However, Makimattila et al. have reported that plasma Vitamin C in Type II diabetic patients was not significantly different from those of the control groups.
This study specifically focussed on the correlation of HbA1c with the antioxidant Vitamin C level and the activity of the antioxidant enzyme, SOD. The study findings showed a significant inverse correlation of HbA1C with Vitamin C level and SOD activity 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 hyperglycaemia and hyperinsulinaemia may increase oxidative stress and consequently production of more free radicals that might lead to increased antioxidant enzymes and vitamins damage. This study also focused on the correlation of MDA with the antioxidant status. The study showed significant inverse correlations of MDA with Vitamin C level and SOD activity 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 lipid peroxidation as indicated by MDA is present in diabetic patients and is even stronger in diabetic patients with different complications; this is consistent with the findings of other studies.,, The findings of the present study clearly demonstrated that oxidative stress occurs early in diabetes and progresses during the course of the disease, leading to diabetic complications. This finding was in line with those of other authors.
In this study, the non-diabetics' ability to tolerate the damage due to the oxidative stress, was found to be almost three times higher than that of the patients exhibiting complications; again, the difference between the diabetic groups with complications and the diabetic group without complications was also found to be statistically highly significant, although both groups are diabetic. These findings are in a good agreement with the findings of a previous study by Opara et al.
The present study also focussed on the correlation of HbA1c with the MDA as tested in many previous studies, and showed significant direct correlation of HbA1c with the MDA in DWC and even stronger significant direct correlation in diabetic patients with different complications. However, this highly significant correlation is not a realistic relationship but a virtual one. HbA1c does not in itself produces MDA, but the free radicals produced during the process of glycation of haemoglobin attack lipid membranes, leading to lipid peroxidation and MDA is formed as a by-product of this process. Therefore, MDA is related to HbA1c by an indirect way.
The present study also showed a 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 et al. and Sundaram et al.
The significant correlation between depletion of antioxidants and high HbA1c levels in diabetic patients with complications in this study confirms that this depletion exposes the target tissues of the body to be attacked by oxygen free radicals and in so doing exposes them to damage and depletion becomes a major risk factor for developing complications, and that antioxidant supplements could lower this risk factor as reported by others.
The present study has assessed the multivariate model and found that the oxidative stress markers; MDA, SOD and Vitamin C were independently associated with diabetic complications when compared to diabetes mellitus. This is may be the increment in the knowledge of diabetes mellitus. Therefore, the overall effect of the individual markers may aggravate the situation and speed up the development of complications. The present study findings have also shown that poor glycaemic control and long duration of diabetes were associated with nephropathy, neuropathy and cardiovascular disease. This is similar to that reported in several other studies.,
| Conclusions|| |
- There is an imbalance between the oxidants and antioxidants in diabetic patients with and without complications as compared to healthy controls
- This study emphasizes the important role of hyperglycaemia and oxidative stress in the pathogenesis of Type II diabetes mellitus and development of its complications, suggesting that controlling the blood glucose levels and reducing of oxidative stress is a critical step for delayed progression of diabetic complications and hence the need for antioxidant supplements to postpone the severity of diabetic complications
- Performing multivariate analysis showed the association between oxidative stress biomarkers and complications in diabetes mellitus; the oxidative stress and other factors were found to independently contribute towards the progression of diabetes mellitus and related to the presence and severity of complications and consequently concluded that measurement of oxidative stress biomarkers is vitally important for early detection of diabetic complications and their prevention.
We are grateful to the staff and all the diabetic patients of Jaber Abu Eliz Diabetic Health Center and People Teaching Hospital of the Heart and Chest in Khartoum State.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]