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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 16  |  Issue : 1  |  Page : 2-5

Association of fasting blood glucose and glycated haemoglobin with some markers of oxidative stress in Ile-Ife, Nigeria


1 Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
2 Department of Medical Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
3 Department of Medicine, Obafemi Awolowo University, Ile-Ife, Nigeria
4 Department of Surgery, Obafemi Awolowo University, Ile-Ife, Nigeria

Date of Web Publication8-Sep-2016

Correspondence Address:
B O Emma-Okon
Department of Medical Biochemistry, Obafemi Awolowo University, Ile-Ife 220005
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1596-4078.189948

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  Abstract 

Background: Increased oxidative stress is a widely accepted contributor to the development and progression of diabetes mellitus (DM). A lot of work is being done to unravel the mechanisms linking oxidative stress with diabetes and its complications.
Objective: The objective was to investigate the relationship between blood glucose levels, glycated haemoglobin and three markers of oxidative stress in DM: catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST).
Methods: Subjects were consecutive patients with Type 2 DM receiving treatment and apparently healthy controls. Fasting blood glucose, HbA1c, CAT, SOD and GST were assayed in the diabetic subjects and controls using standard procedures. Information on the presence of neuropathy and nephropathy was obtained from case notes, whereas screening for retinopathy was carried out at the Ophthalmology Unit of Obafemi Awolowo University Teaching Hospitals Complex. Data were analysed using Statistical Package for the Social Sciences for Windows, version 17.
Results: A total of 115 subjects comprising 65 diabetics and 50 controls were studied. Mean fasting blood glucose, CAT, SOD and GST activities in test and control subjects were 9.12 (0.48) and 4.78 (0.63) mmol/L, 48.20 (2.69) and 63.22 (1.83) units/mg, 53.22 (2.44) and 62.55 (2.68) units/mg and 1.35 (0.09) and 3.47 (0.11) units/mg, respectively. Statistically significant differences were observed when levels of fasting blood sugar, CAT, SOD and GST were compared between diabetic and non-diabetic subjects. Diabetic subjects with one or more microvascular complications were found with significantly higher HbA1c levels than those without apparent complication. There was significant negative correlation between fasting blood glucose and all three antioxidant enzymes.
Conclusion: The results of the present study suggested that fasting blood sugar modulated levels of antioxidant enzymes while protein glycation plays a role in the development of microvascular complications.

Keywords: Catalase, diabetes mellitus, fasting blood glucose, microvascular complications, oxidative stress


How to cite this article:
Odewole T O, Emma-Okon B O, Agboola F K, Kolawole B A, Onakpoya O H, Fagboun O F. Association of fasting blood glucose and glycated haemoglobin with some markers of oxidative stress in Ile-Ife, Nigeria. Niger J Health Sci 2016;16:2-5

How to cite this URL:
Odewole T O, Emma-Okon B O, Agboola F K, Kolawole B A, Onakpoya O H, Fagboun O F. Association of fasting blood glucose and glycated haemoglobin with some markers of oxidative stress in Ile-Ife, Nigeria. Niger J Health Sci [serial online] 2016 [cited 2017 Aug 17];16:2-5. Available from: http://www.chs-journal.com/text.asp?2016/16/1/2/189948


  Introduction Top


The incidence of Type 2 diabetes mellitus (DM) is on the increase globally, with about 382 million people diagnosed as of 2013. [1] It is expected that this number will double by 2030. [1] Although more common in developed countries, the greatest increase in incidence is expected to occur in Asia and Africa following the trend of increased urbanisation and negative lifestyle changes. [2] In Nigeria, there were about 4 million diagnosed cases by 2014 [3] and about the same number, if not more of undiagnosed cases due to insufficient levels of awareness and poor utilisation of health facilities. [3]

Oxidative stress is a pathological condition resulting from either increased production of free radicals or decreased level of antioxidants. High levels of free radicals damage cellular proteins, membrane lipids and nucleic acids and lead to cell death. DM is usually accompanied by increased production of free radicals or impaired antioxidant defences. [4] Sources of free radicals in DM include glucose oxidation, [5] lipid peroxidation [6] and the interaction of glucose with proteins leading to the formation of advanced glycation end products. [7] These products inactivate enzymes and alter their structures and functions, promote free radical formation and quench antiproliferative pathways. [8] While on the one hand, hyperglycaemia engenders free radicals, on the other hand, it also impairs the endogenous antioxidant defence systems in many ways. [9] The enzymes superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) peroxidase and GSH reductase play very crucial roles in the detoxification of free radicals and are regarded as biomarkers of oxidative stress. Alterations in the levels of these enzymes may lead to ineffective scavenging of free radicals in the body. Several authors have reported a link between these biomarkers of oxidative stress and the occurrence of diabetes though most of the findings do not agree. [10],[11],[12] In addition, many studies have reported that some antioxidant vitamins have glucose-lowering effects, [13],[14] but to the best of our knowledge, none has provided information about a direct link between blood glucose levels, glycated haemoglobin (Hb1c) and antioxidant enzyme levels. The aim of the present study was therefore to investigate the relationship between fasting blood sugar, Hb1c and three biomarkers of oxidative stress: CAT, SOD and glutathione transferase (GST) in diabetic patients and controls in Ile-Ife, thus contributing to knowledge on the relationship between hyperglycaemia and oxidative stress and ultimately providing information which may assist in better management of patients with Type 2 DM.


  Patients and Methods Top


The study population consisted of patients with Type 2 DM receiving treatment at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife. Sample size was calculated using a prevalence of 3.9%, an error margin of 0.05 and 95% confidence level. The control subjects were members of staff of Obafemi Awolowo University. Ethical approval was obtained from the Ethical Committee of the OAUTHC, and informed consent form was signed by each participant. Control subjects were screened for diabetes using Accu-chek Active glucometers. Subjects with fasting blood glucose above 100 mg/dl were excluded from the study. Other exclusion criteria included pregnant women and subjects who had one form of illness or the other at the time of the study.

Structured questionnaires were administered to the patients and controls to obtain information on demographic parameters as well as drug and medical histories of the participants. Information about the presence of neuropathy and nephropathy in the patients was obtained from the case notes while the subjects were screened for retinopathy at the Ophthalmology Unit of OAUTHC.

Sample collection and preparation

A fasting venous blood sample was collected from each subject into heparinised tubes for enzyme assay and fluoride oxalate tubes for glucose assay. Erythrocytes were separated by centrifugation at 4000 rpm for 20 min at 4°C. The erythrocytes were washed three times with a buffer solution containing 250 mM Tris hydroxyl methylaminomethane-HCl Tris Hcl pH 7.4, 140 mM NaCl, 1 mM MgCl 2 , 10 mM glucose and re-centrifuged at 4000 rpm. [15] The erythrocytes were resuspended in the buffer and lysed by freezing and thawing, [16],[17] and the haemosylate was diluted with 40 volumes of 0.1M Tris-HCl buffer pH 7.4 and centrifuged at 13500 rpm for 30 min. The supernatant was removed and kept at −80°C before analysis.

Biochemical analyses

Fasting blood glucose level was determined using Randox glucose kits, based on the method of Barham and Trinder. [18] In this method, glucose is oxidised by glucose oxidase to produce gluconate and hydrogen peroxide. The hydrogen peroxide is oxidatively coupled with 4-aminoantipyrene and phenol in the presence of peroxidase to yield a red quinone-amine dye which is measured at 505 nm. Hb1c was measured using clover A1C kits. Protein concentration was determined using the Bradford method [19] in which the dye, Coomassie brilliant blue forms a complex with proteins in solution, which is the maximally absorbed at 595 nm.

SOD activity was measured based on the ability of the enzyme to inhibit auto-oxidation of epinephrine at alkaline pH. [20] Superoxide anion generated by the xanthine oxidase reaction causes oxidation of epinephrine to an adrenochrome which can be monitored at 480 nm. One unit of SOD was expressed as the amount of the enzyme needed to cause 50% inhibition of adrenaline oxidation.

CAT activity was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm. [21] Activity of GST was determined as described by Habig et al. [22] This is a spectrophotometric method based on the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced GSH to form CDNB-S-GSH complex. GST activity is the amount of the complex formed, which is monitored spectrophotometrically at 340 nm. Specific activities of each of the 3 enzymes were calculated for patients and controls by dividing the activities by the protein concentration.

Data analysis

Data were analysed using Statistical Package for Social Sciences (SPSS), IBM Inc, New York for Windows version 17. Mean values were compared using Student's t-test and ANOVA. Pearson's correlation coefficient (r) and regression analyses were used to ascertain the relationship among continuous variables. Statistically significant level was P < 0.05.


  Results Top


A total of 65 Type 2 diabetic patients and 50 apparently healthy controls were studied. Mean age of diabetic patients was 61.0 (2.15) years, whereas that of controls was 60.0 (1.81) years (the difference was not statistically significant). Mean fasting glucose, erythrocyte CAT, SOD and GST activities patients and controls were 9.12 (0.48) and 4.78 (0.63) mmol/L (P = 0.000); 48.20 (2.69) and 63.22 (1.83) units/mg (P = 0.000); 53.22 (2.44) and 62.55 (2.68) units/mg (P = 0.012); and 1.35 (0.09) and 3.47 (0.11) units/mg (P = 0.000), respectively.

A total of thirty patients had one or more of retinopathy, neuropathy and nephropathy.

[Table 1] compares the levels of biochemical parameters between the diabetic patients and controls. The diabetic patients had statistically significantly lower levels of all three antioxidant enzymes compared with the controls, whereas fasting blood glucose was statistically significantly higher in the diabetic patients.
Table 1: Comparison of levels of biochemical parameters between the diabetic patients and controls

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[Table 2] shows the results of correlations between fasting blood sugar, Hb1c and the antioxidant enzymes. There was a significant negative correlation between fasting blood glucose levels and the antioxidant enzymes.
Table 2: Correlation of fasting blood sugar, glycated haemoglobin and antioxidant enzymes

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[Table 3] shows the linear regression analysis of fasting blood glucose levels against the levels of the enzymes; showing how fasting blood glucose predicted antioxidant enzymes. Fasting blood glucose levels were inversely related to the levels of the enzymes.
Table 3: Linear regression showing how fasting blood glucose predicted antioxidant enzymes

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[Table 4] compares the levels of biochemical parameters between patients with and without microvascular complications. Thirty patients had one or more of diabetic nephropathy, neuropathy and retinopathy. Patients with microvascular complications had statistically significantly higher Hb1c levels and lower GST levels than patients without complications.
Table 4: Compares the levels of biochemical parameters between patients with and without microvascular complications

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  Discussion Top


Increasing evidence suggests that oxidative stress plays a major role in the pathogenesis of Type 2 DM. It is however not clear whether increased oxidative stress is a cause of Type 2 DM or a consequence of disease progression. In the present study, in addition to the fact that diabetic patients had significantly lower levels of the three antioxidant enzymes, significant negative correlation was found between fasting blood glucose on the one hand and the levels of the enzymes on the other hand. Regression analyses also showed that high fasting blood glucose levels were significantly associated with low levels of each of the three enzymes shown by negative Pearson's correlation coefficient. It is however noted that there was no significant difference in mean levels of these enzymes when diabetic patients who were recently diagnosed with the disease and were yet to show signs of microvascular complications were compared with those who had one or more of such complications. This would suggest that the low levels of the enzymes found in the diabetic patients were related more to manifestation of diabetes itself than its progression, evidenced by the development of complications. The significantly higher Hb1C levels found in the diabetic patients with complications further buttressed the role of advanced glycation end products in the development of diabetic complications. Glycaemic control is therefore crucial to the prevention of complications of DM.

It has been suggested that oxidative stress acts on signal transduction and via nF-kB affects gene expression, thereby reducing the expression of antioxidant enzymes. [23] Another possibility is that hyperglycaemia inactivates existing enzymes by glycating these proteins. Both postulations suggest that oxidative stress may develop due to insufficient antioxidant activities. [24] However, Hb1c levels did not have a significant correlation with any of the enzymes studied, thus making it unlikely that their levels were reduced as a result of glycation.

Considering the limited sample size in the present study, further studies are desirable, using a larger sample size with adequate representation of diabetic patients at various stages of the disease. Another limitation of the study was the fact that information about the presence of microvascular complications, with the exception of retinopathy was extracted from case histories of the patients and not through biochemical assays or direct observation.


  Conclusion Top


While the present study showed the significant negative relationship between fasting blood glucose levels and the studied markers of oxidative stress, there is a need for better understanding of the mechanisms involved and how this affects the prognosis and outcome of Type 2 DM. In addition, it is suggested that drugs capable of upregulating the synthesis of these enzymes may prove useful as therapeutic agents for the management of diabetes and its complications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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