Journal of Diabetology, February 2013; 1:2

http://www.journalofdiabetology.org/

Original Article:

Relationship of oxidative stress with type 2 diabetes and hypertension

* S .I .R . O k od uw a 1 , 2 , Al . U ma r 2 , S . I brahim 2 , F . Bell o 3 . Abstract Hypertension and diabetes commonly occur together. The aim of this study was to investigate the role of oxidative stress in the link among Northern Nigerians. The study involved 200 subjects (aged 20 to 79 years) divided into four groups of 50 subjects each, viz.: Non Diabetic Normotensive Persons (NDNP) as controls, while Diabetic Normotensive Patients (DNP), Non Diabetic Hypertensive Patients (NDHP) and Diabetic Hypertensive Patients (DHP) as cases. Biomarkers of oxidative stress were correlated with some trace mineral elements in serum samples collected from all the subjects. A significant (p < 0.01) negative correlation was observed between the following: CAT/MDA and Fe2+/MDA in the 3 case groups; SOD/MDA, GSH/MDA in both DNP and DHP groups; Vitamin C/MDA, Se2+/MDA and Zn2+/MDA in both NDHP and DHP groups. The negative correlation of Vitamin E with MDA was found to be significant (p < 0.01) only in DHP group. The relationship between biomarkers of oxidative stress and trace mineral elements obtained in this study is implicated in the changes in antioxidant defense system which resulted in the pathophysiologic mechanisms underlying the complication of type 2 diabetes and associated hypertension. Key words: Oxidative stress, antioxidants, diabetes, hypertension.
1Department

of Biology, Nigerian international Colleges, Abuja - Nigeria.

2Department

Turkish

of Biochemistry, Ahmadu Bello University, Zaria - Nigeria.

3Department

of Medicine, Ahmadu Bello University Teaching Hospital, Shika - Nigeria. *Corresponding author:
(Current Details)

Okoduwa SIR Department of Biochemistry, Ahmadu Bello University, Zaria - Nigeria. E-mail: siroplc1@yahoo.com Introduction Diabetes and hypertension are life style related non communicable diseases that are of global significance, affecting 285 million and 1 billion people, respectively (1,2). These figures are expected to increase to about 438 million and 1.56 billion for diabetes and hypertension respectively, over the next 20 years as a result of increasing population growth, increasing prevalence of obesity, and sedentary life-style (1, 2). In Nigeria, diabetes affect 2.8 million adults between the ages of 20-79 years with gender distribution of 1.5 million male and 1.3

million female (with type 2 diabetes accounting for 90-95% of all diagnosed cases), while over 8 million individuals are affected with hypertension (1-4). Mortality in 80% of the patients with diabetes is due to cardiovascular diseases (3). Globally, cardiovascular diseases are known to be the number one group of killer diseases, accounting for about 12 million deaths annually. The commonest cardiovascular disease is hypertension, and it is a major public health issue affecting 25% of adult population in developed as well as developing countries (2). About 73% of adults with diabetes have blood pressure greater than or equal to 130/80 mmHg or use prescription medications for hypertension (1). Understanding, why these two diseases commonly occur together is the major concern in the scientific community today. Hence, acquaintance of the relationship between these devastating diseases early in their progression would be of great help in preventing their serious complications. Series of clinical and experimental studies have shown that oxidative stress, through free radical generation, plays a major role in the onset of diabetes (5) and hypertension (6). The deleterious effect of these free radicals can be prevented by antioxidants (7,8). But the

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Journal of Diabetology, February 2013; 1:2 effectiveness of antioxidants enzymes in scavenging free radicals depends on their antioxidant cofactors. These cofactors are micronutrients such as trace mineral elements like Zinc, Copper, Molybdenum and Selenium etc, which form the structural basis for the effectiveness of the antioxidant enzyme defense system. These trace mineral elements together with vitamin E and ascorbic acid are collectively responsible for protecting cells from pro-oxidant molecules, especially free radicals (9). Hence, the need to study the status of these traces mineral elements in hypertensive patients and patients with diabetes in Nigeria. Methodology Study Population The study population consisted of 200 subjects, which included 150 patients with diabetes, hypertension and hypertensive-diabetics as case groups from the medical outpatient, cardiac and diabetic clinic of Ahmadu Bello University Teaching Hospital (ABUTH), ZariaNigeria. They were divided into three groups with 50 patients in each group based on the disease condition. The diagnosis of Hypertension was established according to the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure diagnostic criteria: Systolic Blood Pressure above 140 mmHg and Diastolic Blood Pressure above 90 mmHg (10). The diagnosis of diabetes was established based on the World Health Organisation diagnostic criteria of the fasting plasma glucose above 7.0 mmol/l, and/or 2-hours post-prandial plasma glucose above 11.1 mmol/l (11,12,13). The control group consisted of 50 subjects who were nondiabetic/non-hypertensive healthy volunteers, recruited from the Hospital and the University and matched for age, sex and body mass index with the study population. The study was ethically cleared by the ethical committee of the institution. Written informed consent for participation was obtained from all subjects. Inclusion criteria Patients diagnosed with hypertension and/or diabetes on their first attendance of the clinics at ABUTH were included in the study. The

http://www.journalofdiabetology.org/ control subjects were apparently healthy. All subjects were > 20 years but < 79 years of age. Exclusion criteria Patients who were present or past smokers, had hepatic disease or on lipid lowering drugs or antioxidant vitamin supplements, probucol, allopurinol, quinidine, disopyramide, or other drugs known for affecting serum lipid peroxidation and antioxidant values, < 20 or > 79 years of age, or failed to give a written consent were excluded. Sample Collection Aliquot blood samples were collected after an overnight fasting of about 10-14 hours, from all subjects between 08:00 and 09:00 hours, local time, by venipuncture. Part of the blood sample were placed into coagulating tubes and allowed to clot and serum was separated by centrifugation at 3000 rpm for 10 minutes. The serum was stored at -200C until required. Portions (0.5 ml) of the blood collected were placed in sodium oxalate tubes for fasting plasma glucose estimation. Analyses Lipid peroxidation in serum was estimated colorimetrically by measuring malondiadehyde by a modification of the methods of Albro et al., (14) and Das et al., (15). Superoxide dismutase (SOD) activity was measured at 560 nm according to the method described by Martins et al., (16). Reduced glutathione was determined spectrophotometrically at 412nm according to the method described by Tietze (17). Catalase activity was measured spectrophotometrically at 240nm according to the method described by Aebi (18). Vitamin C (ascorbic acid) concentration was measured according to the colorimetric method described by Omaye et al., (19), at 530 nm. Vitamin E (-tocopherol) was estimated by the method of Desai (20) colorimetrically at 520 nm. The FPG was determine using Glucose oxidase assay kits obtained from Randox Laboratories Switzerland according to the modified glucose-oxidase method described by Trinder (21). Serum Iron, Zinc, Copper, Chloride, Calcium were determined by using LABKIT reagent assay

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Journal of Diabetology, February 2013; 1:2 kit (Chemelex, S.A, Spain) according to the manufacturers instructions (22,23). Serum Selenium was determined by a flameless graphite furnace Atomic Absorption Spectrophotometer (AAS), (AA670G, Shimadzu, Japan); the absorbance was measured at 195 nm (22). Statistical Analysis All statistical analysis was done using a computer software program, SPSS.17 version for windows. Data from all groups were compared using analysis of variance (ANOVA). Differences between mean were assessed by Duncan Multiple Range Test (DMRT). The relationships between the various parameters were assessed by Pearsons rank correlation. Data were expressed as mean standard error of mean. P < 0.05 was considered statistically significant. Results The data indicated that the majority of patients that had Diabetes were between the ages of 50-59 years and 60-69 years for those with hypertensive diabetes (Figure 1). It was observed that morbidly obese subjects with body mass index (BMI) > 40 kg/m2 were among

http://www.journalofdiabetology.org/ males in the DHP and females in the DNP groups (Figure 2). Significant correlation exists between the biomarkers of oxidative stress in the cases, however, no significant (p < 0.01) association was observed in the control group (Table 1). CAT and serum Fe were negatively correlated (p < 0.01) with MDA in the case groups (Tables 2, 3 and 4). SOD and GSH were negatively correlated with MDA both in DNP (r = - 0.427 and - 0.500 respectively) and NDHP (r = - 0.025 and - 0.092 respectively) groups (Table 2 & 3). Vitamin C, serum Se and serum Zn were negatively correlated (p < 0.01) with MDA both in NDHP (r = - 0.384, - 0.469, - 0.469 respectively) and DHP (r = - 0.599, - 0.697, - 0.414 respectively) groups (Table 2 and 4). Serum Cu was statistically correlated (r = 0.594) with MDA, only in NDHP group (Table 2); whereas Vitamin E was negatively correlated (r = - 0.609) with MDA, only in DHP group (Table 4) while serum Cu was negatively correlated (r = - 0.388) with serum Se only in DNP group (Table 3). Every other variable considered were correlated with each other positively but some were not significant (Table 2 4).

Figure 1: Age distribution within the groups

DNP: Diabetic normotensive patients DHP: Diabetic hypertensive patients NDNP: Non diabetic normotensive patients NDHP: Non diabetic hypertensive patients

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Journal of Diabetology, February 2013; 1:2

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Figure 2: Percentage variation in BMI within the groups

DNP: Diabetic normotensive patients DHP: Diabetic hypertensive patients M: Male F: Female NDnP: Non diabetic normotensive patients NDHP: Non diabetic hypertensive patients BMI: Body mass index

Table 1: Correlation of trace mineral elements with biomarkers of oxidation stress in non-diabetic normotensive (NDNP group) CAT CAT SOD GSH MDA VC VE SE FE CU ZN 1 0.359* - 0.175 0.054 - 0.181 - 0.265 - 0.031 - 0.276 0.022 - 0.180 1 - 0.009 0.051 - 0.261 - 0.084 0.004 0.082 0.008 - 0.070 1 - 0.036 - 0.16 - 0.134 - 0.004 - 0.089 0.147 - 0.098 1 - 0.211 0.063 - 0.067 0.151 0.130 - 0.156 1 0.207 0.242 0.253 - 0.192 - 0.096 1 0.022 0.284* - 0.170 - 0.143 1 - 0.035 0.123 - 0.263 1 - 0.005 - 0.119 1 - 0.147 1 SOD GSH MDA VC VE SE FE CU ZN

*Correlation is significant at the 0.05 level (2 tailed) CAT : Catalase , SOD : superoxide dismutase , MDA: Malondialdehyde, VC : Vitmain C , Se: serum selenium ion Fe : Serum Iron ion , Zn : Serum Zinc ion

GSH : Reduced glutathione VE : vitamin E Cu : Serum Copper ion

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Journal of Diabetology, February 2013; 1:2

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Table 2: Correlation of trace mineral elements with biomarkers of oxidation stress in hypertension (NDHP group) CAT CAT SOD GSH MDA VC VE SE FE CU ZN 1 0.719 * 0.446 * - 0.379 ** 0.619 ** 0.567 ** 0.326 * 0.481 ** - 0.385 ** 0.507 ** 1 0.539 ** -0.225 0.501 ** 0.451 ** 0.347 * 0.260 - 0.143 0.438 ** 1 - 0.092 0.524 ** 0.611 ** 0.460 ** 0.420 ** - 0.189 0.461 ** 1 0.384 ** - 0.281 ** - 0.469 ** - 0.445** 0.594** - 0.469** 1 0.462 ** 0.476 ** 0.492 ** - 0.707 ** 0.568 ** 1 0.546 ** 0.393 ** - 0.304 ** 0.636 ** 1 0.428 ** 0.283 * 0.725 ** 1 - 0.510 ** 0.397 ** 1 - 0.395 * 1 SOD GSH MDA VC VE SE FE CU ZN

** Correlation is significant at the 0.01 level (2 tailed) * Correlation is significant at the 0.05 level (2 tailed) Green : significantly different in the 3 case groups Yellow : significantly different in DNP And NDHP group Red : significantly different in NDHP and DHP group Pink : significantly differently only in one case group CAT : Catalase , SOD : superoxide dismutase , GSH : Reduced glutathione MDA: Malondialdehyde, VC : Vitamin C , VE : vitamin E Se: serum selenium ion Fe : Serum Iron ion , Cu : Serum Copper ion Zn : Serum Zinc ion

Discussion Results from this study indicated that, the prevalence of hypertension and diabetes is highest in age groups 60-69 and 50-59 years, respectively. This is consistent with earlier studies in Ibadan-Nigeria (24), Bursa-Turkey (25), BeninNigeria (26) and Rajahmundry-India (27). Also it was observed that the prevalence of the diseases increased with age. Similar to the other researchers who reported that the severity of these diseases increase with age (28). For instance, in this study it was observed that hypertension which co-exists with diabetes in about 18% of patients at age 35 years rises to 20% at age 45 years and 28% at age 65 years. As age increases, the prevalence of hypertension in diabetes also increases. This was observed among the DHP group were the prevalence rate was observed to increase from 18.9% among young adults (20-39 years) to 34.6% among the old (60 79 years). This observation is similar to the findings of others researchers that say, hypertension which co-

exists with type 2 diabetes in about 40% at age 45 rises to 60% at age 75 years (29). On these basis someone diagnosed with type 2 diabetes in middle age (40 60 years) stands to lose as much as 10 years of their life expectancy (1,29-31). The risk of death among individuals with diabetes mellitus is almost twice that of individuals without diabetes of similar age (32). For patients diagnosed before the age of 40 years, the average reduction in life expectancy is 12 years for men and 19 years for women (32). Adults aged 65 to 74 years have the highest prevalence of diabetes mellitus; approximately 12 time the prevalence seen in adults younger than 45 years (32). The findings of this study confirmed that diabetes and hypertension are associated with increased oxidative stress, which results in higher serum concentration of lipid peroxidation products like MDA in serum. This is in agreement with the findings of others that, plasma MDA is increased in diabetes (33) and hypertension (34).

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Journal of Diabetology, February 2013; 1:2

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Table 3: Correlation of trace mineral elements with biomarkers of oxidation stress in diabetes (NDP group) CAT CAT SOD GSH MDA VC VE SE FE CU ZN 1 0.771 *** 0.520 ** - 0.465 ** 0.222 0.463 ** 0.272 0.450 ** -0.476 ** 0.777 ** 1 0.524 ** - 0.427 ** 0.221 0.417 ** 0.327 * 0.501 ** -0.358 * 0.584 ** 1 - 0.050 ** 0.189 0.562 ** 0.439 ** 0.498 ** - 0.384 ** 0.338 * 1 - 0.298 ** - 0.26 - 0.225 - 0.430 ** 0.182 -0.199 1 0.192 0.314 ** 0.249 0.315 * 0.269 1 0.668 ** 0.622 ** - 0.535 ** 0.590 ** 1 0.473 ** - 0.388 ** 0.486 ** 1 - 0.328 ** 0.427 ** 1 - 0.594 ** 1 SOD GSH MDA VC VE SE FE CU ZN

** Correlation is significant at the 0.01 level (2 tailed) * Correlation is significant at the 0.05 level (2 tailed) Green : significantly different in the 3 case groups Yellow : significantly different in DNP And NDHP group Red : significantly different in NDHP and DHP group Pink : significantly differently only in one case group CAT : Catalase , SOD : superoxide dismutase , MDA: Malondialdehyde, VC : Vitamin C , Se: serum selenium ion Fe : Serum Iron ion ,

GSH : Reduced glutathione VE : vitamin E Cu : Serum Copper ion Zn : Serum Zinc ion

Table 4: Correlation of trace mineral elements with biomarkers of oxidation stress in Hypertensive diabetes (NHP group) CAT CAT SOD GSH MDA VC VE SE FE CU ZN 1 0.780 ** 0.459 ** - 0.660 ** 0.524 ** 0.574 ** 0.610 ** 0.346 * - 0.295 * 0.517 ** 1 0.564 ** - 0.663 ** 0.596 ** 0.587 ** 0.617 ** 0.538 ** - 0.341 * 0.574 ** 1 - 0.630 ** 0.573 ** 0.496 ** 0.599 ** 0.595 ** - 0.302 ** 0.599 ** 1 - 0.599 ** -0.609 ** - 0.697 ** - 0.489 ** 0.199 - 0.414 ** 1 0.671 ** 0.657 ** 0.742 ** - 0.478 ** 0.410 ** 1 0.427 ** 0.526 ** - 0.500 ** 0.518 ** 1 0.551 ** - 0.310 * 0.398 ** 1 - 0.456 ** 0.507 ** 1 - 0.481 ** 1 SOD GSH MDA VC VE SE FE CU ZN

** Correlation is significant at the 0.01 level (2 tailed) * Correlation is significant at the 0.05 level (2 tailed) Green : significantly different in the 3 case groups Yellow : significantly different in DNP And NDHP group Red : significantly different in NDHP and DHP group Pink : significantly differently only in one case group CAT : Catalase , SOD : superoxide dismutase , GSH : Reduced glutathione MDA: Malondialdehyde, VC : Vitamin C , VE : vitamin E Se: serum selenium ion Fe : Serum Iron ion , Cu : Serum Copper ion
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Zn : Serum Zinc ion

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Journal of Diabetology, February 2013; 1:2 Also, the higher BMI in these diseases could be attributed to increased calorie intake which leads to overweight as observed in the hypertensive / diabetic hypertensive groups. This could possibly be due to sedentary life style, resulting from decreased exercises as the individuals increase in age (35). It has been reported that excessive use of alcohol can contribute to increase BMI (36). However, excessive use of alcohol cannot be acclaimed to the observed high BMI in this study, because use of alcohol is highly prohibited in the area of this study, and consumption of alcohol was one of the exclusion criteria in this study. Malondiadehyde is a highly toxic by product, produced in part by oxidation; derived from free lipid radicals, and studies have shown significantly raised concentrations in diabetes mellitus (37). MDA reacts both irreversibly and reversibly with proteins and phospholipids with profound effects. In this study highly statistically significant differences in the levels of MDA were observed in the cases. MDA was higher in case groups than in the control group; this is in agreement with the findings of Mahreen et al., (37) and Ozdemir et al., (38). The observed high levels of plasma MDA in case groups reflected lipid peroxidation as the consequence of oxidative stress. Non enzymic antioxidants such as GSH, vitamins C and E play an excellent role in protecting the cells from oxidative damage (39). It is well established that GSH in blood keeps up the cellular levels of the active forms of Vitamins C and E by neutralizing the free radicals. The decrease in the GSH level may be related to decreased activity of GPx; as indicated by its cofactor, Selenium (40). The decrease in the activity of catalase in patients with diabetes as compared to the control group is in agreement with the reports of many other researchers who reported decrease in antioxidant enzymes including catalase and an increase in lipid peroxidation (41,42). In erythrocytes, H2O2 is mainly detoxified by GPx and GR. When the activity of GPx decreases due to non-availability of NADPH and reduced glutathione (GSH), the second enzyme catalases rises and disposes off the H2O2. Kumar et al., (43) reported that due to decreased activity of GPx, there is compensatory increase in the catalase activity. However, increased production of hydrogen
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http://www.journalofdiabetology.org/ peroxide possibly led to the depletion of the catalase. Catalase, present in almost all mammalian cells, is responsible for the elimination of H2O2. The decrease in CAT activity could also result from inactivation by glycation of the enzyme. The reduced activities of SOD and CAT in this study are in agreement with what has been observed during diabetes as reported by other researchers (41, 42, 44). This observation may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxides (45). Increase in the products of lipid peroxidation (as indicated by the level of MDA) due to decreased activity of most of the antioxidant enzyme are in line with the reports of Ohtsuki et al., (46), Kumawat, et al., (47) and Salem et al., (48). The increase in the level of the MDA correlated with hyperglycemia in these patients because of auto oxidation of glucose, which causes the generation of free radicals. Hence complications of diabetes may be the result of this elevated level of free radicals (increase in the level of MDA) and the reduction in antioxidant defenses (GSH, GPx, GR and SOD). Conclusion Diabetes mellitus coexisted with hypertension in the patients included in this study and this was more prominent among the elderly. The link between these two conditions in the patients may be the increased oxidative stress due to alteration in the antioxidant defense system. Hence, that resulted in the pathophysiological conditions in uncontrolled diabetes as the individuals grow older. Acknowledgment The authors mention the following with profound gratitude for their immense contributions to the success of this research: The Management of ABUTH, Zaria; Prof. A. Mamma of the Department of Haematology, ABUTH; Prof. J. O. Ayo of Veterinary Pathology, ABU; Dr. J.E. El-Bashir of Chemical Pathology Unit, ABUTH; Dr. T.T. Wakama, National Hospital, Abuja; Dr. E. Akase and Dr. T. Nkoro of Endocrinology Unit, ABUTH; Dr. M.S. Isa of Cardiology Unit, ABUTH; finally, the Management of BioRapid Nigeria Diagnostics and SIRONigeria Global Ventures.

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