Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 17 Estimation of Superoxide Dismutase, Matrix-metalloprotinase-9, and Interleukin -18 in Patients with Type Two Diabetes Mellitus Shatha R. Moustafa *,1 and Shawn A. Omar ** * Academic faculty at College of Pharmacy, Hawler Medical University, Erbil, Iraq. ** Ministry of Heath, Erbil, Iraq. Abstract Antioxidant status imbalance and inflammatory process are cooperative events involved in type 2 diabetes mellitus. This study aimed to investigate superoxide dismutase as a potential biomarkers of antioxidant imbalance, matrix-metaloprotinase-9, and interleukin -18 as biomarkers of inflammation in serum and to estimate the effects of other confounding factors gender, age and finally measuring the relation among the interested biomarkers. This case - control study included 50 patients, and 45 of healthy subjects matched age – gender were also enrolled in this study as a control group. The focused parameters were measured using ELISA technique. There was significant reduction in the serum superoxide dismutase level and significant elevation in serum matrix- metaloptotinase-9 and interleukin -18 levels which have been associated with diabetes. This finding may explain the role of the defect in antioxidants status leading to significant reduction in serum superoxide dismutase levels associated with increased inflammatory process leading to significant elevations of a matrix- metaloptotinase-9 and interleukin -18. These parameters added a diagnostic information and evaluated as potential tools for disease risk prediction. Keywords: Type 2 diabetes mellitus, Superoxide dismutase, Matrix metaloptotinase-9 and Interleukin -18. لدى مزضي81 -واوتزلوكيه 9-ميتالوبزوتيىيزقياس صوبز اوكضايد دصميوتيز, ماتزكش الضكز مه الىوع الثاوي شذى رؤوف مصطفي ،*8 و شوان علي مصطفي ** * كلُت الصُذلت الطبُت ، اربُل ، العزاق . ، جاهعت هىلُز ** وسارة الصحت ، اربُل ، العزاق . الخالصة الخلل المائن فٍ هضاداث األكسذة والعولُت االلخهابُت هٍ أحذاد حشارن فٍ الٌىع الثاًٍ هي هزض السكزٌ. حهذف هذٍ بوثابت الوؤشزاث الحُىَت الوحخولت هي عذم الخىاسى الوضادة لألكسذة، ولُاس superoxide dismutaseالذراست الً لُاس metaloprotinase-9 وinterleukin -18 ؤشزاث حُىَت لاللخهاب، للخحمُك فٍ اِثار الوخزحبت علً غُزها هي العىاهل الجٌس كو والعوز وأخُزا الكشف عي هعاهل االرحباط بُي هذٍ الوعاَُز. هعافً سلُن هطابمت 50هزَضا، و 05هاث الوحذدٍ فٍ حطىر الوزض فٍ ىهذٍ الذراست هصووت لذراست االرحباط بُي الوعل .ELISAفٍ الجٌس والعوز للخحك فٍ هذٍ الذراست كوجوىعت ححكن. حن لُاس الوعلواث الوحذد باسخخذام حمٌُت فٍ هسخىي الوصل احصائًوارحفاع كبُز superoxide dismutase كاى هٌان اًخفاض احصائٍ هلحىظ فٍ هسخىي هصل الخٍ حزحبظ هع حطىر الوزض. interleukin -18و metaloptotinase-9للوعاَُز superoxide هذا االكخشاف لذ َفسز دور خلل فٍ وضع هضاداث األكسذة هوا َؤدٌ إلً اًخفاض كبُز فٍ هسخىي هصل dismutase ُزة فٍ فٍ الذم وسَادة عولُت االلخهاب الخٍ حؤدٌ إلً ارحفاعاث كبmetaloptotinase-9 وinterleukin -18 ٍهذ . الوعاَُز أضافج للوعلىهاث الخشخُصُت وَوكي اى حمُُن كأدواث هحخولت للخٌبؤ بوخاطز الوزض. . 81 -واوتزلوكيه 9-ماتزكش ميتالوبزوتيىيز ،صوبز اوكضايد دصميوتيز ،مزض الضكز مه الىوع الثاوي :الكلمات المفتاحية Introduction The pathogenesis of type 2 diabetes mellitus is associated with the induction of the OS that is induced by hyperglycemia (1) . High blood glucose level induces OS and decrease antioxidant defenses, thus leading to increase free radical (FR) formation so, there was as a significant difference between patients with T2DM and healthy control group concerning the total antioxidant capacity between T2DM patients and healthy control (2) . Several studies have reported the lower concentration of antioxidant enzymes in type 2 diabetes such as superoxide dismutase and glutathione peroxidase (3,4) . Superoxide dismutase (SOD) CuZnSOD EC 1.15.1.1 is one of the most important intracellular antioxidant enzymes (5) . 1 Corresponding author E-mail: shatha003@yahoo.com Received: 15/10/2016 Accepted: 14/3/2017 http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=blood+glucose http://scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=oxidative+stress http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=free+radical mailto:shatha003@yahoo.com Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 18 Matrix metalloproteinases (MMPs) are a family of zinc-binding proteolytic enzymes that were remodeling the extracellular matrix and then as the response of the inflammatory process pathologically attack the substrates (6) . Increased activity of MMPs has been reported in numerous disease processes. Hyperglycemia and DM enhance expression and activity of MMPs, MMP-9 has been investigated to be increased as a result of the stress hyperglycemia (7) . Interleukin -18 (IL-18) is a pro- inflammatory cytokine with multiple biologic functions. Elevated levels of IL-18 has been found in the serum of patients with T2DM (8) . From these information, it has been found that immunological and inflammatory processes have an important roles in an initiation and progression of the disease as the same time it was known that inflammatory process plays in etiologic and pathogenic mechanisms for T2DM. Because of impaired glucose metabolism leads to OS and there is an interrelationship between OS and inflammatory process so, this study was aimed to determine the association of the change in serum levels of SOD, MMP-9, and IL-18 with T2DM development and to assess other confounding risk factors age, gender and finally detect the relation between these studied parameters. Patients and Methods This case-control study was designed to examine the associations between the selected parameters with in T2DM patients that was performed at Hawler Medical University, College of Pharmacy on 50 patients of both genders who were randomly selected and newly diagnosed by specialist using clinical examination and confirmed by laboratory tests at diabetic center (Al-Shahida Layla Qassim) and after exclusion of other diseases by clinical history, laboratory investigations and clinical examination. In addition to 45 age – gender matched subjects have been also enrolled in this study as a control group who were also randomly selected. All participants completed the baseline questionnaire concerning several risk factors. This study has been done during the period between October 2013 to December 2014. The control group was confirmed to be healthy by clinical, biochemical and hematological examinations. All clinicopathological data of the patients have been collected from the clinical files. During the period of this study, the patients and the control groups were not taken anti-inflammatory drugs and antioxidants as dietary supplements. All procedures were in accordance by the established ethical standards. The protocol of this study has been approved by Ethics Committee of Medical Research at College of Pharmacy / Hawler Medical University. Oral consents have been taken from all the participants before starting the study. Sample collection Ten ml of the fasting blood samples have been collected and left for 30 minutes for coagulation purposes and then made centrifugation for 15 minutes at 2500- 3500 rpm. The sera of the participants have been separated and divided into several parts and put them into several plastic plain tubes for the biochemical analysis. The sera of the patients and control groups have been stored at (-80 Cº) at Medical Research Center /Hawler Medical University till the day of the analysis. The sera have been prepared for measurement by thawing the frozen sera at room temperature. The studied parameters have been estimated using enzyme - linked immunosorbent assay (ELISA) technique at Medical Research Center/ Hawler Medical University. Statistical analysis The statistical study has been done using the Statistical Patch for Social Sciences (SPSS vi.18). The results of biochemical tests were expressed as mean ± standard deviation (SD). Furthermore, student t-test was applied to compare between two means. A (p) value of ≤ 0.05 was considered as statistical significance. Correlations between laboratory findings and continuous variables were evaluated using linear regression analysis. Results Subjects characteristics The clinical characteristics of the patients and control groups have been shown in (Table 1). The patients group has been divided into two groups either as poor control and uncontrolled groups (35 patients with mean age 52.9 ± 7.8 and15 patients with mean age 63.3 ± 3 respectively) while the control group has been included 45 healthy adults with mean age was 55.9± 7.5. Effect of controlling the disease on the Serum levels of selected parameters Serum levels of HbA1C% and glucose were founded to be significantly higher in T2DM patients (uncontrolled group) as compared with poor control and healthy subjects (p< 0.001) (Table 1), the serum levels of MMp-9 and IL-18 were increased significantly in uncontrolled group T2DM as compared to poor control and healthy Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 19 individuals (p< 0.001), while the serum level of SOD was decreased significantly in uncontrolled T2DM as compared to poor control and healthy subjects (p< 0.001). Table ( 1): The demographic and clinical characteristics of the studied groups N Mean ±SD p Age (Years) No DM 45 55.933 7.500 < 0.001 Poor control 35 52.857 7.845 Un-control 15 63.267 3.011 Total 95 55.958 7.877 Weight (Kg) No DM 45 74.222 4.188 .168 Poor control 35 73.943 7.372 Un-control 15 70.400 11.319 Total 95 73.516 6.986 HbA1C % No DM 45 5.281 .183 < 0.001 Poor control 35 8.386 .323 Un-control 15 10.487 1.499 Total 95 7.247 2.097 FBG mg/dl No DM 45 92.311 6.338 < 0.001 Poor control 35 231.486 26.983 Un- control 15 297.133 42.963 Total 95 175.926 86.010 MMP-9 ng/ml No DM 45 1.506 .945 < 0.001 Poor control 35 1.804 .943 Un-control 15 4.563 1.669 Total 95 2.098 1.524 IL-18 pg/ml No DM 45 48.400 26.375 < 0.001 Poor control 35 84.240 17.581 Un-control 15 116.760 6.603 Total 95 72.398 32.953 SOD ug/ml No DM 45 501.447 162.781 < 0.001 Poor control 35 357.320 43.149 Un-control 15 171.867 51.409 Total 95 396.308 165.428 p< 0.05 Effect of the type 2 diabetes mellitus on the serum levels of selected parameters: The statistical study has been exhibited that the serum levels of HbA1C% ,glucose, MMp-9 and IL-18 were significantly increased in patients with T2DM as compared with the control group p˂ 0.001, while the serum level of SOD was significantly decreased in patients with T2DM as compared with the control group p˂ 0.001 (Table 2). Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 20 Table ( 2): Comparison between the studied groups regarding the focused parameters p< 0.05 Gender effect Concerning MMP-9, there was a significant difference between males and females in patients group p= 0.049 (Table3). Regarding SOD and IL-18 , there were no significant differences between men and women in patients group p > 0.05. While, in both studied groups, concerning MMP-9, there was a significant difference between males and females p=0.049 as well as there were no significant differences between men and women regarding HbA1C , glucose , SOD and IL-18 p > 0.05 (Table 4). Table (3): Comparison between males and females concerning the focused parametersin patient group Sex N Mean ±SD p MMP-9 ng/ml Men 25 2.512 1.840 .049 Women 25 1.861 1.070 IL-18 pg/ml Men 25 70.906 36.060 .665 Women 25 73.858 29.912 SOD ug/ml Men 25 367.226 128.068 .090 Women 25 424.785 192.370 p< 0.05 Table (4): Comparison between males and females concerning the focused parameters in both groups Sex N Mean SD P Age Years Men 47 55.936 8.641 0.979 Women 48 55.979 7.141 Weight Kg Men 47 74.213 6.504 0.339 Women 48 72.833 7.433 HbA1C % Men 47 7.359 2.348 0.609 Female 48 7.137 1.837 FBG mg/dl Men 47 176.532 90.680 0.946 Women 48 175.333 82.139 MMP-9 ng/ml Male 47 2.409 1.840 0.049 Women 48 1.794 1.070 IL-18 pg/ml Men 47 70.906 36.060 0.665 Women 48 73.858 29.912 SOD ug/ml Men 47 367.226 128.068 0.090 Women 48 424.785 192.370 p<0.005 Group N Mean ±SD SE p Age years patients 50 55.980 8.277 1.171 .977 Control 45 55.933 7.500 1.118 Weight kg patients 50 72.880 8.775 1.241 .337 Control 45 74.222 4.188 .624 HbA1C % patients 50 9.016 1.288 .182 < 0.001 Control 45 5.281 .183 .027 FBG Mg/dl patients 50 251.180 44.227 6.255 < 0.001 Control 45 92.311 6.338 .945 MMP-9 ng/ml patients 50 2.632 1.745 .247 < 0.001 Control 45 1.506 .945 .141 IL-18 pg/ml patients 50 93.996 21.297 3.012 < 0.001 Control 45 48.400 26.375 3.932 SOD ug patients 50 301.684 97.041 13.724 < 0.001 Control 45 501.447 162.781 24.266 Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 21 Age effect There was a significant negative weak correlation between age and SOD in patients group ( r= - 0.35 , p <0.05), as much as the age increase the serum level of SOD decrease, while there were a significant positive weak correlations between age and MMp-9 and IL- 18 ( r= 0.28 ,p<0.05 ) ( r=0.26 , p<0.05) respectively (Table 5). Table ( 5): The correlation between the age with SOD, MMP-9 and IL-18 in diabetic patients X variable Y variable R P value N Age SOD -0.35 <0.05 50 Age MMP-9 0.28 <0.05 50 Age IL-18 0.26 <0.05 50 Correlation coefficient Correlation coefficient between HbA1C and studied parameters There was a strong significant inverse correlation between HbA1C and SOD (p< 0.001) as much as HbA1C increase SOD decrease which showed more OS (r = - 0.88, p< 0.001). Moreover, there was a strong significant positive correlation between HbA1C and MMP-9 (r= 0.76, p< 0.001), as much as HbA1C increased the serum level of MMP-9 increased. In addition , there was a strong significant positive correlation between HbA1C and IL-18 (r= 0.76, p< 0.001), as much as HbA1C increased the serum level of IL-18 increased (Table 6). Table (6): The relation between HbA1C with SOD, MMP-9, IL-18 in diabetic patients Y variable X variable R P N HbA1C SOD - 0.88 <0.001 50 HbA1C MMP-9 0.76 <0.001 50 HbA1C IL-18 0.76 <0.001 50 Correlation coefficient between studied parameters in patients group. In patients group, there were a negative strong correlations between SOD with IL-18 and MMp-9 (r= - 0.89, p ˂0.001; r=-0.7, p ˂0.001) respectively. In addition, there was a positive strong correlations between MMP-9 with IL-18 ( r= 0.7, p ˂0.001) (Table 7). Table (7): Correlations between the focused parameters in the patients group X variable Y variable R P value N SOD IL-18 -0.89 <0.001 50 SOD MMP-9 -0.70 <0.001 50 IL-18 MMP-9 0.70 <0.001 50 Discussion T2DM and its complications are becoming one of the most important health problems in Erbil population. Therefore, it is necessary to confirm the involvement of novel pathogenic way to provide a good chance for early diagnosis and therapy. The current results has exhibited that antioxidant enzyme SOD and inflammatory markers MMP-9 and IL-18 are useful biomarkers for differentiating diabetic patients from healthy individuals. The results were in harmony with the hypothesis of the current study that, there were a statistical relationship between the significant decreased in the serum level of SOD and significant elevation of the serum levels of MMP-9 and IL-18 with development of T2DM. This study has been detected the existence of inflammation with reduction of antioxidant status in patients with T2DM. The results also suggested that the reduction of the serum antioxidant level may be presented consequence as increased OS associated with T2DM. Effect of controlling the disease on the serum levels of selected parameters In uncontrolled patients with T2DM, the serum levels of HbA1C%, glucose, MMP-9 and IL-18 were significantly increased as compared with poor control and healthy individuals (Table 1), while there was a significantly decreased in the serum SOD level in uncontrolled patients with T2DM as compared with poor control and healthy individuals (Table 1). The result of SOD has been exhibited that, OS is severe through uncontrolled stage of the disease which was concordant with the previous findings (4) . Effect of the Type 2 diabetes mellitus on the serum levels of selected parameters The statistical study has been revealed that there was a significant decrease in the serum SOD level in patients with T2DM as compared with the control group (Table 2). It has been published (3) that hyperglycemia may interfere with the defensive mechanism antioxidant enzymes which induces the generation of free radicals (FRs). The statistical decrease in the serum SOD level which was significantly related with Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 22 the development of T2DM and might be used as a necessary biomarker for diagnosis T2DM. The current study has been shown an elevated OS as a consequence of overproduction of FRs in patients with T2DM concomitant with the significant decreased in antioxidant enzymes such as SOD which might be associated to the etiology of T2DM and has been used as an important parameters of antioxidant status imbalance and has an significant role in the occurrence of T2DM. The result of the present study was concordant with the previous findings (3,4) . The explanation for the reduction of antioxidant levels in patients with T2DM because of the that antioxidant enzymes may have been diminished as an attempt to counteract the lipid DNA, protein and lipid damage. Moreover, The increased lipid, protein and DNA oxidation may be as a consequence of a weakened antioxidant defensive system (9) , SOD is considered the first defensive mechanism line against reactive oxygen species (ROS), and it is thought that the activity of SOD might be affected firstly before other antioxidant enzymes as a result of OS (10) . Damaged the activity of SOD is due to over production of superoxide –free radicle in addition to the increased of other ROS and induces lipid peroxidation processes in diabetes (4) , moreover, hyperglycemia is associated with loss of Cu 2+ , which is considered as an important cofactor in SOD activity as well as the activity of SOD is diminished by glycosylation in erythrocytes (10) . The current result indicated that there was a statistical increase in the serum MMP-9 level in patients with T2DM as compared with the control group (Table 2). The result of the present study was consistent with the previous findings (6, 8-13) . The current study also focused the important role of MMP-9 to predict the development of T2DM, in addition, supported the relation between the statistical increase of the serum MMP-9 level with development of the T2DM. The explanation for this significant elevation in the serum level of MMP-9 in patients with T2DM was that, high blood glucose level directly or indirectly as a consequence of OS and increase glycosylation of products like lipid or protein might elevate the level of MMP-9 and it , s activity in large blood vessels (14) , moreover, OS may has a necessary role for the pathogenesis of T2DM as well as MMP-9 is activated as a consequence of the FRs generation, which emphasize that antioxidants have a beneficial effects in T2DM therapy. It is widely accepted that T2DM is related with low grade chronic inflammation (15) . So, increased inflammation is considered as non-dependent risk factor for the development T2DM (16) . This study was designed to detect the IL-18 as an inflammatory diagnostic parameter for T2DM. So, this study emphasized that inflammation is associated with increased level of IL-18 by high blood glucose level. There was a statistical increase in the serum IL-18 level as compared with healthy individuals (Table 2) which was in harmony with the previous studies (8,17,18,) . According to the current study that, IL-18 might be associated with the pathogenesis of the T2DM as well as the data supported the role of the inflammatory process in the pathophysiology of T2DM. So, this result supported the findings of the previous researches that T2DM might be considered as a low - grade chronic inflammatory status. So, the high serum IL- 18 level might expect the development of T2DM and there was a relation between a significant elevation of the serum IL-18 level with the development of T2DM. Gender –effect Concerning MMP-9, there was a significant difference between males and females in patients group (Table3). Regarding SOD and IL-18, there were no significant differences between men and women in patients group (Table3). While, in both studied groups, concerning MMP-9, there was a significant difference between males and females as well as there were no significant differences between men and women regarding HbA1C, glucose, SOD and IL-18 (Table 4). It has been published (4) and illustrated the association between genders and oxidant / antioxidant state, it has been recognized that, the SOD was increased in non-diabetic healthy men compared with non-diabetic healthy women, while in diabetic state, SOD concentration was slightly reduced but there was no statistical significant difference between diabetic men and diabetic women, so the study (4) , has been shown that diabetic patients regardless of the gender, were exposed to an elevated levels of ROS through enhanced lipid peroxidation. Age - factor Effect of aging on glucose serum level in type 2 diabetes mellitus T2DM is a common burden in the elderly (19) . Aging is associated with alterations in body composition, which has implications for the development of insulin resistance and diabetes. These information supported the result of the current study that Iraqi J Pharm Sci, Vol.26(1) 2017 Antioxidant and inflammation in type II diabetes mellitus 23 elderly individuals are at a high risk to develop T2DM. The average age of patients in the current study at diagnosis was 55.98± 8.277years (Table 2). Moreover, the fining of the current study was in harmony with the result of the previous research (20) which has been reported that, the patients with T2DM group with the average age (60-70 years) their mean blood glucose level was 173.4±54.2 mg/dl. Accordingly, there was an association between old age and T2DM. In addition, other study (21) has been emphasized that an elevated incidence of this disease in elderly subjects might be as result of the variations in the homeostatic pathway that regulates the inflammatory parameters such as C- reactive protein (CRP), IL-6, tumor necrosis factor-α (TNF-α) which are associated with the chronic low grade inflammation occurs in T2DM. Effect of aging on studied parameters in type 2 diabetes mellitus There was a significant negative weak correlation between age and SOD in patients group, as much as the age increase the serum level of SOD decrease, while there were a significant positive weak correlations between age and MMp-9 and IL-18 (Table 5). Similarly, (4) reported that, SOD lowers in diabetic age group ≥50 years compared with age groups 30–39 and 40–49 years. So, the results of (4) indicated that OS impacts the aging process that may be caused by a number of factors including increased free radical production, decreased antioxidant defense system, or a decreased removal or repair. Correlation coefficient The current study reinforced the possibility that high blood glucose level might be associated with elevated level of MMP-9 and HbA1C (Table 6). There was a positive strong statistical relationship between HbA1C and MMP-9 when the serum activity of MMP- 9 was increased the amount of HbA1C was also increased. This result was consistent with the finding of the previous research (22). Furthermore, the current result reinforced the possibility that high blood glucose might be associated with reduction in the level of SOD. Table 6 shows, that there was a strong significant negative correlation between HbA1C and SOD, as much as HbA1C increase SOD decrease which showed more OS, this result was shown that antioxidant status are linked with glycemic control, which was consistent with previous result (23). Indeed, there was a significant interaction between elevated glucose and serum IL-18 level. Data from the current study reinforced the possibility that high blood glucose level might be associated with elevated concentration of IL-18, the data has been shown a positive strong statistical relation between HbA1C and IL-18, when the serum activity of IL-18 was increased the amount of HbA1C was also increased. In addition, in patients group, there were a negative strong correlations between SOD with IL-18 and MMp-9. In addition, there was a positive strong correlations between MMP-9 with IL- 18 (Table7). The current study was the first study in Erbil population that investigated the combined effects of antioxidant status by investigating SOD and inflammation by measuring MMP-9 and IL-18 markers in T2DM risk prediction. Variations in the serum concentrations of these biomarkers associated with occurrence of T2DM and it is important to supply diagnostic information which might help in the management and therapy of T2DM. Conclusion The results of the current study detected a statistical decrease in the activity of the SOD in patients with T2DM, accordingly, it was concluded that, T2DM is associated with overproduction of FRs as a consequence of hyperglycemia as well as decreased the activity of the antioxidant enzymes like SOD, which emphasized to supply the therapeutics with antioxidant products. Measuring SOD might be used a parameter to regulate the blood glucose level. In addition, the statistical increase in the concentrations of the MMp-9 and Il-18 as an inflammatory markers emphasized the role of inflammatory process in T2DM risk prediction. Acknowledgments Deep thanks and appreciation to Prof. Dr. Namir Al-Tawil for performing the statistical analysis. References 1. Román-Pintos LM, Villegas-Rivera G, Rodríguez-Carrizalez AD, Miranda-Díaz AG, Cardona-Muñoz EG. Diabetic Polyneuropathy in Type 2 Diabetes Mellitus: Inflammation, Oxidative Stress, and Mitochondrial Function. 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