IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Evaluation of α1-Antitrypsine and Reduced Glutathione in Iraqi Patients of Diabetes Mellitus Type II. Received in : May , 9, 2010 Accepted in : June , 30, 2010 A. F. Al-Tai, , * Y. Y. Z. Farid and W.F. AL- Tai Department of Chemistry, College of Education Ibn AL-Haitham, University of Baghdad * College of Medicine AL-Nahrain, University of AL-Nahrain Abstract In order to investigate the levels of reduced glutathione GSH and α1-antitrypsine in the sera of 20 type 2 diabetic patients and 10 healthy subjects, were enrolled in this study. A significant reduction in GSH level was found in the patient group compared with control. On the other hand a significant elevation in α1-antitrypsine in patient compared with control was observed. Correlation between α1-antitrypsine and reduced glutathion was found to be positive (+Ve) for diabetes mellitus type2 patients and negative (-Ve) for healthy control with r values 0.257 and – 0.339 respectively. In conclusion the depletion of GSH as antioxidant defense insured higher free radical generation in diabetic patients which is conformed by the high α1-antitrypsine level in the sera. Key words: α1-Antitrypsine , Glutathione , Diabetes Mellitus Type 2 Introduction Diabetes mellitus DM is a group of metabolic disorders of carbohydrate metabolism in which glucose in underutilized by the body tissue, producing hyperglycemia. Some patients may develop life threatening conditions like keto-acidosis and coma.[1] Over the last decade, there has been a significant interest in oxidative stress and its role in the development of complications in diabetic patients.[2] Glutathione (GSH) (γ-glutamyl-cysteinyl glycine) is a tripeptide consisting of glutamic acid, cysteine and glycine. A number of potentially toxic electrophilic xenobiotics are conjugated to the nucleophilic GSH in reactions that can be represented as follows: 2R + 2GSH → 2R – S – G +H2 Where R= an electrophilic xenobiotic If the potentially toxic xenobioties were not conjugated to GSH they would be free to combine covalently with DNA, RNA or cell protein and could thus lead to serious cell damage.[2] GSH has other important functions in human cells apart from its role in xenobiotic metabolism. It participates in the decomposition of potentially toxic hydrogen peroxide in the reaction catalyzed by glutathione peroxidase, it is an important intracellular reductant helping to maintain essential SH groups of enzymes in their reduced state. A metabolic cycle involving GSH as a carrier has been implicated in the transport of certain amino acid across membranes in the kidney.[3] IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Alpha 1-antitrypsine is among positive acute phase proteins (Apps) which is a major pathophysiologic phenomenon that accompanies inflammation either acute or chronic[4], the release of inflammatory molecules (cytokines) changes the level of Apps as well as a number of behavioral, physiologic, biochemical and nutritional changes are induced.[5] The aim of the present study is to evaluate the levels of GSH as a major endogenous antioxidant and α1-antitrypsine as a reactant modulated during inflammatory diseases in the sera of type 2 DM Iraqi patients and to correlate both parameters. Experimental Subjects: The study group comprised 20 patients from both sexes with type 2 DM diagnosed by physicians at AL-Kadhymia teaching hospital in addition to ten healthy control matching the age. Blood samples: About five milliliters of venous blood were collected from each subject in the study after a 12-hour fast. The blood samples were collected into blastic tubes, left at room temperature for 15 min then centrifuged at 3000 rpm for 15 min. Serum was separated and aliquoted for subsequent measurement of GSH and α1-antitrypsine.. Laboratory methods: Determination of glutathion concentration GSH. Reduced glutathione was determined according to the method of Ellman[6], based on the reaction of aliphatic thiol compounds with 5,5-dithiobis (2-nitrobenzoic acid)(DTNB) at pH8. The absorbance of the yellow chromagen was measured at 412 nm and is directly proportional to GSH concentration. So one mole of the thiol produces one mole of p-nitro thiophenol anion which is highly coloured (ε=13600 M -1 cm -1 ) Determination of α1-antitrypsine: Alpha one antitrypsine was measured in the sera of patients and healthy control groups using a ready kit from Bindarid UK RNO 34.3. The method invaders antigen diffusing radially from a cylindrical well through an agarose gel containing an appropriate mono-specific antibody. Antibody- Antigen- complexes are formed which, under the right conditions, will form a precipitin ring. The ring size will increase until equilibrium is reached between the formation and breakdown of these complexes, this point is termed completion. At this stage, a linear relationship exists between the square of this ring diameter and the antigen concentration. By measuring the ring diameters produced by a number of samples of known concentration, a calibration curve may be constructed. The concentration of the antigen in an unknown sample may then be determined by measuring the ring diameter produced by that sample and reading off the calibration curve[7]. Statistical analysis: Data are expressed as mean standard deviation of (mean ±S.D.). Statistical significance was determined by unpaired student's, t-test. One way of analysis of variance (ANOVA) is followed by Pearson's correlation (r). P values equal or lower than 0.05 were considered statistically significant. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Results and Discussion Reduced glutathione GSH: Table1 shows the level mean ±SD of GSH in the serum of type 2 DM patient and healthy control, a significant decrease in GSH level in the serum of type 2 DM patient compared with the control was found with values 1.092 ± 0.204 M and 3.4 ± 0.529 M respectively. These results are in agreement with recent study reported that a significant decrease in GSH level in patient with type 2 DM [8]. Reduced glutathione is physiological free radical scavengers.Thus glutathione plays a central role in antioxidant defense [9]. In hyperglycemic condition, glucose is preferentially used in the polyol pathway that consumes NADPH which is necessary for GSH regeneration by the glutathione reductase enzyme [10]. Many reportes showed that diabetic humans have shown increased lipid peroxidation and decreased levels of reduced glutathione and these results suggest that the increase in lipid peroxidation, and the decline in antioxidant defences may appear early in type 2 non-insulin dependent diabetes mellitus patients, before the development of secondary complications [11,12]. α1-antitrypsine: A significant increase in α1-antitrypsine levels was found between DM patients which was 1877.11 ± 222.889 mg/L compared with healthy control which was 950.00 ± 104.88 mg/L The results of the present study is in agreement with reported data claimed an increase in the levels of acute-phase protein including α1-antitrypsine in adult diabetes (principally type (2)) [13], also results proposed a novel biological function for α1-antitrypsine and suggest that it may represent an effective candidate for attempts seeking to prevent or reverse type 1 diabetes [14]. On the other , a study is on α1-antitrypsine in diabetes in both. Type I and II diabetes mellitus are associated with reduced α1-antitrypsine as serine protease inhibitory capacity of plasma [15]. Fig (1) shows correlation relation between α1-antitrypsine and reduced glutathione for DM type II patients and healthy control. The correlation was found to be (+Ve) for DMII patients and (-Ve) for healthy control with r values 0.257 and -0.339 respectively. References 1 Shiranda, N.B. (2008), "Manipal Manual of clinical biochemistry", New Delhi; 3rd edition, Jaypee brothers medical publishers (P) LTD, p70. 2 Shweta, B.; Rimi, S. and sriv, (2003) "clinical biochemistry V.36: Issue 7. 3 Robert, K.; Murray,; Daryl, K. Granner and Victor, W. Rodwell (2006) "Harper's Illustrated biochemistry" 27 th edition. Mc Graw Hill, New York Chicago, p636. 4 Gabay, C. and Kushner, I. (1999) Acute phase proteins and other systemic responses to inflammation- N Engl J" Med. V. 340 :Issue 448. 5 Liyp, Schwartz, R.J. and Waddell, I.D. (1998), Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-Kappa B activation in response to tumor necrosis factor alpha. FASEB J:12:871. 6 Ellman, G.L. (1959): Arch-biochem. Biophys 82:70-77. 7 Protein Reference unit hand book of clinical Immuno chemistry. (1990), Ed, Amilford IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 word, publ, publ. PRU publication sheffie. D.63-65. 8 Nwcke, N.; Cohari, C. Obioma and Cezeala, C. Chinyere. (2009) " Effect of vitamin on malondialdehyde and glutathione levels in type 2 diabetic Nigerians, Internet Journal of pharmacology volume 7 Number1. 9 Lee Ay and Chung, S.S; (1999); "Contrrbutrons of polyol pathway to oxidative stress in diabetrc cataract". FASEB J.; 13:23-30. 10 Paolisso, G. D.; Maro, D. and Pizza, G. D. (1992) Plasma GSH/GSSH affects glucose none ostasis in healthy subjects and non-insulin dependent diabetics. American Journal of physiology; 263:435-440. 11 Sailaja, Y.R.; Baskar, R. and Saralaku mari, D. (2003) The antioxidant erythrocytes in type2 diabeties free radical Biol Med ; 35:133-139. 12 Mahbob, M.; Rahman, M. F. and Grover, P. (2005) Serum lipid peroxidation and antioxidant enzyme levels in male and female diabetic patients Singapore. Med J; 46(7):322. 13 Millon, M.C. D.E. (1989); increased levels of acute phase serum proteins in diabetes; pub Med J. 38(II): 1042-1046. 14 Bin ,Z.; Ymanging, L.U. and Marth compbell; (2007) Alpha 1 antitrypsin protects β-cells from Apoptosis; published online March 14. 15 Hashemi, M.; Naderi. M. and Rashidi (2007) Diabetes Res; clinical pract; 75(2):246-248. Table(1): Shows the level, mean and SD of reduced glutathione in sera of type 2 DM patient and healthy control GSH M N mean SD P value Patient 20 1.092 ± 0.204 Control 10 3.400 ± 0.529 S: significant Table(2): Shows the level, mean and SD of α1 –antitrypsine in the sera of type 2 DM patient and healthy control α1 –antitrypsin mg/L N mean SD P value Patient 20 1877.111 ± 222.889 Control 10 950.000 ± 104.881 S: significant IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L.24 (1) 2011 Fig.(1): The correlation relation between α1 –antitrypsine and reduced glutathione for DM 2 patints and healthy control. 2011) 1( 24المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة للمرضى العراقیین α1 –antitrypsin GSHتقدیر الكلوتاثیون المختزل المصابین بداء السكري من النوع الثاني 2010 ایار 9: استلم البحث في 2010 حزیران 30 :قبل البحث في یحیى یحیى زكي فرید ، أنوار فاروق مسلم الطائي * وفاء فاضل الطائي ، جامعة بغداد ،ابن الھیثم –كلیة التربیة ، قسم الكیمیاء ن جامعة النھری، كلیة الطب الخالصة ــزل ( لدراسـة مسـتویات اثیون المخت فـي مصــل α1 –antitrypsine بوصـفھم والمضـاد للتـربین مــن GSHالكلوـت وع الثـاني ینعشـر ن الـن داء السـكري ـم ن االصـحاء فضـال عــن مصـاباً ـب ي ھــذه ،اذمجموعـة سـیطرة بوصـفھم عشـرة ـم وجـد ـف ــزل ــاثیون المخت ــز لمســتوى الكلوت ــة مــع االصــحاء GSHالدراسـة نقصــان ممی ــاني مقارن .لمرضــى داء الســكري مــن النــوع الث ع α1 –antitrypsineوفي الجانب اآلخر لوحظت زیادة ممیـزة لمسـتوى ك المرضـى بالمقارنـة ـم ن أألولـئ نظـارھم ـم .االصحاء وع الثـاني α1 –antitrypsineلوحظت العالقة بین ن الـن زل لمرضـى داء السـكري ـم -)و (Ve+)والكلوتاثیون المخـت Ve) لالصحاء وكانت قیمة(r) على التوالي) 0,337-(و ) 0,257(مساویة اـ. اثیون المختـزل ق الجـذور الحـرة بنسـبة GSHوقد لوحظ من النتائج ان اسـتھالك الكوـت الحـامي ضـد االكسـدة یؤكـد تخلـی .المصابین في مصل α1 –antitrypsineي عالیة لدى المرضى الذي یكون مطابقاً للمستوى العال ،الكلوتاثایون ، داء السكري النوع الثاني α1مضاد التربسین نوع : الكلمات المفتاحیة