IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Evaluation o f α1-Antitrypsine and Reduced Glutathione in Iraqi Patients of Diabetes Mellitus Type II. Received in : May , 9, 2010 Accepte d in : June , 30, 2010 A. F. Al-Tai, , * Y. Y. Z. Farid and W.F. AL- Tai Departme nt of Chemistry, College of Education Ibn AL-Haitham, Unive rsity of Baghdad * College of Medicine AL-Nahrain, Unive rsity of AL-Nahrain Abstract In order to invest igate the lev els of reduced glutathione GSH and α1- antitrypsine in the sera of 20 type 2 diabetic p atients and 10 healthy subjects, were enrolled in this st udy . A significant reduction in G SH level was found in the patient group comp ared with control. On the other hand a significant elevation in α1-antitryp sine in p atient comp ared with control was observed. Correlation between α1-antitryp sine and reduced glutathion was found to be p ositive (+Ve) for diab etes mellitus typ e2 p atients and negative (- Ve) for healthy control with r valu es 0.257 and – 0.339 resp ectively. In conclusion the depletion of GSH as antio xidant defense insured high er free radical generation in diabetic p atients which is conformed by the high α1-antitryp sine level in the sera. Key words: α1-Antitrypsine , Glutathione , Diabete s Mellitus Type 2 Introduction Diabetes mellitus DM is a group of metabolic d isorders of carbohy drate metabolism in which glucose in underutilized by the body tissue, p roducing hyp erglycemia. Some p atients may develop life threatening conditions like keto-acidosis and coma.[1] Over the last decade, there has be en a si gn ificant interest in oxidative stress and its role in the develop ment of comp lications in diabetic p atients.[2] Glutathione (GSH) (γ-glutamyl-cy steiny l glycine) is a tripep tide consisting of glutamic acid, cy st eine and glycine. A number of p otentially toxic electrop hilic xenobiotics are con jugated to the nucleophilic GSH in reactions that can be represented as follows: 2R + 2GSH → 2R – S – G +H2 Where R= an electrop hilic xenobiotic If the p otentially toxic xenobioties were not conju gated to GSH they would be free to combine covalently with DNA, RNA or cell p rotein and could thus lead to serious cell damage.[2] GSH has other imp ortant functions in human cells apart from its role in xenobiotic metabolism. It p articip ates in the decomposition of p otentially toxic hy drogen p eroxide in the reaction cataly zed by glutathione p eroxidase, it is an imp ortant intracellular reductant help ing to maintain essential SH group s of enzy mes in their reduced st ate. A metabolic cycle involving GSH as a carrier has been imp licated in the transp ort of certain amino acid across membran es in the kidney.[3] IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Alp ha 1-antitryp sine is among p ositive acute p hase p roteins (Apps) which is a major p athop hysiologic p henomenon that accomp anies inflammation either acute or chronic[4], the release of inflammatory molecules (cytokines) changes the level of App s as well as a number of behavioral, p hysiologic, biochemical and nutritional changes are induced.[5] The aim of the p resent study is to evaluate the levels of GSH as a major endogenous antioxidant and α1-antitryp sine as a reactant modulated during inflammatory diseases in the sera of type 2 DM Iraqi p atients and to correlate both p arameters. Experime ntal Subjects: The study group comp rised 20 p atients from both sexes with type 2 DM diagnosed by p hy sicians at AL-Kadhy mia teaching hosp ital in addition to t en healthy control matching the age. Blood sample s: 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 blast ic tubes, left at room temperature for 15 min then centrifuged at 3000 rp m for 15 min. Serum was sep arated and aliquoted for subsequent measurement of GSH and α1-antitryp sine.. La boratory methods: De termination of glutathi on concentration GS H. Reduced glutathione was determined accordin g to the method of Ellman[6], based on the reaction of aliphatic thiol compounds with 5,5-dithiobis (2-nitrobenzoic acid)(DTNB) at p H8. The absorbance of the y ellow chroma gen was measured at 412 nm and is directly p rop ortional to GSH concentration. So one mole of the thiol p roduces one mole of p -nitro thiophenol anion which is hi ghly coloured (ε=13600 M -1 cm-1) De termination of α1-antitrypsi ne: Alp ha one antitrypsine was measured in the ser a of p atients and healthy control group s 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 ap p rop riate mono-sp ecific antibody. Ant ibody - Antigen- comp lexes are formed which, under the right conditions, will form a p recipitin ring. The rin g size will incr ease until equilibrium is reached between the formation and breakdown of these comp lexes, this p oint is termed comp letion. At this st age, a linear relationship exists between the square of this ring diameter and the anti gen con centration. By measuring the ring d iameters p roduced by a number of sa mples 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 p roduced by that sample and readin g off the calibr ation curve[7]. S tatistical anal ysis: Data are exp ressed as mean st andard deviation of (mean ±S.D.). Statist ical significance was determined by unp aired st udent'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 st atist ically significant. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Results and Discussion Reduced glutathione GS H: Table1 shows the level mean ±SD of G SH in the serum of ty p e 2 DM p atient and healthy control, a significant decrease in G SH level in the serum of ty p e 2 DM p atient comp ared with the control was found with values 1.092 ± 0.204 M and 3.4 ± 0.529 M resp ectively. These results are in agreement with recent st udy rep orted that a significant decrease in GSH lev el in p atient with type 2 DM [8]. Reduced glutathione is p hysiological free r adical sc aven gers.Thus glutathione p lay s a central role in antio xidant defense [9]. In hy p erglycemic condition, glucose is p referentially used in the p olyol pathway that consumes NADPH which is necessary for GSH regeneration by the glutathione reductase enzy me [10]. M any reportes showed that diabetic humans have shown increased lip id peroxidation and decreased levels of reduced glutathione and these results suggest that the increase in lip id p eroxidation, and the d eclin e in antio xidant defen ces may app ear early in typ e 2 non-insulin dependent diabetes mellitus p atients, before the develop ment of secondary comp lications [11,12]. α1-anti trypsine : A significant increase in α1- antitrypsine levels was found between DM p atients which was 1877.11 ± 222.889 mg/L compared with healthy control which was 950.00 ± 104.88 mg/L The results of t he present study is in agreement with rep orted data claimed an in crease in the levels of acute-phase protein includin g α1-antitryp sine in adult diabetes (p rincipally type (2)) [13], also results prop osed a novel biological function for α1-antitrypsine and suggest that it may represent an effective candidate for att empts seeking to p revent or reverse type 1 diabetes [14]. On t he other , a study is on α1-antitryp sine in diabetes in both. Type I and II diabetes mellitus are associated with reduced α1-antitrypsine as serine p rotease inhibitory cap acity of p lasma [15]. Fig (1) shows correlation relation between α1-antitryp sine and reduced glutathione for DM type II p atients and healthy control. The correlation was found to be (+Ve) for DM II p atients and (-Ve) for healthy control with r values 0.257 and -0.339 resp ectively. Re ferences 1 Shirand a, N.B. (2008), "M anip al M anual of clinical biochemistry", New Delhi; 3 rd edition, Jaypee brothers medical p ublishers (P) LTD, p 70. 2 Shweta, B.; Rimi, S. and sriv, (2003) "clinical biochemistry V.36: Issue 7. 3 Robert, K.; M urray,; Dary l, K. Granner and Victor, W. Rodwell (2006) "Harp er's Illust rated biochemistry " 27 th edition. M c Graw Hill, New York Chicago, p 636. 4 Gabay, C. and Kushner, I. (1999) Acute p hase p roteins and other systemic resp onses t o inflammation- N En gl J" M ed. V. 340 :Issue 448. 5 Liy p , Schwartz , R.J. and Waddell, I.D. (1998), Skeletal muscle myocytes undergo p rotein loss and reactive o xy gen-mediated NF-Kapp a B activation in resp onse to tumor necrosis factor alp ha. FASEB J:12:871. 6 Ellman, G.L. (1959): Arch-bio chem. Biophys 82:70-77. 7 Prot ein Reference unit hand book of clinical Immuno chemistry . (1990), Ed, Amilford IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 word, p ubl, p ubl. PRU p ublication sheffie. D.63-65. 8 Nwcke, N.; Cohari, C. Obioma and Cezeala, C. Ch inyere. (2009) " Effect of vitamin on malondialdehy de and glutathione levels in ty p e 2 diabetic Nigerians, Internet Journal of p harmacology volume 7 Number1. 9 Lee Ay and Chung, S. S; (1999); "Contrrbutrons of p olyol pathway to oxidative st ress in diabetrc cataract". FASEB J.; 13:23-30. 10 Paolisso, G. D.; M aro, D. and Pizza, G. D. (1992) Plasma GSH/GSSH affects glucose none ostasis in healthy subjects and non-insulin dependent diabetics. American Journal of p hy siology ; 263:435-440. 11 Sailaja, Y.R.; Baskar, R. and Saralaku mari, D. (2003) The antioxidant erythrocytes in type2 diabeties free rad ical Biol M ed ; 35:133-139. 12 M ahbob, M.; Rahman, M . F. and Grover, P. (2005) Serum lip id p eroxidation and antioxidant enzy me levels in male and female diabetic p atients Singapore. M ed J; 46(7):322. 13 M illon, M .C. D.E. (1989); increased levels of acute p hase serum p roteins in diabetes; p ub M ed J. 38(II): 1042-1046. 14 Bin ,Z.; Yman ging, L.U. and M arth compbell; (2007) Alp ha 1 antitryp sin p rotects β-cells from Ap optosis; p ublished online M arch 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 se ra of type 2 DM patient and healthy control GS H M N mean SD P value Patie nt 20 1.092 ± 0.204 Control 10 3.400 ± 0.529 S : si gni ficant Table(2): Shows the level, mean and SD of α1 –antitrypsi ne in the se ra of type 2 DM patient and healthy control α1 –anti trypsin mg/L N mean SD P value Patie nt 20 1877.111 ± 222.889 Control 10 950.000 ± 104.881 S : si gni ficant IBN AL- HAITHAM J. FO R PURE & APPL. SCI. VO L.24 (1) 2011 Fig.(1): The correl ation relation between α1 –antitrypsi ne 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 α1 –antitry بوصـفھم والمضـاد للتـربین مــن GSHالكلوـت p sine ــل فـي مص وع الثـاني ینعشـر ن الـن داء السـكري ـم ن االصـحاء فضـال عــن مصـاباً ـب ي ھــذه ،اذمجموعـة سـیطرة بوصـفھم عشـرة ـم وجـد ـف ــزل ــز لمســتوى الكلوتــاثیون المخت ــان ممی ــحاء GSHالدراسـة نقص ــة مــع االص ــاني مقارن ــن النــوع الث ــكري م ــى داء الس .لمرض α1 –antitryوفي الجانب اآلخر لوحظت زیادة ممیـزة لمسـتوى p sine ع ك المرضـى بالمقارنـة ـم ن أألولـئ نظـارھم ـم .االصحاء α1 –antitryلوحظت العالقة بین p sine وع الثـاني 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 α1 –antitryي عالیة لدى المرضى الذي یكون مطابقاً للمستوى العال p sine المصابین في مصل. ،الكلوتاثایون ، داء السكري النوع الثاني α1مضاد التربسین نوع : الكلمات المفتاحیة