IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Comparative Biochemical Study of Glutathione, Ceruloplasmin and Trace Element in Sera of Control Group and Human Female Patients with Osteoarthritis Nodal in Iraqies Patients B. H.Al – Wihaly Departme nt of Chemistry, College of Education Ibn-Al Haitham,Unive rsity of Baghdad Abstract The p resent st udy conducted on 30 female p atients with ost eoarthritis 0A a att ending Baghdad teaching hosp ital, in addition to 30 healthy females , all subjects were ( 35-65) years old. Some biochemical p arameters were measured in the sera of p atients and healthy group s. T he parameters were Glutathione (GSH). Cerulop lasmin (Cp ) and some trace elements ,including Cop p er (Cu) ,Cu/ Cp ratio and Selenium (Se) were determined . The results revealed a significant reduction in all p arameters of p atients sera comp ared to healthy group . The reduction in GSH and Cu/Cp ratio confirms tissue damage associated with oxidative st ress injury A conclusion was obtained hrer ,that Cu wasn’t an imp ortant element in oxidative st ress in p atients . Introduction Osteoarthritis (OA) is a slow p rogressive disorder of sy novial joints .This joints disorder is characterized by a loss of balance between sy nthesis and degradation of the articular cartilage constituents leading to subsequent erosion of joint cartilage remodeling of the underly ing bone oseop hy te formation and variable degree of sy nop itis [1].When clinical characterist ics of OA (p ain loss of mobility and radiographic narrowing of the joint sp ace ) manifest , the actual changes in articular cartage and subchondrla ,bone have st arted long ago[2] Glutathione (γ-glutamyl cy st einy l glycin) is atrip eptide commonly abbreviated GSH. It is the most common intracellular thiol which is found in most mammalian cells as an important defense mechanism against certain toxic comp ounds such as some drugs and carcinogens. If these toxic electrop hiles (xenobiotics) were not conjugated to GSH, they would be free to combine covalently with DNA, RNA, or cell p rotein and could thus lead to serious cell damage [3]. If the levels of GSH in a tissue are lowered than that tissue can be shown to be more suscep tible to injury by various chemicals t hat would normally be conjugated to GSH [4]. Cerulop lasmin (Cp ) is an alp ha 2 _ glycoprotein with enzy matic activity containing six or seven cop p er atom per molecule ‘ thus Cp considered as antioxidant through its ferroxidase activity by p lay ing a role in the oxidation of ferrous ion and hence releases from cells and loading on to apotransferrin binds largely the ferric form , so Cp is involved p rimarily in maintaining iron homeostasis and p reventing iron mediated free radicals injury [5,6]. Cop p er is an essential trace element p resent as an integral comp onent of many IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 metalloenzy mes involved in oxidation reduction reactions Cop p er is necessary for the formation of the blood cells and connective tissue [7]. Selenium (Se) is an essential element for a human , help s to defend the organism against oxidant st ress and protect and help to p revent some forms of cancers and heart disease , and also boost the immune sy st em[8]. Aim of the study:- The aim of this st udy is to evaluate some biochemical p arameters such as Cerulop lasmin, Glutathione and some trace elements for their imp ortance in oxidant- antioxidant balance in sera of OA female patients which may p lay a role in oxidative st ress . Experimental S ubjects The p resent st udy was p erformed on a group of 30 human females aged (35-65 ) y ears with OA diagnosed by examining the p atients by in the (Baghdad teaching hosp ital )and by x-ray examination. In addition a group of 30 healthy females were enrolled in the st udy as control group . S ampling Blood samp les of 5 ml were drawn all from subjects enrolled in the st udy , and kep t ,in p lain tubes, left to clot at room temp erature for l5 min. Then centrifuged at 3500 rp m for 10 min to sep arate the serum. Glutathi one (GS H) determination :- Glutathione concentration in the sera of all subjects was p erformed according to Ell man( 1959 ) [9] .The method is based on the reaction of aliphatic thiol comp ounds with 5,5- dithiobis (2-nitrobenzoic acid ) (DT NB) at p H 8. The absorbance of the y ellow chromagen was measured at 412 nm and is directly p rop ortional to GSH concentration. So one mole of thiol p roduces one mole of p -nitrot hiophenol anion which is highly colored (€=13600M -4 Cm -1 ) Ce ruloplasmi n (Cp) determination:- The method for determination of Cp concentration in serum based on the cataly tic ability of Cp to oxidize the colorless p -p henylene diamine to a blue-violet oxidized form which has a maximum absorbance at 525 nm , using molar absorbtivity coefficient of 0.68 mol -1 cm -1 for the base[10]. De termination of Copper (Cu) concentration:- Flame atomic absorp tion (Shimadzu-A-A-600atomicabsorp tion/flameless) was used with a hollow cathode lamp of 324.8nm. De termination of S eleni um (S e) concentration :- Flameless atomic absorp tion was used with a hollow cathode lamp of 196.00 nm. S tatisti cal analysi s:- The results were exp ressed as mean ±SD of mean , using st udent-t -test, Significant variation is considered significant when p - values is ≤ 0.05. Re sults and Discussion The results of serum GSH and Cp of OA p atients and healthy females are shown in table (1). The results of GSH concentration in sera of OA p atients were significantly lower than that for control . IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 This results are in agreement with other st udies which reported that GSH is the major non enzy matic antioxidant in p lasma and erythrocytes [11]. So this reduction is an indicative of an increased oxidative st ress in p atients with ost eoarthritis disease , also GSH maintains the sulfhy dry l group s of other molecules including enzy mes in the reduced form for their p rop er function so t he glutathione will oxidized (GSSG) , which will be no longer of great value in p rotection and maintaining other molecules in their native st ructure [12]. It have been reported that oxidative st ress seems to p lay a role in OA, so human cartilage in p atient with OA was significantly deficient in enzy matic antioxidant which are the major free radical scavengers [13] . The life Extension foundation believes that p eople with OA should maintain a healthy intake of antioxidant and other sup p lements t hat sup p ort glutathione levels such as N- acety l cy st eine [14] From table (1)also a significant decrease in Cp sera levels in OA female p atients comp ared to control was found. It had been rep orted that Cp is, classified as one of the acute - phase reactants , its sy nthesis and secretion is altered by inflammation [15]. The reduction in sera Cp levels of OA p atient in this st udy could be due to t hat the p atients are of anti -inflamanrtory OA with moderate to severe p ain [16]. Table (2) shows the Copp er and Selenium levels in sera of OA p atients and control. The significant decrease in Cu concentration in sera of OA p atients comp ared to control was noticed . This reduction in Cu concentration could be exp lained by the fact that generally serum Cu and Cp concentration are usually closely correlated with each other , because cerulop lasmin is the major copp er binding p roteins in serum [17] . The serum Cu/Cp ratio in OA p atient group are significantly decreased in comp arison to that of control group . Since the Cu/Cp ratio reflect the concentration of copp er that is bound to the albumin and free copp er which is the form considered as one of most imp ortant reasons for free radicals formation in the body , so the slight decrease in Cu/Cp ratio may be a defense mechanism against many diseases [18]. The results of lower serum Cu concentration of OA p atients in this st udy reflects t hat the contribution of free copp er in the formation of free radicals in the patient group is not significant . A significant decrease in Se concentration in sera of OA p atients comp ared to control was found. T he result of the present study agrees with ot her rep orted data stated that se deficiency has been correlated with a higher risk and severity of OA [19]. The concentration of Se in serum is highly resp onsive to the Se level in the diet [20], other reason for the decreased Se serum levels in OA p atient is that Se is a comp onent of the enzy me glutathione p eroxidase at t he active site ( as selenocyst eine ) [4]. The cataly zing action of glutathione p eroxidase in removing H2 O2 from the biological sy st em is of great imp ortance , since accumulation of H2O2 may decrease the life sp an of the erythrocytes and imp ortance molecules in human cells by causing oxidative damage to the cell membrane of the biological molecules [21]. From the p resent st udy a conclusion could be st ated , that decreased of Cp concentration in sera of OA p atient is due to oxidative st ress. Also non Cp copp er, as indicated by the ratio Cu/Cp seems t o p lay no role here in the formation of the oxidative st ress. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Re ferences 1-Hegemann,N.B.; kohn.L. Brnnberg, and Schmidt,M .F. (2002). Osteoarthritis cartil, 10:714-721 2-M aty as ,J.R.; At teyl lonescu, M . and Ey re, D.R. (2004). Art hritis Rhenum ,50:543-552 3-M urray, R.K.; Granner, D.K.; Rodwell,V.W.(2006) “Harpers lllustrated Biochemistry ” 27 th . Edition, M CGraw. Hill companies Inc. (U.S.A).P.636 4-Champ e, P.C. and Harvey, R.A(2008) “Lip in cott s illnst rated Reviews Biochemistry “ 4 th Edition Lipp incott Williams Wilkins ,U.S.A P.148 5- Young, S. Fahmy, M . and Golding, S. (1997) : Cerulop lasmin , transferrin and apotransferrin facilitate iron release from human liver cells . FEBS letters, 411: 93-96. 6- David, S. and Patel, B.N. (2000):Ceruloplasmin st ructure and function of an essential Ferroxidase . In "Advance in st ructural Biology " JAI p ress, Inc., 6:211-237. 7- Harries, E.D. (2000): Cellular Cop p er transp ort and metabolism. Annu Rev. Nut r. , 20, 291. 8- Bender, D.A. (2003) : Nut ritional Biochemistry of the Vitamins. 2 nd ed. , Cambridge Univ . Press, 166. 9-Ellman ,G.L., (1959): Tissue Sulfhydral group s . Arch. Biochem Biophy s, 82:70-77 10-M enden, C.E.;Boian, J.17.;M urthy ,lanad petering , H.G.,(1977):Anal lett; 10: 197-204. 11- Fernandez Checa, J.C. ; Kaplowits, N. ; Garcia Ruiz, C . ; Colell, A. ; M iranda, M . ; M ari M . ;Ardite E. and M orates A. (1997) :GSH t ransp ort in mitochondria: defense against TNF_induced oxidative st ress and alcohol_induced defect , Am. J. p hy siol. 273 :7-17. 12-Vina, J . (1990) “ Glutathione metabolism and p hy siological functions” Boca Raton ,CRC p ress ,p .378. 13- Bogdanskg, J.J.; Korneti, P. and Todorova, B. (2003) : Free radical tissue damage : Prot ective role of antioxidant , 104(3) : 108-114. 14-Lozada ,C.J and st eigelfest, E, (2006):”Osteoarthritis p athop hy siology , causes, and treatment” George town university medical center . Section of Rheumatology , Washinglon hosp ital. 15-Cousins, R, J (1985): Absorp tion, transp ort, and hepatic metabolism of copp er and zinc: sp ecial reference to metallothionein and cerulop lasmin Phsy ol.Rev. 65(2): 238-309. 16-Furst ,D.E. and M unst er ,T. (2001):”Non-steroidal anti –inflammatory drugs , disease modify ing antirheumatics drug” ln: Basic and clinical p harmacology , 8 th ed. International Edition ,Lange M edical Books, M cGraw-Hill ; 596-623. 17-Burtis ,C.A. and Ashwood , E.R.(1996) Tetiz Fundamentals of clinical chemistry .”4 th Ed, Philadelp hia, sunders W.B. 18-Fisher ,G.I.; Sp itcer ,L.E. and M ckneil, K.L.(1981):Serum Cop p er and Zinc levels in melanoma p atients .Cancer ,47 :1838_1843 . 19- Devlin M . (2006) : "Textbook of Biochemistry with clinical correlations " 6 th ed. , A John Wiley &Sons, Inc. , USA,1115. 20- Sudhakar M . , and Donald F.K. (2000) : "M icronutrient and Trace elements monitoring in adult Nut rition sup p ort " ,Review: Nutrition in clinical p ractice; 15:120-126. 21-Berg .J.M .; Ty moczko, J.L. and Stry er ,L .(2007) “Biochemistry ” 5 th ed .Free man and comp any ,New York . IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Table (1): Glutathi one and Ceruloplasmin levels in sera of OA patient and control contr ol n=30 OA patient n=30 P value G SH mM/L mean ± SD 2.50± 0.10 1.58±0.01 <0.05 Cp mg/ dl mean ± SD 24.25± 2.35 18.97±3.20 <0.05 Table(2): Copper and Se lenium level in sera of OA patient and control groups Control n=30 OA patient n=30 P value Cu ppm mean ± SD 0.39± 0.04 0.13±0.01 <0.05 Se ppm 0.23 ±0.04 0.17± 0.05 <0.05 Cu/Cp rat io 0.016 0.006 <0.05 IHJPAS 2010) 2( 32المجلد رفة والتطبیقیة مجلة ابن الهیثم للعلوم الص كلوتاثیون والسیریولوبالزمین وبعض لكیمو حیویة للللمتغیرات ا ةن مقار ةدراس لتھاب المفاصل العقدي مجموعة نساء أصحاء ومریضات با صرالنزرة في مصلاالعن في العراق يالو حیل حمید بشرى بغداد جامعة، الھیثم ابن التربیة كلیة،الكیمیاء قسم -: لخالصھا مــن 30 عـن" مـن مستشــفى بغـداد التعلیمـي فضــالبالتهــاب المفاصـل العقـدي اخــذت ةضـمری 30 ةهـذه الدراســتضـمنت .لمجمـوعتین ا فـي مصـل یـةوقیسـت بعـض المتغیـرات الحیو ، ةسـن 65-35وتراوحت اعمار المجموعتین بین ، األصحاءالنساء السـیریولوبالزمین و إلـىنسـبة النحـاس لسـیریولوبالزمین وبعـض المعـادن النـزرة مثـل النحـاس و االكلوتاثیون و هي لمتغیراتاوكانت . السلینیوم نسـب النحـاس علـى ان و .المجمـوعتین في جمیع المتغیرات في مصل نساء محسوس معنوي إحصائي جمیع النتائج عكست فرق .وان النحاس الحر لم یكن عامًال مهمًا في هذا الضرربسب جهد االكسدة ةسجلضرر االن عزویالسیریولوبالزمین IHJPAS