IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Biochemical Study on Splenectomy and Non Splenectomy Iraqi Major Thalassemic Patients M. S. Sabri Departme nt of Chemistry , College of Education I bn – AL- Haitham , Unive rsity of Baghdad. Abstract In this st udy the activities of alanine transaminase (ALT) and asp artate transaminase (AST) were evaluated, in addition to total p rotein and albumins in sera of sixty one subjects whose ages were ranged between(4-16) y ears. These subjects were, twenty eight major thalassemic p atient (12 with sp lenectomy and 16 non sp lenectomy ) and fifteen with minor thalassemia. ei ghteen healty subjecte as control. The result revealed a si gnificant elevation in the activities of both aminotransferases enzy mes (AST and ALT) in the sera of all the alassmic patient group s compared with control. Also a significant increase in the activity of ALT in sera of non-sp lenectomy comp ared to sp lenectomy major thalassemic p atient , which could be an indicative of the severity of liver dy sfunction. No significant differences in the albumine levels of all p atient group s comp ared to control was noticed. A sign ificant low level of total p rotein in non sp lenectomy major thalassemic comp ared to control was found , while no significant difference between total p rotein level in the sera of sep lenectomy major Thalassemic comp ared with control was found. A conclusion could be obtained for the low lev els of t otal p rotein and the normal level of albumin in sera of non sp lenectomy major thalassemia is the reduction in some p rotein fractions of the globulin p art of t he serum. Introduction The thalassemias are her editary hemolytic diseases in which an inbalance occurs in the sy nthesis of globin chains. As a group they are the most common single gene disorders in humans. Normally , sy nthesis of the α and β globin chains are coordinated, so that each α – globin chain h as a β- globin chain p artner.This leads to the formation of α2 β2 (HbA). In the thalassemias , the sy nthesis of either the α- or the β- globin chain is defective. A thalassemia can be caused by a v ariety of mutations, includin g entire gene deletions, or substitutions or deletions of one to many nucleotides in the DNA [1]. In t hese disorders, synthesis of β- globin chains is d ecreased or absent, t yp ically as a result of p oint mutations t hat affect p roduction of functional mRNA, however, α – globin chain sy nthesis is normal. α- Globin chains cannot form st able tetramers and, therefore precipitate, causing the p remature death of cells initially destined to become immature red b lood cells [2] . This defect results in an excess of t he other chain which p recipitates within the red cell membrane causin g h emolytic anemia bringing about cell death within the bone marrow and premature removal of cir culatin g red cells by the sp leen[3]. β- thalassemia is the most familiar typ e, in which the β- globin chain sy nthesis is imp aired. A deficiency of β chains, α chain sy nthesis continuos p roducing an increased p rop ortion of HbF and α chain p roduction increases the amount of HbA2[4]. The severity of the disease dep ends on the amount of HbA and HbF, which p resent [3]. Amino transferses catalyze the transfer of an α- amino group from an α – amino acid to an α- keto acid .These enzy mes , also called transaminases, gener ally funnel α- amino group s from a variety of amino acids to α- ketoglutarate for conversion in to NH4 + . Aspartate IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 amino transferase (AST), one of the most imp ortant of these enzy mes, catalyzes the transfer of the amino group of asp artate to α-ketoglutarate . Aspartate + α- ketoglutarate � oxalo ace tate + glutamate Alanine amino transferase (ALT) cataly zes the transfer of the amino group of alanine to α- ketoglutarate. Alanine + α- keto glutarate � py ruvate + glutamate These transamination reactions are r eversible and can thus be used to sy nthesize amino acids fro m α- ketoacids[5]. The p roteins are substances made up of smaller bu ild ing blocks called amino acid. And are imp ortant constituents of all cells and tissues. Human serum contains more than 125 identified p roteins. So there are many different kinds of p roteins in the body with many different functions[6]. The major site of sy nthesis of the p lasma p roteins is the liver. The second major site is the immune sy st em[7]. The concentration of tot al p rotein in human p lasma is app roximately 7.0 – 7.5 g/dl and comprises the major p art of the solids of the plasma. The proteins of the plasma are actually a comp lex mixture that includes not only simple p roteins but also conjugated p roteins such as glycoprotein and var ious typ es of lip op roteins[8] . T otal p rotein lev el d epends on the balance between their sy nthesis and their catabolism or loss from body . A total serum p rotein test measures total amount of p rotein in blood seru m as we ll as the amounts of albu min and globulin which are the main two group s of p rotein[9] . Albumin has a single p olypeptide chain of 580 amino acids. It is a very stable p rotein with a high net negative char ge at p hy siologic p H with a M .Wt. of 66,000 D. Albumin forms the lar gest p ortion of the serum p roteins it is p roduced and degraded by the liver. It carr ies m any small molecules[10] . It is a collective term, used to refer to p roteins other than albumin with the exception of the immunoglobulin and some comp lement p roteins which are formed by immune sy stem , most of the globulins are produced in the liv er[11]. Experime ntal Part - Blood collection and subject samples : five mL of v enous blood sample was obtained from twenty eight subjects of both sexes , whose β-thalassemia major (sp lenectomy and non sp lenectomy) condition was confirmed by evalution of HbF , HbA and HbA2 usin g the Bio. Rad variant hemo globin testing sy st em from Bio. Rad comp any (Italy ) , at Ibn- AL Balady hosp ital in addition to fifteen minor thalassemic subjects as p athological control were includ ed in the present study . Also eighteen healthy subjects were enrolled as control group . Al l blood samples , which were collected fro m the above subjects were allowed to coagulate at room temp erature within 30 minutes, centrifuged for 10 minutes at 2500g. The r esulting serum was sep arated and divided to p arts and frozen until time of analysis. Information of all subjects enrolled in the study are shown in table (1) - Determination of amino transferases activities the dermination were p erformed using a ready Kit p urctuesed from Randox labor atories , England. - Determination of AST activity . Colorimetic method for determination of serum asp artate amino tranferase by monitoring the concentration of oxaloacetate hy drazine formed with 2,4 dinitrop heny l- hydrazine (DNPH)[12]. - Determination of (ALT) Activity . The Activity of ALT is measured by monitoring the concentration of py ruvate hy drozone formed with (DNPH)[12]. - Determination of tot al p rotein. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 The concentration of total p rotein was determined accordin g to the Rando x Kit[13], in which cupric ions, in an alk aline medium, interact with p rotein p ep tide bonds resulting in the formation of a colour ed complex . - Determination of Albumin levels. Plasma albu min level was determined by the dye-binding technique usin g bromocresol green (BCG), p rovided by a ready kit from Randox laboratories, England. In the assay medium albu min wi ll b ind BCG in p hosp hate buffer ( p H 4.2), thus y ielding a b lue comp lex. The incr ease in the absorbance, at 630 n m , is p rop ortional to the amount of albumin p resent in the samp le. Human albumin is used as a standared[14]. S tatistical anal ysis The results were analyzed by student -t- test with p values ≤ 0.05 considered si gnificant using Excel XP 2000 p rogram. Results and Discussion Table(2): showes the activity of AST and ALT in sera of major thalassemic p atients (sp lenectomy and non sp lenectomy) , minor thalassemia and healthy control. A high significant increase in the activity of AST and ALT for both major thalasemic group comp ared to that for minor thalassemic and control were found. The r esult of the p resent study is in a good agreement with result reported earlier ,the reason for this elevation in the activities are du e to sy mp toms of liver damages. As liver is the major site of iron st orage , the liver is a consp icuous victim of excess iron deposition[15]. However the inciden ce and p revalence of iron- induced or gan injury was not influenced by the ethnicity of the p atients in multiple large thalassemia trials[16]. Plasma ALT is a marker of liver injure which increased in thalassemia and considered to be an indicative of liver dysfunction and leakage of liver metabolites into the plasma[17]. Results of serum tot al p rotein and albumin ar e shown in table (3).From table (3) the mean valu es of albumin in sera of p atient with β-thalassemia are not siginfin gthy different as comp ared to control group . These results agree with a studied p arameter reported that p atient with β- thalassemia major (sp lenectomy and non sp lenectomy ) showed similar serum albumin lev el comp atable to healthy individuals[18]. On t he other hand reports showed lower level of albu min in the thalasseamic p atient due to the p resence of antibody in the sera of p atient under blood transfusion[19]. The total p rotein concentration in sera of all p ataient group s except non sp lenectomy showed non significant differences with that for healthy control while a significant reduction was found in the non sp lenectomy comp ared to control the p ossible cause reported to be secondarily decreased sy nthesis of p rotein by the liver and whi ch imp osed to damages by transfusion therapy leadin g to iron over load[20]. The reduction in tot al p rotein and the near normal valu e of albumin could be du e to the decrease in the globulin sy nthesis because the latter consist of different t yp es (i,e α1 , α2 , β and δ ) globuline so the redu ction in any one of these globulin could r esult in a decrease of total level. Re ferences 1.Clarke,G.m.,Higgins,T.N.(2000) : Laboratory invest igation of hemo globin op athies and thalassemia.Clin.Chem.46 :1284. 2.Champ e,P.C.,Harvey,R.A., and Ferrier , D.R.(2008) “ Lipp incott’s Illustrated Reviews IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Biochemist ry ” 4 th ed , Lippincott Williams & Wilkins Philadelp hia. 3.Lissaure, T., Colay den, G. (2001) “ Illusration text book of p aediatrics, 2 nd , ed., p.305. M osby intevnational lmd. 4.Lanzkowsky ,P.(2000), “ Pediatric Hematology and Oncolo gy ” 3 rd ed.pp:182-193, 305- 306.A cademic p ress. 5.Berg,J.M .,Tymoczko,T.L., and st ry er,L. (2007) “ Biochemistry ” 5 th ed.W.H Freeman and comp any , New York. 6.Stry er,L.(2000) “ Biochemistry ” 4 th ed .New York .W.H.Freeman and Company , 153- 7.Kaneko, J.J., Harvey , J.W., and Brunss., M.C. (1997) “ Clinical Bioch emistry of Domestic Animals ” 5 th ed. Academic press. pp :120-226. 8.M urray ,R.K., Granner, D.K. and Rodwell V.W.(2006) “ HARPER’S Illust rated Biochemist ry ”, M c Grow Hill, 27 th ed .U.S.A. 9.M ickael, L., Janet T ,L., and Edwar, P.(2002) “ Clinical chemist ry ”4 th ed. Lippincott Williams & wi lkin g, p .172. 10.Carl, A.,Edward, R., and Ray mond ,E.(1991) “ Tiets t ext Book of clinical chemistry ” W.B. Sannders Company. 1: 151-155, 482-496. 11.Bishop , M .L., Fody , E.P., and schoff, L.(2005)“Clinical chemistry p rinciples, p rocedures, correlations 5 th ed.Lip p incott Williams & wilkins , p p : 105 -106, 194-212. 12.Reitman,S. and Grankel,S.(1957) : A colorimetric method for t he determination of serum glutamic o xalacetic and glutamic p y ruric transamination , Am.J.clin pathol.28:26-63. 13.Weich e selbaum,T.E.(1946): An accurate and rap id method for t he determination of p roteins in small amounts of blood, serum and p lasma. Amer.J. clin.patho., 16:40-49. 14.Rodkey,E.L.(1965): Direct Spectrop hotometic determination of albu min in human serum, clin. ch em. .1:478. 15.Wood,J.C, Tyszra, J.M ; Carson, s, Nelson, M .D, coots, T.D. : My ocadial iron load ing in transfusion dependent thalassemia and sickle cell disease blood 103: 1954-1936 [pub M ed] 16.Vich insky , E.P., M acklin, E.A., Way e, J.s., Lorey, F; Olivieri, N.F.(2005) chan ges in the epidemiology of thalassaemia in north America a new minority disease.p ediatrics. b; 116.e 818- e 825 [p ub M ed] 17.Walter,P B., Fung, E.B. , Killilea, D.W., Harmatz, P.(2008) :Oxidative st rees and inflammation in iron- ov erloaded patients with β- thalassaemia or overlo aded p atients with β- thalassaemia or sickle cell dise ase, p1-14 Bro Haematal ,135(2):254-263. 18.Aitxala,M .T., and sarandria , C.N.(2001):M icrosferocitosis Erythroid profik and its relation with different lab. Test s. M edicina. 61(4):417-23. 19.Wanach iwanawin, W., Leun grojana- Kul, P.,and Fucharocn, S.(2003) : Prevalence and clinical si gn ificance of hepatitis C virus infection thai p atients with thalassemia .Int.J.Heamatol., 78(4):374-8. 20.Hentze,M .W., M uckenthaler, M .V. and Andrews, N.C. (2004): Balancin g Acts : M oleculor control of M ammalian Iron metabolism cell, 117 :285-297. Table (1): information of thal assemi c patients and healthy control. Groups No. M ean age (year) range of age (year) M ajor thalassemic (sp lenectomy) 12 9 ± 2.2 6-11 M ajar thalassimic (non–sp lenectomy) 16 8 ± 1.8 4-12 M inor thalassemia 15 14 ± 3.7 9-16 Healthy control 18 12 ± 2.3 7-16 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Table(2) : AS T and ALT actives i n sera of all groups . Groups No. AST mean ±SD(U/L) P ALT mean ±SD(U/L) P M ajor thalassemic (sp lenectomy) 12 46.8 ± 19.1 < 0.05 S 51.8±13.1 < 0.05 S M ajor thalassemic ( non sp lenectomy) 16 48.7 ± 11.7 < 0.05 S 58.7 ± 21.3 < 0.05 S M inor thalassemic 15 10.3 ± 2.3 S < 0.05 8.4 ± 3.1 < 0.05 S Health control 18 7.9 ± 1.8 < 0.05 S 5.6 ± 1.9 p * >0.05 NS p * < 0.05S * represent p value between both major thalassemic p atients (sp lenectomy and non sp lenectomy). Table (3): serum total protein and albumin in sera of all studie d groups. Groups Total p rotein (gm/dL)± SD p Albumin (gm/dL)± SD p M ajor thalassemic (sp lenectomy) 7.12 ± 0.39 > 0.05 NS 4.08 ± 0.41 > 0.05 NS M ajor thalassemic ( non sp lenectomy) 6.21 ± 0.81 < 0.05 S 4.03 ± 0.46 > 0.05 NS M inor thalassemic 7.09 ± 0.37 > 0.05 NS 4.12 ± 0.51 > 0.05 NS Health control 7.18 ± 0.28 > 0.05 NS 4.01 ± 0.31 > 0.05 NS p * <0.05 S p * < 0.05 NS 2010) 1( 23ة المجلدمجلة ابن الھیثم للعلوم الصرفة والتطبیقی دراسة كیموحیویة لمرضى فقر دم البحر االبیض المتوسط لكل من الخاضعین وغیر الخاضعین لرفع الطحال في العراق میادة سمیر صبري جامعة بغداد ، ابن الهیثم –كلیة التربیة ، قسم الكیمیاء الخالصة فضال عن (AST)واالسبارتیت ترانزامنیر ، (ALT)مینز اس فعالیة انزیمي االنین ترانز قی ةتمت في هذه الدراس ة ) 16-4(تتراوح اعمارهم من انموذجأتقدیر مستوى البروتین وااللبومین في مصل الدم واحد وستون سنة في هذه الشریح ر ،ثمانیة وعشرون یعانون من مرض فقر دم البحر االبیض المتوسط منهم اثنا عشر خضعوا لعملیة رفع الطحال وستة عش ط الخفیف كما شملت الدراسة ثمانیة . لم یخضعوا لها وهناك خمسة عشر مریضًا یعاني من فقر دم البحر االبیض المتوس .الصحاء كمجموعة سیطرة انموذجأشر ع في مصل دم كل من مجامیع المرضى مقارنة مع ALT و ASTإظهرت النتائج زیادة معنویة في فعالیة انزیمي في مصل دم المجموعة التي لم تخضع لرفع الطحال نسبة ALTمجموعة السیطرة كذلك وجدت زیادة معنویة في فعالیة .عة للرفع والتي یمكن ان تكون داللة على شدة التلف الحاصل في الكبد الى المجموعة الخاض ظهر انخفاض لم تظهر اختالفات معنویة في مستوى االلبومین بین مجامیع المرضى مقارنة بمجموعة السیطرة ا لم تظهر فروقات بینم ،معنوي في البروتین الكلي في المرضى غیر خاضعین لعملیة رفع الطحال مقارنة مع االصحاء .معنویة في المرضى الخاضعین لعملیة رفع الطحال مقارنة مع االصحاء كلي وبقاء مستویات االلبومین في مصل دم المرضى غیر الخاضعین لرفع خیمكن ان نستنتج من ان فاض البروتین ال .في بعض اجزاء من الكلوبیولینات في مصل الدم االطحال بأن هناك انخفاض