IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 Effect Of Some Enzymes Activity In Liver Diseases From Patients Of Salmonella Paratyphi A With Iraqi Woman. N. M. J. Al-Shammaa , , B. H .Al- Wihaly , E. A.A. Abass. Departme nt of Chemistry ,College of Education I bn-Al- Haitham,Unive rsity of Baghdad Received in : 25 April 2010 Accepte d in : 8 February 2011 Abstract This p resent st udy demonst rated that liver was involved in 14 %of ty p hoid p atients manifest ing with hepatomegaly . Elevation of serum enzy mes in ty p hoid fever was p resumably of a muscular origin, while elevation of liver enzy me was relatively less common. This st udy was p erformed on 30 female p atients diagnosed by ultrasound (US) of abdomen, with p araty p hoid A, ranged between (20-40) y ears comp ared with 30 healthy control .Patients volunteers were treated with app rop riate antibiotics for 14 days and invest igations were repeated 2-3 week after comp letion of treatment. Patients had clinical and biochemical evidence of hepatic dy sfunction. The sp ectrum of hepatic involvement included hepatomegaly , jaundice, derangement of various hepatic functions and abnormal US abdomen. The results revealed a highly significant values increase in serum AST and ALT comp ared to control was found while a significant increase of serum ALP comp ared to control was noticed and a significant decrease in serum LDH compared to control was obt ained. Key Words: Enzy mes Activity , Liver Diseases and Salmonella Paraty p hi A Introduction Salmonella paraty p hi A t ends to p roduce an illness clinically to p roduced by S.ty p hi with p rolonged fever and a tendency to relapse .This is the commonest p araty p hoid fever in India and Asia[1] .There are three sp ecies of salmonella that cause p araty p hoid salmonella p araty p hi A, S.p araty p hi B (or S .Schotmulleri) and S.p araty p hi C(S.hirschfeldil) [2].Salmonella Ty p hi can sp ecifically only att ack humans,So the infection nearly alway s comes from contact another human, either an ill p erson or a healthy carrier of the bacterium. The bacterium is p assed on with water and foods and can withst and both dry ing and refrigeration but by keep ing food refrigerated correctly this minimizes the p roduction of the bacterium .Significantly unclean food from st reet vendors and flooding help distribute the disease from a p erson to p erson[3].Because of p overty and p oor hy giene and sanitary conditions the disease is more common in less-industraized ,p rincipally owing to the p roblem of unsafe drinking water ,inadequate sewage disp osal and flooding [4]. Paraty p hoid fever is caused by any of three st rains of salmonella paraty p hoid [5,6]. Paraty p hoid fever is found in large part of Asia, Africa, Central and South America. There are 16 million cases a y ear ,which result in a bout 25,000 daths world wide[7] Paraty p hoid fever resembles Ty p hoid fever but p resents with a more abrupt onsent,milder sy mptoms and a short er course. Infection is charactetrized by a sust ained fever IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 ,headache,abdominal p ain, malaise,anorexia, a non p roduction cough(in early st age of illness),a relative Brady cardia(slow heart rate),and Hep ato Sp lenomegaly (an enlargement of the liver or Sp leen).In adults,Constipation is more common than diarrhea only20 % to 40%of p eople will initially have abdominal p ain[8]. Aft er ingestion of contaminated food or water,S.ty p hiparaty p hi bacilli reach the small Intestine, p enetrate the mucosa and then remain viable within the macrophages which ingest them. The bacilli reach the blood stream via the lymphatic sy st em, and are then dissemiated to the organ of the reticuloendothelial sy st em within the first 24 hours of infection .Aft er the incubation p eriod(3 days-1-months), usually 8-14 days which is affected by the infective dose, the clinical sy ndrome app ears[1]. Fulminant hepatic is a dramatic clinical sy ndrome characterized by massive necrosis of liver cells[9]. Although salmonella hepatitis is a rare p resentation of ty p hid fever, fulminant hepatic failure is extremely uncommon, few cases were repoted with such p resentation the first reported case of fulminant hep atic failure in the st ate of Qater, associated with salmonella p araty p hi A infection.Enteric fever, that is ty p hoid and p araty p hoid fever , is the common name for infections caused by salmonella enterica seroty p es ty p hi and p araty p h. Of t he three ty p es of S. p araty p hi (A,B and C),B is t he most common[10]. Alanine Transaminase (ALT)Serum is p resent in high concentration in liver and to a lesser extent in skeletal muscle ,kindney and heart 11]. Alkaline p hosp hatase(ALP) are a group of enzy mes with hy drolyse p hosp hates at an enzymes which hy drolyse p hosp hates at alkaline PH.The activity measured by routine methods includes that of several isoenzy mes. They are found in bone, liver, kidney ,intest inal wall, Lactating mammary gland and p lacenta. In bone the enzy me is found in ost eoblasts and is p robably imp ortant for normal bone formation. In adults, the normal levels of alkaline p hosp hatase are derived largely from the liver. Pregnancy raises the normal rangs because of the production of a heat-stable alkaline phosp hatase by the placenta[12]. Lactate dehydrogenase (LDH) cataly ses the reversible inter conversion of lactate to p y ruvate. It is widely distributed, with high concentrations in the heart, skeletal muscle, liver, kidney , brain , erythrocytes and measurement of total LDH is therefore a non-sp ecific index of cell damage. The aim of the present study was t o evaluate the major source of increased serum enzy me level sp ecify these enzy mes because liver and serum contain of enzy mes in p araty p hoid fever and to determine the most relevant clinical entity , hepatitis or myop athy , during p araty p hoid fever [13]. Experimental part S ubjects: This st udy p erformed on 30 female patients diagnosed by ultrasound (US) of abdomen, with p araty p hoid A ranged between (20-40) y ears, who were selected from p atients att ending Baghdad Teaching Hosp ital in addition to 30 female healthy control . Col lection of Blood sample s: Six ml of venous blood was withdrawn from all subjectes enrolled in study .The blood were transferred to p lain tubes, left to clot at room temp reture for 15 min.,centrifuged at 3500 rp m for 10 min. The resulted serum was sep arated and kept frozen. S tatisti cal Analysi s Statist ical analysis was p erformed using st udent t-test for comp arison of variables. The p value <0.05considered significant and p <0.01considered highly signif icant. All data in the table (1) are M ean ± SD [14]. AST LD IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 De termination of Se rum Aspartate Transaminase (AS T) Activity Serum (AST) activity was measured using colorimetric method according to (Reitman and Frankel, 15) utilizing a ready made kit for determination of serum asp artate aminotransferase. In this method, the following reaction represents measurement of AST activity -Oxoglutarate +L- Asp artate L-glutamat+ Oxaloacetate Glutamate-Oxaloocetate transaminase is measured by monitoring the concentration of Oxaloacetate hydrazone formed with 2, 4-dinitrop henyl-hydrazine. The absorbance was read at 540 nm after 5 min. Cal culation :Expresse d equati on i s : AST Activity =Test-Blank test/Standard Solution-Blank Standard×67 =µml/M in/Liter blood serum and change to International Unite Per Liter(IU/L) Normal valu e:AST Activity in Serum up to 12U/L [15]. De termination of S erum Alani ne Transami nase( ALT) Activity: The (ALT) activity was measured using colormetric method according to (Reitman and Frankel, 15) Ut ilizing a ready made kit for t he determination of serum alanine aminotransferase. In this method , it rep resents measurement of ALT activity -Oxoglutarate +L-alanine L-glutamate+ p y ruvate Glutamate-p y ruvate transaminase is measured by monitoring the concentration of p y ruvate hy drazone formed with 2, 4-dinitrop henyl-hydrazine . The absorbance was read at 540 nm after 5 min. Cal culation :Expresse d equati on i s: ALT Activity =Test- Blank test/Standard Solution-Blank Standard×133 =µml/M in/Liter blood serum and change to International Unite Per Liter(IU/L) Normal value: ALT Activity in Serum up to 12U/L [15]. De termination of S erum Lactate Dehydrogenase Activity (LDH) Serum ( LDH) activity was measured using enzy matic colorimetric method where a r eady made kit is used. The method is based on the reduction of py ruvate to located in the p resence of NADH by the action of the lactate dehydrogenase: Py ruvate +NADH+H + Lactate +NAD + ALT IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 The Py ruvate that remains unchanged reacts with 2, 4- dinitrop heny l-hy drazine which is determined by calorimetric. Calculation:Enzy me activity was determined by reference to t he calibration curve. M ixed and allow t o st and for 20min M ixed and allow to st and at room temperature for 10 minutes, read absorbance of all tubes against distilled water at 520nmThe corresp onding values in U/L are shown in the followin g table: Ordinate= absorbance Abscissa= activity in U/L Normal valu e: 80-190 U/L [16]. De termination of Alkaline Phosphates Activity Colorimetric determination of ALP activity was p erformed using a kit from Biomerieux, France according to the following reaction: PH=10 Pheny l phosp hate Phenol + Phosp hate The phenol liberated was measured in the p resence of amino-4-antipy rine and p otassium ferricyanide. The p resences of sodium arsenate in the reagent stop the enzy matic reaction. The absorbance of the sample was measured at 510 nm against reagent blank [17]. Re sults and Discussions The results of serum AST, ALT, Alkaline p hosp hate (ALP) and Lactate dehydrogenase (LDH) in p araty p hoid p atients and their control group are shown in table (1).A highly significant values increase in serum AST and ALT comp ared to control was found while a significant increase of serum ALP comp ared to control was noticed and a significant values decrease in serum LDH comp ared to control was found. The diagnosis of fulminant hepatic failure due to S. Paraty p hoid was p assed mainly on two facts: First, p ositive blood culture for S. p araty p hoid A elevated alkaline p hosp hatase level that agreed with this st udy [18]. The p athogenesis of severe hepatic involvement in Salmonella infection may be multifactorial, involving end toxin, local inflammatory and or host immune reactions [19]. Alkalines p hosp hatase 150.6 U/L normal(21-92) also had increased levels of AST serum similar observations were made with ALT serum, SA salmonella endotoxin induced consumptive coagulopatt y , damage to hepatocy tes arteritis [20], direct invast ion of the hepatocy tes by the organisms, immune comp lexes and consumption of comp lement are believed to contribute to hepatic insult [21]. The clinical p resentation and extent of hepatic dy sfunction in ty p hoid fever would, therefore,dep end up on these contributory . Factors and may or may not be associated with hepatomegaly ty p hoid [22]. It is concluded that p resence of high fever, Jaundic and tender hep atomegaly should arouse susp icion of ty p hoid hep atitis IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 .Hep atic dysfunction in these cases, desp ite its high incidence and serious nature, is transient and resp onds favorably to app rop riate antibiotic therap y . M y op athy during the course of ty p hoid fever is reported increasingly [23]. Both clinical entities, my op athy and hepatitis, are usually diagnosed by serum enzy mes such as asp artate aminotransferase(AST), alanine aminotranst erase(ALT), alkaline p hosp hatase (ALP),lactate dehydrogenase(LDH),AST and LDH are elevated in myop athy as well as hepatitis, whereas ALT are relatively sp ecific for liver and muscle, resp ectively[24] . According to the original definition of ty p hoid hepatitis, it is p ossible that an over diagnosis for ty p hoid hepatitis occurred and ty p hoid my op athy was missed [25].The invasion of intestinal ly mphatic tissue is suggested to result in a host reaction with hy p erplasia of the liver reticuloendothelial sy st em and infiltration of p ortal sp aces as well as the reduction of the microcirculation causing necrosis[26].Hepatic damage by salmonella ty p hi app ears to be mediated by bacterial endotoxin.The p resence of intact bacilli has been demonst rated in the hep atic tissue that may be related to p resence of Salmonella bacteria in reticuloendothelial sy st em and hyp erplasia of kup ffer cell (ty p hoid nodule) without significant liver injury [27]. In the p resent st udy , the p att ern of serum enzy mes is more comp arable to whate is found in my op athy (60 % ) rather than liver disease (22%) ,as the association of AST and liver enzy mes [28].The higher level of serum LDH in all other age group s a mong cases is more likely due to mycobacterial induced tissue injury as well as other p athop hy siological conditions , which remained undiagnosed during this st udy [29]. The results clearly indicate that adult have higher p revalence and suscep tibility to mycobacterium infection as comp ared to control .Thus, serum LDH st ill remains as one of the imp ortant p arameters to a ssess the cell/tissue sp ecific st ress or p athology , caused by mycobacterium tuberculosis the observed correlation between serum LDH level and sp utum mycobacterial load alone or in combination with its individual isoformes, esp ecially LDH3 and LDH5 can thus be used as an index for diagnosis of tissue/organ affected by the disease (clinical). It also indicates the severe p atho-p hy siological conditions of the liver of p atients [30]. Hep atic dysfunction detected by clinical and or biochemical p arameters was noticed in as many as 64.5%of cases in this st udy which is higher than that reported by others[31]. Hep atomegaly is usually observed in enteric fever after the first week of illness, most often p ersists throughout the p eriod of marked elevation of temp erature, becomes less evident as defervescence p rogresses and usually last s for 3-4 week. Incidence of hepatomegaly is believed to be 2-3 times more common in ty p hoid fever than p araty p hoid fever [22]. Tender hepatomegaly observed in 2 of our cases, suggested a more severe hepatocellular involvement as both of these cases p resented with jaundice. Even IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 though majority of cases with jaundice had hepatomegaly , Liver was not enlarged in one case suggesting that significant hepatic dy sfunction can occur in ty p hoid fever without hepatomegaly . Jaundice associated with ty p hoid fever tends to occur at the p eak of fever which differentiates it from viral hepatitis in which case fever usually comes down after the app earance of jaundice .Jaundice in most of these cases is due to ty p hoid hep atitis. However, hemolysis resulting in jaundice is a recognized comp lication of ty p hoid fever in p atients with G-6-P deficiency or thalassemia [32].Ot her causes of Jaundice include ascending cholangitis, Salmonella liver abscess, sup p urative py elop hlebitis and cholecy st itis [21]. Conclusions From the results of p resent st udy , conclusions could be drown that liver is involved in 14 %of ty p hoid patients manifest ing with hepatomegaly . Elevation of serum enzy mes in ty p hoid fever is p resumably of a muscular origin, while elevation of liver enzy me is relatively less common. Re ferences 1-World Health Organization.Background document.(2003)The diagnosis,t reatment and prevention of typ hoid fever.WHO/V&B/03.07. 2-Bhan, M .K.,Bahal. R; and Bhatnagar, S.(2005)Ty p hoid and p aratyphoid fever lancet 366:749-762.A comp rehensive review of typ hoid and p aratyphoid fever. 3-Bhan ,M .K;Rajiv, Bahl; and,Shinjini, Bhatnagar.(2005)"Typ hoid and p aratyphoid fever."The lancent 366.9487.August 27:794(14). 4-Water-related Diseases (2008) Paraty p hoid fever is caused by any of three st rains of Salmonella Paraty p hoid. 5-Frey , J.Rebecca.(2008)Paratyp hoid fever (1999) Encyclop edia of M edicine. 6-Chin J(ed).( 2000) Control of communicable Diseases M anual,17 th edition. American Pup lic Health Association. 7-Rubin,Rap hael;David, S. Strayer. Emanuel; Rubin ,Tay . M ;and M c Donald Rubins.(2007) Pathology .5 th ed. 8-Water-related dise ases communi-cable diseases.( 2001) World Haelth. 9-Khan, F.Y;Kamha, AA;and Alo mary IY.(2006)Fulmin an hep atic failur e caused by Salmonella Paray tp hoid A infection. World J Gastroenterol;12(32) :5253-5255. 10-Crump, JA; Luby ,SP;and M int,Z. ED.(2004)The global burden of typ hoid fever . Bull World He alth Organ,82:346-353. 11-Schei g, R.(1996) Evaluation of tests used to screen p atients with liver disorders p rim care;23 :551-60. 12-Adams ,E.B. Typ hoid and p aratyphoid fever In.(1987)Weatherall DJ ,Ledin gh am JGG,Warrell DA, eds.Oxford Textbook of M edicine, vol 2,2nd Ed.Oxford : Oxford Univ p ress, 5:223-5.225. 13-Ginsberg , A.L.(1970) Very high levels of SGOT and LDH in patients with extrahep atic biliary tract obst ruction. Am J Dig Dis;15:803-7. 14-Sorlie,D.E.(1995)M edical biost atist ics &epidemiology :Examination& board r eview :First ed. Norwalk,Connecticut,App leton&Lange:47-88. 15-Reitman S, and Frankel S Amer(1957) J Clin.Path.,28:56 16-Wroblewsk. F; La, Due. J;and Proc.(1955) Soc Exp .Biol.90:210. 17-A. J. Pesce; and L. A .Kaplan.(1987) M ethods in .,M osby Comp any , Clinical Ch emistry ,< "New Dalhi, 527:543-590. 18-Kamath, P.S; Talihal, A ; and Chakr aborty ,A.(2000)Differentation of typhoid fever from IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 fulminant hepatic failur e in p atients p resenting with Joundice and encephalop athy M ay o Clin Proc, 75:426-466. 19-Forsyth ,JRL.Typhoid and p aratyphoid:In collier L, B alows A,Sussman M .Top lex and Wilsons. (1998)M icrobiology and M icrobial inf ections9 th ed. London:Arnold Press :456-478. 20-Gupt a, SP; Gupt a, M S;Bharadwaj, S;and Chu gh, T.D.(1985)Current clinical p att erns of typhoid fever:A p rosp ective st udy .J T rop M ed Hy g.88:377-381. 21-Kumar ,A;Kap oor, R. Chop ra. K;and et al.(1989)Unusual hep atic manifestions in ty p hoid fever. Clinical Pediatr .2:99-100. 22-Khosla ,SN; Singh, R;Singh, GP;and et al.(1988) The sp ectrum of hep atic injury in enteric fever AM J Gastroenterol. 4:413-416. 23-M ody ,GM ; Gathiram, V; and Abdulla, EA.(1989)Severe reversible M y op athy due to ty p hoid. J.Trop M ed Hy g;92:102-3. 24-Scheig, R.( 1996)Evaluation of tests used to screen patients with liver disorders. Prim Care;23:551-60. 25-M irsadraee, M ; ,Shirdel, A;and Rokneef.(2007)Typ hoid my op athy or typhoid hep atitis:A matter of debate. Indian Journal of M edical M icrobiolo gy , Vol.25, No.4, October- Deceber.pp 351-353. 26-Khosia, SN;Singh, R;Singh, GP;and Trehan VK.(1988)T he sp ectrum of hep atic injury in enteric fever. Am J Gastroenterol. 83:413-6. 27-Pramoolsinasp , C. and Viraanuvatt i, V.(1998)Salmon ella hep atitis. J Gasteroenterol Heptol.13:745-51. 28-Pandy , CK; Singh, N; Kumar ,V; A garwal, A; and Singh, PK.(2002) Typ hoid, hep atitis E or typhoid and hepatitis E:The cause of fulminant hep atic failure::Adiagnost ic dilemma.Crit Care M ed.30:376-8. 29-Nusslnovitch ,M Finkelst ein .Y; Elishkevitz , KP; Volvoitz, B; Harel, D; Klinger, G ;and et al (2009)Cerebrosp inal fluid lactate dehy drogenase isoenzy mes in children with bacteriala nd asep meningitis.T ranslational Res;154(4):214-8. 30-Yun,S.J;Cho i, M .S;Piao,M S;Lee, JB;Kin, SJ; Won, YH;and Lee,SC.(2008) Serum Lactate Dehy drogenase is a novel mark er for the evaluation of disease necroiy sis Dermatol;217:254-9. 31-Khosla ,SN.(1990)T y p hoid hep atitis. Postgrad M ed J,66:923-925. 32-Thisy akorn, U;M ansuwan, P;and Tay lor, DN.(1987)Typhoid and p aratyphoid fever in 192 hosp italized children in Thailand.AM J Dis Child,141:862-865. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (2) 2011 Table (1): AS T, ALT, ALP an d LDH levels in se ra of controls and patients Parameter Control n=30 Patient n=30 P Value AST(IU/L) M ean±SD 13 ±1.05 28.7± 2.27 P< 0.01 Highly Sig ALT(IU/L) M ean±SD 12±0.52 23.2±1.25 P< 0.01 Highly Sig ALP(IU/L) M ean± SD 49.2 ±3.04 150.6± 1.32 P< 0.05 Sig. LDH (IU/L) M ean± SD 205± 4.67 159± 3.11 P< 0.05 Sig. 2011) 2( 24مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد باراتیفوئیدتأثیر بعض فعالیة أنزیمات أمراض الكبد في مرضى سالمونیال A لدى المرأةالعراقیة أیمان عبد علي عباس،بشرىحمیدالوحیلى،نوال محمد جوادالشماع جامعة بغداد، أبن الهیثم_كلیة التربیة،قسم الكیمیاء 2010نیسان 25: استلم البحث في 2011شباط 8:قبل البحث في الخالصة مریضات التیفوئید الالتي لدیهن تضخم الكبد وأرتفاع أمصال أنزیمـات من% 14للكبد عند أظهرت الدراسة الحالیة شــملت . الحمـى التیفوئیدیـة ٍوعلــى مـا یبــدو مـن منشـأ عضــلي، بینمـا أرتفــاع أنـزیم الكبــد نسـبیا أدنـى بدرجــة أقـل مــن الشـائع وكانـت عالمـات (US)وتیةللجـنس األنثـوى الالتـي شخصـن بأسـتعمال األمـواج فـوق الصـ Aمریضـا بالباراتیفوئیـد30الدراسة ســنة وتـــم ) 40-20(مـــن األصــحاء بوصــفهن ســـیطرة ضــبط تتــراوح أعمـــارهن بــین 30المــرض ظــاهرة علـــیهن فضــال عــن أســابیع بعـد أكمـال العـالج للتحقـق مـن الشـفاء برهنــت 3- 2یومـا وتكرارهـا 14معـالجتهن بتنـاولهن المضـادات الحیاتیـة مـدة تضـمن الطیـف الكبـدي تضـخم الكبـد ،الیرقـان، ومختلـف . نـدهن أختالالوظیفیـا فـي الكبـدجمیع الحاالت سریریا وكمیوحیویـه ع . (US)الوظائف المشوشة للكبدوالصفة الشاذة للبطن من خالل أمواج فوق الصوتیة فـي أمصــال مرضــى (ALT) ، وAST)(أظهـرت نتــائج الدراسـة ان هنــاك زیـاده معنویــة عالیــة فـي مســتوى الــ مقارنــــة (ALP)إذ لـــوحظ زیـــادة معنویـــة فــــي مستوىمصـــل الفوســـفاتیزالقاعدي ،مقارنـــة بمجموعـــة الســــیطرة Aوئیـــدالباراتیف مقارنـة بمجموعــة (LDH) بمجموعـة السـیطرة، بینمـا وجـد أن هنـاك أنخفاضـا معنویــا فـي مصـل الكتیـت دي هیـدروجینیز .السیطرة Aفعالیة األنزیمات،أمراض الكبد ،سالمونیال باراتیفوئید: الكلمات المفتاحیة