IBN AL- HAITHAM J. FOR PURE & APPL. SCI. 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. Department of Chemistry ,College of Education Ibn-Al- Haitham,University of Baghdad Received in : 25 April 2010 Accepted in : 8 February 2011 Abstract This present study demonstrated that liver was involved in 14 %of typhoid patients manifesting with hepatomegaly. Elevation of serum enzymes in typhoid fever was presumably of a muscular origin, while elevation of liver enzyme was relatively less common. This study was performed on 30 female patients diagnosed by ultrasound (US) of abdomen, with paratyphoid A, ranged between (20-40) years compared with 30 healthy control .Patients volunteers were treated with appropriate antibiotics for 14 days and investigations were repeated 2-3 week after completion of treatment. Patients had clinical and biochemical evidence of hepatic dysfunction. The spectrum 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 compared to control was found while a significant increase of serum ALP compared to control was noticed and a significant decrease in serum LDH compared to control was obtained. Key Words: Enzymes Activity , Liver Diseases and Salmonella Paratyphi A Introduction Salmonella paratyphi A tends to produce an illness clinically to produced by S.typhi with prolonged fever and a tendency to relapse .This is the commonest paratyphoid fever in India and Asia[1] .There are three species of salmonella that cause paratyphoid salmonella paratyphi A, S.paratyphi B (or S .Schotmulleri) and S.paratyphi C(S.hirschfeldil) [2].Salmonella Typhi can specifically only attack humans,So the infection nearly always comes from contact another human, either an ill person or a healthy carrier of the bacterium. The bacterium is passed on with water and foods and can withstand both drying and refrigeration but by keeping food refrigerated correctly this minimizes the production of the bacterium .Significantly unclean food from street vendors and flooding help distribute the disease from a person to person[3].Because of poverty and poor hygiene and sanitary conditions the disease is more common in less-industraized ,principally owing to the problem of unsafe drinking water ,inadequate sewage disposal and flooding [4]. Paratyphoid fever is caused by any of three strains of salmonella paratyphoid [5,6]. Paratyphoid fever is found in large part of Asia, Africa, Central and South America. There are 16 million cases a year ,which result in a bout 25,000 daths world wide[7] Paratyphoid fever resembles Typhoid fever but presents with a more abrupt onsent,milder symptoms and a shorter course. Infection is charactetrized by a sustained fever IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 ,headache,abdominal pain, malaise,anorexia, a non production cough(in early stage of illness),a relative Brady cardia(slow heart rate),and Hepato Splenomegaly(an enlargement of the liver or Spleen).In adults,Constipation is more common than diarrhea only20 % to 40%of people will initially have abdominal pain[8]. After ingestion of contaminated food or water,S.typhiparatyphi bacilli reach the small Intestine, penetrate the mucosa and then remain viable within the macrophages which ingest them. The bacilli reach the blood stream via the lymphatic system, and are then dissemiated to the organ of the reticuloendothelial system within the first 24 hours of infection .After the incubation period(3 days-1-months), usually 8-14 days which is affected by the infective dose, the clinical syndrome appears[1]. Fulminant hepatic is a dramatic clinical syndrome characterized by massive necrosis of liver cells[9]. Although salmonella hepatitis is a rare presentation of typhid fever, fulminant hepatic failure is extremely uncommon, few cases were repoted with such presentation the first reported case of fulminant hepatic failure in the state of Qater, associated with salmonella paratyphi A infection.Enteric fever, that is typhoid and paratyphoid fever , is the common name for infections caused by salmonella enterica serotypes typhi and paratyph. Of the three types of S. paratyphi (A,B and C),B is the most common[10]. Alanine Transaminase (ALT)Serum is present in high concentration in liver and to a lesser extent in skeletal muscle ,kindney and heart 11]. Alkaline phosphatase(ALP) are a group of enzymes with hydrolyse phosphates at an enzymes which hydrolyse phosphates at alkaline PH.The activity measured by routine methods includes that of several isoenzymes. They are found in bone, liver, kidney ,intestinal wall, Lactating mammary gland and placenta. In bone the enzyme is found in osteoblasts and is probably important for normal bone formation. In adults, the normal levels of alkaline phosphatase are derived largely from the liver. Pregnancy raises the normal rangs because of the production of a heat-stable alkaline phosphatase by the placenta[12]. Lactate dehydrogenase (LDH) catalyses the reversible inter conversion of lactate to pyruvate. 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-specific index of cell damage. The aim of the present study was to evaluate the major source of increased serum enzyme level specify these enzymes because liver and serum contain of enzymes in paratyphoid fever and to determine the most relevant clinical entity , hepatitis or myopathy , during paratyphoid fever [13]. Experimental part Subjects: This study performed on 30 female patients diagnosed by ultrasound (US) of abdomen, with paratyphoid A ranged between (20-40) years, who were selected from patients attending Baghdad Teaching Hospital in addition to 30 female healthy control . Collection of Blood samples: 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 tempreture for 15 min.,centrifuged at 3500 rpm for 10 min. The resulted serum was separated and kept frozen. Statistical Analysis Statistical analysis was performed using student t-test for comparison of variables. The p value <0.05considered significant and p<0.01considered highly signif icant. All data in the table (1) are Mean ± SD [14]. AST LD IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Determination of Serum Aspartate Transaminase (AST) Activity Serum (AST) activity was measured using colorimetric method according to (Reitman and Frankel, 15) utilizing a ready made kit for determination of serum aspartate aminotransferase. In this method, the following reaction represents measurement of AST activity -Oxoglutarate +L- Aspartate L-glutamat+ Oxaloacetate Glutamate-Oxaloocetate transaminase is measured by monitoring the concentration of Oxaloacetate hydrazone formed with 2, 4-dinitrophenyl-hydrazine. The absorbance was read at 540 nm after 5 min. Calculation :Expressed equation is : AST Activity=Test-Blank test/Standard Solution-Blank Standard×67 =µml/Min/Liter blood serum and change to International Unite Per Liter(IU/L) Normal value:AST Activity in Serum up to 12U/L [15]. Determination of S erum Alanine Transaminase( ALT) Activity: The (ALT) activity was measured using colormetric method according to (Reitman and Frankel, 15) Utilizing a ready made kit for the determination of serum alanine aminotransferase. In this method , it represents measurement of ALT activity -Oxoglutarate +L-alanine L-glutamate+ pyruvate Glutamate-pyruvate transaminase is measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4-dinitrophenyl-hydrazine . The absorbance was read at 540 nm after 5 min. Calculation :Expressed equation is: ALT Activity=Test- Blank test/Standard Solution-Blank Standard×133 =µml/Min/Liter blood serum and change to International Unite Per Liter(IU/L) Normal value: ALT Activity in Serum up to 12U/L [15]. Determination of S erum Lactate Dehydrogenase Activity (LDH) Serum (LDH) activity was measured using enzymatic colorimetric method where a ready made kit is used. The method is based on the reduction of pyruvate to located in the presence of NADH by the action of the lactate dehydrogenase: Pyruvate +NADH+H + Lactate +NAD + ALT IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 The Pyruvate that remains unchanged reacts with 2, 4- dinitrophenyl-hydrazine which is determined by calorimetric. Calculation:Enzyme activity was determined by reference to the calibration curve. Mixed and allow to stand for 20min Mixed and allow to stand at room temperature for 10 minutes, read absorbance of all tubes against distilled water at 520nmThe corresponding values in U/L are shown in the following table: Ordinate= absorbance Abscissa= activity in U/L Normal value: 80-190 U/L [16]. Determination of Alkaline Phosphates Activity Colorimetric determination of ALP activity was performed using a kit from Biomerieux, France according to the following reaction: PH=10 Phenyl phosphate Phenol + Phosphate The phenol liberated was measured in the presence of amino-4-antipyrine and potassium ferricyanide. The presences of sodium arsenate in the reagent stop the enzymatic reaction. The absorbance of the sample was measured at 510 nm against reagent blank [17]. Results and Discussions The results of serum AST, ALT, Alkaline phosphate (ALP) and Lactate dehydrogenase (LDH) in paratyphoid patients and their control group are shown in table (1).A highly significant values increase in serum AST and ALT compared to control was found while a significant increase of serum ALP compared to control was noticed and a significant values decrease in serum LDH compared to control was found. The diagnosis of fulminant hepatic failure due to S. Paratyphoid was passed mainly on two facts: First, positive blood culture for S. paratyphoid A elevated alkaline phosphatase level that agreed with this study [18]. The pathogenesis of severe hepatic involvement in Salmonella infection may be multifactorial, involving end toxin, local inflammatory and or host immune reactions [19]. Alkalines phosphatase 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 coagulopatty, damage to hepatocytes arteritis [20], direct invastion of the hepatocytes by the organisms, immune complexes and consumption of complement are believed to contribute to hepatic insult [21]. The clinical presentation and extent of hepatic dysfunction in typhoid fever would, therefore,depend upon these contributory. Factors and may or may not be associated with hepatomegaly typhoid [22]. It is concluded that presence of high fever, Jaundic and tender hepatomegaly should arouse suspicion of typhoid hepatitis IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 .Hepatic dysfunction in these cases, despite its high incidence and serious nature, is transient and responds favorably to appropriate antibiotic therapy. Myopathy during the course of typhoid fever is reported increasingly [23]. Both clinical entities, myopathy and hepatitis, are usually diagnosed by serum enzymes such as aspartate aminotransferase(AST), alanine aminotransterase(ALT), alkaline phosphatase (ALP),lactate dehydrogenase(LDH),AST and LDH are elevated in myopathy as well as hepatitis, whereas ALT are relatively specific for liver and muscle, respectively[24] . According to the original definition of typhoid hepatitis, it is possible that an over diagnosis for typhoid hepatitis occurred and typhoid myopathy was missed [25].The invasion of intestinal lymphatic tissue is suggested to result in a host reaction with hyperplasia of the liver reticuloendothelial system and infiltration of portal spaces as well as the reduction of the microcirculation causing necrosis[26].Hepatic damage by salmonella typhi appears to be mediated by bacterial endotoxin.The presence of intact bacilli has been demonstrated in the hepatic tissue that may be related to presence of Salmonella bacteria in reticuloendothelial system and hyperplasia of kupffer cell (typhoid nodule) without significant liver injury[27]. In the present study, the pattern of serum enzymes is more comparable to whate is found in myopathy (60 % ) rather than liver disease (22%) ,as the association of AST and liver enzymes [28].The higher level of serum LDH in all other age groups a mong cases is more likely due to mycobacterial induced tissue injury as well as other pathophysiological conditions , which remained undiagnosed during this study[29]. The results clearly indicate that adult have higher prevalence and susceptibility to mycobacterium infection as compared to control .Thus, serum LDH still remains as one of the important parameters to a ssess the cell/tissue specific stress or pathology, caused by mycobacterium tuberculosis the observed correlation between serum LDH level and sputum mycobacterial load alone or in combination with its individual isoformes, especially 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 patho-physiological conditions of the liver of patients [30]. Hepatic dysfunction detected by clinical and or biochemical parameters was noticed in as many as 64.5%of cases in this study which is higher than that reported by others[31]. Hepatomegaly is usually observed in enteric fever after the first week of illness, most often persists throughout the period of marked elevation of temperature, becomes less evident as defervescence progresses and usually lasts for 3-4 week. Incidence of hepatomegaly is believed to be 2-3 times more common in typhoid fever than paratyphoid fever [22]. Tender hepatomegaly observed in 2 of our cases, suggested a more severe hepatocellular involvement as both of these cases presented with jaundice. Even IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 though majority of cases with jaundice had hepatomegaly, Liver was not enlarged in one case suggesting that significant hepatic dysfunction can occur in typhoid fever without hepatomegaly. Jaundice associated with typhoid fever tends to occur at the peak of fever which differentiates it from viral hepatitis in which case fever usually comes down after the appearance of jaundice .Jaundice in most of these cases is due to typhoid hepatitis. However, hemolysis resulting in jaundice is a recognized complication of typhoid fever in patients with G-6-P deficiency or thalassemia [32].Other causes of Jaundice include ascending cholangitis, Salmonella liver abscess, suppurative pyelophlebitis and cholecystitis [21]. Conclusions From the results of present study, conclusions could be drown that liver is involved in 14 %of typhoid patients manifesting with hepatomegaly. Elevation of serum enzymes in typhoid fever is presumably of a muscular origin, while elevation of liver enzyme is relatively less common. References 1-World Health Organization.Background document.(2003)The diagnosis,treatment and prevention of typhoid fever.WHO/V&B/03.07. 2-Bhan, M.K.,Bahal. R; and Bhatnagar, S.(2005)Typhoid and paratyphoid fever lancet 366:749-762.A comprehensive review of typhoid and paratyphoid fever. 3-Bhan ,M.K;Rajiv, Bahl; and,Shinjini, Bhatnagar.(2005)"Typhoid and paratyphoid fever."The lancent 366.9487.August 27:794(14). 4-Water-related Diseases (2008) Paratyphoid fever is caused by any of three strains of Salmonella Paratyphoid. 5-Frey, J.Rebecca.(2008)Paratyphoid fever (1999) Encyclopedia of Medicine. 6-Chin J(ed).( 2000) Control of communicable Diseases Manual,17 th edition. American Puplic Health Association. 7-Rubin,Raphael;David, S. Strayer. Emanuel; Rubin ,Tay. M;and Mc Donald Rubins.(2007) Pathology.5 th ed. 8-Water-related diseases communi-cable diseases.( 2001) World Haelth. 9-Khan, F.Y;Kamha, AA;and Alomary IY.(2006)Fulminan hepatic failure caused by Salmonella Paraytphoid A infection. World J Gastroenterol;12(32) :5253-5255. 10-Crump, JA; Luby ,SP;and Mint,Z. ED.(2004)The global burden of typhoid fever . Bull World Health Organ,82:346-353. 11-Scheig, R.(1996) Evaluation of tests used to screen patients with liver disorders prim care;23:551-60. 12-Adams ,E.B. Typhoid and paratyphoid fever In.(1987)Weatherall DJ ,Ledingham JGG,Warrell DA, eds.Oxford Textbook of Medicine, vol 2,2nd Ed.Oxford : Oxford Univ press, 5:223-5.225. 13-Ginsberg , A.L.(1970) Very high levels of SGOT and LDH in patients with extrahepatic biliary tract obstruction. Am J Dig Dis;15:803-7. 14-Sorlie,D.E.(1995)Medical biostatistics &epidemiology:Examination& board review :First ed. Norwalk,Connecticut,Appleton&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) Methods in .,Mosby Company, Clinical Chemistry,< "New Dalhi, 527:543-590. 18-Kamath, P.S; Talihal, A ; and Chakraborty ,A.(2000)Differentation of typhoid fever from IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 fulminant hepatic failure in patients presenting with Joundice and encephalopathy Mayo Clin Proc, 75:426-466. 19-Forsyth ,JRL.Typhoid and paratyphoid:In collier L, Balows A,Sussman M.Toplex and Wilsons. (1998)M icrobiology and Microbial infections9 th ed. London:Arnold Press :456-478. 20-Gupta, SP; Gupta, MS;Bharadwaj, S;and Chugh, T.D.(1985)Current clinical patterns of typhoid fever:A prospective study .J Trop Med Hyg.88:377-381. 21-Kumar ,A;Kapoor, R. Chopra. K;and et al.(1989)Unusual hepatic manifestions in typhoid fever. Clinical Pediatr .2:99-100. 22-Khosla ,SN; Singh, R;Singh, GP;and et al.(1988) The spectrum of hepatic injury in enteric fever AM J Gastroenterol. 4:413-416. 23-Mody ,GM ; Gathiram, V; and Abdulla, EA.(1989)Severe reversible Myopathy due to typhoid. J.Trop Med Hyg;92:102-3. 24-Scheig, R.( 1996)Evaluation of tests used to screen patients with liver disorders. Prim Care;23:551-60. 25-Mirsadraee, M; ,Shirdel, A;and Rokneef.(2007)Typhoid myopathy or typhoid hepatitis:A matter of debate. Indian Journal of Medical Microbiology, Vol.25, No.4, October- Deceber.pp 351-353. 26-Khosia, SN;Singh, R;Singh, GP;and Trehan VK.(1988)The spectrum of hepatic injury in enteric fever. Am J Gastroenterol. 83:413-6. 27-Pramoolsinasp, C. and Viraanuvatti, V.(1998)Salmonella hepatitis. J Gasteroenterol Heptol.13:745-51. 28-Pandy, CK; Singh, N; Kumar ,V; Agarwal, A; and Singh, PK.(2002) Typhoid, hepatitis E or typhoid and hepatitis E:The cause of fulminant hepatic failure::Adiagnostic dilemma.Crit Care Med.30:376-8. 29-Nusslnovitch ,MFinkelstein .Y; Elishkevitz, KP; Volvoitz, B; Harel, D; Klinger, G ;and et al (2009)Cerebrosp inal fluid lactate dehydrogenase isoenzymes in children with bacteriala nd asep meningitis.Translational Res;154(4):214-8. 30-Yun,S.J;Choi, M.S;Piao,MS;Lee, JB;Kin, SJ; Won, YH;and Lee,SC.(2008)Serum Lactate Dehydrogenase is a novel marker for the evaluation of disease necroiysis Dermatol;217:254-9. 31-Khosla ,SN.(1990)Typhoid hepatitis. Postgrad Med J,66:923-925. 32-Thisyakorn, U;Mansuwan, P;and Taylor, DN.(1987)Typhoid and paratyphoid fever in 192 hosp italized children in Thailand.AM J Dis Child,141:862-865. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Table (1): AST, ALT, ALP and LDH levels in sera of controls and patients Parameter Control n=30 Patient n=30 P Value AST(IU/L) Mean±SD 13 ±1.05 28.7± 2.27 P< 0.01 Highly Sig ALT(IU/L) Mean±SD 12±0.52 23.2±1.25 P< 0.01 Highly Sig ALP(IU/L) Mean± SD 49.2 ±3.04 150.6± 1.32 P< 0.05 Sig. LDH (IU/L) Mean± 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 د، أبن الهیثم_كلیة التربیة،قسم الكیمیاء جامعة بغدا 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 ا بالباراتیفوئیـد30الدراسة وكانـت عالمـات (US)وتیةللجـنس األنثـوى الالتـي شخصـن بأسـتعمال األمـواج فـوق الصـ Aمریضـ ـارهن بــین 30المــرض ظــاهرة علـــیهن فضــال عــن ــیطرة ضــبط تتــراوح أعمــ ســنة وتـــم ) 40-20(مـــن األصــحاء بوصــفهن سـ ة مـدة أســابیع بعـد أكمـال العـالج للتحقـق مـن الشـفاء برهنــت 3- 2یومـا وتكرارهـا 14معـالجتهن بتنـاولهن المضـادات الحیاتیـ 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 ة فـي مســتوى الــ ـاك زیـاده معنویــة عالیــ فـي أمصــال مرضــى (ALT) ، وAST)(أظهـرت نتــائج الدراسـة ان هنـ ــیطرة Aوئیـــدالباراتیف ــة الســ ة بمجموعـ مقارنــــة (ALP)إذ لـــوحظ زیـــادة معنویـــة فــــي مستوىمصـــل الفوســـفاتیزالقاعدي ،مقارنـــ ة بمجموعــة (LDH) بمجموعـة السـیطرة، بینمـا وجـد أن هنـاك أنخفاضـا معنویــا فـي مصـل الكتیـت دي هیـدروجینیز مقارنـ .السیطرة Aفعالیة األنزیمات،أمراض الكبد ،سالمونیال باراتیفوئید: الكلمات المفتاحیة