مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Estimation of ALP, GPT and GOT Activities in Iraqi Patients Female With Breast Cancer Z. I. AL-Mashhadani , A. J. A. Mukhlis,*A. S. A-Razaq AL-Faraji Department of Chemistry, College of Education Ibn- ALhaitham, University of Baghdad. *Center for Market Research & Consumer Protection, University of Baghdad Received in : 24 July 2011 Accepted in : 18 October 2011 Abstract To investigate the activity and role of certain enzyme markers in 30 patients female with breast cancer (non-treated, treated, and treatment with recovered).The serum activity of enzyme tumor markers (ALP, GPT and GOT) of (30) patients with breast cancer, and (7) healthy control subjects by using statistical analysis: There is significant difference higher in activity of serum enzyme tumor markers (ALP, GPT, and GOT) in all patients as compared with healthy control. Key word: Breast Cancer, Enzyme tumor markers. Introduction Breast Cancer: Cancer is a disease in which cells become abnormal and form more cells in an uncontrolled way[1]. With breast cancer, the cancer begins in cells that make up the breast (usually in the tubes that carry milk to nipple or the glands that make milk). The cancerous cells form a mass of tissue called a malignant tumor that starts from cells in the breast. Sometimes, the cancer spreads to other parts of the body[1,2]. The disease occurs mostly in women, but men can get breast cancer as well. Breast cancer is the most common cancer among women, other than skin cancer. It is the second leading cause of cancer death in women, after lung cancer[1-4]. About one in eight women will be diagnosed with breast cancer during their lifetime. Breast cancer also strikes men but in much lower numbers (1 in 100)[4]. There are several types of breast cancer, although some of them are quite rare. In some cases a single breast tumor can have a combination of these types or have a mixture of invasive and in situ cancer which are[5]: 1- Ductal carcinoma in situ. (DCIS). 2- Lobular carcinoma in situ. (LCIS). 3- Invasive (or infiltrating) ductal carcinoma. (IDC). 4- Invasive (or infiltrating) lobular carcinoma. (ILC). Alkaline phosphatase (ALP) (Ec: 3.1.3.1): Alkaline phosphatase (ALP) is ahydrolase enzyme responsible for removing phosphate group from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation[6]. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase[7]. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 In humans, alkaline phosphatase (ALP) is found in many tissues, including bone, liver, intestine, kidney, and placenta [8]. High (ALP) usually means that the bone or liver been damaged[9]. The normal range is 20 to 140 IU/L[10]. Alanine Transaminase (ALT) or (GPT) (Ec: 2.6.1.2): Alanine transaminase or ALT is a transaminase enzyme. It is also called serum glutamic pyruvic transaminse (SGPT) or alanine aminotransferase (ALAT). ALT is found in serum and in various bodily tissues, but is most commonly associated with the liver. It catalyzes the two parts of the alanine cycle[11]. Significantly elevated levels of ALT often suggest the existence of other medical problems such as viral hepatitis, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy[11]. Reference range of ALT : 6-37 U/L[10]. Aspartate Transaminse (AST) or (GOT) (Ec: 2.6.1.1): Aspartate transaminase (AST) also called serum glutamic oxaloacetic transaminase (SGOT) or aspartate aminotransferase (ASAT/AAT/AspAT) is similar to alanine transaminase (ALT) in that it is another enzyme associated with liver parenchymal cells[12]. ALT is found predominately in the liver, with lesser quantities found in the kidneys, heart, and skeletal muscle. As a result the ALT is a more specific indicator of liver inflammation than the AST, as the AST may also be elevated in diseases affecting other organs, such as the heart or muscles in myocardial infarction, also in acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, musculoskeletal diseases, and trauma[13]. AST (SGOT) is commonly measured clinically as a part of diagnostic liver function tests, to determine liver health. Reference ranges of AST:6-34 IU/L[14]. The aim of our study is to evaluate some biochemical parameters like enzymes as tumor biomarker (i.e. ALP, GPT, GOT). In Iraqi patients suffering from breast cancer and compared that with the same parameters in normal healthy Iraqi control. Material and Methods Patients: Blood samples were collected from a thirty breast cancer (females) (Non- treated (8) female, treated (12) female, treated and recovered (10) female), their age was range (19-60) years at the Medical City Hospital in Baghdad. Seven apparently healthy individuals (females) were selected with age range (20-43) years. Ten milliliters (ml) of venous blood were collected into plain tubes from each patient and healthy individuals after 12 hours fast. The blood samples were allowed to stand for 15 minutes (min) then centrifuged at 3500 rpm for 10 min. Serum was frozen at -20 ºC till used for the estimation of ALP, GPT, GOT. Determination of Some Enzyme Markers: Determination of Alkaline Phosphatase (ALP Activity): Colorimetric determination[15,16] of the ALP activity which reaction scheme is as follows: Phenyl phosphate Alkaline Phosphatase Phenol + Phosphate Free phenol librated by hydrolysis of the substrate reacts then with 4-amino-antipyrine in the presence of alkaline potassium ferricyanide to form a red-coloured complex which absorbance measured at 510nm is directly proportional to the ALP activity in the specimen. Sodium arsenate in corporated in the reagent abolishes further enzyme activity and prevents the dilution of the colour inherent in earlier methods. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Calculation (kind and k ing units/100ml) . - . = 20 . standard A bs Assay A bs specimen blank ALP activity A bs  Determination of Aminotransferases (ALT) or (GPT) Activity: Colorimetric determination[17-19] for GPT activity according to the following reactions: GPT Alanine ketoglutarate Pyruvate glutamate    The pyruvate formed is measured in its derivated form, 2,4-dinitrophenylhydrazone at 505nm. Calculation number of GPT units/ml in serum were calculated using the standard curve. Determination of Aminotransferases (AST) or (GOT) Activity: Colorimetric determination[17-19] of GOT activity according to the following reactions: GOT L A spartate ketoglutarate Oxaloacetic L glutamate      The oxaloacetate formed is measured in its derivated form, 2,4- dinitrophenylhydrazone at 505nm. Calculation The number for GOT units/ml in serum were calculated using the standard curve. Results and Discussion Results Serum Enzyme Markers Levels: Serum Alkaline Phosphatase (ALP) Activity: Data in Table (1) and Fig. (1) show that, the mean ± SD of ALP activity in serum of control was (16.6565 ± 5.7695 IU/L). While the mean ± SD of ALP activity in serum of patient with breast cancer ( No treatment) was (38.4583 ± 27.4304IU/L) and for (treatment and treatment & recancer) were (56.8010 ± 23.6085IU/L) and (47.6151 ± 29.0696IU/L) respectively. The results showed no significant difference (P  0.05) in serum of ALP activity of Breast Cancer patient's no treatment, treatment and treatment & recancer as compared with healthy control. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig.(1) of our results shows that no treatment breast cancer patients female was two times high in ALP activity, while treatment patients high four times and treatment with recancer was three times high activity than healthy control. Serum Alanine Transaminase (ALT) or (GPT) Activity: Table (2) and Fig. (2) shows that, the Alanine transaminase (ALT) or (GPT) activity in serum of control is (19.2500 ± 2.4749 units/ml). While the (ALT) activity in serum of breast cancer patient no treatment is (24.7000 ± 13.9803 units/ml) and treatment and treatment & recancer patient's breast cancer were (28.9143 ± 22.0185 units/ml) and (21.8200 ± 12.4333 units/ml) respectively. The results showed that there is no significant difference (P  0.05) in serum ALT activity of Breast Cancer patient compared with that of healthy control. Our results from the Fig.(2) show that no treatment was high one time in ALT activity , while treatment female Breast cancer patients was two times higher in activity , and treatment with recancer half time higher than healthy control. Serum Aspartate Transaminase (AST) or (GOT) Activity Data in Table (3) and Fig. (3) show that the mean ± SD of aspartate transaminase (AST) or (GOT) activity in serum of control was (28.75 ± 3.8891units/ml). While the mean ± SD of AST activity in serum of patient with breast cancer (No treatment, treatment, and treatment & recancer) were (38.3667±17.5342units/ml), (30.0750 ± 5.6076 units/ml), and (50.6167 ± 25.6691units/ml) respectively. The results showed no significant difference (P  0.05) in serum of AST activity of all breast cancer patients as compared with healthy control. Our study from the Fig.(3) shows that the no treatment patients of breast cancer was higher AST activity one time than the control and high in treatment, while in treatment with recancer is higher two times compared with healthy control. Discussions Alkaline phosphatase (ALP) Activity: Our results demonstrate that none of our patients with total-ALP activities lower than the control presented metastases. On the contrary, patients with bone metastases show a significantly increased total-ALP activity. This finding is in agreement with data from other authors[20]. It has been demonstrated that total-ALP is highly sensitive to detect hepatic metastases[20]. Our patients with hepatic or bone metastases show the highest total-ALP activity and there are no significant differences of total-ALP activity between them. According to our results, total-ALP could not differentiate between hepatic and bone metastases and it is necessary to take into account bone-ALP values that, in bone metastases[21]. Alanine transaminase (ALT) or (GPT) Activity: Alanine transaminase was significantly high in patients with lymph node metastasis at disease p resent at ion as comp ared t o cont rol. T his sugges t s t he role of alanine aminotransferase in relation to axillary lymph node metastasis and disease prognosis. The pathogenesis of drug induced liver disease usually involve the participation of the parent drug or its metabolites that either directly effect the cell biochemistry or elicit an immune response. Susceptibility to drug induced hepatotoxicity is also influenced by genetic and environmental risk fact ors. Unpredict able, low frequency, iodiosyncratic react ions often occur on a background of a higher rate of mild asymptomatic liver injury, it is very difficult to detect t hem but t hey may be detected by monitoring serum aminotransferase levels[22]. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Some trials suggest biochemical evaluation as the better tool for the screening of metastatic disease at the time of diagnosis of breast cancer[23,24]. In these trials ALT is suggested as first-line examination to detect liver or bone metastases. Aspartate Transaminase (AST) or (GOT) Activity: Aspartate transaminase (AST) is familiar markers of liver function and to ascertain the non-involvement of systemic toxicity , the activities of marker enzyme like Aspartate transaminase (AST) was assayed. In our study, the AST levels is higher in cancer when compared to normal control[24]. As a marker for liver metastases in breast cancer patients and also as a marker for hepatotoxicity , aspartate transaminase was found to be increased. It was concluded that the elevations in the activities could be due to the decreased synthesis of degradative products which could have resulted in the elevation in the circulation. Elevations in liver transaminases following tamoxifen administration have been observed. Tissue damage is the sensitive feature in the cancerous conditions so any deterioration or destruction of the membrane can lead to the leakage of these enzymes from the tissues. Hence elevation of these liver specific enzymes observed in breast cancer condition may be due to the progression of tumor growth[25]. Conclusion Our results suggest that ALP, GPT, and GOT may play an important role in breast cancer. A significant elevation in the activity of ALP, GPT, and GOT in female breast cancer. Recommendation Although, some of our data were statistically no significant, our interpretation from thes results indicate that the chemotherapy are unusefull in treatment of breast cancer. References 1. Singh, S.P., (2008). Text book of Biochemistry, 4th Ed., CBS publishers & Distributors, New Delhi, Bangalore. 2. Harris, L.; Fritsche, H.; Mennel, R.; (2007). American society of clinical Oncology 2007 up date of recommendations for the use of tumor markers in breast cancer. J. clin. Oncol., 25:5287-5312. 3. Foubert, E.; Craene, B. and Berx, G.(2010) The Snail 1- Twist 1 Conspiracy in malignant breast Cancer progression. Breast Cancer Research, 12:206. 4. Diedrich, J.; Depke, J. and Engel, J. (2007) An overview and update of breast cancer Screening, diagnosis, and treatment. American Nurse Today 2(10): 32-37. 5. Diamandis, E.P.; Hoffman, B.R. and Sturgeon, C.M. (2008). National Academy of clinical Biochemistry Laboratory Medicine Practical Guidelines for the use of Tumor Markers. Clin. Chem. 54:1935-1939. 6. Kim, E.E.and Wyckoff, H.W. (March 1991). “Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis”. J. Mol. Biol. 218(2): 449-464. 7. Tamas, L.; Huttova, J.; Mistrk, I.and Kogan, G. (2002). “Effect of carboxymethyl chitin- Glucan on the Activity of Some Hydrolytic Enzymes in Maize Plants”. Chem. Pap. 56(5): 326-329. 8. Enders, D.B.and Rude, R.K. (1999) Mineral and Bone metabolism. In Burtis CA, Ashwood E.R.eds. Tietz text book of clinical chemistry, 3 rd ed. Philadelphia, W.B., Saunders, P: 1395-1457. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 9. Posen, S. and Doherty, E. (2010). The measurement of serum alkaline phosphates in clinical medicine. Adv. Clin. Chem., 22:165. 10. Bishop, M.L.; Fody, E.P.and Schoeff, L.E. (2010). Clinical chemistry, techniques, principles, correlations by lippincott Williams & Wilkins. 11. Paul, T.and Giboney M.D. (2010). Mildly Elevated liver Transaminase levels in the Asymptomatic Patient. American Family physician. 12. Almo, S.C.; Smith, D.L.; Danishefsky, A.T.and Ringe, D. (M arch 1994). “ The structural basis for the altered substrate specificity of the R292D actine site mutant of aspartate aminotransferase from E.Coli ”. Protein Eng. 7(3):405-12. 13. Lott, J.A. and Stang, J.M . (2010). Serum enzymes and isoenzymes in the diagnosis and differential diagnosis of myocardial ischemia and necrosis. Clin. Chem., 26:1241. 14. Remaley, A.T. and Wilding, P. (2009). Macroenzymes: biochemical characterization, clinical significance and laboratory detection. Clin. Chem., 35:2261-2270. 15. Kind P.R.N., King E.J.,(1954). Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-anti-antipyrine, J. clin. Path., 7:.322-326. 16. Belfield, A.and Goldberg, D.M . (1971)Revised assay for serum phenyl phosphatase activity using 4-amino-antipyrine, Enzyme, 12: 561-573. 17. Britman, S.and Frankel, S. (1957) Am.J.clin-Path., 28:56. 18. Cabaud et al. (1956). Am. J. Clin. Path, 26:1101. 19. Karmen, A. (1955)J. clin. Invest. 34:141,. 20. Moss, D.W. (1989). Alkaline phosphatase in hepatobiliary disease. Progr. Clin. Biochem. Med. 8:47-62. 21. Yorioo, M.A.; Sembaj, A.and Sanz, E. (2000). Alkaline phosphatase isoenzymes for the diagnosis of metastatic tumors and lymphomas of liver and bone. Medicina, 60:311-315. 22. Raza, V.; Khanam, A.; Naimatullah, S. (2010). Evaluation of Non-Heamatological and Hepatic Toxicities Developed During Chemotherapy Treatment And Role of Liver Enzymes In Disease Prognosis. Journal of Biochemistry, 16(1): 10-16. 23. Ravaioli, A.; Tassinari, D.and Pasini, G. (1998). Staging of breast cancer: what standard should be used in research and clinical practice? Ann. Oncol. 9:1173-1177. 24. Alcazar, J.L.; Rolle, A.and Murcia, J.M . (1995). Bone scanning in preoperative staging of early breast cancer patients:shouldit be aroutine procedures? Breast Dis, 8:7-11. 25. Vennila, R.; Thirunavukkarasu, S.V. and Muthumary, J. (2010). In vivo studies on Anticancer Activity of Taxol Isolated from An Endophytic fungus pestalotiopsis pauciseta SACC. VM1. Asian Journal of pharmaceutical and clinical research 3(4):30-34. Table(1): Alkaline phosphatase (ALP) Activity (IU/L) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. Groups N Mean SD t-test No treatment 8 38.4583 27.4304 P  0.05 Treatment 12 56.8010 23.6085 P 0.05 Treatment & recancer 10 47.6151 29.0696 P  0.05 Control 7 16.6565 5.7695 P<0.05 مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Table(2): Alanine transaminase (ALT) or (GPT) Activity (units/ml) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. Table(3): Aspartate transaminase (AST) or (GOT) Activity (units/ml) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. Groups N Mean SD t-test No treatment 8 38.3667 17.5342 P  0.05 Treatment 12 30.0750 5.6076 P 0.05 Treatment & reccancer 10 50.6167 25.6691 P  0.05 Control 7 28.7500 3.8891 P<0.05 Groups N Mean SD t-test No treatment 8 24.7000 13.9803 P  0.05 Treatment 12 28.9143 22.0185 P 0.05 Treatment & recancer 10 21.8200 12.4333 P  0.05 Control 7 19.2500 2.4749 P<0.05 Fig. (1): Alkaline phosphatase (ALP) Activity (IU/L) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig.(2):Alanine transaminase (ALT) or (GPT) Activity (units/ml) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. Fig.(3):Aspartate transaminase (AST) or (GOT) Activity (units/ml) in serum of Patients with Breast Cancer (no treatment, treatment, and treatment & recancer) and healthy control. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 في GOT و GPT واالمینوترانسفیرز ALPتقییم الفوسفات القاعدي ت المصابات بمرض سرطان الثديالمریضات العراقیا علیاء سعدون عبد الرزاق الفراجي*،عبد الجبار عبد القادر مخلص ،زهیر ابراهیم المشهداني جامعة بغداد، ابن الهیثم –كلیة التربیة ، قسم الكیمیاء د، مركز بحوث السوق وحمایة المستهلك * جامعة بغدا 2011 تشرین األول 18:حث في قبل الب2011 تموز 24:استلم البحث في الخالصة .لغرض بیان دور الفعالیة وبعض األنزیمات الواسمة في المرضى المصابین بسرطان الثدي فعالیــة تقیـسو). بعـالج مـع رجـوع المـرض، بعـالج ، بـدون عـالج (مـصابة بـالمرض ) 30( عینـات تتـألف مـن ت اخـذ نـساء ) 7(مریـضة و ) 30(فـي مـصل ) GOT و GPT واالمینـو ترانـسفیرز ALPعدي الفوسفات القا(األنزیمات الواسمة كانت نتـائج البحـث للمجـامیع المـذكورة باسـتخدام التحلیـل اإلحـصائي . مجموعة سیطرة خالیة من أي مرضبوصفهن تطوعن فـي جمیــع المرضــى مقارنــة (GOT, GPT, ALP)حــدوث ارتفـاع معنــوي فــي فعالیــة األنزیمـات الواســمة : يأتكمـا یــ . باألصحاء .أنزیمات واسمة ورمیة ، سرطان الثدي : الكلمات المفتاحیة مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012