Need for the Study s1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT
FOR DISSERTATION
1. / NAME OF THE CANDIDATEAND ADDRESS / Dr. SHANMUKA H. R.
POST GRADUATE STUDENT ,
DEPARTMENT OF BIOCHEMISTRY ,
J.J.M. MEDICAL COLLEGE ,
DAVANGERE – 577004 ,
KARNATAKA
2. / NAME OF THE INSTITUTION / J.J.M. MEDICAL COLLEGE,
DAVANGERE- 577004.
3. / COURSE OF STUDY AND SUBJECT / M.D. BIOCHEMISTRY
4. / DATE OF ADMISSION TO COURSE / 31-05-2011
5. / TITLE OF THE TOPIC / “SIGNIFICANCE OF SERUM ADENOSINE DEAMINASE AND 5'-NUCLEOTIDASE
IN ACUTE HEPATITIS”
6. / BRIEF RESUME OF THE INTENDED WORK
6.1 Need for the study :
Liver, being the largest internal organ in the body plays a central role in many essential physiologic processes including glucose homeostasis, synthesis of plasma proteins, lipids, bile acids and vitamin storage. Liver is vital in biotransformation, detoxification and excretion of a vast variety of exogenous and endogenous compounds.1
The basic physiologic response of hepatocytes to all forms of liver injury is inflammation, which is termed as hepatitis: Hepatitis simply denotes inflammation of liver applied to a broad category of clinicopathologic conditions that result from the hepatocyte damage produced by viral, toxic, metabolic, pharmacologic and/or immune mediated attack on the liver. Acute hepatitis implies a condition lasting less than 6 months, cumulating either in complete resolution of the liver damage with return to normal liver function and structure or a rapid progression of the acute injury towards extensive necrosis and a fatal outcome.2
Liver diseases are a major cause of morbidity and mortality. In India 23 people of every 1 lakh population die of liver diseases and worldwide 1 in every 40 deaths is due to liver diseases. The major cause of liver diseases in developing countries is viral hepatitis and in developed countries it is alcohol abuse & related complications.3
To detect liver diseases clinical laboratories routinely measure serum (or plasma) total bilirubin, alanine aminotransferase(ALT), aspartate aminotransferase(AST), total protein and albumin; collectively termed as liver function tests(LFT). Although termed liver function tests they are of little value in assessing the liver function per se because, they lack sensitivity as well as specificity; e.g., LFT may be normal in conditions like cirrhosis, non-cirrhotic portal fibrosis and congenital hepatic fibrosis. Conversely aminotransferases may be raised in both hepatic and cardiac conditions.4
Hence many new biochemical parameters are being investigated for their value in liver diseases. Adenosine deaminase and 5'-nucleotidase are two such parameters which have been investigated by many researchers and have been found to be useful.
Adenosine deaminase (ADA)[EC 3.5.4.4] is an enzyme that catalyses the hydrolytic cleavage of adenosine to inosine and ammonia, irreversibly. It is widely distributed in human tissues, highest being in the lymphoid tissue. The enzyme levels are significantly elevated in hepatitis especially acute viral hepatitis, active cirrhosis, infectious mononucleosis, tuberculosis and acute lymphoid leukaemia and decreased in patients with severe combined immunodeficiency.5
5'-nucleotidase (5'-NT) [EC 3.1.3.5] is a glycoprotein, present as an ectoenzyme in a variety of mammalian cells that catalyse hydrolysis of nucleotides. Its levels are elevated primarily in hepatobiliary diseases. Raised levels are found in 92% cases of obstructive jaundice, in 70% cases of parenchymal liver disease and in 81% cases of hepatic metastases.6
Irrespective of the cause the ultimate result of disease affecting liver would be damage, necrosis and cell death of hepatocytes. The present study is an attempt to investigate the significance of adenosine deaminase (ADA) and 5'-nucleotidase(5'-NT) in the evaluation of liver status in patients of acute hepatitis irrespective of the cause.
6.2 Review of literature :
A study has shown that estimation of serum adenosine deaminase and 5'-nucleotidase would be more sensitive to diagnose the patients with acute infective hepatitis in the initial stages when routine liver function tests were within the normal range.6
In older age groups, it was shown that serum adenosine deaminase appears to be a better biochemical marker for hepatitis as compared to serum ALT.7
Studies have shown that the raise in levels of serum adenosine deaminase was highest in acute viral hepatitis and active hepatic cirrhosis.8,9
In a study it was concluded that 5'-nucleotidase in alcoholic liver disease contributed as an encouraging boost along with other laboratory findings and clinical data. 5'-nucleotidase was superior to serum alkaline phosphatase as far as its sensitivity and specificity was concerned.10
Studies have shown that the activity of serum 5'-nucleotidase is consistently higher in alcohol consumers; proportionate to the extent of liver damage, hepatobiliary damage and biliary stasis.11
6.3 Objectives of the study :
a. To determine the activity of adenosine deaminase and 5'-nucleotidase in acute hepatitis.
b. To evaluate the usefulness of adenosine deaminase and 5'-nucleotidase in the diagnosis of acute hepatitis.
c. To compare the efficacy of adenosine deaminase and 5'-nucleotidase as diagnostic tools with the routine liver function tests.
7. / MATERIALS AND METHODS
7.1. Source of data
The cross sectional study will be carried out for a period of one year. The patients will be selected from Bapuji hospital and Chigateri general hospital, Davangere, both of which are attached to J.J.M. Medical College, Davangere.
These cases will be selected during the study period consecutively as and when they are presented with the following inclusion and exclusion criteria.
Inclusion Criteria
Cases: This group will include clinically proven cases of acute hepatitis in the age group of 25-60 years due to any cause.
Controls: This group will include normal age and sex matched individuals without any major illness and not on any long term medication.
Exclusion Criteria
Patients with the following diseases will be excluded.
· Infectious mononucleosis.
· Acute lymphoid leukaemia.
· Tuberculosis.
· Bone disorders.
· Diseases of ovaries in females.
· Any patient with history of drug intake that are known to be hepatotoxic.
· Diabetes, hypertension and any other long term systemic illness.
Based on inclusion and exclusion criteria a minimum of 50 cases of acute hepatitis and equal number of controls of age & sex matched individuals will be included in the present study after obtaining informed consent. A pre-tested proforma will be used to record relevant information (patient data , investigation reports, etc.,)
7.2. Method of data collection:
Under all aseptic precautions about 6 ml of venous blood will be collected in a sterile fluoride-EDTA vacutainer bulb by venepuncture using an appropriate sterile syringe. The blood samples will be allowed to clot and serum will be separated after centrifugation and analysed immediately for levels of adenosine deaminase, 5'-nucleotidase, bilirubin, AST, ALT, total protein and albumin.
A]. Estimation of serum adenosine deaminase by the method of Giusti and Galanti12
Principle: Enzyme adenosine deaminase catalyses the hydrolytic cleavage of adenosine to inosine and ammonia. The ammonia produced is determined by Chaney and Marbach modification of Berthelot’s reaction. Ammonia forms intensely blue coloured indophenol with sodium hypochlorite and phenol in alkaline solution. Sodium nitroprusside is the catalyst. The ammonia concentration is directly proportional to absorbance of indophenol, which is measured by spectrophotometry at 620 nm against water.
adenosine deaminase
Adenosine + H2O Inosine + NH4+
B]. Estimation of 5'-nucleotidase by kinetic method13
Principle: The 5'-nucleotidase assay is based on the enzymatic hydrolysis of Inosine 5'-monophosphate (5'-IMP) to form inosine which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then converted to uric acid and hydrogen peroxide (H2O2) by xanthine oxidase(XOD). H2O2 is further reacted with N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to generate quinone dye which is monitored in a kinetic manner. The entire enzymatic reaction scheme is shown below.
Inosine monophosphate + H2O Inosine + Pi
Inosine + Pi Hypoxanthine + Ribose-1-phosphate
Hypoxanthine + 2H2O + 2O2 Uric acid + 2H2O2
2H2O2 + 4-AA + EHSPT 4H2O + Quinone dye
One unit of 5'-nucleotidase is defined as the amount of 5'-nucleotidase that generates 1 µmole of inosine from inosine monophosphate per minute at 37°C.
C]. Estimation of serum total, direct and indirect bilirubin14
i. Serum total bilirubin by photometry
Principle: Total bilirubin in serum is determined using Jendrassik and Grof method by coupling with diazotised sulfanilic acid after addition of caffeine, sodium benzoate and sodium acetate. A blue azobilirubin is formed in alkaline Fehling’s solution ΙΙ, which is measured photometrically.
ii. Serum Direct bilirubin by photometry
measured as a red azo dye at 546 nm by the method of Schellong and Wende without the addition of alkali.
iii. Serum Indirect bilirubin
is calculated from the difference between total and direct bilirubin.
D]. Estimation of serum Alanine aminotransferase(ALT) by U.V. kinetic method15
Principle: ALT catalyses the transfer of an amino group between L-alanine and α-ketoglutarate to form Pyruvate and L-Glutamate. The Pyruvate then reacts with NADH in the presence of malate dehydrogenase to form NAD.
L-Alanine + α-Ketoglutarate Pyruvate + L-Glutamate
Pyruvate + NADH + H+ L-Lactate + NAD+
The rate of NADH oxidation is directly proportional to the catalytic activity of ALT. It is determined by measuring the decrease in absorbance at 340 nm.
E]. Estimation of serum Aspartate aminotransferase(AST) by U.V. kinetic method15
Principle: AST catalyses the transfer of an amino group between L-aspartate and α-ketoglutarate to form oxaloacetate and L-glutamate. The Oxaloacetate then reacts with NADH in the presence of Malate dehydrogenase to form NAD.
L-Aspartate + α-Ketoglutarate Oxaloacetate + L-Glutamate
Oxaloacetate + NADH +H+ L-Malate + NAD+
The rate of NADH oxidation is directly proportional to the catalytic activity of AST. It is determined by measuring the decrease in absorbance at 340 nm.
F]. Estimation of serum Total protein by Biuret method16
Principle: Serum proteins form a coloured complex in the presence of Copper salt in alkaline solution.
Divalent copper reacts with the peptide bonds of proteins under alkaline conditions to form a characteristic violet blue coloured complex. Sodium-potassium tartarate prevents copper hydroxide precipitation and potassium iodide prevents the autoreduction of copper.
Proteins + Cu2+ Coloured complex
The colour intensity is directly proportional to the protein concentration. It is determined by measuring increase in absorbance at 540nm.
G]. Estimation of serum albumin by colorimetric bromocresol green method17
Principle: Albumin at a pH of 4.2 is sufficiently cationic to bind to anionic dye bromocresol green (BCG) to form a blue green coloured complex.
Albumin + Bromocresol green Albumin-BCG complex
The intensity of the blue green colour is directly proportional to the concentration of albumin in the sample. It is determined by monitoring the increase in absorbance at 546 nm.
H]. Calculation of albumin/globulin ratio
Globulin value is calculated by the formula, Globulin = Total protein albumin.
Using the values so obtained albumin/globulin ratio is calculated.
STATISTICAL ANALYSIS :
Results will be subjected for appropriate statistical analysis. Unpaired-t test will be used to compare the different parameters between two groups. Diagnostic validity tests will be performed to assess the diagnostic value of adenosine deaminase and 5'-nucleotidase for acute hepatitis.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes
Estimation of serum adenosine deaminase, 5'-nucleotidase, bilirubin, ALT, AST, total protein and albumin using human venous blood sample in patients and healthy controls.7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes
8. / LIST OF REFERENCES :
1. Gary A. Abrams, Michael B., Laboratory tests in liver diseases. In Thomas E. Andreoli, Charles C.J. Carpenter, Robert C. Griggs, Ivor J. Benjamin. Andreoli and Capenter’s Cecil essentials of Medicine.7thed. New York. Saunders Elsevier publishers ;2008;40;p433
2. Aasim M. Sheikh, Michael B. Fallon, Acute and chronic hepatitis. In Thomas E. Andreoli, Charles C.J. Carpenter, Robert C. Griggs, Ivor J. Benjamin. Andreoli and Capenter’s Cecil essentials of Medicine. 7thed. New York. Saunders Elsewier publishers; 2008;42;p441.
3. G. Webster , J. D. Collier, Liver and Biliary Tract disease. In Nickie R. Colledge, Brian R. Walker, Stuart H. Ralston, Davidson’s Principles and Practice of Medicine. 21st ed. China. Churchill Livingstone Elsvier.2009;23;p922.
4. Neil MeIntyre, Sidney rosalki, Biochemical investigations in the management of liver disease. In Neil MeIntyre, Jean-Pierre Benhamon, Oxford Textbook of Clinical Hepatology.ed. Oxford. Oxford University press.(vol-1);1991;6.1; 293.
5. J.P.J. Ungerer, H.M. Oosthuizen, S.H. Bissbort, W.J.H. Vermaak, Serum adenosine deaminase: Isoenzymes and diagnositc application. Clin. Chem;1992;38/7;1322-26.
6. K. Pratibha, Usha Anand, Rajni Agarwal. Serum adenosine deaminase, 5'-nucleotidase and malondyaldehyde in acute infective hepatitis. Indian Journal of Clinical Biochemistry. 2004; 19 (2); 128-131.
7. K.C. Vasudha, A. Nirmal Kumar, T. Venkatesh. Studies on the age dependent changes in serum adenosine deaminase activity and its changes in Hepatitis. Indian Journal of Clinical Biochemistry. 2006 (1); 116-120.
8. Selcuk Kaya, EmelSesli, Cetin, Buket cicoglu aridogan, Salih arikan, Muatafa demirci. Adenosine deaminase activity in serum of patients with hepatitis-a useful tool in monitoring clinical status. Journal of Microbiology, Immunology and Infection. 2007;40;288-292.
9. Sanchez Rodriguez A, Hueso Perez J, et al., An Medica Interna,1989, Jun 6(6);300-4.
10. Anil Batta, Comparative study of serum of serum 5' nucleotidase, alkaline phosphatase, aminotransferases and bilirubin in alcoholic liver disease, International Journal of Biological and Medical Research. 2011; 2(1);439-42.
11. Tarannum. F. Subhani, Mohammed. A. Nasar, et. al., 5'-Nucleotidase, Oxidative stress and antioxidant status in alcohol consumers and cirrhotic patients.2009;19(3):277-286.
12. Giusti. G (1974). Adenosine deaminase In :H.U. Bergmeyer(ed). Methods of enzymatic analysis, 2nd edition, New York. Verlag Chemie,Weinheim and academic press;1092-1099.
13. Victor B. Bethune, Martin Fleisher and Morton K. Schwartz., Automated method in determination of serum 5'-nucleotidase activity. In Clinical chemistry, vol-18; no 12; 1972.
14. Trefor Higgins, Ernest Beutler and Basil T. Doumas., Haemoglobin, Iron and Bilirubin. In Carl A. Burtis, Edward R. Ashwood and Davis E. Bruns. Teitz textbook of Clinical chemistry and molecular diagnostics.4th ed. New York. Saunders Elsevier publishers;2005;31;p1195-1198.
15. Mauro Panteghini, RenzeBais and Wouter W. van soling., Enzymes. In Carl A. Burtis, Edward R. Ashwood and Davis E. Bruns. Teitz textbook of Clinical chemistry and molecular diagnostics.4thed. New York. Saunders Elsevier publishers;2005;21;p604-607.