RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT

FOR DISSERTATION

1. NAME OF THE CANDIDATE Dr. SHANMUKA H. R.

AND ADDRESS 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 M.D. BIOCHEMISTRY
SUBJECT
4. DATE OF ADMISSION TO 31-05-2011
COURSE

“SIGNIFICANCE OF SERUM ADENOSINE

5. TITLE OF THE TOPIC
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.

MATERIALS AND METHODS

7.
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.

5'-nucleotidase
Inosine monophosphate + H2O Inosine + Pi

purine nucleoside phosphorylase

Inosine + Pi Hypoxanthine + Ribose-1-phosphate

xanthine oxidase
Hypoxanthine + 2H2O + 2O2 Uric acid + 2H2O2

peroxidase
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.

alanine transaminase
L-Alanine + α-Ketoglutarate Pyruvate + L-Glutamate
lactate dehydrogenase
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.

aspartate transaminase
L-Aspartate + α-Ketoglutarate Oxaloacetate + L-Glutamate
malate dehydrogenase
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 :
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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. 7 thed. 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.
16. A. Myron Johnson., Amino acids, peptides and proteins. 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;20;p586-588.
17. A. Myron Johnson., Amino acids, peptides and proteins. In Carl A. Burtis, Edward
R. Ashwood and Davis E. Bruns. Teitz textbook of Clinical chemistry and
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publishers;2005;20;p546-548.
 9. SIGNATURE OF THE CANDIDATE

10. REMARKS OF THE GUIDE The study helps in the early diagnosis of
acute hepatitis.

11. NAME & DESIGNATION.

Dr.MANJUNATH M . TEMBAD
11.1 GUIDE M.B.B.S.,M.D.

PROFESSOR,
DEPARTMENT OF BIOCHEMISTRY,
J.J.M. MEDICAL COLEGE,
DAVANGERE.

11.2 SIGNATURE

11.3 CO-GUIDE Dr. GURUSHANTHAPPA S .

M.B.B.S.,M.D.
PROFESSOR,
DEPARTMENT OF MEDICINE,
J.J.M. MEDICAL COLLEGE,
DAVANGERE

11.4 SIGNATURE

11.5 HEAD OF THEDEPARTMENT Dr. D.S. JAYAPRAKASH MURTHY

B.Sc., M.B.B.S.,M.D.

PROFESSOR & HEAD,

DEPARTMENT OF BIOCHEMISTRY,
J.J.M. MEDICAL COLLEGE,
DAVANGERE.

11.6 SIGNATURE

12. REMARKS OF THE CHAIRMAN &

PRINCIPAL

12.1 SIGNATURE