AST and ALT belong to the group called
Transaminase / Aminotransferase
AST – Aspartate Transaminase
SGOT – Serum Glutamate Oxaloacetate
Transaminase
ALT – Alanine Transaminase
SGPT – Serum Glutamate Pyruvate
Transaminase
ESTIMATION OF AST
Aim of the experiment –
To estimate the amount of AST present in the
supplied serum sample by UV kinetic
method.
Apparatus required-
Test tubes
Micropipette
semiautomated analyser
Chemicals required-
Reagents
Supplied serum sample
Composition of reagent
REAGENT 1: R1
Tris-buffer, pH 7.80
L-Aspartate
LDH
MDH
Sodium Azide
REAGENT:R2
Alpha ketoglutarate
NADH
Sodium Azide
PRINCIPLE
AST
L-aspartate + α –ketoglutarate <------------->
Oxaloacetate + L-glutamate
MDH
Oxaloacetate +NADH +H+<---------->L-malate
+ NAD+
PRINCIPLE
α –ketoglutarate reacts with L-aspartate in presence of
aspartate transaminase to form oxaloacetate and L-glutamate.
The increase in oxaloacetate is determined in an indicator
reaction catalyzed by malate dehydrogenase.
The conversion of NADH to NAD+ at 340nm is proportional to the
activity of AST in serum/plasma and is determined kinetically as
rate of decrease in absorbance.
PROCEDURE
RESULT
The supplied serum sample contains
___________ U/L AST.
INTERPRETATION
Normal level of AST : 8-40 U/L
INCREASE AST LEVEL
Myocardial infarction
Acute and chronic hepatitis
Liver cirrhosis
Hepatoma
NASH(Non Alcoholic Steato Hepatitis)
Non alcoholic fatty liver
Muscular dystrophy
DECREASE AST LEVEL – Vit B6 deficiency
ESTIMATION OF ALT
Aim of the experiment –
To estimate the amount of ALT present in the
supplied serum sample by UV kinetic
method.
Apparatus required-
Test tubes
Micropipette
semiautomated analyser
Chemicals required-
Reagents
Supplied serum sample
PRINCIPLE
➢ α –ketoglutarate reacts with L-alanine in presence
of ALT to form pyruvate & L-glutamate.
➢ The increase in pyruvate is determined in an
indicator reaction catalyzed by lactate
dehydrogenase.
➢ The conversion of NADH to NAD+ at 340nm is
proportional to the activity of ALT in serum/plasma
and is determined kinetically as rate of decrease in
absorbance.
PROCEDURE
RESULT
The supplied serum sample contains
___________ U/L ALT.
INTERPRETATION
Normal level of ALT : MEN < OR equal to 45 U/L
WOMEN < OR equal to 34 U/L
Increase ALT
Alcoholic liver disease
-Liver disease –(viral- acute and chronic
hepatitis)
-Toxic hepatis- drugs like ccl4, NSAID,
-ATT like ISONIAZID , Antibiotics like
-AMOXYCILLIN AND ERYTHROMYCIN
-Cirrhosis of liver
Clinical Significance of AST/ALT Ratio
Normal AST: ALT ratio is 0.8. A ratio >2 is seen in:
o Alcoholic hepatitis
o Hepatitis with cirrhosis
o Nonalcoholic steatohepatitis (NASH)
o Liver metastases
o Myocardial infarction
o Erythromycin treatment
ALT higher than AST is seen in:
o Acute hepatocellular injury
o Toxic exposure
ALKALINE PHOSPHATASE
(ALP)
Removes phosphate group
Hydrolyses aliphatic, aromatic or
heterocyclic compounds
pH : 9-10
Activated by Mg, Mn
Zn is an important constituent of ALP
ESTIMATION OF SERUM ALP BY KINETIC
METHOD
AIM : To estimate the amount of alkaline
phosphatase(ALP) present in the supplied serum
sample.
APPARATUS REQUIRED : Test tube, micropipette,
semiautomated analyser
CHEMICAL REQUIRED : Working reagent, serum sample
PRINCIPLE
➢ ALP cleaves p-nitrophenyl phosphate (p-NPP)
into p-nitrophenol & phosphate.
➢ p-nitrophenol is a yellow colour compound
formed in alkaline medium & absorbs light at
405nm.
➢ The rate of increase in absorbance at 405nm is
proportional to alkaline phosphatase activity in
serum.
p-nitrophenyl phosphate ------->
ALP p-nitrophenol + inorganic
phosphate
PROCEDURE
RESULT
The supplied serum sample contains
___________ U/L ALP.
INTERPRETATION
Normal level of ALP : MEN- <270U/L
WOMEN- <240U/L
CHILDREN - <645U/L (below 14 yrs of
age)
Causes of ↑ ALP :
Physiological – growing children, old age, late pregnancy
Pathological – Rickets
Osteomalacia
Paget’s disease of bone
Healing phase of bone fracture
Obstructive jaundice
Hepatitis
ESTIMATION OF TOTAL AND
DIRECT BILIRUBIN
Aim:
To estimate total and direct bilirubin in the given serum sample using
Malloy-Evelyn modified, end point method.
PRINCIPLE: -Sulfanilic acid reacts with sodium nitrite to
form diazotized sulfanilic acid.
- In the presence of accelerator cetrimide, conjugated
and unconjugated bilirubin react with diazotized sulfanilic
acid to form azobilirubin (Bilirubin total)
-In the absence of accelerator ,only conjugated bilirubin
reacts(bilirubin direct).
-The increase of absorbance at 550 nm is proportional to
bilirubin concentration
PROCEDURE
NORMAL RANGE/
REFERENCE RANGE
Direct bilirubin- 0.1- 0.4 mg/dl
Indirect bilirubin- 0.2-0.7 mg/dl
Total bilirubin- 0.3 to1.1 mg/dl
Total bilirubin > 2.0 mg/dl - Jaundice
Latent jaundice 1-2 mg/dl
