Estimation of AST, ALT

Estimation of enzymes
What do we measure ??????????????
Concentration Vs Catalytic activity
How do we measure enzymes
 Estimation of enzymes
End point method Rate of reaction method

Accuracy and precision are not good Accuracy and precision are good

Lengthy procedures requires 30 Very short procedures requires 2 to 3

minutes to 2 hours minutes
Can be done in ordinary colorimeters Requires special equipments such as
spectrophotometers ,semiauto or
autoanalyzers
Reagents can be prepared in Diagnostic kits required
laboratory
cheap expensive
 Estimation of enzymes
How do we measure enzymes ????
End point method Vs Rate of reaction method
 Aspartate transaminase
• Enzyme that catalyses the transfer of amino groups
between amino and keto acids.
• Also known as SGOT (serum glutamate oxaloacetate
transaminase)
• Found in greater amounts in heart and liver and
skeletal muscles
• Found in lesser amounts in kidneys, pancreas, RBC.
• PLP acts as a coenzyme
• Present in two forms- mitochondrial and cytoplasmic
• Half life 17 hours
 Estimation of AST
• Aim: to estimate the amount of AST in the
given sample
• Method: Karmen method (IFCC Method)
• Principle:
Specimen handling:
 Avoid hemolysis
 Stable for :
4 days at room temperature
10 days at 2-8 degree celsius
Two weeks at -20 degree celsius
Working reagent :
 L-aspartic acid, α-ketoglutaric acid, LDH, MDH,
NADH, buffer,Stabilizers.
• Procedure : (kinetic method using factor)
Adding sequence Test
Working reagent 1 ml
sample 0.1 ml

Mix well and incubate

 find the initial absorbance and change in
absorbance every 1,2,3 minutes at 340 nm
Calculation:
AST activity in U/L
= Δ A/minute X 1768

Δ A/minute= change in absorbance per minute

1768= factor given by kit manafacturers
Result:The amount of AST present in the given sample………U/L

Reference range: adults- 5 to 35 U/L

AST activity in neonates and infants is approximately twice that
in adults, but these decline to adult levels by approximately 6
months of age
clinical significance:
AST levels in acute myocardial infarction:
Rise within 6 to 8 hours, peak at 24 hours and return to normal
within 5 days.
Increased levels of AST:
 liver diseases.
 muscular dystrophy,
 acute pancreatitis
 Hemolysis
Decreased levels of AST:
 uremia,
 vitamin B deficiency,
 some drugs
Other methods:
 Dinitrophenyl hydrazine coupling method
 Diazonium dye coupling method
 ALT
• Enzyme that catalyses the transfer of amino
groups between amino and keto acids
• Also known as SGPT (serum glutamate Pyruvate
transaminase)
• Found in greater amounts in liver and therefore
liver specific.
• Lesser amount found in kidney
• Found primarily in cytoplasm
• Half life 47 ( + / – 10 hours)
 Estimation of ALT
• Aim: to estimate the amount of ALT in the
given sample
• Method: Wrobleski method (IFCC Method)
• Principle:
Specimen handling:
 Avoid hemolysis
 Stable for :
3 days at room temperature
7 days at 2-8 degree celsius
Decrease in ALT following freeze thaw cycles

Working reagent :
 L-alanine, α-ketoglutaric acid, LDH, NADH, tris
buffer,Stabilizers.
• Procedure: : (kinetic method using factor)
Adding sequence Test
Working reagent 1 ml
sample 0.1 ml

Mix well and incubate

 find the initial absorbance and change in
absorbance every 1,2,3 minutes at 340 nm
Calculation:
ALT activity in U/L
= Δ A/minute X 1738

Δ A/minute= change in absorbance per minute

1738= factor given by kit manafacturers

Result:The amount of ALT present in the given sample………

U/L
Reference range:
Adult
male: 0-31 U/L
Female: 0-40 U/L
ALT activity in neonates and infants is approximately
twice that in adults due to seepage through
hepatocyte membrane, but these decline to adult
levels by approximately 3 months of age
clinical significance
 De ritis ratio: AST/ALT ratio elevated in liver
disease normal < 1
 ALT specific marker for liver disease.
Other methods:
 Dinitro phenyl hydrazine coupling method