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Estimation of transaminase in serum as ALT , AST – liver function tests

بواسطة ALM3ML 196

ALT , AST test
    Liver Function Tests (LFT): are group of clinical biochemistry laboratory blood assays designed to give information about the state of patients’liver.
    Estimation of transaminase in serum as ALT , AST
    • Introduction
    ALT test :
    • ALT is an enzyme normally found inside liver cells 
    • The function of ALT is to convert alanine (A.As) into pyruvate
    • ALT is more spesific for the liver disease
    • In healthy individuals , ALT levels in the blood is low
    • When the liver is damaged , ALT is released into the blood usually before more signs of liver damage occur.this makes ALT a useful test for early detection of liver damage but it can’t show how much liver damage there is or how much fibrosis or scarring is present
    • AlT test may also performed to :
    1. Monitor the progression of liver diseases as hepatitis
    2. Assess whether treatment for liver disease should be started
    3. Evaluate how well treatment is working
    ↚
    Estimation of transaminase in serum as ALT , AST

     AST test :
    • AST is an enzyme found in cells throughout the body but mostly in the heart , liver and to a lesser extent in the kidneys and muscles so, it is less specific for liver disease
    • The function of ALT is to catalyze the reversible transfer of an amino group between aspartate and glutamate
    • To catalyze the inter conversion of aspartate and 𝜶 – keto glutarate to oxaloacetate and glutamate
    ↚
    Principle
    •                                                                                         GPT
    • Alanine + 𝜶-  keto glutarate ⥨ Pyruvate + Glutamate                                 
    •                                                                                               GoT            
    • Aspartate +𝜶-  keto glutarate ⥨ Oxaloacetic acid + Glutamate
    • The pyruvate produced by transamination by GPT reacts with 2,4-dinitrophenyl hydrazine to give a brown colored complex (hydrazone) which is measured by the colorimeter at 505 nm
    • The oxaloacetate formed in the reaction with GOT decarboxylated spontaneously to pyruvate which converted to hydrazone also 
    • The transaminase activity is proportional to the amount of oxalate to pyruvate formed over a definite period of time and is measured by 2,4-dinitrophenyl hydrazine in alkaline solution
    ↚
    Normal range
    • ALT (GPT) up to 45 units / ml
    • AST (GOT) up to 40 units / ml
    Reagents 
    • Phosphate buffer :PH 7.5 (optimum PH of enzyme activity)
    • ASTsubstrate 
    • ALT substrate
    • Color reagent 
    • Standard pyruvate
    • NaoH 
    ↚
    Procedure
    • Depend on colorimetric technique
    Reagent
    test
    St1
    St2
    St3
    St4
    St5
    Dist H2o
    –
    0.05 ml
    0.05 ml
    0.05 ml
    0.05 ml
    0.05 ml
    Alt or Ast substrate
    0.25 ml
    0.25 ml
    0.225 ml
    0.2 ml
    0.175 ml
    0.15 ml
    Serum
    0.25 ml
    –
    –
    –
    –
    –
    Standards
    –
    –
    0.25 ml
    0.05 ml
    0.075 ml
    0.1 ml
    Color reagent
    0.25 ml
    0.25 ml
    0.25 ml
    0.25 ml
    0.25 ml
    0.25 ml

    Mix well , stand for 20 min at R.T
    Naoh
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml

    Mix well , wait for 5 min then read at 505 nm aginst dist H2o


    ↚
    Serum ALT and AST levels are increased in :
    • Acute hepatitis 
    • Exposure to drugs or toxic substances
    • Obstruction of bile ducts
    • Cirrhosis
    • Heart damage
    • liver tumers
    Serum ALT and AST levels are decreased in :
    • low levels of ALT and AST in blood are expected and normal in healthy individuals , healthy liver 
    Procedure with kits in lab :
    • Depend on kinetic technique
    • 500 µ AST or ALT reagent or (400µ R1 + 100µ R2),  incubate for 5 min at 37ͦ  C ,then add 50 µ  serum , Mix well and read .
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