Estimation of Serum SGPT

Introduction

• ALT stands for Alanine Aminotransferase.
• ALT is also called SGPT (Serum Glutamate Pyruvate Transaminase).
• It is an important intracellular enzyme involved in amino acid metabolism.
• ALT belongs to the group of transferase enzymes.
• It catalyzes transfer of amino group from alanine to alpha-ketoglutarate.
• This reaction forms pyruvate and glutamate.
• ALT is present mainly in the cytoplasm of liver cells.
• Highest concentration is found in liver tissue.
• Small amounts are present in kidney, heart, skeletal muscle, pancreas, spleen, and lungs.
• Because liver contains maximum ALT, this enzyme is considered highly specific for liver injury.
• ALT estimation is one of the most important liver function tests in clinical biochemistry.
• It helps detect hepatocellular damage even before clinical symptoms appear.
• ALT rises significantly when hepatocytes are damaged and enzyme leaks into circulation.
• ALT is commonly estimated together with AST for better interpretation of liver disease.
• In acute liver injury, ALT usually rises more than AST.
• In chronic liver disease, AST may become higher than ALT.
• ALT testing is routinely performed in liver profile investigations.
• It is useful for diagnosis, prognosis, and monitoring of treatment response.

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Principle

• ALT estimation is based on IFCC kinetic enzymatic method.
• The reaction occurs in two sequential steps.

Primary Reaction

• ALT catalyzes transfer of amino group from L-alanine to 2-oxoglutarate.
• Pyruvate and L-glutamate are produced.

Reaction

L-Alanine + 2-Oxoglutarate → Pyruvate + L-Glutamate

Secondary Reaction

• Pyruvate formed in first reaction reacts with NADH.
• LDH enzyme catalyzes this reaction.
• Pyruvate is reduced to lactate.
• NADH is oxidized to NAD⁺.

Reaction

Pyruvate + NADH → Lactate + NAD⁺

Principle of Measurement

• NADH absorbs ultraviolet light strongly at 340 nm.
• NAD⁺ does not absorb at 340 nm.
• During reaction NADH concentration decreases continuously.
• This causes gradual fall in absorbance.
• Rate of absorbance decrease per minute is proportional to ALT activity in sample.
• Therefore ALT is measured kinetically.

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Specimen

Type of Sample

• Serum is preferred specimen.
• Plasma can also be used.
• Heparin plasma is acceptable.
• EDTA plasma can also be used.

Sample Requirements

• Sample should be fresh.
• Non-hemolyzed specimen is essential.
• Hemolyzed sample should be avoided because hemoglobin interferes.
• Lipemic sample should be avoided if possible.
• Icteric sample may affect reading if severe.

Sample Handling

• Separate serum quickly after clotting.
• Avoid bacterial contamination.
• Store refrigerated if delayed.

Stability

• Stable up to 3 days at 2–8°C
• Stable for months at −20°C

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Reagents

Reagent 1 (Buffer Reagent)

• Tris buffer (pH 7.5)
• L-alanine
• LDH enzyme

Reagent 2 (Substrate Reagent)

• CAPSO
• 2-oxoglutarate
• NADH

Role of Each Component

• Tris Buffer – Maintains proper pH for enzyme activity
• L-Alanine – Provides substrate for ALT reaction
• LDH – Catalyzes secondary reaction
• 2-Oxoglutarate – Amino group acceptor
• NADH – Indicator molecule for kinetic measurement
• Storage
  • Store at 2–8°C
  • Protect from light
  • Do not freeze repeatedly

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Materials Required

• Test tubes
• Micropipette
• Pipette tips
• Semi-auto analyzer
• Spectrophotometer
• Cuvette
• Timer
• Water bath at 37°C
• ALT reagent kit
• Serum sample

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Procedure

Two Reagent Method

First Incubation

• Mix properly
• Incubate at 37°C for 5 minutes

Then Add

Second Incubation

• Mix again
• Incubate at 37°C for 1 minute

Reading

• Read absorbance at 340 nm
• Record absorbance at 1 minute, 2 minute, and 3 minute
• Calculate average change in absorbance per minute

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Calculation

Formula

ALT (U/L) = ΔA/min × Factor

Factor at 37°C and 340 nm

1745

Example

• Absorbance at 1 minute = 0.650
• Absorbance at 2 minute = 0.630
• Absorbance at 3 minute = 0.610

ΔA/min

• Difference = 0.020

Final Calculation

• ALT = 0.020 × 1745
• ALT = 34.9 U/L

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Normal Reference Values