Introduction
- Amylase is an important digestive enzyme that catalyzes the hydrolysis of starch and glycogen into smaller carbohydrates such as maltose and oligosaccharides.
- It is mainly produced by the pancreas and salivary glands.
- Small amounts are also present in other tissues.
- Serum amylase estimation is an important biochemical test used mainly for diagnosis of pancreatic disorders.
- Because amylase is a small molecule, it is rapidly filtered by kidneys and appears in urine.
- Serum amylase rises rapidly in acute pancreatic injury and therefore has important diagnostic value.
- Elevated serum amylase is commonly seen in acute pancreatitis, but it may also increase in salivary gland disease and other abdominal conditions.
Principle
- Serum amylase estimation is based on CNPG kinetic method.
- The substrate used is 2-chloro-4-nitrophenyl-β-D-galactopyranosylmaltotrioside (CNPG).
- Amylase hydrolyzes CNPG and releases 2-chloro-4-nitrophenol (CNP).
- The rate of CNP formation is directly proportional to amylase activity.
- Increase in absorbance is measured kinetically at 405 nm.
Reaction
CNPG + H₂O → 2-Chloro-4-nitrophenol + Galactosyl maltotrioside
- The increase in yellow color intensity is directly proportional to serum amylase concentration.
Specimen
Sample Type
- Serum is preferred specimen
- Plasma may also be used
- Heparin plasma acceptable
- EDTA plasma acceptable
- Urine can also be used
Precautions
- Use non-hemolyzed sample
- Avoid saliva contamination
- Fresh specimen preferred
Stability
Serum / Plasma
- 7 days at 20–25°C
- 7 days at 4–8°C
- 1 year at −20°C
Urine
- 2 days at 20–25°C
- 10 days at 4–8°C
- 3 weeks at −20°C
Reagents
Reagent Composition
- MES buffer
- Calcium chloride
- Sodium chloride
- Potassium thiocyanate
- Sodium azide
- CNPG substrate
Important Reagent Function
CNPG – Specific substrate for amylase reaction
Calcium Chloride – Required for enzyme activity
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Semi-auto analyzer / spectrophotometer
- Cuvette
- Timer
- Amylase reagent kit
Procedure
| Components | Test |
|---|---|
| Working reagent | 1000 µL |
| Sample | 20 µL |
Procedure Steps
- Mix properly
- Incubate at 37°C for 1 minute
- Measure initial absorbance
- Read absorbance after 1 minute, 2 minutes, and 3 minutes
- Calculate ΔA/min
Reading
- Wavelength: 405 nm
- Cuvette: 1 cm
Calculation
Formula Using Factor
Amylase activity (U/L) = ΔA/min × 3128
Formula Using Calibrator
Amylase activity (U/L) = ΔA sample / ΔA calibrator × Calibrator concentration
Normal Reference Values
| Sample | Normal Value |
|---|---|
| Serum | Up to 80 U/L |
| Urine | Up to 500 U/L |
Clinical Significance
Increased Serum Amylase Seen In
- Acute pancreatitis
- Chronic pancreatitis
- Salivary gland disorders
- Mumps
- Biliary tract disease
- Intestinal obstruction
- Ruptured ectopic pregnancy
Acute Pancreatitis
- Serum amylase begins to rise within 4 hours after onset of pain
- Peak occurs at 24 hours
- Remains elevated for 3–7 days
Diagnostic Importance
- Important marker for pancreatic injury
- Used along with serum lipase for pancreatitis diagnosis
- Helps monitor acute abdominal disorders
