Enzyme kinetic assays (ALT, AST, ALP)

Principle

• Enzyme activity is measured as the rate of reaction under zero-order conditions (excess substrate), so the rate depends only on enzyme amount.
• Change in absorbance per minute is measured in the linear phase after a short lag phase. Results in U/L (µmol substrate converted per minute per litre); IFCC methods at 37 °C.
• ALT: alanine + 2-oxoglutarate → pyruvate + glutamate; lactate dehydrogenase reduces pyruvate using NADH. Decrease in absorbance at 340 nm.
• AST: aspartate + 2-oxoglutarate → oxaloacetate + glutamate; malate dehydrogenase reduces oxaloacetate using NADH. Decrease at 340 nm.
• IFCC methods add pyridoxal-5'-phosphate (coenzyme) for ALT and AST.
• ALP: hydrolyses p-nitrophenyl phosphate to yellow p-nitrophenol at alkaline pH (about 10.4, AMP buffer, Mg2+ activator). Increase at 405 nm.

Specimen

• Serum or heparin plasma. Do not use EDTA, citrate or oxalate for ALP: they chelate Mg2+ and Zn2+ and inhibit activity.
• Avoid haemolysis, especially for AST (much higher in red cells).

Results

• Typical adult upper limits: ALT and AST about 35–45 U/L; ALP about 40–130 U/L. Values vary widely by method, age and sex; local reference ranges apply.
• ALP is physiologically higher in growing children and in late pregnancy (placental isoenzyme).

Quality control and pitfalls

• Very high activity can exhaust substrate or NADH within the lag phase (substrate depletion), giving falsely low or non-linear results; dilute and repeat.
• Check reaction linearity flags; maintain accurate temperature.

Clinical use

• ALT is more liver-specific; AST also rises in muscle and cardiac injury.
• AST:ALT ratio above 2 suggests alcoholic liver disease.
• Raised ALP with raised GGT points to cholestasis; raised ALP with normal GGT suggests bone origin (Paget disease, bone metastases, osteomalacia).