Diagnostic enzymology is routinely used to discriminate between several forms of liver disease, including:
• Hepatitis: General inflammation of the liver most commonly caused by viral infection, but which may also be a consequence of blood poisoning (septicaemia) or glandular fever. Acute hepatitis can show a marked increase (60-fold) in aminotransferases over a few days before returning to normal.
• Cirrhosis: A general destruction of the liver cells and their replacement by fibrous tissue. It is most commonly caused by excess alcohol intake, but is also a result of prolonged hepatitis, various autoimmune diseases and genetic conditions. Enzyme activity may only be mildly elevated when chronic liver damage is present.
• Malignancy: Primary and secondary tumours.
• Cholestasis: The prevention of bile from reaching the gut either due to blockage of the bile duct by gallstones or tumours, or to liver cell destruction as a result of cirrhosis or prolonged hepatitis. This gives rise to obstructive jaundice (presence of bilirubin, a yellow metabolite of haem, in the skin).
• Obesity: Serum aminotransferases can be mildly but persistently elevated in obese subjects reflecting non-alcoholic fatty liver disease.
Patients with these various liver diseases often present to their doctor with similar symptoms and a differential diagnosis needs to be made on the basis of a range of investigations, including imaging techniques, especially ultrasonography (ultra sound), magnetic resonance imaging (MRI), computerised tomography (CT) scanning, microscopic examination of biopsy samples and liver function tests. Four enzymes are routinely assayed to aid differential diagnosis:
• Aspartate aminotransferase (AST) and alanine aminotransferase (ALT): As previously stated, these enzymes are widely distributed, but their ratios in serum are characteristic of the specific cause of liver cell damage. For example, an AST/ALT ratio of less than 1 is found in non-alcoholic fatty liver disease, a ratio of about 1 in obstructive jaundice caused by viral hepatitis and a ratio of greater than 1 in alcohol abuse.
• Aspartate aminotransferase activity: This is assessed by a coupled assay with malate dehydrogenase and the measurement of the decrease in absorbance at 340 nm:
L-aspartate + 2-oxoglutarate ⇌ oxaloacetate + L-glutamate
oxaloacetate + NADH + H+ ⇌ malate + NAD+
• Alanine aminotransferase activity: This is also assessed by a coupled assay, in this case with lactate dehydrogenase and the measurement of the decrease in absorbance at 340 nm:
L-alanine + 2-oxoglutarate ⇌ pyruvate + L-glutamate
pyruvate + NADH + H+ ⇌ L-lactate + NAD+
• γ-Glutamyl transferase (GGT): This enzyme transfers a γ-glutamyl group between substrates and may be assayed by the use of γ-glutamyl-3-carboxy-4-nitroanilide as substrate and glycylglycine as acceptor. The amount of 5-amino-2-nitrobenzo ate released is proportional to GGT activity. GGT is widely distributed and is abundant in liver, especially bile canaliculi, kidney, pancreas and prostate. Raised enzyme activities are found in cirrhosis, secondary hepatic tumours and cholestasis, and tend to parallel increases in the enzyme activity of alkaline phosphatase, especially in cholestasis. GGT enzyme activity in the serum is raised by alcohol and some drugs; for this reason, it is not a particularly useful marker.
• Alkaline phosphatase (ALP): This enzyme is found in most tissues, but is especially abundant in the bile canaliculi, kidney, bone and placenta. It may be assayed using 4-nitrophenyl phosphate as substrate and monitoring the release of 4-nitrophenol at 400 nm. Its enzyme activity is raised in obstructive jaundice and, when measured in conjunction with ALT, can be used to distinguish between obstructive jaundice and hepatitis since ALP enzyme activity is raised more than that of ALT in obstructive jaundice. Decreasing serum activity of ALP is valuable in confirming an end of cholestasis. Raised serum ALP levels can also be present in various bone diseases and during growth and pregnancy.
