Biomarkers Used in Detection of Liver Disease

This article throws light upon the top five biomarkers used in detection of liver disease. The biomarkers are: 1. Alanine Transaminase 2. Aspartate Transaminase 3. Alkaline Phosphatase 4. Bilirubin 5. Gamma Glutamyl Trans Peptidase.

Biomarker # 1. Alanine Transaminase:

Alanine transaminase (ALT), also called Serum Glutamic Pyruvate Transaminase (SGPT) is an enzyme present in hepatocytes. When a cell is damaged, it leaks this enzyme into the blood, where it is measured. ALT rises dramatically in acute liver damage.

Principle of Estimation:

ALT Catalyses the following Reaction:

α-ketoglutarate + L- Alanine↔ Oxaloacetate + L- Glutamate.

Pyruvate reacts with 2,4 di-nitro phenyl hydra­zine (DNPH) to give pyruvate hydrazone, which gives brown color in alkaline condition and is measured colorimetrically at 505nm (Rietman and Frankel, 1957).

Biomarker # 2. Aspartate Transaminase:

Aspartate transaminase (AST) also called Serum Glutamic Oxaloacetic Transaminase (SGOT) is similar to ALT in that it is another enzyme associ­ated with liver parenchymal cells. It is raised in acute liver damage. The ratio of AST to ALT is sometimes useful in differentiating between caus­es of liver damage. Elevated AST levels are not specific for liver damage, and AST has also been used as a cardiac marker.

Principle of Estimation:

AST catalyses the following reaction:

Α-ketoglutarate + L-Aspartate ↔ Oxaloacetate + L-Glutamate

Oxaloacetate so formed reacts with 2,4 di-nitro-phenyl hydrazine (DNPH) to give oxaloacetate hydrazone, which gives brown color in alka­line condition and is measured colorimetrically at 505nm (Rietman and Frankel, 1957).

Biomarker # 3. Alkaline Phosphatase:

Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP lev­els in plasma will rise with large bile duct ob­struction, intrahepatic cholestasis or infiltrative diseases of the liver. ALP is also present in bone and placental tissue, so it is higher in growing children (as their bones are being re-modelled) and elderly patients with Paget’s disease.

Principle of Estimation:

Alkaline phosphatase from serum converts Phenyl phosphate to inorganic phosphate and phenol at pH 10. Phenol so formed reacts in al­kaline medium with 4-amino-antipyrine in pres­ence of oxidizing agent potassium ferricyanide and forms an orange red complex. This colored complex is estimated colorimetrically at 510nm (Kind and King, 1954).

Biomarker # 4. Bilirubin:

Bilirubin is a breakdown product of heme. The liver is responsible for clearing bilirubin. It does this by the following mechanism: bilirubin is tak­en up into hepatocytes, conjugated (modified to make it water-soluble), and secreted into the bile, which is excreted into the intestine. Increased total bilirubin causes jaundice, and can signal a number of problems:

1. Pre-hepatic:

Increased bilirubin production. This can be due to a number of causes, in­cluding hemolytic anemia and internal he­morrhage.

2. Hepatic:

Problems with the liver, which are reflected as deficiencies in bilirubin metabo­lism (e.g. reduced hepatocyte uptake, im­paired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin). Some ex­amples would be cirrhosis and viral hepati­tis.

3. Posthepatic:

Obstruction of the bile ducts, re­flected as deficiencies in bilirubin excretion. (Obstruction can be located either within the liver or in the bile duct.)

Principle of Estimation:

Direct or conjugated bilirubin couples with diazotized sulfanilic acid, forming azobilirubin, a red purple colored product in acidic medium, whereas unconjugated bilirubin is diazotized only in presence of methanol. The intensity of the red purple colour so developed above is measured colorimetrically at 540nm.

Biomarker # 5. Gamma Glutamyl Trans Peptidase:

Gamma glutamyl trans peptidase (GGT) may be elevated with even minor, sub-clinical levels of liver dysfunction. It can also be helpful in identi­fying the cause of an isolated elevation in ALP. GGT is raised in alcohol toxicity (acute and chron­ic).

Histopathological Examinations:

Liver pieces are preserved in 10% formaldehyde solution for histopathological study. The pieces of liver were dehydrated with a sequence of ethanol solution and embedded in paraffin wax. Sec­tions of about 4-6 µm thickness are cut.

They are stained with hematoxylin and eosin and photo­graphed using a photomicroscope. The haematoxylin and eosin stained section of paracetamol treated liver tissue with intense lymphocytic in­filtration around central vein and centrilobular necrosis (zone 3 necrosis). Dilated sinusoids and hepatocytes with ballooning degeneration are also observed.

Cellular boundary and normal archi­tecture of hepatic lobule is also damaged.

The haematoxylin and eosin stained section of CCl₄ shows distorted hepatic lobule with extensive fatty changes (steatosis i.e., accumulation of fat drop­lets), sinusoidal congestion, extensive lym­phocytic infiltration around central vein and ex­tensive vacuolization with loss of nuclei in hepa­tocytes and normal hepatic lobular architecture.

The liver of d-galactosamine treated animals showed inflammation of lymphocytes and Kupffer cells and hyaline degeneration around the portal vein area.