Dr. Samah Kotb Nasr Eldeen

 1. Total and conjugated bilirubin  2. ALT  3. AST  4. Total protein  5. GGT  6. ALP  7. 5′-nucleotidase  8. Albumin  9. prothrombin time

 Aminotransferase was estimated according to the method of (Reitman and Frankel, 1957).

 Transaminase activity is measured by determining the concentration of pyruvate hydrazone formed with 2,4- Dinitrophenylhydrazine under controlled pH and temperature conditions. ALT enzyme  α-Ketoglutrate + Alanine Glutamate + pyruvate.  Color appeared with the aid of developer (NaOH) and measured calorimetrically.

 ALT Buffer substrate : Phosphate buffer : 100 mmol/L,The pH 7.5. L.alanine : 200 mmol/L α-Ketoglutarate : 2.0 mmol/L  2,4-Dinitrophenylhydrazine solution: 1 mmol/L  Sodium hydroxide solution (0.4M): 16 g of sodium hydroxide were dissolved in distilled water to make 1 liter.

 Aspartate aminotransferase activity is measured by determining the concentration of oxalacetate hydrazone formed with 2,4- Dinitrophenylhydrazine respectively under controlled pH and temperature conditions. AST enzyme  α-Ketoglutrate + Aspartate Glutamate + oxalacetate.

 AST Buffer substrate: Phosphate buffer : 100 mmol/L,The pH 7.5. L.aspartate : 100 mmol/L α-Ketoglutarate : 2.0 mmol/L  2,4-Dinitrophenylhydrazine solution: 1 mmol/L  Sodium hydroxide solution (0.4M): 16 g of sodium hydroxide were dissolved in distilled water to make 1 liter.

 ml of ALT substrate was added into unknown test, 0.5 ml of distalled water was added into blank test, then 0.1 ml of serum were added to unknown. The contents were mixed well, incubated at 37 °C for 30 min. after incubation then 0.5 ml of 2,4- dinitrophenyl hydrazine was added into unknown and blank tube.

2- The contents of each tube were mixed well, let to stand at 25 °C for 20 min. and then 5 ml of sodium hydroxide were added into each tube. The tubes were mixed by inversion, let to stand for 5 min. and the absorbance read at 546 nm.

3- Blank was used for zero adjustment 4- The concentration of test sample was derived from the standard curve (Fig. 1).

( Fig.1): Standard curve of ALT

 Procedure of AST activity determination had the same steps of ALT except AST substrate using instead of ALT substrate (Fig. 2).

( Fig. 2): Standard curve of AST

The liver may be assessed by measurement of total and conjugated bilirubin because the liver is the site for the conjugation of bilirubin. Liver function tests are very useful to see if there is active damage in the liver (hepatitis) or slow bile flow (cholestasis). Assessment of Liver Function

 Hepatitis  Cirrhosis  Tumors

Increased levels of liver enzymes,bilirubin and lowered protein Liver disease Increased alkaline phosphatase and both total and direct bilirubin Obstructive Jaundice