Transaminases (Aminotransferases)

Transamination process in which amino group is transferred from a-amino acid to an a-oxo acid.

As a result a different a-amino acid and a different  a-oxo acid are formed.

Two clinically important examples are :

• L-aspartate: (aspartate aminotransferase, AST)
• L-alanine: (alanine amino transferase, ALT)

Assay of Alanine transaminase

ALT (SGPT)

Reagents:

1. Substrate:  0.2 M L-alanine  + 2 mM a-oxoglutarate  + 100 mM phosphate buffer, adjust pH to 7.4

2. Color reagent:  mM 2,4-dinitrophenyl hydrazine in HCl

3.Color developer: 0.5 N NaOH

4. Calibrator: Solution of sodium pyruvate in 100 mM phosphate buffer at   pH 7.4  (70 IU/L)

Technique:

1. Transfer 0.5 ml substrate to each tube and place in a 37 °C  heating bath for 3-5 minutes.
2. Add 100 ml of sample (plasma) to the tube , mix and keep it at 37 °C for 30 minutes.
3. Add 0.5 ml color reagent to each tube and keep them for 10 minutes.
4. Add color developer (2 ml) and keep it for 5 minutes.
5. Zero the spectrometer with the reagent blank at 505 nm.
6. Blank (substrate + color reagent  + developer)
7. Read and record absorbance of all tubes at 505 nm.