Serum biochemical parameters(ALT) (AST) assay

Biochemistry Clinical practiceCLS 432

Dr. Samah KotbLecturer of Biochemistry

2015

What do you know about liver function tests ????

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

Liver Function tests

• 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 Principle

• 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.

ALT Reagents

• 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 Principle

• 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.

AST Reagents

• 1- 0.5 ml of ALT Buffer 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.

Procedure of ALT assay

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).

0 10 20 30 40 50 60 70 80 90 1000

0.1

0.2

0.3

0.4

0.5

0.6

ALT activity (U/l)

Ab

so

rba

nc

e a

t 5

46

nm

(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).

0 10 20 30 40 50 60 70 80 90 1000

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

AST activity (U/l)Ab

so

rba

nc

e a

t 5

46

nm

(Fig. 2): Standard curve of AST

• It is recommended that each laboratory establish its own reference values.

• The following value may be used as guide line.• Serum up to: 46 U/L.

Normal range

Disorders of Liver

• Hepatitis• Cirrhosis• Tumors

• 1) The primary clinical application of serum AST and ALT measurement is the detection of hepatic disease.

• 2) Hepatic cell injury is manifested by elevated serum transaminase activity prior to the appearance of clinical symptoms and signs (such as jaundice).

Clinical significance of ALT and AST

• 3) Comparable elevations of both AST and ALT are highly characteristic of acute viral, toxic, or non-ethanol drug-induced hepatitis.

• 4) In chronic hepatitis and cirrhosis, serum AST levels are higher than ALT; this may reflect hepatic cell necrosis with release of mitochondrial AST. In alcohol hepatitis, AST is more significantly increased than ALT.

Clinical significance Of ALT and AST

Increased levels of liver enzymes

,bilirubin and lowered protein

Liver disease

Increased alkaline phosphatase and

both total and direct bilirubin

Obstructive Jaundice

Clinical significance of ALT and AST