Transcript Serum biochemical parameters (ALT) (ast) assay

Dr. Samah Kotb Nasr Eldeen
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1. Total and conjugated bilirubin
2. ALT
3. AST
4. Total protein
5. GGT
6. ALP
7. 5′-nucleotidase
8. Albumin
9. prothrombin time
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Aminotransferase was estimated according to the method of
(Reitman and Frankel , 1957).
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Transaminase activity is measured by determining the
concentration of pyruvate hydrazone formed with 2,4Dinitrophenylhydrazine under controlled pH and temperature
conditions.
ALT enzyme
α-Ketoglutrate + Alanine
Glutamate + pyruvate.
Color appeared with the aid of developer (NaOH) and measured
calorimetrically.
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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.
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Aspartate aminotransferase activity is measured by determining
the concentration of oxalacetate hydrazone formed with 2,4Dinitrophenylhydrazine respectively under controlled pH and
temperature conditions.
AST enzyme
α-Ketoglutrate + Aspartate
Glutamate + oxalacetate.
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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.
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1- 0.5 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 2,4dinitrophenyl 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).
Absorbance at 546 nm
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
70
ALT activity (U/l)
(Fig.1):
Standard curve of ALT
80
90
100
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Procedure of AST activity determination had the same steps
of ALT except AST substrate using instead of ALT substrate
(Fig. 2).
Absorbance at 546 nm
0.25
0.2
0.15
0.1
0.05
0
0
10
20
30
40
50
60
70
AST activity (U/l)
(Fig.
2): Standard curve of AST
80
90
100
Assessment of Liver Function
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).
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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