Download 1. First , calculate the amount of pyruvic acid ( µg )

Experiment 8:
Assay The Activity of ALT
in Serum
ALT: Alanine transaminase
GTP: Glutamate pyruvate
transaminase
Objective
To understand the principle and significance
of detecting ALT in serum.
Principle
ALT can catalyze the transamination
between Ala and α－ketoglutatrate , the
equation is :
Ala+α－ketoglutatrate
pyruvic acid + L-Glu
ALT
Glutamic acid
pyacid
ALT
α－ketoglutarate
pyruvic acid
pyruvic acid
Alanine
pyruvic acid + 2，4-dinitrophenylhydrazine
pyruvic acid 2，4-dinitrophenylhydrabenzone
（marron）
Procedure
1. Take 4 dry and clean test tubes.
2. Manipulate according to the table as follow.
Reagents (ml)
Pyruvate (100µg/ml)
Serum
Substrate buffer
Phosphate buffer
(pH 7.4)
Control-1 Control-2
0.1
--0.5
---
----0.5
D-1
--0.1
0.5
D-2
--0.1
---
0.1
----37℃ water bath for 30min
2,4-dinitrophenylhydrazine 0.5
0.5
0.5
0.5
Substrate buffer
------0.5
37℃ water bath for 20min
NaOH(0.4mol/L)
5
5
5
5
room temperature for 10min
Control 1
10μg pyruvate
OD1
α－ketoglutarate
Control 2
α－ketoglutarate
10μg pyruvate
OD1-OD2
OD2
D-1
X μg pyruvate
OD3
serum
D-2
serum
X μg pyruvate
OD3-OD4
OD4
OD1-OD2
10 μg
OD3-OD4
＝
X μg
OD1-OD2
× 10
X (μg) ＝
OD3-OD4
3. Measure the absorbance of each tube at
500nm with the spectrophotometer.
Using distilled water as the blank tube to
adjust the OD value to 0.
Record these values on your paper.
Result and Calculate
blank
OD1
(C1)
OD2
(C2)
OD3
(D1)
OD4
(D2)
0
The Definition of ALT activity: ALT in 1ml
serum catalyzes the substrate to produce 2.5µg
pyruvic acid at 37℃ for 30min which is
defined as one ALT unit.
1. First , calculate the amount of pyruvic acid ( µg )
which produced in enzymatic-catalyzed reaction of
0.1 millilitre serum
OD1-OD2
× 10
X (μg) ＝
OD3-OD4
2. Then , according to the definition of ALT
activity, calculate the ALT units of serum sample.
ALT units
per millilitre serum =
pyruvic acid ( µg )
×
2.5
REFERENCE RANGE
2－40 Karman units
1
0.1
Clinical significance
The ALT test detects liver injury. ALT values are usually
compared to the levels of other enzymes, such as alkaline
phosphatase (AKP) and aspartate transaminase (AST), to
help determine which form of liver disease is present.
What do abnormal results mean?
Greater-than-normal levels may indicate:
hepatitis (viral, autoimmune)
use of hepatotoxic drugs
hepatic (liver) ischemia (blood deficiency)
cirrhosis
hepatic tumor
Very high levels of ALT (more than 10 times the highest
normal level) are usually due to acute hepatitis, often due
to a virus infection. In acute hepatitis, ALT levels usually
stay high for about 1–2 months, but can take as long as 3–6
months to come back to normal.
ALT levels are usually not as high in chronic hepatitis,
often less than 4 times the highest normal level: in this case,
ALT levels often vary between normal and slightly
increased, so doctors typically will order the test frequently
to see if there is a pattern. In some liver diseases, especially
when the bile ducts are blocked, when a person has
cirrhosis, and when other types of liver cancer are present,
ALT may be close to normal levels.
Discussion