experiment 8: assay the activity of alt in serum
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Experiment Experiment 8:8:
Assay The Activity of ALT Assay The Activity of ALT in Serumin Serum
ALT: Alanine transaminase
GTP: Glutamate pyruvate
transaminase
ObjectiveObjective
To understand the principle and significance To understand the principle and significance
of detecting ALT in serum.of detecting ALT in serum.
ALT ALT can catalyze the transamination betwecan catalyze the transamination between Ala and αen Ala and α -- ketoglutatrate , the equatioketoglutatrate , the equation is :n is :
Ala+α- ketoglutatrateALT
pyruvic acid + L-Glu
PrinciplePrinciple
α- ketoglutarate
pyruvic acid Glutamic acid
pyruvic acid
Alanine
pyacid ALT
pyruvic acid + 2, 4-dinitrophenylhydrazine
pyruvic acid 2, 4-dinitrophenylhydrabenzone
(marron)
ProcedureProcedure
1. Take 4 dry and clean test tubes.1. Take 4 dry and clean test tubes.
2. Manipulate according to the table as follow.2. Manipulate according to the table as follow.
Reagents (ml) Control-1 Control-2 D-1 D-2 Reagents (ml) Control-1 Control-2 D-1 D-2
Pyruvate (100µg/ml) 0.1 --- --- ---Pyruvate (100µg/ml) 0.1 --- --- ---Serum --- --- 0.1 0.1Serum --- --- 0.1 0.1Substrate buffer 0.5 0.5 0.5 ---Substrate buffer 0.5 0.5 0.5 ---Phosphate buffer Phosphate buffer (pH 7.4) --- 0.1 --- ---(pH 7.4) --- 0.1 --- --- 37 water bath for 30min℃37 water bath for 30min℃2,4-dinitrophenylhydrazine 0.5 0.5 0.5 0.52,4-dinitrophenylhydrazine 0.5 0.5 0.5 0.5Substrate buffer --- --- --- 0.5 Substrate buffer --- --- --- 0.5 37 water bath for 20min℃37 water bath for 20min℃NaOH(0.4mol/L) 5 5 5 5NaOH(0.4mol/L) 5 5 5 5 room temperature for 10minroom temperature for 10min
Control 2
10μg pyruvate
α - ketoglutarate
OD1
Control 1
10μg pyruvate OD1-OD2
α - ketoglutarate
OD2
D-2
X μg pyruvate
serumOD3
D-1
X μg pyruvate OD3-OD4
serum OD4
OD1-OD2 OD3-OD4
10 μg X μg =
OD1-OD2
OD3-OD410X (μg) = ×
3. Measure the absorbance of each tube at 3. Measure the absorbance of each tube at
500nm with the spectrophotometer.500nm with the spectrophotometer.
Using distilled water as the Using distilled water as the blank tubeblank tube to to
adjust the OD value to 0.adjust the OD value to 0.
Record these values on your paper.Record these values on your paper.
The The Definition of Definition of ALT activity:ALT activity: ALT in 1ml se ALT in 1ml se
rum catalyzes the substrate to produce 2.5µg prum catalyzes the substrate to produce 2.5µg p
yruvic acid at 37 for 30min which is defined ℃yruvic acid at 37 for 30min which is defined ℃as one ALT unit.as one ALT unit.
Result and CalculateResult and Calculate
blank blank OD1OD1
(C1)(C1)OD2OD2
(C2)(C2)OD3OD3
(D1)(D1)OD4OD4
(D2)(D2)
00
OD1-OD2
OD3-OD410X (μg) = ×
1. First , calculate the amount of pyruvic acid ( µg
) which produced in enzymatic-catalyzed reaction
of 0.1 millilitre serum
REFERENCE RANGE
2 - 40 Karman units
2. Then , according to the definition of ALT
activity, calculate the ALT units of serum sample.
ALT units ALT units per millilitre serum per millilitre serum ==
pyruvic acid ( µg )
2.5×
10.1
Clinical significanceClinical 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.
DiscussionDiscussion
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