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