1. Experiment 8: Assay The Activity of ALT in Serum

2. ALT: Alanine transaminase GTP: Glutamate pyruvate transaminase

3. Objective To understand the principle and significance of detecting ALT in serum.

4. ALT can catalyze the transamination between Ala and α － ketoglutatrate, the equation is : ALT can catalyze the transamination between Ala and α － ketoglutatrate, the equation is : Ala+α － ketoglutatrate ALT pyruvic acid + L-Glu Principle

5. α － ketoglutarate pyruvic acid Glutamic acid pyruvic acid Alanine pyacid ALT

6. pyruvic acid + 2 ， 4-dinitrophenylhydrazine pyruvic acid 2 ， 4-dinitrophenylhydrabenzone （ marron ）

7. Procedure 1. Take 4 dry and clean test tubes. 2. Manipulate according to the table as follow.

8. Reagents (ml) Control-1 Control-2 D-1 D-2 Pyruvate (100µg/ml) 0.1 --- --- --- Serum --- --- 0.1 0.1 Substrate buffer 0.5 0.5 0.5 --- Phosphate buffer (pH 7.4) --- 0.1 --- --- 37 ℃ water bath for 30min 37 ℃ water bath for 30min 2,4-dinitrophenylhydrazine 0.5 0.5 0.5 0.5 Substrate buffer --- --- --- 0.5 37 ℃ water bath for 20min 37 ℃ water bath for 20min NaOH(0.4mol/L) 5 5 5 5 room temperature for 10min room temperature for 10min

9. Control 2 10μg pyruvate α － ketoglutarate OD1 Control 1 10μg pyruvate OD1-OD2 α － ketoglutarate OD2

10. D-2 X μg pyruvate serum OD3 D-1 X μg pyruvate OD3-OD4 serumOD4

11. OD1-OD2OD3-OD4 10 μgX μg ＝ OD1-OD2 OD3-OD4 10 X (μg) ＝ ×

12. 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. Using distilled water as the blank tube to adjust the OD value to 0. Record these values on your paper. Record these values on your paper.

13. 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. Result and Calculate blankOD1(C1)OD2(C2)OD3(D1)OD4(D2) 0

14. OD1-OD2 OD3-OD4 10 X (μg) ＝ × 1. First, calculate the amount of pyruvic acid ( µg ) which produced in enzymatic-catalyzed reaction of 0.1 millilitre serum

15. 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 = pyruvic acid ( µg ) 2.5 × 1 0.1

16. 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.alkaline phosphatase (AKP) aspartate transaminase (AST)liver disease What do abnormal results mean? Greater-than-normal levels may indicate: hepatitishepatitis (viral, autoimmune) use of hepatotoxic drugs hepatichepatic (liver) ischemia (blood deficiency) cirrhosis hepatic tumor

17. 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.hepatitis 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.liver diseases

18. Discussion