According to this experiment, trainees can understand the reagent preparation of the experimental program and master its principle and measurement method. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.
Operation method
Serum aspartate aminotransferase (AST) assay test
Principle
AST catalyzed the amino conversion reaction of aspartic acid and α-ketoglutaric acid to produce oxaloacetic acid and glutamic acid.L-aspartic acid ten α-ketoglutaric acid = oxaloacetic acid ten L-glutamic acid after 60min reaction, add 2,4 dinitrophenylhydrazine to terminate the reaction, and with the two α-keto acids in the reaction solution to produce the corresponding 2,4 dinitrophenylhydrazone, under the alkaline condition, the two kinds of benzene hydrazones absorption spectral curves have a difference. Under alkaline conditions, the absorption spectra of the two phenylhydrazones differed, with the largest difference at 500-520 nm, and the color intensity of the phenylhydrazone generated from oxalic acid was significantly greater than that of the α-ketoglutarate-based hydrazone, which can be used to determine the activity of AST by colorimetric method. Move I. Experimental reagents: Caveat l. The disadvantage of this method is that when the specimen AST activity is high, oxaloacetic acid inhibits the AST reality counterlocking, which makes the result low, and acetoacetic acid and β-hydroxybutyric acid in ketonemia will not cause the result to be falsely high because of the absence of a control tube. 2. If L-menthane is used weigh 1.33g. 3. When the serum enzyme activity exceeds 15O Kammen units, the serum should be diluted 5-fold or 10-fold with saline before measurement. 4. α-ketoglutaric acid and 2,4-dinitrophenylhydrazine in the substrate are color-developing substances, the weighing must be accurate, and the fluctuation range of absorbance in the blank tube of each batch of reagent should not exceed 0.015 A. If it is out of the range, check the reagents and instrumentation. For more product details, please visit Aladdin Scientific website.
l. 0.1 mol/L phosphate buffer, pH 7.4
2. 1 mmol/L 2,4 dinitrophenylhydrazine solution
3. 0.4 mol/L sodium hydroxide solution
4. 2mmol/L pyruvic acid standard solution
5. AST substrate solution (DL-aspartic acid 200mmol / L. α-ketoglutarate 2mmol / L): weighing α-ketoglutarate 29.2mg and DL-aspartic acid 2.66g, placed in a small beaker, add lmol / L sodium hydroxide solution of 1.5ml, after dissolution, add 0.lmol / L phosphate buffer 80ml, with lmol / L Sodium hydroxide to PH7.4, then transfer the solution to a 100ml volumetric flask, diluted to scale with phosphate buffer and stored in a refrigerator.
Experimental operation: 
After mixing the tubes, placed in a 37 ℃ water bath box insulation for 20min, and then each tube to add 0.4mol / L sodium hydroxide solution 5ml, placed at room temperature for 5min, with a spectrophotometer at a wavelength of 505nm with distilled water to adjust the zero point, read the absorbance of each tube, the measurement of the tube absorbance subtracted from the absorbance of the control tube of the sample from the standard curve set of the AST activity unit.
The standard curve was plotted: 
Reference value: 8-28 kammon units.