Assay of serum glutamate transaminase (SGPT).

1. Understand the
of the determination of
activity of glutamate
transaminase.
2. Master the method of determining the activity of glutamate transaminase.Serum
Glutamate Transaminase (SGPT) catalyzes alanine and α-ketone diacine to produce glutamate and acetone acid. Acetone acid can be reduced with 2,4-dinitrobenzene to produce acetone dinitrobenzeneunder acidic conditions, which is brownish-red under alkaline conditions and has maximum absorption at 520nm.
according to the color of the depth, the enzyme activity can be calculated by color matching method. GPT is most high in the liver, and when a drug damages the liver in the morning or the acute stage of viral hepatitis, the GPT is released into the bloodstream due to damage to liver cells, resulting in a significant increase in the level of this enzyme in the serum. Therefore, the activity of serum glutamate transaminase can be used as an important index to diagnose liver disease."
Reagents
Equipment"
(i) Reagents
1. 0.1M Phosphate buffer (pH7.4)
2. Acetone acid standard liquid (2 Mol/ml)
accurately named 22 mg of pure sodium acetone, with 0.1M pH7.4 phosphate buffer to 100 ml.
3. Glutamate transaminase substrate solution
accurately named α-ketone diacid 29.2mg (2mM), DL -alanine 1.78g (200mM), dissolved at 50 Ml 0.1M pH7.4 phosphate buffer, with 20% NaOH to adjust pH to 7.4, then add phosphate buffer to 100 ml, add a few drops of chloroform anticorrosion, refrigerator storage can be put for one week.
4. 2,4-dinitrobenzene solution (1mM) is called 2,4-dinitrobenzene 20mg, placed in 100 ml
capacity bottle
, first dissolved with 10 ml of thick hydrochloric acid, then diluted with water to the scale.
5. 0.4M sodium hydroxide.
(ii) equipment . Test tube
and test tube rack, 0.1, 0.5, 1 and 5 ml straw,
ringing temperature
water bath, 722 type
hydrometer
.Method:
(i) Standard curve drawing
with absorbent value as the ordinate, the number of enzyme activity units as horizontal coordinates, drawing the standard curve on the coordinate paper.
(ii) Serum SGPT vitality determination . Note:
1. When measuring SGPT, the substrate and serum should be heated in a water bath at 37 degrees C in advance, and then the substrate should be added to the serum tube to accurately remember the time.
2. The value on the standard curve is accurate and reliable at 20 to 500U, and the sample needs to be diluted when it exceeds 500U.
3. Transaminase can only act on α-L-
amino acids
and have no effect on D-amino acids. Laboratory multi-α-DL-amino acids (cheaper than L-amino acids), if L-amino acids, the dosing is halved.
4. Hemolytic specimens should not be used, because of the high vitality of transaminase in blood cells, will affect the determination effect.
5. The determination of serum samples needs to be completed within 30 minutes of coloration. .