Biochemistry laboratory
Lecture 7
Activity Determination of Serum Glutamate Pyruvate
Transaminase
Biochemistry laboratory
Understand the principle and method of serum glutamate
pyruvate transaminase (SGPT) activity determination
Understand the clinical significance of SGPT activity
determination
Aims
Biochemistry laboratory
• The SGPT stands for Serum Glutamate Pyruvate Transaminase. This enzyme is found in many body tissues in small amounts, but it is very concentrated in the liver. It is also commonly known as Alanine Transaminase, abbreviated as ALT.
• In a normally healthy individual, the level of SGPT is measurablein the blood. When there is acute liver damage, the level of SGPT tends to rise dramatically.
• The normal GPT range for adults in most labs is 0 to 45 units per liter (U/L). The normal range may vary slightly from lab to lab.
Clinical significance
Biochemistry laboratory
The SGPT test is done to:
• Identify liver disease, especially cirrhosis and hepatitis caused by alcohol, drugs, or viruses.
• Help check for liver damage.• Find out whether jaundice was caused by a blood disorder or
liver disease.• Keep track of the effects of cholesterol-lowering medicines
and other medicines that can damage the liver.
Clinical significance
Biochemistry laboratory
SGPT
The principle
L-alanine+α-oxoglutarate pyruvate+L-glutamate
pyruvate 2, 4-dinitrophenylhydrazine (DNPH) pyruvate hydrazone
Biochemistry laboratory
• The SGPT activity is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine.
• α-oxoglutarate can also react with 2,4-dinitrophenylhydrazine to form α-oxoglutarate hydrazone whose absorption maximumin alkaline solution is different to that of pyruvate hydrazone.
Biochemistry laboratory
Pyruvate hydrazone turns into
brown color in the NaOH
solution and it is light
absorption is at wavelength 510
nm. The light absorption is
correlated with the pyruvate
concentration.
Biochemistry laboratory
Reagents and equipments
small test tubes, micropipette, constant temperature water bath, spectrophotometer
Substrate buffer ( pH 7.4 )
2, 4–dinitrophenylhydrazine
100u/L (3.0mmol/L) Standard SGPT 0.4mol/L NaOH
Biochemistry laboratory
Hazardous materials
• NaOH is caustic. Do not swallow, and avoid contact with the skin and mucous membranes.
• Human serum is a potential biohazard. In case of skin contact, wash off immediately with plenty of water
Biochemistry laboratory
Procedure
Blank Standard Sample
Sample (μl) —— —— 50
Standard (μl) —— 50 ——
ddH2O (μl) 50 —— ——
Substrate buffer ( μl )
250 250 250
Incubation at 37 for℃ 30 min
chromogenic reagent (μl)
250 250 250
Incubation at 37 for℃ 20 min
0.4M NaOH (ml) 2.5 2.5 2.5
Stand for 3min , measure light absorption at 510 nm
Take 3 small test tubes and add following solutions
Biochemistry laboratory
Prevent from contaminating the
substrate buffer
Use the “blank” not distilled water for
zero adjustment before every
measurements
Notes
Biochemistry laboratory
•Beer's Law The absorbance varies linearly with both the cell path length and the analyte concentration.
A=ε·C·LA-absorbance ε-molar absorptivity (a measurement of how strongly a chemical species absorbs light at a given wavelength)C-concentration of the absorbing molecule L-cell path length (1.00 cm)
Biochemistry laboratory
Csample = Asample / Astandard × Cstandard
( Cstandard : 100u/L )
Comparing with the standard
Normal range : 0 ~ 50 u/L
Asample =ε·Csample·L Astandard =ε·Cstandard·L
Biochemistry laboratory
Spectrophotometry
A spectrophotometer is employed to measure the amount of light
that a sample absorbs. The instrument operates by passing a
beam of light through a sample and measuring the intensity of
light reaching a detector. Visible region 400–700 nm
spectrophotometry is used extensively in colorimetry
Biochemistry laboratory
cuvette Made of optical grade quartz
Try to touch the cuvettes only on the opaque sides avoiding touching the clear sides especially on the lower half of the cuvette. Opaque sides are held between two finger.
Rinse the cuvette with water and followed by
distilled water; wipe the sides with a kimwipe
The total volume of the solution in the cuvette should be around 3/4 (1cm from the of the) total volume
Biochemistry laboratory
返回
Light path
Spectrophotometer
Biochemistry laboratory
Lab report
• The title of an experiment• The aim of an experiment• The principle of an experiment• The reagents and equipments• The experimental procedure• Results• Answers to the assignment questions
Please turned in the lab report next week
Biochemistry laboratory
1. What is the clinical significance of SGPT test ?
2. SGPT is found in many tissues. Which tissue contains the highest SGPT activity?
Questions