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

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• 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

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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

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SGPT

The principle

L-alanine+α-oxoglutarate pyruvate+L-glutamate

pyruvate 2, 4-dinitrophenylhydrazine (DNPH) pyruvate hydrazone

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• 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.

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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.

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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

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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

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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

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Prevent from contaminating the

substrate buffer

Use the “blank” not distilled water for

zero adjustment before every

measurements

Notes

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•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)

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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

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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

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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

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返回

Light path

Spectrophotometer

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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