Colorimetric assay for serum lactate dehydrogenase (LDH) assay

Summary

This experiment enables students to master the principle of colorimetric determination of lactate dehydrogenase, the preparation of reagents, and learn the method of LDH colorimetric determination. This experiment is from the laboratory guide of Mudanjiang Medical College undergraduate 5-year testing program.

Operation method

Colorimetric assay for serum lactate dehydrogenase (LDH) assay

Principle

Lactate dehydrogenase catalyzes the dehydrogenation of L-lactate to produce pyruvate, which reacts with 2,4-dinitrophenylhydrazine to produce pyruvate, dinitrophenylhydrazone, which is brownish-red in magnetic solution, and whose shade of color is proportional to the concentration of pyruvate, from which the enzyme's unit of activity is calculated.

Move

I. Experimental reagents:

1. substrate buffer (containing 0.3 mol/L lactic acid, pH 8.8):

Weigh 2.1g of diethanolamine, add about 80ml of distilled water, adjust to PH8.8 with 1mol/L hydrochloric acid and add water to 100ml.

2. 11.3mmol/L NAD solution: weigh 15mg of NAD (content of 70%, then weigh 21.4mg) dissolved in 2ml of distilled water, 4 ℃ storage at least 2 weeks available.

3. lmmol/L 2,4-dinitrophenylhydrazine solution: weigh 2,4-dinitrophenylhydrazine 198mg plus 10mol/L hydrochloric acid 100ml, to be dissolved and then add distilled water to 1000ml, placed in a brown glass bottle, stored at room temperature.

4. 0.4mol/L NaOH solution

5. 0.5mmol/L pyruvate standard solution: accurately weigh 11mg of sodium pyruvate (standard grade MW=110.06), dissolved in substrate buffer, transferred to a 200m1 volumetric flask, add substrate buffer diluted to the scale, prepared before use.

Laboratory operation: take 16mm × 100mm test tube, according to the table operation

After 5 minutes at room temperature, the wavelength is 440 nm, the optical diameter of the colorimeter is 1.0 cm, and the absorbance of each tube is read with solution B. The standard curve is plotted with the corresponding units of enzyme activity.

Definition of King's unit: 100 ml of serum, acted at 37℃ for 15 min, producing 1 micromole of pyruvate as a unit.

Reference value: 190 - 437 Gin units.

Caveat

1. LDH only acts on L-lactic acid, such as using sodium L-lactate, only DL-lactate2. oxalate can only inhibit the activity of lactate dehydrogenase, so you can not use potassium oxalate as an anticoagulant anticoagulation to determine the method.3. LDH activity in red blood cells is about 100 times higher than that in serum, so hemolyzed specimens cannot be measured.4. When the result is higher than 2500 units, the blood should be diluted and measured, and the result should be multiplied by the dilution factor.5. Colorimetry should be completed within 5-15 minutes, otherwise the absorbance decreases.

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