Experimental effects of sodium phenobarbital and actinomycin D on cytochrome P-450 content in mouse liver

P-450 belongs to hemoglobin protein, and its reduced form combines with CO and shows an absorption peak at 450 nm. Therefore, the content of P-450 can be determined by the method of oscillometric spectrometry. After passing CO through the liver homogenate samples, the reducing agent sodium dithionite (Na2S2O4) was added, and then the absorbance was measured at 450 nm and 490 nm, and the difference was substituted into the formula to calculate the content of cytochrome P450.

Operation method

aberration spectroscopy

Principle

P-450 belongs to hemoglobin protein, and its reduced form combines with CO and shows an absorption peak at 450 nm. Therefore, the content of P-450 can be determined by the method of oscillometric spectrometry. After CO was passed through the liver homogenate sample, the reducing agent sodium dithionite (Na2S2O4) was added, and then the absorbance was measured at 450 nm and 490 nm, and the difference was substituted into the formula to calculate the content of cytochrome P450.

Materials and Instruments

Mouse
Saline Sodium phenobarbital Actinomycin D Sucrose solution Tris-HCl buffer
Spectrophotometer Torsion balance Ice maker Glass homogenizer Funnel Test tubes Filter paper Pipettes Ice box

Move

The animals were divided into 3 groups: saline group, phenobarbital sodium group and actinomycin D group.

1. Sodium phenobarbital and actinomycin D i.p. for three days to induce or inhibit hepatic enzymes.

2. Head-breaking and blood-letting: mice were head-breaking and blood-letting on the third day (the day of the experiment) (must be cleaned, because hemoglobin will affect the results of the experiment);

3. Preparation of liver homogenate: pre-cool 0.25M sucrose solution and 0.05M Tris-HCl buffer in ice; cut off the liver (liver tissue should not be less than 400mg; do not destroy the gallbladder, because bile pigment interferes with the results of the experiment; absorb the blood stains with filter paper, because blood pigment interferes with the results of the experiment); torsion balance weight (padded with tinfoil; each time when adding or subtracting samples or codes, you must turn off the shut-off balance); place the liver tissue in the homogenizer. Place the liver tissue in a homogenizer, add pre-cooled 0.25M sucrose (0.5ml/100mg of liver tissue); grind under an ice bath until the tissue turns into a light pink homogenate; take 1ml of this homogenate, add pre-cooled 9ml of 0.05M Tris-HCl buffer, and mix thoroughly; fill with CO under an ice bath, 1~2 bubbles/second, and ventilate for 2 minutes.

4. After aeration, pour the solution into two colorimetric cups, one as a reference cup and the other as a sample cup. Add 5 mg of sodium dithionite to the sample cup and mix thoroughly. After zeroing the reference cup, measure the absorbance of the sample cup at 450 nm and 490 nm, and calculate the amount of P-450 according to the following formula:

Caveat

1. operate quickly (to ensure enzyme activity);2. ice bath (in order to ensure enzyme activity);3. blood should be drained (P-450 is a heme protein; heme affects P-450);4. bilirubin affects the measurement of P-450 when the gallbladder is ruptured;5. do not pass CO too fast or too slow;6. grind until the liver tissue turns from red to white homogenate7. when colorimetrically measuring on a spectrophotometer, measure A.D, then N.S, and finally Pheno (P-450 from low to high concentrations).

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