This experiment is from the official website of the Fourth Military Medical University

Operation method

Determination of hepatic drug metabolizing enzyme CYP450 content

Principle

The in vivo metabolism of drugs can be divided into phase I (catabolism) and phase II (anabolism) reactions.Phase I reactions include oxidation, reduction and hydrolysis, which are mainly catalyzed by cytochrome P450 (CYP450) enzymes, which are mainly found in the liver, and are therefore also known as hepatic enzymes.The induction and inhibition of CYP450 enzymes are of great clinical importance, and can lead to clinical drug-drug interactions. Interactions. Phenobarbital sodium is an inducer of CYP450 enzymes, while actinomycin D is an inhibitor of CYP450 enzymes. In this experiment, one of the screening methods for hepatic drug enzyme inducers and inhibitors was introduced by determining the effects of sodium phenobarbital and actinomycin D on hepatic CYP450 enzyme content.Determination of CYP450 enzyme content:CYP450 is a hemoglobin protein, and its reduced form binds to CO and then an absorption peak occurs at a wavelength of 450 nm, so it can be reflected in the solution by determining the absorbance value of the solution. Therefore, the absorbance value of the solution can be measured to reflect the content of CYP450 enzyme in the solution.

Materials and Instruments

Phenobarbital sodium solution Actinomycin D solution 0.25mol L sucrose solution Tris-HCl buffer Sodium dithionite physiological saline Spectrophotometer Balance Low-temperature centrifuge Ice machine CO gas bottle Glass homogenizer Funnel Pipette Ice box

Move

1. Grouping The mice were first weighed one by one and sorted according to body weight from small to large (or from large to small). According to the principle of simplified randomization, the mice were divided into 3 groups (A, B, C), with 3 mice in each group.

2. Intraperitoneal administration Group A was 0.75% phenobarbital sodium solution, Group B was 0.002% actinomycin D solution, and Group C was saline. Each group of mice was administered for 3 consecutive days.

3. Preparation of liver homogenate The mice were executed and the livers were removed, taking care not to destroy the gallbladder. After weighing, the liver tissue was placed in a glass homogenizer, and pre-cooled 0.25 mol/L sucrose solution (0.5 ml/100 mg of liver tissue) was added and ground to a light pink homogenate under an ice bath. The supernatant was then retained after centrifugation at 10,000 g for 20 min at 4 degrees Celsius.

4. Determination of CYP450 enzyme content 1 ml of supernatant after centrifugation was taken, and 9 ml of pre-cooled 0.05 mol/L Tris-HCl buffer was added and mixed thoroughly. Under the ice bath, the solution was aerated with CO gas, 1~2 bubbles/s, for 2 min. The aerated solution was divided into two halves and placed in two test tubes: one of them was used as the reference cup for the determination of absorbance, and the other was added with 5 mg of sodium dithionite, and mixed thoroughly, which was the sample cup to be tested. Determine and record the absorbance value of the sample cup at 450nm and 490nm (Table 23-4).

5. Calculation of CYP450 enzyme content

According to the formula A = E-C-L, where

A: absorbance

E: the extinction coefficient of the spectroscopic difference from 490 nm to 450 nm, in this experiment E = 104L/cm-mmol

C: CYP450 concentration

L: thickness of the colorimetric cup (length of the optical path), the colorimetric cup was 1cm in this experiment.

Therefore, CYP450 (nmol/g liver) was obtained = [(A450 - A490) / E-L] × 50000

6 Induction and inhibition of CYP450 enzyme

(1) Percentage of CYP P450 elevation:

[(Phenobarbital group - saline control group) / saline control group] x 100 percent

(2) Percentage of CYP P450 reduction:

[(saline control group - actinomycin D group) / saline control group] × 100%

Caveat

1. The preparation of liver homogenate should be done on ice, and the solutions used should be pre-cooled in ice to avoid the loss and destruction of CYP450 enzyme during operation.

2. When removing the liver, be careful not to destroy the gallbladder to avoid the influence of bilirubin on the CYP450 hemoglobin protein measurement.

3. The formula for CYP450 (nmol/g liver) = [(A450 - A490) / E-L] × 50000, where 50000 is the dilution factor during the preparation of liver homogenate for this experiment.

4. Determination of CYP450 enzyme content in liver homogenate is a simple and crude assay. Liver CYP450 enzyme mainly exists in the endoplasmic reticulum of the smooth surface of hepatocytes, so the CYP450 content of liver microsomes can be determined after extracting liver microsomes by ultracentrifugation. The extracted liver microsomes can also be further co-incubated with CYP450 enzyme substrate probes to determine the metabolic activity of CYP450 enzyme on the substrate drugs. The method of liver microsomes extraction is briefly described as follows: centrifuge the supernatant of liver homogenate at 105,000 g for 60 min at 4°C, retain the centrifuged precipitate, and add liver microsomes to resuspension (0.25 M sucrose-0.1 M potassium phosphate buffer, 1 mM EDTA, pH 7.

4， 1 ml/g of liver), and then store it at -80°C for later use.

5. The unit of CYP450 enzyme content in this experiment is nmol/g liver. If liver microsomes are extracted, the CYP450 enzyme content per unit protein concentration can be found by determining the protein concentration of liver microsomes (e.g., Bradford method).

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