Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity

A technology of liver tissue and enzyme activity, which is applied in the field of detection of CYP450 enzyme activity by in vitro incubation of trace liver tissue, which can solve the problems of expensive equipment, large amount of liver tissue, and long centrifugation time, and achieve the effect of saving cost and time

Inactive Publication Date: 2008-11-19
CENT HOSPITAL XUHUI DISTRICT SHANGHAI CITY
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Problems solved by technology

[0003] The liver is the main organ for drug metabolism in organisms. The study of liver metabolism of drugs is of great significance to the study of drug metabolism in vivo. At present, the research on the relationship between cytochrome P450 and drug metabolism is increasing. Using liver microsomes as an in vitro incubation system for The study of cytochrome P450 enzyme sy

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  • Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity
  • Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity
  • Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity

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Embodiment 1

[0032] 1. Solution preparation

[0033] 1.1 Homogenate: add KCl 6g Tris 3.0g to 500ml distilled water;

[0034] 1.2 Incubation solution: add K to 500ml distilled water 2 HPO 4 11g, then adjust the pH to 7.4 with HCl;

[0035] 1.3 Precipitating agent: TCA was added with distilled water to make a 10% (W / V) solution for later use.

[0036] 1.4 Stock solutions of probe substrates and metabolites: Weigh an appropriate amount of PN and ACE respectively, dissolve them in methanol and dilute to a methanol solution with a required concentration of 1-10 mg / ml.

[0037] 2. Sample

[0038] 2.1 20 healthy Kunming rats, male, were randomly divided into four groups, 5 rats in each group were weighed, fed to the stomach after fasting for 12 hours on the first day, the control group was given normal saline, and the other three groups were given drug C (such as berberine derivatives ) High, medium and low doses of drugs, low dose group: 0.3mg / ml, medium dose group: 0.6mg / ml, high dose gro...

Embodiment 2

[0074] Confirmation experiment of the change of CYP1A2 enzyme activity after gavage of high, medium and low doses of drug C to mice:

[0075] 1. Thirty-two healthy Kunming rats, male, were randomly divided into four groups, with 8 rats in each group, and weighed.

[0076] 2. Blank control group (physiological saline), 0.64 mg / ml low dose group, 1.6 mg / ml medium dose group, and 4 mg / ml high dose group.

[0077] 3. Gastrointestinal administration after fasting for 12 hours on the first day, the control group was given normal saline, and the other groups were given high, medium and low doses of drugs.

[0078] 4. Gastrointestinal administration every day and fasting at night for 8 days. On the 8th day, the mice were sacrificed and the liver was taken out.

[0079] 5. The liver was incubated with phenacetin probe drug in vitro, and the effect of drug C on liver CYP1A2 was evaluated by LC-MS / MS detection.

[0080] Solution preparation and incubation methods, and LC-MS / MS detectio...

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Abstract

Disclosed is a method for detecting CYP450 enzymatic activity by means of micro-liver tissue incubation in vitro, which is characterized in that: a liver trace puncturing method used in clinic takes micro-liver tissues of 0.1 to 0.2g, or kills a mouse to take out the liver in an animal experiment, and builds a micro-liver tissue in vitro incubation system, then uses a one-probe medicine to perform the liquid phase chromatography tandem mass spectrometry for detecting probe substrates and concentration of corresponding metabolites, and obtains the activity of P450 enzyme according to metabolite ratios thereof. The method has the advantages of: 1. micro scale: only micro-liver tissues of 0.1 to 0.2g are needed for detecting cytochrome P450enzyme activity, and can be used for animal experiment and checking clinic micro-liver tissue liver drug enzyme activity; 2. time saving and convenience: costly ultracentrifugation equipment used in the conventional method is saved, costs and time for detection are greatly saved, and liver enzyme metabolic condition simulation is better; and 3. establishing indexes for evaluating the enzyme metabolic activity: metabolite ratios.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and relates to a method for detecting enzyme activity, in particular to a method for detecting CYP450 enzyme activity by incubating a trace amount of liver tissue in vitro Background technique [0002] Cytochrome P450 is a class of enzymes involved in the metabolism of endogenous and exogenous compounds. This enzyme mainly exists in the endoplasmic reticulum of organisms and belongs to one of the mixed function oxidase systems. ) drug biotransformation by enzymes is an important part of drug metabolism, and many drugs can inhibit or induce the activity of CYP450 enzymes. When a subtype of the drug enzyme CYP450 is induced or inhibited, drug interactions can be induced, such as taking cimetidine and terfenadine at the same time, because terfenadine is mainly metabolized by cytochrome CYP3A (CYP450 subenzyme), and Cimetidine has a strong inhibitory effect on CYP3A, and combined medica...

Claims

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Application Information

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IPC IPC(8): G01N30/00G01N33/48C12Q1/26
Inventor 余琛周菊珍周红李春霞李雪宁施孝金张渊陈伟力王书云
Owner CENT HOSPITAL XUHUI DISTRICT SHANGHAI CITY
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