Application of serum as reagent for dissolving halogenated organic pollutants in in-vitro liver microsome metabolism system

A technology for organic pollutants and liver microsomes, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, instruments, etc., to achieve the effect of simplifying operation steps, avoiding inhibition, and being suitable for large-scale operations

Active Publication Date: 2022-07-22
广东省农业科学院农业质量标准与监测技术研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One of the purposes of the present invention is to provide the use of serum as a reagent for dissolving halogenated organic pollutants in the in vitro liver microsome metabolism system, to solve the problem of organic solvents inhibiting the activity of metabolic enzymes in the prior art, and to make the in vitro liver microsomes metabolize halogenated organic pollutants. The reaction of organic pollutants can more realistically simulate the metabolic transformation of liver microsomes in vivo, and can more truly reflect the metabolic characteristics of halogenated organic pollutants in animals

Method used

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  • Application of serum as reagent for dissolving halogenated organic pollutants in in-vitro liver microsome metabolism system
  • Application of serum as reagent for dissolving halogenated organic pollutants in in-vitro liver microsome metabolism system

Examples

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Embodiment 1

[0041] In vitro metabolism of medium-chain chlorinated paraffins (MCCPs) by chicken liver microsomes: a comparative experiment between serum substrate systems and organic solvent substrate systems

[0042] (1) Serum substrate system: Take 200 μL of MCCPs standard solution (100 μg / mL, isooctane) in a 1.5 mL glass injection bottle, blow the organic solvent nitrogen to dryness with mild nitrogen, and then add 200 μL fetal bovine serum, sonicated for 20 min (ultrasonic frequency is 40KHz), vortexed to obtain the substrate solution dissolved in serum;

[0043]Acetonitrile substrate system: Take 200 μL of MCCPs standard solution (100 μg / mL, isooctane) into a 1.5 mL glass injection bottle, blow isooctane nitrogen to dryness with mild nitrogen, and then add 200 μL of acetonitrile , vortex to obtain the substrate solution dissolved in acetonitrile;

[0044] (2) Under ice bath conditions, take two 1.5 mL disposable centrifuge tubes, add 5 μL of the above two substrate solutions respect...

Embodiment 2

[0049] In vitro metabolism of triclosan by chicken liver microsomes: a comparative experiment between a serum substrate system and an organic solvent substrate system

[0050] (1) Serum substrate system: Take 10 μL of triclosan standard solution (100 μg / mL, methanol) in a 1.5 mL glass injection bottle, blow the organic solvent nitrogen to dryness with mild nitrogen, and then add 100 μL porcine serum, sonicated for 5 min, and vortexed to obtain a serum-dissolved substrate solution;

[0051] Methanol substrate system: take triclosan standard solution (100 μg / mL, methanol) as the methanol-dissolved substrate solution;

[0052] (2) Under ice bath conditions, take two 1.5 mL disposable centrifuge tubes, add 5 μL of the above two substrate solutions, and then add 455 μL potassium phosphate buffer solution (pH 7.4) and 12.5 μL chicken liver microsomes in turn. , 25 μL of Solution A, 5 uL of Solution B, shake well, and quickly place in a 40°C water bath for 30 min of shaking reaction...

Embodiment 3

[0056] In vitro metabolism of MCCPs by chicken liver microsomes: a comparative experiment between serum substrate system and organic solvent-serum substrate system

[0057] (1) Serum substrate system: Take 200 μL of MCCPs standard solution (100 μg / mL, isooctane) in a 1.5 mL glass injection bottle, blow the organic solvent nitrogen to dryness with mild nitrogen, and then add 200 μL chicken serum, sonicated for 20 min (ultrasonic frequency of 40KHz), vortexed to obtain serum-dissolved substrate solution; 1 μL of substrate solution was placed at the bottom of a 1.5 mL disposable centrifuge tube to obtain serum substrate system;

[0058] Acetonitrile-serum substrate system: Take 1 μL of MCCPs-acetonitrile solution and place it at the bottom of a 1.5 mL disposable centrifuge tube, add 1 μL chicken serum, vortex slightly, and let it stand at 4°C for 12 hours to equilibrate to obtain an acetonitrile-serum substrate system ;

[0059] (2) Under ice bath conditions, add 955 μL potassiu...

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Abstract

The invention discloses an application of serum as a reagent for dissolving halogenated organic pollutants in an in-vitro liver microsome metabolism system, and also provides a method for metabolizing halogenated organic pollutants by in-vitro liver microsome. According to the invention, the problem that an organic solvent inhibits the activity of metabolic enzymes in the prior art is solved, so that the reaction of the in-vitro liver microsome for metabolizing the halogenated organic pollutants can simulate the in-vivo metabolic transformation condition of the liver microsome more truly, and the metabolic characteristics of the halogenated organic pollutants in animal bodies can be reflected more truly.

Description

technical field [0001] The present invention particularly relates to the use of serum as a reagent for dissolving halogenated organic pollutants in an in vitro liver microsomal metabolic system. Background technique [0002] In the prior art, metabolizing halogenated organic pollutants by in vitro animal liver microsomes is an important means to study their metabolic transformation mechanism in animals. The current method for metabolizing halogenated organic pollutants by liver microsomes in vitro includes the following steps: 1. Dissolving the substrate (halogenated organic pollutants) with an organic solvent to form a substrate solution, the organic solvent is acetonitrile, methanol or dimethyl 2. Add the substrate solution, potassium phosphate buffer solution, liver microsomes to the reaction system, and place the reaction system in a water bath at a suitable temperature for pre-incubation for 5 minutes, then add the NADPH enzyme regeneration system , the reaction starts...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N30/02G01N30/72
CPCG01N33/5067G01N33/5038G01N30/02G01N30/72
Inventor 黄晓梅王旭丁晨红王威利许开航
Owner 广东省农业科学院农业质量标准与监测技术研究所
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