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Method for in-vitro metabolism of chlorinated paraffin by liver microsome

A technology of chlorinated paraffin and liver microsomes, which is applied in the field of in vitro metabolism of chlorinated paraffins by liver microsomes, can solve the problems of limited metabolic capacity, low sensitivity, and reduced metabolic clearance rate, so as to improve metabolic clearance rate and reduce reaction cost , suitable for the effect of batch operation

Active Publication Date: 2021-10-22
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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Problems solved by technology

However, it is difficult to determine the metabolic clearance rate of chlorinated paraffins by using the current method of liver microsomes to metabolize halogenated organic pollutants in vitro
This is due to the relatively low sensitivity of the current method for detecting the concentration of chlorinated paraffin, and it is necessary to use a higher concentration of chlorinated paraffin as the substrate for experiments to meet the needs of subsequent detection; but due to the limited metabolic capacity of liver microsomes, its effect on substrate The metabolic clearance rate of the substance usually decreases with the increase of the substrate concentration. When referring to the liver microsomal metabolic conditions of other halogenated organic pollutants, even if the lowest concentration of 0.2 μg / mL that can meet the follow-up detection method of chlorinated paraffin is adopted, It is also difficult to observe the consumption of chlorinated paraffins

Method used

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  • Method for in-vitro metabolism of chlorinated paraffin by liver microsome
  • Method for in-vitro metabolism of chlorinated paraffin by liver microsome
  • Method for in-vitro metabolism of chlorinated paraffin by liver microsome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Metabolism of short-chain chlorinated paraffins (SCCPs) by chicken liver microsomes in vitro

[0029] (1) Take 1 μL of SCCPs-acetonitrile solution and place it at the bottom of a 1.5 mL disposable centrifuge tube, add 10 μL of chicken serum, shake, vortex slightly, and let it stand at 4°C for 12 hours;

[0030] (2) Add 345 μL of potassium phosphate buffer solution with a pH value of 7.4, 12.5 μL of chicken liver microsomes, and 25 μL of A solution and 5 μL of B solution in the NADPH enzyme regeneration system, shake well, and quickly place in a 37°C water bath for shaking reaction 0.5h;

[0031] (3) Add 400 μL of ice-cold acetonitrile to terminate the reaction, centrifuge at 10,000 rpm / min for 5 min at 4°C, then take 500 μL of the supernatant to an injection bottle, and use UPLC-ESI-HRMS to measure the content of SCCPs molecular formula homologues. See Table 1. Table 1 shows the metabolic clearance rates of SCCPs with different carbon chains with 7 chlorine atoms and ...

Embodiment 2

[0037] Metabolism of medium-chain chlorinated paraffins (MCCPs) by chicken liver microsomes in vitro

[0038] (1) Take 12 μL of MCCPs acetonitrile solution and place it at the bottom of a 1.5 mL disposable centrifuge tube, add 30 μL of chicken serum, shake, vortex slightly, and let it stand at room temperature for 3 hours;

[0039] (2) Add 415 μL of potassium phosphate buffer solution with a pH value of 7.4, 12.5 μL of chicken liver microsomes, and 25 μL of A solution and 5 μL of B solution in the NADPH enzyme regeneration system, shake well, and quickly place in a 37°C water bath for shaking reaction 6h;

[0040] (3) Add 500 μL of ice-cold acetonitrile to terminate the reaction, centrifuge at 10,000 rpm / min for 5 min at 4°C, then take 500 μL of the supernatant to an injection bottle, and use UPLC-ESI-HRMS to determine the content of MCCPs molecular formula homologues. See Table 2. Table 2 shows the metabolic clearance rates of MCCPs with different carbon chains with 7 chlor...

Embodiment 3

[0045] Metabolism of medium-chain chlorinated paraffins (LCCPs) by chicken liver microsomes in vitro

[0046] (1) Take 25 μL of LCCPs acetonitrile solution and place it at the bottom of a 1.5 mL disposable centrifuge tube, add 25 μL of chicken serum, shake, vortex slightly, and let stand at 4°C for 6 hours;

[0047] (2) Add 510 μL of potassium phosphate buffer solution with a pH value of 7.4, 12.5 μL of chicken liver microsomes, and 25 μL of A solution and 5 μL of B solution in the NADPH enzyme regeneration system, shake well, and quickly place in a 37°C water bath for shaking reaction 2h;

[0048] (3) Add 600 μL of ice-cold acetonitrile to terminate the reaction, centrifuge at 10,000 rpm / min for 5 min at 4°C, then take 500 μL of the supernatant to an injection bottle, and use UPLC-ESI-HRMS to determine the content of LCCPs molecular formula homologues. See Table 3. Table 3 shows the metabolic clearance rates of LCCPs with different carbon chains with 7 chlorine atoms and LC...

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Abstract

The invention discloses a method for in-vitro metabolism of chlorinated paraffin by liver microsomes, which comprises the following steps: (1) dissolving chlorinated paraffin in an organic solvent to obtain a chlorinated paraffin solution, adding serum into the chlorinated paraffin solution, and standing for balancing to obtain a substrate system; (2) sequentially adding a potassium phosphate buffer solution, liver microsome and an NADPH enzyme regeneration system into the substrate system, uniformly shaking to obtain a final reaction system, and then putting the final reaction system into a water bath at 37 DEG C for oscillating reaction; and (3) adding an ice-colded organic solvent into the reaction system in the step (2) to terminate the reaction, then centrifuging the solution, taking a supernatant liquid, and measuring the content of chlorinated paraffin. The serum is adopted as a carrier of the chlorinated paraffin, and reaction of the chlorinated paraffin and the liver microsome is promoted, so that the metabolic clearance rate of the chlorinated paraffin is remarkably increased, and the measurement of the metabolic clearance rate of the liver microsome for in-vitro metabolism of the chlorinated paraffin is realized.

Description

technical field [0001] The invention relates to a method for liver microsomes to metabolize chlorinated paraffin in vitro. Background technique [0002] Chlorinated paraffins (CPs) are a class of chlorinated alkanes mixtures, mainly used in the metal processing industry and the plastics industry. It is one of the most serious halogenated organic pollutants that pollute the environment and agricultural products in our country. According to the carbon chain length, chlorinated paraffins can be divided into short-chain chlorinated paraffins (SCCPs, C 10-13 ), medium chain chlorinated paraffins (MCCPs, C 14-17 ) and long-chain chlorinated paraffins (LCCPs, C 18-20 ). Studies have shown that chlorinated paraffins are reproductive and developmental toxic to animals. [0003] In the prior art, the in vitro metabolism of halogenated organic pollutants by animal liver microsomes is an important means to study their metabolic transformation mechanism in animals, and the latter is...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/04
CPCG01N30/04G01N30/02G01N2030/067
Inventor 黄晓梅王旭殷秋妙丁晨红王威利苏秋权王英崔泽锋
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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