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Overall level characterization method of COMT enzyme activity and application thereof

An enzyme activity and level technology, applied in biochemical equipment and methods, microbial measurement/inspection, material inspection products, etc., can solve problems such as difficult COMT enzyme activity evaluation, undetermined methyl products, complex metabolic pathways, etc., to achieve Good specificity, suitable for widespread application, good detection sensitivity

Active Publication Date: 2021-12-17
THE AFFILIATED HOSPITAL OF SOUTHWEST MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, although there have been research reports on COMT enzyme probes in vivo, it is difficult to evaluate COMT enzyme activity at the overall level due to the wide distribution of COMT enzymes in tissues and organs in vivo.
However, most of the existing in vivo COMT probes are catecholamine compounds with high water solubility and complex metabolic pathways, such as figure 1 As shown, it is difficult to detect
The existing Chinese patent CN103193746A discloses a specific probe substrate for a class of catechol-O-methyltransferase and its application. The final methyl product cannot be determined, and only the amount of 7-O-methyl metabolite To measure the activity of COMT enzyme, the error is very large. In addition, the biological samples used in the test of COMT can only be the tissues of organs such as liver cells, which cannot reflect the overall level of COMT enzyme activity in the organism as a whole.

Method used

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  • Overall level characterization method of COMT enzyme activity and application thereof
  • Overall level characterization method of COMT enzyme activity and application thereof
  • Overall level characterization method of COMT enzyme activity and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The present embodiment determines to use 3-aminocoumarin as IS, and the specific operations are as follows:

[0040] High signal intensity of the target analyte is necessary for an MS-based analysis method. The chromatographic separation conditions and mass spectrometry parameters were optimized.

[0041] Based on the analytes (daphnetin and daphnetin-Me 100ng / mL) of Q1 full-scan mass spectrometry, it was found that the strong signal of daphnetin-Me in positive ion mode was significantly higher than that in negative ion mode, while that of daphnetin in positive and negative ion modes The comparison of the MS signals of the above results shows that the positive ion mode is beneficial for the determination of daphnetin and 8-methyl metabolites, such as figure 2 shown. Based on this observation, 3-aminocoumarin was finally selected as the proton-accepting ability of 3-aminocoumarin, as well as its similarity with daphnetin and the 8-methyl metabolite in terms of structu...

Embodiment 2

[0043] This embodiment provides the hydrolysis treatment process of daphnetin metabolites, the specific operations are as follows:

[0044] (1) Select SD rats (male, 200±20g), and place the rats in a standard environment with a temperature of 25±2° C. and a humidity of 50±5% for 2 weeks (12 / 12h dark / light cycle) before the experiment. Fast overnight before administration;

[0045] (2) Dissolve daphnetin with Tween-80, pour it into rats, and collect rat urine for a period of time;

[0046] (3) Take the same amount of urine, add sulfotransferase and uridine diphosphate glucuronyltransferase to hydrolyze it;

[0047] (4) Daphnetin and its 8-methyl metabolites remained in the final urine.

Embodiment 3

[0049] The present embodiment provides the analytical conditions of liquid chromatography and liquid phase mass spectrometry detection system, specifically as follows:

[0050] The detection system includes liquid chromatography and liquid chromatography mass spectrometry. The analysis conditions are as follows: All analyzes are carried out by LC-MS / MS system, which includes LC-30AD×2 infusion pump, DGU-20A5 online degasser, SIL-30AC automatic sampling device, CTO-20A column furnace, CBM- 20A system controller and 8045 triple quadrupole mass spectrometer. The computer is equipped with LC-MS / MS solution software (V version 5.85). Acchrom Xcharge C18 column (2.1×150mm, 5μm) was used for chromatographic separation, and the column temperature was 40°C. The mobile phase composition was a mixture of 0.1% formic acid-acetonitrile (63:37, v / v). The flow rate was 0.3 mL / min, and the temperature of the autosampler was maintained at 10°C. The injection volume was 10 μL, and the run t...

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Abstract

The invention discloses an overall level characterization method of COMT enzyme activity. The overall level characterization method is characterized in that daphnetin or a derivative thereof is used as a substrate, the substrate is subjected to a C-8-site methyl substitution reaction under the action of COMT enzyme and is treated by sulfotransferase and glucuronic acid hydrolase to generate an 8-O-methylated product, and the activity of the COMT enzyme in each biological sample is determined by quantitatively detecting the contents of the substrate and 8-O-methyl metabolite thereof in unit time. According to the method provided by the invention, the activity level of COMT enzyme in a living body can be represented from the overall level, daphnetin or the derivative thereof can only be converted into 8-methyl metabolite thereof, the specificity is good, and the detection accuracy is high; the detection sensitivity is good, and the safety is relatively high; and when the method is applied to a kit, the activity of the COMT enzyme can be accurately detected, the activity of the COMT enzyme in a biological sample is determined according to the ratio of the content of the substrate to the content of the 8-O-methylated product, and a definite evaluation standard for the activity of the COMT enzyme is given.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a method for characterizing the overall level of COMT enzyme activity and its application. Background technique [0002] COMT enzyme is an important methyltransferase widely distributed in mammals. Its main physiological function is to be responsible for endogenous neurotransmitters (such as adrenaline, norepinephrine and its metabolites, dopamine, etc.) and Metabolism of exogenous catechol compounds (such as carbidopa, apomorphine, dobutamine, etc.). [0003] The activity of COMT enzyme in the human body is a genetic polymorphism, which can be divided into high, medium and low metabolic groups according to the different genotypes. According to the analysis of a large number of clinical samples, the in vivo activity of COMT enzyme is closely related to pain, alcoholism, breast cancer and mental diseases. For example, high or low activity of COMT enzyme in human brain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/34G01N33/15
CPCC12Q1/48C12Q1/34G01N33/15G01N2333/924G01N2333/91011G01N2333/914G01N2800/2835G01N2800/52
Inventor 齐晓怡梁思成梁可白睿然赵文静邓明明熊霞吕沐瀚
Owner THE AFFILIATED HOSPITAL OF SOUTHWEST MEDICAL UNIV
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