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Method for detecting activity of catechol-oxygen-methyltransferase in blood

A technology of methyltransferase and catechol, which is applied in the field of biomedicine, can solve the problems of poor chemical stability and achieve the effects of stable properties, good fluorescence emission spectrum characteristics, good reliability and selectivity

Inactive Publication Date: 2018-09-04
沈阳北创医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the commonly used COMT enzyme probe substrates mainly include catecholamine compounds such as levodopa, dopamine, and adrenaline, but these substrates have poor chemical stability, and need to add reducing agents and dark operations, and require mass spectrometry. Technology completes the detection of trace COMT activity in biological samples

Method used

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  • Method for detecting activity of catechol-oxygen-methyltransferase in blood
  • Method for detecting activity of catechol-oxygen-methyltransferase in blood
  • Method for detecting activity of catechol-oxygen-methyltransferase in blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Synthesis of 3-benzothiazole-7,8-dihydroxycoumarin: Weigh 1.0 g of 2,3,4-trihydroxybenzaldehyde and dissolve it in 25 mL of methanol, add 2-(2-benzothiazole) ethyl acetate Ester 1.5 mL, piperidine 0.1 mL, stirred at room temperature for 20 minutes, heated to 65 °C, reacted for 4 h, TLC detected the end of the reaction. After cooling the reaction solution, add 10 mL of water, filter, and collect the filter cake, which is the crude product. The crude product was added to 20 mL of acetonitrile and refluxed for 1 h, filtered after cooling, and dried in vacuo to obtain 1.8 g of the product 3-benzothiazole-7,8-dihydroxycoumarin as a brownish-yellow solid with a yield of 88%. 1 H NMR spectrum and 13 C NMR spectrum as figure 2 and image 3 shown;

[0031] 1 H NMR (400 MHz, DMSO- d 6 ) δ: 10.59 (s, 1H), 9.68 (s, 1H), 9.11 (s, 1H), 8.16 (d, J= 7.5 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.56 (td, J =7.2Hz, J =1.2Hz, 1H), 7.45 (td, J =8.0Hz, J =0.8 Hz, 1H), 7.44 (d, J ...

Embodiment 2

[0035] Determination of fluorescence absorption and emission spectra of substrates and products: Accurately weigh 3-benzothiazole-7,8-dihydroxycoumarin and dissolve in chromatographic grade dimethyl sulfoxide solution to prepare a 50 mmol / L standard The mother liquor was then diluted with 0.5% formic acid in acetonitrile solution and deionized water (1:1) to obtain a standard solution with a final concentration of 1.0 mmol / L, and its fluorescence absorption and emission spectra were measured using a Synergy H1 microplate reader. 3-Benzothiazole-7-hydroxy-8-methoxycoumarin was prepared into a standard solution of the same concentration by the same method, and its fluorescence absorption and emission spectra were measured by using a Synergy H1 microplate reader. The fluorescence absorption and emission spectra of 3-benzothiazole-7,8-dihydroxycoumarin and 3-benzothiazole-7-hydroxy-8-methoxycoumarin are attached Figure 4 with attached Figure 5 shown.

Embodiment 3

[0037] Establishment of high performance liquid chromatography-fluorescence detection and analysis conditions: liquid phase system Shimadzu 20A XR system, equipped with system controller CBM-20A, two LC-20AD pumps, CTO-20A column thermostat, automatic sampler SIL- 20ACHT, one DGU-20A3 vacuum degasser, one RF-20A xs fluorescence detector. The analytical column was a Hedera C18 reverse-phase chromatographic column (150.0 mm×2.1 mm, 3 μm), and the temperature of the column oven was set at 40°C. Mobile phase A was acetonitrile, B was water (containing 0.2% formic acid), and the flow rate was 0.4 ml / min. The gradient elution conditions are: 0-10.0 min, 90% B-5% B; 10.0-13.0 min, 5% B; 13.0-16.0 min, equilibrated to 90% B. The fluorescence detection conditions are: the excitation wavelength is 390 nm, and the emission wavelength is 520 nm.

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Abstract

The invention relates to a method for detecting the activity of catechol-oxygen-methyltransferase (COMT) in blood and belongs to the technical field of biological medicine. According to the invention,3-benzothiazole-7,8-dihydroxycoumarin (3-BTD) with high chemical stability is selected as the fluorescence substrate of COMT enzyme by virtue of the advantages of a high performance liquid chromatography and fluorescence detection combined instrument on biological matrix interference resistance and detection sensitivity; meanwhile, a high-efficiency and high-sensitivity measuring method capable of quantitatively analyzing the activity of a trace amount of COMT in the blood is developed by combining a high performance liquid chromatography-fluorescence detection technology. A fluorescence probe substrate adopted by the method can be obtained through chemical synthesis, the synthesis process is simple and practical, and the performance is stable. The method has the advantages of high sensitivity, high accuracy degree, high biological matrix interference resistance, high repeatability and the like, can be applied to detection on the activity of the COMT enzyme in the blood, and also canbe applied to quantitative measurement on the activity of the COMT enzyme in various mammal-derived cells or tissue preparation objects as well as screening and evaluation of a COMT inducer / activator.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a high-performance liquid chromatography-fluorescence detection method for detecting the activity of catechol-oxygen-methyltransferase in blood. Background technique [0002] Catechol-O-methyltransferase (Catechol-O-methyltransferase, COMT) is an important phase II transferase in the human body, widely distributed in human tissues such as liver, kidney, lung, breast, red blood cells and brain middle. In the human body, COMT is responsible for the removal of active and toxic catechol compounds, such as neurotransmitters dopamine, epinephrine and norepinephrine, and catechol estrogen compounds such as estradiol. In addition, COMT enzymes are also involved in exogenous catechol drugs (such as carbidopa, benserazide, apomorphine, dobutamine, fenoldopam, α-methyl-L-DOPA, Metabolic clearance of isoproterenol and rimiterol, etc.). [0003] The COMT enzyme in the human ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
CPCC12Q1/48G01N2333/91017
Inventor 高明宇
Owner 沈阳北创医学检验所有限公司
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