Method for detecting acetyl choline content in peripheral blood mononuclear cells

A technology of acetylcholine and detection method, applied in the field of biochemistry, can solve problems such as lack of a method for detecting the content of acetylcholine in human peripheral blood mononuclear cells, and achieve the effects of good linearity and sensitivity, high recovery rate and guaranteed accuracy

Inactive Publication Date: 2016-08-24
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0007] The present invention aims at the technical defects of the prior art, and provides a method for detecting the content of acetylcholine in human peripheral blood mononuclear cells, so as to solve the problem that the relevant methods in the prior art are generally only used to detect the content of ACh in the nervous system, and lack a specific method. Technical problems of a method for detecting the content of acetylcholine in human peripheral blood mononuclear cells

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  • Method for detecting acetyl choline content in peripheral blood mononuclear cells
  • Method for detecting acetyl choline content in peripheral blood mononuclear cells
  • Method for detecting acetyl choline content in peripheral blood mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 (a kind of detection method of acetylcholine content in human peripheral blood mononuclear cells)

[0066] 1.1 Experimental objects

[0067] Randomly selected 22 healthy adults in the Health Examination Center of Tianjin Medical University General Hospital from March 1, 2013 to April 1, 2013, including 12 males, aged (50.2±3.8) years old, and 10 females, aged (49.1±3.6) years old. Take 2 tubes of 5ml of peripheral venous blood (EDTA-K2 anticoagulant) on an empty stomach in the morning. Peripheral blood mononuclear cells were extracted at 4°C or 25°C within 1 hour after blood collection. All operating procedures were approved by the Ethics Committee of Tianjin Medical University General Hospital. All selected subjects signed the informed consent.

[0068] 1.2 Instruments and materials

[0069] 1.2.1 Main Instruments

[0070]

[0071] 1.2.2 Main reagents

[0072]

[0073]

[0074] 1.3 Experimental method

[0075] 1.3.1 Extraction of human peri...

Embodiment 2

[0107] Embodiment 2 (optimization process and experimental ideas of analyte and analytical column in the detection method of embodiment 1)

[0108] 2.1 Optimization of analyte and analytical column

[0109] 2.1.1 Selection of internal standard

[0110] Quantitative analysis by liquid chromatography-mass spectrometry mostly uses the internal standard method. ACh and iso-ACh are isomers, and the endogenous content in the cell is very low, and the d9-ACh isotope is used as the internal standard, because their physical and chemical properties are the same, it can minimize The analysis error caused by the loss of the sample improves the accuracy of the determination result. As for choline, the intracellular content is relatively high, using the isotope internal standard and methacholine (similar to the chemical structure of choline) used in this experiment as the internal standard can also obtain stable results and save costs .

[0111] 2.1.2 Selection of chromatographic column...

Embodiment 3

[0115] Embodiment 3 (optimization process and experimental thinking of mass spectrometry conditions in the detection method of embodiment 1)

[0116] Positive ion electrospray ionization (ESI) mode can dissociate molecules by high voltage to form ionized [M] + and [M+H] + . We found that in Waters TQD mass spectrometry, when the electrospray voltage range is 2.0-3.0kV and the cone voltage is between 15-20V, most small molecular organic compounds can be efficiently dissociated into [M+H] + ion. Since ACh, iso-ACh and Cho all have quaternary ammonium functional groups and carry cations under acidic conditions, the application of ESI mode on TQD mass spectrometry results in good sensitivity and optimal MRM conditions.

[0117] ACh, iso-ACh and d 9 - ACh molecules only produce two product ions (fragment ions) after energy collision: m / z 87 is a common product ion, and there are corresponding product ions m / z 60 (ACh and iso-ACh), m / z 69 ( d 9 -ACh), dissociation modes such a...

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Abstract

The invention provides a method for detecting the acetyl choline content in peripheral blood mononuclear cells. The method provided by the invention lays a foundation for accurately detecting the ACh (acetyl choline) content by separating an ACh ingredient from a sample to be detected by adopting a UPLC (ultra performance liquid chromatography) technology. According to the technical scheme, an HILIC (hydrophilic interaction chromatography) column is found to be more suitable for separation of a polar compound by adopting experimental measures, can be used for effectively separating ACh from iso-ACh, and has a better separation effect on Cho. On this basis, chromatographic conditions and internal standard substance categories are further designed, and d9-ACh of isotope is taken as national standard substances of ACh and iso-ACh at the same time to furthest reduce the analysis error caused by sample damage and effectively ensure the accuracy of a measurement result. According to the method provided by the invention, on the basis of effectively separating ACh by using the UPLC, ACh signal detection is performed by a liquid chromatography-mass spectrometry system, and the ACh is accurately quantified by optimizing mass spectrometer conditions with a stable and accurate result and favorable linearity and sensitivity; meanwhile, the method has relatively low detection limit and relatively high recovery rate and also has a broad application prospect.

Description

technical field [0001] The invention relates to the technical field of biochemistry, and further relates to the quantitative analysis of trace substances in human body, in particular to a method for detecting the content of acetylcholine in human peripheral blood mononuclear cells. Background technique [0002] Acetylcholine (ACh), a classic cholinergic neurotransmitter, plays an important role between nerve cells or between nerve cells and effector cells by binding to specific M-type or N-type cholinergic receptors. The role of electrical information transmission. Acetylcholine has a wide range of physiological functions, and plays a key role in regulating muscle strength, sleep, body temperature, blood pressure, especially advanced cognitive functions such as learning, memory, and judgment. Traditionally, most people think that acetylcholine mainly exists in the nervous system, and the research on acetylcholine and its receptors mainly focuses on their role in the nervous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 郝峻巍张超夏义平蒋薇王春阳
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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