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A liquid-mass spectrometry method for the determination of acetyl-CoA in animal liver

A technology of acetyl coenzyme and animal liver, which is applied in the field of analysis and detection, and can solve the problems of lowering the stability, accuracy, reproducibility of the determination, high requirements for detection methods and processing procedures, and inability to accurately indicate the content in the liver, etc. , to achieve the effect of simplified operation steps, easy operation and fast method

Active Publication Date: 2020-12-01
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the liver tissue is obtained, if it is not processed in a reasonable and timely manner, the acetyl-CoA in the tissue will be degraded rapidly, and the content in the liver cannot be accurately indicated, which obviously increases the difficulty of detecting acetyl-CoA. and processing requirements are also higher
[0004] At present, enzyme-linked immunoassay (ELISA) is commonly used for the determination of acetyl-CoA in the liver. This method involves cumbersome operations and requires a series of kits. Finally, it is determined by a microplate reader, and many operating steps reduce the Its assay stability, accuracy, reproducibility

Method used

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  • A liquid-mass spectrometry method for the determination of acetyl-CoA in animal liver
  • A liquid-mass spectrometry method for the determination of acetyl-CoA in animal liver
  • A liquid-mass spectrometry method for the determination of acetyl-CoA in animal liver

Examples

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Effect test

Embodiment 1

[0048] Example 1 Verification of the detection methodology of acetyl-CoA in the liver

[0049] The concentration range of the standard curve is set to 0.05-5μg / mL (not limited to this concentration range, the lower limit of quantitation can be set to a lower concentration, such as 0.01μg / mL), which can meet the determination requirements of acetyl-CoA in the liver , so the lower limit of quantitation was set at 0.05 μg / mL, and the quality control concentrations were set at 0.1 μg / mL, 0.5 μg / mL, and 4 μg / mL. The specificity in the method meets the requirements, and the endogenous acetyl-CoA in the inactivated liver homogenate will not significantly interfere with the determination of acetyl-CoA (such as Figure 1-3 Shown), the retention time of acetyl-CoA is about 0.60min, and the retention time of the internal standard is about 1.78min.

[0050] Accurately draw 5 μL of STD1-STD7 (STD0 does not add analyte) series acetyl-CoA standard curve working solution and place it in a 1....

example 2

[0075] Determination of Acetyl-CoA in Example 2 Normal Mouse Liver Sample

[0076] ICR mice weighing 18-22 g were provided by Shanghai Xipro-Bikay Experimental Animal Co., Ltd., and adapted to a normal diet for 1 week. Fasted overnight before the test, during which time the rats were allowed to drink water freely. After the rats were anesthetized at a specific time, the liver was washed with cold normal saline from the hepatic portal vein until it was khaki, and the rat liver was obtained immediately. And put it on ice immediately to avoid the rapid degradation of acetyl-CoA. Weigh about 0.1g of liver tissue, add 900μL of cold 10% perchloric acid aqueous solution (containing 0.2-0.5% dithiothreitol DTT, 20-50mM phenylmethylsulfonyl fluoride PMSF), under ice bath condition Homogenize at high speed to obtain liver tissue homogenate. Add a certain volume of internal standard (20 μL of diazepam solution with a concentration of 200 ng / mL) to the homogenate, and vortex the obtaine...

example 3

[0079] Example 3 Determination of Acetyl-CoA in Liver Samples of Mice Fed with High Fat Diet

[0080] ICR mice weighing 18-22 g were provided by Shanghai Xipro-Bikay Experimental Animal Co., Ltd., and were fed with a high-fat diet after 1 week of adaptation to a normal diet. Before the specific modeling time node, the mice were fasted overnight, and they could drink water freely during the period. After the rats were anesthetized at a specific time, the liver was washed with cold normal saline from the hepatic portal vein until it was khaki, and the large animals were obtained immediately. mouse liver. And put it on ice immediately to avoid the rapid degradation of acetyl-CoA. Weigh about 0.1g of liver tissue, add 900μL of cold 10% perchloric acid aqueous solution (containing 0.2-0.5% dithiothreitol DTT, 20-50mM phenylmethylsulfonyl fluoride PMSF), under ice bath condition Homogenize at high speed to obtain liver tissue homogenate. Add 20 μL of diazepam solution (the intern...

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Abstract

The invention discloses a liquid chromatography-mass spectrometry measuring method of an acetyl coenzyme A in animal livers. The method specifically comprises the steps that firstly, the fresh animallivers are taken, and blood in the animal livers is washed off; then the liver sample is mixed with pretreatment liquid, homogenizing is carried out, and thus tissue homogenate is prepared, wherein the pretreatment liquid is a perchloric acid aqueous solution containing dithiothreitol and phenylmethylsulfonyl fluoride; an internal standard substance is added into the homogenate and mixed evenly, then centrifugation is carried out, and supernatant is taken as a sample to be detected; and finally, the sample is sent to a liquid chromatography-mass spectrometry detector to be detected, data are collected, and after standard curve regression calculation, the concentration of the acetyl coenzyme A in the animal livers is obtained. Compared with an existing enzyme-linked immunoassay labeling method, the liquid chromatography-mass spectrometry method adopted by the invention is rapid, reliable and simple in operation step and suitable for measuring the content of the acetyl coenzyme A in thelivers in batches.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for measuring acetyl coenzyme A in animal liver by liquid chromatography-mass spectrometry. Background technique [0002] Acetyl-CoA is the product of β-oxidation of fatty acids in the body and oxidative decarboxylation of pyruvate produced after glycolysis, and is an intermediate metabolite common to the three major nutrients of sugar, fat and protein. The main outlet of acetyl-CoA includes complete oxidation to carbon dioxide and water through the tricarboxylic acid cycle and oxidative phosphorylation, releases ATP and provides energy; synthesizes energy precursors such as fatty acids and ketone bodies, and synthesizes physiologically active substances such as cholesterol. The liver is the main site for the synthesis, metabolism and transformation of acetyl-CoA. The dynamic changes of acetyl-CoA in the liver have a necessary relationship wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 邱志霞李宁黄芳
Owner CHINA PHARM UNIV
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