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Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue

A metabolite and lipid group technology, applied in the field of analytical chemistry, can solve the problems of large tissue sample consumption and multiple extractions, and achieve the effects of reducing potential harm, reducing dosage, and facilitating data integration

Inactive Publication Date: 2014-05-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the shortcomings of the current commonly used tissue extraction methods, such as the large amount of tissue samples used and the need for multiple extractions, the present invention has developed a new method for simultaneously extracting metabolites and lipids from trace tissues. Tissues are obtained to cover different types of metabolites, followed by metabolomics and lipidomics analysis of microtissues by liquid chromatography-mass spectrometry

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  • Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue
  • Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue
  • Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Simultaneous extraction and analysis of metabolome and lipidome in liver samples

[0035] 1. Sample Collection

[0036] Liver tissues were collected from anesthetized and sacrificed C57Bl / 6J mice, washed with phosphate buffered saline and frozen in liquid nitrogen. Frozen tissues were lyophilized in a freeze dryer and ground through a tissue grinder. Liver tissues were stored in a -80°C freezer before and after lyophilization.

[0037] 2. MTBE system to extract metabolites and lipids

[0038] Weigh 10 mg of liver tissue into a 2 mL Eppendorf tube, add 400 μL of 75% methanol and sonicate for 2 min. Add 1 mL MTBE and shake for 1 h. Add 250 μL of water to separate the extract into upper and lower layers. After standing at room temperature for 10 minutes, centrifuge at 14000 g for 15 minutes.

[0039] Pipette 220 μL of the upper layer extract into a 1.5 mL Eppendorf tube for freeze-drying, and record it as the upper layer extract; pipette 110 μL of the lowe...

Embodiment 2

[0057] Example 2: Simultaneous extraction and analysis of target metabolite group and lipid group in liver tissue with different sample sizes For rat liver tissue, the sample was extracted by the method in Example 1, and target acylcarnitine (mixed layer extract) analysis and lipidomics (upper layer extract) analysis were performed. The volume of extract reconstitution solvent increased proportionally with the sample volume, allowing the results of acylcarnitine metabolites and lipid analysis in tissues with different sample volumes to be comparable.

[0058] It can be seen from the analysis results that, compared with 10mg tissue, most of the acylcarnitine metabolites in 1mg, 2.5mg, 5mg and 25mg tissues ( Figure 6 ) and representative lipids ( Figure 7 ) extraction efficiencies are close to 100%, indicating that the method established by the present invention is applicable to a wide range of tissue samples (1-25 mg).

Embodiment 3

[0059] Example 3: Simultaneous extraction and analysis of target metabolites and lipid groups in soleus muscle samples

[0060] The above-mentioned MTBE extraction-liquid chromatography-mass spectrometry analysis method was used for the target metabolites and non- Analysis of target lipidome (top extract).

[0061] Carnitine and 42 acylcarnitine metabolites ( Figure 8 ), 24 free fatty acids ( Figure 9 ) and hundreds of lipids (representative lipids such as Figure 10 shown). So many metabolites and lipids can be detected from such a small amount of muscle tissue (dry weight 2.5 mg), which fully demonstrates the superiority of the method established by the present invention.

[0062] schedule

[0063] Table 1 Comparison of acylcarnitine recovery between liver tissue MTBE mixed layer extract and 80% methanol extract

[0064] Table 2 Comparison of recovery rates of free fatty acids between liver tissue MTBE mixed layer extract and 80% methanol extract

[0065] Schedule 1...

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Abstract

The invention discloses a method for simultaneous extraction and analysis of a metabolite group and a lipid group in microtissue. The method comprises the following steps: freeze drying to-be-analyzed microtissue, accurately weighing 1 to 25 mg of the freeze-dried microtissue and adding solvents like methanol (MeOH), methyl tert butyl ether (MTBE) and water in certain proportion for extraction; allowing a solution obtained after completion of extraction to be divided into two layers, wherein an upper layer mainly contains nonpolar metabolites and lipids, and the lower layer mainly comprises polar and medium-polar metabolites; and subjecting the upper-layer solution and the lower-layer solution to mixing in proportion and freeze-drying, then carrying out redissolving and then metabonomical analysis based on liquid chromatography-mass spectrometry, subjecting the upper-layer solution to freeze-drying and then to redissolving and carrying out lipidomical analysis based on liquid chromatography-mass spectrometry. The method has the following advantages: metabolites and lipids are extracted as many as possible through one extraction of a small amount of tissue, and through metabonomical and lipidomical analysis, the amount of a tissue sample is saved, which benefits other biochemical analysis of the tissue sample.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a new method for simultaneous extraction of metabolites and lipid groups in trace tissues and performing metabolomics and lipidomics analysis based on liquid chromatography-mass spectrometry. Background technique [0002] Metabolic disorders of liver and muscle tissue are involved in the occurrence and development of many diseases. Liquid chromatography-mass spectrometry-based analysis of off-target and targeted metabolomics and lipidomics is a useful tool for studying metabolism in healthy and diseased tissues. The limited amount of tissue available at the time of human biopsy and the small amount of tissue available in commonly used mouse models necessitated both an ethical and a practical need to develop an efficient, tissue-saving metabolite extraction process . Human muscle biopsies routinely yield tissues with a wet weight of about 50 mg, equivalent to 15 mg dry weight...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 许国旺陈世礼李艳杰李佳赵欣捷路鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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