WO2014045932A1 - 代謝物の抽出方法 - Google Patents

代謝物の抽出方法 Download PDF

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Publication number
WO2014045932A1
WO2014045932A1 PCT/JP2013/074319 JP2013074319W WO2014045932A1 WO 2014045932 A1 WO2014045932 A1 WO 2014045932A1 JP 2013074319 W JP2013074319 W JP 2013074319W WO 2014045932 A1 WO2014045932 A1 WO 2014045932A1
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Prior art keywords
extracting
water
organic solvent
metabolite
extraction
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Ceased
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PCT/JP2013/074319
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English (en)
French (fr)
Japanese (ja)
Inventor
一謹 佐々木
基 佐藤
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Human Metabolome Technologies Inc
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Human Metabolome Technologies Inc
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Publication of WO2014045932A1 publication Critical patent/WO2014045932A1/ja
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction

Definitions

  • the present invention relates to a method for extracting a metabolite, and particularly suitable for use in qualitative and quantitative analysis of a hydrophilic metabolite in cultured cells and microorganisms or for extracting a product substance, while suppressing oxidation.
  • the present invention relates to a method for extracting a metabolite capable of quickly stopping metabolism and efficiently extracting a hydrophilic substance.
  • Metabolomic analysis is a method for comprehensive analysis of metabolites in the living body. However, in order to grasp metabolism more accurately, it is possible to stop metabolism quickly and suppress oxidation, thereby efficiently analyzing metabolites. A method of extraction is needed.
  • Non-patent Document 1 As a simple method for extracting metabolites in cells, there is a method of extraction with an organic solvent such as methanol or acetonitrile or a mixture of organic solvent and water (Non-patent Document 1).
  • Patent Documents 1 to 3 There are also methods for extracting metabolites by liquid-liquid extraction using three liquids of methanol, water and chloroform (Patent Documents 1 to 3, Non-Patent Document 2).
  • Non-Patent Document 3 reports a two-stage extraction method in which extraction is performed by adding water to the extraction residue after extraction with methanol.
  • the present invention has been made to solve the above-mentioned conventional problems, and efficiently extracts a hydrophilic metabolite by a simple method while rapidly stopping the progress of metabolism while suppressing oxidation. Is an issue.
  • Non-Patent Document 3 The method described in Non-Patent Document 3 is similar to the present invention in that it operates in two steps, but extraction is performed every time a solvent is added, and the method is performed in two steps as in the present invention. Extraction is not performed once by adding a solvent.
  • the organic solvent added in the first step can be methanol or acetonitrile.
  • the water added in the second step can be ultrapure water or pure water.
  • the organic solvent added in the first step can be a first solution containing a predetermined ratio or less of water.
  • the water added in the second step can be a second solution containing an organic solvent in a predetermined ratio or less.
  • the ratio of the organic solvent in the second solution can be made smaller than the ratio of the organic solvent in the first solution.
  • the ratio of the total amount of the organic solvent in the first solution and the organic solvent in the second solution to the total amount of the first solution and the second solution can be a predetermined value or more.
  • the supernatant can be ultrafiltered.
  • a thiol compound can be extracted using the extraction method described above.
  • metabolites can be extracted from adherent cells using the extraction method described above.
  • the enzyme since an organic solvent is first added, the enzyme can be reliably deactivated while suppressing oxidation, and the progress of metabolism can be stopped rapidly. Subsequently, since water is added, hydrophilic metabolites that are hardly soluble in organic solvents can be dissolved in the liquid phase, and the liquid phase can be recovered.
  • a substance having a thiol group that is easily oxidized, acetyl CoA, and pyruvic acid have a large improvement effect. Therefore, it is possible to quickly stop the metabolism while suppressing oxidation by a simple method, efficiently extract a hydrophilic metabolite, and improve reproducibility.
  • FIG. 1 is a flowchart showing the procedure of this embodiment.
  • an organic solvent for example, methanol, acetonitrile, etc.
  • methanol 14 in FIG. 2 eg, methanol 14 in FIG. 2
  • the organic solvent to be added is desirably 100%, but water may be mixed, for example, about 10%.
  • the hydrophilic metabolite is difficult to dissolve in the organic solvent, the hydrophilic metabolite is dissolved in the liquid phase by adding water, for example, ultrapure water 16, as shown in FIG. After the state is made easy, the liquid phase is separated and collected in step 130.
  • water for example, ultrapure water 16
  • the water added here is preferably 100%, but an organic solvent may be mixed. Furthermore, the water added here is not limited to ultrapure water as long as there is no problem of impurities, and may be pure water or tap water.
  • the ratio of the organic solvent added in step 120 needs to be kept lower than the ratio of the organic solvent added in step 110.
  • step 130 the liquid phase is collected and, for example, ultrafiltered through the filter 18 as illustrated in FIG.
  • the ultrafiltration is performed in order to completely remove the protein during measurement with a capillary electrophoresis mass spectrometer (CE-MS).
  • CE-MS capillary electrophoresis mass spectrometer
  • filtration with a normal filter may be used.
  • the enzyme can be reliably deactivated while inhibiting oxidation, and a hydrophilic metabolite can be efficiently extracted. .
  • the operation is simple.
  • methanol 800 ⁇ L is added to 2 ⁇ 10 7 cultured cells on a 90 mm diameter petri dish, methanol is infiltrated into the whole cultured cell on the petri dish, the enzyme is inactivated while inhibiting oxidation, and metabolism is stopped. I let you.
  • liquid-liquid detection using 1000 ⁇ L of methanol, 1000 ⁇ L of chloroform, 400 ⁇ L of water and ultrasonic waves was performed on the same cultured cells.
  • Fig. 3 shows the results of comparison with the extraction amount and reproducible liquid-liquid extraction method.
  • the oxidation does not proceed and the extraction amount of the compound having a thiol group such as reduced glutathione and CoA is high.
  • oxidation of the thiol group is suppressed, and disulfide compounds formed by oxidation of the compound having a thiol group are reduced. That is, the conversion from thiol to disulfide is suppressed.
  • the liquid-liquid extraction method has a large variation.
  • acetyl CoA and pyruvic acid which are considered to continue the enzymatic reaction.
  • Table 1 shows the quantitative values of CoA and acetyl CoA.
  • the relative standard deviation RSD of acetyl CoA is large, but the RSD is small when the sum of acetyl CoA and CoA is taken. That is, conversion from acetyl CoA to CoA occurs in the liquid-liquid extraction method, which is considered to be suppressed in the method of the present invention.
  • CoA has a reaction that it is consumed during lipid metabolism and acetyl CoA is produced, and in the liquid-liquid extraction method, the enzyme is not completely inactivated, and it is considered that such a reaction occurs.
  • pyruvic acid is a metabolite immediately after acetyl-CoA, and pyruvic acid is considered to be affected by it.
  • FIG. 4 shows a comparison result of the extraction amount and reproducibility between the 60% methanol aqueous solution extraction method and Example 1 of the present invention.
  • the methanol aqueous solution extraction method was performed by adding 1300 ⁇ L of 60% methanol to the same cultured cells.
  • NADPH is enzymatically oxidized to NADP +, but the reaction is suppressed in the method of the present invention.
  • the RSD of O-acetylhomoserine, thioproline, pyruvic acid, glyceraldehyde 3-phosphate is remarkably reduced.
  • the method of the present invention enables extraction with high reproducibility while stopping oxidation and metabolism as compared with the conventional method.
  • acetonitrile aqueous solution (v / v) is added to 2 ⁇ 10 7 cultured cells on a 90 mm diameter petri dish, and the whole cultured cells on the petri dish are infiltrated with 90% acetonitrile aqueous solution to suppress oxidation. While inactivating the enzyme, the metabolism was stopped.
  • Fig. 5 shows the results of comparison with the extraction amount and reproducible liquid-liquid extraction method.
  • the variation in thiol-related substances is reduced, and cysteine glutathione disulfide is reduced. Compared with the liquid-liquid extraction method, oxidation of the thiol compound is suppressed as in Example 1. The variation of acetyl CoA is also reduced, and the metabolism of acetyl CoA is suppressed as in Example 1, compared to the liquid-liquid extraction method.
  • 650 ⁇ L of methanol is added to 2 ⁇ 10 7 cultured cells on a 90 mm diameter petri dish, methanol is infiltrated into the entire cultured cell on the petri dish, the enzyme is deactivated while suppressing oxidation, and metabolism is stopped. I let you.
  • FIG. 6 shows the comparison results of the extraction amounts and reproducibility in Examples 1, 3 and 4.
  • the method of the present invention suppressed the oxidation of thiols and the metabolism of substances related to acetyl CoA regardless of the solvent.
  • the method of the present invention when compared with the methanol aqueous solution extraction method, the method of the present invention was improved in reproducibility for substances with large variations, and it was confirmed that thiol oxidation and NADPH oxidation were suppressed.
  • the method of the present invention was able to realize high extraction efficiency while efficiently stopping the progress of metabolism while suppressing the oxidation of substances.
  • the sample prepared by the method of the present invention can be applied to analysis using CE, liquid chromatography, gas chromatography, MS, thin layer chromatography, nuclear magnetic resonance, spectroscopic techniques, and the like.
  • the sample prepared by the method of the present invention can also be used as a substrate for enzyme reaction.
  • the present invention can also be used as an efficient method for extracting a target substance in substance production using cultured cells or microorganisms.
  • the method of the present invention can be applied to microorganisms such as Escherichia coli and yeast, cultured animal cells, cultured plant cells, blood cells, sperm, ova, other single cell samples, and living tissues such as liver, brain and muscle.
  • microorganisms such as Escherichia coli and yeast, cultured animal cells, cultured plant cells, blood cells, sperm, ova, other single cell samples, and living tissues such as liver, brain and muscle.

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  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Extraction Or Liquid Replacement (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/JP2013/074319 2012-09-20 2013-09-10 代謝物の抽出方法 Ceased WO2014045932A1 (ja)

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JP2012-206518 2012-09-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104614212A (zh) * 2015-01-20 2015-05-13 济宁神康生物科技有限公司 一种贴壁细胞定量代谢组学分析样品预处理的方法
EP3348988A4 (en) * 2015-09-11 2019-05-22 Keio University PROCESS FOR EXTRACTION OF A SUBSTANCE FROM A CHAIR TEST

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007192746A (ja) * 2006-01-20 2007-08-02 Keio Gijuku 酸化ストレスの判定方法
WO2011003945A1 (en) * 2009-07-08 2011-01-13 Basf Plant Science Company Gmbh Methods for analyzing polar metabolites of the energy metabolism
WO2011158590A1 (ja) * 2010-06-18 2011-12-22 学校法人 慶應義塾 肝臓疾患マーカー、その測定方法、装置及び医薬品の検定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007192746A (ja) * 2006-01-20 2007-08-02 Keio Gijuku 酸化ストレスの判定方法
WO2011003945A1 (en) * 2009-07-08 2011-01-13 Basf Plant Science Company Gmbh Methods for analyzing polar metabolites of the energy metabolism
WO2011158590A1 (ja) * 2010-06-18 2011-12-22 学校法人 慶應義塾 肝臓疾患マーカー、その測定方法、装置及び医薬品の検定方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104614212A (zh) * 2015-01-20 2015-05-13 济宁神康生物科技有限公司 一种贴壁细胞定量代谢组学分析样品预处理的方法
EP3348988A4 (en) * 2015-09-11 2019-05-22 Keio University PROCESS FOR EXTRACTION OF A SUBSTANCE FROM A CHAIR TEST
US10927401B2 (en) 2015-09-11 2021-02-23 Keio University Method for extracting substance from feces sample

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JP2014061459A (ja) 2014-04-10

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