WO2018062204A1 - Procédé de mesure de la phosphoéthanolamine - Google Patents

Procédé de mesure de la phosphoéthanolamine Download PDF

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WO2018062204A1
WO2018062204A1 PCT/JP2017/034796 JP2017034796W WO2018062204A1 WO 2018062204 A1 WO2018062204 A1 WO 2018062204A1 JP 2017034796 W JP2017034796 W JP 2017034796W WO 2018062204 A1 WO2018062204 A1 WO 2018062204A1
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sample
pea
ammonia
phosphoric acid
enzyme
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PCT/JP2017/034796
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Japanese (ja)
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一謹 佐々木
佐藤　俊
かおり 阿部
香織 塙
綾 星
崇臣 福原
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ヒューマン・メタボローム・テクノロジーズ株式会社
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Priority claimed from PCT/JP2016/079088 external-priority patent/WO2018061194A1/fr
Application filed by ヒューマン・メタボローム・テクノロジーズ株式会社 filed Critical ヒューマン・メタボローム・テクノロジーズ株式会社
Priority to JP2018505750A priority Critical patent/JP6467110B2/ja
Publication of WO2018062204A1 publication Critical patent/WO2018062204A1/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B99/00—Subject matter not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for measuring ethanolamine phosphate (PEA) present in a sample.
  • PEA ethanolamine phosphate
  • Depression is a kind of mood disorder, and its main symptoms are “depressed mood” and “loss of interest / joy”. According to a survey of medical institutions in Japan, the number of patients with depression in 2008 is estimated to be over 700,000. According to a 2014 survey by the Ministry of Health, Labor and Welfare, the number of mood disorders (emotional disorders) is 1,212,000, and the number of patients is increasing.
  • the diagnosis of depression largely depends on the subjectivity of doctors and psychologists about the mental aspect of the patient, or the subjectivity and reporting of the patient itself, and therefore it cannot be said that an objective judgment is made. Thus, in order to objectively diagnose depression, attempts have been made in recent years to use components in body fluids of patients as a standard for diagnosing depression.
  • Patent Documents 1 and 2 Conventionally, it has been reported that tryptophan or its degradation product in body fluids or the expression level of a specific gene is used as a predictive marker for depression (Patent Documents 1 and 2).
  • PDA ethanolamine phosphate
  • the present inventors further developed a method for measuring PEA in a sample by generating acetaldehyde, phosphate, and ammonia from PEA using ⁇ -aminobutylaminotransferase (GabT) (Patent Document). 5).
  • the present inventors have found that the threshold value for the amount of PEA in blood for determining whether a patient suffers from depression is about 1.5 ⁇ M (Patent Document 4). ).
  • the conventional PEA measurement method (Patent Document 5) cannot measure a value of less than 1.5 ⁇ M.
  • acetaldehyde, phosphoric acid, and ammonia are present in the sample before the measurement, and it has been difficult to accurately measure PEA with the conventional PEA measurement method (Patent Document 5). Therefore, there is a need for a method for measuring PEA with higher sensitivity than conventional methods.
  • a problem to be solved by the present invention is to provide a method for measuring PEA in a sample with high sensitivity, and a kit, a program, and an apparatus for performing the method.
  • the present inventors have established a method for measuring PEA with higher sensitivity than the conventional PEA measurement method, and completed the present invention.
  • the present invention provides the following: [1] A method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity, (A) removing ammonia and / or phosphoric acid in the sample; (B) adding an enzyme to the sample obtained in step (a) to produce ammonia and / or phosphoric acid from PEA contained in the sample; (C) adding an ammonia detection composition and / or a phosphate detection composition to the sample obtained in step (b); and (d) ammonia and / or in the sample obtained in step (c).
  • a method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity A method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity, (A) removing ammonia and / or phosphoric acid in the sample; (B) adding an enzyme to the sample obtained in step (a) to produce ammonia and / or phosphoric acid from PEA contained in the sample; (C) adding an ammonia detection composition and
  • a method comprising detecting phosphate; [2] The method according to [1], wherein steps (b) and (c) are performed simultaneously; [3] The method according to [1] or [2], wherein the enzyme is PEA lyase or ⁇ -aminobutylaminotransferase (GabT); [4] The enzyme is (I) human-derived ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1), (Ii) a protein having a GabT domain derived from Pantoea ananatis (SEQ ID NO: 2), (Iii) a protein having a GabT domain derived from E.
  • [6] The method according to [9] The method according to any one of [1] to [8], wherein at least one or more of steps (b) to (d) are performed in an environment substantially free of ammonia and / or phosphoric acid. the method of; [10] A method for determining whether a subject is suffering from depression, comprising: (A) measuring PEA in the sample by the method according to any one of [1] to [9]; and (b) when the measured value of PEA in the sample is less than 1.5 ⁇ M.
  • a method for measuring PEA in a sample with high sensitivity and a kit, a program, and an apparatus for performing the method. Also provided are methods for determining whether a subject suffers from depression, and kits, programs, devices, etc. for performing the methods.
  • FIG. 1 is a flowchart showing an example of a method for measuring PEA of the present invention. An example in which plasma is used as a measurement target is shown. In this example, PEA is converted to ammonia, and the amount of PEA is measured.
  • reagent 1 is a reagent containing an enzyme that converts PEA into ammonia
  • reagent 2 is an enzyme cycling solution
  • reagent 3 is a detection solution.
  • FIG. 2 is an example of a flowchart showing processing performed by the apparatus of the present invention.
  • FIG. 3 shows the residual ratio of the components in the sample after the treatment with the cation exchange resin. NH3: ammonia, PEA: ethanolamine phosphoric acid.
  • FIG. 1 is a flowchart showing an example of a method for measuring PEA of the present invention. An example in which plasma is used as a measurement target is shown. In this example, PEA is converted to ammonia, and the amount of PEA is measured.
  • reagent 1 is a
  • FIG. 4 shows the residual ratio of the components in the sample after the treatment by the enzyme method and the precipitation method.
  • PEA ethanolamine phosphoric acid.
  • FIG. 5 is a graph showing the results of examining the influence of the presence or absence of sealing with oil on the permeation of ammonia into the sample.
  • the present invention relates to a method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity.
  • the method includes (1a) removing ammonia and / or phosphoric acid in a sample, (1b) adding an enzyme to the sample obtained in step (1a), and removing ammonia and / or from PEA contained in the sample. (1c) a step of adding an ammonia detection composition and / or a phosphoric acid detection composition to the sample obtained in step (1b), and (1d) obtained in step (1c). Detecting ammonia and / or phosphoric acid in the sample.
  • PDA ethanolamine phosphate
  • the term “highly sensitive” refers to a lower limit of quantification than the conventional PEA measurement method (Patent Document 5). Specifically, it means that PEA of less than 1.5 ⁇ M can be quantified.
  • measuring PEA not only confirms the presence of PEA in a sample and measures its amount, but also the absence of PEA in the sample (ie, the PEA Confirmation that the amount is below the limit of detection).
  • the method for measuring PEA of the present invention utilizes a hydrolysis reaction represented by the following formula (1).
  • step (1a) any known means and method for removing ammonia may be used.
  • ammonia is removed by the resin in step (1a).
  • resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical), Dowex 50Wx8 200-400 mesh, strongly acidic cation exchange resin (H form).
  • ammonia may be removed by enzymatic treatment in step (1a).
  • enzyme treatment include, but are not limited to, treatment with glutamate dehydrogenase, glutamine synthase, or nicotinamide adenine dinucleotide synthase.
  • about removal of ammonia it describes in Unexamined-Japanese-Patent No. 2003-334099 (patent document 6) and patent 4127767 (patent document 7), for example.
  • any enzyme that catalyzes the reaction represented by the above formula (1) is used.
  • examples of such enzymes include, but are not limited to, PEA lyase and ⁇ -aminobutylaminotransferase (GabT).
  • a human-derived ethanolamine phosphate phospholyase (SEQ ID NO: 1)
  • a protein having a GabT domain from Pantoea ananatis (SEQ ID NO: 2)
  • E. coli One or more enzymes selected from the group consisting of a protein having a GabT domain (SEQ ID NO: 3) are used.
  • a protein having a GabT domain derived from Pantoea ananatis (SEQ ID NO: 2) or a protein having a GabT domain derived from E. coli (SEQ ID NO: 3) may be used.
  • the reaction represented by the above formula (1) catalyzes the reaction represented by the above formula (1) and is 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96, with any amino acid sequence of SEQ ID NO: 2 or 3. Proteins comprising amino acid sequences with homology of%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9% may be used.
  • a protein containing an amino acid sequence in which a tag is added to any amino acid sequence of SEQ ID NOs: 1 to 3 may be used.
  • Exemplary tags, His 6 - tag, FLAG tag, myc tag, and GST but tags include, but are not limited to.
  • the tag may be added to the N terminus of the amino acid sequence or may be added to the C terminus.
  • an enzyme having a GabT domain is used.
  • GabT domain is GXXXBADEBQXGFZRXG [where X is any amino acid, B is isoleucine (I), leucine (L) or valine (V), and Z is glycine (G) or alanine (A) ] (SEQ ID NO: 4).
  • Japanese Patent No. 5688163 Patent Document 5 describes sequences of GabT domains in various biological species.
  • An exemplary enzyme having an exemplary GabT domain is also described in US Pat. No. 5,688,163.
  • it catalyzes the reaction represented by the above formula (1) and is 50% or more, for example, 52%, 58%, 64%, 70%, 76, with the amino acid sequence of GabT domain (SEQ ID NO: 4). Proteins containing amino acid sequences with%, 82%, 88%, or 94% homology are used.
  • an amino acid that catalyzes the reaction represented by the above formula (1) and in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence of the GabT domain (SEQ ID NO: 4).
  • a protein containing the sequence is used. Such a protein may have a tag as described above.
  • the enzyme used in step (1b) is a human-derived ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1) or a protein having a GabT domain from Pantoea ananatis (SEQ ID NO: 2). is there.
  • the enzyme used in step (1b) is human-derived ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1).
  • any enzyme that catalyzes the reaction of formula (1) above such as PEA lyase or ⁇ -aminobutylaminotransferase (GabT) is added to the sample.
  • the method may further include performing the enzyme reaction.
  • the “ammonia detection composition” used in step (1c) includes any composition, reagent, and kit for detecting ammonia present in a sample.
  • a commercially available ammonia detection composition, reagent, or kit may be used. Examples include, but are not limited to, Shikari Liquid NH 3 (Kanto Chemical Co., Inc.), Ammonia-L (Cerotech Co., Ltd.), and F-Kit Ammonia (JK International Co., Ltd.).
  • the combination of Reagent I and Reagent II listed in the following table may be used to measure the amount of ammonia present in the sample.
  • the pH of the above reagents I and II is adjusted using a suitable buffer. Examples of the buffer used for pH adjustment include, but are not limited to, potassium phosphate and Tris-HCl.
  • the reagent may further contain a salt such as sodium chloride.
  • steps (1b) and (1c) are performed simultaneously. That is, the enzymes, compositions, reagents, and / or kits required at each stage may be used simultaneously. By performing the steps (1b) and (1c) at the same time, the chance that the sample is exposed to the outside air can be reduced, and more accurate measurement is possible.
  • any known means and method for removing phosphoric acid may be used in step (1a).
  • methods for removing phosphoric acid include, but are not limited to, enzymatic methods and precipitation methods.
  • purine nucleotide phosphorylase PNP
  • xanthosine and PNP are added to a sample containing phosphoric acid
  • phosphoric acid reacts with xanthosine by the action of PNP to produce xanthine and ribose 1-phosphate (Ri1P), thereby enabling removal of phosphoric acid.
  • a PNP inhibitor may be added to stop the action of PNP after the reaction.
  • sucrose phosphorylase (SP) may be used.
  • sucrose and SP When sucrose and SP are added to a sample containing phosphoric acid, phosphoric acid reacts with sucrose by the action of SP to produce ⁇ -glucose 1-phosphate ( ⁇ -Glc-1-P and fructose, and removal of phosphoric acid.
  • a buffer having a pH of about 9 to about 10 for example, CHES buffer
  • an SP inhibitor such as fructose
  • rare earth metal ions or alkaline earth metal ions are added to a sample containing phosphoric acid. Thereby, a hardly soluble phosphate (MPO 4 ) is generated, and phosphoric acid can be removed.
  • MPO 4 alkaline earth metal ions
  • the pH of the solution may be made higher than 9, or carbonate ions (CO 3 2 ⁇ ) may be added to remove excess metal.
  • the reagent used for the precipitation method include water-soluble calcium salts such as CaCl 2 .
  • an enzymatic method and a precipitation method are used in combination. By using these methods in combination, phosphoric acid can be removed more reliably.
  • any composition, reagent, and / or kit for detecting phosphoric acid present in the sample is used as the phosphoric acid detection composition. It is done.
  • a commercially available phosphoric acid detection composition, reagent, or kit may be used.
  • An exemplary phosphate detection reagent is a malachite green (MG) reagent.
  • the sample may be subjected to ultrafiltration before adding the MG reagent.
  • MG malachite green
  • PEA may be measured using a hydrolysis reaction represented by the following formula (2).
  • the present invention is a method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity, and (2a) removing ethanolamine in a sample.
  • (2c) adding ethanolamine to the sample obtained in step (2b) A method is provided comprising the steps of adding a detection composition and (2d) detecting ethanolamine in the sample obtained in step (2c).
  • phosphoric acid may be removed and detected together with ethanolamine or instead of ethanolamine.
  • step (2a) any known means and method for removing ethanolamine may be used.
  • ethanolamine is removed by the resin in step (2a).
  • resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical).
  • ethanolamine may be removed by enzymatic treatment in step (2a).
  • step (2b) any enzyme that catalyzes the reaction represented by the above formula (2) is used.
  • examples of such enzymes include, but are not limited to alkaline phosphatase.
  • alkaline phosphatase or a variant thereof is used in step (2b).
  • the enzyme used in step (2b) is alkaline phosphatase.
  • the method further includes the step of adding any enzyme that catalyzes the reaction represented by the above formula (2) to the sample, for example, alkaline phosphatase, and performing the enzyme reaction. May be.
  • the “ethanolamine detection composition” used in step (2c) includes any composition, reagent, and kit for detecting ethanolamine present in a sample.
  • a commercially available composition, reagent, or kit for detecting ethanolamine may be used.
  • steps (2b) and (2c) are performed simultaneously. That is, the enzymes, compositions, reagents, and / or kits required at each stage may be used simultaneously. By performing the steps (2b) and (2c) at the same time, the chance of the sample touching the outside air can be reduced, and more accurate measurement can be performed.
  • MG malachite green
  • the method for measuring PEA of the present invention includes a step of removing ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine from a sample as a first step.
  • Ammonia, acetaldehyde, phosphoric acid, and ethanolamine can be naturally present, so they are likely already present in the sample. In addition, these may be mixed into the sample at the sample acquisition stage. Therefore, by removing ammonia, acetaldehyde, phosphoric acid and / or ethanolamine present in the sample before carrying out the reaction of the above formula (1) or (2), it is more accurate than the conventional method.
  • Ammonia, acetaldehyde, phosphoric acid, or ethanolamine generated from PEA can be detected with high sensitivity.
  • the present invention provides a method for measuring PEA with high sensitivity by using instrumental analysis.
  • PEA is measured by quantification by post-column fluorescence derivatization of PEA utilizing ion exchange chromatography (IC) and a fluorescence detector (FLD).
  • PEA is measured using CE (capillary-electrophoresis) and MS (mass spectrometer). In this case, a triple quadrupole (QqQ type) MS device may be used.
  • the above-described method for measuring PEA of the present invention is performed under an environment substantially free of ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine (ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine-free environment). May be implemented.
  • An environment that is substantially free of ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine refers to an environment in which the inflow of these substances from the outside is reduced or prevented.
  • Substantially absent means not only the state in which these substances are not completely present but also a state in which these substances are below the detection limit.
  • Such an environment refers to, for example, a state in which contact with the atmosphere is blocked.
  • the contact between the reaction system and the atmosphere may be blocked by forming a film on the reaction solution.
  • the substance used for forming the coating include colorless, non-volatile, hydrophobic, and non-polar substances.
  • oil or an organic solvent is used.
  • the oil used include mineral oil and plant-derived oil, but are not limited thereto.
  • the organic solvent used include octanol and dodecanol, but are not limited thereto.
  • Reduced pressure means a pressure lower than normal pressure.
  • the pressure is reduced to the ambient pressure, and the pressure is JIS standard low vacuum defined as industrially usable pressure. .86 ⁇ 10 ⁇ 4 atm or more, 9.86 ⁇ 10 ⁇ 4 to 9.99 ⁇ 10 ⁇ 1 atm, 7.0 ⁇ 10 ⁇ 4 to 9.99 ⁇ 10 ⁇ 1 atm.
  • the atmosphere in the environment (for example, a container) in which the method for measuring PEA of the present invention is performed may be replaced with an atmosphere substantially free of ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine.
  • substantially free of ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine not only means that these substances are not completely contained, but also means that these substances are below the detection limit. .
  • At least one of the steps (1b) to (1d) is performed in an environment substantially free of ammonia, phosphoric acid, and / or acetaldehyde. To be implemented. In other embodiments, at least two of steps (1b)-(1d) are performed in such an environment. In another embodiment, steps (1b)-(1d) are all performed in such an environment.
  • steps (2b) to (2d) are performed in an environment substantially free of ethanolamine and / or phosphoric acid.
  • steps (2b)-(2d) are performed in such an environment.
  • steps (2b) to (2d) are all performed in such an environment.
  • the present invention relates to a method for determining whether a subject is suffering from depression.
  • the method comprises (i) a step of measuring PEA in a sample by a method of measuring PEA of the present invention with high sensitivity; and (ii) when the measured value of PEA in the sample is less than 1.5 ⁇ M Determining that the subject is suffering from depression.
  • sample used in the method of the present invention described above is any sample that contains or may contain PEA.
  • samples include, but are not limited to, biological sample samples such as whole blood, serum, plasma, tissue, cerebrospinal fluid, and urine.
  • the sample used in the present invention is a whole blood, serum or plasma sample, preferably a plasma sample.
  • the samples used in the present invention may be pre-processed to make them suitable for subsequent stages.
  • the sample used in the present invention is a whole blood or plasma sample that has been pre-treated with an anticoagulant.
  • anticoagulants include, but are not limited to, EDTA, citric acid, oxalic acid, sodium fluoride, heparin.
  • the present invention relates to a kit for performing the method of the present invention described above.
  • the kit of the invention includes a means for removing ammonia.
  • a resin used to remove ammonia is included.
  • resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical), Dowex 50Wx8 200-400 mesh, strongly acidic cation exchange resin (H form).
  • an enzyme that converts ammonia into another substance is included. Examples of such enzymes include, but are not limited to, glutamate dehydrogenase, glutamine synthase, and nicotinamide adenine dinucleotide synthase.
  • the kit of the present invention includes a means for removing acetaldehyde, phosphoric acid, and / or ethanolamine together with or in place of the means for removing ammonia.
  • a means for removing acetaldehyde, phosphoric acid, and / or ethanolamine together with or in place of the means for removing ammonia.
  • a resin used to remove acetaldehyde, phosphate, or ethanolamine or an enzyme that converts acetaldehyde, phosphate, or ethanolamine to another substance.
  • the kit of the present invention comprises an ammonia detection composition.
  • any composition, reagent, and / or kit for detecting ammonia present in the sample is included.
  • commercially available ammonia detection compositions, reagents, or kits are included.
  • Shikarikiddo NH 3 Kanto Chemical Co., Inc.
  • ammonia -L Serotec Ltd.
  • F- kit ammonia manufactured J.K. International
  • an improved product of the above-mentioned commercial product may be used.
  • a combination of reagent I and reagent II described above may be used (see Tables 1 and 2).
  • the kit of the present invention includes an acetaldehyde, phosphoric acid, and / or ethanolamine detection composition together with or in place of the ammonia detection composition.
  • an acetaldehyde, phosphoric acid, and / or ethanolamine detection composition together with or in place of the ammonia detection composition.
  • commercially available acetaldehyde, phosphoric acid, and / or ethanolamine detection compositions, reagents, or kits are included.
  • phosphate detection reagents include, but are not limited to, malachite green (MG) reagents.
  • the kit may contain any reagent used in the method of the present invention.
  • an appropriate reagent for simplifying the measurement such as a sample diluent, a reaction diluent, a buffer, and a cleaning agent may be included.
  • the reagent is provided in a suitable container.
  • materials such as instructions necessary for carrying out the method of the present invention may be included.
  • the kit of the present invention is a research-only (RUO) kit. In another embodiment, the kit of the present invention is an investigative use only (IUO) kit. In another embodiment, the kit of the present invention is a clinical diagnostic kit.
  • the present invention relates to a program for causing a computer to execute the above-described method of the present invention.
  • the program of the present invention may be recorded on a computer-readable recording medium, or may be recorded on a computer recording medium attached to the apparatus. Examples of the recording medium include, but are not limited to, a hard disk, a CD, a DVD, a USB memory, and a floppy (registered trademark) disk.
  • the invention relates to an apparatus for measuring PEA.
  • the device of the invention comprises a kit of the invention as described above.
  • the program of the present invention described above is incorporated in the apparatus of the present invention. An example of a flowchart showing the processing performed by the apparatus of the present invention is shown in FIG.
  • Ammonia removal by cation exchange resin Plasma was used as a sample.
  • the column was filled with cation exchange resin MCI GEL AFR2 (Mitsubishi Chemical Corporation, catalog number 1-033-01) and washed with 400 ⁇ L of ultrapure water. 50 ⁇ L of the sample was filled in the washed column and passed through to collect the solution. The residual ratio of ammonia and PEA in the collected solution was measured.
  • Ammonia was measured by measuring absorbance at 450 nm using Shikari Liquid NH 3 (Kanto Chemical Co., Inc.).
  • PEA was measured by the CE-MS instrument method described later.
  • the results are shown in FIG.
  • the residual ratio of ammonia was 1.5%.
  • the residual ratio of PEA was 100%. From this, it was shown that selective removal of ammonia with respect to PEA is possible by using a cation exchange column.
  • Phosphoric acid was measured by measuring the absorbance at 620 nm using Malachite Green Phosphate Assay Kit (Funakoshi Co., Ltd.). PEA was measured by the CE-MS instrument method described in Example 1. As shown in FIG. 4, the residual rate of phosphoric acid was below the detection limit, and the residual rate of PEA was about 100%. From this, it was shown that the phosphoric acid can be selectively removed with respect to PEA by using the precipitation method and the enzyme method.
  • the present invention a method and kit for measuring PEA with higher sensitivity than conventional methods are provided. Therefore, the present invention can be used in the fields of research reagents, test agents, diagnostic agents and the like.
  • SEQ ID NO: 1 Ethanolamine-phosphate phospho-lyase Sequence number 2: Ethanolamine phosphate catalytic enzyme Sequence number 3: Ethanolamine phosphate catalytic enzyme Sequence number 4: GabT domain

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
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  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Medical Informatics (AREA)
  • Evolutionary Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Theoretical Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Sustainable Development (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de mesure, avec une sensibilité élevée, de la phosphoéthanolamine (PEA) dans un échantillon, le procédé comprenant : (a) une étape d'élimination d'ammoniac et/ou d'acide phosphorique dans un échantillon; (b) une étape d'ajout d'une enzyme à l'échantillon obtenu à l'étape (a) en vue de produire de l'ammoniac et/ou de l'acide phosphorique à partir de la PEA contenue dans l'échantillon; (c) une étape d'ajout d'une composition de détection d'ammoniac et/ou d'une composition de détection d'acide phosphorique à l'échantillon obtenu à l'étape (b); et (d) une étape de détection d'ammoniac et/ou d'acide phosphorique dans l'échantillon obtenu à l'étape (c). L'invention concerne également un kit, un programme et un dispositif d'exécution dudit procédé.
PCT/JP2017/034796 2016-09-30 2017-09-26 Procédé de mesure de la phosphoéthanolamine WO2018062204A1 (fr)

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JP2018505750A JP6467110B2 (ja) 2016-09-30 2017-09-26 エタノールアミンリン酸の測定方法

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PCT/JP2016/079088 WO2018061194A1 (fr) 2016-09-30 2016-09-30 Procédé de mesure de la phosphoéthanolamine
JPPCT/JP2016/079088 2016-09-30
JP2017-070496 2017-03-31
JP2017070496 2017-03-31

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WO2018062204A1 true WO2018062204A1 (fr) 2018-04-05

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Cited By (1)

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WO2020122231A1 (fr) * 2018-12-13 2020-06-18 キッコーマン株式会社 Procédé de quantification de phosphate d'éthanolamine, d'oxyde réductase pour quantification, composition de quantification, kit de quantification et capteur pour quantification

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4170041A1 (fr) * 2020-06-17 2023-04-26 Kikkoman Corporation Procédé de quantification de phosphate d'éthanolamine, oxyde réductase pour quantification, composition de quantification, kit de quantification, puce de capteur et capteur

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JPS5066951A (fr) * 1973-10-22 1975-06-05
JPS5460285A (en) * 1977-10-21 1979-05-15 Toray Ind Inc Medical adsorbent
JPS5771693A (en) * 1980-10-21 1982-05-04 Katayama Chem Works Co Ltd Method of removing phosphate ion contained in liquid
JPS62294097A (ja) * 1986-06-13 1987-12-21 Takara Shuzo Co Ltd 生体成分の定量方法
JPH0191798A (ja) * 1987-09-30 1989-04-11 Shimadzu Corp クレアチニン分析法及び前処理カラム
JPH11225793A (ja) * 1997-11-12 1999-08-24 Oriental Yeast Co Ltd アンモニア測定用液状試薬
JP2000189196A (ja) * 1998-12-25 2000-07-11 Iatron Lab Inc 尿素窒素測定方法および尿素窒素測定用試薬
WO2013069645A1 (fr) * 2011-11-10 2013-05-16 ヒューマン・メタボローム・テクノロジーズ株式会社 Procédé pour la mesure d'éthanolamine phosphate
WO2016047677A1 (fr) * 2014-09-26 2016-03-31 ヒューマン・メタボローム・テクノロジーズ株式会社 Méthode de prédiction d'alternatives aux médicaments de traitement de la dépression

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JPS622157A (ja) * 1985-06-28 1987-01-08 Tokyo Zoki Kagaku Kk 臨床検査液中のリン酸除去試薬ならびに該試薬で処理するステロイド抱合体の加水分解方法
JP5327578B2 (ja) * 2008-02-06 2013-10-30 日東紡績株式会社 ホスファターゼの測定方法

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JPS49130364A (fr) * 1973-04-19 1974-12-13
JPS5066951A (fr) * 1973-10-22 1975-06-05
JPS5460285A (en) * 1977-10-21 1979-05-15 Toray Ind Inc Medical adsorbent
JPS5771693A (en) * 1980-10-21 1982-05-04 Katayama Chem Works Co Ltd Method of removing phosphate ion contained in liquid
JPS62294097A (ja) * 1986-06-13 1987-12-21 Takara Shuzo Co Ltd 生体成分の定量方法
JPH0191798A (ja) * 1987-09-30 1989-04-11 Shimadzu Corp クレアチニン分析法及び前処理カラム
JPH11225793A (ja) * 1997-11-12 1999-08-24 Oriental Yeast Co Ltd アンモニア測定用液状試薬
JP2000189196A (ja) * 1998-12-25 2000-07-11 Iatron Lab Inc 尿素窒素測定方法および尿素窒素測定用試薬
WO2013069645A1 (fr) * 2011-11-10 2013-05-16 ヒューマン・メタボローム・テクノロジーズ株式会社 Procédé pour la mesure d'éthanolamine phosphate
WO2016047677A1 (fr) * 2014-09-26 2016-03-31 ヒューマン・メタボローム・テクノロジーズ株式会社 Méthode de prédiction d'alternatives aux médicaments de traitement de la dépression

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020122231A1 (fr) * 2018-12-13 2020-06-18 キッコーマン株式会社 Procédé de quantification de phosphate d'éthanolamine, d'oxyde réductase pour quantification, composition de quantification, kit de quantification et capteur pour quantification
CN113166794A (zh) * 2018-12-13 2021-07-23 龟甲万株式会社 磷酸乙醇胺的定量方法、定量用的氧化还原酶、定量用组合物、定量用试剂盒和定量用传感器
US11879149B2 (en) 2018-12-13 2024-01-23 Kikkoman Corporation Quantification method of ethanolamine phosphate, oxidoreductase for quantification, composition for quantification, kit for quantification and sensor for quantification

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JP6868649B2 (ja) 2021-05-12
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JPWO2018062204A1 (ja) 2018-09-27

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