WO2020067437A1 - Method for measuring phosphoethanolamine - Google Patents

Method for measuring phosphoethanolamine Download PDF

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WO2020067437A1
WO2020067437A1 PCT/JP2019/038190 JP2019038190W WO2020067437A1 WO 2020067437 A1 WO2020067437 A1 WO 2020067437A1 JP 2019038190 W JP2019038190 W JP 2019038190W WO 2020067437 A1 WO2020067437 A1 WO 2020067437A1
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sample
acid
pea
ion
phosphoric acid
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PCT/JP2019/038190
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French (fr)
Japanese (ja)
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進 大澤
弥季 箕輪
一謹 佐々木
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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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    • 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
    • C12Q1/30—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving catalase
    • 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
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • 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
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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

Definitions

  • the present invention provides a method for measuring ethanolamine phosphate (PEA) present in a sample, the method including a step of detecting color development by a chelating reagent, a kit for performing the method, a program for performing the method,
  • the present invention relates to a computer-readable recording medium storing the program, and an apparatus for measuring PEA including the kit or incorporating the program.
  • Depression is a type of mood disorder, the main symptoms of which 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 was over 700,000. According to a 2014 survey by the Ministry of Health, Labor and Welfare, the number of patients with mood disorders (emotional disorders) was 11.22,000, and the number of patients is increasing.
  • the diagnosis of depression largely depends on the subjective opinion of the physician or psychologist on the mental aspect of the patient, or on the subjective opinion or declaration of the patient, and therefore, it cannot be said that objective judgment has been made. Therefore, in order to objectively diagnose depression, to observe the course of treatment, and to predict prognosis, in recent years, attempts have been made to use components in body fluids of patients as a guide for the diagnosis of depression. .
  • Non-Patent Document 1 ethanolamine phosphate (PEA) in blood is useful as a biomarker for diagnosing depression
  • PDA ethanolamine phosphate
  • the measured value of the PEA concentration is considered to be useful for objective diagnosis and follow-up of depression
  • Patent Document 5 The present inventors have further developed a method for measuring PEA in a sample by generating acetaldehyde, phosphoric acid, and ammonia from PEA using ⁇ -aminobutylaminotransferase (GabT)
  • the present inventors have also developed a method for more accurately measuring PEA by removing ammonia and / or phosphoric acid before producing acetaldehyde, phosphoric acid, and ammonia from PEA (Patent Document 6). .
  • the conventional method for measuring PEA uses a mass spectrometer, for example, a capillary mass spectrometer.
  • mass spectrometers are expensive and cannot be analyzed using ordinary equipment, such as a general hospital or medical institution. Further, the sensitivity of the conventional enzymatic PEA measurement method is not sufficient. Therefore, there is a need for a method that can easily and highly sensitively measure PEA by using a device generally used in ordinary facilities, for example, an automatic biochemical analyzer.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a step of producing ammonia, acetaldehyde, and phosphoric acid from PEA by acting O-phosphoethanolamine phospholyase (Phosphoethanolamine Phospho-Lyase), Reacting an enzyme to generate reduced nicotinamide adenine dinucleotide (NADH 2 ) from phosphoric acid, reacting a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion, and
  • the present inventors have found a method for measuring ethanolamine phosphate (PEA) in a sample, which includes a step of detecting color development by a chelating reagent, and have completed the present invention.
  • Phosphoethanolamine Phospho-Lyase O-phosphoethanolamine phospholyase
  • NADH 2 nicotinamide adenine dinucle
  • the present invention provides: [1] A method for measuring ethanolamine phosphate (PEA) in a sample, (A) removing phosphoric acid in the sample; (B) reacting the sample obtained in step (a) with O-phosphoethanolamine Phospho-Lyase to produce ammonia, acetaldehyde and phosphoric acid from PEA contained in the sample; (C) reacting the sample obtained in step (b) with an enzyme to generate NADH 2 from phosphoric acid contained in the sample; (D) reacting the sample obtained in step (c) with a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion; and (e) the sample obtained in step (d).
  • PEA ethanolamine phosphate
  • step (d) The method according to [1], wherein in the step (d), the metal ion is a trivalent iron (Fe 3+ ) ion, and the reduced ion of the metal ion is a divalent iron (Fe 2+ ) ion; [3] The method of [1] or [2], wherein step (d) is performed under conditions comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA); [4] The method according to any of [1] to [3], wherein step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA); [5] The method according to any one of [1] to [4], wherein in step (d), the chelating reagent is Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5-B
  • a method for measuring ethanolamine phosphate (PEA) in a sample by detecting color development by a chelating reagent a kit for performing the method, a program for executing the method, and the program
  • a computer-readable recording medium that stores the program and a device for measuring PEA, which includes the kit or incorporates the program.
  • a device generally used in a normal facility for example, a general hospital or medical institution, for example, a biochemical automatic analysis
  • PEA can be measured easily and with high sensitivity.
  • a large amount of a sample can be measured quickly, for example, in about 10 minutes per test using an automatic biochemical analyzer.
  • a step of generating NADH 2 phosphate of the present method by using a step of generating a plurality moles of NADH 2 from 1 mole phosphoric acid, by detecting the color development by sensitized chelating agent , It is possible to measure PEA with higher sensitivity.
  • FIG. 1 shows the upper diagram (first reaction: removal of endogenous inorganic phosphorus) at the stage of removing phosphoric acid in the sample
  • the lower diagram shows the O-phospho- ethanolamine phosphotransferase lyase (phosphoethanolamine Phospho-lyase) by the action of the step of generating ammonia, acetaldehyde and phosphoric acid from PEA, by the action of enzymes, generating a NADH 2 from phosphoric acid contained in the sample
  • metal 5 shows an exemplary measurement of the step of applying a chelating reagent capable of forming a complex with the ion and the reduced ion of the metal ion.
  • FIG. 2 shows a calibration curve of ethanolamine phosphate based on the absorbance (570 nm).
  • FIG. 3 shows the ability of a protective chelating agent for iron trivalent ions to reduce iron divalent ions by a coenzyme NADH.
  • FIG. 4 shows a comparison between the stability constant ratio of the protected chelating reagent to iron trivalent ions and divalent ions and the NADH reducing power. The stability constant ratio of 0.47 to 0.56 shows the reducing power most efficiently.
  • the invention is directed to a method for measuring ethanolamine phosphate (PEA) in a sample.
  • the method comprises the steps of: (a) removing phosphoric acid in a sample; and (b) reacting the sample obtained in step (a) with O-phosphoethanolamine phospholyase to cause the sample to react with the sample.
  • ammonia PEA contained generating a acetaldehyde and phosphoric acid
  • measure PEA means not only confirming the presence of PEA in a sample and measuring its amount, but also the absence of PEA in the sample (ie, the absence of PEA). The amount is below the detection limit).
  • step (a) any of known means and methods for removing phosphoric acid may be used.
  • methods for removing phosphoric acid include, but are not limited to, enzymatic methods and precipitation methods.
  • the means and method for removing phosphoric acid are described, for example, in WO2018 / 062204 (Patent Document 6).
  • an enzymatic method and a precipitation method are used in combination to remove phosphoric acid. By using these methods together, phosphoric acid can be more reliably removed.
  • the method for measuring PEA of the present invention includes, as step (a), a step of removing phosphoric acid from a sample. Since phosphates can occur naturally, it is likely that they are already present in the sample. Also, at the sample acquisition stage, there is a possibility that these may be mixed into the sample. Therefore, by removing the phosphoric acid present in the sample before performing the reaction of the above formula (1), the phosphoric acid generated from PEA can be detected accurately and with high sensitivity.
  • step (b) a hydrolysis reaction represented by the following formula (1) is used.
  • ethanolamine phosphate phospholyase from any species is used.
  • the enzyme used in step (b) is human ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1).
  • Step (b) is performed in an environment where phosphoric acid is substantially absent (phosphoric acid-free environment).
  • the environment in step (b) may be not only an environment in which phosphoric acid in the sample is not completely present, but also an environment in which phosphoric acid is below the detection limit.
  • step (c) may be used any known means and methods one step or plural steps of generating NADH 2 phosphate by the action of enzymes.
  • Enzymes used in step (c) is one or more of any known enzyme capable of generating NADH 2 phosphate.
  • step (c) one mole of phosphoric acid is converted to multiple moles, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 5.5, generating a 6,6.5,7,7.5,8,8.5,9,9.5 or 10 moles of NADH 2.
  • step (c) produces 3 or 4 moles of NADH 2 from 1 mole of phosphoric acid.
  • reaction represented by the following formula (2) is used in the step (c).
  • step (c) When the reaction represented by the above formula (2) is used, the enzymes used in step (c) include inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine oxidase), uricase, methanol or ethanol, catalase, and Contains formaldehyde dehydrogenase.
  • step (c) utilizes a reaction in which inosine in formula (2) above is replaced with another purine nucleoside, such as adenosine or guanosine.
  • the purine nucleoside is preferably inosine.
  • reaction represented by the formula (2) acetaldehyde obtained by the above formula (1) is reacted with NAD, it may further include a reaction for obtaining a NADH 2.
  • a reaction represented by the following formula (3) is used.
  • step (c) the enzymes used in step (c) include inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine oxidase), uricase, methanol or ethanol, catalase, and Includes formaldehyde dehydrogenase and acetaldehyde dehydrogenase.
  • step (c) utilizes a reaction in which inosine in formula (3) above is replaced with another purine nucleoside, such as adenosine or guanosine.
  • the purine nucleoside is preferably inosine.
  • any known metal ion reduced by the reducing power of NADH 2 obtained in step (c) and a known ion capable of forming a complex with the reduced ion of the metal ion to form a color may be utilized.
  • Metal ions include, but are not limited to, for example, iron ions, cobalt ions, and copper ions.
  • the metal ions to be reduced include, but are not limited to, for example, iron trivalent ions, cobalt trivalent ions, and copper divalent ions.
  • Metal ions that are reduced to form a complex include, but are not limited to, for example, iron divalent ions, cobalt divalent ions, and copper monovalent ions.
  • the metal ion is an iron ion.
  • the chelating reagent can be appropriately selected by those skilled in the art. Chelating reagents include, for example, Nitroso-PSAP (Mikihiko Saito, Daikichi Horiguchi, Kanae Kina: Analytical Chemistry 30; 635-639, 1981), Nitro-PAPS (Noriko Ohno, Tadao Sakai: Analytical Chemistry 46; 937-942, 1997) , 5-Br-PAPS or 5-Br-PSAA.
  • NADH 2 The reducing power of NADH 2 is equivalent to the divalent reducing power.
  • monovalent reduced metal ions such as iron trivalent ions, cobalt trivalent ions, and copper divalent ions
  • one molecule of NADH 2 produces a metal ion that forms a complex of two molecules. can do.
  • the reaction represented by the above formula (2) or (3) proceeds at about pH 7 to about 8, so that a color can be formed by forming a complex with a metal ion under alkaline conditions.
  • Nitroso-PSAP and Nitro-PAPS are used as chelating reagents. Nitroso-PSAP and Nitro-PAPS can particularly detect iron divalent ions with high sensitivity.
  • step (d) is performed under conditions comprising a carboxylic acid such as citric acid, L-tartaric acid, oxalic acid or nitrilotriacetic acid (NTA) or an aromatic acid such as sulfosalicylic acid.
  • step (d) is performed under conditions comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA).
  • step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA).
  • a carboxylic acid such as citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA).
  • step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA).
  • iron ions can be prevented from turning into iron hydro
  • step (e) color development by the chelating reagent in the sample obtained in step (d) is detected.
  • the detection of color development can be performed by a device generally used in ordinary facilities, for example, an automatic biochemical analyzer.
  • the detected color development generally varies depending on the chelating reagent used.
  • a person skilled in the art can appropriately adjust equipment generally used in an ordinary facility for detecting color development, for example, an automatic biochemical analyzer, depending on the chelating reagent used.
  • color development can be detected at an absorbance of about 570 nm.
  • step (e) when generating a plurality moles of NADH 2 from 1 mole of phosphoric acid, step (e) can detect the color produced by a multiple of sensitized chelating agent.
  • the reaction represented by the above formula (2) 3 moles of NADH 2 can be produced from 1 mole of phosphoric acid, and the step (e) is a step in which color development by the chelating reagent which is 3 times sensitized is performed. Can be detected.
  • step (e) shows the color development by the 4-fold sensitized chelating reagent. Can be detected.
  • step (d) if in step (d) one metal NADH 2 is used with a metal ion such as an iron ion, a cobalt ion, a copper ion which forms a complex of two molecules, then step (e) is performed It is possible to detect the color development due to the chelating reagent sensitized twice.
  • a metal ion such as an iron ion, a cobalt ion, a copper ion which forms a complex of two molecules
  • a combination of steps by one mole of generating a plurality moles of NADH 2 from phosphoric acid (c) and 1 NADH 2 molecules to form a complex of two molecules (d), and further a sensitizer Color development due to the chelating reagent can be detected.
  • the reaction represented by the above formula (2) and the step (d) of forming a complex of two molecules by one molecule of NADH 2 such as an iron ion, a cobalt ion, and a copper ion are used, the step (e) is performed. Can detect color development due to the chelating reagent sensitized 6 times.
  • the step (e) is performed.
  • the reaction represented by the above formula (3) and the step (d) of forming a complex of two molecules by one molecule of NADH 2 such as iron ion, cobalt ion, and copper ion are used, the step (e) is performed.
  • the step (e) is performed.
  • step (e) comprises determining the concentration of PEA from the color developed by the detected chelating reagent. Determining the concentration of PEA from color development may be performed by any known means and method. For example, the concentration of PEA can be determined using a calibration curve between the absorbance of color development by the chelating reagent and the PEA concentration.
  • steps (b), (c), (d) and / or (e) are performed simultaneously. That is, enzymes, compositions, reagents, and / or kits required in each step may be used simultaneously.
  • steps (b), (c), (d) and / or (e) are performed simultaneously.
  • the step (a) may include a step of removing each substance generated in the step (c) from the sample.
  • the sample is subjected to the reaction represented by the above formula (2) or the formula (3) from the sample. It may include the step of removing hypoxanthine, uric acid and / or hydrogen peroxide formed.
  • inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine) Oxidase), uricase and / or catalase can be used. As shown in FIG. 1, for example, hydrogen peroxide can be removed with catalase.
  • the sample prior to step (a), is supplemented with any enzyme that catalyzes the reaction of formula (1) above, such as PEA lyase or ⁇ -aminobutylaminotransferase (GabT). And performing the enzymatic reaction may further be included.
  • any enzyme that catalyzes the reaction of formula (1) above such as PEA lyase or ⁇ -aminobutylaminotransferase (GabT).
  • GabT ⁇ -aminobutylaminotransferase
  • the present invention relates to a method for determining whether a subject has depression.
  • the method comprises the steps of (i) measuring PEA in a sample by the method of measuring PEA of the present invention; and (ii) treating the subject with depression when the PEA in the sample is less than 1.5 ⁇ mol / L. And determining that the subject is affected.
  • sample used in the method of the present invention described above is any sample containing or possibly containing PEA.
  • samples include, but are not limited to, biological 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 have been 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 pretreated 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 above-described method of the present invention.
  • the kit of the invention comprises a means for removing phosphate.
  • the kit of the present invention comprises O-phosphoethanolamine phosphorylase.
  • the kit of the invention further comprises a metal.
  • the metal is trivalent iron.
  • the kit of the invention further comprises citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA), and Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5- Contains Br-PSAA.
  • the kit of the present invention may contain any reagent used in the method of the present invention. It may also contain appropriate reagents for simplifying the measurement, such as sample diluents, reaction diluents, buffers, detergents, and the like. Usually, the reagent is provided in a suitable container. Still further, it may include materials such as instructions necessary for performing the method of the present invention.
  • the kit of the present invention is a Research Only Use Only (RUO) kit. In another embodiment, the kit of the present invention is an Investigational 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 recording medium of a computer attached to the device. 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 above-described program of the present invention is incorporated in the device of the present invention.
  • Phosphate removal 5 ⁇ L of plasma was used as a sample.
  • 120 ⁇ L of the first reagent described in Table 1 below was added to the sample to remove phosphoric acid, hypoxanthine, uric acid and hydrogen peroxide in the sample, and then after 5 minutes at 37 ° C., a Hitachi 7180 automatic analyzer ( The absorbance was measured at 570 nm (manufacturer: Hitachi High-Technologies) (absorbance 1).
  • Iron trivalent ion (Fe (III)) coordinates more strongly with citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA) than iron divalent ion (Fe (II)) I do.
  • Iron trivalent ions become iron hydroxide in an alkaline, may interfere with reduction by NADH 2.
  • NADH reduced by NADH to form iron divalent ions
  • the stability constant decreases, so that a ligand substitution reaction occurs with Nitroso-PSAP or Nitro-PAPS to develop color.

Abstract

Provided is a method for measuring phosphoethanolamine (PEA). Provided is a method that is for measuring phosphoethanolamine (PEA) and that comprises a step for producing ammonia, acetaldehyde, and phosphoric acid from PEA by causing O-phosphoethanolamine phospho-lyase to act on PEA, a step for causing a metal ion and a chelating agent capable of forming a complex together with a reduced ion of the metal ion to act on each other, and a step for detecting a color exhibited by the chelating reagent.

Description

エタノールアミンリン酸の測定方法Method for measuring ethanolamine phosphoric acid
　本発明は、キレート試薬による発色を検出する段階を含む、サンプル中に存在するエタノールアミンリン酸（ＰＥＡ）を測定する方法、その方法を実施するためのキット、その方法を実行させるためのプログラム、そのプログラムを保存したコンピュータで読み取り可能な記録媒体、およびそのキットを含む、またはそのプログラムが組み込まれた、ＰＥＡを測定するための装置に関する。 The present invention provides a method for measuring ethanolamine phosphate (PEA) present in a sample, the method including a step of detecting color development by a chelating reagent, a kit for performing the method, a program for performing the method, The present invention relates to a computer-readable recording medium storing the program, and an apparatus for measuring PEA including the kit or incorporating the program.
　うつ病は気分障害の一種であり、その主な症状は「抑うつ気分」と「興味・喜びの喪失」である。日本の医療機関に対する調査によると、２００８年のうつ病の患者数は７０万人以上とされている。また、２０１４年の厚生労働省の調査によると、気分障害（感情障害）患者数は１１２．２万人であり、患者数が増加してきている。しかし、うつ病の診断は、患者の精神面についての医師や心理士の主観、または患者自身の主観や申告に依存する面が大きく、そのため、客観的な判断がなされているとは言い難い。そこで、うつ病を客観的に診断するため、治療の経過を観察するため、および予後を予測するために、近年、患者の体液中の成分をうつ病の診断の目安として用いる試みがなされている。 Depression is a type of mood disorder, the main symptoms of which 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 was over 700,000. According to a 2014 survey by the Ministry of Health, Labor and Welfare, the number of patients with mood disorders (emotional disorders) was 11.22,000, and the number of patients is increasing. However, the diagnosis of depression largely depends on the subjective opinion of the physician or psychologist on the mental aspect of the patient, or on the subjective opinion or declaration of the patient, and therefore, it cannot be said that objective judgment has been made. Therefore, in order to objectively diagnose depression, to observe the course of treatment, and to predict prognosis, in recent years, attempts have been made to use components in body fluids of patients as a guide for the diagnosis of depression. .
　従来、うつ病の予測マーカーとして、体液中のトリプトファンまたはその分解物、あるいは特定の遺伝子の発現量などを用いることが報告されていたが（特許文献１、２）、本発明者らは以前に、血液中のエタノールアミンリン酸（ＰＥＡ）がうつ病を診断するためのバイオマーカーとして有用であることを見出した（特許文献３、４）。ＰＥＡ濃度の測定値は、うつ病の客観的な診断や経過観察に有用であるとされている（非特許文献１）。本発明者らはさらに、γ－アミノブチルアミノ基転移酵素（ＧａｂＴ）を用いてＰＥＡからアセトアルデヒド、リン酸、およびアンモニアを生成させることにより、サンプル中のＰＥＡを測定する方法を開発した（特許文献５）。本発明者らはまた、ＰＥＡからアセトアルデヒド、リン酸、およびアンモニアを生成させる前に、アンモニアおよび／またはリン酸を除去することによって、ＰＥＡをより正確に測定する方法を開発した（特許文献６）。 Conventionally, it has been reported that tryptophan or its degradation product in a body fluid or the expression level of a specific gene is used as a predictive marker for depression (Patent Documents 1 and 2). Have found that ethanolamine phosphate (PEA) in blood is useful as a biomarker for diagnosing depression (Patent Documents 3 and 4). The measured value of the PEA concentration is considered to be useful for objective diagnosis and follow-up of depression (Non-Patent Document 1). The present inventors have further developed a method for measuring PEA in a sample by generating acetaldehyde, phosphoric acid, and ammonia from PEA using γ-aminobutylaminotransferase (GabT) (Patent Document 5). The present inventors have also developed a method for more accurately measuring PEA by removing ammonia and / or phosphoric acid before producing acetaldehyde, phosphoric acid, and ammonia from PEA (Patent Document 6). .
国際公開第２００６／１０５９０７号International Publication No. WO 2006/105907 特開２００８－２５３２５８号公報JP 2008-253258 A 国際公開第２０１１／０１９０７２号International Publication No. 2011/019072 国際公開第２０１６／０４７６７７号WO 2016/047677 特許第５６８８１６３号明細書Patent No. 5688163 国際公開第２０１８／０６２２０４号International Publication No. WO2018 / 062204
　従来のＰＥＡを測定する方法は、質量分析装置、例えばキャピラリー質量分析装置を使用する。しかしながら、質量分析装置は高価であり、通常の施設、例えば一般的な病院または医療機関ではこのような装置を用いて分析を行うことができない。また、従来の酵素的ＰＥＡ測定法は感度が十分でない。そのため、通常の施設で一般的に使用されている機器、例えば生化学自動分析装置によって、簡便かつ高感度にＰＥＡを測定することができる方法が必要とされている。 The conventional method for measuring PEA uses a mass spectrometer, for example, a capillary mass spectrometer. However, mass spectrometers are expensive and cannot be analyzed using ordinary equipment, such as a general hospital or medical institution. Further, the sensitivity of the conventional enzymatic PEA measurement method is not sufficient. Therefore, there is a need for a method that can easily and highly sensitively measure PEA by using a device generally used in ordinary facilities, for example, an automatic biochemical analyzer.
　本発明者らは、上記課題を解決するため鋭意研究を行った結果、Ｏ－ホスホエタノールアミンホスホリアーゼ（Phosphoethanolamine Phospho-Lyase）を作用させて、ＰＥＡからアンモニア、アセトアルデヒドおよびリン酸を生成する段階、酵素を作用させて、リン酸から還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ２）を生成する段階、金属イオンおよび当該金属イオンの還元イオンと錯体を形成することができるキレート試薬を作用させる段階、およびキレート試薬による発色を検出する段階を含む、サンプル中のエタノールアミンリン酸（ＰＥＡ）を測定する方法を見いだし、本発明を完成させるに至った。 The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a step of producing ammonia, acetaldehyde, and phosphoric acid from PEA by acting O-phosphoethanolamine phospholyase (Phosphoethanolamine Phospho-Lyase), Reacting an enzyme to generate reduced nicotinamide adenine dinucleotide (NADH 2 ) from phosphoric acid, reacting a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion, and The present inventors have found a method for measuring ethanolamine phosphate (PEA) in a sample, which includes a step of detecting color development by a chelating reagent, and have completed the present invention.
　すなわち、本発明は以下を提供する：
［１］サンプル中のエタノールアミンリン酸（ＰＥＡ）を測定する方法であって、
　（ａ）サンプル中のリン酸を除去する段階、
　（ｂ）段階（ａ）で得られたサンプルにＯ－ホスホエタノールアミンホスホリアーゼ（Phosphoethanolamine Phospho-Lyase）を作用させて、サンプルに含まれるＰＥＡからアンモニア、アセトアルデヒドおよびリン酸を生成する段階、
　（ｃ）段階（ｂ）で得られたサンプルに酵素を作用させて、サンプルに含まれるリン酸からＮＡＤＨ２を生成する段階、
　（ｄ）段階（ｃ）で得られたサンプルに金属イオンおよび当該金属イオンの還元イオンと錯体を形成することができるキレート試薬を作用させる段階、および
　（ｅ）段階（ｄ）で得られたサンプル中のキレート試薬による発色を検出する段階
を含む、方法；
［２］段階（ｄ）において、金属イオンが三価の鉄（Ｆｅ３＋）イオンであり、金属イオンの還元イオンが二価の鉄（Ｆｅ２＋）イオンである、［１］に記載の方法；
［３］段階（ｄ）がクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる、［１］または［２］に記載の方法；
［４］段階（ｄ）がＬ－酒石酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる、［１］～［３］のいずれかに記載の方法；
［５］段階（ｄ）において、キレート試薬がNitroso-PSAP、Nitro-PAPS、5-Br-PAPSまたは5-Br-PSAAである、［１］～［４］のいずれかに記載の方法；
［６］段階（ｃ）が、
　（ｃ－１）リン酸にイノシンおよびプリンヌクレオチドホスホリラーゼを作用させて、ヒポキサンチンを生成する段階、
　（ｃ－２）ヒポキサンチンにキサンチン脱水素酵素を作用させて、尿酸を生成する段階、
　（ｃ－３）尿酸にウリカーゼを作用させて、過酸化水素を生成する段階、
　（ｃ－４）過酸化水素にメタノールまたはエタノールおよびカタラーゼを作用させて、ホルムアルデヒドまたはアセトアルデヒドを生成する段階、および
　（ｃ－５）ホルムアルデヒドまたはアセトアルデヒドにＮＡＤおよびホルムアルデヒド脱水素酵素を作用させて、ＮＡＤＨ２を生成する段階
を含む、［１］～［５］のいずれかに記載の方法；
［７］段階（ａ）が、サンプル中のリン酸、ヒポキサンチン、尿酸および過酸化水素を酵素で除去する段階である、［１］～［６］のいずれかに記載の方法；
［８］サンプルが生体試料サンプルである、［１］～［７］のいずれかに記載の方法；
［９］サンプルが、全血、血清、または血漿サンプルである、［８］に記載の方法；
［１０］対象がうつ病に罹患しているかどうかを判定するための方法であって、
　（ａ）［１］～［９］のいずれかに記載の方法により、対象由来のサンプル中のＰＥＡを測定する段階；および
　（ｂ）サンプル中のＰＥＡが１．５μｍｏｌ／Ｌ未満である場合に、対象がうつ病に罹患していると判定する段階
を含む、方法；
［１１］Ｏ－ホスホエタノールアミンホスホリアーゼを含む、［１］～［１０］のいずれかに記載の方法を実施するためのキット；
［１２］さらに金属を含む、［１１］に記載のキット；
［１３］金属が三価の鉄である、［１２］に記載のキット；
［１４］さらにクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）、およびNitroso-PSAP、Nitro-PAPS、5-Br-PAPSまたは5-Br-PSAAを含む、［１１］～［１３］のいずれかに記載のキット；
［１５］コンピュータに、［１］～［１０］のいずれかに記載の方法を実行させるためのプログラム；
［１６］［１５］に記載のプログラムを保存したコンピュータで読み取り可能な記録媒体；
［１７］［１１］～［１４］のいずれかに記載のキットを含む、または［１５］に記載のプログラムが組み込まれた、ＰＥＡを測定するための装置。 That is, the present invention provides:
[1] A method for measuring ethanolamine phosphate (PEA) in a sample,
(A) removing phosphoric acid in the sample;
(B) reacting the sample obtained in step (a) with O-phosphoethanolamine Phospho-Lyase to produce ammonia, acetaldehyde and phosphoric acid from PEA contained in the sample;
(C) reacting the sample obtained in step (b) with an enzyme to generate NADH 2 from phosphoric acid contained in the sample;
(D) reacting the sample obtained in step (c) with a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion; and (e) the sample obtained in step (d). Detecting color development by the chelating reagent therein;
[2] The method according to [1], wherein in the step (d), the metal ion is a trivalent iron (Fe 3+ ) ion, and the reduced ion of the metal ion is a divalent iron (Fe 2+ ) ion;
[3] The method of [1] or [2], wherein step (d) is performed under conditions comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA);
[4] The method according to any of [1] to [3], wherein step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA);
[5] The method according to any one of [1] to [4], wherein in step (d), the chelating reagent is Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5-Br-PSAA;
[6] Step (c)
(C-1) reacting inosine and purine nucleotide phosphorylase with phosphate to produce hypoxanthine;
(C-2) producing xanthine dehydrogenase on hypoxanthine to produce uric acid;
(C-3) a step of causing uricase to act on uric acid to generate hydrogen peroxide;
(C-4) reacting hydrogen peroxide with methanol or ethanol and catalase to produce formaldehyde or acetaldehyde; and (c-5) reacting formaldehyde or acetaldehyde with NAD and formaldehyde dehydrogenase to form NADH 2 The method according to any one of [1] to [5], comprising the step of:
[7] The method according to any one of [1] to [6], wherein the step (a) is a step of removing phosphoric acid, hypoxanthine, uric acid and hydrogen peroxide in the sample with an enzyme;
[8] The method according to any of [1] to [7], wherein the sample is a biological sample;
[9] The method according to [8], wherein the sample is a whole blood, serum, or plasma sample;
[10] A method for determining whether a subject is suffering from depression,
(A) measuring PEA in a sample derived from a subject by the method according to any one of [1] to [9]; and (b) when the PEA in the sample is less than 1.5 μmol / L. Determining that the subject is suffering from depression;
[11] a kit for performing the method according to any one of [1] to [10], which comprises O-phosphoethanolamine phosphorylase;
[12] the kit of [11], further comprising a metal;
[13] the kit of [12], wherein the metal is trivalent iron;
[14] further including citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA), and Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5-Br-PSAA [11]. A kit according to any one of to [13];
[15] A program for causing a computer to execute the method according to any one of [1] to [10];
[16] A computer-readable recording medium storing the program according to [15];
[17] An apparatus for measuring PEA, comprising the kit according to any one of [11] to [14], or incorporating the program according to [15].
　本発明によれば、キレート試薬による発色を検出することによってサンプル中のエタノールアミンリン酸（ＰＥＡ）を測定する方法、その方法を実施するためのキット、その方法を実行させるためのプログラム、そのプログラムを保存したコンピュータで読み取り可能な記録媒体、およびそのキットを含む、またはそのプログラムが組み込まれた、ＰＥＡを測定するための装置等が提供される。 According to the present invention, a method for measuring ethanolamine phosphate (PEA) in a sample by detecting color development by a chelating reagent, a kit for performing the method, a program for executing the method, and the program And a computer-readable recording medium that stores the program and a device for measuring PEA, which includes the kit or incorporates the program.
　したがって、本発明の方法等を使用することによって、質量分析装置を使用せずに、通常の施設、例えば一般的な病院または医療機関にて一般的に使用されている機器、例えば生化学自動分析装置によって、簡便かつ高感度にＰＥＡを測定することができる。 Therefore, by using the method or the like of the present invention, without using a mass spectrometer, a device generally used in a normal facility, for example, a general hospital or medical institution, for example, a biochemical automatic analysis With the device, PEA can be measured easily and with high sensitivity.
　生化学自動分析装置によって、本発明の方法を使用することによって、大量の試料を迅速に、例えば１テスト約１０分で測定することが可能である。 (4) By using the method of the present invention, a large amount of a sample can be measured quickly, for example, in about 10 minutes per test using an automatic biochemical analyzer.
　また、本発明の方法のリン酸からＮＡＤＨ２を生成する段階として、１モルのリン酸から複数モルのＮＡＤＨ２を生成する段階を使用することによって、増感したキレート試薬による発色を検出して、より高感度にＰＥＡを測定することが可能である。 Further, as a step of generating NADH 2 phosphate of the present method, by using a step of generating a plurality moles of NADH 2 from 1 mole phosphoric acid, by detecting the color development by sensitized chelating agent , It is possible to measure PEA with higher sensitivity.
図１は、上図（第一反応：内因性無機りんの除去）がサンプル中のリン酸を除去する段階、ならびに、下図（第二反応：エタノールアミンリン酸のリン酸測定）がＯ－ホスホエタノールアミンホスホリアーゼ（Phosphoethanolamine Phospho-Lyase）を作用させて、ＰＥＡからアンモニア、アセトアルデヒドおよびリン酸を生成する段階、酵素を作用させて、サンプルに含まれるリン酸からＮＡＤＨ２を生成する段階、および金属イオンおよび当該金属イオンの還元イオンと錯体を形成することができるキレート試薬を作用させる段階の例示的な測定法を示す。FIG. 1 shows the upper diagram (first reaction: removal of endogenous inorganic phosphorus) at the stage of removing phosphoric acid in the sample, and the lower diagram (second reaction: phosphoric acid measurement of ethanolamine phosphate) shows the O-phospho- ethanolamine phosphotransferase lyase (phosphoethanolamine Phospho-lyase) by the action of the step of generating ammonia, acetaldehyde and phosphoric acid from PEA, by the action of enzymes, generating a NADH 2 from phosphoric acid contained in the sample, and metal 5 shows an exemplary measurement of the step of applying a chelating reagent capable of forming a complex with the ion and the reduced ion of the metal ion. 図２は、吸光度（５７０ｎｍ）に基づくエタノールアミンリン酸の検量線を示す。FIG. 2 shows a calibration curve of ethanolamine phosphate based on the absorbance (570 nm). 図３は、鉄三価イオンの保護キレート試薬と補酵素ＮＡＤＨによる鉄二価イオンへの還元力能力を示す。FIG. 3 shows the ability of a protective chelating agent for iron trivalent ions to reduce iron divalent ions by a coenzyme NADH. 図４は、鉄三価イオンと二価イオンに対する保護キレート試薬の安定度定数比とＮＡＤＨ還元力の比較を示す。安定度定数比０．４７～０．５６が最も効率的に還元力を示す。FIG. 4 shows a comparison between the stability constant ratio of the protected chelating reagent to iron trivalent ions and divalent ions and the NADH reducing power. The stability constant ratio of 0.47 to 0.56 shows the reducing power most efficiently.
　一つの実施態様では、本発明は、サンプル中のエタノールアミンリン酸（ＰＥＡ）を測定する方法に関する。当該方法は、（ａ）サンプル中のリン酸を除去する段階、（ｂ）段階（ａ）で得られたサンプルにＯ－ホスホエタノールアミンホスホリアーゼ（Phosphoethanolamine Phospho-Lyase）を作用させて、サンプルに含まれるＰＥＡからアンモニア、アセトアルデヒドおよびリン酸を生成する段階、（ｃ）段階（ｂ）で得られたサンプルに酵素を作用させて、サンプルに含まれるリン酸からＮＡＤＨ２を生成する段階、（ｄ）段階（ｃ）で得られたサンプルに金属イオンおよび当該金属イオンの還元イオンと錯体を形成することができるキレート試薬を作用させる段階、および（ｅ）段階（ｄ）で得られたサンプル中のキレート試薬による発色を検出する段階を含む。 In one embodiment, the invention is directed to a method for measuring ethanolamine phosphate (PEA) in a sample. The method comprises the steps of: (a) removing phosphoric acid in a sample; and (b) reacting the sample obtained in step (a) with O-phosphoethanolamine phospholyase to cause the sample to react with the sample. ammonia PEA contained, generating a acetaldehyde and phosphoric acid, (c) phase obtained by the action of the enzyme to the sample in (b), generating a NADH 2 from phosphoric acid contained in the sample, (d A) reacting the sample obtained in step (c) with a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion; and (e) in the sample obtained in step (d). Detecting the color developed by the chelating reagent.
　本明細書で用いる場合、「ＰＥＡを測定する」とは、サンプル中にＰＥＡが存在することを確認し、その量を測定することだけでなく、サンプル中にＰＥＡが存在しない（すなわち、ＰＥＡの量が検出限界以下である）ことを確認することも包含する。 As used herein, “measure PEA” means not only confirming the presence of PEA in a sample and measuring its amount, but also the absence of PEA in the sample (ie, the absence of PEA). The amount is below the detection limit).
　段階（ａ）では、リン酸を除去する公知の手段および方法のいずれを用いてもよい。リン酸を除去する方法の例として、酵素法および沈殿法が挙げられるが、これらに限定されない。リン酸を除去する手段および方法については、例えば、国際公開第２０１８／０６２２０４号（特許文献６）に記載されている。 In step (a), any of known means and methods for removing phosphoric acid may be used. Examples of methods for removing phosphoric acid include, but are not limited to, enzymatic methods and precipitation methods. The means and method for removing phosphoric acid are described, for example, in WO2018 / 062204 (Patent Document 6).
　一実施形態では、リン酸を除去するために、酵素法および沈殿法が併用される。これらの方法を併用することで、より確実にリン酸を除去することができる。 で は In one embodiment, an enzymatic method and a precipitation method are used in combination to remove phosphoric acid. By using these methods together, phosphoric acid can be more reliably removed.
　上述のとおり、本発明のＰＥＡを測定する方法は、段階（ａ）として、サンプルからリン酸を除去する段階を含む。リン酸は天然に存在し得るため、サンプル中にこれらが既に存在している可能性が高い。また、サンプル取得段階で、これらがサンプル中に混入する可能性もある。したがって、上記式（１）の反応を実施する前にサンプル中に存在しているリン酸を除去することにより、正確かつ高感度に、ＰＥＡから生成したリン酸を検出することができる。 As described above, the method for measuring PEA of the present invention includes, as step (a), a step of removing phosphoric acid from a sample. Since phosphates can occur naturally, it is likely that they are already present in the sample. Also, at the sample acquisition stage, there is a possibility that these may be mixed into the sample. Therefore, by removing the phosphoric acid present in the sample before performing the reaction of the above formula (1), the phosphoric acid generated from PEA can be detected accurately and with high sensitivity.
　段階（ｂ）では、下記式（１）で示される加水分解反応が利用される。
Figure JPOXMLDOC01-appb-C000001
 In the step (b), a hydrolysis reaction represented by the following formula (1) is used.
Figure JPOXMLDOC01-appb-C000001
　この反応系では、あらゆる種由来のエタノールアミンリン酸ホスホリアーゼが用いられる。好ましい実施形態では、段階（ｂ）で用いられる酵素は、ヒト由来エタノールアミンリン酸ホスホリアーゼ（ＡＧＸＴ２Ｌ１）（配列番号１）である。 エ タ ノ ー ル In this reaction system, ethanolamine phosphate phospholyase from any species is used. In a preferred embodiment, the enzyme used in step (b) is human ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1).
　段階（ｂ）は、リン酸が実質的に存在しない環境（リン酸フリーの環境）下で実施される。段階（ｂ）の環境は、サンプル中のリン酸が完全に存在しない環境だけでなく、リン酸が検出限界以下である環境でもよい。 Step (b) is performed in an environment where phosphoric acid is substantially absent (phosphoric acid-free environment). The environment in step (b) may be not only an environment in which phosphoric acid in the sample is not completely present, but also an environment in which phosphoric acid is below the detection limit.
　段階（ｃ）では、酵素を作用させてリン酸からＮＡＤＨ２を生成する１段階または複数段階の公知の任意の手段および方法を用いてよい。段階（ｃ）で使用される酵素は、リン酸からＮＡＤＨ２を生成することができる１つまたは複数の公知の任意の酵素である。 In step (c), may be used any known means and methods one step or plural steps of generating NADH 2 phosphate by the action of enzymes. Enzymes used in step (c) is one or more of any known enzyme capable of generating NADH 2 phosphate.
　好ましい実施形態では、段階（ｃ）では、１モルのリン酸から複数モル、例えば、１．５、２、２．５、３、３．５、４、４．５、５、５．５、６、６．５、７、７．５、８、８．５、９、９．５または１０モルのＮＡＤＨ２を生成する。好ましい実施形態では、段階（ｃ）では、１モルのリン酸から３または４モルのＮＡＤＨ２を生成する。 In a preferred embodiment, in step (c), one mole of phosphoric acid is converted to multiple moles, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 5.5, generating a 6,6.5,7,7.5,8,8.5,9,9.5 or 10 moles of NADH 2. In a preferred embodiment, step (c) produces 3 or 4 moles of NADH 2 from 1 mole of phosphoric acid.
　一実施形態では、段階（ｃ）では、下記式（２）で示される反応が利用される。
Figure JPOXMLDOC01-appb-C000002
 In one embodiment, the reaction represented by the following formula (2) is used in the step (c).
Figure JPOXMLDOC01-appb-C000002
　上記式（２）で示される反応が利用される場合、段階（ｃ）で使用される酵素は、イノシン、プリンヌクレオチドホスホリラーゼ、キサンチン脱水素酵素（キサンチンオキシダーゼ）、ウリカーゼ、メタノールまたはエタノール、カタラーゼ、およびホルムアルデヒド脱水素酵素を含む。この反応系では、１モルのリン酸から３モルのＮＡＤＨ２を生成する。別の実施形態では、段階（ｃ）では、上記式（２）中のイノシンを他のプリンヌクレオシド、例えばアデノシンまたはグアノシンで置き換えた反応が利用される。プリンヌクレオシドは、好ましくはイノシンである。 When the reaction represented by the above formula (2) is used, the enzymes used in step (c) include inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine oxidase), uricase, methanol or ethanol, catalase, and Contains formaldehyde dehydrogenase. In this reaction system, to produce a 3 mole of NADH 2 from 1 mole of phosphoric acid. In another embodiment, step (c) utilizes a reaction in which inosine in formula (2) above is replaced with another purine nucleoside, such as adenosine or guanosine. The purine nucleoside is preferably inosine.
　上記式（２）で示される反応が利用される場合、上記式（１）で得られるアセトアルデヒドをＮＡＤと反応させ、ＮＡＤＨ２を得る反応をさらに含んでもよい。例えば、段階（ｃ）では、下記式（３）で示される反応が利用される。
Figure JPOXMLDOC01-appb-C000003
 If the reaction represented by the formula (2) is used, acetaldehyde obtained by the above formula (1) is reacted with NAD, it may further include a reaction for obtaining a NADH 2. For example, in the step (c), a reaction represented by the following formula (3) is used.
Figure JPOXMLDOC01-appb-C000003
　上記式（３）で示される反応が利用される場合、段階（ｃ）で使用される酵素は、イノシン、プリンヌクレオチドホスホリラーゼ、キサンチン脱水素酵素（キサンチンオキシダーゼ）、ウリカーゼ、メタノールまたはエタノール、カタラーゼ、およびホルムアルデヒド脱水素酵素およびアセトアルデヒド脱水素酵素を含む。この反応系では、１モルのリン酸から４モルのＮＡＤＨ２を生成する。別の実施形態では、段階（ｃ）では、上記式（３）中のイノシンを他のプリンヌクレオシド、例えばアデノシンまたはグアノシンで置き換えた反応が利用される。プリンヌクレオシドは、好ましくはイノシンである。 When the reaction represented by the above formula (3) is used, the enzymes used in step (c) include inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine oxidase), uricase, methanol or ethanol, catalase, and Includes formaldehyde dehydrogenase and acetaldehyde dehydrogenase. In this reaction system, to produce a 4 mol of NADH 2 from 1 mole of phosphoric acid. In another embodiment, step (c) utilizes a reaction in which inosine in formula (3) above is replaced with another purine nucleoside, such as adenosine or guanosine. The purine nucleoside is preferably inosine.
　段階（ｄ）では、段階（ｃ）で得られたＮＡＤＨ２の還元力により還元される公知の任意の金属イオンおよび当該金属イオンの還元イオンと錯体を形成して、発色することができる公知の任意のキレート試薬が利用される。金属イオンは、例えば鉄イオン、コバルトイオンおよび銅イオンを含むが、これらに限定されない。還元される金属イオンは、例えば鉄三価イオン、コバルト三価イオンおよび銅二価イオンを含むが、これらに限定されない。還元され錯体を形成する金属イオンは、例えば鉄二価イオン、コバルト二価イオンおよび銅一価イオンを含むが、これらに限定されない。好ましくは、金属イオンは鉄イオンである。キレート試薬は、当業者が適宜選択することができる。キレート試薬は、例えばNitroso-PSAP（斎藤幹彦、堀口大吉、喜納兼勇：分析化学　30；635-639,1981）、Nitro-PAPS（大野典子、酒井忠雄：分析化学　46；937-942,1997）、5-Br-PAPSまたは5-Br-PSAAを含むが、これらに限定されない。 In step (d), any known metal ion reduced by the reducing power of NADH 2 obtained in step (c) and a known ion capable of forming a complex with the reduced ion of the metal ion to form a color. Any chelating reagent may be utilized. Metal ions include, but are not limited to, for example, iron ions, cobalt ions, and copper ions. The metal ions to be reduced include, but are not limited to, for example, iron trivalent ions, cobalt trivalent ions, and copper divalent ions. Metal ions that are reduced to form a complex include, but are not limited to, for example, iron divalent ions, cobalt divalent ions, and copper monovalent ions. Preferably, the metal ion is an iron ion. The chelating reagent can be appropriately selected by those skilled in the art. Chelating reagents include, for example, Nitroso-PSAP (Mikihiko Saito, Daikichi Horiguchi, Kanae Kina: Analytical Chemistry 30; 635-639, 1981), Nitro-PAPS (Noriko Ohno, Tadao Sakai: Analytical Chemistry 46; 937-942, 1997) , 5-Br-PAPS or 5-Br-PSAA.
　ＮＡＤＨ２の還元力は、二価分の還元力に相当する。よって、鉄三価イオン、コバルト三価イオンおよび銅二価イオンのような一価分還元される金属イオンが使用される場合、１分子のＮＡＤＨ２によって、２分子の錯体を形成する金属イオンを生成することができる。 The reducing power of NADH 2 is equivalent to the divalent reducing power. Thus, when monovalent reduced metal ions, such as iron trivalent ions, cobalt trivalent ions, and copper divalent ions, are used, one molecule of NADH 2 produces a metal ion that forms a complex of two molecules. can do.
　一実施形態では、段階（ｄ）では、上記式（２）または（３）で示される反応が約ｐＨ７～８前後で進行するため、アルカリ性で金属イオンと錯体を形成して発色することができるNitroso-PSAPやNitro-PAPSが、キレート試薬として使用される。Nitroso-PSAPやNitro-PAPSは、特に鉄二価イオンを高感度に検出することができる。 In one embodiment, in the step (d), the reaction represented by the above formula (2) or (3) proceeds at about pH 7 to about 8, so that a color can be formed by forming a complex with a metal ion under alkaline conditions. Nitroso-PSAP and Nitro-PAPS are used as chelating reagents. Nitroso-PSAP and Nitro-PAPS can particularly detect iron divalent ions with high sensitivity.
　一実施形態では、段階（ｄ）は、クエン酸、Ｌ－酒石酸、シュウ酸またはニトリロ三酢酸（ＮＴＡ）などのカルボン酸またはスルホサリチル酸などの芳香族酸を含む条件下で行われる。好ましい実施形態では、段階（ｄ）は、クエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる。さらに好ましい実施形態では、段階（ｄ）は、Ｌ－酒石酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる。例えば、段階（ｄ）がカルボン酸または芳香族酸を含む条件下で行われるとき、鉄イオンが水酸化鉄となることを防止することができる。 In one embodiment, step (d) is performed under conditions comprising a carboxylic acid such as citric acid, L-tartaric acid, oxalic acid or nitrilotriacetic acid (NTA) or an aromatic acid such as sulfosalicylic acid. In a preferred embodiment, step (d) is performed under conditions comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA). In a more preferred embodiment, step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA). For example, when step (d) is performed under conditions containing a carboxylic acid or an aromatic acid, iron ions can be prevented from turning into iron hydroxide.
　段階（ｅ）では、段階（ｄ）で得られたサンプル中のキレート試薬による発色を検出する。発色の検出は、通常の施設で一般的に使用されている機器、例えば生化学自動分析装置によって行うことができる。検出される発色は、一般的に、使用されるキレート試薬に依存して変化する。当業者は、使用されるキレート試薬に依存して、発色を検出する通常の施設で一般的に使用されている機器、例えば生化学自動分析装置を適宜調整することができる。例えば、本発明の方法において、金属イオンとして鉄イオンを、キレート試薬としてNitro-PAPSを用いたとき、約５７０ｎｍの吸光度で発色を検出することができる。 In step (e), color development by the chelating reagent in the sample obtained in step (d) is detected. The detection of color development can be performed by a device generally used in ordinary facilities, for example, an automatic biochemical analyzer. The detected color development generally varies depending on the chelating reagent used. A person skilled in the art can appropriately adjust equipment generally used in an ordinary facility for detecting color development, for example, an automatic biochemical analyzer, depending on the chelating reagent used. For example, in the method of the present invention, when iron ions are used as metal ions and Nitro-PAPS is used as a chelating reagent, color development can be detected at an absorbance of about 570 nm.
　一実施形態では、段階（ｃ）で、１モルのリン酸から複数モルのＮＡＤＨ２を生成する場合、段階（ｅ）は、複数倍の増感したキレート試薬による発色を検出することができる。上記式（２）で示される反応が利用される場合、１モルのリン酸から３モルのＮＡＤＨ２を生成することができ、段階（ｅ）は、３倍の増感したキレート試薬による発色を検出することができる。上記式（３）で示される反応が利用される場合、１モルのリン酸から４モルのＮＡＤＨ２を生成することができ、段階（ｅ）は、４倍の増感したキレート試薬による発色を検出することができる。 In one embodiment, in step (c), when generating a plurality moles of NADH 2 from 1 mole of phosphoric acid, step (e) can detect the color produced by a multiple of sensitized chelating agent. When the reaction represented by the above formula (2) is used, 3 moles of NADH 2 can be produced from 1 mole of phosphoric acid, and the step (e) is a step in which color development by the chelating reagent which is 3 times sensitized is performed. Can be detected. When the reaction represented by the above formula (3) is used, 4 moles of NADH 2 can be produced from 1 mole of phosphoric acid, and step (e) shows the color development by the 4-fold sensitized chelating reagent. Can be detected.
　他の実施態様では、段階（ｄ）で、１分子のＮＡＤＨ２によって、２分子の錯体を形成する鉄イオン、コバルトイオン、銅イオンのような金属イオンが使用される場合、段階（ｅ）は、２倍の増感したキレート試薬による発色を検出することができる。 In another embodiment, if in step (d) one metal NADH 2 is used with a metal ion such as an iron ion, a cobalt ion, a copper ion which forms a complex of two molecules, then step (e) is performed It is possible to detect the color development due to the chelating reagent sensitized twice.
　別の実施形態では、１モルのリン酸から複数モルのＮＡＤＨ２を生成する段階（ｃ）および１分子のＮＡＤＨ２によって２分子の錯体を形成する段階（ｄ）を組み合わせて、さらに増感したキレート試薬による発色を検出することができる。例えば、上記式（２）で示される反応および鉄イオン、コバルトイオン、銅イオンのような１分子のＮＡＤＨ２によって２分子の錯体を形成する段階（ｄ）が利用される場合、段階（ｅ）は、６倍の増感したキレート試薬による発色を検出することができる。例えば、上記式（３）で示される反応および鉄イオン、コバルトイオン、銅イオンのような１分子のＮＡＤＨ２によって２分子の錯体を形成する段階（ｄ）が利用される場合、段階（ｅ）は、８倍の増感したキレート試薬による発色を検出することができる。 In another embodiment, a combination of steps by one mole of generating a plurality moles of NADH 2 from phosphoric acid (c) and 1 NADH 2 molecules to form a complex of two molecules (d), and further a sensitizer Color development due to the chelating reagent can be detected. For example, when the reaction represented by the above formula (2) and the step (d) of forming a complex of two molecules by one molecule of NADH 2 such as an iron ion, a cobalt ion, and a copper ion are used, the step (e) is performed. Can detect color development due to the chelating reagent sensitized 6 times. For example, when the reaction represented by the above formula (3) and the step (d) of forming a complex of two molecules by one molecule of NADH 2 such as iron ion, cobalt ion, and copper ion are used, the step (e) is performed. Can detect color development due to the chelating reagent sensitized 8-fold.
　一実施形態では、段階（ｅ）は、検出されたキレート試薬による発色からＰＥＡの濃度を決定することを含む。発色からＰＥＡの濃度を決定することは、公知の手段および方法のいずれを用いてもよい。例えば、キレート試薬による発色の吸光度とＰＥＡ濃度との検量線を用いて、ＰＥＡの濃度を決定することができる。 In one embodiment, step (e) comprises determining the concentration of PEA from the color developed by the detected chelating reagent. Determining the concentration of PEA from color development may be performed by any known means and method. For example, the concentration of PEA can be determined using a calibration curve between the absorbance of color development by the chelating reagent and the PEA concentration.
　別の実施形態では、段階（ｂ）、（ｃ）、（ｄ）および／または（ｅ）が同時に行われる。すなわち、各段階で必要な酵素、組成物、試薬、および／またはキットを同時に用いてもよい。段階（ｂ）、（ｃ）、（ｄ）および／または（ｅ）を同時に行うことで、サンプルのロスを減らすことができ、より正確な測定が可能となる。 In another embodiment, steps (b), (c), (d) and / or (e) are performed simultaneously. That is, enzymes, compositions, reagents, and / or kits required in each step may be used simultaneously. By performing steps (b), (c), (d) and / or (e) simultaneously, sample loss can be reduced and more accurate measurements can be made.
　上記式（１）に示されるとおり、加水分解されるＰＥＡと、生成されるアセトアルデヒド、アンモニア、およびリン酸の比率は、ＰＥＡ：アセトアルデヒド：アンモニア：リン酸＝１：１：１：１である。このことから、生成されたリン酸の量を測定することで、元のサンプル中に存在していたＰＥＡの量を測定することができる。 と お り As shown in the above formula (1), the ratio of PEA to be hydrolyzed to acetaldehyde, ammonia, and phosphoric acid to be produced is PEA: acetaldehyde: ammonia: phosphoric acid = 1: 1: 1: 1. From this, by measuring the amount of generated phosphoric acid, the amount of PEA existing in the original sample can be measured.
　さらに、段階（ａ）では、サンプルから段階（ｃ）によって生成される各物質を除去する段階を含むことができる。 段 階 Furthermore, the step (a) may include a step of removing each substance generated in the step (c) from the sample.
　例えば、段階（ｃ）として上記式（２）または式（３）で示される反応が利用される場合、段階（ａ）では、サンプルから上記式（２）または式（３）で示される反応によって生成されるヒポキサンチン、尿酸および／または過酸化水素を除去する段階を含むことができる。例えば、段階（ｃ）として上記式（２）または式（３）で示される反応が利用される場合、段階（ａ）では、あらゆる生物由来の、イノシン、プリンヌクレオチドホスホリラーゼ、キサンチン脱水素酵素（キサンチンオキシダーゼ）、ウリカーゼおよび／またはカタラーゼを使用することができる。図１に示されているように、例えば、過酸化水素は、カタラーゼで除去することができる。 For example, when the reaction represented by the above formula (2) or (3) is used as the step (c), in the step (a), the sample is subjected to the reaction represented by the above formula (2) or the formula (3) from the sample. It may include the step of removing hypoxanthine, uric acid and / or hydrogen peroxide formed. For example, when the reaction represented by the above formula (2) or (3) is used as step (c), in step (a), inosine, purine nucleotide phosphorylase, xanthine dehydrogenase (xanthine) Oxidase), uricase and / or catalase can be used. As shown in FIG. 1, for example, hydrogen peroxide can be removed with catalase.
　別の実施形態では、段階（ａ）の前に、サンプルに上記式（１）で示される反応を触媒する任意の酵素、例えばＰＥＡリアーゼまたはγ－アミノブチルアミノ基転移酵素（ＧａｂＴ）を添加し、その酵素反応を実施する段階がさらに含まれてもよい。 In another embodiment, prior to step (a), the sample is supplemented with any enzyme that catalyzes the reaction of formula (1) above, such as PEA lyase or γ-aminobutylaminotransferase (GabT). And performing the enzymatic reaction may further be included.
　別の実施態様では、本発明は、対象がうつ病に罹患しているかどうかを判定するための方法に関する。当該方法は、（ｉ）本発明のＰＥＡを測定する方法により、サンプル中のＰＥＡを測定する段階；および（ｉｉ）サンプル中のＰＥＡが１．５μｍｏｌ／Ｌ未満である場合に、対象がうつ病に罹患していると判定する段階を含む。 In another embodiment, the present invention relates to a method for determining whether a subject has depression. The method comprises the steps of (i) measuring PEA in a sample by the method of measuring PEA of the present invention; and (ii) treating the subject with depression when the PEA in the sample is less than 1.5 μmol / L. And determining that the subject is affected.
　上述した本発明の方法で用いられるサンプルは、ＰＥＡを含む、または含んでいる可能性がある任意のサンプルである。かかるサンプルの例としては、全血、血清、血漿、組織、脳髄液、および尿などの生体試料サンプルが挙げられるが、これらに限定されない。一実施形態では、本発明で用いられるサンプルは全血、血清、または血漿サンプルであり、好ましくは血漿サンプルである。 The sample used in the method of the present invention described above is any sample containing or possibly containing PEA. Examples of such samples include, but are not limited to, biological samples such as whole blood, serum, plasma, tissue, cerebrospinal fluid, and urine. In one embodiment, the sample used in the present invention is a whole blood, serum, or plasma sample, preferably a plasma sample.
　他の実施形態では、本発明で用いられるサンプルは、後続の段階に適した状態にするために、事前に処理されていてもよい。例えば、本発明で用いられるサンプルは、抗凝固剤で事前に処理された全血または血漿サンプルである。抗凝固剤の例としては、ＥＤＴＡ、クエン酸、シュウ酸、フッ化ナトリウム、ヘパリンが挙げられるが、これらに限定されない。 In other embodiments, the samples used in the present invention may have been pre-processed to make them suitable for subsequent stages. For example, the sample used in the present invention is a whole blood or plasma sample that has been pretreated with an anticoagulant. Examples of anticoagulants include, but are not limited to, EDTA, citric acid, oxalic acid, sodium fluoride, heparin.
　別の実施態様では、本発明は、上述した本発明の方法を実施するためのキットに関する。一実施形態では、本発明のキットは、リン酸を除去する手段を含む。この手段の例として、本発明のキットは、Ｏ－ホスホエタノールアミンホスホリアーゼを含む。他の実施形態では、本発明のキットは、さらに金属を含む。例えば、金属は三価の鉄である。他の実施形態では、本発明のキットは、さらにクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）、およびNitroso-PSAP、Nitro-PAPS、5-Br-PAPSまたは5-Br-PSAAを含む。 In another embodiment, the present invention relates to a kit for performing the above-described method of the present invention. In one embodiment, the kit of the invention comprises a means for removing phosphate. As an example of this means, the kit of the present invention comprises O-phosphoethanolamine phosphorylase. In another embodiment, the kit of the invention further comprises a metal. For example, the metal is trivalent iron. In another embodiment, the kit of the invention further comprises citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA), and Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5- Contains Br-PSAA.
　本発明のキットは、本発明の方法に用いられる任意の試薬を含んでもよい。また、測定の実施を簡便にするための適当な試薬、例えばサンプル希釈液、反応希釈液、バッファー、洗浄剤などを含んでもよい。通常、試薬は適切な容器に入れて提供される。またさらに、本発明の方法の実行に必要な説明書などの資材を含んでもよい。 キ ッ ト The kit of the present invention may contain any reagent used in the method of the present invention. It may also contain appropriate reagents for simplifying the measurement, such as sample diluents, reaction diluents, buffers, detergents, and the like. Usually, the reagent is provided in a suitable container. Still further, it may include materials such as instructions necessary for performing the method of the present invention.
　一実施形態では、本発明のキットは、研究専用（Ｒｅｓｅａｒｃｈ　Ｕｓｅ　Ｏｎｌｙ：ＲＵＯ）キットである。他の実施形態では、本発明のキットは、調査専用（Ｉｎｖｅｓｔｉｇａｔｉｏｎａｌ　Ｕｓｅ　Ｏｎｌｙ：ＩＵＯ）キットである。別の実施形態では、本発明のキットは、臨床診断キットである。 In one embodiment, the kit of the present invention is a Research Only Use Only (RUO) kit. In another embodiment, the kit of the present invention is an Investigational Use Only (IUO) kit. In another embodiment, the kit of the present invention is a clinical diagnostic kit.
　別の実施態様では、本発明は、コンピュータに、上述した本発明の方法を実行させるためのプログラムに関する。本発明のプログラムは、コンピュータで読み取り可能な記録媒体に記録させてもよく、装置に付属するコンピュータの記録媒体に記録してもよい。記録媒体の例としては、ハードディスク、ＣＤ、ＤＶＤ、ＵＳＢメモリ、およびフロッピー（登録商標）ディスクが挙げられるが、これらに限定されない。 In another embodiment, 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 recording medium of a computer attached to the device. 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.
　別の実施態様では、ＰＥＡを測定するための装置に関する。一実施形態では、本発明の装置は上述した本発明のキットを含む。別の実施形態では、本発明の装置に上述した本発明のプログラムが組み込まれている。 In another embodiment, the invention relates to an apparatus for measuring PEA. In one embodiment, the device of the invention comprises a kit of the invention as described above. In another embodiment, the above-described program of the present invention is incorporated in the device of the present invention.
　以下に実施例を示して本発明をさらに具体的かつ詳細に説明するが、本発明の範囲がこれらの実施例に限定されると解すべきではない。 The present invention will be described more specifically and in detail with reference to examples below, but it should not be understood that the scope of the present invention is limited to these examples.
リン酸除去
　サンプルとして５μＬの血漿を用いた。サンプルに以下の表１に記載の１２０μＬの第一試薬を加え、サンプル中のリン酸、ヒポキサンチン、尿酸および過酸化水素を除去し、次に３７℃で５分後に、日立7180自動分析装置（製造業者：日立ハイテクノロジース）を使用して５７０ｎｍで吸光度（吸光度１）を測定した。
Figure JPOXMLDOC01-appb-T000004
 Phosphate removal 5 μL of plasma was used as a sample. 120 μL of the first reagent described in Table 1 below was added to the sample to remove phosphoric acid, hypoxanthine, uric acid and hydrogen peroxide in the sample, and then after 5 minutes at 37 ° C., a Hitachi 7180 automatic analyzer ( The absorbance was measured at 570 nm (manufacturer: Hitachi High-Technologies) (absorbance 1).
Figure JPOXMLDOC01-appb-T000004
ＰＥＡからのアンモニア、アセトアルデヒドおよびリン酸の生成、リン酸からＮＡＤＨ２の生成、およびキレート試薬による発色の検出
　リン酸、ヒポキサンチン、尿酸および過酸化水素除去後の第一試薬が混合されたサンプルに、以下の表２に記載の６０μＬの第二試薬を加えた。次に３７℃で５分後に、日立7180自動分析装置（製造業者：日立ハイテクノロジース）を使用して５７０ｎｍで吸光度（吸光度２）を測定した。
Figure JPOXMLDOC01-appb-T000005
 Production of ammonia, acetaldehyde and phosphoric acid from PEA, production of NADH 2 from phosphoric acid, and detection of color development with a chelating reagent Phosphoric acid, hypoxanthine, uric acid and a sample mixed with the first reagent after removal of hydrogen peroxide 60 μL of the second reagent described in Table 2 below was added. Next, after 5 minutes at 37 ° C., the absorbance (absorbance 2) was measured at 570 nm using a Hitachi 7180 automatic analyzer (manufacturer: Hitachi High-Technologies).
Figure JPOXMLDOC01-appb-T000005
ＰＥＡ濃度の測定
　０．６２５、３．２５、７．５０、１５．０、３０．０および６０．０μｍｏｌ／Ｌ濃度のＰＥＡを用いて、吸光度とＰＥＡ濃度との検量線を図３として作成した。得られた吸光度２から得られた吸光度１を容量補正して差し引き、酵素反応による吸光度の変化から図３を使用してＰＥＡ濃度を換算した。結果を以下の表３に示す。
Figure JPOXMLDOC01-appb-T000006
 Measurement of PEA Concentration Calibration curves of absorbance and PEA concentration were prepared as FIG. 3 using PEAs having concentrations of 0.625, 3.25, 7.50, 15.0, 30.0, and 60.0 μmol / L. . The absorbance 1 obtained from the obtained absorbance 2 was volume-corrected and subtracted, and the PEA concentration was converted from the change in absorbance due to the enzyme reaction using FIG. The results are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000006
安定度定数
　鉄三価イオン(Fe(III))は、鉄二価イオン(Fe(II))よりも、クエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）と強固に配位する。鉄三価イオンは、アルカリ性で水酸化鉄となり、ＮＡＤＨ２による還元を妨げ得る。しかし、ＮＡＤＨで還元されて鉄二価イオンになると安定度定数は低下するため、Ｎｉｔｒｏｓｏ－ＰＳＡＰやＮｉｔｒｏ－ＰＡＰＳと配位子置換反応を起こして発色する。測定されたクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）での安定度定数比の結果を以下の表４に示す。
Figure JPOXMLDOC01-appb-T000007
　安定度定数の二価と三価の比が、０．４７～０．５６で最もＮＡＤＨ２の還元力が伝達される。 Stability constant Iron trivalent ion (Fe (III)) coordinates more strongly with citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA) than iron divalent ion (Fe (II)) I do. Iron trivalent ions, become iron hydroxide in an alkaline, may interfere with reduction by NADH 2. However, when reduced by NADH to form iron divalent ions, the stability constant decreases, so that a ligand substitution reaction occurs with Nitroso-PSAP or Nitro-PAPS to develop color. The results of the measured stability constant ratios for citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid, or nitrilotriacetic acid (NTA) are shown in Table 4 below.
Figure JPOXMLDOC01-appb-T000007
When the ratio between the divalent and trivalent stability constants is 0.47 to 0.56, the reducing power of NADH 2 is transmitted most.
様々な条件下での、キレート試薬による作用および発色
　様々な条件下での、鉄三価イオンの保護キレート試薬として上記第二試薬中のＬ－酒石酸の代わりにクエン酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）を用いて、ＮＡＤＨ２による鉄二価イオンへの還元能力を実施例２と同様に、５７０ｎｍで吸光度（吸光度２）を測定した。次に、実施例３と同様に、ＰＥＡ濃度を換算した。結果を以下の表５に示す。
Figure JPOXMLDOC01-appb-T000008
 Action and color development by chelating reagents under various conditions Citric acid, oxalic acid, sulfosalicylic acid or nitrilo acid in place of L-tartaric acid in the second reagent as a protective chelating agent for iron trivalent ions under various conditions Using triacetic acid (NTA), the absorbance at 570 nm (absorbance 2) was measured for the ability of NADH 2 to reduce iron divalent ions in the same manner as in Example 2. Next, as in Example 3, the PEA concentration was converted. The results are shown in Table 5 below.
Figure JPOXMLDOC01-appb-T000008

Claims (17)

  1. 　サンプル中のエタノールアミンリン酸（ＰＥＡ）を測定する方法であって、
    　（ａ）サンプル中のリン酸を除去する段階、
    　（ｂ）段階（ａ）で得られたサンプルにＯ－ホスホエタノールアミンホスホリアーゼ（Phosphoethanolamine Phospho-Lyase）を作用させて、サンプルに含まれるＰＥＡからアンモニア、アセトアルデヒドおよびリン酸を生成する段階、
    　（ｃ）段階（ｂ）で得られたサンプルに酵素を作用させて、サンプルに含まれるリン酸からＮＡＤＨ２を生成する段階、
    　（ｄ）段階（ｃ）で得られたサンプルに金属イオンおよび当該金属イオンの還元イオンと錯体を形成することができるキレート試薬を作用させる段階、および
    　（ｅ）段階（ｄ）で得られたサンプル中のキレート試薬による発色を検出する段階
    を含む、方法。     A method for measuring ethanolamine phosphate (PEA) in a sample, comprising:
    (A) removing phosphoric acid in the sample;
    (B) reacting the sample obtained in step (a) with O-phosphoethanolamine Phospho-Lyase to produce ammonia, acetaldehyde and phosphoric acid from PEA contained in the sample;
    (C) reacting the sample obtained in step (b) with an enzyme to generate NADH 2 from phosphoric acid contained in the sample;
    (D) reacting the sample obtained in step (c) with a metal ion and a chelating reagent capable of forming a complex with a reduced ion of the metal ion; and (e) the sample obtained in step (d). Detecting color development by a chelating reagent therein.
  2. 　段階（ｄ）において、金属イオンが三価の鉄（Ｆｅ３＋）イオンであり、金属イオンの還元イオンが二価の鉄（Ｆｅ２＋）イオンである、請求項１に記載の方法。 The method according to claim 1, wherein in step (d), the metal ion is trivalent iron (Fe3 + ) ion and the reduced ion of the metal ion is divalent iron (Fe2 + ) ion.
  3. 　段階（ｄ）がクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる、請求項１または２に記載の方法。 方法 The method according to claim 1 or 2, wherein step (d) is performed under conditions comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA).
  4. 　段階（ｄ）がＬ－酒石酸またはニトリロ三酢酸（ＮＴＡ）を含む条件下で行われる、請求項１～３のいずれかに記載の方法。 方法 The method according to any one of claims 1 to 3, wherein step (d) is performed under conditions comprising L-tartaric acid or nitrilotriacetic acid (NTA).
  5. 　段階（ｄ）において、キレート試薬がNitroso-PSAP、Nitro-PAPS、5-Br-PAPSまたは5-Br-PSAAである、請求項１～４のいずれかに記載の方法。 方法 The method according to any one of claims 1 to 4, wherein in step (d), the chelating reagent is Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5-Br-PSAA.
  6. 　段階（ｃ）が、
    　（ｃ－１）リン酸にイノシンおよびプリンヌクレオチドホスホリラーゼを作用させて、ヒポキサンチンを生成する段階、
    　（ｃ－２）ヒポキサンチンにキサンチン脱水素酵素を作用させて、尿酸を生成する段階、
    　（ｃ－３）尿酸にウリカーゼを作用させて、過酸化水素を生成する段階、
    　（ｃ－４）過酸化水素にメタノールまたはエタノールおよびカタラーゼを作用させて、ホルムアルデヒドまたはアセトアルデヒドを生成する段階、および
    　（ｃ－５）ホルムアルデヒドまたはアセトアルデヒドにＮＡＤおよびホルムアルデヒド脱水素酵素を作用させて、ＮＡＤＨ２を生成する段階
    を含む、請求項１～５のいずれかに記載の方法。     Step (c) comprises:
    (C-1) reacting inosine and purine nucleotide phosphorylase with phosphate to produce hypoxanthine;
    (C-2) producing xanthine dehydrogenase on hypoxanthine to produce uric acid;
    (C-3) a step of causing uricase to act on uric acid to generate hydrogen peroxide;
    (C-4) reacting hydrogen peroxide with methanol or ethanol and catalase to produce formaldehyde or acetaldehyde; and (c-5) reacting formaldehyde or acetaldehyde with NAD and formaldehyde dehydrogenase to form NADH 2 A method according to any of the preceding claims, comprising the step of:
  7. 　段階（ａ）が、サンプル中のリン酸、ヒポキサンチン、尿酸および過酸化水素を酵素で除去する段階である、請求項１～６のいずれかに記載の方法。 (7) The method according to any one of (1) to (6), wherein step (a) is a step of removing phosphoric acid, hypoxanthine, uric acid and hydrogen peroxide in the sample with an enzyme.
  8. 　サンプルが生体試料サンプルである、請求項１～７のいずれかに記載の方法。 The method according to any one of claims 1 to 7, wherein the sample is a biological sample.
  9. 　サンプルが、全血、血清、または血漿サンプルである、請求項８に記載の方法。 9. The method of claim 8, wherein the sample is a whole blood, serum, or plasma sample.
  10. 　対象がうつ病に罹患しているかどうかを判定するための方法であって、
    　（ａ）請求項１～９のいずれかに記載の方法により、対象由来のサンプル中のＰＥＡを測定する段階；および
    　（ｂ）サンプル中のＰＥＡが１．５μｍｏｌ／Ｌ未満である場合に、対象がうつ病に罹患していると判定する段階
    を含む、方法。     A method for determining whether a subject is suffering from depression, comprising:
    (A) measuring PEA in a sample derived from the subject by the method according to any one of claims 1 to 9; and (b) controlling the PEA in the sample when the PEA in the sample is less than 1.5 μmol / L. Determining that the subject is suffering from depression.
  11. 　Ｏ－ホスホエタノールアミンホスホリアーゼを含む、請求項１～１０のいずれかに記載の方法を実施するためのキット。 A kit for performing the method according to any one of claims 1 to 10, comprising O-phosphoethanolamine phosphorylase.
  12. 　さらに金属を含む、請求項１１に記載のキット。 The kit according to claim 11, further comprising a metal.
  13. 　金属が三価の鉄である、請求項１２に記載のキット。 The kit according to claim 12, wherein the metal is trivalent iron.
  14. 　さらにクエン酸、Ｌ－酒石酸、シュウ酸、スルホサリチル酸またはニトリロ三酢酸（ＮＴＡ）、およびNitroso-PSAP、Nitro-PAPS、5-Br-PAPSまたは5-Br-PSAAを含む、請求項１１～１３のいずれかに記載のキット。 Claims 11 to 13 further comprising citric acid, L-tartaric acid, oxalic acid, sulfosalicylic acid or nitrilotriacetic acid (NTA), and Nitroso-PSAP, Nitro-PAPS, 5-Br-PAPS or 5-Br-PSAA. The kit according to any one of the above.
  15. 　コンピュータに、請求項１～１０のいずれかに記載の方法を実行させるためのプログラム。 A program for causing a computer to execute the method according to any one of claims 1 to 10.
  16. 　請求項１５に記載のプログラムを保存したコンピュータで読み取り可能な記録媒体。 A computer-readable recording medium storing the program according to claim 15.
  17. 　請求項１１～１４のいずれかに記載のキットを含む、または請求項１５に記載のプログラムが組み込まれた、ＰＥＡを測定するための装置。 (4) An apparatus for measuring PEA, comprising the kit according to any one of (11) to (14), or incorporating the program according to (15).
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KAWANO MIKI: "Development of the highly sensitive assay of ethanol phosphoric acid which is a depression marker.", vol. 66, 2017 *
KONO MIKI: "Development of high-sensitive measurement of ethanolamine phosphate", JAPANESE JOURNAL OF CLINICAL LABORATORY AUTOMATION, vol. 43, 1 September 2018 (2018-09-01) *

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