JPWO2018062204A1 - Method for measuring ethanolamine phosphate - Google Patents

Abstract

The present invention is a method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity, wherein (a) removing ammonia and / or phosphate in the sample, (b) in step (a) A step of adding an enzyme to the obtained sample to produce ammonia and / or phosphoric acid from PEA contained in the sample; (c) an ammonia detection composition and / or phosphorous in the sample obtained in step (b); A method is provided comprising adding an acid detection composition, and (d) detecting ammonia and / or phosphoric acid in the sample obtained in step (c). In addition, a kit, a program, and an apparatus for carrying out the method are provided.

Description

  The present invention relates to a method for measuring ethanolamine phosphate (PEA) present in a sample.

  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. However, 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.

  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). The present inventors have found that ethanolamine phosphate (PEA) in blood is useful as a biomarker for diagnosing depression (Patent Documents 3 and 4). The present inventors further developed a method for measuring PEA in a sample by generating acetaldehyde, phosphoric acid, and ammonia from PEA using γ-aminobutylaminotransferase (GabT) (Patent Literature). 5).

International Publication No. 2006/105907 JP 2008-253258 A International Publication No. 2011/019072 International Publication No. 2016/047677 Japanese Patent No. 5688163 JP 2003-334099 A Japanese Patent No. 4127767 Japanese Patent No. 5327578 Japanese Patent No. 5458487

  Based on the results of previous studies, 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 μM (Patent Document 4). ). However, the conventional PEA measurement method (Patent Document 5) cannot measure a value of less than 1.5 μM. In addition, 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.

  Therefore, the 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.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have established a method for measuring PEA with higher sensitivity than the conventional method for measuring PEA, and completed the present invention.

That is, 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). Or 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 γ-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. coli (SEQ ID NO: 3),
(Iv) GXXXBADEBQXGFZRXG [where X is any amino acid, B is isoleucine (I), leucine (L) or valine (V), and Z is glycine (G) or alanine (A)] ( An enzyme comprising the amino acid sequence defined by SEQ ID NO: 4),
(V) the tag or the enzyme or protein according to any one of (iv) and (vi) the enzyme or protein variant according to any one of (ii) to (iv) The method according to [3], which is one or more enzymes selected from the group;
[5] The method according to any one of [1] to [4], wherein in step (a), ammonia is removed using a resin;
[6] The method according to any one of [1] to [5], wherein the sample is a biological sample;
[7] The method according to [6], wherein the sample is a whole blood, serum, or plasma sample;
[8] The sample is a whole blood or plasma sample that has been pretreated with one or more anticoagulants selected from the group consisting of EDTA, citric acid, oxalic acid, sodium fluoride (NaF), and heparin. [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 from ammonia and / or phosphoric acid. the method of;
[10] A method for determining whether a subject is suffering from depression, comprising:
(A) a step of 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 μM. Determining that the subject is suffering from depression;
[11] A kit for carrying out the method according to any one of [1] to [10], comprising means for removing ammonia and / or means for removing phosphoric acid;
[12] A kit for carrying out the method according to any one of [1] to [10], comprising an ammonia detection composition and / or a phosphate detection composition;
[13] A program for causing a computer to execute the method according to any one of [1] to [10];
[14] A computer-readable recording medium storing the program according to [13]; and a kit according to [15] [11] or [12] or including the program according to [13] A device for measuring PEA.

  According to the present invention, a method for measuring PEA in a sample with high sensitivity, and a kit, a program, an apparatus, and the like for carrying out the method are provided. 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. In FIG. 1, reagent 1 is a reagent containing an enzyme that converts PEA into ammonia, reagent 2 is an enzyme cycling solution, and 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. 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.

  In one embodiment, 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.

  As used herein, the term “highly sensitive” refers to a lower limit of quantification than a conventional PEA measurement method (Patent Document 5). Specifically, it means that PEA of less than 1.5 μM can be quantified.

  As used herein, “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).

In one embodiment, the method for measuring PEA of the present invention utilizes a hydrolysis reaction represented by the following formula (1).

  In step (1a), any of the known means and methods for removing ammonia may be used. In one embodiment, ammonia is removed by the resin in step (1a). Examples of such resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical), Dowex 50Wx8 200-400 mesh strong acid cation exchange resin (H form). Alternatively, ammonia may be removed by enzymatic treatment in step (1a). Examples of such 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.

  When the reaction represented by the above formula (1) is used, in the step (1b), 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 γ-aminobutylaminotransferase (GabT).

In one embodiment, in step (1b), a human-derived ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1), a protein having a GabT domain from Pantoea ananatis (SEQ ID NO: 2), and E. coli One or more enzymes selected from the group consisting of a protein having a GabT domain (SEQ ID NO: 3) are used. Alternatively, 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. For example, it 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. Alternatively, a protein that catalyzes the reaction represented by the above formula (1) and includes an amino acid sequence in which one or several amino acids in the amino acid sequence of SEQ ID NO: 2 or 3 are substituted, deleted, and / or added. May be used. Or you may use the protein containing the amino acid sequence by which the tag was added to the amino acid sequence in any one of sequence number 1-3. Exemplary tags include, but are not limited to, His ₆ -tags, FLAG tags, myc tags, and GST tags. The tag may be added to the N terminus of the amino acid sequence or may be added to the C terminus. Regarding human-derived AGXT2L1, IUBMB Life. 2013 Jul; 65 (7): 645-50.

  In another embodiment, in step (1b), 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. In another embodiment, 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. In still another embodiment, 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.

  In a preferred embodiment, the enzyme used in step (1b) is a human ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1) or a protein having a GabT domain from Pantoea ananatis (SEQ ID NO: 2). is there. In a more preferred embodiment, the enzyme used in step (1b) is human-derived ethanolamine phosphate phospholyase (AGXT2L1) (SEQ ID NO: 1).

  In another embodiment, prior to step (1a), any enzyme that catalyzes the reaction of formula (1) above, such as PEA lyase or γ-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. For example, 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.).

上記の試薬ＩおよびＩＩのｐＨは、適切なバッファーを用いて調整される。ｐＨ調整のために用いられるバッファーには、例えばリン酸カリウムやＴｒｉｓ−ＨＣｌが挙げられるが、これに限定されない。上記の試薬は、例えば塩化ナトリウムなどの塩をさらに含んでもよい。Or you may use the improvement of said commercial item. For example, 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.

  In another embodiment, 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.

  As shown in the above formula (1), the ratio of PEA to be hydrolyzed and acetaldehyde, ammonia and phosphoric acid produced is PEA: acetaldehyde: ammonia: phosphoric acid = 1: 1: 1: 1. From this, the amount of PEA present in the original sample can be measured by measuring the amount of acetaldehyde, ammonia, or phosphoric acid produced. Therefore, in the method for measuring PEA of the present invention in which the reaction represented by the above formula (1) is used, acetaldehyde or phosphoric acid may be employed together with ammonia or instead of ammonia.

  When phosphoric acid is used as the measurement object, any known means and method for removing phosphoric acid may be used in step (1a). Examples of methods for removing phosphoric acid include, but are not limited to, enzymatic methods and precipitation methods.

  In the enzyme method, for example, purine nucleotide phosphorylase (PNP) is used. When 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 the removal of phosphoric acid. In this case, a PNP inhibitor may be added to stop the action of PNP after the reaction. Alternatively, sucrose phosphorylase (SP) may be used. When sucrose and SP are added to a sample containing phosphoric acid, phosphoric acid reacts with sucrose by the action of SP, α-glucose 1-phosphate (α-Glc-1-P and fructose are generated, and phosphoric acid is removed. In this case, in order to stop the action of SP after the reaction, a buffer having a pH of about 9 to about 10 (for example, CHES buffer) or an SP inhibitor such as fructose may be added.

In the precipitation method, rare earth metal ions or alkaline earth metal ions (M ^{X +} ) are added to a sample containing phosphoric acid. Thereby, a hardly soluble phosphate (MPO ₄ ) is generated, and phosphoric acid can be removed. After adding M ^{X +} , the pH of the solution may be made higher than 9, or carbonate ions (CO ₃ ²⁻ ) may be added to remove excess metal. Examples of the reagent used for the precipitation method include water-soluble calcium salts such as CaCl ₂ .

  In one embodiment, an enzymatic method and a precipitation method are used in combination. By using these methods in combination, phosphoric acid can be removed more reliably.

  When using phosphoric acid as the measurement object, in the step (1c), 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. For example, 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. About the measurement of phosphoric acid, it is described in the patent 5327578 specification (patent document 8) and the patent 5458487 specification (patent document 9), for example.

In another embodiment, PEA may be measured using a hydrolysis reaction represented by the following formula (2).

  When the reaction represented by the above formula (2) is used, the present invention is a method for measuring ethanolamine phosphate (PEA) in a sample with high sensitivity, and (2a) removing ethanolamine in a sample. (2b) adding an enzyme to the sample obtained in step (2a) to produce ethanolamine from PEA contained in the 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). Alternatively, in the method, phosphoric acid may be removed and detected together with ethanolamine or instead of ethanolamine.

  In step (2a), any of the known means and methods for removing ethanolamine may be used. In one embodiment, ethanolamine is removed by the resin in step (2a). Examples of such resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical). Alternatively, ethanolamine may be removed by enzymatic treatment in step (2a).

  When the reaction represented by the above formula (2) is used, any enzyme that catalyzes the reaction represented by the above formula (2) is used in the step (2b). Examples of such enzymes include, but are not limited to alkaline phosphatase. In one embodiment, in step (2b), alkaline phosphatase or a variant thereof is used. In a preferred embodiment, the enzyme used in step (2b) is alkaline phosphatase.

  In another embodiment, prior to step (2a), 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. For example, a commercially available composition, reagent, or kit for detecting ethanolamine may be used.

  In another embodiment, 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.

  As shown in the above formula (2), the ratio of PEA to be hydrolyzed and ethanolamine and phosphoric acid produced is PEA: ethanolamine: phosphoric acid = 1: 1: 1. From this, the amount of PEA present in the original sample can be measured by measuring the amount of ethanolamine or phosphoric acid produced. Therefore, in the method for measuring PEA of the present invention in which the reaction represented by the above formula (2) is used, phosphoric acid may be employed together with ethanolamine or instead of ethanolamine. In that case, in the step (2a), any of the known means and methods for removing phosphoric acid as described above may be used. In step (2c), any composition, reagent, and kit for detecting phosphoric acid present in the sample are used as the phosphoric acid detection composition. For example, a commercially available phosphoric acid detection composition, reagent, or kit may be used. An exemplary phosphate detection reagent is a malachite green (MG) reagent.

  As described above, the method for measuring PEA of the present invention includes the 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.

  In another embodiment, the present invention provides a method for measuring PEA with high sensitivity by using instrumental analysis. In one embodiment, PEA is measured by quantification by post-column fluorescence derivatization of PEA utilizing ion exchange chromatography (IC) and a fluorescence detector (FLD). In other embodiments, 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 where contact with the atmosphere is blocked. For example, the contact between the reaction system and the atmosphere may be blocked by forming a film on the reaction solution. Examples of the substance used for forming the coating include colorless, non-volatile, hydrophobic, and non-polar substances. For example, oil or an organic solvent is used. Examples of the oil used include mineral oil and plant-derived oil, but are not limited thereto. Examples of the organic solvent used include octanol and dodecanol, but are not limited thereto.

Or you may implement the method of measuring PEA of this invention under pressure reduction or a vacuum. “Reduced pressure” means a pressure lower than normal pressure. For example, 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 higher, 9.86 × 10 ^{−4 to} 9.99 × 10 ⁻¹ atm, 7.0 × 10 ^{−4 to} 9.99 × 10 ⁻¹ atm.

  Alternatively, the atmosphere in the environment (eg, 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. .

  When the reaction represented by the above formula (1) is utilized, in one embodiment, in an environment in which at least one of steps (1b) to (1d) is 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.

  When the reaction represented by the above formula (2) is utilized, in one embodiment, at least one of steps (2b) to (2d) is performed in an environment substantially free of ethanolamine and / or phosphoric acid. The In other embodiments, at least two of steps (2b)-(2d) are performed in such an environment. In another embodiment, steps (2b)-(2d) are all performed in such an environment.

  In another embodiment, the invention relates to a method for determining whether a subject is suffering from depression. The method includes (i) a step of measuring PEA in a sample by a method for 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 μM, Determining that the subject is suffering from depression.

  The sample used in the method of the present invention described above is any sample that contains or may contain PEA. Examples of such samples include, but are not limited to, biological sample 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.

  In other embodiments, the samples used in the present invention may be 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 pre-treated with an anticoagulant. Examples of anticoagulants include, but are not limited to, EDTA, citric acid, oxalic acid, sodium fluoride, heparin.

  In another embodiment, the present invention relates to a kit for performing the method of the present invention described above. In one embodiment, the kit of the invention includes a means for removing ammonia. For example, a resin used to remove ammonia is included. Examples of such resins include, but are not limited to, cation exchange resins such as MCI GEL AFR2 (Mitsubishi Chemical), Dowex 50Wx8 200-400 mesh strong acid cation exchange resin (H form). Alternatively, 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.

  In other embodiments, the kit of the present invention includes a means for removing acetaldehyde, phosphoric acid, and / or ethanolamine in conjunction with, or in place of, means for removing ammonia. For example, a resin used to remove acetaldehyde, phosphate, or ethanolamine, or an enzyme that converts acetaldehyde, phosphate, or ethanolamine to another substance.

In another embodiment, the kit of the present invention comprises an ammonia detection composition. In this case, any composition, reagent, and / or kit for detecting ammonia present in the sample is included. For example, commercially available ammonia detection compositions, reagents, or kits are included. 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.).

  Or you may use the improvement of said commercial item. For example, a combination of reagent I and reagent II described above may be used (see Tables 1 and 2).

  In another embodiment, the kit of the present invention comprises an acetaldehyde, phosphoric acid, and / or ethanolamine detection composition together with or in place of the ammonia detection composition. For example, commercially available acetaldehyde, phosphoric acid, and / or ethanolamine detection compositions, reagents, or kits are included. Examples of 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. In addition, an appropriate reagent for simplifying the measurement, such as a sample diluent, a reaction diluent, a buffer, and a cleaning agent may be included. Usually, the reagent is provided in a suitable container. Furthermore, materials such as instructions necessary for carrying out the method of the present invention may be included.

  In one embodiment, the kit of the present invention is a Research Use 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.

  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 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.

  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 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.

  EXAMPLES The present invention will be described more specifically and in detail below with reference to examples. However, it should not be understood that the scope of the present invention is limited to these examples.

(I) PEA measurement method 1 using ammonia
1. Ammonia removal by cation exchange resin Plasma was used as a sample. The column was packed with cation exchange resin MCI GEL AFR2 (Mitsubishi Chemical Corporation, catalog number 1-033-01) and washed with 400 μL of ultrapure water. 50 μL 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.

2. Production of Ammonia from PEA To 50 μL of the sample after removal of ammonia, 50 μL of PEA enzyme solution (500 μg / mL) was added and mixed, and allowed to stand at 25 ° C. for 20 minutes. 100 μL of enzyme cycling solution (reagent A) was added and mixed, and allowed to stand at 25 ° C. for 5 minutes. 50 μL of a detection solution (reagent B) was added and mixed. The composition of Reagents A and B is as shown in the table below. Ammonia was produced from PEA in solution by the above procedure.

3. Measurement of PEA concentration Absorbance at 450 nm was measured 10 minutes and 12 minutes later to detect ammonia. The obtained result was converted into PEA concentration. In addition, the measurement using the instrument method (Thermo Fisher Scientific IC-FLD and Agilent Technologies CE-MS) was also performed. The equipment and equipment used are as follows: IC-FLD (ICS5000); column (AS20); CE (G1600); and MS (6460). According to the manufacturer's instructions, the peak of the standard substance (2-aminoethyl dihydrogen phosphate, SIGMA-ALDRICH, product number 292869) was compared with the peak of the sample. The results are shown in the table below.

(II) PEA measurement method 2 using ammonia
1. PEA removal in sample Plasma was used as a sample. To 100 μL of sample (1), 10 μL of 1M TAPS (pH 8.5) and 25 μL of ultrafiltered PEA enzyme solution (4 mg / mL) were added and mixed, and allowed to stand at 37 ° C. for 20 minutes. To 100 μL of sample (2), 10 μL of 1M TAPS (pH 8.5) and 25 μL of PEA enzyme solution (4 mg / mL) were added and mixed, and allowed to stand at 37 ° C. for 20 minutes. Thereafter, samples (1) and (2) were each subjected to ultrafiltration. The ultrafiltration conditions were as follows: exclusion limit 3 kDa, 14000 g, 4 ° C.

2. Ammonia removal by cation exchange resin Cation exchange resin MCI GEL AFR2 (Mitsubishi Chemical Corporation, catalog number 1-033-01) was packed in a column and washed with 400 μL of ultrapure water. 100 μL of each of samples (1) and (2) was packed into a washed column and passed through to recover the solution.

3. Production of ammonia from PEA Samples (1) and (2) after removal of ammonia were mixed with 67.5 μL of 12.5 μL of PEA enzyme solution (4 mg / mL) and 10 μL of 1M TAPS (pH 8.5). It was allowed to stand at 37 ° C. for 20 minutes. Ammonia was produced from PEA in solution by the above procedure.

４分後と６分後に４５０ｎｍの吸光度を測定し、アンモニアを検出した。サンプル（１）の結果からサンプル（２）の結果を差し引き、サンプル中のＰＥＡ濃度を求めた。なお、機器法（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製ＩＣ−ＦＬＤ）を用いる測定も行った。使用した機器および装置は以下のとおりである：ＩＣ−ＦＬＤ（ＩＣＳ５０００）；カラム（ＡＳ−２０）。結果を以下の表に示す。

4). Measurement of PEA concentration 100 μL of enzyme cycling solution (reagent A) and 100 μL of detection solution (reagent B) were added to and mixed with 50 μL of the enzyme reaction solution obtained above. The composition of Reagents A and B is as shown in the table below.

After 4 and 6 minutes, the absorbance at 450 nm was measured to detect ammonia. The result of sample (2) was subtracted from the result of sample (1) to determine the PEA concentration in the sample. In addition, the measurement using the instrument method (Thermo Fisher Scientific IC-FLD) was also performed. The equipment and equipment used are as follows: IC-FLD (ICS5000); column (AS-20). The results are shown in the table below.

(III) PEA measurement method using phosphoric acid Phosphate removal with calcium chloride (precipitation method)
Serum was used as a sample. After adding 20 μL of 1M calcium chloride to 180 μL of the sample and mixing, the solution was ultrafiltered.

2. Phosphate removal by sucrose phosphorylase (enzymatic method)
To 110 μL of the ultrafiltered sample, 10 μL of 500 mM MOPS (pH 7.5), 10 μL of 1M sucrose, and 10 μL of sucrose phosphorylase (200 U / mL) were added and mixed, and allowed to stand at 37 ° C. for 10 minutes. The enzyme reaction was stopped by adding 20 μL of 1 M CHES buffer (pH 9.0) to 140 μL of the obtained sample after removing phosphoric acid. Here, the result of separately measuring the residual ratio of phosphoric acid and PEA in the solution before adding the CHES buffer is shown in FIG. 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.

3. Production of phosphoric acid from PEA To 150 μL of the sample after addition of CHES buffer, 40 μL of PEA enzyme solution (2.5 mg / mL) was added and mixed, and allowed to stand at 37 ° C. for 20 minutes. The above procedure produced phosphoric acid from PEA in solution.

4). Measurement of PEA concentration To 160 μL of the enzyme reaction solution obtained above, 40 μL of Reagent MIX of Malachite Green Phosphate Assay Kit (Funakoshi Co., Ltd.) was added and mixed, and allowed to stand at room temperature for 20 minutes. Absorbance at 620 nm was measured to detect phosphoric acid. The PEA concentration determined from the detected phosphoric acid concentration was 2.32 μM. In addition, the measurement using the instrument method (Thermo Fisher Scientific IC-FLD) was also performed. The equipment and equipment used are as follows: IC-FLD (ICS5000); column (AS-20). The result by the instrument method was 2.33 μM.

(IV) Inhibitory effect of oil on permeation of atmospheric ammonia into sample Ultra pure water (for Wako Pure Chemical Industries, Ltd. LC / MS (CAS. No. 7732-18-5)) and human plasma (application) Prepared by human). Mineral oil was used as the oil. In a 96-well plate, a system sealed with mineral oil and a system not sealed with mineral oil were prepared. Experiments were performed under air. After the start of measurement, the ammonia concentration in the sample was measured every 2 minutes from 4 to 20 minutes. Ammonia was measured by measuring absorbance at 450 nm using Shikari Liquid NH ₃ (Kanto Chemical Co., Inc.).

  The results are shown in FIG. In both plasma and pure water samples, the ammonia concentration changed greatly with and without oil. From these results, the effect and significance of carrying out the method of the present invention in an “environment substantially free of ammonia, acetaldehyde, phosphoric acid, and / or ethanolamine” were confirmed.

  According to 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-phospho phospho-lyase
Sequence number 2: Ethanolamine phosphate catalytic enzyme
Sequence number 3: Ethanolamine phosphate catalytic enzyme
Sequence number 4: GabT domain

Claims (15)

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). Or a method comprising detecting phosphoric acid.   The method of claim 1, wherein steps (b) and (c) are performed simultaneously.   The method according to claim 1 or 2, wherein the enzyme is PEA lyase or γ-aminobutylaminotransferase (GabT). The enzyme
(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. coli (SEQ ID NO: 3),
(Iv) GXXXBADEBQXGFZRXG [where X is any amino acid, B is isoleucine (I), leucine (L) or valine (V), and Z is glycine (G) or alanine (A)] ( An enzyme comprising the amino acid sequence defined by SEQ ID NO: 4),
(V) the tag or the enzyme or protein according to any one of (iv) and (vi) the enzyme or protein variant according to any one of (ii) to (iv) 4. The method of claim 3, wherein the method is one or more enzymes selected from the group.   The process according to any one of claims 1 to 4, wherein in step (a) ammonia is removed using a resin.   The method according to claim 1, wherein the sample is a biological sample.   The method of claim 6, wherein the sample is a whole blood, serum, or plasma sample.   The sample is a whole blood or plasma sample pretreated with one or more anticoagulants selected from the group consisting of EDTA, citric acid, oxalic acid, sodium fluoride (NaF), and heparin. 6. The method according to 6.   The method according to any one of claims 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. A method for determining whether a subject has depression, comprising:
(A) a step of measuring PEA in the sample by the method according to any one of claims 1 to 9; and (b) when the measured value of PEA in the sample is less than 1.5 μM. Determining that is suffering from depression.   The kit for performing the method as described in any one of Claims 1-10 containing the means to remove ammonia, and / or the means to remove phosphoric acid.   The kit for performing the method as described in any one of Claims 1-10 containing the composition for ammonia detection and / or the composition for a phosphoric acid detection.   The program for making a computer perform the method as described in any one of Claims 1-10.   A computer-readable recording medium storing the program according to claim 13.   An apparatus for measuring PEA comprising the kit according to claim 11 or 12, or incorporating the program according to claim 13.

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