JP2005052034A - Method for analyzing ethanolamine-containing phospholipid and composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a reagent for analyzing an ethanolamine phospholipid by an enzymatic method. <P>SOLUTION: This method for analyzing the concentration of the ethanolamine phospholipid is characterized by using a reagent comprising at least phospholipase and an enzyme acting on ethanolamine. Thereby, the ethanolamine phospholipid can accurately and simply be determined at a low cost without needing a complicated operation. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for analyzing ethanolamine-containing phospholipids by an enzymatic method and an analytical reagent.

Ethanolamine-containing phospholipids, which are one type of phospholipid, are widely present in living bodies such as food materials and blood. The method of analyzing the phospholipid content and concentration is a method of separating the phospholipid in advance by thin layer chromatography operation using silica gel or alumina as a support for a long time, then coloring the phospholipid and measuring the concentration according to the degree, Although a method using an HPLC method and a measurement method using an enzyme are known (Non-patent Document 1), the color development reaction time using the enzyme is as long as 15 minutes, and it has been desired to shorten the time.
History of Medicine, Vol. 127, No. 2, 1983, pp. 114-120

  An object of the present invention is to provide an enzymatic analysis method for analyzing ethanolamine-containing phospholipids in a test solution easily and accurately in a short time, and an ethanolamine-containing phospholipid analysis reagent used in the method. .

  The present invention has been made in order to achieve the above object, and as a result of extensive studies by the present inventors, an enzyme that acts on phospholipase, which is a phospholipid hydrolase, and ethanolamine as a test solution. A simple method for analyzing glycated phospholipids was devised and the present invention was completed.

That is, the present invention
1) Analytical method of ethanolamine-containing phospholipid characterized by using at least phospholipase and tyramine oxidase 2) A test solution containing ethanolamine-containing phospholipid in the first reagent (R1) containing at least tyramine oxidase In addition, after the reaction is performed, the reaction is performed by adding the second reagent (R2) containing at least an enzyme that hydrolyzes the ethanolamine-containing phospholipid, and the reaction is performed 3) The method for analyzing an ethanolamine-containing phospholipid according to claim 1, wherein the enzyme that hydrolyzes the ethanolamine-containing phospholipid is phospholipase D. 4) The ethanolamine-containing phospholipid characterized by containing at least phospholipase and tyramine oxidase. 5) At least tyramine oxy A composition 6 for analyzing ethanolamine-containing phospholipids, comprising a first reagent (R1) containing a dase and a second reagent (R2) containing at least an enzyme that hydrolyzes ethanolamine-containing phospholipids 6 5. The composition for analyzing ethanolamine-containing phospholipid according to claim 4, wherein the enzyme that hydrolyzes ethanolamine-containing phospholipid is phospholipase D.

Hereinafter, preferred embodiments of the present invention will be described in more detail.
The phospholipase used in the present invention may be any enzyme that can hydrolyze ethanolamine-containing phospholipids. Phospholipases A, B, C, and D are known as enzymes that hydrolyze phospholipids, and these enzymes are widely present in nature such as animals and plants and microorganisms. Any of these enzymes can be used to measure ethanolamine-containing phospholipids, but the reagent raw material is preferably phospholipase C or phospholipase D, and particularly preferably phospholipase D. This enzyme is known to exist in plant tissues such as cabbage, peanuts, carrots, and microorganisms such as actinomycetes, and any of these enzymes can be used, but the stability of the enzyme itself, the stable supply of the enzyme From this aspect, an enzyme derived from a microorganism is preferred. Among them, phospholipase D derived from Streptomyces chromofuscus, which is one of actinomycetes, is particularly preferable because of its excellent reactivity and stability. This phospholipase D has a property of acting widely on phospholipids such as phosphatidylcholine (lecithin), lysolecithin, phosphatidylserine and the like other than ethanolamine-containing phospholipids, but there is no problem in analyzing ethanolamine-containing phospholipids. The concentration of the enzyme in the reagent is 0.05 to 100 units / ml, preferably 0.5 to 50 units / ml. The activity of the enzyme was defined as 1 unit of activity of decomposing the substrate phosphatidylcholine by 1 micromole per minute at 37 ° C.

  As an enzyme that acts on ethanolamine that can be used in the present invention, any enzyme that acts on ethanolamine may be used, and examples thereof include oxidase, kinase, dehydrogenase and the like. Among them, oxidase is well known and is preferable because it can be obtained at low cost because it is manufactured for industrial use. As an oxidase that acts on ethanolamine, any enzyme may be used as long as it works well on ethanolamine and generates hydrogen peroxide. An example of an enzyme that oxidizes ethanolamine is an enzyme derived from the genus Bacillus, but when this enzyme is used, a reaction time of at least 15 minutes is required (Medical History, Vol. 127, No. 2). 1984, pp. 114-120).

  Compared with this, tyramine oxidase derived from the genus Arthrobacter can efficiently perform the color reaction in 5 minutes. Furthermore, tyramine oxidase derived from Arthrobacter is particularly preferable from the viewpoints of enzyme stability and stable enzyme supply. The concentration in the reagent is 0.1 to 50 U / ml, preferably 1 to 20 units / ml. The activity of the enzyme acting on tyramine was defined as 1 unit of activity that oxidizes 1 micromole per minute at 37 ° C. using tyramine as a substrate. Moreover, tyramine oxidase, monoamine oxidase, and diamine oxidase can be used in combination for the purpose of eliminating monoamine and diamine contained in the test solution. Enzymes derived from porcine kidney, porcine plasma, bovine plasma, leguminous seedlings, and Aspergillus niger are known as enzymes that oxidize monoamines and diamines. Aspergillus niger derived enzymes are preferred.

  Next, an analysis reagent for ethanolamine-containing phospholipid will be described. In order to maintain the storage stability of the analytical reagent, the analytical reagent is divided into two types of reagents. In addition, since free amines may be present in biological components such as serum, there is a risk of causing a positive error. By adding the test solution to Reagent 1 (R1) containing ethanolamine oxidase (tyramine oxidase), catalase or peroxidase and a chromogen of a tender reagent such as a phenol derivative, aniline derivative, or toluidine derivative, and pre-heating. This component can be eliminated. Reagent 2 (R2) is composed of a component composed of a coupler such as phospholipase D, 4 aminoantipyrine, or 3-methyl-2-benzothiazolinone hydrazone (MBTH). Hydrogen peroxide produced by the ethanolamine oxidase reaction simultaneously with the mixing of R1 and R2 produces a dye by oxidative condensation between the chromogen of the Trinder reagent and the coupler. The absorbance of this dye can be measured spectroscopically.

  As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives, etc. can be used, and specific examples include N, N dimethylaniline, N, N diethylaniline, 2,4 dichlorophenol, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5 dimethylaniline (MAPS), N-ethyl-N- (2- Hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N-sulfopropyl -m- Anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxy Aniline (HDAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl- N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS) (manufactured by Dojindo Laboratories) and the like. Hydrogen peroxide can be colored using a leuco reagent in the presence of peroxidase. Specific examples of this reagent include o-dianisidine, o-tolidine, 3,3 diaminobenzidine, 3,3,5,5-tetramethylbenzidine; N- (carboxymethylaminocarbonyl) manufactured by Doujin Chemical Laboratory Co., Ltd. -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67);

  Further, as a method for quantifying hydrogen peroxide, it can also be analyzed by a fluorescence method or an electrode method. In the fluorescence method, compounds that fluoresce upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivatives and the like, and in the chemiluminescence method, luminol, lucigenin, isoluminol, Pyrogallol or the like can be used. Examples of a method for producing aldehyde from alcohol using catalase or the like and quantifying the generated aldehyde include a method using a hunch reaction, a method of developing a color by a condensation reaction with MBTH, or a method using an aldehyde dehydrogenase. .

  In addition, when measuring hydrogen peroxide using an electrode, the electrode is not particularly limited as long as it is a material that can exchange electrons with hydrogen peroxide, and examples thereof include platinum, gold, and silver. As an electrode measurement method, known methods such as amperometry, potentiometry, coulometry, etc. can be used, and further, an oxidation or reduction current obtained by interposing an electron carrier in the reaction between oxidase or substrate and electrode. Alternatively, the amount of electricity may be measured. Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Alternatively, the oxidation or reduction current obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode, or the amount of electricity thereof may be measured. For the analytical reagent of the present invention, a buffer, an enzyme stabilizer, a preservative, and the like are appropriately used as necessary.

In appropriate additives, surfactants include polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbite fatty acid esters, polyoxyethylene alkylphenyl formaldehyde condensates , Polyoxyethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine / fatty acid amides, polyoxyethylene alkyl ether phosphates / phosphates, Polyoxyethylene alkyl ether sulfates, polyglycerin fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters Sorbitan fatty acid esters, N acylamino acid salts, alkyl ether carboxylates, alkyl phosphates, N acyl taurates, sulfonates, alkyl sulfates, betaine acetate amphoteric surfactants, imidazoline amphoteric surfactants, lecithin Derivatives, polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctyl phenyl ether, polypropylene glycol, polyvinyl alcohol, etc. 0.01-10%, preferably 0.05-5%, various metal salts such as lithium chloride, sodium chloride, Potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride, etc. 1 mM to 5 M, preferably 10 mM to 1 M, various buffers such as Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good 10mM to 2M, such as buffer solution, suitable The 20MM～1M, various preservatives such 0.01% to 10% of sodium azide, preferably may be appropriately added 0.05 to 1%.
As a stabilizer for ethanolamine oxidase, various saccharides represented by sorbitol are appropriately used. These may be added in the range of 0.1 to 10%, preferably 0.5 to 5%.

  As a method for quantifying ethanolamine-containing phospholipid that can be used in the present invention, 0.01 to 0.05 ml of a test solution is added to 1 ml of the ethanolamine-containing phospholipid analysis composition of the present invention, and reacted at a temperature of 37 ° C. When performing a rate assay, several minutes to several tens of minutes between two points after the start of the reaction, for example, 1 minute after 3 minutes and 4 minutes, or 5 minutes after 3 minutes and 8 minutes The amount of dissolved oxygen, hydrogen peroxide or other product changed in the above can be measured directly or indirectly by the above-described method. In the case of an endpoint assay, the amount of dissolved oxygen changed after a certain time from the start of the reaction, The amount of hydrogen peroxide or other products may be measured in the same way. In this case, the amount of glycated protein in the test solution can be determined by comparing with changes in absorbance and the like when measured using a known concentration of ethanolamine phospholipid.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
Reagent 1 (R1) was prepared by mixing each component so as to have the following final concentration.
50 mM Tris-HCl buffer (pH 8.0)
6U / ml tyramine oxidase (Asahi Kasei Corporation)
10U / ml peroxidase (Sigma)
0.1% Triton X100
0.1% TOOS

[Example 2]
Reagent 2 (R2) was prepared by mixing each component so as to have the following final concentration.
50 mM Tris-HCl buffer (pH 8.0)
Phospholipase D (Asahi Kasei Corporation; 7U / ml)
2 mM calcium chloride 0.1% 4-aminoantipyrine 0.1% Triton X100

Example 3
500 microliters of each of the reaction solutions described in Examples 1 and 2 were mixed, 20 microliters of a 0.1 to 1 mM phosphatidylethanolamine solution was added, and the mixture was reacted at 37 ° C. for 5 minutes, and then the absorbance at 550 nm was measured. The results at that time are shown in FIG.
As can be seen from FIG. 1, phosphatidylethanolamine could be measured using an enzyme that acts on phospholipase and ethanolamine. Similar results were obtained when lysophosphatidylethanolamine was used instead of phosphatidylethanolamine.

Example 4
To 500 μl of the reaction solution described in Example 1, 20 microliters of 0.5 mM phosphatidylethanolamine solution containing 0-100 μM phenylethylamine was added to carry out an amine elimination reaction at 37 ° C. for 5 minutes, followed by Example 2. After adding 500 μl of the reaction solution and reacting at 37 ° C. for 5 minutes, the absorbance at 550 nm was measured. The results at that time are shown in FIG.

As can be seen from FIG. 2, since a constant measurement value of glycated phosphatidylethanolamine can be obtained regardless of the concentration of phenylethylamine, it is possible to eliminate only the free amine and accurately measure only phosphatidylethanolamine. there were. Similar results were obtained when lysophosphatidylethanolamine was used instead of phosphatidylethanolamine.

  The analysis method and analysis composition of the present invention can be suitably used for phospholipid analysis and clinical diagnosis in foods.

It is a calibration curve of the ethanolamine phospholipid measurement based on Example 3 of this invention. It is an influence test result in the ethanolamine phospholipid measurement of the free amine based on Example 4 of this invention.

Claims (6)

  Method for analyzing ethanolamine-containing phospholipid, characterized by using at least phospholipase and tyramine oxidase   After the reaction is performed by adding a test solution containing ethanolamine-containing phospholipid to the first reagent (R1) containing at least tyramine oxidase, at least a second reagent containing an enzyme that hydrolyzes ethanolamine-containing phospholipid ( A method for analyzing ethanolamine-containing phospholipids, characterized in that the reaction is carried out by adding R2)   The method for analyzing ethanolamine-containing phospholipid according to claim 1, wherein the enzyme that hydrolyzes ethanolamine-containing phospholipid is phospholipase D.   Composition for analysis of ethanolamine-containing phospholipid characterized by containing at least phospholipase and tyramine oxidase   Composition for analysis of ethanolamine-containing phospholipids, comprising at least a first reagent (R1) containing tyramine oxidase and a second reagent (R2) containing at least an enzyme that hydrolyzes ethanolamine-containing phospholipids object The composition for analyzing ethanolamine-containing phospholipid according to claim 4, wherein the enzyme that hydrolyzes ethanolamine-containing phospholipid is phospholipase D.

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