JP2004215552A - Method for analyzing saccharified phospholipid and analysis reagent therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an analysis reagent for saccharified phospholipids utilizing an enzymic method. <P>SOLUTION: A method for analyzing the saccharified phospholipids using the reagent is provided, which comprises determining the concentration of the saccharified phospholipids using an enzyme acting on a phospholipase and ketamine. Thereby, the saccharified phospholipids can be quantified accurately, easily and inexpensively without the need of any complicated operations. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for analyzing glycated phospholipids by an enzymatic method and a reagent for analysis.
[0002]
[Prior art]
It is known that phosphatidylethanolamine and phosphatidylserine, which are one of the phospholipids, react with various sugars such as glucose and mannose to form stable Amadori-type phospholipids, that is, glycated phospholipids via an unstable Schiff base. Have been. In a living body, various compounds are saccharified with an increase in blood sugar, and various saccharified products are produced.
Currently, a diabetes test for quantifying glycated proteins such as hemoglobin and albumin among various glycated substances present in blood has been put to practical use. Since these glycated proteins represent the average of past blood sugar levels, they are widely used as indicators of blood sugar control.
[0003]
On the other hand, in recent years, it has been suggested that glycated lipids, particularly glycated phospholipids, rather than glycated proteins, contribute to the development of the Maillard reaction in tissues and cells as a factor of diabetes complications. Measurement of glycated lipids, particularly glycated phospholipids, is expected to be useful as a measure of risk factors for progression to complications and for confirming the therapeutic effect of Maillard reaction inhibitors.
Methods for analyzing glycated phospholipids include liquid chromatography-MASS (Non-patent Document 1) and UV-labeled liquid chromatography (Non-patent Document 2), but currently require a large-sized dedicated analytical instrument. And the analysis operation is complicated and it takes a long time to obtain a result. Further, a method for measuring using an enzyme has not been known so far.
[0004]
[Non-patent document 1]
Lipids 1995 Oct; 30 (10): 885-91.
[Non-patent document 2]
J. Lipid Res 2002 Mar; 43 (3): 523-9
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide an enzymatic analysis method for easily and accurately analyzing glycated phospholipids in a test solution in a short time and a glycated phospholipid analysis reagent used in the method.
[0006]
[Means for Solving the Problems]
The present invention has been made in order to achieve the above-mentioned object, and as a result of diligent studies from various aspects, the present inventor has found that phospholipase, which is a phospholipid hydrolase, and an enzyme that acts on ketoamine are used. The present inventors have found a simple and accurate method for analyzing glycated phospholipids, and have completed the present invention.
That is, the present invention
1) a method for analyzing a glycated phospholipid, which comprises treating a test solution with an enzyme acting on phospholipase and ketoamine;
2) the method, wherein the phospholipase is phospholipase D;
3) a method wherein the enzyme acting on ketoamine is ketoamine oxidase;
4) a method wherein the enzyme acting on phospholipase and ketoamine is a microorganism-derived enzyme;
5) an analytical reagent for glycated phospholipid analysis, which comprises an enzyme acting on phospholipase and ketoamine;
6) an assay reagent wherein the phospholipase is phospholipase D;
7) an analytical reagent in which the enzyme acting on ketoamine is ketoamine oxidase;
8) an analytical reagent in which the enzyme acting on phospholipase and ketoamine is an enzyme derived from a microorganism;
9) a glycated phospholipid analysis kit comprising at least a first reagent containing an enzyme acting on ketoamine and a second reagent containing phospholipase;
10) a method characterized by adding the first reagent of 9) to the test solution and then adding the second reagent;
About.
[0007]
According to the present invention, glycated phospholipids in a test solution produce low-molecular-weight ketoamine by the action of phospholipase. Next, an enzyme acting on the ketoamine acts on the produced ketoamine. As a result, the glycated phospholipid can be analyzed with high accuracy by quantifying the generated substance or quantifying the consumed coenzyme or oxygen.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in more detail.
The phospholipase used in the present invention may be any enzyme capable of hydrolyzing glycated phospholipid. Further, the enzyme may be a purified product or a non-purified product as long as the desired activity is expressed. Phospholipases A, B, C and D are known as enzymes that hydrolyze phospholipids, and these enzymes are widely found in animals, plants and microorganisms. Any of these enzymes can be used to measure glycated phospholipids, but phospholipase C or phospholipase D is preferable as a reagent material, and phospholipase D is particularly preferable. This enzyme is known to be present in plant tissues such as cabbage, peanuts and carrots, and in microorganisms such as actinomycetes, and any of these enzymes can be used. In view of the above, enzymes derived from microorganisms are preferred. Among them, phospholipase D derived from Streptomyces chromophus sox, which is one of actinomycetes, is particularly preferable because of its excellent reactivity and stability. The present phospholipase D has a property of widely acting on phospholipids other than glycated phosphatidylethanolamine, such as phosphatidylcholine (lecithin), lysolecithin, and phosphatidylserine, but does not cause any problem in analyzing glycated phospholipids. The concentration of this enzyme in the reagent is 0.05 to 100 U / ml, preferably 0.5 to 50 U / ml. The activity of the enzyme was 1 U (unit) for decomposing the substrate phosphatidylcholine by 1 μmol per minute at 37 ° C.
[0009]
The “ketoamine” in the present invention is represented by the following general formula (1)
-(CO) -CHR-NH-
(R represents a hydrogen atom or a hydroxyl group)
A compound having a ketoamine structure represented by the following formula: specifically, an Amadori compound or a decomposition product of the Amadori compound. Examples of the decomposition product of the Amadori compound having a ketoamine structure include saccharified ethanolamine and saccharified serine.
[0010]
As used herein, the term "enzyme acting on ketoamine" means an enzyme that specifically recognizes and acts on a ketoamine structure. That is, any enzyme that acts on ketoamine that can be used in the present invention may be any enzyme that specifically recognizes and acts on the ketoamine structure. Among the enzymes, enzymes that act on saccharified ethanolamine are preferred, and enzymes that act on saccharified ethanolamine and saccharified serine are particularly preferred. Further, the enzyme may be a purified product or a non-purified product as long as the desired activity is expressed. Examples of the above enzymes 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.
[0011]
As an oxidase acting on ketoamine, a coenzyme or an enzyme consuming oxygen that acts well on ketoamine and produces a substance that can be measured by a known method, or that can measure a change in amount by a known method Is fine.
Most of these enzymes act on low-molecular-weight ketoamines, and have extremely low effects on high-molecular-weight glycated phospholipids. Therefore, it is preferable to decompose high-molecular-weight glycated phospholipids to low molecules with lipase, preferably phospholipase, most preferably phospholipase C or D, and then act on an enzyme that acts on ketoamine.
[0012]
Examples of enzymes that act on ketoamines include the genus Gibberella, the genus Aspergillus, the genus Candida, the genus Penicillium, the genus Fusarium, and the genus Acremonium mycerium or Acremonium mysium. And the like. Ketoamine oxidase derived from the genus, and the like, and Fusarium-derived enzymes are particularly preferable from the viewpoint of enzyme stability and stable supply of enzymes. The concentration in the reagent is 0.01 to 300 U / ml, preferably 0.1 to 100 U / ml. The activity of the enzyme acting on ketoamine was determined by glycated Z-lysine (α-carbobenzoxy-ε-D-deoxyfructosyl-L-lysine; synthesized and purified based on the method of Hashiba et al., Hashiba H, J. Agric. Food Chem. 24:70, 1976) at 37 ° C. for 1 minute (1 U).
As the glycated phospholipid to be measured in the present invention, any phospholipid may be used as long as it is a glycated phospholipid, but a glycated phospholipid such as phosphatidylethanolamine or phosphatidylserine is preferred.
[0013]
As a method for quantifying glycated phospholipids in the present invention, the amount of a substance produced by the action of an enzyme acting on a known ketoamine or the amount of coenzyme or oxygen consumed from a ketoamine produced by the action of phospholipase is determined by a known method. Should be measured. For example, when an oxidase acting on ketoamine is used, 0.001 to 0.5 ml of a test solution is added to the glycated phospholipid analysis composition of the present invention, and the mixture is reacted at a temperature of 37 ° C. to perform a rate assay. For example, a change in the solubility between two points at a certain time after the start of the reaction for several minutes to several tens of minutes, for example, 1 minute after 3 minutes and 4 minutes, or 5 minutes after 3 minutes and 8 minutes The amount of oxygen, hydrogen peroxide or other products may be measured directly or indirectly.In the case of an endpoint assay, the amount of dissolved oxygen, hydrogen peroxide or other products changed after a certain time after the start of the reaction Should be measured. In this case, the amount of the glycated phospholipid in the test liquid can be determined by comparing the change in absorbance or the like when measured using a glycated phospholipid of a known concentration.
[0014]
The analysis reagent containing the glycated phospholipid analysis composition of the present invention may include an enzyme acting on phospholipase and ketoamine, and may further contain a substance produced or consumed by the action of the enzyme acting on ketoamine. A reagent for measuring the amount of coenzyme or oxygen by a known method may be contained in advance. Hereinafter, as an example, a case where ketoamine oxidase is used as an enzyme acting on ketoamine and hydrogen peroxide is measured as a substance produced by the action of the enzyme acting on ketoamine will be described, but the present invention is limited to the following examples. is not.
Examples of the reagent for measuring hydrogen peroxide include a combination of a chromogen of a Trinder-type reagent and a coupler reagent. Hydrogen peroxide produced by the ketoamine oxidase reaction produces a dye by oxidative condensation of the chromogen of the Trinder reagent with the coupler reagent. The absorbance of this dye can be measured spectrophotometrically (WO02061119 International Patent Publication).
[0015]
Examples of the chromogen of the Trinder-type reagent include a toluidine derivative, an aniline derivative and a phenol derivative, and a toluidine derivative is preferred. Examples of toluidine derivatives include N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) and N-ethyl-N-sulfopropyl-m-toluidine (TOPS). TOOS is preferred. As the aniline derivatives, N, N-dimethylaniline, N, N-diethylaniline, 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-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- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), -Ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline ( HALPS). Examples of the phenol derivative include 2,4-dichlorophenol. All the chromogens of the above-mentioned Trinder-type reagents are available from Dojindo Chemical Laboratory.
[0016]
Examples of the coupler reagent include 4-aminoantipyrine and 3-methyl-2-benzothiazolinone hydrazone (MBTH), but 4-aminoantipyrine is preferable.
In addition, hydrogen peroxide can be colored using a leuco-type reagent in the presence of peroxidase. Specific examples of the leuco-type reagent include o-dianisidine, o-tolidine, 3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine; N- (carboxymethylamino; Carbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67); Can be
[0017]
Compounds that emit fluorescence by oxidation, compounds that emit chemiluminescence as a catalyst, and catalase are also useful as reagents for quantifying hydrogen peroxide.
Examples of the compound that emits fluorescence upon oxidation include homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, and diacetylfluorescin derivative, which can be quantified by a fluorescence method.
Examples of the chemiluminescent catalyst include luminol, lucigenin, isoluminol, pyrogallol, and the like, which can be quantified using a chemiluminescence method.
Further, there is a method in which an aldehyde is generated from an alcohol using catalase or the like, and the generated aldehyde is quantified. Specific examples include a method using a Hunt reaction, a method in which a color is formed by a condensation reaction with MBTH, or a method in which an aldehyde is formed. Examples include a method using dehydrogenase.
[0018]
When hydrogen peroxide is measured 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. Can be As the electrode measuring method, known methods such as amperometry, potentiometry, and coulometry can be used. Further, an oxidase or reduction current or the amount of electricity obtained may be measured by interposing an electron carrier in the reaction between the oxidase or the substrate and the electrode. 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, oxidation, reduction current or the amount of electricity obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode may be measured.
[0019]
Additives may be added to the analytical reagent of the present invention, if necessary. Examples of the additives include surfactants, buffers, preservatives, and enzyme stabilizers. As surfactants, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbite fatty acid esters, polyoxyethylene alkyl phenyl formaldehyde condensate, polyoxyethylene castor oil, Polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolin, polyoxyethylene alkylamine / fatty acid amides, polyoxyethylene alkyl ether phosphoric acid / phosphate, polyoxyethylene alkyl ether sulfate Salts, polyglycerin fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters , N acyl amino acid salts, alkyl ether carboxylates, alkyl phosphates, N acyl taurates, sulfonates, alkyl sulfates, betaine acetate amphoteric surfactants, imidazoline amphoteric surfactants, lecithin derivatives, Examples thereof include polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctyl phenyl ether, polypropylene glycol, and polyvinyl alcohol, and the concentration is 0.01 to 10%, and preferably 0.05 to 5%. Examples of the buffer include Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good's buffer, and the like, and the concentration is 10 mM to 2 M, preferably 20 mM to 1 M. Examples of the preservative include sodium azide, and the concentration is 0.01 to 10%, preferably 0.05 to 1%. Examples of ketoamine oxidase stabilizers include various sugars represented by sorbitol, various amino acids represented by glutamic acid, and the like. These may be added in the range of 0.1 to 10%, preferably 0.5 to 5%. As the phospholipase stabilizing agent, any additive can be used as long as the phospholipase is stably present in the reagent, and examples thereof include salts containing calcium and magnesium. 0.01 mM to 1 M, preferably 0.1 mM to 500 mM. In addition, metal salts, for example, lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride and the like are also preferred additives. Its concentration is between 1 mM and 5M, preferably between 10 mM and 1M.
[0020]
The analysis reagent of the present invention can be used as one composition containing all necessary reagents, but two reagents are required to maintain the storage stability of the analysis reagent, namely, the first reagent and the second reagent. It is preferable to use by dividing.
The first reagent is preferably a reagent containing an enzyme acting on ketoamine, and examples include a reagent containing a chromogen of ketoamine oxidase and a Trinder-type reagent. As the second reagent, a reagent containing phospholipase is preferable, and examples thereof include a reagent containing phospholipase and a coupler.
The test liquid may be added after the first reagent and the second reagent are mixed in advance, or the test reagent may be added to the first reagent before the second reagent is added. Conversely, the first reagent may be added after the test liquid is added to the second reagent. In short, the order of adding both reagents to the test solution may be any order.
[0021]
By the way, there is a possibility that a glycated amino acid is rarely present in a biological component such as serum, so that there is a risk that a correct error occurs.However, by appropriately selecting the composition of the first reagent and the second reagent, an appropriate By acting in order, the risk of a positive error can be avoided, and the accuracy of the analysis can be significantly improved. From such a viewpoint, a reagent containing a chromogen of ketoamine oxidase and a Trinder-type reagent, that is, a peroxidase or catalase is contained in the first reagent, and a test solution is first added to the first reagent, for example, Heat at 5-50 ° C for 0.1-60 minutes. At this stage, the glycated amino acids are eliminated. Thereafter, a second reagent containing a phospholipase and a coupler reagent may be added for analysis.
Further, the present reagent can be supplied in the form of a liquid, a lyophilized product, a frozen liquid reagent, or the like.
[0022]
[Formulation example]
Hereinafter, Formulation Examples will be specifically described, but the present invention is not limited to the following Examples.
[Formulation Example 1]
Reagent A was prepared by mixing the components so as to have the following final concentrations.
50 mM Tris-HCl buffer (Wako Pure Chemical Industries, Ltd.) (pH 8.0)
6U / ml ketoamine oxidase (Asahi Kasei Corporation)
10U / ml peroxidase (Sigma)
0.1% Triton X100 (manufactured by Wako Pure Chemical Industries, Ltd.)
0.1% TOOS (manufactured by Wako Pure Chemical Industries, Ltd.)
[0023]
[Formulation Example 2]
Reagent B was prepared by mixing the components so as to have the following final concentration.
50 mM Tris-HCl buffer (pH 8.0)
7U / ml phospholipase D (T-07, manufactured by Asahi Kasei Corporation)
20 mM calcium chloride (Wako Pure Chemical Industries, Ltd.)
0.1% 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.)
0.1% Triton X100
[0024]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples.
[Example 1]
500 μl of each of the reagents A and B described in Formulation Examples 1 and 2 were mixed, 20 μl of a 0.1 to 1 mM glycated phosphatidylethanolamine solution was added, and after reacting at 37 ° C. for 5 minutes, the absorbance at 550 nm was measured. The result at that time is shown in FIG. As can be seen from FIG. 1, it was possible to measure glycated phosphatidylethanolamine using enzymes that act on phospholipase and ketoamine. Similar results were obtained by using glycated phosphatidylserine instead of glycated phosphatidylethanolamine.
[0025]
[Example 2]
20 μl of a 0.5 mM glycated phosphatidylethanolamine solution containing 0 to 100 μM of a glycated amino acid (glycated Z-lysine) was added to 500 μl of the reagent A described in Formulation Example 1, and a glycated amino acid was eliminated at 37 ° C. for 5 minutes. After adding 500 μl of the reagent B described in Formulation Example 2 and reacting at 37 ° C. for 5 minutes, the absorbance at 550 nm was measured. The result at that time is shown in FIG.
As can be seen from FIG. 2, since a constant measurement value of glycated phosphatidylethanolamine is obtained regardless of the concentration of glycated Z-lysine, low-molecular-weight ketoamine (glycated amino acid in this case) existing in the test solution from the beginning is obtained. And it was possible to accurately measure only glycated phosphatidylethanolamine. Similar results were obtained by using glycated phosphatidylserine instead of glycated phosphatidylethanolamine.
[0026]
【The invention's effect】
According to the present invention, there is provided a method for analyzing glycated phospholipid concentration by treating with an enzyme acting on phospholipase and ketoamine.
Further, the present invention provides an analytical reagent and a kit for glycated phospholipid analysis, which contain an enzyme acting on phospholipase and ketoamine.
[Brief description of the drawings]
FIG. 1 is a calibration curve for glycated phospholipid measurement based on Example 1 of the present invention.
FIG. 2 shows the results of an influence test of glycated amino acids present in a test solution from the beginning in the measurement of glycated phospholipids based on Example 2 of the present invention.

Claims (10)

A method for analyzing glycated phospholipids, comprising treating a test solution with an enzyme that acts on phospholipase and ketoamine. 2. The method according to claim 1, wherein the phospholipase is phospholipase D. 3. The method according to claim 1, wherein the enzyme acting on ketoamine is ketoamine oxidase. The method according to claim 1, wherein the enzyme acting on phospholipase and ketoamine is a microorganism-derived enzyme. An analytical reagent for glycated phospholipid analysis, comprising an enzyme acting on phospholipase and ketoamine. The analysis reagent according to claim 5, wherein the phospholipase is phospholipase D. 7. The reagent according to claim 5, wherein the enzyme acting on ketoamine is ketoamine oxidase. The analytical reagent according to any one of claims 5 to 7, wherein the enzyme acting on phospholipase and ketoamine is a microorganism-derived enzyme. A glycated phospholipid analysis kit comprising at least a first reagent containing an enzyme acting on ketoamine and a second reagent containing phospholipase. The method according to any one of claims 1 to 4, wherein the first reagent according to claim 9 is added to the test solution, and then the second reagent is added.

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