JP2000333696A - Measurement of glycosylated amine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method enabling a glycosylated amine in a specimen to be measured with a high precision. SOLUTION: This method is to perform the oxidative decomposition of a glycosylated amine with a glycosylated amine-oxidizing and decomposing enzyme into a saccharide and an amine, to subject a saccharide to oxidative decomposition with a saccharide-oxidizing and decomposing enzyme, to measure the quantity of the same reaction product or the same consumed substance in both oxidative decomposition reactions and to determine the quantity of glycosylated amine from the measured value. For example, when the glycosylated amine is a glycosylated protein, a sample is decomposed with a protease, is treated with a fructosyl amino acid oxidase and then is treated with a glucose oxidase. By adding a peroxidase and a chromogenic substrate to the obtained solution and by measuring its coloring, sensitivity of measurement is improved in comparison with a conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring the amount of glycated amine such as glycated protein, glycated peptide or glycated amino acid present in a sample.

[0002]

2. Description of the Related Art Glycated proteins in blood, for example, glycated albumin and glycated hemoglobin in erythrocytes (hereinafter, referred to as glycated hemoglobin).
“HbA1c”) and the like reflect the past history of the blood glucose level in the living body, and are therefore used as important indexes in the diagnosis and treatment of diabetes.

[0003] As a method for measuring such glycated proteins, for example, high performance liquid chromatography (HPL)
C) method, mini-column method, immunization method, enzyme method and the like. Among them, the enzymatic method using fructosyl amino acid oxidase (FAOD) is more effective than other measurement methods.
This is a method that can accurately and quickly measure glycated proteins.

A method for measuring a glycated protein using FAOD is, for example, a method of treating a glycated protein with a protease so that FAOD acts easily, and then reacting the glycated product, which is a decomposed product, with FAOD to cause a catalytic reaction of the FAOD. Is a method of measuring the amount of hydrogen peroxide generated by the method described above, and obtaining the amount of glycated protein and the saccharification rate from the measured value.

However, it has been difficult to measure low-concentration glycated proteins, particularly HbA1c, by the above-mentioned enzymatic method. For the measurement of HbA1c, hemolysis of erythrocytes is necessary, and the sample is diluted by the addition of the reagent, so that the concentration of HbA1c reaches the detection limit, which sometimes makes the measurement difficult. Further, in the measurement of HbA1c by the conventional method, for example, when the concentration value of HbA1c between samples changes only by 0.1%, to detect the concentration difference based on the amount of each generated hydrogen peroxide, There was a problem with its accuracy.

[0006] These problems are presumed to be due to the following causes. Glycated proteins usually have sugars added to the N-terminal α-amino group, lysine (Lys) side chain group, arginine (Arg) side chain group and the like in the peptide chain. Is measured. In particular, HbA1c is characterized in that the N-terminal valine residue (α-amino group) of two β-chains usually present in hemoglobin is glycated, and the amount thereof is determined by measuring the glycated portion. Can be When the amount is actually represented by a numerical value, the blood of a healthy person usually contains about 130 g / liter (2 mm) of hemoglobin (Hb) having a molecular weight of about 65,000.
ol / liter) and 4% of the Hb
(80 μmol / liter) is HbA1c, and the concentration of β-chain containing a saccharified moiety involved in the measurement is 160 μm
ol / liter. This β-chain concentration corresponds to the concentration of the saccharified moiety (glycated α-amino group). When measuring this, it is necessary to dilute the sample 100 times or more, so that the concentration becomes very low.

[0007]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a saccharified amine capable of setting the detection limits of the amount of saccharified amine and the saccharification rate lower than those of the conventional method and obtaining a highly reliable measurement value. It is an object of the present invention to provide a method for measuring an amine.

[0008]

In order to achieve the above object, a measuring method of the present invention is a method for measuring a glycated amine in a sample, wherein the glycated amine is converted to a saccharide by a glycated amine oxidase. It is oxidatively decomposed to an amine, and the sugar is oxidatively decomposed by a sugar oxidase, and the amount of the same product or the same consumable by the both oxidative decomposition reactions is measured, and the amount of the saccharified amine is determined from the measured value. It is characterized in that it is determined.

As described above, when the saccharified amine oxidase and the saccharoxidase are used in combination, two identical products are produced and two identical consumables are consumed for one saccharified moiety. Therefore, the measurement method of the present invention can theoretically achieve twice the detection sensitivity as compared with the conventional method, and can more accurately measure saccharified amine. Further, even if the dilution ratio of the sample is increased, the detectable concentration of the product or the concentration of the consumable product can be maintained, so that there is an advantage that the influence of the coexisting substance in the sample can be reduced by dilution.

In the measuring method according to the present invention, it is preferable that the same product is hydrogen peroxide. Preferably, the same consumable is oxygen.

[0011] In the measurement method of the present invention, the glycated amine to be measured is preferably a glycated amino acid, a glycated protein or a glycated peptide. When the saccharified amine is a saccharified protein or saccharified peptide, the saccharified amine is preferably decomposed with a protease in advance, and then the degraded product is reacted with the saccharified amine oxidase.

In some cases, a substrate of a glucose oxidase such as glucose is present in a sample from the beginning, and the hydrogen peroxide generated by the reaction between the substrate and the sugar oxidase and the consumed oxygen are measured accurately. May be affected. In order to avoid this, in the measurement method of the present invention, it is preferable to eliminate the substrate of the saccharide oxidase present in the sample before reacting the saccharified amine with the saccharified amine oxidase. Preferably, the sugar oxidase is added to the sample.

In the measurement method of the present invention, the sugar oxidase is glucose oxidase (hereinafter referred to as "GOD").
), And at least one oxidase selected from the group consisting of β-galactosidase and pyranose oxidase.

When the glycated amine is a glycated protein or the like, it is often saccharified with glucose.
It is preferred that the sugar produced by oxidative degradation of the saccharified amine is glucosone, and in this case, the GOD is particularly preferred as the sugar oxidase.

In the measuring method of the present invention, in order to oxidatively degrade the sugar, it is preferable to add the sugar oxidase to the reaction solution so as to have a concentration of 1,000 to 5,000,000 U / liter. , Particularly preferably,
It is in the range of 10,000 to 1,000,000 U / liter.

In the method of the present invention, the saccharified amine oxidase is a fructosyl amino acid oxidase (F
(AOD).

In the measurement method of the present invention, the measurement of the amount of hydrogen peroxide is preferably a measurement using peroxidase and a substrate oxidized by the peroxidase.

The substrate to be oxidized is preferably a substrate that develops color by oxidation (hereinafter referred to as a “chromogenic substrate”). Examples of the chromogenic substrate include sodium N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine (for example, DA-6
4: a high-sensitivity color forming agent such as Wako Pure Chemical Industries, Ltd., but is not particularly limited thereto. In addition, for example, orthophenylenediamine (OPD), a Trinder reagent and aminoantipyrine A chromogenic substrate or the like can be used.

In these measurement methods of the present invention, the object to be measured is not particularly limited as long as it is a glycated amine, but is preferably a glycated protein, glycated peptide or glycated amino acid as described above. Examples of the glycated protein include HbA1c, glycated albumin, glycated globulin, and glycated casein. In addition, since measuring glycated protein in blood is useful for diagnosing diabetes, the sample to be measured is preferably blood. However, for example, glycated proteins are also included in biological samples such as whole blood, plasma, serum, blood cells and urine, and drinking water such as juice, soy sauce, and foods such as sauces.
The sample to be measured is not particularly limited.

The measuring method of the present invention is preferably applied to, for example, a measuring kit, whereby the measuring method of the present invention can be carried out simply and quickly.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The FAOD which can be used in the measuring method of the present invention is preferably a FAOD which catalyzes a reaction represented by the following formula (Formula 1).

[0022]

Embedded image R ¹ —CO—CH ₂ —NH—R ² + H ₂ O +
O ₂ → R ¹ -CO-CHO + NH ₂ -R ² + H ₂ O
_Two

In the above formula (1), R ¹ represents a hydroxyl group or a residue (sugar residue) derived from a sugar before the saccharification reaction. The sugar residue (R ¹ ) is an aldose residue when the sugar before the reaction is aldose, and is a ketose residue when the sugar before the reaction is ketose. For example, when the sugar before the reaction is glucose, the structure after the reaction takes a fructose structure by Amadori rearrangement. In this case, the sugar residue (R
¹ ) is a glucose residue (aldose residue). This sugar residue (R ¹ ) can be represented by, for example,-[CH (OH)] _n -CH ₂ OH, where n is an integer of 0-6.

In the above formula (1), R ² is not particularly limited, but when the glycated amine is a glycated amino acid, a glycated peptide or a glycated protein, when the α-amino group is glycated, It differs when the group is saccharified.

In the above formula (1), when the α-amino group is saccharified, R ² is an amino acid residue or a peptide residue represented by the following formula (2).

[0026]

Embedded image —CHR ³ —CO—R ⁴

In the above formula (Formula 2), R ³ represents an amino acid side chain group, and R ⁴ represents a hydroxyl group, an amino acid residue or a peptide residue, and can be represented by the following formula (Formula 3). In the following formula (Formula 3), n is an integer of 0 or more, and R ³ represents an amino acid side chain group as described above, and the amino acid side chain groups may be all the same or different.

[0028]

## STR3 ##-(NH-CR ³ H-CO) _n -OH

In the above formula (Chem. 1), when an amino group other than the α-amino group is saccharified (the amino acid side chain group is saccharified), R ² is represented by the following formula (Chem. 4). be able to.

[0030]

Embedded image —R ⁵ —CH (NH—R ⁶ ) —CO—R ⁷

In the above formula (4), R ⁵ represents a portion of the amino acid side chain other than the glycated amino group. For example, when the glycated amino acid is lysine, R ⁵
Is —CH ₂ —CH ₂ —CH ₂ —CH ₂ —. For example, when the saccharified amino acid is arginine, R ⁵ is —CH ₂ —CH ₂ —CH ₂ —NH—CH (NH ₂ ) —. It is.

In the above formula (Formula 4), R ⁶ is
It is hydrogen, an amino acid residue or a peptide residue, and can be represented by, for example, the following formula (Formula 5). In the following formula (Formula 5), n is an integer of 0 or more, and R ³ is
An amino acid side chain group is shown in the same manner as described above, and all the amino acid side chain groups may be the same or different.

[0033]

## STR5 ##-(CO-CR ³ H-NH) _n -H

Further, in the above formula (Formula 4), R ⁷ is
It is a hydroxyl group, an amino acid residue or a peptide residue, and can be represented by, for example, the following formula (Formula 6). In the following formula (Formula 6), n is an integer of 0 or more, and R ³ is
An amino acid side chain group is shown in the same manner as described above, and all the amino acid side chain groups may be the same or different.

[0035]

[Of 6] ^{- (NH-CHR 3 -CO)} n -OH

In the measuring method of the present invention, the sugar oxidatively decomposed by the sugar oxidase is represented by the following formula (Formula 1):
It is a sugar represented by R ¹ -CO-CHO. However, in this case, R ¹ represents a sugar residue and does not include a hydroxyl group. The reaction between the sugar and the sugar oxidase is presumed to be a reaction represented by the following formula (Formula 7). In the following formula (Formula 7), R ¹ is a sugar residue as described above, for example,-[C
H (OH)] _n —CH ₂ OH, where n is 0
6 to an integer.

[0037]

[Image Omitted] R ¹ -CO-CHO + H ₂ O + O ₂ → R ¹
_{-CHO + CO 2 + H 2 O} 2

Next, the measurement method of the present invention will be described using an example of measuring glycated proteins in blood cells using FAOD as the saccharified amine oxidase and GOD as the saccharoxidase.

First, a blood cell fraction is separated from whole blood by a conventional method such as centrifugation, and the blood cell is lysed. The hemolysis method is not particularly limited, and for example, a method using a surfactant, a method using ultrasonic waves, a method using a difference in osmotic pressure, and the like can be used. Among them, a method using a surfactant is preferable for the reason of simplicity of operation and the like.

As the surfactant, for example, Tri
tonX-100, Tween-20, Brij35 and the like can be used. The treatment conditions with the surfactant are usually such that when the blood cell concentration in the treatment solution is 1 to 10% by volume,
The surfactant may be added so that the concentration in the treatment solution is 0.1 to 1% by weight, and the mixture may be stirred at room temperature for about 5 seconds to 1 minute.

Next, protease treatment is performed on the hemolyzed sample. The type of the protease is not particularly limited, and for example, protease K, subtilisin, trypsin, aminopeptidase, papain and the like can be used. In addition, glycated amino acid releasing enzyme and the like can be used. The protease treatment is usually performed in a buffer, and the treatment conditions are appropriately determined depending on the type of protease to be used, the type of glycated protein, the concentration thereof, and the like.

Using trypsin as a protease,
When the hemolyzed sample is treated, usually, the protease concentration in the reaction solution is 100 to 6,000 U / liter, the blood cell concentration in the reaction solution is 0.2 to 5% by volume, the reaction temperature is 20 to 50 ° C,
The reaction time ranges from 10 minutes to 20 hours, and the pH ranges from 6 to 9.
The type of the buffer is not particularly limited. For example, Tris-HCl buffer, phosphate buffer, EPPS buffer, PI
A PES buffer or the like can be used.

Next, the degradation product obtained by the protease treatment is treated with FAOD. By the FAOD treatment, the reaction represented by the above formula (Chem. 1) is catalyzed.

This FAOD treatment is preferably carried out in a buffer as in the case of the protease treatment. The buffer is not particularly limited, and the same buffer as in the protease treatment can be used.

Next, the FAOD product is processed by GOD. As a result, the sugar produced together with the hydrogen peroxide in the FAOD treatment is oxidatively decomposed to further generate hydrogen peroxide. By the FAOD treatment and the GOD treatment, 2 mol of hydrogen peroxide is theoretically generated from 1 mol of the saccharified amino acid residue.

This GOD processing is performed by the GOD processing as described above.
Is added to the reaction solution at a concentration of 1,000 to 5,000,000 U /
It is preferable to add so as to be in the range of liter,
The reaction conditions are usually in a range of a temperature of 15 to 37 ° C and a reaction time of 1 to 30 minutes. Further, it is preferable to carry out the treatment in a buffer solution as in the protease treatment or the FAOD treatment. As the buffer, for example, a buffer such as Tris-HCl buffer, phosphate buffer, EPPS buffer, PIPES buffer, citrate buffer and the like can be used. Processing and FAO
It is preferable to use the same buffer as for D treatment.

Then, the hydrogen peroxide generated by the FAOD process and the hydrogen peroxide generated by the GOD process are
The measurement is performed using a redox reaction using POD and a chromogenic substrate for the POD.

The oxidation-reduction reaction is usually performed in a buffer, and the conditions are appropriately determined depending on the concentration of hydrogen peroxide and the like. Further, the buffer is not particularly limited, and the FA
The same buffer and the like as used in the OD treatment and the GOD treatment can be used.

The amount of hydrogen peroxide can be measured by, for example, an electric method in addition to the enzymatic method using peroxidase or the like.

When the chromogenic substrate is used, the concentration of hydrogen peroxide can be measured by measuring the color development (absorbance of the reaction solution) with a spectrophotometer, and the concentration of glycated protein in the sample can be determined from this. Can be. As described above, according to the measurement method of the present invention, the measurement target can be measured with high accuracy.

In this measurement, each processing step may be performed separately as described above, but there are processing steps that may be performed simultaneously in the following combinations, for example. 1: hemolysis treatment + protease treatment 2: protease treatment + FAOD treatment 3: FAOD treatment + GOD treatment 4: FAOD treatment + GOD treatment + POD redox treatment 5: protease treatment + FAOD treatment + GOD treatment + P
OD redox treatment

The FAOD, GOD, POD
Also, the order of adding the chromogenic substrate is not particularly limited.

When a substance (GOD substrate) that produces hydrogen peroxide by reaction with GOD is present in the sample, the sample (for example, a hemolyzed sample in the case of blood cells) is prepared before performing the FAOD treatment or the like. As a pre-treatment, GOD may be previously added to the sample to eliminate the GOD substrate. Further, if a sufficient amount of GOD is added to the sample in this pretreatment, it is not necessary to add GOD again in the GOD treatment of the FAOD-treated product performed later. In this case, GOD is added to the reaction solution at a concentration of 1,000 to 5,000,
It is preferable to add so as to be in the range of 000 U / liter.

The hydrogen peroxide produced by the pretreatment of the sample may be eliminated by, for example, catalase existing in the hemolyzed sample from the beginning, or may be eliminated by adding catalase separately. When the sample is pretreated and the hydrogen peroxide generated by the pretreatment is eliminated by the catalase, the catalase eliminates the hydrogen peroxide produced by the subsequent FAOD treatment and GOD treatment. To prevent this, it is preferable to add an excess amount of POD and a chromogenic substrate together with the addition of FAOD. In this case, it is preferable to add the activity (U) of the POD 5 to 100 times the amount (U) of the catalase.

[0055]

EXAMPLES (Example 1 and Comparative Example 1) 10 μl of fructosyl valine (hereinafter referred to as “FV”) solution at each concentration
Was added to each of the following GOD solutions (40 μl). Then, 50 μl of the following redox reaction solution A was added to these solutions.
Was added to start the redox reaction, and the absorbance of each reaction solution at a main wavelength of 694 nm and a sub-wavelength of 884 nm was measured using an automatic biochemical analyzer JCA-BM8 (manufactured by JEOL Ltd.). In addition, Comparative Example 1 was obtained by measuring the absorbance in the same manner as described above except that water (DW) was used instead of the GOD solution. These results are shown in Table 1 and the graph of FIG. FIG.
5 is a graph showing the relationship between the final concentration of V and the absorbance.
A sample without D treatment (D.W added) (Comparative Example 1) is shown.

(FV solution) FV was prepared by a conventional method using valine (manufactured by Peptide Research Laboratories) and glucose. Then, the FV is adjusted to a predetermined density (62.5, 12
5, 250, 500, 1000 μmol / liter) to distilled water.

(Buffer) A 100 mM NaH ₂ PO ₄ solution was mixed with a 100 mM K ₂ HPO ₄ solution to form a mixture.
An mM phosphate buffer (pH 8.0) was prepared.

(Composition of Redox Reaction Solution A) DA64 (manufactured by Wako Pure Chemical Industries, Ltd.) 40 μmol / liter POD (Type III: manufactured by Toyobo Co., Ltd.) 200 KU / liter FAOD (manufactured by Asahi Kasei Corporation) 10 KU / liter Buffer solution 100 mmol / liter

(GOD Solution) GOD (manufactured by Toyobo Co., Ltd.) was dissolved in purified water to a concentration of 2,000 KU / liter.

(Example 2 and Comparative Example 2)
In the redox reaction solution A used in the above, instead of the phosphate buffer, a 100 mM Tris-HCl buffer (pH
The absorbance was measured in the same manner as in Example 1 except that 7.0) was used. Comparative Example 2 was measured in the same manner as in Example 1 except that water was used instead of the GOD solution and the Tris-HCl buffer was used instead of the phosphate buffer. These results are shown in Table 2 and the graph of FIG. The figure is a graph showing the relationship between the final concentration of FV and the absorbance. In FIG.
D processed (Example 2) is GOD unprocessed (D.W
Addition) (Comparative Example 2).

[0061]

Table 1 FV final concentration Example 1 Comparative Example 1 (μmol / liter) (Abs.) (Abs.) 0 0 0 0.625 0.045 0.038 1.25 0.098 0.076 2.5 0.208 0.151 5.0 0.444 0.304 10.0 0.950 0.623

[0062]

[Table 2] FV final concentration Example 1 Comparative Example 1 (μmol / liter) (Abs.) (Abs.) 00 00 0.625 0.021 0.010 1.25 0.078 0.047 2.5 0.204 0.126 5.0 0.476 0.292 10.0 1.097 0.662

As shown in Tables 1 and 2 and FIGS. 1 and 2, in the examples, the GOD treatment increased the absorbance indicating the amount of generated hydrogen peroxide as compared with the comparative example. The detection sensitivity was about 1.5 times in Example 1 and about 1.7 times in Example 2 as compared with the comparative example.
From these results, it is understood that the measurement method according to the present invention can improve the measurement accuracy.

[0064]

As described above, according to the measuring method of the present invention, saccharified amine in a sample can be measured simply and with high accuracy. For this reason, if the measurement method of the present invention is applied, for example, to the measurement of HbA1c in red blood cells, the measurement accuracy is improved as compared with the conventional method, and the importance of HbA1c as an indicator for diabetes diagnosis and the like is further improved. .

[Brief description of the drawings]

FIG. 1 is a graph showing the correlation between the FV concentration in a sample and the absorbance in one example of the measurement method of the present invention.

FIG. 2 shows another embodiment of the measuring method of the present invention.
5 is a graph showing the correlation between the FV concentration in a sample and the absorbance.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/72 G01N 33/72 A

Claims (2)

[Claims] 1. A method for measuring a saccharified amine in a sample, wherein the saccharified amine is oxidatively decomposed into sugar and an amine by a saccharified amine oxidase, and
A measuring method in which the sugar is oxidatively decomposed, the amount of the same product or the same consumable by the both oxidative decomposition reactions is measured, and the amount of the saccharified amine is determined from the measured value. 2. A sugar oxidase is added to a reaction solution at a concentration of 1,
The method according to claim 1, wherein the addition is performed so as to be in a range of 000 to 5,000,000 U / liter.