JPH06237794A - Determination of 1,5-anhydroglucitol - Google Patents

Abstract

PURPOSE:To accurately and rapidly measure 1,5-AG by adding pyranose oxidase or L-sorbose oxidase to a specimen which has been subjected to a specific pretreatment and subsequently determining the 1,5-AG. CONSTITUTION:(a) Glucokinase and/or hexokinase, (b) adenosine triphosphate, (c) glucose-6-phosphate dehydrogenase, (d) NAD(p) and/or NAD(p)H, (e) lactic acid dehydrogenase and (f) pyruvic acid are added to a specimen to eliminate contaminating saccharides contained in the specimen. Subsequently, 1,5-AG in the specimen is determined with pyranose oxidase or L-sorbose oxidase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is applied to an accurate, rapid and automatic analyzer for 1,5-anhydroglucitol (hereinafter abbreviated as 1,5-AG) used as a diagnostic marker for diabetes. It concerns a possible measurement method.

[0002]

2. Description of the Related Art 1,5-AG is a cyclic polyol having a structure similar to glucose. This 1,5-AG is present in the blood of healthy people at the next highest concentration (after glucose has a constant value for each individual) in glucose, but in diabetic patients, 1,5-AG is present.
Since the concentration of AG specifically decreases and the rate of decrease is remarkable, it has come to be used for the diagnosis of diabetes.

Conventionally, a separation analysis method such as gas chromatography and liquid chromatography has been used to measure 1,5-AG, but it has not been put to practical use because it is complicated. On the other hand, recently, an enzyme that oxidizes 1,5-AG (hereinafter referred to as 1,5-AG oxidase) has been found, and a measurement method (Japanese Patent Publication No. 3-24200) using this enzyme has been developed. . In addition, although it is 1,5-AG oxidase, pyranose oxidase or L-sorbose oxidase whose substrate specificity is not strict is used, and sugars other than 1,5-AG having reactivity with these enzymes (hereinafter, contaminated saccharides). Measuring method), which is combined with one of several methods for selectively removing (see Japanese Patent Application Laid-Open No. 63-185397).
Issue) was developed.

In Japanese Patent Laid-Open No. 63-185397, as a method for removing contaminant saccharides (mainly glucose in human blood), (1) adsorption with an ion exchange resin (minicolumn), (2) decomposition with hydrochloric acid , (3) sodium borohydride reduction, (4) glucose oxidase oxidation, and (5) hexokinase phosphorylation. Of these methods, the method (1) is the most rigorous and simple, and currently a 1,5-AG assay kit using this method is developed and put on the market as a diagnostic agent. However, considering a busy clinical site, a kit of a technique requiring column manipulation is not always simple, and strong expectations are placed on the development of an automated method.

[0005] Therefore, attention is paid to the enzymatic phosphorylation method of contaminating sugars (mainly glucose) of (5), which does not require column operation and can be applied to a general-purpose automatic analyzer commercially available for biochemical examination. JP-A-1-320998, JP-A-2-104298, and JP-A-3-27.
An improved method is proposed in Japanese Patent Publication No. 299.

[0006]

By the way, glucose in the blood is controlled to an amount of 800 to 1,200 mg / L on an empty stomach and about 1,400 mg / L after a meal.
However, when it becomes diabetic, it rises to more than 3,000.
It is not uncommon to increase from 1 to 4,000 mg / L. 1
It is possible to increase to 50,000 mg / L. On the other hand, 1,
The amount of 5-AG is distributed in the range of 0 to 50 mg / L, and an average of 23 to 25 mg / L in healthy subjects (about 1/4 of glucose)
0), much lower in diabetic patients 4-7 mg on average
/ L, which may be 1 mg / L or less (1 in thousands of glucose). Therefore, it cannot be said that 1,5-AG can be measured with sufficient accuracy unless it is a method capable of completely removing 10,000 mg / L of glucose. That is, in the blood of a diabetic patient with a glucose concentration of 10,000 mg / L, even if 99.99% of glucose can be removed, 1 mg / L of glucose remains and the measured value of 1,5-AG increases.
It cannot be measured accurately.

[0007] Therefore, 1,5-AG automatic which can be measured by a general-purpose biochemical analyzer by using pyranose oxidase or L-sorbose oxidase which has a poor specificity of 1,5-AG and rather strongly reacts with glucose. In order to develop a measurement method, a method for completely removing contaminating sugars mainly containing glucose is required without using column treatment.

However, in the method (5) described in JP-A-63-185397, that is, the method in which hexokinase is used to remove glucose and the remaining 1,5-AG is measured with pyranose oxidase, the hexokinase is used. However, there are problems that glucose cannot be completely removed, that the reaction time of pyranose oxidase is long, and accurate quantification cannot be performed quickly.

Further, in JP-A-1-320998 and JP-A-3-27299, glucose 6-position phosphatase (glucokinase or hexokinase) is used for treating glucose, and the reaction is further completed. It has been devised to combine glucose-6-phosphate dehydrogenase to completely shift the equilibrium of glucose phosphorylation reaction to glucose-6-phosphate. However, with these methods 1,5 in human serum or plasma
When -AG is measured, coexisting serum proteins cause interference, which requires complicated deproteinization operations in advance, which is not suitable for automated methods.

Further, in Japanese Unexamined Patent Publication (Kokai) No. 2-104298, glucokinase or hexokinase is used for the treatment of contaminating saccharides mainly containing glucose, and pyruvate kinase and its substrate phosphoenolpyruvate are used as an ATP supply system. A method of working is proposed,
As a result, ATP required for phosphorylation of glucose at the 6-position
Decrease the concentration of (adenosine triphosphate) and excess ATP on the oxidation reaction of 1,5-AG by pyranose oxidase.
It is said that it has become possible to avoid the inhibition due to the above, and to enable short-time processing that can be fitted to a general-purpose automatic analyzer. However, looking at the measurement accuracy of the sample compared with gas chromatography, there is a problem that it is still insufficient for clinical application.

[0011]

Means for Solving the Problems The present inventors have studied a simple and practical method for measuring 1,5-AG, that is, a method applicable to a general-purpose automatic analyzer, and completed the present invention. did.
That is, the present invention, when measuring 1,5-anhydroglucitol in a sample using pyranose oxidase or L-sorbose oxidase, a glucokinase and / or hexokinase and adenosine triphosphate are previously added to the sample. Glucose-6-phosphate dehydrogenase and NAD
(P) and / or NAD (P) H, lactate dehydrogenase, and pyruvic acid are added and treated 1,
A method for quantifying 5-anhydroglucitol. In the present invention, "NAD (P) and / or NA
"D (P) H" means nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine.・ Dinucleotide phosphate (NADP
It means one or more selected from H).

The 1,5-AG quantification method of the present invention will be described in detail below. In the present invention, 1,5 in the sample
Prior to measuring -AG with pyranose oxidase or L-sorbose oxidase, the sample was (a) glucokinase and / or hexokinase, (b) adenosine triphosphate (ATP), (c) glucose-6- Phosphate dehydrogenase, (d) NAD (P) and / or NAD
(P) H, (e) lactate dehydrogenase, and (f) pyruvic acid are added and treated.

Various kinds of samples can be used,
There is no particular limitation as long as 5-AG is to be quantified, and examples thereof include body fluids such as cerebrospinal fluid, plasma, serum and urine, extracts from plants and animal tissues, and deproteinized products thereof. Glucokinase, hexokinase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase are designated by IUPAC-IUB as EC2.7.1.2 and EC2.7.
1.1, EC 1.1.1.49, EC 1.1.1.17
There is no particular limitation as long as it can be classified into, and commercially available products can be used.

The amount of each enzyme and reagent used varies depending on the amount of glucose in the sample, etc., but is usually as follows. Glucokinase and / or hexokinase: 0.5-
20 U / ml ATP: 0.1-50 mM Glucose-6-phosphate dehydrogenase: 1-100 U / m
l NAD (P) and / or NAD (P) H: 0.1-2
0 mM lactate dehydrogenase: 0.2 to 20 U / ml pyruvate: 0.2 to 20 mM These enzymes and reagents may be added simultaneously or in any order.

By adding the above-mentioned enzymes and reagents to a sample, they act on the sample and, for example, glucose is converted into glucose-6-phosphate by glucokinase and / or hexokinase, and at the same time ATP is converted into ADP. It Further, glucose-6-phosphate dehydrogenase converts glucose-6-phosphate into 6-phosphogluconate, and at the same time, NAD (P) is converted into NAD (P) H, and pyruvate In the presence, a reaction system in which NAD (P) H is converted to NAD (P) by lactate dehydrogenase is established, and contaminant saccharides in the sample are eliminated.

Japanese Unexamined Patent Publication No. 2-104298 discloses an AT.
A method of acting pyruvate kinase and its substrate phosphoenolpyruvate as a P supply system has been proposed, but in the case of this method, the reaction is inhibited by the accumulation of glucose-6-phosphate, Contaminant sugars in the sample could not be completely removed. According to the present invention, glucose-6-phosphate is converted to 6-phosphogluconic acid, so that glucose-6 of glucose by glucokinase
-Conversion to phosphoric acid is efficiently performed, and contaminating sugars in the sample are efficiently removed.

In the case of the reagent using phosphoenolpyruvate, the measurement of 1,5-AG was impossible with 2000 mg / dL of glucose, but according to the present invention, it can be measured normally.

The above-mentioned reaction (the above-mentioned treatment in the present invention) is usually carried out in a buffer of 1 to 400 mM and pH 5 to 10.
It is carried out at 0 to 60 ° C. for about 1 to 60 minutes. Examples of the buffer include phosphate buffer, citrate buffer, citrate phosphate buffer, acetic acid buffer, Tris-hydrochloric acid buffer, Good's buffer, imidazole buffer, triethanolamine buffer and the like.
During the reaction, it is preferable to coexist with salts, and examples of the salts include magnesium salts such as magnesium chloride and magnesium acetate, potassium salts such as potassium chloride and potassium sulfate, and the like. It is preferable to use about 1 to 100 mM of these salts, but it is not particularly limited.

After the contaminating sugars in the sample are eliminated by the above reaction, 1,5-AG is pyranose oxidase or L-.
Quantify using sorbose oxidase. This 1,5
-A method for quantifying AG is known, and is disclosed in JP-A-3-24200.
JP-A-63-185397, JP-A-1-3209
It can be carried out according to the method described in Japanese Patent Publication No. 98, etc.

For example, it can be performed as follows.
That is, glucokinase and / or hexokinase, adenosine triphosphate, glucose-6-phosphate dehydrogenase, NAD (P) and / or NAD (P) H were previously added to the sample.
And lactate dehydrogenase and pyruvate are added to eliminate contaminating saccharides in the sample by their actions, pyranose oxidase or L-sorbose oxidase is added to the sample, and then in the presence of an electron acceptor such as oxygen. To 50 ° C, preferably 25 to 40 ° C, 30 seconds to 3 hours, preferably 2 minutes to
After incubating for 1 hour, a reduction product of an electron acceptor such as hydrogen peroxide produced is measured, and the 1,5-AG amount may be determined from a separately prepared calibration curve.

Pyranose oxidase and L-sorbose oxidase are not particularly limited as long as they can be classified into EC 1.1.3.10 and EC 1.1.3.11 by the nomenclature of IUPAC-IUB, respectively. You can use one. These enzymes are usually 0.5-500 U / mL
Preferably 1 to 50 U / mL is used.

A specific example is as follows. For example, when the reduced form of the electron acceptor is hydrogen peroxide, the method for detecting hydrogen peroxide may be any method as long as it can be detected with high sensitivity, and many methods can be used. The most commonly used method among these is to oxidize various HRP substrates with hydrogen peroxide using horseradish peroxidase (HRP) as a catalytic enzyme. The light substance and chemiluminescence may be measured by absorbance measurement, fluorescence measurement and luminescence measurement, respectively.

As a substrate for HRP which produces a dye,
2'-azinobis (3-ethylbenzothiazoline-6-
Sulfonic acid) (ABTS), o-phenylenediamine (OPD), 5-aminosalicylic acid (5-AS), 3,
3 ', 5,5'-tetramethylbenzidine (TMB),
N- (carboxymethylaminocarbonyl) -4,4 '
-Bis (dimethylamino) -diphenylamine sodium salt (DA-64), 4-aminoantipyrine and phenols, N-ethyl-N-sulfopropyl-m-toluidine or N-ethyl-N- (2-hydroxy-3-)
There is a so-called Trinder-based color-developing agent which is a combination of sulfopropyl) -m-toluidine (TOOS) and the like. As a substrate for HRP that produces a fluorescent substance, p-
Examples include hydroxyphenylacetic acid and 3- (p-hydroxyphenyl) propionic acid (HPPA). Luminol, isoluminol, and the like are known as substrates for chemiluminescent HRP.

Several methods for chemiluminescence detection without HRP are also known. For example, a method of causing luminol to emit light with hydrogen peroxide in the presence of ferricyan ion, a method of emitting lucigenin with hydrogen peroxide in the presence of metal ion, and an allyl oxalic acid such as bis (2,4,6-trichlorophenyl) oxalate. There is known a method of reacting an ester compound with hydrogen peroxide in the presence of a fluorescent substance to excite the fluorescent substance with the decomposition energy of oxalate ester to emit light. Further, as a method for directly detecting hydrogen peroxide, a hydrogen peroxide electrode may be used.

According to the present invention, since the reaction of glucose to glucose-6-phosphate can be carried out almost completely, the quantification of 1,5-AG can be carried out with high accuracy. Further, compared with the method described in Japanese Patent Application Laid-Open No. 2-104298, the glucose processing ability is higher and more accurate measurement results can be obtained. Thus, according to the present invention,
It is possible to quantify 1,5-AG with high accuracy by a quick and simple method, and automatic measurement is possible with a general-purpose biochemical analyzer.

[0026]

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

Comparative Example 1 (Phosphoenolpyruvate-Pyruvate Kinase System) A 1,5-AG standard solution was diluted twice with distilled water and a 10,000 mg / L concentration glucose aqueous solution to give a 1,5-AG concentration of 0. , 1.5, 3.1, 6.3, 12.5, 25, 5
A dilution series of 0 mg / L standard solution was prepared. To these samples 0.05 mL, glucokinase 2 U / mL, ATP 1 mM, 4
-Aminoantipyrine 1.2 mM, N-ethyl-N- (2
-Hydroxy-3-sulfopropyl) -m-toluidine (hereinafter abbreviated as TOOS) 1.2 mM, horseradish peroxidase (hereinafter abbreviated as HRP) 5 U / mL, M
Add 1.15 mL of 50 mM imidazole buffer (pH 7.8) containing 30 mM of gCl ₂ and 75 mM of KCl, and add 3 at 30 ° C.
The reaction was allowed for 0 minutes.

Further, a pyranose oxidase (hereinafter abbreviated as PROD) solution 5 having a concentration of 100 U / mL was added to this reaction solution.
After adding 0 μL and reacting at 30 ° C. for another 30 minutes,
The absorbance at 550 nm was measured. 10,000 mg / L
The results of the calibration curve of the system containing the glucose aqueous solution and the calibration curve of the system containing no glucose are shown in FIG.

Reference Example 1 (calibration curve according to the present invention) 1,5-AG concentration of 0, prepared in the same manner as Comparative Example 1
2U / m of glucokinase was added to 0.05mL dilution series of 1.5, 3.1, 6.3, 12.5, 25, 50mg / L standard solution.
L, ATP 1 mM, glucose-6-phosphate dehydrogenase 1
2 U / mL, NAD 1 mM, lactate dehydrogenase 2 U / mL, pyruvate 2 mM, 4-aminoantipyrine 1.2 mM, TOO
S1.2 mM, HRP 5 U / mL, MgCl ₂ 30 mM, K
HEPES containing 75 mM Cl (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid)
1.15 mL of a buffer solution (pH 7.0) was added and reacted at 30 ° C for 30 minutes.

Further, 50 μL of PROD solution having a concentration of 100 U / mL was added to the reaction solution, and the mixture was reacted at 30 ° C. for another 30 minutes, and then the absorbance at 550 nm was measured. 1
The results of the calibration curve of the system containing the aqueous solution of 10,000 mg / L glucose and the calibration curve of the system containing no glucose are shown in FIG.

As is clear from the comparison between FIG. 1 and FIG. 2, the efficiency of elimination of glucose immediately after preparation of the enzyme reagent is higher than that of the phosphoenolpyruvate-pyruvate kinase system by the method of the present invention glucose-6-phosphorus. Acid dehydrogenase, NA
The system of D, lactate dehydrogenase, and pyruvate was better.

Example 1 (Measurement of 1,5-AG in serum) (1) When glucokinase is used as a glucose phosphatase Reagent R-1 4-aminoantipyrine 1.5 mM MgCl ₂ 7.5 mM KCl 80 mM ATP 1 mM HRP 3.75 U / mL glucokinase 1 U / mL ascorbate oxidase 5 U / mL glucose-6-phosphate dehydrogenase 12 U / mL lactate dehydrogenase 2 U / mL pyruvate 2 mM NAD 1 mM HEPES buffer 50 mM (pH 8 .0) Reagent R-2 TOOS 4.5 mM Pyranose oxidase 100 U / mL HEPES buffer 50 mM (pH 8.0)

Reagent R-1 1.3 in 0.05 mL of human serum
mL was added and it was made to react at 37 degreeC for 5 minutes. Then reagent R
-2 was added in an amount of 0.65 mL, and after 5 minutes, the absorbance at 550 nm was measured. At the same time, a blank using purified water instead of human serum was also measured. 1,5 made in advance
When the 1,5-AG concentration in serum was measured using a -AG calibration curve, it was 31.2 mg / L.

(2) When hexokinase is used as a glucose phosphatase The procedure was the same as (1) except that 20 U / mL hexokinase was used instead of glucokinase. The 1,5-AG concentration in serum was 30.9 mg / L. (3) When NADP is used (1) except that NAD in reagent R-1 is changed to NADP
The same operation was performed. The serum 1,5-AG concentration was 31.
It was 0 mg / L. (4) When NADH is used (1) except that NAD in reagent R-1 is changed to NADH
The same operation was performed. The serum 1,5-AG concentration was 31.
It was 5 mg / L.

Example 2 (Comparison between Adsorption and Removal Method of Glucose by Ion Exchange Resin and Method of the Present Invention) For 30 human serum samples, a conventional mini-column method using an ion exchange resin (Rana AG kit: 1,5-A using the method of Example 1 (1) and manufactured by Nippon Kayaku Co., Ltd.
The G concentration was measured. The measurement of Example 1 (1) was carried out by Hitachi 71
Using a 50 type automatic analyzer, add 10 μL of the sample to the reagent R-1
Was added to the reaction mixture at 37 ° C. for 5 minutes, 130 μL of reagent R-2 was further added, and the reaction was performed at 37 ° C. for 5 minutes.
The change in absorbance was measured at a main wavelength of 546 nm and a sub wavelength of 700 nm.

A correlation diagram of the results obtained by the minicolumn method using an ion exchange resin and the method of the present invention is shown in FIG. The correlation coefficient was 0.99, and a high correlation was observed between the two measurement methods.

[0039]

EFFECTS OF THE INVENTION According to the present invention, 1,5-AG in a sample can be quantitatively determined by a quick and simple method with high accuracy and without being affected by contaminant saccharides, and measurement by an automatic analyzer is possible. Is.

[Brief description of drawings]

FIG. 1 1,5-AG calibration curve (glucose treatment performed with phosphoenolpyruvate-pyruvate kinase system)

FIG. 2 1,5-AG calibration curve (glucose treated by the method of the present invention)

FIG. 3: 1,5-A by the minicolumn method and the method of the present invention
Correlation diagram of G quantitative results

Claims (1)

[Claims] 1. When measuring 1,5-anhydroglucitol in a sample using pyranose oxidase or L-sorbose oxidase, glucokinase and / or hexokinase and adenosine triphosphate are previously added to the sample. Glucose-6-phosphate dehydrogenase and NAD
(P) and / or NAD (P) H, lactate dehydrogenase, and pyruvic acid are added and treated 1,
Method for quantifying 5-anhydroglucitol.