JP3415873B2 - Determination of 1,5-anhydroglucitol - Google Patents

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. . Further, 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 referred to as contaminating saccharides). ) In combination with one of several methods for the selective removal of
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 can be measured by a general-purpose biochemical analyzer using pyranose oxidase or L-sorbose oxidase, which has a low specificity for 1,5-AG and reacts strongly with glucose. In order to develop an automatic measurement method, a method for completely removing contaminating sugars mainly containing glucose is required without using a 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 the treatment of glucose in order to further complete the reaction. , Glucose-6-phosphate dehydrogenase are combined to make the equilibrium of the glucose phosphorylation reaction completely toward 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 composed of glucose, and pyruvate kinase and its substrate, phosphoenolpyruvate, are used as an ATP supply system. Has been 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 is: (1) When measuring 1,5-anhydroglucitol in a sample, glucokinase and / or hexokinase, adenosine triphosphate and / or adenosine diphosphate are previously added to the sample; A method for quantifying 1,5-anhydroglucitol, which comprises adding and treating 3-phosphoglycerate, phosphoglycerate mutase, enolase, and pyruvate kinase. (2) The quantification method according to (1) above, wherein 1,5-anhydroglucitol is measured with pyranose oxidase or L-sorbose oxidase. Regarding

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
-Before assaying AG with, for example, pyranose oxidase or L-sorbose oxidase, the sample is (a) glucokinase and / or hexokinase, (b) adenosine triphosphate (ATP) and / or adenosine diphosphate. Acid (ADP), (c) 3-phosphoglycerate, (d) phosphoglycerate mutase, (e) enolase, and (f) pyruvate kinase 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, phosphoglycerate mutase, enolase and pyruvate kinase are IUPAC-
The IUB nomenclature is EC 2.7.1.2, EC respectively.
2.7.1.1, EC 5.4.2.1, EC 4.2.
There is no particular limitation as long as it can be classified into 1.11, EC 2.7.1.10, 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: 0.5-2
0 U / ml ATP and / or ADP: 0.1-10 mM 3-phosphoglycerate: 1-20 mM Phosphoglycerate mutase: 0.5-10 U / ml Enolase: 0.5-10 U / ml pyruvate kinase : 0.5 to 20 U / ml These enzymes and reagents may be added at the same time 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. In addition, a reaction system in which ADP is converted to ATP by phosphoglycerate mutase, enolase, and pyruvate kinase in the presence of 3-phosphoglycerate is established, and contaminant saccharides in the sample are eliminated.

Japanese Unexamined Patent Publication No. 2-104298 discloses an AT.
A method has been proposed in which pyruvate kinase and its substrate, phosphoenolpyruvate, act as a P-supplying system, and contaminant saccharides in a sample are efficiently removed, but the stability of phosphoenolpyruvate is poor. Therefore, the reagent cannot be stored for a long time. According to the present invention, 3-
Since phosphoglyceric acid has good stability, it can be stably used for measurement for a long time.

Further, in the case of a reagent using phosphoenolpyruvate, deterioration is observed after about 1 week of storage in the refrigerator,
The reagent of the present invention can be used for 2 weeks.

The above-mentioned reaction (the above-mentioned treatment in the present invention) is usually carried out in a buffer of 1 to 200 mM, 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, Tris-hydrochloric acid buffer, Good's buffer, imidazole 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 and adenosine triphosphate and / or adenosine diphosphate and 3-phosphoglycerate, phosphoglycerate mutase, and enolase are added to the sample in advance, and these effects cause contamination in the sample. After eliminating the sugar, pyranose oxidase or L-sorbose oxidase is added to this,
In the presence of an electron acceptor such as oxygen, 4 to 50 ° C., preferably 2
After incubating at 5 to 40 ° C. for 30 seconds to 3 hours, preferably 2 minutes to 1 hour, the reductant of the electron acceptor such as hydrogen peroxide produced is measured, and 1,5 is obtained from a separately prepared calibration curve. -The amount of AG should be calculated.

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, the amount of ATP present in the reaction system can be controlled within the optimum range, and therefore, the reaction is not suppressed when quantifying 1,5-AG. Further, according to the present invention, the reaction of glucose to glucose-6-phosphate can be carried out almost completely, so that 1,5-AG can be quantified 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. As described above, according to the present invention, 1,5-AG can be accurately quantified by a quick and simple method, and automatic measurement can be performed by 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 0.05 mL of these samples, 2 U / mL of glucokinase, 1 mM of ATP, 6 mM of phosphoenolpyruvate, 1 U of pyruvate kinase
/ ML, 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) 5U
/ ML, MgCl ₂ 30 mM, KCl 75 mM 50 mM
Add 1.15 mL of imidazole buffer (pH 7.8) and add 3
The reaction was carried out at 0 ° C for 30 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.

Comparative Example 2 In Comparative Example 1, an enzyme stored in a refrigerator for 7 days was used, and a calibration curve was prepared in the same manner as in Comparative Example 1. The results of the calibration curve of the system containing the 10,000 mg / L 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, pyruvate kinase 4 U / mL, 3-phosphoglycerate 6 mM, phosphoglycerate mutase 3 U / mL, enolase 3 U / mL, 4-aminoantipyrine 1.2 mM, TOOS 1.2 mM, HRP 5 U / mL, M
HEPES containing ₂ mM of gCl ₂ and 75 mM of KCl (2
-[4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) buffer (pH 7.0) 1.15
mL was added and it was made to react at 30 degreeC 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 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.

Reference Example 2 In Reference Example 1, a calibration curve was prepared in the same manner as in Reference Example 1 except that the enzyme was stored in a refrigerator for 7 days. 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 not containing glucose are shown in FIG.

As is clear from the comparison between FIG. 1 and FIG. 3, the efficiency of glucose elimination immediately after preparation of the enzyme reagent was almost unchanged. However, as is clear from the comparison of FIG. 2 and FIG. 4, the efficiency of glucose elimination after storage is higher than that of the phosphoenolpyruvate-pyruvate kinase system by the method of the present invention. The glycerate mutase-enolase-pyruvate kinase system was better.

Example 1 (Measurement of 1,5-AG in serum) (1) When glucokinase is used as a glucose phosphatase Reagent R-14 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 pyruvate kinase 4 U / mL ascorbate oxidase 5 U / mL phosphoglycerate mutase 3 U / mL enolase 3 U / mL 3-phosphoglycerate 6 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 28.8 mg / L.

(2) When hexokinase is used as a glucose phosphatase (2-1) The same operation as (1) was performed except that 20 U / mL hexokinase was used instead of glucokinase.
The 1,5-AG concentration in serum was 28.3 mg / L. (2-2) The same operation as (2-1) was performed except that ATP1 mM in the reagent R-1 was changed to ADP1 mM. 1, in the serum
The 5-AG concentration was 28.7 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 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 0.97, and 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 accurately quantified by a quick and simple method and can be measured by an automatic analyzer.

[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 (using an enzyme solution stored in a refrigerator for 7 days. Glucose treatment was performed with a phosphoenolpyruvate-pyruvate kinase system)

FIG. 3 1,5-AG calibration curve (glucose treated by the method of the present invention)

[Fig. 4] 1,5-AG calibration curve (using an enzyme solution stored in a refrigerator for 7 days; subjected to glucose treatment by the method of the present invention)

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

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-2-104298 (JP, A) Agric. Biol. Chem. , Vol. 48, p. 355-364 (1984) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12Q 1/26 C12Q 1/48 C12Q 1/527 BIOSIS / CA / MEDLINE / W PIDS (STN)

Claims (2)

(57) [Claims] 1. When measuring 1,5-anhydroglucitol in a sample, glucokinase and / or hexokinase, adenosine triphosphate and / or adenosine diphosphate, and 3-phosphine are previously added to the sample. A method for quantifying 1,5-anhydroglucitol, which comprises adding and treating foglycerate, phosphoglycerate mutase, enolase, and pyruvate kinase. 2. The assay method according to claim 1, wherein the measurement of 1,5-anhydroglucitol is performed with pyranose oxidase or L-sorbose oxidase.