JP3402487B2 - Determination of 1,5-anhydroglucitol - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to 1,5-anhydroglucitol (hereinafter referred to as 1,5-A) used for diagnosis of diabetes.
Abbreviated as G).

[0002]

2. Description of the Related Art It has been reported that 1,5-AG is present in human body fluids, and the amount of serum and plasma is decreased in certain diseases, especially in diabetes, and the quantification of 1,5-AG can diagnose diabetes. Possible compounds.

Conventionally, a gas chromatography method has been used as a method for quantifying 1,5-AG, but it is difficult to process a large amount of a sample, and therefore, a measuring method using an enzyme specific to 1,5-AG. (JP-A-62-79780) or pyranose oxidase (EC1,1,3,10) or L-sorbose oxidase (EC1,1,3,1)
A measuring method using 1) (Japanese Patent Laid-Open No. 185397/1988) was developed.

In the measuring method using pyranose oxidase or L-sorbose oxidase, since the specificity to 1,5-AG is low, it is necessary to measure after removing sugars such as glucose coexisting in serum or the like. A method for efficiently removing the coexisting glucose and the like has also been proposed (JP-A-2-104298). In the method using this pyranose oxidase or L-sorbose oxidase, 1,5-AG is oxidized by these enzymes and the hydrogen peroxide produced at the same time is quantitatively measured.

[0005]

Since the structure of 1,5-AG is similar to glucose in that it does not have a hydroxyl group at the 1-position but has a similar three-dimensional structure, it acts selectively on 1,5-AG. A specific enzyme that can withstand practical use is not known. Therefore, pyranose oxidase or L-sorbose oxidase having low specificity is used for the quantification of 1,5-AG, and therefore, saccharides such as glucose are removed by pretreatment, and 1,5-AG is oxidized. And quantified. However, in the conventional methods using these pyranose oxidases or L-sorbose oxidases, there are rare specimens in which the measured value becomes an abnormally high value.
Cannot measure AG.

[0006]

[Means for Solving the Problems] Pyranose oxidase or L-sorbose oxidase, which is an oxidase for 1,5-AG, has low specificity as an enzyme and therefore oxidizes various monosaccharides. In particular, since the reactivity with a large amount of glucose present in blood is large, an accurate quantitative value must be obtained after removing glucose by some method when measuring 1,5-AG in serum or plasma. Can't get However, even after measurement after removing glucose, the measured value shows an abnormally high value, and it was found that there are samples in which the measured value fluctuates because reproducibility is not obtained, and these causative substances select sugars in the sample. It was found to be maltose (a dimer of glucose) that cannot be completely removed by the conventional method of removing it.

As an infusion solution for inpatients including diabetic patients, an infusion solution containing maltose may be used, and after the infusion, blood is collected and used as a sample for 1,5-AG measurement. The value is greatly deviated, and it is disclosed in JP-A-63-18.
It was found that the pretreatment with the column described in 5397 (sugar removal column) does not provide reproducibility of measurement and the measured value fluctuates.

The present inventors have investigated the cause of maltose affecting the measurement, and have earnestly studied a method for avoiding the effect. Pyranose oxidase or L-sorbose oxidase hardly reacts with maltose, which interferes with the measurement.
It was found that glucose was regenerated from maltose by α-glucosidase contained as an impurity in sorbose oxidase. That is, this glucose is oxidized by pyranose oxidase or L-sorbose oxidase, resulting in a large measurement error.

Therefore, one of the sample specimens containing maltose
In the measurement of 5-AG, it was thought that interference can be avoided by controlling α-glucosidase well, and the method was studied thoroughly, and the present invention was completed.

That is, the present invention provides (1) selective removal of saccharides from a sample or 1,5-A
G and glucose are converted into a compound that does not react with an enzyme having an ability to oxidize or dehydrogenate, and
5-AG is allowed to act with an enzyme having the ability to oxidize or dehydrogenate 1,5-AG and glucose and is substantially free of α-glucosidase, or in the presence of an α-glucosidase inhibitor. , 5-AG and an enzyme having an ability to oxidize or dehydrogenate glucose are allowed to act on the assay method for 1,5-AG,

(2) The sample is treated with α-glucosidase, and then saccharides in the sample are selectively removed or 1,5
-Converting AG and glucose into a compound that does not react with an enzyme that has the ability to oxidize or dehydrogenate, and has the ability to oxidize or dehydrogenate 1,5-AG and glucose to 1,5-AG in the obtained sample A method for quantifying 1,5-AG characterized by allowing an enzyme to act,

(3) In a sample obtained by selectively converting saccharides in a sample in the presence of α-glucosidase into a compound that does not react with an enzyme capable of oxidizing or dehydrogenating 1,5-AG and glucose 1,5-AG to 1,5-A
A method for quantifying 1,5-AG, which comprises reacting an enzyme having the ability to oxidize or dehydrogenate G and glucose,

(4) The above-mentioned (1), (2), wherein the enzyme is pyranose oxidase or L-sorbose oxidase.
Or a quantification method of 1,5-AG according to (3),

(5) The present invention relates to the method for quantifying 1,5-AG according to the above (1), wherein the content of α-glucosidase in the enzyme is 1 × 10 ⁻³ unit / unit or less.

In the above method (1), if maltose is contained in the sample so that it does not change to glucose,
Since it can be measured without interference, it is a method of using an enzyme substantially free of α-glucosidase, or in the presence of an inhibitor of α-glucosidase, that is, a condition under which α-glucosidase does not act. .

In the above method (2), maltose contained in a sample is converted into glucose by α-glucosidase in advance to prepare a sample containing no maltose, and then saccharides are removed or converted from the sample, and then 1,5 -A method of measuring AG.

In the above method (3), when glucose in a sample is converted to a compound other than glucose by enzymatic conversion or the like, α-glucosidase is allowed to coexist to convert maltose into glucose, and at the same time glucose is converted into a compound other than glucose. It is a method of measuring 1,5-AG after changing.

By adopting these methods, the influence of maltose mixed in the sample on the measured value can be avoided, and the true 1,5-AG value can be measured.

The samples used in the present invention are 1,5
-If AG is to be quantified, there is no particular limitation.
Examples include cerebrospinal fluid, plasma, serum and urine.

In the quantification methods (1) to (3), the method for selectively removing the saccharides in the sample is not particularly limited,
Various methods can be used. For example, JP-A-63-1853
97, that is, a method of treating an analyte with a strongly basic anion exchange resin, a method of treating an analyte with boric acid itself or a resin to which boric acid is bound, 1,5
-A method of utilizing the chemical stability of AG, a method of modifying a saccharide such as glucose with an enzyme as a catalyst, and the like.

In the method (2), when the sample is treated with α-glucosidase, the concentration of α-glucosidase is preferably 1 unit / ml or less, and particularly 1 uni.
t / ml to 1 × 10 ⁻³ unit / ml is preferable, and
The treatment temperature is preferably 15 to 40 ° C, particularly 25 to 37 ° C.
It is preferable to carry out.

In the above (1), (2) or (3),
As a method of converting glucose in a sample into a compound that does not react with 1,5-AG used in the present invention and an enzyme having an ability to oxidize or dehydrogenate glucose, glucose is converted into glucose using an enzyme such as glucokinase or hexokinase. There is a method of changing to glucose 6-phosphate and the method is not particularly limited. When α-glucosidase is present during this conversion, the concentration of α-glucosidase is preferably 1 unit / ml or less, particularly 1 u.
It is preferably nit / ml to 1 × 10 ⁻³ unit / ml.
The temperature at which glucose is converted in the presence or absence of α-glucosidase is generally 15 to 40 ° C.
The degree is preferable and it is most preferable to carry out at 25 to 37 ° C.

Examples of the enzyme having the ability to oxidize or dehydrogenate 1,5-AG and glucose used in the above methods (1) to (3) include, for example, pyranose oxidase, L-sorbose oxidase, and 1,5 anhydro. Examples thereof include glucitol dehydrogenase.

Enzymes capable of oxidizing 1,5-AG and glucose (eg pyranose oxidase or L
-Sorbose oxidase), these enzymes can act on 1,5-AG in a sample in the presence of oxygen, and 1,5-AG can be quantified from the amount of hydrogen peroxide and the like produced. .

Various producing bacteria of pyranose oxidase or L-sorbose oxidase are known, for example, strains of the genus Polyporus, the genus Coriolus, the genus Daedaleopsis, the genus Proirotas, the genus Basidiomycetaus, etc. are known. These strains are cultivated and purified using various purification methods, but most strains have α-glucosidase and cannot be excluded unless the purification purity is increased. Therefore, practically used pyranose oxidase or L-sorbose oxidase contains a very small amount of α-glucosidase, and maltose is hydrolyzed into glucose by the small amount of α-glucosidase, and the produced glucose is pyranose. It is oxidized by oxidase or L-sorbose oxidase to generate hydrogen peroxide, which develops color to increase the quantitative value.

The α-glucosidase in pyranose oxidase or L-sorbose oxidase can be confirmed by allowing pyranose oxidase or L-sorbose oxidase to act in a maltose solution for 1 hour and then detecting hydrogen peroxide. . If hydrogen peroxide is detected at this time, it means that maltose is hydrolyzed into glucose by α-glucosidase in pyranose oxidase or L-sorbose oxidase, and hydrogen peroxide is produced by pyranose oxidase or L-sorbose oxidase. Therefore, in the method (1) using an enzyme that does not substantially contain α-glucosidase, it is necessary to use an enzyme such as pyranose oxidase or L-sorbose oxidase, under which hydrogen peroxide is hardly detected. .

In commercially available pyranose oxidase or L-sorbose oxidase, α-glucosidase contains 1 × 10 ⁻³ to 1 × 10 ⁻² unit / unit.
Many of them are mixed with (pyranose oxidase or L-sorbose oxidase), but this amount completely pushes up the 1,5-AG measurement value. 1,5-A
The content of α-glucosidase that does not substantially affect the measured value of G is preferably 1 × 10 ⁻³ unit / unit or less, and more preferably 1 × 10 ⁻⁴ unit / unit or less. At such an amount, almost no increase in the measured value is observed within 1 hour of the reaction time for colorimetric determination.

The enzymatic activity of α-glucosidase is p
This is calculated from the amount of change in absorbance at 400 nm at 37 ° C. for 15 minutes using -nitrophenyl-α-D-glucopyranoside as a substrate. The enzyme activity of pyranose oxidase or L-sorbose oxidase is calculated from the change in absorbance at 500 nm for 10 minutes using 4-aminoantipyrine and phenol at 37 ° C. with glucose as a substrate. The specific enzyme activity of α-glucosidase is divided by the specific enzyme activity of pyranose oxidase or L-sorbose oxidase to obtain α-glucosidase uni
t / unit (pyranose oxidase or L-sorbose oxidase) can be calculated. Here, 1 unit is defined as the amount of enzyme that converts 1 μmole of substrate in 1 minute.

When an enzyme having the ability to dehydrogenate 1,5-AG and glucose (eg, 1,5-anhydroglucitol dehydrogenase described in JP-A-2-268679) is used, these are used. The enzyme acts on 1,5-AG in the sample in the presence of an electron acceptor such as a coenzyme, and from the amount of the reduced form of the electron acceptor produced,
1,5-AG can be quantified (JP-A-62-7)
9780, etc.).

In the method (1) using an enzyme substantially free of α-glucosidase, 1,5
-As an enzyme having the ability to oxidize or dehydrogenate AG and glucose, an enzyme that does not substantially contain α-glucosidase is used, but the content of α-glucosidase in the enzyme is 1 x 10 ^-3 unit / unit or less. Is preferable, and particularly preferably 1 × 10 ⁻⁴ unit / unit or less. In the method using the α-glucosidase inhibitor in the method (1), 1,5-AG
As the enzyme having the ability to oxidize or dehydrogenate glucose, it is not particularly necessary to use an enzyme that does not substantially contain α-glucosidase, and there is no problem even if an enzyme that substantially contains α-glucosidase is used. Of course, it is also possible to use a substance that does not substantially contain α-glucosidase.

In the methods (1) to (3) of the present invention, the enzyme capable of oxidizing or dehydrogenating 1,5-AG and glucose is usually used in a solution dissolved in a buffer solution. Enzymes immobilized by a conventional method such as capsules, those covalently bound to or adsorbed on resins and polysaccharides can also be used. Although the amount used varies depending on the amount of the sample and the like, the concentration during the reaction may be 1 unit / ml or more in order to make the reaction time by the enzyme a suitable short time. It goes without saying that the higher the specific activity of the enzyme used, the better the reaction.

In the method using the α-glucosidase inhibitor in the above (1), various ones can be used as the α-glucosidase inhibitor, for example, those with competitive inhibition of a substrate analog and general enzyme inhibitors. There are heavy metals such as For example, castanospermine, (+)-1 deoxynojirimycin and the like. The usage amount has an optimum range for each. For example, in the case of castanospermine or (+)-1 deoxynojirimycin, 0.1
The range from μM to 100 μM is preferred.

The quantification method of the present invention can be carried out according to a known method, for example, as follows. That is, an electron acceptor such as oxygen and 1,5-
An enzyme having an ability to oxidize or dehydrogenate AG and glucose is present at 0.5 to 100 unit, preferably 1 to 50 unit per 1 ml of a sample, and 4 to 50 ° C., preferably 25 to 40 ° C., for 30 seconds to 3 hours, Preferably 1 minute to 1
After incubating for a period of time, the amount of reductant of the electron acceptor such as the amount of hydrogen peroxide produced, etc. is measured, and the 1,5-AG amount may be determined from a separately prepared calibration curve. A specific example is as follows.

For example, when the reduced form of the electron acceptor is hydrogen peroxide, any method can be used for detecting hydrogen peroxide 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. Absorbance measurement of light substances and chemiluminescence,
Fluorescence measurement and luminescence measurement may be performed.

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 type color-developing agent which is a combination of sulfopropyl) -m-toluidine (TOOS) and the like. Substrates for HRP that produce a fluorescent substance include p-hydroxyphenylacetic acid and 3- (p-hydroxyenyl) propionic acid (HPPA). Luminol, isoluminol, and the like are known as substrates for chemiluminescent HRP.

There are several known methods for chemiluminescence detection without HRP. 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.

For example, when the reductant of the electron acceptor is the reductant of the coenzyme, the 1,5-AG amount can be measured by measuring the absorbance of the reductant of the coenzyme and the fluorescence. It is possible to generate hydrogen peroxide from the reductant of the coenzyme, and measure this amount in the same manner as above to obtain 1,5-A
The amount of G can also be measured.

According to the present invention, the amount of 1,5-AG in body fluids such as serum and plasma can be accurately measured, and it is a very useful method which can be used for discrimination of diabetes.

[0039]

EXAMPLES The present invention will be described in more detail below with reference to Examples and Comparative Examples.

Example 1 250 units of pyranose oxidase containing 8.0 × 10 ⁻⁸ units of α-glucosidase per unit of pyranose oxidase, 6 units of horseradish peroxidase and 2,2′-azinobis (3-e)
thylbenothiazolin-6-sulfo
nic acid) 0.1 mmole was dissolved in 25 ml of 0.2 M phosphate buffer (pH 6.0) to give a color developing solution. Maltose 300mg / dl and 1,5-AG 25μ
Aqueous solution containing g / ml (standard sample), maltose 10
% Infusion solution containing 25% of maltose (25 g of maltose) was taken immediately after instillation for about 1 hour, and serum A and B (serum A (maltose content 200.2 mg / dl), serum B
(Maltose content 128.8 mg / dl) was used to measure 1,5-AG. 5 of standard sample or each serum
0 μl was passed through a pretreatment column Lana AG (manufactured by Nippon Kayaku) consisting of a strongly basic anion exchange resin, and then purified water 0.5
Elute twice with ml.

To 1.05 ml of this eluate, 200 μl of the coloring solution prepared above was added, stirred and reacted at room temperature for 1 hour. Absorbance at 420 nm was measured using distilled water as a control, and quantification was performed using a calibration curve prepared from the absorbance of an aqueous solution of 1,5-AG having a known concentration and simultaneously subjected to column treatment. The results were as follows. Standard sample 24.9 μg / ml Serum A 2.1 μg / ml Serum B 8.4 μg / ml

Example 2 250 units of pyranose oxidase containing 4.7 × 10 ^-3 units of α-glucosidase per unit of pyranose oxidase, 6 units of horseradish peroxidase, 2,2'-azinobis (3-eth)
ylbenzothiazolin-6-sulfon
ic acid) 0.1 mmole and (+)-1 deoxynojirimycin 0.5 mmole were dissolved in 25 ml of 0.2 M phosphate buffer (pH 6.0) to give a color developing solution. The results of measuring the amount of 1.5-AG in the same manner as in Example 1 using the color developing solution and the same standard sample and each serum as in Example 1 are shown below. Standard sample 25.4 μg / ml Serum A 2.6 μg / ml Serum B 8.5 μg / ml

Example 3 Pyranose oxidase 250uni used in Example 2
t, horseradish peroxidase 6 unit, 2, 2 '
-Azinobis (3-ethylbenzothi
azolin-6-sulfonic acid) 0.
1 mmole and castanospermine 0.5 mmole
e was dissolved in 25 ml of 0.2 M phosphate buffer (pH 6.0) to give a color developing solution. Using this color-developing solution, the results of measuring the amount of 1,5-AG in the same standard sample as in Example 1 and each serum in the same manner as in Example 1 are shown below. Standard sample 25.8 μg / ml Serum A 2.7 μg / ml Serum B 8.9 μg / ml

Example 4 To the standard sample used in Example 1 and 0.9 ml of each serum,
Using 0.1 ml of a phosphate buffer containing 2.5 units of α-glycosidase and treating at 37 ° C. for 30 minutes, each was used and passed through a pretreatment column in the same manner as in Example 1 to obtain an eluate for each. It was The standard for the calibration curve was diluted with purified water to 9: 1 (1,5-AG standard solution: water), and similarly treated with a pretreatment column to obtain an eluate. The color forming liquid was α used in Comparative Example 1 shown below.
The amount of 1,5-AG was measured in the same manner as in Example 1 except that the coloring solution containing glycosidase was used. The results are shown below. Standard sample 25.1 μg / ml Serum A 2.2 μg / ml Serum B 8.2 μg / ml

Example 5 To the standard sample used in Example 1 and 50 μl of each serum,
Glucokinase 4.6 unit, pyruvate kinase 1.2 unit, phosphoenolpyruvate 4.6 μm
ole, adenosine triphosphate 0.6 μmole and 1
1.0 ml of 0.05 M Tris-hydrochloric acid buffer solution (pH 7.4) containing 6.5 μmole of MgCl ₂ was added, and the mixture was reacted at 25 ° C. for 30 minutes. Using the color-developing liquid used in Example 1 for each of the obtained samples, a color-forming reaction was carried out in the same manner as in Example 1 to measure the amount of 1.5-AG. The results are shown below. Standard sample 25.8 μg / ml Serum A 2.9 μg / ml Serum B 9.1 μg / ml

Example 6 In Example 5, the color developing solution used in Example 2 was used instead of the color developing solution used in Example 1, and the amount of 1,5-AG was changed in the same manner as in Example 5. It was measured. The results are shown below. Standard sample 26.2 μg / ml Serum A 3.3 μg / ml Serum B 9.2 μg / ml

Example 7 50 μl of the standard sample and each serum used in Example 1 were added with glucokinase 4.6 unit and pyruvate kinase 1.
2 unit, 0.05 unit of α-glucosidase, 4.6 μmole of phosphoenolpyruvate, 0.6 μmole of adenosine triphosphate, and 0.05 M tris-hydrochloric acid buffer (pH: 0.6 μmole) containing MgCl ₂ of 16.5 μmole.
7.4) 1.0 ml was added and reacted at 25 ° C. for 30 minutes. For the obtained sample, using the color-developing liquid containing α-glucosidase used in Comparative Example 1 shown below,
A color reaction was performed in the same manner as in Example 1 to measure the amount of 1,5-AG. The results are shown below. Standard sample 25.6 μg / ml Serum A 3.0 μg / ml Serum B 8.5 μg / ml

Example 8 To the standard sample used in Example 1 and 0.9 ml of each serum,
Phosphate buffer solution 0.1 ml containing α-glucosidase 2.5 unit was added and treated at 37 ° C. for 30 minutes, and to 50 μl of the obtained treatment solution, 1.0 ml of enzyme solution containing glucokinase used in Example 5 was added. The mixture was further reacted at 25 ° C for 30 minutes. With respect to each of the obtained samples, a color forming reaction was performed in the same manner as in Example 1 using the color forming liquid used in Comparative Example 1 described below, and the 1,5-AG amount was measured. The results are shown below. Standard sample 25.5 μg / ml Serum A 3.2 μg / ml Serum B 8.8 μg / ml

Examples 9 to 13 In Examples 1 to 3 and 5 to 6, instead of 250 unit of pyranose oxidase containing 8.0 × 10 ^-8 unit of α-glucosidase per unit of pyranose oxidase as a component of the color developing solution, An experiment was conducted in the same manner as in Examples 1 to 3 and 5 to 6, except that 250 units of L-sorbose oxidase containing 7.3 × 10 ^-6 units of α-glucosidase per unit of L-sorbose oxidase was used. Results similar to those of Examples 1-3 and 5-6 were obtained.

Comparative Example 1 250 units of pyranose oxidase containing 4.7 × 10 ⁻³ units of α-glucosidase per unit of pyranose oxidase, 6 units of horseradish peroxidase and 2,2′-azinobis (3-)
ethylbenzothiazolin-6-sul
0.1 mmole of fonic acid) was dissolved in 25 ml of 0.2 M phosphate buffer (pH 6.0) to give a coloring solution. Using this coloring solution, the amount of 1,5-AG was measured in the same manner as in Example 1 for the same standard sample and each serum as in Example 1. The results were as follows. Standard sample 125.5 μg / ml (scale-over) Serum A 34.1 μg / ml Serum B 78.5 μg / ml (scale-over)

Comparative Example 2 With respect to each sample obtained by pretreatment in Example 5, a coloring reaction was carried out in the same manner as in Example 5 using the coloring reagent used in Comparative Example 1, and the 1,5-AG amount was adjusted. It was measured. The results were as follows. Standard sample 220.9 μg / ml (scale-over) Serum A 52.3 μg / ml (scale-over) Serum B 103.4 μg / ml (scale-over)

Example 14 Next, a method using an automatic analyzer 7150 type (manufactured by Hitachi Ltd.) will be shown. The same standard sample as in Example 1 and 1 for each serum
260 μl of the following reagent R-1 was added to 0 μl, and the mixture was reacted at 37 ° C. for 5 minutes.
After adding 1 liter and reacting at 37 ° C. for 5 minutes, the main wavelength was 546n
m, change in absorbance at subwavelength 700 nm
-AG concentration of 0 μg / ml saline) and 50 μg / ml
From the calibration curve obtained from the ml standard solution, the following 1,5-AG
A quantitative value of was obtained.

Reagent R-1 4-Aminoantipyrine 1.5 mM MgCl ₂ 15 mM KCl 80 mM ATP 1 mM Glucokinase 2 U / ml Pyruvate kinase 3 U / ml Phosphoenolpyruvate 4 mM Ascorbate oxidase 5 U / ml HEPES buffer 50 mM (p
H8.0)

Reagent R-2 TOOS 4.5 mM Pyranose oxidase 100 U / ml (used in Example 1) HRP 3.75 U / ml HEPES buffer 50 mM (p
H8.0) where ATP is adenosine triphosphate and HEPES is 2
-[4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid.

Quantitative value of 1,5-AG Standard sample 24.9 μg / ml Serum A 2.3 μg / ml Serum B 8.6 μg / ml

Example 15 As samples R-1 and R-2, the following were used, and other tests were carried out in the same manner as in Example 14, and the following results were obtained. Reagent R-1 4-aminoantipyrine 1.5 mM MgCl ₂ 15 mM KCl 80 mM ATP 1 mM Glucokinase 2 U / ml Pyruvate kinase 3 U / ml Phosphoenolpyruvate 4 mM Ascorbate oxidase 5 U / ml HEPES buffer 50 mM (p
H8.0)

Reagent R-2 TOOS 4.5 mM (+)-1 Deoxynojirimaincin 10 mM Pyranose oxidase 100 U / ml (used in Comparative Example 1) HRP 3.75 U / ml HEPES buffer 50 mM (p
H8.0)

Quantitative value of 1,5-AG Standard sample 24.8 μg / ml Serum A 2.2 μg / ml Serum B 8.8 μg / ml

Example 16 As samples R-1 and R-2, the following were used, and other tests were conducted in the same manner as in Example 14, and the following results were obtained. Reagent R-1 4-Aminoantipyrine 1.5 mM MgCl ₂ 15 mM KCl 80 mM ATP 1 mM Glucokinase 2 U / ml Pyruvate kinase 3 U / ml Phosphoenolpyruvate 4 mM Ascorbate oxidase 5 U / ml α-Glucosidase 1 U / ml HEPES buffer 50 mM (p
H8.0)

Reagent R-2 TOOS 4.5 mM Pyranose oxidase 100 U / ml (used in Comparative Example 1) HRP 3.75 U / ml HEPES buffer 50 mM (p
H8.0)

Quantitative value of 1,5-AG Standard sample 25.1 μg / ml Serum A 2.4 μg / ml Serum B 8.3 μg / ml

Example 17 The same standard sample as in Example 1 and 0.9 ml of each serum were treated with α
Measurement was carried out in the same manner as in Example 14 by using 0.1 ml of a phosphate buffer containing 2.5 units of glucosidase and treating at 37 ° C. for 30 minutes as each sample. The results were as follows.

Reagent R-1 4-Aminoantipyrine 1.5 mM MgCl ₂ 15 mM KCl 80 mM ATP 1 mM Glucokinase 2 U / ml Pyruvate kinase 3 U / ml Phosphoenolpyruvate 4 mM Ascorbate oxidase 5 U / ml HEPES buffer 50 mM (p
H8.0)

Reagent R-2 TOOS 4.5 mM Pyranose oxidase 100 U / ml (used in Comparative Example 1) HRP 3.75 U / ml HEPES buffer 50 mM (p
H8.0)

Quantitative value of 1,5-AG Treatment standard sample 25.0 μg / ml Treated serum A 2.2 μg / ml Treated serum B 8.2 μg / ml

Comparative Example 3 As samples R-1 and R-2, the following were used, and other tests were conducted in the same manner as in Example 14, and the following results were obtained. Reagent R-1 4-aminoantipyrine 1.5 mM MgCl ₂ 15 mM KCl 80 mM ATP 1 mM Glucokinase 2 U / ml Pyruvate kinase 3 U / ml Phosphoenolpyruvate 4 mM Ascorbate oxidase 5 U / ml HEPES buffer 50 mM (p
H8.0)

Reagent R-2 TOOS 4.5 mM Pyranose oxidase 100 U / ml (used in Comparative Example 1) HRP 3.75 U / ml HEPES buffer 50 mM (p
H8.0)

Quantitative value of 1,5-AG Standard sample 208.4 μg / ml Serum A 60.2 μg / ml Serum B 81.5 μg / ml

[0069]

As shown in the examples and comparative examples, the conventional method causes a large error when the serum of a patient who has received an infusion containing maltose is used as a sample. By using the method according to the present invention, the amount of 1,5-AG can be accurately measured without knowing the origin of a sample such as serum. Therefore, when 1,5-AG is used for the determination of diabetes or the progress of treatment, accurate diagnosis can be performed.

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Claims (4)

(57) [Claims] 1. A saccharide in a sample is selectively removed or
5-anhydroglucitol and glucose are converted into a compound that does not react with an enzyme having the ability to oxidize or dehydrogenate, and 1,5-anhydroglucitol in the obtained sample is converted to 1,5-anhydroglucitol. An enzyme having the ability to oxidize or dehydrogenate sitol and glucose and having an α-glucosidase content of 1 × 10 ⁻³ unit / unit or less is allowed to act, or in the presence of an α-glucosidase inhibitor. A method for quantifying 1,5-anhydroglucitol, which comprises reacting with 1,5-anhydroglucitol and an enzyme capable of oxidizing or dehydrogenating glucose. 2. A compound which does not react with an enzyme having the ability to treat a sample with α-glucosidase and then selectively remove saccharides in the sample or to oxidize or dehydrogenate 1,5-anhydroglucitol and glucose. 1,5-anhydroglucitol in the obtained sample is converted into 1,5
-A method for quantifying 1,5-anhydroglucitol, which comprises causing an enzyme having an ability to oxidize or dehydrogenate anhydroglucitol and glucose to act. 3. A saccharide in a sample is selectively converted into a compound that does not react with an enzyme having an ability to oxidize or dehydrogenate 1,5-anhydroglucitol and glucose in the presence of α-glucosidase, 1,5-anhydroglucitol in a sample to be treated with an enzyme having an ability to oxidize or dehydrogenate 1,5-anhydroglucitol and glucose. Glucitol quantification method. 4. The method for quantifying 1,5-anhydroglucitol according to claim 1, 2 or 3, wherein the enzyme is pyranose oxidase or L-sorbose oxidase.