JPH0549500A - Method for decomposing hydrogen peroxide - Google Patents

Abstract

PURPOSE:To decompose hydrogen peroxide into water for pretreatment of 1,5-anhydrogluycitol determination for diacrisis of diabetics diagnosis by treating hydrogen peroxide with a peroxidase using a specific phenoxanzine, etc., as an electron donor. CONSTITUTION:In treating 1,5-anhydrogluycitol in a specimen with a pyranose oxidase or L-sorbose oxidase and determining 1,5anhydrogluycitol from an amount of hydrogen peroxide formed, in order to previously remove glucose to be reacted with these enzymes from the specimen, the specimen is previously treated with glucose oxidase and formed hydrogen peroxide is decomposed into water in the presence of an electron donor compound such as a phenoxazine shown by formula I (X is 0 or S; R<1> is alkyl, alkenyl, etc.; R<2> and R<3> are H, halogen or alkyl), a phenothiazine derivative and a 4-aminoantipyrine derivative shown by formula II (R<4> is phenyl, etc.).

Description

Detailed Description of the Invention

The present invention relates to a method for decomposing hydrogen peroxide into water by peroxidase in the presence of an electron donating compound.

A method of decomposing hydrogen peroxide into water by peroxidase in the presence of an electron-donating compound is, for example, 1,5-anhydroglucitol which is known as a diagnostic marker for diabetes in the medical diagnostic field. It can be used when measuring (hereinafter, referred to as 1,5-AG).

1,5-AG exists in human urine, blood, etc., and in the case of patients with certain diseases, especially in diabetic patients, the amount of 1,5-AG in blood is higher than that in normal people. It is reported that the amount decreases. Therefore, 1,5-AG in blood
Diabetes can be diagnosed by measuring the amount of.

Conventionally, 1,5-AG in blood has been measured mainly by gas chromatography ("Diabetes", Vol. 25, pp. 1115 to 1118, 1982).
However, this method requires treatment of the test solution and labeling of 1,5-AG, and also requires a long time for measurement, making it difficult to measure a large number of samples at the same time. However, there are drawbacks such as the need for advanced technology for the maintenance and management of the, and it was inconvenient to apply it to actual clinical practice.

On the other hand, pyranose oxidase and L-sorbose oxidase are known as enzymes for detecting sugars, and these enzymes are used to oxidize 1,5-AG, which is one of sugar alcohols, to produce it. A method has been developed for quantifying 1,5-AG in a sample from the amount of hydrogen peroxide used (Japanese Patent Laid-Open No. 185397/1988 and Japanese Patent Laid-Open No. 2-1 / 1993).
No. 04298, etc.). In this method, pyranose oxidase and L-sorbose oxidase are 1,
Since it acts not only on 5-AG but also on other sugars such as glucose, in order to accurately quantify 1,5-AG in the sample, other sugars such as glucose are selectively removed from the sample beforehand. Or it must be converted into a substance inactive to the above-mentioned enzyme, and as a method therefor, a method utilizing ion exchange column chromatography (Japanese Patent Laid-Open No. 63-63242).
No. 185397), a method in which a reagent containing glucokinase or hexokinase and adenosine triphosphate, pyruvate kinase, phosphoenolpyruvate is allowed to act on a sample in advance (Japanese Patent Application Laid-Open No. 2-104298) and the like have been proposed. ..

The main object of the present invention is to obtain 1,5-
In measuring AG, it is possible to provide a method for effectively decomposing and removing hydrogen peroxide generated when a sample is previously treated with glucose oxidase, without disturbing the subsequent quantitative measurement of the target substance. is there.

Another object of the present invention is to provide a method for quantifying 1,5-AG in a sample by using such a method.

According to the present invention, therefore, in the method of decomposing hydrogen peroxide into water by peroxidase in the presence of an electron donating compound, the electron donating compound is represented by the general formula

[0009]

[Chemical 3]

In the formula, X represents an oxygen or sulfur atom,
R ¹ represents a substituted or unsubstituted lower alkyl group or a substituted or unsubstituted lower alkenyl group, and R ² and R ³
Each represents a hydrogen atom, a halogen atom, a lower alkoxy group, a lower alkyl group or a lower alkenyl group, and a phenoxazine or phenothiazine derivative represented by

[0011]

[Chemical 4]

In the formula, R ⁴ represents a substituted or unsubstituted phenyl group, which is a compound selected from the group consisting of 4-aminoantipyrine derivatives represented by:

Peroxidase (EC 1.11.1.
7) is an enzyme that catalyzes the reaction of decomposing hydrogen peroxide into water in the presence of an electron-donating compound, and the electron-donating compound used in this reaction is conventionally pyrogallol,
Oxidizing dyes such as benzidine, 2,2'-azino-di (3-ethylbenzthiazoline) -6'-sulfonic acid (ABTS) and guaiacol (The Enzymes, Vo
l. 8, Academic press (1963) 1
227; Enzyme HandBook, Sprin
ger-Verlag, Berlin (1969),
P.234) and the like are known, but these compounds are used for the quantification of hydrogen peroxide or the quantification of POD, because they are oxidized by the action of POD and hydrogen peroxide to form a pigment. It is the current situation. Using an electron-donating compound that normally forms a dye for the decomposition of hydrogen peroxide,
It could not be used for subsequent measurements.

Therefore, the inventors of the present invention have conducted extensive studies as to an electron donating compound which does not cause such a problem. As a result, the phenoxazine or phenothiazine derivative represented by the general formula (I) and the general formula (II) have been investigated. Shown 4-
The inventors have found that aminoantipyrine derivatives are particularly suitable and have completed the present invention.

In the present specification, the term "lower" means that the group or compound to which this term is attached has 6 or less carbon atoms, preferably 4 or less carbon atoms.

The "lower alkyl group" may be linear or branched, and includes, for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-
Butyl, n-pentyl and the like can be mentioned. Further, the "lower alkenyl group" may be of any type of straight chain or branched chain, and includes, for example, ethynyl, propenyl, butenyl, pentenyl, hexenyl group and the like.

"Substituted or unsubstituted lower alkyl group"
And examples of the substituent of the alkyl group or alkenyl group in the “substituted or unsubstituted lower alkenyl group” include a hydroxyl group, a carboxyl group, a sulfonic acid group, an amino group, and an acid or alkali salt thereof.
Specific examples of the lower alkyl group and lower alkenyl group substituted with such a group include 2-hydroxyethyl group and 3-hydroxyethyl group.
Hydroxypropyl group, 3-carboxypropyl group, 2
- hydroxy-3-sulfopropyl group, 3-sulfopropyl group, 3-aminopropyl group, sodium 2-hydroxy-3-sulfopropyl group, sodium 3-sulfopropyl group _{(NaSO 3 - (CH 2)} 3 -) , etc. Is included.

"Halogen atom" includes fluorine, chlorine, bromine and iodine atoms, and "lower alkoxy group" means a (lower alkyl) -O- group in which the lower alkyl moiety has the above-mentioned meaning, For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy groups and the like can be mentioned.

Examples of the substituent of the phenyl group in the "substituted or unsubstituted phenyl group" include amino group,
A halogen atom etc. are mentioned.

The following are typical examples of the phenoxazine or phenothiazine of the above formula (I).

10-methylphenothiazine, 10-methylphenoxazine, 10-ethylphenothiazine, 10
-Ethylphenoxazine, 2- (10-phenothiazinyl) ethanol, 2- (10-phenoxazinyl) ethanol, 3- (10-phenothiazinyl) propanoic acid,
3- (10-phenoxazinyl) propanoic acid, 4- (1
0-phenothiazinyl) butanoic acid, 4- (10-phenoxazinyl) butanoic acid, sodium 3- (10-phenothiazinyl) propanoate, sodium 3- (10-phenoxazinyl) propanoate, 4- (10-phenothiazinyl) butane Sodium acid salt, sodium 4- (10-phenoxazinyl) butanoate, sodium 3- (10-phenothiazinyl) propanesulfonate, 3- (2,7)
-Sodium dimethyl-10-phenothiazinyl) propane sulfonate, sodium 3- (3,6-dimethyl-10-phenothiazinyl) propane sulfonate, 3-
Sodium (2,5-dimethyl-10-phenothiazinyl) propanesulfonate, 3- (4,6-dimethyl-1)
Sodium 0-phenothiazinyl) propane sulfonate, sodium 3- (2,8-dimethyl-10-phenothiazinyl) propane sulfonate, sodium 3- (3,7-dimethyl-10-phenothiazinyl) propane sulfonate, 3- Sodium (3,5-dimethyl-10-phenothiazinyl) propane sulfonate, 3- (3,3
Sodium 7-diethyl-10-phenothiazinyl) propanesulfonate, sodium 3- (2,7-diethyl-10-phenothiazinyl) propanesulfonate, 3-
Sodium (1-methyl-10-phenothiazinyl) propanesulfonate, Sodium 3- (2-methyl-10-phenothiazinyl) propanesulfonate, 3- (3-
Sodium methyl-10-phenothiazinyl) propanesulfonate, sodium 3- (4-methyl-10-phenothiazinyl) propanesulfonate, sodium 3- (1-ethyl-10-phenothiazinyl) propanesulfonate, 3- (2 -Ethyl-10-phenothiazinyl) propane sulfonate sodium, 3- (3-ethyl-10)
-Phenothiazinyl) propane sulfonate sodium,
Sodium 3- (4-ethyl-10-phenothiazinyl) propanesulfonate, 3- (3,7-dioctyl-1)
Sodium 0-phenothiazinyl) propanesulfonate, sodium 3- (2,8-dioctyl-10-phenothiazinyl) propanesulfonate, sodium 3- (10-phenothiazinyl-1-isopropyl) propanesulfonate, 3- (2 -Ethenyl-10-phenothiazinyl) propanesulfonic acid sodium salt, 3- (3,7-
Sodium diethenyl-10-phenothiazinyl) propane sulfonate, sodium 3- (3-ethenyl-10-phenothiazinyl) propane sulfonate, 3- (3-
Sodium t-butyl-10-phenothiazinyl) propane sulfonate, 3- (3,7-di-t-butyl-10)
-Phenothiazinyl) propane sulfonate sodium,
Sodium 3- (3,7-bis (1,1,3,3-tetramethylbutyl) -10-phenothiazinyl) propanesulfonate, Sodium 3- (10-phenoxazinyl) propanesulfonate, 3- (2,7) -Dimethyl-10
-Phenoxazinyl) propane sulfonate sodium,
Sodium 3- (3,6-dimethyl-10-phenoxazinyl) propane sulfonate, Sodium 3- (2,5-dimethyl-10-phenoxazinyl) propane sulfonate, 3- (4,6-Dimethyl-10-phenoxazinyl) propane Sodium sulfonate, sodium 3- (2,8-dimethyl-10-phenoxazinyl) propane sulfonate, sodium 3- (3,7-dimethyl-10-phenoxazinyl) propane sulfonate, 3- (3,3
Sodium 5-dimethyl-10-phenoxazinyl) propane sulfonate, sodium 3- (3,7-diethyl-10-phenoxazinyl) propane sulfonate, 3-
Sodium (2,7-diethyl-10-phenoxazinyl) propane sulfonate, Sodium 3- (1-methyl-10-phenoxazinyl) propanesulfonate, 3-
Sodium (2-methyl-10-phenoxazinyl) propanesulfonate, Sodium 3- (3-methyl-10-phenoxazinyl) propanesulfonate, 3- (4-
Sodium methyl-10-phenoxazinyl) propanesulfonate, sodium 3- (1-ethyl-10-phenoxazinyl) propanesulfonate, sodium 3- (2-ethyl-10-phenoxazinyl) propanesulfonate, 3- (3-ethyl- Sodium 10-phenoxazinyl) propane sulfonate, 3- (4-ethyl-10)
-Phenoxazinyl) propane sulfonate sodium,
3- (3,7-dioctyl-10-phenoxazinyl)
Sodium propane sulfonate, 3- (2,8-Dioctyl-10-phenoxazinyl) sodium sulfonate, 3- (10-phenoxazinyl-1-isopropyl) sodium propane, 3- (2-ethenyl-10-phenoxazinyl) Sodium propanesulfonate, 3- (3-ethenyl-10-phenoxazinyl) sodium propanesulfonate, 3- (3,7-
Sodium diethenyl-10-phenoxazinyl) propanesulfonate, sodium 3- (3-t-butyl-10-phenoxazinyl) propanesulfonate, 3-
(3,7-di-t-butyl-10-phenoxazinyl)
Sodium propanesulfonate, sodium 3- (3,7-bis (1,1,3,3-tetramethylbutyl) -10-phenoxazinyl) propanesulfonate, and the like.

Specific examples of the 4-aminoantipyrine derivative of the above formula (II) include the following.

4-amino-1,2-dihydro-1,5-
Dimethyl-2-phenyl-3H-pyrazol-3-one (4-aminoantipyrine), 4-amino-1,2-dihydro-1,5-dimethyl-2- (2,4,6-trichlorophenyl) -3H -Pyrazol-3-one, 4-amino-2- (3-chlorophenyl) -1,2-dihydro-1,5-dimethyl-3H-pyrazol-3-one, 4
-Amino-2- (4-aminophenyl) -1,2-dihydro-1,5-dimethyl-3H-pyrazol-3-one and the like.

The compounds of the formulas (I) and (II) described above are known per se, or can be produced according to the methods for producing the known compounds.

The reaction of decomposing hydrogen peroxide into water in the presence of a compound as described above as an electron donor can be carried out according to a method known per se. For example, a glucose oxidase is allowed to act on serum to selectively convert glucose present in the serum into hydrogen peroxide, and a solution containing hydrogen peroxide such as a sample solution obtained by diluting the solution appropriately with a buffer aqueous solution. Then, the action pH of peroxidase is pH 4 to
After adjusting the pH within the range of 10, the formula (I) or (I
The compound of I) and peroxidase can be added and reacted. Examples of the buffer aqueous solution that can be used here include a phosphate buffer solution and a Tris-succinate buffer solution, and the concentration of hydrogen peroxide in the reaction solution is generally 0.
It can be set to 01 to 1000 μmol / l.

As peroxidase, those of various origins are known, and commercially available products can be obtained and used. The amount used may vary depending on the reaction conditions (temperature, time, pH) and the like, but is generally 500 to 100,000.
A range of U / l is suitable.

The amount of the compound of the formula (I) or (II) used is not strictly limited and can be changed according to the reaction conditions and the like, but is generally 50 to 1,000.
It is suitable to use at a concentration within the range of μmol / l.

The above reaction can be carried out usually at a temperature of about 5 to about 40 ° C., preferably about 25 to about 40 ° C. for about 1 to about 60 minutes, preferably about 1 to about 10 minutes.

Since the reaction mixture after the reaction is substantially colorless, the method of the present invention is carried out by reacting 1,5-AG in a sample such as serum with pyranose oxidase or L-sorbose oxidase. To be used advantageously in pretreatment of a sample when a coloring reagent is added to hydrogen peroxide and the amount of color development is measured by a colorimetric method, a fluorescence method, a luminescence method or the like to quantify 1,5-AG in the sample You can

That is, when 1,5-AG in a sample is quantified by the above method, other saccharides such as glucose present in the sample collected will interfere with the above quantification.
Must be removed in advance. Therefore, glucose oxidase (EC 1.1.3.4) is added to the sample to selectively decompose sugars such as glucose, and peroxidase and It is decomposed into water by the action of the compound of formula (I) or (II).

In this case, the treatment with glucose oxidase and the treatment with peroxidase according to the present invention and the compound of formula (I) or (II) may be carried out sequentially in this order, or the sample may be treated with glucose oxidase, peroxidase and 3 of compounds of formula (I) or (II)
People may be added together and performed simultaneously.

In the sample pretreated in this way,
The quantification of 5-AG is performed by a method known per se, for example, Cli.
neural Chemistry 35 , (10), 2
039-2043 (1989), Japanese Patent Publication No. 3-24200
It can be carried out by the method described in Japanese Patent Publication.

As described above, the electron-donating compound represented by the general formula (I) or (II) can be advantageously used in the decomposition of hydrogen peroxide into water by peroxidase. , Peroxyboric acid and CH ₃
It can also be used for the decomposition reaction of peroxides such as OOH by peroxidase.

Further, the electron-donating compound represented by the above general formula (I) or (II) can be used also in the pretreatment of the sample when quantifying the ester type cholesterol or triglyceride in the sample. For example, in the determination of ester type cholesterol, cholesterol in a sample must be removed in advance. In this case, the sample must be supplemented with cholesterol oxidase and formula (I) or (I
The compounds of I) can act. When quantifying triglyceride, glycerol can be decomposed and removed by allowing glycerol in the sample to act with glycerol reductase, NADH oxidase and the compound of formula (I) or (II). In addition, glycerol kinase, glycerol-3-phosphate oxidase and the compound (I) or (II) can be allowed to act to decompose and remove glycerol.

[0035]

EXAMPLES The present invention will be described in more detail with reference to reference examples and examples.

Reference Example 1 2.0 g (10 mM) of phenothiazine and 3.9 g (30 mM) of propane sultone were placed in a container at 80 to 120 ° C.
The mixture was heated and stirred for 1 hour. After the reaction, cool the reaction product and add 5 with water.
It was diluted to 10 times and then neutralized with an alkaline aqueous solution. Then, water was distilled off with an evaporator, the residue was dissolved in a small amount of ethanol, and left at room temperature for recrystallization. Do this operation 2
Repeatedly to obtain 2.5 g of sodium 3- (10-phenothiazinyl) propanesulfonate (yield: 72%).

Analytical data ¹ H-NMR (300 MHz, CD ₃ OD): δ 2.25 (2H, tt, J = 7.0,
7.0Hz), 2.94 (2H, t, J = 7.0Hz), 4.08 (2H, t, J = 7.0H
z), 6.87 to 7.20 (8H, m) Reference Example 2 Synthesis of N-ethylphenoxazine 1.83 g (10 mM) phenoxazine, 4.7 g (30 mM) ethyl iodide, 50 mg trimethylbenzyl chloride, 50% sodium hydroxide 10 ml of the solution is placed in an eggplant-shaped flask and heated under reflux at 80 ° C. for 2 hours.

After the reaction, the reaction product was cooled and chloroform 50 was added.
Add ml to extract the desired product. Remove the solvent chloroform with an evaporator and dissolve in a minimum amount of ethyl alcohol. The ethyl alcohol solution is left in a refrigerator at room temperature or 10 ° C. to crystallize. Repeat this operation twice,
1.75 g of N-ethylphenoxazine was obtained. (Yield 7
7%) Analytical data ¹ H-NMR (300 MHz, CD ₃ OD): δ 6.82 to 6.56 (8H, m),
6.67 ~ 3.60 (2H, q, J = 7.0Hz), 1.23 ~ 1.18 (3H, t, J =
(7.0 Hz) Reference Example 3 Synthesis of N-methylphenothiazine 2 g (10 mM) of phenothiazine, 4.5 g (30 mM) of methyl iodide, 50 mg of trimethylbenzyl chloride, and 10 ml of 50% sodium hydroxide solution were placed in an eggplant-shaped flask and heated at 80 ° C. for 2 hours. Heat to reflux for hours.

After the reaction, the reaction product was cooled and chloroform 50 was added.
Add ml to extract the desired product. Remove the solvent chloroform with an evaporator and dissolve in a minimum amount of ethyl alcohol. The ethyl alcohol solution is left in a refrigerator at room temperature or 10 ° C. to crystallize. Repeat this operation twice,
1.9 g of N-methylphenothiazine was obtained. (Yield 89
%) Analytical data ¹ H-NMR (300 MHz, CD ₃ OD): δ 7.18 to 6.88 (8 H, m), 3.
36 (3H, s) Reference Example 4 Synthesis of N-ethylphenothiazine 1.83 g (10 mM) of phenothiazine, 4.7 g (30 mM) of ethyl iodide, 50 mg of trimethylbenzyl chloride, and 10 ml of 50% sodium hydroxide solution were placed in an eggplant flask. Heat at reflux for 2 hours at 80 ° C.

After the reaction, the reaction product was cooled and chloroform 50 was added.
Add ml to extract the desired product. Remove the solvent chloroform with an evaporator and dissolve in a minimum amount of ethyl alcohol. The ethyl alcohol solution is left in a refrigerator at room temperature or 10 ° C. to crystallize. Repeat this operation twice N
-1.6 g of ethylphenothiazine were obtained. (Yield 75
%) Analytical data ¹ H-NMR (300 MHz, CD ₃ OD): δ 7.18 to 6.86 (8 H, m),
3.97 to 3.90 (2H, q, J = 6.9Hz), 1.39 to 1.34 (3H, t, J =
6.9 Hz) Reference Example 5 Synthesis of N-methylphenoxazine 1.83 g (10 mM) of phenoxazine, 4.7 g (30 mM) of methyl iodide, 50 mg of trimethylbenzyl chloride, and 10 ml of 50% sodium hydroxide solution were placed in an eggplant flask. Heat at reflux for 2 hours at 80 ° C.

After the reaction, the reaction product was cooled and chloroform 50 was added.
Add ml to extract the desired product. Remove the solvent chloroform with an evaporator and dissolve in a minimum amount of ethyl alcohol. The ethyl alcohol solution is left in a refrigerator at room temperature or 10 ° C. to crystallize. Repeat this operation twice,
1.4 g of N-methylphenoxazine was obtained. (Yield 71
%) Analytical data ¹ H-NMR (300 MHz, CD ₃ OD): δ6.86 to 6.58 (8H, m),
3.01 (3H, s) Example 1 Elimination of hydrogen peroxide 1-mM sodium 3- (10-phenothiazinyl) propanesulfonate and 20000 units peroxidase /
Phosphate buffer containing L (pH = 7.3, 50 mM) 1 m
50 μl of 0-100 mM hydrogen peroxide was added to 1 l, and the temperature was 37 ° C.
And allowed to stand for 3 minutes. After that, N-ethyl-
Phosphate buffer (pH = 7.3, 50 mM) containing N- (2-hydroxy-3-sulfopropyl) -m-toluicin sodium salt dihydrate (TOOS) 2 mM and 4-aminoantipyrine 0.5 mM. Add 1 ml and bring to 37 ° C for 3
After standing for a minute, the absorbance was measured at a wavelength of 555 nm. The measurement result is shown in FIG.

As shown in FIG. 1, no increase in absorbance was observed even at a final concentration of 2 mM hydrogen peroxide, indicating that hydrogen peroxide was sufficiently eliminated.

Example 2 Measurement of 1,5-AG Sodium 3- (10-phenothiazinyl) propanesulfonate 1 mM, peroxidase 2000 units / L,
Phosphate buffer containing glucose oxidase 6000 units / L and mutarolase 10,000 units / L (pH =
7.3, 50 mM) 1 ml glucose (100 mg /
dl, or 500 mg / dl) and 1,5-AG
Add 50 μl of test solution containing (0 to 2 mg / dl), and add 3
It was left still at 7 ° C. for 10 minutes.

Thereafter, TOOS 2 mM, 4-
Aminoantipyrine 0.5 mM and pyranose oxida
Phosphate buffer containing 30000 units / L of enzyme (pH =
7.3, 50 mM) 1 ml, and left still at 37 ° C for 5 minutes
did. Absorbance was measured at a wavelength of 555 nm. The result
As shown in FIG. As shown in FIG. 2,
To measure 1,5-AG specifically without being affected
You can see that Example 3 Hydrogen peroxide using 4-aminoantipyrine
Elimination of 4-aminoantipyrine 1 mM and peroxidase 6
Phosphate buffer containing 000 units / L (pH = 7.3, 5
0 mM) 1 ml with 0-100 mM hydrogen peroxide 50 μl
In addition, the mixture was allowed to stand at 37 ° C for 3 minutes. Then, in this reaction solution
Phosphate buffer containing 2 mM TOOS (pH = 7.3, 5
0 mM) 1 ml was added, and this was left still at 37 ° C. for 3 minutes.
Post absorbance was measured at a wavelength of 555 nm. Figure 3 shows the measurement results.
Shown in.

Example 4 Measurement of ester type cholesterol Sodium 3- (10-phenothiazinyl) propanesulfonate 1 mM, cholesterol oxidase 500 U
/ L and peroxidase 6000 U / L in phosphate buffer (100 mM, pH = 7.0, containing 0.1% Triton X-100) 1 ml, body fluid (serum) 10 μl, and standard solution (cholesterol ester 50 mg / dl)
10 μl was added to each test tube and left at 37 ° C. for 2 minutes. Next, cholesterol esterase 200U /
L, 4-aminoantipyrine 0.5 mM and N, N-dimethylaniline 1.0 mM phosphate buffer (100
2 ml of mM, pH = 7.0, containing 0.1% Triton X-100) was added, and the mixture was allowed to stand at 37 ° C. for 5 minutes.
Absorbance was measured at 45 nm. By comparing the results with the standard solution, only the esterified cholesterol in the body fluid can be determined.

FIG. 4 shows standard curves for measuring ester cholesterol at various concentrations. Furthermore, the measuring method of the present invention and commercially available determiner TC-S and determiner FC-
FIG. 5 shows the comparison results obtained by calculating the amount of ester cholesterol (conventional method) using 555 (manufactured by Kyowa Medix Co., Ltd.).

Example 5 Measurement of 1,5-AG Sodium 3- (10-phenoxazinyl) propanesulfonate 1 mM, peroxidase 3000 units / L,
Phosphate buffer containing glucose oxidase 6000 units / L and mutarotase 10,000 units / L (pH = 7.
Glucose (100 mg / d in 1 ml of 3, 50 mM)
1, or 500 mg / dl), 1,5-AG (0 to
50 μl of test liquid containing 2 mg / dl) and specimen 5 containing glucose (100 mg / dl, 500 mg / dl)
Add 0 μl and leave at 37 ° C. for 20 minutes.

Then, N-ethyl-N- (2
-Hydroxy-3-sulfopropyl) -m-toluidine (TOOS) 2 mM, 4-aminoantipyrine 0.5 m
M, 1 ml of phosphate buffer (pH = 7.3, 50 mM) containing 30,000 units / L of pyranose oxidase / L is added. This is left at 37 ° C. for 5 minutes. After standing, the absorbance is measured at a wavelength of 555 nm. As shown in FIG. 6, 1,5-AG could be specifically measured without being affected by coexisting glucose.

Example 6 Measurement of triglyceride 10-ethylphenoxazine 150 μM, peroxidase 5000 units / L, glycerol kinase 2000
Unit / L, glycerophosphate oxidase 1000 Unit /
L, ATP 1.2 mM, lipoprotein lipase 200
Tris-hydrochloric acid buffer solution containing 100 units / L (100 m
M, pH = 7.8) 2 ml, specimen containing triglyceride 20 μl, and glycerol (50 mg / dl, 10
Add 20 μl of the sample containing 0 mg / dl)
Let stand for a minute. Then 4-aminoantipyrine 1.5
Tris-hydrochloric acid buffer solution (10 mM) containing 3 mM of N-ethyl-N-sulfopropyl-m-anisidine (ADPS).
Add 1 ml of 0 mM, pH = 7.8) and let stand at 37 ° C. for 10 minutes. Then, the absorbance is measured at a wavelength of 540 nm. Triglyceride in the sample is determined using the standard solution measured according to the above method as a control. The result is shown in FIG. 7.

[Brief description of drawings]

FIG. 1 is a diagram showing a result of quantifying H ₂ O ₂ remaining after a H ₂ O ₂ erasing reaction according to Example 1.
As is clear from this figure, it was found that H ₂ O ₂ up to ₂ mM was completely decomposed and erased, and no color was developed at all.

FIG. 2 is a graph showing the effect of glucose, which is an interfering substance, in quantifying 1,5-AG according to Example 2. As is clear from this figure, even in the presence of a large amount of glucose, it is shown that the same result as the sample containing no glucose was obtained.

FIG. 3 is a diagram showing the quantitative results of H ₂ O ₂ remaining after the elimination reaction of H ₂ O ₂ according to Example 3.

FIG. 4 is a diagram showing that free cholesterol present in a standard solution is completely removed by the elimination method according to Example 4.

FIG. 5 is a diagram showing a correlation with a conventional book (total cholesterol value-free cholesterol value = ester cholesterol). As is clear from this figure, the present invention showed a good correlation.

FIG. 6 is a diagram showing the influence of glucose when quantifying 1,5-AG in Example 6.

FIG. 7 is a graph of triglyceride standard solution with 50, 1
It is the figure which compared the sample which added glycerol to each dilution series so that it might be set to 00 mg / dl, and the standard solution without addition. As shown in this figure, a graph was obtained in which the unadded sample and the added sample were almost overlapped.

Claims (3)

[Claims] 1. A method of decomposing hydrogen peroxide into water by peroxidase in the presence of an electron-donating compound, wherein the electron-donating compound has the general formula: In the formula, X represents an oxygen or sulfur atom, R ¹ represents a substituted or unsubstituted lower alkyl group or a substituted or unsubstituted lower alkenyl group, and R ² and R ³ are each a hydrogen atom, a halogen atom, a lower alkoxy. Group, a lower alkyl group or a lower alkenyl group, and a phenoxazine or phenothiazine derivative represented by the formula: In the formula, R ⁴ represents a substituted or unsubstituted phenyl group, and is a compound selected from the group consisting of 4-aminoantipyrine derivatives represented by: 2. A compound selected from the group consisting of a phenoxazine or phenothiazine derivative represented by the general formula (I) and a 4-aminoantipyrine derivative represented by the general formula (II) according to claim 1. Electron donor for peroxide decomposition by peroxidase. 3. The amount of hydrogen peroxide produced by reacting 1,5-anhydroglucitol in a sample with pyranose oxidase or L-sorbose oxidase is 1,5.
In the method for quantifying anhydroglucitol, the sample is glucose oxidase, peroxidase, and a phenoxazine or phenothiazine derivative represented by the general formula (I) or a compound represented by the general formula (II) according to claim 1. -A method for quantifying 1,5-anhydroglucitol in a sample, which comprises pretreatment with an aminoantipyrine derivative.