JP2954661B2 - Determination of hydrogen peroxide - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for quantifying hydrogen peroxide, and is used in a clinical laboratory.

2. Description of the Related Art Conventionally, in the field of clinical testing, a substance to be measured is converted into hydrogen peroxide using an oxidase, and this is led to a color-forming compound in the presence of peroxidase, and the substance to be measured is colorimetrically converted. Quantitation methods are widely used. Examples of the method using such a coloring compound include a method of condensing 4-aminoantipyrine and a phenol derivative, an aniline derivative or a toluidine derivative (“Development of biochemistry and a new diagnostic agent”, published by CMC Corporation), Leuco. Method using methylene blue derivative (JP-A-57-29297)
JP-A-59-74713), a method using a triarylmethane derivative (JP-A-59-71313), and a method using a triarylmethane derivative (JP-A-59-74713).
JP-A-56-31641, JP-A-62-296, JP-A-60-18
No. 4400) is known.

Problems to be Solved by the Invention In recent years, the needs in the field of clinical examinations have been reduced to small amounts of specimen samples and quantification of smaller amounts of biological components. On the other hand, it is becoming impossible to satisfy the demand.
That is, even if a leucomethylene blue derivative, which is one of the coloring reagents having the highest detection sensitivity, is used, it is extremely difficult to quantify a biological component on the order of μmol / order.

There is a strong demand for a color-forming compound having higher detection sensitivity.

Means for Solving the Problems The present invention provides a method for reacting hydrogen peroxide with a chromogen represented by the following general formula (I) or a salt thereof in the presence of peroxidase to form a reaction mixture in the visible part of a colored reaction solution. Provided is a method for quantifying hydrogen peroxide, which comprises measuring absorption.

In the formula, R represents a group represented by the following general formulas (II), (III), and (IV).

Any _two of P ₁ , P ₂ , P ₃ , P ₄ and P ₅ in the general formula (II) are And the remaining four are the same or different and represent hydrogen, hydroxyl, methyl, methoxy, sulfo, carboxyl, or halogen.

P ₁ , P ₂ , P ₃ in the general formulas (III) and (IV)
Any two of P ₄ , P ₅ , P ₆ , P ₇ And the remaining five are the same or different and represent hydrogen, hydroxyl, methyl, methoxy, sulfo, carboxyl, or halogen.

 Hereinafter, the present invention will be described in detail.

In the definitions of the general formulas (II), (III) and (IV), halogen includes a fluorine atom, a chlorine atom and a bromine atom.

Further, as the salt of the chromogen represented by the general formula (I),
Sulfate, phosphate, hydrochloride, acetate, sodium salt, potassium salt, lithium salt and the like are exemplified.

According to the present invention, the hydrogen peroxide in the sample and the general formula (I)
The hydrogen peroxide can be quantified by measuring the absorption of the compound produced by reacting with the chromogen represented by the formula near the maximum absorption wavelength (λmax).

In practicing the present invention, a buffer having a pH of 2 to 11, such as phosphoric acid, Tris-HCl, succinate, oxalate, acetate, Good, etc.
A reagent solution containing 0 IU / ml of peroxidase and a chromogen is equimolar to hydrogen peroxide in the sample, usually 10 to 1000 times the molar amount, and this is added to the hydrogen peroxide-containing sample.
The reaction is carried out at 5050 ° C., preferably at 25-40 ° C. The absorption by the reaction solution after the reaction is measured at the maximum absorption wavelength of the produced dye using a reagent blank as a control, and the amount of hydrogen peroxide in the sample is quantified using a calibration curve previously obtained from a test for a known amount.

A surfactant such as Triton X-100 is used as necessary in the reaction system.

The method of the present invention can be suitably applied to the determination of a reactant involved in a reaction system for producing hydrogen peroxide or an enzyme activity as described later. In particular, when the hydrogen peroxide generating system is an enzymatic reaction, the hydrogen peroxide generating reaction and the dye forming reaction can be performed in the same system, so that the amount can be determined in a short time.

If the target to be measured is a substrate, an enzyme that decomposes it to generate hydrogen peroxide is added.If the target has enzymatic activity, the substrate of the enzyme is added to the reagent solution together with the chromogen, The substrate or enzyme activity can be measured by adding the reagent solution to the sample and reacting the sample, and measuring the absorption of the colored reaction solution.

Representative chromogens used in the present invention are exemplified in Table 1. A in the table is Represents

The maximum absorption wavelength (λma
x) and sensitivity are shown in Table 2.

 The measurement was performed by the following method.

10 U / ml peroxidase, 1 mg / ml Triton X-10
0, 0.2 mg / ml of a Good buffer solution (pH 6.5) containing the compound represented by the general formula (I) was prepared, and 20 μl of this solution was added to 3 ml of the buffer.
0.25 mg / dl H ₂ O ₂ solution was added and reacted.
Measure the OD at x.

4-aminoantipyrine and N-ethyl- as chromogens
Table 2 shows the relative values when the OD is 100 when N- (3-methylphenyl) -N'-acetylethylenediamine (4AA-EMAE color developing system) is used.

Chromogens with higher sensitivity are more suitable for quantification of trace components.

The compound represented by the general formula (I) used in the present invention can be obtained by subjecting a benzhydrol and a phenol or a naphthol to a condensation reaction using sulfuric acid. The reaction is completed in a few hours at 30-100 ° C. The reaction solution is added to an organic solvent capable of crystallizing the target and the compound, for example, n-octanol, the crystallized product is dissolved in an organic solvent such as methanol, and the target product can be isolated by silica gel chromatography.

Production examples of the compounds shown in Table 1 are shown below.

(1) Compounds Nos. 1 to 6 were obtained by combining 4,4'-bis (dimethylamino) benzhydrol with the compound shown in Table 3 below:
At a molar ratio of 1, it is added to 5 times the amount of 50% sulfuric acid of 4,4'-bis (dimethylamino) benzhydrol, and the reaction is carried out at 80 ° C with stirring for 4 to 5 hours. The reaction solution is added to n-butanol to crystallize the desired product, and the crystals are dissolved in a small amount of ethanol. A column chromatography purification using silica gel is performed, and a fraction containing the target substance is collected. Elution is performed by adjusting the concentration gradient of ethyl acetate to chloroform so that chloroform: ethyl acetate = 90: 10 v / v is finally obtained. The target fraction is obtained by concentrating and drying the obtained fraction.

(2) Compound No. 7 comprises 4,4'-bis (dimethylamino) benzhydrol and 1,5-dihydroxynaphthalene in a molar ratio of 2: 1 to 4,4'-bis (dimethylamino) benzhydrol. Add 50% sulfuric acid 5 times that of hydrol and add 2 to 3 at 80 ° C.
The reaction is carried out with stirring for hours. The reaction solution is added to n-butanol to crystallize the desired product, and the crystals are dissolved in a small amount of ethanol. Column chromatography purification using silica gel is performed under the same conditions as in the above (1), fractions containing the target substance are collected, and concentrated to dryness to obtain the target substance.

(3) Compound No. 8 is 4,4'-bis (dimethylamino)
Benzhydrol and 2,3-dihydroxynaphthalene
A 2: 1 molar ratio is added to 40% sulfuric acid, 5 times the amount of 4,4'-bis (dimethylamino) benzhydrol, and the mixture is reacted at 80 ° C. with stirring for 6 to 7 hours. The reaction solution is added to n-butanol to crystallize the desired product, and the crystals are dissolved in a small amount of ethanol. Column chromatography purification using silica gel is performed under the same conditions as in the above (1), fractions containing the target substance are collected, and concentrated to dryness to obtain the target substance.

In the above (1), (2) and (3), the general formula (I)
The detection of the chromogenic compound represented by can be performed by subjecting each fraction collected by silica gel column chromatography to thin layer chromatography, and collecting the fractions having a UV absorption that develops a blue color on the thin layer. .

The method of the present invention can be applied to measurement of a reactant or an enzyme activity in a reaction system that produces stoichiometrically H ₂ O ₂ by a reaction.

Enzymatic reactions using various oxidases are known as such a reaction system, and the method of the present invention is suitable for quantification of an oxidase substrate.

Uric acid, cholesterol, neutral fatty acids, free fatty acids, glycerol, sialic acid, pyruvic acid, glucose, inorganic phosphate, phospholipids, creatine,
Creatinine, polyamine, lactic acid and the like can be mentioned, and monoamine oxidase, cholinesterase and the like can be mentioned as enzymes whose enzyme activities are to be measured.

 Below, these reaction systems are shown schematically.

The H ₂ O ₂ determination composition used in the present invention comprises peroxidase and a compound represented by the general formula (I).
A composition or kit to which surfactants and buffers are added or combined as necessary is convenient for the determination of H ₂ O ₂ .

Further, a composition to which an enzyme necessary for decomposing a substrate to be measured or a substrate of the enzyme to be measured is added is convenient for quantification of a target substance.

 Examples will be described below.

Example 1 (Quantification of hydrogen peroxide) 10 U of peroxidase was added to 50 mM phosphate buffer (pH 6.5).
/ ml, Triton X-100 1 mg / ml, Compound No. 1, Compound No. 2, Compound No. 3, Compound No. 4, Compound No. 5,
Compound No. 6, Compound No. 7, Compound No. 8 at 0.1 mg / ml
And eight kinds of color reagents were prepared.

To each of the color reagent 3 ml, the H ₂ O ₂ solution 1.0 mg / dl 5 to 100
After addition of μ, the mixture was heated in a 37 ° C. constant temperature bath for 10 minutes, and the absorbance at each λmax was measured for the reagent blank. FIG. 1 shows a calibration curve.

Example 2 (Quantification of creatine) 1,750 U of sarcosine oxidase, 420 U of peroxidase and 100 mg of Triton X-100 were dissolved in 100 ml of a 50 mM Good buffer solution (pH 7.4) to prepare a first reagent solution.

Separately, 13,250 U of creatinase, 100 mg of Triton X-100 and 100 mg of compound No. 1 as a chromogen, 100 mg of compound Nos. 5 and 10 in 100 ml of 50 mM Good buffer (pH 8.0)
0 mg of compound No. 6, 100 mg of compound No. 7, 100 mg of compound No. 8, 100 mg of bis (4-N, N-diethylaminophenyl) -3,4-disodiumsulfopropoxyphenylmethane (JP-A-6060) -184400) was dissolved in each solution to obtain a second test solution.

10 μl of serum and 2.25 ml of the first test solution were placed in a test tube, left at 37 ° C. for 5 minutes, 0.75 ml of the second test solution was added, and left at 37 ° C. for 5 minutes. Next, the absorbance of the reaction solution at λmax was measured using a reagent blank as a control, and the creatine concentration in the serum was calculated from a previously prepared calibration curve.

For comparison, the same measurement was performed using "Determiner CR633" (manufactured by Kyowa Medex). In this case, 20 µ of serum was used. The results are shown in Table 4.

Example 3 (Lactic acid quantification) 100 U of lactate oxidase, 300 U of peroxidase, and Triton X-100 were added to 100 ml of 100 mM phosphate buffer (pH 6.25).
100 mg and 25 mg as a chromogen Compound No. 1, 25 m
g of compound No. 2, 25 mg of compound No. 3, 25 mg of compound N
o.4 were each dissolved to give a reagent solution.

Take 5μ of serum and 3ml of reagent solution in a test tube, leave at 37 ° C for 5 minutes, measure the absorbance of the reaction solution at λmax using the reagent blank as a control, and calculate the lactate concentration in serum from the calibration curve prepared in advance. did.

For comparison, measurement was similarly performed using "Determiner LA" (manufactured by Kyowa Medex). In this case, the serum is 20μ
Using. The results are shown in Table 5.

Example 4 (Quantification of pyruvic acid) In 100 ml of 100 mM Good buffer (pH 6.0), 140 mg of potassium dihydrogen phosphate, 23 mg of thiamine pyrophosphate, 27 mg of Mg
Cl ₂ · 6H ₂ O, pyruvate oxidase 300 U, peroxidase 100 U, Triton X-100 100 mg and chromogenic compounds of the compounds No.3,25mg compounds No.2,25mg compounds No.1,25mg of 25mg as body No .4 were each dissolved to give a reagent solution.

Take 10 µ of serum and 3 ml of reagent solution in a test tube, leave at 37 ° C for 5 minutes, measure the absorbance of the reaction solution at λmax using the reagent blank as a control, and calculate the lactate concentration in serum from the calibration curve created in advance. did.

For comparison, the same measurement was performed using "Determiner PA" (manufactured by Kyowa Medex). In this case, the serum is 50
μ was used. The results are shown in Table 6.

As is evident from Example 2, bis (4-N, N-diethylaminophenyl) -3,4-disodiumsulfopropoxyphenylmethane gave significantly different measured values as compared to the control method. Although it cannot be used, as shown in Examples 2, 3, and 4, the value measured by the compound of the present invention is not limited to the fact that the sample amount (serum) is 1/2 to 1/5 of the conventional method. However, since the chromogen is extremely sensitive, it shows a good correlation with the control method, and can be said to be an excellent quantitative method.

Effect of the Invention According to the present invention, hydrogen peroxide can be quantified with extremely high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a calibration curve measured using a chromogen represented by the general formula (I). In the figure, ●-● indicates Compound No. 1, ■-■ indicates Compound No. 2, △-
△: Compound No. 3, □-□: Compound No. 4, ○-○: Compound
No. 5 and ×-× represent compound No. 6, and ▲-▲ represent compound No. 7 and compound No. 8, respectively.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C12Q 1/28 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A method comprising reacting hydrogen peroxide with a chromogen represented by the following general formula (I) or a salt thereof in the presence of peroxidase, and measuring the absorption of a colored reaction solution in the visible part. Characteristic method for determining hydrogen peroxide. In the formula, R represents a group represented by the following general formulas (II), (III), and (IV). Any _two of P ₁ , P ₂ , P ₃ , P ₄ and P ₅ in the general formula (II) are And the remaining three are the same or different and represent hydrogen, hydroxyl, methyl, methoxy, sulfo, carboxyl, or halogen. P ₁ , P ₂ , P ₃ , P ₄ , P in the general formulas (III) and (IV)
_5, any two of P _6, P ₇ is And the remaining five are the same or different and represent hydrogen, hydroxyl, methyl, methoxy, sulfo, carboxyl, or halogen. 2. A composition for the determination of hydrogen peroxide comprising (a) peroxidase and (b) a compound represented by the general formula (I) or a salt thereof.