JPH06197795A - Determination of hydrogen peroxide - Google Patents

Abstract

PURPOSE:To determine hydrogen peroxide in high sensitivity in a clinical examination, etc., with an oxidase by using a peroxidase and a specific triphenylmethane derivative and reacting the compound with hydrogen peroxide in the presence of a superoxide dismutase. CONSTITUTION:A specimen liquid containing hydrogen peroxide is added to a peroxidase and a triphenylmethane derivative of formula [R<1> and R<3> are H or (substituted) 1-4C alkyl; R<2> and R<4> are 1-4C alkyl having substituent such as sulfo and hydroxyl group; R<5> is H, Cl, Br, I, 1-4C alkyl, etc.; R<6> to R<8> are H, 1-4C alkyl or 1-3C alkoxy] {e.g. 4,4',4''-tri[N,N-di(3-sulfopropyl) amino]triphenylmethane hexasodium salt} in the presence of a superoxide dismutase, the components are made to react with each other at 37 deg.C for 10min and the absorbance at 595nm wavelength is determined by an absorptiometer. Hydrogen peroxide generated by the reaction of the substrate with the oxidase can be determined by this process to enable the determination of the hydrogen peroxide in a specimen for clinical examination, etc., in high sensitivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring hydrogen peroxide. More specifically, peroxidase (hereinafter PO
D) and general formula

[Chemical 2] (In the formula, R ¹ and R ³ are each a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or a sulfo group, a substituted alkyl group having 1 to 4 carbon atoms having a substituent of an alkali metal base such as a hydroxyl group or a sulfo group, R ² and R ⁴ are each a sulfo group, a hydroxyl group or a C1-C1 substituent having a substituent of an alkali metal base of the sulfo group.
4 is a substituted alkyl group, R ⁵ is a hydrogen atom, a chlorine atom, a bromine atom, an iodine atom, an alkyl group having 1 to 4 carbon atoms, or-
R ( ⁹ ) is a group represented by N (R ⁹ ) R ¹⁰ , wherein R ⁹ has a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a sulfo group, a hydroxyl group, or a substituent of an alkali metal base such as a sulfo group, 4 substituted alkyl group, R ¹⁰ is a sulfo group, a hydroxyl group or a substituted alkyl group having 1 to 4 carbon atoms having a substituent of an alkali metal base of a sulfo group, R ⁶ , R ⁷ and R ⁸
Is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or 1 carbon atom
~ 3 alkoxy groups). In the method for measuring hydrogen peroxide using the triphenylmethane derivative represented by the formula, superoxide dismutase (hereinafter referred to as SOD)
And a method for measuring hydrogen peroxide in the presence of.

[0002]

2. Description of the Related Art Heretofore, as a method for measuring hydrogen peroxide, a method has been known in which a Trinder reagent and 4-aminoasothipyrin are reacted in the presence of POD to measure an oxidative condensation dye produced (K. .Tamaoku; Ana
l. Chim. Acta. 136 , 121-127
(1982)). With the method using the Trinder reagent, only a sample having a hydrogen peroxide concentration up to about 1 μM can be measured, and the advent of a highly sensitive measurement reagent has been desired. In order to solve this problem, a method using POD and a triphenylmethane derivative as a color-developing reagent has been found and used for the measurement of hydrogen peroxide (Japanese Patent Application No. 3-3).
51199).

[0003]

However, although the hydrogen peroxide concentration of up to about 0.3 μM can be measured by using the triphenylmethane derivative, it is much higher in the analysis in the field of clinical examination. The current situation is that sensitivity is required. And the appearance of an effective measurement method was expected for a long time.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to find a method for measuring highly sensitive hydrogen peroxide, and as a result, in a solution containing hydrogen peroxide, POD and the above general formula (I) were used. In the method using the represented triphenylmethane derivative, the present invention has been completed by finding a measuring method performed by further adding SOD.

In the measuring method of the present invention, first, a triphenylmethane derivative represented by the above-mentioned general formula (I), POD and SOD, is used as a coloring reagent in a solution containing a sample containing hydrogen peroxide.
It can be carried out by adding and reacting with each other and measuring the resulting dye. The triphenylmethane derivative represented by the general formula (I) used for the measurement is 10 to 500.
It may be μM, and preferably 100 to 200 μM can be used. The amount of POD added is 100 to 1
50,000 U / L, preferably 1,000 to 3,
It is 000 U / L. The amount of SOD added is 10,000
Although it is ~ 1,000,000 U / L, it is 60,000-200,000 U / L for efficient performance.

The reaction is preferably carried out in a buffer solution having a pH of 5 to 8, and the buffer solution used is, for example, Tris-
Examples thereof include succinate buffer solution and Tris-HCl buffer solution. The reaction can be carried out at 10 to 50 ° C,
Usually, it is preferably carried out at around 37 ° C. The dye after the reaction can be measured by reacting the reaction solution for a certain period of time and then measuring the absorbance, or by adding a reaction stop solution and then measuring the absorbance. Further, the measurement can be performed using a rate method for measuring the rate of change in absorbance. Furthermore, the activity of POD can also be measured in place of hydrogen peroxide, which is the measurement object of the present invention.

The hydrogen peroxide, which is the substance to be measured according to the present invention, can be measured in addition to hydrogen peroxide contained in a sample, for example, hydrogen peroxide produced by an enzymatic reaction or the like.
Examples of the reaction for producing hydrogen peroxide include a reaction between a substrate and an oxidase. For example, a reaction between glucose and glucose oxidase, cholesterol and cholesterol oxidase, uric acid and uricase, galactose and galactose oxidase, lactic acid and lactate oxidase, etc. Is. That is, by measuring the hydrogen peroxide produced by the enzymatic reaction, it is possible to measure the substrate mass or the oxidase activity.

As the SOD used in the present invention, a commercially available or easily available SOD can be used.
u-SOD, Zn-SOD, EC-SOD (Extra
Cellular), Mn-SOD, Fe-SOD, and the like.

The triphenylmethane derivative represented by the general formula (I) is not only a commercially available compound having a triphenylmethane skeleton, but also Japanese Patent Application No. 3-35119.
Examples thereof include triphenylmethane derivatives shown in No. 9. As the triphenylmethane derivative,
For example, 4,4 ', 4 "-tri [N, N-di (3-sulfopropyl) amino] triphenylmethane hexasodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) amino- 2-methoxyphenyl)] [4- (N, N
-Diethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) aminophenyl)] [4- (N, N-diethylamino) phenyl] methane disodium salt, bis [ 4-
(N-ethyl-N- (2-hydroxy-3-sulfopropyl) amino-2-methylphenyl)] [4- (N, N
-Diethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) amino-2-methoxyphenyl)] [4- (N, N
-Dimethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) aminophenyl)] [4- (N, N-dimethylamino) phenyl] methane disodium salt, Screw [4-
(N-ethyl-N- (2-hydroxy-3-sulfopropyl) amino-2-methylphenyl)] [4- (N, N
-Dimethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) aminophenyl)] (4-chlorophenyl) methane disodium salt, bis [4- (N-ethyl) -N- (3
-Sulfopropyl) aminophenyl)] (4-hydroxyphenyl) methane disodium salt, bis [4- (N
-Ethyl-N- (3-sulfopropyl) aminophenyl)] (2-sulfophenyl) methane trisodium salt and the like can be mentioned.

[0010]

The present invention will be described in more detail with reference to the following examples.

Example 1 Measurement of hydrogen peroxide SOD 120,000 U / L, POD 1,500 U /
Tris-succinate buffer (pH = 150 μM of L and 4,4, '4 "-tri [N, N-di (3-sulfopropyl) amino] triphenylmethane hexasodium salt (pH =
Hydrogen peroxide (0 to 2
10 μl of a test solution containing 50 μM) was added and left at 37 ° C. for 10 minutes. After standing, the absorbance was measured with an absorptiometer using a test solution containing no hydrogen peroxide at a wavelength of 595 nm as a control. Similarly, it was compared with that measured by removing SOD from the test solution, and the result is shown in FIG.

Example 2 Measurement of glucose (1) Glucose oxidase (derived from Aspergius, manufactured by Amano Pharmaceutical Co., Ltd.) 50,000 U / L, mutarotase (derived from porcine kidney, manufactured by Amano Pharmaceutical Co., Ltd.) 10,000 U / L, SOD12
50,000 U / L, POD 1,500 U / L, 4,
4, '4 "-tri [N, N-di (3-sulfopropyl)
Amino] triphenylmethane hexasodium salt 150μ
Tris-succinate buffer containing M (pH = 6.5, 50
10 μl of a test solution containing glucose (0 to 7 mg / dl) was added to 2 ml of a test solution of mM), and the mixture was allowed to stand at 37 ° C. for 10 minutes. After standing, the absorbance was measured at a wavelength of 595 nm with 0 mg / dl glucose as a control. Similarly, it was compared with that measured by removing SOD from the test solution, and the result is shown in FIG.

Example 3 Measurement of glucose (2) 4,4 ', 4 "-tri [N, N-di (3-sulfopropyl) amino] triphenylmethane used in Example 2
Bis [4-[(N-ethyl-N
-(3-Sulfopropyl) amino] phenyl] [4-
Glucose was measured and absorbance was measured in the same manner as in Example 2 using a test solution containing (N, N-diethylamino) phenyl] methane disodium salt. The result is shown in FIG.

Example 4 Measurement of glucose (3) 4,4 ', 4 "-tri [N, N-di (3-sulfopropyl) amino] triphenylmethane used in Example 2
Bis [4-[(N-ethyl-N
Using a reagent solution containing -sulfopropyl) amino] phenyl] (4-chlorophenyl) methane disodium salt, glucose was measured and absorbance was measured in the same manner as in Example 2.
The result is shown in FIG.

Example 5 Measurement of cholesterol Cholesterol oxidase (derived from Streptomyces, manufactured by Asahi Kasei) 50,000 U / L, cholesterol esterase (derived from Pseudomonas, manufactured by Asahi Kasei) 1
50,000U / L, SOD120,000U / L, PO
D1,500U / L, 4,4 ', 4 "-Tri [N, N-
Di (3-sulfopropyl) amino] triphenylmethane hexasodium salt containing 150 μM Tris-succinate buffer (pH = 6.5, 50 mM, Triton X-10)
Cholesterol (0% is included) in 2 ml of test solution (0%)
10 μl of a test solution containing 10 mg / dl) was added and left at 37 ° C. for 5 minutes. After standing, the absorbance was measured at a wavelength of 595 nm with 0 mg / dl cholesterol as a control. Similarly, it was compared with that measured by removing SOD from the test solution, and the result is shown in FIG.

Example 6 Measurement of uric acid Uricase (derived from yeast, manufactured by Toyobo Co., Ltd.) 50,000 U
/ L, SOD120,000U / L, POD1,500
U / L, 4,4 ′, 4 ″ -tri [N, N-di (3-sulfopropyl) amino] triphenylmethane tris-succinate buffer solution (pH = 150) containing 150 μM hexasodium salt
Uric acid (0 to 5 mg / dl) in 2 ml of the test solution (8.5, 50 mM)
10 μl of a test solution containing the above was added and left at 37 ° C. for 5 minutes. After standing, the absorbance was measured at a wavelength of 595 nm with 0 mg / dl uric acid as a control. Similarly, it was compared with that measured by removing SOD from the test solution, and the result is shown in FIG.

Example 7 Measurement of galactose Galactose oxidase (derived from dactylium, manufactured by Toyobo Co., Ltd.) 1000 U / L, SOD 120,000 U /
Tris-containing L, POD 1,500 U / L, 4,4 ′, 4 ″ -tri [N, N-di (3-sulfopropyl) amino] triphenylmethane hexasodium salt 150 μM
2 ml of succinate buffer solution (pH = 7.0, 50 mM)
10 μl of test solution containing galactose (0-5 mg / dl)
Was added and the mixture was allowed to stand at 37 ° C. for 5 minutes. Wavelength after standing 595n
Absorbance was measured at m with 0 mg / dl galactose as a control. Similarly, it was compared with the one obtained by removing SOD from the test solution, and the result is shown in FIG. 7.

Example 8 Measurement of Lactic Acid Lactic acid oxidase (derived from dactylium, manufactured by Asahi Kasei Corp.) 1
000U / L, SOD120,000U / L, POD
1,500 U / L, 4,4 ', 4 "-tri [N, N-di (3-sulfopropyl) amino] triphenylmethane
Lactic acid (0 to 0 ml) was added to 2 ml of a Tris-succinate buffer solution (pH = 7.0, 50 mM) containing 150 μM hexasodium salt.
10 μl of a test solution containing 5 mg / dl) was added and left at 37 ° C. for 5 minutes. After standing, the absorbance was measured at a wavelength of 595 nm with 0 mg / dl lactic acid as a control. Similarly, from the test solution to SO
The results are shown in FIG. 8 in comparison with those measured with the exception of D.

Example 9 Measurement of glucose oxidase activity 140 mM glucose, SOD 120,000 U / L,
POD 2,000 U / L, 4,4 ', 4 "-Tri [N,
3 ml of tris-succinate buffer solution (pH = 5.5, 50 mM) containing 150 μM of N-di (3-sulfopropyl) amino] triphenylmethane hexasodium salt was placed in a 37 ° C. constant temperature cell (1 cm square), and 20 Leave for a minute. If the temperature in the cell becomes constant, glucose oxidase (0
˜200 U / L) was added to the test solution (50 μl), and the increase in absorbance was measured at a wavelength of 595 nm. Similarly, the measurement results were compared with those measured using a reagent solution containing no SOD, and the results are shown in FIG.

[0020]

INDUSTRIAL APPLICABILITY The method of the present invention uses POD and the triphenylmethane derivative represented by the general formula (I) as shown in the examples, and is carried out in the presence of SOD to obtain a hydrogen peroxide concentration of 0. It is possible to measure up to about 1 μM. It can be performed with 2 to 4 times the sensitivity as compared with the conventional method.

[Brief description of drawings]

FIG. 1 is a measurement diagram of hydrogen peroxide according to the present invention.

FIG. 2 shows 4,4 ′, 4 ″ -as a color developing reagent according to the present invention.
It is a measurement diagram of glucose using tri [N, N-di (3-sulfopropyl) amino] triphenylmethane hexasodium salt.

FIG. 3 is a bis [4-[(N
-Ethyl-N-sulfopropyl) amino] phenyl]
[4- (N, N-diethylamino) phenyl] methane
It is a measurement diagram of glucose using a disodium salt.

FIG. 4 is a bis [4-[(N
-Ethyl-N-sulfopropyl) amino] phenyl]
It is a measurement diagram of glucose using (4-chlorophenyl) methane disodium salt.

FIG. 5 is a measurement diagram when measuring cholesterol according to the present invention.

FIG. 6 is a measurement diagram when measuring uric acid according to the present invention.

FIG. 7 is a measurement diagram when measuring galactose according to the present invention.

FIG. 8 is a measurement diagram when measuring lactic acid according to the present invention.

FIG. 9 is a measurement diagram when measuring the activity of glucose oxidase according to the present invention.

Claims (1)

[Claims] 1. A peroxidase and a general formula: In the method for measuring hydrogen peroxide using the triphenylmethane derivative represented by the formula 1, the method is carried out in the presence of superoxide dismutase. (In the formula, R
¹ and R ³ are a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or a sulfo group, a substituted alkyl group having 1 to 4 carbon atoms having a substituent of an alkali metal base such as a hydroxyl group or a sulfo group, R ² and R ⁴ are A sulfo group, a substituted alkyl group having 1 to 4 carbon atoms having a substituent of a hydroxyl group or an alkali metal base of a sulfo group, R ⁵ represents a hydrogen atom, a chlorine atom, a bromine atom, an iodine atom,
An alkyl group having 1 to 4 carbon atoms or a group represented by -N (R ⁹ ) R ¹⁰ , wherein R ⁹ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a sulfo group, a hydroxyl group or an alkali metal sulfo group. A substituted alkyl group having 1 to 4 carbon atoms having a base substituent, R ¹⁰ is a sulfo group, a substituted alkyl group having 1 to 4 carbon atoms having an alkali metal base substituent of a hydroxyl group or a sulfo group, R ⁶ ,
R ⁷ and R ⁸ are hydrogen atom, an alkyl group or an alkoxy group having 1 to 3 carbon atoms having 1 to 4 carbon atoms).