JPS63291594A - Determination of hydrogen peroxide - Google Patents

Abstract

PURPOSE:To accurately and easily determine hydrogen peroxide, by adding a water-soluble compound of manganese and/or cobalt to a system forming hydrogen peroxide by the action of XOD and adding a metal chelating agent to the mixture. CONSTITUTION:A system forming hydrogen peroxide by the action of XOD (xanthine oxidase) is added with a water-soluble compound of preferably Mn(II) and/or Co(II) in an amount of >=0.01mM, preferably 0.1-30mM. A metal chelating agent such as EDTA is added to the mixture in an amount equal to or higher than the molar concentration of the water-soluble metal compound. The hydrogen peroxide generated by the action of XOD is determined by colorimetry using POD and an oxidizable color-developing reagent.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to peracid produced through the action of xanthine oxidase (hereinafter abbreviated as XOD) [Background of the Invention] An enzyme involved in nucleic acid metabolism. Certain guanase, adenosine deaminase, purine nucleoside phosphorylase, etc.
In the field of clinical testing, inosine, hypoxanthine, xanthine, etc., which are metabolites of these enzymes, and electrophosphorus, which is the substrate of purine nucleoside bosphorylase, are important measurement items in the field of clinical testing. , the establishment of accurate and simple measurement methods has recently become an important issue.

As a method for measuring these substances that contribute to nucleic acid metabolism, for example, in the case of guanase, the decrease in the substrate purine is measured by changes in ultraviolet absorbance (Arch, Biochem,
2Q, p. 124, 1950), a method for measuring the product ammonia (Cancer Res, 12, 5
p. 24, +952), XOD to the product xanthine
(J, Biol,
Chelg,, 167.461 pages.

For the measurement of adenosine deaminase, a method for measuring the product ammonia (J, Biol, Che.
■,, +67.445 pages, +947), a method of converting the product inosine into uric acid and hydrogen peroxide through purine nucleoside phosphorylase and XOD, and measuring the hydrogen peroxide produced (Che+g, Pharn
+, Bul 1, . 29,426 pages, +981), etc. are known.

However, methods that measure ammonia, methods that measure the decrease in substrate purines by changes in ultraviolet absorbance, or methods that measure the product uric acid are affected by endogenous ammonia, ultraviolet absorbing substances, or uric acid in the sample. Therefore, even if a sample blank value is measured, the measurement result may be inaccurate.

On the other hand, in the method of measuring hydrogen peroxide produced by the action of XOD using hyboxanthin (or xanthine) as a substrate, superoxide anion (0 = 2) is simultaneously produced in addition to hydrogen peroxide, Moreover, the production ratio of hydrogen peroxide and superoxide anion varies depending on the pH or substrate concentration, J, Biol, Chem, υ
15, p. 4053, +970), hydrogen peroxide and superoxide anion react directly to decompose hydrogen peroxide, or hydrogen peroxide and peroxidase (hereinafter referred to as
It is abbreviated as POD. ) Since the superoxide anion reduces the dye produced through the reaction of This reaction may be lower than the theoretical yield or the amount of dye produced may not be proportional to the theoretical yield of hydrogen peroxide, and these phenomena vary depending on the measurement conditions. When quantifying substances, there were problems such as the reliability of measurements only in low concentration ranges.

Therefore, in order to solve these problems in the method of measuring hydrogen peroxide produced by the action of XOD, various methods are used, such as superoxide dismutase (SOD).
A method of making the liquid coexist and making the liquid acidic after the reaction (Tokuko Kokko 5th
9-66899 (Japanese Patent Publication No. 59-2280), or a method in which colorimetric determination is carried out by adding a coloring reagent after once making the reaction liquid acidic (Japanese Patent Publication No. 59-2280) has been reported. However, these methods require complicated operations due to the increased number of measurement steps, and have not been applicable to the so-called late method for measuring enzyme activity. In addition to these, there are also reports (Japanese Patent Publication No. 55-10829
8), but the enzymes (XOD) that can be used for this are limited, and further improvements have been desired.

In addition, as a method for measuring phosphorus on a screen, Fiske-5u
bbaRow method (J, Biol, Chem, J6,3
75, 1925) is commonly used, but since it uses a strong acid, there are concerns that it may affect the materials used in the automatic analyzer, so a new automatic analysis! It has been desired to establish a measurement method that can be applied to a position.

[Object of the invention] The present invention was made in view of the above-mentioned circumstances, and
The purpose of the present invention is to provide an accurate and simple method for quantifying hydrogen peroxide produced through the action of OD, which solves all the various problems that have conventionally been sought for improvement.

[Configuration of the Invention] In order to achieve the object of the present invention, the present invention has the following configuration.

A method for measuring hydrogen peroxide produced through the action of rXOD, which is characterized by adding a water-soluble compound of manganese and/or cobalt to the hydrogen peroxide production system. Quantification method. In other words, when the present inventors measure hydrogen peroxide produced by the action of XOD on hyboxanthin or xanthine, for example, various problems arise due to superoxide anions produced simultaneously with hydrogen peroxide. In order to solve this problem, we conducted intensive research on substances that quantitatively convert superoxide anions into hydrogen peroxide, and discovered that water-soluble compounds of manganese and cobalt could achieve this purpose. The invention was completed.

Examples of the water-soluble manganese or cobalt compound used in the present invention include manganese acetate.

Acetylacetone manganese JC #Ammonium manganese, manganese benzoate, manganese p-aminobenzoate, manganese borate, manganese n-butyrate, manganese carbonate, manganese chloride, manganese 4-cyclohexylbutyrate, 2-ethylhexane manganese, manganese formate, manganese nitrate , manganese phosphate (basic).

Manganese sulfate, MrrEDTA, manganese hexafluoroacetylacetone, manganese trifluoroacetylacetone, manganese bromide, manganese gluconate, and, for example, cobalt chloride, cobalt bromide.

Water-soluble salts (including complex salts) of manganese or cobalt, such as cobalt sulfate, cobalt nitrate, cobalt acetate), ammonium cobalt M acid, and hexaammine cobalt chloride.
and water-soluble chelate compounds, but especially divalent manganese ions (Mn2+) and divalent cobalt ions (C
o2") is more preferably used. These manganese or cobalt water-soluble compounds may be used alone or in combination of two or more, and the amount used is the total amount in the system. The minimum requirement is for the presence of at least 0.01 mm, but usually 0.
A range of 1 to 30 m is preferably used.

One of the preferred embodiments of the present invention is a method of using a water-soluble compound of manganese or/and cobalt in combination with a metal chelating agent. That is, the metal chelating agent is manganese or/
By using it together with a water-soluble compound of cobalt,
Hydrogen peroxide produced under the action of XOD can be measured with higher sensitivity, and substances related to nucleic acid metabolism can be measured with higher sensitivity.

Metal chelating agents used for this purpose include:
Examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid fi (DTPA), etc., and substances that can form chelates with manganese or cobalt. Either is fine. These metal chelating agents may be used alone or in combination of two or more, and their concentration may be equivalent to or higher than the molar concentration of the water-soluble metal compound used.

Examples of substances that can be measured by the method of the present invention include X
Xanthine, hyboxanthin, etc., which are substrates for OD, or adenosine, inosine, guanine, etc., which are converted into these substances by appropriate enzymatic reactions, or enzymes such as adenosine deaminase, purine nucleoside phosphorylase, guanase, etc., which produce the above-mentioned substances. , inorganic phosphorus, etc., but there is no particular limitation as long as it can lead to a reaction system that ultimately generates hydrogen peroxide by the action of XOD.

The XOD used in the present invention may be derived from milk or microorganisms.

The quantitative method of the present invention does not require the addition of a water-soluble manganese or/and cobalt compound to the hydrogen peroxide production system, or the addition of a metal chelating agent to the hydrogen peroxide production system. This can be carried out according to a method known per se in which hydrogen oxide is colorimetrically fixed using POD and an oxidizable coloring reagent, and the measuring reagent used can be a reagent used in a method known per se. Enough. That is, POD may be derived from plants, animals, or microorganisms, but it is usually preferably derived from horseradish, and the oxidizable coloring reagent is usually hydrogen peroxide-P.
3-Methylbenzothiazolinone hydrazone (MBTH) is an oxidizable coloring reagent used in OD systems, such as a combination of 4-aminoantipyrine and a phenol compound or an N,N-disubstituted aniline compound. and aniline compound, triphenylmethane leuco dye, benzidine derivative, 0-tolidine derivative,
Examples include, but are not limited to, diphenylamine derivatives, triallylimidazole readers, leucomethylene blue derivatives, 0-phenylenediamine, 2,2'-aminobis(3-ethylhendithiazoline-6-sulfonic acid) or salts thereof, etc. It's not a thing.

Furthermore, the buffer used to adjust the liquid properties of the measurement reagent is not particularly limited as long as it is commonly used, and examples thereof include phosphate buffer, Tris buffer, Gud buffer, and the like. However, it goes without saying that for the measurement of inorganic phosphorus, a buffer other than the phosphate buffer must be used. The liquid property (pH) of the measurement reagent is determined by the substance to be measured, XOD
Although it is necessary to consider the optimum 1) H of the enzyme used in conjugation with the enzyme, a pH range of 6 to 10 is usually preferably used. The temperature at the time of measurement is not particularly limited as long as it is a temperature at which the desired reaction proceeds, but it is usually between 25 and 40°C.
A range of 0.degree. C. is preferably used. It goes without saying that the measurement reagent used in the present invention contains necessary substrates and conjugated enzymes at a required concentration depending on the substance to be measured.

In the method of the present invention, when the sample contains substances that interfere with measurement such as ascorbic acid and bilirubin, methods using enzymes such as ascorbic acid oxidase and bilirubin oxidase, or methods using iodate and periodate are used. It may be processed by a conventionally well-known method such as a method using
Also, the method described in JP-A No. 60-262599,
That is, the treatment may be performed by using copper ions and POD in combination with aniline compounds, phenol compounds, 4-aminoantipyrine, etc., and the effects of these can be reduced by treatment according to an appropriate method among these. Can be done.

The method of the present invention can normally be measured in one step, but it may also be a two-step operation by adding a pretreatment operation to remove interfering substances, or when the target substance to be measured is an enzyme. This may be carried out as a two-step operation using a reaction stop solution, or as a three-step operation by adding a pretreatment operation as described above.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these in any way.

[Example] Example 1. Quantification of Hyboxanthin (Measurement Reagent) A measurement reagent was prepared by dissolving the following substance in a 0.1M N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)/NaOH buffer to the following concentration.

XOD
0.04U/s+IP OD
4U/m1N-ethyl-N-(2
-Hydroxy-3-sulfopropyl)-s-toluidine (TOO5) 260g/14-aminoantipyrine 90g/IMnC12114H
201mM (sample) Hyboxanthin 3.2.6.4.9.6.12.
An aqueous solution containing 8■- was prepared and used as a sample.

(Procedure) A 10 μm sample was taken, a measuring reagent 2.51 was added thereto, mixed well, and subjected to a five-park reaction at 37° C., and then the absorbance Eq at 555 n− was measured.

A similar operation was performed using purified water instead of the sample to measure the reagent blank FBI.

(Results) The absorbance (E
A calibration curve obtained by connecting the points plotted along the vertical axis is shown in FIG. 1 (-Δ-).

Example 2 C instead of MnC12.4H20 in the measurement reagent of Example I
Example 1 was carried out using the same measurement reagent as in Example 1, except that the measurement reagent was a 1mM solution of oCl2.6H20.
Measurement was performed on the same sample as in Example 1 using the same procedure as in Example 1.

(Results) The absorbance obtained for the hypoxanthin concentration on the horizontal axis (
A calibration curve obtained by connecting the points plotted along the vertical axis (-0-) is also shown in FIG.

As is clear from FIG. 1, the calibration curves according to the present invention obtained in Examples 1 and 2 were both straight lines passing through the origin, indicating good quantitative properties.

Example 3. ' (Measurement reagent) EDTA-2Na 1mM was added to the measurement reagent of Example 1.
The mixture to which was added was used as the measurement reagent.

(sample) Same as Example 1.

(Operation method) Same as Example I.

(Results) The luminous intensity obtained for each hypoxanthin concentration on the horizontal axis (
A calibration curve obtained by connecting the points plotted along the vertical axis of Es-EBt) is shown in FIG. 2 (-〇-).

Comparative Example 1゜MnCl2・4H20 and ED from the measurement reagent of Example 3
The measurement was performed on the same sample as in Example 3 using the same measurement reagent as in Example 3, except that TA-2Na was omitted, and in the same manner as in Example 3.

The results are also shown in Figure 2 (-).

Comparative Example 2゜ Superoxide dismutase (
The same measurement reagent as in Example 3 was used, except that 50Ul of SOD) was added to the measurement reagent.
The same sample as in Example 3 was measured by the same operation as in Example 3.

The results are also shown in FIG. 2 <-X->.

As is clear from FIG. 2, Example 3 according to the method of the present invention
The calibration curve obtained in Comparative Example 1 or 2 was superior in measurement sensitivity and linearity. It can be seen that the combined use of -2Na) resulted in a much higher measurement sensitivity than the calibration curves obtained in Examples 1 and 2 in which no metal chelating agent was used.

Reference example 1゜ (Measurement reagent) Same as Example 3.

(Sample) Add hydrogen peroxide to 6.4. I2.8. +9.2,25.
I8 solution containing 6mM was prepared and used as a sample.

<Pk etiquette) Same as Example 3.

(Results) The obtained absorbance (Es-Eat) is shown in Table 1 together with the results obtained in Example 3.

Margin Table-1 Theoretically, 1 equivalent of hypoxanthin produces 2 equivalents of hydrogen peroxide by reaction with XOD. That is, as is clear from Table 1, it can be seen that according to the method of the present invention, the theoretical amount of hydrogen peroxide is produced from hyboxanthin.

Real Ai! fIJ4a,j! ! (Measurement reagent) Dissolve the following substances in 0.1M N-(2-7cetamido)-2-aminoethanesulfonic acid (ACES) Na0ll buffer solution to the following concentration to prepare the measurement reagent. And so.

X OD O,04U/a+
1POD 4υ1IN-
Ethyl-N-(2-hydroxy-3-sulfopropyl)
1-Toluidine (TOOS) 260mg/
l-doaminoantipyrine 9g/IMn
C12・4H201mM EDTA-2Na Im purine nucleoside phosphorylase 0.4U/ml inosine
2sM (sample) An aqueous solution containing 10, 20, 30, and 40 grams of inorganic phosphorus, respectively, was prepared as an AS1 sample.

(Procedure) A 10 μm sample was taken, a measurement reagent 2.51 was added thereto, mixed well, reacted at 37° C. for 5 minutes, and then the absorbance Es at 555 nm was measured.

A similar operation was performed using purified water instead of the sample to measure reagent blank E81.

(Results) FIG. 3 shows a calibration curve obtained by connecting the points obtained by plotting the obtained absorbance (Es EBI) along the vertical axis against the inorganic phosphorus concentration (l1g/old) on the horizontal axis.

As is clear from FIG. 3, the obtained calibration curve was a straight line passing through the origin, indicating good quantitative properties.

Example 5. Determination of inorganic phosphorus in serum (Measurement reagent) Same as Example 4.

(Trial I'4) Ten human serum samples were used as samples.

(Procedure) A 108 m sample was taken, 2.5 ml of the measurement reagent was added thereto, mixed well, and reacted at 37° C., after which the absorbance ES at 555 nm was measured.

Purified water and agricultural machinery phosphorus standard solution (denki phosphorus IO) can be used instead of the sample.
++8/old content), the reagent blank Ea+ and the standard solution absorbance E s+,l were measured.

Using the obtained absorbance, the agricultural machinery phosphorus concentration P (+s3/dl) in the sample was calculated using the following formula.

P (mg/dl)”((Es-Es+)÷(Es+d
-EBI))XIO (Results) The measurement results are shown in Table-2.

Comparative example 3. Quantification of Wt organophosphorus in serum by conventional method Quantification of inorganic phosphorus was carried out for the same sample as in Example 5 using a commercially available inorganic phosphorus measuring reagent [Inorganic phosphorus C-Test Fuco (manufactured by Wako Pure Chemical Industries, Ltd.)]. went.

The procedure was carried out according to the instruction manual for the detection reagent.

(result) The measurement results are also shown in Table-2.

Table 2 Correlation coefficient: γ: 0.999 Regression linear equation: Y = 1.003
There was good agreement with the values obtained in , and no significant difference was observed between them.

[Effects of the Invention] As described above, the present invention provides a method for measuring hydrogen peroxide produced via XOD that is accurate and simple and can be applied to an automatic analyzer. This method has a remarkable effect in that it has become possible to easily and more accurately measure substances involved in nucleic acid metabolism.

[Brief explanation of drawings]

Figure 1 shows the calibration curve obtained in Examples 1 and 2, connecting the points obtained by plotting the absorbance obtained against the hypoxanthin concentration (mM) on the horizontal axis and along the vertical axis. be. Here, -1 indicates the results obtained in Example 1, and -0- indicates the results obtained in Example 2. Figure 2 shows the calibration curves obtained in Example 3, Comparative Example 1, and Comparative Example 2, with the horizontal axis representing the hyboxanthin concentration (mM).
It connects the points obtained by plotting the absorbance obtained for the vertical axis along the vertical axis. Here, -0- is the result obtained in Example 3, -Δ- is the result obtained in Comparative Example 1, -×
- indicates the results obtained in Comparative Example 2. Figure 3 shows the calibration curve obtained in Example 4, which connects the points obtained by plotting the absorbance obtained against the noodle machine phosphorus concentration (sg/dl) on the horizontal axis and along the vertical axis. be. Patent Applicant: Aiko Pure Chemical Industries, Ltd. Figure 1 Hypoxa〉Kibunashi concentration (rr, M) Figure 2 Hypoxa〉 Tishidan degree (rnM) M3 diagram Inorganic/1 degree (rngn1)

Claims (2)

[Claims] (1) A method for quantifying hydrogen peroxide produced through the action of xanthine oxidase, which is characterized by adding a water-soluble compound of manganese or/and cobalt to the hydrogen peroxide production system. Method for quantifying hydrogen oxide. (2) The quantitative method according to claim 1, in which a water-soluble compound of manganese or/and cobalt and a metal chelating agent are used in combination.