JPH05336998A - Method for measuring hydroxy radical - Google Patents

Abstract

PURPOSE:To provide the method for readily measuring hydroxy radicals with high speciality and high precision. CONSTITUTION:A specimen containing active oxygen is preliminarily treated with superoxide dismutase and catalase. Superoxide anions, hydrogen peroxide, etc., are thereby removed, and subsequently the concentration of the hydroxyl radicals is measured by methional method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring active oxygen, and more particularly to a method for measuring hydroxy radical.

[0002]

2. Description of the Related Art Recently, in the field of clinical medicine, reports on active oxygen and diseases have been popular in magazines. However, active oxygen is generally highly reactive, and even if it is generated in the living body, there is almost no method for quickly capturing it and taking it to the measurement.

Therefore, it is no exaggeration to say that it is mainly the reaction that progresses in a test tube, which is a closed system, that can measure active oxygen, and it is expected that it will be difficult to measure active oxygen in vivo ¹⁾ .

Generally, the following are known as active oxygen.

[0005] (a) superoxide anion oxygen triplet state occurs undergo one-electron reduction (O ₂ ^-) (b) hydrogen peroxide caused by receiving oxygen two-electron reduction of the triplet state (H ₂ O ₂₎ (c) is O-O bond of hydrogen peroxide, Note hydroxy radicals generated by receiving one electron reduction (· OH), there is a ¹ 0 ₂ in the active oxygen other than (a) ~ (c),
Since it has a short life in liquid and can be ignored ⁴⁾ , it does not cause an error in the above measurement method.

The method of measuring each active oxygen will be described below.

The superoxide anion is 2-methyl-6-phenyl-3,7-dihydroimidazo (1,2-
a) It can be specifically measured using a P-methoxy derivative of pyrazin-3-one.

Hydrogen peroxide is more stable than superoxide anion and is almost in a hydrogen peroxide state in a neutral solution. Various measurement methods such as the peroxidase and titanium methods have been reported.

Chemical measurement methods such as the p-nitrodimethylaniline method and the methional method are known for the hydroxy radical. However, these measurement methods have a fatal defect that the specificity is low, so that ESR (electron spin resonance) is used. ) Method is used ³⁾ .

However, the ESR method is not generally popular because the measuring device is expensive and requires skill in operation.

In the biological system, the hydroxy radical is mainly generated by the decomposition reaction (Fentou reaction) of hydrogen peroxide with Fe ²⁺ .

Since the reaction between the hydroxy radical and the organic compound proceeds at a rate almost equal to the diffusion-controlling rate, it rapidly disappears at the place where the hydroxy radical is generated, and the detection of this radical species is not easy. Therefore, the detection of hydroxy radicals is possible only in an in vitro (extracorporeal) system and in a state of not containing much organic matter.

As a chemical method of this hydroxyl radical, there are a p-nitrosodimethylaniline method and a methional method. The feature of the former is that the hydroxyl radical reacts with p-nitrosodimethylaniline (NMA: yellow substance) to change from yellow to colorless. The expected reaction mechanism of this reaction is shown in FIG.

The P-nitrosodimethylaniline method is
It is a method of knowing the generation of hydroxy radicals by adding NMA to the reaction system and following the decrease of A ₄₄₀ with time.
Assuming that 1 mol of MA reacts with 1 mol of hydroxy radicals, the amount of hydroxy radicals produced is expressed in mol.

On the other hand, the characteristic of the latter is that methional and a hydroxy radical react to produce ethylene by the reaction shown in FIG. Generated ethylene was collected and quantified by gas chromatography to obtain 1 mol of hydroxyl radical
More ethylene will be produced. The conditions of gas chromatography are: column: Chromosorb-102, column temperature: 70-80 ° C., carrier gas: nitrogen.

However, the specificity of hydroxy radicals is low in both measurement methods ^{3) 4)} .

Currently, the most reliable method for detecting hydroxyl radicals is PBN (α-phenyl-Nt-butylniy).
orone) or DMPO (5,5-dimethyl-1-pyrroline
-N-oxide) and other spin trapping agents
This is a method of analyzing OH adduct or DMPO-OH adduct (both radicals) by ESR spectroscopy ^{3) 5)} . ¹⁾ (Minaka Nakano, respiration, 9-9, P1158, 1990) ²⁾ (Nisida A.et, Clin.Chem.Acta, 179, p177, 1989) ³⁾ (Minaka Nakano, protein nucleic acid enzyme, 33-16, p2684, 198
8) ⁴⁾ (Naoshi Kaneda et al., Lipid Peroxide Experimental Method, p127, 1983) ^{5) 4)} , p2693

[0018]

It has been reported that NMA of the p-nitrosodimethylaniline method does not react with superoxide anion ^{4) 6),} but this NMA seems to react with hydrogen peroxide as well as hydroxyl radicals.

In the methional method, not only hydroxy radicals but also various organic peroxy radicals oxidize to produce ethylene ^{4) 7)} . As described above, both of the above-described conventional chemical methods for measuring hydroxyl radicals have fatal drawbacks in terms of the specificity of detecting hydroxyl radicals.

The ESR method, which is the most reliable measurement method for detecting hydroxy radicals, has the drawbacks that (i) the apparatus is expensive and the penetration rate is low. (Ii) Skilled in analysis. Therefore, there has been a demand for a specific simple measurement method for hydroxy radicals.

The present invention has been made based on the above background, and an object thereof is to provide a method for measuring a hydroxy radical which has high specificity, is easy, and is highly accurate.

⁶⁾ (Rose W., et., Eur. J. Biochem., 95, p62
1,1979) ⁷⁾ (Pryor WA, et., Biochem.Biophys.Res.Commun., 78,
418,1977)

[0023]

Means and Actions for Solving the Problems In order to solve the above problems, the present invention is characterized in that after adding superoxide dismutase and catalase to a sample containing active oxygen, hydroxy radicals are measured by the methional method. And

As described above, superoxide anion and hydrogen peroxide are removed by pretreatment by adding superoxide dismutase (SOD) and catalase to a sample containing active oxygen.

Further, active oxygen has ¹⁰ ₂ but it has a short life in a liquid and can be neglected ^4), so that it does not cause an error in the above measuring method.

Therefore, by measuring the hydroxy radical concentration by ordinary chemical measurement after the above-mentioned pretreatment, the true hydroxy radical concentration can be measured without being affected by other active oxygen. ..

In particular, it is possible to easily and highly accurately determine the true hydroxy radical concentration, which does not include superoxide anion, hydrogen peroxide concentration, etc., by detecting a hydroxy radical using the methional method as the above-mentioned chemical measurement method. You can

[0028]

EXAMPLE In this example, samples containing various active oxygens were previously treated with superoxide dismutase (SOD) and catalase to remove superoxide anion and hydrogen peroxide, and then hydroxy radicals were detected by the methional method. did.

Next, as a comparative example, the methional method, which is a conventional measuring method, was used to measure the above-mentioned various active oxygen-containing specimens and compare the measurement results to determine the chemistry of the hydroxy radical according to this example. The specificity and effectiveness of the dynamic assay were verified. The details are shown below.

First, a sample which has been confirmed to contain superoxide anion, hydrogen peroxide, and hydroxyl radical by the ESR method is prepared.

Next, 0.3 ml of the above sample and 1 × 10 ^-6 MSO
D, 1 × 10 ^-4 M catalase, including 1 × 10 ^-4 MEDTA 0.0
3 ml of 5M potassium phosphate buffer (pH 7.8) is sequentially added to the test tube, and pretreatment is performed at 37 ° C. for 15 minutes.

Next, methional is added to the pretreated sample to react with the hydroxy radical. As shown in FIG. 2, 1 mol of ethylene was produced from 1 mol of hydroxy radical by this reaction, and thus the produced ethylene was sampled and quantified by gas chromatography to determine the concentration of hydroxy radical.

The conditions for gas chromatography are as follows: column: Chromosorb-102, column temperature: 70-80.
C, carrier gas: nitrogen.

Three types of specimen samples 1, 2, and 3 are prepared by the above method.
Table 1 shows the results of measuring the hydroxyl radical concentration
Shown in.

[0035]

[Table 1]

Next, the same specimen sample 1 as in the above embodiment,
Two or three were measured by the methional method without pretreatment to determine the hydroxy radical concentration. The results are shown in Table 1 above.
Is also shown.

From this table, it can be seen that the concentration of hydroxy radicals in the measuring method of this example is lower than that in the conventional measuring method of the comparative example.

Here, as described above, the above 3 by the ESR method is used.
As a result of measuring the samples, it was confirmed that the above three samples contained superoxide anion, hydrogen peroxide, and hydroxyl radical.

Further, as a result of measuring the samples pretreated with SOD and catalase by the ESR method, superoxide anions and hydrogen peroxide were removed from the samples subjected to these pretreatments, and hydroxy radicals were used as active oxygen. It was confirmed that only exist.

From these results, the conventional measuring method has low specificity, and not only hydroxy radicals but also superoxide anion, hydrogen peroxide, etc. are detected, and as a result, values higher than the true hydroxy radical concentration are obtained. It can be seen that is obtained.

On the other hand, in this embodiment, the true hydroxy radical concentration can be obtained by applying the p-nitrosodimethylaniline method after the above pretreatment.

[0042]

As described above, in the method for measuring hydroxy radicals according to this embodiment, superoxide anion, hydrogen peroxide, etc. are removed by performing the above pretreatment.

Therefore, by applying a chemical measurement method such as the p-nitrosodimethylaniline method after performing the above-mentioned pretreatment, the hydroxy radical can be specifically determined, and the hydroxy radical concentration can be easily and highly accurately determined. You can ask.

[Brief description of drawings]

FIG. 1 is an explanatory view of a reaction mechanism of p-nitrosodimethylaniline and a hydroxy radical.

FIG. 2 is an explanatory diagram of a reaction mechanism of methional and a hydroxy radical.

Claims (1)

[Claims] 1. A method for measuring hydroxyl radicals, which comprises adding superoxide dismutase and catalase to a sample containing active oxygen and then measuring the hydroxyl radicals by the methional method.