JPH02245198A - Measurement of superoxide dismutase and reagent for measuring - Google Patents

Abstract

PURPOSE:To measure the title enzyme for clinical test, etc., in high accuracy by reacting xanthine, etc., with xanthine oxidase in the presence of a phenol and treating the resulting substance with superoxide dismutase and measuring the amount of formed hydrogen peroxide. CONSTITUTION:Xanthine or hypoxanthine is reacted with xanthine oxidase in the presence of a phenol compound (e.g. sodium sallcylate) to form a superoxide anion, which is used as a substrate and treated with superoxide dismutase in a test specimen, formed hydrogen peroxide is decomposed with peroxidase, generated enzyme is reacted with a hydrogen donor such as 4 aminoantipyrin-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine base and absorbance at 550nm wavelength is measured to determine the aimed enzymatic activity.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for measuring the activity of superoxidase (hereinafter abbreviated as SOD) contained in body fluids, such as serum, and a reagent used therein.

(Prior Art) In recent years, it has been revealed that superoxide anion (hereinafter referred to as 0□-), which is a type of active oxygen produced in living bodies, causes inflammation, aging, and cancer. On the other hand, the existence of SOD, which is involved in the reaction that disproportionates 02- in the body, has also been revealed, and it is believed that this enzyme protects the body from oxygen exposure. Based on this idea, S in serum
The development of a simple method for measuring OD activity is eagerly awaited from the viewpoints of clinicopathology and clinical testing.

Conventional methods for measuring SOD activity include the nitro blue tetrazolium method and the cytochrome C method. These methods use xanthine and xanthine oxidase in the 0°-generation system, and reduce the reduced forms of nitroblue tetrazolium and cytochrome C due to the dismutation of 0□- to hydrogen peroxide by SOD. SOD expressed as inhibition rate
I'm looking for activation.

The nitroblue 5-tetrazolium method will be described below as a conventional technique.

When xanthine oxidase acts on xanthine, 02
- is generated [Formula (I)]. The generated 02- reduces the coexisting nitroblue tetrazolium (NET) to form diformazane [Formula 2] contains superoxide dismutase (S) in the reaction solution.

In the presence of D), a portion of 02- is disproportionated to hydrogen peroxide and oxygen [(3), reducing the formation of diformazan. Therefore, while the SOD activity in the sample can be measured by determining the degree of inhibition of the diformazan form based on the reaction between 02'' and NBT, the cytochrome C method also requires that nitroblue tetrazolium is replaced with cytochrome C. The principle is the same.

However, these methods require high reagent blinding and have poor measurement accuracy. Problems such as not directly measuring SOD activity were pointed out.

There have also been attempts to measure hydrogen peroxide by utilizing the disproportionation of N O2- into hydrogen peroxide by SOD, but the xanthine-xanthine oxidase system used for the 0°-generation system is , 02- as well as hydrogen peroxide are generated from the reaction system, the reagent blindness is high and the accuracy of SOD activity measurement is very poor.

(Problems to be Solved by the Invention) The purpose of the present invention is to solve the drawbacks of the conventional SOD activity measurement method that measures the phenomenon of sample absorbance against reagent blinding, such as high reagent blinding, poor measurement accuracy, and measurement range. An object of the present invention is to provide a novel, highly accurate and simple method for measuring SoD activity that improves the narrowness of the method, and a reagent for the same.

(Means for Solving the Problems) That is, the present invention involves reacting xanthine or hypoxanthine with xanthine oxidase, causing superoxide dismutase to act using superoxide anion as a substrate, and measuring the amount of hydrogen peroxide produced. In the method for measuring the activity of superoxide dismutase, the method for measuring superoxide dismutase is characterized by carrying out a xanthine oxidase reaction in the presence of a phenolic compound, and i) xanthine or hypoxanthine, i) xanthine oxidase, A reagent for measuring superoxide dismutase characterized by containing ii) a phenol compound, iv) a hydrogen donor, and V) peroxidase.

The present invention prevents the production of hydrogen peroxide from the xanthine or hypoxanthine-xanthine oxidase system by adding a phenol compound when using a xanthine or hypoxanthine-xanthine oxidase system in the 0□-generating system, and also prevents the production of hydrogen peroxide from the xanthine or hypoxanthine-xanthine oxidase system by The present invention provides a novel method for measuring SOD activity and its reagent, which is characterized in that excess 02- that is not converted to hydrogen peroxide is eliminated by a reaction between peroxidase and hydrogen donor.

In the present invention, reagent blinding for SOD activity measurement is reduced;
Furthermore, the activity of SOD can now be determined by measuring the amount of hydrogen peroxide produced.

The xanthine oxidase used in the present invention is not particularly limited as long as it is an enzyme that acts on hypoxanthine or xanthine to produce superoxide anions. Examples of such enzymes include those purified from milk and the liver of mammals.

In the present invention, the phenol compound may be any substance as long as it prevents the production of hydrogen peroxide from the xanthine or hypoxanthine-xanthine oxidase system, and includes, for example, salicylic acid, salts thereof, and derivatives thereof.

Examples of salicylic acid salts include sodium salicylate, potassium salicylate, and magnesium salicylate. Derivatives of salicylic acid include salicyl alcohol, salicylaldehyde, salicylamide, salicylanilide, methyl salicylate, and salicyl sulfuric acid. The effective concentration of the phenol compound is not particularly limited, but is preferably 0.01% to 10% (w/v) in order to suitably measure SOD activity.

Examples of the hydrogen donor in the present invention include 4-aminoantipyrine and a phenol compound, 4-aminoantipyrine and an N,N-disubstituted aniline compound, and a benzothiazolinone hydrazone compound and an aniline compound.

Specific examples of phenolic compounds include p-chlorophenol, p-bromophenol, 3,5-dichlorophenolsulfonic acid, 3-hydroxy-2,4,E3-triiodobenzoic acid, and the like.

Specific examples of aniline compounds include N-methyl-N-
Hydroxymethyl-3-methylaniline, N-ethyl-
N-hydroxyethyl-3-methylaniline, N-ethyl-N-hydroxyethyl-3-ethylaniline, N-
Methyl-N-hydroxyethyl-3-methylaniline,
N-methyl-N-hydroxypropyl-3-methylaniline, N-ethyl-N-hydroxypropyl-3-methylaniline, N-methyl-N-hydroxyethyl-3-
Ethylaniline, N-propyl-Nhydroxyethyl-
3-Thylaniline, N-methyl-N-hydroxyethyl-3-propylaniline, N,N-bis(β-hydroxyethyl)-3-methylaniline, N,N-bis(β-
hydroxypropyl)-3-methylaniline, N,N-
Dimethyl-3-methylaniline, N,N-dimethyl-3
-ethylaniline, N,N-dimethyl-3-propylaniline, N,N-diethyl-3-methylaniline, N,
N-diethyl-3-ethylaniline, N,N-dipropyl-3-methylaniline, N-ethyl-N-(β-methanesulfonamidoethyl)-m-)luidine, N-ethyl-N-(β-acetamide ethyl)-3-methylaniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diisopropylaniline, N-methyl-N-hydroxyethylaniline, N-ethyl-N-hydroxyethylaniline, N -ethyl-N-sulfopropyl-m-)luidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-)luidine, N-ethyl-N-(2-bitroxy-3-sulfopropyl) ,
m-anisidine, N-ethyl-N-(2-hydroxy-
3-sulfopropyl)aniline, 3,5-dimethoxy-
N-ethyl-N-(2-71 idroxy-3-sulfopropyl)aniline, 3,5-dimethyl-N-ethyl-N
-(2-hydroxy-3-sulfopropyl)aniline,
N-ethyl-N-sulfopropyl-m-aniline, N-
Examples include ethyl-N-sulfopropyl-3,5-dimethylaniline and N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline.

There is no particular restriction on the concentration of the aniline compound and the phenol compound used, but approximately 0.1 to 20 mM is optimal. The concentration of 4-aminoantipyrine used is 1~
About 20mM is preferable.

The concentration of peroxidase used in the present invention is 1 unit/-
If it is 100 units/if, SOD activity can be suitably measured.

When the present invention is to be carried out suitably, it is necessary to carry out the work in a buffer solution. The pH of the reagent of the present invention is 5.0 to 9.
．． Any amount is acceptable as long as it is 0, but preferably 6.0~
It is 9.0.

In the present invention, xanthine or hypoxanthine is reacted with xanthine oxidase in the presence of a phenol compound, SOD is applied using the produced 02- as a substrate, and the amount of hydrogen peroxide produced is measured according to a conventional method. As a method for measuring hydrogen peroxide, for example, there is a method of developing color using peroxidase and a hydrogen donor and measuring absorbance. Reagents for measuring hydrogen peroxide, such as peroxidase and a hydrogen donor, are preferably present together with a phenol compound in the xanthine oxidase reaction system.

The reagent for measuring SOD of the present invention contains i) xanthine or hypoxanthine, i) xanthine oxidase, ii) a phenol compound, iv) a hydrogen donor, and V) peroxidase.

(Example) EXAMPLES The present invention will be described in detail below with reference to Examples.

Example 1゜Effect of phenolic compound First reagent xanthine oxidase 0.1 unit/-, peroxidase 10 units/m1.4-aminoantipyrine 0
．． 5 mol/no, N-ethyl-N-(2-hydroxy-3
-sulfopropyl)-m-toluidine 1.0 mmol/
11 Sodium salicylate was added to 50mMBS (Gud buffer) pH 6 to a concentration of 0.05% (W/V).
, 3.

A second reagent, hypoxanthine, was dissolved in distilled water to a concentration of 20 μmol/no.

third reagent A reagent obtained by removing sodium salicylate from the first reagent.

Procedure Place the second reagent 1.5- in a test tube and incubate for 5 minutes in a 37°C water bath.
After prewarming for 1 minute, the first reagent is 1.511L1! were added to a test tube and mixed, and the absorbance at 550 nm was measured 5 minutes after mixing. In order to examine the effect of sodium salicylate, the above operation was repeated in the same manner, replacing the first reagent with the third reagent. The measurement results are shown in Figure 1. In the figure, the vertical axis is 550 nm
The absorbance at , the horizontal axis indicates the measurement time (minutes). ■: A phenol compound was added, and ■: a phenol compound was not added. Even if the first reagent to which sodium salicylate is added and the second reagent are mixed and reacted, the absorbance of the reagent blind test does not increase. On the contrary, when the third reagent to which no sodium salicylate was added was mixed with the second reagent, the reagent blindness clearly increased due to the formation of hydrogen peroxide from hypoxanthine.

From the above, it can be seen that sodium salicylate inhibits the two-electron transfer of xanthine oxidase.

Example 2 Measurement of SOD Using the first reagent and second reagent of Example 1, SOD was measured.

Sample Toyobo - 600 units of SOD (derived from bovine blood groove)
d in distilled water.

Place 50μ of the operation sample in a test tube, prewarm it in a 37°C water bath for 5 minutes, add 1.5 parts of the first reagent to the test tube, mix,
Subsequently, 1.5 J of the second reagent was added and mixed. Absorbance at 550 nm was measured 5 minutes after mixing the second reagent. Reagent blind testing was performed in the same manner as above, using distilled water instead of the sample. The measurement results are shown in Figure 2. In the figure, the vertical axis is 550
Absorbance in nm, horizontal axis indicates measurement time (minutes). ■
is measured on a sample of 5OD600 units/+L1, ■
is measured using distilled water as a sample. The difference in absorbance between the reagent blind measurement and the measurement of the sample (SOD 800 units/1L1) is clear, indicating that hydrogen peroxide produced by SOD is being measured.

Example 3 SOD activity measurement First reagent xanthine oxidase 0.4 units/+d, peroxidase 30 units/-14-aminoantipyrine 0
．． 5 mmol/je, N-ethyl N-(2-hydroxy-
1.0 mol/3-sulfopropyl)-m-toluidine
Sodium salicylate 2.0% (W/V)
50 m Tris-HC buffer pH to a concentration of
It was dissolved at 8.0.

A second reagent, hypoxanthine, was dissolved in distilled water to a concentration of 50 μmol/l.

Sample SOD (derived from bovine serum) manufactured by Toyobo M Co., Ltd. at 0°20.4
It was dissolved in distilled water to a concentration of 0,60,100 units/-.

Place 0.15ml of the operational sample in a test tube and incubate for 5 minutes in a 37°C water bath.
After prewarming for minutes, first reagent 1.5. , J were added to the test tube and mixed, and then 1.5 d of the second reagent was added and mixed. After mixing the second reagent, the rate of increase in absorbance at 550 nm (Δ
0D550/min) was measured. Reagent blind testing was performed in the same manner as above, using distilled water instead of the sample. The measurement results are shown in Figure 3. The vertical axis shows the amount of change in absorbance per minute at 550 nm, and the horizontal axis shows the concentration of SOD (unit/d).

As the sample concentration increased, the rate of increase in absorbance at 550 nm (Δ0D550/min) also increased. 5OD10
High linearity up to 0 units/+n1, and reagent-blind 55
The rate of increase in absorbance at 0 nm (Δ0D550/min) is also very low.

From the above, it can be seen that the present invention is a highly practical SOD measurement method.

(Effects of the Invention) According to the SOD activity measuring method and its reagent of the present invention, the addition of a phenol compound prevents the production of hydrogen peroxide by xanthine oxidase, and also eliminates excess 02- by peroxidase and a hydrogen donor. Koki became possible. As a result, only hydrogen peroxide disproportionated by SOD is measured, making it possible to provide a highly accurate SOD measurement method with low reagent blinding and its reagent.

[Brief explanation of drawings]

FIG. 1 is a graph showing the effect of the phenol compound in Example 1. FIG. 2 is a graph of SOD measurement in Example 2. FIG. 3 is a graph of SOD measurement in Example 3.

Claims (2)

[Claims] (1) The activity of superoxide dismutase is measured by acting on superoxide dismutase using the superoxide anion produced by reacting xanthine or hypoxanthine with xanthine oxidase as a substrate and measuring the amount of hydrogen peroxide produced. A method for measuring superoxide dismutase, which comprises carrying out a xanthine oxidase reaction in the presence of a phenolic compound. (2) i) xanthine or hyposanthin, ii) xanthine oxidase, iii) phenolic compound, iv)
A reagent for measuring superoxide dismutase, comprising a hydrogen donor and v) peroxidase.