JP2004518433A - How to measure the antioxidant capacity of cells - Google Patents

Abstract

The present invention relates to a method for measuring the antioxidant capacity of a cell. The method for measuring the antioxidant capacity of the cells of the present invention comprises the following steps: a step of preparing a sample by removing and homogenizing an animal tissue whose antioxidant capacity is to be measured; 2 ′, 7 ′ -Adding dichlorofluorescin diacetate (DCFH-DA) to start the reaction; adding ascorbic acid and iron ions to the reaction mixture and measuring the fluorescence; and measuring the fluorescence measurement value as a standard curve of fluorescence versus DCF concentration. And measuring the antioxidant ability of the cells by converting the concentration into 2 ', 7'-dichlorofluorescein (DCF) concentration. The method of the present invention can simultaneously measure and compare the antioxidant capacity of multiple samples, and can easily measure total oxidative stress in a sample. Thus, it can be used in practice for studies on radioactivity at the biological level.

Description

[0001]
(Background of the Invention)
(Technical field)
The present invention relates to a method for measuring the antioxidant capacity of cells, and more particularly, to the use of 2 ′, 7′-dichlorofluorescin diacetate (DCFH-DA) when free radicals are generated by ascorbic acid and iron ions. The present invention relates to a method for measuring oxidizing ability.
[0002]
(Background technology)
Cells under normal physiological conditions continuously produce free radicals, and various factors such as disease, drugs, and radiation can cause enormous increases in free radicals. Although the cell type may vary, all cells have enzymatic / non-enzymatic protection mechanisms against free radicals, so the amount of free radicals in the cells may be reduced by prooxidants and antioxidants. It is represented by the sum of the functions of the oxidants. In general, the quantification of free radicals in tissues has been used as an indicator of the susceptibility of tissues to irradiation, since a tremendous amount of free radicals are generated after irradiation of animals.
[0003]
On the other hand, the amount of free radicals with high reactivity easily changes over time. For that reason, the only available means for measuring free radicals have hitherto been electron spin resonance spectroscopy (ESR) or electron paramagnetic resonance spectroscopy (ESR), or electron paramagnetic resonance spectroscopy (ESR). The method used. However, the method using ESR has shown difficulty in measuring highly reactive free radicals such as superoxide and hydroxy radicals. Therefore, the spin trapping method is commonly used in the art, but it converts free radicals in a sample into 5,5-dimethylpyrroline-N-oxide. A method for quantifying relatively stable free radicals produced by reacting with a trapping agent such as DMPO) or α-phenyl-tert-butylnitrone (PBN); It is. Although the method is accurate and can identify the type of free radicals by using different trapping agents, it is satisfactory in the sense that the equipment required is very expensive and only ESR spectrometer experts can analyze the results. Proved not to be.
[0004]
To overcome the above problems, indirect methods have been used in the art. One of them is a method in which a free radical is reacted with a specific marker such as salicylic acid to produce a product such as dihydroxybenzoic acid, which is separated by HPLC to indirectly determine the amount of the free radical. Although this method is more widely used than the ESR method, analysis using HPLC is time-consuming and expensive.
[0005]
In another indirect method used, chemiluminescence is generated during the free radical generation process. Chemiluminescence has been reported to be found, for example, in singlet oxygen, active triplet oxygen such as carbonyl groups, ONOO-reactions, lipoxygenase reactions, Fenton reactions, and the like. However, the intensity of chemiluminescence is too low to measure directly and is amplified by reaction with a suitable compound. One example of an amplification method uses luminol or lucigenin, which reacts with radicals, which is simpler and easier than ESR or HPLC methods. However, it has disadvantages such as low sensitivity, susceptibility to reaction environment such as pH, and high background noise.
[0006]
To overcome the above problem of chemiluminescence, the conversion of non-chemiluminescent 2 ', 7'-dichlorofluorescin (DCFH) to chemiluminescent 2', 7'-dichlorofluorescein (DCF) is useful for detecting free radicals. Used. However, since the amount of free radicals in cells is the sum of the functions of prooxidants and antioxidants, such a method that can only measure the amount of free radicals in cells or tissues is not enough to quantify the antioxidant capacity of cells. Not appropriate.
[0007]
Under such circumstances, there is strong motivation to seek and develop a method to measure the antioxidant capacity of cells, which indicates the ability of the cells to reduce the amount of free radicals.
[0008]
(Disclosure of the Invention)
The present inventors have conducted intensive studies to develop a method for measuring the antioxidant capacity of cells, and found that free radicals that were suddenly generated in various tissues and cells by ascorbic acid and iron ions were 2 ′, 7 ′. -Dichlorofluorescin diacetate (DCFH-DA); and found that the relative antioxidant capacity of various samples could be measured by comparing the measured values.
[0009]
Accordingly, it is a primary object of the present invention to provide a method for measuring the antioxidant capacity of cells using ascorbic acid, iron ions and DCFH-DA.
[0010]
The above and other objects and features of the present invention will become more apparent from the following description given in conjunction with the accompanying drawings.
[0011]
(Embodiment of the invention)
The method for measuring the antioxidant capacity of cells according to the present invention comprises the following steps: a step of preparing a sample by removing and homogenizing an animal tissue whose antioxidant capacity is to be measured; 2 ′, 7′- adding dichlorofluorescin diacetate (DCFH-DA) to start the reaction; adding ascorbic acid and iron ions to the reaction mixture and measuring the fluorescence; and measuring the fluorescence measurement value with a standard curve of fluorescence versus DCF concentration. And measuring the antioxidant ability of the cells by converting to 2 ', 7'-dichlorofluorescein (DCF) concentration.
[0012]
The method for measuring the antioxidant capacity of a cell according to the present invention is further described by the following steps.
[0013]
Step 1 : Preparation of sample A sample is prepared by removing and homogenizing an animal tissue whose antioxidant ability is to be measured: liver, stomach, lung, heart, brain and kidney can be used as the tissue. However, the present invention is not limited to these. The homogenization of the tissue is preferably, but not limited to, carried out in a buffer using a glass homogenizer with previously frozen-fixed tissue.
[0014]
Step 2 : Addition of DCFH-DA The reaction is started by adding DCFH-DA to the sample: DCFH-DA is preferably added to a final concentration of 35 to 45 μM, and the reaction is performed at 25 to 35 ° C. for 10 minutes. It is preferably performed for 20 to 20 minutes.
[0015]
Step 3 : Measurement of Fluorescence Ascorbic acid and iron ions are added to the reaction mixture, and the generated fluorescence is measured: Ascorbic acid is preferably added to a final concentration of 0.05 to 0.3 mM, and is limited. However, it is preferable to add Fe ^{2+ so} that the final concentration becomes 3 to 10 μM. Although not limited, it is preferable that the fluorescence is measured using a microplate fluorometer at intervals of 1 to 3 minutes.
[0016]
Step 4 : The antioxidant capacity of the cells is determined by converting the measured values of free radicals and comparative fluorescence between samples to DCF concentrations using a standard curve of fluorescence versus DCF concentration, where the standard curve is 0. It is made by measuring fluorescence in the range of 2 nmol DCF.
[0017]
Because it is difficult to introduce DCFH into cells, we used its acetate, 2 ', 7'-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA has high cell permeability and easily translocates to cells. In the cell, DCFH-DA is hydrolyzed into charged DCFH remaining in the cell. Non-fluorescent DCFH is oxidized into highly fluorescent DCF that can be easily detected in cells (excitation wavelength: 480 nm, emission wavelength: 530 nm).
[0018]
The measurement method of the present invention can be applied to various types of samples, and it is preferable to use a 96-well plate or a microfluorometer.
[0019]
The principle of the present invention is as follows: The amount of free radical measured in a cell is expressed by the sum of the functions of prooxidant and antioxidant. Within cells, certain levels of prooxidants are present, but vary by cell type and condition. To determine the antioxidant capacity of a cell, the tissue or cell is placed under equal oxidative stress and then the amount of free radicals is measured. Here, the measured value of free radicals indicates the free radicals remaining after the cells have exerted their antioxidant capacity, which is regarded as an indicator of the relative antioxidant capacity of the cells. That is, after adding equal amounts of ascorbic acid and iron ions to a sample containing equal amounts of proteins, the remaining free radicals are measured. Here, the smaller the amount of the remaining free radicals, the more excellent the cell has the antioxidant ability.
[0020]
The invention is further described in the following examples, which should not be construed as limiting the scope of the invention.
[0021]
Example 1 Measurement of Free Radicals Using DCFH-DA The validity of the method for measuring free radicals of the present invention using DCFH-DA, which is an essential component of the relative quantification method of the antioxidant ability of cells, was examined.
[0022]
Mouse liver tissue was homogenized in a buffer (40 mM Tris-HCl, 5 mM EDTA, pH 7.4) to a concentration of 20 mg / μl, and then diluted 40-fold with the same buffer. DCFH-DA was added to the diluted homogenate to a final concentration of 125 μM, after which the mixture was incubated at 37 ° C. for 15 minutes. Divide the reaction mixture into two portions, a final concentration of ascorbic acid and FeSO ₄ are added to one portion such that the 0.1mM and 5μM, respectively, were added to buffer an equal volume of another part. The fluorescence is measured at 485 nm for excitation and 530 nm for emission using a microplate fluorometer at 2 minute intervals for 20 minutes, and the slope of the straight line within the linear increase range is measured. (See FIG. 1). FIG. 1 is a graph showing changes in fluorescence over time. Here, (●) indicates a portion to which ascorbic acid and FeSO ₄ were added, and (○) indicates a portion to which a buffer was added. As shown in FIG. 1, a remarkable increase in fluorescence was observed in the portion where ascorbic acid and FeSO ₄ were added.
[0023]
A standard curve of fluorescence versus DCG was generated in the range of 0 to 2 nmol (see FIG. 2). The measured fluorescence was converted to the amount of DCF by applying the slope obtained in FIG. 1 to the standard curve of FIG. 2, and the amount of free radical was measured therefrom. The amount of free radicals divided by the amount of protein in the sample was expressed in units of nmol (DCF) / mg (protein) / min.
[0024]
For the reliability of the free radical quantification method according to the present invention, the change in fluorescence depending on the amount of liver tissue was measured in the same manner as described above (see FIG. 3). FIG. 3 is a graph showing a change in fluorescence depending on the amount of liver tissue. Here, (●) indicates a portion to which ascorbic acid and FeSO ₄ were added, and (○) indicates a portion to which a buffer was added. As shown in FIG. 3, the fluorescence increased in proportion to the amount of liver tissue added.
[0025]
As clearly described and demonstrated above, the present invention provides a method for measuring the antioxidant capacity of a cell. The method for measuring the antioxidant capacity of the cells according to the present invention includes a step of preparing a sample by removing and homogenizing an animal tissue whose antioxidant capacity is to be measured; adding DCFH-DA to the sample to perform a reaction. Initiating; adding ascorbic acid and iron ions to the reaction mixture and measuring fluorescence; and converting the measured fluorescence value into DCF concentration using a standard curve of fluorescence versus DCF concentration to convert the antioxidant activity of the cells. Is measured. The method of the present invention has advantages over prior art methods. That is, the method of the present invention can simultaneously measure and compare the antioxidant capacity of multiple samples, and can easily measure the total oxidative stress in the samples. Thus, it can be used in practice for studies on radioactivity at the biological level.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in fluorescence in liver tissue over time.
FIG. 2 is a graph showing a standard curve of DCF.
FIG. 3 is a graph showing a standard curve of a change in fluorescence with an increase in liver tissue.

Claims (4)

A method for measuring the antioxidant capacity of a cell, comprising the following steps:
(I) a step of preparing a sample by extracting and homogenizing an animal tissue whose antioxidant ability is to be measured;
(Ii) adding 2 ′, 7′-dichlorofluorescin diacetate (DCFH-DA) to the sample to start a reaction;
(Iii) adding ascorbic acid and iron ions to the reaction mixture and measuring the fluorescence; and (iv) measuring the fluorescence measurement using a standard curve of fluorescence versus DCF concentration in 2 ′, 7′-dichlorofluorescein (DCF). A) measuring the antioxidant capacity of the cells in terms of concentration;
The above method comprising: 2. The method of claim 1, wherein said DCFH-DA is added to said sample to a final concentration of 35-45 [mu] M. The method according to claim 1, wherein the reaction of the step (ii) is performed at a temperature of 25 to 35 ° C for 10 to 20 minutes. The method according to claim 1, wherein the ascorbic acid is added to the reaction mixture to a final concentration of 0.05 to 0.3 mM.

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