WO2002061422A1

WO2002061422A1 - Method for measuring antioxidative activity of cell

Info

Publication number: WO2002061422A1
Application number: PCT/KR2001/000117
Authority: WO
Inventors: Young-Mee Park; Mi-Young Han; Eun-Mi Choi; Jong-Hoon Park; Ji-Sun Park; Yun-Jeong Kim
Original assignee: Young-Mee Park; Mi-Young Han; Eun-Mi Choi; Jong-Hoon Park; Ji-Sun Park; Yun-Jeong Kim
Priority date: 2001-01-29
Filing date: 2001-01-29
Publication date: 2002-08-08
Also published as: JP2004518433A

Abstract

The present invention relates to a method for measuring antioxidative activity of cell. The method for measuring antioxidative activity of cell of the present invention comprises the steps of: preparing a sample by extracting and homogenizing animal tissue of which antioxidative activity is to be measured; adding 2´, 7´-dichlorofluorescin diacetate(DCFH-DA) to the sample to initiate reaction; adding ascorbic acid and iron ion to the reaction mixture and measuring fluorescence; and, converting the fluorescence measurement into 2´, 7´-dichlorofluorescein(DCF) concentration using a standard curve of fluorescence vs. DCF concentration to measure antioxidative activity of cell. The invented method has merits over the prior art methods, that is, the antioxidative activity of multiplicity of samples can be measured and compared simultaneously and total oxidative stress in the sample can be measured easily, which makes possible its practical application in the studies on radioactivity at the level of an organism.

Description

METHOD FOR MEASURING ANTIOXIDATIVE ACTIVITY OF CELL

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a method for measuring antioxidative activity of cell, more particularly, to a method for measuring antioxidative activity against free radicals using 2 ' , 7 ' -dichlorofluorescin diacetate (DCFH-DA) when the free radicals are generated in the cells by ascorbic acid and iron ion.

Background of the Invention

The cells under normal physiological condition continuously generate free radicals, and various factors such as diseases, drugs, and irradiation may cause vast increase of free radicals. Since all cells have enzymatic/nonenzymatic protection mechanisms against free radicals, which may vary depending on cell types, the amount of free radicals in the cells is represented by the sum of functions of prooxidants and antioxidants. Generally, tremendous amount of free radicals are generated upon irradiation of animals, thus, quantitative measurement of free radicals in tissues has been used as an index of sensitivity of tissues to irradiation.

Meanwhile, the amount of free radicals, having a high reactivity, easily changes with time. By such reason, only available means for measuring free radicals, until now, is a method employing electron spin resonance spectroscopy (ESR) or electron paramagnetic resonance spectroscopy (EPR) . The said method using ESR, however, has revealed difficulties in measuring highly reactive free radicals such as superoxide and hydroxyl radicals. Thus, spin trapping technique has been conventionally employed in the art, which quantitates relatively stable free radicals produced by reacting free radicals in the sample and trapping agents such as 5, 5-dimethylpyrroline-N-oxide (DMPO) or α-phenyl-tert-butylnitrone (PBN) . Although the said method is accurate and is able to identify types of free radicals by using different trapping agents, it is proven to be less satisfactory in the senses that the required equipment is very expensive and only an expert on ESR spectrophotometer can analyze the results. To overcome the problems mentioned above, indirect methods have been used in the art. One of those is that free radicals react with a specific marker such as salicylic acid to generate a product such as dihydroxybenzoic acid which is separated with HPLC and quantitated to measure amount of free radicals indirectly. The said method is used more widely than ESR method, however, it also has disadvantages that it is time consuming and expensive to analyze with HPLC.

Another indirect method used the chemiluminescence generated in the course of free radical formation. Chemiluminescence has been reported to be found in singlet oxygen, active triplet oxygen of such as carbonyl group, 0N00- reaction, lipoxigenase reaction, Fenton reaction, etc. However, the intensity of chemiluminescence is too low to measure directly, it is amplified through the reaction with proper compounds. One example of amplification method employs luminol or lucigenin to react with radicals and produce amplified chemoluminescence, which is simpler and easier method than ESR or HPLC methods. However, it has disadvantages of low sensitivity, susceptibility to the reaction environment such as pH and high background noise.

To overcome problems of the said chemiluminescence method, the conversion of nonchemiluminescent 2 ' , 7 ' - dichlorofluorescin (DCFH) to chemiluminescent 2 ' , 7 ' - dichlorofluorescein (DCF) is used to detect free radicals. However, the said methods which can merely measure the amount of free radicals in cells or tissues are not appropriate for assaying the antioxidative activity of cell, since the amount of free radicals in cells is the sum of functions of prooxidants and antioxidants.

Under the circumstances, there are strong reasons for exploring and developing a method for measuring antioxidative activity of cell which represents ability of the cell to reduce the amount of free radicals.

Summary of the Invention

The present inventors have made an effort to develop a method for measuring antioxidative activity of cell, and found that: free radicals generated abruptly in various tissues and cells by ascorbic acid and iron ion can be measured by employing 2 ', 7 ' -dichlorofluorescin diacetate (DCFH-DA) ; and, the relative antioxidative activity of various samples can be measured by comparing the measurements.

The primary object of the present invention is, therefore, to provide a method for measuring antioxidative activity of cell using ascorbic acid, iron ion and DCFH- DA.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present invention will become apparent from the following descriptions given in the conjunction with the accompanying drawings, in which:

Figure 1 is a graph showing the changes in fluorescence in liver tissue with time.

Figure 2 is a graph representing a standard curve of DCF.

Figure 3 is a graph representing a standard curve of changes in fluorescence with increment of liver tissue. DETAILED DESCRIPTION OF THE INVENTION

The method for measuring antioxidative activity of cell of the present invention comprises the steps of: preparing a sample by extracting and homogenizing animal tissue of which antioxidative activity is to be measured; adding DCFH-DA to the sample to initiate reaction; adding ascorbic acid and iron ion to the reaction mixture and measuring fluorescence; and, converting the fluorescence measurement into DCF concentration using a standard curve of fluorescence vs. DCF concentration to measure antioxidative activity of cell.

The method for measuring antioxidative activity of cell of the invention is further illustrated by the following steps.

Step 1: Preparation of samples

Samples are prepared by extracting and homogenizing animal tissues of which antioxidative activity is to be measured: the tissues include, but without limitation, liver, stomach, lung, heart, brain and kidney. Homogenization of the tissues is preferably, but without limitation, carried out by homogenizing the tissues frozen and stored ahead in a buffer solution using a glass-glass homogenizer .

Step 2: Addition of DCFH-DA

DCFH-DA is added to the samples to initiate reaction: a final concentration of DCFH-DA is preferably 35 to 45 μM, and the reaction is carried out preferably at 25 to 35°C for 10 to 20 minutes.

Step 3: Detection of fluorescence Ascorbic acid and iron ion are added to the reaction mixture and measured fluorescence generated: a final concentration of ascorbic acid is preferably 0.05 to 0.3mM and Fe²⁺ is preferably, but without limitation, added to a final concentration of 3 to lOμM. The fluorescence is preferably, but without limitation, measured by the aid of a microplate fluoremeter at an interval of 1 to 3 minutes.

Step 4 : Detection of free radicals and comparison between samples

Antioxidative activity of cells is measured by converting the fluorescence measurement into DCF concentration using a standard curve of fluorescence vs. DCF concentration, where the standard curve is prepared by measuring the fluorescence in the range of 0 to 2nmol DCF.

Since it is difficult to introduce DCFH into cells, the inventors used its acetate salt, 2'7'- dichlorofluorescin diacetate (DCFH-DA) . DCFH-DA has a high cell permeability and is easily transferred into cells. In the cell, DCFH-DA is hydrolyzed into charged DCFH which remains in the cell. Nonfluorescent DCFH is oxidized into highly fluorescent DCF in the cell which is readily detectable (excitation wave length: 484nm, emission wave length: 530 nm) .

The invented measurement method may be applied to various types of samples, where 96 well plate or microplate fluorometer is preferably employed.

The principle of the invention is as follows: the amount of free radicals measured in cells is represented by the sum of functions of prooxidants and antioxidants. In cells, there exists a constant level of prooxidant which varies depending on the types and the states of cells. To measure antioxidative activity of cells, the tissues or the cells are placed under equal strength of oxidation stress and then measured the amount of free radicals. Herein, measured values of free radicals represent remained free radicals after exhibiting antioxidation ability of the cells, which is regarded as an index of relative antioxidative ability of the cells. That is, following equal amount of ascorbic acid and iron ion are added to the samples containing equal amount of protein, the amount of remained free radicals are measured, where the less amount of free radicals remained, the better antioxidative activity the cells have.

The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

Example 1: Measurement of free radicals employing DCFH-DA

The appropriateness of measurement of free radicals of the invention employing DCFH-DA which is an essential component of the relative assay method for antioxidative activity of cell was examined.

The mouse liver tissue was homogenized in a buffer solution (40mM Tris-HCl, 5mM EDTA, pH 7.4) to a concentration of 20mg/μl, which was followed by 40 fold dilution with the same buffer. To the diluted homogenate, DCFH-DA was added to a final concentration of 125uM and then the mixture was incubated at 37 °C for 15 minutes. The reaction mixture was divided into two portions, and then, ascorbic acid and FeS0₄ were added to one portion to final concentrations of 0. litiM and 5uM respectively, and equal volume of the buffer solution was added to the other portion. The fluorescence was measured at 485nm for excitation and 530nm for emission employing microplate fluorometer for 20 minutes at an interval of 2 minute, and then a slope of the line within a range of linear increase of fluorescence was measured (see : Figure 1). Figure 1 is a graph showing the changes in fluorescence with time, where (•) represents the portion with ascorbic acid and FeS0₄, and (O) represents the portion with buffer solution. As shown in Figure 1, the remarkable increase of fluorescence in the portion with ascorbic acid and FeS0₄ was observed.

A standard curve of fluorescence vs. DCF in the range of 0 to 2nmol has been prepared (see: Figure 2). Fluorescence, determined by applying the slope obtained in Fig.l to the standard curve of Fig.2, was converted into amount of DCF, from which amount of free radical was measured. Amount of free radical divided by amount of protein in the sample was represented as an unit of nmol (DCF) /mg (protein) /min.

For reliability of the free radical assay method of the invention, change in fluorescence depending on amount of liver tissue was determined by the identical method used above (see: Figure 3). Figure 3 is a graph showing changes in fluorescence depending on the amount of liver tissue, where (•) represents the portion with ascorbic acid and FeS0₄, and (O) represents the portion with buffer solution. As shown in Figure 3, fluorescence increased proportionally to the amount of liver tissue added.

As clearly illustrated and demonstrated above, the present invention provides an assay method of antioxidative activity of cell. The method for measuring antioxidative activity of cell of the present invention comprises the steps of: preparing a sample by extracting and homogenizing animal tissue of which antioxidative activity is to be measured; adding DCFH-DA to the sample to initiate reaction; adding ascorbic acid and iron ion to the reaction mixture and measuring fluorescence; and, converting the fluorescence measurement into DCF concentration using a standard curve of fluorescence vs. DCF concentration to measure antioxidative activity of cell. The invented method has merits over the prior art methods, that is, the antioxidative activity of multiplicity of samples can be measured and compared simultaneously and total oxidative stress in the sample can be measured easily, which makes possible its practical application in the studies on radioactivity at the level of an organism.

Claims

WHAT IS CLAIMED IS:

1. A method for measuring antioxidative activity of cell which comprises the steps of: (i) preparing a sample by extracting and homogenizing animal tissue of which antioxidative activity is to be measured;

(ii) adding 2' , 7' -dichlorofluorescin diacetate (DCFH- DA) to the sample to initiate reaction; (iϋ) adding ascorbic acid and iron ion to the reaction mixture and measuring fluorescence; and,

(iv) converting the fluorescence measurement into 2' , 7' -dichlorofluorescein (DCF) concentration using a standard curve of fluorescence vs. DCF concentration to measure antioxidative activity of cell.

2. The method of claim 1, wherein DCFH-DA is added to the sample to reach a final concentration of 35 to 45 μM.

3. The method of claim 1, wherein the reaction of step(ii) is carried out at the temperature of 25 to 35°C for 10 to 20 minutes.

4. The method of claim 1, wherein the ascorbic acid is added to the reaction mixture to reach a final concentration of 0.05 to 0.3mM.