WO2005103282A1

WO2005103282A1 - Method of measuring active oxygen

Info

Publication number: WO2005103282A1
Application number: PCT/JP2005/007948
Authority: WO
Inventors: Hideyuki Majima
Original assignee: Mitochondria Research Institute Co., Ltd.
Priority date: 2004-04-20
Filing date: 2005-04-20
Publication date: 2005-11-03
Also published as: TW200540408A; JPWO2005103282A1

Abstract

A method of accurately measuring the amount of active oxygen generated by intracellular mitochondria. There is provided a method of measuring active oxygen generated by mitochondria, characterized by including the first step of applying a fluorescent reagent capable of specific reaction with intracellular active oxygen to a biosample; the second step of obtaining a fluorescent image containing one or two or more cells in its visual field by subjecting to micrography and photoelectric conversion treatment the biosample resulting from the first step and having the contained fluorescent reagent held in an excited state; the third step of determining the sum of luminance values of constituent pixels within a given region of the fluorescent image obtained in the second step; and the fourth step of identifying the amount of active oxygen generated by intracellular mitochondria on the basis of the sum of luminance values of constituent pixels determined in the third step.

Description

Description Method for measuring active oxygen Technical field

The present invention relates to a method for measuring active oxygen generated from mitochondria, and a method for quantifying the antioxidant ability of an antioxidant using the method. Background art

With respect to aging, the importance of telomeres has been pointed out by the importance of the ゃ kuroto (k 1 o t ho) gene. However, it was difficult for the telomere (k 1 o t ho) gene alone to fully explain health and aging. Recently, it has been revealed that the causes of many nervous diseases and incurable diseases of unknown origin, such as aging and Alzheimer's, Parkinson's, ALS (amyotrophic lateral sclerosis), are actually diseases of reactive oxygen species. It is getting. These diseases are often accompanied by chronic rather than acute symptoms. Apoptosis (cell death) is said to be involved in these diseases, and it has been reported that mitochondria play an important role in apoptosis. Mitochondria contain 165969 base pairs of DNA, and oxidative stress causes many mutations in this mitochondrial DNA, causing aging and many neurological diseases such as Alzheimer, Parkinson, and ALS. It is said that the relationship between stress oxide and mitochondrial disorders is highlighted.

An electron transport system exists in the inner mitochondrial membrane, and ATP (adenosine triphosphate) synthesis is performed under oxygen. This electron transport system is a complex (C om p 1 ex) 1: -IV, composed of ATP synthase (synthase) and ATP / ADP (adenosine diphosphate) translocator (trans 1 ocator). While this complex biomachine is the largest energy-producing organ in a cell, it is not a perfect machine and is known to leak electrons. This electron leakage is most abundant from complexes (Comp 1 ex) I and III. Normally, even under normal conditions, 2-3% of the electrons leak, which is thought to be the source of superoxide. That is, the leaked electrons are captured by oxygen, and the oxygen becomes superoxide. Mitochondria have a unique manganese superoxide, but it is easily suggested that the role of this enzyme is important in the role of capturing this superoxide. Recently, a large number of diseases caused by this electron transport system abnormality are being reported. For example, in Parkinson's disease, the abnormalities are concentrated in the electron transport complex (Comp 1 ex) I. As described above, this part causes the largest leakage of electrons, and it is considered that the cause is that superoxide is generated by this leakage of electrons. In addition to Parkinson's disease, mutations in mitochondrial DNA have been reported in many neurological disorders. Similarly, the occurrence of a large amount of superoxide from mitochondria is likely to occur in these diseases as well, and this is likely to be the etiology. In studies on aging, it is known that mitochondrial DNA deficiency occurs with the aging of cells, so-called common ionization (commonde 1 etion). In neuropathic diseases, it is known that this common duration starts to occur early. In the mitochondrial DNA common dilation, this part, in particular, partially encodes the ATPase protein of the electron transport system, and as described above, a larger amount of superoxide may be released. Great nature. Fig. 1 shows a part of the in vivo reaction of active oxygen. In the figure As shown, oxygen radical (〇 ₂ - ') and hydrogen peroxide (Eta ₂ 0 ₂₎ is by reaction, hydroxycarboxylic radical (Omikuron'ita ·) is generated. The oxygen radical (〇 ₂ - ·) and nitrogen monoxide radicals (vo-) and is .nu.0 ₃ radicals by reacting (Nyu_〇 ₃ ·) is generated. Both the hydroxyl radical and the Ν03 radical are highly reactive substances.

Oxygen radicals (〇 _2- ·) form super 'oxide' dimutase

By reacting with (S OD), it becomes hydrogen peroxide (H ₂ 0 ₂ ), and by reacting this hydrogen peroxide with catalase (CAT), it becomes water (H ₂ 0) and oxygen ( ₂ ). Moreover, by hydrogen peroxide and Dar glutathione peroxide O Kishida over peptidase (GPX) are reacted water (H ₂ 0) and oxidized glutathione (GS SG) and is generated. Daltathione reductase (GR) transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized daltathione, which converts oxidized daltathione to reduced daltathione (GSH) and reduces it. Nicotinamide dodenine dinucleotide phosphate is converted to oxidized nicotinamide dodenine dinucleotide phosphate (NADP). Glucose 6-phosphate dehydratase (G6PD) converts oxidized nicotinamide amide adenine into prostaglandin (PG) in the pentose phosphate cycle during the process of converting prostaglandin (PG) to dalcose-6-phosphate (G6P). The dinucleotide phosphate is converted back to reduced nicotinamide dodenine dinucleotide phosphate.

For Super O genius de described above, of the active oxygen Supaokisa Lee de (0 ₂ · -) is what, there is a theory to say that is an important substance responsible for the first of the active oxygen group series. In 1969, Dr. Frid ο Vich discovered a Superox de dismutase (SOD) that eliminates superoxides. Superoxide and carbon monoxide easily reacted with each other and turned into peroxinitrite, a substance with extremely high reaction activity. Applicants 1 In 1998, it was shown that cells transfected with the manganese superoxide dismutase gene exhibited resistance to apoptosis induced by reactive oxygen species (Majima et al., JBC, 1989). ). In other words, controlling the amount of superoxide in mitochondria to be reduced means that it is possible to control a series of subsequent in vivo reactions.

It is highly likely that active oxygen generated from mitochondria is the cause of aging and neuropathy. Therefore, the antioxidant capacity of mitochondrial-derived active oxygen should be examined by examining the extent to which antioxidant drugs, foods, etc. (hereinafter referred to as antioxidants) suppress active oxygen derived from mitochondria. Is possible.

Currently, detection of intracellular reactive oxygen using reagents using methods such as 2,7 dich 1 o r o f 1 u o r e s c e in d i a c e t a t e (DCF), d i h y d r o r h i d am i n e 1 2 3 (DHR), and h y d r o e t h i d e n (H E).

As a method of measuring the antioxidant ability of an antioxidant having an effect of suppressing active oxygen, a method of measuring the antioxidant ability by a chemical reaction in a test tube is mainly used. As one of such measurement methods, Japanese Patent Application Laid-Open No. 08-189929 (hereinafter referred to as Patent Document 1) discloses a method for measuring the amount of 8-OhdG, which is oxidized DNA in a living body. Have been. Also, “Free Rad. Res. Comms., Vol. 14, No. 3, pp. 173-178, 1991” (hereinafter referred to as Non-Patent Document 1) states that active oxygen or antioxidant against cells, animals, and humans is described. Methods for evaluating the effects of substances and cytotoxicity on active oxygen and hydrogen peroxide are disclosed.

[Patent Document 1] Japanese Patent Application Laid-Open No. 08-1898929

[Non-Patent Document 1] Free Rad. Res. Comms., Vol. 14, No. 3, pp. 173-178,

1991

However, activated acids using conventional reagents such as DCF, DHR, HE, etc. The elemental detection method could measure intracellular active oxygen, but could not verify which organelle it originated from. In addition, some reagents generate a positive or negative charge when dyeing active oxygen, and the active oxygen stained with such a reagent has a negative charge on the cell membrane plus the negative charge. It was extremely difficult to identify only the mitochondrial-derived active oxygen and measure the amount of active oxygen generated, because it was drawn to the outer membrane of the tochondria and the original generation site was unknown.

Regarding the method of measuring the antioxidant capacity, the conventional measuring methods include a method of removing cells from a living body after the administration of the antioxidant, measuring the antioxidant ability of the antioxidant, a method of measuring by a chemical reaction in a test tube, According to the former, there is a possibility that accurate measurement cannot be performed due to the time lag from the moment of administration, and the latter cannot be quantitatively expressed. The present invention has been made based on the above technical background, and an object of the present invention is to provide a method capable of accurately measuring the amount of mitochondrial-derived active oxygen in a cell. It is in.

Another object of the present invention is to provide a method for accurately quantifying the antioxidant ability of various antioxidants.

Another object of the present invention is to provide a scientific basis for antioxidants from the viewpoint of preventing aging and promoting health by accurately quantifying the antioxidant ability of various antioxidants. It is to provide an evaluation method.

Still other objects, functions and effects of the present invention will be easily understood by those skilled in the art by referring to the following description. Disclosure of the invention

In order to achieve the above object, the method for measuring active oxygen generated from mitochondria according to the present invention comprises a method of applying a fluorescent reagent having a property of specifically reacting with active oxygen in cells to a biological sample. Steps and the first step In a state where the fluorescent reagent contained in the biological sample obtained in the step is excited, the biological sample is microscopically photographed and subjected to photoelectric conversion processing to obtain a fluorescent image containing one or more cells in the field of view. A second step, a third step of calculating the total brightness of constituent pixels within a predetermined area of the fluorescent image acquired in the second step, and a total brightness of the constituent pixels obtained in the third step. And identifying the amount of active oxygen generated from mitochondria in the cells. Here, the biological sample may be any of a human cell, an animal cell, and a plant cell, or may be a cell at any site.

According to such a configuration, it is possible to specify the amount of mitochondrial-derived active oxygen generated.

Here, as the fluorescent reagent, a reagent capable of measuring hydroxy radical, singlet oxygen, or superoxide may be used. According to such a configuration, the active oxygen in the cells is stained using a fluorescent reagent that specifically acts on the hydroxyl radical, singlet oxygen, and superoxide among the active oxygens. Active oxygen derived from mitochondria can be trapped in the active oxygen.

In addition, if HPF or APF is used among the fluorescent reagents that can measure hydroxy radical, singlet oxygen, or superoxide, the selective use of HPF and APF to selectively stain the hydroxyl radical among the active oxygen is also possible. When DMAX is used as the fluorescent reagent, singlet oxygen can be selectively fluorescently stained. Since the fluorescent reagents of HPF, APF, and DMAX do not have a positive or negative charge, they are attracted to the oppositely charged site like the conventional fluorescent reagents, and the original active oxygen generating organs There is no problem of not knowing.

Next, the method for quantifying the antioxidant ability of the antioxidant according to the present invention comprises the following steps: a first step of preparing two sets of biological samples having the same active oxygen generation characteristics; The antioxidant to be quantified for antioxidant capacity is applied to one of the two sets of biological samples prepared in the first step, while the other set of biological samples is In the second step, an antioxidant is used as a standard for quantification of antioxidant capacity, and the same set of active oxygen inducers are used for the two sets of biological samples to which the antioxidant is applied in the second step. The third step of applying a stimulus and the property of reacting specifically with the active oxygen in the cell to each of the two sets of biological samples to which the active oxygen-induced stimulus was applied in the third step Fourth step of applying the fluorescent reagent and microscopic imaging and photoelectric conversion of each of the two sets of biological samples to which the fluorescent reagent has been applied in the fourth step, with the fluorescent reagent being excited To see one or two or more cells each A fifth step of obtaining two sets of fluorescent images including: a sixth step of obtaining a sum of luminance of constituent pixels in a predetermined area for each of the two sets of fluorescent images obtained in the fifth step; For the two sets of luminance sums determined in step 6, the antioxidant material to be quantified for the antioxidant capacity is the sum of the luminance on the side where the antioxidant material is applied, which is the standard for quantifying the antioxidant capacity. And a seventh step of calculating a ratio of the sum of luminance on the side to which is applied.

Here, the term “antioxidant” refers to chemicals, foodstuffs, etc. having (or possibly having) antioxidant ability.

According to such a configuration, the antioxidant capacity can be easily determined from the total brightness of the images taken by the microscope. In addition, the amount of active oxygen generated significantly increases when a biological sample is stimulated with active oxygen, and the difference between the antioxidant used as the measurement reference and the antioxidant of the antioxidant used as the measurement target becomes clearer. become. It is possible to relatively express the antioxidant ability of the antioxidant to be measured by administering the antioxidant to be measured and the antioxidant to be measured to biological samples under the same conditions and comparing them. It is. In addition, the amount of active oxygen generated can be measured more accurately than the method of removing cells from the body after administering antioxidants. It is possible to

The active oxygen-induced stimulus may be given either before or after administration of the antioxidant.

In a preferred embodiment of the present invention, the active oxygen-induced stimulus may be X-ray irradiation. Further, the reactive oxygen-induced stimulus may be application of a site force-in.

Here, HPF, APF, or DMAX may be used as a fluorescent reagent having a property of specifically reacting with active oxygen in cells. If HPF or APF is used as a fluorescent reagent, it is possible to selectively fluorescently stain hydroxy radicals, especially among active oxygens.If DMAX is used, it is possible to selectively fluorescently stain singlet oxygen. It is.

Vitamin E may be used as an antioxidant used as a standard for quantifying antioxidant capacity. According to this configuration, vitamin E, which is relatively easy to obtain, is used as an antioxidant capacity measurement standard, resulting in “two times the antioxidant power of vitamin E” and “five times the antioxidant power of vitamin E” It is possible to show the antioxidant power of various antioxidants in an easy-to-understand expression like this.

A biological sample having the same active oxygen generation characteristics may be a biological sample obtained from a previously prepared standard individual.

Here, the “standard individual” refers to one sample included in a certain sample group such as a cell line, a lineage animal and a plant, and a subject.

According to such a configuration, by using one individual in a certain set as a standard individual, it is possible to predict the effect of the antioxidant on the sample group.

Biological samples having the same active oxygen generation characteristics may be biological samples obtained from specific individuals who should take the antioxidant.

Here, the “specific individual” refers to one individual, one individual, a mutant strain, a plant or animal, a human, a gene or a protein treated with a drug, etc. A specific individual in a sample group of cells, transgenic animals and plants, cloned animals and plants, etc.

According to such a configuration, by quantifying the antioxidant ability of a specific individual, for example, it is possible to measure the effect on an individual, and a test can be performed in which individual differences are taken into account.

In the method for quantifying the antioxidant ability of the antioxidant of the present invention, the antioxidant to be quantified is orally administered to a specific individual to whom the antioxidant is to be taken, and then the specific individual A biological sample is collected from the sample to measure the amount of antioxidant taken into cells, and the amount of antioxidant applied when quantifying antioxidant capacity is adjusted according to the measured value of intake. Is also good.

According to such a configuration, it is possible to know how much antioxidant is taken into the body when a specific individual takes the antioxidant by oral administration. When quantifying antioxidant capacity, by administering an antioxidant to a biological sample with reference to this intake, it is possible to conduct experiments with the same intake as when taken orally, so that more specific individuals can be treated. It is easier to determine the impact.

According to the method for measuring active oxygen generated from mitochondria according to the present application, active oxygen generated from mitochondria among active oxygen in cells is identified, and the amount of active oxygen generated from mitochondria is measured to visually determine It is possible to confirm this.

Further, according to the method for quantifying the antioxidant ability of the antioxidant according to the present invention, it is possible to measure the antioxidant ability of various foods and drugs and visually recognize the antioxidant ability. In addition, by performing image processing on images obtained by microscopic photographing, it is possible to quantify and compare the antioxidant ability of foods, drugs, and the like. Brief Description of Drawings

FIG. 1 is a diagram showing a reaction formula of an antioxidant enzyme. FIG. 2 is a diagram showing the progress of active oxygen suppression.

FIG. 3 is a diagram schematically showing a mitochondrial membrane surface.

FIG. 4 is a diagram showing the structure of HPF.

FIG. 5 is a diagram showing the structure of DMAX.

FIG. 6 is a diagram showing the fluorescence activity of HPF.

FIG. 7 is a diagram showing the fluorescence activity by DMAX.

Fig. 8 is a graph (1) showing the fluorescence sensitivity.

FIG. 9 is a graph (part 2) showing the fluorescence sensitivity.

FIG. 10 is a graph (part 3) showing the fluorescence sensitivity.

FIG. 11 is a graph (part 4) showing the fluorescence sensitivity.

FIG. 12 is a graph (part 1) showing the antioxidant ability of the antioxidant. Figure 13 is a graph (part 2) showing the antioxidant capacity of antioxidants. Figure 14 is a graph (part 3) showing the antioxidant capacity of antioxidants. FIG. 15 is a diagram illustrating the first embodiment of the present application.

FIG. 16 is a diagram illustrating a second embodiment of the present application.

FIG. 17 is a diagram illustrating a third embodiment (part 1) of the present application. FIG. 18 is a view for explaining the third embodiment (No. 2) of the present application. FIG. 19 is a photomicrograph of 144 B cells. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings.

As described above, the method for measuring active oxygen according to the present invention comprises: a first step of applying a fluorescent reagent having a property of specifically reacting with active oxygen in cells to a biological sample; In a state where the fluorescent reagent contained in the biological sample obtained in step 1 is excited, the biological sample is subjected to confocal microscopy imaging and photoelectric conversion processing, so that one or more cells can be seen in the field of view. A second step of obtaining a fluorescent image including the fluorescent image, a third step of obtaining a luminance sum of constituent pixels in a predetermined area of the fluorescent image obtained in the second step, and a constituent pixel obtained in the third step And a fourth step of identifying the amount of active oxygen generated from mitochondria in the cell based on the total luminance of the cells.

Mitochondria have long been suspected as a source of active oxygen in living cells.However, even though conventional methods can prove that active oxygen exists in cells, any of these active oxygen sources can It was not possible to identify whether it originated from a small organ. The present inventors have verified active oxygen generated from mitochondria by the following method, and have verified that most of active oxygen in cells is generated from mitochondria.

FIG. 19 shows a micrograph of a fluorescent reagent applied to 144 B cells. In the same figure, 1a is a state in which the active oxygen in the cell is fluorescently stained with HE, 1b is a state in which the mitochondria in the cell are stained with mitochondrial green, and 1c is a superimposition of 1a and 1b. Things. 2a is a state in which the active oxygen in the cell is fluorescently stained with HPF, 2b is a state in which the mitochondrial in the cell is stained with mitochondrial, and 2c is a state in which 2a and 2b are superimposed. is there. Although the stained area of HE does not completely overlap with the mitochondrial image (lc), it is clear from the figure that the stained area of HPF completely matches the mitochondria.

The structures of the fluorescent reagents HPF and APF are shown in FIG. HPF is Hydroxyphenyl Fluorescein ^ APe is Aminophenyl Fluorescein, and both are fluorescent reagents that specifically detect hydroxyl radical (OH) and peroxynitrite (ONOO-). By adding HPF or APF to cells that produce active oxygen such as hydroxy radicals and peroxynitrite and exciting them with laser light, only the sites that generate active oxygen are stained. Before reacting with active oxygen (a) In the state of (a), almost no fluorescence is observed in both HP F and APF, but in the state of (b) after reacting with active oxygen, high fluorescence is observed.

The structure of another fluorescent reagent, DMAX, is shown in FIG. DMAX is 9_ [2- (3-carboxy-9,10_dimethyl) anthryl] 6-hydroxy-3H-xanthen-3, which is a fluorescent reagent having high reactivity with singlet oxygen. When DMAX is administered to cells that produce singlet oxygen and then excited by laser light, the site that produces singlet oxygen emits a fluorescent color. DMAX-DA (a) is hydrolyzed or reacted with esterase to obtain DM AX (b), and DMAX (b) reacts with -doublet oxygen to form DMAX-EP (c). Become. No fluorescence is observed in the state of DMAX-DA (a), but it tends to be low in the case of DMAX (b), and high in the case of DMAX-EP (c) after reacting with singlet oxygen.

The present inventor has discovered that by fluorescently staining a biological sample using a fluorescent reagent such as HPF or DMAX, active oxygen generated from mitochondria can be fluorescently colored. Based on this finding, it became possible to measure the change in the amount of active oxygen before and after administration of the antioxidant, and to quantitatively measure the antioxidant ability of the antioxidant.

A first embodiment of the present application is shown in FIG. A biological sample (A) is collected from a standard individual 100 and divided into two, and one of the biological samples contains an antioxidant (B x) to be measured, and the other has an antioxidant ( B ref). A biological sample to which an antioxidant has been administered is given an active oxygen-induced stimulus (C) and a fluorescent reagent (E) for staining active oxygen, and allowed to stand for a predetermined time (for example, about 2 hours). Then take a picture with a confocal microscope. Image processing is performed on the obtained microscopic images, and the total brightness is calculated for each image. The total brightness (D x) of the antioxidant to be measured and the total brightness (D ref) of the antioxidant to be measured are determined. Calculate the remaining amount of active oxygen and determine the antioxidant capacity of the antioxidant to be measured. Antioxidant capacity of antioxidant to be measured = (Dx) / (Dref) A second embodiment of the present invention is shown in FIG. A biological sample (A) is collected from a standard individual 100, divided into two, and an active oxygen-inducing stimulus (C) and a fluorescent reagent (E) for staining active oxygen are added to each of the biological samples. The biological sample (A) to which the active oxygen-induced stimulus (C) was added, the antioxidant (Bx) to be measured in one biological sample, and the antioxidant as the measurement reference in the other.

(Bref) is added, and the mixture is allowed to stand for a predetermined time. Thereafter, the administered fluorescent reagent is excited, and imaging is performed with a confocal microscope. Image processing is performed on the obtained microscope images, and the luminance sum is calculated for each image. The luminance sum (Dx) of the antioxidant to be measured and the luminance sum (Dref) of the antioxidant to be measured are determined. Calculate the remaining amount of active oxygen and determine the antioxidant ability of the antioxidant to be measured.

A third embodiment (No. 1) of the present application is shown in FIG. First, an antioxidant to be measured is orally administered to a specific individual 200, and then blood, cells, etc. are taken to measure the amount of the antioxidant taken into the body. Determine the dose of antioxidant (Bx, Bref) to the biological sample (A) according to the measured body intake. As a result, the subsequent antioxidant ability test can be performed under the same conditions as when the antioxidant is actually administered orally, so that the test can be performed under almost the same conditions as when the antioxidant is actually ingested.

A biological sample (A) is collected from a specific individual 200, and a biological sample A is collected and divided into two parts. One of the biological samples contains an antioxidant (B x) to be measured, and the other has an antioxidant as a measurement reference. Add material (B ref). The biological sample to which the antioxidant has been added is given an active oxygen-induced stimulus (C) and a fluorescent reagent (E) for staining active oxygen, and allowed to stand for a predetermined period of time. Take a picture. The obtained microscope images are image-processed, and the total brightness is calculated for each image. The total brightness (D x) of the antioxidant to be measured and the total brightness (D ref) of the antioxidant to be measured are measured. The remaining amount of active oxygen is calculated from the calculated value, and the antioxidant capacity of the antioxidant to be measured is determined. A third embodiment (No. 2) of the present application is shown in FIG. As in the previous example, first, an antioxidant to be measured is orally administered to a specific individual 200, and then blood, cells, etc. are taken to measure the amount of antioxidant taken into the body, and the amount taken into the body Determine the dose of antioxidant (B x, B ref) to biological sample (A) according to. A biological sample (A) is collected from a specific individual 200 and divided into two, and each of the divided biological samples is given an active oxygen-induced stimulus (C) and a fluorescent reagent (E) for staining active oxygen. Biological sample given reactive oxygen-stimulated stimulus (C)

To (A), an antioxidant (B x) to be measured is added to one biological sample, and an antioxidant (B ref) to be a measurement reference is added to the other biological sample, and the mixture is added for a predetermined time (for example, about 2 hours). ) Leave still, then excite the administered fluorescent reagent, and take a picture with a confocal microscope. Image processing is performed on the obtained microscopic images, and the luminance sum is calculated for each image. The activity is calculated from the luminance sum (D x) of the antioxidant to be measured and the luminance sum (D ref) of the antioxidant to be measured. Calculate the remaining amount of oxygen and determine the antioxidant capacity of the antioxidant to be measured.

In the examples of Figs. 15 to 18, the comparison was made between the antioxidant to be measured and the reference antioxidant, but the biological sample was divided into three or more samples, and multiple antioxidants were used. The antioxidant ability may be measured simultaneously, or a blank sample without any antioxidant may be used. The context of the timing of the administration of the fluorescent reagent (E) and the timing of the application of the antioxidant (B x, B ref) / reactive oxygen-stimulating stimulus (C) is not specified, but the fluorescent reagent (E) is altered. If it is a type that is easy to administer, the timing of administration should be considered.

In the method for measuring antioxidant capacity according to the present application, it is desirable that stress is applied to a biological sample before or after administration of a substance having antioxidant capacity to generate more active oxygen. By generating more active oxygen, the difference in antioxidant ability between the reference antioxidant and the antioxidant to be measured becomes more pronounced. Here, as a method of applying stress to a biological sample, X-ray irradiation, site power-in administration, anticancer drug administration, etc. are conceivable.

FIG. 2 shows a state in which active oxygen is suppressed by a substance having antioxidant ability. Figure 2 (a) shows the cells irradiated with X-rays and observed 2 hours after. In the figure, the colored area is where active oxygen derived from mitochondria is generated. From this figure, it can be seen that a large amount of active oxygen is generated from mitochondria by X-ray irradiation. Next, Fig. 2 (b) shows the cells exposed to X-rays, vitamin E immediately after, and the cells observed two hours later. It is clear that the amount of generated active oxygen is suppressed as compared with Fig. 2 (a).

This measurement is performed according to the following procedure.

1. Cell culture

2. X-ray irradiation

3. Culture medium replacement

4. Fluorescent staining (HP F)

5. Excitation by laser light and observation of fluorescence (Fig. 2)

Figure 2 (a) shows the cells exposed to X-rays and the hydroxy radicals observed 2 hours after. A comparison with the photograph in which only mitochondria was stained showed that more active oxygen was generated than in mitochondria. On the other hand, FIG. 2 (b) shows the state of the hydroxy radical 2 hours after vitamin E administration immediately after X-ray irradiation. It was found that the degree of light emission was clearly smaller than in the case of Fig. 2 (a), and less active oxygen was generated. From these results, it was proved that the administration of vitamin E suppressed the generation of active oxygen. FIG. 12 is a graph comparing the degree of fluorescence emission of the cells shown in FIG. In the same figure, (a) shows the fluorescence sensitivity when only X-ray (18.8 Gy) irradiation was performed on the biological sample, and (b) shows the case when vitamin E was administered 3 hours before X-ray irradiation. (C) shows the fluorescence sensitivity immediately after X-ray irradiation. This is the fluorescence sensitivity when Min E was administered. HPF was used for fluorescent staining of active oxygen. From the figure, it can be seen that the fluorescence was different between the administration of the antioxidant vitamin E two hours before the X-ray irradiation (b) and the administration of the antioxidant vitamin E immediately after the X-ray irradiation (c). Sensitivity is almost the same value, while (a) does not use any antioxidant only by X-ray irradiation, (b),

It can be seen that the amount of active oxygen generated is more than 10% higher than (c). This indicates that vitamin E suppresses the generation of active oxygen, and that vitamin E is hardly affected by X-ray irradiation. In other words, FIG. 12 shows that the administration of vitamin E suppresses the generation of active oxygen regardless of before and after X-ray irradiation. Reactive oxygen inducement The timing of applying a stimulus can be determined as appropriate depending on the type of antioxidant. Next, FIG. 13 shows an example in which DMAX was used as the fluorescent stain in the case of FIG. DMAX is a fluorescent stain that selectively stains singlet oxygen, as opposed to HPF, which selectively stains hydroxyl radicals. In the figure, (a) shows the fluorescence sensitivity when only X-ray (18.8 Gy) irradiation was performed, and (b) shows the fluorescence sensitivity when vitamin E was administered 3 hours before X-ray irradiation. (C) shows the fluorescence sensitivity when vitamin E was administered immediately after X-ray irradiation. Even when staining with DMAX, there was no difference in fluorescence sensitivity depending on the timing of administration of vitamin E as in the case of HPF (see (b) and (c)). On the other hand, in (a) where only X-ray irradiation was performed, the amount of active oxygen generated was more than 10% higher than in (b) and (c), indicating that the amount of active oxygen generated was large.

Figure 14 shows a graph comparing the apoptosis incidence after irradiating a biological sample with X-rays (18.8 Gy). As in the case of Figs. 12 and 13, (a) When only X-ray (18.8 Gy) irradiation is performed,

(b) When vitamin E was administered 3 hours before X-ray irradiation, and ( _c ) When vitamin E was administered immediately after X-ray irradiation. As a fluorescent stain, Hoechst 3 3 342 was used. The incidence of apoptosis in X-irradiation alone (a) was significantly different from the incidence of apoptosis in vitamin E administered (b) and (c). Vitamin E was administered (b), ( In c), the incidence of apoptosis is reduced to about 1/5 of that in the case without vitamin E (a). From this, it is predicted that the apoptosis generation rate will decrease due to the suppression of active oxygen.

FIG. 6 shows a state in which various cells were fluorescently stained with HPF, and FIG. 7 shows a state in which various cells were stained with DMAX. In Figures 6 and 7, (a) is a human cell (143B), (b) is an mtDNA-deficient cell (p0), and (c) is a cell in which normal mitochondria have been returned to p0 cells ( 8 7

(WT), (d) shows immortalized human carcinoma cells (He1a), (e) shows (BH5), (f) shows (BH50), and (g) shows (BH3 # 12). Each was fluorescently stained with HP F. The procedure for fluorescent staining is the same as the method described above. From the figure, it can be seen that a large amount of active oxygen is generated in 143 B cells and 87 WT cells, etc., but that the amount of active oxygen is small in p0 cells lacking mtDNA.

Graphs of the fluorescence intensities of the photographs shown in FIGS. 6 and 7 are shown in FIGS. FIGS. 8 and 9 show the fluorescence sensitivities of the cells stained with HPF by fluorescence, and quantify the fluorescence sensitivities (a) to (f) in FIG. Similarly, FIGS. 10 and 11 show the fluorescence sensitivities of the cells fluorescently stained using DMAX, and numerically represent the fluorescence sensitivities of (a) to (f) in FIG. is there.

The action of mitochondria in cells is briefly described below because of the well-known force.

Figure 3 shows a schematic diagram showing the movement of internal and external substances on the mitochondrial membrane surface. In the figure, 1 is the outer membrane, 2 is the complex ( _CO mplex, the same applies hereinafter) I, 3 is the complex II, 4 is the complex III, 5 is the complex IV, 6 is the ATP Synthase, 7 is ATP / ADP Translocator, 8 is inner membrane, 9 is cytochrome C, 10 is coenzyme (3,), and the side on which outer membrane 1 is provided (that is, the lower part of the figure) The outer side of the mitochondria and the side where the intima 8 is provided (ie, the upper part of the figure) is the inner side of the mitochondria.

Figure 3 shows a schematic diagram of the reaction on the mitochondrial membrane surface. As explained earlier, 1 is outer membrane, 2 is complex I, 3 is complex II, 4 is complex I II, 5 is complex IV, 6 is ATP synthase, 7 is ATP ADP translocator, 8 Is the inner membrane, 9 is cytochrome C, 10 is coenzyme Q, and the process from complex I to IV is the electron transport system.

Hydrogen carried as NADH ₂ + moves from complex I to the transmembrane space, and at the same time two electrons are passed to the capture enzyme Q. On the other hand, the electrons carried as FADH ₂ are also passed from complex へ to coenzyme Q. Hydrogen of reduced ubiquinone is released as 2 H + in the chain with complex III and moves to the intermembrane space. At the same time, the electrons are passed to complex III. The electrons passed to complex III are sent to complex IV via cytochrome C, which is superficial on the mitochondrial membrane. Complex IV oxidizes reduced cytochrome C, the resulting electrons are passed to o ₂ molecule. 1 2 0 molecules ₂ are attached water two H + Upon binding to one molecule of the matrix.

In the present application, HPF or DMAX is preferred as the fluorescent reagent used for staining active oxygen. Conventional fluorescent reagents such as DCF, DHR, and HE sometimes become positively or negatively charged after reacting with active oxygen, and are sometimes biased toward sites having the opposite charge. In other words, with these fluorescent reagents, it was very difficult to specify where the stained active oxygen originated because it did not stop at that spot after staining with active oxygen. On the other hand, according to the HPF and DMAX used in the present application, the charge is not biased to a specific side unlike the conventional fluorescent reagent, and the generation site of the active oxygen can be reliably specified. [Example 1]

Cell culture and reactive oxygen measurement>

In this example, human cells (143B) were used (other cells may be used). Human cells were cultured at 37 ° C in air containing 5% carbon dioxide using Dulbecco's MEM culture solution containing 10% fetal bovine serum. The cells are seeded on a glass bottom dish (MatTek Corp., Ashland, Mass., USA) containing the above culture solution and cultured for approximately one day under the above conditions. Explanation This culture Ha Nkusu solution (HB SS) (1 0. OmM HE PES, 1. 0 mM Mg C 12, 2 mM C a CJ 2, and 2. 7 mM glucose, p H 0. 0 5) to Replace. For the observation of active oxygen, HPF, which reacts relatively specifically with hydroxyl radicals among active oxygen species and emits fluorescence, was used. (Similar results have been obtained with DMAX for singlet oxygen observation.) Using a confocal laser microscope, 500 W of 488 nm excitation light was irradiated for 0.4 seconds, and 5 15 Observe using a nm parier-free filter. The images are stored in a computer, and the fluorescence intensity is determined on a computer using the created program.

Antioxidant capacity test>

Cells are seeded in a glass bottom dish and cultured as described above. One day after the start of the culture, 15 Gy irradiation is performed using an X-ray irradiator (MBR-1505R (Hitachi)). Then, immediately return the cells to a 5% CO 2 incubator, and after 2 hours, replace the culture medium with 1 ml of HBSS, and adjust the concentration of 1 IX 1 HPF to a final concentration of 10 μg / m 1. In addition, observe the fluorescence with a confocal laser microscope, store it in a computer, and then calculate the intensity using a program that creates the fluorescence intensity on a computer. Following this irradiation, 1 μl of vitamin E dissolved in DMSO is added to the culture solution to a final concentration of 10 μg / m 1 immediately after the irradiation, and the mixture is returned to the incubator for 2 hours. Thereafter, the fluorescence is measured as described above. Vitamin Ε administration, non The ratio is determined from the fluorescence intensity of the administration.

The antioxidant extracted from foods is added to the culture medium in the same manner as vitamin E, and the fluorescence intensity of HPF is examined. The antioxidant ability is compared with the case of vitamin E administration. Reactive oxygen detection using HPF was detected near the mitochondria and matched the distribution of the mitochondria-specific detection reagent (Mitotracker Red) .Using HPF, active oxygen generated from mitochondria was detected. It was verified that. Industrial applicability

As is clear from the above, according to the present application, it is possible to specify active oxygen generated from mitochondria among active oxygen in cells. Further, according to the present invention, it is possible to measure the amount of active oxygen generated from the mitochondria simply by integrating the luminance of a predetermined area image of the image uniformly. Reduces processing time.

Further, according to the method for quantifying the antioxidant ability of the antioxidant according to the present invention, it is possible to measure the antioxidant ability of various foods and drugs and visually recognize the same. In addition, by performing image processing on images obtained by microscopic photographing, it is possible to quantify and compare the antioxidant ability of foods, drugs, and the like.

Claims

1. a first step of applying a fluorescent reagent having a property of specifically reacting with intracellular active oxygen to a biological sample;

The guest who excited the fluorescent reagent contained in the biological sample obtained in the first step

In this state, the biological sample is microscopically photographed and subjected to photoelectric conversion processing to obtain a fluorescence image including one or more cells in the field of view.

Steps and

Luminance of constituent pixels in a predetermined area of the fluorescence image acquired in the second step

A third step for summation,

A fourth step of identifying the amount of active oxygen generated from the mitochondria in the cell based on the sum of luminance of the constituent pixels obtained in the third step. How to measure active oxygen.

2. The active oxygen generated from the mitochondrial according to claim 1, wherein the active oxygen in the cell is a hydroxy radical, a singlet oxygen, or a superoxide. Measurement method.

3. The method for measuring active oxygen generated from mitochondria according to claim 2, wherein the fluorescent reagent is HPF.

4. The method for measuring active oxygen generated from mitochondria according to claim 2, wherein the fluorescent reagent is DMAX.

5. The method for measuring active oxygen generated from mitochondria according to claim 2, wherein the fluorescent reagent is APF.

6. A first step of preparing two sets of biological samples having the same active oxygen generation characteristics,

One set of biological samples from the two sets of biological samples prepared in the first step A second step of applying an antioxidant which is a target of the antioxidant capacity quantification to the sample while applying an antioxidant which is a reference of the antioxidant capacity quantification to the other set of biological samples;

A third step of providing the same active oxygen-induced stimulus to the two sets of biological samples to which the antioxidant has been applied in the second step;

A fourth step of applying a fluorescent reagent having a property of specifically reacting with intracellular active oxygen to each of the two sets of biological samples to which the active oxygen-induced stimulus has been given in the third step;

Each of the two biological samples to which the fluorescent reagent has been applied in the fourth step is subjected to microscopic photography and photoelectric conversion processing in a state where the fluorescent reagent has been excited, so that one or more cells can be visualized. A fifth step of acquiring two sets of fluorescence images included in

A sixth step of calculating the sum of the luminance of the constituent pixels in the predetermined area for each of the two sets of fluorescent images acquired in the fifth step;

For the two sets of luminance sums obtained in the sixth step, the antioxidant material to be quantified for the antioxidant capacity is the sum of the luminance on the side where the antioxidant material, which is the standard for antioxidant capacity quantification, is applied. A seventh step of calculating the ratio of the sum of luminance on the side where

A method for quantifying the antioxidant ability of an antioxidant material, comprising:

7. A first step of preparing two sets of biological samples having the same active oxygen generation characteristics,

A second step of applying the same active oxygen-induced stimulus to the two sets of biological samples prepared in the first step;

In the second step, one of the two biological samples to which the same active oxygen-induced stimulus was given was applied with an antioxidant to be quantified for antioxidant ability, Criteria for quantification of antioxidant capacity in the other set of biological samples A third step of applying an antioxidant,

A fourth step of applying a fluorescent reagent having a property of specifically reacting with intracellular active oxygen to each of the two sets of biological samples to which the antioxidant has been applied in the third step;

Each of the two biological samples to which the fluorescent reagent was applied in the fourth step was microscopically photographed and subjected to photoelectric conversion while the fluorescent reagent was excited, whereby one or more cells were obtained. A fifth step of acquiring two sets of fluorescence images included in the field of view,

8. The method for quantifying the antioxidant ability of an antioxidant according to claim 5 or 6, wherein the active oxygen-induced stimulus is X-ray irradiation. '

9. The method for quantifying the antioxidant ability of an antioxidant according to claim 5 or 6, wherein the reactive oxygen-induced stimulus is application of a site force-in.

10. The antioxidant ability of the antioxidant according to claim 5 or 6, wherein the fluorescent reagent having a property of specifically reacting with active oxygen in cells is HPF. How to quantify.

11. The antioxidant of the antioxidant according to claim 5 or 6, wherein the fluorescent reagent having a property of specifically reacting with active oxygen in cells is DMAX. How to quantify performance.

12. The antioxidant used as the standard for quantifying the antioxidant capacity is vitamin E, and the antioxidant ability of the antioxidant according to claim 5 or 6 is quantified. how to.

13. The antioxidant according to claim 5 or claim 6, wherein the biological samples having the same active oxygen generation characteristics are biological samples obtained from a standard specimen prepared in advance. A method for quantifying the antioxidant capacity of wood.

14. The biological sample having the same active oxygen generating property is a biological sample obtained from a specific individual to be taken the antioxidant, wherein the biological sample has the same active oxygen generating property. A method for quantifying the antioxidant ability of the described antioxidant.

15. Antioxidant whose oral antioxidant capacity is to be quantified is orally administered to a specific individual who is to take the antioxidant, and then a biological sample is collected from the specific individual and the antioxidant is injected into cells. The method according to claim 13, wherein the intake amount is measured, and the amount of the antioxidant applied when quantifying the antioxidant ability is adjusted in accordance with the measured intake amount. A method for quantifying the antioxidant capacity of an antioxidant.