JP6285691B2 - Method for evaluating the activity of neutrophil cells - Google Patents

Description

  The present invention relates to a method for evaluating the activity of neutrophil cells. The present invention also relates to a method for evaluating the oxidative stress state of a living body and a method for evaluating the antioxidant activity of food and drink in vivo.

  Neutrophil cells exhibit migratory activity against inflammatory cytokines and bacteria, etc., thereby gathering in the inflamed area and having a function of phagocytosing bacteria and the like, playing an important role in defense of the body . As an action mechanism for phagocytosing bacteria and the like, production of active oxygen (superoxide, hydrogen peroxide, etc.) and production of hypochlorous acid by myeloperoxidase (MPO) are known.

  On the other hand, it has been pointed out that the production of active oxygen due to excessive activation of neutrophil cells causes oxidative stress, causes cell and tissue damage, and is involved in various pathological conditions (for example, non-patent literature). 1). In recent years, it has been found that MPO released from neutrophil cells is also involved in lipid peroxidation, which is another cause of oxidative stress (for example, Non-Patent Document 1).

  As a method for measuring the function of neutrophil cells and the like, for example, Patent Document 1 discloses an unevenness having a center line average roughness Ra value of 0.2 μm to 10 μm and a bumpy average interval Sm value of 5 μm to 200 μm. There is disclosed a method for measuring cell function, characterized in that the production of myeloperoxidase is induced by contacting a material having a surface with blood and the production of myeloperoxidase is measured.

JP 9-206099 A

Biological sample analysis, Vol. 35, No2, 2012, pp. 133-139.

  Oxidative stress is a cause of various diseases including lifestyle-related diseases. If there is a technique for evaluating the activity of neutrophil cells, it is possible to detect a dangerous state in front of the oxidative stress state, so that it is earlier than the conventional technique for evaluating the oxidative stress state. It becomes possible to deal with.

  In the conventional technique for evaluating the activity of neutrophil cells, it is necessary to separate neutrophil cells from a blood sample, which requires complicated operations and specialized techniques, and lacks simplicity. Moreover, there is a possibility that the activity of neutrophil cells may be reduced or unnecessary activation may occur during the separation process, and it cannot be said that accurate evaluation can be performed. In addition, although patent document 1 describes performing a cell function measurement using whole blood, it is only evaluating the production amount of MPO. There has been no known example in which MPO activity has been evaluated using whole blood.

  Therefore, an object of the present invention is to provide a method capable of evaluating the activity of neutrophil cells without separating neutrophil cells from a blood sample.

The present invention is a method for evaluating the activity of neutrophil cells, comprising measuring myeloperoxidase activity and superoxide (O ₂ ⁻ ) production activity using the same sample containing whole blood, At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on fluorescence detection, and the fluorescence detection is a sample container containing the excitation light output from an excitation light source. And the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are arranged on the same side with respect to the excitation light irradiation surface of the sample container. The present invention provides a method for evaluating the activity of neutrophil cells based on the measured myeloperoxidase activity and superoxide production activity.

  Since this method evaluates based on the measurement data of myeloperoxidase activity and superoxide production activity, it directly evaluates the activity of neutrophil cells compared to the case based on the amount of MPO production. Is possible. In addition, because the sample containing whole blood is used without separating neutrophil cells, in addition to simple operation, the kinetics in vivo including interaction with various humoral factors contained in blood Can be obtained.

  Conventionally, fluorescence detection and chemiluminescence detection are used to measure myeloperoxidase activity and superoxide production activity. For this reason, when a sample containing whole blood is used, the influence of light absorption by red blood cells (hemoglobin) or the like in the blood is large (see FIG. 3), and in the conventional method, fluorescence and its excitation light are particularly absorbed by the sample. As a result, the detection sensitivity was remarkably reduced, and measurement was almost impossible. According to the method of the present invention, since the sample is irradiated with excitation light and fluorescence is detected on the same side of the sample container with the excitation light irradiation surface, the influence of light absorption by red blood cells (hemoglobin) or the like in blood. Can be greatly reduced, and evaluation is possible even when at least one of measurement of myeloperoxidase activity and measurement of superoxide production activity is based on fluorescence detection. This also enables evaluation with a small blood volume of about several μL.

  The method for evaluating the activity of the neutrophil cells may include adding a neutrophil stimulating agent to the sample. By adding a neutrophil stimulating agent, a quasi-stimulus is given to the neutrophil cells in the sample to induce an innate immune reaction (biological defense reaction), and the neutrophil cells have an anti-inflammatory ability (antioxidant ability or The ability to prevent oxidative stress can be evaluated.

  The present invention is also a method for evaluating the oxidative stress state of a living body, comprising measuring myeloperoxidase activity and superoxide producing activity using the same sample containing whole blood derived from the living body, At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on fluorescence detection, and the fluorescence detection is a sample container containing the excitation light output from an excitation light source. And the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are arranged on the same side with respect to the excitation light irradiation surface of the sample container. A method for evaluating the oxidative stress state of the living body based on the measured myeloperoxidase activity and superoxide production activity .

  Since the method for evaluating the oxidative stress state of the living body is based on the measurement data of myeloperoxidase activity and superoxide production activity, the oxidative stress state and oxidative state of the living body due to excessive activation of neutrophil cells. It is possible to evaluate a dangerous state before a stress state. In addition, since a sample containing whole blood is used without separating neutrophil cells, in addition to simple operation, the state of the living body can be accurately evaluated.

  The method for evaluating the oxidative stress state of the living body is a method of collecting data for evaluating the oxidative stress state of the living body, wherein the data includes data on myeloperoxidase activity in blood and superoxide in blood Production activity data, including measuring the myeloperoxidase activity and the superoxide production activity using the same sample containing the whole blood derived from the living body, and measuring the myeloperoxidase activity, and At least one of the measurement of the superoxide production activity is based on fluorescence detection, and the fluorescence detection is performed by irradiating the sample container containing the sample with the excitation light output from the excitation light source and releasing the emitted fluorescence. Is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are the sample container. The irradiation surface of the electromotive light, are disposed on the same side, or a method.

  The present invention further relates to a method for evaluating the in vivo antioxidant activity of a food or drink, the step of causing the food or drink to be evaluated to be ingested by the living body, the myeloperoxidase activity and the superoxide production activity, Measuring using the same sample containing whole blood derived from a living body, and at least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on the detection of fluorescence, The detection of the fluorescence is to irradiate the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by the fluorescence detector, and the excitation light source and the fluorescence detector are Measured myeloperoxidase activity and superoxide production, arranged on the same side of the excitation light irradiation surface of the sample container Also provides a method of evaluating the antioxidant activity in vivo of the food products on the basis of sex.

  Since the method for evaluating the antioxidant activity of the food and drink in vivo is based on measurement data of myeloperoxidase activity and superoxide production activity, as described above, the oxidative stress state and oxidation of the organism It is possible to evaluate a dangerous state before a stress state. Therefore, the antioxidant activity of food and drink can be evaluated by evaluating the oxidative stress state after ingesting the food and drink. In addition, since the evaluation reflects the stress state of the living body, in contrast to conventional methods (DPPH method, ORAC method, etc.) for evaluating the antioxidant activity of food and drink as chemical substances, It is possible to evaluate the true antioxidant activity in the living body after absorption. Furthermore, since a sample containing whole blood is used without separating neutrophil cells, the operation can be easily performed and an evaluation reflecting the state of the living body more accurately can be performed.

  In any of the methods according to the present invention described above, the amount of whole blood contained in the sample may be more than 0 μL and not more than 10 μL. Further, the sample container may be a plate-type container.

  Furthermore, the measurement of myeloperoxidase activity may be based on detection of fluorescence, and the measurement of superoxide production activity may be based on detection of chemiluminescence. In this case, the detection of the fluorescence is to irradiate the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by a fluorescence detector. The emitted light is detected by a chemiluminescence detector, and the excitation light source, the fluorescence detector, and the chemiluminescence detector are arranged on the same side with respect to the excitation light irradiation surface of the sample container. And the detection ranges of the fluorescence detector and the chemiluminescence detector may be the same. The fluorescence detector and the chemiluminescence detector may be a single detector.

  ADVANTAGE OF THE INVENTION According to this invention, the method which can evaluate the activity of a neutrophil cell is provided, without isolate | separating a neutrophil cell from a blood sample. In addition, according to the present invention, since a dangerous state (non-disease state) before the oxidative stress state can be detected, it is possible to cope with an earlier stage than the conventional method for detecting the oxidative stress state. Become.

  In 2010, the proportion of elderly people aged 65 and over accounted for 23.3% of the total population in 2010, leading to a super-aging society ahead of the world, and also expected to reach 39.9% in 2060. Has been. It is well known that the incidence of age-related diseases such as arteriosclerotic diseases, metabolic diseases, neurodegenerative diseases, and malignant tumors increases with age. In addition, with the westernization of food, lifestyle-related diseases have increased and now account for about 60% of causes of death. It is known that active oxygen is greatly involved in oxidative stress, which is a cause of lifestyle-related diseases. It is expected to prevent these diseases and extend their healthy lifespan by the bioregulatory function (for example, antioxidant activity) that is the tertiary function of food and drink at an unaffected stage before the onset of these diseases. .

  As an index for uniformly evaluating the antioxidant activity of foods and drinks, the AOU (Antioxidant-unit) study group is the center, and the AOU value is defined. The AOU value is determined by H-ORAC (Hydrophilic Oxygen Radical Absorbance Capacity) method for evaluating water-soluble substances, L-ORAC (Lipophilic Oxygen Radical Absorbance Capacity) method for evaluating water-soluble substances, and Singe method for S The three values obtained by the three methods of Oxygen Absorption Capacity) are added together. On the other hand, a means for evaluating the true antioxidant activity in the living body after ingesting food and drink and undergoing digestion and absorption has not yet been prepared. In the above-mentioned AOU study group, the indexing of the antioxidant activity in the living body is taken up as an issue to be tackled with the highest priority in the future.

  According to the present invention, it is possible to evaluate the true antioxidant activity in a living body after ingesting a food or drink and undergoing digestion and absorption.

  The method according to the present invention can be widely disseminated in general because it does not require complicated operations and specialized techniques, and can be widely used for individual constitution and physical condition management tools, food suitable for individuals, so-called tailor-made food selection It can be used as a tool and a tool for improving dietary habits.

It is a schematic diagram which shows an example of the detection (preparation) of fluorescence. It is a schematic diagram which shows an example of the detection (cuvette) of fluorescence. It is a graph which shows the result of the absorption spectrum measurement of whole blood. It is a graph which shows the result of simultaneous measurement (cuvette) of fluorescence and chemiluminescence. It is a graph which shows the result of simultaneous measurement (preparation) of fluorescence and chemiluminescence. It is a schematic diagram which shows the detection (cuvette) of the conventional fluorescence.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings as necessary. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

The method for evaluating the activity of neutrophil cells according to this embodiment includes the step of measuring myeloperoxidase activity and superoxide (O ₂ ⁻ ) production activity using the same sample containing whole blood. In the method, at least one of measurement of myeloperoxidase activity and measurement of superoxide production activity is based on detection of fluorescence. In addition, the fluorescence is detected by irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by the fluorescence detector, and the excitation light source and the fluorescence detector are the sample. It arrange | positions on the same side with respect to the irradiation surface of the excitation light of a container. Based on the data of myeloperoxidase activity and superoxide production activity thus obtained, the activity of neutrophil cells is evaluated.

Neutrophil cells are a type of white blood cell. The main role of neutrophil cells is to prevent bacteria and fungi that have entered the body by phagocytosis and prevent infection. When the neutrophil cell comes into contact with bacteria and fungi, it is encapsulated in the neutrophil plasma membrane to form a phagosome. The phagosome then fuses with the granule and the granule contents are released into the vesicle. Active oxygen (superoxide, hydrogen peroxide) is generated by the NADPH oxidase system formed in the cell membrane (the phagocytic membrane), and bacteria and fungi are sterilized. Further, hypochlorous acid (HOCl) is converted from hydrogen peroxide (H ₂ O ₂ ) and chloride ion (Cl ⁻ ) by enzymatic reaction of myeloperoxidase (EC No. 1.11.2.2.2) contained in the granule contents. (Or its halogen equivalent) is produced and kills bacteria and fungi. Therefore, the activity of neutrophil cells can be evaluated using myeloperoxidase activity and superoxide production activity as indices.

  The sample contains whole blood. “Whole blood” means blood itself collected from a living body. The sample may be whole blood itself or may be whole blood diluted with physiological saline, buffer solution or the like. In the case of dilution, the dilution ratio may be set as appropriate, but from the viewpoint of improving detection sensitivity, it is preferably diluted 10 to 200 times, more preferably 20 to 180 times, and more preferably 50 to 150. It is more preferable to dilute it twice, it is still more preferable to dilute it 80-120 times, and it is especially preferable to dilute 90-110 times.

  The amount of whole blood used as a sample (blood volume) is not particularly limited. On the other hand, according to the method of the present embodiment, it is possible to greatly reduce the influence of light absorption by red blood cells (hemoglobin) or the like in blood, so that evaluation with a small blood volume of about several μL is also possible. Therefore, the amount of whole blood contained in the sample may be more than 0 μL and 10 μL or less. Further, the amount of whole blood contained in the sample may be more than 0 μL and 8 μL or less, may be more than 0 μL and 6 μL or less.

  For example, a small amount of peripheral blood (2 to 3 μL) may be collected from a fingertip or the like using a blood collection device (for example, a lancet) used by a diabetic patient when measuring daily blood glucose levels. Since such a small amount of blood has a small load, the activity of neutrophil cells can be evaluated on a daily basis.

  The sample container may be a cuvette-type container having a cubic shape or a rectangular parallelepiped shape, or may be a plate-type container (for example, a preparation) capable of thinly extending the sample. From the viewpoint of improving measurement efficiency, a plate-type container is preferable. Since the possibility that neutrophil cells exist near the surface of the sample container increases in the plate-type container, the problem of absorption of excitation light and fluorescence by red blood cells (hemoglobin) can be solved as described later. The sample container is preferably a transparent sample container from the viewpoint of improving the detection efficiency of fluorescence or the like.

  The myeloperoxidase activity and the superoxide production activity in the sample are measured by, for example, detecting fluorescence, luminescence, or absorbance over time using a fluorescence indicator, a luminescence (eg, chemiluminescence) indicator, or an absorbance indicator. be able to. As the indicator, a commercially available indicator may be used, or a commercially available measurement kit may be used.

  The myeloperoxidase activity can be measured using, for example, a fluorescent indicator, a luminescent indicator, or an absorbance indicator that reacts with hypochlorous acid (or its halogen equivalent) produced by myeloperoxidase. Examples of such an indicator include aminophenylfluorescein (APF), taurine / TNB (see J. Clin. Invest., Vol. 70, pp. 598-607, 1982), 8-amino-5 Chloro-7-phenylpyrido [3,4-d] pyridazine-1,4- (2H, 3H) dione (see L-012: Anal Biochem., Vol. 271 (1), pp. 53-58, 1999) Is mentioned. These indicators are preferably those with poor reactivity with superoxide.

  The reaction between APF and HOCl can be detected by fluorescence (for example, excitation wavelength 490 nm, fluorescence wavelength 515 nm). The reaction between taurine / TNB and HOCl can be detected by absorption (for example, wavelength 412 nm). The reaction between L-012 and HOCl has low specificity but can be detected by chemiluminescence (for example, wavelength 455 nm).

  The superoxide production activity can be measured using, for example, a fluorescent indicator, a luminescence indicator, or a light absorption indicator that reacts with superoxide. Examples of such indicators include 2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (CLA), 2-methyl-6- (4-methoxyphenyl) -3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA), 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC), indocyanine type imidazopyranodine compound ( NIR-CLA), 2- [2,4,5,7-tetrafluoro-6- (2-nitro-4,5-dimethoxyphenylsulfonyloxy) -3-oxo-3H-xanthen-9-yl] benzoic acid (BES-So). These indicators are preferably poor in reactivity with hypochlorous acid (or its halogen equivalent) produced by myeloperoxidase.

  The reaction between CLA and superoxide can be detected by chemiluminescence (for example, maximum emission wavelength 380 nm). The reaction between MCLA and superoxide can be detected by chemiluminescence (for example, maximum emission wavelength 465 nm). The reaction between MPEC and superoxide can be detected by chemiluminescence (for example, maximum emission wavelength of 430 nm). The reaction between NIR-CLA and superoxide can be detected by chemiluminescence (for example, maximum emission wavelength of 800 nm). The reaction between BES-So and superoxide can be detected by fluorescence (for example, excitation wavelength 505 nm, fluorescence wavelength 544 nm).

  At least one of measurement of myeloperoxidase activity and measurement of superoxide production activity is based on fluorescence detection. From the viewpoint of improving the detection sensitivity, it is preferable to measure the myeloperoxidase activity by detecting fluorescence and to measure the superoxide production activity by detecting chemiluminescence. The myeloperoxidase activity is preferably measured by a fluorescent indicator (for example, APF). More preferably, the superoxide production activity is measured using a chemiluminescent indicator (for example, MCLA).

  The myeloperoxidase activity and superoxide production activity are measured using the same sample containing whole blood. Moreover, you may measure myeloperoxidase activity and superoxide production activity substantially simultaneously. Thereby, both activities under the same conditions can be measured, and the activity of neutrophil cells can be more accurately evaluated.

  The fluorescence is detected by irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the fluorescence from the sample is detected by a fluorescence detector arranged on the same side of the excitation light irradiation surface of the sample container. It is to detect. The case of measuring myeloperoxidase activity and superoxide production activity almost simultaneously by fluorescence and chemiluminescence will be described as an example, and the detection of fluorescence will be described more specifically.

  1 and 2 are schematic diagrams illustrating an example of fluorescence detection. FIG. 6 is a schematic diagram showing conventional fluorescence detection.

  FIG. 6 shows a sample container in a conventional apparatus for measuring fluorescence and chemiluminescence at the same time (for example, described in JP-A-2004-61438, JP-A-2000-13234, etc.). In FIG. 6, the excitation light 30 output from the excitation light source (not shown) is irradiated to the sample container (cuvette) 10 containing the sample, and the emitted fluorescence 40 is detected by the fluorescence detector (not shown). However, the fluorescence 40 is detected by a fluorescence detector in which the excitation light source and the fluorescence detector are arranged on a side different from the excitation light source with respect to the irradiation surface of the excitation light 30 of the sample container 10.

  Since the sample stored in the sample container 10 contains whole blood, neutrophil cells 20 and red blood cells (hemoglobin) 50 exist. Usually, the number of neutrophil cells 20 contained in whole blood is about 1/1000 of the number of red blood cells 50. When fluorescence and chemiluminescence are measured simultaneously, hemoglobin contained in red blood cells 50 emits chemiluminescence indicator (for example, 380 to 450 nm), fluorescence indicator excitation light (for example, 488 nm), and fluorescence (for example, 500 to 530 nm). And there is a problem of absorbing strongly. FIG. 3 shows the results of measuring the absorption spectrum of whole blood. For this reason, in the conventional method shown in FIG. 6, especially in the case of fluorescence, the attenuation of the excitation light until reaching the neutrophil cell 20 and the attenuation of the fluorescence until reaching the fluorescence detector are remarkable. Is extremely difficult to measure.

  In the method according to the present embodiment shown in FIGS. 1 and 2, the excitation container 30 (preparation in FIG. 1 and cuvette in FIG. 2) 10 containing the sample is irradiated with the excitation light 30 output from the excitation light source (not shown). The emitted fluorescence is detected by a fluorescence detector (not shown), and the excitation light source and the fluorescence detector are arranged on the same side with respect to the irradiation surface of the excitation light 30 of the sample container 10. . By such a technique, the problem of absorption of excitation light and fluorescence by the red blood cells (hemoglobin) 50 can be solved at least for the neutrophil cells 20 in contact with the sample container surface. As shown, the myeloperoxidase activity and the superoxide production activity can be measured almost simultaneously by fluorescence and chemiluminescence.

  When measuring by fluorescence and chemiluminescence, the excitation light source, the fluorescence detector and the chemiluminescence detector are arranged on the same side with respect to the excitation light irradiation surface of the sample container, and the fluorescence detector and the chemiluminescence detection The detector detection ranges may be the same. In addition, the fluorescence detector and the chemiluminescence detector may be a single detector.

  The method for evaluating the activity of neutrophil cells according to the present embodiment may include adding a neutrophil stimulant to the sample.

  The neutrophil stimulating agent may be any substance that activates the function of neutrophil cells (for example, migration, phagocytosis). Examples of the neutrophil stimulant include formylmethionyl leucylphenylalanine (fMLP: neutrophil migratory peptide), phorbol 12-myristic acid 13-acetate (PMA), and opsonized zymon (OZ). You may use these individually by 1 type or in combination of 2 or more types.

  By adding a neutrophil stimulating agent, a quasi-stimulus is given to the neutrophil cells in the sample to induce an innate immune reaction (biological defense reaction), and the neutrophil cells have an anti-inflammatory ability (antioxidant ability or The ability to prevent oxidative stress can be evaluated. On the other hand, when the pseudo stimulus is not given, the intact state of the neutrophil cells present in the peripheral blood can be evaluated. For example, the neutrophil cells are activated by intense exercise, smoking, etc. It is possible to evaluate a state in which active oxygen is released (oxidative stress state).

  Derivation of myeloperoxidase activity and superoxide production activity will be described with reference to FIG. FIG. 5 shows an example in which myeloperoxidase activity and superoxide production activity are detected by fluorescence and chemiluminescence. A neutrophil stimulant is added at the time indicated by the arrow “stimulation”. Before the addition of the neutrophil stimulant, for example, the myeloperoxidase activity and the superoxide production activity can be derived by calculating the integrated value or the average value of the luminescence amount (or fluorescence amount). On the other hand, after the addition of the neutrophil stimulant, for example, the myeloperoxidase activity and the superoxide production activity can be derived by calculating the peak area.

  Neutrophil cell activity can be assessed based on measured myeloperoxidase activity and superoxide production activity. As described above, when the myeloperoxidase activity and the superoxide production activity are enhanced, it means that the activity of neutrophil cells is enhanced. For example, when the myeloperoxidase activity and the superoxide production activity exceed a certain predetermined reference value, it may be evaluated that the activity of neutrophil cells is enhanced. Similarly, when the myeloperoxidase activity and the superoxide production activity are below a certain predetermined reference value, it may be evaluated that the activity of neutrophil cells is reduced.

  What is necessary is just to set a reference value suitably according to the objective. For example, the reference value may be derived from an average value or the like from the myeloperoxidase activity and the superoxide production activity measured for a large number of human specimens. There may be a plurality of reference values depending on, for example, sex, age, and the like. In addition, for a specific individual, a measurement value in a healthy state may be used as a reference value. Furthermore, in the method for evaluating the antioxidant activity of foods and drinks in vivo, the measured value before the intake of foods and drinks can be used as a reference value.

  The ability to prevent inflammation when a neutrophil stimulant is added can be similarly evaluated. Moreover, when the inflammatory defense ability falls below a certain reference value, it may be evaluated that the activity of neutrophil cells is reduced for some reason and the innate immunity is reduced.

  The method for evaluating the activity of neutrophil cells according to this embodiment can also be referred to as a method for collecting data for evaluating the activity of neutrophil cells.

  The method for evaluating the oxidative stress state of the living body according to the present embodiment is a step of measuring myeloperoxidase activity and superoxide production activity using the same sample containing whole blood derived from the living body for evaluating the oxidative stress state. including. The measurement of myeloperoxidase activity and the measurement of superoxide production activity are as described above.

  The oxidative stress state of a living body can be evaluated based on the measured myeloperoxidase activity and superoxide production activity. As described above, when the myeloperoxidase activity and the superoxide production activity are enhanced, the activity of the neutrophil cell is enhanced, and the oxidative stress state or oxidative stress of the living body due to the overactivation of the neutrophil cell It means that it is in a dangerous state before the state. For example, when a predetermined reference value is exceeded, an oxidative stress state or a dangerous state before the oxidative stress state may be evaluated. Similarly, when it falls below a certain predetermined reference value, it may be evaluated that it is not an oxidative stress state or a dangerous state before the oxidative stress state. The reference value can be set as described above.

  The method for evaluating the oxidative stress state of the living body according to this embodiment can also be referred to as a method of collecting data for evaluating the oxidative stress state of the living body.

  The method for evaluating the in vivo antioxidant activity of a food or drink according to this embodiment includes the step of causing a living body to ingest the food or drink to be evaluated, the myeloperoxidase activity, and the superoxide production activity. Measuring using the same sample containing whole blood. The measurement of myeloperoxidase activity and the measurement of superoxide production activity are as described above.

  The in vivo antioxidant activity of food and drink can be evaluated based on the measured myeloperoxidase activity and superoxide production activity. As described above, an oxidative stress state or a dangerous state before the oxidative stress state can be evaluated using myeloperoxidase activity and superoxide production activity as indices. Therefore, after ingesting food / beverage products, when myeloperoxidase activity and superoxide production activity fall, it can be evaluated that the said food / beverage products have antioxidant activity in the living body.

  The method for evaluating the in vivo antioxidant activity of a food or drink according to the present embodiment uses the same sample containing whole blood derived from the living body before the step of ingesting the food or drink to be evaluated into the living body. And measuring the myeloperoxidase activity and the superoxide production activity. A more quantitative evaluation becomes possible by comparing the myeloperoxidase activity and the superoxide production activity before and after ingesting the food / beverage products to be evaluated.

  The method for evaluating the in vivo antioxidant activity of the food and drink according to this embodiment can also be referred to as a method of collecting data for evaluating the in vivo antioxidant activity of the food and drink.

  Hereinafter, the present invention will be described more specifically based on test examples.

As a sample container, a preparation and a cuvette (10 mm square) were used. In the case of a preparation, 2 μL of blood was collected from a healthy person with a lancet (manufactured by Becton Dickinson). This was diluted 100 times with RH buffer (10 mM HEPES, 154 mM NaCl, 5.6 mM KCl, pH 7.4) kept at 37 ° C., and MCLA represented by the following formula (1) (MCLA trade name, Tokyo Chemical Industry Co., Ltd.) APF (APF trade name, manufactured by Sekisui Medical Co., Ltd.) represented by the following formula (2) is added so as to be 3 μM, and the sample is placed on the slide for 1 minute. After incubating at 37 ° C., measurement was started using a fluorescence and luminescence simultaneous measurement apparatus as shown in FIG. In the case of a cuvette, 20 μL of blood is diluted 100 times with RH buffer kept at 37 ° C., MCLA is added to 9 μM, APF is added to 10 μM, the sample is transferred to the cuvette, and incubated for 5 minutes. Then, measurement was started using a fluorescence and luminescence simultaneous measurement apparatus as shown in FIG.

  While the LED (488 nm) was blinking at 125 msec intervals as excitation light, irradiation of the above-described sample was started. After 1.25 minutes (in the case of a preparation) and 2.5 minutes (in the case of a cuvette), 5 μM (in the case of a preparation) of a neutrophil migratory peptide (formylmethionyl leucylphenylalanine: neutrophil stimulant). ) 1 μM (in the case of cuvette) was added to the sample (at the time indicated by the arrow “Stimulation” in FIGS. 4 and 5). The measurement results of the fluorescence intensity of APF and the chemiluminescence intensity of MCLA are shown in FIGS.

  FIG. 4 is a graph showing the results of simultaneous measurement (cuvette) of fluorescence and chemiluminescence. FIG. 5 is a graph showing the results of simultaneous measurement (preparation) of fluorescence and chemiluminescence. As is apparent from the results of FIGS. 4 and 5, fluorescence and chemiluminescence can be detected simultaneously even when a sample containing whole blood is used. In particular, when a preparation was used, detection with higher sensitivity was possible.

  DESCRIPTION OF SYMBOLS 10 ... Sample container, 20 ... Neutrophil cell, 30 ... Excitation light, 40 ... Fluorescence, 50 ... Red blood cell (hemoglobin).

Claims (9)

A method for evaluating the activity of neutrophil cells, comprising:
Measuring myeloperoxidase activity and superoxide production activity using the same sample containing whole blood,
Adding a neutrophil stimulant to the sample,
At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on detection of fluorescence,
The detection of the fluorescence involves irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are , Arranged on the same side with respect to the excitation light irradiation surface of the sample container,
A method for evaluating the activity of neutrophil cells based on the measured myeloperoxidase activity and superoxide production activity. A method for evaluating the oxidative stress state of a living body,
Measuring myeloperoxidase activity and superoxide production activity using the same sample containing whole blood derived from the living body,
Adding a neutrophil stimulant to the sample,
At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on detection of fluorescence,
The detection of the fluorescence involves irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are , Arranged on the same side with respect to the excitation light irradiation surface of the sample container,
A method for evaluating the oxidative stress state of the living body based on the measured myeloperoxidase activity and superoxide production activity. A method for collecting data for evaluating the oxidative stress state of a living body,
The data is data of myeloperoxidase activity in blood and data of superoxide production activity in blood,
Measuring the myeloperoxidase activity and the superoxide production activity using the same sample containing whole blood derived from the living body,
Adding a neutrophil stimulant to the sample,
At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on detection of fluorescence,
The detection of the fluorescence involves irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are The method of arrange | positioning on the same side with respect to the irradiation surface of the said excitation light of the said sample container. A method for evaluating the antioxidant activity of food and drink in vivo,
A step of causing a living body to consume a food or drink to be evaluated;
Measuring myeloperoxidase activity and superoxide production activity using the same sample containing whole blood derived from the living body,
Adding a neutrophil stimulant to the sample,
At least one of the measurement of the myeloperoxidase activity and the measurement of the superoxide production activity is based on detection of fluorescence,
The detection of the fluorescence involves irradiating the sample container containing the sample with the excitation light output from the excitation light source, and the emitted fluorescence is detected by a fluorescence detector, and the excitation light source and the fluorescence detector are , Arranged on the same side with respect to the excitation light irradiation surface of the sample container,
A method for evaluating the in vivo antioxidant activity of the food based on the measured myeloperoxidase activity and superoxide production activity. The amount of the whole blood contained in the sample, beyond the 0MyuL, and is less than 10 [mu] L, The method according to any one of claims 1-4. The sample container is a plate-shaped container, the method according to any one of claims 1-5. The measurement of myeloperoxidase activity are those based on the detection of fluorescence, and the superoxide-producing activity of the measurement is based on the detection of chemiluminescence A method according to any one of claims 1 to 6 . The fluorescence detection is to irradiate a sample container containing the sample with excitation light output from an excitation light source, and detect the emitted fluorescence with a fluorescence detector,
The chemiluminescence detection is to detect the emitted light with a chemiluminescence detector,
The excitation light source, the fluorescence detector, and the chemiluminescence detector are arranged on the same side with respect to the irradiation surface of the excitation light of the sample container, and the fluorescence detector and the chemiluminescence detector The method according to claim 7 , wherein the detection ranges are matched. The method of claim 8 , wherein the fluorescence detector and the chemiluminescence detector are a single detector.

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