JP5253494B2 - Sample solution for electron spin resonance measurement, dried product thereof, and measurement method using them - Google Patents

Description

  The present invention relates to a sample solution for electron spin resonance measurement, a dried product thereof, and a measurement method using them, which can stably store a spin adduct for a long time.

  Electron spin resonance (hereinafter referred to as ESR) is a spectroscopic method that detects unpaired electrons using an electron spin resonance spectrometer (hereinafter referred to as ESR apparatus), and performs selective and quantitative free radical measurement. It is the only way you can do it. Free radicals are generally highly reactive and have a short lifetime, and are therefore difficult to detect directly. Therefore, a method that uses a relatively stable radical adduct (hereinafter referred to as a spin adduct) generated by trapping free radicals with a spin trap agent for measurement, that is, a spin trap method is frequently used.

  Conventional spin trapping agents are unstable and are particularly susceptible to decomposition in aqueous solutions. Moreover, although it is more stable than free radicals before trapping, the lifetime of the spin adduct is at most several minutes and cannot be stored for a long time. The measurer had to sequentially and quickly collect or prepare a sample and capture and measure free radicals for each sample in the vicinity of the ESR apparatus. Furthermore, since the ESR device is expensive and the measurement environment needs to be strictly controlled, its installation is not easy and only a limited number of people can use it.

  Various compounds have been developed with the aim of stabilizing the spin trap agent itself and the spin adduct. For example, a novel spin having a phosphinyl group, including a compound represented by the formula (II) (hereinafter referred to as DPPMDPO) A trapping agent is disclosed (for example, see Patent Document 1). DPPMDPO does not decompose even when the aqueous solution is stored at room temperature for several months, has a high 1-octanol / water partition coefficient, and is easily extracted into an organic solvent (for example, see Non-Patent Document 1).

Among free radical, superoxide anion radical ^(· O 2 ^_-) and hydroxyl radicals ^(· OH) is a reactive oxygen species, plays an important role in signal transduction and the immune system in vivo. In vivo, there is a mechanism for producing or eliminating reactive oxygen species, and the amount of reactive oxygen species is controlled by them. However, when the balance is lost and reactive oxygen species exceeding the antioxidant capacity of the in vivo antioxidant system are generated, that is, when subjected to oxidative stress, various diseases such as cancer, arteriosclerosis and acute lung injury develop or It is pointed out that it worsens. These diseases are considered to be caused by oxidative damage of nucleic acids, proteins, and the like by reactive oxygen species and lipid-derived radicals generated by reactive oxygen species.

  So far, methods of collecting blood, urine and other biological samples and measuring oxidative stress have been studied, but all were not free radicals themselves, but detected various oxidative stress markers produced by them. . According to these methods, oxidative stress cannot be measured directly and quickly, artifacts are large, and measurement with high reproducibility cannot be performed. Although it is desirable to detect free radicals themselves, biological samples have complex compositions containing metal ions, reducing substances, proteins, dissolved oxygen, etc., producing artifacts, or spin adducts undergo secondary reactions and decomposition. There is a problem that disappears in a short time.

  The spin adduct of the spin trap agent DMPO (5,5-Dimethyl-4,5-dihydro-3H-pyrrole N-oxide) and the oxygen-centered radical is inherently short-lived. It is disclosed that a lifetime exceeding the above is observed (for example, see Non-Patent Document 2). A hypothesis is stated that the spin adduct of DMPO and oxygen-centered radical is avoided from being extinguished by the attack of other coexisting radicals.

JP 2006-335738 A Masahiro Nishizawa and 4 others, Bull. Chem. Soc. Jpn. , 2007, 80, No. 3, p. 495-497 Steven Yue Qian and 4 others, Free Radical Biology & Medicine, 2000, Vol. 29, No. 6, p. 568-579

  Although the spin trapping agents described in Patent Document 1 and Non-Patent Document 1 are stable in an aqueous solution, the spin adduct is not necessarily stable. For example, the half-life of DPPMDPO superoxide anion radical adduct or hydroxyl radical adduct is at most 8.3 min or 13.2 min, so when measuring these free radicals, radical scavenging in the vicinity of the ESR apparatus And forced to measure.

  Patent Document 1 and Non-Patent Document 1 do not disclose a technique for capturing radicals in a sample having a complex composition such as a biological sample and stably storing a spin adduct for a long time.

  The method described in Non-Patent Document 2 is a complicated treatment method in which a DMPO spin adduct solution extracted with an organic solvent is concentrated in an inert gas atmosphere and then redissolved in an organic solvent saturated with the inert gas. Since the processing method takes a long time, the spin adduct may disappear during the processing.

Although the spin adduct has a lifetime of more than 10 hours after extraction, the spin adduct disappears steadily. Although it is possible to quickly capture and measure free radicals in the vicinity of the ESR apparatus, it is impossible to stably transport and measure the spin adduct obtained at a remote location.
Patent Document 2 describes the use of cultured cells, but does not disclose the use of a material having a complicated composition such as a biological sample.

  As described above, none of the above-mentioned prior arts can stably store the spin adduct for a long period of time that allows measurement with a remote ESR device. Further, there is no disclosure of a technique for measuring ESR while capturing free radicals in a biological sample or other sample having a complicated composition and avoiding the problem of disappearance of spin adducts and artifacts.

  In view of these problems, it is an object of the present invention to provide a method for stably storing a spin adduct for a long period of time that enables transportation to a remote place and measuring by ESR. It is another object of the present invention to provide a method of measuring by ESR while applying a spin trap method to free radicals in a sample having a complicated composition and avoiding problems of disappearance of spin adducts and artifacts. It is another object of the present invention to provide a method for measuring oxidative stress by directly detecting free radicals in a biological sample.

The inventor of the present invention has intensively studied in view of the above problems, and as a result, has found a method for stably storing a spin adduct and has completed the present invention.
That is, the first invention for solving the above problems is
(A) Step (B) of dissolving a spin trap agent represented by the following general formula (I) in a test solution in the form of an aqueous solution or water dispersion: (B) At least one type of solution obtained in step (A) (C) Adding an extraction solvent containing a water-insoluble organic solvent (C) ESR measurement obtained by a method comprising the step of separating the organic layer obtained in step (B) from the aqueous layer to obtain a sample solution for ESR measurement This is a sample solution.

[In Formula (I), each of R ¹ and R ² independently represents a linear or branched alkyl group having 1 to 6 carbon atoms, or a linear or branched chain having 1 to 6 carbon atoms. Represents an alkoxyl group, a phenyl group, or a substituted phenyl group,
Alternatively, an alkylene group or an alkylenedioxy group having a linear or branched chain having 2 to 6 carbon atoms bonded to each other is represented. ]

A second invention is a sample solution for ESR measurement according to the first invention, and in Formula (I), R ¹ and R ² are both phenyl groups.

  A third invention is a sample solution for ESR measurement according to the first or second invention, wherein the test solution is a biological sample.

  A fourth invention is a sample solution for ESR measurement according to any one of the first to third inventions, wherein the extraction solvent is a chloroform / methanol mixed solvent.

  The fifth invention is a sample solution for ESR measurement according to any one of the first to fourth inventions, which is further cooled and stored.

  A sixth invention is a dried product obtained by evaporating and removing the solvent from the sample solution for ESR measurement according to any one of the first to fifth inventions.

  The seventh invention is a dried product of the sample solution for ESR measurement according to the sixth invention, which is further stored in a cooled state.

The eighth invention
(A) The step (B) step of dissolving the spin trap agent represented by the above general formula (I) in a test solution in the form of an aqueous solution or water-dispersed liquid. Step of adding extraction solvent containing water-insoluble organic solvent (C) Separating the organic layer obtained in step (B) from the aqueous layer to obtain a sample solution for ESR measurement (D) Obtained in step (C) This is a measurement method for determining the amount of free radicals in a test solution by a method including a step of measuring the amount of a spin adduct contained in a sample solution for ESR measurement using an electron spin resonance apparatus.

The ninth invention
(A) The step (B) step of dissolving the spin trap agent represented by the above general formula (I) in a test solution in the form of an aqueous solution or water-dispersed liquid. In the step of adding an extraction solvent containing a water-insoluble organic solvent (C) The organic layer obtained in step (B) is separated from the aqueous layer to obtain a sample solution for ESR measurement (D ′) in step (C) Step of removing the solvent from the obtained sample solution for ESR measurement by removing the solvent to obtain a dried product (E) Re-dissolving the dried product obtained in step (D ′) to obtain a solution ( F) A measuring method characterized in that the amount of free radicals in the test solution is determined by a method including a step of measuring the amount of spin adduct contained in the solution obtained in step (E) with an electron spin resonance apparatus. It is.

  Free radicals present in the test solution or generated after dissolution of the spin trap agent react with the spin trap agent represented by the general formula (I) to generate a spin adduct, and the ESR measurement sample solution of the present invention Extracted into In the solution, the spin adduct exists stably for a long time that cannot be achieved by the prior art, and the spin adduct generated in a remote place can be stably transported and measured by ESR.

  Even when the test solution has a complex composition, the sample solution for ESR measurement separated from the test solution has few impurities, and the generated spin adduct disappears in a short time due to secondary reaction or decomposition, or an artifact. Is avoided. Therefore, the amount of free radicals present in the test solution can be measured by ESR using a test solution having a complicated composition as a measurement target.

The sample solution for ESR measurement of the present invention in which R ¹ and R ² are both phenyl groups is prepared using a spin trap agent having a large 1-octanol / water partition coefficient. Spin trap agents and their spin adducts can be easily extracted into organic solvents, and even with test solutions with low free radical content, a spin adduct solution with a concentration sufficient to perform ESR measurement can be obtained, allowing high-precision measurements. It is.

Since R ¹ and R ² are the same substituent, spin adduct diastereomers that give different ESR spectra are not produced. Since it is avoided that the ESR spectrum becomes complicated, analysis is easy.

  Since the spin trapping agent has lower cytotoxicity than the known spin trapping agent, even if the test solution contains cells, the measurement can be performed while suppressing the cell damage. In addition, since the spin trap agent has cell membrane permeability and is well dispersed in the cells in the test solution, free radicals in the cells can be captured. Therefore, it is suitable for measurement using a test solution containing cells.

  By using a biological sample as a test solution, it is possible to measure free radicals in the biological sample with high reproducibility. Furthermore, the oxidative stress of an individual can be measured by measuring the free radical. When a biological sample of an individual administered with food is used, knowledge about the antioxidant activity exhibited by the food in vivo can be obtained.

  The sample solution for ESR measurement of the present invention in which the extraction solvent is a mixed solvent of chloroform / methanol can store the dissolved spin adduct more stably for a longer time. Spin adducts generated at remote locations can be transported more stably and measured by ESR.

  Furthermore, according to the sample solution for ESR measurement of the present invention that is stored in a cooled state, the concentration of the spin adduct can be maintained by suppressing the volatilization of the organic solvent, so that highly accurate measurement is possible. Further, since the spin adduct can be stored stably for a long time, the spin adduct generated in a remote place can be transported more stably and measured by ESR.

  According to the dried product of the sample solution for ESR measurement of the present invention, the spin adduct can be stably stored for a longer time than the sample solution for ESR measurement. Spin adducts generated at remote locations can be transported more stably and measured by ESR.

  Furthermore, according to the dried product of the sample solution for ESR measurement of the present invention which is stored in a cold state, the spin adduct can be stored stably for a longer period of time. It can be measured by ESR.

3 is a graph showing the ESR signal relative intensity indicated by the sample solution for ESR measurement of Example 1. FIG. 6 is a graph showing the ESR signal relative intensity indicated by the sample solution for ESR measurement of Example 2. It is a graph which shows stability of the spin adduct in the sample solution for ESR measurement of this invention. 10 is a graph showing the ESR signal relative intensity indicated by the sample solution for ESR measurement of Example 6.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, but the present invention is not limited thereto.
First, main elements constituting the present invention will be described.
The spin trap agent according to the present invention is not particularly limited as long as it is a compound represented by the above general formula (I). In addition to DPPMDPO, for example, compounds represented by formula (III), formula (IV) or formula (V) can be mentioned.

  In the formula (I), the substituted phenyl group is a group in which an arbitrary substituent is bonded to an arbitrary carbon on the phenyl group, and the type, position or number of the substituent is not particularly limited. It is preferably a substituent that does not lose the lipid solubility of the spin adduct, and is a linear or branched alkyl group having 1 to 6 carbon atoms, or an alkoxyl having a linear or branched chain having 1 to 6 carbon atoms. A group or a halogen group is preferred.

R ¹ and R ² are preferably the same substituents in order to avoid giving a complex and difficult-to-separate ESR spectrum due to the coexistence of diastereomers. It is more preferable that both R ¹ and R ² are phenyl groups so that they can be efficiently extracted into an organic solvent.

  The spin adduct is not particularly limited as long as it is a spin adduct of a spin trap agent represented by the general formula (I). Preferably, a spin adduct generated from one or more free radicals selected from the group consisting of a superoxide anion radical, a hydroxyl radical, and a lipid-derived radical is included.

  In the present invention, the test solution in the form of an aqueous solution is not particularly limited as long as water is the main solvent, and the pH, temperature, type of solute, and the like are not particularly limited. For example, drinking water, fresh water, seawater, hot spring water, buffer solution, physiological saline, liquid culture medium, plasma, serum, or one or more simple substances, compounds, nucleic acids, soluble proteins or coenzymes dissolved therein Things.

  The water-dispersed test solution is a solution in which insoluble components are dispersed in the aqueous test solution, and the type of insoluble components is not particularly limited. Examples of the insoluble component include simple substances, compounds, polymers, colloidal particles, insoluble proteins, or cells such as animal cells, plant cells, and microbial cells.

  The water-dispersed liquid test solution may be a tissue or cell homogenate, whole blood, or a solution in which a solute is dissolved or an insoluble component is dispersed. These can be further diluted with an aqueous solution or water such as a buffer solution, physiological saline, or a liquid medium.

  Examples of the water-insoluble organic solvent include hydrocarbons such as hexane and heptane, ethers such as diethyl ether and ethyl methyl ether, ketones such as ethyl methyl ketone and diethyl ketone, 1-octanol, 1-hexadecanol and the like. Alcohols, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and bromoform can be used, but are not particularly limited thereto.

  The extraction solvent according to the present invention is not particularly limited as long as it contains at least one water-insoluble organic solvent, and may consist of only one water-insoluble organic solvent, A seed or a mixture of two or more water-insoluble or water-soluble organic solvents may be used. When a mixed solvent is used as the extraction solvent, it does not mean that the mixing ratio matches the mixing ratio of the solvent contained in the organic layer after extraction, and only a part of the solvent in the extraction solvent. May be separated from the aqueous phase to provide an organic layer.

  As a solvent for extraction, a chloroform / methanol mixed solvent, ethyl acetate, a chloroform / ethanol mixed solvent, or a chloroform / isopropyl alcohol mixed solvent is preferably used, and a chloroform / methanol mixed solvent that can store a spin adduct most stably is particularly preferable. Used for.

  The inert gas is not limited to a specific chemical substance as long as it is a chemically stable gas that hardly causes a chemical reaction with the spin trap agent, spin adduct, or solvent according to the present invention. Rare gas or nitrogen gas is preferably used.

  In the present invention, the biological sample refers to a sample collected from a living individual. For example, bodily fluids such as blood, lymph, ascites, sweat, urine, tissue homogenate, or an aqueous solution such as buffer solution, physiological saline, liquid medium, or the like diluted with water.

  Next, a method for preparing a sample solution for ESR measurement according to the present invention will be described, but various modifications can be made within a range that can be understood by those skilled in the art, such as concentration, volume, time, temperature, instrument or apparatus. The conditions are not particularly limited.

  The spin trap agent according to the present invention is added and dissolved in a test solution in the form of an aqueous solution or water suspension. The spin trap agent itself may be added directly to the test solution, or a stock solution prepared in advance may be added, but the stock solution should be used so that free radical scavenging starts immediately after the addition. Is preferred. Although the solvent of a stock solution is not specifically limited, For example, an aqueous solution, a buffer solution, a physiological saline, or a liquid medium is used suitably.

  When the test solution contains free radicals or generates free radicals after the spin trap agent is added, the free radicals are trapped by the spin trap agent to generate a spin adduct.

  After trapping free radicals, extraction solvent is added and extraction is performed. The amount of the extraction solvent is not particularly limited, but is preferably equal to or larger than the test solution in order to perform efficient extraction. Moreover, in order to avoid that a measurement precision falls by dilution or to make it easy to dry, it is preferable that it is 4 times or less volume of a test liquid. Although the extraction solvent may be simply added, it is preferable to perform extraction by sufficiently bringing the aqueous layer and the organic layer into contact by pipetting, inversion mixing, or the like.

  Then, it is made to phase-separate by leaving still or centrifuging. The separated organic layer is transferred to another container to obtain a sample solution for ESR measurement. The material of the container is not particularly limited. For example, a synthetic resin such as polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, or glass can be used. A difficult chemical-resistant synthetic resin or glass is preferable, and glass that is not easily damaged by cooling is more preferable.

  The sample solution for ESR measurement is subjected to ESR measurement immediately or after refrigerated storage. The dried product obtained by volatilizing and removing the organic solvent from the sample solution for ESR measurement can be redissolved immediately or after being stored in a cold state and used for ESR measurement. The method for removing the organic solvent is not particularly limited. For example, the organic solvent may be concentrated under normal pressure or reduced pressure, and the solvent may be volatilized by blowing a gas. In the case of blowing a gas, an inert gas is preferably used.

  When the spin adduct of a lipid-derived radical is used as a measurement target, it is preferable that the ESR measurement sample solution is not measured immediately, but is measured after cooling and storage. The spin adduct of the superoxide anion radical and the spin adduct of the hydroxyl radical are more unstable than the spin adduct of the lipid-derived radical, and thus gradually disappear even under cooling. Therefore, by cooling and storing after extraction, only the lipid-derived radical spin adduct remains and can be selectively measured.

  When the sample solution for ESR measurement or a dried product thereof is stored in a cold state, it is preferable to use a sealed container in order to avoid the occurrence of artifacts or disappearance of the spin adduct due to mixing of free radicals or other substances. More preferably, the inside is filled with an inert gas. The storage temperature is not particularly limited, but is preferably cooled in order to enable more stable storage. By using a mixed solvent of chloroform and methanol mixed at a volume ratio of 2: 1 as a solvent for extraction and storing at about -30 ° C., the sample solution for ESR measurement is stable for 2 weeks or more and the dried product is stable for 1 month or more. Can be saved.

  Examples will be described below. In all the examples, the reagent used was a grade higher than the special grade. The concentration represents the final concentration unless otherwise specified. As the spin trap agent, a 500 mM to 1 M aqueous solution was prepared in advance and used as a stock solution. The present invention is not limited to these examples.

(Measurement of free radicals released from phagocytic cells in human blood)
Heparin (manufactured by Mochida Pharmaceutical Co., Ltd .; Novo-Heparin 5000 units for injection) Human blood was collected in a syringe containing about 1 mL. Lipopolysaccharide (manufactured by Wako Pure Chemical Industries, Ltd .; Lipopolysaccharide, from E-coli O55; hereinafter abbreviated as LPS) 0.01 mg / mL, linoleic acid (manufactured by Nacalai Tesque) 20 μL, DPPMDPO (stock) (Trade name diphenyl-PMPO) 5 mM was added. This mixture was incubated at 37 ° C. for 2 hours. Superoxide anion radicals and hydroxyl radicals released from blood phagocytic cells by lipopolysaccharide stimulation react with linoleic acid to produce lipid-derived radicals. The lipid-derived radicals, superoxide anion radicals and hydroxyl radicals are captured by DPPMDPO to produce spin adducts.

After incubation, ferroxamine (manufactured by Sigma Chemicals; hereinafter abbreviated as DFX), which is an iron chelator, is metal-catalyzed to produce superoxide anion radicals and hydroxyl from hydrogen peroxide by adding 0.4 mM. The production of radicals was stopped. 2 mL of a mixed solvent in which chloroform and methanol were mixed at a volume ratio of 2: 1 was added, pipetted, and then centrifuged at 1500 × g for 10 minutes. The organic layer was separated, and after 30 minutes, it was placed in a 160 μL cell and subjected to ESR measurement. For the measurement, an ESR apparatus JES-TE200 (manufactured by JEOL Ltd.) was used, and the following measurement conditions were adopted (the same conditions were adopted in all measurements below).
Microwave frequency: 9.431 GHz
Central magnetic field: 335.3 ± 5mT
Microwave output: 40mW
Magnetic field modulation width: 0.25mT
Amplification ratio: 320
Response time: 0.3 seconds Sweep time: 2 minutes

  As a result, only the ESR spectrum of the lipid-derived radical adduct was observed. The ESR signal was integrated to determine the signal intensity. Using other commonly used spin trapping agents POBN [α- (4-pyridyl-1-oxide) -N-tert-butylnitrone] (Alexis) or DMPO (Dojindo Laboratories) Was measured. The relative intensity of the obtained ESR signal is shown in FIG.

  When DPPMDPO is used, the signal intensity is about 5 times as high as that when POBN or DMPO is used. Since the reaction rate constant of free radical trapping of DPPMDPO is large and free radicals are easily trapped, and since the extraction efficiency of spin adduct is high and the organic layer is easily extracted, a high concentration spin adduct solution can be obtained.

  In this example, Trolox (manufactured by Calbiochem Novabiochem Novagen), ascorbic acid (manufactured by Wako Pure Chemical Industries, Ltd.), Superoxide dismutase (manufactured by Sigma Chemicals), which is a substance or enzyme having radical scavenging ability; Cu, Zn-SOD When further added to the form bovine erythrocyte (hereinafter abbreviated as SOD) or glutathione (Wako Pure Chemical Industries, Ltd.), the signal intensity was significantly attenuated in any case.

  Further, ESR measurement was performed by the same procedure with the final concentration of DPPMDPO set to 10 mM or 20 mM. Although the signal intensity increased depending on the concentration as the DPPMDPO concentration increased, a sufficiently analyzable ESR spectrum was obtained at any concentration, and no cytotoxicity was observed. When DMPO is used, the signal intensity increases in a concentration-dependent manner up to a concentration of 100 mM, but obvious cytotoxicity is observed at 20 mM or more. In order to obtain an ESR spectrum equivalent to 5 mM DPPMDPO, the concentration of DMPO needs to be about 25 mM, and the influence of cytotoxicity cannot be avoided (data not shown).

(Measurement of t-Butyl radical)
Human blood was collected in a syringe containing approximately 1 mL of heparin. To 1 mL of the blood, 10 mM t-Butyl hydroxide (manufactured by Sigma Chemicals) and 5 mM DPPMDPO were added and incubated at room temperature for 30 minutes. t-Butyl hydroxide reacts with blood hemoglobin to produce a t-Butyl radical, which is captured by DPPMDPO to produce a spin adduct.

  After the incubation, 2 mL of a mixed solvent in which chloroform and methanol were mixed at a volume ratio of 2: 1 was added, pipetted, and centrifuged at 1500 × g for 10 minutes. The organic layer was separated, and after 30 minutes, it was placed in a 160 μL cell and subjected to ESR measurement. Similar to Example 1, when DPPMDPO was used, signal intensity significantly higher than that of POBN or DMPO was obtained.

  Moreover, the signal intensity was significantly attenuated by adding Trolox, ascorbic acid or glutathione. On the other hand, even when SOD was added, no significant signal intensity attenuation was observed. This is understood because SOD can erase the superoxide anion radical but cannot remove the t-Butyl radical generated in this example.

(Measurement of free radicals produced by Xanthine oxidase)
In 1 mL of phosphate buffer (pH 7.4), Xanthine oxidase (EC 1.17.3.2; manufactured by Roche Diagnostics; hereinafter abbreviated as XO) 0.1 U / mL, hypoxanthine (manufactured by Sigma Chemicals) Hereinafter referred to as HPX) 0.1 mg / mL, 20 μL of linoleic acid, Fe ³⁺ 100 μM, and 10 mM DPPMDPO were added and incubated at 37 ° C. for 30 minutes. XO and HPX produce superoxide anion radicals, some of which react with linoleic acid to produce lipid-derived radicals. Superoxide anion radicals and lipid-derived radicals are trapped in DPPMDPO and produce spin adducts.

  After incubation, 2 mL of a mixed solvent in which chloroform and methanol were mixed at a volume ratio of 2: 1 was added, pipetted, and centrifuged at 1500 × g for 10 minutes. The organic layer was separated, and after 30 minutes, it was placed in a 160 μL cell and subjected to ESR measurement.

  As a result, only the ESR spectrum of the lipid-derived radical adduct was observed. The ESR signal was integrated to determine the ESR signal intensity. The result is shown in FIG. The signal intensity obtained by integrating the ESR signal was significantly higher than that of the control to which HPX was not added. Further, when Cu, Zn-SOD (EC 1.15.1.1; manufactured by Sigma Chemicals) 500 U / mL was further added to SOD and HPX, a significantly low signal intensity was shown. The disappearance of the superoxide anion radical showed that the amount of spin adduct produced decreased and the amount of spin adduct extracted into the organic solvent also decreased.

(Evaluation of storage stability of spin adduct in sample solution for ESR measurement)
The sample solution for ESR measurement obtained in Example 2 was cooled and stored at −30 ° C. for 2 weeks in a sealed glass container. ESR measurement of the solution was performed, and the signal intensity obtained by integrating the signal was compared with the signal intensity immediately after preparation of the solution. The results of a similar comparison using POBN or DMPO are shown in FIG. FIG. 3 is a graph showing the stability of the spin adduct in the spin adduct solution of the present invention. The signal intensity after 2 weeks is shown as a relative value to the signal intensity immediately after preparation of the solution.

  The spin adduct was stably present in the solution of the spin adduct according to the present invention, and 98.33 ± 2.52% remained even after 2 weeks. Similarly, the residual rate of the spin adduct of POBN prepared was 81.67 ± 3.21%, and the residual rate of the spin adduct of DMPO was 73.33 ± 8.08%. According to the present invention, the spin adduct can be stably stored as compared with POBN or DMPO which are widely used. Moreover, if it is within at least 2 weeks after preparation, the solution just after the preparation and the storage solution can be almost quantitatively compared.

  In addition, the type and volume of the organic solvent to be added were also examined. Extraction is possible with other organic solvents such as ethyl acetate, chloroform / ethanol mixed solvent, chloroform / isopropyl alcohol mixed solvent, etc., but mixed solvent in which chloroform and methanol are mixed at a volume ratio of 2: 1 has the highest extraction efficiency and storage stability. showed that. Extraction is possible even if 3 mL or 4 mL of chloroform / methanol mixed solvent is used for 1 mL of blood, and the amount of spin adduct that is extracted increases as the volume increases. I was not able to admit.

(Evaluation of storage stability of spin adduct in ESR measurement sample solution in dried product)
250 μL of the spin adduct solution obtained in Example 2 was put in a glass container, and the organic solvent was evaporated by blowing nitrogen gas to obtain a dried solid product. The dried product was cooled and stored at −30 ° C., then redissolved in 250 μL of a mixed solvent in which chloroform and methanol were mixed at a volume ratio of 2: 1, and ESR measurement was performed immediately after dissolution. In the dried product of this example, the spin adduct was present more stably than in the solution of Example 2, and no significant disappearance was observed for at least one month.

(ESR measurement using blood of LPS-induced acute lung injury mice)
Ten-week-old male C57BL / 6N mice (weight 19-26 g; manufactured by Charles River Laboratories) were anesthetized with chloral hydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5 μg / kg, and LPS 2.5 mg / LPS-induced acute lung injury mice were created by administering kg intrabronchially to induce lung injury. Further, a treatment experimental mouse was prepared by intraperitoneal administration of 0.1 mL of 30 mg / mL DFX. In the control group, a corresponding amount of physiological saline was administered intrabronchially and intraperitoneally.

  Twenty-four hours after administration of LPS, anesthetized with chloral hydrate 0.5 μg / kg, 200 μL of blood was collected from the inferior vena cava. This blood was diluted to 1 mL with physiological saline, and subjected to ESR measurement by spin trapping and organic solvent extraction in the same manner as in Example 1. In acute lung injury mice, LPS increased free radicals and associated spin adduct production, thereby observing significantly higher ESR signal intensity than in control mice. In the experimental experimental mice administered with DFX, an iron chelator, the signal intensity was reduced by about 25% as a result of the suppression of free radical production.

  It was shown that the present invention is also effective for blood collected from a living body. Moreover, it was shown that this invention is effective in the diagnosis of the disease relevant to oxidative stress.

Claims (5)

(A) a step of dissolving a spin trap agent represented by the following general formula (I) in an aqueous solution or a water-dispersed test solution;
(B) adding an extraction solvent containing at least one water-insoluble organic solvent to the solution obtained in step (A);
(C) The step of separating the organic layer obtained in step (B) from the aqueous layer to obtain a sample solution for electron spin resonance measurement, and (D) The sample for electron spin resonance measurement obtained in step (C). A dried product of a sample solution for electron spin resonance measurement obtained by a method comprising a step of evaporating and removing a solvent from a solution to obtain a dried product,
A long-term storage- dried product of an electron spin resonance measurement sample solution, wherein both R ¹ and R ² in formula (I) are phenyl groups.

The test solution is a biological sample, the long-term storage dry-solid substance of the sample solution for electron spin resonance measurement according to claim 1. The extraction solvent is a chloroform / methanol mixed solvent, the long-term storage dry-solid substance of the sample solution for electron spin resonance measurement according to any one of claims 1 and 2. 4. The long-term dry product of the sample solution for electron spin resonance measurement according to any one of claims 1 to 3, which is further stored by cooling. (E) A step of redissolving the long-term storage dry matter according to any one of claims 1 to 4 to obtain a solution, and (F) an amount of spin adduct contained in the solution obtained in step (E). A method for measuring the amount of free radicals in a test solution by a method comprising a step of measuring the above with an electron spin resonance apparatus.

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