WO2009110585A1 - Solution échantillon pour la mesure de la résonance paramagnétique des électrons, solide séché de cette solution et procédé de mesure utilisant ces solution et solide - Google Patents

Solution échantillon pour la mesure de la résonance paramagnétique des électrons, solide séché de cette solution et procédé de mesure utilisant ces solution et solide Download PDF

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WO2009110585A1
WO2009110585A1 PCT/JP2009/054252 JP2009054252W WO2009110585A1 WO 2009110585 A1 WO2009110585 A1 WO 2009110585A1 JP 2009054252 W JP2009054252 W JP 2009054252W WO 2009110585 A1 WO2009110585 A1 WO 2009110585A1
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solution
sample solution
electron spin
spin resonance
resonance measurement
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PCT/JP2009/054252
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Japanese (ja)
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圭創 佐藤
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国立大学法人熊本大学
株式会社同仁化学研究所
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Priority to JP2010501972A priority Critical patent/JP5253494B2/ja
Publication of WO2009110585A1 publication Critical patent/WO2009110585A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/10—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/60—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using electron paramagnetic resonance

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  • 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.
  • a spin adduct relatively stable radical adduct
  • 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.
  • DPPMDPO 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 (see, for example, Non-Patent Document 1).
  • 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, but extraction with an organic solvent requires 10 hours after extraction. It is disclosed that a lifetime exceeding the above is observed (for example, see Non-Patent Document 2).
  • JP 2006-335738 A Masahiro Nishizawa and 4 others, Bull. Chem. Soc. Jpn. 2007, Vol. 80, No. 3, p. 495-497 Steven Yue Qian and 4 others, Free Radical Biology & Medicine, 2000, Vol. 29, No. 6, p. 568-579
  • the spin adduct is not necessarily stable.
  • 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 a radical in a sample having a complicated composition such as a biological sample and stably storing a spin adduct for a long time.
  • 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 re-dissolved 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.
  • 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.
  • 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.
  • 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.
  • the third invention is a sample solution for ESR measurement according to the first or second invention, and the test solution is a biological sample.
  • the 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.
  • the 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 cooled and stored.
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the spin trapping agent Since the spin trapping agent has lower cytotoxicity than the known spin trapping agent, even if the test solution contains cells, it can be measured while suppressing 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.
  • free radicals in the biological sample can be measured with high reproducibility. Furthermore, the oxidative stress of an individual can be measured by measuring the free radical.
  • 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 chloroform / methanol mixed solvent can stably store the dissolved spin adduct for a longer time.
  • Spin adducts generated at remote locations can be transported more stably and measured by ESR.
  • the concentration of the spin adduct can be maintained by suppressing the volatilization of the organic solvent, so that high-precision 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.
  • 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.
  • the spin adduct can be stored stably for a longer period of time, so that the spin adduct generated at a remote location can be transported more stably. It can be measured by ESR.
  • FIG. 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.
  • 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).
  • DPPMDPO for example, compounds represented by formula (III), formula (IV) or formula (V) can be mentioned.
  • 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 the spin trap agent represented by the general formula (I).
  • 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.
  • the test solution in the form of an aqueous solution is not particularly limited in terms of pH, temperature, type of solute, etc., as long as water is the main solvent.
  • water 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.
  • the insoluble component include simple substances, compounds, polymers, colloidal particles, insoluble proteins, or cells such as animal cells, plant cells, and microbial cells.
  • the aqueous dispersion liquid test solution may be 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.
  • water-insoluble organic solvents examples 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.
  • 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.
  • 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.
  • 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.
  • a biological sample refers to a sample collected from a living individual.
  • 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.
  • the spin trap agent according to the present invention is added to the test solution in the form of an aqueous solution or suspension and dissolved.
  • 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.
  • 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.
  • the free radicals are trapped by the spin trap agent to generate a spin adduct.
  • 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.
  • 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.
  • phase separation is performed by standing 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.
  • 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.
  • 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.
  • the spin adduct of lipid-derived radicals When the spin adduct of lipid-derived radicals is to be measured, it is preferable not to measure the sample solution for ESR measurement immediately but to store it after refrigerated 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.
  • 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.
  • a reagent of a grade higher than special grade was used.
  • the concentration represents the final concentration unless otherwise specified.
  • 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.
  • Heparin Measurement of free radicals released from phagocytic cells in human blood
  • Heparin Measurement of free radicals released from phagocytic cells in human blood
  • 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
  • LPS Lipopolysaccharide 0.01 mg / mL
  • linoleic acid manufactured by Nacalai Tesque
  • DPPMDPO stock
  • DPPMDPO stock
  • DPPMDPO stock
  • diphenyl-PMPO trade name
  • the lipid-derived radicals, superoxide anion radicals and hydroxyl radicals are captured by DPPMDPO to produce spin adducts.
  • ferroxamine manufactured by Sigma Chemicals; hereinafter abbreviated as DFX
  • DFX iron chelator
  • DFX iron chelator
  • the signal intensity is about 5 times that of 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.
  • Trolox Calbiochem Novabiochem Novagen
  • ascorbic acid Wako Pure Chemical Industries, Ltd.
  • Superoxide dismutase Sigma Chemicals, Inc.
  • Cu Zn-D
  • SOD bovine erythrocyte
  • glutathione manufactured by Wako Pure Chemical Industries, Ltd.
  • 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.
  • 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.
  • the concentration of DMPO needs to be about 25 mM, and the influence of cytotoxicity cannot be avoided (data not shown).
  • 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 ⁇ L of linoleic acid, Fe 3+ 100 ⁇ M, and 10 mM DPPMDPO were added and incubated at 37 ° C. for 30 minutes.
  • XO Xanthine oxidase
  • HPX hypoxanthine
  • 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.
  • 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.
  • the residual rate of the spin adduct of POBN prepared was 81.67 ⁇ 3.21%
  • the residual rate of the spin adduct of DMPO was 73.33 ⁇ 8.08%.
  • spin adducts 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.
  • ESR measurement using blood of LPS-induced acute lung injury mice 10-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 ⁇ g / 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.
  • 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.

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Abstract

L'invention concerne une solution échantillon destinée à la mesure de la résonance paramagnétique des électrons. La solution échantillon peut stocker de manière stable un produit d'addition de spin en ESR pendant une longue période de temps. Un agent de piégeage de spin représenté par la formule (A) est dissous dans une solution de test sous la forme d'une solution aqueuse ou d'une dispersion dans l'eau. Un solvant d'extraction contenant au moins un solvant organique insoluble dans l'eau est ajouté à la solution. La couche organique et la couche aqueuse sont séparées et ensuite une mesure de ESR est effectuée. Conformément à la constitution ci-dessus, le produit d'addition de spin peut être stocké de manière stable pendant une longue période de temps. Par conséquent, une solution échantillon, qui a été préparée à un emplacement à distance, peut être transportée, et la quantité de radicaux libres dans la solution de test peut être déterminée. Les radicaux libres dans les solutions de test ayant une composition compliquée comprenant des échantillons biologiques peuvent être détectés, et l'application de la technique à la mesure du stress oxydatif peut également être réalisée.
PCT/JP2009/054252 2008-03-07 2009-03-06 Solution échantillon pour la mesure de la résonance paramagnétique des électrons, solide séché de cette solution et procédé de mesure utilisant ces solution et solide WO2009110585A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06340646A (ja) * 1991-07-30 1994-12-13 Kei Onishi プロスタグランジン誘導体
JP2002179563A (ja) * 1989-10-17 2002-06-26 Oklahoma Medical Res Found 酸化的損傷に伴う疾患を阻止する組成物
JP2008050295A (ja) * 2006-08-24 2008-03-06 Univ Fukuoka ジフェニルホスフィニル基をもつ新規直鎖型スピントラップ剤

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002179563A (ja) * 1989-10-17 2002-06-26 Oklahoma Medical Res Found 酸化的損傷に伴う疾患を阻止する組成物
JPH06340646A (ja) * 1991-07-30 1994-12-13 Kei Onishi プロスタグランジン誘導体
JP2008050295A (ja) * 2006-08-24 2008-03-06 Univ Fukuoka ジフェニルホスフィニル基をもつ新規直鎖型スピントラップ剤

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MASAHIRO NISHIZAWA ET AL.: "Spin-Trapping Properties of 5-(Diphenylphosphinoyl)-5- methyl-4,5-dihydro-3H-pyrrole N-Oxide (DPPMDPO)", BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, vol. 80, no. 3, 2007, pages 495 - 497 *
STEVEN YUE QIANA ET AL.: "EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations", FREE RADICAL BIOLOGY AND MEDICINE, vol. 29, no. 6, 2000, pages 568 - 579 *
TOMOKO SHIMAMURA ET AL.: "Electron Spin Resonance Analysis of Superoxide Anion Radical Scavenging Activity with Spin Trapping Agent, Diphenyl-PMPO", ANALYTICAL SCIENCES, vol. 23, no. 10, 2007, pages 1233 - 1235 *

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