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WO1994002865A1 - Detection de l'oxyde nitrique par resonance de spin electronique - Google Patents

Detection de l'oxyde nitrique par resonance de spin electronique Download PDF

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Publication number
WO1994002865A1
WO1994002865A1 PCT/US1993/006868 US9306868W WO9402865A1 WO 1994002865 A1 WO1994002865 A1 WO 1994002865A1 US 9306868 W US9306868 W US 9306868W WO 9402865 A1 WO9402865 A1 WO 9402865A1
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Prior art keywords
spin
epr
nitric oxide
concentration
fusinite
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PCT/US1993/006868
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English (en)
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David A. Wink, Jr.
Robert B. Clarkson
Marc F. Desrosiers
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The United States Of America, Represented By The Secretary, Department Of Health And Human Services
The Board Of Trustees Of The University Of Illinois
THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF COMMERCE
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Priority to AU46867/93A priority Critical patent/AU4686793A/en
Publication of WO1994002865A1 publication Critical patent/WO1994002865A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/20—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations containing free radicals, e.g. trityl radical for overhauser
    • 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
    • G01R33/28—Details of apparatus provided for in groups G01R33/44 - G01R33/64
    • G01R33/281—Means for the use of in vitro contrast agents
    • 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

Definitions

  • This invention relates to a nondestructive method of detecting nitric oxide.
  • this invention enables the measurement of nitric oxide specifically and quantitatively in aqueous and nonaqueous solutions of biological media, both in vitro and in vivo, and chemical media.
  • Nitric oxide is a key bioregulatory molecule that plays critical roles in the regulation of various biological processes, including the normal physiological control of blood pressure, macrophage-induced cytostasis and cytotoxicity, inhibition of platelet aggregation, and neurotransmission (Moncada et al.. Pharmacological Reviews 43(2): 109-142. 1991.). Many tissues in the body endogenously release NO in different amounts (Marietta, Chem. Res. in Toxicology 1(5): 249-257. 1988.; Biochemistry 27: 8706-8711. 1988.) but the actual amounts released are very difficult to quantify.
  • the ability to nondestructively measure NO concentrations specifically and quantitatively in aqueous and nonaqueous solutions of biological media, both in vitro and in vivo, and chemical media would be highly advantageous.
  • the ability to measure the concentration of NO by a nondestructive method, e.g., in a manner which does not consume NO is an important requirement for further investigation of the mode of action of NO as a key bioregulatory molecule and for the development of therapeutic applications of NO-releasing compounds.
  • Several techniques have been employed to determine the concentration of NO in aqueous solution.
  • One method employs an automated system that analyzes nitrate by reduction with a high-pressure cadmium column to determine amounts of nitrate and/or nitrite in urine, saliva, deproteinized plasma, gastric juice, and milk samples (Green et al.. Analytical Biochemistry 126: 131- 138. 1982.).
  • the lower limit of detection of the method is said to be 1.0 n ol N0 3 ⁇ or N0 2 ⁇ /ml.
  • the system reportedly allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in biological fluids. Most samples may be prepared by simple dilution with distilled water, and 30 samples reportedly may be analyzed in an hour.
  • the disadvantage of such a technique in measuring NO is that it does so indirectly, by measuring NO byproducts, which also can be generated from other sources. Accordingly, such a method is not very accurate in determining NO concentration.
  • Dithionite is used to treat the samples of human plasma to convert nitrite to nitric oxide, with the treated samples being passed over bovine hemoglobin columns. NO is allowed to bind the hemoglobin in columns of bovine hemoglobin covalently bound to agarose. An excess of dithionite is used to ensure that the hemoglobin is reduced to a ferrous, nonoxygenated state. The NO bound to the hemoglobin forms a complex on the column, and the column is then subjected to electron paramagnetic resonance spectroscopy, i.e., the column is subjected to a magnetic field and microwave radiation to obtain a characteristic electron paramagnetic resonance spectrum. This method suffers from the same disadvantages as the previously described method. NO concentration is determined indirectly, through the measurement of nitrite. Also, the NO is modified by binding to hemoglobin covalently bound to agarose.
  • NO has been quantified by the chemiluminescence resulting from the product of NO and ozone (Palmer et al., Nature 327: 524-526. 1987.; Maragos et al., J. Med. Chem. 34: 3242-3247. 1991.). This method also involves modification of NO, in this case by reaction with ozone.
  • a modified oxygen electrode has be used to detect NO (Shibuki et al., Neuroscience Res. 9 69-76. 1990.; Nature 349: 326-328. 1991.).
  • the electrochemical microprobe was developed to detect the release of NO in brain tissue.
  • the output current of probe was found to correlate linearly with the concentration of NO at the tip.
  • the sensitivity of th probe was reportedly between 3.5 and 106 pA/ ⁇ M change NO concentration.
  • the validity of this technique has been questioned due to the small current that has been observed ( ⁇ 0.5 pA) and the lack of use o standards at submicromolar concentrations of NO.
  • the technique measures NO by its oxidation to nitrites and those who developed the modified oxygen electrode claim that NO is spontaneously released from sodium nitroprusside and that the release is accurately measured by the electrode.
  • a nondestructive method e.g., nonconsuming method
  • the methods described above result in the destruction of NO, necessitate the modification of NO, e.g., its consumption through, for example, modification of NO, in order to measure NO concentration, are invasive procedures, require extrapolation to an earlier event in order to determine the concentration of NO.
  • the present invention not only provides a method that enables the detection and measurement of NO concentration specifically, quantitatively, and repro ⁇ ucibly in a nondestructive manner, but it enables the ability to nondestructively assay NO concentration over time. The other methods do not allow such a real time assay to both readily and reliably determine NO concentration.
  • the present inventive method employs electron spin resonance.
  • Electron spin resonance has been used to measure oxygen concentration.
  • fusinite a naturally occurring polymeric solid component of many coals, has been shown to give an ESR signal upon L-band irradiation. Rich in unpaired electrons, fusinite has been shown to generate a single electron paramagnetic resonance (EPR) signal that broadens in the presence of oxygen (Clarkson et al., Fuel 69: 1405-1411. 1990.). The line broadens as a function of oxygen concentration in a reproducible fashion.
  • fusinite When properly isolated from whole coal, purified, and ground to a fine powder (d ⁇ 5 ⁇ m) , fusinite provides a useful probe for EPR measurement of oxygen concentration.
  • the utility of fusinite in the EPR measurement of oxygen concentration in vivo has been demonstrated in cells and animals (Swartz et al.. Magnetic Resonance Medicine 20: 333-339. 1991.).
  • the advantages of fusinite in such a technique are very low toxicity, excellent chemical stability, and sensitivity to concentrations as low as 0.1 ⁇ M. In fact, fusinite can be easily placed inside a cell and can be retained within an organism for as long as a year without loss of signal or development of toxic side effects.
  • fusinite and EPR also could be used to measure NO concentration, particularly in a reliably specific and quantitative manner.
  • oxygen and NO are both paramagnetic gases, the two gases differ in at least one very significant respect.
  • Oxygen is not a radical species
  • NO is a radical species.
  • radicals are known to be short-lived and highly reactive and that fusinite is a coal derivative that has many potential reaction sites, one who is skilled in the art would have predicted that a coal derivative, such as fusinite, and EPR could not be used to measure NO concentration.
  • Such reactions include radical couplings, which would result in the formation of diamagnetic species, thereby resulting in the loss of the EPR signal from the fusinite. The loss of the EPR signal would prevent the determination of the concentration of NO in a sample.
  • fusinite does not chemically react with NO, and, therefore, it is possible to obtain an EPR signal for NO using fusinite and EPR spectroscopy, which enables the measurement of the EPR linewidth, its comparison with EPR linewidths for known signals, and the subsequent determination of NO concentration.
  • the ability to measure successfully NO concentration using EPR and a coal derivative, such as fusinite, as provided by the present invention is an unexpected result, which was neither taught nor suggested in the art.
  • the present inventive method overcomes the deficiencies of the methods currently being used to measure NO concentration by providing a nondestructive, e.g., nonconsuming, method that enables the measurement of NO concentration specifically, quantitatively, and reproducibly, in aqueous and nonaqueous solutions of biological media, both in vitro and in vivo, and chemical media. It also enables real time assay of NO concentration.
  • the present invention provides a nondestructive method, which utilizes a spin-labeled material, particularly a derivative of coal, such as fusinite, and EPR, for the detection of nitric oxide.
  • the present inventive method of detecting nitric oxide comprises contacting a sample of unknown nitric oxide concentration with a spin-labeled material, subjecting the unknown sample and spin-labeled material to EPR spectroscopy under hypoxic conditions to obtain an EPR signal having a linewidth, and comparing the EPR linewidth for the unknown sample and spin-labeled material to the EPR linewidth of an EPR signal for the spin-labeled material in the presence of a sample or samples of known nitric oxide concentration in order to detect the presence, and determine the concentration, of nitric oxide in the unknown sample.
  • the method enables the measurement of NO specifically and quantitatively in aqueous and nonaqueous solutions of biological, both in vitro and in vivo, and chemical media in a reproducible manner.
  • the present invention also enables the real time assay of NO concentration.
  • the present invention further provides a means of monitoring NO production or inhibition by drugs, both in vitro and in vivo, in the design of drugs for the treatment of diseases related to defects in NO regulation and/or production as well as a means of detecting and quantifying defects in NO regulation and/or production, both in vitro and in vivo, which result from disease, injury, and mutation.
  • the present invention additionally provides a means of monitoring pollution of which NO is a component.
  • Figure 1 is a graph of EPR linewidth (milligauss) versus NO concentration ( ⁇ M) that shows the variation in the EPR linewidth for fusinite powder suspended in a 0.1 M phosphate buffered saline at pH 7.4 and 35°C subjected to varying pressures of NO. Measurements were made in an EPR tube at X-band (8.9-9.6 GHz).
  • Figure 2 is a graph of NO concentration ( ⁇ M) versus time (seconds) that shows the variation in NO concentration with time as measured using EPR and fusinite for the NO-releasing compound DEANO in 0.1 M phosphate buffered saline at pH 7.4 and 35°C.
  • Figure 3 is a graph of NO concentration ( ⁇ M) versus time (seconds) that shows the variation in NO concentration with time as measured using EPR and fusinite for the NO-releasing compound SPERNO in 0.1 M phosphate buffered saline at pH 7.4 and 35°C.
  • Figure 4 is a graph of signal amplitude (A.U.) versus magnetic field (Gauss) that shows the immediate narrowing of the EPR linewidth for fusinite upon injection of pure oxygen gas into the reaction cell following the conclusion of the DEANO reaction, i.e., when no further change in the linewidth was observed.
  • Figure 5 is a graph of EPR linewidth (Gauss) versus time (minutes) that shows the initial decrease and subsequent increase in the EPR linewidth for fusinite in the presence of NO over time as measured in Chinese hamster ovary cells in vitro.
  • Figure 6 is a graph of EPR linewidth (milligauss) versus NO concentration ( ⁇ M) that shows the variation in the EPR linewidth for 15 N-perdeutero TEMPONE suspended in 0.1 mM phosphate buffered saline at pH 7.4 and 35°C subjected to varying pressures of NO. Measurements were made in an EPR tube at X-band (8.9-9.6 GHz).
  • the present invention is predicated on the discovery that EPR and a coal derivative, such as fusinite, may be used to detect nondestructively and measure specifically and quantitatively the concentration of NO in aqueous and nonaqueous solutions of biological media, both in vitro and in vivo, and chemical media, in a reproducible manner with a limit of detection ⁇ 0.2 ⁇ M.
  • the present inventive method does not result in the consumption of NO, e.g., through modification of NO, and, therefore, is a technique, thereby rendering the present inventive method quite suitable for in vivo, as well as in vitro. detection and measurement of NO.
  • the present inventive method utilizes EPR spectroscopy, which is a technique that is well-known to those who are skilled in the art. It was found that the EPR linewidth for fusinite is sensitive to the presence of NO and that the variation in the EPR linewidth for fusinite can be used to determine the concentration of NO in a sample under test.
  • the present inventive method of detecting NO specifically comprises contacting a sample of unknown NO concentration with a spin-labeled material, subjecting the unknown sample and spin-labeled material to EPR spectroscopy under hypoxic conditions to obtain an EPR signal having a linewidth, and comparing the EPR linewidth for the unknown sample and spin-labeled material to the EPR linewidth of an EPR signal of the spin-labeled material in the presence of a sample or samples of known NO concentration in order to detect the presence, and determine the concentration, of NO in the unknown sample.
  • the method of the present invention can be used to merely detect the presence of NO in an unknown sample, for example by comparison of the observed EPR linewidth to the EPR linewidth for the spin-labeled material in the absence of NO, or to determine the concentration of NO in the unknown sample, for example by comparison of the EPR linewidth to the EPR linewidths for the spin-labeled material in the presence of different NO concentrations.
  • spin-labeled materials which do not chemically react with NO, may also be used in the context of the present invention.
  • Such other spin-labeled materials are coal derivatives that have characteristic features in common with fusinite, burnt or partially combusted cellulose, irradiated gas-permeable plastics, and spin-labeled molecules, such as TEMPO (2,2,6,6-tetramethylpiperidine-N-oxy1) , DOXYL (4,4-dimethyloxazolidine-N-oxyl) , and PROXYL (2,2,5,5-tetramethylpyrrolidine-N-oxyl) (all available from Sigma, St.
  • TEMPO 2,2,6,6-tetramethylpiperidine-N-oxy1
  • DOXYL 4,4-dimethyloxazolidine-N-oxyl
  • PROXYL 2,2,5,5-tetramethylpyrrolidine-N-oxyl
  • spin-labeled molecules that have characteristic features in common with the spin-labeled molecule(s) of fusinite. Moreover, the spin-labeled molecule(s) of fusinite could be isolated for use in the present inventive method.
  • Fusinite, TEMPO, and derivatives thereof are most preferably utilized as the spin-labeled materials in the context of the present invention.
  • spin-labeled compounds which have been modified by use of isotopes with lower spin numbers e.g., the use of 15 N for 14 N and deuteration
  • 15 N-perdeutero TEMPONE 15 N-perdeutero 4-0x0-2,2,6,6-tetramethylpiperidine-N-oxy1 or 4-OXO-TEMPO
  • This greater sensitivity is a result of the use of 15 N leading to a two line, rather than three line, EPR pattern and deuteration leading to a narrower EPR linewidth in the absence of a paramagnetic species.
  • EPR in combination with fusinite has been demonstrated to have utility in the detection and measurement of oxygen, it is important that the use of EPR in combination with fusinite in the detection and measurement of NO be carried out under hypoxic conditions so that the variation in the EPR linewidth for fusinite accurately reflects the concentration of NO in the sample under test.
  • the chemical reactivity of NO and 0 2 may offer the opportunity to follow the titration of NO by physiological oxygen levels in cells and tissues.
  • the method of the present invention may be used to detect the presence of NO or measure the concentration of NO in a wide variety of samples.
  • the method may be used to test aqueous and nonaqueous solutions of biological and chemical media.
  • the testing of biological media may be carried out in vitro or in vivo.
  • the present inventive method may be used to detect and/or measure the presence of NO in a biological fluid, such as blood, urine, or saliva, cells, tissues, or even an intact organism.
  • a biological fluid such as blood, urine, or saliva, cells, tissues, or even an intact organism.
  • the use of the present inventive method to measure the presence of NO in an intact organism would enable imaging of tumor cells, which are known to produce large quantities of NO.
  • the application of the present inventive method to the testing of intact cells or tissues in vivo or an intact living organism would require utilization of a modified ESR spectrometer, for example, in which the magnets that generate the magnetic field are positioned in an upright manner so as to form a tabletop for positioning of the cells, tissues, or intact organism for testing.
  • a modified ESR spectrometer for example, in which the magnets that generate the magnetic field are positioned in an upright manner so as to form a tabletop for positioning of the cells, tissues, or intact organism for testing.
  • the present inventive method provides for the rapid determination of NO concentration, as well as being nondestructive, e.g., nonconsuming of NO, it is well suited for use in real time procedures, particularly on living organisms such as humans in need of either constant monitoring or emergency medical diagnosis and treatment.
  • the temperature and band of irradiation at which the testing is done may necessarily vary with the sample under test. Generally, however, testing in the temperature range from about 4°C to about 90°C, preferably from about 20°C to about 40°C, and more preferably from about 20°C to about 37°C, and irradiation from about L-band (1-2 GHz) to about X-band (8.9-9.6 GHz) is preferred. Higher frequency irradiation may be used. However, penetration decreases and heat generation increases with an increase in frequency. For example, at Q-band (34-36 GHz), it is believed that an ESR signal still can be obtained but that the sensitivity decreases significantly. Also, the ability to penetrate fusinite also decreases.
  • irradiation at such high frequencies requires the use of finer particles of fusinite or the like and also necessitates cooling of the material under test, since irradiation at such high frequencies generates much heat.
  • L-band is preferred for intact organisms in order to avoid tissue breakdown and cell death.
  • the variation in or the broadening of the EPR linewidth for fusinite or the like, such as a spin-labeled molecule, such as TEMPO or a spin-labeled molecule isolated from fusinite, in the presence of NO may be measured.
  • the concentration of NO is determined by extrapolation from the standard curve generated during calibration of the system.
  • the present inventive method is also expected to have utility in monitoring pollution of which NO is a component, in monitoring NO production or inhibition by drugs, both in vitro and in vivo, in the design of drugs for the treatment of diseases related to defects in NO regulation and/or production, and in detecting and quantifying defects in NO regulation and/or production, both in vitro and in vivo, which result from disease, injury, and mutation.
  • NO NO regulation
  • septic shock may be distinguished from trauma by high and low NO concentration, respectively.
  • EXAMPLE 1 This example describes the preparation of fusinite. Large, pure fusinite lenses were hand-selected from an Illinois No. 5 coal. The hand-selected lenses were washed in hot, dilute hydrochloric acid and rinsed in triply distilled water. The washing and rinsing of the lenses was repeated twice more. The resulting powder, upon L-band irradiation, gave a single EPR resonance signal, free from inorganic paramagnetic ion contamination.
  • EXAMPLE 2 This example describes the calibration of the EPR linewidth for the fusinite powder as a function of NO concentration.
  • the EPR linewidth for the fusinite powder was calibrated as a function of NO concentration utilizing a • dynamic vacuum system that allowed a small quantity of fusinite to be placed in an X-band (8.9-9.6 GHz) EPR cavity.
  • Figure 1 shows the variation in EPR linewidth for a fusinite powder subjected to varying concentrations of NO.
  • the nonlinear behavior of ⁇ B__ as a function of NO is similar to that observed for oxygen (Swartz et al., Magnetic Resonance Medicine 20: 333-339. 1991.).
  • Other paramagnetic species in solution such as nitroxide radicals and Fe +3 ions, have been reported to have no effect on the linewidth of fusinite, presumably due to size exclusion by the fusinite pore system and the rather hydrophobic character of the interior surfaces.
  • Samples of two NO-releasing compounds, specifically the secondary amine ⁇ Et 2 N-N(N 0)-0 ⁇ Na, referred to as DEANO, and the polyamine
  • fusinite powder (d ⁇ 10 ⁇ m) was suspended in 10 ml of phosphate buffered saline at pH 7.4 in a glass cell fitted with a septum cap.
  • the fusinite suspension was purged with nitrogen gas to remove dissolved oxygen until no further reduction in the EPR linewidth for the fusinite was observed (about 30 minutes) .
  • the cell containing the purged fusinite suspension was then placed on a loop-gas resonator surface coil connected to an EPR spectrometer operating at L-band (1-2 GHz) as described previously (Nilges et al., Phys. Med. 2: 195-201. 1989.; Bacic et al., Magnetic Resonance Medicine 10: 266-272. 1989.).
  • Figure 2 shows the measurement of the variation in NO concentration with time for DEANO.
  • the excellent agreement between the data obtained with fusinite and EPR and the data obtained by optical absorbance suggests that the fusinite/EPR method accurately follows the rate of production of NO. It appears that during the initial 100 seconds following injection of DEANO into the reaction cell, scavenging of residual adsorbed oxygen by the first NO molecules produced in the reaction may be taking place.
  • fusinite powder (d ⁇ 10 ⁇ m) was suspended in 10 ml of phosphate buffered saline at pH 7.4 in a glass cell fitted with a septum cap. Then, 0.2 ml of DEANO in deoxygenated, phosphate buffered saline at pH 11 was injected into the cell, and, while the system was maintained at 20°C, the EPR linewidth was monitored as a function of time. The EPR linewidth decreased due to the reaction of N0 « with the dissolved oxygen. The reaction is believed to be 4NO* + 2H 2 0 + 0 2 ⁇ 4HN0 2 (Schwartz et al. In: Trace Atmospheric Constituents. J.
  • Nitrites e.g., N0 2 ⁇ dissociated from HN0 2 in aqueous media, are nonparamagnetic species.
  • the rate-determining step is, therefore, the production of NO* from the decomposition of DEANO.
  • the rate constant for this step is believed to be 2 x 10 ⁇ 3 /sec.
  • CHO cells Chinese hamster ovary (CHO) cells were allowed to endocytose fusinite in culture. The cells were then placed in phosphate buffered saline at pH 7.4 in a glass cell fitted with a septum cap. An initial decrease in the EPR linewidth for fusinite was observed before the introduction of DEANO solution. The decrease in linewidth is believed to reflect cellular respiration, i.e., the consumption of oxygen by the cells. The cells were allowed to respire until the linewidth was about 0.8 G, which indicates that almost all of the oxygen in the system had been consumed.
  • EXAMPLE 5 This example describes the use of fusinite and EPR to measure NO in an intact organism. Fine particles of fusinite may be suspended in phosphate buffered saline at pH 7.4 and injected into a live mouse. The injection may be localized or systemic. If systemic, the fusinite should be allowed to circulate throughout the body. A surface coil, operating at L-band, then may be placed in contact with a localized region of the body or the entire body. In connection with an EPR spectrometer calibrated to measure the variation in the EPR linewidth for fusinite in the presence of NO, the location of NO production in the mouse may be determined by detecting variation in the EPR linewidth for the fusinite present in the body of the mouse. Such a technique also may be used to quantify the NO produced and to image tumors, which are known to produce NO and which may be present in the body.
  • EXAMPLE 6 This example illustrates the effect on EPR linewidth for 15 N-perdeutero TEMPONE as a function of NO concentration.
  • EXAMPLE 7 This example describes an appropriate procedure for the use of TEMPO and EPR in the measurement of NO concentration in solution as a function of time.
  • An EPR system can be initially calibrated for TEMPO in the presence of NO in a manner similar to that described in Example 2 for fusinite in the presence of NO.
  • a solution containing 1 mM TEMPO in phosphate buffered saline at pH 7.4 would be then placed in a glass cell fitted with a septum cap.
  • the TEMPO solution would then be purged with nitrogen gas to remove dissolved oxygen until no further reduction in the EPR linewidth for the TEMPO was observed.
  • the cell containing the purged TEMPO solution would be then placed on a loop-gas resonator surface coil connected to an EPR spectrometer operating at L-band.
  • a suitable portion, e.g., 0.2 ml, of a sample containing an unknown concentration of NO would be injected into separate TEMPO solutions.
  • EPR spectra of the TEMPO would be collected at set intervals, utilizing a computer-controlled data acquisition system. During the collection of the EPR spectra, the temperature should be monitored with a thermocouple and stabilized at an appropriate temperature, e.g., about room temperature.
  • the EPR linewidth for TEMPO would be expected to broaden in the presence of NO in the same manner as that of fusinite, thereby allowing for measurement of the NO concentration in the sample.

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Abstract

Procédé de détection de l'oxyde nitrique présent dans un échantillon par spectroscopie à résonance paramagnétique électronique, consistant à utiliser une matière à marquage de spin telle que la fusinite pour détecter des changements dans la largeur de raie du signal de résonance paramagnétique électronique de la matière à marquage de spin en présence d'oxyde nitrique, afin d'établir une corrélation entre ces changements et la quantité d'oxyde nitrique dans l'échantillon.
PCT/US1993/006868 1992-07-23 1993-07-21 Detection de l'oxyde nitrique par resonance de spin electronique WO1994002865A1 (fr)

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Cited By (7)

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EP0646808A1 (fr) * 1993-10-01 1995-04-05 Commissariat A L'energie Atomique Procédé de contrôle de la teneur en monoxyde d'azote d'un milieu par résonance paramagnétique électronique en utilisant une phtalocyanine de lithium
WO1997024145A1 (fr) * 1995-12-28 1997-07-10 Daiichi Radioisotope Laboratories, Ltd. Medicaments d'aide au diagnostic
WO2004002536A1 (fr) * 2002-06-28 2004-01-08 Pharmacia Corporation Agents de contraste des plus utiles pour quantifier de l'oxyde nitrique et methodes a cet effet
WO2005081622A1 (fr) * 2004-02-17 2005-09-09 Pharmacia & Upjohn Company Llc Procedes et compositions de detection de l'oxyde nitrique
US7662362B2 (en) 2003-09-05 2010-02-16 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
US8066973B2 (en) 2003-09-05 2011-11-29 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
WO2017011393A1 (fr) * 2015-07-10 2017-01-19 Stc.Unm Spectromètre à résonance magnétique

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MAGNETIC RESONANCE IN MEDICINE. vol. 20, no. 2, 1 August 1991, DULUTH,MN US pages 333 - 339 H.M. SWARTZ ET AL. 'MEASUREMENTS OF PERTINENT CONCENTRATIONS OF OXYGEN IN VIVO' cited in the application *
PHYSICS IN MEDECINE AND BIOLOGY. vol. 34, no. 9, 1 September 1989, LONDON GB pages 1317 - 1323 S. ISHIDA ET AL. 'IN VIVO ANALYSIS OF NITROXIDE RADICALS INJECTED INTO SMALL ANIMALS BY L-BAND ESR TECHNIQUE' *
SCIENCE vol. 227, no. 4686, 1 February 1985, LANCASTER, PA US pages 517 - 518 L.J. BERLINER ET AL. 'MAGNETIC RESONANCE IMAGING OF BIOLOGICAL SPECIMENS BY ELECTRON PARAMAGNETIC RESONANCE OF NITROXIDE SPIN LABELS' *
TETRAHEDRON LETTERS vol. 27, no. 39, 1986, OXFORD GB pages 4795 - 4798 M. GY\R ET AL. 'SPIN TRAPPING REACTIONS WITH NITRIC OXIDES. V. REACTIONS WITH UNSATURATED MACROMOLECULAR CHAINS- A NEW SPIN LABELING METHOD' *

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EP0646808A1 (fr) * 1993-10-01 1995-04-05 Commissariat A L'energie Atomique Procédé de contrôle de la teneur en monoxyde d'azote d'un milieu par résonance paramagnétique électronique en utilisant une phtalocyanine de lithium
WO1997024145A1 (fr) * 1995-12-28 1997-07-10 Daiichi Radioisotope Laboratories, Ltd. Medicaments d'aide au diagnostic
WO2004002536A1 (fr) * 2002-06-28 2004-01-08 Pharmacia Corporation Agents de contraste des plus utiles pour quantifier de l'oxyde nitrique et methodes a cet effet
US7662362B2 (en) 2003-09-05 2010-02-16 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
US8066973B2 (en) 2003-09-05 2011-11-29 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
US8568694B2 (en) 2003-09-05 2013-10-29 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
US8569482B2 (en) 2003-09-05 2013-10-29 The Ohio State University Research Foundation Nanoparticulate probe for in vivo monitoring of tissue oxygenation
WO2005081622A1 (fr) * 2004-02-17 2005-09-09 Pharmacia & Upjohn Company Llc Procedes et compositions de detection de l'oxyde nitrique
WO2017011393A1 (fr) * 2015-07-10 2017-01-19 Stc.Unm Spectromètre à résonance magnétique
US10914800B2 (en) 2015-07-10 2021-02-09 Stc.Unm Magnetic resonance spectrometer

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