WO2008009867A2 - Epr-based detection, identification and quantification of free radicals, sampling device and kit - Google Patents

Abstract

The invention relates to a method for detecting, identifying or quantifying free radicals in a biological fluid, in which a biological fluid is sampled, said biological fluid is immediately mixed with a reactive composition containing a compound for trapping free radicals, a stabilizer and a nonoxidizing chelating agent, and then the mixture thus obtained is analysed using an analytical apparatus that detects a compound comprising a free electron, to a device for taking a sample of a biological fluid so as to immediately introduce it into a mixture with said reactive composition, and to kits for implementing the above method.

Description

 METHOD FOR DETECTION, IDENTIFICATION AND QUANTIFICATION FREE RADICAL PE. SAMPLING DEVICE AND KIT FOR IMPLEMENTING IT

The present invention relates to a novel method for the detection, identification and quantification of free radicals, a sampling device and a kit for its implementation.

Dikalova et al describe in PNAS 98: 13549 et seq. (2001) the detection of CO 2 - radicals in the bile of rats after their sodium formate intoxication. The nitrone alpha (4-pyridyl-1-oxide) -N-t-butyl nitrone (POBN) is administered directly to the animal intraperitoneally at a dose of 1.5 g / kg. The scavenger administered in vivo causes cytotoxicity and interference with free radical processes. In addition, the in vitro control tests did not make it possible to detect the adduct formed between the nitrone and the desired free radical-carrying species. The radical adduct was no longer detected in the presence of horseradish peroxidase or catalase. Kadiiska et al study in The Journal of Clinical Investigation 96, 1653-1657 (1995) the in vivo formation of radical species. They describe in particular the detection of hydroxyl radicals in the bile of rats after iron overload poisoning and directly intraperitoneal administration of alpha phenyl-N-tert-butyl nitrone (PBN) to the animal at a dose of 70 mg / kg mM.

There are currently indirect methods for the detection, identification and quantification of free radicals. For example, high performance liquid chromatography (HPLC) and UV spectrophotometry can be mentioned, two techniques for the determination of oxidized secondary products formed during the degradation of lipids or proteins by free radicals. It should be known that these by-products are not necessarily specific for damage of radical origin, which can be obtained by other means.

Electron paramagnetic resonance (EPR) is the only technique for direct detection of free radicals that, under certain conditions and in a relatively short time (1 to 5 minutes), to obtain a result on the nature and concentration of free radicals present. in one mixing, by obtaining a resonance signal (spectrum). The only possibility of observing radicals centered on oxygen or carbon, highly unstable in an aqueous medium at room temperature, is to use the spin trapping technique of reacting the unstable radical R * with a molecule. trap P initially introduced into the medium. This reaction gives rise to a new radical (PR) * sufficiently stable to lead to an observation by RPE; it is called a trap adduct and belongs to the chemical family of nitroxides. The analysis of the RPE spectrum of Padduit and the knowledge of the chemical structure of P will then go back to the chemical nature of R ^" .

Among the many traps that have been developed for the specific capture of oxygenated free radicals in biological medium, the most successful belong to the chemical family of nitrones. 5-Diethoxyphosphoryl-5-methyl-1-pyrroline / V-oxide (DEPMPO) and 2-ethoxycarbonyl-2-3,4-dihydro-2W-pyrrole-1-oxide (EMPO) have been remarkably effective in superoxide radical entrapment, key intermediary of radical processes in vivo).

However, under the usual conditions encountered in biology, especially in the presence of many reducing components of the cell (ascorbate, thiols) and oxygen, the nitroxide adducts are rapidly reduced to non-radical species (diamagnetic) undetectable by EPR.

It would therefore be desirable to have a method for the direct detection, identification and quantification of free radicals which gives results that are in conformity with reality. However after long research the applicants have developed such a method and a device for its implementation in view of the need to perform very quickly one of the stages of the process.

This is why the present application relates to a method for detecting, identifying or quantifying free radicals in a biological fluid in which:

a biological fluid is taken, said biological fluid is immediately mixed with a reactive composition containing a free radical scavenging compound, a stabilizing agent and a non-oxidizing chelating agent, and then

the mixture thus obtained is analyzed with the aid of an analysis apparatus detecting a compound comprising a free electron, for example using the electron paramagnetic resonance (EPR) technique.

In the present application and in what follows, the term "free radical scavenging compound" designates a compound capable of reacting with a free radical and of fixing it, for example a nitrone, that is to say a / V-oxide imine also called azomethine oxide, especially a cyclic nitrone, preferably an analogue of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and particularly 5-diethoxyphosphoryl 5-methyl-1-pyrroline N -oxide (DEPMPO) or 2-ethoxycarbonyl-2-3,4-dihydro-2H-pyrrole-1-oxide (EMPO).

The concentration of free radical scavenger compound during the analysis is advantageously between 50 and 300 mM, preferably between 50 and 200 mM, especially between 50 and 150 mM, most preferably between 80 and 100 mM.

The term "stabilizing agent" refers to a compound having a cavity allowing the formation of inclusion complexes, for example a mineral compound such as a zeolite or a kaolinite, or organic such as a crown ether, a cyclophane, a cyclic oligomer of a dose such as glucose, preferably a cyclic oligomer of an ose such as glucose (for example a cyclodextrin) or a crown-ether, particularly a cyclodextrin or a crown-ether and especially a β-cyclodextrin such as 2-hydroxypropyl-2-hydroxypropyl ether. beta-cyclodextrin or methyl-beta-cyclodextrin.

The concentration of the stabilizing agent during the analysis is advantageously between 50 and 300 mM, preferably between 50 and 200 mM, in particular between 50 and 150 mM, most particularly between 80 and 100 mM. It will depend on the compound used and will preferably be between 50 and 200 mM, in particular between 50 and 150 mM, especially between 80 and 100 mM for methyl-β-cyclodextrin (Randomly Methylated-beta-cyclodextrin, RM-beta -CD). The term "immediately" means as quickly as possible, and advantageously in less than 10 seconds, preferably in less than 5 seconds, especially in less than 3 seconds, particularly in less than 2 seconds and especially in less than 1 second after the sample. The chelating agent is, for example, deferoxamine, and especially diethylene triaminepentaacetic acid (DTPA). For example, ethylene diamine tetraacetic acid, which is oxidizing, is not suitable.

The concentration of the chelating agent is advantageously between 0.1 and 2 mM, preferably between 0.1 and 1.5 mM, especially between 0.2 and 1 mM, especially between 0.5 and 1 mM. , O mM.

For example, biological fluids that may be mentioned include biological fluids of mammals, for example equines, in particular humans, in particular blood, urine, sweat, sperm, effluents of biological origin. as organ perfusates or cell culture media, preferably plasma, blood and sperm, and especially the latter, especially the latter.

Depending on the biological fluid, the reactive composition may contain other compounds. For example, if the biological fluid is blood, it also contains an anticoagulant such as citrate ions as in citrate dextrose acid or heparin.

The amount of the anticoagulant agent is advantageously between 10 and 100 IU, preferably between 10 and 50 IU, especially between 10 and 25 IU for heparin. It will depend on the compound used and will for example be between doses producing an anticoagulant effect equivalent to the above.

Under preferred conditions of implementation of the method described above, the reactive composition also contains a buffer.

The buffer used is then advantageously a pH 7 buffer such as the phosphate buffer, the most conventionally used. It may additionally contain an acid or a base to adjust the pH of the medium. In still other preferred conditions of implementation of the method described above, the mixture of the sample and reagents of the reactive composition is frozen before analysis using an analysis apparatus detecting a compound comprising a free electron. For example, after taking the sample, in particular blood, the sample can be transferred to a cryotube and said tube placed in liquid nitrogen.

To proceed with the analysis, the mixture is quickly thawed and immediately analyzed. For example, the mixture is thawed in 30 seconds and is carried out in the 30-60 seconds after thawing at the filling of the measuring cell and preferably at the analysis.

As seen above, the mixing of the sample and reagents of the reactive composition must be carried out immediately, as quickly as possible.

There is no device for easily implementing the above method in the case in particular of a blood sample. It has therefore been specially designed for the implementation of the above method a sampling device may include a syringe, allowing the collection of a sample for example the in vivo sampling of a blood sample for the immediately send a mixture with the reactive composition without risk of injection back of the sample mixed with reagents.

This is why the present application also relates to a device for taking a sample of a biological fluid to send it immediately in a mixture with a reactive composition above, characterized in that it comprises a chamber of aspirating said parameterized sample to sample the amount of sample needed for the reaction with a pre-established amount of reactive composition above, and a non-return device. Under preferred conditions of implementation of the invention, the above sampling device comprises a syringe provided with a non-return device.

The present application also relates to a kit for carrying out a method for detecting, identifying or quantifying free radicals in a biological fluid, characterized in that it comprises a device for sampling a sample of a above biological fluid and a container containing a predetermined amount of reactive composition above, and optionally, a container containing a pre-established amount of a free radical scavenging compound.

The process which is the subject of the present invention has very interesting qualities. It allows in particular to detect, identify or directly quantify free radicals in a biological fluid, in vitro.

These qualities are illustrated below in the experimental part. They justify the use of the methods, devices and kits described above, in the determination of free radicals such as radicals centered on oxygen.

(superoxide, hydroxyl, alkoxyl ...) or carbon (alkyl ..) in the blood. For example, in humans, information is obtained on the presence of inflammation, ischemia, septic shock, the effect of intensive sports training, etc.

There may also be mentioned the possibility of testing the efficacy of drugs or biological products or compositions such as transplants transport or conservation media.

This is why the present application also relates to a process for the determination of free radicals such as radicals centered on oxygen (superoxide, hydroxyl, alkoxyl, etc.) or carbon (alkyl) in the blood, in which the above method is carried out on a blood sample.

Thanks to the sampling device above, an analysis is possible both in the analysis laboratory and in the hospital and it is not necessary to be near the analyzer to benefit from it. . A sample ready for analysis may wait before the analysis is done.

The preferred conditions of implementation of the methods for detecting, identifying or quantifying free radicals in a biological fluid described above also apply to the other objects of the invention referred to above, in particular to the sampling devices of a sample of a biological fluid to send immediately in mixture with a reactive composition and kits for the implementation of said method.

Figure 1 shows the results obtained on blood samples taken on a swimmer (30 min, 3 times / day for 3 months) at the end of the exercise phase, trapping with the DEPMPO (100 mM) A) in the presence of methyl-beta-cyclodextrin (100 mlVl), spectrum recorded 35 sec after sampling; B) in the presence of methyl-beta-cyclodextrin (100 mM) and after freezing the sample in liquid nitrogen for 3 days, spectrum recorded 35 sec after thawing; C) in the absence of methyl-beta-cyclodextrin, spectrum recorded 35 sec after thawing.

Figure 2 shows the results obtained on blood samples taken on a swimmer (30 minutes, 3 times / day for 3 months) at the end of the exercise phase. Trapping with DEPMPO (100 mM) in the presence of methyl-beta-cyclodextrin (100 mM). Freezing of the sample in liquid nitrogen for 3 days, A) spectrum recorded 35 sec after thawing; B) spectrum recorded 1min 30 sec after thawing; C) spectrum recorded 2 min 30 sec after thawing; D) spectrum recorded 3 min 30 sec after thawing.

Figure 3 shows the results obtained on blood samples taken on a rat heart reperfused with blood after preservation of the graft for 5 hours in cardioplegic fluid. A) Stopping and Preservation in Ringer's Lactate; B) Stopping and conservation in Celsior®. Trapping with DEPMPO (150 mM) in the presence of methyl-beta-cyclodextrin (120 mM).

Figure 4 shows the results obtained on venous blood samples taken in an elderly patient, suffering from Alzheimer's disease and showing signs of dementia, recruited in a clinical trial.

Trapping with DEPMPO (100 mM) in the presence of methyl-beta-cyclodextrin

(100 mM); A) spectrum recorded 35 sec after thawing; B) spectrum recorded 2 min 30 sec after thawing; C) spectrum recorded 3 min 30 sec after thawing.

The following examples illustrate the present application.

Example 1: Tube for analysis A blood test tube was prepared by installing in a cryotube of 1.8 ml an aliquot of 0.25 ml of a composition having the following molar composition:

The pH of the phosphate buffer was previously adjusted to 7 with an aqueous solution of potassium hydroxide.

A vial containing 5 ml of DEPMPO (600 mM in distilled water) was provided separately.

_.. Example 2; Tube for analysis ready to use

0.25 ml of the DEPMPO solution in distilled water is added to the blood test tube of Example 1. We therefore have the following final reactive composition:

Example 3: Syringe for analysis A syringe was made for blood analysis by installing in a syringe whose suction chamber is 2 ml and provided with a non-return device, 0.25 ml of a composition dosed as indicated in example 1.

Example 4 Kit for analysis A kit for blood analysis was prepared by providing in a package, a syringe for blood analysis of Example 3 and a vial containing 0.25 ml of DEPMPO (600 mM in distilled water).

Example 5 Determination of Free Radicals in an In Vivo Blood Sample in the Rat

The contents of the ready-to-use test tube of Example 2 are aspirated into a 2 ml syringe. We collect with the same syringe

1 ml of blood in the femoral artery of an anesthetized rat following a 30-minute sports training period (swimming) as part of an intensive program (30 min, 3 times / day) during 3 months).

The final composition of the reagents in this sample is as follows:

The mixture is transferred to a quartz flat cell and then to the air gap of the magnet of an EPR spectrometer (Bruker ESP 300).

The analysis is carried out immediately (within 30-60 seconds after the sampling and filling of the quartz measuring cell) at 25 ^° C. under the following conditions:

Microwave frequency: 9.79 GHz

Microwave power: 10 mW Amplitude of modulation: 0,198 Gauss

Modulation frequency: 100 kHz

Receiver gain: 1 x 10 ⁵

Time constant: 40.96 ms

Scanning range in field: 130 Gauss Scanning speed in field: 3.1 Gauss / s

The spectrum of Figure 1A is obtained.

In the sample analyzed by RPE, it can be observed that intensive physical exercise results in an intense production of free radicals, which had already been reported by other, indirect methods. Example 6: Determination of free radicals in a blood sample in vivo after freezing in liquid nitrogen and thawing.

The procedure is as in Example 5, but after taking the blood, the contents of the syringe are transferred to a cryotube and the tube is placed in liquid nitrogen.

The mixture is defrosted (in 30 seconds) 3 days after being placed in liquid nitrogen and the analysis is carried out immediately (within 30-60 seconds after thawing and filling of the quartz measuring cell). The conditions of Example 5 give a spectrum shown in Figure 1B, qualitatively similar to that of Figure 1A. It is specified that the comparisons between the spectra can be made only when these spectra were recorded at the same time after sampling or thawing. Figure 2 (A to D) shows that signal decay is, however, low within a reasonable time (3 min 30 seconds minimum) after thawing of the sample, allowing reproducible recording.

Surprisingly, it is possible to freeze the sample before analysis.

Comparative Example: Use of a Reactive Composition Without a Stabilizing Agent in an In Vivo Blood Sample

The procedure is as in Example 5, but the blood test tube of Example 1 does not contain methyl-β-cyclodextrin. The spectrum of FIG. 1C is obtained,

It can be seen that no EPR signal can be detected under these conditions.

Stabilization is therefore necessary. Example 7; Determination of free radicals in a blood sample obtained in an in vitro model.

The contents of the ready-to-use test tube of Example 2 are aspirated into a 2 ml syringe. 0.5 ml of blood are drawn from the same syringe after cannulation and aspiration into the coronary sinus of a rat heart under the following conditions.

The blood sample is taken in the coronary sinus of a rat heart which has been previously isolated and perfused and then subjected to the following experimental protocol: a normoxic control period of 30 min with a Krebs-Henseleit solution (37 ^° C., pH 7.35, 2.5 mM CaCl ₂ , 10 mM glucose),

- a cardioplegic arrest at 4 ° C performed on the one hand by the classic cardioplegic solution of St. Thomas Hospital on the other hand by a Celsior® type solution containing antiradical agents (see Pietri et al (1994) Transplantation 58 (6): 739, Menasche et al (1994) Eur J. Cardiothorac, Surg 8: 207).

- a storage period of 5 hours in cardioplegic solution.

a 1 minute reperfusion with rat blood, during which the sample is taken from the coronary sinus, added to the scavenger + stabilizer mixture (syringe of Example 2), and then frozen in expectation of the experiment of RPE.

The final composition of the reagents in this sample is as follows;

The mixture is immediately frozen in liquid nitrogen. After thawing (carried out in 30 seconds), the mixture is transferred to a quartz flat cell and then to the air gap of the magnet of an EPR spectrometer (Bruker ESP 300).

The analysis is carried out immediately (within 30-60 seconds after thawing and filling of the quartz measuring cell) under the following conditions;

Microwave frequency: 9.79 GHz

Microwave power; 10 mW

Amplitude of modulation: 0,198 Gauss

Modulation frequency: 100 kHz Receiver gain: 1 x 10 ⁵

Constant of time; 40.96 ms

Scanning range in field: 130 Gauss

Scanning rate in the field: 3.1 Gauss / s

The spectra of Figure 3 (A and B) are obtained.

The spectrum of FIG. 3A corresponds to a sample taken from a rat heart which has been stopped and then preserved in the cardioplegic solution of St. Thomas; the spectrum of FIG. 3B corresponds to a sample taken from a rat heart which has been stopped and then stored in the cardioplegic solution of Celsior® type. Both spectra were recorded 35 seconds after thawing. It is observed that radicals are produced at the post-ischemic reperfusion in the presence of blood (FIG. 3A) and that the preservation of the graft in an antiradical solution inhibits this production.

In addition, previous results have shown that the presence of antiradical agents significantly improves post-cardiac cardiac recovery (see Pietri et al (1994) Transplantation 58 (6): 739;

Ménasché et al. (1994) Eur. J. Cardiothorac. Surg. 8: 207), thus emphasizing the interest of the detection of free radicals.

Example 8 Determination of free radicals in a blood sample in vivo in humans.

Venous blood sampling was performed in elderly patients with Alzheimer's disease who had signs of dementia enrolled in a clinical trial.

The contents of the ready-to-use test tube of Example 2 are aspirated into a 2 ml syringe. Using a separate sterile syringe, 3 ml of venous blood were removed from a patient above, and the blood removed was immediately transferred to a heparin hemolysis tube.

Immediately, 1 ml of this blood is sucked into the 2 ml syringe containing the pre-installed scavenger + stabilizer mixture and then frozen in liquid nitrogen pending the EPR experiment. The final composition of the reagents in this sample is as follows:

After thawing, the mixture is transferred to a quartz flat cell and then to the air gap of the magnet of an EPR spectrometer (Bruker ESP 300).

Immediately (within 30-60 seconds after thawing and filling of the quartz measuring cell) the RPE analysis is performed under the following conditions:

Microwave frequency: 9.79 GHz

Microwave power: 10 mW Amplitude of modulation: 0,198 Gauss

Modulation frequency; 100 kHz

Receiver gain: 1 x 10 ⁵

Constant of time; 40.96 ms

Scanning range in field: 130 Gauss Scanning speed in field: 3.1 Gauss / s

The spectra of Figure 4 (A, B, C) recorded at different times after thawing are obtained (35 sec, 2 min 30 sec, 3 min 30 sec)

The spectra obtained show the existence of free radicals in the samples from this patient population and underlines the interest of this detection technique for clinical and therapeutic studies.

Other tests in rats have shown the possibility of sperm dosing, which can be used to study the problem of male infertility.

Claims

1. A method for detecting, identifying or quantifying free radicals in a biological fluid in which: - a biological fluid is taken, said biological fluid is immediately mixed with a reactive composition containing a free radical scavenging compound, a stabilizing agent and a non-oxidizing chelating agent, then the analysis thus obtained is carried out using an analysis apparatus detecting a compound comprising a free electron, 2. A method according to claim 1, characterized in that the analysis of the mixture is carried out by electron paramagnetic resonance (EPR). 3. A process according to claim 1 or 2, characterized in that the scavenger compound of free radicals is a nitrone. 4. A process according to claim 1 or 2, characterized in that the scavenger compound of free radicals is a nitrone selected from 5-diethoxyphosphoryl 5-methyl-1-pyrroline N-oxide (DEPMPO) and 2-ethoxycarbonyl-2 -3,4-dihydro-2H-pyrrole-1 -oxide (EMPO). 5. A method according to one of claims 1 to 4, characterized in that the concentration of the scavenger free radical compound during the analysis is between 50 and 200 mM. 6, A method according to one of claims 1 to 5, characterized in that the stabilizing agent is a cyclodextrin. 7. A method according to one of claims 1 to 6, characterized in that the concentration of the stabilizing agent in the analysis is between 50 and 200 mM. 8. A method according to one of claims 1 to 7, characterized in that the biological fluid is mixed with the reactive composition containing a scavenger free radical compound, a stabilizing agent and a non-oxidizing chelating agent in less than 10. seconds after the sampling. 9. A process according to one of claims 1 to 8, characterized in that the chelating agent which is not oxidizing is diethylene triamine pentacetique acid. 10. A method according to one of claims 1 to 9, characterized in that the concentration of the chelating agent is between 0.1 and 1.5 mM. 11. A method according to one of claims 1 to 10, characterized in that the biological fluid is selected from blood, urine, sweat, organ perfusates and human cell culture media. 12. A method according to one of claims 1 to 11, characterized in that the reactive composition further contains an anticoagulant. 13. A method according to one of claims 1 to 12, characterized in that the reactive composition further contains a buffer. 14. A method according to one of claims 1 to 12, characterized in that the mixture of the sample and reagents of the reactive composition is frozen before analysis using the analysis apparatus. 15. A device for taking a sample of a biological fluid to send it immediately in a mixture with a reactive composition as defined in one of claims 1 to 14, characterized in that it comprises a chamber of aspirating said parameterized sample to sample the amount of sample needed for the reaction with a pre-established amount of reactive composition above, and a non-return device. 16. A kit for carrying out a method as defined in one of claims 1 to 14 for the detection, identification or quantification of free radicals in a biological fluid, characterized in that it comprises a sampling device. a sample of a biological fluid as defined in claim 14 and a container containing a pre-established amount of reactive composition as defined in one of claims 1,3,4,5,6,7,8 , 9, 10, 1261 13. 17. A kit according to claim 16, characterized in that it further comprises a container containing a pre-established amount of a scavenger free radical compound. 18. A method according to one of claims 1 to 12, characterized in that the biological fluid is taken and immediately mix the biological fluid with the reactive composition using a sampling device as defined in the claim 15.