Method and device for measuring the status of oxidative stress in a biological matrix

Abstract

The invention relates to the use of a compound AB selected from NADH, NADPH, Cyt C (Fe2+) or H2O2, to measure the oxidative stress status in a biological matrix. The compound AB is available in two forms A and B that do not interact with each other, and are capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or within said biological matrix.

Description

Title of the invention: Method and device for measuring the status of oxidative stress in a biological matrix

[0001] The present invention relates to the field of methods and devices for measuring the status of oxidative stress on the surface or in a biological matrix.

[0002] Oxidative stress corresponds to a breakdown in the balance between the production of free radicals, which are oxidizing species, and the capacity of the body's protective mechanisms, represented by antioxidants, to neutralize these free radicals before they cause damage to the matrix.

[0003] With regard more particularly to living organisms, and in particular the human species, oxidative stress is suspected as being the cause of cellular aging and numerous pathologies, such as Alzheimer's and Parkinson's diseases in particular.

[0004] As a result, some manufacturers in various sectors, such as cosmetics, food processing, and medicine, are seeking to develop new molecules or new compounds with antioxidant properties, with the aim of reducing the effects of oxidative stress. These antioxidant compounds are also incorporated into certain food, pharmaceutical, nutraceutical, or cosmetic products, in order to increase their shelf life while having a beneficial effect on the health of the consumer.

[0005] It may therefore prove useful to be able to reliably and reproducibly measure the effects on an organism of the administration of an antioxidant substance or, on the contrary, of an oxidizing substance. Prior art

[0006] Patent application WO2017077237A1 is known from the prior art, which presents a device and a method for measuring the total antioxidant and oxidative capacity of any matrix. The measurements are carried out by reacting a mediator system comprising a compound A and a compound B, which are different, with the matrix to be tested, and by measuring the electrochemical potential of said mediator system. Summary of the invention

[0007] The inventors have developed a mediator system particularly suitable for biological matrices composed of a single compound AB available in two forms A and B. Form A can react and / or form complexes with an antioxidant and form B can react and / or form complexes with an oxidant, but forms A and B do not interact with each other.

[0008] Advantages of the invention

[0009] The AB compounds according to the invention have the advantage of being molecules that are non-toxic to the skin, unlike the compounds used in the prior art such as ferric ammonium sulfate, cerium or iodine monobromide. Thus, the AB compounds prove to be particularly suitable for use in systems allowing the analysis of biological matrices such as the skin.

[0010] Furthermore, unlike the methods of the prior art operating at pH 2 and temperatures above 50°, the matrix analysis methods according to the invention using a compound AB according to the invention make it possible to obtain stable analyses at pH and body temperatures (pH between 5 and 8, and T° between 23 and 40°C), making the use of such compounds particularly effective for in vivo analyses such as for example on the skin, but also to carry out stable analyses on biological samples taken from samples, without risking degrading the sample during the analysis.

[0011] A compound AB according to the invention has the advantage of offering a potential difference close to that of the skin, which is both high enough to be observable (at least 20mV potential difference between A and B), and not too high so as not to create a danger for the skin. Thus, compound AB proves to be particularly suitable for carrying out electrochemical analyses on the skin.

[0012] An AB compound according to the invention also has the advantage of causing a color change depending on the oxidation state of the matrix, and this without adding colored indicators. This makes the AB compounds particularly suitable for spectrophotometric analyses. In addition, since it is not necessary to add a colored indicator, which is often toxic, the AB compounds are particularly suitable for spectrophotometric analysis in systems comprising biological matrices.

[0013] An AB compound according to the invention also has the advantage of being visible by fluorescence spectroscopy (fluorimetry) without having to add a fluorescent compound. This makes AB compounds particularly suitable for analyses by fluorimetry. DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention relates to the use of a compound AB chosen from NADH, NADPH, Cyt C (Fe2+) or H2O2, for measuring the status of oxidative stress in a biological matrix. The compound AB is available in two forms A and B which do not interact with each other, and are capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in said biological matrix.

[0015] The term “AB compound” or “AB mediator” or “mediator” according to the invention means a compound chosen from NADH, NADPH, Cyt C (Fe2+) or H2O2.

[0016] This AB mediator has the advantage of being dissociated into two forms:

[0017] - on the one hand form A, “oxidant” form, will attract antioxidant molecules, but does not react with form B, the “antioxidant” form of compound AB;

[0018] - on the other hand, form B, “antioxidant” form, will attract the molecules oxidants (also called free radicals), but does not react with form A, the “oxidant” form of compound AB.

[0019] The compounds AB according to the invention are presented in forms A and B as presented in Table 1.

[0020] [Tables 1] Mediator Mediator N ADH Mediator NADPH Mediator Cyt C(Fe2+) Mediator H2O2 Form A “oxidant” NAD+ NADP Cyt C(Fe3+) HO- Form B “antioxidant” NADH NADPH Cyt C(Fe2+) H2O2

[0021] Thus, the two different states, A and B of the compound AB do not balance each other, they only balance in contact with the oxidizing and antioxidant molecules of the matrix, this guarantees the accuracy of the measurement carried out, which will not be distorted by interactions between A and B.

[0022] The term "biological matrix" means a matrix originating from the living world. Such a matrix may originate from a sample (e.g., blood, urine, sweat, hair, seminal and follicular fluids, etc.), or may concern a part of a living organism (e.g., a tissue such as skin, etc.).

[0023] The term "oxidative stress status" refers to the balance between oxidants and antioxidants in the matrix to be analyzed. This status indicates whether the matrix is ​​predominantly made up of oxidizing molecules, predominantly made up of antioxidant molecules, or in balance between antioxidants and oxidants.

[0024] The term "measurement of the status of oxidative stress" means the measurement of the electrochemical potential, of the absorbance by spectrophotometry or of the intensity of the fluorescence by fluorimetry to obtain one of the following parameters: total antioxidant / oxidant power, anti-radical effect, reducing effect or protective effect; in order to deduce a general status concerning the state of oxidative stress of the matrix.

[0025] The status of oxidative stress can be presented in the form of a numerical value scale whose values:

[0026] - The weakest corresponds to the case where there is little or no oxidative stress

[0027] - The intermediate values ​​correspond to the case where oxidative stress can lead to risks for biological matrices.

[0028] - High values ​​correspond to severe oxidative stress.

[0029] The term "total antioxidant / oxidant power" refers to the ratio between the total antioxidant power and the total oxidant power. Thus, total antioxidant / oxidant power can also be written as total antioxidant power / total oxidant power or even ratio of total antioxidant / oxidant power.

[0030] The total antioxidant / oxidant power of the matrix is ​​positively correlated with the oxidative stress status of the matrix.

[0031] Thus, a high total antioxidant / oxidant power corresponds to an imbalance in the antioxidant / oxidant balance in favor of an increased presence of antioxidant molecules and therefore corresponds to an oxidative stress status: “no or little oxidative stress”.

[0032] Conversely, a low total antioxidant / oxidant power corresponds to an imbalance in the antioxidant / oxidant balance in favor of an increased presence of oxidizing molecules and therefore corresponds to a status of oxidative stress: “severe oxidative stress”.

[0033] The anti-radical effect of the matrix is ​​negatively correlated with the oxidative stress status of the matrix.

[0034] Thus, a high anti-radical effect corresponds to an increased presence of antioxidant molecules and therefore corresponds to a status of oxidative stress: “no or little oxidative stress”.

[0035] Conversely, a low anti-radical effect corresponds to an imbalance in the antioxidant / oxidant balance in favor of an increased presence of oxidizing molecules and therefore corresponds to the status of oxidative stress: “severe oxidative stress”.

[0036] The reducing effect of the matrix is ​​negatively correlated with the oxidative stress status of the matrix.

[0037] Thus, a high reducing effect corresponds to an imbalance in the antioxidant / oxidant balance in favor of the increased presence of antioxidant molecules and therefore corresponds to a status of oxidative stress: “no or little oxidative stress”.

[0038] Conversely, a low reducing effect corresponds to an imbalance in the antioxidant / oxidant balance in favor of an increased presence of oxidizing molecules and therefore corresponds to the status of antioxidant / oxidative stress: “severe oxidative stress”.

[0039] The protective effect of the matrix is ​​negatively correlated with the oxidative stress status of the matrix.

[0040] Thus, a high protective effect corresponds to an imbalance in the antioxidant / oxidant balance in favor of the increased presence of antioxidant molecules and therefore corresponds to a status of oxidative stress: “no or little oxidative stress”.

[0041] Conversely, a low protective effect corresponds to an imbalance in the antioxidant / oxidant balance in favor of an increased presence of oxidizing molecules and therefore corresponds to the status of oxidative stress: “severe oxidative stress”.

[0042] A second subject of the invention relates to a device for measuring the total antioxidant / oxidant power of a biological matrix comprising a measuring medium in which one or more working electrodes and one or more reference electrodes are immersed, characterized in that said medium comprises a compound AB available in two forms A and B, capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in the matrix, and not interacting with each other.

[0043] The term "measurement of the total antioxidant / oxidant power" means the measurement of the ratio between the total antioxidant power and the total oxidant power. This measurement is carried out mainly by measuring the electrochemical potential of the measurement medium.

[0044] The measurement of total antioxidant / oxidant power makes it possible to monitor oxidative stress in order to detect and prevent possible pathologies, it also makes it possible to monitor the efficacy and safety of a treatment and its timely adjustments, as well as to optimize the formulation and manufacturing of pharmaceutical, cosmetic, food and nutraceutical products.

[0045] The present device is characterized by the fact that said means for measuring the antioxidant / oxidant power consists of a means for measuring the electrochemical potential of the measuring medium, the latter incorporating at least one compound AB present in two forms A and B, capable of reacting and / or forming a complex respectively with the antioxidant and oxidant species present on the surface or in said matrix, so as to modify the electrochemical potential of said measuring medium.

[0046] A compound AB according to the invention has the advantage of offering a potential difference close to that of the skin, which is both high enough to be observable (at least 20 mV potential difference between A and B), and not too high so as not to create a danger for the biological matrix. Thus, compound AB proves to be particularly suitable for electrochemical analyses.

[0047] The term “measuring medium”, which may also be called a mediator system, means any medium which can be used to measure the total antioxidant / oxidant power of the matrix.

[0048] In a preferred embodiment of the invention, the measuring medium is in liquid or gel form.

[0049] In an even more preferred embodiment, the liquid or gelled measuring medium is a solution composed of KCl. Such a composition makes it possible to stabilize the measurement.

[0050] In a preferred embodiment, the KC1 solution is at a concentration of between 0.5 and 2.5 mmol / L.

[0051] The specificity of the measuring media according to the invention is that they always comprise the compound AB according to the invention, available in two forms A and B, not interacting not between them, but capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in the matrix.

[0052] In a particular embodiment, the measuring medium is in liquid form. A liquid measuring medium is particularly useful for measuring the total antioxidant / oxidant power of liquid biological matrices such as blood or urine.

[0053] In another alternative embodiment, the measuring medium is in gelled form. A gelled measuring medium is particularly useful for measuring the total antioxidant / oxidant power at the surface of biological matrices such as skin.

[0054] For the purposes of the invention, the term “electrode” means an electronic or ionic conductor capturing electrons.

[0055] The device for measuring the total antioxidant / oxidant power of the matrix comprises two types of electrodes. On the one hand, the reference electrode(s), the potential of which does not vary during the measurements, and on the other hand, the working electrode(s), the potential difference of which relative to the reference electrode will be measured.

[0056] In a preferred embodiment of the invention the electrodes are made of metal.

[0057] In a preferred embodiment, the electrodes are composed of a metal mixture.

[0058] The term "mixture of metal" means either a combination of metals within a single electrode, or the use of different metals each forming a part of an electrode considered as a whole.

[0059] In one embodiment, the reference electrode is Ag+ / Ag (silver chloride electrode)

[0060] In another embodiment, the working electrode is a mixture of tungsten, platinum and gold.

[0061] In a preferred embodiment, the reference electrode is Ag+ / Ag and the working electrode is a mixture of tungsten, platinum and gold.

[0062] A third subject of the invention relates to a method for measuring the oxidative stress status of a biological matrix comprising the steps of:

[0063] - Bringing said matrix into contact with a measuring medium comprising a compound AB available in two forms A and B, capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in said matrix,

[0064] - Measurement of the electrochemical potential between one or more working electrodes and one or more reference electrodes placed on the matrix by electrochemistry, or measuring a variation in absorbance by spectrophotometry.

[0065] The term “measuring medium”, also called “mediator system” or “mediator medium”, means any medium that can be used to measure the oxidative stress status of the matrix.

[0066] Advantageously, the mediator medium according to the invention comprises the mediator AB, and allows measurement by electrochemistry by spectrophotometry or by fluorimetry, without having to add reagents specific to the measurement method in the medium.

[0067] In one embodiment of the invention, the measuring medium is in liquid or gel form.

[0068] In a preferred embodiment, the liquid or gelled measuring medium is composed in particular of a KC1 solution. Such a composition makes it possible to stabilize the measurement.

[0069] In a preferred embodiment, the KC1 solution is at a concentration of between 0.5 and 2.5 mmol / L

[0070] The matrix can be brought into contact with a measuring medium:

[0071] - In the case where the matrix to be tested is solid, by applying a measuring medium gelled on the surface of the matrix, this contact method is particularly useful for carrying out “in vivo” type analyses directly on a tissue such as the skin.

[0072] - In the case where the matrix to be tested is liquid, by dissolving the matrix in a liquid mediating medium, this contact method is particularly useful for carrying out analyses on biological samples such as blood or urine. For example, for a blood sample, the measurement is carried out by putting 200ql of blood into a volume of 2.5ml of mediator system.

[0073] The term "measurement of the status of oxidative stress" means the measurement of the electrochemical potential, of the absorbance by spectrophotometry or of the intensity of the fluorescence by fluorimetry, of one of the following parameters: total antioxidant / oxidant power, anti-radical effect, reducing effect or protective effect of a matrix in order to deduce a general status concerning the state of oxidative stress of the matrix.

[0074] In a particular embodiment of the invention, the method is characterized by the fact that said means for measuring the status of oxidative stress consists of a means for measuring the total antioxidant / oxidant power of the matrix, based on the analysis of the electrochemical potential of the measuring medium. The latter incorporating at least one compound AB present in two forms A and B, capable of reacting and / or forming a complex respectively with the antioxidant and oxidant species present on the surface or in said matrix, so as to modify the electrochemical potential of said measuring medium.

[0075] The use of a compound AB according to the invention in an electrochemical measurement has the advantage of offering a potential difference close to that of the skin, which is both sufficiently high to be observable (at least 20 mV potential difference between A and B), and not too high so as not to create a danger. for the biological matrix. Thus, compound AB is particularly suitable for carrying out electrochemical analyses.

[0076] The measurement of the electrochemical potential also allows the analysis of the effect of a product on a biological matrix, for this purpose the difference is taken between the value of the potential difference between the working electrode(s) and the reference electrode(s) taken at tO and the value of the potential difference between these same electrodes taken at a time t to be tested. The time tO corresponding to a time before the intake or the action of the product whose effect on the biological matrix is ​​to be verified. It is thus possible to measure different total antioxidant / oxidant powers corresponding to different times t, and to carry out the corresponding kinetics.

[0077] In an alternative embodiment of the invention, the method is characterized by the fact that said means for measuring the status of oxidative stress consists of a means for measuring the anti-radical effect and the reducing effect of the matrix, based on the study of the absorbance of the measuring medium. The latter incorporating at least one compound AB present in two forms A and B, capable of reacting and / or forming complexes respectively with the antioxidant and oxidizing species present on the surface or in said matrix, so as to modify the value of the absorbance of said measuring medium.

[0078] The use of an AB compound according to the invention has the advantage of causing a change in color of the medium according to the oxidation state of the matrix brought into contact with it, and this without adding colored indicators. This makes the AB compounds particularly suitable for spectrophotometric analyses. In addition, since it is not necessary to add a colored indicator, which is often toxic, the AB compounds are particularly suitable for spectrophotometric analysis in systems comprising biological matrices.

[0079] Spectrophotometry also allows the analysis of the effect of a product on a biological matrix, for this purpose the difference is taken between the absorbance value at tO, and the absorbance value at a time t to be tested. The time tO corresponds to a time before the intake of the product whose effect on the biological matrix is ​​to be verified. It is thus possible to measure different anti-radical and reducing effects, corresponding to different times t, and to carry out the corresponding kinetics.

[0080] In an alternative embodiment of the invention, the method is characterized by the fact that said means for measuring the status of oxidative stress consists of a means for measuring the fluorescence of the measuring medium, the latter incorporating a molecule of at least one compound AB present in two forms A and B, capable of reacting and / or forming complexes respectively with the antioxidant and oxidizing species present at the surface or in said matrix, so as to modify the value of the fluorescence of said measuring medium.

[0081] In an advantageous embodiment, the measurement of the oxidative stress status also allows the analysis of the effect of a product on a biological matrix, for this purpose the difference is taken between the value of the oxidative stress status at tO and the value at a time t to be tested. The time tO corresponding to a time before the intake or the action of the product whose effect on the biological matrix is ​​to be verified. It is thus possible to measure different oxidative stress statuses corresponding to different times t, and to carry out the corresponding kinetics.

[0082] The study of the kinetics of the status of oxidative stress is particularly useful for determining the effect of a product on the antioxidant / oxidant balance of the biological matrix over time.

[0083] A fourth subject of the invention relates to a method for determining the oxidative score of a biological matrix which comprises the combination of the results of at least two measurements chosen from:

[0084] - An electrochemical measurement measuring the total antioxidant / oxidant power of said matrix,

[0085] - A measurement by spectrophotometry measuring the anti-radical effect and / or the effect reducer of said matrix,

[0086] - A measurement by fluorimetry measuring the protective effect of said matrix.

[0087] The term “oxidative score” means an index giving precise information on the antioxidant and oxidative state of the biological matrix.

[0088] Such an index differs from the status of oxidative stress in that it is based on at least two informative parameters concerning the oxidative stress of the matrix, obtained by at least two different measurement methods. Using two different measurement methods chosen from electrochemistry, spectrophotometry or fluorimetry makes it possible to refine the precision of the index.

[0089] As stated previously, each measurement method will allow one or more different parameters to be measured. Manufacturers have often played on this multiplicity of parameters to highlight the parameter that benefits them most in the eyes of customers.

[0090] The oxidative score makes it possible to avoid this type of bias, by combining the results of different parameters from different measurements into a single score, thus providing a clear indication of the oxidative state of the biological matrix, while offering better guarantees as to the relevance of the result.

[0091] In a preferred embodiment of the invention, the oxidative score is calculated by combining the measurement of the total antioxidant / oxidative power, the anti-radical effect, the reducing effect and the protective effect. Such an oxidative score offers the most accurate measurement of the oxidative state of the biological matrix because it combines a large number of parameters relating to the oxidative state of the biological matrix.

[0092] In a specific embodiment, the measurement of the oxidative score also allows the analysis of the effect of a product on a biological matrix, for this purpose the difference is taken between the value of the oxidative score taken at t0 and the value taken at a time t to be tested. The time t0 corresponding to a time before the intake or the action of the product whose effect on the biological matrix is ​​to be verified. It is thus possible to measure different oxidative scores corresponding to different times t, and to carry out the corresponding kinetics.

[0093] The study of the kinetics of the oxidative score is particularly useful for determining the effect of a product on the oxidative state of the biological matrix over time. BRIEF DESCRIPTION OF THE FIGURES

[0094] [Fig.l] [Fig.l] shows the electrochemical curve obtained after 10 minutes of recording using the mediator system and the microelectrodes applied to the skin. EXAMPLES EXAMPLE 1#: Measurement of oxidative stress status.

[0095] The status of oxidative stress is measured on the skin (forearm) of a 26-year-old woman, fasting 12 hours before the start of the measurement, at a temperature and humidity of respectively (20°C <T<23°C et 50%<HR<60%).

[0096] Before analysis, the skin was cleaned with water and dried with absorbent paper (the analysis area is 2 cm2).

[0097] The device is composed of: - A gelled mediator system comprising the mediator compound NADH according to the invention (dissociating into two forms NAD+ and NADH reacting respectively with an antioxidant and an oxidant and not reacting with each other) as well as a solution of KC1 at a concentration of 2mmol / L. - Microelectrodes fixed to the arms, in contact with the mediator system: a silver reference electrode, and a working electrode in gold (50%), platinum (27%) and tungsten (33%).

[0098] The status of oxidative stress is observed by measuring the total antioxidant / oxidant power.

[0099] The measurement of the total antioxidant / oxidant power consists of electrochemical monitoring of the changes in the concentration of the oxidized (NAD+) and reduced (NADH) forms of the mediator (NADH) during the reaction with the antioxidants and oxidants present in the matrix.

[0100] [Fig.l] shows the electrochemical curve obtained after 10 minutes of recording using the mediator system and the microelectrodes applied to the skin.

[0101] The total oxidizing power and the total antioxidant power are measured according to the following formulas:

[0102] [Math.l] . -s / t^PeouiirPÊamtralto) 1 i ru, ni -, Total oxidizing power = I---pp------- xlOO = 3.12 ' \ r ^control t0 /

[0103] [Math.2] Total antioxidant power = ( )x!00 = 15.62

[0104] Where PEPeauioCorresponds to the electrochemical potential measured at time 10 min.

[0105] PEcontroi ^corresponds to the electrochemical potential measured at time tO.

[0106] And PEpeau 3 min corresponds to the highest electrochemical potential.

[0107] The total antioxidant / oxidant power is equal to the total antioxidant power divided by the total oxidant power, i.e. 15.62 / 3.12 = 5.00

[0108] The total antioxidant / oxidant power of the patient's skin is therefore 5.00.

[0109] The patient therefore has an oxidative stress status value of 5.00; this corresponds to the cases where there is very little oxidative stress.

Claims

Claims

1. Device for measuring the total antioxidant / oxidant power of a biological matrix comprising a measuring medium in which one or more working electrodes and one or more reference electrodes are immersed, characterized in that said medium comprises a compound AB which is non-toxic to the skin and available in two forms A and B, capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in the matrix, and not interacting with each other, and in that the working electrode(s) comprise a mixture of gold, platinum and tungsten.

2. Device according to claim 1, characterized in that said measuring medium is in liquid form or in gel form.

3. Measuring device according to claim 2, characterized in that said measuring medium is a solution composed of KC1.

4. Method for measuring the oxidative stress status of a biological matrix characterized in that it comprises the steps of bringing said matrix into contact with a measuring medium comprising a compound AB which is non-toxic to the skin and available in two forms A and B, capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in said matrix, measuring the electrochemical potential between one or more working electrodes and one or more reference electrodes placed on the matrix by electrochemistry, or measuring a variation in absorbance by spectrophotometry, characterized in that the working electrode(s) comprise a mixture of gold, platinum and tungsten.

5. Method for measuring the status of oxidative stress according to claim 4, characterized in that said measuring medium is in liquid form or in gelled form.

6. Method for determining the oxidative score of a biological matrix characterized in that it comprises the combination of the results of at least two measurements chosen from: a measurement by electrochemistry measuring the total antioxidant / oxidant power of said matrix, a measurement by spectrophotometry measuring the anti-radical effect and the reducing effect of said matrix, a measurement by fluorimetry measuring the protective effect of said matrix and in that it comprises a non-toxic AB compound for the skin available in two forms A and B, capable of interacting and / or forming complexes respectively with an antioxidant and an oxidant present on the surface or in said matrix, characterized in that the working electrode(s) comprise a mixture of gold, platinum and tungsten.

7. Method for determining the oxidative score according to claim 6, characterized in that it comprises both the measurement of the total antioxidant / oxidant power, the anti-radical effect, the reducing effect and the protective effect.