EP3278094A1

EP3278094A1 - Method for measuring the recovery and/or rate of recovery of the antioxidant power of biological fluids after exercising

Info

Publication number: EP3278094A1
Application number: EP15722256.3A
Authority: EP
Inventors: Philippe Tacchini
Original assignee: Edel-For-Life SA
Current assignee: Edel-For-Life SA
Priority date: 2015-04-02
Filing date: 2015-04-02
Publication date: 2018-02-07
Also published as: WO2016156924A1

Abstract

The present disclosure describes a method, biomarkers, its associated electrochemical device and sensors for measuring the individual recovery and/or the rate of recovery of the antioxidant capacity of a biological fluid of an individual human or animal, for predicting its evolution over time and for determining the physical activity and life-style conditions for managing it in a verifiable manner.

Description

METHOD FOR MEASURING THE RECOVERY AND/OR RATE OF

RECOVERY OF THE ANTIOXIDANT POWER OF BIOLOGICAL FLUIDS AFTER EXERCISING

FI E LD O F TH E I NVE NT ION

The present invention concerns a method for assessing the recovery and/or the recovery rate of the antioxidant power of a biological fluid of an individual human or animal after said individual has exercised. In the present disclosure, "exercising" means any physical activity as for example: exercise and training as leisure activities, exercise and training for fitness, or exercise and training for competition, during which the consumption of oxygen and the cardiac frequency are increased. The present invention further concerns an electrochemical device and its associated sensors adapted to be used for implementing the method of the invention.

BACKGROUND ART

The antioxidant power of a biological fluid can be defined as the available antioxidant capacity for preventing and/or controlling oxidation, as measured from a sample of the fluid. A low antioxidant power corresponds to a weak resistance to oxidation, while a high antioxidant power corresponds to a high resistance to oxidation. Likewise, when several samples of the biological fluid are collected at different times and then measured, the antioxidant power can display a stable, reductive or oxidative trend, corresponding to a relatively stable antioxidant defence system, an increasing or decreasing antioxidant activity, respectively.

The antioxidant power of a biological fluid is an indicator of its capacity to resist oxidation, to prevent oxidative stress and to maintain its proper redox balance (1 , 2, 3). Such balance is critical because, both extra- and intra- cellular over-oxidation (oxidative stress) or overreduction (reducing stress) are responsible for structural and functional damages of the oxidized or reduced structures, leading to impaired signalling (4) and resulting in the alteration or loss of biological and physiological properties and functions (5, 6).

The antioxidant power of a biological fluid, such as capillary blood for example, is tightly regulated and depends on several biological, physical, biochemical, physiological, internal and external parameters. Among several thousands of parameters, physical activity and exercising play an important role. Exercising increases both cardiac frequency and the consumption of oxygen, resulting in the concomitant production of free radicals, also increasing oxidative stress (7, 8). Such an increase can have different consequences on the antioxidant power of capillary blood depending on the personal response to exercising, in relationship with the starting status of the capillary blood antioxidant power, its ability to react to an oxidative or reductive challenge and the intensity and frequency of exercising (9). The antioxidant power of capillary blood taken at different times, before and after exercising, reflects the systemic antioxidant defence system. The antioxidant power will either remain stable, decrease or increase, depending on the individual response to exercising. Such a response is personal and needs to be measured to be established (10).

Several methods have been described for measuring antioxidant activity, including either the measurement of a global antioxidant activity or the dosage of specific antioxidants components (1 1 , 12). In the first category, several methods have been used for measuring the global antioxidant activity of a sample, including the FRAP (13), TEAC (14), ORAC (15) and electrochemically based assays (16). In the second category, the dosage of specific vitamins, such as vitamin C (17) or E (18), specific enzymes such as the superoxide dismutase (19) or catalase (20) or specific metabolites such as glutathione (21 ) have been described. However, there are several disadvantages of the prior art because recording multiple parameters is complicated and requires the implementation of several different assays, requiring very large volume of sample. Moreover, the collection of large volume of blood cannot be repeated over time. Furthermore, none of these methods and markers allow the measurement of the recovery and/or the rate of recovery of the antioxidant power of capillary blood. Neither does any of these methods provide the necessary information for predicting the evolution of the antioxidant power, so as to make it possible to control external conditions, including life-style and exercising, in such a way as to be in a position to manage the level of antioxidant power in a verifiable manner as described in this disclosure.

To date, no simple method, nor its associated markers, device and sensors have been described for measuring the recovery and/or rate of recovery of the antioxidant power of biological fluids after exercising, for assessing and establishing its stable, reductive or oxidative trend.

As reliable methods, markers, electrochemical device and sensors for effectively measuring the positive or negative impact of exercising and life-style conditions upon the antioxidant power of biological fluid, including capillary blood are lacking, the monitoring of such effects, including the measurement of its recovery, recovery rate, the prediction of its evolution and the determination of the individual physical activity and life-style conditions for managing it in a verifiable manner cannot be achieved by the current and available art.

Hence, there is a need for a reliable, easy, practical, personal and predictive method, biomarker, associated device and sensors for measuring the impact of physical activity and life-style conditions on the antioxidant power of biological fluids in a verifiable manner, so that negative consequences of over-oxidation or over-reduction can be prevented.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention meets the above mentioned need by providing a method for assessing the recovery and/or the rate of recovery after exercising of the antioxidant power of a biological fluid of an individual human or animal after said individual has exercised, the method comprising:

(i) collecting at least three biological fluid samples over a fixed period of time, including one or more first biological fluid samples collected before exercising (resting samples RS), one or more second biological fluid samples collected during or immediately after exercising (after exercising samples AES), and one or more third biological fluid samples collected at a predefined elapsed time since exercising (after recovery samples (ARS);

(ii) using voltammetry or amperometry so as to obtain a primary electrochemical signature for each biological fluid sample collected in step (i);

(iii) mathematically treating each of the primary electrochemical signatures obtained in step (ii) so as to obtain substantially bell-shaped secondary electrochemical signal curves; (iv) integrating each of the secondary electrochemical signal curves obtained in step (iii) so as to extract from each curve a scalar quantity, the value of which corresponds to the area below the bell-shaped curve;

(v) calculating at least three mean values for sets of scalar quantities obtained in step (iv), the mean values including a first value (RV) equal to the mean value of one or more scalar quantities corresponding to the one or more first biological samples respectively, a second value (AEV) equal to the mean value of one or more scalar quantities corresponding to the one or more second biological samples respectively, and a third value (ARV) equal to the mean value of one or more scalar quantities corresponding to the one or more third biological samples respectively;

(vi) assessing the recovery and/or the rate of recovery of the antioxidant power based on the at least three mean values calculated in step (v).

An advantage of the present invention is that it makes it possible to predict the evolution of the antioxidant power and to determine the appropriate physical activity and life-style conditions for managing the antioxidant power in a verifiable manner.

According to a particular implementation of the invention, the biological fluid of an individual human or animal is selected from the group consisting of capillary blood, whole blood, arterial blood, venous blood, plasma, serum, saliva, urine, sweat and tears. Preferably, the biological fluid is capillary blood.

According to another particular implementation, the form of voltammetry or amperometry used in the method of the invention is selected from the group consisting of cyclic voltammetry, differential voltammetry, and linear sweep voltammetry. Preferably, linear sweep voltammetry with a voltage increasing inside a range comprised between -0.5 and +1 .5 volts.

According to yet another particular implementation, the recovery of the antioxidant power is assessed by determining whether or not the difference between the first value (RV) and the third value (ARV) amounts to more than 10% of the first value.

According to still another particular implementation, the rate recovery of the antioxidant power is assessed by computing the slope between the second value (AEV) and the third value (ARV).

According to still another particular implementation, the succession of method steps that precedes the step of assessing the recovery and/or rate of recovery of the antioxidant power is performed periodically, and the successive mean values of the at least three values computed during each performance of the succession of steps are plotted. Preferably, the step of assessing the recovery of the antioxidant power further includes predicting a stable, reductive or oxidative trend in recovery based on the means plot.

According to still another particular implementation, the step of assessing the recovery comprises comparing the first, second and third calculated mean values with reference values obtained from previous monitored individuals, and/or with theoretical reference values.

According to still another particular implementation, the method of the invention comprises an additional step of establishing a programme for managing external conditions for an individual (including life-style and exercising) in such a manner as to control the recovery and rate of recovery of the antioxidant power of a biological fluid of the individual in a verifiable manner. According to a second aspect, the present invention meets the above mentioned need by providing an electrochemical device and its associated sensors adapted to be used for implementing the method of the invention.

According to particular embodiment of the second aspect of the invention, the sensors associated with the electrochemical device comprise a working electrode, a reference electrode and a counter electrode, the working, reference and counter electrodes being combined so as to form a single disposable interface (strip) for one sample.

BRIEF DESCRIPITON OF THE DRAWINGS

Other features and advantages of the present invention will appear upon reading the following description, given solely by way of non-limiting example, and made with reference to the annexed drawings, in which:

- figure 1 illustrates the applied mathematical treatment of a measured electrochemical signal for obtaining the antioxidant power of the measured sample under the form of a single number;

- figure 2 illustrates the antioxidant power differences between capillary blood samples taken before exercising, after exercising and after a recovery period of 2 hours thereafter, demonstrating the use of the present disclosure for identifying such differences;

- figure 3 illustrates three distinct zones, including the recovery, reductive and oxidative ones and the measurement of the rate of recovery of the antioxidant power corresponding to the slope between the value of the second (after exercising) and the third measurement (after a recovery period); - figure 4 illustrates the use of the cumulated mean of the antioxidant power taken regularly and once a day during a 5 days period and before any physical activity for predicting its evolution over time. Series 2 and 4 shows a reductive and oxidative trend, respectively;

- figure 5 illustrates the use of the method described herein for comparing the effect of different exercising and resting conditions for controlling the evolution of the antioxidant power of capillary blood in a verifiable manner.

DETAILLED DESCRIPTION

Figure 1 shows the measured signal of a capillary blood sample of one volunteer, the modulation curve corresponding to a Fermi-Dirac derived dimension less function used for the mathematical treatment of the measured signal (dotted line), and the resulting modulated bell shape curve and the calculated antioxidant capacity, corresponding to the surface below the modulated curve taken between 0.2 and 1 volt.

Figure 2 shows three modulated curves from three capillary blood samples taken from one volunteer a three different times, before (140 nW) and after (163 nW) exercising, and after a recovery period of 2 hours (153 nW).

Figure 3 shows the antioxidant power of the capillary blood of one volunteer, measured from three samples taken before and after 30 minutes of exercising and after a recovery period of 2 hours. The recovery (+/-10% of the resting value), reductive and oxidative zone are indicated. The slope between the second and third measurement defines the rate of recovery of the antioxidant power of capillary blood.

Figure 4 shows the daily results (series 1 and 3) and their corresponding cumulated mean (series 2 and 4) of the antioxidant power of capillary blood samples of 2 volunteer taken before any physical activity and measured once a day over a period of 5 days. The slope of series 2 and 4 is used to predict the stable, reductive or oxidative evolution of the antioxidant power.

Figure 5 shows the results obtained before, after exercising and after a period of recovery from capillary blood samples of 2 volunteers. Volunteer 1 (series 1 ) practiced a mild physical activity during the recovery period, while volunteer 2 (series 2) observed a full rest during the same time. Depending on the intensity of the physical activity or resting, the recovery value of the antioxidant power differs significantly, exemplified by the comparison between series 1 (medium intensity) and series 2 (full rest). Within this disclosure, the antioxidant power refers to the measurement of the rate of oxidation of the sample measured by linear sweep with a potential comprised between -0.5 and +1 .5 volts. Such antioxidant power corresponds to the resistance of the sample to an electrochemically forced oxidation. A low antioxidant power corresponds to a low resistance to oxidation, while a high antioxidant power corresponds to a high resistance to oxidation. Likewise, electrochemical signatures correspond to original voltamograms plots generated by linear sweep voltammetry. Their mathematical processing includes, but is not limited to treatment of the entire or fraction of the original signal with a dimension less Fermi- Dirac or Fermi-Dirac derived mathematical function so that the final result can be expressed as a bell shape curve in a first step, and as single number, by integrating the so obtained bell curve, in a second step.

In the present disclosure, exercising includes, but is not limited to any form of physical activity, exercising, sport, training and/or competition resulting in an increase of oxygen uptake and cardiac frequency. This disclosure is based on the finding that the electrochemical signature of a biological sample, including but not limited to the one of a capillary blood sample can be used for monitoring the impact of physical activity on its antioxidant power in man and animals. Such results can then be used for measuring its recovery by comparing its value before, after exercising and after recovery as well as for predicting its evolution by plotting its cumulated mean over a fixed period of time and with the same number of measurements for each time splits within this period.

The present disclosure provides methods, biomarkers and its associated device and sensors for measuring the recovery and rate of recovery of the antioxidant power of biological fluids after exercising and for predicting its evolution in man and animals requiring a minimum of 3 measurements or sets of measurements. The first one before physical activity, the second one after having stopped the physical activity and the third one anytime thereafter. In agreement with the above description, it includes the following steps.

The recording of the electrochemical signature of a biological sample is peformed in agreement with electrochemical procedures, involving the use of an electrochemical recording unit and a corresponding sample interface, harbouring 3 electrodes, working, counter and reference ones combined into a single sensor. The electrochemical signature of the biological sample is obtained by linear sweep with an increasing potential ranging from -0.5 to +1 .5 volts.

Once the original electrochemical signal has been obtained, part or the entire signal is processed with a mathematical treatment, including, but not limited to a dimension less Fermi-Dirac or Fermi- Dirac derived function so that the final result can be expressed as a bell shape curve in a first step, and a single number, by integrating the surface below the bell shape curve in a second step. A minimum of three single or sets of measurements must be obtained. The first one before exercising, the second one after exercising and the third one after a fixed period of time thereafter.

A positive or negative difference between the first and third results smaller than 10% of the first result indicates the recovery of the antioxidant power of the measured sample. The rate of recovery is obtained by calculating the slope between the second and third measurements. A slope comprises between minus and plus 5% indicates a stable trend, while a slope larger than plus 5% indicates a reductive trend and a slope below minus 5%, an oxidative one.

The prediction of the evolution of the antioxidant power of capillary blood is obtained by calculating the slope of the plotted cumulated mean of the antioxidant power over a fixed period of time, including the same number of measurements per time spits within this period. A positive or negative slope predicts an increasing, respectively decreasing evolution of the antioxidant power of capillary blood over time, corresponding to a reductive or oxidative trend, respectively. The comparison of the obtained recovery, rate of recovery and predictive results with equivalent results and the corresponding data bank, containing verified and theoretical references is also used. The comparison of recovery, rate of recovery and prediction is then used, in relationship with physical activity and life style conditions, for managing the recovery in a verifiable manner.

The present disclosure provides a method for measuring the recovery, the rate of recovery and for predicting the evolution of the antioxidant power of the measured sample, so that oxidative or reductive stress, leading to increased risks of injuries and poor performance can be prevented. Accordingly, the efficacy of specific physical activity, exercising and recovery and life style conditions that modify and control the recovery of the antioxidant power of the measured sample can also be verified.

Example:

The use of this disclosure for measuring the recovery, recovery rate, predicting the evolution and managing the conditions that control the antioxidant power of capillary blood of one volunteer is performed according to the following sequence:

1 . Measurement of a capillary blood sample with an electrochemical measuring unit and its corresponding sensor, comprising three integrated electrodes.

2. The voltamogram of the capillary blood sample shown in figure 1 is obtained by linear sweep with a potential comprised between -0.5 and +1 .5 volts.

3. The obtained original voltamogram is processed with a dimensionless and Fermi-Dirac derived mathematical function so that the end result, the antioxidant power of the capilary blood sample, can first be expressed as a bell shape curve (figure 1 ), and then as a single number, by integrating the surface below the bell shape curve.

4. A minimum of three measurements, one before exercising, the second one after exercising and the third one within a fixed period of time thereafter are performed (figure 2). Each time point can then show either a stable, decreasing or increasing result over time.

5. The difference between the first and third measurement is calculated. A positive or negative difference smaller than 10% of the resting score, measured before exercising corresponds to its recovery (figure 3). The slope between the second and third measurement is calculated for obtaining the rate of recovery of the antioxidant power of capillary blood (figure 3). The slope of the plotted cumulated mean of the antioxidant power over a fixed period of time, including the same number of measurement per unit of time within this period is calculated with the measured results, in this case measured before physical activity and used for predicting its evolution, showing an increasing and reductive trend or a decreasing and oxidative trend (figure 4). Exercising and/or resting period can be modified, so that the recovery, recovery rate and prediction of the evolution of the antioxidant power of capillary blood can be optimized, based on the comparison of the obtained results with equivalent reference ones (figure 5).

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Claims

CLAI MS

1 . A method for assessing the recovery and/or the rate of recovery after exercising of the antioxidant power of a biological fluid of an individual human or animal after said individual has exercised, the method comprising:

(iii) mathematically treating each of the primary electrochemical signatures obtained in step (ii) so as to obtain substantially bell-shaped secondary electrochemical signal curves;

(iv) integrating each of the secondary electrochemical signal curves obtained in step (iii) so as to extract from each curve a scalar quantity, the value of which corresponds to the area below the bell-shaped curve;

2. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to claim 1 , wherein the biological fluid of an individual human or animal is selected from the group consisting of capillary blood, whole blood, arterial blood, venous blood, plasma, serum, saliva, urine, sweat and tears.

3. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to claim 1 or 2, wherein step (ii) is performed using a form of voltammetry selected from the group consisting of cyclic voltammetry, differential voltammetry, and linear sweep voltammetry.

4. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to claim 3, wherein step (ii) is performed using linear sweep voltammetry with a potential increasing inside a range comprised between -0.5 and +1 .5 volts.

5. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein step (iii) consists in multiplying each of the functions corresponding to the primary electrochemical signatures by a Fermi-Dirac or a Fermi-Dirac derived dimensionless function.

6. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein the mathematical treatment of step (iii) is applied only to a selected portion of each of the primary electrochemical signatures.

7. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein step (vi) comprises determining whether or not the difference between the first value (RV) and the third value (ARV) amounts to more than 10% of the first value.

8. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein step (vi) comprises determining a rate of recovery by calculating a ratio of the difference between the second value (AEV) and the third value (ARV) over said predefined elapsed time.

9. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein the succession of steps (i) to (v) is performed periodically, and wherein step (vi) comprises plotting the successive mean values of the at least three mean values computed in step (v) during each performance of the succession of steps (i) to (v).

10. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to claim 9, wherein step (vi) comprises predicting a stable, reductive or oxidative trend in recovery based on the means plot.

1 1 . The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to claim 10, wherein a stable trend is predicted when the slope of the means plot is comprised between plus or minus 5%, an oxidative trend when the slope is below minus 5%, and finally a reductive trend when the slope is above plus 5%.

12. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein step (vi) comprises comparing the first, second and third values calculated in step (v) with reference values obtained from previous monitored individuals, or with theoretical reference values.

13. The method for assessing the recovery and/or the rate of recovery of the antioxidant power according to any one of the preceding claims, wherein it comprises an additional step of establishing a programme for managing external conditions for the individual (including life-style and exercising) in such a manner as to control the recovery and rate of recovery of the antioxidant power of a biological fluid of the individual in a verifiable manner.

14. A complete electrochemical measuring system for the implementation of the method of any one of claims 1 to 13, the system comprising a wireless measuring unit, a reference electrode, a counter electrode and a working electrode.

15. The complete electrochemical measuring system of claim 14, wherein the reference electrode, the counter electrode and the working electrode are combined into a single disposable sensor.