WO2010045986A1 - Method to measure total antioxidant capacity and total oxidative power - Google Patents

Abstract

A method for measuring oxidative resistibility in sample materials involves adding an oxidative challenge to the sample, and after a certain time measuring the residual total antioxidant capacity by means of a chemiluminescent horseradish peroxidase catalyzed reaction or by measuring reduced oxygen products like hydrogen peroxide by means of a chemiluminescent reaction. The invention also includes measuring oxidative power of an oxidative challenger in a sample by adding one or more antioxidants to the sample and measuring the residual total antioxidant capacity by means of a chemiluminescent horseradish peroxidase catalyzed reaction or by measuring oxygen reduction products like hydrogen peroxide by means of a chemiluminescent reaction. The method is useful to assess oxidative stress effects of e.g. particulate matter and other nanomaterials and also to assess the resistibility of e.g. biological materials against these challenges.

Description

METHOD TO MEASURE TOTAL ANTIOXIDANT CAPACITY AND TOTAL OXIDATIVE

POWER

VERFAHREN ZUR BESTIMMUNG DER TOTALEN ANTIOXIDATIVE KAPAZITAT UND DER TOTALEN OXIDATIVE LEISTUNG

METHODE POUR LA MESURE DE LA CAPACITE ANTIOXYDANTE TOTALE ET DE

LA PUISSANCE OXYDATIVE TOTALE

Description of the Invention

This invention relates to a method to assess the oxidative stress induced by a sample towards a reductive system. The invention also relates to a method to assess the oxidative resistibility of a sample towards an oxidative challenge.

Background of the invention related art

Oxidative stress in biological systems relates to the way oxygen is used as an energy source.

Oxygen is hereby reduced to water, a process which involves the transfer of four electrons and four protons. Intermediate oxygen reduction products, e.g. superoxide, hydrogen peroxide and hydroxyl radical, are reactive species capable of reacting with biological molecules like protein, DNA, and cell membrane.

To counteract these effects the biological system responds to these challenges for instance by enzymatic degradation or by antioxidants. Oxidative stress can be seen as the result of an imbalance between oxidative challengers and antioxidative resistibility.

Oxidative stress is believed to be an important factor in the development of several human diseases

(cancer, heart attack and stroke, auto-immune and neurodegenerative diseases, asthma, and in the aging process) and also in the exposure to particulate matter (PM).

Therefore it is desirable to be able to assess the oxidative resistibility of a biological system and the power of the oxidative challenges.

Currently, oxidative stress is assessed by measurement of so-called biomarkers. These are compounds that indicate the result of an oxidation reaction, like oxidized biomolecules. These biomarkers are measured using common analytical techniques like chromatography and immunoassay. To give a more descriptive estimate of oxidative stress a panel of biomarkers should be measured. All these methods have in common that oxidative stress is measured in the form of oxidized biomolecules as a result of a challenge. This approach necessitates the use of a rather large panel of biomarkers to be determined. WO 2006/083293 teaches the use of five tests including one in vivo test to characterize the oxidative stress protective capacity of an antioxidant substance.

A well known assay to assess oxidative stress is the so called DTT depletion assay in which the depletion of DTT is measured photometrically after a challenge of dithiothreitol (DTT) with e.g. particulate matter. Oxidative stress induced by for instance diesel exhaust particulates (DEP) can then be expressed as depletion of DTT per microgram of DEP per minute (nmol/ug/min). Cellular studies with murine macrophages have shown a correlation of this activity with hemeoxygenase-1 induction ability. (Li N, Sioutas C, Cho AK, Schmitz D, Misra C, Sempf J, Wang M, Oberley T, Froines J, NeI A: Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Persp 2003, 111:455-460.)

It has been shown that redox-active compounds catalyze the reduction of oxygen to superoxide by DTT, which is oxidized to its disulfide. The remaining thiol is allowed to react with DTNB, generating the mixed disulfide and 5-mercapto-2-nitrobenzoic acid which is determined by its absorption at 412nm. The PM-dependent DTT consumption is measured under conditions such that the rate is linear, i.e., when less than 20% is depleted. Catalytic activity is expressed as the rate of DTT consumption per minute per microgram of sample less the activity observed in the absence of PM. The DTT-assay is not affected by metal ions like Fe3+ and Cu2+. It is well known that metal ions like Fe3+ and Cu2+ are involved in redox reactions leading to the production of deleterious ROS. The DTT depletion assay being insensitive towards these ions is therefore underestimating oxidative stress by samples like PM, that also contain metals. The DTT assay is described in greater detail by Cho et al. (Cho AK, Sioutas C, Miguel AH,

Kumagai Y, Schmitz DA, Singh M, Eiguren-Fernandez A, Froines JR: Redox activity of airborne particulate matter at different sites in the Los Angeles Basin. Environ Res 2005, 99:40-47). This assay provides a measure of the overall redox activity of the sample based on its ability to catalyze electron transfer between DTT and oxygen in a simple chemical system. Other assays used for this purpose include the consumption of ascorbate ( Mudway I, Stenfors N, Blomberg A, Helleday R, Dunster C, Marklund S, Frew A, Sandstrom T, Kelly F: Differences in basal airway antioxidant concentrations are not predictive of individual responsiveness to ozone: a comparison of healthy and mild asthmatic subjects. Free Radic Biol Med 2001, 31:962-974), oxidation of dichlorofluorescin (Venkatachari P, Hopke P, Grover B, Eatough D: Measurement of particle- bound reactive oxygen species in rubidoux aerosols. J Atmos Chem 2005, 50:49-58.) and an ESR procedure (Shi T, Schins R, Knaapen A, Kuhlbusch T, Pitz M, Heinrich J, Borm P: Hydroxyl radical generation by electron paramagnetic resonance as a new method to monitor ambient particulate matter composition. J Environ Monitor 2003, 5:550-556) to monitor the levels of free radical species.

70

Some time ago a method was published to measure total antioxidant capacity that was based on an enhanced HRP catalyzed chemiluminescent reaction involving acridan ester. (Zomer et al, Development of an assay for antioxidant activity using acridan ester GZ-Il. in Bioluminescence and Chemiluminescence Progress and Current Applications. Eds Stanley and Kricka. World

75 Scientific Singapore 2002: 309-312). This method was subsequently improved by fine tuning the start and trigger reagent as described in example 1. It was anticipated that this approach could also be used for the assessment of oxidative capacity of e.g. PM. An advantage of this approach would be the universal detection system that could be used with all kinds of antioxidants. Thus it would be possible to measure for instance antioxidant depletion caused by an oxidative challenge by a single

80 method. Another advantage associated with the use of acridan ester is its use at physiological conditions. Moreover, the assay can be performed in microtiter format allowing for high throughput of samples.

Preliminary experiments with metal ions gave some unexpected results: When ascorbate solutions 85 were incubated with ferric chloride or copper sulphate solutions, Cu2+ ions were capable to deplete very efficiently ascorbate in a catalytic process while Fe3+ ions gave only moderate depletion of ascorbate (example 3). At the same time Fe3+ ions when incubated with ascorbate were capable of producing hydrogen peroxide more efficiently than Cu2+ ions (example 4). The generated reactive oxygen species (ROS) was measured using chemiluminescence (example 2). 90

Both of these aspects, the depletion of antioxidant defenses and the production of deleterious products needs to be measurable in order to truly assess oxidative stress. Current methods focus only on one aspect (DTT or ascorbate depletion) or measurement of biomarkers which can be seen as compounds that are formed after an oxidative challenge (like oxidation products from fatty acids, 95 DNA, proteins). When an oxidative challenge like by Diesel Exhaust Particulates (DEP) is assessed this can be done by measuring the depletion of Total Antioxidant Capacity (TAOC). This is exemplified in examples 5 and 7. On the other hand it can also be measured be the hydrogen peroxide production as shown in example 6. The proposed method is also capable to measure the TotalAntiOxidant Capacity (TAOC) of mixtures of antioxidants allowing for assessment of 100 oxidative resistibility of mixtures towards oxidative challenge (example 9). The invention

It is an objective of the present invention to overcome the aforementioned deficiencies in the prior art and to present a measurement system that allows the assessment of the capacity to resist

105 oxidative challenge (the oxidative resistibility or Total Antioxidant Capacity) as well as the capacity to generate reactive oxygen species (the Total Oxidative Capacity). Hence, the measurement system consists of an oxidative part and a reductive part. In this, total antioxidant capacity is the capability of a mixture of antioxidants to exert their antioxidant properties and total oxidative capacity is the capability of a mixture of oxidants to exert their oxidative properties.

110 If oxidative resistibility is to be measured the oxidative part consists of an oxidative challenger while the reductive part consists of one or more antioxidative entities. The oxidative challenger can be a single compound like hydrogen peroxide, a synthetic mixture e.g. ROS producing systems, or also a mixture of unknown composition like particulate matter. Antioxidative entities comprise one or more antioxidants like ascorbate dithiothreitol, glutathione, vitamin E, or a mixture e.g. lung

115 lining fluid, plasma or serum.

If the power of oxidative challenge is to be measured the reductive part consists of one or more antioxidants like ascorbate dithiothreitol, glutathione, urate, vitamin E.

The measurement system measures either the depletion of the total antioxidant capacity (TAOC) or 120 the formation of hydrogen peroxide during the reaction of the oxidative part with the reductive part. The measurement of hydrogen peroxide can be accomplished in a number of ways known in the art. A useful method involves the reaction of hydrogen peroxide with acridinium ester yielding light emission which is proportional to the amount of hydrogen peroxide (example 2). An additional advantage of using acridinium esters like GYl 1 is the improvement in signal/background ratio 125 when working with reductants like DTT (or in general reductants with a strong nucleophilic character (RSH). This is due to the competition reaction that takes place between the two nucleophiles H2O2 and RSH in their reaction with acridinium ester. The reactions (exemplified for the PM catalyzed oxidation of RSH) that occur are depicted in Figure 1 :

Insert Figure 1

130 Incubating PM and RSH yields hydrogen peroxide while at the same time the concentration of RSH gets diminished. In the second step hydrogen peroxide and RSH will compete for the available acridinium ester. In the absence of PM no H2O2 will be formed, therefore no RSH will be consumed (apart from the uncatalyzed slow reaction with molecular oxygen). These two effects will give raise to low background values for the relative light units. When there is PM present H2O2

135 will be formed while RSH concentration will drop giving a higher signal relative to the background. This unexpectedly yields an assay that is very sensitive down to the detection of less than 15 ng/well (example 12). The short analysis time justifies the approach to a continuous monitoring system.

140 Examples

Example 1: Measurement of Total Antioxidant Capacity (TAOC)

TAOC signal reagent is prepared by mixing a 175 uL of a solution of GZl 1 see: US6030803/EP0915851 (5 mg/mL) with 25 uL of a solution of 4-phenylphenol (5 mg/mL). 20 uL of this solution is diluted with 10 mL of PBS containing 0.01% Tween20.

145 TAOC trigger reagent is prepared by mixing 2.5 uL of an HRP solution (10ug/mL) with 0.003% of H2O2 in PBS.

In wells of a microtiterplate ascorbate dilutions (0-8uM, 50 uL/well) in water were pipetted. Subsequently, TAOC signal reagent was added, followed by trigger reagent. The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about

150 forty minutes. The chemiluminescent signal was plotted against time yielding graphs like the one shown in Figure 2.

Insert Figure 2 From the Figure it is clear that increasing ascorbate concentrations result in larger lag times

Example 2: Measurement of H2O2

155 Preparation of HTOOTO signal reagent:

Dilute a solution of GYU see: US6030803/EP0915851 (lmg/mL) 1:10000 in 0.01M HN03

Preparation of HTOOTO trigger reagent:

Prepare a carbonate buffer pH 9.6 10OmM

Prepare a dilution series of H2O2 in water in the range 0.1-100 uM starting from a 3% H2O2

160 solution. Add 100 uL of the H2O2 dilutions to wells of a microtiterplate. To each well add lOuL of the HTOOTO signal reagent. Place the plate inside a plate luminometer. Using the microtiterplate luminometer dispense 50 uL of the HTOOTO trigger reagent. The resulting chemiluminescent signal is followed kinetically during 5 seconds. Alternatively, the signal can be integrated for 5 seconds. The plot of the signal against the H2O2 concentration gives a calibration line (Figure 3)

165 from which unknowns can be calculated.

Insert Figure 3 From the Figure it is clear that hydrogen peroxide can be measured over a wide concentration range with excellent linearity.

Example 3: Depletion of ascorbate by CuSO4 and FeC13

170 In wells of a microtiterplate mixtures of FeC13 (0-10OuM) and CuSO4 dilutions (0-1OuM) in water were incubated with equal volumes of ascorbate (lOuM) during 15 minutes. Subsequently, TAOC signal reagent (example 1) was added, followed by trigger reagent (example 1). The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about half an hour. The chemiluminescent signal was plotted against time yielding graphs (see Figure 4 and 5):

175 Insert Figure 4

Insert Figure 5 From the Figures it is clear that ascorbate is depleted much more efficiently by Cu2+ than by Fe3+.

Example 4: H2O2 production by ascorbate and CuSO4 and FeC13

In wells of a microtiterplate mixtures of FeC13 (0-10OuM) and CuSO4 dilutions (0-1OuM) in water 180 were incubated with equal volumes of ascorbate (lOuM) during 15 minutes. Subsequently, a solution containing H2O2 signal reagent (lOuL/well) was added to each well. The plate was put inside a plate luminometer and H2O2 trigger reagent was dispensed into each well, followed by measurement of the chemiluminescent signal during 5 seconds. A graph of the results is shown in Figure 6:

185 Insert Figure 6

From Figure 6 it is clear that ascorbate with Fe3+ is capable of producing hydrogen peroxide much more efficiently than with Cu2+ whereas Cu2+ depletes ascorbate preventing Fe3+ from generating H2O2.

Example 5: Depletion of ascorbate by Diesel Exhaust Particles

190 In wells of a microtiterplate DEP extract dilutions (0-1350 ng/well) in water were incubated with equal volumes of ascorbate (lOuM) during 15 minutes. Subsequently, TAOC signal reagent (example 1) was added, followed by trigger reagent (example 1). The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about half an hour. The chemiluminescent signal was plotted against time yielding graphs as shown in Figure 7:

195 Insert Figure 7 It is clear from the Figure that DEP is depleting ascorbate in a dose-response correlated way.

Example 6: H2O2 production by DTT and Exhaust Particles

In wells of a microtiterplate DEP extract dilutions (0-45 ug/mL) in water were incubated with equal volumes of dithiothreitol (DTT) (25uM) during 5 minutes. Subsequently, a solution containing 200 H2O2 signal reagent (example 2) (lOuL/well) was added to each well. The plate was put inside a plate luminometer and H2O2 trigger reagent was dispensed into each well, followed by measurement of the chemiluminescent signal during 5 seconds. A graph of the results is shown in Figure 8:

Insert Figure 8

205 Example 7: Depletion of DTT by Diesel Exhaust Particles

In wells of a microtiterplate DEP extract dilutions (0, 193, 225, 270, 338, 450, 675, and 1350 ng/well) in water were incubated with equal volumes of DTT (25uM) during 15 minutes. Subsequently, TAOC signal reagent (example 1) was added, followed by trigger reagent (example 210 1). The plate was put inside a plate luminometer and the chemiluminescent signal from each well was measured for about half an hour. The chemiluminescent signal was plotted against time yielding graphs as shown in Figure 9:

Insert Figure 9 It is clear from the Figure that DEP is depleting DTT in a dose-response correlated way.

215 Example 8: Comparison of the depletion of DTT and ascorbate by DEP

An experiment according to example 7 was set up in such a way that different concentrations of DTT (40, 50, 60, 70, 80, 90, and 100 uM, respectively) were incubated with methanolic DEP extract (5 ug/mL) during 25 minutes. In the same experiment different concentrations of ascorbate (0, 4, 8, 12, 16, 20, 24, and 28 uM, respectively) were incubated with the same methanol DEP 220 extract (5 ug/mL) during 25 minutes. Subsequently, TAOC signal reagent (example 1) was added, followed by trigger reagent (example 1, with 5uL HRP (lOug/mL) in 10 mL 0.003% H2O2 in PBS). The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about one and a half hour. The chemiluminescent signal was plotted against time yielding graphs as shown in Figure 10 for lOOuM DTT without and with DEP (5ug/mL):

225 Insert Figure 10 An example of data plots involving ascorbate are shown in Figure 11 :

Insert Figure 11

From these Figures it is clear that DEP depletes DTT more efficiently than ascorbate. The data were also fitted to a theoretical curve obtained from the reaction pairs (1) and (2): 230

A -> B -> C [1] B + QH -> A + Q [2]

Reaction [1] describes the light vs time curve (dB/dt) of the signal without antioxidant (QH). In the 235 presence of antioxidant (QH) B reacts with QH forming A which causes a delay in the production of B and hence of light. Fitting consists of a first run without antioxidants to give reaction rate constants kl and k2 for reaction [1] Next the curves involving the presence of known concentrations of antioxidants were fitted using kl and k2 of the first run. This yields values for QH and k3. QH values were fitted using linear regression with the known concentrations of QH. From 240 this calibration line QH concentrations of the depletion experiments can be calculated. From this analysis DTT depletion by DEP was calculated to be 230 pmol/ug/min, while ascorbate depletion by DEP was calculated to be 20 pmol/ug/min.

Example 9: Depletion of DTT, ascorbate and DTT-Ascorbate mixtures by DEP

In wells of a microtiterplate antioxidant dilutions (50 uL/well) were pipetted according to the layout 245 of Figure 12:

Insert Figure 12

Columns 3,4,7,8,11, and 12 received 5 ug/mL of a DEP extract while columns 1,2,5, 6,9,and 10 received blank extraction fluid. The plate was allowed to incubate for 25 minutes.

Subsequently, TAOC signal reagent (example 1) was added, followed by trigger reagent (example

250 1, with 5uL HRP (lOug/mL) in 10 mL 0.003% H2O2 in PBS). The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about one and a half hour. The kinetic curves were analyzed using the method as described in example 8. Results are shown in Figure 12. It can be deduced from these results that both DTT and ascorbate are depleted. Interestingly, the mixture of DTT and Ascorbate is depleted more than is expected from the

255 individual antioxidants. Example 10: DTT and Ascorbate depletion by phenanthraquinone

In wells of a microtiterplate antioxidant dilutions of phenanthraquinone (PQ) (50 uL/well, uM) were pipetted according to the layout of Figure 13.

Insert Figure 13

260 Into the wells of every row solutions (50 uL/well) containing PQ (PQ, 0-10OuM) were pipetted.

After 25 minutes TAOC reagent was added to all wells followed by trigger reagent (see example 1). The plate was put inside a plate luminometer and the chemiluminescent signal from each well measured for about one and a half hour. The resulting kinetic curves were analyzed using the TAOC program as described in example 8.

265 From this analysis remaining Total Antioxidant Capacity could be calculated. Comparison with starting TAOC-values resulted in depletion data. For Ascorbate and DTT these data are plotted against the PQ concentration as shown in Figures 14 and 15, respectively.

Insert Figure 14

270 Insert Figure 15

It is clear from the Figures that DTT is depleted much more by PQ than ascorbate is.

Example 11: H2O2 generation by DEP

275 In wells of a microtiterplate 10 uL of a solution of GY 11 ( 150 ng/mL in 0.1 M HN03) was added. From an extract of 0.1 mg/mL DEP in water serial dilutions were prepared resulting in seven diluted samples ranging from 0.3125 to 20 ug/mL. To each mL of sample 40 uL of a lOOuM DTT solution in PBS was added. After incubating for five minutes 50 uL from each sample was added to the wells of the microtiterplate containing the GYl 1 solution. The plate was put into the plate

280 luminometers. Automatic dispensing of 50 uL of a trigger solution (0.4M carbonate buffer pH 9.6) resulted in flashes of light that were integrated during 5 seconds. The results are shown in Figure 16.

Insert Figure 16

285 From the Figure it is clear that there is a linear response over the entire range of the sample dilutions. As little as 0.31 ug/mL (15 ng/well) of DEP can be measured.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method to measure the interaction between total antioxidant capacity of antioxidants and total oxidant capacity of oxidants.

2. A method according to claim 1 wherein the interaction is measured in the form of a depletion of total antioxidant capacity.

3. A method according to claim 1 wherein the interaction is measured in the form of the generation of reduced oxygen products.

4. A method according to claim 2 wherein total antioxidant capacity is measured using a chemiluminescence reaction.

5. A method according to claim 4 wherein the chemiluminescence reaction involves an enhanced chemiluminescence horseradish peroxidase catalyzed reaction.

6. A method according to claim 5 wherein the chemiluminescence reaction involves an acridan ester or organic hydrazide.

7. A method according to claim 3 wherein reduced oxygen product is hydrogen peroxide.

8. A method according to claim 7 wherein reduced oxygen product is measured using a chemiluminescence reaction.

9. A method according to claim 8 wherein the chemiluminescence reaction involves the use of acridinium ester.

10. A method according to claim 1 wherein antioxidants include one or more naturally occurring antioxidants or artificial ones.

11. A method according to claim 10 wherein naturally occurring antioxidants include vitamin C and E, urate, glutathione, cysteine, and etcetera.

12. A method according to claim 10 wherein artificial antioxidants include dithiothreitol, Trolox, butylated hydroxytoluene (BHT), and etcetera.

13. A method according to claim 1 wherein oxidants include metal ions, hydrogenperoxide, quinones, and chemical and biological systems inducing oxidative properties, such as xanthine oxidase, haloperoxidase, and etcetera.

14. A method according to claim 13 wherein chemical systems include particulate matter and nanomaterials.

15. A method according to claim 1 consisting of one or more oxidants which are incubated with sample material during a certain time. Thereafter either TotalAntiOxidant Capacity (TAOC) or TotalOxidantCapacity (TOC) is measured. Sample material without oxidants serves as controls.

16. A method according to claim 15 wherein oxidants include but are not limited to hydrogen peroxide, Cu- salts, Fe-salts, quinone, ozone or mixtures thereof.

17. A method according to claim 15 wherein sample material is biological material like serum, saliva, tissue material, synthetic and natural lung lining fluid, and etcetera.

18. A method according to claim 1 consisting of one or more antioxidants which are incubated with one or more oxidants during a certain time. Thereafter, either TotalAntiOxidant Capacity (TAOC) or TotalOxidantCapacity (TOC) is measured. Sample material without antioxidants serve as controls.

19. A method according to claim 18 wherein sample material is particulate matter or extracts thereof.

20. Kits according to claim 2 consisting of TAOC signal reagent, trigger reagent, and standard oxidants capable of assessing a sample's oxidative resistibility.

21. Kits according to claim 20 wherein TAOC signal reagent consists of acridan ester and enhancer.

22. Kits according to claim 21 wherein acridan ester is GZ-11 and enhancer is 4-phenylphenol.

23. Kits according to claim 21 wherein trigger reagent consists of a mixture of HRP and hydrogen peroxide.

24. Kits according to claim 3 consisting of HTOOTO signal reagent, trigger reagent, and one or more antioxidants.

25. Kits according to claim 24 wherein HTOOTO signal reagent is an acridinium ester

26. Kits according to claim 25 wherein the acridinium ester is GY-11.

27. Kits according to claim 24 wherein trigger reagent is an aqueous solution with pH higher than 7.