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WO2010045986A1 - Procédé pour mesurer la capacité antioxydante totale et la puissance oxydative totale - Google Patents

Procédé pour mesurer la capacité antioxydante totale et la puissance oxydative totale Download PDF

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Publication number
WO2010045986A1
WO2010045986A1 PCT/EP2008/064486 EP2008064486W WO2010045986A1 WO 2010045986 A1 WO2010045986 A1 WO 2010045986A1 EP 2008064486 W EP2008064486 W EP 2008064486W WO 2010045986 A1 WO2010045986 A1 WO 2010045986A1
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Prior art keywords
oxidative
antioxidants
measured
oxidants
reagent
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Ceased
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PCT/EP2008/064486
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English (en)
Inventor
Bert Zomer
Henk Bloemen
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ZOMERBLOEMEN BV
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ZOMERBLOEMEN BV
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Priority to PCT/EP2008/064486 priority Critical patent/WO2010045986A1/fr
Publication of WO2010045986A1 publication Critical patent/WO2010045986A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase

Definitions

  • 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.
  • 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.
  • 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
  • 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.
  • DTT depletion assay 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.
  • DTT dithiothreitol
  • 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).
  • DEP diesel exhaust particulates
  • 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+.
  • 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.
  • 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.
  • acridan ester 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.
  • the measurement system consists of an oxidative part and a reductive part.
  • total antioxidant capacity is the capability of a mixture of antioxidants to exert their antioxidant properties
  • total oxidative capacity is the capability of a mixture of oxidants to exert their oxidative properties.
  • 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
  • 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).
  • 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.
  • TAOC trigger reagent is prepared by mixing 2.5 uL of an HRP solution (10ug/mL) with 0.003% of 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):
  • Reaction [1] describes the light vs time curve (dB/dt) of the signal without antioxidant (QH).
  • QH antioxidant
  • Fitting consists of a first run without antioxidants to give reaction rate constants kl and k2 for reaction [1]
  • 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
  • Example 10 DTT and Ascorbate depletion by phenanthraquinone
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé permettant de mesurer la capacité de résistance à l'oxydation dans des matériaux échantillons. Ce procédé consiste à ajouter à l'échantillon, un test de provocation oxydatif, et après un certain temps, à mesurer la capacité antioxydante résiduelle totale au moyen d'une réaction de chimioluminescence catalysée par la peroxydase de raifort ou par la mesure des produits de réduction de l'oxygène tels que le peroxyde d'hydrogène, au moyen d'une réaction de chimioluminescence. L'invention concerne également la mesure de la puissance oxydative d'un test de provocation oxydatif dans un échantillon par adjonction à l'échantillon d'un ou de plusieurs antioxydants, et la mesure de la capacité antioxydante résiduelle totale par une réaction de chimioluminescence catalysée par la peroxydase de raifort ou par la mesure des produits de réduction de l'oxygène tels que le peroxyde d'hydrogène au moyen d'une réaction de chimioluminescence. Ce procédé permet d'évaluer les effets du stress oxydatif, par exemple de matières particulaires ou d'autres nanomatériaux, et également d'évaluer la capacité de résistance, par exemple, de matières biologiques confrontées à ces tests de provocation.
PCT/EP2008/064486 2008-10-24 2008-10-24 Procédé pour mesurer la capacité antioxydante totale et la puissance oxydative totale Ceased WO2010045986A1 (fr)

Priority Applications (1)

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PCT/EP2008/064486 WO2010045986A1 (fr) 2008-10-24 2008-10-24 Procédé pour mesurer la capacité antioxydante totale et la puissance oxydative totale

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2008/064486 WO2010045986A1 (fr) 2008-10-24 2008-10-24 Procédé pour mesurer la capacité antioxydante totale et la puissance oxydative totale

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WO2010045986A1 true WO2010045986A1 (fr) 2010-04-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012028167A3 (fr) * 2010-05-20 2012-05-18 Innohyphen Bv Chimioluminescence d'ester d'acridinium lors du déclenchement réducteur
IT201700064897A1 (it) * 2017-06-12 2018-12-12 Francesco Violi Valutazione dell’attività scavenger in un campione biologico e relativi metodi
RU2821319C1 (ru) * 2023-10-02 2024-06-20 Павел Вадимович Логинов Способ оценки антиоксидантной активности природных соединений

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Bioluminescence and Chemiluminescence Progress and Current Applications", 2001, STANLEY AND CRICKA EDS. WORLD SCIENTIFIC SINGAPORE, XP009116586 *
GHISELLI ANDREA ET AL: "Total antioxidant capacity as a tool to assess redox status: Critical view and experimental data", FREE RADICAL BIOLOGY AND MEDICINE, vol. 29, no. 11, 1 December 2000 (2000-12-01), pages 1106 - 1114, XP002527329, ISSN: 0891-5849 *
GIROTTI STEFANO ET AL: "Chemiluminescent determination of total antioxidant capacity during winemaking", LUMINESCENCE (CHICHESTER), vol. 21, no. 4, July 2006 (2006-07-01), pages 233 - 238, XP002527408, ISSN: 1522-7235 *
OSMAN A M ET AL: "COMPARATIVE STUDIES OF THE CHEMILUMINESCENT HORSERADISH PEROXIDASE-CATALYSED PEROXIDATION OF ACRIDAN (GZ-11) AND LUMINOL REACTIONS: EFFECT OF PH AND SCAVENGERS OF REACTIVE OXYGEN SPECIES ON THE LIGHT INTENSITY OF THESE SYSTEMS", LUMINESCENCE, WILEY, CHICHESTER, GB, vol. 15, 1 January 2000 (2000-01-01), pages 189 - 197, XP007900758 *
TOKLU ET AL: "Silymarin, the Antioxidant Component of Silybum marianum, Prevents Sepsis-Induced Acute Lung and Brain Injury", JOURNAL OF SURGICAL RESEARCH, ACADEMIC PRESS INC., SAN DIEGO, CA, US, vol. 145, no. 2, 22 October 2007 (2007-10-22), pages 214 - 222, XP022514646, ISSN: 0022-4804 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012028167A3 (fr) * 2010-05-20 2012-05-18 Innohyphen Bv Chimioluminescence d'ester d'acridinium lors du déclenchement réducteur
IT201700064897A1 (it) * 2017-06-12 2018-12-12 Francesco Violi Valutazione dell’attività scavenger in un campione biologico e relativi metodi
RU2821319C1 (ru) * 2023-10-02 2024-06-20 Павел Вадимович Логинов Способ оценки антиоксидантной активности природных соединений

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