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WO2004068140A1 - Procede de determination de la capacite antioxydante totale (tac) et de la capacite antioxydante totale corrigee (corrtac) dans des fluides - Google Patents

Procede de determination de la capacite antioxydante totale (tac) et de la capacite antioxydante totale corrigee (corrtac) dans des fluides Download PDF

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Publication number
WO2004068140A1
WO2004068140A1 PCT/IB2004/000150 IB2004000150W WO2004068140A1 WO 2004068140 A1 WO2004068140 A1 WO 2004068140A1 IB 2004000150 W IB2004000150 W IB 2004000150W WO 2004068140 A1 WO2004068140 A1 WO 2004068140A1
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Prior art keywords
tac
crocin
reagent
determination
sample
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Elias Castanas
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MEDICON HELLAS SA
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MEDICON HELLAS SA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • TAC Total Antioxidant Capacity
  • corrTAC Corrected Total Antioxidant Capacity
  • the present invention relates to a new method for the determination of Total Antioxidant Capacity (TAC) and Corrected Total Antioxidant Capacity (corrTAC) in fluids. More specifically the present invention relates to a new automated method for the deter- mination of TAC and corrTAC. This method may be used for investigational and diagnostic purposes.
  • TAC Total Antioxidant Capacity
  • corrTAC Corrected Total Antioxidant Capacity
  • the present invention also relates to a crocin reagent, and to the use of said crocin reagent for the determination of TAC in fluids.
  • the pre- sent invention relates to a kit for use in the determination of TAC.
  • ROS Reactive oxygen species
  • RNS reac- tive nitrogen species
  • the relative sufficiency of the organism antioxidant defences is critical in the development of oxidative stress in patients affected by a number of diseases, including HIV infections [8, 9], neurode- generation [10] , diabetes [11-14] , angina [15-18] , certain forms of cancer [19-27] , and probably ageing [28-30] .
  • These diseases are characterized by an overproduction of free radicals, i.e. when the antioxidant defence of an organism is overwhelmed or are established when a deficit of defences of the organism against oxidation occurs.
  • the primary defence against oxidative stress in extracellular fluids results from a number of low molecular weight antioxidant molecules either water- (ex. ascorbic acid) or lipid-soluble (ex. Vitamin E) .
  • antioxidants can also be generated during nor- mal metabolism (ex. uric acid, bilirubin, albumin, thiols) or introduced in the body by the consumption of dietary products rich in antioxidants (olive oil, fruits and vegetables, tea, wine, etc) [7] .
  • the sum of endogenous and food-derived antioxidants repre- sents the total antioxidant capacity of the extracellular fluid.
  • the levels of these antioxidants are suitable not only as a protection against oxidation, but could also reflect their consumption during acute oxidative stress states. The cooperation among different antioxidants provides a greater protection against attack by reactive oxygen or nitrogen radicals, than any single compound alone.
  • the overall antioxidant capacity may give more relevant biological information compared to that ob- tained by the measurement of individual parameters, as it considers the cumulative effect of all antioxidants present in plasma and body fluids [31] .
  • Recently a theory has been proposed, taking into ac- count the redox potentials of exogenous and endogenous antioxidants, and the construction of a chained reaction, in which a given antioxidant, after oxidation is regenerated through a number of reactions in- volving a number of other, more potent antioxidants. Through this cascade, interactions among the lipid and the aqueous phases could be established [32] :
  • antioxidant activi ty corresponds to the rate constant of a single antioxidant against a given free radical.
  • Antioxidant capaci ty is the measure of moles of a given free radical scavenged by a test solution, independently of the capacity of any one antioxidant present in the mixture [31] . Therefore, for plasma being a heterogeneous so- lution of diverse antioxidants, the antioxidant status is better reflected by antioxidant capacity rather than activity.
  • This capacity is a combination of all the redox chain antioxidants, including different analytes such as thiol bearing proteins, and uric acid. Therefore, the plasma antioxidant capacity is rather a concept than a simple analytical determination. Indeed, an increase of the antioxidant capacity of plasma indicates absorption of antioxidants and an improved in vivo antioxidant status [33] , or an adaptation mechanism to an increased oxidative stress. Nevertheless, due to the participation of diverse metabolites to the antioxidant capacity of hu- man plasma, its increase may not be necessarily a desirable condition.
  • One aspect of the present invention relates to a method for the determination of Total Antioxidant Capacity (TAC) performed at a preset temperature and comprising the steps of: a. mixing a sample with a crocin reagent, b. incubating the mixed sample obtained in step a for a predetermined period of time, c. adding an oxidizing agent to the incubated sample obtained in step b, d. measuring the absorbance of the sample obtained in step c at a preset wavelength after a predetermined period of time from the addition of the oxidizing agent .
  • the method further comprises the steps of: e.
  • the method even further comprises the steps of: g. determination of endogenous antioxidants and their active metabolites in the sample in parallel with the determination of TAC, and h. calculating corrTAC by subtracting the TAC deriving from endogenous antioxidants and their active metabolites determined in step g from the TAC ob- tained in step f.
  • Another aspect of the present invention relates to a crocin reagent for use in the method mentioned above obtainable by a process comprising the steps Of: A. isolating crocin from saffron,
  • step B adjusting pH of the isolated sample obtained in step A with a buffer having a specific pH
  • step B mixing the sample obtained in step B with an inert filler to obtain a certain final concentration of the filler
  • step D lyophilising an aliquot of the mixed sample obtained in step C.
  • step E sealing the aliquot obtained in step D under vacuum
  • step F reconstitution of the aliquot obtained in step E with buffer prior to the use of said crocin reagent in the method mentioned above.
  • Yet another aspect of the present invention relates to the use of said crocin reagent for the de- termination of TAC and/or corrTAC in fluid samples by use of the above-mentioned method.
  • Still another aspect of the present invention relates to a kit for use in the determination of TAC and/or corrTAC by use of the above-mentioned method.
  • the following terminology will be used in accordance with the definitions set out below.
  • automated method as used herein is meant any method using an autoanalyzer in order to perform the TAC determinations.
  • autoanalyzer is meant any automated apparatus used in clinical laboratories to measure analytes in any kind of fluids .
  • Antioxidant is de- fined as a substance, which, at low concentrations compared to that of an oxidizable substrate, significantly delays or prevents oxidation of the substrate".
  • Antioxidant Capacity is defined as the integrated capacity of a given fluid to scavenge, on a molar basis, a test solution, independently of the capacity of any antioxidant present in the mixture.
  • Total Antioxidant Capacity is meant the overall antioxidant capacity, which is the antioxidant capacity de- riving from both endogenous and exogenous antioxidants as well as their active metabolites.
  • endogenous antioxidants and their active metabolites refers to antioxi- dants generated as a result of metabolism e.g. uric acid, bilirubin, albumin, and thiols. It will be obvious for a person skilled in the art to supplement the list of endogenous antioxidants and their metabo- lites.
  • exogenous antioxidants and their active metabolites refers to antioxidants deriving from food and beverages and their metabolites produced as the food is digested.
  • corrTAC Corrected Total Antioxidant Capacity
  • standard curve is defined as the correlation between the concentration of a given antioxidant in various samples and the measured absorbance of said samples when subjected to the method according to the present invention.
  • any antioxidant may be employed when preparing said standard curve.
  • the water- soluble synthetic antioxidant Trolox is used in a concentration ranging from 0-3 mmol/1.
  • fluid sample any fluid in which the TAC and/or corrTAC may be measured.
  • biological fluids in humans and other mammals such as plasma, urine, and cerebrospinal fluid
  • cell culture media such as cell culture media
  • extracts from food and beverages . It will be ob- vious for a person skilled in the art to extend the list of fluids in which TAC may be determined.
  • incubating is meant leaving a sample on standing for a specified or unspecified period of time at specified or unspecified conditions.
  • sample blank is a sample consisting of distilled water, which is subjected to the method according to the present inven- tion.
  • the absorbance measured in the sample blank is determined in order to correct the measured absorbance of the sample for the contribution of absorbance originating from the reagents used when performing the method.
  • oxidizing agent as used herein is defined as an agent capable of oxidizing a substance. It will be obvious for a person skilled in the art that any oxidizing agent may be used in step c of the method according to the present invention.
  • a reagent comprising the oxidizing agent 2,2'- azobis- (2-amidinopropane) dihydrochloride (ABAP) reagent is used in the present invention.
  • ABAP 2,2'- azobis- (2-amidinopropane) dihydrochloride
  • the method according to the present invention is performed at a tempera- ture in the range of 15 to 50°C, more preferred in the range of 30 to 45°C, even more preferred in the range of 35 to 39°C, and most preferred at 37°C.
  • the predetermined period of time in step b in the method accord- ing to the present invention is usually in the range of 1 to 5000 seconds, more preferred in the range of 20 to 500 seconds, even more preferred in the range of 100 to 200 seconds, and most preferred 160 seconds .
  • the predetermined period of time in step d in the method accord- ing to the present invention is usually in the range of 10 to 10000 seconds, more preferred in the range of 100 to 1000 seconds, even more preferred in the range of 200 to 300 seconds, and most preferred 256 seconds .
  • the preset wavelength at which the absorption is measured in step d of the method according to the present invention is set in the range of 300 to 600 nm, more preferred in the range of 400 to 500 nm, even more pre- ferred in the range of 440 to 460 nm, and most preferred at 450 nm.
  • crocin is obtained by isolating it from saffron.
  • a person skilled in the art will understand that the way of obtaining crocin - whether it is purchased, extracted from a plant or prepared synthetically - does not change the inventive idea of the present invention.
  • reagent any substance that participates in the reaction of interest .
  • the reagent may be in a form of a solution ready-to-use or it may be a solid, a concentrate or a lyophilising composition requiring dilution prior to use.
  • a person skilled in the art may easily extend the list of states for reagents.
  • buffer as used herein is defined as a solution that resists changes in pH. Most buffers consist of either a weak base and its conjugate acid or a weak acid and its conjugate base. A buffer is used whenever there is a need to maintain the pH of a solution at a constant and predetermined level . In the present invention a phosphate buffer having a pH of 7.4 is preferably used.
  • the term "filler” as used herein is defined as an inactive substance, which does not interfere with the TAC determination in the method according to the present invention.
  • the buffer used in step B when obtaining the crocin reagent according to the invention is preferably a buffer having a specific pH in the range of 5.0 to 9.0, more preferred in the range of 6.8 to 8.0, even more preferred in the range of 7.3 to 7.5. Most preferably, the buffer is a phosphate buffer having a pH of 7.4.
  • the filler used in step C when obtaining the crocin reagent according to the invention is preferably present in a concentration from 5 to 15% by weight, more preferred from 6 to 10% by weight, even more preferred from 7 to 8% by weight, and most preferred 7.5% by weight.
  • the method for determining TAC and/or corrTAC according to the present invention may be performed on any fluid in the liquid state, but preferably the method is performed on body fluids, cell culture media, food and beverages samples and extracts.
  • kits are used in the determination of TAC and/or corrTAC.
  • Said kit is a set of key parts needed for the determination of TAC and/or corrTAC.
  • the kit may comprise crocin reagent, ABAP reagent, Trolox reagent, buffers, and other components.
  • the kit may comprise any other tool (as for example glass equipment) , which may become helpful when performing the determination of TAC and/or corrTAC.
  • Each reagent and buffer may be in a form ready-to-use, or in a concentrated form (either in solid or liquid state) requiring dilution prior to use.
  • TAC and/or corrTAC is determined in extracts of food and beverages .
  • the extraction method used for obtaining the extracts includes any mixing method in which one or more compound (s) is transferred from a substance to a liquid by mixing for a given period of time.
  • the resulting extract is the liquid phase into which the compound (s) is transferred. Two or more consecutive mixings may be necessary in order to obtain a sufficient transfer of the compound (s) .
  • TAC and/or corrTAC is determined in food and beverage extracts, but it will be obvious for a person skilled in the art to complement the list of extracts in which TAC and/or corrTAC may be determined.
  • Caffeic (97%), fer- ulic (99%) and protocatechuic (99%) acids were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO).
  • Sinapic (98%), syringic (98%), 3 , 4-dihydroxy- phenylacetic acids and epigalocatechin were from Sigma Chemical Co. (St. Louis, MO), while quercetine was a kind gift from Prof. J. Vercauteren (University of Bordeaux I, France) .
  • Spectrophotometric determinations were performed with a Kontron Uvicon 860 Spectrophotometer (Paris, France) .
  • An Athos 2001 microplate reader (Vi- enna, Austria) , with a filter at 450 nm was used for initial microplate assay.
  • the automated microplate apparatus Triturus (Grifols, Barcelona, Spain) , equipped with a pipettor, an incubator and a reader station, connected and driven from a PC station was used.
  • the Olympus AU 400 autoana- lyzer was used for the automated assay.
  • the above method was adapted to the Triturus microplate automate, using exactly the above- described protocol .
  • the adaptation of the method to the Olympus autoanalyzer was based on the measurement of the inhibition that is caused by total antioxidants on the bleaching of crocin from ABAP.
  • the procedure was as follows: A concentration of crocin isolated from saffron as previously described [38] , was adjusted at 25 ⁇ M with 10 mM phosphate buffer, pH 7.4 (A) and mixed with an inert filler at a final concentration of 7.5% (w/w) .
  • the assay was performed at 37°C in the follow- ing steps: Two ⁇ l of sample, calibrator or control were mixed with 250 ⁇ l of crocin reagent (Rl) and this mixture was incubated for 160 s. Thereafter, 250 ⁇ l ABAP reagent R2 were added and the decrease in absorbance at 450nm was measured 256 s later.
  • An analogous reaction was performed for the sample blank assay using blank reagents, as mentioned above. The difference between the two signals for the reaction and the reagent blank reaction (the reaction using deionised water as sample) was used to establish the standard curve and to calculate values of controls and serum samples. The result was always negative, indicating an inhibition in the development of color compared to the reaction in the absence of antioxidants i.e., sample.
  • Conjugated Bilirubin Conjugated bilirubin (CalbioChem, La Jolla, CA) was dissolved into a very small quantity of water. Turbidity: Intralipid 10% (Pharmacia (Hellas) SA, Ath- ens, Greece) was used with no further treatment. Ascorbate: (Merck, Darmstadt, Germany) was dissolved into distilled water. Bovine serum albumin (protease free) : Serological Products (IL) .
  • Blood samples were usually collected on K 3 - EDTA, and immediately centrifuged in a refrigerated centrifuge. They were aliquoted, and stored at -80°C until use.
  • Figure 1 presents the antioxidant capacity of a number of phenolic acids (A) and polyphenols (B) on the TAC assay.
  • the indicated concentrations of different phenolic acids (A) and purified polyphenols (B) were assayed for their inhibitory effect of cro- cin oxidation, over the range 0-10 ⁇ g/ml for phenolic acids and 10 "7 -10 " ⁇ M for polyphenols.
  • a Trolox standard curve is presented for comparison.
  • Figure 2 shows the effect of freeze/thawing cycles on the plasma concentration of TAC. Ten different plasmas, collected on K-EDTA were assayed for TAC following the manual method described in the Methods section. In A, different plasma volumes were assayed after 1-4 freezing cycles.
  • FIG. 4 shows the correlation between TAC and TEAC. Correlation of the TAC assay and the Randox Total Antioxidant Status assay (TAS) , based on the TEAC method, initially described by Miller and Rice-Evans [35-37] . Both assays were performed serially on the same autoanalyzer on 44 human plasma samples . Diamonds show four outliers.
  • TAS Randox Total Antioxidant Status assay
  • Figure 5 indicates the effect of dilution on the TAC assay.
  • Figure 6 shows the correlation of TAC and Randox TAS assays with uric acid concentration of human plasmas. The assay was performed on 44 human plasma samples, serially using the two assays, on the same autoanalyzer (Olympus AU400) . Squares represent TAS results, while diamonds represent TAC results. The straight lines and the correlation equations are also shown .
  • Figure 7 shows the effect of different analytes on the TAC assay
  • Figure 8 indicates the effect of diet on the TAC values in human plasma.
  • Figure 10 presents the corrected TAC levels during antineoplasmatic treatment of children with malignancies .
  • Figure IB presents the cro- cin inhibition in the presence of two polyphenols: quercetine and epigallocatechin. As shown, both polyphenols produce a dose-dependent inhibition of crocin oxidation, indicating that the assay could be used for the determination of antioxidants in a number of natural products, such as wine, olive oil or tea.
  • the inventors have linearized the Trolox curve through a Log/Normal plot, calculated the equation of the straight line, and reported directly the obtained inhibitions of plasma to the equation.
  • the coefficient of correlation (r 2 ) of the Trolox linearized assay was >0.996.
  • the inventors have assayed 10 different plasma samples, collected on K 3 -EDTA, for their antioxidant activity, immediately after collection and after 1-4 freeze-thawing cycles. After one freeze/thawing cycle results were identical (see also Table 1) .
  • Results are presented as mean+SEM of ten different assays, and expressed as mmol/L.
  • FIG. 2B presents the calculated TAC expressed as Trolox equivalent per ⁇ l of plasma.
  • mi- nor changes in the calculated TAC/ ⁇ l were found when 3.3-5 ⁇ l of plasma were used in the assay and the number of freeze cycles was ⁇ 3.
  • a linear decrease of the calculated TAC/ ⁇ l was found when 2 ⁇ l of plasma were used.
  • the TAC per unit of volume calculated and presented in Figure 2B (0.37+0.07 ⁇ g of Trolox equivalent) is not significantly different from the one calculated from the IC 50 s presented in Figure 2A (0.38+0.04).
  • TAC total antioxidant status
  • a concentration of 4.0 mmol/L of Trolox in normal saline was diluted serially, and assayed 10 times.
  • the same dilution of Trolox was diluted in a plasma sample (target value 0.86 mmol/L) 1/1 (v/v) , and assayed 10 times, performing serial dilutions in normal saline.
  • the results, presented in Figure 5, show that the TAC assay show a linear relation of values in log/log coordinates, indicating that dilution of plasmas do not introduce an interference in the TAC assay.
  • FIG. 6 shows the correlation of TAC and TAS assays on uric acid concentration of human plasmas. As shown, straight lines crossed the ordinate axes at 0.57 and 0.82 mmol/L of TAC and TAS, respectively. This result indicates that about 49% of the measured TAC activity is due to uric acid, significantly lower than the contribution of TAS on uric acid, calculated to 68%. The slope of the straight line obtained between TAC and uric acid indicates that 0.11 mmol/L of TAC correspond to 1 mg/dl of uric acid.
  • the corrected TAC assay may be of value in a variety of circumstances: For example, as presented in Figure 8, exclusion of animal products, and a diet rich in fruits, vegetables and olive oil, dramatically increases the TAC, and much more the corrected TAC values. Furthermore, as shown in Figure 9, al- though the Total TAC is decreased significantly in hemodialyzed patients, the corrected TAC increases. This result makes questionable the common dogma of vitamin substitution of this group of patients.
  • corrTAC assay may be its substantial increase in ibese patients, during and after weight reduction. Indeed, in malignant obesity status (body weight >150 kg) , corrTAC is dramatically decreased ( ⁇ 0.2 mmol/L). After a weight loss of 35 kg, its levers return to normal, indicating a normalization of the redox state of the organism.

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Abstract

L'invention concerne un procédé automatisé permettant de déterminer la capacité antioxydante totale (TAC) et la capacité antioxydante totale corrigée (corrTAC) dans des fluides. De plus, l'invention concerne un réactif de crocine et l'utilisation de celui-ci aux fins de détermination de la TAC dans des fluides. Ce procédé présente un caractère avantageux en ce qu'il peut être utilisé dans un milieu médical, du fait qu'il permet d'obtenir des résultats stables pendant au moins trois cycles de résistance au gel/dégel et qu'il présente une sensibilité plus élevée comparativement à d'autres analyses de la TAC.
PCT/IB2004/000150 2003-01-27 2004-01-22 Procede de determination de la capacite antioxydante totale (tac) et de la capacite antioxydante totale corrigee (corrtac) dans des fluides Ceased WO2004068140A1 (fr)

Applications Claiming Priority (2)

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GR20030100034A GR1004535B (el) 2003-01-27 2003-01-27 Αυτοματοποιημενη μεθοδος προσδιορισμου της ολικης αντιοξειδωτικης ικανοτητος (total antioxidant capacity -tac-), και της διορθωμενης ολικης αντιοξειδωτικης ικανοτητος (corr tac) σε βιολογικα υγρα καιεκχυλισματα τροφων και ποτων βασισμενη στην αναστολη οξειδωσεως της κροκινης
GR20030100034 2003-01-27

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006022558A1 (fr) * 2004-08-26 2006-03-02 Syft Technologies Limited Procede d'evaluation de l'activite antioxydante d'huiles
EP1701158A1 (fr) 2005-03-10 2006-09-13 Chimar S.r.l. Procédé pour la détermination de la capacité de réduction de produits ou de mélanges contenant des substances antioxydantes
WO2008003170A3 (fr) * 2006-07-07 2008-02-21 1692124 Ontario Inc Additif pour carburant
US8057557B2 (en) 2006-07-07 2011-11-15 1692124 Ontario Inc. Fuel additive
US9063070B2 (en) 2007-05-18 2015-06-23 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US10036723B2 (en) 2007-05-18 2018-07-31 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US8512548B2 (en) 2007-05-18 2013-08-20 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9423372B2 (en) 2007-05-18 2016-08-23 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US8709709B2 (en) 2007-05-18 2014-04-29 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
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