WO2004068140A1 - A method for the determination of total antioxidant capacity (tac) and corrected total antioxidant capacity (corrtac) in fluids - Google Patents

Abstract

The present invention relates to a new automated method for the determination of Total Antioxidant Capacity (TAC) and Corrected Total Antioxidant Capacity (corrTAC) in fluids. Furthermore, the present invention relates to a crocin reagent for the determination of TAC in fluids. This method is especially advantageous for the use in clinical practice, as it produces stable results during at least three freeze thawing cycles, and is more sensitive compared to other TAC assays.

Description

A method for the determination of Total Antioxidant Capacity (TAC) and Corrected Total Antioxidant Capacity (corrTAC) in fluids

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.

The present invention also relates to a crocin reagent, and to the use of said crocin reagent for the determination of TAC in fluids. Finally, the pre- sent invention relates to a kit for use in the determination of TAC.

Background of the invention

Reactive oxygen species (ROS) as well as reac- tive nitrogen species (RNS) are produced as a consequence of normal aerobic metabolism in animal species [1-3] . These "free radicals" are removed and/or inactivated in vivo by a battery of antioxidants [1-5] . A biological antioxidant is defined as a substance, which, at low concentrations compared to that of the oxidizable substrate, significantly delays or prevents this oxidation [4, 6, 7] . Individual members of the antioxidant defence team are employed to prevent the generation of free ROS and RNS, to destroy poten- tial antioxidants and to scavenge ROS and RNS. However, 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) . These 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. In addition, 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. Thus, 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] :

A great variety of methods have been proposed for the assay of total antioxidant activity or capac- ity of serum or plasma [reviewed extensively and critically in 7, 31] . In these reviews a clear distinction among antioxidant activi ty and capaci ty is made: Antioxidant activi ty corresponds to the rate constant of a single antioxidant against a given free radical. Antioxidant capaci ty, on the other hand, 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. Indeed, in some cases, such as renal failure (uric acid) , icteric status (bilirubin) , hepatic damage (hypoalbuminemia) the variation of several metabolites falsely modifies the plasma antioxidant capacity, a situation returning to normal values after correction of the underlying disease [34] .

A new automated method for the determination of TAC has been developed. This method is especially advantageous for the use in clinical practice, as it produces stable results during at least three freeze thawing cycles, and is more sensitive compared to other TAC assays.

Description of the invention

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 . In a preferred embodiment the method further comprises the steps of: e. subtracting the absorbance of a sample blank ob- tained by subjecting a sample of distilled water to the steps from a to d from the absorbance obtained in step d, f . determining TAC in the sample by comparing the cal- culated absorbance obtained in step e to a standard curve .

In another preferred embodiment 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,

B. adjusting pH of the isolated sample obtained in step A with a buffer having a specific pH,

C. mixing the sample obtained in step B with an inert filler to obtain a certain final concentration of the filler,

D. lyophilising an aliquot of the mixed sample obtained in step C.

In a preferred embodiment the process for obtaining the crocin reagent further comprises the steps of:

E. sealing the aliquot obtained in step D under vacuum, and

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. In the present description the following terminology will be used in accordance with the definitions set out below.

By the term "automated method" as used herein is meant any method using an autoanalyzer in order to perform the TAC determinations. By the term "autoanalyzer" is meant any automated apparatus used in clinical laboratories to measure analytes in any kind of fluids .

As used herein, the term "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.

By the term "Total Antioxidant Capacity" ("TAC") as used herein 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.

The term "endogenous antioxidants and their active metabolites" as used herein 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.

As used herein the term "exogenous antioxidants and their active metabolites" refers to antioxidants deriving from food and beverages and their metabolites produced as the food is digested. The term "Corrected Total Antioxidant Capacity" ("corrTAC") as used herein is defined by subtracting the antioxidant capacity deriving from endogenous antioxidants and their active metabolites from the TAC, and thereby obtaining a value representing the con- tribution deriving from exogenous antioxidants and their active metabolites in the total antioxidant capacity.

The term "standard curve" as used herein 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. A person skilled in the art will acknowledge that any antioxidant may be employed when preparing said standard curve. However, in the present description the water- soluble synthetic antioxidant Trolox is used in a concentration ranging from 0-3 mmol/1.

By the term "fluid sample" as used herein is meant any fluid in which the TAC and/or corrTAC may be measured. Of special interest are biological fluids in humans and other mammals (such as plasma, urine, and cerebrospinal fluid) , cell culture media, and 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.

By the term "incubating" as used herein is meant leaving a sample on standing for a specified or unspecified period of time at specified or unspecified conditions.

The term "sample blank" as used herein 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. The term "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. Prefera- bly, a reagent comprising the oxidizing agent 2,2'- azobis- (2-amidinopropane) dihydrochloride (ABAP) reagent is used in the present invention.

In a preferred embodiment 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.

In another preferred embodiment 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 .

In another preferred embodiment 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 . In yet another preferred embodiment 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.

In one aspect of the present invention crocin is obtained by isolating it from saffron. However, 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.

By the term "reagent" as used herein is meant 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.

The term "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.

In one aspect of the present invention a kit is 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. In other words, the kit may comprise crocin reagent, ABAP reagent, Trolox reagent, buffers, and other components. Furthermore, 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.

In another aspect of the present invention 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) . In the description 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.

The invention is now further described with reference to the following experimental work, which should not be construed as limiting for the invention.

Experimental

Chemicals and biochemicals

2,2' -Azobis- (2-amidinopropane) dihydrochloride (ABAP) and 6-hydroxy-2 , 5 , 7, 8-tetramethylchroman-2- carboxylic acid (Trolox C) were purchased from Sigma-

Aldrich (St Louis, MO) . ABAP was dissolved just before use with a 10 mM Phosphate buffer (pH 7.4) at concentrations varying from 4-10 mg/ml . The usual concentration used was 5 mg/ml . Saffron (Greek red saffron of the COUPE class) was purchased from the Association of Saffron producers (Krokos, Kozani, GR) . All other chemicals and biochemicals were from Sigma (St Louis, MO) , or Merck (Darmstad, Germany) .

Crocin was isolated from saffron by wa- ter/methanol extraction after repeated extraction with ethyl-ether, as described previously [38] . After extraction, crocin was tested for purity (absorbance peak at 440 and a shoulder at 464) , diluted in 30% methanol in water, diluted fivefold with phosphate buffer (10 mM, pH 7.4), and the concentration of crocin was adjusted to 20 μM with buffer, using the mo- lecular absorbance coefficient of crocin e ₄₃=89000 M^{" 1}cm^_1 [39] . Aliquots of crocin, protected from light, were stored at -20°C, until use. 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) .

Instruments

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. Later, 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. Finally, the Olympus AU 400 autoana- lyzer was used for the automated assay.

Methods

1. Comparative example: Colorimetric method for the determination of the TAC of human plasma

The method for manual TAC plasma determination was previously described by Lusignoli et al [38] . In brief, in each well of the microplate 100 μl of crocin and 50 μl of the plasma sample, diluted in phosphate buffer were pipetted. The reaction was initiated with the addition of 100 μl of prewarmed (37°C) ABAP (5 mg/ml) , and crocin bleaching was made by incubating the plate at a humidified thermostated oven at 37°C, for 60-75 min. Blanks consisted of crocin, plasma samples, and phosphate buffer (100, 50 and 100 μl respectively) and were run in parallel. The ab- sorbance was measured at 450 nm. The specific absorbance was then calculated by subtracting the corresponding blanc value, and the antioxidant activity was calculated as the ratio: 100 x (Abs₀- Abs_sampi_e) /Abs₀, in which Abs₀ was the absorbance in the absence of antioxidants, and Abs_sapi_e was the absorbance in the presence of sample. A standard curve of the water-soluble synthetic antioxidant Trolox, prepared prior to use, ranging from 0-10 μg/ml was equally assayed under the same conditions. 2. Automation of TAC method

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 μM with 10 mM phosphate buffer, pH 7.4 (A) and mixed with an inert filler at a final concentration of 7.5% (w/w) . Three ml of the above solution were dispensed into glass vials, lyophilized on BOC Edwards Calumatic Lyoflex 0.8 lyophilizer and sealed under vacuum after the end of lyophilization. Each vial was reconstituted with 7.5ml of buffer A prior to use. The reconstituted solution was Rl of the final assay while R2 was a ready-to-use liquid reagent containing 50mg/ml ABAP in buffer A. For the auto- mated procedure a blank reagent was run together with the above-described reagent. The blank reagent was consisted of buffer A as Rl_bi_ank and 50mg/ml ABAP in buffer A as R2_bι_ank.

The assay was performed at 37°C in the follow- ing steps: Two μl of sample, calibrator or control were mixed with 250 μl of crocin reagent (Rl) and this mixture was incubated for 160 s. Thereafter, 250 μl 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.

All biochemical parameters were assayed on an Olympus AU400 autoanalyzer. Reagent for the measure- ment of Uric acid was from OLYMPUS Diagnostica GmbH, Lismmehan, Ireland and the reagent for TAS activity was from RANDOX Laboratories Ltd, United Kingdom. All other reagents were from Sigma (St Louis, MO) except where indicated. For the interference studies the following materials were used: Hemoglobin: Erythrocytes were washed with physiological saline, and a hemoglobin solution was prepared through hemolysis, by adding distilled water. Bilirubin: Crystallized bilirubin was dissolved into a very small quantity of weak alkaline (0.1N NaOH) solution. 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) .

All biochemical parameters were assayed on an Olympus AU 400 autoanalyzer, with Olympus reagents provided from Medicon Hellas (Gerakas, Greece) . (Albumin OSR6102, total bilirubin 0SR6112, iron 0SR6123 uric acid 0SR6136) . 3. Collection of blood samples

Forty healthy blood donors, aged 21-52 years (28 males, and 12 females) from the region of Herak- lion, Crete, were used for the determination of the reference interval of the TAC assay. They were on a normal diet, while the inventors had little information of their nutritional and smoking habits. In addition, one hundred samples from a hematology labora- tory, with no indication of the underlying pathology were further used for the correlation of TAC assay with the uric acid, bilirubin, iron and protein concentrations. Finally, 17 nuns, from an orthodox monastery in the region of Heraklion were assayed, after a 40 days ritual fasting before Easter. This fasting consisted in the abolishment of all animal food from their diet .

Blood samples were usually collected on K₃- EDTA, and immediately centrifuged in a refrigerated centrifuge. They were aliquoted, and stored at -80°C until use.

Results

Description of the drawings 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 μg/ml for phenolic acids and 10^"7-10^"Ξ 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. In B, the calculated TAC concentration per μl of plasma was reported over the number of freezing cycles. Mean+SEM of 10 different plasma samples, from normal individuals. Figure 3 depicts the linearity of the automated TAC assay. A standard curve of 0-3 mmol/L of Trolox was assayed on an Olympus AU400 auto-analyzer. The results of the inhibition of crocin oxidation are presented as the absorbance at 450 nm. A linear fit of the data is also presented.

Figure 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.

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. TAC assay of 17 samples taken from nuns, from a local monastery, after 40 days of ritual fasting. Their diet, during this period, was exempted of any animal food, and rich in fruits, vegetables, and extra-virgin olive oil, and compared the obtained results with 12 normal blood donors. Each dot presents an individual. Mean val- ues+SEM are also shown

Figure 9 shows the assay of TAC and corrected TAC during hemo-dialysis (n=45)

Figure 10 presents the corrected TAC levels during antineoplasmatic treatment of children with malignancies .

1. Validation of the assay 1.1 Reagents and antioxidants As indicated in the original paper for the assay [38] , crocin spectra in the oxidized and the reduced form present an absorption maximum at 440 nm. This was also verified, with similar results. In addition, different concentrations of ABAP, in the pre- sence of 5μM crocin, were assayed. Concentrations of 4-5 mg/ml produced a very sensitive curve, while the sensitivity was lost at ABAP concentrations >6 mg/ml . In view of these results, an ABAP concentration of 5 mg/ml was chosen. On the other hand, an optimum cro- cin concentration of 5 μM was chosen, after a series of different dilutions, ranging from 2 to 20 μM, concentrations which, after 60-75 min incubations generated similar curves as in the original publication [38] .

A series of antioxidant phenolic acids, found in a number of foods was tested for inhibition of crocin, in the assay generated by the use of the above concentrations of ABAP and crocin. The results are presented in Figure 1A. As shown, all phenolic acids produced a dose-dependent inhibition of crocin oxidation. Caffeic acid was the most potent inhibitor of crocin oxidation, followed by sinapic and ferulic acid. Protocatechuic, siringic and dihydrophenyl acetic acids exhibited the lower antioxidant activity, a result reported previously in other in vitro systems [40] . Figure IB on the other hand, 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.

1.2 Plasma

In the original assay, serial dilutions of plasma were tested for their inhibitory activity on crocin oxidation by ABAP, the antioxidant capacity of the plasma was calculated by the sigmoidal fitting of the resulting curve, and the calculation of the IC₅₀, reported finally to the Trolox inhibitory curve. In the present work, the inhibitory activity of human plasma on crocin oxidation was directly reported to the Trolox curve, and the equivalent Trolox activity was directly calculated. Long incubation times (60-75 min) were used, in order to minimize the lag time of antioxidant activity reported in the initial kinetic assay of crocin bleaching [41] . For this purpose, 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 (r²) of the Trolox linearized assay was >0.996. The inventors have assayed 10 different plasma samples, collected on K₃-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) .

Table 1.

Effect of different anticoagulants and blood preservation on the TAC assay.

Results are presented as mean+SEM of ten different assays, and expressed as mmol/L.

The same result was obtained from one to three freezing cycles, as shown in Figure 2A, for plasma volumes from 2 to 5 μl . In contrast, a complete anni- hilation of the plasma antioxidant capacity was observed after a fourth freezing cycle. The calculated IC₅₀s, through sigmoidal fitting of the data presented in Figure 2A gave values of 3.36+0.44, and 3.06+0.39 μl of plasma, corresponding to 1.23+0.09 μg of Trolox for 1 or two freezing cycles, not different from tho- se reported in the original publication (2.70+0.49 μl of plasma, corresponding to 1.38 Trolox equivalents) . In contrast, after three freezing cycles, IC₅₀ was significantly reduced to 1.87+0.21 μl of plasma, and anihilated after four freezing cycles. It was there- fore concluded that plasma must be used either immediately, or after a maximum of two freeze/thawing cycles .

Figure 2B presents the calculated TAC expressed as Trolox equivalent per μl of plasma. As shown, mi- nor changes in the calculated TAC/μl were found when 3.3-5 μl of plasma were used in the assay and the number of freeze cycles was <3. In contrast, a linear decrease of the calculated TAC/μl was found when 2 μl of plasma were used. As expected from the data pre- sented in Figure 2A, a dramatic decrease of the TAC was observed after four freezing cycles. The TAC per unit of volume calculated and presented in Figure 2B (0.37+0.07 μg of Trolox equivalent) is not significantly different from the one calculated from the IC₅₀s presented in Figure 2A (0.38+0.04). In view of this result, it was therefore concluded that a plasma volume between 3 and 5 μl could be used, and calculate directly the TAC from the linearized Trolox curve. This method was further used in the following experiments. 2. Validation of automated TAC assay

Based on the above results, an automated assay has been developed, as described in the Methods section. This assay was further validated at various ex- perimental conditions:

2.1 Linearity

As presented above, a major disadvantage of the manual method is non-linear Trolox standard curves. Therefore, a Log/Linear plot is needed, in order to calculate the results. In order to simplify the assay, and to make it suitable for an end-point determination on different auto-analyzers, the inventors have modified the assay volumes and reagents, as de- scribed in the Methods section. The obtained curve, assayed on an auto-analyzer was linear, over the range of 0-3 mmol/L (Figure 3) , a concentration including the majority of the obtained values (see later results) .

2.2 Correlation between TAC and TEAC

The only fully automated method of plasma antioxidant activity in the market is the Total Antioxidant Status assay by Randox (Antrtim, UK) , based on the TEAC method, initially described by Miller and Rice-Evans [35-37] . Therefore, the inventors have performed both assays on a number of 44 human plasma samples. The results are presented in Figure 4. As shown, with the exception of four outliers (marked as diamonds) a very good linear correlation (r=0.8478, p<0.0001) was found between the two assays. Nevertheless, the intercept of the linear fit is 0.51 mmol/L, indicating that at low concentrations TAC results tend to be lower than the TAS assays, while the coefficient of the linear fit is 1.4, suggesting that these two assays based on the oxidation of different substances, might measure a slightly different number of circulating antioxidants.

2.3 Reference values

Based on a number of 44 human blood donors (freezed plasmas) , the reference values of TAC were estimated to be 1.175+0.007 mmol/L, similar to the RAS (Randox) assay of the total antioxidant status, which, estimated on the same samples was found to be 1.209+0.005 mmol/L.

2.4 Dilutions

A concentration of 4.0 mmol/L of Trolox in normal saline was diluted serially, and assayed 10 times. In addition, 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. In addition, the target value of plasma, in which a reference concentration of Trolox was introduced (2.53 mmol/L) was not significantly different form the one assayed by the TAC assay 2.705±0.170 mmol/L) . 2.5 Effect of anticoagulants and storage

A number of anticoagulants have been tested for blood collection, under different conditions. The obtained results are presented in Table 1. As shown, heparin and K₃-EDTA gave similar results, in all experimental conditions. In contrast, citrates decreased the TAC by about 20%. Serum values, in the same samples, showed a 5% more elevated results.

2.6 Effect of freezing

A number of ten different plasma samples from normal individuals, collected on K-EDTA, were assayed after a number of 1-5 freeze/thawing cycles. As shown in Table 2, one to three freezing cycles did not produce any difference in plasma TAC. In contrast, and inversely with the results of the manual assay, a progressive increase of TAC values is found after 3 repeated freezing cycles. A possible explanation for the discrepancy of results between the manual and the automated method might be the fact that automated method, using smaller volumes of plasma, might be more sensitive to repeated freeze thawing, or alternatively, to plasma modifications produced by re- peated freezing. Nevertheless, the fact that the automated method produces stable results during three freeze thawing cycles, makes it a robust technique for use in clinical practice. Table 2

Effect of repeated freezing cycles on TAC values

Ten different plasma samples from normal individuals, collected on K₃-EDTA or heparin, were assayed after a number of 1-5 freeze/thawing cycles, by the automated TAC assay. Values are presented as the mean±SEM (units mmol/L) . The target value of non- freezed plasma, collected in a K₃-EDTA tube was 1.03±0.05 mmol/L.

2.7 Inter- and Intra-assay coefficient of variation

A number of ten different plasma samples, were assayed 10 times in the same run, after one freeze/thawing cycle, and in consecutive days, on a new aliquot, for the determination of intra- and inter- assay coefficient of variation. A 2.4% intra-assay CV and a 3.2% inter-assay CV were found.

2.8 Effect of endogenous substances As indicated above, a number of investigators have proposed that uric acid, ascorbate or plasma proteins could interfere with the measurement of TAC, or they may represent the major component of TAC [5, 14, 42, 43] . In addition, as the measurement of ab- sorbance of crocin bleaching is performed at 450 nm, bilirubin could interfere with the assay [38] . The inventors have therefore tested these contributions in the automated TAC assay.

2.8.1 Uric Acid

Figure 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.

2.8.2 Other endogenous substances

In order to evaluate the interference of a number of other endogenous substances, the inventors have performed the introduction in given samples of varying concentrations of ascorbate, lipids, bilirubin (total and conjugated) and hemoglobin. Our results (Figure 7) , presented as an increased TAC over the sample concentration, show that ascorbate shows a statistically significant relationship of TAC values (r=0.86, p<0.0001). Analysis of the results indicates that there is a slope of 0.07 mmol/L of TAC per mg/dl of ascorbate. Bilirubin represents a possible interference to the TAC assay [38] , due to its antioxidant capacity and to the fact that it presents an absorption maximum at 450 nm, a wavelength used in the TAC assay. Indeed, as shown in Figure 7, a significant correlation could be observed with increasing bilirubin concentrations varying from 0-20 mg/dl (r=0.98, p<0.0001). Analysis of the linear fit indicated an increase of 0.14 mmol/L of TAC per mg/dl of biliru- bin. Taking into account that normal bilirubin values are lower than 1 mg/dl , it was concluded that about 12% of TAC might be attributed to this analyte . Nevertheless, a higher interference might be expected in icteric patients. The same linear correlation is also found with the water-soluble conjugated bilirubin concentration. In this case, the interference was 0.11 mmol/L of TAC per mg/dl of conjugated bilirubin. In contrast, hemoglobin did not show any significant relationship in the TAC assay. Albumin is one of the oxidant scavenger systems in plasma. Nevertheless, as shown in Figure 7, a minor interference of albumin (ranging from 0 to 20 mg/dl) was found on the TAC assay. A significant correlation (r=0.8992, p<0.0004) was observed, while an interference of 0.01 mmol/L of TAC was observed per mg albumin/dl. Finally, total lipids show a strong correlation with TAC (r= 0.99, p<0.0001) with an increase in TAC of 0.18 per 100 mg/dl of lipids, indicating a participation of 30% in the TAC assay. Summarizing therefore the different contributions found, it is concluded that about 85% of TAC, in normal subjects, is due to endogenous antioxidants, and only about 15%, under normal circumstances is due to exogenously (dietary provided) antioxidant substances. 2.9 Effect of diet

The above conclusion indicates that exogenously provided antioxidants might modify the TAC of human plasma. In order to investigate this possibility, the inventors have measured the TAC of 17 samples taken from nuns, from a local monastery, after 40 days of ritual fasting. Their diet, during this period, was exempted of any animal food, and was rich in fruits, vegetables, and extra-virgin olive oil. The results of TAC assay are presented in Figure 8. As shown, the distribution of TAC in the nuns' group is broader, and the mean value higher than in samples from normal individuals (mean values+SEM 2.48+0.10 as compared to 1.20+0.01 mmol/L, t-value 12.8, p<0.0001). This high increase of TAC was attributed to the fasting diet followed by this group, as all other analyte measurements were comparable between the two groups .

3. Determination and clinical significance of the Corrected TAC concept

In view of the above results, it becomes obvious that a fraction of the observed TAC activity in the human plasma is due to endogenous substances, not always presenting a beneficial effect in the human physiology. As examples are stated cases of renal failure, in which elevation of uric acid drifts the TAC assay to very high values. In order to provide a meaningful measurement of the TAC in human plasma, the inventors have introduced the concept of the Cor- rected Total Antioxidant Capacity (corrTAC) . In the corrTAC assay, the TAC is measured, in parallel with total proteins, bilirubin, uric acid and total lip- ids. According to our previous findings (see Figures 6 and 7), the above metabolites account for 0.07 0.14, 0.11, and 0.18 mmol Trolox equivalent/L of the TAC assay. Subtracting these interactions from the TAC assay (calculations are automatically performed by modern autoanalyzers) a new value is obtained, named corrTAC, and representing the contribution of exogenous antioxidants and their active metabolites in the total TAC value. Further validation of the corrTAC assay are presented below:

3.1 Normal Reference values

Reference values for the corrected TAC, in a sample of normal blood donors, varied from 0.43-0.654 mmol/L of Trolox.

3.2 Variation of corrTAC assay in different pathologies

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.

In cancer, a recent finding is that the TAC and corrected TAC is decreased, as compared to the levels of matching controls. Indeed, values of TAC are 1.056+0.047 and 1.229+0.037 for children with malig- nant diseases and matching controls, respectively, while corrected TAC was 0.586+0.047 and 0.709+0.027 respectively. An interesting finding is presented in Figure 10. It was found that corrected TAC levels de- crease during therapy. The above results provide another insight in the possible role of antioxidants in cancer. Indeed, the decrease of antioxidant capacity in neoplasias suggests a new possible role of antioxidant rich diets in malignancies. In addition, the dramatic decrease of TAC during cancer therapy suggests a possible need for substitution, especially through dietary interventions, of circulating active antioxidant levels in cancer therapy.

Finally, a very recent indication, with possi- ble clinical relevance, of the 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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Claims

P A T E N T C L A I M S 1. A method for the determination of Total Antioxidant Capacity (TAC) , wherein said method is performed at a preset temperature and comprises 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, and 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 .

2. A method according to claim 1, wherein said method further comprises the steps of: e. subtracting the absorbance of a sample blank obtained by subjecting a sample of distilled water to the steps from a to d from the absorbance obtained in step d, and f . determining TAC in the sample by comparing the calculated absorbance obtained in step e to a standard curve .

3. A method according to claim 1 or 2 , wherein the preset temperature is in the range of 15 to 50°C.

4. A method according to any of the claims 1 to 3, wherein the predetermined period of time in step b is in the range of 1 to 5000 seconds.

5. A method according to any of the claims 1 to 4, wherein the oxidizing agent in step c is a 2,2'- azobis- (2-amidinopropane) dihydrochloride (ABAP) reagent .

6. A method according to any of the claims 1 to 5, wherein the predetermined period of time in step d is in the range of 10 to 10000 seconds.

7. A method according to any of the claims 1 to 6, wherein the preset wavelength in step d is in the range of 300 to 600 nm.

8. A method according to any of the claims 1 to

7, wherein the preset temperature is 37°C, the predetermined period of time in step b is 160 seconds, the oxidizing agent in step c is a 2 , 2 ' -azobis- (2- amidinopropane) dihydrochloride (ABAP) reagent, the predetermined period of time in step d is 256 seconds, and the preset wavelength in step d is 450 nm.

9. A method according to any of the claims 1 to 8 for the determination of corrTAC, wherein the method 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 antioxidant capacity deriving from endogenous antioxidants and their active metabolites determined in step g from the TAC obtained in step f .

10. A method according to claim 9, wherein the endogenous antioxidants and their active metabolites comprises of one or more of: total proteins, bilirubin, uric acid and total lipids.

11. A method according to any of the claims 1 to 10, wherein the method is an automated method.

12. A crocin reagent for use in the method according to any of the claims 1 to 11 obtainable by a process comprising the steps of : A. isolating crocin from saffron,

B. adjusting pH of the isolated sample obtained in step A with a buffer having a specific pH,

C. mixing the sample obtained in step B with an inert filler to obtain a certain final concentration of the filler,

D. lyophilising an aliquot of the mixed sample obtained in step C,

E. sealing the aliquot obtained in step D under vac- uu , and

F. reconstitution of the aliquot obtained in step E with buffer prior to the use of said crocin reagent in the method according to any of the claims 1 to 11.

13. A crocin reagent according to claim 12, wherein the buffer in step B is a buffer having a pH in the range of 5.0 to 9.0.

14. A crocin reagent according to claim 12 or 13, wherein the concentration of the filler in step C is in the range of 5 to 15% by weight.

15. A crocin reagent according to any of the claims 12 to 14, wherein the buffer in step B is a phosphate buffer having a pH of 7.4 and the final concentration of the filler in step C is 7.5% by weight.

16. Use of the crocin reagent according to any of the claims 12 to 15 in the method according to any of the claims 1 to 11 for the determination of TAC or corrTAC in fluid samples.

17. Use of the crocin reagent according to claim 16, wherein the fluid samples are selected from a group comprising body fluids, cell culture media, food and beverages samples and extracts.

18. Use of the crocin reagent according to claim 16 for the determination of TAC in body fluids, cell culture media, food and beverages samples and extracts for investigational and diagnostic use.

19. A kit comprising crocin reagent according to any of the claims 12 to 15, an oxidizing agent, an antioxidant for preparing the standard curve, buffers, and other components for use in the determination of TAC or corrTAC according to any of the claims 1 or 11 in fluid samples.