A new automated method for the determination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay
© Kampa et al; licensee BioMed Central Ltd. 2002
Received: 9 May 2002
Accepted: 28 August 2002
Published: 28 August 2002
Antioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product.
In the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay), based on the crocin bleaching (oxidation) method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L), and performed using an end point measurement.
The TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS) test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM), while a diet rich in antioxidants more than doubled this value.
The proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and/or exogenous metabolites. The antioxidant capacity of plasma thus calculated can be used as a useful indicator of the antioxidant value of foods and beverages in the daily diet.
Evidence from biochemical and animal models suggests that nutritional antioxidants protect against the development of chronic diseases, including coronary heart disease, atherosclerosis and certain forms of cancers. However, this hypothesis is not clearly corroborated by intervention studies in human subjects, due, in part, to inadequacies in current analytical methodologies. Indeed, although in vitro assays may give useful information on the attributes required by a compound to act as an antioxidant, results have limited nutritional relevance , since plasma and other biological fluids abound in antioxidant molecules, which scavenge free radical species thus reducing oxidative damage of either macromolecules, membrane lipids or lipoproteins. Bilirubin, uric acid and protein thiols are major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbenes, flavonoids and phenolic acids are the main classes of nutritional antioxidants [2–5].
Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. These assays give a wide range of results, should never be used in isolation, and results should be interpreted with caution . To counteract these limitations, several methods have been proposed for determination of total plasma antioxidant capacity (TAC). They can be divided in two main classes: Either distinct antioxidant components are assayed (ex. Vitamin E, ascorbic acid, etc), or the total antioxidant potency is estimated by the combined reducing activities of a given body fluid (especially plasma). A number of methods have been developed for this later estimation, given the considerable interest in antioxidants as bioactive components of food and as nutritional agents with a role in the maintenance of health and in disease prevention . Indeed, there are situations in which knowledge of the individual levels of specific antioxidant components might be less useful than the total antioxidant potency of the medium concerned. Such situations might be in the understanding of structure-activity relationships of pure antioxidant compounds, in the determination of the antioxidant contributions of specific dietary components and how this relates to the antioxidant composition and activities of the individual constituents, and in the study of decreases in plasma antioxidant activity in individuals under oxidative stress in specific disease states . The total antioxidant activity of plasma has been assayed by a number of different methods including oxygen consumption during lipid peroxidation , luminol-enhanced chemiluminescence , measurement of R-phycoerythrin bleaching [ORAC method [10–12]], sensitivity of erythrocytes to hemolysis , ferric reducing activity , lipid peroxides generation [15, 16], and finally carotenoid (crocin) bleaching . This latter method has been used in food chemistry for assaying the antioxidant capability of complex food mixtures. A recent development of this method, based on serial dilutions of human plasma, has been recently reported, allowing the determination of the plasma antioxidant activity in humans . The present work builds on these data, proposes additional modifications and attempts to automate it, in order to meet the requirements and capacities of modern autoanalyzers.
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 (Milwaukee, WI). 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, De). Crocin was isolated from saffron by water/methanol extraction after repeated extraction with ethyl-ether, as described previously . 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 molecular absorbance coefficient of crocin ε443 = 89000 M-1cm-1. Aliquots of crocin, protected from light, weres to redat -20°C, until use. Caffeic (97%), ferulic (99%) and protocatechuic (99%) acids were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Sinapic (98%), syringic (98%), 3,4-dihydroxyphenylacetic acids and epigalocatechin were from Sigma Chemical Co. (St. Loui, 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 (Vienna, 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 autoanalyzer was used for the automated assay.
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 . 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) were run in parallel. The absorbance was measured at 450 nm. The specific absorbance was then calculated by subtracting the corresponding blank value, and the antioxidant activity was calculated as the ratio: 100 × (Abs0-Abssample)/Abs0, in which Abs0 was the absorbance in the absence of antioxidants, and Abssample 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.
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 , 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 7.5 ml of buffer A prior to use. The reconstituted solution was R1 of the final assay while R2 was a ready-to-use liquid reagent containing 50 mg/ml ABAP in buffer A. For the automated procedure a blank reagent was run together with the above-described reagent. The blank reagent was consisted of buffer A as R1blank and 50 mg/ml ABAP in buffer A as R2blank.
The assay was performed at 37°C in the following steps: Two μl of sample, calibrator or control were mixed with 250 μl of crocin reagent (R1) and this mixture was incubated for 160 s. Thereafter, 250 μl ABAP reagent R2 were added and the decrease in absorbance at 450 nm 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 measurement 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 we used the following materials: 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 (0.1 N 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, Athens, 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 OSR6112, iron OSR6123 uric acid OSR6136).
Forty healthy blood donors, aged 21–52 years (28 males, and 12 females) from the region of Heraklion, Crete, were used for the determination of the reference interval of the TAC assay. They were on a normal diet, while we had little information of their nutritional and smoking habits. In addition, one hundred samples from the hematology laboratory of the Heraklion University Hospital, 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.
The statistical analysis of the results was performed using parametric methods, with the aid of the Origin v 5.0 (Microcal Software, Northampton, MA) and the Systat v 10.0 (SPSS, Chicago, IL) microcomputer programs.
Validation of the assay
Reagents and antioxidants
As indicated in the original paper for the assay , crocin spectra in the oxidized and the reduced form present an absorption maximum at 440 nm. This was also verified in the present work (not shown). In addition, different concentrations of ABAP, in the presence of 5 μM 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 crocin 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 .
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.
Immediate plasma separation
Storage at room temperature for 4 hours
Storage at 4°C for 4 hours
1.09 ± 0.04
1.10 ± 0.03
1.09 ± 0.03
1.03 ± 0.05
1.02 ± 0.03
1.01 ± 0.04
0.82 ± 0.04
0.81 ± 0.03
0.83 ± 0.03
1.04 ± 0.05
1.08 ± 0.04
1.04 ± 0.04
Figure 2B presents the calculated TAC expressed as Trolox equivalent per μl of plasma. As shown, minor 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 presented 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, we have therefore concluded that we could use a plasma volume between 3 and 5 μl, and calculate directly the TAC from the linearized Trolox curve. This method was further used in the following experiments.
Validation of automated TAC assay
Based on the above results, an automated assay has been developed, as described under Material and Methods. This assay was further validated in various experimental conditions:
TAC of human plasma
Correlation between TAC and TEAC
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.
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.
Effect of freezing
Effect of repeated freezing cycles on TAC values
Number of freeze/thawing cycles
1.05 ± 0.02
1.00 ± 0.02
1.05 ± 0.03
1.13 ± 0.03
1.22 ± 0.04
1.05 ± 0.03
0.95 ± 0.02
1.00 ± 0.03
1.24 ± 0.04
1.32 ± 0.05
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.
Effect of endogenous substances
As indicated in the Introduction, 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 [2–5]. In addition, as the measurement of absorbance of crocin bleaching is performed at 450 nm, bilirubin could interfere with the assay . We have therefore tested these contributions in the automated TAC assay.
Other endogenous substances
Summarizing therefore the different contributions found, we have 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.
Effect of diet
Reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) are produced as a consequence of normal aerobic metabolism in animal species [24–26]. These "free radicals" are removed and/or inactivated in vivo by a battery of antioxidants [4, 24–27]. A biological antioxidant is defined as a substance, which, at low concentrations compared to that of the oxidisable substrate, significantly delays or prevents this oxidation [6, 27, 28]. Individual members of the antioxidant defense team are employed to prevent the generation of free ROS and RNS, to destroy potential antioxidants and to scavenge ROS and RNS. However, the relative sufficiency of the organism antioxidant defenses is critical in the development of oxidative stress in patients affected by a number of diseases, including HIV infections [29, 30], neurodegeneration , diabetes [3, 32–34], angina [35–38], certain forms of cancer [39–47], and probably ageing [48–50]. These diseases are characterized by an overproduction of free radicals, i.e. when the antioxidant defense of an organism is overwhelmed or are established when a deficit of defenses of the organism against oxidation occurs.
The primary defense 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 normal 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) . The sum of endogenous and food-derived antioxidants represents the total antioxidant activity 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 obtained by the measurement of individual parameters, as it considers the cumulative effect of all antioxidants present in plasma and body fluids . A theory has recently be proposed, taking into account 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 involving a number of other, more potent antioxidants. Through this cascade, interactions among the lipid and the aqueous phases could be established .
A great variety of methods have been proposed for the assay of total antioxidant activity or capacity of serum or plasma [reviewed extensively and critically in [6, 51]]. In these reviews a clear distinction among antioxidant activity and capacity is made: Antioxidant activity corresponds to the rate constant of a single antioxidant against a given free radical. Antioxidant capacity, 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 . Therefore, for plasma, being a heterogeneous solution 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 , or an adaptation mechanism to an increased oxidative stress. Nevertheless, due to the participation of diverse metabolites (see Figures 6 and 7) to the antioxidant capacity of human 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 .
As derived from the definition of antioxidant capacity, and the heterogeneity of antioxidant substances in human plasma, all methods used for its determination are by definition indirect . The crocin bleaching method, used in the present paper, initially described by Tubaro et al , and Lusignoli et al  uses crocin oxidation by peroxyl radicals produced by ABAP. By comparing the inhibition of bleaching (oxidation) of crocin by plasma, to an artificial antioxidant (Trolox C), either kinetically , or at end point , a standard antioxidant capacity of plasma can be derived, expressed as Trolox equivalent. As discussed by Prior and Cao , a serious problem of the crocin method is the lag time phase, when lipids and proteins act as antioxidants, a result not encountered in the modification proposed here (Figure 7), at analyte concentrations exceeding by far the reference values in human plasma. In addition, concerning ascorbic acid (that the previous kinetic method provides values exceeding all other reported, 7.7 Trolox equivalents) was not a problem in the current assay. Indeed, ascorbic acid accounted (on a molar basis) only for 1.22 Trolox equivalents. Compared to another commercialized antioxidant capacity determination (Total Antioxidant Status by Randox) (see Figure 4), a significant linear correlation was observed, while TAC assay tends to assay lower AC by 0.5 mmol/L, expressed as Trolox equivalents. The TAS assay is based on the TEAC (Trolox Equivalent Antioxidant Capacity) method, reported by Miller and Rice-Evans [21–23]. It is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) formed by the interaction of ABTS with ferrylmyoglobin radical species. This, also indirect, method gave similar reference values as the TAC assay, on 44 healthy blood donors (TAS: 1.209 ± 0.005 mmol/L; TAC: 1.175 ± 0.007 mmol/L of Trolox equivalents), measured serially on the same autoanalyzer.
As discussed elegantly by Prior et al , different metabolites interfere with all indirect antioxidant capacity methods. These endogenous analytes include uric acid, ascorbate, albumin, bilirubin and lipoproteins. During the validation of the TAC assay we performed analysis of the above metabolites on the antioxidant capacity. We have found that uric acid, bilirubin, ascorbate and lipoproteins accounted for 0.11 mmol/mg, 0.14 mmol/mg, 0.07 mmol/mg, and 0.18 mmol/100 mg respectively. Taking into account the normal concentrations of these analytes, it was concluded that about 1 mmol/L (i.e. about 85% of the TAC) is due to endogenous analytes, and only 15% of the observed TAC might be due to exogenously provided antioxidants. Of course, as our reference subjects were blood donors, we could not have precise evaluation of their dietary and smoking habits. Indeed, it is well established that smoking habits reduce the TAC of human plasma, a reduction which is reversed after stopping smoking . Non-smoking nuns, following a diet rich in antioxidant substances, increase dramatically their TAC of plasma, demonstrating the importance of dietary antioxidants (Figure 8).
As discussed above, the AC of plasma might be considered as a concept rather than a pure analytical determination. Taking into consideration the participation of major analytes in the bleaching of crocin, we propose the measurement of TAC in relation to these metabolites, in order to estimate the contribution of exogenous substances to the total antioxidant capacity. In this way, the possible interference of endogenous and exogenous antioxidants can be evaluated and tested in diverse physiological and pathological situations. The TAC assay, fully automated, stable and reliable could, in such a way be of value in the estimation of the AC of plasma, and the role of different kind of foods and beverages, with presumed or definite role in human health, could be evaluated.
Total Antioxidant Capacity
Total Antioxidant Status
Work partially supported by the University of Crete, Research Committee. Professor A. Margioris is deeply acknowledged for the stimulating discussions and for reviewing a version of the present work.
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