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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200009 

Methodologies for evaluation of total antioxidant
activities in complex mixtures. A critical review

SUSANA LLESUY,1 PABLO EVELSON,1 ANA MARIA CAMPOS,2 and EDUARDO
LISSI3

1Cátedra de Química General e Inorgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires; Argentina;
2 Facultad de Química, Universidad Católica de Valparaíso; Chile;
3Departamento de Química, Facultad de Química y Biología, Universidad de Santiago de Chile

Received: June 14, 2001. Accepted: June 26, 2001.

There is increased evidence that reactive oxygen species and their promoted oxidative damage are involved in a large number of pathologies, as well as in the aging process (Valdez et al, 2000). The oxidative stress experienced by a tissue, organelle or organ results from the balance between the production and removal of potentially damaging reactive oxygen species (ROS). Since the ROS removal rate is mostly controlled by a variety of low molecular weight antioxidants, there is a great interest in determining their levels and the way they are related to pathological states and whether they can be controlled by an antioxidant-rich diet and/or by the ingesta of an antioxidant supplementation (Urquiaga and Leighton, 2000; Crozier et al, 2000). In order to assess the capacity to remove the oxidative species of a given tissue, organ or physiological fluid, the concentration of a variety of antioxidants present in the medium must be determined. Due to their different hydrophobicities, these antioxidants will be distributed among all the cellular compartments. A complete analysis of all the antioxidants is precluded by the large number of molecules that play (or can play) this role, even in a single organelle. Therefore, in these types of studies only the antioxidants considered to be the most important are analyzed. This group includes glutathione, uric acid, thiol groups, ascorbic acid, vitamin E, bilirubin, and ubiquinol-10. In the case of supplementation or a phenol rich diet, the main components of the supplementation are also considered. An alternative approach involves the evaluation of a parameter that is able to give information regarding the total charge of antioxidants present in the tissue or fluid considered. The index thus obtained is then considered a measure of the system's ability to regulate the damage associated to the ROS production. Despite the various arguments that can be immediately raised against this approach (Kohen and Berry, 1997), particularly that there are a large number of ROS and that their relative reactivity toward a series of compounds can vary, the parameter obtained is frequently considered a useful indicator of the system's ability to regulate the damage due to ROS. Some authors even consider them to be more valuable than the evaluation of several of the individual compounds (Ghiselli et al, 2000). Additionally, scores comprising both antioxidant and pro-oxidant indicators (Sharma et el, 1999) have been proposed as even more useful indicators in pathological states and/or as predictors of the outcome in critical situations (Ceriello et al, 1997a; 1997b; Cowley et al, 1996; Luukkainen et al, 1999).

The above considerations lead to the proposal of a great variety of procedures aimed at evaluating the total charge of antioxidants present in a complex mixture of compounds. These methodologies and the information that they provide have been partially discussed in a series of review articles (Miller and Rice-Evans, 1996; Romay et al, 1996a; Alho and Leinonen, 1999; Arnao et al, 1999; Cao and Prior, 1999, Ghiselli et al, 2000; Perez et al, 2000; Rice-Evans, 2000; Abuja and Albertini, 2001). Most of the proposed procedures are listed in Table I. These procedures can be classified in six categories:

i) Procedures in which the consumption of a stable free radical is measured following the addition of the tested compound(s);

ii) Procedures in which the time required to consume all the antioxidants present in the considered sample is measured. This time is usually equated to the time during which the additives are able to significantly reduce the rate of an ongoing free radical process. This category includes the original TRAP (Total Reactive Antioxidant Potential) assay proposed by Wayner et al, (1985) and most of the presently employed methodologies. This type of experiment therefore requires a source of radicals, a target molecule and an easily observable parameter related to the free radical modification of the target molecule. The different procedures proposed differ in at least one of these three factors. An ideal radical source is that whose rate can be considered constant, independent of the complexity of the tested sample. This can be verified by measuring the induction time elicited by the addition of a given amount of a reference compound both in the absence and the presence of the tested sample.

iii) Procedures in which the rate of a given free radical process is observed and the way this rate decreases after addition of the tested sample is evaluated. If the observed rate is proportional to the steady state free radical concentration, the observed effect can be related to the efficiency of the additive to trap the active radicals and/or their precursors. These procedures also require a reliable source of radicals, the target molecule and a parameter related to the steady state free radical concentration. Ideally, the source of radicals must be metal-independent (i.e.: azo-compounds) in order to evaluate only the sample's ability to scavenge radicals and not how it affects the initiation rate. If the source of radicals is metal dependent, efficient chelators will strongly reduce the initiation rate and hence the steady state radical concentration. This happens, for example, in brain homogenates autoxidation, where desferrioxamine appears as one of the most efficient antioxidants (Lissi et al, 1986). In these systems, it is difficult to establish whether the reduction elicited by a given compound is due to its capacity to trap radicals and/or to reduce the initiation rate. However, methods have been proposed to decide between these two possibilities (Llesuy et al, 1998).

iv) Methodologies based on equating the total amount of antioxidants to the reducing capacity of the samples (Benzie and Strain, 1999; Kohen et al, 2000) . The reducing capacity of the tested sample can be assessed either by chemical methods (Benzie and Strain, 1999) or by cyclic voltametry (Chevion et al, 2000; Kohen et al, 2000). In some senses, these methodologies measure the total amount of active compounds, as do i) and ii).

v) Procedures that do not conform to any of the four previously described categories. Some measure the property at a single time (Miller et al, 1993) or integrate the value up to the total target molecule consumption (Cao et al, 1993; 1995). These indices measure a composite of the quantity and reactivity of the antioxidants present in the sample and the relevance of each factor depending upon the substrate(s) reactivity. In particular, they tend to report stoichiometric factors for highly reactive compounds and are related to the reactivity for poorly reactive compounds that are not able to define an induction time.

vi) Miscellaneous methods, based on different principles.

Any one of the procedures included in the categories i) to v) has advantages and disadvantages. In particular, it must be considered that a given index can be an indication of the total amount of antioxidants (without a discrimination regarding their reactivity), or be an indication of the quantity and reactivity of the antioxidants present in the tested sample. This has been discussed previously by Romay et al, (1996a). Procedures i) and ii) tend to give information on the quantities of antioxidants present and, when applied to individual compounds, they are only determined by stoichiometric factors. In fact, it only provides an indication of the number of radicals trapped per each antioxidant molecule introduced into the system (Miller et al, 1996; Re et al, 1999). Another drawback of measurements based on induction times is that with complex mixtures, recovering the signal is sometimes slow and does not allow the evaluation of an induction time. This is particularly true when there are significant amounts of compounds of low reactivity present. One example of this behavior was found in the analysis of the antioxidant capacity of a gingival crevicular fluid (Chapple et al, 1997). In this system, the recovery is slow and biphasic, precluding the evaluation of an induction time. This behavior is frequently observed when there is a significant contribution of the proteins to the measured AO activity. In some of these systems, it has been proposed that the antioxidant capacity of the sample can be estimated from the time required for the measured property to reach a given fraction (up to 40 or 50 %) of their initial value (Visolli and Galli, 1997, Perez et al, 2000). This introduces a fair degree of arbitrariness into the measurements, since the recovering profiles are generally much sharper for the reference compound. In these procedures, the measured AO capacity of the sample increases when the percentage of recovery at which it is measured increases.

Type iii) procedures tend to give information regarding the amount and reactivity of the added sample. When applied to a single compound, they provide information regarding their reactivity toward the trapped radicals. In this sense, they are better indices than those based on type ii) procedures. However, it has to be considered that they are strongly dependent upon the radical being trapped (Regoli and Winston, 1999) and, in complex systems, more than one radical can be present, and frequently it is not easy to determine which one is being trapped (Lissi et al, 1992; Boveris et al, 2000). Furthermore, it must also be considered that in order to obtain values related to the ability of a compound (or mixture) to trap radicals, the tested compounds must not modify the rate of the free radical generation process. This is one disadvantage of procedures that use metals and/or hemoproteins as free radical sources, as they can be affected by the added compounds (Strube et al, 1997). In this sense, the most reliable sources of radicals are the azo-compounds, whose decomposition rates are independent of the presence of additives, which is the reason why these types of compounds, particularly water-soluble 2,2-azobis(2-amidinopropone) dihydrochloride (AAPH), are the free radical source employed in most of the proposed procedures.

Type i) procedures have the advantage of being very simple to carry out. Their primary disadvantage is that the radicals involved are very far from those relevant in oxidative stress situations. Furthermore, the kinetics of the process is complex (Campos and Lissi, 1997, Aliaga and Lissi, 2000) and can be computed as antioxidant compounds that are pro-oxidant in biological systems, such as hydrogen peroxide or organic hydroperoxides (Aliaga and Lissi, 1998). Furthermore, when the consumption of the free radical is measured at a single (long) reaction time, the result obtained is related to stoichiometric factors and gives no information regarding the reactivity of the tested compounds. This lack of kinetic information is particularly observed when the ABTS derived radical is employed (Campos and Lissi, 1997; Lissi et al, 1999). In this case, the index obtained by this procedure presents characteristics similar to those based on procedure ii) (Perez et al, 2000). On the other hand, the interpretation of data obtained by employing DPPH as the stable free radical is less straightforward. When applied to a fixed time at a pure compound, the method provides stoichiometric factors for highly reactive compounds and/or a mixture of reactivity and stoichiometry factors for low reactivity compounds (Lissi et al, 1999; Pekkarinen et al, 1999).

Similar considerations apply to methods based on the reducing power of the sample, although cyclic voltametry measurements could discriminate between different types of compounds (Chevion et al, 2000). If we accept that there is a direct relationship between the oxidation potential of a compound and its reactivity toward free radicals, the voltamogram can give information regarding the proportion of highly reactive compounds.

When applied to complex mixtures, such as those present in plant extracts, beverages, infusions, or biological samples, the above-mentioned procedures can provide a quantitative estimation of the total charge of antioxidants. This total charge is expressed in terms of the equivalent concentration of a standard antioxidant. This equivalent concentration corresponds to the concentration of the reference antioxidant that produces the same effect as those presented in the tested sample. For example, if a type ii) procedure is employed, the equivalent concentration of antioxidants in the tested sample (Eq. Conc.) in micromolar concentration is given by


Eq. Conc. = f tsample / t ref
(1)

 

where tsample is the induction time elicited by an aliquot of the tested sample, tref is the induction time elicited by the reference inhibitor at a 1 µM concentration, and 'f'' is a dilution factor defined by the ratio between the volume of the solution where the measurements are carried out and the tested aliquot. This formula requires a lineal relationship between the measured property (the induction time in this case) and the antioxidant(s) concentration. This condition is fulfilled in most of the proposed procedures, but it must be checked in any new experimental setting.

Several of the proposed methodologies are based on the bleaching of pre-formed ABTS radical cations (Campos and Lissi, 1996; Romay et al, 1996b; Miller and Rice-Evans, 1997) or in the lag time prior to their formation when ABTS is exposed to a free radical source (Arnao et al, 1996; Bartosz et al 1998; Yu and Ong, 1999). All these methodologies improve the original proposal (Miller et al, 1993) that has been criticized for the possibility of interferences and the fact that it involves a mixture of possible processes (Romay et al, 1996b; Strube et al, 1997). However, they still have the limitation that they are only titrating all the antioxidants and those molecules with reducing power and/or hydrogen donating capabilities (Aliaga and Lissi, 1998; 2000). Of the proposed methods based on induction times, the most reliable seems to be that employing AAPH as radical source due to the fact that its rate of radical production is unaffected by the presence of additives, including metal ions.

The value of the index obtained will depend upon the reference antioxidant considered. For water-soluble antioxidants, the reference inhibitor most frequently employed is Trolox, a water-soluble compound with an active moiety similar to that of a-tocopherol. The latter compound is the reference antioxidant employed when lipid-soluble antioxidants are tested. Equivalent concentrations of antioxidants measured in biological samples are collected in Table II.Regarding the stability of the samples, it has been shown that serum samples, stored for 9 weeks at -20 ºC lose 27 % of their antioxidant capacity (Chapple et al, 1997). Samples (blood serum and saliva) stored at liquid nitrogen temperature maintain their AO capacity for several months.

The data collected in Table II show that, in biological samples, most methodologies have been applied to water-soluble AO present in plasma or serum. However, some of the methodologies have been adapted for the evaluation of lipid-soluble antioxidants (Popov and Lewin; 1996 Escobar et al, 1997) and or modified to allow the evaluation of the AO present in tissue homogenates and/or in their cytosolic fractions (Di Meo et al, 1996; Shohami et al, 1997; Winston et al, 1998; Chevion et al, 2000; Evelson et al, 2001). An interesting development is the non-invasive, in vivo determination of AO in tissues, such as skin (Kohen et al, 2000).

Values determined in different plant extracts, infusions and beverages are collected in Table III. Extensive studies have been performed on a variety of vegetables (Velioglu et al, 1998; Vinson et al, 1998; Paganga et al, 1999), fruits (Wang et al, 1996), and beverages. Attempts have been made for the estimation of the average reactivity of the measured compounds by a comparison of antioxidant related indexes with the total content of phenolic compounds (Vinson et al, 1998). Also, an estimation of the average reactivity of the measured compounds can be obtained from a comparison of the values of indexes obtained applying methodology types ii) and iii) (Lissi et al, 1999;Perez et al, 2000). In particular, comparison of the amounts of antioxidant titrated employing ABTS derived radicals and DPPH could provide a rough estimate of the average reactivity of the antioxidants present in the tested sample (Lissi et al, 1999).

The data given in Table III show that wines are the most tested type of alcoholic beverage, although spirits (Goldberg et al, 1999) and beers (Bright, 1999; Liegeois et al, 2000) have also been considered. In all the studies regarding the antioxidant capacity of wines, it has been found that red wines have nearly 7 times more AO than white wines (Campos and Lissi, 1996; Kondo et al, 1999; Perez et al, 2000). On the other hand, rosé wine values are very close to those of white wines (Campos and Lissi, 1996; Kondo et al, 1999). The antioxidants present in red wines increase during maturation (Pellegrini et al, 2000), and correlates with phenol content (Simonetti et al, 1997; Campodonico et al, 1998). Anthocyanins (Lapidot et al, 1999), particularly and proanthocyanidines extracted from the seeds, make a major contribution to the measured AO capacity (Rigo et al, 2000).

The values collected in Table III for a given beverage, such as wine, show noticeable differences. This can be due to both the different methodologies employed and to the different samples considered. For example, the high AO capacities measured for Chilean red wines employing ABTS based methodologies (Campos and Lissi, 1996) can reflect the high content of phenolic compounds present in Chilean red wines (Sato et al, 1996).

The pro-oxidant / antioxidant balance is perturbed in several pathologies and can be modified by the diet or antioxidant supplementation. In pathological situations, the imbalance in antioxidants can be related to the causes of the pathology and/or contribute to the damage associated with it. This is particularly so when there is a significant decrease in AO related indexes. However, the data in Table IV show that in some situations an increase in AO levels is observed. This, as will be mentioned later, can be due to an increase in uric acid levels or a response to diminish the effects of an increased ROS production.

Great effort has therefore been devoted evaluating the changes in the antioxidant defenses in different pathologies and to determine whether this can be related to its severity and/or to its outcome (Table IV). There are also several studies in which the effect of antioxidant supplementation has been evaluated to determine whether it can up-regulate the defense levels with a diminution of the adverse effects of an increased oxidative stress. Most of the results obtained in these studies are also summarized in Table IV. The data presented in this table include pathologies, supplementations and dependence with living habits, such as diet, smoking and exercise. Most of these studies have been performed in plasma and/or serum, although some evaluations have also been carried out in other fluids (Lonnrot, 1996; Smith et al, 1996; Meucci et al, 1998; Kondakova et al, 1999; Sharma et al, 1999; Barbieri et al, 2000) or in homogenates (Child et al, 1999). In particular, data obtained in seminal plasma show very large differences between normal and pathological conditions (Smith et al, 1996; Sharma et al, 1999; Barbieri et al, 1999).

Results obtained in blood plasma or serum samples have the drawback that they are dominated by changes in uric acid levels. For example, after streneous physical exercise a significant increase in TRAP-related indices may occur due to an increase in uric acid, although the opposite effect might have been anticipated due to an increase in ROS production (Ghiselli et al, 2000). Similarly, a strong correlation TAS/uric acid has been observed in critically-ill patients, associated mainly with renal failure (MacKinnon et al, 1999; Molnar et al, 1998). In these situations, it is best to use indices such as TAR (Lissi et al, 1995), which are less influenced by uric acid or, when possible, to carry out evaluations after uric acid removal (Evelson et al, 2001) and/or in fluids (such as seminal plasma) or tissues whose total AO levels are less influenced by uric acid. In this regard, the kinetic method (Tubaro et al, 1998) shows a significant response to changes in ascorbic acid levels due to the large coefficient (7.7 times that of Trolox) of this vitamin in this particular assay. This high value contrasts with that obtained in other TRAP type assays, where a molecule of ascorbic acid corresponds to ca. 0.3 Trolox equivalents (Wayner et al, 1987; Romay et al, 1996a). It is thus interesting to point out that the data given in Table IV show that supplementation with vitamin E or C has no effect on TRAP levels measured in plasma, despite significant increases in the plasmatic levels of both vitamins (Mulholland and Strain, 1993). This is the expected result given the small contribution of vitamin E to the total TRAP (Wayner et al, 1987) and the poor contribution of ascorbic acid to this type of indexes. It is likely that differences could be observed if other indexes, more sensitive to vitamin C, such as TAR or the kinetic method, were employed. These considerations emphasize the point that the methodology employed must consider the type of change expected using the methodology most sensitive to these changes (Romay et al, 1996a; Severin et al, 1999).

The experimentally determined TRAP like values, based on methodology types i) and ii) have frequently been compared with the "theoretical" value calculated from the relationship

TRAPcalc = S ni [XH]i / 2

(2)

where ni is the number of free radicals trapped by each ith molecule, and [XH]i is the micromolar concentration of this component. Since TRAP values are usually expressed in Trolox equivalents, the factor two has been introduced to take into account that each Trolox molecule is able to remove two radicals. This procedure assumes additivity in the effects, an assumption implicit in the use of these methodologies for the evaluation of total antioxidant capacities in complex mixtures. Frequently, experimentally determined values are considerably higher than those calculated taking in consideration the more relevant individual compounds present in the sample. These differences are usually related to "synergism" and/or to the contribution of "unknown" antioxidants (Uotila et al, 1994; Ceriello et al, 1997b).

However, the most likely source of the difference is that the stoichiometric factors (the n coefficients in Eqn. 2) can depend, in an unknown fashion, on the composition of the tested sample. Despite the fact that there is no clear consensus regarding the source of the discrepancy, the total amount of unknown antioxidants, defined as the difference between experimentally determined values and those predicted by Eqn. 2 has been related to the occurrence of pathologies or to the prognosis regarding the possible outcome of the patients (Uotila et al, 1994; Luukkainen et al, 1999).

Similarly, TAR-type indices provided by methodologies type iii) can be predicted by Eqn. (3):


TARcalc = Sai [XH]i

(3)

where ai is the ratio between the efficiencies of the considered compound and Trolox in decreasing the steady state concentrations of the radicals involved in the process. In general, if there is only one type of radical involved in the process, we can expect to find experimentally determined indices that are very close to those predicted by Eqn. 3.

Regarding the validity of these indexes as indicators of the quality of a beverage and/or as useful indicators of pathological situations, there are several fundamental questions:

a) Is there any relationship between the measured index in a food or beverage and the impact it has on the AO status of the organism?

b) Can these indices, measured in biological samples, provide information regarding the existence of oxidative stress conditions? and

c) Can these indices provide information that will lead to a patient's prognosis?

As expected, there is no clear-cut yes-or-no answer to these broad questions and it strongly depends on the system considered. For example, using a DPPH-based method (Yokozawa et al, 1998) it was found that green tea was 6.5 times more powerful as an AO than black tea. However, in preventing the lipid peroxidation of renal homogenates induced by hydrogen peroxide and Fe(II), green tea was only 1.5 time more efficient. Evidently, other factors, such as the distribution and metal chelating characteristics, preclude a quantitative relationship between the amount of antioxidants present in a food or beverage and their biological effect. In in vivo evaluations, the relationship between ingesta and observed levels is still more indirect, since factors such as absorption at the gut level and metabolization can be completely different for the different antioxidants present in the samples. It should further be considered that part of the polyphenols present in a beverage or plant extract can be removed by the prolin-rich proteins present in saliva, compounds that present a high affinity for tannin-like compounds. In this regard, we have determined that 12.2 ± 3.4% of red wine antioxidant capacity is lost when the sample is shaken with human saliva (5:1 V:V). Taking all these complexities into account, it is not surprising that despite the considerably higher quantity of antioxidants in red wines, it has been reported that in some situations its effect is less than that of white wine (Struck et al, 1994).

The relevance of these indices in pathological situations has also been questioned, at least in inflammatory diseases (Tsai et al, 2000). It has also been concluded that the TAC index does not appear to be a reliable parameter for assessing the oxidative susceptibility of critical renal failure patients (Bergesio et al, 1998). Furthermore, doubts have been cast regarding the real effect of AO levels in controlling oxidative stress damage (Cao and Cutler, 1993). On the other hand, other authors (Cowley et al, 1996; Shohami et al, 1997) have concluded that in several pathologies, there is a positive correlation between the ability of a tissue, organ (for example brain following close head injury) or whole individual to increase the levels of low molecular weight antioxidants and the clinical outcome of the patient.

ACKNOWLEDGMENTS

This work has been supported by DICYT (Universidad de Santiago de Chile).

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