Quantification, characterization and description of synergy and antagonism in the antioxidant response
Introduction
An important and characteristic problem of any system (as defined in the Bertalanffy's theory: a set of interacting elements) is to determinate whether the joint effect of two or more elements on the system behavior is directly deducible from the individual effects of the elements. This issue has a long history of controversy whose first known attempt to solve it dates back to Aristotle, and it is frequently stated by replacing the expression “directly deducible from” with “the sum of”, which significantly changes the focus. Thus, in the field of antioxidant action, the concepts of synergy and antagonism are often characterized as those interactions of two (or more) antioxidants that are greater (synergy) or lesser (antagonism) than the sum of the individual effects (Jia et al., 1998, Marinova et al., 2008, Parker et al., 2010, Yang et al., 2009). Such a characterization is not acceptable for two reasons.
First, it postulates that the joint effect in the absence of interactions is the sum of the individual effects, which is an especially simplistic case and not applicable to asymptotic responses, such as those involved in the action of anti- and pro-oxidant agents. Indeed, the sum of two individual responses is meaningless if it exceeds the asymptotic response of the system. In fact, the referent of any phenomenon that perturbs the joint action of two agents is that joint action in the absence of perturbation (not the individual actions), a situation that is often called as the “null interaction”. Consequently, the first condition to decide the possible presence of synergistic or antagonistic effects is to define the null interaction. A second difficulty arises from the common tools applied to characterize the antioxidant action. Despite abundant criticism (Labuza and Dugan, 1971, Murado and Vázquez, 2010, Özilgen and Özilgen, 1990, Prieto et al., 2012, Prieto, Vázquez and Murado, 2012a, Terpinc and Abramovič, 2010), such a characterization frequently disregards the kinetic aspects of the oxidation process and its inhibition. Although this objection has a less theoretical significance than the first one, its practical consequence is that the results may be poorly suited to discern the joint effect of two antioxidants.
This paper pursues a solution for each of these objections by using concentration–time response models applied to the β-carotene (βCM) (Marco, 1968, Miller, 1971) and crocin bleaching (CM) (Bors, Michel, & Saran, 1984) methods – extensively used to quantify antioxidant and prooxidant activities – to assess the synergistic or antagonistic interactions between several pairs of well-known antioxidants. Their respective protocols have been repeatedly revised and improved, and they are optimized at present (Prieto et al., 2012, Prieto et al., 2013). They are appropriate for lipophilic and hydrophilic matrices and can provide useful complementary information in the study of complex natural extracts containing components with a variable degree of polarity (Prieto, Murado, Vázquez, Anders, & Curran, 2013). β-carotene is a lipophilic oxidizable substrate that can join the system of lipidic micelles in which the oxidation reaction is accomplished. The method is especially sensitive to oxidation modifying agents in a lipidic environment, and it produces a very low response with hydrophilic antioxidants, even powerful ones (polar paradox). Complementarily, crocin is a hydrophilic oxidizable substrate, and lipophilic oxidation modifiers, even powerful ones, produce very low responses in the aqueous system that characterize the application of this method (apolar paradox). These assays were selected because they provide an optimized response system that is fairly representative of the lipidic and hydrophilic oxidation processes, especially accurate, reproducible and yields a low experimental error.
The first problem, which consists of distinguishing between null interaction and synergistic or antagonistic effects was studied by generalizing the classical approaches (Berenbaum, 1985a, Berenbaum, 1985b, Bliss, 1937, Bliss, 1939, Greco et al., 1995, Loewe and Muischnek, 1926) applied in the dose–response area (not free either of debate about the interactive effects) and others (Baldwin and Roling, 2009, Gessner, 1988, Hewlett and Plackett, 1964, Qin et al., 2011, Rovati and Nicosia, 1994). The second difficulty was solved by defining the response of the system to the simultaneous action of two antioxidants through a single value obtained from a kinetic description as previously discussed (Dávalos et al., 2004, Huang et al., 2008, Naguib, 2000, Prieto et al., 2012).
The proposed generalized procedures for the joint action of several well-known antioxidants produced consistent results in all cases. In addition, it provided some evidence of a more basic character, which could be transferable to the general field of the in vivo dose–response relationships.
Section snippets
Equipment and reagents
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Equipment: Multiskan spectrum microplate photometer using polypropylene plates with 96 wells.
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Antioxidants: butyl-hydroxyanisole (BHA); propyl 3,4,5-trihydroxybenzoate (Propyl gallate; PG); butyl-hydroxytoluene (BHT); 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Ethoxyquin; ETO); 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; TRO); and (2R)-2,5,7,8-tetramethyl-2-[(4R,8R)-(4,8,12-trimethyltridecyl)]-6-chromanol (α-tocopherol; TOC); manganese sulfate (Mn);
Meaning of the synergy and antagonism notions
Once Eqs. (10), (15) were accepted as generalized models for IA and CA hypotheses, respectively, an algebraic framework was established that characterizes synergy and antagonism through the specific variations imposed by the perturbations to the parameters and the response.
In a broad sense, an interaction is synergistic or antagonistic as it increases or decreases the expected response in the null interaction. In the IA model (10), a synergistic interaction raises at least one Ki parameter,
Discussion
Synergy and antagonism are controversial characteristic behaviors of very diverse systems. Despite their importance, the common characterization of these phenomena in the context of the antioxidant action is often questionable due to some problematic definitions and the type of data used. The models proposed here showed a good ability to describe the joint action of several pairs of antioxidant under both aqueous and lipid emulsions. Their application allows one to: 1) typify a joint
Conclusions
In this paper, a methodological procedure has been developed for the joint action of several pairs of antioxidants in both aqueous and lipid emulsions, which enables the determination and quantification of the synergistic and antagonistic interactive effects. Although the approach could be directly expanded to other types of classical antioxidant methods, the methods selected are fairly representative of the most complex scenarios that can be found in the oxidation process. Unfortunately, the
Acknowledgments
The authors wish to thank CSIC (Intramural project: 200930I183) and Ministerio de Ciencia e Innovación (project CTM2010-18411, co-financed with FEDER funds by European Union) for financial support. Miguel Angel Prieto Lage was awarded one grant from the JAE predoctoral program co-financed by the CSIC and European Social Fund (ESF).
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