Analytical criteria to quantify and compare the antioxidant and pro-oxidant capacity in competition assays: The bell protection function

Highlights

• Simple non-linear dose–time criteria to test the anti- and pro-oxidant activities

• Two robust values that are obtained allows one to perform easily comparisons.

• The criteria were validated with data from common anti- and pro-oxidant assays.

• Further capabilities of the criteria are illustrated to more complex responses.

Abstract

The development of a convenient mathematical application for testing the antioxidant and pro-oxidant potential of standard and novel therapeutic agents is essential for the research community and food industry in order to perform more precise evaluations of products and processes. In this work, a simple non-linear dose–time tool to test the effectiveness of compounds for competitive assays is presented. The model helps to describe accurately the antioxidant and pro-oxidant response as a function of time and dose by two criteria values and allows one to perform easily comparisons of both capacities from different compounds. The quantification procedure developed was applied to two well known in vitro competition assays, the β-carotene and crocin bleaching asymptotic reactions. The dose–time dependency of the response of commercial antioxidants and some expected pro-oxidant compounds was evaluated in this study and the results showed low experimental error. In addition, as an illustrative example of the capabilities of the criteria proposed, the quantification of the combined effect of an antioxidant and a pro-oxidant was analyzed. Afterwards, the model was verified for other relevant competitive methods, using available experimental data from the bibliography. Its application is simple, it provides parametric estimates which characterize the response, and it facilitates rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data tested, the calculated parameters were always statistically significant (Student's t-test, α = 0.05), the equations were consistent (Fisher's F-test) and the goodness of fit coefficient of determination was higher than 0.98.

Introduction

Antioxidants and pro-oxidants are compounds that can delay or accelerate oxidation processes. Living organisms have developed a complex network (Kalyanaraman, 2004) of antioxidants (enzymes such as superoxide dismutase, catalase, glutathione peroxidase or non-enzymatic compounds such as uric acid, bilirubin, albumin, metallothioneins); they are essential for a healthy life in order to counteract various harmful (Hussain, Hofseth, & Harris, 2003) pro-oxidants or reactive species (i.e. O₂, H₂O₂, ROO, OH). Apart from these endogenous antioxidants, there are exogenous ones that can derive from natural sources (vitamins, flavonoids, anthocyanins, some mineral compounds), or from synthetic compounds (such as butylhydroxyanisole, butylhydroxytoluene, etc.). There are also exogenous compounds such as metal ions that can promote or accelerate the oxidation processes (Carocho & Ferreira, 2013). Clinical trials and epidemiological studies have established an inverse correlation between the intake of natural exogenous antioxidants and the occurrence of oxidative stress diseases such as inflammation, cardiovascular problems, cancer, and aging-related disorders (Gutteridge & Halliwell, 2010). Thus, the analysis of natural antioxidants for disease prevention (Chatterjee et al., 2005, Notas et al., 2005) and the identification of possible pro-oxidant substances have become topics of increasing interest.

Several in vivo and in vitro methods have been developed for determining the total antioxidant and pro-oxidant (oxidation modifiers, OM) capacity of compounds. The capacity of OM is frequently determined in competition assays, in which the OM and indicators of the reaction (in general another OM) compete for the reactive species. Competition assays are performed to describe OM capacity and to rank the affinity of OM to counteract or increase the action of reactive species against an indicator. In general, these assays differ in the mechanism of generation of different radical species and/or target molecules and in the way end-products are measured. At present, there is no convenient assay that enables the evaluation of the OM capacity (Halliwell, 2013, Naguib, 2000, Tsuchihashi et al., 1995) for different compounds. The current methods used to test the OM capacity still have left many open questions (Frankel and Meyer, 2000, Halliwell, 2012). The in vitro assays can only rank OM capacity for their particular reaction system and their relevance to in vivo activities is uncertain. Thus, it is logical that in the last decade, researchers have claimed unity of the approaches (Frankel and Finley, 2008, Murado and Vázquez, 2010) and have tended to standardize the protocols to increase the effectiveness of methods for in vitro and in vivo responses (Dawidowicz and Olszowy, 2010, Frankel, 1993, Frankel, 1994, Ordoudi and Tsimidou, 2006, Prior and Cao, 1999, Prior et al., 2005).

Additionally, the arbitrary use of simple analytical procedures to calculate molecular properties, occasionally without a validation study, as well as a lack of statistical significance, has caused much controversy (Frankel, 1993, Frankel, 1994, Huang et al., 2005, Koleva et al., 2002, Laguerre et al., 2007, Naguib, 2000, Roginsky and Lissi, 2005). Commonly, the mathematical determinations of the OM capacity are based on a fixed endpoint without proper considerations of the kinetic behavior. The most typical and incorrect practice is to use the single-time dose–response of one commercial OM as a calibration curve (normally focusing on the linear range), and afterwards to compute the equivalent OM capacity of any type of sample by testing it only at one single-time–dose, assuming too many false aspects as true.

In the current study, a simple non-linear mathematical application for competitive OM assays, in which the responses have one common asymptote (majority of ones) is presented. It helps to describe accurately the response as a function of time and dose by two criteria values and facilitates convenient comparisons of the capacity of different compounds. The model was validated in well known in vitro competition assays, evaluating the dose–time-dependency of the response of OM compounds.