In order to maintain a correct balance between free radicals and antioxidant systems, it is important to constantly provide the body with an adequate supply of molecules with antioxidant properties from the outside, to prevent the natural defenses against radicals, constituted by the antioxidant barrier, from running out. leaving the biomolecules exposed to the aggression of reactive species that compromise their functionality.
The molecules with antioxidant action, which can be taken with the diet through the consumption of foods rich in these substances, or through targeted supplementation, are numerous and include polyphenols, vitamins, carotenoids and many other substances. These compounds are able to react with free radicals reducing their reactivity and generating less dangerous molecules that can be easily eliminated by the body.
It is also important to take into account that antioxidants act with different mechanisms and with different efficiency depending on the type of radical involved in the reaction. Each antioxidant, in fact, is able to carry out its own contrasting action on a few specific radicals, so it is necessary that the supply of exogenous antioxidants is as varied as possible, so that the different molecules can act in a complementary way or in synergy in protecting biomolecules from oxidation caused by radical species of different nature.
In this regard, the focus of the research has been on investigating the mechanisms by which antioxidants protect cells. In particular, the possibility of measuring the amount of antioxidants introduced with the diet or the effectiveness of the antioxidant barrier is of great importance, so as to be able to correct any risky situations in a targeted manner.
The main difficulty in measuring the antioxidant efficacy of a substance is due to the fact that the free radical species involved in determining oxidative stress are numerous and react with biomolecules with different speeds and mechanisms. Due to the different nature of free radicals, it is extremely difficult to identify a method of analysis that allows to univocally measure the ability of a compound to counteract the oxidizing action of reactive species, especially when dealing with complex matrices such as blood, food or plant extracts. In fact, free radicals differ in reactivity, in the type of target biomolecule, in the biological matrix in which they act and in chemical-physical affinity (lipophilic or hydrophilic environment), as well as in the mechanism by which they are generated.
Furthermore, in order to compare the measured data for different substances, it is important to try to standardize the methods used as much as possible. An ideal analytical method should first of all be simple and easily reproducible, in order to guarantee good repeatability of results. Furthermore, it should employ significant biological radicals, which react with clear and known mechanisms, so as to simulate as much as possible in vitro what happens in the body, minimizing interference. Finally, an ideal assay should be versatile to allow measurement of both hydrophilic and lipophilic substances.
Currently there is no single valid method for measuring the antioxidant power of a compound, which responds to the described characteristics. It is therefore necessary to use the combination of the results of several essays based on mechanisms and on different radical species, in order to reach a compromise, which also takes into account the final use of the results themselves.
Establish what we want to measure and why it is important not only for the choice of the most suitable measurement methods, but also for the use of the most suitable extraction protocol, since antioxidants represent a very large family of compounds with very different chemical-physical characteristics and there is no extraction technique capable of extracting all the antioxidants present in a complex matrix, at the same time minimizing the presence of potential interferers that can distort the results.
ANALYTICAL METHODS
The most direct way to assess the ability of a compound to protect cells and tissues from oxidative stress is to measure the antioxidant capacity of the blood after taking the compound itself, ie the effectiveness in strengthening the antioxidant barrier, which includes all the antioxidant substances present in the blood. The tests developed generally have very specific characteristics and are able to measure the action of a particular type of antioxidant in well-defined conditions. However, the different antioxidants present in the blood do not act separately, but carry out a strictly interconnected action to create a synergy that allows to obtain optimal protection against aggression by free radicals. Therefore, the actual measurement of the total antioxidant capacity cannot be reduced to the mere sum of the antioxidant capacity of the individual components, and it is impossible to determine the overall action of the antioxidant systems in biological fluids by means of a single assay.
An alternative way consists in the in vitro measurement of the antioxidant power of the exogenous substances that are taken with the diet (foods and supplements). In this case, however, it must be borne in mind that this is a measure of the antioxidant potential of a compound, which provides only an approximation of its ability to exert a real protective action in the biological compartments against the aggression of free radicals, since it assesses quantitatively the antioxidants present, but does not give any information on their bioavailability and their effectiveness once introduced into the body.
The methods for measuring antioxidant capacity can be divided into two categories based on the mechanism by which they react with free radicals to inactivate their reactivity:
- HAT (Hydrogen Atom Transfer) methods, which are based on the ability of a substance to exert its antioxidant action by transferring a hydrogen atom to the radical species;
- SET (Single Electron Transfer) methods, which evaluate the ability of a substance to reduce free radicals by electron transfer.
Some of the analytical methods used are able to act with both mechanisms.
Based on what has been said so far, it is clear that the number of assays developed for the determination of antioxidant and antiradical capacity is very high, so below we will limit ourselves to briefly illustrating the most widespread and significant ones, trying to highlight their strengths and limitations .
