**3. Need and difficulties of carrying out bioavailability studies of polyphenols**

In this section, we will discuss some common mistakes that can be made when bioavailability studies are carried out, the difficulties that the analyst can find and the limitations of some of the studies made so far. **Figure 1** gives a general idea of the most important topics commented in the section.

Metabolism and Bioavailability of Olive Oil Polyphenols 349

of the data from humans presented in the literature on the bioavailability refer only to the release of the polyphenols from the food matrix and their consequent absorption

To address the bioavailability of olive oil phenols, we should exclude studies without a control diet and studies in which the amount of ingested phenols is not reported or could not be estimated (Miró-Casas et al., 2001a; Visioli et al., 2000a; Vissers et al., 2004). In other words, it is essential to characterize in depth the polyphenolic extract of the olive oil before starting bioavailability studies to assure their usefulness; since this fraction is quite complex and heterogeneous, it represents another requirement which difficults the whole process.

Advances in the understanding of olive oil polyphenols metabolism have been made possible by improvements in the analytical methodologies used, particularly high-resolution chromatographic systems with mass spectrometry as detector (Bai et al., 1998; Del Boccio et al., 2003; García-Villalba et al., 2010; Khymenets et al., 2011; Miró-Casas et al., 2003b). Performing metabolomic studies is challenging and requires measurements of a very high quality using powerful platforms. Even if the analyst uses proper tools, the fully structural assignment of the metabolites under study is sometimes very difficult due to the lack of the metabolite standards; fact which makes difficult the correct quantification too (D'Archivio et al., 2010; García-Villalba et al., 2010). The amount of information about the sample under study achieved in metabolomic studies is considerable, that is why for meaningful interpretation the appropriate statistical tools must be employed to manipulate the large raw data sets in order to provide understandable and workable information (Manach et al.,

A very interesting review written by D'Archivio et al. (2010) gives a critical overview about the difficulties and the controversies surrounding the studies aimed at determining the bioavailability of polyphenols. Summarizing, there are some essential steps to be followed to establish conclusive evidences for the effectiveness of polyphenols in disease prevention and in human health improvement: 1) determination of the distribution of these compounds in our diet, estimating their content in each food; 2) identification of which of the existing polyphenols are likely to provide the greatest effects in the context of preventive nutrition, and 3) assesment of the bioavailability of polyphenols and their metabolites, to evaluate

Even though the bioavailability studies are properly designed, we have to be aware of how many different endogenous and exogenous variables are involved and the difficulties that have to be faced. The main factors recognized as affecting olive oil polyphenols bioavailability can be grouped in the following categories: factors related to the polyphenol characteristics, food/food processing related factors, external factors and factors related to the host, as it can be observed in **Figure 1.** An in-depth discussion of every factor influencing the bioavailability of olive oil polyphenols has been made by Manach et al.

To explore and understand the mechanism of action of olive oil polyphenols and their role in disease prevention and human health improvement, extensive studies of absorption, metabolism, excretion, toxicity, and efficacy are needed. Although *in vitro* studies can be

(D'Archivio et al., 2010; Vissers et al., 2004).

their biological activity in target issues.

(2004) and Cicerale et al. (2009).

**4. Conclusions** 

2009).

Fig. 1. Definition of bioavailability and the limits affecting bioavailability studies of polyphenols

Since intervention studies are very difficult to carry out, in many cases the researchers have to turn to *in vitro* or animal studies. *In vitro* studies are a pillar of pharmacological research and build the bases for future *in vivo* assays; however, the interpretation and extrapolation of the achieved data have to be made very carefully (Kroon et al., 2004). When the biological activity of polyphenols is assessed by using culture cells as tissue models, in most of the cases, cells are treated with aglycones or polyphenols-rich extract derived from plants or, in this case, from olive oil, and data are reported at concentrations which elicited a response. It is absolutely necessary to bear in mind that plasma and tissues are not exposed *in vivo* to polyphenols in these forms: the molecular forms reaching the peripheral circulation and tissues are different from those present in the olive oil (Day et al., 2001). Moreover, the polyphenols concentration tested should be of the same order as the maximum plasma concentration attained after a polyphenol-rich meal (0.1-10 µmol/l).

Matters of practicality determine the use of rats rather than humans as the model of choice for *in vivo* studies, although interspecies variability renders comparisons between the model species (animals, humans) complex and sometimes questionable (Visioli et al., 2003), since the rats and rodents in general are not the best model for the study of dietary problem of human metabolism.

When *in vivo* studies are carried out, we can say that most of researches have investigated the kinetics and extent of polyphenol absorption by measuring plasma concentrations and/or urinary excretion among adults after the ingestion of a single dose of polyphenol, provided as pure compound, plant extract, or whole food/beverage. Using this "singledose" design, the increase in the blood concentration is transitional and reflects mainly the ability of the organism to take up the polyphenol from the food matrix. Consequently, most

Rigorous determination of

bioavailability of polyphenols?

the studies carried out so far Bioavailability




Since intervention studies are very difficult to carry out, in many cases the researchers have to turn to *in vitro* or animal studies. *In vitro* studies are a pillar of pharmacological research and build the bases for future *in vivo* assays; however, the interpretation and extrapolation of the achieved data have to be made very carefully (Kroon et al., 2004). When the biological activity of polyphenols is assessed by using culture cells as tissue models, in most of the cases, cells are treated with aglycones or polyphenols-rich extract derived from plants or, in this case, from olive oil, and data are reported at concentrations which elicited a response. It is absolutely necessary to bear in mind that plasma and tissues are not exposed *in vivo* to polyphenols in these forms: the molecular forms reaching the peripheral circulation and tissues are different from those present in the olive oil (Day et al., 2001). Moreover, the polyphenols concentration tested should be of the same order as the maximum plasma

Matters of practicality determine the use of rats rather than humans as the model of choice for *in vivo* studies, although interspecies variability renders comparisons between the model species (animals, humans) complex and sometimes questionable (Visioli et al., 2003), since the rats and rodents in general are not the best model for the study of dietary problem of

When *in vivo* studies are carried out, we can say that most of researches have investigated the kinetics and extent of polyphenol absorption by measuring plasma concentrations and/or urinary excretion among adults after the ingestion of a single dose of polyphenol, provided as pure compound, plant extract, or whole food/beverage. Using this "singledose" design, the increase in the blood concentration is transitional and reflects mainly the ability of the organism to take up the polyphenol from the food matrix. Consequently, most

Fig. 1. Definition of bioavailability and the limits affecting bioavailability studies of

concentration attained after a polyphenol-rich meal (0.1-10 µmol/l).




*From the characterization of olive oil-polyphenols fraction*

polyphenols

human metabolism.

*Fraction of an ingested nutrient or compound that reaches the systemic circulation and the specific sites where it can exert its biological action*

> -Release from the carrier matrix -Intestinal absorption -Tissue uptake

Three main steps are implicit

Limits/difficulties of some of

*In-vitro* 


*In-vivo*

**Food matrix**

**Metabolomic studies**

*To achieve evidence about health benefits and to understand their metabolism* of the data from humans presented in the literature on the bioavailability refer only to the release of the polyphenols from the food matrix and their consequent absorption (D'Archivio et al., 2010; Vissers et al., 2004).

To address the bioavailability of olive oil phenols, we should exclude studies without a control diet and studies in which the amount of ingested phenols is not reported or could not be estimated (Miró-Casas et al., 2001a; Visioli et al., 2000a; Vissers et al., 2004). In other words, it is essential to characterize in depth the polyphenolic extract of the olive oil before starting bioavailability studies to assure their usefulness; since this fraction is quite complex and heterogeneous, it represents another requirement which difficults the whole process.

Advances in the understanding of olive oil polyphenols metabolism have been made possible by improvements in the analytical methodologies used, particularly high-resolution chromatographic systems with mass spectrometry as detector (Bai et al., 1998; Del Boccio et al., 2003; García-Villalba et al., 2010; Khymenets et al., 2011; Miró-Casas et al., 2003b). Performing metabolomic studies is challenging and requires measurements of a very high quality using powerful platforms. Even if the analyst uses proper tools, the fully structural assignment of the metabolites under study is sometimes very difficult due to the lack of the metabolite standards; fact which makes difficult the correct quantification too (D'Archivio et al., 2010; García-Villalba et al., 2010). The amount of information about the sample under study achieved in metabolomic studies is considerable, that is why for meaningful interpretation the appropriate statistical tools must be employed to manipulate the large raw data sets in order to provide understandable and workable information (Manach et al., 2009).

A very interesting review written by D'Archivio et al. (2010) gives a critical overview about the difficulties and the controversies surrounding the studies aimed at determining the bioavailability of polyphenols. Summarizing, there are some essential steps to be followed to establish conclusive evidences for the effectiveness of polyphenols in disease prevention and in human health improvement: 1) determination of the distribution of these compounds in our diet, estimating their content in each food; 2) identification of which of the existing polyphenols are likely to provide the greatest effects in the context of preventive nutrition, and 3) assesment of the bioavailability of polyphenols and their metabolites, to evaluate their biological activity in target issues.

Even though the bioavailability studies are properly designed, we have to be aware of how many different endogenous and exogenous variables are involved and the difficulties that have to be faced. The main factors recognized as affecting olive oil polyphenols bioavailability can be grouped in the following categories: factors related to the polyphenol characteristics, food/food processing related factors, external factors and factors related to the host, as it can be observed in **Figure 1.** An in-depth discussion of every factor influencing the bioavailability of olive oil polyphenols has been made by Manach et al. (2004) and Cicerale et al. (2009).
