**2.1 Absorption and disposition**

Direct evidence on bioavailability of olive oil phenolic compounds has been obtained by measuring the concentration of the polyphenols and their metabolites in biological fluids, mostly plasma and urine, after ingestion of pure compounds or of olive oil, either pure or enriched with the phenolics under study. The majority of research regarding the bioavailability of olive oil polyphenols has been focused on three major phenolics: Hyty, Ty and Ol, as can be seen in **Tables 1** and **2**.

After ingestion, olive oil polyphenols can be partially modified in the acidic environment of the stomach. The effect of such environment on aglycone secoiridoids has been examined *in vitro* by incubating the compounds at 37 ºC in simulated gastric pH conditions and during normal physiological time frames (Corona et al., 2006; Pinto et al., 2011). Although hydrolysis takes place releasing free phenolic alcohols, a significant amount remains intact and thus, enters the small intestine unmodified. Ol Agl and its dialdehydic form, however, are likely not absorbed as such in the small intestine; the major metabolites detected using the perfused rat intestine model were the glucuronide conjugates of the reduced form of both compounds (Pinto et al., 2011).

Manna et al. (2000) carried out studies on the transport kinetics of radiolabeled Hyty using differentiated Caco-2 cells. The only metabolite found in the culture medium was the methylated derivative (i.e. homovanillic alcohol - HVAlc). They also demonstrated that Hyty was transported across the membrane of the human enterocytes by a bidirectional passive diffusion mechanism. Caco-2/TC7 cell monolayers have been used to study the metabolism of other olive oil polyphenols, such as Ty, *p*-coumaric acid, pinoresinol, luteolin (Soler et al., 2010) and Hyty acetate (Mateos et al., 2011). Results showed that the methylated conjugates are the main metabolites and that the acetylation of Hyty significantly increases its transport across the small intestinal epithelial cell barrier, enhancing the delivery of Hyty to the enterocytes.

To study the potential hepatic metabolism of olive oil phenols, human hepatoma HepG2 cells were incubated for 2 and 18 h with Ty, Hyty and Hyty acetate (Mateos et al., 2005). Extensive uptake and metabolism of Hyty and Hyty acetate were observed, with scarce metabolism of Ty. Hyty acetate was converted into free Hyty and then metabolized;

Metabolism and Bioavailability of Olive Oil Polyphenols 337

glucurono- and methyl-, but no sulfo-conjugates, were found. Olive oil phenols are

The colonic metabolism of olive oil polyphenols is scarcely reported. Corona et al. (2006) demonstrated that secoiridoids, which appear not to be absorbed in the small intestine, suffer bacterial catabolism in the large intestine with Ol undergoing rapid degradation by

It is essential to establish whether olive oil phenolics are absorbed in the intestine *in vivo* and how they are distributed in the organism. **Table 1** shows the *in vivo* bioavailability studies of olive oil polyphenols carried out so far. For practical reasons, rats are used as the model of choice for *in vivo* studies. Bai et al. (1998) studied the absorption and pharmacokinetics of Hyty in rats, finding that the absorption of Hyty after the ingestion of a single dose is very fast. The metabolic fate of Hyty and Ty in rats has been also evaluated by administration of the radiolabeled polyphenols. Hyty appeared in plasma at maximum levels 5 min after oral administration, although the proportion of free aglycones in some tissues differed to that observed in plasma (D'Angelo et al., 2001). In all of the investigated tissues, Hyty was enzymatically converted in oxidized and/or methylated derivatives, whereas the major urinary products were sulfo-conjugates. Tuck et al. (2001) compared the elimination of Hyty and Ty in rat urine within 24 h after administration, both orally (in oil- and water-based solutions) and intravenously (in saline). When orally administrated, polyphenols will be subjected to first-pass metabolism, so that the contribution of intestinal metabolism will be quite relevant. If the administration is intravenous, only hepatic contribution to its disposition will be seen. Results showed that Hyty and Ty can be absorbed into the systemic circulatory system after oral dosing and that their bioavailability when administered as an olive oil solution is almost complete. Later, urine samples were re-examined and Hyty and five of its metabolites were detected (Tuck et al., 2002). Three were conclusively identified as monosulfate and 3-*O*-glucuronide conjugates of Hyty, and homovanillic acid (HVA), and one was tentatively identified as *O*-glucuronide conjugate of HVA. Although there is no disagreement between studies, a major limitation is that they were done with rats and some researches suggest that comparisons between the model species might not be adequate. Visioli et al. (2003) observed a 25 fold higher basal excretion of Hyty and of its main

In a well-designed approach, Vissers et al. (2002) measured the absorption and urinary excretion of olive oil polyphenols in healthy ileostomy subjects and subjects with a colon after the ingestion of increasing doses of extracted phenols. Only a small amount of the ingested compounds was recovered in the urine, supporting the hypothesis that humans absorb a major fraction of the olive oil phenols consumed. Furthermore, the comparison between the absorbed polyphenols in normal and ileostomy subject showed similar results, which implies that the small intestine is the major site for the absorption of those compounds. Free Hyty and Ty and their glucurono-conjugates were the only metabolites detected in the urine samples. Another study carried out in human subjects assessed quantitatively the uptake of phenolics from olive oils containing different amounts of Ty and Hyty (Visioli et al., 2000). It was observed that these compounds were absorbed in a dose-dependent manner, that they were excreted in urine as glucuronide conjugates and that, as the concentration of phenols administered increased, the proportion of conjugation with glucuronic acid also increased. Upon re-examination of samples two more metabolites

of Hyty were identified: HVA and HVAlc (Caruso et al., 2001).

metabolized by the liver as well, as suggested by these results.

the colonic microflora producing Hyty as the major end product.

metabolites in rats than humans.


a Caco-2 cells: model system of the human intestinal epithelium; HepG2 cells: model system of the human liver; TC7 cells: spontaneously differentiating clone derived from the original Caco-2 cell population.

*Abbreviations:* AP: apical; BL: basolateral; gluc: glucuronide; Hyty: hydroxytyrosol; Hyty-Ac: hydroxytyrosol acetate; HVA: homovanillic acid; HVAlc homovanillic alcohol; Ol: oleuropein; Ol Agl: oleuropein aglycone; Papp: apparent permeability coefficient; Ty: tyrosol.

Table 1. *In vitro* studies carried out with olive oil polyphenols.

Detected

Hyty mono-gluc and methyl-gluc, HVA, Ty gluc, Hyty-Ac mono-

Hyty and Ty gluc, HVAlc, Hyty glutathionylated

*Hyty:* methyl, sulfate, methylsulfate. *Ty:* methyl, sulfate. *p-Coumaric acid:* disulfate, methyl. *Pinoresinol:* gluc, sulfate. *Luteolin:* gluc, methyl, methyl-

gluc

gluc,

a Caco-2 cells: model system of the human intestinal epithelium; HepG2 cells: model system of the human liver; TC7 cells: spontaneously differentiating clone derived from the original Caco-2 cell

hydroxytyrosol acetate; HVA: homovanillic acid; HVAlc homovanillic alcohol; Ol: oleuropein; Ol Agl:

*Abbreviations:* AP: apical; BL: basolateral; gluc: glucuronide; Hyty: hydroxytyrosol; Hyty-Ac:

*Hyty:* HVAlc. *Hyty-Ac:* Hyty, HVAlc, mono-gluc.

Hyty, HVAlc, Hyty and HVAlc gluc, gluc conjugates of the reduced forms of tested compounds

Study Outcome Ref.

Manna et al., 2000

Edgecombe et al., 2000

Mateos et al., 2005

Corona et al., 2006

Soler et al., 2010

Mateos et al., 2011

Pinto et al., 2011

passive diffusion mechanism, bidirectionally and in a dosedependent manner. Hyty is quantitatively absorbed in the

Ol in aqueous solution can be absorbed, albeit poorly, from isolated perfused rat intestine. The Papp of Ol in hypotonic conditions is significantly

Extensive uptake and hepatic metabolism of Hyty and Hyty-Ac with scarce metabolism of Ty; main derivatives formed: glucuronidated and methylated

Hyty and Ty were transferred across the cell monolayers and rat segments of intestine and were subjected to classic phase I/II biotransformation. No absorption of Ol

Limited intestinal metabolism. Major metabolites: methylated conjugates. Time-dependent efflux of various free and conjugated forms, showing preferential AP to BL transport after 24 h of incubation

Hyty-Ac is better absorbed than free Hyty and serves to enhance delivery of Hyty to the enterocytes for subsequent metabolism and BL efflux)

Caco-2 cells expressed limited metabolic activity. Major metabolites using the perfused rat intestine model: gluc of the reduce forms. Secoiridoids in the parental form were little absorbed in the small intestine

HVAlc Hyty transport occurs via a

intestine

higher

conjugates

Methods Metabolites

Transport kinetics: incubation with increasing concentrations (50-500 μM) at 37 and 4 ºC for 2 min. Transepithelial transport: incubation with 100 μM Hyty, glucose and mannitol

In situ intestinal perfusion technique: infusion of aqueous solution (1 mM, 50 μl/min) at 37 ºC during 40 min in both iso-osmotic and hypotonic luminal conditions

Cell uptake and metabolism of phenols: incubation with 100 μM at 37 ºC for 2 and 18 h

Phenols metabolism: incubation with 40, 50 and 100 μM at 37 ºC for 1, 6 and 24 h. Transport experiments in the AP, cellular and BL compartments: AP loading of phenol at 100 μM

Metabolism experiments and transport experiments in the AP and BL compartments: incubation with 50 μM at 37 ºC for 1, 2

Transport experiments using Caco-2 cells: incubation with 50, 100 and 200 μM at 37 ºC for 2 h; AP loading. Transport experiments using rat intestine: perfusion of methanol solution (100 μM) at 37 ºC during 80 min

oleuropein aglycone; Papp: apparent permeability coefficient; Ty: tyrosol. Table 1. *In vitro* studies carried out with olive oil polyphenols.

and 4 h

Tested Phenol

Hyty, Ty, Hyty-Ac

Hyty, Ty, *p*-coumaric acid, pinoresinol, luteolin

Hyty, Hyty-Ac

Ol Agl, dialdehydic form of Ol Agl

population.

Model systema

monolayers

intestine

Hepatoma HepG2 cells

monolayers and rat segments of jejunum and ileum

Caco-2/TC7 cell monolayers

Caco-2/TC7 cell monolayers

Human Caco-2 cell monolayers and isolated lumen of rat intestine (jejunum and ileum)

[14C] Hyty Caco-2 cell

Ol glycoside Isolated rat

Hyty, Ty, Ol Caco-2 cell

glucurono- and methyl-, but no sulfo-conjugates, were found. Olive oil phenols are metabolized by the liver as well, as suggested by these results.

The colonic metabolism of olive oil polyphenols is scarcely reported. Corona et al. (2006) demonstrated that secoiridoids, which appear not to be absorbed in the small intestine, suffer bacterial catabolism in the large intestine with Ol undergoing rapid degradation by the colonic microflora producing Hyty as the major end product.

It is essential to establish whether olive oil phenolics are absorbed in the intestine *in vivo* and how they are distributed in the organism. **Table 1** shows the *in vivo* bioavailability studies of olive oil polyphenols carried out so far. For practical reasons, rats are used as the model of choice for *in vivo* studies. Bai et al. (1998) studied the absorption and pharmacokinetics of Hyty in rats, finding that the absorption of Hyty after the ingestion of a single dose is very fast. The metabolic fate of Hyty and Ty in rats has been also evaluated by administration of the radiolabeled polyphenols. Hyty appeared in plasma at maximum levels 5 min after oral administration, although the proportion of free aglycones in some tissues differed to that observed in plasma (D'Angelo et al., 2001). In all of the investigated tissues, Hyty was enzymatically converted in oxidized and/or methylated derivatives, whereas the major urinary products were sulfo-conjugates. Tuck et al. (2001) compared the elimination of Hyty and Ty in rat urine within 24 h after administration, both orally (in oil- and water-based solutions) and intravenously (in saline). When orally administrated, polyphenols will be subjected to first-pass metabolism, so that the contribution of intestinal metabolism will be quite relevant. If the administration is intravenous, only hepatic contribution to its disposition will be seen. Results showed that Hyty and Ty can be absorbed into the systemic circulatory system after oral dosing and that their bioavailability when administered as an olive oil solution is almost complete. Later, urine samples were re-examined and Hyty and five of its metabolites were detected (Tuck et al., 2002). Three were conclusively identified as monosulfate and 3-*O*-glucuronide conjugates of Hyty, and homovanillic acid (HVA), and one was tentatively identified as *O*-glucuronide conjugate of HVA. Although there is no disagreement between studies, a major limitation is that they were done with rats and some researches suggest that comparisons between the model species might not be adequate. Visioli et al. (2003) observed a 25 fold higher basal excretion of Hyty and of its main metabolites in rats than humans.

In a well-designed approach, Vissers et al. (2002) measured the absorption and urinary excretion of olive oil polyphenols in healthy ileostomy subjects and subjects with a colon after the ingestion of increasing doses of extracted phenols. Only a small amount of the ingested compounds was recovered in the urine, supporting the hypothesis that humans absorb a major fraction of the olive oil phenols consumed. Furthermore, the comparison between the absorbed polyphenols in normal and ileostomy subject showed similar results, which implies that the small intestine is the major site for the absorption of those compounds. Free Hyty and Ty and their glucurono-conjugates were the only metabolites detected in the urine samples. Another study carried out in human subjects assessed quantitatively the uptake of phenolics from olive oils containing different amounts of Ty and Hyty (Visioli et al., 2000). It was observed that these compounds were absorbed in a dose-dependent manner, that they were excreted in urine as glucuronide conjugates and that, as the concentration of phenols administered increased, the proportion of conjugation with glucuronic acid also increased. Upon re-examination of samples two more metabolites of Hyty were identified: HVA and HVAlc (Caruso et al., 2001).




a Percentage of administered amount after 24 h, unless otherwise indicated

*Abbreviations:* apo-B: apolipoprotein-B; COO: common olive oil; DAD: diode array detector; DOPAC: 3,4-dihydroxyphenylacetic acid; DOPAL: 3,4-

dihydroxyphenylacetaldehyde; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EA: elenolic acid; EVOO: extra virgin olive oil; gluc: glucuronide; GC: gas

chromatography; GI: gastrointestinal; HPLC: high performance liquid chromatography; Hyty: hydroxytyrosol; HVA: homovanillic acid; HVAlc:

homovanillic alcohol; IV: intravenous; LC: liquid chromatography; LDL: low-density lipoproteins; Lig Agl: ligstroside aglycone; MS: mass

spectrometry; Ol: oleuropein; Papp: apparent permeability coefficient; ROO: refined olive oil; Ty: tyrosol; UV: ultraviolet; VOO: virgin olive oil

Table 2. Bioavailability of olive oil polyphenols in animals and humans.


a Percentage of administered amount after 24 h, unless otherwise indicated *Abbreviations:* apo-B: apolipoprotein-B; COO: common olive oil; DAD: diode array detector; DOPAC: 3,4-dihydroxyphenylacetic acid; DOPAL: 3,4dihydroxyphenylacetaldehyde; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EA: elenolic acid; EVOO: extra virgin olive oil; gluc: glucuronide; GC: gas chromatography; GI: gastrointestinal; HPLC: high performance liquid chromatography; Hyty: hydroxytyrosol; HVA: homovanillic acid; HVAlc: homovanillic alcohol; IV: intravenous; LC: liquid chromatography; LDL: low-density lipoproteins; Lig Agl: ligstroside aglycone; MS: mass spectrometry; Ol: oleuropein; Papp: apparent permeability coefficient; ROO: refined olive oil; Ty: tyrosol; UV: ultraviolet; VOO: virgin olive oil

Table 2. Bioavailability of olive oil polyphenols in animals and humans.

Metabolism and Bioavailability of Olive Oil Polyphenols 343

A notable metabolic pathway for Hyty is the methylation, giving rise to the formation of HVAlc (Caruso et al., 2001; Bazoti et al., 2010; Manna et al., 2000; Visioli et al, 2003). Oxidation and methylation-oxidation, rendering 3,4-dihydroxyphenilacetic acid (DOPAC) and HVA, respectively, have been also proposed (D'Angelo et al., 2001). It is noteworthy that many of the reported metabolites of Hyty are also the major molecular species deriving from dopamine metabolism (HVA, DOPAC, 3,4-dihydroxyphenyl acetaldehyde - DOPAL);

Besides, olive oil phenolic compounds and most of their corresponding phase I metabolites can be subsequently subjected to phase II reactions, preferentially glucuronoconjugation (García-Villalba et al., 2010). The presence of glucuronoconjugates of phenolic compounds belonging to most of chemical classes families described in olive oil has been widely detected in both urine and plasma, whereas the presence of sulfated metabolites has scarcely

The metabolism of olive oil lignans has not been reported in detail so far and one of the few references appeared only recently (Soler et al., 2010). In this study, pinoresinol glucuronide and sulfate conjugates were identified after incubation of free pinoresinol using

As far as flavonoids are concerned, products of methylation and glucuronidation have been observed (Soler et al., 2010; Suárez et al., 2009). Methyl-monoglucuronides of apigenin and

Several reports converge on the *in vitro* ability of olive oil phenolic compounds to bind low density lipoproteins (LDL) and to protect them against oxidation (Covas et al., 2000; Visioli et al., 1995). Moreover, both animal and human *in vivo* studies (Coni et al., 2000; Marrugat et al., 2004) have provided evidence on the effects of olive oil ingestion on LDL composition and the incorporation of olive oil phenolics and their metabolites in LDL. In one of the first studies, Bonanome et al. (2000) determined the presence of Hyty and Ty in human lipoprotein fractions after olive oil ingestion. Both compounds were recovered in all of the fractions, except in the very low density lipoproteins one; concentrations peaked between 1 and 2 h. Covas et al. (2006) demonstrated that the postprandial oxidative stress can be modulated by the olive oil phenolic content and that the degree of LDL oxidation decreases in a dose-dependent manner with the phenol concentration of the olive oil ingested. They arrived to these conclusions administering a single dose of olive oil, but similar results were obtained in studies using sustained doses; olive oil consumption for 1 week led to an increase in the total phenolic content of LDL (Gimeno et al., 2002). In a later study, volunteers were requested to ingest virgin, common or refined olive oil daily for 3 weeks (Gimeno et al., 2007). The concentration of total phenolic compounds in LDL was directly correlated with the phenolic concentration of the oils and with the resistance of LDL to their

De la Torre-Carbot et al. (2006, 2007) developed a rapid method for the determination in LDL of Ty, Hyty and several of their metabolites. The presence of these compounds in LDL strengthens claims that these compounds can act as *in vivo* antioxidants. The effect of the intake of virgin and refined olive oils after long-term ingestion of real-life doses on the

in fact, Hyty can be also called DOPET, a well-known dopamine metabolite.

been reported in literature.

*in vitro* oxidation.

differentiated Caco-2/TC7 cell monolayers.

luteolin have been identified as well (García-Villalba et al., 2010).

**2.3 Binding of olive oil polyphenols to lipoproteins** 

A major limitation of the commented human studies is that they used phenolics extracts or olive oil samples artificially enriched with phenolics extracts, and therefore extrapolation of these results to typical olive oil consumption may not be realistic. Further studies have been performed administering VOO at doses close to that used in the Mediterranean countries (30-50 g/day) (Bonanome et al., 2000; de la Torre-Carbot et al., 2006, 2007; García-Villalba et al., 2010; Khymenets et al., 2011; Miró-Casas et al., 2001a, 2001b, 2003a, 2003b; Suárez et al, 2009). Results confirmed that Hyty and Ty are mainly excreted in their glucuronoconjugated form; in fact, the role of glucuronidation in metabolism of main olive oil phenols can be evaluated in about 65-75% of totally recovered in urine after dietary VOO consumption (Khymenets et al., 2011; Miró-Casas et al., 2003b), which suggests an extensive first-pass intestinal/hepatic metabolism of the compounds ingested. Suárez et al. (2009) considered for the first time the absorption and disposition of flavonoids and lignans after the ingestion of VOO. Besides the presence of those VOO polyphenols in their conjugated forms, an important variability in the concentrations was observed between the plasma samples obtained from different volunteers. This variability may be attributed to differences in the expression of metabolizing enzymes due to genetic variability within the population. The most comprehensive study regarding the identification of metabolites in human urine of practically all the olive oil polyphenols described was reported by García-Villalba et al. (2010). These authors were able to achieve the tentative identification of 60 metabolites; the most abundant were those containing a catechol group, such as Hyty and the secoiridoids Ol Agl and deacetoxy-Ol Agl. Phenolic compounds were subjected to various phase I and phase II reactions, mainly methylation and glucuronidation. The report suggests that most of the olive oil polyphenols are absorbed to a greater or lesser extent, although absorption and metabolism seems to differ greatly among the different compounds.

#### **2.2 Conjugation and nature of metabolites**

Low doses of polyphenols are delivered through human diet and, generally, do not escape first-pass metabolism. As a result, most olive oil polyphenols undergo structural modifications, i.e. conjugation process; in fact, conjugates are the predominant forms in plasma. Once absorbed, olive oil polyphenols are subjected to three main types of conjugation: methylation, glucuronidation and, to a lesser extent, sulfation, through the respective action of catechol-O-methyl transferases (COMT), uridine-5'-diphosphate glucuronosyltransferases (UDPGT) and sulfotransferases (SULT) (Manach et al., 2004).

Recently, García-Villalba et al. (2010) carried out a broad study of the metabolites of most olive oil phenolic compounds excreted in human urine, showing that most polyphenols were absorbed, metabolized and excreted to a lesser or greater extent. It was initially suggested in literature that Ol Agl and Lig Agl were hydrolyzed in the gastrointestinal tract (GI) tract and then, the resulting polar phenols, Hyty and Ty, were absorbed and metabolized (Vissers et al., 2002). Nevertheless, the results obtained in later experiments with Caco-2 cells (Pinto et al., 2011) and humans (García-Villalba et al., 2010), showed that, at least, part of the secoiridoids can be absorbed and metabolized; reduction (hydrogenation) is the most probable metabolic pathway of these compounds. Hydroxylation and hydration are also possible pathways for the secoiridoids. In fact, they can precede or follow the action of COMT on compounds such as Hyty, deacetoxy-Ol Agl, and Ol Agl. Some compounds can even suffer a double hydroxylation before or after the methylation (García-Villalba et al., 2010).

A major limitation of the commented human studies is that they used phenolics extracts or olive oil samples artificially enriched with phenolics extracts, and therefore extrapolation of these results to typical olive oil consumption may not be realistic. Further studies have been performed administering VOO at doses close to that used in the Mediterranean countries (30-50 g/day) (Bonanome et al., 2000; de la Torre-Carbot et al., 2006, 2007; García-Villalba et al., 2010; Khymenets et al., 2011; Miró-Casas et al., 2001a, 2001b, 2003a, 2003b; Suárez et al, 2009). Results confirmed that Hyty and Ty are mainly excreted in their glucuronoconjugated form; in fact, the role of glucuronidation in metabolism of main olive oil phenols can be evaluated in about 65-75% of totally recovered in urine after dietary VOO consumption (Khymenets et al., 2011; Miró-Casas et al., 2003b), which suggests an extensive first-pass intestinal/hepatic metabolism of the compounds ingested. Suárez et al. (2009) considered for the first time the absorption and disposition of flavonoids and lignans after the ingestion of VOO. Besides the presence of those VOO polyphenols in their conjugated forms, an important variability in the concentrations was observed between the plasma samples obtained from different volunteers. This variability may be attributed to differences in the expression of metabolizing enzymes due to genetic variability within the population. The most comprehensive study regarding the identification of metabolites in human urine of practically all the olive oil polyphenols described was reported by García-Villalba et al. (2010). These authors were able to achieve the tentative identification of 60 metabolites; the most abundant were those containing a catechol group, such as Hyty and the secoiridoids Ol Agl and deacetoxy-Ol Agl. Phenolic compounds were subjected to various phase I and phase II reactions, mainly methylation and glucuronidation. The report suggests that most of the olive oil polyphenols are absorbed to a greater or lesser extent, although absorption

and metabolism seems to differ greatly among the different compounds.

Low doses of polyphenols are delivered through human diet and, generally, do not escape first-pass metabolism. As a result, most olive oil polyphenols undergo structural modifications, i.e. conjugation process; in fact, conjugates are the predominant forms in plasma. Once absorbed, olive oil polyphenols are subjected to three main types of conjugation: methylation, glucuronidation and, to a lesser extent, sulfation, through the respective action of catechol-O-methyl transferases (COMT), uridine-5'-diphosphate glucuronosyltransferases (UDPGT) and sulfotransferases (SULT) (Manach et al., 2004).

Recently, García-Villalba et al. (2010) carried out a broad study of the metabolites of most olive oil phenolic compounds excreted in human urine, showing that most polyphenols were absorbed, metabolized and excreted to a lesser or greater extent. It was initially suggested in literature that Ol Agl and Lig Agl were hydrolyzed in the gastrointestinal tract (GI) tract and then, the resulting polar phenols, Hyty and Ty, were absorbed and metabolized (Vissers et al., 2002). Nevertheless, the results obtained in later experiments with Caco-2 cells (Pinto et al., 2011) and humans (García-Villalba et al., 2010), showed that, at least, part of the secoiridoids can be absorbed and metabolized; reduction (hydrogenation) is the most probable metabolic pathway of these compounds. Hydroxylation and hydration are also possible pathways for the secoiridoids. In fact, they can precede or follow the action of COMT on compounds such as Hyty, deacetoxy-Ol Agl, and Ol Agl. Some compounds can even suffer a double hydroxylation

**2.2 Conjugation and nature of metabolites** 

before or after the methylation (García-Villalba et al., 2010).

A notable metabolic pathway for Hyty is the methylation, giving rise to the formation of HVAlc (Caruso et al., 2001; Bazoti et al., 2010; Manna et al., 2000; Visioli et al, 2003). Oxidation and methylation-oxidation, rendering 3,4-dihydroxyphenilacetic acid (DOPAC) and HVA, respectively, have been also proposed (D'Angelo et al., 2001). It is noteworthy that many of the reported metabolites of Hyty are also the major molecular species deriving from dopamine metabolism (HVA, DOPAC, 3,4-dihydroxyphenyl acetaldehyde - DOPAL); in fact, Hyty can be also called DOPET, a well-known dopamine metabolite.

Besides, olive oil phenolic compounds and most of their corresponding phase I metabolites can be subsequently subjected to phase II reactions, preferentially glucuronoconjugation (García-Villalba et al., 2010). The presence of glucuronoconjugates of phenolic compounds belonging to most of chemical classes families described in olive oil has been widely detected in both urine and plasma, whereas the presence of sulfated metabolites has scarcely been reported in literature.

The metabolism of olive oil lignans has not been reported in detail so far and one of the few references appeared only recently (Soler et al., 2010). In this study, pinoresinol glucuronide and sulfate conjugates were identified after incubation of free pinoresinol using differentiated Caco-2/TC7 cell monolayers.

As far as flavonoids are concerned, products of methylation and glucuronidation have been observed (Soler et al., 2010; Suárez et al., 2009). Methyl-monoglucuronides of apigenin and luteolin have been identified as well (García-Villalba et al., 2010).

#### **2.3 Binding of olive oil polyphenols to lipoproteins**

Several reports converge on the *in vitro* ability of olive oil phenolic compounds to bind low density lipoproteins (LDL) and to protect them against oxidation (Covas et al., 2000; Visioli et al., 1995). Moreover, both animal and human *in vivo* studies (Coni et al., 2000; Marrugat et al., 2004) have provided evidence on the effects of olive oil ingestion on LDL composition and the incorporation of olive oil phenolics and their metabolites in LDL. In one of the first studies, Bonanome et al. (2000) determined the presence of Hyty and Ty in human lipoprotein fractions after olive oil ingestion. Both compounds were recovered in all of the fractions, except in the very low density lipoproteins one; concentrations peaked between 1 and 2 h. Covas et al. (2006) demonstrated that the postprandial oxidative stress can be modulated by the olive oil phenolic content and that the degree of LDL oxidation decreases in a dose-dependent manner with the phenol concentration of the olive oil ingested. They arrived to these conclusions administering a single dose of olive oil, but similar results were obtained in studies using sustained doses; olive oil consumption for 1 week led to an increase in the total phenolic content of LDL (Gimeno et al., 2002). In a later study, volunteers were requested to ingest virgin, common or refined olive oil daily for 3 weeks (Gimeno et al., 2007). The concentration of total phenolic compounds in LDL was directly correlated with the phenolic concentration of the oils and with the resistance of LDL to their *in vitro* oxidation.

De la Torre-Carbot et al. (2006, 2007) developed a rapid method for the determination in LDL of Ty, Hyty and several of their metabolites. The presence of these compounds in LDL strengthens claims that these compounds can act as *in vivo* antioxidants. The effect of the intake of virgin and refined olive oils after long-term ingestion of real-life doses on the

Metabolism and Bioavailability of Olive Oil Polyphenols 345

acid sulfate could be formed as products of alcohol dehydrogenase and aldehyde dehydrogenase activities. Suárez & co-workers also found hydroxybenzoic acid in all the plasma samples. The glucuronide metabolite of apigenin was tentatively quantified in all the samples analyzed, but showing a considerable inter-individual variation. Lignans (pinoresinol and acetoxypinoresinol) could not be detected in the plasma samples even in

Once the polyphenols reach the bloodstream, they are able to penetrate tissues, particularly those in which they are metabolized. The nature of the tissular metabolites may be different from that of blood metabolites; data are still very scarce, even in animals, and their ability to accumulate within specific target tissues needs to be further investigated. An article written by D'Angelo et al. (2001) studied the fate of radiolabelled 14C Hyty intravenously injected in rats in different biological fluids (plasma, urine and feces) and tissues (brain, heart, kidney, liver, lung, skeletal muscle and GI content). The pharmacokinetic analysis indicated a fast and extensive uptake of the molecule by the organs and tissues investigated, with a preferential renal uptake. Over 90% of the administered radioactivity was excreted in urine after 5 h and about 5% was detectable in feces and GI content. Less than 8% of the administered radioactivity was still present in the blood stream 5 min after injection. Regarding tissues, the time course analysis indicated that the highest level of radioactivity was detected 5 min after injection, followed by a rapid decrease. It is worth noting that Hyty is able to cross the blood-brain barrier, even though its brain uptake is lower compared with other organs. In all the investigated tissues, Hyty was enzimatically converted in four oxidized and/or methylated derivatives (HVAlc, HVA, DOPAC, DOPAL) and sulfoconjugated derivatives. Enzymatic methylation is presumably operative in the brain, HVAlc representing 41.9% of the detected, labeled species. This reflects the key role of COMT in the central nervous system. The occurrence in the analyzed organs of both labeled DOPAL and DOPAC implies a sequential oxidation of Hyty ethanol side chain catalyzed by alcohol, and aldehyde dehydrogenase, respectively. Labeled HVA, the product of both methylation and oxidation, was also identified. Sulfoconjugated metabolites were mainly

As data on plasma concentration of olive oil phenols are still scarce, an alternative is to look at olive oil phenols excreted in urine; these may provide information on the form in which

The amount and form in which the olive oil phenols are excreted in urine may give an insight into their metabolism in the human body. The first experimental evidence of the absorption of Ty and Hyty from olive oil in humans was obtained by Visioli et al. (2000) from a single oral dose of 50 ml of phenolic-enriched olive oil. The proportions of Hyty and Ty recovered in glucuronidase-hydrolyzed urine, with respect to ingested dose, were in the ranges of 30–60% and 20–22%, respectively. This paper postulated that Hyty and Ty were dose-dependently absorbed in humans and excreted in urine as glucuronide

Miró-Casas et al. (2001a) measured the urinary recovery of administered Ty during the 24 h after EVOO ingestion. Maximal Ty values were obtained in the 0-4 h urine samples and

glucuronide or sulfate conjugated forms.

found in plasma (43.3%) and urine (44.1%).

phenols are present in plasma.

**2.5 Elimination** 

conjugates.

content of the metabolites in LDL was examined as well (de la Torre-Carbot et al., 2010). The phenols in VOO modulated the LDL content of 3 phenolic metabolites, Hyty, Ty, and HVA sulfates; the concentration of these compounds increased significantly after the ingestion of VOO, in contrast to the refined one. In parallel, the ingestion of VOO significantly reduced LDL and plasma oxidative markers, which suggests that the metabolic activities of phenols can be related to the capacity of these compounds to remain bound to LDL.
