**2.4 Plasma concentration and tissue uptake**

In 1998, Bai et al. reported for the first time the absorption of Hyty into the bloodstream. Hyty was administered orally to rats and appeared in plasma 2 min after, reaching the highest level at 5-10 min. Its concentration was low compared to the administered amount. The experiment, however, did not take into account the presence of metabolites.

After this first approach, different methods for the simultaneous detection of Ol and Hyty in rat plasma have been optimized. Ruiz-Gutiérrez et al. (2000) determined Hyty in overnightfasted rat plasma after its oral administration. Ol and Hyty plasma concentrations were measured after oral administration of a single dose of Ol to rats using soya oil and distilled water as administration vehicle (Del Boccio et al., 2003). Analysis of plasma showed the presence of unmodified Ol, reaching a peak value within 2 h, with a small amount of Hyty. In another study, Ol and Hyty plasma levels were monitored in rats after intravenous dosing of Ol (Tan et al., 2003). The dosing profile showed that at 10 min both Ol and Hyty were rapidly distributed.

Studies in which phenolic ingestion is closer to typical dietary patterns may be more appropriate for estimating bioavailability than the administration of pure compounds. Recently (Bazoti et al., 2010), the simultaneous quantification of Ol and its major metabolites in rat plasma was carried out after a control diet of 80 days supplemented with Ol or with EVOO. Basal levels of HVAlc were detected in the blood stream after the enzymatic treatment of the samples with *β*-glucuronidase. Before the enzymatic treatment, HVAlc was detected below the limits of quantification in plasma samples of rats supplement with Ol. Hyty was not detected, which indicates that it was metabolized to HVAlc. Ol was detected below the LOQ before and after the enzymatic treatment. These results are in accordance with the study made by Del Boccio et al. (2003), who demonstrated that Ol was rapidly metabolized and eliminated.

Miró-Casas et al. (2003b) quantified Hyty and HVAlc in human plasma and urine after reallife doses of VOO. Both compounds appeared rapidly in plasma mainly as glucuronides, with peak concentrations at 30 min for Hyty and 50 min. for HVAlc after the ingestion, supporting the premise that the small intestine is the major site of absorption for these compounds (Vissers et al., 2002).

Most of the studies described so far have centered their attention on Hyty, Ty and Ol derivatives. In a recent work, the absorption and disposition of other olive oil polyphenols (flavonoids and lignans) have been considered (Suárez et al., 2009). Samples were obtained from healthy humans 1 and 2 h after the ingestion of VOO. The major compounds identified and quantified in plasma corresponded to metabolites of Ty, although Ty sulfate was only detected in one subject, and especially Hyty, as glucuronide and sulfate conjugates. HVA sulfate could be the direct product of the Hyty methylation, and vanillin sulfate and vanillic

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

In 1998, Bai et al. reported for the first time the absorption of Hyty into the bloodstream. Hyty was administered orally to rats and appeared in plasma 2 min after, reaching the highest level at 5-10 min. Its concentration was low compared to the administered amount. The experiment,

After this first approach, different methods for the simultaneous detection of Ol and Hyty in rat plasma have been optimized. Ruiz-Gutiérrez et al. (2000) determined Hyty in overnightfasted rat plasma after its oral administration. Ol and Hyty plasma concentrations were measured after oral administration of a single dose of Ol to rats using soya oil and distilled water as administration vehicle (Del Boccio et al., 2003). Analysis of plasma showed the presence of unmodified Ol, reaching a peak value within 2 h, with a small amount of Hyty. In another study, Ol and Hyty plasma levels were monitored in rats after intravenous dosing of Ol (Tan et al., 2003). The dosing profile showed that at 10 min both Ol and Hyty

Studies in which phenolic ingestion is closer to typical dietary patterns may be more appropriate for estimating bioavailability than the administration of pure compounds. Recently (Bazoti et al., 2010), the simultaneous quantification of Ol and its major metabolites in rat plasma was carried out after a control diet of 80 days supplemented with Ol or with EVOO. Basal levels of HVAlc were detected in the blood stream after the enzymatic treatment of the samples with *β*-glucuronidase. Before the enzymatic treatment, HVAlc was detected below the limits of quantification in plasma samples of rats supplement with Ol. Hyty was not detected, which indicates that it was metabolized to HVAlc. Ol was detected below the LOQ before and after the enzymatic treatment. These results are in accordance with the study made by Del Boccio et al. (2003), who demonstrated that Ol was rapidly

Miró-Casas et al. (2003b) quantified Hyty and HVAlc in human plasma and urine after reallife doses of VOO. Both compounds appeared rapidly in plasma mainly as glucuronides, with peak concentrations at 30 min for Hyty and 50 min. for HVAlc after the ingestion, supporting the premise that the small intestine is the major site of absorption for these

Most of the studies described so far have centered their attention on Hyty, Ty and Ol derivatives. In a recent work, the absorption and disposition of other olive oil polyphenols (flavonoids and lignans) have been considered (Suárez et al., 2009). Samples were obtained from healthy humans 1 and 2 h after the ingestion of VOO. The major compounds identified and quantified in plasma corresponded to metabolites of Ty, although Ty sulfate was only detected in one subject, and especially Hyty, as glucuronide and sulfate conjugates. HVA sulfate could be the direct product of the Hyty methylation, and vanillin sulfate and vanillic

can be related to the capacity of these compounds to remain bound to LDL.

**2.4 Plasma concentration and tissue uptake** 

were rapidly distributed.

metabolized and eliminated.

compounds (Vissers et al., 2002).

however, did not take into account the presence of metabolites.

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 glucuronide or sulfate conjugated forms.

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

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 phenols are present in plasma.

#### **2.5 Elimination**

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 conjugates.

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

Metabolism and Bioavailability of Olive Oil Polyphenols 347

The urinary excretion of HVAlc and HVA in humans was reported for the first time by Caruso et al. (2001) after the intake of different VOOs. HVAlc contributes to 22% of the total excretion of Hyty and its metabolites, and HVA 56%. The excretion of both metabolites correlated with the administered dose of Hyty. Even at low doses, HVAlc and HVA were excreted. In a later study, Miró-Casas et al. (2003b) observed how urinary amounts of Hyty and HVAlc increased in response to VOO ingestion, reaching the maximum peak at 0-2 h. Urinary recovery 12 h after olive oil ingestion was rather different depending on the hydrolytic treatment applied. Under acidic conditions recoveries were higher for both Hyty and HVAlc than with enzymatic hydrolysis. It was apparent that 65% of Hyty was in its

To understand the impact of glucuronidation on the metabolic pathway of olive oil phenols, the simultaneous determination of Ty, Hyty, HVAlc and their corresponding *O*glucuronides in human urine was carried out by Khymenets et al. (2011). It was the first time that the glucuronides of these compounds have been directly identified and quantified in urine samples of volunteers supplemented with EVOO, because previous methods measured either free or total phenols after hydrolysis. The maximum excretion of Hyty and Ty occurred during the first 6 h after administration, which is in agreement with earlier reported data. The free Ty and Hyty, as well as HVAlc, were detected at significant concentrations in all urine samples collected 6 h after EVOO acute intake. Concentrations of *O*-glucuronide metabolites (4-*O*-gluc-Ty, 4-*O*-gluc-Hyty, 3-*O*-gluc-Hyty, and 4-*O*-gluc-HVAlc) were substantially higher in 6 h postprandial samples when compared to their parent compounds. About 13% of the consumed olive oil polyphenols were recovered in 24 h urine, 75% of which were in the form of glucuronides and 25% as

In another recent work, specific information about the type of conjugates in human urine was provided (García-Villalba et al., 2010). The authors were able to indentify more than 60 metabolites. This was the first report in which metabolites of simple phenols, flavonoids, lignans and secoiridoids have been found in human urine samples. Phenolic compounds were subjected to different phase I (hydrogenation, hydroxylation, methylation) and phase II (mainly glucuronidation) reactions. Ten metabolites were identified as possible biomarkers of olive oil intake and their levels in urine after the olive oil ingestion were

In summary, data on urinary excretion indicate that at least 5% of ingested olive oil phenols is recovered in urine mainly as glucuronidated Ty and Hyty. The remaining phenols are metabolized into other compounds, such as *O*-methylated Hyty. Monosulfate conjugates might be other metabolites, as shown in two rat studies; however, if olive oil phenols are

**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

monitored, finding the highest level of most of them 2 h after administration.

also metabolized into these conjugates in humans remains to be elucidated.

important topics commented in the section.

glucuronoconjugated form and 35% in other conjugated forms.

free compounds.

decrease to reach basal values after 8-12 h. Ty was excreted in urine mainly in its glucuroconjugated form, with only 6–11% excreted in the free form. In a later study (Miró-Casas et al., 2001b), the simultaneous determination of Hyty and Ty in human urine after the intake of VOO was reported. Like the previous study, Hyty and Ty levels in urine rose after VOO consumption, reaching a peak at 0–4 h and returning to basal values at 12–24 h. After hydrolytic treatment, the amount of total compounds recovered in 24 h urine was also determined for Hyty and Ty. Recoveries ranged between 32-98% for Hyty and 12.1-52% for Ty. Both compounds were mainly excreted in conjugated form since only 5.9% Hyty and 13.8% Ty of the total amount excreted were in free form. The hydrolysis procedure applied in this study was limited because it did not provide specific information about the type of conjugation involved. This paper also postulated that Ol is not the main source of Hyty after ingestion of olive oil. The absorption of Hyty and Ty was later confirmed in an experiment using single and sustained doses of VOO (Miró-Casas et al., 2003a). Urinary recoveries of Ty were similar for both cases; however, mean recovery values for Hyty after ingestion of 25 ml/day VOO for one week, were 1.5-fold of those obtained after a 50 ml single dose.

Vissers et al. (2002) studied the absorption of Hyty, Ty and, for the first time, Ol and Lig Agl, in ileostomy subjects and in volunteers with a colon. The results showed that 55-66% of the ingested olive oil phenols were absorbed in ileostomy subjects, which implies that most phenols are absorbed in the small intestine. Excreted phenolics, mainly in the form of Hyty and Ty, were determined to be 5–16% of the total ingested. Similar levels of Hyty and Ty were found in the urine of subjects with and without a colon, confirming that olive oil phenols are absorbed mainly in the small intestine. The obtained values, lower than those reported by others, could be underestimations because metabolites of olive oil phenols were not considered. In this work it is also suggested that an important step in the metabolism of the Ol glycoside and Ol and Lig-aglycones is their transformation into Hyty or Ty. This was supported by finding that 15% of an Ol glycoside supplement administered to healthy human subjects was excreted in urine as Hyty and Ty.

Tuck et al. (2001) investigated the *in vivo* fate of tritium labeled Hyty and Ty after intravenous (in saline, tail vein) and oral dosing (in oil- and water-based solutions) to rats. For both Hyty and Ty, the elimination of radioactivity in urine within 24 h for the intravenously and orally administered oil-based dosing was significantly greater (95 and 75%, respectively) than the oral aqueous dosing method (74 and 53%, respectively). The majority of the excreted dose was eliminated from the body within 2 h, when intravenously dosed, and within 4 h for both oral dosings. Later, urine samples collected after 24 h were re-examined (Tuck et al., 2002). After oral oil dosing Hyty represented 4.10% of compound eliminated, monosulfate conjugate 48.42%, glucuronide conjugate 9.53%, HVA 10.26% and other possible metabolites 20.27%. Other study with rats supplemented with Ol (Ol rats) or with EVOO (EVOO rats) was developed for the simultaneous determination of Hyty, Ty and EA in rat urine (Bazoti et al., 2005). The urinary levels of free Ty and Hyty were higher in EVOO rats than in Ol rats. When the urine sample were treated with β-glucuronidase, the total amount of metabolites measured for the EVOO rat was higher for Ty but lower for Hyty than in Ol rats. EA was not detected, probably because of its further metabolism to simpler molecules. Nevertheless, as already mentioned, caution should be taken interpreting the results achieved from rats (Visioli et al., 2003).

decrease to reach basal values after 8-12 h. Ty was excreted in urine mainly in its glucuroconjugated form, with only 6–11% excreted in the free form. In a later study (Miró-Casas et al., 2001b), the simultaneous determination of Hyty and Ty in human urine after the intake of VOO was reported. Like the previous study, Hyty and Ty levels in urine rose after VOO consumption, reaching a peak at 0–4 h and returning to basal values at 12–24 h. After hydrolytic treatment, the amount of total compounds recovered in 24 h urine was also determined for Hyty and Ty. Recoveries ranged between 32-98% for Hyty and 12.1-52% for Ty. Both compounds were mainly excreted in conjugated form since only 5.9% Hyty and 13.8% Ty of the total amount excreted were in free form. The hydrolysis procedure applied in this study was limited because it did not provide specific information about the type of conjugation involved. This paper also postulated that Ol is not the main source of Hyty after ingestion of olive oil. The absorption of Hyty and Ty was later confirmed in an experiment using single and sustained doses of VOO (Miró-Casas et al., 2003a). Urinary recoveries of Ty were similar for both cases; however, mean recovery values for Hyty after ingestion of 25

ml/day VOO for one week, were 1.5-fold of those obtained after a 50 ml single dose.

human subjects was excreted in urine as Hyty and Ty.

interpreting the results achieved from rats (Visioli et al., 2003).

Vissers et al. (2002) studied the absorption of Hyty, Ty and, for the first time, Ol and Lig Agl, in ileostomy subjects and in volunteers with a colon. The results showed that 55-66% of the ingested olive oil phenols were absorbed in ileostomy subjects, which implies that most phenols are absorbed in the small intestine. Excreted phenolics, mainly in the form of Hyty and Ty, were determined to be 5–16% of the total ingested. Similar levels of Hyty and Ty were found in the urine of subjects with and without a colon, confirming that olive oil phenols are absorbed mainly in the small intestine. The obtained values, lower than those reported by others, could be underestimations because metabolites of olive oil phenols were not considered. In this work it is also suggested that an important step in the metabolism of the Ol glycoside and Ol and Lig-aglycones is their transformation into Hyty or Ty. This was supported by finding that 15% of an Ol glycoside supplement administered to healthy

Tuck et al. (2001) investigated the *in vivo* fate of tritium labeled Hyty and Ty after intravenous (in saline, tail vein) and oral dosing (in oil- and water-based solutions) to rats. For both Hyty and Ty, the elimination of radioactivity in urine within 24 h for the intravenously and orally administered oil-based dosing was significantly greater (95 and 75%, respectively) than the oral aqueous dosing method (74 and 53%, respectively). The majority of the excreted dose was eliminated from the body within 2 h, when intravenously dosed, and within 4 h for both oral dosings. Later, urine samples collected after 24 h were re-examined (Tuck et al., 2002). After oral oil dosing Hyty represented 4.10% of compound eliminated, monosulfate conjugate 48.42%, glucuronide conjugate 9.53%, HVA 10.26% and other possible metabolites 20.27%. Other study with rats supplemented with Ol (Ol rats) or with EVOO (EVOO rats) was developed for the simultaneous determination of Hyty, Ty and EA in rat urine (Bazoti et al., 2005). The urinary levels of free Ty and Hyty were higher in EVOO rats than in Ol rats. When the urine sample were treated with β-glucuronidase, the total amount of metabolites measured for the EVOO rat was higher for Ty but lower for Hyty than in Ol rats. EA was not detected, probably because of its further metabolism to simpler molecules. Nevertheless, as already mentioned, caution should be taken The urinary excretion of HVAlc and HVA in humans was reported for the first time by Caruso et al. (2001) after the intake of different VOOs. HVAlc contributes to 22% of the total excretion of Hyty and its metabolites, and HVA 56%. The excretion of both metabolites correlated with the administered dose of Hyty. Even at low doses, HVAlc and HVA were excreted. In a later study, Miró-Casas et al. (2003b) observed how urinary amounts of Hyty and HVAlc increased in response to VOO ingestion, reaching the maximum peak at 0-2 h. Urinary recovery 12 h after olive oil ingestion was rather different depending on the hydrolytic treatment applied. Under acidic conditions recoveries were higher for both Hyty and HVAlc than with enzymatic hydrolysis. It was apparent that 65% of Hyty was in its glucuronoconjugated form and 35% in other conjugated forms.

To understand the impact of glucuronidation on the metabolic pathway of olive oil phenols, the simultaneous determination of Ty, Hyty, HVAlc and their corresponding *O*glucuronides in human urine was carried out by Khymenets et al. (2011). It was the first time that the glucuronides of these compounds have been directly identified and quantified in urine samples of volunteers supplemented with EVOO, because previous methods measured either free or total phenols after hydrolysis. The maximum excretion of Hyty and Ty occurred during the first 6 h after administration, which is in agreement with earlier reported data. The free Ty and Hyty, as well as HVAlc, were detected at significant concentrations in all urine samples collected 6 h after EVOO acute intake. Concentrations of *O*-glucuronide metabolites (4-*O*-gluc-Ty, 4-*O*-gluc-Hyty, 3-*O*-gluc-Hyty, and 4-*O*-gluc-HVAlc) were substantially higher in 6 h postprandial samples when compared to their parent compounds. About 13% of the consumed olive oil polyphenols were recovered in 24 h urine, 75% of which were in the form of glucuronides and 25% as free compounds.

In another recent work, specific information about the type of conjugates in human urine was provided (García-Villalba et al., 2010). The authors were able to indentify more than 60 metabolites. This was the first report in which metabolites of simple phenols, flavonoids, lignans and secoiridoids have been found in human urine samples. Phenolic compounds were subjected to different phase I (hydrogenation, hydroxylation, methylation) and phase II (mainly glucuronidation) reactions. Ten metabolites were identified as possible biomarkers of olive oil intake and their levels in urine after the olive oil ingestion were monitored, finding the highest level of most of them 2 h after administration.

In summary, data on urinary excretion indicate that at least 5% of ingested olive oil phenols is recovered in urine mainly as glucuronidated Ty and Hyty. The remaining phenols are metabolized into other compounds, such as *O*-methylated Hyty. Monosulfate conjugates might be other metabolites, as shown in two rat studies; however, if olive oil phenols are also metabolized into these conjugates in humans remains to be elucidated.
