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The Bioavailability of Olive Oil Phenolic Compounds
Chapter 73
The Bioavailability of Olive Oil Phenolic Compounds María-Isabel Covas, Montserrat Fitó, Olha Khymenets and Rafael de la Torre Cardiovascular Risk and Nutrition and Human Pharmacology and Clinical Neurosciences Research Groups, Institut Municipal d´Investigació Mèdica (IMIM-Hospital del Mar), Barcelona, Spain. CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN)
73.1 Introduction The beneficial effects of olive oil on cardiovascular risk factors are now recognized and often only attributed to its high levels of monounsaturated fatty acids (MUFA). On November 2004, the Federal Drug Administration of the USA permitted a claim on olive oil labels concerning: ‘the benefits on the risk of coronary heart disease of eating about 2 tablespoons (23 grams) of olive oil daily, due to the MUFA in olive oil’ (US FDA Press Release P04100. November 1, 2004. http://www.fda.gov/bbs/topics/ news/2004/NEW01129.htlm; accessed on May 2, 2008). Olive oil is, however, more than a MUFA fat. Olive oil is a functional food which besides having a high level of MUFA contains other minor components with biological properties (Covas et al., 2006c). The content of the minor components of an olive oil varies, depending on several conditions such as the cultivar, climate, ripeness of the olives at harvesting, and the processing system employed to produce the olive oil. Three types of olive oil are currently present on the market: virgin, ordinary, or pomace (Gimeno, 2002). Virgin olive oils are produced by direct pressing or centrifugation of the olives, among them those with an acidity greater than or equal to 3.3 degrees (2 degrees in the European Union) are submitted to a refining process in which some components, mainly phenolic compounds, and to a lesser degree squalene, are lost (Owen et al., 2000a). By mixing virgin and refined olive oil, an ordinary olive oil (olive oil, UE 1991) is produced and marketed. After virgin olive oil production, the rest of the olive drupe and seed is processed and submitted to a refining process, resulting in pomace olive oil, to which a certain quantity of virgin olive oil is added before marketing. The minor components of virgin olive oil are classified into two types: the unsaponificable fraction, defined as the fraction extracted with solvents after the saponification of the oil, and the soluble fraction which includes the phenolic compounds (Covas et al., 2006c). Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
The major phenolic compounds in olive oil are: (1) simple phenols (e.g., hydroxytyrosol, tyrosol, vanillic acid); (2) secoiridoids: oleuropein glucoside, and SIDs which are the dialdehydic form of oleuropein (SID-1) and ligstroside (SID-2) lacking a carboxymethyl group, and the aglycone form of oleuropein glucoside (SID-3) and ligstroside (SID4); and (3) polyphenols: lignans (e.g., ()-pinoresinol and ()-1-acetoxypinoresinol) and flavonols (Owen, 2000; Covas et al., 2006c) (Figure 73.1). Tyrosol, hydroxytyrosol, and their secoiridoid derivatives make up around 90% of the total phenolic content of a virgin olive oil.
73.2 Bioavailability of olive oil phenolic compounds 73.2.1 Absorption and Disposition After olive oil ingestion, tyrosol (Tyr) and hydroxytyrosol (OH-Tyr), as well as their glucosides and aglycones, such as oleuropein, undergo rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of Tyr and OH-Tyr free forms which enter the small intestine (Corona et al., 2006). In in vitro models, both OH-Tyr and Tyr are able to cross human Caco-2 cell monolayers and rat segments of jejunum and ileum (Manna et al., 2000; Corona et al., 2006). Data from experiments performed in Caco-2 cell monolayers using 14C–OH-Tyr, showed the transport of the phenolic compound occurs via a bidirectional passive diffusion mechanism (Manna et al., 2000). In animal models, when Tyr and OH-Tyr were orally administered in oil or water solutions, the orally administered oil dosing promoted a recovery of the phenolics in 24-h urine greater (25%) than that obtained with the oral aqueous dose (Tuck et al., 2001). When Tyr and OH-Tyr were administered intravenously in saline solution no significant differences were observed
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Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
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A
C
OH
HO
D
O
HO
O OH
OH
O
HO
COOCH3
E
O
COOCH3
HO
O O
COOCH3
OH O
O
O O
B
HO
O
O
O
OH
HO
HO OH
F
G
O
HO
O
CH2OH
COOCH3
HO
O
O COOCH3
OH O
O OH
OH
Figure 73.1 Structures of tyrosol, hydroxytyrosol and derivatives: (A): hydroxytyrosol; (B): tyrosol; (C): oleuropein glucoside; (D): SID-1; (E): SID-2; (F): SID-3; (G): SID-4.
in the amount of phenolic compounds recovered in urine between the intravenous and the oral oil doses for either Tyr or OH-Tyr. Similar results were obtained in humans when the bioavailability of OH-Tyr was compared by administering this compound in different matrices (olive oil, spiked refined oil, or yoghurt) (Visioli et al., 2003). Urinary OHTyr recovery was higher after virgin olive oil administration (44.2% of the OH-Tyr administered) than after addition of OH-Tyr to a refined olive oil (23% of the OH-Tyr administered), or to a yoghurt (5.8% of dose or approximately, 13% of that recorded after virgin olive oil intake) (Visioli et al., 2003). Concerning oleuropein, data from animal models show it can be absorbed, albeit poorly, from isolated perfused rat intestine. Thus, the most plausible way for oleuropein to exert its biological activities seems to be through its conversion to OH-Tyr (Edgecombe et al., 2000). This idea is supported by the results of bioavailability studies in rats, in which peak plasma concentrations reached after ingestion of high doses of oleuropein (100 mg kg1) were in the nanogram range, whereas those of OH-Tyr were highly increased (Del Boccio et al., 2003; Bazoti et al., 2005). These observations have been further confirmed in humans (Vissers et al., 2002; Visioli et al., 2003). In the process of crossing epithelial cells of the gastro intestinal tract, phenolic compounds from olive oil are subject to a biotransformation phase and, therefore, subjected to an important first-pass metabolism. According to data of in vitro studies, about 10% of OH-Tyr is converted in
homovanyl alcohol by the catechol-O-methyltransferase (Manna et al., 2000). In addition to the O-methylated derivative of OH-Tyr, the glucuronides of OH-Tyr and Tyr have also been described (Corona et al., 2006). In contrast, there was no absorption of oleuropein as it was rapidly degraded by the colonic microflora resulting in OH-Tyr formation (Corona et al., 2006). The hepatic metabolism of the olive oil phenols has been studied in human hepatoma HepG2 cells. After incubation, culture media and cell lysates were hydrolyzed with -glucuronidase and sulfatase. Methylated and glucuronidated forms of OH-Tyr were detected after 18 h of incubation, together with methyl-glucuronidated metabolites. Hydroxytyrosyl acetate was largely converted into free OH-Tyr and subsequently metabolized, although small amounts of glucuronidated hydroxytyrosyl acetate were detected. Tyrosol was poorly metabolized, with 10% of the phenol glucuronidated after 18 h. Minor amounts of free or conjugated phenols were detected in cell lysates. No sulfated metabolites were found (Mateos et al., 2005). The pharmacokinetics of OH-Tyr intravenously administered to rats indicates a fast and extensive uptake of the molecule by the organs and tissues, with a preferential renal uptake (D’Angelo et al., 2001). Hydroxytyrosol was recovered mainly in the sulfo-conjugated forms. The recovery of OH-Tyr in urine was about 6% of the dose administered: 0.3% recovered as 3-methyl-4-hydroxy-phenylethanol (HVAL or MOPET), 12.3% as 3,4-dihydroxy-phenylacetic acid (DOPAC), 23.6%
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Chapter | 73 The Bioavailability of Olive Oil Phenolic Compounds
as homovanillic acid (3-methyl-4-hydroxy-phenylacetic acid, HVA), and 26% as 3,4-dihydroxy-phenylacetaldehyde (DOPAL) (D’Angelo et al., 2001). It has been suggested that non-absorbable phenolic compounds can exert local antioxidant activities in the gastro intestinal tract (Ursini et al., 1998), an idea supported by the capacity of phenolic compounds isolated from olive oil to scavenge the free radicals generated by the fecal matrix (Owen et al., 2000b) and those induced in intestinal epithelium cells (Manna et al., 1996). However, one of the prerequisites to assess their in vivo physiological significance is to determine their absorbability and presence in human plasma. The first report on the bioavailability and disposition of olive oil phenolic compounds in humans was provided by Visioli et al. (2000). In this experiment, Tyr and OH-Tyr were spiked to a refined olive oil (very low phenolic content) and administered to healthy volunteers. Phenolic compounds were dose-dependently absorbed in humans, most phenolic compounds being recovered in biological fluids as conjugates in a dose-dependent manner with the phenolic content of the olive oil administered (Visioli et al., 2000). Also, in human studies it was demonstrated that tyrosol, hydroxytyrosol and oleuropein were absorbed at the small intestine level. Oleuropein was not quantified in plasma nor in urine but it was shown that it was metabolized in the body and recovered in urine mainly in the form of hydroxytyrosol (Vissers, 2000). Phenolic compounds, particularly those bearing a catechol group, are typically biotransformed by three enzymatic systems: catechol-O-methyl-transferase, sulfatases, and glucuronosyltransferases. Depending on the dose and the availability of co-factors the proportion of methyl, sulfate, and glucuronide conjugates varies among subjects. Further studies on the olive oil phenolic compound bioavailability (Miró-Casas et al., 2001, 2003a, b) were performed with virgin olive oil in its
natural form. After administering 25 mL of virgin oil (with an estimated content 1.2 mg of OH-Tyr), OH-Tyr plasma concentrations peaked at 30 minutes and those of its methylated metabolite, HVAL at 50 min. Plasma peak concentrations were around 25 ng mL1 for OH-Tyr and 4 ng mL1 for HVAL. The estimated half-life for OH-Tyr was 3 hours, reaching baseline concentrations after 8 hours of the virgin olive oil ingestion (Figure 73.2). More than 98% of both OH-Tyr and HVAL were in their conjugated forms, mainly glucuronates, confirming previous findings. In urine, OH-Tyr and HVAL concentrations peaked in the collection period 0–2 h (MiróCasas et al., 2003a). Despite the short half-life of Tyr and OH-Tyr, sustained consumption promotes an increase of olive oil phenolic compounds in biological fluids (Figure 73.2). Plasma and urinary levels of OH-Tyr and Tyr increase in a dose-dependent manner with the phenolic content of the olive oil administered (Miró-Casas et al., 2003b; Marrugat et al., 2004; Weinbrenner et al., 2004; Covas et al., 2006a, b) (Figure 73.3). Table 73.1 shows the urinary recoveries of OH-Tyr, Tyr, and HVAL after olive oil of medium (164 mg kg1) and high (466 mg kg1) phenolic content. The fact that a dose-dependent increase of Tyr and OH-Tyr with the phenolic content of the olive oil administered exists, at real-life olive oil doses, confirms the usefulness of these compounds as biomarkers of compliance in clinical trials (Covas et al., 2006b). With regard to the dose–effect relationship, 24-h urinary Tyr seems to be a better biomarker of sustained and moderate doses of virgin olive oil consumption than OH-Tyr (Miró-Casas et al., 2003b). Both OH-Tyr and Tyr urinary concentrations have been used, and are currently in use, in nutritional intervention studies as biomarkers of virgin olive oil ingestion Covas et al., 2006a, b; Fitó et al., 2007, 2008). Most bioavailability studies on olive oil phenols have measured total Tyr and OH-Tyr concentrations in blood or
9 8
LPC MPC HPC
Conc(ng/mL−1)
7 6 5 4 3 2 1 0
0 Without supplementation
24
48 Hours
72
96 After supplementation
Figure 73.2 Plasma hydroxytyrosol concentrations after ingestion of olive oil with low (LPC, 10 mg kg1), medium (133 mg kg1), and high (486 mg kg1) phenolic content. A single 25 mL dose was ingested before and after 4 days of olive oil supplementation (25 mL day1). Adapted from Weinbrenner, 2004.
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in some physiopathological states, such as inflammation or cancer, with a concomitant in situ deconjugation of phenol metabolites. Data are still very scarce and further studies are needed to elucidate the role and bioactivity of the human biological metabolites of olive oil phenolic compounds.
Percentage (%) of change
250 200
Tyrosol Hydroxytyrosol
150 100
73.2.2 Binding of Olive Oil Phenolic Compounds and their Metabolites to Human Lipoproteins
50 0
Low Medium High Olive oil phenolic content
Figure 73.3 Changes in urinary tyrosol and hydroxytyrosol after 3 weeks of sustained olive oil (range of phenolic compounds from 0 to 150 mg kg1) ingestion. Adapted from Marrugat et al., 2004.
Table 73.1 Urinary recoveries (mol) of olive oil phenolic compounds. Olive oil
Hydroxytyrosol
Tyrosol
HVAL
MPC
1.21 0.07
1.33 0.11
0.38 0.04
HPC
3.12 0.21
2.69 0.19
0.78 0.06
Values are expressed as Mean SD. HPC, high phenolic content olive oil (466 mg kg1); MPC, medium phenolic content olive oil (164 mg kg1); HVAL,3-methyl-4-hydroxy-phenylethanol, the methylated biological metabolite of hydroxytyrosol.
urine after acidic or enzymatic treatment of the samples. There is a lack of studies in which glucuronide and sulfate conjugates of Tyr and OH-Tyr in biological samples have been measured. It could be hypothesized that some conjugates of phenolic compounds may behave as carriers of the free forms of the phenolic compounds to target tissues, the ‘depot hypothesis’. Within this hypothesis, the determination of the bioavailability of phenol metabolites in plasma or tissues may be more relevant than that in urine. The proportion of free aglycones in some tissues can differ from that observed in blood (D’Angelo et al., 2001); this may be explained by a specific uptake of the aglycone or intracellular deconjugation. This last hypothesis implies that anionic conjugates could be transported across plasma membranes via carrier systems, as has been shown for acyl-glucuronides (Sallustio et al., 2000). Furthermore, -glucuronidase is located in the lumen of the endoplasmic reticulum in various organs, which could be reached by phenol glucuronides. -Glucuronidase is also present in the lysosomes of several cells, from which the enzyme can be released under some particular conditions such as an oxidative stress situation. -Glucuronidase activity can increase
Phenolic compounds which can bind low-density lipoproteins (LDL) are likely to perform their peroxyl scavenging activity in the arterial intima, where full LDL oxidation occurs in microdomains sequestered from the richness of antioxidants present in plasma (Witzum, 1994). Olive oil phenolic compounds can bind lipoproteins in vivo in humans. Tyrosol and hydroxytyrosol were recovered in all human lipoprotein fractions after virgin olive oil ingestion, except in very low-density lipoproteins, with concentrations peaking between 1 and 2 hours after olive oil ingestion (Bonanome et al., 2000). Tyrosol and hydroxytyrosol, as well as their biological metabolites hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate, and homovanillic acid sulfate, have been identified in LDL after virgin olive oil ingestion (De la Torre-Carbot et al., 2006, 2007). In addition, the concentration of total phenolic compounds in LDL has been shown to be directly correlated with the phenolic concentration of the olive oils ingested and with the resistance of LDL to their in vitro oxidation (Gimeno et al., 2007). After ingestion of 40 mL of virgin olive oil, the phenolic content of the LDL directly correlated with the plasma concentrations of Tyr and OH-Tyr (Covas et al., 2006a). The susceptibility of the LDL to oxidation depends not only on their fatty acid content, but also on the LDL antioxidant content (i.e., vitamin E and polyphenols) bound to the LDL (Fuller and Jialal, 1994). Further studies are required to establish the nature of the bond between the LDL and the phenolic compounds, including olive oil phenolic compounds and their metabolites, due to the physiopathological implications involved.
References Bazoti, F.N., Gikas, E., Puel, C., Coxam, V., Tsarbopoulos, A., 2005. Development of a sensitive and specific solid phase extraction-gas chromatography-tandem mass spectrometry method for the determination of elenolic acid, hydroxytyrosol, and tyrosol in rat urine. J. Agric. Food Chem. 53, 6213–6221. Bonanome, A., Pagnan, A., Caruso, D., Toia, A., Xamin, A., Fedeli, E., Berra, B., Zamburlini, A., Ursini, F., Galli, G., 2000. Evidence of postprandial absorption of olive oil in humans. Nutr. Metab. Cardiovas. Dis. 10, 111–120. Corona, G., Tzounis, X., Assunta-Dessa, M., Deiana, M., Debnam, E.S., Visoli, F., Spencer, J.P., 2006. The fate of olive oil polyphenols in the
Chapter | 73 The Bioavailability of Olive Oil Phenolic Compounds
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Manna, C., Galletti, P., Cucciolla, V., 1996. The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J. Nutr. 127, 286–292. Manna, C., Galletti, P., Maisto, G., Cucciolla, V., D’Angelo, S., Zappia, V., 2000. Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 470, 341–344. Marrugat, J., Covas, M.I., Fitó, M., Schröder, H., Miró-Casas, E., Gimeno, E., López-Sabater, M.C., de la Torre, R., Farré, M. and the SOLOS Investigators, 2004. Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation. A randomized controlled trial. Eur. J. Nutr. 43, 140–147. Mateos, R., Goya, L., Bravo, L., 2005. Metabolism of the olive oil phenols hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate by human hepatoma HepG2 cells. J. Agric. Food Chem. 53, 9897–9905. Miró-Casas, E., Farré-Albadalejo, M., Covas Planells, M.I., Fitó Colomer, M., Lamuela Raventós, R.M., de la Torre, R., 2001. Tyrosol bioavailability in humans after ingestion of virgin olive oil. Clin. Chem. 47, 341–343. Miró-Casas, E., Covas, M.-I., Farré, M., Fitó, M., Ortuño, J., Weinbrenner, T., Roset, P., de la Torre, R., et al., 2003a. Hydroxytyrosol disposition in humans. Clin. Chem. 49, 945–952. Miró-Casas, E., Covas, M.I., Fitó, M., Farré-Albaladejo, M., Marrugat, J., de la Torre, R., 2003b. Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur. J. Clin. Nutr. 57, 186–190. Owen, R.W., Mier, W., Giacosa, A., Hule, W.E., Spiegelhalder, B., Bartsch, H., 2000a. Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoroids, lignans and squalene. Food Chem. Toxicol. 38, 647–659. Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Spigelhalder, B., Bartsch, H., 2000b. The antioxidant/anticancer potential of phenolic compounds from olive oil. Eur. J. Cancer 36, 1235–1247. Sallustio, B.C., Sabordo, L., Evans, A.M., Nation, R.L., 2000. Hepatic disposition of electrophilic acyl glucuronide conjugates. Curr. Drug. Metab. 1, 163–180. Tuck, K.L., Freeman, M.P., Hayball, P.J., Stretch, G.L., Stupans, I., 2001. The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats. J. Nutr. 131, 1993–1996. Ursini, F., Zamburlini, A., Cazzolato, G., Maiorino, M., Bon, G.B., Sevanian, A., 1998. Postprandial plasma lipid hydroperoxides: a possible link between diet and atherosclerosis. Free Radic. Biol. Med. 25, 250–252. Vissers, M.N., Zock, P.L., Roodenburg, A.J., Leenen, R., Katan, M.B., 2002. Olive oil phenols are absorbed in humans. J. Nutr. 132, 409–417. Visioli, F., Galli, C., Bornet, F., Mattei, A., Patelli, R., Galli, G., Caruso, D., 2000. Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett. 468, 159–160. Visioli, F., Galli, C., Grande, S., Colonelli, K., Patelli, C., Galli, G., Caruso, D., 2003. Hydroxytyrosol excretion differs between rats and humans and depends on the vehicle of administration. J. Nutr. 133, 2612–2615. Weinbrenner, T., Fitó, M., de la Torre, R., Saez, G.T., Rijken, P., Tormos, C., Coolen, S., Farré-Albaladejo, M., Abanades, S., Schröder, H., Marrugat, J., Covas, M.I., 2004. Olive oils high in phenolic compounds modulate oxidative/antioxidative status in men. J. Nutr. 134, 2314–2321. Witzum, J.L., 1994. The oxidation hypothesis of atherosclerosis. Lancet 344, 793–795.
The Bioavailability of Olive Oil Phenolic Compounds María-Isabel Covas, Montserrat Fitó, Olha Khymenets and Rafael de la Torre Cardiovascular Risk and Nutrition and Human Pharmacology and Clinical Neurosciences Research Groups, Institut Municipal d´Investigació Mèdica (IMIM-Hospital del Mar), Barcelona, Spain. CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN)
73.1 Introduction The beneficial effects of olive oil on cardiovascular risk factors are now recognized and often only attributed to its high levels of monounsaturated fatty acids (MUFA). On November 2004, the Federal Drug Administration of the USA permitted a claim on olive oil labels concerning: ‘the benefits on the risk of coronary heart disease of eating about 2 tablespoons (23 grams) of olive oil daily, due to the MUFA in olive oil’ (US FDA Press Release P04100. November 1, 2004. http://www.fda.gov/bbs/topics/ news/2004/NEW01129.htlm; accessed on May 2, 2008). Olive oil is, however, more than a MUFA fat. Olive oil is a functional food which besides having a high level of MUFA contains other minor components with biological properties (Covas et al., 2006c). The content of the minor components of an olive oil varies, depending on several conditions such as the cultivar, climate, ripeness of the olives at harvesting, and the processing system employed to produce the olive oil. Three types of olive oil are currently present on the market: virgin, ordinary, or pomace (Gimeno, 2002). Virgin olive oils are produced by direct pressing or centrifugation of the olives, among them those with an acidity greater than or equal to 3.3 degrees (2 degrees in the European Union) are submitted to a refining process in which some components, mainly phenolic compounds, and to a lesser degree squalene, are lost (Owen et al., 2000a). By mixing virgin and refined olive oil, an ordinary olive oil (olive oil, UE 1991) is produced and marketed. After virgin olive oil production, the rest of the olive drupe and seed is processed and submitted to a refining process, resulting in pomace olive oil, to which a certain quantity of virgin olive oil is added before marketing. The minor components of virgin olive oil are classified into two types: the unsaponificable fraction, defined as the fraction extracted with solvents after the saponification of the oil, and the soluble fraction which includes the phenolic compounds (Covas et al., 2006c). Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
The major phenolic compounds in olive oil are: (1) simple phenols (e.g., hydroxytyrosol, tyrosol, vanillic acid); (2) secoiridoids: oleuropein glucoside, and SIDs which are the dialdehydic form of oleuropein (SID-1) and ligstroside (SID-2) lacking a carboxymethyl group, and the aglycone form of oleuropein glucoside (SID-3) and ligstroside (SID4); and (3) polyphenols: lignans (e.g., ()-pinoresinol and ()-1-acetoxypinoresinol) and flavonols (Owen, 2000; Covas et al., 2006c) (Figure 73.1). Tyrosol, hydroxytyrosol, and their secoiridoid derivatives make up around 90% of the total phenolic content of a virgin olive oil.
73.2 Bioavailability of olive oil phenolic compounds 73.2.1 Absorption and Disposition After olive oil ingestion, tyrosol (Tyr) and hydroxytyrosol (OH-Tyr), as well as their glucosides and aglycones, such as oleuropein, undergo rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of Tyr and OH-Tyr free forms which enter the small intestine (Corona et al., 2006). In in vitro models, both OH-Tyr and Tyr are able to cross human Caco-2 cell monolayers and rat segments of jejunum and ileum (Manna et al., 2000; Corona et al., 2006). Data from experiments performed in Caco-2 cell monolayers using 14C–OH-Tyr, showed the transport of the phenolic compound occurs via a bidirectional passive diffusion mechanism (Manna et al., 2000). In animal models, when Tyr and OH-Tyr were orally administered in oil or water solutions, the orally administered oil dosing promoted a recovery of the phenolics in 24-h urine greater (25%) than that obtained with the oral aqueous dose (Tuck et al., 2001). When Tyr and OH-Tyr were administered intravenously in saline solution no significant differences were observed
699
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
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Section | II General Aspects and Changes in Food Processing
A
C
OH
HO
D
O
HO
O OH
OH
O
HO
COOCH3
E
O
COOCH3
HO
O O
COOCH3
OH O
O
O O
B
HO
O
O
O
OH
HO
HO OH
F
G
O
HO
O
CH2OH
COOCH3
HO
O
O COOCH3
OH O
O OH
OH
Figure 73.1 Structures of tyrosol, hydroxytyrosol and derivatives: (A): hydroxytyrosol; (B): tyrosol; (C): oleuropein glucoside; (D): SID-1; (E): SID-2; (F): SID-3; (G): SID-4.
in the amount of phenolic compounds recovered in urine between the intravenous and the oral oil doses for either Tyr or OH-Tyr. Similar results were obtained in humans when the bioavailability of OH-Tyr was compared by administering this compound in different matrices (olive oil, spiked refined oil, or yoghurt) (Visioli et al., 2003). Urinary OHTyr recovery was higher after virgin olive oil administration (44.2% of the OH-Tyr administered) than after addition of OH-Tyr to a refined olive oil (23% of the OH-Tyr administered), or to a yoghurt (5.8% of dose or approximately, 13% of that recorded after virgin olive oil intake) (Visioli et al., 2003). Concerning oleuropein, data from animal models show it can be absorbed, albeit poorly, from isolated perfused rat intestine. Thus, the most plausible way for oleuropein to exert its biological activities seems to be through its conversion to OH-Tyr (Edgecombe et al., 2000). This idea is supported by the results of bioavailability studies in rats, in which peak plasma concentrations reached after ingestion of high doses of oleuropein (100 mg kg1) were in the nanogram range, whereas those of OH-Tyr were highly increased (Del Boccio et al., 2003; Bazoti et al., 2005). These observations have been further confirmed in humans (Vissers et al., 2002; Visioli et al., 2003). In the process of crossing epithelial cells of the gastro intestinal tract, phenolic compounds from olive oil are subject to a biotransformation phase and, therefore, subjected to an important first-pass metabolism. According to data of in vitro studies, about 10% of OH-Tyr is converted in
homovanyl alcohol by the catechol-O-methyltransferase (Manna et al., 2000). In addition to the O-methylated derivative of OH-Tyr, the glucuronides of OH-Tyr and Tyr have also been described (Corona et al., 2006). In contrast, there was no absorption of oleuropein as it was rapidly degraded by the colonic microflora resulting in OH-Tyr formation (Corona et al., 2006). The hepatic metabolism of the olive oil phenols has been studied in human hepatoma HepG2 cells. After incubation, culture media and cell lysates were hydrolyzed with -glucuronidase and sulfatase. Methylated and glucuronidated forms of OH-Tyr were detected after 18 h of incubation, together with methyl-glucuronidated metabolites. Hydroxytyrosyl acetate was largely converted into free OH-Tyr and subsequently metabolized, although small amounts of glucuronidated hydroxytyrosyl acetate were detected. Tyrosol was poorly metabolized, with 10% of the phenol glucuronidated after 18 h. Minor amounts of free or conjugated phenols were detected in cell lysates. No sulfated metabolites were found (Mateos et al., 2005). The pharmacokinetics of OH-Tyr intravenously administered to rats indicates a fast and extensive uptake of the molecule by the organs and tissues, with a preferential renal uptake (D’Angelo et al., 2001). Hydroxytyrosol was recovered mainly in the sulfo-conjugated forms. The recovery of OH-Tyr in urine was about 6% of the dose administered: 0.3% recovered as 3-methyl-4-hydroxy-phenylethanol (HVAL or MOPET), 12.3% as 3,4-dihydroxy-phenylacetic acid (DOPAC), 23.6%
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Chapter | 73 The Bioavailability of Olive Oil Phenolic Compounds
as homovanillic acid (3-methyl-4-hydroxy-phenylacetic acid, HVA), and 26% as 3,4-dihydroxy-phenylacetaldehyde (DOPAL) (D’Angelo et al., 2001). It has been suggested that non-absorbable phenolic compounds can exert local antioxidant activities in the gastro intestinal tract (Ursini et al., 1998), an idea supported by the capacity of phenolic compounds isolated from olive oil to scavenge the free radicals generated by the fecal matrix (Owen et al., 2000b) and those induced in intestinal epithelium cells (Manna et al., 1996). However, one of the prerequisites to assess their in vivo physiological significance is to determine their absorbability and presence in human plasma. The first report on the bioavailability and disposition of olive oil phenolic compounds in humans was provided by Visioli et al. (2000). In this experiment, Tyr and OH-Tyr were spiked to a refined olive oil (very low phenolic content) and administered to healthy volunteers. Phenolic compounds were dose-dependently absorbed in humans, most phenolic compounds being recovered in biological fluids as conjugates in a dose-dependent manner with the phenolic content of the olive oil administered (Visioli et al., 2000). Also, in human studies it was demonstrated that tyrosol, hydroxytyrosol and oleuropein were absorbed at the small intestine level. Oleuropein was not quantified in plasma nor in urine but it was shown that it was metabolized in the body and recovered in urine mainly in the form of hydroxytyrosol (Vissers, 2000). Phenolic compounds, particularly those bearing a catechol group, are typically biotransformed by three enzymatic systems: catechol-O-methyl-transferase, sulfatases, and glucuronosyltransferases. Depending on the dose and the availability of co-factors the proportion of methyl, sulfate, and glucuronide conjugates varies among subjects. Further studies on the olive oil phenolic compound bioavailability (Miró-Casas et al., 2001, 2003a, b) were performed with virgin olive oil in its
natural form. After administering 25 mL of virgin oil (with an estimated content 1.2 mg of OH-Tyr), OH-Tyr plasma concentrations peaked at 30 minutes and those of its methylated metabolite, HVAL at 50 min. Plasma peak concentrations were around 25 ng mL1 for OH-Tyr and 4 ng mL1 for HVAL. The estimated half-life for OH-Tyr was 3 hours, reaching baseline concentrations after 8 hours of the virgin olive oil ingestion (Figure 73.2). More than 98% of both OH-Tyr and HVAL were in their conjugated forms, mainly glucuronates, confirming previous findings. In urine, OH-Tyr and HVAL concentrations peaked in the collection period 0–2 h (MiróCasas et al., 2003a). Despite the short half-life of Tyr and OH-Tyr, sustained consumption promotes an increase of olive oil phenolic compounds in biological fluids (Figure 73.2). Plasma and urinary levels of OH-Tyr and Tyr increase in a dose-dependent manner with the phenolic content of the olive oil administered (Miró-Casas et al., 2003b; Marrugat et al., 2004; Weinbrenner et al., 2004; Covas et al., 2006a, b) (Figure 73.3). Table 73.1 shows the urinary recoveries of OH-Tyr, Tyr, and HVAL after olive oil of medium (164 mg kg1) and high (466 mg kg1) phenolic content. The fact that a dose-dependent increase of Tyr and OH-Tyr with the phenolic content of the olive oil administered exists, at real-life olive oil doses, confirms the usefulness of these compounds as biomarkers of compliance in clinical trials (Covas et al., 2006b). With regard to the dose–effect relationship, 24-h urinary Tyr seems to be a better biomarker of sustained and moderate doses of virgin olive oil consumption than OH-Tyr (Miró-Casas et al., 2003b). Both OH-Tyr and Tyr urinary concentrations have been used, and are currently in use, in nutritional intervention studies as biomarkers of virgin olive oil ingestion Covas et al., 2006a, b; Fitó et al., 2007, 2008). Most bioavailability studies on olive oil phenols have measured total Tyr and OH-Tyr concentrations in blood or
9 8
LPC MPC HPC
Conc(ng/mL−1)
7 6 5 4 3 2 1 0
0 Without supplementation
24
48 Hours
72
96 After supplementation
Figure 73.2 Plasma hydroxytyrosol concentrations after ingestion of olive oil with low (LPC, 10 mg kg1), medium (133 mg kg1), and high (486 mg kg1) phenolic content. A single 25 mL dose was ingested before and after 4 days of olive oil supplementation (25 mL day1). Adapted from Weinbrenner, 2004.
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Section | II General Aspects and Changes in Food Processing
in some physiopathological states, such as inflammation or cancer, with a concomitant in situ deconjugation of phenol metabolites. Data are still very scarce and further studies are needed to elucidate the role and bioactivity of the human biological metabolites of olive oil phenolic compounds.
Percentage (%) of change
250 200
Tyrosol Hydroxytyrosol
150 100
73.2.2 Binding of Olive Oil Phenolic Compounds and their Metabolites to Human Lipoproteins
50 0
Low Medium High Olive oil phenolic content
Figure 73.3 Changes in urinary tyrosol and hydroxytyrosol after 3 weeks of sustained olive oil (range of phenolic compounds from 0 to 150 mg kg1) ingestion. Adapted from Marrugat et al., 2004.
Table 73.1 Urinary recoveries (mol) of olive oil phenolic compounds. Olive oil
Hydroxytyrosol
Tyrosol
HVAL
MPC
1.21 0.07
1.33 0.11
0.38 0.04
HPC
3.12 0.21
2.69 0.19
0.78 0.06
Values are expressed as Mean SD. HPC, high phenolic content olive oil (466 mg kg1); MPC, medium phenolic content olive oil (164 mg kg1); HVAL,3-methyl-4-hydroxy-phenylethanol, the methylated biological metabolite of hydroxytyrosol.
urine after acidic or enzymatic treatment of the samples. There is a lack of studies in which glucuronide and sulfate conjugates of Tyr and OH-Tyr in biological samples have been measured. It could be hypothesized that some conjugates of phenolic compounds may behave as carriers of the free forms of the phenolic compounds to target tissues, the ‘depot hypothesis’. Within this hypothesis, the determination of the bioavailability of phenol metabolites in plasma or tissues may be more relevant than that in urine. The proportion of free aglycones in some tissues can differ from that observed in blood (D’Angelo et al., 2001); this may be explained by a specific uptake of the aglycone or intracellular deconjugation. This last hypothesis implies that anionic conjugates could be transported across plasma membranes via carrier systems, as has been shown for acyl-glucuronides (Sallustio et al., 2000). Furthermore, -glucuronidase is located in the lumen of the endoplasmic reticulum in various organs, which could be reached by phenol glucuronides. -Glucuronidase is also present in the lysosomes of several cells, from which the enzyme can be released under some particular conditions such as an oxidative stress situation. -Glucuronidase activity can increase
Phenolic compounds which can bind low-density lipoproteins (LDL) are likely to perform their peroxyl scavenging activity in the arterial intima, where full LDL oxidation occurs in microdomains sequestered from the richness of antioxidants present in plasma (Witzum, 1994). Olive oil phenolic compounds can bind lipoproteins in vivo in humans. Tyrosol and hydroxytyrosol were recovered in all human lipoprotein fractions after virgin olive oil ingestion, except in very low-density lipoproteins, with concentrations peaking between 1 and 2 hours after olive oil ingestion (Bonanome et al., 2000). Tyrosol and hydroxytyrosol, as well as their biological metabolites hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate, and homovanillic acid sulfate, have been identified in LDL after virgin olive oil ingestion (De la Torre-Carbot et al., 2006, 2007). In addition, the concentration of total phenolic compounds in LDL has been shown to be directly correlated with the phenolic concentration of the olive oils ingested and with the resistance of LDL to their in vitro oxidation (Gimeno et al., 2007). After ingestion of 40 mL of virgin olive oil, the phenolic content of the LDL directly correlated with the plasma concentrations of Tyr and OH-Tyr (Covas et al., 2006a). The susceptibility of the LDL to oxidation depends not only on their fatty acid content, but also on the LDL antioxidant content (i.e., vitamin E and polyphenols) bound to the LDL (Fuller and Jialal, 1994). Further studies are required to establish the nature of the bond between the LDL and the phenolic compounds, including olive oil phenolic compounds and their metabolites, due to the physiopathological implications involved.
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Chapter | 73 The Bioavailability of Olive Oil Phenolic Compounds
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