acetylation of hydroxytyrosol enhances its transport across differentiated caco-2 cell monolayers

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Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers R. Mateos a,b , G. Pereira-Caro a,c , S. Saha a , R. Cert d , M. Redondo-Horcajo e , L. Bravo b , P.A. Kroon a,a Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK b CSIC, Instituto del Frío-ICTAN, C/José Antonio Novais 10, Ciudad Universitaria, E-28040-Madrid, Spain c IFAPA, Centro Venta del Llano, Bailen-Motril, Km 18.5, E-23620-Mengíbar (Jaén), Spain d CSIC, Instituto de la Grasa, Avda. Padre García Tejero 4, E-41012-Sevilla, Spain e CSIC, Centro de Investigaciones Biológicas, C/Ramiro Maeztu 9, Ciudad Universitaria, E-28040-Madrid, Spain article info Article history: Received 30 June 2010 Received in revised form 10 August 2010 Accepted 14 September 2010 Keywords: Phenols Antioxidants Trans-epithelial transport Metabolism Phase-2 conjugation Caco-2 cells LC-MS analysis abstract Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is depen- dent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosyl acetate and the extent to which they are conjugated and metabolised during transfer across intestinal Caco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification and identification of metabolites. Further evidence was obtained by observing metabolite susceptibility to b-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authentic phenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detected as a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in the basolateral compartment after 2 h of incubation. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition, hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate (37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred across human Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability (Papp AP?BL /Papp BL?AP ) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugates were preferentially transported to the basolateral side. These results show that the acetylation of hydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier, and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhanced bioavailability. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction There is considerable evidence that part of the health benefits associated with habitual consumption of a Mediterranean diet are attributable to the intake of virgin olive oil, the main source of fat in the Mediterranean diet. The health benefits of virgin olive oil consumption are partly attributed to its constituent phenolic compounds. Phenolics are a major group of natural antioxidants that have been shown to possess multiple biological activities that are in keeping their consumption providing benefits in terms of re- duced risk of disease (Bendini, Cerretani, Carrasco-Pancorbo, & Gomez-Caravaca, 2007; Trípoli, Giammanco, Tabacchi, & Di Majo, 2005). The main phenolic compounds in virgin olive oil are seco- iridoid derivatives of 2-(3,4-dihydroxyphenyl)ethanol (hydroxyty- rosol) (HTy, 1)(Fig. 1) and of 2-(4-hydroxyphenyl)ethanol or tyrosol (Ty), and 2-(3,4-dihydroxyphenyl)ethyl acetate (hydroxy- tyrosyl acetate) (HTy-Ac, 2). Minor amounts of HTy, Ty, 2-(4-dihy- droxyphenyl)ethyl acetate (tyrosyl acetate), vanillin and phenolic derivatives of benzoic and cinnamic acids, together with some flavonoids and lignans, constitute the antioxidant fraction of virgin olive oil (Mateos et al., 2001). Several randomised, cross-over, controlled, human studies have been performed in order to elucidate the effect of chronic con- sumption of phenolic compounds from virgin olive oil. Protective effects of virgin olive oil phenols on in vivo circulating oxidised LDL and DNA oxidation, but not on plasma F2-isoprostanes, were found in healthy male subjects (Marrugat et al., 2004; Weinbrenner et al., 2004; Covas & de la Torne et al., 2006). A clinical trial per- formed in 200 individuals from five Europeans countries, the EUROLIVE study, provided evidence that the phenolic components of virgin olive oil were incorporated into plasma low-density 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.054 Corresponding author. E-mail address: [email protected] (P.A. Kroon). Food Chemistry 125 (2011) 865–872 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

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Food Chemistry 125 (2011) 865–872

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Acetylation of hydroxytyrosol enhances its transport across differentiatedCaco-2 cell monolayers

R. Mateos a,b, G. Pereira-Caro a,c, S. Saha a, R. Cert d, M. Redondo-Horcajo e, L. Bravo b, P.A. Kroon a,⇑a Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UKb CSIC, Instituto del Frío-ICTAN, C/José Antonio Novais 10, Ciudad Universitaria, E-28040-Madrid, Spainc IFAPA, Centro Venta del Llano, Bailen-Motril, Km 18.5, E-23620-Mengíbar (Jaén), Spaind CSIC, Instituto de la Grasa, Avda. Padre García Tejero 4, E-41012-Sevilla, Spaine CSIC, Centro de Investigaciones Biológicas, C/Ramiro Maeztu 9, Ciudad Universitaria, E-28040-Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 June 2010Received in revised form 10 August 2010Accepted 14 September 2010

Keywords:PhenolsAntioxidantsTrans-epithelial transportMetabolismPhase-2 conjugationCaco-2 cellsLC-MS analysis

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.09.054

⇑ Corresponding author.E-mail address: [email protected] (P.A. Kroo

Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidantproperties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is depen-dent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosylacetate and the extent to which they are conjugated and metabolised during transfer across intestinalCaco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification andidentification of metabolites. Further evidence was obtained by observing metabolite susceptibility tob-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authenticphenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detectedas a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in thebasolateral compartment after 2 h of incubation. Hydroxytyrosyl acetate was largely converted into freehydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition,hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate(37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred acrosshuman Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability(PappAP?BL/Papp BL?AP) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugateswere preferentially transported to the basolateral side. These results show that the acetylation ofhydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier,and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhancedbioavailability.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

There is considerable evidence that part of the health benefitsassociated with habitual consumption of a Mediterranean dietare attributable to the intake of virgin olive oil, the main sourceof fat in the Mediterranean diet. The health benefits of virgin oliveoil consumption are partly attributed to its constituent phenoliccompounds. Phenolics are a major group of natural antioxidantsthat have been shown to possess multiple biological activities thatare in keeping their consumption providing benefits in terms of re-duced risk of disease (Bendini, Cerretani, Carrasco-Pancorbo, &Gomez-Caravaca, 2007; Trípoli, Giammanco, Tabacchi, & Di Majo,2005). The main phenolic compounds in virgin olive oil are seco-iridoid derivatives of 2-(3,4-dihydroxyphenyl)ethanol (hydroxyty-

ll rights reserved.

n).

rosol) (HTy, 1) (Fig. 1) and of 2-(4-hydroxyphenyl)ethanol ortyrosol (Ty), and 2-(3,4-dihydroxyphenyl)ethyl acetate (hydroxy-tyrosyl acetate) (HTy-Ac, 2). Minor amounts of HTy, Ty, 2-(4-dihy-droxyphenyl)ethyl acetate (tyrosyl acetate), vanillin and phenolicderivatives of benzoic and cinnamic acids, together with someflavonoids and lignans, constitute the antioxidant fraction of virginolive oil (Mateos et al., 2001).

Several randomised, cross-over, controlled, human studies havebeen performed in order to elucidate the effect of chronic con-sumption of phenolic compounds from virgin olive oil. Protectiveeffects of virgin olive oil phenols on in vivo circulating oxidisedLDL and DNA oxidation, but not on plasma F2-isoprostanes, werefound in healthy male subjects (Marrugat et al., 2004; Weinbrenneret al., 2004; Covas & de la Torne et al., 2006). A clinical trial per-formed in 200 individuals from five Europeans countries, theEUROLIVE study, provided evidence that the phenolic componentsof virgin olive oil were incorporated into plasma low-density

R1

R2

R1=R2=R3=OHR1=R2=OH; R3=COOCH3

R1=R3=OH; R2=OCH3

R1=OH; R2=OGlucAc; R3=COOCH3

R3

Hydroxytyrosol (1)Hydroxytyrosyl acetate (2)Homovanillyl alcoholMonoglucuronide conjugate of 2

Fig. 1. Chemical Structure of HTy (1), HTy-Ac (2) and their metabolites.

866 R. Mateos et al. / Food Chemistry 125 (2011) 865–872

lipoproteins (LDL) and inhibited LDL oxidation (Covas & Nyyssonenet al., 2006). In addition, orally administered olive oil phenolics havebeen shown to be effective in reducing the ecosanoid inflammatorymediators derived from araquidonic acid (Bogani, Galli, Villa, &Visioli, 2007; Leger et al., 2005; Visioli et al., 2005). Further, a signif-icant reduction in systolic blood pressure was demonstrated follow-ing consumption of a phenolic-rich olive oil stable hypertensivecoronary heart disease (CHD) patients (Fito et al., 2005).

Mean dietary intake of virgin olive oil polyphenols has beenestimated to be around 9 mg in the Mediterranean population,which is derived from the consumption of 25–50 ml of virgin oliveoil per day, and where at least 1 mg of them is derived from freeHTy and Ty and 8 mg are related to their secoiridoid and acetatederivatives. It is well known that biological properties of thesecompounds in vivo will depend on their gastrointestinal stability,and the extent of their absorption and metabolism. Thus, althoughHTy and Ty levels are relatively low in olive oil, they may increaseduring the digestive process by the partial hydrolysis suffered bysecoiridoid derivatives present in olive oil (Corona et al., 2006).Several human (Miró-Casas et al., 2003; Visioli et al., 2003; Vissers,Zock, Roodenburg, Leenen, & Katan, 2002) and animal (Tuck,Hayball, & Stupans, 2002) studies have shown that the main virginolive oil phenols, HTy and Ty, are bioavailable and as such a frac-tion of the oral dose can be recovered in urine, with >90% of theurinary excretion in the form of metabolites, mainly O-glucuronid-ed conjugates (Visioli et al., 2000,2003; Caruso, Visioli, Patelli, Galli,& Galli, 2001). O-methylated forms such as homovanillyl alcohol orhomovanillyl acid, and monosulphated conjugates have been alsoidentified in plasma and urine after virgin olive oil administration(Caruso et al., 2001; Miró-Casas et al., 2003; Tuck et al., 2002;Visioli et al., 2003). In addition, in a human ileostomy study itwas shown that up to 66% of the ingested virgin olive oil phenolswere absorbed from the small intestine (Vissers et al., 2002), indi-cating effective intestinal absorption of these compounds.

The bioavailability studies of phytochemicals carried out in ani-mals or human subjects are complex, expensive and do not distin-guish between the contribution of intestinal, hepatic and otherorgans in the biotransformation of polyphenols. Moreover, it isessential to know the structural form of virgin olive oil polyphenolswithin the peripheral circulation (and other target tissues) in orderto obtain more detailed information about their mechanism of ac-tion in vivo (Kroon et al., 2004). The differentiated Caco-2 celltranswell monolayer model is a well established small intestinaltransport model that is frequently used to mimic the transportand metabolism of xenobiotics. Currently, existing data concernedwith the efficiency of transport and the extent and products ofmetabolism of virgin olive oil phenolics are rather limited andinconsistent (Corona et al., 2006; Manna et al., 2000; Soler et al.,2010). The aim of this study was to evaluate the transport andmetabolism of HTy-Ac (2) in comparison with HTy (1) using differ-entiated human Caco-2/TC7 cells on transwells inserts as a modelsystem of the human intestine.

2. Materials and methods

2.1. Materials

All cell culture media and reagents were from Invitrogen(Paisley, UK) unless otherwise stated. Enzymes (catechol-O-meth-yltransferase, b-glucuronidase from Helix pomatia, sulphatasetype V from limpets), S-adenosyl-L-methionine chloride, UDP-glucuronic acid, and adenosine-3’-phospho-50-phosphosulphate,homovanillyl alcohol, methanol and formic acid were purchasedfrom Sigma–Aldrich (Poole, Dorset, UK). Hydroxytyrosol (HTy, 1)was recovered with 95% purity from olive oil wastewaters(Fernandez-Bolaños et al., 2005), and further purified by columnchromatography. Hydroxytyrosyl acetate (HTy-Ac, 2) was obtainedfrom HTy in ethyl acetate after incubation with p-toluenesulfonicacid and purification by column chromatography following a pat-ented procedure (Alcudia, Cert, Espartero, Mateos, & Trujillo,2004). All reagents were of analytical or chromatographic grade.

2.2. Human colon adenocarcinoma cells culture

The human Caucasian colon adenocarcinoma cell line Caco-2(TC7 clonal cells) was a kind gift from Dr. Monique Rousset(INSERM, Paris, France). Cells were grown in 75 cm2 flasks andmaintained in Dulbelco modified Eagle’s medium DMEM contain-ing 20% fetal calf serum, 1% (v/v) nonessential amino acids, 2 mML-glutamine, 100 IU/ml penicillin, and 100 lg/ml streptomycin(pH 7.4) in a incubator at 37 �C with 5% CO2 and 95% air. Cells usedwere cultured between passages 51 and 62.

2.3. Cell proliferation assay

The effect of HTy (1) and HTy-Ac (2) on the Caco-2/TC7 cell pro-liferation was evaluated using a WST-1 cell proliferation kit (RocheApplied Science, Germany). The WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) reagentwas used according to the kit specifications. Caco-2/TC7 cells wereplated in 96-well culture plates in a final volume of 100 ll/wellculture medium in a humidified atmosphere (37 �C, 5% CO2). Cellswere allowed to adhere to the plate surface for 36 h before beingexposed to various concentrations of HTy (1) and HTy-Ac (2) (1–500 lM; n = 6 per concentration), for 24 h. WST-1 reagent (10 ll)was added to each well and incubated for 90 min in a humidifiedatmosphere (37 �C, 5% CO2). Quantification of the formazan dyeproduced by metabolically active cells was performed by a scan-ning multiwell spectrophotometer, measuring absorbance at450 nm by micro-plate ELISA reader (ELx808, Ultra Micro-plateReader; BIO-TEK Instruments, Inc., Winooski, VT). Results are ex-pressed as percentages of cell viability calculated based on theabsorbance measured relative to the absorbance of cells exposedto the control vehicle.

2.4. Transport and metabolism experiments

Caco-2/TC7 cells were grown in Transwell inserts with thesemipermeable membrane first coated with type I collagen(12-mm diameter and 0.4 lm pore size, Corning Costar, NY). Thecells were seeded at a density of 2–4 � 104 cells/cm2, and the mon-olayers were formed after culturing for 21 days post confluent. Theintegrity of the cell monolayer was evaluated by measurement oftrans-epithelial electrical resistance (TEER) with Millicel-ERS voltohmmeter equipment (Millipore Corporation, Billerica, MA, USA).Monolayers with a TEER equal or higher than 200 X cm2 were usedfor the trans-epithelial transport experiments. Additionally, TEERmeasurements were taken before and after experiments to ensure

R. Mateos et al. / Food Chemistry 125 (2011) 865–872 867

adequate monolayer integrity. Moreover, barrier integrity of Caco-2/TC7 monolayers was determined by quantitating the transfer of asolution of 100 lM of phenolsulfonaphthalein (phenol red), a mar-ker of paracellular flux, from the apical compartment to the baso-lateral compartment and vice versa, after incubation for 60 min at37 �C. Wells that supported <0.1% of phenol red transport underthese conditions were considered suitable for use.

Stocks solutions of polyphenols were dissolved in 10% of DMSOin deionised water and diluted with free serum DMEM supple-mented with 200 lM of ascorbic to prepare test solutions(50 lM; 0.1% DMSO). The 0.1% resulting final concentration ofDMSO present in test solutions was equivalent to that used in con-trol and did not affect the transport and metabolism experiments.

2.5. Olive oil phenols metabolism experiments

HTy (1) and HTy-Ac (2) test solution (50 lM; 0.1% DMSO) wasapically added to each well, with the equivalent volume of DMSOas control. All solutions contained 200 lM of ascorbic acid as a pro-tective antioxidant. Control and treated cells were incubated at37 �C for 1, 2 and 4 h. Media from apical and basolateral compart-ments were removed and kept at �20 �C until analysis. Cells werewashed twice with PBS (0.01 M phosphate buffered saline solution,pH 7.4) and harvested by scraping. After centrifugation at1500 rpm for 5 min at 4 �C, the supernatant was removed andthe cell pellet resuspended in 200 ll of PBS. Cells were sonicatedfor 10 min at room temperature to break down the cell membraneand to release the total amount of metabolites. After centrifugationat 5000 rpm for 10 min at 4 �C, the supernatant was transferredinto an eppendorf vial and kept frozen at �20 �C. Media and cyto-plasmatic content were separately subjected to HPLC analysis bothdirectly (non-hydrolysed) and following incubation with sulpha-tase and b-glucuronidase enzymes (enzyme hydrolysed). Enzy-matic hydrolyses of conjugates in media samples were carriedout by the addition of 0.23 mg (150 units) b-glucuronidase(645,200 units/g) or 10 mg (150 units) of sulphatase type V fromlimpets (15,000 units/g) per sample. Subsequently, 1 ml of en-zyme-treated samples were mixed with 50 ll of glacial acetic acidand 50 ll of methanol and centrifuged at 13,000 g (10 min) beforebeing injected onto the HPLC. In parallel, in vitro glucuronidation,methylation and sulphatation of HTy-Ac (2) were carried out usingpure enzyme (catechol-O-methyltransferase, COMT) or a rat livermicrosomal fraction that contained both UDP-glucuronosyltrans-ferase (UGT) and sulphotransferase (ST) (Mateos, Goya, & Bravo,2005).

2.6. Olive oil phenols transport experiments

To measure the apical-to-basolateral permeability, 0.6 ml of thetest solution containing HTy (1) or HTy-Ac (2) (50 lM; 0.1% DMSO,200 lM ascorbic acid) was added to the apical side while the baso-lateral chamber of the Transwell insert was filled with 1.5 ml ofserum-free DMEM with ascorbic acid (200 lM). However, to mea-sure the basolateral-to-apical transport, 1.5 ml of the test solutioncontaining the polyphenols (HTy (1) or HTy-Ac (2)) and 200 lMascorbic acid was added to the basolateral side while the apicalside was filled with 0.6 ml of serum-free DMEM with ascorbic acid(200 lM). Plates were incubated in a humidified atmosphere of 5%CO2 at 37 �C during 1, 2 and 4 h. Two controls without DMSO andwith the equivalent volume of DMSO (0.1%) were monitored. Med-ia from apical and basolateral side and harvested cells were kept at�20 �C until their later analysis by HPLC. The apparent permeabil-ity coefficient (Papp, cm/s) for parent compounds (HTy and HTy-Ac) after 1 h of incubation with cells was calculated according to:Papp = (dQ/dt) (1/A Co) where dQ/dt is the permeability rate, Cothe initial concentration in the donor chamber and A the surface

area of the monolayer. Besides, the rate of individual conjugate ef-flux for both the apical and basolateral compartments was calcu-lated, and these rates used to determine an apical to basolateralratio. This ratio is a measure of the favoured direction of efflux,with a value of over 1.0 indicating an apically favoured efflux, val-ues below 1.0 symbolising a basolaterally favoured efflux, and val-ues of 1.0 symbolising an equal distribution of conjugate efflux.Percentage of parent compound or metabolites of HTy (1) andHTy-Ac (2), found in apical and basolateral side after 1, 2 and 4 hof incubation with Caco-2/TC7 cells was also offered in order toknow its behaviour over time.

2.7. HPLC analysis

HTy (1) and HTy-Ac (2) and the metabolites formed after incu-bation of the chemically pure compounds with Caco-2/TC7 cellswere analysed on an Agilent 1100 liquid chromatographic system(Agilent Stockport, UK) comprising two pump units, autosampler,mixer and a diode-array detector. Portions (50 ll) of sample wereinjected onto a Gemini C18 narrow bore reversed-phase column(Phenomenex, Macclesfield, UK) at a rate of 1 ml/min at 37 �C.The mobile phase consisted of a mixture of 0.1% formic acid indeionised water (solvent A) and a mixture of 0.1% formic acid inmethanol (solvent B). The solvent gradient changed according tothe following conditions: from 100% A to 90% A in 5 min; 85% Ain 5 min; 65% A in 10 min; 60% A in 10 min; 55% A in 5 min; 50%A in 5 min; 30% A in 5 min; 0% A in 5 min, followed by 5 min ofmaintenance and 100% A in 5 min. Chromatograms were acquiredat 280 nm. HTy (1) recovered from olive oil wastewaters(Fernandez-Bolaños et al., 2005), synthetic HTy-Ac (2) (Alcudiaet al., 2004) and commercial homovanillyl alcohol authentic stan-dards were used for quantification purposes. Besides, HTy-Ac (2)was used for the quantification of its monoglucuronide. Standardof HTy (1), HTy-Ac (2) and homovanillyl alcohol were preparedin free serum DMEM culture media in a range of concentrationsfrom 2.15 lM to 50 lM obtaining a linear response for all standardcurves, as checked by linear regression analysis with R2 valuesgreater than 0.98. The recoveries from standards added to the cul-ture medium were ranging from 95.3 to 99.5% and detection andquantification limits ranging from 0.2 to 0.6 lM and 0.8 to1.5 lM, respectively. The precision of the assay was excellent be-tween 99.2 and 99.6% (as the coefficient of intra-assay variation)allowing the quantification of HTy (1) and HTy-Ac (2) and theirmetabolites as equivalents of the respective parent molecule, ex-cept homovanillyl alcohol, which had its own reference.

2.8. Liquid chromatography-mass spectrometry

Following HPLC-DAD analysis, a subset of samples were then in-jected onto an HPLC-electrospray ionisation (ESI)-MS system forstructural elucidation, using an Agilent 1100 series HPLC-DAD cou-pled to an Agilent 1100 series Agilent LC/MSD SL single quadrupolemass spectrometer (Micromass, Manchester, U.K.). ESI was per-formed in full scan mode (mass range, 100–1000 Da) with the fol-lowing spray chamber conditions: drying gas (N2) flow of 13 l/min;nebulizer pressure of 50 Psi; and drying gas temperature of 350 �C.Positive mode ionisation was used to analyse all olive oil phenolsspecies at a capillary voltage of 4000 V and a fragmentor settingof 100. HPLC conditions (eluents, column, flow rate, gradient,etc.) were as stated above.

2.9. Statistical analysis

Results are expressed as means ± standard deviation (SD) offour single measurements obtained from four independent exper-iments. Data were subjected to a one-way analysis of variance

868 R. Mateos et al. / Food Chemistry 125 (2011) 865–872

(ANOVA) using Statistix 8.0. Differences were considered signifi-cant when p < 0.05.

3. Results

3.1. General

Because the in vivo biological activity of polyphenols is depen-dent on their intestinal absorption and metabolism, the absorptionof HTy (1) and HTy-Ac (2) and the extent to which they are conju-gated and metabolised during transfer across Caco-2/TC7 cell mon-olayers, was investigated. First, the potential deleterious effects ofthe treatments on normal Caco-2 cell growth was measured in or-der to ensure that the concentrations of phenolics used were nottoxic to the cultured cells.

3.2. Antiproliferative effects of HTy (1) and HTy-Ac (2) after treatmentof Caco-2/TC7 cells

Results obtained from the WST-1 proliferation assay indicatedthat HTy (1) had no significant effect on Caco-2/TC7 cell viabilityafter 24 h at concentrations up to 50 lM. HTy-Ac (2) was even lesstoxic and did not affect cell viability at concentrations up to200 lM (Fig. 2). At the highest concentration tested (= 500 lM),both compounds had a substantial inhibitory effect on Caco-2/TC7 viability (�50% and 70% reduction for HTy (1) and HTy-Ac(2), respectively). For both compounds, no cytotoxic effects wereobserved at 50 lM treatment concentration, and this concentra-tion was subsequently used to carry out the transport and metab-olism experiments.

3.3. Identification of HTy (1) and HTy-Ac (2) conjugates

When HTy (1) and HTy-Ac (2) were incubated with Caco-2/TC7cells to a final concentration of 50 lM, analysis of media samplesresulted in the appearance of a number of peaks of absorbance at280 nm that were not present in the untreated control media sam-ples. At the same time, the concentration of HTy (1) and HTy-Ac (2)decreased, suggesting that the additional peaks were metabolitesof the added polyphenols (Fig. 3). All of the new peaks were suffi-ciently separated from peaks corresponding to the culture med-ium, to allow identification and later quantification of allindividual metabolites present in the medium after incubationwith cells. Comparison of retention time and spectra characteris-tics with synthetic standard and confirmation of structures by elec-trospray ionisation mass spectrometry in positive mode wascarried out. Furthermore, the in vitro conjugation of pure standards

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C 1 2 5 10 20Concentration

Cel

l via

bilit

y (%

)

i i i

i iia a

a aab ab

Fig. 2. The effects of HTy (1) and HTy-Ac (2) treatment for 24 h on the cell viability of Cacsuperscript letter are significantly different, p < 0.05.

and the enzymatic hydrolysis with glucuronidase and sulphataseenzyme of metabolites was carried out to identify putativephase-II conjugate peaks.

3.4. Identification of HTy (1) conjugates

Media from HTy (1) treated cells yielded a new peak at 19.5 minlabeled as M1 together with the original compound (1) at 12.3 min,as can be observed in Fig. 3 (Traces a and b). The UV spectrum ofM1 contained two absorption bands around 230–240 and 270–280 nm characteristic of o-diphenolic compounds. Comparing thespectral characteristics and retention time of M1 with an authenticreference standard indicated that M1 was homovanillyl alcohol.Furthermore, the methyl conjugate of HTy (1) was confirmed byLC/MS which indicated molecular ion [M + H]+ at m/z 169.1 plusa fragment ion at m/z 151.1, corresponding to the protonatedmolecular ion and dehydrated homovanillyl alcohol, respectively(Table 1).

3.5. Identification of hydroxytyrosyl acetate (2) conjugates

A number of new peaks from HTy-Ac (2) treated media sampleswere identified as shown in Fig. 3 (Traces c and d). Besides a peakat 24.8 min corresponding to the HTy-Ac (2) standard, a number ofadditional peaks appeared in the UV trace that were not present inthe control assay, and were tentatively identified as HTy-Acmetabolites.

The first chromatographic peak at 12.3 min was coincident inretention time and spectra characteristics with HTy (1). LC-MSanalysis confirmed the presence of free HTy (1) (molecularion = m/z 155.1 together with a fragment ion at m/z = 137.1 corre-sponding to dehydrated hydroxytyrosyl [M-H2O + H]+). The secondputative conjugate, eluted after 19.5 min, had the same retentiontime, UV spectrum and mass spectrum as the homovanillyl alcoholstandard (= M1). Finally, the chromatographic peak at 24.4 min, la-belled M2, gave a molecular ion of m/z = 373.1 (consistent withthat of a hydroxytyrosol acetate-glucuronide) and a fragment ionm/z = 197.1 (consistent with that of hydroxytyrosol acetate (2)after the loss of glucuronic acid moiety (176)) (Table 1). Enzymatichydrolysis of media from HTy-Ac (2) treated cell samples usingglucuronidase resulted in a significant reduction in the size of peakM2 and a concomitant increase in the size of the peak correspond-ing to the parent aglycone, HTy-Ac (2). Together, these data indi-cate that peak M2 is a monoglucuronide conjugate of HTy-Ac (2).Following hydrolysis with sulphatase, none of the peaks observedin the chromatogram for the culture media from the incubations

50 100 200 500 (µM)

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HTy-Ac

i ii

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o-2/TC7 cells measured by the WST-1 assay. All values within a series with different

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HTy-Ac

(M2) HTy-Ac-Gluc HTy-Ac

Fig. 3. A typical profile of the chromatographic separation of different molecular species detectable in medium from Caco-2/TC7 in culture after 1 h of incubation with 50 lMconcentration of the following compounds: hydroxytyrosol (HTy) (a) apical side, (b) basolateral side; hydroxytyrosyl acetate (HTy-Ac) (c) apical side, (d) basolateral side. Forpeak identification, see Table 1; Homovanillyl alcohol (HMV); monoglucuronide of HTy-Ac (HTy-Ac-gluc). AP is the apical side as loading compartment and BL is thebasolateral side as receiving compartment.

Table 1Chromatographic and spectroscopic characteristics of HTy (1) and HTy-Ac (2) and the formed metabolites after incubation with Caco-2/TC7 cell monolayer.

Compound MW RT (min) kmax [M + H]+ (m/z) Fragment ions (m/z) Proposed structure

HTy (1) 154 12.3 280 155.1 137.1 Hydroxytyrosol (1)HMV (M1) 168 19.5 280 169.1 151.1 Monomethyl of 1HTy-Ac (2) 196 24.8 282 197.1 137.1 Hydroxytyrosyl acetate (2)HTy-Ac-Gluc (M2) 372 24.4 280 373.1 197.1 Monoglucuronide of 2

R. Mateos et al. / Food Chemistry 125 (2011) 865–872 869

of HTy (1) or HTy-Ac (2) with Caco-2/TC7 cells were altered. Thisobservation is consistent with the absence of sulphate conjugates.

3.6. Transport of HTy (1) and HTy-Ac (2) across differentiated Caco-2/TC7 cell monolayers

A preliminary study showed that HTy (1) and HTy-Ac (2) werestable (>98% recovery) for up to 24 h at 37 �C when incubated inserum-free DMEM media containing 200 lM of ascorbic acid inthe absence of cells. After confirming the stability of the two com-pounds, their transport across the monolayers was evaluated after

both apical and basolateral addition in independent experiments,which facilitated calculations of their apical (AP) to basolateral(BL) and their BL to AP transport rates. The apparent permeabilitycoefficients (Papp, cm/s) were calculated – this value is an estimateof the rate of transport in the direction indicated – and for apical tobasolateral transport is an indicator of the trans-intestinal barriertransport to the portal blood (Artursson & Karlsson, 1991).

The Papp values for AP to BL transport and for BL to AP trans-port, as well as the Efflux Ratio (PappAP?BL/PappBL?AP), determinedafter 1 h of incubation of HTy (1) and HTy-Ac (2) with Caco-2/TC7cells, are provided in Table 2. AP to BL transport was higher than

Table 2Papp (cm/s) and Efflux Ratio (AP:BL) values for HTy (1) and HTy-Ac (2). AP?BLindicates apical to basolateral transport of the parent compounds after 1 h ofincubation. BL?AP indicates basolateral to apical transport of the parent compoundsafter 1 h of incubation. Efflux Ratio values are from (PappAP?BL/PappBL?AP). Values areexpressed as means ± SD of four determinations.

Compound AP?BL(Papp, � 10�6 cm/s)

BL?AP(Papp, � 10�6 cm/s)

Effluxratio

HTy (1) 16.2 ± 4.2 14.6 ± 1.3 1.1 ± 0.2HTy-Ac (2) 33.6 ± 1.9 21.1 ± 2.3 1.6 ± 0.4

Table 3Transport of individual HTy (1) and HTy-Ac (2) conjugates in Caco-2/TC7 cellmonolayers to the apical and basolateral media. Efflux Ratio values are from (APtransport/BL transport). Values are expressed as means ± SD of four determinations.

Compound Apical transport(pmol/min)(average ± SD)

Basolateral transport(pmol/min)(average ± SD)

Efflux ratio

HTy (1) metabolismHMV (M1) 0.9 ± 0.1 20.4 ± 0.9 0.044TPM (pmol/min)* 0.9 (4.2%) 20.4 (95.8%)

HTy-Ac (2) metabolismHTy (1) 16.8 ± 0.9 127.4 ± 0.9 0.132HMV (M1) 1.0 ± 0.2 32.9 ± 0.1 0.030HTy-Ac-Gluc (M2) 1.2 ± 0.5 64.0 ± 0.5 0.019TPM (pmol/min)* 28.1 (11.1%) 224.3 (88.9%)

* TCP: Total polyphenol metabolite.

870 R. Mateos et al. / Food Chemistry 125 (2011) 865–872

that for BL to AP (indicated by AP: BL efflux ratios >1.0) confirmingthe good permeability of HTy (1) and HTy-Ac (2) across Caco-2monolayers. However, HTy-Ac (2) exhibited a higher Papp ratiothan free HTy (1), indicating that it was better absorbed.

The rates of efflux of individual HTy (1) and HTy-Ac (2) conju-gates from the confluent Caco-2/TC7 cells monolayers were alsodetermined following 60 min incubation (Table 3). Data are pro-vided as individual efflux rates (apical, basolateral) and as the api-cal to basolateral transport ratio. For HTy (1), total conjugateproduction was 21.3 pmol/min. Apical efflux accounted for 4.2%(0.9 pmol/min) of conjugate efflux, with basolateral efflux account-ing for 95.8% (20.4 pmol/min) of efflux. For HTy-Ac (2), total conju-gate production was 252.4 pmol/min, with apical and basolateralefflux accounting for 11.1% and 88.9% of total conjugate efflux,respectively. Conjugates retained within the cells accounted forless than 0.1% of the total conjugates within the monolayer forboth compounds. For all the conjugates, basolateral efflux was sub-stantially higher than apical efflux, indicated by AP: BL ratios < 1.

0%

50%

100%

1 2 1 2 1 2

Parent compound (AP) Metabolites (AP)

Parent compound (BL) Metabolites (BL)

1h 2h 4h

1

APICAL LOADING

Fig. 4. Percentage of parent compound or metabolites of HTy (1) and HTy-Ac (2), found

The proportion of the total recovered HTy (1) or HTy-Ac (2) madeup by either un-metabolised aglycone or metabolites in the apical orbasolateral compartments, following incubation of the CaCo-2monolayers with 50 lM compound for 1, 2 or 4 h, is shown inFig. 4. Since the total recovery of each compound (= un-metabolisedplus metabolised material) was >98%, the relative heights of each ofthe fill types in each bar stack can be compared between bar stacks(treatments). For both compounds, time-dependent transport wasobserved up to 4 h. Total production of conjugates was substantiallygreater for either polyphenol (1–2) when added apically comparedto basolaterally. In addition, AP to BL transfer of aglycones (HTy(1), HTy-Ac (2)) was greater than BL to AP. This observation is consis-tent with the notion that the basolateral efflux of the un-metabolisedcompounds is faster across the basolateral membrane comparedwith the apical membrane, possibly due to the presence of basolat-eral membrane transporters with affinity for the compounds. ForAP to BL transport, the total amount of material transported washigher for HTy-Ac (2) (41–75% over 1–4 h) than for HTy (1) (13–59% over 1–4 h). It is noteworthy that a high proportion [91–82%of HTy (1) and 50–32% of HTy-Ac (2)] reaching the basolateral com-partment were un-metabolised, which indicates the potential forhepatic transformation.

4. Discussion

There is increasing evidence that polyphenolics present in vir-gin olive oils are partly responsible for the health benefits associ-ated with habitual consumption of virgin olive oil, for example aspart of a Mediterranean diet. The major phenolic in virgin oliveoil is HTy (1) and derivatives of this compound, including an acetylester, HTy-Ac (2). The principal objectives of this research were toinvestigate small intestinal epithelial transport characteristics ofHTy (1) and HTy-Ac (2) and their intestinal metabolism. The datapresented here show that both compounds are efficiently takenup from the apical (= gut luminal) side of differentiated CaCo-2 cellmonolayers and transferred to the basolateral compartmentalthough the rate of apical to basolateral transport of HTy-Ac (2)was significantly greater than that of HTy (1). Besides, while homo-vanillyl alcohol was the only conjugated metabolite detected as aproduct of HTy metabolism by Caco-2/TC7 cells, resulting frommethylation of the parent compound, HTy-Ac glucuronide togetherwith free HTy (1) and homovanillyl alcohol were identified afterHTy-Ac (2) metabolism by the enterocytes. For both compounds,a substantial portion of the material reaching the basolateral com-partment was un-metabolised, indicating that hepatic metabolismmay be important for these compounds.

0%

50%

00%

1 2 1 2 1 2

Parent compound (AP) Metabolites (AP)

Parent compound (BL) Metabolites (BL)

1h 2h 4h

BASOLATERAL LOADING

in apical and basolateral side after 1, 2 and 4 h of incubation with Caco-2/TC7 cells.

R. Mateos et al. / Food Chemistry 125 (2011) 865–872 871

One of the main findings of this research was that HTy-Ac (2)acted as a more absorbable source of HTy (1). The principal metab-olite of the metabolism of both HTy (1) and HTy-Ac (2) metabolismwas methylated HTy (homovanillyl alcohol). Taking these observa-tions together, it is most likely that the higher trans-epithelialtransport observed for HTy-Ac (2) is due to the uptake of HTy-Ac(2) being substantially faster than HTy (1). The data are also consis-tent with intracellular de-esterification of HTy-Ac (2), presumablycatalysed by one of the numerous esterases known to be expressedin small intestinal epithelial cells (Prueksaritanont, Gorham,Hochman, Tran, & Vyas, 1996), because the alternative (extracellu-lar de-esterification) is not in keeping with the faster uptake andmetabolism of the acetate form. In this respect, the esterase(s) en-zymes present in Caco-2 cells were noted as responsible for thehydrolysis of the major dietary hydroxycinnamates (ferulate, sina-pate, p-coumarate, and caffeate) and of diferulates in the humansmall intestinal mucosa (Kern et al., 2003). Indeed, since secoirid-oid derivatives of HTy (1) have never been detected in blood afteringestion of virgin olive oil in human intervention studies, andthere are several reports consistently illustrating the high bioavail-ability of virgin olive oil phenolic compounds (Caruso et al., 2001;Visioli et al., 2000, 2003; Vissers et al., 2002), it is highly likely thathydrolysis of secoiridoid ester derivatives to yield free HTy (1) byesterases is a significant source of HTy that is subsequently ab-sorbed. If our novel observation that the higher apparent perme-ability (PappAP-BL) of an ester derivative of HTy (1) (= HTy-Ac (2))also extended to other secoiridoid ester derivatives, this couldexplain the high bioavailability described in intervention studiesfor virgin olive oil phenolic compounds.

However, it is important to note that these derivatives of HTy(1), secoiridoid derivatives and HTy-Ac (2), could suffer some alter-ation in the gastrointestinal tract, decreasing the amount of esterderivatives reaching the gut. Thus, under the markedly acidic gas-tric conditions a rapid hydrolysis of secoiridoid derivatives has beenreported (Corona et al., 2006) although by contrast a really high sta-bility of these components was observed in the same acidic enviro-ment present in the stomach (Romero, Medina, Vargas, Brenes, &De Castro, 2007; Soler et al., 2010). In this sense, preliminary resultsobtained by our group (Pereira-Caro et al., 2009) showed also thatHTy (1) and HTy-Ac (2) were stable in conditions mimicking thosein the stomach, observing a slight hydrolysis of HTy-Ac (2) into freeHTy (1). However, these compounds were more sensitive to themild alkaline conditions in the small intestine enhancing theamount of free HTy (1) released into the gut lumen althoughapproximately one third of HTy-Ac (2) remained unaltered.

Regarding the nature of the metabolites detected in the presentmanuscript, previous investigations have shown the presence ofHTy glucuronides in urine following ingestion of olive oil polyphe-nols (Miró-Casas et al., 2003; Visioli et al. 2000); however no HTyglucuronide was identified in our experiments performedin Caco-2/TC7 cells while small amounts of HTy-Ac glucuronidewere identified after the incubation of HTy-Ac (2) in Caco-2/TC7cells. Human Caco-2 monolayers exhibit many morphologicaland biochemical features of adult human enterocytes includingthe expression of phase II enzymes such as the UDP-glucuronosyltransferases UGT1 and UGT2 (Gregory, Lewinsky, Gardner-Stephen, & Mackenzie, 2004; Liu, Tam, & Hu, 2007), the sul-fotransferases SULT1 and SULT2 (Chen, Huang, Zhou, & Chen,2008; Meinl, Ebert, Glatt, & Lampen, 2008) and methyl transferases(Bonifacio & Soares-Da-Silva, 2004). Several reports concernedwith the absorption and metabolism of dietary polyphenols acrossCaco-2 cell monolayers (Barrington et al., 2009; Kaldas, Walle, &Walle, 2003; Kern et al., 2003), reported efficient sulphation, meth-ylation and glucuronidation by these cells. Therefore, our data sup-port the notion that the human small intestine is not the major siteof glucuronidation of HTy (1), and that since these are major forms

of this compound in human blood following consumption, the liveris likely the principal site of glucuronidation of HTy (1).

No sulphate metabolites were observed in any of the incubationsof either compound. These data are in agreement with previouslyreported results from similar incubations of CaCo-2 cells with HTy(1) (Corona et al., 2006; Manna et al., 2000). Small quantities of sul-phated and methyl-sulphated conjugates of HTy were reported fol-lowing longer incubations with a higher concentration of HTy(100 lM, 6 h) or very long incubation periods (24 h, 50 lM) (Soleret al., 2010). The preferential methylation of HTy (1) by CaCo-2 cellsreported here is consistent with data from studies reported else-where (Corona et al., 2006; Manna et al., 2000; Soler et al., 2010).In contrast, we observed no methylation of HTy-Ac (2).

Once taken up by the enterocytes, a portion of the HTy is conju-gated, and then the HTy or its metabolites may be effluxed to viathe apical or basolateral membranes. The data presented hereshow that HTy and its intestinal metabolites are preferentially eff-luxed across the basolateral membrane; this observation is inkeeping with the high bioavailability of HTy from virgin olive oilin humans.

Apart from a very efficient de-esterification of HTy-Ac (2) toyield HTy (1) by the CaCo-2 cells, there was only limited metabo-lism of HTy (1) to yield homovanillyl alcohol. The transport ofun-metabolised HTy (1) and HTy-Ac (2) to the basolateral compart-ment was 85% and 40%, respectively (2 h incubation). This observa-tion indicates that for both HTy (1) and HTyAc (2), there isconsiderable potential for hepatic transformation to yield the glu-curonide, sulphate and other conjugates that are observed in hu-man plasma. It is interesting to note that the enhancedabsorption of HTy-Ac (2), combined with efficient ester hydrolysis,served to enhance delivery of HTy (1) to the enterocytes for subse-quent metabolism and basolateral efflux. The limited bioavailabil-ity of dietary polyphenols, which is largely due to a combination ofunfavourable physico-chemical properties, extensive first-passmetabolism and significant apical efflux, provides a rationale forseeking strategies that can be applied to improve the absorptionof polyphenols and hence increase their potential biological activ-ity. The data reported here support further research into the use ofHTy-Ac (2) as a HTy (1) prodrug with enhanced HTy bioavailability.

In conclusion, these data indicate that HTy (1) and HTy-Ac (2)are absorbed across intestinal epithelial cell monolayers. Of partic-ular interest, HTy-Ac (2) was significantly better absorbed thanfree HTy (1) and essentially served to enhance delivery of HTy(1) to the enterocytes for subsequent metabolism and basolateralefflux. Apart from hydrolysis of HTy-Ac (2) to yield free HTy (1),partial metabolism of this olive oil phenolic compound was ob-served in the Caco-2/TC7 cells, and methylation was the principalmodification.

Acknowledgements

This work was supported by projects AGL2007-64042, AGL2007-66373-C04/ALI and CSD2007-00063 as part of the Consolider-Ingenio Program from the Spanish Ministry of Science andInnovation (CICYT), Grant RTA2007-000036-00-00 from the Re-search National Institute of Food, Agriculture and Technology(INIA),a contract (110105090014) with CSIC-IFAPA, and a core grant fromthe Biotechnology and Biological Sciences Research Council (toPAK and SS). G.P.-C. is a predoctoral fellow of the National ResearchInstitute of Food, Agriculture and Technology (INIA).

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