European Journal of Clinical Nutrition (2003) 57, 186–190 ß 2003 Nature Publishing Group All rights reserved 0954–3007/03 $25.00 www.nature.com/ejcn SHORT COMMUNICATION and are absorbed from moderate and sustained doses of virgin oil in humans

E Miro´-Casas1, M-I Covas2, M Fito´ 2, M Farre´-Albadalejo1, J Marrugat2 and R de la Torre1

1Unitat de Recerca en Farmacologia, Institut Municipal d’Investigacio´ Me`dica, Barcelona, Spain; and 2Unitat de Lipids I Epidemiologia Cardiovascular, Institut Municipal d’Investigacio´ Me`dica, Barcelona, Spain

Objective: To investigate the absorption of tyrosol and hydroxytyrosol from moderate and sustained doses of virgin consumption. The study also aimed to investigate whether these phenolic compounds could be used as biomarkers of virgin olive oil intake. Design and interventions: Ingestion of a single dose of virgin olive oil (50 ml). Thereafter, for a week, participants followed their usual diet which included 25 ml=day of the same virgin olive oil as the source of raw fat. Setting: Unitat de Recerca en Farmacologia. Institut Municipal d’Investigacio´ Me`dica (IMIM). Subjects: Seven healthy volunteers. Results: An increase in 24 h urine of tyrosol and hydroxytyrosol, after both a single-dose ingestion (50 ml) and short-term consumption (one week, 25 ml=day) of virgin olive oil (P < 0.05) was observed. Urinary recoveries for tyrosol were similar after a single dose and after sustained doses of virgin olive oil. Mean recovery values for hydroxytyrosol after sustained doses were 1.5- fold those obtained after a single 50 ml dose. Conclusions: Tyrosol and hydroxytyrosol are absorbed from realistic doses of virgin olive oil. With regard to the dose – effect relationship, 24 h urinary tyrosol seems to be a better biomarker of sustained and moderate doses of virgin olive oil consumption than hydroxytyrosol. Sponsorship: This work was supported by grants ALI97-1607-CO2-01 from CICYT, 98=9562 FPI from FIS and by Federacio´ de Cooperatives Agra`ries de Catalunya. European Journal of Clinical Nutrition (2003) 57, 186 – 190. doi:10.1038=sj.ejcn.1601532

Descriptors: olive oil; tyrosol; hydroxytyrosol; ; phenolic compounds

Introduction Fito´ et al, 2000; Owen et al, 2000). Other biological proper- The Mediterranean diet, in which olive oil is the major fat ties of olive oil phenolic compounds include anti-platelet component, has been associated with a lower incidence of aggregation, and apoptosis induction in HL-60 cells (Manna coronary heart disease and certain cancers (Renaud et al, et al, 1999). 1995). Virgin olive oil is rich in phenolic compounds Tyrosol and hydroxytyrosol are two characteristic olive oil which have been shown to delay in vitro metal-induced phenolic compounds (Tsimidou et al, 1992) which are present and radical-dependent LDL oxidation (Visioli et al, 1995; in olive oils as free or conjugated forms as secoroids or aglycones. Although the content of both total and individual phenolics varies among cultivars and harvests, free forms of tyrosol and hydroxytyrosol and their secoroids derivatives *Correspondence: MI Covas, Unitat de Lipids I Epidemiologia Cardiovascular, Institut Municipal d’Investigacio´ Me`dica, Carrer have been described as representing around 30%, and other Dr Aiguader 80, 08003 Barcelona, Spain. conjugated forms such as oleuropeine and ligstroside agly- E-mail: [email protected] cones representing almost half, of the total phenolic content Guarantor: MI Covas. of a virgin olive oil (Owen et al, 2000). Tyrosol has been shown Received 22 March 2002; revised 3 May 2002; to be effective in inhibiting the oxidation of cholesterol in accepted 17 May 2002 LDL and preventing the modification of the apoproteic Tyrosol and hydroxytyrosol E Miro´-Casas et al 187 moiety (Caruso et al, 1999). Tyrosol has also been effective in 253 mg=kg measured by colorimetric method (Singleton inhibiting leukocyte 5-lipooxygenase (De la Puerta et al,1999) & Ross, 1965). Phenolic compounds were extracted from and protecting the Caco-2 intestinal mucosa cells against the 19 ml of virgin olive oil with 90 ml of methanol:water cytostatic and cytotoxic effects produced by oxidized LDL (80:20, v=v). Methanol:water mixture contained 1 mM (Giovannini et al, 1999). However, due to its lack of ortho- ascorbic and 250 mM EDTA. Extraction was carried out diphenolic structure, the in vitro activity of during 90 min using a shaker. After extraction, methanol tyrosol is weak, in contrast to that of tyrosol (Giovannini was removed from olive oil extracts under vacuum at et al, 1999). Hydroxytyrosol was highly protective against the 40C. Sodium bisulphite (100 M final concentration) was peroxynitrite-dependent nitration of and DNA added in order to prevent catechol losses due to oxidation damage by peroxynitrite in vitro (Deiana et al, 1999). The during the analytical procedure. Aliquots of the methano- inhibitory effect of hydroxytyrosol on the formation of F2- lic extract (5 ml, three replicates) were treated with con- isoprostanes during in vitro LDL oxidation has also been centrated hydrochloric acid at 3 mM per aliquot. reported (Salami et al, 1995). Incubation was carried out at 37C during 30 min. After Data from cell culture models suggest that hydroxytyrosol hydrolysis, samples were adjusted to pH 2 – 3 and dis- is quantitatively absorbed at the intestinal level (Manna et al, solved in methanol up to 10 ml. Samples were stored at 2000). Orally administered oil-based dosing in rats resulted in 780C until their analysis. For the quantification of significantly greater elimination of tyrosol and hydroxytyr- hydroxytyrosol and tyrosol in each olive oil extract, stan- osol in 24 h urine than aqueous dosing (Tuck et al, 2001). The dards of both phenolic compounds were prepared by first experimental evidence of the absortion of tyrosol and spiking 28 and 20 mg, respectively, in a methanol:water hydroxytyrosol from olive oil in humans was obtained by mixture (80:20, v=v), and submitted to the same hydro- Visioli et al (2000) from a single oral dose of 50 ml of phenolic- lytic treatments used in oil extracts. Aliquots of each enriched olive oils. After ingestion of supplements containing control and olive oil extract were analysed, after derivati- 100 mg of olive oil phenols, a large part of olive oil phenols zation during 60 min at 60C with 100 ml of reaction was absorbed, being the absorbed olive oil phenols exten- mixture of MSTFA:NH4I:2-mercaptoethanol (1000:2:6, sively modified in the body (Vissiers et al, 2002). The bio- v=w=v), by gas chromatography (GC-MS) (Miro´-Casas et al, availability of tyrosol and hydroxytyrosol in humans from a 2001b). Concentrations of both phenolic compounds were single oral dose of 50 ml of virgin olive oil in its natural form 38.6 and 30.8 mg=kg of olive oil for tyrosol and hydro- has also been reported (Miro´-Casas et al, 2001a,b). Tyrosol xytyrosol, respectively. and hydroxytyrosol levels in urine rose after virgin olive oil consumption reaching a peak at 0 – 4 h and returning to basal values at 12 – 24 h (Miro´-Casas et al, 2001a,b). Study design This study is aimed on determining whether tyrosol and The experiment lasted 12 days. Volunteers followed an hydroxytyrosol may be absorbed from moderate and olive oil and low antioxidant diet during 4 days (washout sustained doses of virgin olive oil consumption. The study period). A nutritionist instructed them on excluding sev- is also aimed to investigate whether these phenolic com- eral foods from their diet (coffee, tea, fruit, vegetables, pounds could be used as biomarkers of virgin olive oil intake. wine and olive oil). At 8 am on day 5, 50 ml (44.5 g) of virgin olive was administered to the participants in a single dose either ingested directly or with some bread. Methods Thereafter for a week, all participants followed their usual Subjects diet which included 25 ml (22.5 g)=day of the same virgin The study was done in accordance with the Helsinki Declara- olive oil as ingested on day 5 as the source of raw fat. tion of 1975, as revised in 1989. The protocol was approved Twenty-four-hour urine was collected at baseline (day 4), by the local ethics committee (CEIC-IMAS). The subjects gave after the administration of 50 ml of virgin olive oil (day 5), their written informed consent. Seven healthy volunteers and after a week of 25 ml=day virgin olive oil consumption (four women and three men), aged between 22 and 63, were (day 12). Urine samples were stored at 780C until their recruited from the laboratory staff. Subjects had a weight of analysis. Twenty-four-hour urine volumes ranged from 0.8 64.1Æ 14.2 kg (meanÆ s.d.; men 75.7Æ 14.0 kg and women to 2.8 l, from 0.98 to 2.55 l, and from 0.78 to 3 l at days 4, 55.5Æ 6.4 kg) and body mass index of 22.8Æ 3.0 (meanÆ 5 and 12, respectively. Participants were requested to s.d.; men 24.7Æ 3.8 and women 21.1Æ 0.95). They were avoid high intakes of foods containing phenolic com- considered healthy on the basis of results obtained after pounds. Daily dietary records were obtained from each physical examination and routine laboratory tests. volunteer.

Olive oil characteristics Dietary assessment Virgin olive oil from an Arbequina cultivar was used in Nutrient intakes were calculated from seven daily dietary the study. Total phenolic content of olive oil was records with the software Diet Analysis Nutritionist IV

European Journal of Clinical Nutrition Tyrosol and hydroxytyrosol E Miro´-Casas et al 188 (N Squared Computing, San Bruno, CA). The daily energy intake of the subjects during the week of sustained olive oil ingestion ranged from 1465 to 2284 kcal (mean 1748Æ 331 kcal) and consisted of lipids 37.5Æ 3% (PUFA 4.8Æ 1.2%, MUFA 17.9Æ 2.1%, and SFA 14.7Æ 1.58%), carbohy- drate 44.2Æ 5.1%, and protein 18.3Æ 2.7%.

Laboratory measurements Tyrosol and hydroxytyrosol analyses in urine were per- formed as previously described (Miro´-Casas et al, 2001b). Briefly, sample hydrolysis was performed using hydrochloric acid (0.6 N of final concentration) and incubation for 20 min at 100C. After subsequent extraction and derivatization, quantitative determination of tyrosol and hydroxytyrosol in human urine was performed by capillary GC-MS. Syn- thetic urine was used as a blank urine in the preparation of Figure 1 Tyrosol and hydroxytyrosol in 24-hour urine after a single dose reference materials, and for the dilution of human urine (50 ml) and sustained doses (one week, 25 ml=day) of virgin olive oil samples with high levels of phenolic compounds. Synthetic consumption. * P < 0.05 versus baseline. urine was prepared using a modified method described by " P < 0.05 versus single dose intervention. Hedelin et al (1986). Synthetic urine contained 10 solutes with the pH adjusted to 6.5. The solutes with the concen-

trations in brackets (g=l) were as follows: CaCl2 Á 2H2O Results (0.65), MgCl2 Á H2O (0.65), NaCl (4.6), Na2SO4 (2.3), Na3 The urinary levels of tyrosol and hydroxytyrosol at baseline, citrate Á 2H2O (0.65), KH2PO4 (2.8), KCl (1.6), NH4Cl (1.0), after a single dose (50 ml), and after sustained doses (one urea (25) and creatinine (1.1). The solution was filtered and week, 25 ml per day) of virgin olive oil are reported in stored at 720C until use. All solutes were purchased from Figure 1. Urinary tyrosol and hydroxytyrosol levels increased Merck (Darmstadt, Germany). Calibration curves and urine after the single dose administration (P < 0.05), in the case of control samples were prepared by spiking the suitable tyrosol decreasing after one week of sustained doses of virgin volume of each working solution (10 mg=ml for tyrosol and olive oil consumption (P < 0.05). After one week of sustained 7 mg=ml for hydroxytyrosol) in synthetic urine. Both calibra- doses of virgin olive oil urinary amounts of tyrosol and tion curves and control samples were included in each hydroxytyrosol were higher than those obtained at baseline analysis batch of human urine samples collected throughout (P < 0.05). the study. Table 1 shows the 24 h urinary recoveries of tyrosol and hydroxytyrosol as a percentage of the estimated adminis- tered amount at both intervention periods. Urinary recov- eries of tyrosol were similar. However, mean recovery values Statistical analyses for hydroxytyrosol after one week of 25 ml=day sustained Differences between groups were assessed by the Wilcoxon doses of virgin olive oil were 1.5-fold of those obtained after test for paired samples. Statistical significance was defined as a single dose of 50 ml. Also, at the end of the sustained P < 0.05 for a two-sided test. Statistical analyses were period the mean amount of urinary hydroxytyrosol in 24 h performed using the SPSS statistical package (SPSS for urine (822 mg) was higher than the daily administered dose Windows 9.0.1). (685 mg).

Table 1 Urinary recoveries of tyrosol and hydroxytyrosol as a percentage of the administered amount after a single and sustained doses of virgin olive oil consumption

Single dose (50 ml) Sustained doses (one week, 25 ml=day)

Compound Intake (mg) Urinary recovery (%) Intake (mg=day) Urinary recovery (%)

Tyrosol 1720 16.9Æ 4.8 860 19.4Æ 12.2 Hydroxytyrosol 1370 78.5Æ 14.1 685 121.5Æ 45.9

Values are expressed as meanÆ s.d.

European Journal of Clinical Nutrition Tyrosol and hydroxytyrosol E Miro´-Casas et al 189 Discussion periods and using virgin olive oils with differences in their Data obtained in the present study confirm the absorption of phenolic content are needed to confirm these results. tyrosol and hydroxytyrosol after virgin olive oil consump- tion. An important finding of this investigation is that urinary tyrosol and hydroxytyrosol are absorbed from a sustained and moderate dose of virgin olive oil in its natural References form. This dose is close to that used as daily intake in a Caruso D, Berra B, Giavarini F, Cortesi N, Fedeli E & Galli G (1999): Effect of virgin olive oil compounds on in vitro oxidation of typical Mediterranean diet. human low density lipoproteins. Nutr. Metab. Cardiovasc. Dis. 9, Nevertheless, it seems that differences in the phenolic 102 – 107. compounds metabolism exist. Levels of urinary tyrosol Deiana M, Aruoma OI, Spencer JP, Kaur H, Halliwell B, Aeschbach R, obtained after one week of sustained doses (25 ml=day) of Dessi MA & Corongiu FP (1999): Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by virgin olive oil were lower than those obtained after a single extra virgin olive oil-derived antioxidant hydroxytyrosol. Free 50 ml dose. Urinary recoveries of tyrosol were similar in both Rad. Biol. Med. 26, 762 – 769. intervention periods. Urinary hydroxytyrosol levels, how- De la Puerta R, Ru´ız-Gutierrez V & Hoult JR (1999): Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. ever, were similar at both intervention periods, being the Biochem. Pharmac. 57, 445 – 449. estimated recovery of hydroxytyrosol in urine after sustained Fito´ M, Covas MI, Lamuela-Ravento´s RM, Vila J, Torrents J, de la doses of 25 ml of virgin olive oil greater than 100%. Torre C & Marrugat J (2000): Protective effect of olive oil and its At present, we do not know which metabolic or dietary phenolic compounds against low density lipoprotein oxidation. Lipids 35, 633 – 638. factors could be involved. We could discard analytical meth- Giovannini C, Straface E, Modesti D, Coni E, Cantafora A, odological differences given that inter-day coefficients of de Vincenzi M, Malorni, W & Masella R (1999): Tyrosol, the variation (CV%) and the accuracy (error%) are only slightly major olive oil biophenol, protects against oxidized-LDL induced better for tyrosol than for hydroxytyrosol (CV, 2.8 vs 6%, and injury in Caco-2 cells. J. Nutr. 129, 1269 – 1277. Goldstein DS, Swoboda KJ, Miles JM, Coppack SW, Nemman A, error 2.7 vs 5%, respectively; Miro´-Casas et al, 2001b). After a Holmes C, Lamensdorf I & Eisenhofer G (1999): Sources and single dose of virgin olive oil ingestion the half-life of tyrosol physiological significance of plasma sulfate. J. 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V Zappia, F Della Ragione, A Barbarisi, GL Russo and R Dello ( and glycosides of hydroxytyrosol) in other foods Iacovo, pp 115 – 130. New York: Kluwer Academic=Plenum has not been reported. Alternatively, dihidroxyphenyletha- Publishers. Manna C, Galletti P, Maisto G, Cucciolla V, D’Angelo S & Zappia V nol has been identified as a metabolite from dopamine (2000): Transport mechanism and metabolism of olive oil hydro- metabolism in dopaminergic cell lines (Lamensdorf et al, xytyrosol in Caco-2 cells. FEBS Lett. 470, 341 – 344. 2000). Other in vitro studies have demonstrated the forma- Mardh G & Vallee BL (1986): Human class I lcohol dehydrogenases tion of hydroxytyrosol as a result of 3,4-dihidroxyphenyl- catalyze the interconversion of and aldehydes in the metabolism of dopamine. Biochemistry 25, 7279 – 7282. acetaldehyde (DOPAL), a dopamine metabolite, reduction by Miro´-Casas E, Farre´ Albadalejo M, Covas Planells MI, Fito´ Colomer M, the human liver dehydrogenase isoenzyme (Mardh Lamuela Ravento´s R & de la Torre R (2001a): Tyrosol bioavail- & Vallee, 1986). Tyrosine derived from the breakdown of ability in humans after ingestion of virgin olive oil. Clin. Chem. 47, dietary protein could be a precursor of endogenous forma- 341 – 343. Miro´-Casas E, Farre´ Albadalejo M, Covas MI, Ortun˜o Rodriguez J, tion of dopamine in cells containing the tyrosine hydroxy- Menoyo Colomer E, Lamuela Ravento´s R & de la Torre R (2001b): lase enzyme (Goldstein et al, 1999). Therefore, endogenous Capillary gas chromatography-mass spectrometry quantitative formation of hydroxytyrosol could be derived from the determination of hydroxytyrosol and tyrosol in human urine human metabolism of dietary tyrosine. It cannot be dis- after olive oil intake. Anal. Biochem. 294,63– 72. 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