Hydroxytyrosol Disposition in Humans Elisabet Miro-Casas, 1 Maria-Isabel Covas, 2 Magi Farre, 1,3 Montserrat Fito, 2 Jordi Ortun ˜ o, 1 Tanja Weinbrenner, 2 Pere Roset, 1,3 and Rafael de la Torre 1,4 Background: Animal and in vitro studies suggest that phenolic compounds in virgin olive oil are effective antioxidants. In animal and in vitro studies, hydroxyty- rosol and its metabolites have been shown to be strong antioxidants. One of the prerequisites to assess their in vivo physiologic significance is to determine their pres- ence in human plasma. Methods: We developed an analytical method for both hydroxytyrosol and 3-O-methyl-hydroxytyrosol in plasma. The administered dose of phenolic compounds was estimated from methanolic extracts of virgin olive oil after subjecting them to different hydrolytic treat- ments. Plasma and urine samples were collected from 0 to 12 h before and after 25 mL of virgin olive oil intake, a dose close to that used as daily intake in Mediterra- nean countries. Samples were analyzed by capillary gas chromatography–mass spectrometry before and after being subjected to acidic and enzymatic hydrolytic treatments. Results: Calibration curves were linear (r >0.99). Ana- lytical recoveries were 42– 60%. Limits of quantification were <1.5 mg/L. Plasma hydroxytyrosol and 3-O-meth- yl-hydroxytyrosol increased as a response to virgin olive oil administration, reaching maximum concentrations at 32 and 53 min, respectively (P <0.001 for quadratic trend). The estimated hydroxytyrosol elimination half- life was 2.43 h. Free forms of these phenolic compounds were not detected in plasma samples. Conclusions: The proposed analytical method permits quantification of hydroxytyrosol and 3-O-methyl-hy- droxytyrosol in plasma after real-life doses of virgin olive oil. From our results, 98% of hydroxytyrosol appears to be present in plasma and urine in conjugated forms, mainly glucuronoconjugates, suggesting exten- sive first-pass intestinal/hepatic metabolism of the in- gested hydroxytyrosol. © 2003 American Association for Clinical Chemistry Epidemiologic studies support the beneficial effects of the Mediterranean diet in human health, particularly in the prevention of cardiovascular diseases (1–4). The Mediter- ranean diet includes, as distinctive components, high intake of fiber, fruit, legumes, and vegetables, with olive oil being the main source of fat. The beneficial effects of olive oil could be linked to both its monounsaturated fatty acid and its antioxidant content. In several human and animal dietary studies, virgin olive oil and oleic acid-rich diets have been shown to reduce LDL susceptibility to oxidation (5–8). Virgin olive oil is rich in phenolic com- pounds, which have been shown to delay in vitro metal- induced and radical-dependent LDL oxidation (9, 10). Other biological properties of the phenolic compounds in olive oil include activity against platelet aggregation and apoptosis induction in HL-60 cells (11 ). The main phenolic compounds in olives are the glyco- sylated forms of oleuropein and ligstroside (12, 13). The glucose residue is removed by enzymatic hydrolysis, giving rise to the aglycone forms of both compounds. In olive oil under acidic conditions, both oleuropein and ligstroside give rise to the polar phenolic compounds hydroxytyrosol (HT) 5 and tyrosol (14 ). HT may also be a product of the enzymatic hydrolysis of its own corre- sponding glycoside (15 ). Free forms of tyrosol and HT and their secoroid derivatives have been described as representing 30%, and other conjugated forms, such as oleuropein and ligstroside aglycones, represent almost one-half of the total phenolic content of virgin olive oil (16 ). In animal and in vitro studies, HT and its metabolites have shown to be strong antioxidants (17, 18). Bioavail- 1 Unitat de Farmacologia de l’Institut Municipal d’Investigacio ´ Me `dica (URAF-IMIM) and 2 Unitat de Lı ´pids i Epidemiologia Cardiovascular de l’Institut Municipal d’Investigacio ´ Me `dica (ULEC-IMIM), Doctor Aiguader No. 80, 08003 Barcelona, Spain. 3 Universitat Auto ´ noma de Barcelona (UAB), 08003 Barcelona, Spain. 4 Universitat Pompeu Fabra (CEXS-UPF), 08003 Barcelona, Spain. *Address correspondence to this author at: Unitat de Farmacologia Institut Municipal d’Investigacio ´ Me `dica (IMIM), Carrer Doctor Aiguader, 80, 08003 Barcelona, Spain. Fax 34-932213237; e-mail rtorre@imim.es. Received December 11, 2002; accepted March 18, 2003. 5 Nonstandard abbreviations: HT, hydroxytyrosol; 3-O-methyl-HT, 3-O- methyl-hydroxytyrosol; ISTD, internal standard; MSTFA, N-methyl-N-trim- ethylsilyltrifluoroacetamide; GC-MS, gas chromatography–mass spectrometry; LOD, limit(s) of detection; LOQ, limit(s) of quantification; AUC, area under the curve; c max , maximum concentration; and t max , time corresponding to c max . Clinical Chemistry 49:6 945–952 (2003) Nutrition 945