Metabolite profiling of olive oil and thyme phenols after a sustained intake of two phenol-enriched olive oils by humans: Identification of compliance markers Laura Rubió a , Marta Farràs b , Rafael de La Torre c,d , Alba Macià a , Maria-Paz Romero a , Rosa M. Valls e , Rosa Solà e , Magí Farré c,d , Montserrat Fitó b , Maria-José Motilva a, ⁎ a Food Technology Department, UTPV-XaRTA, University of Lleida-Agrotecnio Center, Alcalde Rovira Roure 191, 25198 Lleida, Spain b Cardiovascular Risk and Nutrition Research Group, CIBER de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), IMIM-Institut Hospital del Mar d'Investigacions Mèdiques, Doctor Aiguader 88, 08003 Barcelona, Spain c Human Pharmacology and Clinical Neurosciences Research Group, IMIM-Institut Hospital del Mar d'Investigacions Mèdiques, Doctor Aiguader 88, 08003 Barcelona, Spain d Universitat Pompeu Fabra (CEXS-UPF), Doctor Aiguader 80, 08003 Barcelona, Spain e Unitat de Recerca en Lípids i Arteriosclerosis, CIBERDEM, St. Joan de Reus University Hospital, IISPV, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, C/Sant Llorenç 21, 43201 Reus, Spain abstract article info Article history: Received 23 January 2014 Received in revised form 25 April 2014 Accepted 3 May 2014 Available online xxxx Keywords: Compliance biomarkers Human intervention Olive oil Phenolic metabolites Thyme An understanding of causal relations between phenol intake and its beneficial effects on health is hindered by the lack of robust biological markers of its exposure. This is particularly relevant in mid/long-term nutritional inter- vention studies. An analytical methodology based on UPLC–MS/MS has been developed to determine the metab- olites of the phenolic compounds from olive oil and thyme in biological fluids after a sustained intake of two phenol-enriched olive oils for their further use as compliance biomarkers. In a randomized, double-blind, con- trolled, cross-over trial, 33 hypercholesterolemic volunteers received during 3 weeks 25 mL/day of (1) raw Vir- gin Olive Oil with a low phenolic content as a control (80 mg total phenols / kg oil; VOO), (2) Functional Virgin Olive Oil enriched with its own phenolics (500 mg total phenols / kg oil; FVOO), and (3) Functional Virgin Olive Oil enriched with its own phenolics plus complementary phenolics from Thyme (500 mg total phenols / kg oil, 50% from olive oil and 50% from thyme respectively; FVOOT). Plasma and 24 h-urine samples were collected. The results showed that some hydroxytyrosol (HT) metabolites presented low specificity as biomarkers of intake. However, hydroxytyrosol sulfate and hydroxytyrosol acetate sulfate appeared to be suitable biomarkers for mon- itoring compliance with olive oil intake as their values in plasma or/and 24-h urine were significantly higher after FVOO compared to baseline pre-intervention concentrations. They were also significantly correlated with the monitored level of compliance. On the other hand, metabolites derived from thyme were more specific, thymol sulfate and hydroxyphenylpropionic acid sulfate being the metabolites with the largest increase in both plasma and 24-h urine, whereas urinary p-cymene-diol glucuronide presented the greatest increase post-treatment. Their urinary excretion values also displayed significant correlations with the level of compliance and they were defined as FVOOT compliance biomarkers. This study enabled robust quantitative and qualitative compli- ance biomarkers after the ingestion of two phenol-enriched olive oils to be determined and provided a thorough analysis of the true phenolic exposure after a sustained consumption that could be further related to expected biological effects. © 2014 Elsevier Ltd. All rights reserved. Introduction The European Food Safety Authority Scientific and Technical Guid- ance has recently provided the criteria for authorizing health claims for foods. This clearly establishes that relevant human interventional studies have to be presented to substantiate any claim. While studies in animal or in vitro models may provide supporting evidence (e.g. in support of a mechanism), human data are essential to substantiate the health claim (EFSA Panel on Dietetic Products, 2011). Over the past de- cade, a significant number of human nutrition intervention studies have been conducted with the goal of establishing the exact bioefficacy of various subclasses of polyphenols as protection against chronic degen- erative diseases (Del Rio et al., 2013; Kay, Hooper, Kroon, Rimm, & Cassidy, 2012). Nevertheless, few validated biomarkers of polyphenol exposure are available, which hinders establishing any relationship be- tween exposure and effects (Kay, 2010). This link is essential to decide whether the negative outcome of a controlled trial (i.e., a lack of func- tional change in response to supplementation) can be related to the Food Research International xxx (2014) xxx–xxx ⁎ Corresponding author. Tel.: +34 973 702817; fax: +34 973 702596; FRIN-05251; No of Pages 10 http://dx.doi.org/10.1016/j.foodres.2014.05.009 0963-9969/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres Please cite this article as: Rubió, L., et al., Metabolite profiling of olive oil and thyme phenols after a sustained intake of two phenol-enriched olive oils by humans: Identification of compliance markers, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.05.009