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Gender differences in plasma and urine metabolites from Sprague–Dawley rats after oral administration of normal and high doses of hydroxytyrosol, hydroxytyrosol acetate, and DOPAC Raúl Domínguez-Perles, David Auñón, Federico Ferreres & Angel Gil-Izquierdo

European Journal of Nutrition ISSN 1436-6207 Volume 56 Number 1 Eur J Nutr (2017) 56:215-224 DOI 10.1007/s00394-015-1071-2

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Author's personal copy Eur J Nutr (2017) 56:215–224 DOI 10.1007/s00394-015-1071-2


Gender differences in plasma and urine metabolites from Sprague–Dawley rats after oral administration of normal and high doses of hydroxytyrosol, hydroxytyrosol acetate, and DOPAC Raúl Domínguez‑Perles1 · David Auñón2 · Federico Ferreres1 · Angel Gil‑Izquierdo1 

Received: 26 June 2015 / Accepted: 5 October 2015 / Published online: 13 October 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Purpose  To date, several in vitro and in vivo studies have shown phenolic compounds occurring naturally in olives and olive oil to be beneficial to human health due to their interaction with intracellular signaling pathways. However, the bioavailability of the most important of these compounds, hydroxytyrosol (HT), and its transformation into derivatives within the organism after oral intake are still not completely understood, requiring further in vivo research. This study deals with the differential bioavailability and metabolism of oral HT and its derivatives in rats. Methods  Hydroxytyrosol (HT), hydroxytyrosol acetate (HTA), and 2,3-dihydroxyphenylacetic acid (DOPAC) were administered at doses of 1 and 5 mg/kg to Sprague–Dawley rats (n = 9 per treatment) by oral gavage. Their plasma kinetics and absorption ratio, assessed as their excretion in 24-h urine, were determined by UHPLC/MS/MS. Results  Plasma and urine levels indicated that although the three compounds are efficiently absorbed in the gastrointestinal tract and show similar metabolism, the bioavailability is strongly dependent on the derivative considered, dosage, and gender. Inter-conversion among them has been described also, suggesting an interaction with internal routes. Microbiota metabolites derived from these phenolics were also taken into account; thereby, homovanillic alcohol and tyrosol were identified and quantified in urine

* Angel Gil‑Izquierdo [email protected] 1

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Espinardo, Murcia, Spain


Department of Research and Development, Seprox BIOTECH S.L., Madrid, Spain

samples after enzymatic de-conjugation, concluding the metabolic profile of HT. Conclusions  Our results suggest that different dosages of HT, HTA, and DOPAC do not provide a linear, dosedependent plasma concentration or excretion in urine, both of which can be affected by the saturation of first-phase metabolic processes and intestinal transporters. Keywords  Hydroxytyrosol · Hydroxytyrosol acetate · DOPAC · Oral administration · Bioavailability · Plasma · Urine

Introduction During the last few years, the health benefits attributed to the dietary consumption of olives and olive oil, essential ingredients of the Mediterranean diet, have been related to their contents of bioactive phytochemicals, including glycosylated forms of oleuropein and ligstroside [1, 2], which—after subsequent modifications—give rise to hydroxytyrosol (HT) and tyrosol (Tyr) [3]. HT in its free form has been described as representing 6.0 % of the total phenolics present in virgin olive oil [4]. The dietary intake of foods containing these bioactive phytochemicals has been related to several human health benefits concerning the oxidative status, neuroprotective effects, and cardiovascular disease prevention [5–11]. In this context, data from epidemiological studies showed a lower incidence of cardiovascular events in Mediterranean countries, where olive oil is the major source of dietary fat. The scientific substantiation of health claims in relation to polyphenols in olive is based on the information received by the European Food Safety Authority (EFSA) from the member states. According to this information, it is


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considered that polyphenols, concretely HT and its derivatives in olives and olive oil, are sufficiently characterized in relation to the expected health benefits [12]. To gain a better understanding of the biological effects of these compounds and the cellular pathways on which they act after dietary intake, bioavailability studies in humans and other animals are essential. In this sense, previous investigations revealed dose-dependent absorption of HT in the small intestine and colon and its urinary excretion, after its ingestion in virgin olive oil as well as in oily and aqueous preparations [13–18]. Despite this, the digestive effect on the isolated compounds remains poorly understood, although this information constitutes a crucial element for the bioactive evaluation of olives and olive oil polyphenols, which is essential for the development of new pharmaceutical and nutraceutical commodities. Some clinical trials and dietary intervention studies supported the oral administration of isolated molecules, providing evidences of a positive absorption ratio [19, 20]. However, the low concentrations of these phytochemicals in biological fluids suggest that further analyses of their bioavailability and pharmacokinetics, applying highly sensitive analytical tools, would provide valuable and accurate information on the scope for the oral administration of “healthy” compounds, concerning their actual biological activity. Additionally, some HT derivatives have been evaluated already, showing promising biological activities in vitro [21]—thus increasing the interest in their bioavailability and metabolism within the organism. To fill the currently existing gaps, the present work is focused on the ratio of absorption, plasma levels, and metabolism of HT, hydroxytyrosol acetate (HTA), and 3,4-dihydroxyphenylacetic acid (DOPAC) in male and female Sprague–Dawley rats over the hours following the administration of aqueous preparations of the single compounds at distinct doses. The identification and quantification of the main compounds, metabolites of direct absorption, and gut microflora metabolites derived from HT, HTA, and DOPAC in peripheral blood and in 24-h urine were performed using a sensitive method: ultra-high performance liquid chromatography coupled to a mass spectrometer equipped with an electrospray ionization (ESI) chamber and triple quadrupole mass analyzer for tandem analysis (UHPLC-ESI-QqQ-MS/MS).

Materials and methods Reagents Hydroxytyrosol (HT), HTA, and DOPAC were provided by SEPROX BIOTECH S.L. Tyr and homovanillic alcohol (HVA) were purchased from Sigma-Aldrich (Spain). Hydrolytic β-glucuronidase, type H2, from Patella vulgata


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(Sigma-Aldrich St. Louis, MO, USA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All LC–MS grade solvents were obtained from J.T. Baker (Phillipsburg, New Jersey, USA), and Bis–Tris (bis(2-hydroxyethyl) amino-tris(hydroxymethyl)methane) from Sigma-Aldrich. Reagents such as formic acid and hydrochloric acid were purchased from Panreac (Castellar del Vallés, Barcelona, Spain). The solid-phase extraction (SPE) cartridges used in this study (Strata X-AW, 100 mg/3 mL) were obtained from Phenomenex (Torrance, California, USA). Study design and sample collection The study was carried out on 7-week-old male (n  = 60, body weight 212 ± 7 g) and female (n = 60, body weight 187 ± 6 g) Sprague–Dawley rats, provided by Harlan Interfauna Ibérica S.L. (Barcelona, Spain). The rats were fed with a global diet purchased from Harlan Teklad (Blackthorn, UK) and maintained in plastic cages at room temperature (22 ± 2 °C) and 30–70 % relative humidity with a 12-h light/12-h dark cycle, for 1 week, in agreement with European Union regulations. After food deprivation for 12 h, with free access to water, the rats of each gender were randomly separated into six groups (n  = 9, each group). Solutions of HT, HTA, or DOPAC in 5 % ethanol (v/v) in milliQ water (vehicle) at doses of 1 (equivalent to the normal human dose recommended by the EFSA) and 5 mg/kg (high dose) were administered by oral gavage to animals from the different experimental groups. Two additional control groups constituted by males (n  = 6) and females (n = 6) Sprague–Dawley rats received an equal volume of the vehicle (Table 1), and allowed to monitor its effect on the plasma and urine level of target compounds with comparative purposes. The rats were then placed in metabolic cages. After inhalation anesthesia by the rats with isoflurane, blood samples were collected by sublingual puncture at 0, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h. Immediately after blood collection, citrated blood volumes of 400 μL were transferred to polypropylene tubes and 40 μL of 10 % L-ascorbic acid and 0.58 M acetic acid were added to each sample. The plasma was then separated and stored in UVprotected tubes. Urine was also collected for 24 h, in tubes containing 10 % L-ascorbic acid, and the total volumes were recorded. The urine and plasma samples were stored at −80 °C until extraction and analyses. Immediately after the last blood sampling, all animals were killed by CO2 overdosing. This experimental protocol was approved by the Experimental Animal Ethical Research Committee, in accordance with the European and Spanish Guidelines for Animal Care and Use of Laboratory Animals with reference to the provisions of the European Directives 86/609/ EEC and 2010/63/EC and the Spanish Regulation RD 1201/2005.

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Table 1  Pharmacokinetic study design


Sex (number of animals) Administered compound

Dose (mg/kg) Urine sampling schedule

Group 1a Group 2 Group 3 Group 4b

Male (6) Male (9) Male (9)

Vehicle Hydroxytyrosol Hydroxytyrosol

0 1 5

Female (6) Female (9) Female (9) Male (6) Male (9) Male (9)

Vehicle Hydroxytyrosol Hydroxytyrosol Vehicle Hydroxytyrosol acetate Hydroxytyrosol acetate

0 1 5 0 1 5

Group 11 Group 12 Group 13a Group 14

Female (6) Female (9) Female (9) Male (6) Male (9)

Vehicle Hydroxytyrosol acetate Hydroxytyrosol acetate Vehicle 3,4-Dihydroxyphenylacetic acid

0 1 5 0 1

Group 15

Male (9)

3,4-Dihydroxyphenylacetic acid


Group 16b Female (6) Group 17 Female (9)

Vehicle 3,4-Dihydroxyphenylacetic acid

0 1

Group 18

3,4-Dihydroxyphenylacetic acid


Group 5 Group 6 Group 7a Group 8 Group 9 Group 10b

Female (9)

Blood sampling schedule

Before dosing and 0.5, 1.0, Before dosing and total 2.0, 4.0, 8.0, 24.0 h posturine excreted throughout dosing the 24 h post-dosing

Before dosing and 0.5, 1.0, Before dosing and total 2.0, 4.0, 8.0, 24.0 h posturine excreted throughout dosing the 24 h post-dosing

Before dosing and 0.5, 1.0, Before dosing and total 2.0, 4.0, 8.0, 24.0 h posturine excreted throughout dosing the 24 h post-dosing

a   Groups 1, 7, and 13 are constituted by a single group of male Sprague–Dawley rats receiving an equal volume of vehicle than treated groups with comparative purposes b

  Groups 4, 9, and 16 are constituted by a single group of female Sprague–Dawley rats receiving an equal volume of vehicle than treated groups with comparative purposes

Sample preparation Rat plasma samples were thawed at room temperature and centrifuged (11,000×rpm for 5 min). The supernatants (100 μL) were hydrolyzed by incubating with 5 μL (300 UI) of β-glucuronidase from P. vulgata for 2 h at 37 °C, clarified with 200 μL of MeOH/HCl (200 mM), and centrifuged at 11,000×rpm for 5 min. The supernatants were cleaned up by SPE, using Strata X-AW cartridges according to the manufacturer’s instructions. Briefly, the cartridges were conditioned and equilibrated with 2 mL of MeOH/formic acid (98:2, v/v) and 2 mL of distilled water/

formic acid (98:2, v/v), respectively. Urine samples were diluted with 2 mL of distilled water/formic acid (98:2, v/v) and applied to the column. After that, the SPE cartridges were washed with distilled water/formic acid (98:2, v/v) and aspirated until dryness. The target analytes were eluted with 1 mL of MeOH/formic acid (98:2, v/v) and dried totally using a SpeedVac concentrator (Savant SPD121P; Thermo Scientific, Waltham, MA). The extracts were reconstituted with 200 μL of solvent A/B (90:10, v/v), used for the UHPLC/MS/MS analyses. The urine samples collected were thawed at room temperature and centrifuged (11,000×g for 5 min). The


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supernatants (400 μL) were hydrolyzed by incubating with 25 μL (1500 UI) of β-glucuronidase from P. vulgata for 2 h at 37 °C, clarified with 200 μL of MeOH/HCl (200 mM), and centrifuged at 11,000×rpm for 5 min. Afterward, they were cleaned up by SPE using Strata X-AW cartridges according to the manufacturer’s instructions. Briefly, samples diluted in 1.25 mL of MeOH and 2 mL of 0.02 M BIS–TRIS buffer at pH 7 were applied to a previously conditioned and equilibrated cartridge with 2 mL of MeOH and distilled water, respectively. Retained compounds were eluted with 1 mL of MeOH. The eluted compounds were dried completely using a SpeedVac concentrator (Savant SPD121P, Thermo Scientific, MA, USA). The extracts were reconstituted with the mobile phase A/B (90:10, v/v), solvent A being H2O/formic acid (99.9/0.1, v/v) and solvent B MeOH, to avoid the influence of the solvents on the ionization of the sample at the electrospray interface and to make comparable the extraction results. The concentrations of the target compounds in urine and plasma were calculated from standard curves freshly prepared each day by applying the dilution factors corresponding to the SPE process. UHPLC‑ESI‑QqQ‑MS/MS analysis Plasma and urinary metabolites were analyzed using a UHPLC–MS/MS (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was carried out on a ZORBAX Eclipse Plus C-18 Rapid Resolution HD column (2.1 × 50 mm, 1.8 µm) (Agilent Technologies), using water/ formic acid (99.9:0.1, v/v) (A) and MeOH (B) as solvents. The flow rate was 0.4 mL/min, using a linear gradient (t; %B): (0; 20), (6; 95), (7; 100), (10; 20), and the injection volume was 10 μL. For qualitative analyses, the target analytes were identified according to the MRM transitions Table 2  UHPLC/MS/MS parameters for the quantification of and confirmation of DOPAC, HTA, and HT, in urine and plasma of male and female rats

referred to in Table 2, in order to discern the metabolites present in the plasma at the considered time points as well as the total amount excreted in 24-h urine. For quantification of the total HT derivatives in urine, the capillary exit voltage and collision energy were previously optimized for the administered compounds (HT, HTA, and DOPAC) and for the other two gut microflora metabolites considered, Tyr and homovanillic alcohol. The optimal ESI conditions for maximal detection of the analytes were: gas temperature, 225 °C; sheath gas temperature, 375 °C; capillary voltage, 3000 V; nozzle voltage, 1000 V; sheath gas flow, 11 %; gas flow, 10; nebulizer, 40. The MS parameters fragmentor (ion optics capillary exit voltage) and collision energy were optimized for each compound to generate the most abundant product ions for the MRM negative and positive ion modes. Statistics The quantitative data are presented as mean ± SD. A multifactorial analysis of variance (ANOVA) and Tukey’s multiple range tests were carried out. All the statistical analyses were performed using SPSS19.0 software (LEAD Technologies, Inc., Chicago, IL). The level of statistical significance was set at p  123b 169 > 110c 195 > 91 195 > 123 153 > 123 153 > 95 169 > 151 169 > 91

40 40 73 73 90 90 52 52

0 0 20 20 9 9 0 0




139 > 121




139 > 91



a   Tyr Tyrosol, DOPAC 3,4-Dihydroxyphenylacetic acid, HTA hydroxytyrosol acetate, HT hydroxytyrosol, HVAlc homovanillic alcohol b c

  Quantification transition

  Confirmation transition


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as their conversion into related molecules. The retention times (min) of the analytes were consistently identified and recorded for the standard solutions and biological samples (plasma and urine) analyzed (0.535, 0.542, 1.214, 1.603, and 1.848 for DOPAC, HTA, HT, HVA, and Tyr, respectively) (Table  2). The ESI mass spectra of the DOPAC, HTA, HT, HVA, and Tyr standard solutions exhibited intense protonated/deprotonated molecular signals at m/z [M]+ 169, 195, 169, and 139 amu, corresponding to DOPAC, HTA, HVA, and Tyr, respectively, and at m/z [M-H]− 153 amu, corresponding to HT. The monitoring of the fragmentation patterns of the identified protonated or deprotonated molecular ions as their specific fragments allowed us to record the following MRM transitions: 169 > 123, 110 (DOPAC), 195 > 91, 123 (HTA), 153 > 123, 95 (HT), 169 > 151, 91 (HVA), and 139 > 121, 91 (Tyr). These were selected based on their

sensitivity and specificity as quantification and confirmation transitions, respectively.

Fig. 1  Plasma pharmacokinetic profile of HT derivatives in male rats, showing samples as an overlap of the control group and the two dosages for each compound, expressed as ng/mL, at different sampling time points (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h). The pharma-

cokinetic study was carried out in Sprague–Dawley rats after single oral gavage of HT hydroxytyrosol (a, d, g), HTA hydroxytyrosol acetate (b, e, h), and DOPAC 2,3-dihydroxyphenylacetic acid (c, g, i) at doses of 1 and 5 mg/kg (n = 9 rats per treatment)

Plasma kinetics of the administered compounds The UHPLC-ESI-QqQ-MS/MS-based qualitative and quantitative determination of the administered analytes (DOPAC, HTA, and HT) allowed us to establish accurately the diverse plasma occurrence and concentrations for the compounds ingested and their potential inter-conversion. To clarify the bioavailability of HT, HTA, and DOPAC, two distinct doses, 1 and 5 mg/kg (corresponding to the equivalent normal human dose, according to the EFSA, and a higher dose, respectively), were administered orally to Sprague–Dawley rats. To contrast the effect of the vehicle, analysis of plasma/urine was performed on an additional


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group (n  = 6) treated with an equal volume of vehicle (Table 1). The results obtained with respect to the plasma concentrations of HT, HTA, and DOPAC after the oral administration of the separate compounds showed a similar pharmacokinetic behavior in both male and female rats (Figs. 1, 2), relative to the total concentrations obtained by enzymatic hydrolysis of plasma and urine samples, which allowed quantification of the total amounts of HT, HTA, DOPAC, Tyr, and HVA in their free forms. The separate analyses of the plasma pharmacokinetics in both males and females gave values higher than the quantification limit [1 ng/mL, S/N 10/1 (ICH, 1994)]. Hence, the plots in Figs. 1 and 2 show that the highest plasma concentrations of HT, HTA, and DOPAC occurred between 0.5 and 2.0 h after their oral administration. After having reached their maximum concentrations, the compounds were rapidly removed from the

plasma and had been eliminated almost completely and/ or metabolized after 4.0 h in both male and female rats, as evidenced by the virtual disappearance of the target compounds in plasma samples collected 4.0, 8.0, and 24 h after administration. Concerning genders, the analysis of the administered compounds in terms of bioavailability showed that the different plasma concentrations recorded in males and females were strongly dependent on the compound administered, the dose, and the metabolite monitored. Concretely, with respect to HT, HTA, and DOPAC, in males the higher dose resulted in almost 1.5, 1.6, and 1.3-fold higher plasma levels, respectively, on average, while in females the corresponding values were 2.4, 0.9, and 1.1-fold (for HT, HTA, and DOPAC, respectively), with respect to the lower dose. One-way ANOVA with Tukey’s multiple range tests showed significant differences at p