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1 Unitat de Farmacologia de l’Institut Municipal d’Investigació Mèdica (URAF-IMIM) and
2 Unitat de Lípids i Epidemiologia Cardiovascular de l’Institut Municipal d’Investigació Mèdica (ULEC-IMIM), Doctor Aiguader No. 80, 08003 Barcelona, Spain.
3 Universitat Autónoma de Barcelona (UAB), 08003 Barcelona, Spain.
4 Universitat Pompeu Fabra (CEXS-UPF), 08003 Barcelona, Spain.
aAddress correspondence to this author at: Unitat de Farmacologia Institut Municipal d’Investigació Mèdica (IMIM), Carrer Doctor Aiguader, 80, 08003 Barcelona, Spain. Fax 34-932213237; e-mail rtorre{at}imim.es.
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Abstract |
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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 treatments. 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 Mediterranean 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). Analytical recoveries were 42–60%. Limits of quantification were <1.5 mg/L. Plasma hydroxytyrosol and 3-O-methyl-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-hydroxytyrosol 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 extensive first-pass intestinal/hepatic metabolism of the ingested hydroxytyrosol.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main phenolic compounds in olives are the glycosylated 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)
1
and tyrosol (14). HT may also be a product of the enzymatic hydrolysis of its own corresponding 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). Bioavailability studies in animals and humans administered virgin olive oil or olive oil supplemented with phenolic compounds have demonstrated that HT is absorbed and excreted in urine as a response to the intake of virgin olive oil as well as to intake of oily and aqueous preparations (19)(20)(21)(22)(23)(24). One of the prerequisites, however, for assessing the relationship between the in vivo antioxidant effect of HT and its concentration in food is to determine HT concentrations in human plasma. To our knowledge, plasma concentrations of HT and its metabolites after virgin olive oil ingestion have not been described previously. The difficulty in developing sensitive methods to measure these phenolic compounds in human plasma could account for this fact.
In the present work, we examined HT disposition in humans. From our knowledge, this is the first time a validated method for the quantitative determination of HT and 3-O-methyl-hydroxytyrosol (3-O-methyl-HT), a HT metabolite, in human plasma has been described. This method has been used successfully to measure these phenolic compounds after consumption of a single dose of raw virgin olive oil (25 mL).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HCl, NaOH, NH4I, sodium acetate, acetic acid, and 2-mercaptoethanol were purchased from Merck. Methanol, acetonitrile (HPLC grade), 700 g/L perchloric acid, and ethyl acetate were purchased from Scharlau. N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was purchased from Macherey-Nagel. Sodium metabisulfite and ß-glucuronidase (type H-2; 131 000 units/mL of ß-glucuronidase activity and 3.180 units/mL of sulfatase activity) were supplied by Sigma. Ultrapure water was obtained with a MilliQ purification system (Millipore). The virgin olive oil selected for the study was from the cultivar "Picual" from Jaen.
phenolic compounds in virgin olive oils
HT and 3-O-methyl-HT were measured in virgin olive oil as described previously (23) with only minor modifications. Briefly, phenolic compounds were extracted from 4 mL of virgin olive oil by shaking for 60 min with 40 mL of a methanol–water (80:20 by volume) mixture containing 1 mmol/L ascorbic acid. After centrifugation, the organic phase was distributed into four aliquots, which were evaporated under a nitrogen stream at 40 °C. After methanol evaporation, sodium metabisulfite (final concentration, 100 mmol/L) was added to each aliquot to prevent catechol oxidation during the sample preparation procedure. Aliquots containing olive oil aqueous extracts were used for the determination of HT and 3-O-methyl-HT in olive oil in their free form. To quantify conjugated forms of the phenolic compounds, other aliquots were treated with concentrated HCl (1.5 mmol of HCl/tube). The hydrolytic treatment was used to mimic the gastrointestinal conditions during virgin olive oil digestion (26)(27). All tubes were incubated at 37 °C for 30 min. For quantification of HT and 3-O-methyl-HT in each olive oil extract, calibration curves were prepared by adding different amounts of concentrated pure reference substances to 1 mL of refined olive oil (range, 350-2800 ng). Calibration samples were treated in the same way as virgin olive oil samples.
recovery of ht from oleuropein
Oleuropein pure reference substance (280 nmol/tube) was added to tubes containing 1 mL of ultrapure water. Four different hydrolytic treatments, similar to those that would be used for the biological samples, were evaluated: (a) 1 mL of 0.5 mol/L HCl; (b) 250 µL of 6 mol/L HCl; (c) 50 µL of ß-glucuronidase in 1 mL of acetate buffer; and (d) 1 mL of pure water (hydrolysis step omitted). All tubes were processed as described previously for plasma and urine samples. Eight replicates were assayed for each experimental condition.
calibration curves and control samples
Concentrated solutions of the pure reference substances (140 mg/L for HT, and 100 mg/L for 3-O-methyl-HT and the ISTD) were prepared in methanol. Working solutions at concentrations of 1.4 and 7 mg/L (HT), 1 and 10 mg/L (3-O-methyl-HT), and 10 mg/L (ISTD) were prepared by dilution of the concentrated solutions with methanol.
Plasma calibration curves and control samples were prepared by adding a suitable volume of each working solution (1.4 and 1 mg/L) to 1 mL of blank plasma, free of the tested compounds. We then added 15 ng of ISTD to each tube. Working ranges for calibration curves were 2.1–42 µg/L for HT and 1.5–30 µg/L for 3-O-methyl-HT. Control plasma samples (10.5, 21, and 35 µg/L for HT and 7, 15, and 25 µg/L for 3-O-methyl-HT) were included in each analytical batch. Urine calibration curves and control samples were prepared by adding a suitable volume of each working solution (7 and 10 mg/L) to 2.5 mL of synthetic urine. We then added 200 ng of ISTD to each tube. The final concentrations for the calibration curves were 14–112 µg/L for HT and 10–120 µg/L for 3-O-methyl-HT. Control urine samples were prepared at the same time as the calibration curve (21, 42, and 98 µg/L for HT and 16, 30, and 60 µg/L for 3-O-methyl-HT) and included in each analytical batch. Control plasma and urine samples containing HT and 3-O-methyl-HT were prepared from solutions other than those used in the preparation of calibration curves.
participants and sample collection
We recruited six healthy volunteers (three men and three women), 25–47 years of age, for this study; they were considered healthy on the basis of physical examination and routine biochemical and hematologic tests. The volunteers had a mean (SD) weight of 67.3 (19) kg and body mass index of 24.7 (6.5) kg/m2. The study was done in accordance with the Helsinki Declaration of 1975, as revised in 1996. The protocol was approved by the local ethics committee (CEIC-IMAS, Ref. 2002/1326/I). The volunteers gave written informed consent before their inclusion in the study.
After 12 h of fasting, volunteers ingested 25 mL of virgin olive oil in a single dose with bread (20–25 g). Plasma samples were collected at 0 (predose) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, and 8 h after ingestion of the virgin olive oil. Urine samples were collected at the following time periods: 0 (predose), 0–2, 2–4, 4–6, 6–8, and 8–12 h after ingestion of the virgin olive oil. Sodium metabisulfite (final concentration, 25 mmol/L) was added to samples, which were then stored at -80 °C until analysis.
preanalytical treatment of plasma samples
To evaluate the conjugated forms of the phenolic compounds, samples were subjected to acidic (HCl) or enzymatic (ß-glucuronidase) hydrolysis. For acidic hydrolysis, 15 ng of ISTD and 100 µL of 1 mol/L sodium metabisulfite were added to each glass tube containing 1 mL of plasma, and 60 µg of 4-methylcatechol was added to each tube to prevent the binding of phenolic compounds to proteins. One milliliter of 0.5 mol/L HCl was then added to each tube. Samples were incubated in a dry bath at 100 °C for 20 min. Samples were allowed to reach room temperature, and 25 µL of 700 g/L perchloric acid was added to precipitate the proteins. After protein precipitation, the pH was adjusted to 3–3.5 with 1 mol/L NaOH. After centrifugation for 5 min at 300g, the supernatant was transferred to a glass tube. Liquid–liquid extraction was carried out by the addition of 4 mL of an acetonitrile–ethyl acetate mixture (1:4 by volume) to each sample. Samples were mixed for 30 min on a rocking mixer. After centrifugation for 5 min at 300g, the organic phase was evaporated to dryness under a nitrogen stream at 25 °C. Residues were derivatized with 75 µL of MSTFA–NH4I–2-mercaptoethanol reaction mixture (2 g of NH4I and 6 mL of 2-mercaptoethanol per liter of MSTFA) for 60 min at 60 °C, and 2 µL of the sample was injected into the gas chromatography–mass spectrometry (GC-MS) system.
Enzymatic hydrolysis was performed by addition of 25 µL of ß-glucuronidase and 1 mL of 1.1 mol/L sodium acetate buffer (pH 5.2) to 1 mL of the plasma, control, and calibration samples. Samples were incubated overnight (17 h) at 37 °C. After hydrolysis, the sample preparation procedure was similar to that described above for acidic hydrolysis. Plasma samples were also analyzed, omitting the hydrolytic step to determine the free (nonconjugated) fraction of phenolic compounds.
preanalytical treatment of urine samples
To evaluate the conjugated forms of the phenolic compounds, samples were subjected to acidic (HCl) or enzymatic (ß-glucuronidase) hydrolysis. Acidic hydrolysis was performed as described previously (23). Briefly, 2.5 mL of urine was hydrolyzed with 6 mol/L HCl in the presence of sodium metabisulfite (final concentration, 100 mmol/L). After hydrolysis, the pH was adjusted to 3–3.5 by the addition of NaOH, and the phenolic compounds were extracted with 6 mL of acetonitrile–ethyl acetate (1:4 by volume). After evaporation of the organic phase, residues were derivatized with 100 µL of MSTFA–NH4I–2-mercaptoethanol (2 g of NH4I and 6 mL of 2-mercaptoethanol per liter of MSTFA) for 60 min at 60 °C, and 2 µL of the sample was injected into the GC-MS system.
Enzymatic hydrolysis was performed by addition of 50 µL of ß-glucuronidase and 1 mL of 1.1 mol/L sodium acetate buffer (pH 5.2) to 2.5 mL of the urine, control, and calibration samples. Samples were incubated overnight (17 h) at 37 °C. After hydrolysis, the sample preparation procedure was similar to that described for acid hydrolysis. Two aliquots of each urine sample were analyzed, omitting the hydrolytic step to determine the nonconjugated fraction of phenolic compounds.
gc-ms analyses of phenolic compounds
HT and 3-O-methyl-HT in plasma and urine were measured by GC-MS. The gas chromatograph was coupled to a mass spectrometer detector system consisting of a HP6890N Network gas chromatography system, a HP5973 Network mass-selective detector, and a HP7683 series injector (Agilent Technologies). The phenolic compounds in olive oil were separated on a Zebron ZB-5 5% phenylpolysiloxane capillary column [15 m x 0.25 mm (i.d.); 0.25 µm film thickness; Phenomenex]. The column head pressure of helium, which was used as a carrier gas, was set at 1 mL/min, measured at 180 °C. The GC oven temperature was programmed from 80 to 200 °C at 15 °C/min and from 200 to 280 °C at 25 °C/min, and was held there for 3 min. Total run time was 15 min. Plasma samples were injected in splitless mode (30 s of purge off time). Urine samples were injected in split mode with a split ratio 1:15. The detector was in the single-ion monitoring mode, and compounds were ionized by electron impact. Ions at m/z 206, 296, 281, and 191 for bis-trimethylsilyl-3-(4-hydroxyphenyl)-propanol (ISTD), m/z 370, 267, 179, and 193 for tris-trimethylsilyl-HT, and m/z 312, 209, 297, and 179 for bis-trimethylsilyl-3-O-methyl-HT were recorded. After verifying the ion ratios for each compound analyzed in each determination, we used the ions at m/z 206 (ISTD), 370 (HT), and 312 (3-O-methyl-HT) for quantitative determinations.
method characteristics
Selectivity was assessed by measuring 10 plasma samples and testing for the presence of interfering substances at the retention times of HT and 3-O-methyl-HT. Recoveries for HT, 3-O-methyl-HT, and the ISTD were calculated by comparing the peak areas of the calibration samples with those obtained after the same amounts of the reference substances and the ISTD were added to plasma blank samples. Three different concentrations were tested for each compound (n = 4 replicates): 3.5, 14, and 42 µg/L for HT, and 2.5, 10, and 30 µg/L for 3-O-methyl-HT. Recovery for the ISTD was estimated with samples to which 15 µg/L of the ISTD had been added. The linearity of the method was determined by analyzing different calibration curves at five different concentrations (n = 10) on 4 consecutive days. The concentrations tested were 3.5–42 µg/L for HT and 1.5–30 µg/L for 3-O-methyl-HT. Peak-area ratios between the phenolic compounds and the ISTD were used for calculations. A weighted least-squares regression analysis was used (SSPS for Windows, Ver. 9.0.1). The limits of detection and quantification (LOD and LOQ, respectively) were calculated with use of four replicates of blank control samples to which 2.1 µg/L HT or 1.5 µg/L 3-O-methyl-HT had been added. The SD of these determinations was used as an estimation of the "noise" of the analytical system for the calculation of the LOD (3.3 SD) and LOQ (10 SD). Precision and accuracy were evaluated with use of three different concentrations of HT and 3-O-methyl-HT, with three replicates per run, for 9 days (Table 1
).
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pharmacokinetic calculations
From plasma concentrations obtained after virgin olive oil ingestion, we determined the following parameters for both HT and 3-O-methyl-HT: maximum concentration (cmax; maximum plasma concentration of compound), time to reach cmax (tmax), and the area under the curve (from t = 0 to the last time with a concentration equal to or above the LOQ (AUC0–8h). Plasma half-life (t1/2) and the elimination constant (Ke) were also estimated. Pharmacokinetic parameters were calculated with use of specific functions in a spreadsheet (PK Functions for Microsoft Excel).
statistical analyses
A weighted least-squares regression analysis was used to obtain the correlation coefficients and slope values. Data are expressed as the mean (SD). A general linear model for repeated measurements, with the Tukey tests for multiple comparisons, was fitted to assess the effect on HT and 3-O-methyl-HT concentrations after virgin olive oil ingestion. Statistical significance was defined as P <0.05. SPSS statistical software was used (SPSS for Windows 9.0.1).
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Results |
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recovery of ht from oleuropein
The amounts of HT recovered from oleuropein that had been subjected to the two acid hydrolysis procedures were similar: 32% (90.7 nmoles for treatment 1) and 35% (99.6 nmoles for treatment 2) of the initial amount of oleuropein (280 nmoles). After enzymatic hydrolysis (treatment 3) or spontaneous hydrolysis of oleuropein without hydrolysis (treatment 4), the recovery of HT was negligible (0.12%, or 0.33 nmoles).
method characteristics
Optimum separation, responses, and peak shapes were obtained for the compounds studied independent of the hydrolysis procedure used (Fig. 2
). The ISTD used showed physicochemical properties similar to those of the analytes, eluting at a retention time very close to that of the phenolic compounds of interest in olive oil, indicating the usefulness of 3-(4-hydroxyphenyl)-propanol as ISTD.
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Recoveries were 42 (2.6)% for HT, 54 (4.0)% for 3-O-methyl-HT, and 60 (2.1)% for the ISTD. They were similar, independent of the hydrolysis procedure used. The mean (SD) coefficients of determination (r2) for the calibration curves were 0.992 (0.004) for HT and 0.996 (0.004) for 3-O-methyl-HT. The LOD were 0.46 and 0.12 µg/L for HT and 3-O-methyl-HT, respectively, and the LOQ were 1.39 and 0.36 µg/L for HT and 3-O-methyl-HT, respectively.
The within- and between-day precision and accuracy are shown in Table 1
. We found no differences in terms of accuracy and precision as a function of the hydrolytic treatment used.
phenolic compounds in human plasma
Plasma concentrations of HT and 3-O-methyl-HT increased in response to virgin olive oil ingestion, reaching maximum concentrations at 32 min for HT and 53 min for 3-O-methyl-HT (P <0.001 for quadratic trend). Plasma concentrations of the olive oil phenolic compounds decreased gradually throughout the next 6 h, reaching in some cases concentrations around the LOQ 8 h after virgin olive oil ingestion (Fig. 3
). The free forms of these phenolic compounds were not detected in plasma samples.
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The results obtained in plasma samples after acidic treatment are summarized in Table 2
. Results obtained after enzymatic hydrolysis of plasma samples were different from those obtained after acidic treatment. The AUC0–8h values for HT and 3-O-methyl-HT after enzymatic hydrolysis were 65.6% and 88.3%, respectively, of those obtained after acid hydrolysis. The cmax values for HT and 3-O-methyl-HT after enzymatic hydrolysis were 68.5% and 84.5%, respectively, of those observed after acid hydrolysis. tmax values were similar for both hydrolytic procedures.
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olive oil-derived phenolic compounds in urine
Urinary amounts of HT and 3-O-methyl-HT increased in response to virgin olive oil ingestion, reaching a peak at 0–2 h (P <0.002 for quadratic trend; Fig. 4
). The measured amounts of the free forms were 3.6–9.1 µg [mean (SD), 7.1 (2.0) µg] for HT and 5.7–9.1 µg [7.5 (1.5) µg] for 3-O-methyl-HT.
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Urinary recovery 12 h after olive oil ingestion was rather different depending on the hydrolytic treatment applied to the samples. When urine samples were subjected to acidic conditions, recoveries were 714.7 (159.2) µg (range, 558.1–1015.3 µg) for HT, and 188.0 (54.7) µg (range, 143.3–287.3 µg) for 3-O-methyl-HT. When urine samples were subjected to enzymatic hydrolysis, recoveries were 479.6 (100.0) µg (range, 302.8–575.2 µg) for HT and 122.9 (32.0) µg (range, 81.4–172.8 µg) for 3-O-methyl-HT.
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Discussion |
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Plasma samples were subjected to two types of hydrolysis: enzymatic hydrolysis for the determination of phenolic compounds in olive oil as their glucuronoconjugate metabolites, and chemical hydrolysis to allow the determination of these compounds conjugated not only to glucuronic acid, but also to sulfate and other cofactors, such as glutathione. Given that the phenolic compounds in their free forms were undetectable in plasma samples, a preliminary conclusion from these studies is that independent of the type of hydrolysis studied, phenolic compounds are the subject of an extremely extensive first-pass intestinal/hepatic metabolism. Considering that the LOD and cmax values for HT were 0.46 and 25.83 µg/L, respectively, the concentration of HT in its free form will never be >1.6% compared with its conjugated forms. Thus, the biological activity of HT most probably derives from its metabolites. This concept is supported by animal and in vitro studies in which the 3-O-glucuronide of HT had higher activity as a radical scavenger than did HT itself (28).
When we compared the HT results observed in plasma and urine after the two hydrolysis protocols, it was apparent that 65% of HT seems to be in its glucuronoconjugated form and 35% in other conjugated forms. As stated earlier, there are three main sources of HT from olive oil. HT may be present in its free form, 10% of the dose of HT when compared with values obtained after acid hydrolysis of olive oil. A second source is the HT glucoside (HT-4-ß-D-glucoside), and the last one is oleuropein. We found that HT is released from oleuropein in response to acidic hydrolysis treatment, with a mean HT recovery of 33% compared with the initial amount of oleuropein in the experiment. In a very recent and elegant approach, it was demonstrated that HT and oleuropein are absorbed in the small intestine (24). In this study, oleuropein was not quantified in plasma or urine, but oleuropein has been shown to be metabolized in the body and recovered in urine, mainly in the form of HT (24). Thus, the fraction of conjugated HT (not including the glucuronoconjugated form), which was estimated to be 35% of plasma and urine concentrations, may come not only from HT conjugates but also from the breakdown of oleuropein under acidic hydrolytic conditions. This could mean that the percentage of metabolic conversion of HT to its glucuronoconjugate may be >65%.
Evaluation of the plasma concentration-vs-time curves for HT and 3-O-methyl-HT showed that their pharmacokinetics may fit into a bicompartmental model. Some additional plasma samples between 2 and 4 h after ingestion of olive oil would be valuable for a better pharmacokinetic model of the experimental data. The reported elimination half-life (2.4 h) assumes a monocompartmental model. Previous estimations from urinary data suggested that the half-life was 8 h (23). Discrepancies in the percentages of 3-O-methyl-HT glucuronoconjugate in plasma and urine (88% and 64%, respectively) were observed. This may be explained in light of enterohepatic recirculation, in which after reabsorption from the gut the newly formed conjugate was different from the one excreted through bile. HT appears rapidly in plasma, reaching maximum concentrations 30 min after virgin olive oil ingestion. In some volunteers, peak concentrations were reached at 15 min after olive oil ingestion. Peak plasma concentrations of 3-O-methyl-HT occurred 50 min after olive oil ingestion, confirming previous reports in which this compound has been described as one of the main metabolites of HT (21).
In this as well as in our other previous studies (23)(29), a major unresolved drawback in the evaluation of HT disposition is the fact that after strict dietary control (i.e., diet free of foods containing the phenolic compounds of interest) as well as after hours of fasting, it is not possible to eliminate HT in biological fluids. One explanation that could account for this fact is that HT may be renamed as 3,4-dihydroxyphenylethanol, a well-known metabolite of dopamine. It is not surprising, therefore, to find it in biological fluids, despite a strict washout in the dietary protocol. In fact, homovanillic acid, one of the main metabolites of dopamine, has also been reported as a major metabolite of Ht (30)(31). These observations also raise questions concerning the extent HT from the diet and dihydroxyphenylethanol from dopamine metabolism may participate jointly as an antioxidant system in the body.
In summary, the present work is the first description of plasma concentrations of HT and 3-O-methyl-HT concentrations in plasma after ingestion of virgin olive oil. The methodology developed permits the detection and quantification of these phenolic compounds in plasma after ingestion of real-life doses of virgin olive oil. From our results, 98% of HT is present in plasma and urine in conjugated forms, mainly glucuronoconjugates, suggesting extensive first-pass intestinal/hepatic metabolism of the ingested HT.
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Acknowledgments |
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