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Research Communication

The In Vivo Fate of Hydroxytyrosol and Tyrosol, Antioxidant Phenolic Constituents of Olive Oil, after Intravenous and Oral Dosing of Labeled Compounds to Rats

Centre for Pharmaceutical Research, School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Adelaide, 5000, Australia

¹To whom correspondence should be addressed. E-mail: kellie.tuck{at}unisa.edu.au

	ABSTRACT

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In vitro studies have shown phenolics in olive oil to be strong radical scavengers. The absorption and elimination of two radiolabeled phenolic constituents of olive oil, hydroxytyrosol and tyrosol were studied in vivo using rats. Compounds were administered intravenously (in saline) and orally (in oil- and water-based solutions). For both compounds, the intravenously and orally administered oil-based dosings resulted in significantly greater elimination of the phenolics in urine within 24 h than the oral, aqueous dosing method. There was no significant difference in the amount of phenolic compounds eliminated in urine between the intravenous dosing method and the oral oil-based dosing method for either tyrosol or hydroxytyrosol. Oral bioavailability estimates of hydroxytyrosol when administered in an olive oil solution and when dosed as an aqueous solution were 99% and 75%, respectively. Oral bioavailability estimates of tyrosol, when orally administered in an olive oil solution and when dosed as an aqueous solution were 98% and 71%, respectively. This is the first study that has used a radiolabeled compound to study the in vivo biological fates of hydroxytyrosol and tyrosol.

KEY WORDS: • olive oil • antioxidants • elimination • bioavailability • phenols • rats

	INTRODUCTION

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The Mediterranean diet, of which a notable feature is the use of olive oil as the principal source of fat, has been shown to lead to a lower incidence of coronary heart disease (CHD)² (1) . The composition of olive oil is primarily triacylglycerols and 0.5%–1.0% nonglyceridic constituents (2) . Among these minor constituents are various phenolic compounds, which by their chemical nature, act as antioxidants. Numerous in vitro studies have shown these phenolics to possess strong radical scavenging activity at least equal in potency with other important dietary antioxidants, such as ascorbic acid and -tocopherol (3 ,4) . Uncontrolled production of free radicals contributes to the pathogenesis of CHD and various tumors. Phenolic compounds are also potent in vitro inhibitors of low-density lipoprotein oxidation and can break peroxidative chain reactions (5) . Peroxidative chain reactions have been linked to the pathogenesis of CHD and cancer (3) . It has been recently found that these phenolic compounds decrease the amount of isoprostane excreted in urine (6) and the principal phenolic compound in olive oil decreases the oxidative stress in rats exposed to passive smoking (7) .

Despite the wide body of evidence linking the in vitro properties of olive oil phenolics with positive health outcomes, there are limited data on the absorption and excretion of these compounds. In part, this could be due to the low concentrations of such constituents and, accordingly, the difficulty in detecting the presumptively low concentrations of these compounds in biological systems. We have examined the absorption and excretion of hydroxytyrosol [HT; 2-(3,4-dihydroxyphenyl)ethanol], the principal phenolic compound found in olive oil (2) . The absorption and excretion of tyrosol, another quantitatively important phenolic compound found in olive oil, was also investigated. HT is one of a very limited number of phenolic compounds found in olive oil whose absorption and/or disposition has been assessed, although only briefly. Bai et al. (8) measured plasma levels of HT by gas chromatography mass spectrometry in samples from rats orally administered large doses of HT in aqueous solutions. Rapid appearance of the parent compound in plasma was observed with maximal HT levels attained 10 min after dosing. In a recent human study, the urinary recovery of HT and tyrosol were assessed by HPLC coupled with mass spectrometry after oral dosing with HT- and tyrosol-enriched extra virgin olive oil (EVO) (9) . The proportions of HT and tyrosol recovered in glucuronidase-hydrolyzed urine, with respect to ingested dose, were in the ranges of 30%–60% and 20%–22%, respectively. Aside from glucuronide metabolism of HT [as shown by (9) ] there is limited information on the biological fate of HT and tyrosol after oral and intravenous (IV) dosing.

	MATERIALS AND METHODS

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Materials.

HT-[ring 2,5,6-³H] and tyrosol-[ring 3,5-³H] were synthesized and purified by previously published procedures (sp. ac. HT 66 Ci/mol; Tyrosol 13 Ci/mol) (10 ,11) . Water used in all experiments was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). Italian EVO (Colvita, Italy) was used and its HT concentration (before spiking with radiolabeled HT) was determined to be 13.2 mg/kg by a specific HPLC procedure (2) . ß-Glucuronidase type VII-A (bacterial from Escherichia coli, 1000 U per vial) and sulfatase type VI (from Aerobacter aerogenes, 50 U/3.5 mL) were obtained from Sigma Chemical (Sydney, Australia). Halothane was purchased from Laser Animal Health (Salisbury, Australia). All other reagents were analytical grade or higher and used without purification. Samples were analyzed for radioactivity by a Packard Tri-Carb 2000CA liquid scintillation counter (Meriden, CT). HPLC analysis was performed on a Hewlett Packard 1100 Series system consisting of a 1100 series isocratic pump, 1100 series autosampler and 1100 series variable wavelength detector (Palo Alto, CA) and with an analytical DuPont (Wilmington, DE) phenyl zorbax (4.6 mm x 25 cm) column, mobile phase [99.5 v/v H₂O (containing 0.2 v/v acetic acid)/0.5 v/v methanol, 1 mL/min]. The compounds were detected at 281 nm. HPLC radiometric analysis was performed with a Radiomatic 150TR-flow scintillation analyzer (Meriden, CT; scintillint flow, 2.5 mL/min; HPLC flow, 1 mL/min).

Preparation of oral dosing solutions

    Oil solutions. Italian EVO (Colvita, 1300 mg) was added to 23.5 mg of H³-HT (sp. ac. 17 mCi/mmol). Italian EVO (Colvita, 1300 mg) was added to 14.7 mg of H³-tyrosol (sp. ac. 13 mCi/mmol). The mixtures were thoroughly mixed immediately before dosing to ensure that the phenols were uniformly distributed throughout the solution.

    Aqueous solutions. Water (1300 mg) was added to 25.5 mg of H³-HT (sp. ac. 12 mCi/mmol). Water (1300 mg) was added to 14.4 mg of H³-tyrosol (sp. ac. 12 mCi/mmol).

    Preparation of intravenous dosing solutions. H³-HT (6.5 mg, sp. ac. 33 mCi/mmol) was added to 5 mL of 9 g/L sodium chloride for injection. Radiolabeled tyrosol (9.8 mg, 5 mCi/mmol) was added to 5 mL of 9 g/L sodium chloride for injection.

    Animals and animal experiments. Written ethics approval was obtained from the local committee (IMVS, South Australia, Australia). Healthy male Sprague-Dawley rats (350 g) (Mouse Breeder Cubes, Ridley Agriproducts, Murray Bridge, Australia) were allowed free access to a commercial rat diet and water for at least 2 d before the dosing experiment. They were deprived of food for 2 h before being administered solutions of radiolabeled HT or radiolabeled tyrosol.

Each rat was orally dosed with either 225 mg olive oil solution or 225 mg aqueous solution via a gavage needle or IV-dosed (tail vein) with 950 mg of the saline solution while lightly anesthetized (inhaled Haloethane; Laser Animal Health, Salisbury, Australia). The rats were then placed in individual metabolic cages and allowed free access to food and water. Urine samples were collected (where possible) at 1-, 2-, 3-, 4-, 8- and 24-h time intervals.

    Treatment of urine samples. Urine was collected in sample tubes containing acetic acid (80 µL) to minimize degradation of HT and tyrosol. Once collection was complete, 100 µL of each urine sample was analyzed for total radioactivity by liquid scintillation counting. Samples were also analyzed by HPLC radiometric detection for the presence of metabolites and tritiated water.

    Analysis for metabolites. Urine (50 µL) was diluted with mobile phase (500 µL). The pH was adjusted to 5.7 and ß-glucuronidase (50 µL) or sulfatase (30 µL) was added. The sample was incubated at 37°C for 1 h and then analyzed by HPLC radiometric detection.

    Treatment of feces samples. Feces samples were extracted and analyzed for radioactivity using a standard method (12) .

    Bioavailability estimates. Bioavailability estimates were determined to be the ratio of dose normalized excreted in 24 h of oral (oil or water) versus IV ^{(Eq. 1)} .

(1)

    Statistics. Differences in the mean percentage of radiolabeled HT and radiolabeled tyrosol eliminated in urine within 24 h were analyzed separately using one-way ANOVA with dosing method as the effect. When significant results (P < 0.05) were found from the ANOVA, group differences were analyzed further using the posthoc Tukey-Kramer honestly significant difference test (JMP; SAS Institute, Cary, NC). Data for HT were transformed before analysis using a natural log transform to achieve homogeneous variances. However, only the original untransformed values are presented for ease of interpretation.

	RESULTS

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The tritium label in HT was incorporated at all unsubstituted positions on the aromatic ring. For tyrosol, the tritium label was incorporated at C3 and C5 on the aromatic ring. The stability of the tritium label in HT and tyrosol was investigated in urine; no exchange of the label was observed by radiometric HPLC after 24 h. The tritium label of HT (11) and tyrosol was also stable in aqueous solutions (pH 7).

The percentages of compound eliminated over time by rats after radiolabeled HT and tyrosol were administered are shown in Tables 1 and 2 , respectively. All results are normalized with respect to the amount of radioactivity administered. The majority of the excreted dose was eliminated from the body within 2 h when dosed intravenously and within 4 h for both methods of oral dosing for both HT and tyrosol.

View larger version (20K): [in this window] [in a new window]   Figure 1. A typical radiometric chromatogram of a urine sample from a rat orally administered HT in oil, 2 h after administration (M = metabolite; M4 is HT). The corresponding table shows the presence of metabolites (M1-M6) in urine and whether these metabolites are cleaved with ß-glucuronidase and sulfatase.

View larger version (24K): [in this window] [in a new window]   Figure 2. A typical radiometric chromatogram of a urine sample from a rat orally adminstered tritium-labeled tyrosol in oil, 2 h after administration (M = metabolite; M2 is tyrosol). The corresponding table shows the presence of metabolites (M1, M2) in urine and whether these metabolites are cleaved with ß-glucuronidase and sulfatase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We examined the absorption and elimination of radiolabeled HT and radiolabeled tyrosol (and their metabolites) in the urine of rats dosed orally (dispersed in EVO and water) and dosed intravenously (aqueous solution). Based on previous studies in the literature, it was known that HT is rapidly eliminated from plasma (8) , and the rapid appearance of HT [including its metabolites (9) ] into urine implies that it is rapidly eliminated from the body. Although it would have been desirable to define the plasma clearance of HT, our chief consideration was the absorption and excretion of HT and its metabolites.

To mimic the absorption and excretion of HT and tyrosol, when consumed as part of a Mediterranean diet, rats were orally administered the compounds in an olive oil matrix. These results were compared with orally dosing the compounds as an aqueous solution and IV dosing of the compounds as a saline solution.

The estimated bioavailability values of HT and tyrosol were significantly lower when administered as an aqueous solutions than when they were administered as olive oil solutions. These bioavailability values do not exclude the possibility of intestinal metabolism of either phenolic compound followed by absorption of their biotransformation products. This difference in the bioavailability of compounds, administered as aqueous and oil solutions, has previously been noted (13 ,14) . The increased bioavailability of these compounds when administered as an olive oil solution could be due to other antioxidants present in olive oil preventing the breakdown of the investigated phenolic compounds in the gastrointestinal tract before absorption.

Our findings differ from those previously found with humans (9) . This could be for two reasons. First, our study used rats and these compounds could be handled differently in humans than in rats. Alternatively, this study may be a more accurate method for assessing the absorption and excretion of HT and tyrosol, because the presence of numerous labeled conjugates of HT and tyrosol could be detected, not just those hydrolyzed to the parent compound in ß-glucuronidase-hydrolyzed urine.

Initially, it was of concern that the tritium label of HT or tyrosol could be exchanged with water in the body. When urine samples were analyzed by HPLC radiometric detection, the first peak to elute was discernible from tritiated water (for both compounds), and it eluted after the void time of the column. No tritiated water peak was observed in the urine samples for HT or tyrosol. Furthermore, the first peak to elute disappeared after reaction of the urine sample with sulfatase (the parent compound enlarged accordingly, see Figs. 1 and 2 ). However, we did not focus on the identification of metabolites because the aim of this study was to examine the absorption and excretion of HT and tyrosol in rats dosed orally and IV.

In conclusion, we have shown that HT and tyrosol can be absorbed into the systemic circulatory system after oral dosing. Their bioavailability when administered as an olive oil solution is almost complete. Accordingly, phenolic compounds, such as HT and tyrosol, in olive oil are likely to be systemically available and, thus, able to exert a direct antioxidant action.

	ACKNOWLEDGMENTS

 
We thank Anthony Lucas for his assistance with the dosing experiments.

	FOOTNOTES

 
² Abbreviations used: CHD, coronary heart disease; HT, hydroxytyrosol; EVO, extra virgin olive oil; IV, intravenous.

Manuscript received January 26, 2001. Initial review completed February 21, 2001. Revision accepted April 16, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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2. Montedoro G., Servili N., Baldioli M., Miniati E. Simple and hydrolyzable phenolic compounds in virgin olive oil: their extraction, separation, and quantification and semiquantitative evaluation by HPLC. J. Agric. Food Chem. 1992;40:1571-1576

3. Manna C., Galletti P., Cucciolla V., Moltedo O., Leone A., Zappia V. The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells. J. Nutr. 1997;127:286-292[Abstract/Free Full Text]

4. Visioli F., Bellomo G., Galli C. Free radical-scavenging properties of olive oil polyphenols. Biochem. Biophys. Res. Commun. 1998;247:60-64[Medline]

5. Visioli F., Bellomo G., Montedoro G., Galli C. Low density lipoprotein oxidation is inhibited in vitro by olive oil constituents. Atherosclerosis 1995;117:25-32[Medline]

6. Visioli F., Caruso D., Galli C., Viappiani S., Galli G., Sala A. Olive oil rich in natural catecholic phenols decrease isoprostane excretion in humans. Biochem. Biophys. Res. Commun. 2000;278:797-799[Medline]

7. Visioli F., Caruso D., Galli C., Plasmati E., Viappiani S., Hernandez A., Colombo C., Sala A. Olive phenol hydroxytyrosol prevents passive smoking-induced oxidative stress. Circulation 2000;102:2169-2171[Abstract/Free Full Text]

8. Bai C., Yan X., Takenaka M., Sekiya S., Nagata T. Determination of synthetic hydroxytyrosol in rat plasma by GC-MS. J. Agric. Food Chem. 1998;46:3998-4001

9. Visioli F., Galli C., Bornet F., Mattei A., Patelli R., Galli G., Caruso D. Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett 2000;468:159-160[Medline]

10. Tuck K. L., Tan H., Hayball P. J. A simple procedure for the deuteriation of phenols. J. Labeled Compd Radiopharm. 2000;43:817-823

11. Tuck K. L., Tan H., Hayball P. J. Synthesis of tritiated hydroxytyrosol. J. Agric. Food Chemistry 2000;48:4087-4090[Medline]

12. Riska P., Lamson M., MacGregor T., Sabo J., Hattox S., Pav J., Keirns J. Disposition and biotransformation of the antiretroviral drug nevirapine in humans. Drug Metab. Dispos 1999;27:895-901[Abstract/Free Full Text]

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14. Lyons K. C., Charman W. N., Miller R., Porter C.J.H. Factors limiting the oral bioavailability of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion. Int. J. Pharmaceutics 2000;199:17-28

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