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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2612-2615, August 2003

Nutrient Metabolism
Research Communication

Hydroxytyrosol Excretion Differs between Rats and Humans and Depends on the Vehicle of Administration

Francesco Visioli2, Claudio Galli, Simona Grande, Katia Colonnelli, Cristian Patelli, Giovanni Galli and Donatella Caruso

Department of Pharmacological Sciences, University of Milan, 20133 Milan, Italy

2To whom correspondence should be addressed. E-mail: francesco.visioli{at}unimi.it.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Interest in the in vivo biological activities of olive oil phenolics is rapidly growing, and different models and vehicles of administration are used worldwide. Matters of practicality determine the use of rats rather than humans as the model of choice. Also, growing interest in nutraceuticals is leading to the formulation of compounds containing olive oil phenols. In this study, we compared metabolism and urinary excretion of hydroxytyrosol [(HT), the most representative phenol of olive oil] between rats and humans by evaluating excretion of HT and its major metabolite, homovanillyl alcohol. Also, we compared human excretion of HT when consumed as a natural component of extra virgin olive oil, when added to refined olive oil, or when added to yogurt (as an approximation of functional food). Urinary excretion of HT was greater in humans than in rats, a species with a high basal excretion of HT and its metabolites. The high (234% of HT administered) excretion of free HT suggests that hydrolysis of oleuropein administered in humans (still an unresolved issue) occurs in vivo. Moreover, human HT excretion was much higher after its administration as a natural component of olive oil (44.2% of HT administered) than after its addition to refined olive oil (23% of HT administered) or yogurt (5.8% of dose or ~13% of that recorded after virgin olive oil intake). These data suggest that the rat is not the appropriate model for the study of HT metabolism and that HT-containing functional foods should be carefully formulated.

KEY WORDS: • hydroxytyrosol • oleuropein • nutraceuticals • polyphenols • antioxidants

Hydroxytyrosol (HT), a simple phenol (Fig. 1A), and oleuropein (OE), a complex phenol, are the most representative catecholic components of olives and their derivatives, including extra virgin olive oil and olive mill waste water (1,2). Hydroxytyrosol is also a constituent of the OE moiety, which is normally found in olive oil in its aglyconic form (OEa), and represents the bioactive portion of the OE molecule (3). In vitro, both compounds exhibit potent antioxidant and enzyme-modulating activities (4,5). Recently, several in vivo studies proved that olive oil phenolics are dose-dependently absorbed after oral administration to rats and humans, in which they are metabolized and excreted in the urine mostly as glucuronide conjugates (6–13). Published data show that HT retains its antioxidant activities following ingestion (7,13–16).



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  		FIGURE 1 Human urinary excretion of hydroxytyrosol (HT) depends on the vehicle of administration. (A) Chemical structures of HT (left) and homovanillyl alcohol (HValc) (right). (B) Percent excretion of HT and HValc when HT was provided to men either as a natural component of extra virgin olive oil (EVOO), after enrichment of a refined, phenol-free olive oil (OO), or when added to low-fat yogurt, providing 3, 7 or 20 mg of total HT, respectively. (C) 24-h urinary excretion of HT and HValc. Values are means ± SD, n = 6. *Different from EVOO, P < 0.05.

 
Matters of practicality determine the use of rats rather than humans as the model of choice for in vivo studies. Interspecies variability, however, renders comparisons between the model species (animals, humans) complex and sometimes questionable. Moreover, increasing industrial interest in the formulation of functional foods and/or nutraceuticals (17) requires that different means of olive phenolics administration should be compared. In the current investigation, we sought to compare the metabolism and excretion of HT between rats and humans.

In the second part of the study, we assessed the metabolic fate of HT when administered either as a natural component of virgin olive oil, or when added to olive oil or yogurt, another matrix of potential commercial interest.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Chemicals were purchased from Sigma (Milan, Italy), except for HT and deuterated HT, which were synthesized according to Bai et al. (12). Briefly, (3,4-dihydroxyphenyl)acetic acid was reduced with lithium aluminum hydrure or lithium aluminum deuteride. After extraction and purification, the identity of the synthesized compounds was verified by both NMR and MS. Hydroxytyrosol was quantified by GC-MS (6,18).

The extra virgin olive oil used in these studies was carefully selected by analyzing several southern Italian oils. In the second part of this study, an olive oil sample was prepared by adding an olive oil phenolic extract to a phenol-free oil (6).

Characterization of the phenolic fraction of the oils used in the study was carried out by GC- and liquid chromatography–MS, as previously described (18,19), in order to quantify HT, OEa and homovanillyl alcohol (HValc). The administered extra virgin olive oil contained 200 mg OEa/L (providing 81 mg free HT/L) and 19 mg free HT/L. HValc concentrations, if any, were below the detection limit (i.e., <=1 µg/L).

Low-fat (i.e., 0.1% fat) yogurt used in the experiments was HT free, as determined by MS analysis. Hydroxytyrosol was added to yogurt immediately before its administration to volunteers.

These studies conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (20) and with the principles outlined in the Declaration of Helsinki, and were approved by the local ethics committee.

Rat study: excretion of hydroxytyrosol when given as a component of olive oil.

Six male Wistar rats (250–275 g) were purchased from Charles River (Calco, Italy) and were housed in the local animal care facility, with free access to food (4RF21; good laboratory practice certified; Mucedola; Settimo Milanese, Italy) and tap water. Rats were placed in metabolic cages for 24-h collection of basal urine.

The day of the experiment rats were given by oral gavage 500 µL of extra virgin olive oil containing 100 µg of OEa (which provides 40.8 µg of HT as a component of OEa) and 9.5 µg of free HT (total HT, 50.3 µg), as assessed by GC-MS. We chose this medium of administration of HT (i.e., as naturally present in olive oil), rather than giving the pure compound dissolved in vehicles such as water or ethanol to better approximate the habitual dietary ingestion of HT. After gavage, rats were placed in metabolic cages for 24-h collection of urine, which was extracted for the quantitation of HT and metabolites as previously described (7,19).

Human studies.

Six healthy, normolipidemic men (age, 24–28 y; BMI, 22.36–24.42 g/m2) were recruited from within our department and submitted informed consent for the study. Subjects were asked to abstain from olive oil consumption for 5 d prior to the beginning of the experiment and until its completion. Basal urine was collected over a period of 24 h in an appropriate container with BHT (40 µmol/L, final concentration) added as an antioxidant. After measurement of the total urinary volume, aliquots were stored at -80°C.

Excretion of hydroxytyrosol when administered as a natural component of or when added to olive oil.

Fasting subjects were given 30 mL of the same oil that was administered to rats. This provided 3.2 mg of HT (2.4 mg as component of OEa and 0.6 mg as free HT). They also consumed 50 g of bread with no added fat. After 2 wk, the same men were given 30 mL of refined (phenol-free) olive oil that was enriched with a phenolic extract, providing 2 mg of free and 7 mg of total HT. In both experiments, urine was collected over a period of 24 h after administration of the samples and was processed as above.

Quantitation of HT and its metabolites was performed as described previously (6,19).

Hydroxytyrosol excretion when given in yogurt.

The day of the experiment, the same fasting subjects were administered 20 mg of synthetic HT added to 125 g of low-fat yogurt. Urine (24 h) was then collected, divided into aliquots and extracted as described previously (6,19).

Statistical analysis.

When appropriate, Student’s t test for paired data was used to test for statistical differences. Differences in excretion after HT intake in different vehicles were analyzed by the Friedman test for related observations. Differences with P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Human-rat comparison study.

Administration of HT to rats tended to increase (16.5%; P = 0.089) its urinary excretion (Table 1). Total HT excretion, which included HValc (Fig. 1A), also tended to increase (16.5%; P = 0.091) (Table 1). These figures correspond to 7.6% of total HT administered and 38% of free HT administered, respectively, which is consistent with previous reports (7,21). The high basal excretion of HT in rats confirmed unpublished data from our laboratories and might be derived from active endogenous metabolism of catecholamines, of which HT is one of the end-point metabolites (22). Because of the high basal excretion of HT and its metabolites, we chose to administer rats a fourfold amount (based on body weight) of HT, compared with that of humans, to more accurately register any eventual increase in urinary excretion following HT administration.


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  		TABLE 1 Absolute and relative excretions of hydroxytyrosol (HT) and homovanillyl alcohol (HValc) in urine of rats and humans, before and after administration of HT as a natural component of extra virgin olive oil1, 2

 
Administration of HT to humans increased (P = 0.006) the 24-h urinary excretion of HT + HValc (Table 1). The HT + HValc recovered in the urine was 44% of the total administered HT, consistent with previous data (6), and 234% of free HT administered (Fig. 1B).

When excretion in rats and humans was normalized to body weight, basal HT excretion in rats was ~23-fold that recorded in humans (Table 1). Furthermore, basal HValc excretion was ~28-fold higher in rats than in humans. In humans, but not in rats, ingestion of HT in the doses we chose activated the catechol-O-methyl-transferase (COMT) system, which is responsible for the metabolic conversion of HT to HValc (19,23).

Comparisons among vehicles.

Administration of extra virgin olive oil to humans was associated with a 44.2% urinary recovery of the administered free HT (Fig. 1B). Conversely, when HT was administered after its addition to refined olive oil, the urinary recovery of HT and HValc dropped to 23% of free and to 6.7% of total HT administered. The administration of 20 mg of HT added to yogurt was associated with a 5.8% excretion of the total administered HT, i.e., ~13% of the excretion recorded after extra virgin olive oil intake. Similar profiles of excretion were observed when data were calculated as absolute amounts (µmol/L excreted in 24 h) (Fig. 1C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The first part of this investigation concerned the comparative administration of HT, as a natural component of extra virgin olive oil, to rats and humans. The results suggested that caution should be used in the interpretation of data obtained in rats as the in vivo model of absorption and excretion of HT and related compounds. In fact, rats had a high basal excretion of HT and of its main metabolites (Table 1), and when given extra virgin olive oil, they appeared to absorb and/or excrete HT less than do humans (Table 1). Differences might be due to the absence of a gall bladder in rats, which results in the presentation of lipid-soluble or amphiphilic molecules such as HT to the intestinal flora. Similar conclusions were recently reached by Natsume et al. (24), who reported different excretions of (-)epicatechin metabolites in rats and humans.

To our knowledge, the contribution of OE to HT excretion has never been investigated. The high (234% of free HT) excretion we recorded in humans indicated that hydrolysis of the OE molecule did occur in vivo and that the subsequent release of HT contributed to the total amount found in urine. This hypothesis is corroborated by the observation that ratios of percent excretions referred to as the administered free and total HT were similar in humans (234/44 = 5.3) and rats (38/7.6 = 5.0).

Interestingly, the HValc/HT ratio was not significantly changed in rats and humans by the administration of HT, suggesting that the COMT system was not saturated under these conditions. Also, exogenous HT might be diluted with the endogenous pool after administration and, in turn, not increase excretion, because it does not easily reach cellular metabolic compartments.

The second part of our study concerned the metabolic fate of HT when administered to humans in extra virgin olive oil, enriched olive oil or yogurt as vehicles of potential commercial interest. An important finding was that addition of HT to low-fat yogurt, which approximates formulation of a consumer-appealing functional food, was associated with a lower (i.e., 5.8% of dose) excretion of this catechol, similar to that observed when HT was exogenously added to refined olive oil (Fig. 1B). Lower excretion occurred even though the administered dose (20 mg) was much higher than that administered through the other vehicles. These data suggest different bioavailabilities of both HT and OEa when administered as a natural component of a food such as olive oil, or when added to a refined (phenol-free) olive oil. Interpretations include binding of HT to yogurt proteins, which might render HT less bioavailable. This hypothesis, however, is in contrast with the findings that catechins are less bioavailable when provided with wine than through other solid matrices such as apples or onions (25,26). Possibly, catechins and other amphiphilic phenols are ordinarily dissolved in the aqueous compartments of food in which they are naturally present (similar to HT in olive oil). Conversely, addition of synthetic HT to yogurt might be rapidly followed by its binding to proteins. Support to the hypothesis of better absorption of oligonutrients through food is also given by the observed high absorption of vitamin E after its dispersion in milk, as opposed to administration in capsules (27). Finally, the use of a low fat product, which may be necessary to formulate a consumer-appealing functional food, might reduce bioavailability simply because of the low fat content of the matrix.

While future investigations will concern the fate of the proportion of HT that was not recovered in the urine (which might be nonabsorbed and/or stored in tissues and circulating cells), these and other results suggest that the formulation of functional foods or nutraceutical preparations should approximate as closely as possible the natural environment in which active molecules are found.


   ACKNOWLEDGMENTS
 
Giorgio Bergamini helped with olive oil selection. The oils were gifts from Oleificio Cooperativo (Sannicandro, Italy) and Olearia Italiana (M. Renna) (Monopoli, Italy).


   FOOTNOTES
 
1 Supported in part by European Union Project FAIR CT 97 3039 and Carapelli s.p.a. Back

3 Abbreviations used: COMT, catechol-O-methyl-transferase; HT, hydroxytyrosol; HValc, homovanillyl alcohol; OE, oleuropein; OEa, OE aglycone. Back

Manuscript received 4 March 2003. Initial review completed 1 April 2003. Revision accepted 5 May 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Visioli, F. & Galli, C. (1998) The effect of minor constituents of olive oil on cardiovascular disease: new findings. Nutr. Rev. 56:142-147.[Medline]

2. Blekas, G., Vassilakis, C., Harizanis, C., Tsimidou, M. & Boskou, D. G. (2002) Biophenols in table olives. J. Agric. Food Chem. 50:3688-3692.[Medline]

3. Boskou, D. (2000) Olive oil. World Rev. Nutr. Diet. 87:56-77.[Medline]

4. Visioli, F. & Galli, C. (2002) Biological properties of olive oil phytochemicals. Crit. Rev. Food Sci. Nutr. 42:209-221.[Medline]

5. Stark, A. H. & Madar, Z. (2002) Olive oil as a functional food: epidemiology and nutritional approaches. Nutr. Rev. 60:170-176.[Medline]

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

7. Visioli, F., Caruso, D., Plasmati, E., Patelli, R., Mulinacci, N., Romani, A., Galli, G. & Galli, C. (2001) Hydroxytyrosol, as a component of olive mill waste water, is dose-dependently absorbed and increases the antioxidant capacity of rat plasma. Free Radical Res. 34:301-305.[Medline]

8. Vissers, M. N., Zock, P. L., Roodenburg, A. J., Leenen, R. & Katan, M. B. (2002) Olive oil phenols are absorbed in humans. J. Nutr. 132:409-417.[Abstract/Free Full Text]

9. Miro-Casas, E., Farre, A. M., Covas, M. I., Rodriguez, J. O., Menoyo, C. E., Lamuela, R. R. & de la Torre, R. (2001) Capillary gas chromatography-mass spectrometry quantitative determination of hydroxytyrosol and tyrosol in human urine after olive oil intake. Anal. Biochem. 294:63-72.[Medline]

10. Miro-Casas, E., Covas, M. I., Fito, M., Farre-Albadalejo, M., Marrugat, J. & De La Torre, R. (2003) Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur. J. Clin. Nutr. 57:186-190.[Medline]

11. D’Angelo, S., Manna, C., Migliardi, V., Mazzoni, O., Morrica, P., Capasso, G., Pontoni, G., Galletti, P. & Zappia, V. (2001) Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil. Drug Metab. Dispos. 29:1492-1498.[Abstract/Free Full Text]

12. Bai, C., Yan, X., Takenaka, M., Sekiya, K. & Nagata, T. (1998) Determination of synthetic hydroxytyrosol in rat plasma by GC-MS. J. Agric. Food Chem. 46:3998-4001.

13. Bonanome, A., Pagnan, A., Caruso, D., Toia, A., Xamin, A., Fedeli, E., Berra, B., Zamburlini, A., Ursini, F. & Galli, G. (2000) Evidence of postprandial absorption of olive oil phenols in humans. Nutr. Metab. Cardiovasc. Dis. 10:111-120.[Medline]

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

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

16. Wiseman, S. A., Tijburg, L. B. & van de Put, F. H. (2002) Olive oil phenolics protect LDL and spare vitamin E in the hamster. Lipids 37:1053-1057.[Medline]

17. Katan, M. B. & de Roos, N. M. (2003) Public health. Toward evidence-based health claims for foods. Science 299:206-207.[Abstract/Free Full Text]

18. Caruso, D., Colombo, R., Patelli, R., Giavarini, F. & Galli, G. (2000) Rapid evaluation of phenolic component profile and analysis of oleuropein aglycon in olive oil by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). J. Agric. Food Chem. 48:1182-1185.[Medline]

19. Caruso, D., Visioli, F., Patelli, R., Galli, C. & Galli, G. (2001) Urinary excretion of olive oil phenols and their metabolites in humans. Metabolism 50:1426-1428.[Medline]

20. National Institutes of Health (1996) NIH Guide for the Care and Use of Laboratory Animals. NIH Publication No. 85-23. 1996 National Institutes of Health Bethesda, MD.

21. Tuck, K. L., Freeman, M. P., Hayball, P. J., Stretch, G. L. & Stupans, I. (2001) The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats. J. Nutr. 131:1993-1996.[Abstract/Free Full Text]

22. Lamensdorf, I., Eisenhofer, G., Harvey-White, J., Hayakawa, Y., Kirk, K. & Kopin, I. J. (2000) Metabolic stress in PC12 cells induces the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde. J. Neurosci. Res. 60:552-558.[Medline]

23. Manna, C., Galletti, P., Maisto, G., Cucciolla, V., D’Angelo, S. & Zappia, V. (2000) Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 470:341-344.[Medline]

24. Natsume, M., Osakabe, N., Oyama, M., Sasaki, M., Baba, S., Nakamura, Y., Osawa, T. & Terao, J. (2003) Structures of (-)-epicatechin glucuronide identified from plasma and urine after oral ingestion of (-)-epicatechin: differences between human and rat. Free Radical Biol. Med. 34:840-849.[Medline]

25. Hollman, P. C. & Katan, M. B. (1999) Health effects and bioavailability of dietary flavonols. Free. Radical Res. 31(suppl.):S75-S80.

26. de Vries, J. H., Hollman, P. C., van Amersfoort, I., Olthof, M. R. & Katan, M. B. (2001) Red wine is a poor source of bioavailable flavonols in men. J. Nutr. 131:745-748.[Abstract/Free Full Text]

27. Visioli, F., Rise, P., Plasmati, E., Pazzucconi, F., Sirtori, C. R. & Galli, C. (2000) Very low intakes of N-3 fatty acids incorporated into bovine milk reduce plasma triacylglycerol and increase HDL-cholesterol concentrations in healthy subjects. Pharmacol. Res. 41:571-576.[Medline]




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