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Vol. 29, Issue 11, 1492-1498, November 2001
Department of Biochemistry (S.D'A., C.M., V.M., G.P., P.G., V.Z.) and Chair of Nephrology (G.C.), Medical School, Second University of Naples, Naples, Italy; and Department of Pharmaceutical and Toxicological Chemistry (O.M., P.M.), University of Naples Federico II, Naples, Italy
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
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3,4-Dihydroxyphenylethanol (DOPET) is the major o-diphenol detectable in extra virgin olive oil, either in free or esterified form. Despite its relevant biological effects, mainly related to its antioxidant properties, little data have been reported so far on its toxicity and metabolism. The aim of the present work is to evaluate DOPET toxicity and to investigate its molecular pharmacokinetics by using the 14C-labeled diphenol. When orally administered to rats, the molecule does not show appreciable toxicity up to 2 g/kg b.wt. To identify and quantify its metabolites, [14C]DOPET has been synthesized and intravenously injected in rats. The pharmacokinetic analysis indicates a fast and extensive uptake of the molecule by the organs and tissues investigated, with a preferential renal uptake. Moreover, 90% of the administered radioactivity is excreted in urine collected up to 5 h after injection, and about 5% is detectable in feces and gastrointestinal content. The characterization of the labeled metabolites, extracted from the organs and urine, has been performed by high-pressure liquid chromatography analysis. In all the investigated tissues, DOPET is enzymatically converted in four oxidized and/or methylated derivatives. Moreover, a significant fraction of total radioactivity is associated with the sulfo-conjugated forms, which also represent the major urinary excretion products. On the basis of the reported results, an intracellular metabolic pathway of exogenously administered DOPET, implying the involvement of catechol-O-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, and phenolsulfotransferase, has been proposed.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Several
epidemiological studies suggest that olive oil significantly
contributes to the well known effects of the Mediterranean diet in
lowering the incidence of degenerative pathologies, including coronary
heart disease and cancer (Keys, 1970
; Martin-Moreno et al., 1994;
Trichopoulou et al., 1995; Willett et al., 1995; Lipworth et al., 1997;
Willett, 1997; Braga et al., 1998). In this respect, converging
evidence indicates that the protective effects of olive oil could be
ascribed not only to its high oleic acid content but also to the
antioxidant properties of its polyphenols, absent in seed oil (Bravo,
1998; Visioli and Galli, 1998). Their concentration differs greatly
among olive oils, depending on various conditions, such as the
cultivar, the soil composition, the climate, the degree of ripeness of
the olives, and the extraction procedures (Montedoro and Servili,
1992a). In this respect, virgin olive oils can be classified on the
basis of their polyphenol content, ranging from 100 mg up to 1000 mg/kg
(Montedoro et al., 1992b,c).
The 3,4-dihydroxyphenylethanol (hydroxytyrosol;
DOPET1) is the major component of olive oil
phenolic fraction, either as simple phenol or esterified with elenolic
acid to form oleuropein aglycone (Capasso et al., 1996). This
hydrosoluble and liposoluble molecule is an efficient scavenger of
peroxyl radicals (Aeschbach et al., 1994; Visioli et al., 1998), and it
greatly contributes to determine the shelf life of the oil, preventing
its auto-oxidation (Papadopoulos and Boskou, 1991; Baldioli et al.,
1996). The biological activities of DOPET have been explored by several
groups, as recently reviewed by Manna et al. (1999a). DOPET in vitro
prevents LDL oxidation (Grignaffini et al., 1994; Salami et al., 1995),
platelet aggregation (Petroni et al., 1995), and inhibits 5- and
12-lipoxygenases (Kohyama et al., 1997; de la Puerta et al., 1999). It
also exerts an inhibitory effect on peroxynitrite dependent DNA base
modifications and tyrosine nitration (Deiana et al., 1999). Experiments
from our laboratory demonstrated that DOPET effectively counteracts the
cytotoxic effects of reactive oxygen species in various human
cellular systems (Manna et al., 1997, 1999b). Preincubation of
intestinal Caco-2 cells with DOPET prevents the damages of oxidative
stress, such as lipid peroxidation and alterations of cell permeability
and viability (Manna et al., 1997). Similarly, DOPET exerts a
protective effect against the
H202 induced oxidative
hemolysis and malondialdehyde formation in the red blood cells (Manna
et al., 1999b). Moreover, we have recently demonstrated that the
diphenol exerts an antiproliferative effect, inducing apoptosis in
HL-60 cells and in resting and activated peripheral blood lymphocytes
(Della Ragione et al., 2000). Finally, it has been recently shown that
the molecule permeates cell membranes of human intestinal cells via a
passive diffusion mechanism (Manna et al., 2000).
Despite the variety of interesting DOPET biological effects, little
data have been reported on its bioavailability and metabolism. Preliminary reports indicate that orally administered DOPET can be
absorbed both in rats (Bai et al., 1998) and in humans (Visioli et al.,
2000), whereas no information is available on its toxicity, organ
distribution, and metabolism. The studies reported in this article
represent the first systematic analysis of these aspects: the synthesis
of the labeled DOPET allowed us to carry out a molecular pharmacokinetic analysis and to elucidate its metabolic pathways.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Chemicals. [14C]DOPET (2.25 mCi/mmol) was custom synthesized by PerkinElmer Life Sciences (Cologno Monzese, Italy). The radiochemical purity of [14C]DOPET was 98.9%, according to thin layer chromatography on Silica Gel GF using benzene/dioxane/acetic acid (90:25:4) as eluent.
Ready organic, Ready gel, and BTS-450 (a quaternary ammonium hydroxide in toluene) were obtained from Beckman Coulter, Inc. (Fullerton, CA). Type L-II
-glucuronidase, type VI sulfatase, homovanillic
alcohol (3-hydroxy-4-methoxyphenylethanol; MOPET), homovanillic acid
(HVA, 4-hydroxy-3-methoxyphenylacetic acid; HVA),
3,4-dihydroxyphenylacetic acid (DOPAC), and horse alcohol dehydrogenase
were purchased from Sigma Chemical Co. (St. Louis, MO).
Syntehtic Procedures.
Preparation and purification of
DOPET
The chemical synthesis of DOPET was performed according to Baraldi et
al. (1983), with some modifications. A solution of
3,4-dihydroxyphenylacetic acid (3.5 g, 0.018 mol) in anhydrous
tetrahydrofuran (50 ml) was added dropwise to an ice-cooled stirred
suspension of LiAlH4 (2 g, 0.054 mol) and Pt on
activated carbon in anhydrous tetrahydrofuran (150 ml). The resulting
mixture was heated 6 h at reflux temperature, cooled, and the
excess of hydride was destroyed by addition of 150 ml of 1 N HCl. To
the final mixture, 100 ml of ethyl acetate were added. The organic
layer was separated, and the aqueous phase was extracted with ethyl
acetate (3 × 100 ml). The combined organic phases were dried with
sodium sulfate and evaporated "in vacuum", and the residue was
purified on a silica gel column (petroleum ether/ethyl acetate, 1:1
v/v) to give a colorless oil (2.4 g). The yield was about 70%. DOPET
was identified by 1H and
13C NMR. 1H NMR in
CD3OD: 6.75 (1H, d; J = 8.1 Hz),
6.73 (1H, d; J = 2.0 Hz), 6.59 (1H, dd; J + 8.1, J = 2.0 Hz), 3.75 (2H, t; J = 7.5 Hz). 13C NMR: 146.0 (s), 144.4 (s), 131.7 (s),
121.2 (d), 117.0 (d), 116.2 (s), 64.5 (2C, t), 39.5 (2C, t). Infrared
(film); 3400, 1600 cm
1. The infrared spectra
were taken on a PerkinElmer 1760-X IFT spectrophotometer in film
(PerkinElmer Optoelectronics, Santa Clara, CA). The
1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a Fourier-transform Bruker spectrometer AMX 500 equipped with a Bruker X-32 computer (Bruker, Newark, DE), using the UXNMR software package. To prevent oxidation, the compound was routinely stored under vacuum.
DOPAL synthesis.
3,4-Dihydroxyphenylacetaldehyde (DOPAL) was obtained by enzymatic
synthesis as follows: DOPET (100 µM) was incubated at 25°C in 0.1 M
sodium phosphate buffer, pH 7.4, in the presence of 2.5 mM
NAD+ and alcohol dehydrogenase (0.2 U/ml) (Mardh
and Vallee, 1986). Absorbance at 340 nm was then monitored
spectrophotometrically. Aldehyde formation was followed by HPLC
analysis as indicated below. To prevent oxidation, the compound was
routinely stored under vacuum.
Animal Studies.
Animals Young adult Sprague-Dawley rats were routinely used for the in vivo experiments. The animal treatments were carried out by RBM-Laboratories & Clinics Group (Colleretto Giacosa, Italy).
Toxicity test. Six male and six female rats, about 3-months old and weighing 210 to 262 g, were used for the experiment. They were acclimatized at least 5 days before starting the test and fasted about 16 h before the experiment. A single dose of 2 g/kg b.wt. DOPET was administered by gavage. Three hours after treatment diet was available "ad libitum". During the study period, rats were housed under controlled environmental conditions. Animals were maintained and handled according to the Directive 86/609/EEC, enforced by the Italian D.L. No. 116 January 1992. The rats were observed and weighed daily, after administration of DOPET, until day 14. At the end of the test, rats were sacrificed, and gross pathological changes in main organs were evaluated. Toxicity was determined from the death/survival ratio of treated animals.
Pharmacokinetics of [14C]DOPET.
Twelve young adult male rats, weighing 204.5 ± 6.4 g, were
used for this investigation. During the study period, the rats were
housed under controlled environmental conditions. A single intravenous
dose of [14C]DOPET (1.5 mg/kg-87.5 µCi/kg)
was administered to animals, which had been fasted about 16 h
before treatment. To prevent DOPET oxidation, the radioactive molecule
was kept lyophilized under vacuum, and the solution was prepared just
before the injection. Immediately after administration, the animals
were placed in individual plastic metabolic cages. At the selected
time-intervals (5, 10, 30, 60, 120, and 300 min after intravenous
administration), two rats were anesthetized with diethyl ether and
killed by exsanguination from the abdominal aorta. Blood (5 ml) was
collected and frozen at 
80°C. The other whole blood (5 ml) was
centrifuged for 10 min at 5000g. The plasma and the cell
fraction were separated and then both preserved at 80°C. Brain,
heart, kidney, liver, lung, skeletal muscle, and gastrointestinal
content were promptly frozen at 80°C. This last sample was obtained
by dissecting stomach and intestine and scraping away the content with
a spatula. Feces and urine were collected from animals sacrificed after
300 min from [14C]DOPET administration.
Preparation of biological samples for radioactivity measurement.
Biological samples were prepared according to Galletti et al. (1985).
Tissues were homogenized in water 1:3 (w/v), and aliquots corresponding
to 100 mg of tissue were incubated with 1 ml of BTS-450 at 40°C in a
water bath, until complete solubilization (4-5 h). The samples were
decolorized with a few drops of 30% H2O2, and glacial acetic
acid (70 µl) was added to eliminate chemiluminescence (Galletti et
al., 1985). Ready organic (10 ml) was then added.
.
BTS-450/isopropanol (750 µl; 1:2 v/v) was added to 250 µl of
sample. After 1 h of incubation at 40°C, 30% hydrogen peroxide (500 µl) was added dropwise. Following 15 min at room temperature, samples were incubated 30 min at 40°C. Ready gel (10 ml), containing 7 ml/liter glacial acetic acid, was added. For urine analysis, Ready gel (10 ml) was directly added to 10 µl of sample.
Radioactivity was measured in a Beckman LS 7800 -counter equipped
with an automatic quench correction system. The standardization was
performed by using samples containing
[14C]DOPET (1 × 105
dpm) and increasing aliquots of solubilized liver tissues as a quencher.
HPLC Analysis. Thirty percent TCA was added (1:1 v/v) to aliquots of tissue samples, prepared as above reported. After centrifugation at 13,000g for 15 min, the pellet was washed once with 10% TCA, and the resulting supernatants, accounting for 100% of total tissue radioactivity, were combined and used for HPLC analysis. HPLC separation of DOPET and its metabolites was performed by reversed-phase chromatography on 150- × 4.6-mm C18 5-µm column (Kromasil), using a Beckman Apparatus (Gold-126) equipped with an UV detector fixed at 278 nm. The column was eluted at a flow rate of 1.0 ml/min with acetic acid 0.2%, pH 3.1, (A)/methanol (B) as the mobile phase; the gradient was changed as follow: 95% A/5% B for 2 min, 75% A/25% B in 22 min, 0% A/100% B in 5 min, 0% A/100% B for 5 min, and 95% A/5% B in 5 min. Labeled species were identified on the basis of the retention times of authentic standard references (DOPET, MOPET, HVA, DOPAC, and DOPAL).
Enzymatic Hydrolysis and Identification of Conjugated Forms.
To verify the presence of radioactivity associated with conjugated
forms of DOPET and/or its metabolites, plasma and urine samples were
treated with both -glucuronidase and sulfatase.
Glucuronides.
Plasma and urine aliquots were acidified to pH 5.0 with 0.1 volume of
0.58 mol/l acetic acid solution (Manach et al., 1999) and incubated at
37°C for different times in the presence of 8 × 105 U/l -glucuronidase from Patella
vulgata in which the contaminating sulfatase activity is inhibited
by 0.1 M phosphate.
Sulfates.
Plasma and urine aliquots were incubated at 37°C for different times,
in 5 mM Tris-HCl, pH 7.4, containing 1 × 105 U/l sulfatase from Aerobacter
aerogenes, devoid of any detectable -glucuronidase activity. At
the end of the incubation, the samples were treated with TCA and
analyzed by HPLC, as reported above.
In Vitro Experiments with Human Blood. Whole blood and plasma from rats and humans were used to analyze in vitro metabolism of [14C]DOPET. Samples (200 µl) were incubated at 37°C with 50,000 dpm of [14C]DOPET. After 30 min, 30% TCA was added (1:1 v/v) to the samples. After centrifugation at 13,000g for 15 min, the pellet was washed once with 10% TCA, and the resulting supernatants, accounting for 100% of total tissue radioactivity, were combined and used for HPLC analysis.
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Results and Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Toxicity Test.
A preliminary study on in vitro toxicity was performed using cultured
intestinal Caco-2 cells. These cells undergo spontaneous differentiation to form a polarized monolayer closely resembling, both
morphologically and functionally, the human small intestinal epithelium
(Hidalgo et al., 1989). No decrease in cell viability, measured by the
neutral red assay (Manna et al., 1997), has been observed up to 900 µM DOPET (data not reported).
Radioactivity Tissue Distribution.
The pharmacokinetic analysis was carried out in rats intravenously
injected with [14C]DOPET (1.5 mg/kg b.wt.,
38 × 106 dpm). As shown in Fig.
1, less than 8% of the administered
radioactivity is still present in the blood stream 5 min after
injection (6% associated with plasma and 1.9% with cell fraction).
The time course analysis shows a further gradual decrease of blood
radioactivity; only 0.1% of the administered dose is detectable 5 h after the treatment (Fig. 1). Furthermore, DOPET is rapidly
metabolized both by blood cells and by different rat tissues, and its
radioactive metabolites are already detectable in the blood stream 5 min after the injection, as it will be discussed under DOPET
Metabolism. The rapid decrease of radioactivity in blood stream
prevented an accurate evaluation of the blood half-life of DOPET, which is probably within 1 
2-min range. These data further support our previous finding on the absence of biological barriers hampering DOPET distribution within the cell (Manna et al., 2000). It should be
noted, however, that its absorption and tissue distribution could be
different when the natural occurring esterified precursors of DOPET,
oleuropein and its aglycone, are administered. As a matter of fact,
Coni et al. (2000) report a significant protective effect of
oleuropein-rich diets on LDL oxidation in rabbit.
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Renal and Intestinal Excretion. Ninety percent of the administered radioactivity is detected in urine collected up to 5 h from the injection, indicating that renal excretion represents the preferential route for the disposition of DOPET and/or its metabolites. Moreover, 9% of administered radioactivity is detectable in the gastrointestinal content 5 min after injection (Fig. 3). This value remains almost constant during the other time intervals, decreasing to 2.5% at 5 h. Finally, 3.2% of the injected dose is recovered into the feces 300 min after the administration.
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demonstrated that [14C]DOPET transport through a monolayer of
differentiated intestinal cells occurs via a passive diffusion
mechanism and that it is bi-directional.
DOPET Metabolism.
Little data are available in the literature on pharmacokinetics and
metabolism of exogenously administered DOPET or oleuropein. A
preliminary study from our laboratory, using labeled DOPET in cultured
intestinal cells, indicated that the only labeled metabolite detectable
in the culture medium is MOPET, the methylation being supposedly
catalyzed by catechol-O-methyltransferase (COMT) (Manna et
al., 2000).
), although a
transport of MOPET from other tissues cannot be ruled out. Moreover,
the occurrence, in all analyzed organs, of both labeled DOPAL and DOPAC
implies a sequential oxidation of DOPET ethanol side chain catalyzed by
alcohol and aldehyde dehydrogenase, respectively. Labeled HVA, the
product of both methylation and oxidation, has also been identified.
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-glucuronidase from P. vulgata (containing sulfatase activity inhibited by 0.1 M
phosphate) or with sulfatase from A. aerogenes
(devoid of any detectable -glucuronidase activity).
The HPLC analysis of sulfatase-treated plasma samples shows the
quantitative disappearance of radioactivity associated with peak 1 and
the parallel increase of DOPET and its metabolites, mainly the
methylated derivative MOPET. Conversely, the treatment with
-glucuronidase does not result in any appreciable modification of
HPLC profile. These data allow to identify sulfo-conjugated derivatives
of DOPET and its metabolites as the only components of peak 1 and
confirm the well established role of sulfation in the metabolism of
catechols. These conjugated metabolites represent the major labeled
molecular species excreted in urine (Fig.
5). It is worth noting that Visioli et
al. (2000) reported that in humans, after a dietary intake of olive
oil, this phenol is dose dependently absorbed and excreted in urine,
presumably as glucuronide conjugates. The absence of these derivatives
in rat samples, after intravenous administration of DOPET, is in
agreement with literature data stating that glucuronidation of orally
administered epicatechin in rat occurs at the level of intestinal
mucosa, whereas its sulfation mainly occurs in liver (Piskula and
Terao, 1998).
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). Superimposable results were obtained with total human
blood, suggesting that a similar metabolic pathway is probably
operative in humans.
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). Moreover, Goldstein and Gerber (1963)
demonstrated the conversion of [14C]DOPAC to
[14C]MOPET in rat brain. More recently,
Lamensdorf et al. (2000a,b) reported an increased level of DOPAL and
DOPET in catecholaminergic PC12 cells incubated in the presence of
inhibitors of mitochondrial respiratory chain. The rapid metabolic
conversion of DOPET raises the question whether its metabolites are
still endowed with the variety of biological effects exerted by the
molecule, which are presumably related to its antioxidant activity.
Although no data are available on the biological activity of the
methoxy derivatives, previous studies in different in vitro model
systems (Kohyama et al., 1997; Manna et al., 1997) suggest that the
antioxidant activity of DOPET is strictly related to the integrity of
orthodiphenolic moiety. Methylation of the phenolic hydroxyl group
should result in the loss of antioxidant activity. Moreover, we have
recently demonstrated (Della Ragione et al., 2000) that DOPET induces
apoptosis in white blood cells, as a consequence of a rapid cytochrome
c release from intermembrane mitochondrial space. The
presence of the orthodiphenol group is again a structural requirement
for biological activity. In fact, tyrosol, the structural analog which presents only one hydroxyl group on the phenyl ring, did not induce the
programmed cell death (Della Ragione et al., 2000). On the other hand,
it is still questionable whether the molecular basis of all the
biological effects of DOPET (and its metabolites) is strictly related
to its antioxidant properties. In some instances, there has been
reported a low pro-oxidant activity of the molecule (Aeschbach et al.,
1994).
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; Trichopoulou et al., 1995; Willett,
1997), colorectal (Braga et al., 1998), and prostate (Willett, 1997)
cancer. These observations, together with our recent data on the
ability of DOPET to arrest cell proliferation and induce apoptosis in
cultured human cells (Della Ragione et al., 2000), support the view
that cancer prevention exerted by olive oil could be ascribed to its
high content of DOPET and its precursor, oleuropein aglycone.
On the other hand, according to the "oxidation hypothesis" of
atherosclerosis (Witztum, 1994), it is possible to speculate that the
reported delayed progression of atheromatous plaque due to olive oil
intake (Parthasarathy et al., 1990) can be, in part, attributed to the
antioxidant properties of its phenolic component (Visioli and Galli,
2001). The inhibition of platelet aggregation (Petroni et al., 1995)
and prevention of radical-induced LDL oxidation (Grignaffini et al.,
1994; Salami et al., 1995) by DOPET and oleuropein may be critical in
this respect.
In conclusion, literature data on biological effects of DOPET, together
with our findings on the lack of toxicity and the fast uptake of the
molecule, make this natural occurring phenol a good candidate for novel
therapeutic strategies.
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Footnotes |
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Received March 5, 2001; accepted August 14, 2001.
This work was supported in part by a research grant from the International Olive Oil Council.
Prof. Vincenzo Zappia, Istituto di Biochimica delle Macromolecole, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Via Costantinopoli, 16, 80138, Napoli, Italy. E-mail: vincenzo.zappia{at}unina2.it
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Abbreviations |
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Abbreviations used are: DOPET, 3,4-dihydroxyphenylethanol; LDL, low-density lipoprotein; MOPET, 4-hydroxy-3-methoxyphenylethanol (homovanillic alcohol); HVA, 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid); DOPAC, 3,4-dihydroxyphenylacetic acid; DOPAL, 3,4-dihydroxyphenylacetaldehyde; HPLC, high performance liquid chromatography; TCA, trichloroacetic acid; COMT, catechol-O-methyltransferase.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues.
J Nutr
128:
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