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Centre for Pharmaceutical Research, University of South Australia, North Terrace, Adelaide, South Australia, 5000, Australia
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • oleuropein • olive oil • absorption • Mediterranean diet • bioavailability • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 1995
) and certain cancers
(especially prostate and colon; Martin-Moreno et al. 1994
) is lower in the Mediterranean region. This has been
attributed to the Mediterranean diet, which is largely vegetarian in
nature and includes the consumption of large quantities of olive oil
(Keys 1995
). In addition to being the predominant source
of fat in the Mediterranean diet (Petroni et al. 1995
),
olive oil is a source of at least 30 phenolic compounds, many of which
have antioxidant properties (Kohyama et al. 1997
,
Manna et al. 1997
, Saija et al. 1998
,
Visioli et al. 1995
, Visioli and Galli 1994
, Wiseman et al. 1996
).
Many in vitro studies have been performed to elucidate mechanisms by
which polyphenolic compounds may act to confer positive health effects.
A number of these studies (Beecher et al. 1999
,
Manna et al. 1997
, Visioli et al. 1998
)
have shown that polyphenolic compounds possess strong radical
scavenging activity and appear to be at least, as if not more,
effective than other important dietary antioxidants, such as vitamin C
and 
-tocopherol. Because the uncontrolled production of free
radicals has been hypothesized as contributing to the pathogenesis of
diseases such as coronary heart disease and cancer (Visioli et al. 1998
), the ability of polyphenols to scavenge free radicals
could be important in explaining how polyphenols may play a role in
preventing these diseases. Studies have also demonstrated that
polyphenolic compounds are potent inhibitors of LDL oxidation in vitro
(De Whalley et al. 1990
, Visioli 1995
,
Visioli and Galli 1994
). Inhibition of LDL oxidation is
important because the in vivo oxidation of LDL is strongly linked to
the formation of atherosclerotic plaques, which in turn contribute to
the development of coronary heart disease. Polyphenols have also been
found to have activity in breaking peroxidative chain reactions and
preventing metal ion chelation, processes that have also been linked to
the pathogenesis of heart disease and cancer (Manna et al. 1997
). In addition to their antioxidant properties,
polyphenolic compounds have been shown to exhibit a range of indirect
actions that may be beneficial to health, including the inhibition of
enzymes involved in the inflammatory process (Kohyama et al. 1997
, Laughton et al. 1991
, Middleton and Kandeswami 1992
), the inhibition of platelet aggregation
(Petroni et al. 1995
) and inhibition of the metabolic
activation of procarcinogens (Stavric 1994
).
Surprisingly, despite the myriad of potential health benefits of the
olive oil polyphenolics, there are virtually no data relating to their
biological fate after oral ingestion. Bai et al. (1998)
studied the oral fate of hydroxytyrosol (an olive oil polyphenolic) by
measuring its concentration in plasma after oral dosing of rats and
found that this compound was rapidly but poorly absorbed after a single
oral dose. Another study (Visioli et al. 1999
)
investigated the absorption of two olive oil phenolic constituents,
tyrosol and hydroxytyrosol, after ingestion in humans and found that
they were absorbed in a dose-dependent fashion and excreted in the
urine as glucuronide conjugates. In consideration of this dirth of
information and the current topical nature of polyphenolic antioxidants
and their putative health benefits, we decided to investigate the
absorption of one of the major olive oil polyphenols, oleuropein
(Fig. 1
), using an isolated perfused rat intestine. This is an ideal tool for
investigation of the intestinal absorption of compounds because it
bypasses the influence of metabolism (hepatic and renal) and other
factors that usually make the estimation of absorption difficult
(Stretch 1999
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oleuropein was obtained from the Indofine Chemical Company, Inc. (Belle Mead, NJ). p-Coumaric acid was purchased from ICN BioMedicals Inc. (Aurora, OH). The radiolabeled (14C) marker polyethylene glycol (PEG)2 [average molecular weight (MW) 4000], which was used as an indicator of water flux across the intestine during in situ studies, was purchased from NEN Life Science Products (Boston, MA). Verapamil and mannitol were obtained from Sigma Chemical Co. (St. Louis, MO). All salts used in the preparation of perfusion media were of analytical grade and were purchased from BDH Chemicals (Poole, UK). All solvents used were HPLC grade and were obtained from BDH Laboratory Supplies (Poole, UK). Sulfuric acid was purchased from Ajax Chemicals (New South Wales, Australia). All water was freshly produced with a Milli-R/Q water purifier system from Millipore/Waters (New South Wales, Australia).
Blood-to-plasma concentration ratio of oleuropein.
The blood-to-plasma concentration ratio of oleuropein had to be
determined because an estimate of this variable was required for the
mathematical calculation of the intestinal permeability of oleuropein.
A method similar to that described by Milne et al. (1993)
, was used, in which preheated rat whole blood (37°C)
was spiked to a concentration of 5 µmol/L using an aqueous oleuropein
solution, and samples were mixed using a rotary mixer for 20 min and
then centrifuged for 10 min at 3000 x g. The
resulting plasma layer was removed, and three 500-µL aliquots were
transferred to 10-mL tubes. Samples were then extracted into ethyl
acetate (6 mL), centrifuged and evaporated under a nitrogen stream.
After reconstitution in 200 µL of Milli-Q water, samples were
analyzed with the HPLC method described later.
Octanol-water partition coefficient of oleuropein.
This variable was determined using a modification of the method
described by Henczi et al. (1995)
and was important in
predicting the extent to which oleuropein was likely to permeate the
intestine. After saturation of the aqueous phase with
n-octanol, oleuropein samples (25 µmol/L) were
prepared by spiking the saturated aqueous phase (1 mL) with oleuropein
solution. n-Octanol (1 mL) was added to each sample, and
tubes were agitated for 20 min. After centrifugation, the aqueous layer
was removed, and oleuropein concentrations were analyzed using HPLC.
Intestinal perfusion technique.
The isolated perfused intestine method that we used was a minor
adaptation of the validated method of Blanchard et al. (1990)
from the model described by Windmueller and Spaeth (1981)
. Studies using this technique were approved by
the Animal Ethics Committee of the Institute of Medical and Veterinary
Science, Adelaide, South Australia, in February 1999. The perfusion
medium used was an iso-osmotic medium called GoLYTELY (Davis et al. 1980
), which contained 20 mmol NaHCO3/L, 25
mmol NaCl/L, 10 mmol KCl/L, 40 mmol Na2SO4/L,
80 mmol mannitol/L and 1.25 mmol PEG 4000/L. This medium was chosen
because it is associated with no net movement of fluid or electrolytes
across the intestine, eliminating any effect of solvent flux on the
absorption of oleuropein (Davis et al. 1980
).
The surgical technique is illustrated in Figure 2
. Before the start of the experiment, the GoLYTELY solution was adjusted
to pH 7 with phosphoric acid. Then, 14C-PEG 4000
(250 µL of 0.37 GBq/L), a radiolabeled nonabsorbable marker used to
assess net fluid movement during the experiment, was added to 100 mL of
the GoLYTELY, which was placed in a water bath at 37°C. Collection of
donor blood from two rats (
12-wk-old, 500-g male Sprague-Dawley)
was then carried out, with 15–22 mL of blood typically being collected
from each donor animal. Surgery was carried out in accordance with the
method described by Blanchard et al. (1990)
to prepare
the animal for the perfusion. In contrast to Blanchard et al. (1990)
, who recirculated their analyte-containing
perfusate, flow of perfusate in this experiment was single pass
(out-flowing perfusate discarded), and an aqueous solution of
oleuropein was infused into the flow of blank perfusate just before it
entered the intestinal segment. This technique served to prevent
chemical degradation of the analyte in the perfusate.
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1% absorption from the small intestine. Samples were
then taken for 40 min. The sampling regimen consisted of collecting the
entire mesenteric outflow into preweighed 1.5-mL vials. Blood flow rate
was determined by weighing the tubes and using a blood density of 1.054
kg/L (Blanchard et al. 1990
), and the infusion rate of
donor blood was adjusted accordingly. Blood samples were pooled into
5-min intervals, and at the completion of each interval, the samples
were spun down in a microcentrifuge, and the resulting plasma was
removed and frozen in solid CO2. Both
in-flowing and out-flowing perfusate samples (120 µL) were
also taken every 5 min starting at 2.5 min, with 50 µL placed into a
scintillation vial to allow assessment of the radiolabeled
nonabsorbable marker, and an additional 50 µL placed into a 1.5-mL
vial and frozen in solid CO2 for subsequent
evaluation of oleuropein content. After completion of the perfusion,
the length of the perfused segment of intestine was measured using silk
suture to allow its surface area and volume to be determined. Pharmacokinetic calculations.
The following calculations were made under the assumption that no net
water movement occurred during the perfusion (Kim 1996
).
The apparent permeability coefficient, Papp,
was calculated using the model described by Lennernäs (1995)
:
 |
where dMa/dt is the rate of absorption,
AT is the total epithelial surface area of the
perfused segment of intestine and Cr is the
concentration of compound entering the intestine. The rate of
absorption was calculated as Qm ·
Cm · R, where Qm is the
blood flow rate over the collection period, Cm is
the concentration of compound in mesenteric blood during that period
and R is the blood-to-plasma concentration ratio of the compound.
AT was calculated using the equation for the area
of a cylinder (AT =
2
riLi), where
Li is the length of the perfused segment of
intestine and ri is the internal radius, 0.2 cm
(Kim 1996
).
Given a value for the Papp, a
theoretical oral intestinal bioavailability may be predicted
mathematically using the following equation (Lennernäs 1995
):
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where Fpred is the predicted intestinal
bioavailability, intestinal surface area is calculated using the
formula for a cylinder (2 · · r · l), mean intestinal transit
time is estimated to be 2.5 h (Varga 1976
) and
intestinal volume is calculated as the volume of a cylinder ( ·
r2 · l). The radius of the rat intestine is
estimated at 0.2 cm (Kim 1996
), and the length is
estimated at 103 cm (Hebel and Stromberg 1976
);
therefore, intestinal surface area was calculated at 129.4
cm2, and intestinal volume was calculated at
12.94 cm3.
Study design.
The control series consisted of five male Sprague-Dawley rats, and
it was designed to assess the intestinal absorption of oleuropein using
the iso-osmotic GoLYTELY perfusion medium in single pass through
the intestine. Another series (n = 4) examined the
effect of fluid flux on the absorption of oleuropein. This series of
experiments differed from the control series in that the perfusion
medium used was pure Milli-Q water and the oleuropein was dissolved
in the perfusate reservoir rather than being infused into the stream of
perfusate. A subsequent series of pilot experiments consisting of two
animals investigated the effect of a glucose-transporter inhibitor
on the absorption of oleuropein by incorporating phlorizin (2 mmol/L)
into the oleuropein infusion solution. Finally, the effect of verapamil
(a commonly used inhibitor of P-glycoprotein) on the absorption of
oleuropein was investigated in a series of three pilot experiments. Two
perfusate reservoirs were used: one containing blank GoLYTELY medium
and the other containing GoLYTELY medium with verapamil dissolved at a
concentration of 200 µmol/L (a concentration previously found to be
effective in inhibiting intestinal P-glycoprotein; Aungst and Saitoh 1996
). The first half of the experiment was performed
using the blank GoLYTELY perfusate to establish a control level of
oleuropein absorption; then, the intestine was perfused with the
verapamil-containing perfusate (using a multidirectional tap to
change the perfusate source). Absorption of oleuropein was compared
over the two periods to determine the effect of verapamil.
Analysis of oleuropein in plasma by HPLC.
The lack of in vivo work performed with the olive oil polyphenolics meant that there was no method established in the literature for their determination in biological matrices; therefore, a novel assay was developed. Before extraction into ethyl acetate, 80 µL of internal standard (1 mmol p-oumaric acid/L) was added to each 1-mL plasma sample. Ethyl acetate (6 mL) was added, and each sample was mixed using a rotary mixer. Next, samples were centrifuged, and the organic layer was separated and evaporated to dryness in a vortex evaporator, reconstituted in 200 µL of Milli-Q water and then injected onto the HPLC system.
The HPLC system consisted of an LC-10AS single-piston pump, an
SIL-10A autosampler and an SCL-10A system controller [Shimadzu
Scientific Instruments (Oceania) Pty. Ltd., Adelaide, Australia]. The
detector was a Jasco 821-FP Spectrofluorometer (Japan Spectroscopic
Co., Ltd., Hichioji City, Japan), and peaks were integrated using a
C-R6A Chromatopac Integrator (Shimadzu). HPLC conditions consisted of a
mobile phase of water/acetonitrile (80:20), adjusted to pH 2.9 with
H2SO4, and an Alltima C18 Rocket
column stationary phase [3 µm packing, 53 mm x 7 mm; Alltech
Associates (Aust) Pty. Ltd., New South Wales, Australia). Then, 150
µL of each sample was injected onto the column, with a flow rate of
1.2 mL/min. Oleuropein and p-coumaric acid were detected
by fluorescence, with excitation/emission wavelengths of 280/312 and
319/405 nm, respectively. The compounds eluted at 6.5 min
(p-coumaric acid) and 14 min (oleuropein). A standard
curve was also incorporated into each analytical run, with plasma
standards having nominal oleuropein concentrations of 0.5, 1, 2.5, 5,
10, 50 and 100 µmol/L, whereas aqueous standards had nominal
concentrations of 10, 20, 50, 100 and 200 µmol/L. Quality control
samples of nominal concentrations of 6 and 60 µmol/L were also
incorporated into the plasma standard curve. The assay was found to be
accurate and reproducible over this concentration range, with maximum
interday variability of 17% and 5.9% at the lower and higher ends of
the curve and intraday variability of 2.7% and 6.5% at the lower and
upper ends of the curve (using independent quality control samples of 2
and 80 µmol/L). The limit of quantification was 0.5 µmol/L.
Statistical analysis.
Data were analyzed by one-way ANOVA and by unpaired t tests, assuming equal variance. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean blood-to-plasma concentration ratio was 0.955 ± 0.119,
whereas the octanol-water partition coefficient (D) was 1.57
± 0.052, giving a logD value of 0.196 ± 0.014. Based on
preliminary in vitro stability studies, oleuropein degraded
significantly in GoLYTELY medium but was stable in Milli-Q water.
Logarithmic transformation of the data indicated that the degradation
followed apparent first-order kinetics, with a degradation rate
constant of -0.023 min-1
(r2 = 0.962) (Fig. 3
). Further investigation of the stability of oleuropein in various media
under a range of pH conditions suggested that the degradation was pH
dependent, with degradation occurring at pH 
7 but not at pH 5.2
(Fig. 4
). Assessment of the stability of oleuropein for 2 min followed by
freezing in solid CO2 found that there was no
significant degradation over this time period in GoLYTELY medium,
validating the use of snap freezing to preserve oleuropein
concentrations.
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Assessment of blood flow during each perfusion was important, because
poor or widely fluctuating blood flow could be a source of error using
our model, and could indicate problems with functionality of the
perfused intestinal segment. Both blood flow and absorption rate
(assessed as the absorption rate constant) remained fairly consistent
throughout the perfusions (Fig. 5
), indicating the maintained functionality of the perfusion system. The
concentrations of 14C-PEG 4000 and oleuropein in
both in- and out-flowing perfusate were also plotted together to
assess any trends that may have indicated net water flux during the
experiment. A typical plot is shown in Figure 6
and demonstrates no significant trends indicative of fluid movement
during the perfusions. A summary of results from the in situ studies is
shown in Table 1
.
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Based on control experiments, the Papp
of oleuropein was 1.47 ± 0.13 x 10-6
cm/s (±SE). There was no significant difference
(P 0.05) in permeability between the time points,
indicating that the absorption of oleuropein did not change with time.
The rate of absorption remained fairly constant throughout the control
series, with 0.19 ± 0.2% of the perfused dose being absorbed
during each time interval.
Absorption from a hypotonic lumen.
Absorption of oleuropein under hypotonic luminal conditions was 5.92
± 0.49 x 10- 6 cm/s (±
SE) (Table 1)
, which was significantly higher (P
< 0.001, unpaired t test assuming equal variance) than
that found using the iso-osmotic perfusate in the control series.
The absorption rate throughout the perfusions was again fairly
constant, with 0.49 ± 0.05% of the perfused dose being absorbed
during each time interval.
Effect of verapamil and phlorizin on oleuropein absorption.
The addition of verapamil or phlorizin to the perfusion solution had no significant effect on the intestinal absorption of oleuropein (unpaired t test assuming equal variance) in the in situ model used in our pilot experiments. Mean Papp values for oleuropein before and after the addition of verapamil to the perfusion solution were 2.22 ± 1.71 and 1.91 ± 1.30 x 10-6 cm/s (±SE), respectively, whereas the Papp for oleuropein after perfusion with the phlorizin-containing perfusate was 1.15 ± 0.38 x 10-6 cm/s (±SE).
Mathematical estimation of intestinal bioavailability.
Using the second equation, the intestinal bioavailability of oleuropein under conditions of no water flux across the intestine was calculated to be 0.124 (12.4%). However, this does not take into account the effect of metabolism on the amount of compound that reaches the systemic circulation. When data from the hypotonic perfusate experiments were incorporated into this equation, the intestinal bioavailability was predicted to be 41.3%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Results from this study showed that when the intestine is perfused in
single pass with an iso-osmotic solution (GoLYTELY medium)
containing oleuropein, the compound is absorbed with a
Papp of 1.47 ± 0.13 x 10-6 cm/s (±SE). This value is
similar to values derived experimentally using this in situ model for
clinically used drugs such as frusemide (5.0 ± 4.0 x 10-6 cm/s; Winiwarter et al. 1998
), atenolol (1.95 ± 0.79 x 10-6 cm/s) and hydrochlorothiazide (1.71 ± 0.61 x 10-6 cm/s; Berry 1999
) and classifies oleuropein as a poorly permeable compound.
Because molecular modeling using a computer modeling package called
Mopac (Fujitsu, Tokyo, Japan) found the minimum spherical radius of
oleuropein in aqueous solution (0.828 nm) to be larger than the
estimated radius of the paracellular junctions in the ileum (0.54 nm;
Berry 1999
), it appears that absorption of oleuropein
under these conditions occurs predominantly via the transcellular
route. Given that oleuropein is quite polar (logD 0.196), it seems
unlikely that it would readily diffuse through the lipid bilayer of the
epithelial cell membrane, making absorption via a transporter the most
likely mechanism under these conditions. Being a glycoside, oleuropein
could possibly access a glucose transporter; of which three have been
identified in the epithelial cells of the small intestine. Two of these
are facilitated-diffusion glucose transporters (Glut2 and Glut5),
which act like channels to transport glucose passively, whereas the
other is a sodium-dependent glucose transporter (SGLT1), which uses
active transport to move glucose across a concentration gradient
(Takata 1995
). Both Glut 5 and SGLT1 are found on the
apical side of intestinal epithelial cells, but Glut5 appears to be
specific for the transport of fructose and therefore is unlikely to be
involved in the movement of oleuropein into epithelial cells
(Burant et al. 1992
, Kane et al. 1997
).
The third intestinal glucose transporter, Glut2, has been localized to
the basolateral side of epithelial cells and is likely to mediate the
movement of glucose and like substrates from epithelial cells into the
circulation (Kayano et al. 1990
, Nomoto et al. 1998
).
Given that oleuropein contains a conjugated glucose moiety, it would be
feasible for it to access the SGLT1 transporter and be moved into the
epithelial cell and then move into the blood stream via the Glut2
transporter on the basolateral side of the cell. This theory of glucose
transporter–mediated absorption of oleuropein is supported in the
literature, where Hollman et al. (1995)
observed the
absorption of quercetin glycoside (a similar polyphenolic) after its
oral administration to human ileostomy patients and postulated a
mechanism involving active sugar transporters. Additional work with
quercetin glycosides (Gee et al. 1998
) has shown that
these compounds are capable of interacting with the SGLT1 transporter,
further supporting its role in the absorption of polyphenolic
glycosides from the intestine, whereas work by Ohnishi et al. (1998)
with another glycoside
(p-nitrophenyl-ß-D-glucopyranoside) has shown
that this compound is absorbed from the rat small intestine via a
carrier-mediated transport system.
However, this theory is not supported by preliminary work performed in the present study with an inhibitor of SGLT1, phlorizin, in the in situ perfusion model. At the concentrations used in the present study, phlorizin was observed to have no significant effect on the permeability of oleuropein under iso-osmotic luminal conditions, suggesting that SGLT1 is not involved in the absorption of oleuropein. This observation suggests that under iso-osmotic luminal conditions, oleuropein must move across the intestine either paracellularly (despite its large size) or via transcellular passive diffusion (despite its polarity). However, the involvement of SGLT1 cannot be discounted completely, because studies reported here represent only preliminary investigations. Considering the similarity in the structures of oleuropein and phlorizin, it is possible that oleuropein actually has a higher affinity for the SGLT1 transporter than does phlorizin; thus, the presence of phlorizin would have little effect on the absorption of oleuropein from the intestine in the isolated perfused intestine model. The involvement of the efflux pump P-glycoprotein in the absorption of oleuropein from the intestine was also not supported in pilot experiments performed as part of this study.
Although molecular modeling results from this study suggest that the
paracellular movement of oleuropein under iso-osmotic luminal
conditions is unlikely, it is not entirely unfeasible. Despite the fact
that the minimum spherical radius of oleuropein has been estimated to
be larger than the radius of the paracellular junctions in the ileum,
it may be incorrect to consider the paracellular "pores" as
cylindrical. It is probably more correct to consider this pathway as
having only one truly restrictive dimension, the distance between
adjacent epithelial cells (0.54 nm in the ileum), whereas the other two
dimensions correspond more closely to the length and breadth of the
epithelial cells. Therefore, it may be appropriate to consider the
minimum cuboidal dimensions of oleuropein rather than its spherical
dimensions. These have been estimated using computer modeling (0.861
x 1.25 x 1.16 nm) and reveal that there is one plane in
which oleuropein is smaller than the distance between two epithelial
cells (0.861 nm compared with a distance between cells of 1.08 nm).
Therefore, it may be possible for oleuropein to fit between the cells
if it is orientated correctly. However, because the number of spatial
orientations in which oleuropein could pass through the paracellular
junctions would be much smaller than the number in which oleuropein
could not fit through, the contribution of the paracellular pathway to
the absorption of oleuropein is likely to be minimal. Another possible
explanation for the existence of paracellular absorption of oleuropein
stems from recent work by He et al. (1998)
, who
suggested that a small subpopulation of paracellular junctions exists
that are larger than the normal junctions. It may be that these pores
are large enough to accommodate the ready passage of oleuropein,
facilitating the intestinal permeability observed in the control series
of experiments via the paracellular route.
Absorption from a hypotonic intestinal lumen.
The effect of fluid flux on the absorption of high-molecular-weight
hydrophilic compounds (like oleuropein) from the intestine is a point
of some conjecture. Most studies that used animal models show that the
movement of water across the intestine results in increased absorption
of these compounds (Fine et al. 1993
, Kitazawa et al. 1975
, Lu et al. 1992
, Pappenheimer 1987
, Pappenheimer and Reiss 1987
, See and Bass 1993
). Work by Pappenheimer (1987)
showed that water flux, mediated by glucose or amino acid activation of
the intestine, resulted in a significant increase in the intestinal
transport of molecules ranging in size from creatinine (MW 113) to
inulin (MW 5500). It was noted that this increase was accompanied by a
widening of the paracellular junctions, suggesting that absorption was
occurring by a process called "solvent drag," which is when the
streaming of fluid across the intestine stimulates a concomitant
increase in absorption of solutes via the same pathway (in this case,
via paracellular channels). The positive effect of water flux on
absorption is supported in a study by Fine et al. (1994),
in which the absorption of xylose was found to increase
significantly after glucose activation of the intestine, and by
Noach et al. (1994)
, who found that the use of a
hypotonic medium on a Caco-2 cell monolayer resulted in a significant
increase in transport via the paracellular pathway, including a 15-fold
increase in the absorption of insulin (MW 5800). However, these
findings are contrasted by a human regional intestinal perfusion study
by Lennernäs et al. (1994)
that suggested water
movement had no effect on the intestinal absorption of antipyrine,
atenolol or enalaprilat. These authors concluded that induced net water
absorption in humans has no influence on the absorption of hydrophilic
high-molecular-weight compounds and suggested that this is because an
important component of total water flux across the intestine is made up
of transcellular movement, which is not conducive to the accompanying
movement of the molecules studied.
Results from the present study showed that perfusion with a hypotonic
perfusate caused a significant increase in the absorption of
oleuropein. This increase in absorption is most likely to have occurred
via the mechanism described by Noach et al. (1994)
, who
investigated the absorption of two hydrophilic model compounds,
fluorescein sodium– and fluorescein isothiocyanate–labeled dextran
(MW 376 and 4000, respectively) in Caco-2 cells under the influence of
hypotonic conditions. Significant increases (8- to 12-fold) in
transepithelial transport of the model compounds were noted, and these
increases were attributed predominantly to movement via the
paracellular route, facilitated by cell swelling caused by the
hypotonic medium. The development of this hypothesis was assisted by
the use of confocal laser scanning microscopy and the measurement of
transepithelial electrical resistance. Mechanistically, the use of a
hypotonic medium causes cells to swell to readjust their intracellular
osmotic values. Under normal circumstances, this swelling is overcome
by a response in the cell called the regulatory volume decrease, which
results in enhanced movement of potassium chloride and water out of the
cell to restore its normal volume (Alberts et al. 1989
).
This is mediated by the opening of volume-sensitive chloride
(apical and basolateral membrane) and potassium (basolateral membrane)
ion channels (McEwan et al. 1993
). Noach et al. (1994)
suggested that consistent hypotonic exposure to just the
apical side of epithelial cells may not be sufficient to trigger the
opening of potassium ion channels in the basolateral membrane. This
means that regulatory volume decrease is impeded under these
conditions, resulting in the cells swelling and remaining swollen until
isotonicity of the medium is reestablished. Because the cells cannot
expand laterally, they will tend to take on a columnar shape, in which
there is a slight lateral retraction of the cells, resulting in opening
of the paracellular junctions, which facilitates transport via these
pathways.
Taking into consideration this discussion, it appears that although
under normal iso-osmotic luminal conditions oleuropein is poorly
absorbed, its absorption can be significantly increased by solvent flux
through paracellular junctions, made possible by hypotonic conditions
in the intestinal lumen. The data also suggest that hypotonic
conditions are capable of opening the paracellular junctions to such an
extent that even a molecule 60% larger than the junction can pass
through in significant amounts. The small population of larger
intercellular junctions described by He et al. (1998)
may also play a role in this increased absorption due to solvent drag.
It must be remembered, however, that in the experiments described here,
the hypotonic environment was sustained for the duration of the
perfusion, whereas after the administration of a hypotonic solution
under normal physiological conditions, the body most likely rapidly
adjusts the luminal environment such that isotonicity is reestablished.
Therefore, the observation of increased oleuropein absorption under
hypotonic conditions may be largely irrelevant in vivo. However, it has
been suggested by other authors (Pappenheimer 1987
) that
the presence of glucose or amino acids in the intestinal lumen after a
meal stimulates water flux via the opening of paracellular junctions,
and it may be that this mechanism has a similar effect on the
absorption of oleuropein as does the use of a hypotonic solution.
In conclusion, it appears that oleuropein is capable of permeating the
intestine, but the amount of oleuropein that reaches the systemic
circulation unchanged is likely to be small. It may be that the active
components contributing to a beneficial health outcome are those
released via oleuropein metabolism, in either the intestine or the
liver. Alternatively, oleuropein may be acting locally to protect other
dietary antioxidants, such as vitamin C and -tocopherol, from
degradation in the intestine. This would contribute to a beneficial
increase in the total antioxidant status of the body by enhancing the
bioavailability of these other dietary antioxidants. Finally, it must
be remembered that we examined the absorption of oleuropein from an
aqueous solution rather than from the oily matrix in which it would be
presented to the intestine after a meal. It is possible that absorption
via the lymphatic system may occur when oleuropein is presented in an
oily matrix, although being a hydrophilic compound it is more likely to
move out of the oil into the aqueous medium of the intestine after oral
ingestion. These experiments also excluded bile, which would normally
be present in the intestine after a meal. Bile has been shown to be
capable of opening the paracellular junctions in the intestine
(Fricker et al. 1996
, Swenson et al. 1994
, Yamamoto et al. 1996
), potentially
increasing absorption of oleuropein via the paracellular route.
Therefore, it may be that the absorption of oleuropein after oral
ingestion is actually higher than that predicted in this study using
the in situ intestinal perfusion model described.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received March 30, 2000. Initial review completed May 18, 2000. Revision accepted August 11, 2000.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Alberts B., Bray D., Lewis J., Raff M., Roberts K, Watson J. Molecular Biology of the Cell 2nd ed. 1989:304-310 Garland Publishing New York, NY
2. Aungst B., Saitoh H. Intestinal absorption barriers and transport mechanisms, including secretory transport, for a cyclic peptide, fibrinogen antagonist. Pharmaceut. Res. 1996;13:114-119[Medline]
3. Bai C., Yan X., Takenaka M., Sekiya K., Nagata T. Determination of synthetic hydroxytyrosol in rat plasma by GC-MS. J. Agr. Food Chem. 1998;46:3998-4001
4. Beecher G., Wardern B., Merken H. Analysis of tea polyphenols. Proc. Soc. Exp. Biol. Med. 1999;220:267-270[Medline]
5. Berry A. Development of an In Situ Rat Intestinal Perfusion Technique to Investigate Paracellular Absorption 1999 University of Manchester Doctoral thesis
6. Blanchard J., Tang L., Earle M. Reevaluation of the absorption of carbenoxolone using an in situ rat intestinal technique. J. Pharmaceut. Sci. 1990;79:411-414[Medline]
7.
Burant C., Takeda J., Brot-Laroche E., Bell G., Davidson N. Fructose transporter in human spermatozoa and small intestine is Glut5. J. Biol. Chem. 1992;267:14523-14526
8. Das N. Absorption and metabolism of (+)-catechin in man. Biochem. Pharmacol. 1971;20:3435-3445[Medline]
9. Davis G., Santa Ana C., Morawski S., Fordtran J. Development of a lavage solution associated with minimal water and electrolyte absorption or secretion. Gastroenterology 1980;78:991-995[Medline]
10. De Whalley C., Rankin S., Hoult J., Jessup W., Leake D. Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem. Pharmacol. 1990;39:1743-1750[Medline]
11. Ferro-Luzzi A., Sette S. The Mediterranean diet: An attempt to define its present and past composition. Eur. J. Clin. Nutr. 1989;43(suppl. 2):13-29
12. Fine K., Santa Ana C., Porter J., Fordtran J. Mechanism by which glucose stimulates the passive absorption of small solutes by the human jejunum in vivo. Gastroenterology 1994;107:389-395[Medline]
13. Fricker G., Fahr A., Beglinger C., Kissel T., Reiter G., Drewe J. Permeation enhancement of octreotide by specific bile-salts in rats and human-subjects: In-vitro, in-vivo correlations. Br. J. Pharmacol. 1996;117:217-223[Medline]
14. Gee J., DuPont M., Rhodes M., Johnson I. Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic. Biol. Med. 1998;25:19-25[Medline]
15. Gugler R., Leschik M., Dengler H. Disposition of quercetin in man after single oral and intravenous doses. Eur. J. Clin. Pharmacol. 1975;9:229-234[Medline]
16. He Y., Murby S., Warhurst G., Gifford L., Walker D., Ayrton J., Eastmond R., Rowland M. Species difference in size discrimination in the paracellular pathway reflected by oral bioavailability of poly(ethylene glycol) and D-peptides. J. Pharmaceut. Sci. 1998;87:626-639[Medline]
17. Hebel R., Stromberg M. Anatomy of the Laboratory Rat 1976 Williams & Wilkins Baltimore, MD
18. Henczi M., Nagy J., Weaver D. Determination of octanol-water partition coefficients by an HPLC method for anticonvulsant structure-activity studies. J. Pharm. Pharmacol. 1995;47:345-347[Medline]
19.
Hollman P., de Vries J., van Leeuwen S., Mengelers M., Katan M. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am. J. Clin. Nutr. 1995;62:1276-1282
20. Kane S., Seatter M., Gould G. Functional studies of human Glut5: Effect of pH on substrate selection and an analysis of substrate interactions. Biochem. Biophys. Res. Commun. 1997;238:503-505[Medline]
21.
Kayano T., Burant C., Fukumoto H., Gould G., Fan Y., Eddy R., Byers M., Shows T., Seino S., Bell G. Human facilitative glucose transporters. J. Biol. Chem. 1990;265:13276-13282
22.
Keys A. Coronary heart disease in seven countries. Circulation 1970;41(suppl. 21):1-211
23. Keys A. Mediterranean diet and public health: Personal reflections. Am. J. Clin. Nutr. 1995;61(suppl.):1321S-1323S[Abstract]
24. Kim M. Absorption of polyethylene glycol oligomers (330–1122 Da) is greater in the jejunum than in the ileum of rats. J. Nutr. 1996;126:2172-2178
25. Kitazawa S., Ito H., Sezaki H. Transmucosal fluid movement and its effect on drug absorption. Chem. Pharmaceut. Bull. 1975;23:1856-1865
26. Kohyama N., Nagata T., Fujimoto S., Sekiya K. Inhibition of arachidonate lipoxygenase activities by 2-(3,4-dihydroxyphenyl)ethanol, a phenolic compound from olives. Biosci. Biotechnol. Biochem. 1997;61:347-350[Medline]
27. Kühnau J. The flavonoids: A class of semi-essential food components: their role in human nutrition. World Rev. Nutr. Diet. 1976;24:117-191[Medline]
28. Laughton M., Evans P., Moroney M., Hoult J., Halliwell B. Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Biochem. Pharmacol. 1991;42:1673-1681[Medline]
29. Lennernäs H. Does fluid flow across the intestinal mucosa affect quantitative oral drug absorption?: Is it time for a reevaluation?. Pharmaceut. Res. 1995;12:1573-1582[Medline]
30. Lennernäs H., Ahrenstedt , Ö. & Ungell A. Intestinal drug absorption during induced net water absorption in man: A mechanistic study using antipyrine, atenolol and enalaprilat. Br. J. Clin. Pharmacol. 1994;37:589-596[Medline]
31. Lu H., Thomas J., Fleisher D. Influence of D-glucose-induced water absorption on rat jejunal uptake of two passively absorbed drugs. J. Pharmaceut. Sci. 1992;81:21-25[Medline]
32.
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
33. Martin-Moreno J., Willet W., Gorgojo L., Banegas J., Rodriguez-Artalejo F., Fernandez-Rodriguez J., Maisonneuve P., Boyle P. Dietary fat, olive oil intake and breast cancer risk. Int. J. Cancer 1994;58:774-780[Medline]
34. McEwan G., Brown C., Hirst B., Simmons N. Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells. Pflügers Arch. Eur. J. Physiol. 1993;423:213-220[Medline]
35. Middleton E., Kandaswami C. Effects of flavonoids on immune and inflammatory cell functions. Biochem. Pharmacol. 1992;43:1167-1179[Medline]
36. Milne R., Sloan P., McLean C., Mather L., Nation R., Runciman W., Rutten A., Somogyi A. Disposition of morphine and its 3- and 6-glucuronide metabolites during morphine infusion in the sheep. Drug Metab. Dispos 1993;21:1151-1156[Abstract]
37. Montedoro G., Servili M., Baldioli M., Miniati E. Simple and hydrolyzable phenolic compounds in virgin olive oil: 1. Their extraction, eparation, and quantitative and semiquantitative evaluation by HPLC.. J. Agr. Food Chem. 1992;40:1571-1576
38. Montedoro G., Servili M., Baldioli M., Miniati E. Simple and hydrolyzable phenolic compounds in virgin olive oil: 2. Initial characterization of the hydrolyzable fraction. J. Agr. Food Chem. 1992;40:1577-1580
39.
Noach A., Sakai M., Blom-Roosemalen M., De Jonge H., De Boer A., Breimer D. Effect of anisotonic conditions on the transport of hydrophilic model compounds across monolayers of human colonic cell lines. J. Pharmacol. Exp. Ther. 1994;270:1373-1380
40. Nomoto M., Yamada K., Haga M., Hayashi M. Improvement of intestinal absorption of peptide drugs by glycosylation: Transport of tetrapeptide by the sodium ion-dependent D-glucose transporter. J. Pharmaceut. Sci. 1998;87:326-332[Medline]
41. Ohnishi T., Higashi S., Mizuma T., Awazu S. Transport mechanisms of a glycoside, p-nitrophenyl-ß-D-glucopyranoside, across rat small intestinal brush-border membranes. Biochim. Biophys. Acta 1998;1370:192-198[Medline]
42. Paganga G., Rice-Evans A. The identification of flavonoids as glycosides in human plasma. FEBS Lett 1997;401:78-82[Medline]
43. Pappenheimer J. Physiological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters. J. Membr. Biol. 1987;100:137-148[Medline]
44. Pappenheimer J., Reiss K. Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat. J. Membr. Biol. 1987;100:123-136[Medline]
45. Petroni A., Blasevich M., Salami M., Papini N., Montedoro G., Galli C. Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thromb. Res. 1995;78:151-160[Medline]
46. Saija A., Trombetta D., Tomaino A., Lo Cascio R., Princi P., Uccella N., Bonina F., Castelli F. In vitro evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol. Int. J. Pharmaceut. 1998;166:123-133
47. See N., Bass P. Nutrient-induced changes in the permeability of the rat jejunal mucosa. J. Pharmaceut. Sci. 1993;82:721-724[Medline]
48. Stavric B. Role of chemopreventers in human diet. Clin. Biochem. 1994;27:319-332[Medline]
49. Stretch G., Nation R., Evans A., Milne R. Organ perfusion techniques in drug development. Drug Dev. Res. 1999;46:292-301
50. Swenson E.S., Milisen W.B., Curatolo W. Intestinal permeability enhancement: Efficacy, acute local toxicity, and reversibility. Pharmaceut. Res. 1994;11:1132-1142[Medline]
51. Takata K. Glucose transporters in the transepithelial transport of glucose. J. Electron Microsc. 1995;45:275-284
52. Varga F. Transit time changes with age in the gastrointestinal tract of the rat. Digestion 1976;14:319-324[Medline]
53. Visioli F., Bellomo G., Galli C. Free radical-scavenging properties of olive oil polyphenols. Biochem. Biophys. Res. Commun. 1998;247:60-64[Medline]
54. 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]
55. Visioli F., Galli C. Oleuropein protects low density lipoprotein from oxidation. Life Sci 1994;55:1965-1971[Medline]
56. 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 1999;468:159-160
57. Windmueller H., Spaeth A. Vascular autoperfusion of rat small intestine in situ. Methods Enzymol 1981;77:120-129[Medline]
58. Winiwarter S., Bonham N., Ax F., Hallberg A., Lennernäs H., Karlén A. Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters: A multivariate data analysis approach. J Med. Chem. 1998;41:4939-4949[Medline]
59. Wiseman S., Mathot J., de Fouw N., Tijburg L. Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the resistance of low density lipoproteins to oxidation in rabbits. Atherosclerosis 1996;120:15-23[Medline]
60. Yamamoto A., Uchiyama T., Nishikawa R., Fujita T., Muranishi S. Effectiveness and toxicity screening of various absorption enhancers in the rat small intestine: Effects of absorption enhancers on the intestinal absorption of phenol red and the release of protein and phospholipids from the intestinal membrane. J. Pharm. Pharmacol. 1996;48:1285-1289[Medline]
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