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Department of Pharmacy, Biopharmaceutics Research Group Uppsala University, Uppsala, Sweden (N.P., C.T., H.L.); Department of Surgery, Enkoping Hospital, University of Uppsala, Enkoping, Sweden (L.K., P.F., T.E.); Nestlé Research Center, Lausanne, Switzerland (B.H., G.W.); and Institute of Food Research, Norwich, United Kingdom (B.H., F.A.M., Y.B., G.W.P., J.B., K.A.O., P.A.K., G.W.)
(Received November 1, 2002; Accepted February 27, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionConclusionsReferences |
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), and affect the
development of chronic diseases
(Waladkhani and Clemens, 1998;
Kelloff et al., 2000). In
unprocessed plant material, many phytochemicals occur as glycosides, which are
generally biologically inert. Deglycosylation may occur at different stages of
food processing, mastication, digestion, and metabolism, leading to the
formation of active compounds. The chemical form (glycoside or aglycone) of
phytochemicals affects intestinal absorption and gut wall extraction,
modifying their pharmacokinetics (Hollman
et al., 1999). Sulforaphane, an isothiocyanate derived from a
glucosinolate found in broccoli, has been proposed as a potent dietary
anticarcinogen (Kelloff et al.,
2000). Very little is known about its intestinal absorption, since
there are no specific methods for measuring sulforaphane and its metabolites
in plasma. It is found in the urine as a mercapturate, but the total yield is
low (Shapiro et al., 2001).
The absorption and gut wall extraction of quercetin-3,4'-diglucoside, a
flavonoid glycoside naturally occurring in onion, involves deglycosylation by
ß-glucosidases (Day et al.,
1998,
2000) and conjugation with
glucuronic acid, methyl or sulfate groups
(Gee et al., 2000); although
the sites of these modifications are not clear, the conjugated forms and not
the glycosides are found in plasma (Day et
al., 2001).
For direct investigations of small intestinal absorption and gut wall
extraction in humans, only a limited number of methods are available. As a
"gold standard" for studying the effective intestinal permeability
and metabolism, an intestinal perfusion technique (Loc-I-Gut) has been
developed, validated, and widely applied to investigate drug absorption and
intestinal, presystemic metabolism
(Lennernas et al., 1992;
Lindahl et al., 1996; Lennernas,
1997,
1998). A good correlation
exists between the measured jejunal
Peff2
and the fraction dose absorbed of drugs in humans determined by
pharmacokinetic studies.
Using this technique, the majority of shed human enterocytes collected from
an intestinal perfusion were still functionally active and did not show signs
of apoptosis (Ahrenstedt et al.,
1991; Glaeser et al.,
2002). Therefore we not only investigated the transport and
metabolism of phytochemicals in the human jejunum but also their effect on the
short term changes in mRNA expression of phase II enzymes in shed enterocytes
after perfusion.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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;
Lennernas et al., 1992). All
solutions (37°C) were pumped into the jejunal segment at a flow rate
(Q) of 2.0 ml/min using a calibrated syringe pump. The segment was
first rinsed with isotonic saline (37°C) for 10 to 20 min as a prewash and
the intestinal contents collected. The segment was then perfused with further
isotonic saline (30 min) in a preirrigation step during which the perfusate
(solution leaving the intestinal segment) was collected and used to isolate
cells (see below). After administration of the perfusion solution (onion and
broccoli extract), the perfusate was quantitatively collected on ice in 10 min
fractions and immediately frozen at -20°C pending analysis. The segment
was then perfused again with isotonic saline, and the perfusate in this
postirrigation step was used to isolate cells. The pre and post perfusates for
subsequent cell preparation were collected on ice and immediately centrifuged
at 4°C, 500g for 5 min. The supernatant was removed and the
pellet instantly frozen with liquid nitrogen. The pellet was kept at -70°C
pending analysis.
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To confirm that changes in mRNA expression were only due to compounds in the onion and broccoli extract, a control perfusion was performed in five healthy volunteers using isotonic buffer solution containing volume marker but no plant extracts. As we studied relative changes due to the perfusion, independently from interindividual variations, we did not perform the control study in the same volunteers. Cells were collected as described above. The present study was performed at Uppsala University and was approved by the Ethics Committee of the Medical faculty, Uppsala University.
Perfusion Solution (Onion and Broccoli Extract). For the preparation of the plant extract, all glassware used in the preparation of the extract was autoclaved. The water used was obtained from a Millex Q water filtration system and further filtered through a 0.45-µm filter.
Florets of broccoli (Brassica oleracea var. botrytis subvar. cymosa cv. Marathon F1) and red onions (Allium cepa) were obtained from a local supermarket (Norwich, UK). The broccoli florets were washed, stem tissue removed and cut into pieces of approximately 4 to 5 cm in diameter. The outer skins and dead tissue of the onion were removed and the onions cut into quarters. The samples were frozen at -20°C for 16 h and lyophilized at -60°C and 10-1 mbar. The dried samples were milled to fine powders.
A combined extract was prepared by adding 300 g of onion and 167 g of broccoli powder to 5 liters of 2 mM Na2HPO4 (pH 6.4) in water. After stirring at 20°C for 30 min, the extract was filtered through a muslin cloth and centrifuged (5000g, 4°C, 30 min). The clarified extract was sterile filtered through a Millipore "Pellicon" membrane with a 10 kDa cut-off to remove the majority of proteins in the extract. Two subsamples of the final extract were analyzed by LC/MS for sulforaphane, quercetin-3,4'-diglucoside, and possible metabolites. The remaining extract was aliquoted and frozen at -20°C.
The perfusion buffer was isotonic (about 270–300 mOsm l-1) composed of Na+ (147 mM), (Cl- 156 mM), K+(4 mM), phosphate buffer, pH 6.5, 70 mM. 14C-Labeled polyethylene glycol ([14C]PEG 4000) (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) was added to the perfusion buffer as a nonabsorbable volume marker (2.5 µCi/l). A low concentration of antipyrine (Astra Läkemedel AB, Södertälje, Sweden) was used as a marker for passive transcellular diffusion in all perfusion experiments.
Immediately before the perfusion experiments, the samples were thawed and diluted 1 in 5 with the perfusion buffer.
Data Analysis. Calculations of the net water flux (NWF), the
fraction absorbed (fabs) and Peff,
during the perfusion, were made from the mean of five measurements of
concentrations during steady state in the perfusate fractions. The NWF (ml/cm)
in the perfused jejunal segment was calculated according to eq. 1:
 | (1) |
The amount that disappeared during the perfusion through the jejunal
segment was assumed to have been absorbed (fabs) and was
calculated according to eq. 2:
 | (2) |
;
Lennernas, 1997):
 | (3) |
rL) of the jejunal segment was
calculated using the intestinal radius and length of the segment. The human
jejunal radius is 1.75 cm as determined by perfusion of the jejunal segment
with barium and X-ray analysis (L. Knutson, personal communication)
We assessed if metabolites or conjugates were excreted back into the lumen
during the perfusion. For this purpose, the appearance ratio (Ar) was
calculated, using eq. 4.
 | (4) |
Statistical Analysis. Gene expression data were normalized against GAPDH, and significance of fold-changes (pre-against postperfusion) was analyzed by Student's t test. All data in the present study are presented as mean and standard deviation (± S.D.).
Reference Compounds. The sulforaphane-glutathione,
sulforaphane-l-Cys conjugate, and sulforaphane-mercapturic acid
were synthesized according to the method of Kassahun et al.
(1997) with minor
modifications. Reduced GSH, l-Cys, or
N-acetyl-l-Cys were first dissolved in distilled water and
100% ethanol was added. dl-Sulforaphane was dissolved in 80%
ethanol, and this was added to each of the sulfur compounds. The solutions
were left to react at 20°C for 3 days in sealed round-bottomed flasks. The
dithiocarbamates, formed from the reaction between sulforaphane with either
GSH or l-Cys, precipitated during the reaction. The precipitates
were collected by centrifugation and the pellets were washed with 100%
ethanol. The mercapturic acid formed by the reaction of sulforaphane and
N-acetyl-l-Cys did not precipitate, and the mixture was
rotary evaporated and finally purified chromatographically. A purity of
>95% was assessed by 13C and 1H NMR, Maldi-Tof MS and
LC-positive ion electro-spray MS.
Standards of quercetin glucuronides and of quercetin glucosides were either
synthesized or extracted from plants as described previously
(Price et al., 1998;
Day et al., 2001). All
polyphenolic standards were >97% pure; the identities, including position
of attachment of the conjugate, have been determined using chemical synthesis
and NMR (O'Leary et al.,
2001).
Perfusate Analysis. After thawing at 20°C, each perfusion sample was vortex mixed for 30 s and centrifuged (4000g, 10°C, 10 min) to yield a clear solution.
All the quantification was performed by selected ion monitoring (SIM) LC/MS. Calibration curves were obtained for dl-sulforaphane (from ICN Pharmaceuticals, Biochemicals Division, Aurora, OH), sulforaphane-GSH, sulforaphane-cysteine, sulforaphane-mercapturic acid, quercetin-4'-glucoside, quercetin-3,4'-diglucoside, quercetin-3-glucuronide, quercetin-3-sulfate, glucocheirolin [3-(methylsulfonyl)-propyl glucosinolate; LKT Laboratories, Inc., St. Paul, MN] and quercetin-3-rhamnoglucoside (rutin). All standards were >95% pure.
The concentrations of antipyrine in the perfusion solutions were analyzed
by HPLC/UV using a previously validated method (Lindahl et al., 1996;
Sandstrom et al., 1999). The
total radioactivity of [14C]PEG 4000 in the perfusion solutions
were determined by liquid scintillation counting (Mark III; Searle Analytic
Inc., Des Plaines, IL) and the osmolality by the vapor pressure method (Vescor
osmometer 5500, Wescor Inc., Des Plaines, IL).
SIM-LC/MS Analysis. All SIM-LC/MS analyses were conducted using a Micromass Quattro II triple quadrupole mass spectrometer (Micromass UK Ltd., Manchester, UK) equipped with a Z-spray electrospray ion source, which was coupled to either an Hewlett-Packard 1050 quaternary pump HPLC system (Agilent Technologies, Stockport, UK) or to a Jasco PU-1585 triple pump HPLC equipped with an AS-1559 cooled autoinjector, CO-1560 column oven and UV-1575 UV detector [Jasco (UK) Ltd., Great Dunmow, UK].
HPLC conditions were as follows: solvent A was a 0.1% (v/v) trifluoroacetic/water solution, solvent B was 0.1% (v/v) trifluoroacetic/methanol. The HPLC gradient was 100% A, 0% B at 0.0 min; 80% A, 20% B at 10.0 min; 50% A, 50% B at 25.0 min; 0.0% A 100% B at 40 min; 100% A, 0.0% B at 45 min; equilibrate for 10 min at this composition before next injection. The HPLC column temperature was maintained at 25°C and the autoinjector at 4°C. The 1 ml/min mobile phase flow exiting the HPLC column was split using an ASI 600 fixed ratio splitter valve (Presearch, Hitchin, UK) so that approximately 200 µl/min entered the mass spectrometer (the optimal flow for the Z-spray source); the remainder of the flow was diverted to the diode array detector (UV data were plotted at 227 and 370 nm). A Phenomenex (Phenomenex UK Ltd., Macclesfield, UK) Luna C18 (2) reverse-phase column (250 x 4.6 mm, 5 µM), in combination with a Phenomenex SecurityGuard guard column, was used for all HPLC analyses. All water used in extractions and analyses had been distilled, de-ionized, and filtered (0.45 µm) before use.
The mass spectrometer electrospray source capillary voltage was set to 3.3 to 3.5 kV, cone voltage to 29 V, source block temperature to 120°C and desolvation temperature to 300°C. The nitrogen nebulizing and desolvation gas flows were set to 15 l/h and 450 l/h, respectively. All SIM experiments were performed using dwell times of 0.1 s per mass channel and an inter channel delay of 0.03 s. In most cases, both [M + H]+ and [M + Na]+ ions were monitored. Peak areas were measured, after Savitzky-Golay smoothing, using MassLynx 3.4 (Micromass UK Ltd.) acquisition and processing software.
Caco-2 Cells. Caco-2 cells (human adenocarcinoma cells), obtained from the European Collection of Cell Culture, were maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum, 1% (v/v) nonessential amino acids and 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were grown in an incubator at 5% C02, 95% air, 37°C and passaged every 7 days. The cells were used from passage numbers 41 to 47. Cells were seeded at a density of 2 x 104 cells per cm2 in 55 cm2 dishes and allowed to adhere overnight. Undifferentiated cells were grown to confluency over a period of 7 days, and the medium was changed every second day. After this time, the cells were allowed to differentiate over a period of 14 days, and the medium was changed every day. On day 21, the medium was removed and replaced with Eagle's minimal essential medium supplemented with 10% fetal calf serum containing the following treatments (2.5 µM quercetin, 25 µM quercetin, 10 µM sulforaphane, 1 in 5 dilution of onion/broccoli extract and 1 in 5 dilution of onion/broccoli extract with 25 µM quercetin). The samples were incubated for a further 2 h. Control samples not treated with a flavonoid or sulforaphane were incubated with the equivalent volume of carrier vehicle (methanol <0.1% v/v final concentration, or water).
Measurement of ß-Glucosidase Activity. The ß-glucosidase
activity of the intestinal contents (collected prior to the test perfusion by
elution with saline) was determined according to a method described by Day et
al. (2000) by incubating
samples (20–50 µl) with quercetin 3,4'-diglucoside (197 µM
final concentration, 100 µl of final volume) in 50 mM sodium phosphate
buffer (pH 6.0) at 37°C. Incubations were terminated by the addition of an
equivalent volume of stop solution (0.01% trifluoroacetic acid in 50% aqueous
methanol), filtered and analyzed by HPLC with diode array detection as
described elsewhere (Day et al.,
2000). Peaks were confirmed by matching retention times and
spectra with those of authentic standards (quercetin-3,4'-diglucoside,
quercetin-3-glucoside, quercetin-4'-glucoside, quercetin, see
above).
Preparation of RNA. Total RNA was extracted from cells using a Qiagen RNeasy mini kit according to the protocol described by the manufacturer (QIAGEN, Dorking, Surrey, UK) after homogenization using a QIAshredder column. RNA was eluted from a binding column with RNase-free water, and RNase inhibitor was immediately added (20 U per preparation) and stored at -70°C. The yield of total RNA was determined using Ribogreen RNA Quantitation kit (R11490 [GenBank] ; Molecular Probes, Eugene, OR) against a standard curve of ribosomal RNA (16S and 23S rRNA from Escherichia coli).
TaqMan Real-Time RT-PCR Assay. Target mRNA levels were determined by real-time RT-PCR using the ABI prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The threshold cycle (Ct) was defined by when the fluorescence signal reached to 10 times of noise level (baseline). The TaqMan GAPDH-glyceraldehyde-3-phoshate dehydrogenase control reagents kit (402869) was purchased from Applied Biosystems. Forward and reverse primers, and the fluorogenic TaqMan probes for target genes, were designed using Primer Express Software (Applied Biosystems). Sequence homology of selected oligomers was checked using a National Center for Biotechnology Information BLAST search to ensure that sequences were specific to target genes. Primer and probe sets (Sigma-Genosys, The Woodlands, TX and Applied Biosystems, respectively) were as follows:
GST-
-Human glutathione S-transferase [GST A1–1
(GenBank accession no. M14777
[GenBank]
), A1–2 (GenBank accession no.
M16594
[GenBank]
)].
Forward Primer: CAG CAA GTG CCA ATG GTT GA
Reverse Primer: TAT TTG CTG GCA ATG TAG TTG AGA A
Probe: 5'-FAM-TGG TCT GCA CCA GCT TCA TCC CAT C-3'–TAMRA
UGT1A1 UDP-glucuronosyltransferase
(Basten et al., 2002).
Forward Primer: GGT GAC TGT CCA GGA CCT ATT GA
Reverse Primer: TAG TGG ATT TTG GTG AAG GCA GTT
Probe: 5'-FAM-ATT ACC CTA GGC CCA TCA TGC CCA ATA TG-3'-TAMRA
Real-time RT-PCR reactions were carried out in 96-well plates using TaqMan
one-step RT-PCR master mix reagent kit (Applied Biosystems) in a total of 25
µl per well, consisting of 100 to 200 nM probe, 200 to 400 nM primers, and
1 to 20 ng of total RNA. TaqMan RT-PCR conditions were as follows: 48°C 30
min, 95°C 10 min then 40 cycles of 95°C for 15 s and 60°C for 1
min. Data from reactions in triplicate were analyzed with software provided
with the TaqMan and quantified against a standard curve over a 4-fold
concentration range (correlation coefficients for all quantification assays
were >0.985). Ct values were normalized using -fold induction =
2Ct(control)-Ct(treatment). Ct values were converted to an estimate
of relative copy numbers of each mRNA/ng total RNA based on a universal
standard curve used for TaqMan assays. Expression was normalized by eq. 5:
 | (5) |
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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). This
indicates a phase II metabolism of sulforaphane in the enterocytes. The
sulforaphane conjugate appearance ratio
(Table 1) suggests that a high
proportion of this metabolite is effluxed back into the jejunal lumen.
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Quercetin-3,4'-diglucoside, derived from the onion in the extract,
also rapidly disappeared from the perfused jejunal segment
(Table 1). We have previously
shown that the glucoside is metabolized to an aglycone by lactase phlorizin
hydrolase (LPH) before it is absorbed by the intestine
(Nemeth et al., 2002).
Therefore the permeability for this compound is perhaps better described as a
disappearance rate, which is composed of both brush-border metabolism mediated
by LPH and cellular permeability for the aglycone. LC/MS analysis of samples
obtained at 70 min for quercetin-7-, quercetin-4'-, quercetin-3-, and
quercetin-3'-glucuronides revealed that quercetin-3'-glucuronide
was the only metabolite effluxed back into the lumen. As indicated by the
appearance ratio, the relative amount of metabolite
(quercetin-3'-glucuronide) that was effluxed was lower than for the
sulforaphane conjugate (Table
1). The excretion of the conjugate indicates that at least part of
the glucuronidation of quercetin takes place in the enterocyte. No quercetin
aglycone was found in the effluxed samples, indicating that after
deglycosylation, quercetin enters the cells, probably by passive diffusion,
and is conjugated within the enterocyte. We cannot rule out the possibility
that some changes (degradation and other conjugation reactions) occur to the
aglycone, which would prevent detection by our mass spectrometry methods.
However, at the pH of the perfusion (6.5), quercetin is relatively stable,
although it is unstable at pH 7.4 (Boulton
et al.1999; Day,
2000).
ß-Glucosidase Activity of the Luminal Contents. In the jejunal perfusion described here, the contents of the jejunum were washed out before the perfusion began. To determine whether there were any enzymes in the lumen capable of deglycosylating quercetin-3,4'-diglucoside, we collected the lumen contents and assayed them for ß-glucosidase activity (Table 2). This was compared with the capacity of the perfused segment to deglycosylate quercetin-3,4'-diglucoside as calculated from the change in quercetin-3,4'-diglucoside concentration during the perfusion. The results show that the enterocytes are responsible for the majority (79–100%) of the ß-glucosidase activity and that the lumen contents account for <20% of the activity.
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Modulation of Gene Expression in Human Enterocytes as the Result of the Perfusion. Shed human enterocytes were collected before and after the perfusion.
Table 3 shows the changes in relative mRNA expression of GSTA1 and of UGT1A1 normalized against GAPDH, when compared with cells collected before and after the perfusion. In both cases, perfusion with the onion/broccoli extract led to a significant increase in expression of mRNA encoding for both phase II enzymes. A successful perfusion with buffer alone was carried out in five volunteers. No significant change in expression of mRNA of GST or UGT relative to expression of GAPDH mRNA was seen in these experiments (Table 3).
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Metabolism of Quercetin-3,4'-diglucoside in Caco-2 Cells.
Differentiated Caco-2 cells were extremely slow in metabolizing
quercetin-3,4'-diglucoside, an activity which we have previously shown
to be carried out by LPH (Day et al.,
2000), the brush border ß-glucosidase. We therefore measured
LPH activity in Caco-2 cells and found that the levels are extremely low
compared with human small intestine, which is in accordance with a recent
report showing significant differences in gene expression levels in Caco-2
cells compared with cells of the human duodenum
(Sun et al., 2002).
Differentiated Caco-2 cells contained 0 to 0.12 mU/mg LPH activity in
agreement with published data (0.1–0.3 mU/mg even in the "high
LPH" PD7 Caco-2 clone) (Chantret et
al., 1994), whereas human small intestine from lactose tolerant
and intolerant individuals contained 20 to 80 and 2 to 10 mU/mg, respectively
(Rossi et al., 1997).
Induction in Caco-2 Cells. Since Caco-2 cells do not metabolize quercetin-3,4'-diglucoside, unlike the human jejunum in vivo, we substituted quercetin-3,4'-diglucoside with quercetin (the product of active LPH) in the cell culture medium and measured induction of UGT and GST in Caco-2 cells. Differentiated Caco-2 cells were treated with the onion/broccoli extract, with quercetin and with sulforaphane, for the same length of time (2 h) as the in vivo perfusion (Fig. 3). The onion/broccoli extract showed a significant induction of both GST and UGT mRNA of 1.5- to 1.6-fold. Quercetin alone induced UGT (up to 2.6-fold) but not GST, whereas sulforaphane alone induced GST (3.1-fold) but not UGT.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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; Lennernas,
1998), intestinal transport, and gut wall metabolism in vivo in
humans (Sandstrom et al.,
1999). The major advantage of the current perfusion method is that
it is possible to measure absorption, metabolism, and secretion without
influence of other gastrointestinal factors such as transit time and regional
pH-differences. The total amount of broccoli and onion administered in a
perfusion to each volunteer was equivalent to a dry weight of 2.2 g onion and
1.2 g broccoli.
Because of its lipophilicity [log P (octanol/water) = 0.72]
(Cooper et al., 1997) and
molecular size (mol. wt. = 177), sulforaphane is likely to passively diffuse
into the enterocytes (Winiwarter et al.,
1998). In other experimental systems, sulforaphane was conjugated
(in hepatic cells) with glutathione by GSTs leading to maintenance of the
concentration gradient, thus a fast passive absorption into the cell and a
build up of intracellular sulforaphane-glutathione
(Zhang and Callaway, 2002). In
the perfused small intestine, we have shown that a significant proportion of
the sulforaphane absorbed into enterocytes was effluxed back into the lumen as
a sulforaphane-glutathione conjugate. Based on previous studies in cultured
human cells, P-glyco-protein is likely to be responsible for the efflux of the
sulforaphane conjugate (Dietrich et al.,
2001; Zhang and Callaway,
2002). These results suggest that the excretion of conjugated
sulforaphane could determine the extent of absorption of isothiocyanates
following the mechanism proposed in Fig.
4. Under nonperfusion conditions, GSH is supplied to the
intestinal lumen with the bile and reaches luminal concentrations in rats of
about 250 µM (Samiec et al.,
2000). In the jejunal perfusion model, this glutathione is removed
during washing of the segment in the preperfusion phase, and the two balloons
prevent leakage of intestinal contents into and out of the segment, as
indicated by the high recovery of the nonabsorbable marker PEG 4000
(Table 1). Although during
perfusion conjugation is most likely to occur inside the enterocyte where the
concentration of glutathione is likely to be in the millimolar range
(Baillie and Slatter, 1991),
under nonperfusion conditions, nonenzymatic conjugation of sulforaphane
probably also occurs in the lumen in addition to the conjugation in the
enterocytes. Taking together the luminal conjugation and the high extent of
efflux into the lumen, the transmucosal transport of sulforaphane and
conjugate is not expected to be high, indicating that the bioavailability
might be substantially lower.
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Quercetin-3,4'-diglucoside disappeared readily from the perfused
jejunal segment in humans. However, as quercetin-3,4'-diglucoside is
hydrophilic (log P = -1.3; A. J. Day, personal communication) and
relatively large (mol. wt. = 626), it is expected that the passive
permeability across the apical enterocyte membrane would be low. Unlike
quercetin monoglucosides, quercetin-3,4'-diglucoside only interacts very
poorly with the sugar transporter SGLT1
(Gee et al., 2000), which
means that carrier-mediated transport is unlikely.
However, it is a substrate for ß-glucosidases, which will release the
aglycone from quercetin 3,4'-diglucoside. Based on a log P
value of 1.8 and a mol. wt. of 302, the aglycone is predicted to have a high
passive membrane permeability (Murota et
al., 2000). The intestinal contents may contain
ß-glucosidases originating from the microbial population (very low in the
proximal jejunum; 
104 pfu/ml), food, bile, shed cells from the
upper gastrointestinal tract, or cellular secretions. Enterocytes contain
ß-glucosidases that are capable of deglycosylating quercetin glucosides,
evidenced by deglycosylation of quercetin monoglucosides during absorption in
rats (Day et al., 1998;
Gee et al., 2000;
Nemeth et al., 2002). Our data
demonstrate that the majority of the ß-glucosidase activity is derived
from intact enterocytes (Table
2). A minimum of 79% of the deglycosylation capacity observed
during perfusion was due to intact enterocytes. LPH is expressed on the
outside surface of enterocytes allowing direct contact with luminal contents.
Thus, in the perfusion experiments, it is the most likely enzyme to be
responsible for hydrolysis of quercetin-3,4'-diglucoside. Once inside
the enterocyte, quercetin will be subject to glucuronidation. In the human
perfusion experiment, quercetin-3'-glucuronide was the only metabolite
that was effluxed back into the jejunal lumen. Thus we speculate that the
chemical position of intracellular conjugation of quercetin with glucuronic
acid determines the subsequent metabolic route [i.e., that conjugates in the B
ring (i.e., 3' and 4') are excreted back into the lumen, and
conjugates at other positions (3 and 7) are absorbed into the body]. This is
supported by studies in isolated rat everted intestinal sacs, in which
quercetin glucosides were deglycosylated and appeared at the serosal site as
quercetin-3- and 7-glucuronides (Gee et
al., 2000). In addition, Boersma et al.
(2002) reported glucuronidation
of quercetin in four positions, when they incubated quercetin with human small
intestine microsomes. In their study, the 3'-position was the
preferential site of glucuronide conjugation, followed by the 4'-, 7-
and 3-positions. Using supersomes containing individual UGTs, they also
demonstrated that UGT1A8 (found mainly in the small intestine) selectively
glucuronidated quercetin in the 3'-position. This further indicates that
the glucuronidation of quercetin in the human small intestine is
regioselective for B ring conjugates, causing them to be excreted back into
the lumen. A summary of the proposed mechanism is given in
Fig. 5.
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It is well known that there are significant differences in gene expression
levels between Caco-2 cells and human enterocytes
(Sun et al., 2002). It has
also been reported that these differences in gene expression are consistent
with observed differences in carrier-mediated cellular processes. In this
study we showed additionally that the LPH activity was significantly lower in
the Caco-2 model compared with human jejunum, which leads to pronounced
differences in the absorption/metabolism capability for a range of compounds
and shows the limitations of the Caco-2 cell model.
A similar experimental setup to the Loc-I-Gut has been used to collect and
study shed human enterocytes (Glaeser et
al., 2002). The investigators found that the majority of shed
human enterocytes collected were still functionally active, expressed
drug-metabolizing enzymes and transporters, and did not show signs of
apoptosis. Based on these findings, for the first time we exploited a human
perfusion system to study cellular response of human enterocytes to compounds
applied with the perfusion solution in vivo. Therefore this is the first
report of mRNA modulation encoding metabolic enzymes in human enterocytes in
vivo. GST1A and UGT1A1 were chosen because they are the major enzymes involved
in the metabolism of the compounds considered in the permeability study
(van Lieshout et al., 1998),
they are major phase II enzymes involved in first pass metabolism in
enterocytes, and they are known to be inducible in hepatocytes
(Manson et al., 1997). Control
perfusions of buffer alone showed no induction of either GSTA1 or UGT1A1.
After deglycosylation by ß-glucosidases, quercetin-3,4'-diglucoside
is metabolized by UGT1A1 and also induces UGT1A1 mRNA levels. In Caco-2 cells,
sulforaphane increases GSTA1 mRNA levels, and this is the enzyme that
conjugates sulforaphane. Other compounds have also been reported to modulate
GST in the gastrointestinal tract of animals and in human biopsies. Thus
phenylethylisothiocyanate and the sulforaphane analog compound-30 induced
rGSTT1–1 protein levels in the rat gastrointestinal tract
(van Lieshout et al., 1998).
Cisapride, diuretics, cortisol, analgesics, or a high consumption of
vegetables increased duodenal GST and GST in biopsies from 202
human volunteers (Hoensch et al.,
2002). On the other hand, continuous exposure to 5-fluorouracil
led to a decrease in the activities of drug-metabolizing enzymes [e.g., GSTs
and UGTs in the rat intestinal mucosa
(Yoshisue et al., 2001)].
There is increasing evidence for a coordinated regulation of drug-metabolizing
enzymes and the export pump MRP2 via common cellular mechanisms, most likely
to be related by nuclear hormone receptors [e.g., the pregnane X receptor
(Payen et al., 2001)]. Thus we
speculate that MRP2 may be responsible for efflux of quercetin conjugates from
the enterocyte, especially since this transporter is active on
quercetin-4'-glucoside (Walgren et
al., 2000). The basolateral transporter is unknown. In the future,
a combination of the Loc-I-Gut technique with genomic approaches would enable
us to study gene regulation and to discover common regulatory mechanisms
involved in intestinal absorption and defense.
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Footnotes |
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1 Current address: Toxicology Unit, Section on Clinical Pharmacology,
Imperial College, Hammersmith Campus, DuCane Road, London, W12 ONN, UK. 
2 Abbreviations used are: Peff, effective jejunal
permeability; Q, flow rate; LC/MS, liquid chromatography/mass
spectrometry; PEG; polyethylene glycol; NWF, net water flux;
fabs, fraction absorbed; L, length; C,
concentrations of the analyte; GSH, glutathione; l-Cys,
l-cysteine; SIM, selected ion monitoring; HPLC, high performance
liquid chromatography; EMEM, Eagle's minimal essential medium; RT-PCR, reverse
transcription-polymerase chain reaction; Ct, threshold cycle; LPH, lactase
phlorizin hydrolase; UGT, UDP-glucuronosyltransferase; GST, glutathione
transferase.
Address correspondence to: Dr. Birgit Holst, Nutrient Bioavailability, Nestlé Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland. E-mail: birgit.holst{at}rdls.nestle.com
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