1 Laboratoire des Maladies Métaboliques et des Micronutriments, Institut National de la Recherche Agronomique Centre de Recherche de Clermont-Ferrand/Theix, 63122 Saint Genès-Champanelle; and 2 Laboratoire de Chimie Organique et Macromoléculaire, Unite Propte de Recherche et d'Enseignement Associée 8009, Equipe Polyphénols, Université des Sciences et Technologies de Lille, 59655 Villeneuves d'Ascq, France
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ABSTRACT |
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The absorption and splanchnic metabolism of different flavonoids (namely quercetin, kaempferol, luteolin, eriodictyol, genistein, and catechin) were investigated in rats after an in situ perfusion of jejunum plus ileum (14 nmol/min). Net transfer across the brush border ranged widely according to the perfused compound (from 78% for kaempferol to 35% for catechin). This variation seems linked to the lipophilicity of a given flavonoid rather than to its three-dimensional structure. Except for catechin, conjugated forms of perfused flavonoids were also detected in the intestinal lumen, but the extent of this secretion depended on the nature of the perfused compounds (52% for quercetin to 11% for genistein). For some of the perfused aglycones, biliary secretion was an important excretion route: 30% of the perfused dose for genistein but only 1% for catechin. Thus the splanchnic metabolism of flavonoid is controlled by several factors: 1) the efficiency of their transfer through the brush border, 2) the intensity of the intestinal secretion of conjugates toward the mucosal and serosal sides, respectively, and 3) the biliary secretion of conjugates. These data suggested that the splanchnic metabolism of perfused flavonoids depends on the nature of the compound considered, which in turn influences their availability for peripheral tissues.
absorption; biliary excretion; rats
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INTRODUCTION |
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FLAVONOIDS are phenolic secondary plant metabolites responsible for much of the color and flavor of plant foods. They are ubiquitous in fruits, vegetables, and beverages, with more than 4,000 chemically different flavonoids identified to date. They are assigned to different groups according on their structure: anthocyanins, flavonols, flavones, catechins, isoflavones, and flavonones (1, 8, 38, 43). Owing to their ubiquity in plants, humans are constantly exposed to a wide variety of flavonoids. A wealth of beneficial properties has been reported: antibacterial, antioxidant, anti-inflammatory, and anticarcinogenic effects (2, 21, 29) and estrogenic effects for isoflavones (33). Understanding the absorption and metabolism of dietary flavonoids is fundamental to determining their potential biological activity.
The small intestine is a crucial site for the absorption of dietary flavonoids. In this tissue, aglycones are metabolized by intestinal conjugation enzymes, and the resulting metabolites are then secreted toward the mucosal and/or serosal sides (5, 6, 10, 13, 37). Circulating forms of absorbed flavonoids have been identified as methylated and/or glucuronidated and/or sulfated metabolites (9, 12, 19, 23, 39), resulting from the conjugative activities of both liver and intestine (7). Flavonoid metabolites are then eliminated in urine and by the biliary route (18, 22, 35, 40). When excreted into the bile, flavonoid metabolites flow in the small intestine and reach the hindgut where they can be reabsorbed after their hydrolysis by the microflora (30).
Owing to the difficulties of an in vivo approach, many in vitro methods
have been proposed to estimate the absorption and intestinal metabolism
of flavonoids. Until now, literature data have not allowed a strict
comparison of the fate of the different classes of flavonoids,
essentially because of the diversity of the experimental models used
(Caco-2 cells, everted intestine, intestinal perfusion). Accordingly,
the present study was performed to compare the intestinal absorption
and splanchnic metabolism of different flavonoids, using an in situ
intestinal perfusion model. A representative compound of each major
class of flavonoids was tested, using the same experimental pattern.
All of the chosen compounds were aglycones possessing a catechol group,
quercetin (flavonol), luteolin (flavone), eriodictyol (flavanone), and
catechin (flavanol), except for kaempferol (another flavonol) and
genistein (isoflavone) (Fig. 1).
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MATERIALS AND METHODS |
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Chemicals
Luteolin, chrysoeriol, diosmetin, eriodictyol, homoeriodictyol, genistein, tamarixetin, apigenin, kaempferol, hesperetin, catechin, quercetin, taxifolin, and isorhamnetin were purchased from Extrasynthese (Genay, France).Computational Chemistry
All the calculations were performed on an NT workstation (PIII-650 MHz processor) using Spartan Pro V 1.0.1 software. A conformational analysis was performed for all molecules on all rotatable bonds using the Monte Carlo method implemented in Spartan, and the structures found were then minimized using the molecular mechanics Merck Molecular Force Field MMF94. Then the structures were fully optimized at ab initio level using 3-21G* basis set with one polarization function (*) with the closed-shell Restricted Hartree Fock formalism (RHF/PM3/RHF/3-21G*). Heat of formation energies were calculated from closed-shell semiempirical parameterized method (RHF/PM3) formalization. The log P values rendering the hydrophobic indication of the molecule were calculated using the method described in Ref. 14 implemented in the ab initio Spartan function.Animals and Diets
Wistar rats weighing ~150 g were housed two per cage in temperature-controlled rooms (22°C) with a dark period from 2000 to 0800 and access to food from 1600 to 0800. They were fed a standard semipurified diet (73% wheat starch, 15% casein, 6% mineral mixture, 1% vitamin mixture, 5% corn oil) for 2 wk.Animals were maintained and handled according to the recommendations of the Institut National de la Recherche Agronomique Ethics Committee, as legally required.
Sampling Procedure
Twenty-four-hour fasted rats were anesthetized with pentobarbital sodium (40 mg/kg body wt) and maintained alive throughout the perfusion period. After cannulation of the biliary duct, a perfusion of the jejunal plus ileal segment (from 5 cm distal from the flexura duodenojejununalis to the valvula ileocoecalis) was prepared by introducing cannulas at each extremity. This segment was continuously perfused in situ for 30 min with a physiological thermostated buffer (37°C) supplemented with 14 µM flavonoid aglycone (5 µl of a 140 mM solution in DMSO/50 ml buffer). The composition of buffer was as follows (in mM): 5 KH2PO4, 2.5 K2HPO4, 5 NaHCO3, 50 NaCl, 40 KCl, 10 tripotassium citrate, 2 CaCl2, 2 MgCl2, 8 glucose, and 1 taurocholic acid at pH 6.7. The flow rate was set at 1 ml/min. For all the perfused compounds we have checked beforehand 1) that they were stable in the buffer throughout the perfusion period and 2) that the steady state for their effluent flux was reached after 20 min. The fraction of the effluent collected at the exit of the ileum during the last 5 min was stored until analysis. The bile was collected throughout the perfusion, and the fraction corresponding to the 20- to 30-min period was stored and used for HPLC analysis .At the end of the experiment, blood samples were withdrawn from the
abdominal aorta and sampled in heparinized tubes. Plasma, bile, and
effluent samples were acidified with 10 mM acetic acid (10 µl of 1 M
acetic acid per ml of sample) and stored at 20°C until analysis.
HPLC Analysis
Sample treatment.
Bile, plasma, and perfusate samples were spiked with 3.5 µmol/l of an
appropriate internal standard: apigenin for quercetin, kaempferol, and
eriodictyol experiments; tamarixetin for luteolin experiments;
hesperetin for genistein experiments; and taxifolin for catechin
experiments. All the standards used for the identification of the
compounds were commercially available, except for
3'-O-methyl catechin, which was obtained by chemical
O-methylation of catechin as previously described
(11). The samples were then acidified (to pH 4.9) with 0.1 vol of 0.58 M acetic acid and incubated for 30 min at 37°C in the
absence (unconjugated forms) or in the presence (total forms) of 5 × 106 U/l -glucuronidase and 2.5 × 105 U/l sulfatase. Samples were extracted by adding 2.85 vol of methanol/200 mM HCl and centrifuged for 4 min at 14,000 g. The resulting supernatants were analyzed as described
below. The concentrations of conjugated metabolites were calculated by
the difference between the concentrations of aglycone measured before
and after enzymatic hydrolysis. The absorption flux was calculated by
the difference, initial perfused flux
effluent flux, with
initial perfused flux (nmol/min) = initial flow rate (1 ml/min) × concentration (nmol/ml), and with effluent flux
(nmol/min) = volume recovered (ml)/time of recovery (min) × concentration (nmol/ml).
Chromatographic conditions. To analyze the conjugated metabolites of the flavonoids, other than those of catechin, the HPLC system consisted of an autosampler (Kontron 360), a Diode Array Detector (set at 370 nm for flavonols, 350 nm for flavone, 290 nm for flavanone, and at 280 nm for genistein) and Software system for data recording and processing. The system was fitted with a 5-µm C18 Hypersil BDS analytic column (150 × 4.6 mm; Life Sciences International, Cergy, France). The mobile phases were 15% acetonitrile (solvent A) and 37% acetonitrile (solvent B), each containing 0.5% of H3PO4. The flow rate was set at 1 ml/min, and the chromatographic conditions were 0-2 min, 100% A; 2-22 min, linear gradient from 100% A to 100% B; 22-24 min, 100% B; 24-27 min, return to initial mobile phase conditions, and then equilibration for 8 min.
Catechin and catechin metabolites were analyzed by HPLC coupled to a multielectrode detection using an eight-electrode CoulArray model 5600 system (Eurosep, Cergy, France) with potentials set at 25, 100, 320, 400, 500, 700, 800, and 900 mV. Mobile phase A was 3% acetonitrile in 30 mM NaH2PO4 at pH 3.0, and mobile phase B was 20% acetonitrile in 30 mmol/l NaH2PO4 at pH 3.0. The separation was performed at 35°C. The flow rate was set at 1 ml/min with a linear gradient from 0 to 50% B in 15 min and held at 50% B for 20 min. From 20 to 25 min, the gradient was increased to 100% B and then brought back to 100% A for 40 min. The specifications of the column were similar to those indicated above.Statistics
Values are means ± SE, and the differences between values were determined by one-way ANOVA coupled with the Tukey-Kramer multiple comparisons test. Values of P < 0.05 were considered significant. ![]() |
RESULTS |
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Fate of Flavonoids in the Intestine
Whatever the flavonoid tested, a fraction of the perfused dose was recovered intact in the effluent at the end of the perfusion, indicating its intestinal absorption was not complete (Table 1). The magnitude of this unabsorbed fraction was about 20-40% of the total perfused dose for all the compounds, except for catechin for which it reached 65%. These results suggest that the net transfer into the intestinal wall (Table 1), reflecting the transport through the apical membrane, depended on the nature of the compounds.
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The determination of the net transfer into the intestinal wall shows a specificity of flavonoid uptake (5.25 ± 0.20 nmol/min for catechin and 11.45 ± 0.60 nmol/min for kaempferol). On the basis of these findings, we investigated the structure-activity relationships of the flavonoid pattern. Comparison of the basic chemical structures of the flavonoids tested and the net transfer values indicated that neither the position of the B ring at C2 (flavone/flavonol/flavanone) or C3 (isoflavone) nor the nature of the substitution at 3 (H or OH) affected their transfer through the brush border (Fig. 1, Table 1). However, the absence of a 4-oxo group on ring B (flavanol) was associated with a lower net transfer.
To study the influence of the lipophilicity of the flavonoids on the
efficiency of transfer across the intestinal membrane, log P
was calculated for each of the tested substances (Table 2). The values obtained agree with those
of the literature (32). We found that the most lipophilic
molecules (kaempferol, quercetin, luteolin, eriodictyol, and
genistein) displayed high transfer efficiencies, whereas catechin,
which is much less lipophilic, was less readily transferred.
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Net transfer efficiency was also compared with the three-dimensional
structure of the molecules tested (Table 2). The dihedral angle
indicates the position of the B ring relative to the C ring, i.e., the
planarity of the whole molecule. Moreover, visualizing the molecule in
its most stable conformation shows whether it is elongated or folded
(Table 2). We see that a molecule with an elongated structure like
luteolin is as efficiently transferred across the intestinal wall as
eriodictyol, which nevertheless presents two structures, elongated and
folded (Fig. 2), and which from Boltzman
population analysis represents 62 and 38% of the species in solution,
respectively. In these conditions, it seems that the three-dimensional
structure of flavonoids may not be determinant in their interactions
with the brush border.
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All these data favor a relationship between net transfer and lipophilicity of the molecule, rather than its spatial arrangement.
For all the flavonoids tested, except for catechin, conjugated derivatives were recovered in the effluent (Table 1). These metabolites had an intestinal origin, because the biliary duct was cannulated before starting the perfusion. The rate of the intestinal secretion of conjugates was highly dependent on the nature of the perfused compound (Table 1). For quercetin, it reached 52% of the perfused dose but was markedly lower for the other tested compounds (11-20%). The intestinal metabolism of catechin differed unequivocally from that of the other compounds, since no trace of conjugated forms was found in the lumen at the end of the perfusion (Table 1).
For a given compound, the difference between the net transfer in the intestinal wall and the secretion of conjugates into the lumen has been made to estimate the net absorption (Table 1). Although the net transfer of quercetin in the intestinal wall reached 9.80 ± 0.20 nmol/min, the high rate of secretion of conjugated forms into the lumen (7.70 ± 0.40 nmol/min) resulted in a quite moderate rate of absorption (only 2.1 ± 0.40 nmol/min). This result was also reflected by the fraction of the net transfer in the intestinal wall directed toward the serosal side (21%) (Table 1). By contrast, kaempferol, luteolin, eriodictyol, and genistein presented a moderate intestinal secretion of their conjugates associated with a relatively high level of net absorption: 66 to 86% of the net transfer was directed toward the serosal side (Table 1). It was noteworthy that although kaempferol and quercetin belong to the same class of flavonoid, they presented marked differences in their intestinal metabolism. The net absorption of kaempferol (8.50 ± 0.40 nmol/min) was higher than that of quercetin (2.1 ± 0.40 nmol/min) owing to the less abundant secretion of kaempferol conjugates into the lumen.
For catechin, because no conjugates were secreted into the intestinal lumen, net transfer into the gut corresponded to its net absorption (5.25 ± 0.30 nmol/min) (Table 1).
All these data suggest that in the intestine, the efficiency of flavonoid absorption is modulated by the permeability of the apical membrane toward these compounds and by the extent of secretion of conjugates into the lumen.
Biliary Secretion
The cannulation of the biliary duct at the beginning of the perfusion and the collection of the biliary secretion throughout the experiment enabled us to study the contribution of enterohepatic cycling on flavonoid bioavailability. As shown in Table 3, for each perfused compound, a fraction of the conjugates secreted toward the serosal side was recovered in the bile.
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When kaempferol, luteolin, eriodictyol, or genistein was perfused, the total concentrations of the biliary conjugates measured in the bile were always quite high (160 to 402 µM, which corresponded to biliary fluxes of 1.35 ± 0.40 to 4.21 ± 0.50 nmol/min, respectively) (Table 3). The intensity of this biliary secretion accounted for the role of the enterohepatic cycling in the metabolism of these flavonoids. The biliary secretion of conjugates was also significant for quercetin (0.87 ± 0.10 nmol/min) but quite limited for catechin (0.16 ± 0.05 nmol/min) (Table 3).
Some of the glucuro/sulfoconjugates of quercetin, luteolin, eriodictyol, and catechin present in the bile were also methylated (Table 3). The position and level of methylation largely depended on the nature of the perfused compound. Almost all the biliary metabolites of catechin were methylated, at 3'. By contrast, kaempferol or genistein were only present in hydrolyzed bile samples as aglycone (Table 3), in accordance with their structure, because the presence of a catechol group is crucial for methylation.
The total secretion of conjugated flavonoids can be divided into two
components: intestinal secretion and biliary secretion (Table
4). The flux of quercetin metabolites in
the bile was quite limited compared with that found in the intestine,
and so it contributed to only 10 ± 3% of the total secretion of
conjugates in the intestinal wall (Table 4). For kaempferol and
luteolin, the proportion of metabolites secreted in the bile was
markedly higher than for quercetin, as shown by the high levels of
their biliary fluxes (30 and 38% of the total secretion of conjugates, respectively) (Table 4). Eriodictyol and genistein in the bile were
quite high (59 and 76% of the total secretion of conjugates, respectively) (Table 4), suggesting that biliary secretion constituted a major pathway for their elimination.
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The absence of intestinal secretion of catechin conjugates (Table 4) indicates that the intestinal secretion of metabolites did not constitute a mechanism for catechin elimination. Moreover, the biliary secretion of catechin conjugates was also minor, representing only 1% of the perfused dose. This result indicates that the contribution of the liver to elimination of the metabolites of absorbed catechin was also quite limited.
Plasma Metabolites After Intestinal Perfusion
The analysis of plasma sampled from the aorta at the end of the perfusion indicated that whatever the perfused compound, the circulating metabolites were made up of glucuro- and/or sulfoconjugates, methylated or nonmethylated. This result shows that only conjugated forms of flavonoids absorbed by the gut were delivered to peripheral tissues.The plasma levels measured differed markedly according to the perfused compound: 0.25 ± 0.05 µM for catechin, 0.48 ± 0.04 µM for eriodictyol, 1.04 ± 0.06 µM for luteolin, and 1.34 ± 0.20 µM for genistein. The two flavonols tested, namely quercetin and kaempferol, resulted in quite similar plasma concentrations (0.71 ± 0.06 and 0.95 ± 0.12 µM, respectively).
Estimation of Conjugated Metabolites Available for the Peripheral Tissues
A schematic representation of the splanchnic metabolism of perfused flavonoids, resulting from all the data obtained in the study and from the data presented in Tables 1, 3, and 4, is given in Fig. 3. The difference between the net absorption and the biliary secretion gave an estimate of the proportion of conjugated forms finally available for peripheral tissues (see Fig. 3). Quercetin was distinguishable from the other tested flavonoids by its high rate of intestinal secretion (52%). Thus only a small fraction of absorbed quercetin was finally available for peripheral tissues (9%) (Fig. 3). By contrast, this fraction was quite high for kaempferol (49%), in agreement with its relatively low rate of intestinal and biliary secretion (Fig. 3). These results suggest that the splanchnic metabolism of compounds belonging to the same class of flavonoid can markedly differ. Luteolin, eriodictyol, and genistein, which presented quite similar levels of total excretion into the lumen (20-30% of the perfused doses), exhibited relatively high availabilities for peripheral tissues (Fig. 3).
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Among the tested flavonoids, catechin was a special case, first because no intestinal secretion of catechin conjugates occurred, and second because its biliary secretion was quite low (1%). In these conditions, almost all the catechin entering the intestinal cells, corresponding to 34% of the perfused dose, was available for peripheral tissues.
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DISCUSSION |
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Flavonoid bioavailability is a complex field, and earlier studies on this topic have not simultaneously taken into account parameters such as intestinal absorption and splanchnic metabolism.
It is well established that factors such as the nature of glycosylation and the food matrix modulate the bioavailability of dietary flavonoids (15, 16). However, little is known about how they are transferred as aglycone through the brush border (rate and transport system) or about their intestinal metabolism and secretion. Theoretically, depending on its structure, a compound may cross the epithelial cell layer by several mechanisms: passive diffusion across the membrane, carrier-mediated transport, endocytosis, and paracellular diffusion through tight junctions. The mechanisms involved in flavonoid transport at the intestinal level are the subject of research but have not yet been elucidated. However, because of their structure, flavonoids probably do not cross the phospholipidic membrane by a passive diffusion system, but rather via a mechanism of facilitated diffusion or active transport.
With our in situ perfusion model, the efficiency of the flavonoid transfer through the brush border is inversely related to the level of intact flavonoid recovered in the effluent at the end of the perfusion. Because we failed to found detectable amounts of perfused compounds in intestinal mucosa extract (data not shown), we considered that the difference between the concentration measured in the initial perfusate and that found in the remaining perfusate reflected the magnitude of the transfer of the compounds through the brush border. Quercetin, kaempferol, luteolin, eriodictyol, and genistein were extensively absorbed in the gut (59-78% of the perfused dose), suggesting that their transfer through the brush border is not limiting for bioavailability. Recently, it was shown that genistein entered Caco-2 cells by a transepithelial transporter that was saturable when the concentration of genistein reached 50 µM (25). This result is corroborated by our data, since when perfused at 14 nmol/min (corresponding to a 14 µM dose), genistein was readily transferred in the intestinal wall (73% of the perfused dose).
Catechin is characterized by a relatively low transfer through the gut
(only 35%), indicating that the transfer across the intestinal cells
may be limiting for catechin bioavailability. In a previous study, we
have found that the catechin absorption was directly proportional to
the amount perfused (10). Although the mechanism of
catechin absorption in the small intestine is not established, it
appears probable that catechin enters the enterocytes by a passive
diffusion system or by a transporter that was not saturated at
concentrations up to 100 µM (10). However, in in vitro
experiments, it has been reported that the absorption of genistein is
slightly inhibited by quercetin (50%) and catechin (
20%)
(25). The authors suggested that the uptake of genistein
might occur, at least partly, via a transport system common to other
flavonoids (27). Even if this hypothesis of the existence
of a transport system common to all flavonoids is correct, the affinity
for the transporter and the rate of the transfer may still depend on
the nature of the flavonoids. In our results, the position of the B
ring at C2 or C3 (ring C) and the nature of the
substitution at the 3 position (H or OH) did not influence the net
transfer across the brush border. Thus if an active transport is
involved, then the efficiency of the absorption may depend on the two
hydroxyls at positions 5 and 7 on the A ring. Such a hypothesis could
be tested using selectively synthesized 5- or 7-alkoxy or
benzyloxyflavonoids in our intestinal perfusion model. In addition,
whereas the nature of the three-dimensional structure of the flavonoids
does not seem to be determinant for the efficiency of transfer, the
lipophilicity of the molecule seems important.
Many studies have emphasized the role of the intestinal conjugative enzymes in the metabolism of flavonoids (3, 5, 6, 42). The conjugates produced in the intestine (essentially glucuronides and sulfoconjugates) are then secreted toward the serosal and mucosal sides (5, 6, 42). The recovery of only conjugated metabolites in the mesenteric vein (10, 13) indicated, first, that the intestinal conjugation occurred before the transfer of flavonoid into the serosal side and, second, that the system responsible for the transport of the aglycone is not present on the basolateral membrane.
The present study clearly shows that some of the conjugates synthesized in the enterocytes are secreted back in the intestinal lumen. This process could occur via MRP2, which has been reported to mediate the efflux of conjugated forms of chrysin into the luminal side of Caco-2 cells (42). However, the proportion of the conjugated forms secreted toward the serosal or toward the mucosal side depends markedly on the nature of the flavonoid. According to our data, quercetin conjugates produced in the intestine are copiously secreted into the mucosal side, whereas this secretion appears less abundant for the other perfused compounds. In any case, a high level of secretion of conjugates into the lumen is associated with a reduction of the net absorption of the perfused flavonoids. Thus a high intestinal secretion of flavonoid conjugates could correspond to a mechanism whereby the body reduces the bioavailability of quite reactive compounds such as quercetin (31).
All these data enabled us to define two control steps in the bioavailability of flavonoids: 1) transepithelial transport and 2) secretion of conjugates into the intestinal lumen and into the serosal side.
When flavonoid metabolites resulting from intestinal conjugation are secreted in the mesenteric vein, a proportion of them are taken up and metabolized by the liver (3, 4, 26) and then partially secreted in the bile (20, 34, 44, 45). In our experiments, the presence of flavonoids in the bile for all the tested compounds indicated that whatever their origin, flavonoids undergo enterohepatic cycling. The detection of methylated metabolites of quercetin, luteolin, eriodictyol, and catechin in the bile showed that the liver is an active site for the methylation of flavonoids, when they bear a catechol group. In the present study, we did not determine the precise nature of the metabolites resulting from the respective intestinal and hepatic conjugation, but, as shown by the forms recovered in the bile, they may be quite different. As reported in our previous study, catechin was present in the mesenteric vein as glucuronides (methylated or nonmethylated), whereas it was recovered as glucurono/sulfoconjugates (methylated or nonmethylated) in the bile (10). Taken together, these data strongly suggest that hepatocytes are a site of deconjugation for the circulating forms coming from the mesenteric vein and that the conjugative activity of the liver produced metabolites available for the bile and peripheral tissues.
The nature of the conjugation influences the elimination pathway of the circulating metabolites: the glucuronides are excreted in the bile to a much higher extent than the sulfates, which are preferentially eliminated in urine (24). The nature and the position of the conjugation also probably affect the availability of the circulating metabolites for peripheral tissues, but until now data on this purpose are lacking. The decrease in flavonoid lipophilicity induced by their conjugation does not favor a passive diffusion of the conjugates through the membrane. However, it is quite probable that their uptake by the target cells involved specific transport systems, as it has already been shown for estrogen conjugates (28).
The activity of the biliary secretion strongly depends on the nature of the compounds. Some of them, such as catechin, are sparingly secreted in the bile (1%), whereas others, like genistein and eriodictyol, are extensively recycled (32 and 23% of the perfused dose, respectively). The importance of the biliary secretion in genistein bioavailability was previously suggested in a study reporting that 70-75% of genistein perfused in the small intestine was recovered in the bile over 4 h of perfusion (34). By contrast, the biliary secretion of quercetin and catechin appears quite limited, indicating that the biliary pathway probably plays a minor role in their elimination.
As the rat does not possess a gall bladder and because the liver did not constitute a site for flavonoid accumulation (19), it can be considered that the biliary flux is directly linked to the intestinal absorption flux. Moreover, the plasma concentrations measured after the intestinal perfusion of flavonoids are quite low and, by consequence, cannot constitute a significant pool. In these conditions, the compilation of the data obtained at the intestinal and biliary levels provides information about the fraction of perfused flavonoid ultimately available for peripheral tissues (see Fig. 3).
Because of its high secretion of conjugates in the intestinal lumen (58%), quercetin was poorly available for peripheral tissues (only 9%). By contrast, a relatively high level of perfused catechin (34%) reached peripheral tissues. These results are corroborated by experiments on rats fed a meal containing similar doses of quercetin or catechin showing that 4 h after the beginning of the meal, the plasma concentration of catechin was higher than that of quercetin (19).
It can be noted that quercetin and kaempferol, which belong to the same flavonoid class, presented quite different splanchnic metabolisms. The intestinal metabolism of kaempferol was not limiting for its bioavailability, unlike what was observed for quercetin. In these conditions, the fraction of kaempferol available for peripheral tissues was five times that of quercetin. Kaempferol differs from quercetin in having no catechol group, and this structural difference may be linked to changes in their splanchnic metabolism. However, despite the marked difference in the estimated systemic availability between quercetin and kaempferol, their respective plasma concentrations were quite similar. This could be due to a better elimination of plasma kaempferol conjugates by the kidney, leading to its rapid disappearance from plasma.
The availability of luteolin and eriodictyol for peripheral tissues appeared to depend on the intestinal and biliary secretions, whereas that of genistein was strongly controlled by the biliary secretion alone. It is difficult to compare the present in situ data with the published in vivo studies on luteolin, eriodictyol, and genistein bioavailability, because these are sparse and were conducted in quite different conditions (dose, administration mode) (17, 22, 27, 36). As a direct comparison of the literature data with our findings appears impossible, additional in vivo experiments will have to be performed in conditions that permit a true comparison of the bioavailability of these flavonoids.
In conclusion, the data presented here indicate that flavonoid bioavailability depends on three main factors: 1) efficiency of transfer through the brush border, 2) intensity of intestinal secretion of conjugates toward the mucosal and serosal sides, and 3) activity of biliary secretion. Also, the importance of each of these factors in the control of the splanchnic metabolism varies according to the molecule, making it impossible to state a general rule valid for the splanchnic metabolism of all flavonoids.
Further investigations must now be conducted to determine the mechanisms by which flavonoids enter the cells (intestinal, hepatic, and target cells) and the nature of the molecules responsible for the biological effects inside the cells. Thorough knowledge of these phenomena is crucial for a complete understanding of flavonoid bioavailability.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Morand, Laboratoire des Maladies Métaboliques et des Micronutriments, INRA Centre de Recherche de Clermont-Ferrand/Theix, 63122 Saint Genès-Champanelle, France (E-mail: cmorand{at}clermont.inra.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00223.2002
Received 11 June 2002; accepted in final form 21 January 2003.
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