Thomas Walle

HIGH ABSORPTION BUT VERY LOW BIOAVAILABILITY OF ORAL RESVERATROL IN HUMANS

The dietary polyphenol resveratrol has been shown to have chemopreventive activity against cardiovascular disease and a variety of cancers in model systems, but it is not clear whether the drug reaches the proposed sites of action in vivo after oral ingestion, especially in humans. In this study, we examined the absorption, bioavailability, and metabolism of 14C-resveratrol after oral and i.v. doses in six human volunteers. The absorption of a dietary relevant 25-mg oral dose was at least 70%, with peak plasma levels of resveratrol and metabolites of 491 ± 90 ng/ml (about 2 μM) and a plasma half-life of 9.2 ± 0.6 h. However, only trace amounts of unchanged resveratrol (<5 ng/ml) could be detected in plasma. Most of the oral dose was recovered in urine, and liquid chromatography/mass spectrometry analysis identified three metabolic pathways, i.e., sulfate and glucuronic acid conjugation of the phenolic groups and, interestingly, hydrogenation of the aliphatic double bond, the latter likely produced by the intestinal microflora. Extremely rapid sulfate conjugation by the intestine/liver appears to be the rate-limiting step in resveratrols bioavailability. Although the systemic bioavailability of resveratrol is very low, accumulation of resveratrol in epithelial cells along the aerodigestive tract and potentially active resveratrol metabolites may still produce cancer-preventive and other effects.

Bioavailability of resveratrol

This paper reviews our current understanding of the absorption, bioavailability, and metabolism of resveratrol, with an emphasis on humans. The oral absorption of resveratrol in humans is about 75% and is thought to occur mainly by transepithelial diffusion. Extensive metabolism in the intestine and liver results in an oral bioavailability considerably less than 1%. Dose escalation and repeated dose administration of resveratrol does not appear to alter this significantly. Metabolic studies, both in plasma and in urine, have revealed major metabolites to be glucuronides and sulfates of resveratrol. However, reduced dihydroresveratrol conjugates, in addition to highly polar unknown products, may account for as much as 50% of an oral resveratrol dose. Although major sites of metabolism include the intestine and liver (as expected), colonic bacterial metabolism may be more important than previously thought. Deconjugation enzymes such as β‐glucuronidase and sulfatase, as well as specific tissue accumulation of resveratrol, may enhance resveratrol efficacy at target sites. Resveratrol analogs, such as methylated derivatives with improved bioavailability, may be important in future research.

What Is New for an Old Molecule? Systematic Review and Recommendations on the Use of Resveratrol

Background Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark. Methodology Literature search in databases as PubMed and ISI Web of Science in combination with manual search was used to answer the following five questions: 1Can resveratrol be recommended in the prevention or treatment of human diseases?; 2Are there observed “side effects” caused by the intake of resveratrol in humans?; 3What is the relevant dose of resveratrol?; 4What valid data are available regarding an effect in various species of experimental animals?; 5Which relevant (overall) mechanisms of action of resveratrol have been documented? Conclusions/Significance The overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.

Methylated flavonoids have greatly improved intestinal absorption and metabolic stability.

To better understand the relationship between the chemical structure and biological fate of dietary polyphenols, the hepatic metabolic stability and intestinal absorption of methylated polyphenols, in comparison with unmethylated polyphenols, were investigated in pooled human liver S9 fraction and human colon adenocarcinoma (Caco-2) cells. Consistent with previous in vivo studies, the two well known unmethylated polyphenols resveratrol (3,5,4′-trihydroxystilbene) and quercetin (3,5,7,3′,4′-pentahydroxyflavone) were rapidly eliminated by the S9 fraction in the presence of the appropriate cofactors for conjugation and oxidation. In contrast, the methylated flavones, i.e., 7-methoxyflavone, 7,4′-dimethoxyflavone, 5,7-dimethoxyflavone, and 5,7,4′-trimethoxyflavone, were relatively stable, indicating high resistance to hepatic metabolism. The corresponding unmethylated flavones, i.e., 7-hydroxyflavone, 7,4′-dihydroxyflavone, chrysin (5,7-dihydroxyflavone), and apigenin (5,7,4′-trihydroxyflavone), were rapidly eliminated because of extensive glucuronidation and/or sulfation just as resveratrol and quercetin were. The rate of intestinal absorption was evaluated using Caco-2 cells grown in porous inserts. The methylated flavones showed approximately 5- to 8-fold higher apparent permeability (Papp, 22.6–27.6 × 10–6 cm s–1) of apical to basolateral flux than the unmethylated flavones (Papp, 3.0–7.8 × 10–6 cm s–1). The lower Papp values for the unmethylated flavones correlated with their extensive metabolism in the Caco-2 cells. Thus, combined use of the hepatic S9 fraction and Caco-2 cells will be useful for predicting the oral bioavailability of dietary polyphenols. The higher hepatic metabolic stability and intestinal absorption of the methylated polyphenols make them more favorable than the unmethylated polyphenols to be developed as potential cancer chemopreventive agents.

Transport of the flavonoid chrysin and its conjugated metabolites by the human intestinal cell line Caco-2.

Chrysin (5,7-dihydroxyflavone), a natural product present in our daily diet, is a potent inhibitor of drug-metabolizing enzymes. However, its oral bioavailability is not known. This study examined the intestinal epithelial transport of chrysin (20 microM), using the human colonic cell line Caco-2 as a model of human intestinal absorption. The apical to basolateral flux of chrysin, with an apparent permeability coefficient (P(app)) during the first hour of 6.9 +/- 1.6 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.42 +/- 0.12 x 10(-6) cm x sec(-1). Interestingly, the reverse, basolateral to apical flux of chrysin, P(app) = 14.1 +/- 1.6 x 10(-6) cm x sec(-1), was about 2-fold higher than the apical to basolateral flux (P < 0.01). In transport studies beyond 1 hr, there was a rapid decline in P(app). This correlated with the appearance of two metabolites, M1 (chrysin glucuronide) and M2 (chrysin sulfate), identified by enzymatic hydrolysis procedures and HPLC. Following apical loading of chrysin, as much as 90% of M1 + M2 appeared on the apical side, thus indicating clear efflux of the chrysin metabolites. The addition of the anion transport inhibitor MK-571 (50 microM) on the apical side produced a 71% (P < 0.0001) and 20% (P < 0.05) inhibition of the efflux of M1 and M2, respectively, suggesting the involvement of the multidrug resistance protein MRP2 pump. Indeed, using specific antibodies, MRP2 was in fact detected by western blotting in Caco-2 plasma membranes, whereas MRP1 was not. These observations suggest that chrysin has favorable membrane transport properties but that its intestinal absorption may be seriously limited by surprisingly efficient glucuronidation and sulfation by the enterocytes and almost quantitative efflux by MRP2 of the metabolites formed.

Extensive binding of the bioflavonoid quercetin to human plasma proteins

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma‐protein binding of the most abundant bioflavonoid, quercetin, using 14C‐labelled quercetin.

Transport and metabolism of the Tea flavonoid (-)-epicatechin by the human intestinal cell line Caco-2

AbstractPurpose: Tea flavonoids, including (–)-epicatechin (EC), have been suggested to have chemopreventive properties in cancer. However, there is limited knowledge of the oral bioavailability of these dietary compounds. The purpose of this study was to gain a better understanding of the absorption of EC. Methods: The intestinal epithelial membrane transport of EC was examined using the monolayer of the human Caco-2 cell line grown in Transwells, a common model of intestinal absorption. EC and its metabolites were measured by high performance liquid chromatography with diode array detection. Results: EC showed no apical to basolateral absorption at concentrations ranging from 5 to 50 μM. In contrast, EC demonstrated basolateral to apical efflux with a Papp value of 0.67 ± 0.05 × 10−6 cm/sec, i.e., slightly higher than for mannitol, 0.50 ± 0.30 × 10−6 cm/sec, a paracellular transport marker. There was a 50% reduction in the efflux of EC in the presence of 50 μM MK-571, a competitive inhibitor of the MRP2 transporter expressed in the apical membrane of Caco-2 cells. Most important, the presence of 50 μM MK-571 resulted in clearly measurable apical to basolateral absorption of EC with a Papp of 0.31 ± 0.06 × 10−6 cm/sec. Two polar metabolites, M1 and M2, were formed from EC, both of which appeared exclusively on the apical side. MK-571 (50 μM) dramatically inhibited the transport for both of these metabolites. Incubations with inorganic 35SO42− and hydrolysis by aryl sulfatase strongly suggested that these metabolites were sulfate conjugates. Conclusions: These results suggest an important role for the multispecific organic anion transporter MRP2 in the bioavailability of EC and possibly other tea flavonoids.

Stereoselective delivery and actions of beta receptor antagonists

These studies have revealed that the delivery and actions of beta receptor antagonist drugs are controlled by a cascade of stereoselective processes involving multiple enzymes, transport proteins and receptors. In essence, the free concentration of the pharmacologically active (-)-enantiomer species of these drugs presented to cell surface beta receptors appears to be a function of the stereoselective clearance by hepatic cytochrome P-450 isoenzymes, enantiomer selective binding to alpha 1-acid glycoprotein and albumin and perhaps predominantly by stereoselective sequestration (and release) by the vesicular amine transport protein within adrenergic neurons. Stereoselectivity in the clearance of beta blocking drugs, which can favor either the (+)- or (-)-enantiomer, only appears to be important for the lipophilic drugs which are cleared by hepatic metabolism. Such stereoselectivity is due to differential stereochemical substrate requirements of individual hepatic cytochrome P-450 isoenzymes. Interindividual variations in the stereoselectivity can occur as a result of differences in the amount and expression of cytochrome P-450 isoenzymes due to genetic predisposition or other factors. In the same context, we have observed a significant correlation between the extent and stereoselectivity of binding of beta blocking drugs to plasma proteins. This is another finding which suggests that variability in the expression of individual proteins involved in the beta blocking drug-protein cascade determines the free concentration of the pharmacologically active enantiomer. However, since most observations have been made in young normal subjects, the extent of stereoselectivity in metabolism, binding and other processes is unknown in the general population where steady-state plasma concentrations can vary widely due to multiple biological factors. The observations from neural studies support the concept that adrenergic nerve endings provide a depot for the stereoselective storage and release of the active enantiomer of beta receptor antagonists. The mechanism of this release appears to involve exocytotic secretion of drug that has been stereoselectively accumulated by the neurotransmitter storage vesicles. In terms of this idea, beta receptor antagonists released during nerve stimulation may achieve concentrations of the (-)-enantiomer within the adrenergic synapse greatly in excess of those found in plasma. Such a mechanism could significantly influence both the intensity and duration of beta receptor blockade in the heart, blood vessels, brain and other target tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Resveratrol transport and metabolism by human intestinal Caco-2 cells

Resveratrol is a dietary constituent suggested to have protective effects against cancer as well as cardiovascular disease. The purpose of the study was to learn whether this agent could be absorbed in man and enter the systemic circulation. This was examined by measuring transport and metabolism of resveratrol (5–40 μM) by the human intestinal epithelial cell line Caco‐2 cultured in Transwells. Transport across the Caco‐2 monolayer occurred in a direction‐independent manner with Papp values of ≅ 7 times 10−6 cm s−1, much higher than for the paracellular transport marker mannitol (≅ 0.4 times 10−6 cms−1), suggesting efficient absorption in‐vivo. At the highest resveratrol concentration, the absorption increased, possibly due to saturation of metabolism. In sharp contrast to previous findings in the rat, the metabolism of resveratrol in Caco‐2 cells involved mainly sulfation and, to a minor extent, glucuronidation. At low resveratrol concentrations, most of the sulfate conjugate was exported to the apical side, presumably by MRP2, which is well expressed in these cells. At high concentrations, there was a shift towards the basolateral side, possibly involving MRP3, which was recently shown also to be expressed in Caco‐2 cells. These results indicate that absorption of resveratrol in‐vivo may be high but with limited bioavailability due to efficient sulfate conjugation. Extensive accumulation of resveratrol in the Caco‐2 cells, demonstrated in additional experiments, suggests enterocytes as a major target site for this cancer preventive agent.

Intestinal epithelial cell accumulation of the cancer preventive polyphenol ellagic acid—extensive binding to protein and DNA

Ellagic acid (EA), a polyphenol present in many berries, has been demonstrated to be preventive of esophageal cancer in animals both at the initiation and promotion stages. To be able to extrapolate these findings to humans we have studied the transcellular absorption and epithelial cell accumulation of [14C]EA in the human intestinal Caco-2 cells. The apical (mucosal) to basolateral (serosal) transcellular transport of 10 microM [14C]EA was minimal with a P(app) of only 0.13 x 10(-6)cm/s, which is less than for the paracellular transport marker mannitol. In spite of observations of basolateral to apical efflux, Caco-2 cell uptake studies showed high accumulation of EA in the cells (1054+/-136 pmol/mg protein), indicating facile absorptive transport across the apical membrane. Surprisingly, as much as 93% of the cellular EA was irreversibly bound to macromolecules (982+/-151 pmol/mg protein). To confirm the irreversible nature of the binding to protein, Caco-2 cells treated with 10 microM [14C]EA were subjected to SDS-PAGE analysis. This resulted in radiolabeled protein bands trapped in the stacking gel, consistent with [14C]EA-crosslinked proteins. Treatment of Caco-2 cells with 10 microM [14C]EA also revealed irreversible binding of EA to cellular DNA as much as five times higher than for protein (5020+/-773 pmol/mg DNA). Whereas the irreversible binding to protein required oxidation of EA by reactive oxygen species, this did not seem to be the case with the DNA binding. The avid irreversible binding to cellular DNA and protein may be the reason for its highly limited transcellular absorption. Thus, EA appears to accumulate selectively in the epithelial cells of the aerodigestive tract, where its cancer preventive actions may be displayed.