Anthocyanin-derived phenolic acids form glucuronides following simulated gastrointestinal digestion and microsomal glucuronidation

Scope: Current research indicates that anthocyanins are primarily degraded to form phenolic acid products. However, no studies have yet demonstrated the metabolic conjugation of these anthocyanin‐derived phenolic acids in humans. Methods and results: Within the present study, a simulated gastrointestinal digestion model was used to evaluate the potential degradation of anthocyanins post‐consumption. Subsequently, cyanidin (Cy) and pelargonidin and their degradation products, protocatechuic acid and 4‐hydroxybenzoic acid, were incubated in the presence of human liver microsomes to assess their potential to form hepatic glucuronide conjugates. For structural conformation, phenolic glucuronides were chemically synthesised and compared to the microsomal metabolites. During the simulated gastric digestion, anthocyanin glycosides (200 μM) remained stable however their aglycone derivatives were significantly degraded (20% loss), while during subsequent pancreatic/intestinal digestion only pelargonidin‐3‐glucoside remained stable while cyanidin‐3‐glucoside (30% loss) and Cy and pelagonidin aglycones were significantly degraded (100% loss, respectively). Following microsomal metabolism, pelargonidin formed 4‐hydroxybenzoic acid, which was further metabolised (65%) to form two additional glucuronide conjugates, while Cy formed protocatechuic acid, which was further metabolised (43%) to form three glucuronide conjugates. Conclusions: We propose that following ingestion, anthocyanins may be found in the systemic circulation as free or conjugated phenolic acids, which should be a focus of future dietary interventions.     

Vascular deconjugation of quercetin glucuronide: The flavonoid paradox revealed?

Scope: The dietary flavonoid quercetin exerts protective cardiovascular effects. Because quercetin is rapidly metabolized into less active or inactive glucuronidated metabolites and the plasma concentrations of free quercetin are very low, a huge amount of scientific data generated along decades with the unconjugated compounds in vitro has been questioned. We aimed to determine whether glucuronidated quercetin can deconjugate in situ and whether deconjugation leads to a biological effect. Methods and results: Quercetin and quercetin‐3‐O‐glucuronide (Q3GA) were perfused through the isolated rat mesenteric vascular bed. Quercetin was rapidly metabolized in the mesentery. In contrast, the decay of Q3GA was slower and was accompanied by a progressive increase of quercetin in the perfusate and in the tissue over 6 h, which was prevented by the β‐glucuronidase inhibitor saccharolactone. Incubation of mesenteric arterial rings mounted in a wire myograph with Q3GA for ≥1 h resulted in a significant inhibition of the contractile response which was also prevented by saccharolactone. Moreover, the intravenous administration of Q3GA resulted in a slow onset and sustained blood pressure lowering effect, demonstrating for the first time that Q3GA has effects in vivo. Conclusion: We propose that Q3GA behaves as a quercetin carrier in plasma, which deconjugates in situ releasing the aglycone which is the final effector.     

Absorption,metabolism, and excretion of green tea flavan‐3‐ols in humans with an ileostomy

Green tea containing 634 μmol of flavan‐3‐ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0–24 h after ingestion were analysed by HPLC‐MS. The ileal fluid contained 70% of the ingested flavan‐3‐ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O‐linked sulphates and methyl‐sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan‐3‐ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101–256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan‐3‐ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8±0.6% of total flavan‐3‐ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (?)‐epicatechin and (+)‐catechin.     

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro

Scope: Zearalenone (ZEN) and α‐zearalanol (α‐ZAL, zeranol) were studied in differentiated Caco‐2 cells and in the Caco‐2 Millicell^® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. Methods and results: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14‐hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14‐hydroxyl group, involved the 16‐ and 7‐hydroxyl groups. Using the Caco‐2 Millicell^® system, an efficient transfer of the glucuronides and sulfates of ZEN and α‐ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P_app values) of ZEN, α‐ZAL and the ZEN metabolite α‐zearalenol were determined, using an initial apical concentration of 20 μM and a permeation time of 1 h. Conclusion: According to the P_app values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.     

Glucuronidation of zearalenone,zeranol and four metabolites in vitro: Formation of glucuronides by various microsomes and human UDP‐glucuronosyltransferase isoforms

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α‐zearalanol (α‐ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α‐ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP‐glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14‐hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α‐ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.