Biomedical subjects

Gary Williamson

Publications and source records attributed to Gary Williamson.

At least 37 records · Page 2Linked to original sources

PubMed2004

A critical review of the bioavailability of glucosinolates and related compounds.

Glucosinolates (GLSs) are relatively inert (Z)-N-hydroximinosulfate esters, possessing a sulfur-linked beta-D-glucopyranose moiety and a variable side chain, found almost exclusively in cruciferous vegetables. Following cell disruption, they are hydrolysed by plant myrosinases, forming a group of chemically reactive and biologically active compounds. There is considerable evidence that these breakdown products, when consumed in the diet, may affect the risk of developing chronic diseases. However, in order for any compound to exert an activity in vivo, it is necessary to reach the site of action in an appropriate form and sufficient concentration. Deleterious and toxic effects may be observed at high concentrations: hence, bioavailability is a key factor defining the physiological, beneficial dose window of GLS hydrolysis products (GLS-HPs). For some GLS-HPs, this window can be rather narrow, and therefore is a critical parameter to be considered. In this review we critically evaluate the present state of knowledge on all factors that affect bioavailability of GLS-HPs. This includes liberation from the plant material, absorption from the digestive system, distribution around the body, metabolism and excretion.

Brassicaceae↗

PubMed2004

Interactions between sulforaphane and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells.

The isothiocyanate, sulforaphane and the flavonoid, apigenin modulate gene expression including phase II detoxifying enzymes, such as glutathione S-transferases (GST) and UDP-glucuronosyltransferases (UGT). Using undifferentiated CaCo-2 cells, we have examined the interactions between sulforaphane and apigenin in the regulation of UGT and GST expression. We show that apigenin induces UGT1A1 transcription (4-fold) but not GSTA1, and that sulforaphane induces both UGT1A1 (3.7-fold) and GSTA1 (2.8-fold) transcription in both dose- and time-dependent manners. The combination of sulforaphane and apigenin resulted in a synergistic induction of UGT1A1 mRNA up to 12-fold, although this interaction was not seen for GSTA1. Nuclear factor kappa B (NF-kappaB) mRNA was induced by apigenin and sulforaphane (2.5- and 2-fold, respectively). NF-kappaB translocation inhibitor SN50 and phosphatidylinositol 3-kinase (PI3) inhibitor LY294002 decreased the induction of GSTA1 by sulforaphane almost to baseline level. However, the MEK inhibitor PD98059 enhanced significantly the induction of GSTA1 by sulforaphane. This suggests that NF-kappaB and PI3-kinase signaling pathways play a role in GSTA1 gene expression. Conversely, the induction of UGT1A1 transcription by sulforaphane was totally abolished by PD98059, although PD98059 slightly enhanced (20%) the induction of UGT1A1 by apigenin implying that the induction of UGT1A1 by sulforaphane is mediated by MAPK/extracellular signal-regulated kinase kinase, whereas UGT1A1 induction by apigenin may be associated with NF-kappaB translocation since the NF-kappaB translocation inhibitor, SN50 enhanced the induction of UGT by apigenin. The results show that UGT1A1 and GSTA1 are regulated by sulforaphane through different signal transduction pathways and the differences in the mechanisms of modulation of UGT1A1 transcription by sulforaphane and apigenin resulted in a synergistic effect between these two compounds in the induction of UGT1A1.

Adenocarcinoma↗

PubMed2004

The feruloyl esterase system of Talaromyces stipitatus: production of three discrete feruloyl esterases, including a novel enzyme, TsFaeC, with a broad substrate specificity.

Several extracellular feruloyl esterases were produced by the mesophilic fungus Talaromyces stipitatus when grown on selective carbon sources in liquid media. Type-A and Type-B feruloyl esterases, as defined by their substrate specificity against methyl hydroxycinnamates, were produced during growth on wheat bran and sugar beet pulp, respectively. In addition, Tal. stipitatus produced a new type of esterase (TsFaeC) during growth on sugar beet pulp with a broader spectrum of activity (Type-C) against the (hydroxy)cinnamate esters than those previously described. All three enzymes were purified and N-terminal amino acid sequences and internal peptide sequences determined. The TsFaeC sequences were used to amplify a gene fragment from Tal. stipitatus genomic DNA. The flanking sequences were identified with the aid of RACE-RTPCR, and a full-length clone constructed. The faeC gene is present as a single copy and contains a single intron. The complete cDNA fragment contains an ORF of 1590bp, faeC, which is predicted to encode a 530 amino acid pre-protein, including a 25-residue signal peptide, and to produce a mature protein of M(R) 55 340Da. There was no evidence for a carbohydrate-binding domain in TsFaeC.

Amino Acid Sequence↗

PubMed2004

Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

ATP-Binding Cassette Transporters↗

PubMed2004

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy.

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.

Carbohydrate Sequence↗

PubMed2004

XIP-I, a xylanase inhibitor protein from wheat: a novel protein function.

Endo-(1,4)-beta-xylanases of plant and fungal origin play an important role in the degradation of arabinoxylans. Two distinct classes of proteinaceous endoxylanase inhibitors, the Triticum aestivum xylanase inhibitor (TAXI) and the xylanase inhibitor protein (XIP), have been identified in cereals. Engineering of proteins in conjunction with enzyme kinetics, thermodynamic, real-time interaction, and X-ray crystallographic studies has provided knowledge on the mechanism of inhibition of XIP-I towards endoxylanases. XIP-I is a 30 kDa protein which belongs to glycoside hydrolase family 18, and folds as a typical (beta/alpha)8 barrel. Although the inhibitor shows highest homology with plant chitinases, XIP-I does not hydrolyse chitin; probably due to structural differences in the XIP-I binding cleft. The inhibitor is specific for fungal xylanases from glycoside hydrolases families 10 and 11, but does not inhibit bacterial enzymes. The inhibition is competitive and, depending on the xylanase, the Ki value can be as low as 3.4 nM. Site-directed mutagenesis of a xylanase from Aspergillus niger suggested that the XIP-I binding site was the conserved hairpin loop "thumb" region of family 11 xylanases. Furthermore, XIP-I shows the ability to inhibit barley alpha-amylases of glycoside hydrolase family 13, providing the first example of a protein able to inhibit members of different glycoside hydrolase families (10, 11, and 13), and additionally a novel function for a protein of glycoside hydrolase family 18.

Amino Acid Sequence↗

PubMed2004

Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin.

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3'-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2 microM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3'-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide > quercetin > quercetin-3-glucuronide = quercetin-3-glucoside > catechin > quercetin-4'-glucuronide > isorhamnetin-3-glucuronide > quercetin-3'-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3'-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3'-sulfate > quercetin-7-glucuronide > quercetin-3-glucuronide > quercetin-4'-glucuronide = isorahmnetin-3-glucuronide). Kq values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3'-sulfate (approximately 4 microM)= quercetin > or = quercetin-7-glucuronide > quercetin-3-glucuronide = quercetin-3-glucoside > isorhamnetin-3-glucuronide > quercetin-4'-glucuronide (approximately 20 microM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.

Copper↗

PubMed2004

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes.

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent (12.2 +/- 0.2 and 7.5 +/- 0.2%, respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.

Animals↗

PubMed2004

How should we assess the effects of exposure to dietary polyphenols in vitro?

Human intervention studies have provided clear evidence that dietary polyphenols (eg, flavonoids--eg, flavonols--and isoflavones) are at least partly absorbed and that they have the potential to exert biological effects. Biological activity of polyphenols is often assessed by using cultured cells as tissue models; in almost all such studies, cells are treated with aglycones or polyphenol-rich extracts (derived from plants and foods), and data are reported at concentrations that elicited a response. There are 2 inherent flaws in such an approach. First, plasma and tissues are not exposed in vivo to polyphenols in these forms. Several human studies have identified the nature of polyphenol conjugates in vivo and have shown that dietary polyphenols undergo extensive modification during first-pass metabolism so that the forms reaching the blood and tissues are, in general, neither aglycones (except for green tea catechins) nor the same as the dietary source. Polyphenols are present as conjugates of glucuronate or sulfate, with or without methylation of the catechol functional group. As a consequence, the polyphenol conjugates are likely to possess different biological properties and distribution patterns within tissues and cells than do polyphenol aglycones. Although deconjugation can potentially occur in vivo to produce aglycone, it occurs only at certain sites. Second, the polyphenol concentrations tested should be of the same order as the maximum plasma concentrations attained after a polyphenol-rich meal, which are in the range of 0.1-10 micromol/L. For correct interpretation of results, future efforts to define biological activities of polyphenols must make use of the available data concerning bioavailability and metabolism in humans.

Biological Availability↗

PubMed2004

A review of the health effects of green tea catechins in in vivo animal models.

There is good evidence from in vitro studies that green tea catechins have a role in protection against degenerative diseases. However, the concentrations used in vitro are often higher than those found in animal or human plasma, and so in vivo evidence is required to demonstrate any protective effect of catechins. This article summarizes the most interesting in vivo animal studies on the protective effects of green tea catechins against biomarkers for cancer, cardiovascular disease, and other degenerative diseases. Generally, most studies using animal models show that consumption of green tea (catechins) provides some protection, although most studies have not examined dose response. Tea catechins could act as antitumorigenic agents and as immune modulators in immunodysfunction caused by transplanted tumors or by carcinogen treatment. Green tea has antiproliferative activity in hepatoma cells and hypolipidemic activity in hepatoma-treated rats, and some studies report that it prevents hepatoxicity. It could act as a preventive agent against mammary cancer postinitiation. Nevertheless, the implications of green tea catechins in preventing metastasis have not been clearly established. Long-term feeding of tea catechins could be beneficial for the suppression of high-fat diet-induced obesity by modulating lipid metabolism, could have a beneficial effect against lipid and glucose metabolism disorders implicated in type 2 diabetes, and could also reduce the risk of coronary disease. Further investigations on mechanisms, the nature of the active compounds, and appropriate dose levels are needed.

Animals↗

PubMed2004

Quercetin metabolites downregulate cyclooxygenase-2 transcription in human lymphocytes ex vivo but not in vivo.

Flavonoids have the potential to modulate inflammation by inhibition of cyclooxygenase-2 (COX-2) transcription. In this study, we compared the effect of the human flavonoid plasma metabolites (quercetin 3'-sulfate, quercetin 3-glucuronide and 3'-methylquercetin 3-glucuronide) on expression of COX-2 mRNA in human lymphocytes ex vivo using TaqMan real-time RT-PCR. We show that the flavonoid quercetin metabolites as detected in human plasma at physiologically significant concentrations inhibit COX-2 expression in human lymphocytes ex vivo. To examine the effect in vivo, we measured COX-2 mRNA levels in 8 subjects (5 men and 3 women) participating in a 3-way, single-blind, randomized crossover study after consumption of a single meal of white, low-quercetin onions, compared with yellow, high-quercetin onions. After consumption of high-quercetin onions, quercetin conjugates were detected in plasma (up to a maximum concentration of 4 micro mol/L at approximately 1 h). However, the expression of COX-2 mRNA in lymphocytes was unchanged by the consumption of high-quercetin onions compared with the low-quercetin group. The results show that a single high dose of the flavonoid quercetin from onions does not change COX-2 mRNA expression in human lymphocytes in vivo even though this change occurred in vitro and ex vivo.

Base Sequence↗

PubMed2003

Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes.

The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 micro M. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1-10 micro M), or the flavonoid, quercetin (5-20 micro M), significantly reduced the level of PhIP-DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/ micro g DNA) but at higher PhIP exposure (10 nM and 1 micro M), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP-DNA adduct formation was investigated using real-time RT-PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP-DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase beta. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP-DNA adduct repair. The formation of PhIP-DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.

Anticarcinogenic Agents↗

PubMed2003

Cross-inhibitory activity of cereal protein inhibitors against alpha-amylases and xylanases.

The purification and characterisation of a xylanase inhibitor (XIP-I) from wheat was reported previously. In our current work, XIP-I is also demonstrated to have the capacity to inhibit the two barley alpha-amylase isozymes (AMY1 and AMY2). XIP-I completely inhibited the activity of AMY1 and AMY2 towards insoluble Blue Starch and a soluble hepta-oligosaccharide derivative. A ternary complex was formed between insoluble starch, a catalytically inactive mutant of AMY1 (D180A), and XIP-I, suggesting that the substrate-XIP-I interaction is necessary for inhibition of barley alpha-amylases. K(i) values for alpha-amylase inhibition, however, could not be calculated due to the nonlinear nature of the inhibition pattern. Furthermore, surface plasmon resonance and gel electrophoresis did not indicate interaction between XIP-I and the alpha-amylases. The inhibition was abolished by CaCl(2), indicating that the driving force for the interaction is different from that of complexation between the barley alpha-amylase/subtilisin inhibitor (BASI) and AMY2. This is the first report of a proteinaceous inhibitor of AMY1. BASI, in addition, was demonstrated to partially inhibit the endo-1,4-beta-D-xylanase from Aspergillus niger (XylA) of glycoside hydrolase family 11. Taken together, the data demonstrate for the first time the dual target enzyme specificity of BASI and XIP-I inhibitors for xylanase and alpha-amylase.

Animals↗

PubMed2003

Substrate (aglycone) specificity of human cytosolic beta-glucosidase.

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays beta-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (beta/alpha)(8)-barrel fold characteristic of family 1 beta-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val(168)-->Tyr, Phe(225)-->Ser, Tyr(308)-->Ala and Tyr(308)-->Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val(168)-->Tyr mutation did not affect K (m) on p -nitrophenyl ( p NP)-glycosides, but increased K (m) 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe(225)-->Ser and Tyr(308)-->Ala mutations, and, to a lesser degree, the Tyr(308)-->Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

Amino Acid Sequence↗

PubMed2003

Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson).

A novel class of proteinaceous inhibitors exhibiting specificity towards microbial xylanases has recently been discovered in cereals. The three-dimensional structure of xylanase inhibitor protein I (XIP-I) from wheat (Triticum aestivum, var. Soisson) was determined by X-ray crystallography at 1.8 A (1 A=0.1 nm) resolution. The inhibitor possesses a (beta/alpha)(8) barrel fold and has structural features typical of glycoside hydrolase family 18, namely two consensus regions, approximately corresponding to the third and fourth barrel strands, and two non-proline cis -peptide bonds, Ser(36)-Phe and Trp(256)-Asp (in XIP-I numbering). However, detailed structural analysis of XIP-I revealed several differences in the region homologous with the active site of chitinases. The catalytic glutamic acid residue of family 18 chitinases [Glu(127) in hevamine, a chitinase/lysozyme from the rubber tree (Hevea brasiliensis)] is conserved in the structure of the inhibitor (Glu(128)), but its side chain is fully engaged in salt bridges with two neighbouring arginine residues. Gly(81), located in subsite -1 of hevamine, where the reaction intermediate is formed, is replaced by Tyr(80) in XIP-I. The tyrosine side chain fills the subsite area and makes a strong hydrogen bond with the side chain of Glu(190) located at the opposite side of the cleft, preventing access of the substrate to the catalytic glutamic acid. The structural differences in the inhibitor cleft structure probably account for the lack of activity of XIP-I towards chitin.

Amino Acid Sequence↗

PubMed2003

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy.

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.

Adsorption↗

PubMed2003

Absorption of quercetin-3-glucoside and quercetin-4'-glucoside in the rat small intestine: the role of lactase phlorizin hydrolase and the sodium-dependent glucose transporter.

Two hypotheses on absorption mechanisms of flavonoid glucosides across the small intestine have been proposed: active uptake of the quercetin glucoside by the sodium-dependent glucose transporter (SGLT1) with subsequent deglycosylation within the enterocyte by cytosolic beta-glucosidase, or luminal hydrolysis of the glucoside by lactase phlorizin hydrolase (LPH) and absorption by passive diffusion of the released aglycone. To test the above hypotheses we employed phlorizin (as an inhibitor of SGLT1) and N-(n-butyl)-deoxygalactonojirimycin (as an inhibitor of the lactase domain of LPH) in a rat everted-jejunal sac model. Quercetin-4'-glucoside mucosal hydrolysis was 10 times greater than quercetin-3-glucoside hydrolysis in the absence of inhibitors (449 and 47 nmol g(-1) tissue, respectively), despite the similar amounts (13+/-4 and 9+/-1 nmol g(-1), respectively) being transferred to the serosal compartment during the 15 min incubation. Apical hydrolysis of both quercetin glucosides was significantly reduced in the presence of NB-DGJ (80%), and transfer of quercetin (measured as quercetin metabolites) to the serosal solution was also significantly reduced (40-50%). In the presence of phlorizin, transfer of metabolites to the serosal solution was only reduced in the case of quercetin-4'-glucoside. Evidently the mechanism of absorption of quercetin-4'-glucoside involves both an interaction with SGLT1 and luminal hydrolysis by LPH, whereas quercetin-3-glucoside appears to be absorbed only following hydrolysis by LPH.

Analysis of Variance↗

PubMed2003

Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism.

Quercetin-3- and quercetin-7-glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10(6) cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10(6) cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous beta-glucuronidase followed by sulfation to quercetin-3'-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10(6) cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10(6) cells, over 48 hr). In contrast, quercetin-4'-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by beta-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol-O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3'-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10(6) cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin glucuronides and that human endogenous beta-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

Catechol O-Methyltransferase↗