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Biomedical subjects

G Williamson

Publications and source records attributed to G Williamson.

At least 19 recordsLinked to original sources

In vitro metabolism of anthocyanins by human gut microflora.

BACKGROUND: Only a small part of the dietary anthocyanins are absorbed. Thus large amounts of the ingested compounds are likely to enter the colon. In vitro and in vivo studies have shown that colonic bacteria transform various flavonoids to smaller phenolic acids. However, there is very little information on bacterial transformations of anthocyanins. AIM OF THE STUDY: was to explore if anthocyanin glycosides were deglycosylated,whether the resulting aglycones were degraded further to smaller phenolic compounds by colonic bacteria, and to characterise metabolites. METHODS: Isolated cyanidin-3-glucoside and -rutinoside were fermented in vitro using human faecal microbiota as an inoculum. Metabolites were analysed and characterised by HPLC-DAS and LC-MS. They were identified by comparing their characteristics with those of available standards, and semi-quantified using the amount of substrate analysed from samples at initial timepoint. RESULTS: Cyanidin-3-glucoside and cyanidin aglycone could be identified as intermediary metabolites of cyanidin-3-rutinoside. At early timepoints (before 2 h), the formation of protocatechuic acid as a major metabolite for both cyanidin glycosides and detection of lower molecular weight metabolites show that anthocyanins were converted by gut microflora. Furthermore, reconjugation of the aglycone with other groups, non-typical for dietary anthocyanins, was evident at the later (after 2h) timepoints. CONCLUSION: Bacterial metabolism of anthocyanins involves the cleavage of glycosidic linkages and breakdown of the anthocyanidin heterocycle.

Anthocyanins↗

Watching molecular processes with the atomic force microscope: dynamics of polymer adsorption and desorption at the single molecule level.

The formation of networks is an important step in the synthesis of many biological assemblies. For example, during the synthesis of plant cell walls the factors which dictate the arrangement of the polymeric constituents that make up the cell wall are not yet understood. Factors such as site-directed binding provide a possible theoretical background for beginning to understand the assembly of complex biological structures, but modelling of this process is difficult, time consuming and lacks experimental methods for verification. Through the use of atomic force microscopy (AFM) it has been demonstrated that changes in the binding of a single heterogeneous cell wall polysaccharide to a charged substrate can be followed in real time. Furthermore, subsequent image analysis allows the probability of binding of the molecule to be mapped to produce a real data set which is comparable with those obtained in simulation studies. In addition, these AFM studies have provided new mechanistic clues to the adsorption/desorption process of this polysaccharide.

Adsorption↗

Genomic typing of Pseudomonas aeruginosa isolates by comparison of Riboprinting and PFGE: correlation of experimental results with those predicted from the complete genome sequence of isolate PAO1.

The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P. aeruginosa PAO1. The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII. When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE. These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P. aeruginosa PAO1. Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates. This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence.

Bacterial Typing Techniques↗

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo.

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near future, it has become essential that studies reported in this journal reflect an adequate level of scientific scrutiny. Therefore a set of essential characteristics of studies has been defined. These basic requirements are default properties rather than non-negotiables: deviations are possible and useful, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9) Statistics; and (10) Quality Assurance.

Animals↗

Hydrolysis by lactase phlorizin hydrolase is the first step in the uptake of daidzein glucosides by rat small intestine in vitro.

1. Isoflavones are naturally occurring oestrogenic compounds found in plants, where they exist in the glycosylated form. A proportion of ingested glycosides appears to be absorbed in the upper gastrointestinal tract, where enterocytes play an important role in their metabolism. 2. One hypothesis is that ingestion may involve hydrolysis by the luminally exposed enzyme lactase phlorizin hydrolase (LPH), an enzyme expressed specifically at the small intestinal brush border. 3. Using an everted sac preparation of rat jejunum and an inhibitor of LPH, we investigated the absorption of daidzein-O(7)-glucoside (daidzin) and the effect of LPH inhibition on this process. It was demonstrated that LPH plays a major role in the deglycosylation of daidzin. 4. The hydrolysis product, daidzein, is absorbed by epithelial cells and glucuronidated to daidzein-O(7)-glucuronide, which is subsequently exported primarily to the serosal (vascular) side of the tissue rather than to the luminal side. 5. A small but significant proportion of the intact glycoside is also transferred to the serosal compartment, and in the presence of an LPH inhibitor this was enhanced with a corresponding reduction in deglucosylation and glucuronidation. 6. The results indicate that that LPH plays an important role in the metabolism of glycosylated phytochemicals, and that the expression and activity of this enzyme in the small intestine can modify the profile of metabolites appearing in the circulation.

Animals↗

Development of isothiocyanate-enriched broccoli, and its enhanced ability to induce phase 2 detoxification enzymes in mammalian cells.

Broccoli florets contain low levels of 3-methylsuphinylpropyl and 4-methylsulphinylbutyl glucosinolates. Following tissue disruption, these glucosinolates are hydrolysed to the corresponding isothiocyanates (ITCs), which have been associated with anticarcinogenic activity through a number of physiological mechanisms including the induction of phase II detoxification enzymes and apoptosis. In this paper, we describe the development of ITC-enriched broccoli through the introgression of three small segments of the genome of Brassica villosa, a wild relative of broccoli, each containing a quantitative trait locus (QTL), into a broccoli genetic background, via marker-assisted selection and analysis of glucosinolates in the florets of backcross populations. Epistatic and heterotic effects of these QTLs are described. The ITC-enriched broccoli had 80-times the ability to induce quinone reductase (a standard assay of phase II induction potential) when compared to standard commercial broccoli, due both to an increase in the precursor glucosinolates and a greater conversion of these into ITCs.

Anticarcinogenic Agents↗

Identification and quantification of polyphenol phytoestrogens in foods and human biological fluids.

We review the methods used to measure phytoestrogens (genistein, daidzein, lignans and their derivatives) in foods and biological fluids, and discuss advantages and disadvantages of each. The range of detection limits reported varies widely between individual laboratories, but generally the best reported sensitivity is as follows: immunoassay>HPLC-mass spectrometry=HPLC-multichannel electrochemical detection (coularray)>GC-single ion monitoring-mass spectrometry>HPLC-UV diode array>HPLC-single channel electrochemical detection. The best sensitivity reported so far is 0.002 pmol per assay for daidzein by radioimmunoassay. HPLC with UV diode array detection is the most commonly employed, but is the least sensitive and specific. GC and HPLC coupled with mass spectrometry or electrochemical detection are the most accurate and reproducible methods for a wide variety of analytes. Generally most methods, with the exception of immunoassay, have not been correlated with other methods. Recoveries from extraction methods, limits of detection, nature of compounds analysed and the internal standards used are summarised for more than 90 reports in the literature. From this data, it is clear that an inter-laboratory validation and correlation between a wide range of methods for phytoestrogen analysis is required. One underdeveloped area that requires particular attention is the analysis of plant lignans.

Body Fluids↗

Quercetin derivatives are deconjugated and converted to hydroxyphenylacetic acids but not methylated by human fecal flora in vitro.

By using a batch in vitro anaerobic fecal fermentation model, we have shown that the fecal microflora can rapidly deconjugate rutin, isoquercitrin, and a mixture of quercetin glucuronides. High levels of beta,D-glucosidase, alpha,L-rhamnosidase, and beta,D-glucuronidase were present. Rutin underwent deglycosylation, ring fission, and dehydroxylation. The main metabolite, 3,4-dihydroxyphenylacetic acid, appeared rapidly (2 h) and was dehydroxylated to 3-hydroxyphenylacetic acid within 8 h. The pattern of in vitro fermentation of rutin was not changed by changing the pH (6.0 or 6.9), fermentation scale (10 or 1000 mL), or donors of the inoculum. Hydroxyphenylacetic acids were not methylated by colon flora in vitro. The colonic microflora has enormous potential to transform flavonoids into lower molecular weight phenolics, and these might have protective biological activities in the colon. The site of absorption of flavonoids and the form in which they are absorbed are critical for determining their metabolic pathway and consequent biological activities in vivo.

3,4-Dihydroxyphenylacetic Acid↗

Evidence for consistent patterns between flavonoid structures and cellular activities.

A wide variety of plant-derived compounds, including the polyphenolic flavonoids, is present in the human diet or is consumed for medicinal reasons. Epidemiological and animal studies tend to suggest a protective effect of flavonoids against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological activity are still largely unknown. Antioxidant properties of the flavonoids have been postulated as a mechanism for putative protective effect against cardiovascular disease. Nevertheless, these properties alone are not sufficient to explain the anti-carcinogenic potential of these polyphenols. The mechanisms by which the molecules interact with cells or are absorbed by them are very important for determining the intracellular concentration and distribution of the metabolites to internal organs. With the exception of the cells lining the gastrointestinal tract, all other cells in the body are only exposed to flavonoid metabolites and degradation products. No previous studies have addressed this aspect of cellular exposure, except for some methylated metabolites. Within the last decade, reports on flavonoid activities have been largely associated with enzyme inhibition and anti-proliferative activity. From our recent work on the human colon cancer cell line HT29 and comparison with published studies, structure-function relationships demonstrate that antioxidant, enzyme inhibitor or anti-proliferative activities are dependent on particular structure motifs. The present review also presents a summary of mechanistic data on a few elected compounds.

Animals↗

Antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel.

Gallocatechins and a range of prodelphinidins were purified by high performance liquid chromatography from pomegranate peel. Gallocatechin, gallocatechin-(4-8)-catechin, gallocatechin-(4-8)-gallocatechin and catechin-(4-8)-gallocatechin were all identified, purified and quantified by LC-DAD-MS and MS-MS. The antioxidant properties of these compounds were assessed using two methods: (i) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; and (ii) scavenging of the radical cation of 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonate, ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). The prodelphinidin dimers were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer. However, in the lipid phase, only one of the dimers (gallocatechin-(4-8)-catechin) was significantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. This study represents the first report on the antioxidant properties of prodelphinidins.

Anthocyanins↗

Superoxide scavenging by polyphenols: effect of conjugation and dimerization.

The superoxide anion scavenging capacity of two flavonols (quercetin and kaempferol) and some of their conjugates (quercetin-3-rhamnoglucoside, quercetin-3-sophoroside, quercetin-3-sulphate, quercetin-3-glucuronide, kaempferol-3-sophoroside, kaempferol-3-glucuronide) and of several hydroxycinnamic acids (caffeic acid, ferulic acid, 5-5 diferulic acid, 8-O-4 diferulic acid and 8-8 diferulic acid) were studied. Superoxide anions were generated non-enzymatically in a phenazine methosulphate-NADH system and assayed by reduction of nitro-blue tetrazolium. Among the flavonols examined, the most effective scavengers of superoxide anions were the sophoroside, glucuronide and rhamnoglucoside conjugates. Conversely, quercetin-3-sulphate and the flavonol aglycones, exhibited some pro-oxidant activity at the range of concentrations tested (0.5-10 microM). These results show that conjugation has a marked effect on the scavenging capacity of flavonols and that the type of conjugate at the 3-position determines the final superoxide scavenging capacity. Caffeic acid and ferulic acid showed no effect on the generation of superoxide anions by phenazine methosulphate-NADH. However, dimerization of ferulic acid enhanced the superoxide scavenging capacity of this hydroxycinnamic acid, but this depended on the type of linkage between the monomers. The order, from highest to lowest, of superoxide radical scavenging capacity for the dimers of ferulic acid was: 5-5-diferulic acid > 8-O-4-diferulic acid > 8-8-diferulic acid.

Dimerization↗

Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose.

The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (K(d)) of 17 and 0.95 microM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent K(d) of 23 microM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.

Amino Acid Substitution↗

Flavonoid glucuronides are substrates for human liver beta-glucuronidase.

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.

Chromatography, High Pressure Liquid↗

Intestinal release and uptake of phenolic antioxidant diferulic acids.

Diferulic acids are potent antioxidants and are abundant structural components of plant cell walls, especially in cereal brans. As such, they are part of many human and animal diets and may contribute to the beneficial effect of cereal brans on health. However, these phenolics are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. This study provides the first evidence that diferulic acids can be absorbed via the gastrointestinal tract. The 5-5-, 8-O-4-, and 8-5-diferulic acids were identified in the plasma of rats after oral dosing with a mixture of the three acids in oil. Our study also reveals that human and rat colonic microflora contain esterase activity able to release 5-5-, 8-O-4-, and 8-5-diferulic acids from model compounds and dietary cereal brans, hence providing a mechanism for release of dietary diferulates prior to absorption of the free acids. In addition, cell-free extracts from human and rat small intestine mucosa exhibited esterase activity towards diferulate esters. Hence, we have shown that esterified diferulates can be released from cereal brans by intestinal enzymes, and that free diferulic acids can be absorbed and enter the circulatory system. Our results suggest that the phenolic antioxidant diferulic acids are bioavailable.

Administration, Oral↗

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley.

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.

Chemical Fractionation↗

Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals.

Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye and wheat brans. Hydrolysis by intestinal esterase(s) is very likely the major route for release of antioxidant hydroxycinnamic acids in vivo.

Animals↗

Isolation and characterization of human colonic bacteria able to hydrolyse chlorogenic acid.

AIMS: Conjugated hydroxycinnamates, such as chlorogenic acid (caffeoyl-quinic acid), are widely consumed in a Western diet, coffee being one of the richest sources. Ingested hydroxycinnamate esters can reach the large intestine essentially unaltered, and may then be hydrolysed by esterases produced by the indigenous microflora. This study is aimed at identifying bacterial species responsible for the release of natural antioxidants, such as hydroxycinnamic acids, in the human large intestine. METHODS AND RESULTS: Thirty-five isolates recovered after anaerobic batch culture incubation of human faecal bacteria in a chlorogenic acid-based medium were screened for cinnamoyl esterase activity. Six isolates released the hydroxycinnamate, ferulic acid, from its ethyl ester in a plate-screening assay, and these were identified through genotypic characterization (16S rRNA sequencing) as Escherichia coli (three isolates), Bifidobacterium lactis and Lactobacillus gasseri (two strains). Chlorogenic acid hydrolysing activities were essentially intracellular. These cinnamoyl esterase-producing organisms were devoid of other phenolic-degrading activities. CONCLUSION: The results show that certain gut bacteria, including some already recognized as potentially health-promoting (i.e. species belonging to the genera Bifidobacterium and Lactobacillus), are involved in the release of bioactive hydroxycinnamic acids in the human colon. SIGNIFICANCE AND IMPACT OF THE STUDY: Free hydroxycinnamates, including caffeic, ferulic and p-coumaric acids, exhibit antioxidant and anticarcinogenic properties both in vitro and in animal models. Given that the gut flora has a major role in human nutrition and health, some of the beneficial effects of phenolic acids may be ascribed to the microflora involved in metabolism.

Adult↗