Biomedical subjects
Publications and source records attributed to G Williamson.
Syntheses of the title compounds--commonly known as 'daidzein 7-glucuronide' and 'daidzein 4',7-diglucuronide'--are described. Selective 7-deacetylation of 4',7-di-O-acetyldaidzein is employed.
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.
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.
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.
In this review many examples are given of the complexities involved in using some biomarkers in relation to assessing the effects of dietary exposure, when there is frequently a need to determine changes following long-term low level exposure to dietary components. These range from understanding why the biomarker might be valuable and how best it can be measured, to the pitfalls which can occur in the interpretation of data. Analytical technique is considered in relation to folate and selenium, and flavonoid and carotenoid species are used to illustrate how the metabolism of a compound may alter the validity or adequacy of a marker. Vitamin A is discussed in relation to the difficulties which can arise when there are several biomarkers that may be available to assess exposure to one nutrient. Vitamin B12 is discussed in relation to the dietary choices made by individuals. Possible interactions and the role of measuring total antioxidant capacity is considered in some detail. In contrast to most nutrients, there is a marked lack of biomarkers of either exposure or effect for most non-nutrients. The role of biological effect monitoring is considered for dietary contaminants, fumonisins and polyhalogenated aromatic hydrocarbons. Aflatoxins are discussed to exemplify food contaminants for which the biomarker approach has been extensively studied. Finally some compounds which are deliberately added to foods and some which appear as processing contaminants are each considered briefly in relation to the requirement for a biomarker of exposure to be developed.
Quercetin, a polyphenol with potential health effects, is absorbed by humans and measurement in plasma can be used as a biomarker for intake. However, the chemical nature of the quercetin in blood is still not known, although one possibility is that glucosides are found in an unchanged form from the original food. We propose that the existence of quercetin glucosides in plasma is unlikely, since the metabolic beta-glucosidase capacity of the small intestine and of the liver is too great for quercetin glucosides to escape deglycosylation. We critically examine the limited number of studies which purport to detect quercetin glycosides in blood and the current evidence for the absorption of these compounds from the gastrointestinal tract. We emphasise the need for comprehensive identification of circulating compounds, since polyphenol glucuronides, the expected metabolites in plasma, have almost identical chromatographic properties to the glucosides at acid pH. Studies on the nature of quercetin metabolites in plasma are urgently needed so that the proposed biological activities of quercetin can be re-assessed and that a suitable biomarker of exposure can be established.
The position of conjugation of the flavonoid quercetin dramatically affects biological activity in vitro, therefore it is important to determine the exact nature of the plasma metabolites. In the present study, we have used various methods (HPLC with diode array detection, LCMS, chemical and enzymic synthesis of authentic conjugates and specific enzymic hydrolysis) to show that quercetin glucosides are not present in plasma of human subjects 1.5 h after consumption of onions (a rich source of flavonoid glucosides). All four individuals had similar qualitative profiles of metabolites. The major circulating compounds in the plasma after 1.5 h are identified as quercetin-3-glucuronide, 3'-methylquercetin-3-glucuronide and quercetin-3'-sulfate. The existence of substitutions in the B and/or C ring of plasma quercetin metabolites suggests that these conjugates will each have very different biological activities.
The consumption of cooked meat appears to predispose individuals to colonic cancer and heterocyclic aromatic amines (HA), formed during the cooking of meat, have been suggested as aetiological agents. Consumption of cruciferous vegetables is thought to protect against cancer. To study the effect of cruciferous vegetables on heterocyclic aromatic amine metabolism in man, a three-period, dietary intervention study has been carried out with 20 non-smoking Caucasian male subjects consuming cooked meat meals containing known amounts of these carcinogens. A high cruciferous vegetable diet (250 g each of Brussels sprouts and broccoli per day) was maintained during period 2 but such vegetables were excluded from periods 1 and 3. At the end of each period, subjects consumed a cooked meat meal and urinary excretion of the HA 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was measured. Following a 12 day period of cruciferous vegetable consumption (period 2), induction of hepatic CYP1A2 activity was apparent from changes in the kinetics of caffeine metabolism. Excretion of MeIQx and PhIP in urine at the end of this period of the study was reduced by 23 and 21%, respectively, compared with period 1. This reduction in excretion is probably due to an increase in amine metabolism that might be expected given the observed increase in CYP1A2 activity, since this enzyme has been shown to be primarily responsible for the oxidative activation of MeIQx and PhIP in man. In period 2, urinary mutagenicity was increased relative to period 1 by 52 and 64% in the absence and presence, respectively, of a human liver microsomal activation system, yet no evidence was found of PhIP adduction to lymphocyte DNA, a potential biomarker of the activation process. After another 12 days without cruciferous vegetables (period 3 of the study), the kinetics of caffeine metabolism had returned to original values but excretion of MeIQx and PhIP was still reduced by 17 and 30%, respectively, and urinary mutagenicity (with metabolic activation) was still elevated compared with period 1. This prolonged response of amine metabolism to the cruciferous vegetable diet, shown especially with PhIP, suggests that enzyme systems other than CYP1A2 are involved and affected by a cruciferous vegetable diet.
The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l(-1) were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.
Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4'- > 3'- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The K(i) for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4'- > 3'- > 7- > 3-, with quercetin-4'-glucuronide a particularly potent inhibitor (K(i) = 0. 25 microM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations.
Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.
Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.
Microorganisms able to produce vanillin in excess of 6g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun.
Eight varieties of lettuce (Lactuca sativum) and three varieties of endive (Cichorium endivia) were analyzed for flavonoid composition and content. Total flavonoid contents, expressed as units of aglycon for fresh material, were in the ranges of 0.3-229 microg/g for lettuce and 44-248 microg/g for endive. Five quercetin conjugates [quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-(6-O-malonyl)glucoside, and quercetin 3-O-rhamnoside] and luteolin 7-O-glucuronide were measured in the green-leafed lettuce and an additional two cyanidin conjugates [cyanidin 3-O-glucoside and cyanidin 3-O-[(6-O-malonyl)glucoside]] in the red-leafed varieties. Three kaempferol conjugates [kaempferol 3-O-glucoside, kaempferol 3-O-glucuronide, and kaempferol 3-O-[6-O-malonyl)glucoside]] were measured in each of the endive varieties. The presence and identity of kaempferol 3-O-(6-O-malonyl)glucoside in endive was shown for the first time. Shredding of lettuce leaf followed by exposure to light produced significant losses of the flavonoid moiety in the green oak leaf (94%), red oak leaf (43%), iceberg (36%), green batavia (25%), lollo biondo (24%), and lollo rosso (6%) samples, whereas cos and green salad bowl samples did not show an overall loss. Shredding of endive also produced loss of the flavonoid moiety in escarole (32%), fine frisee (13%), and coarse frisee (8%). Significant demalonation was observed for both the quercetin and cyanidin glucosides in lettuce, whereas a similar degradation of the kaempferol analogue was found in endive tissue. Storage of whole heads of both lettuce and endive in the dark at 1 degrees C and 98% humidity for 7 days resulted in losses of total flavonol glycosides in the range of 7-46%. The identification of the amounts, position of substitution, and nature of the sugars is important for understanding the potential bioavailability and biological activities of flavonoids in salads.
To investigate the etiology of equine dysautonomia (ED), a degenerative polyneuropathy affecting grazing horses, the biochemical composition and antioxidant/prooxidant activities of aqueous extracts of plants collected from ED pastures were determined. Plants collected immediately after an outbreak of ED had reduced antioxidant and weak prooxidant activities when compared with control plants (plants collected from ED pastures out of ED season and control plants from ED pastures that were grown under favorable conditions). ED plants also had significantly increased concentrations of fructose and low molecular weight phenolic compounds, significantly more of one amino acid zone (probably valine), significantly less tartaric acid, and a nonsignificant decrease in ascorbic acid content when compared with control plants from ED pastures that were grown under favorable conditions. These findings suggest that ED plants may be under oxidative stress, possibly due to chilling, drought, or fungal colonization. However, experimental drought and chilling of plants did not reproduce the biochemical alterations identified in ED plants. It is possible that the altered biochemical content of ingested plants may contribute, directly or indirectly, to the development of ED in grazing horses.
Flavonoids and cinnamates are widespread phenolic secondary metabolites synthesized by plants for defensive purposes. Many foods and beverages contain high levels of phenolic compounds. Certain phenolics in the diet are particularly bioactive and have pronounced effects on mammalian cells. These effects, together with epidemiological studies and animal models, have led to the hypothesis that dietary phenolics contribute to the health benefits of a diet rich in fruit and vegetables. This paper examines the biochemistry of the uptake and metabolic route of two groups of plant phenolics, the flavonols and hydroxycinnamates.
An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.
Watercress is an exceptionally rich dietary source of beta-phenylethyl isothiocyanate (PEITC). This compound inhibits phase I enzymes, which are responsible for the activation of many carcinogens in animals, and induces phase II enzymes, which are associated with enhanced excretion of carcinogens. In this study, we show that watercress extracts are potent inducers of quinone reductase (QR) in murine hepatoma Hepa 1c1c7 cells, a widely adopted assay for measuring phase II enzyme induction. However, contrary to expectations, this induction was not associated with PEITC (which is rapidly lost to the atmosphere upon tissue disruption due to its volatility) or a naturally occurring PEITC-glutathione conjugate, but with 7-methylsulfinyheptyl and 8-methylsulfinyloctyl isothiocyanates (ITCs). While it was confirmed that PEITC does induce QR (5 microM required for a two-fold induction in QR), 7-methylsulfinyheptyl and 8-methylsulfinyloctyl ITCs were more potent inducers (0.2 microM and 0.5 microM, respectively, required for a two-fold induction in QR). Thus, while watercress contains three times more phenylethyl glucosinolate than methylsulfinylalkyl glucosinolates, ITCs derived from methylsulfinylalkyl glucosinolates may be more important phase II enzyme inducers than PEITC, having 10 - to 25-fold greater potency. Analysis of urine by liquid chromatography-mass spectroscopy (LC-MS) following consumption of watercress demonstrated the presence of N:-acetylcysteine conjugates of 7-methylsulfinylheptyl, 8-methylsulfinyloctyl ITCs and PEITC, indicating that these ITCs are taken up by the gut and metabolized in the body. Watercress may have exceptionally good anticarcinogenic potential, as it combines a potent inhibitor of phase I enzymes (PEITC) with at least three inducers of phase II enzymes (PEITC, 7-methylsulfinylheptyl ITC and 8-methylsulfinyloctyl ITC). The study also demonstrates the application of LC-MS for the detection of complex glucosinolate-derived metabolites in plant extracts and urine.