Paul W. Needs

Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide

Abstract A comparison of the methylation of cellobiitol by the Hakomori method (using potassium methyl-sulfinylmethanide - “potassium dimsyl” - as base) and by a published or a modified sodium hydroxide-mediated procedure is described. Evidence is presented that the modified sodium hydroxide-catalysed procedure is not prone to the oxidative deficiencies of the original and that, given its reduced tendency towards polysaccharide undermethylation, it should now become the method of choice for the methylation of these polymers.

Flavonoid glucuronides are substrates for human liver β‐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 β‐glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β‐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 β‐glucuronidase inhibitor). Furthermore, pure recombinant human β‐glucuronidase hydrolysed various flavonoid glucuronides, with a 20‐fold variation in catalytic efficiency (k cat/K m=1.3×103 M−1 s−1 for equol‐7‐O‐glucuronide and 26×103 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 β‐glucuronidase from various human cells.

Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate

Background Epidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet. Methods and Findings Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFβ1) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFβ1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFβ1, EGF and insulin peptides to form thioureas, and enhances TGFβ1/Smad-mediated transcription. Conclusions These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease. Trial Registration ClinicalTrials.gov NCT00535977

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 μM) 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 (∼4 μM)=quercetin≥quercetin-7-glucuronide>quercetin-3-glucuronide=quercetin-3-glucoside>isorhamnetin-3-glucuronide>quercetin-4′-glucuronide (∼20 μM). 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.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPE Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. METHODS AND RESULTS Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. CONCLUSION The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

INTERACTION OF POSITIONAL ISOMERS OF QUERCETIN GLUCURONIDES WITH THE TRANSPORTER ABCC2 (CMOAT, MRP2)

The exporter ABCC2 (cMOAT, MRP2) is a membrane-bound protein on the apical side of enterocytes and hepatic biliary vessels that transports leukotriene C4, glutathione, some conjugated bile salts, drugs, xenobiotics, and phytonutrients. The latter class includes quercetin, a bioactive flavonoid found in foods such as onions, apples, tea, and wine. There is no available three-dimensional (3D) structure of ABCC2. We have predicted the 3D structure by in silico modeling, showing that 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) binds most tightly to the putative binding site, and then tested the computational prediction experimentally by measuring interaction with all quercetin monoglucuronides occurring in vivo (quercetin substituted with glucuronic acid at the 3-, 3′-, 4′-, and 7-hydroxyl groups). The 4′-O-β-D-glucuronide is predicted in silico to interact most strongly and the 3-O-β-D-glucuronide most weakly, and this prediction is supported experimentally using binding and competition assays on ABCC2-overexpressing baculovirus-infected Sf9 cells. To test the transport in situ, we examined the effect of two ABCC2 inhibitors, MK571 and cyclosporin A, on the transport into the media of quercetin glucuronides produced intracellularly by Caco2 cells. The inhibitors reduced the amount of all quercetin glucuronides in the media. The results show that the molecular model of ABCC2 agrees well with experimentally determined ABCC2-ligand interactions and, importantly, that the interaction of ABCC2 with quercetin glucuronides is dependent on the position and nature of substitution.

Calcium gelation of pectic polysaccharides isolated from unripe tomato fruit

Abstract Cell-wall material was prepared from unripe tomato fruit, and a pectic polysaccharide extracted with cyclohexanediaminetetraacetic acid. The structure of the purified pectic polysaccharide was examined by sugar and methylation analysis, and was typical of a rhamnogalacturonan from the primary cell wall. The physicochemical properties of the isolated polysaccharide were characterised by viscometry and size-exclusion chromatography. The polysaccharide was polydisperse, but of large molecular size as indicated by an intrinsic viscosity of 810 ml. g −1 . At concentrations above ∼ 0.2–0.6% w/w, coil entanglement was observed as an increase in the dependency of viscosity on concentration. For these concentrated solutions, clear elastic gels were formed on addition of calcium ions. At concentrations in the range 0.6–2.8% w/w the shear modulus of the gel showed a c 1.9 dependence on concentration. The modulus of the gel increased linearly with absolute temperature in a rubberlike way, enabling an estimate of cross-link density to be made.

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.

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.

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery

Background and purpose:  Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery.