No abstract

We have investigated the bacterial-dependent metabolism of (2 )-epicatechin and (þ )-catechin using a pH-controlled, stirred, batch-culture fermentation system reflective of the distal region of the human large intestine. Incubation of (2)-epicatechin or (þ )-catechin (150 mg/l or 1000 mg/l) with faecal bacteria, led to the generation of 5-(3 0 ,4 0 -dihydroxyphenyl)-g-valerolactone, 5-phenyl-g-valerolactone and phenylpropionic acid. However, the formation of these metabolites from (þ )-catechin required its initial conversion to (þ )-epicatechin. The metabolism of both flavanols occurred in the presence of favourable carbon sources, notably sucrose and the prebiotic fructo-oligosaccharides, indicating that bacterial utilisation of flavanols also occurs when preferential energy sources are available. (þ )-Catechin incubation affected the growth of select microflora, resulting in a statistically significant increase in the growth of the Clostridium coccoides -Eubacterium rectale group, Bifidobacterium spp. and Escherichia coli, as well as a significant inhibitory effect on the growth of the C. histolyticum group. In contrast, the effect of (2)-epicatechin was less profound, only significantly increasing the growth of the C. coccoides -Eubacterium rectale group. These potential prebiotic effects for both (þ )-catechin and (2 )-epicatechin were most notable at the lower concentration of 150 mg/l. As both (2)-epicatechin and (þ )-catechin were converted to the same metabolites, the more dramatic change in the growth of distinct microfloral populations produced by (þ )-catechin incubation may be linked to the bacterial conversion of (þ )-catechin to (þ)-epicatechin. Together these data suggest that the consumption of flavanol-rich foods may support gut health through their ability to exert prebiotic actions. Flavanols: Prebiotics: Faecal microflora: Large intestineRepresenting one of the most important lifestyle factors, diet can strongly influence the incidence and onset of CVD (1) , and thus a healthy diet is an essential factor for healthy ageing (2) . A number of dietary intervention studies in human subjects and animals, in particular those using Vitis vinifera (grape), Camellia sinensis (tea) and Theobroma cacao (cocoa) have demonstrated beneficial effects on vascular function (3 -5) . While such foods and beverages differ greatly in chemical composition and macro-and micronutrient content, they have in common that they are amongst the major dietary sources of flavanols. The in vivo effects of flavanols will be dependent on the absorption and metabolism of flavanols in the gastrointestinal tract. Studies have indicated that flavanols are subject to extensive metabolism by phase I and II enzymes to yield O-methylated, sulfated and glucuronidated forms during transfer from the small-intestinal lumen to the portal blood (6) . However, significant amounts of ingested (2 )-epicatechin, (þ)-catechin, and their structurally related oligomeric forms (procyanidins), escape absorption in the small intestine, instead rea...

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H 2 S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H 2 S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO − ), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO − is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO − synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N 2 O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H 2 S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.sulfide | nitric oxide | nitroxyl | redox | gasotransmitter N itrogen and sulfur are essential for all known forms of life on Earth. Our planet's earliest atmosphere is likely to have contained only traces of O 2 but rather large amounts of hydrogen sulfide (H 2 S) (1). Indeed, sulfide may have supported life long before the emergence of O 2 and NO (2, 3).* This notion is consistent with a number of observations: H 2 S is essential for efficient abiotic amino acid generation as evidenced by the recent reanalysis of samples of Stanley Miller's original spark discharge experiments (4), sulfide is an efficient reductant in protometabolic reactions forming RNA, protein, and lipid precursors (5), and sulfide is both a bacterial and mitochondrial substrate (6), enabling even multicellular lifeforms to exist and reproduce under conditions of permanent anoxia (7). Thus, although eukaryotic cells may have originated from the symbiosis of sulfurreducing and -oxidizing lifeforms within a self-contained sulfur redox metabolome (8), sulfide may have been essential even earlier by providing the basic building blocks of ...

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (P app ) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity. Keywords: blood-brain barrier, flavonoids, glucuronidation, hesperetin, naringenin, neuroprotection. There is growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the CNS associated with a number of pathophysiological processes, including Alzheimer's disease, cerebrovascular disease such as strokes or lesions, Parkinson's disease, Creutzfeldt-Jakob disease and certain traumas (Coyle and Puttfarcken 1993;Cantuti-Castelvetri et al. 2000). In addition, changes in the optimal performance of the CNS may occur simply as a function of ageing, possibly exacerbating the motor and cognitive behavioural changes seen in these conditions (Shukitt-Hale 1999). Recent studies have highlighted an important role for the neuroprotective actions of dietary components, including flavonoids found in fruit, vegetables and plant-derived beverages (for a review see Abbreviations used: BBB, blood-brain barrier; b.END5, brain endothelial cell line from mouse; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; C3R, cyanidin-3-rutinoside; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; MTT, 2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OATP, organic anion transport polypeptide; P3G, pelargonidin-3-glucoside; P app , apparent permeability; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; RBE4, brain endothelial cell line from rat; RT, retention time; TEER, transendothelial electrical resistance.

No abstract

Background: The beneficial cardiovascular effects of vegetables may be underpinned by their high inorganic nitrate content.Objective: We sought to examine the effects of a 6-wk once-daily intake of dietary nitrate (nitrate-rich beetroot juice) compared with placebo intake (nitrate-depleted beetroot juice) on vascular and platelet function in untreated hypercholesterolemics.Design: A total of 69 subjects were recruited in this randomized, double-blind, placebo-controlled parallel study. The primary endpoint was the change in vascular function determined with the use of ultrasound flow-mediated dilatation (FMD).Results: Baseline characteristics were similar between the groups, with primary outcome data available for 67 patients. Dietary nitrate resulted in an absolute increase in the FMD response of 1.1% (an ∼24% improvement from baseline) with a worsening of 0.3% in the placebo group (P < 0.001). A small improvement in the aortic pulse wave velocity (i.e., a decrease of 0.22 m/s; 95% CI: −0.4, −0.3 m/s) was evident in the nitrate group, showing a trend (P = 0.06) to improvement in comparison with the placebo group. Dietary nitrate also caused a small but significant reduction (7.6%) in platelet-monocyte aggregates compared with an increase of 10.1% in the placebo group (P = 0.004), with statistically significant reductions in stimulated (ex vivo) P-selectin expression compared with the placebo group (P < 0.05) but no significant changes in unstimulated expression. No adverse effects of dietary nitrate were detected. The composition of the salivary microbiome was altered after the nitrate treatment but not after the placebo treatment (P < 0.01). The proportions of 78 bacterial taxa were different after the nitrate treatment; of those taxa present, 2 taxa were responsible for >1% of this change, with the proportions of Rothia mucilaginosa trending to increase and Neisseria flavescens (P < 0.01) increased after nitrate treatment relative to after placebo treatment.Conclusions: Sustained dietary nitrate ingestion improves vascular function in hypercholesterolemic patients. These changes are associated with alterations in the oral microbiome and, in particular, nitrate-reducing genera. Our findings provide additional support for the assessment of the potential of dietary nitrate as a preventative strategy against atherogenesis in larger cohorts. This trial was registered at clinicaltrials.gov as NCT01493752.

The "trophic level enrichment" between diet and body results in an overall increase in nitrogen isotopic values as the food chain is ascended. Quantifying the diet–body Δ15N spacing has proved difficult, particularly for humans. The value is usually assumed to be +3–5‰ in the archaeological literature. We report here the first (to our knowledge) data from humans on isotopically known diets, comparing dietary intake and a body tissue sample, that of red blood cells. Samples were taken from 11 subjects on controlled diets for a 30-day period, where the controlled diets were designed to match each individual's habitual diet, thus reducing problems with short-term changes in diet causing isotopic changes in the body pool. The Δ15Ndiet-RBC was measured as +3.5‰. Using measured offsets from other studies, we estimate the human Δ15Ndiet-keratin as +5.0–5.3‰, which is in good agreement with values derived from the two other studies using individual diet records. We also estimate a value for Δ15Ndiet-collagen of ≍6‰, again in combination with measured offsets from other studies. This value is larger than usually assumed in palaeodietary studies, which suggests that the proportion of animal protein in prehistoric human diet may have often been overestimated in isotopic studies of palaeodiet. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.