Platelet-mediated metabolism of the common dietary flavonoid, quercetin.

BACKGROUND: Flavonoid metabolites remain in blood for periods of time potentially long enough to allow interactions with cellular components of this tissue. It is well-established that flavonoids are metabolised within the intestine and liver into methylated, sulphated and glucuronidated counterparts, which inhibit platelet function. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate evidence suggesting platelets which contain metabolic enzymes, as an alternative location for flavonoid metabolism. Quercetin and a plasma metabolite of this compound, 4'-O-methyl quercetin (tamarixetin) were shown to gain access to the cytosolic compartment of platelets, using confocal microscopy. High performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that quercetin was transformed into a compound with a mass identical to tamarixetin, suggesting that the flavonoid was methylated by catechol-O-methyl transferase (COMT) within platelets. CONCLUSIONS/SIGNIFICANCE: Platelets potentially mediate a third phase of flavonoid metabolism, which may impact on the regulation of the function of these cells by metabolites of these dietary compounds.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutics

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments (similar to 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Effects of contrasting forage diets on phosphorus utilisation in lactating dairy cows

The effects of different forage mixtures on duodenal flow and faecal output of phosphorus (P) were measured in lactating dairy cows in two experiments. The forages comprised grass silage (GS) alone or mixtures of GS and urea-treated whole crop wheat (WCW). Replacement of GS by high levels of WCW resulted in a significant decrease in total P (TP), water-soluble P (WSP) and acid-soluble P (ASP) intakes, but increased phytate P (PP) and acid-insoluble P (AIP) intakes. Duodenal flows of all P fractions were unaffected by dietary treatments. However, the amounts of all fractions flowing to the intestine were much higher than the amounts consumed-with the exception of AIP, which was unchanged, and PP, which was considerably reduced. Diets did not significantly affect faecal outputs of TP, ASP, PP and AIP; however, faecal outputs of WSP were significantly higher on WCW than GS diets. Inclusion of WCW significantly decreased TP and WSP availabilities in the total tract but did not affect PP and AIP availabilities. A new fraction of P, which was not present in the feeds, was found in significant amounts in the intestines and proved to be soluble in dilute acid but insoluble in water. Estimated annual excretions for a herd of 100 cows ranged from 1550 to 1721 kg for total P of which 337-786 kg was WSP. The results suggest that GS based diets deposit more P in the environment. However, the potential to cause P pollution depends not only on the quantity but also on the nature of faecal P and on soil parameters. © 2004 Elsevier B.V. All rights reserved.

Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7 h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat. (C) 2008 Elsevier Inc. All rights reserved.

Production and metabolic effects of site of starch digestion in lactating dairy cattle

Milk solids yield in modern dairy cows has increased linearly over the last 50 years, stressing the need for maximal dietary energy intake to allow genetic potential for milk energy yield to be realized with minimal negative effects on health and reproduction. Feeding supplemental starch is a common approach for increasing the energy density of the ration and supplying carbon for meeting the substantial glucose requirement of the higher yielding cow. In this regard, it is a long held belief that feeding starch in forms that increase digestion in the small intestine and glucose absorption will benefit the cow in terms of energetic efficiency and production response, but data supporting this dogma are equivocal. This review will consider the impact of supplemental starch and site of starch digestion on metabolic and production responses of lactating dairy cows, including effects on feed intake, milk yield and composition, nutrient partitioning, the capacity of the small intestine for starch digestion, and nutrient absorption and metabolism by the splanchnic tissues (the portal-drained viscera and liver). Whilst there appears to be considerable capacity for starch digestion and glucose absorption in the lactating dairy cow, numerous strategic studies implementing postruminal starch or glucose infusions have observed increases in milk yield, but decreased milk fat concentration such that there is little effect on milk energy yield, even in early lactation. Measurements of energy balance confirm that the majority of the supplemental energy arising from postruminal starch digestion is used with high efficiency to support body adipose and protein retention, even in early lactation. These responses may be mediated by changes in insulin status, and be beneficial to the cow in terms of reproductive success and well-being. However, shifting starch digestion from the rumen impacts the nitrogen economy of the cow as well by shifting the microbial protein gained from starch digestion from potentially absorbable protein to endogenous faecal loss.

Nitrogen recycling through the gut and the nitrogen economy of ruminants: An asynchronous symbiosis

The extensive development of the ruminant forestomach sets apart their N economy from that of nonruminants in a number of respects. Extensive pregastric fermentation alters the profile of protein reaching the small intestine, largely through the transformation of nitrogenous compounds into microbial protein. This process is fueled primarily by carbohydrate fermentation and includes extensive recycling of N between the body and gut lumen pools. Nitrogen recycling occurs via blood and gut lumen exchanges of urea and NH3, as well as endogenous gut and secretory N entry into the gut lumen, and the subsequent digestion and absorption of microbial and endogenous protein. Factors controlling urea transfer to the gut from blood, including the contributions of urea transporters, remain equivocal. Ammonia produced by microbial degradation of urea and dietary and endogenous AA is utilized by microbial fermentation or absorbed and primarily converted to urea. Therefore, microbial growth and carbohydrate fermentation affect the extent of NH3 absorption and urea N recycling and excretion. The extensive recycling of N to the rumen represents an evolutionary advantage of the ruminant in terms of absorbable protein supply during periods of dietary protein deficiency, or asynchronous carbohydrate and protein supply, but incurs a cost of greater N intakes, especially in terms of excess N excretion. Efforts to improve the efficiency of N utilization in ruminants by synchronizing fermentable energy and N availability have generally met with limited success with regards to production responses. In contrast, imposing asynchrony through oscillating dietary protein concentration, or infrequent supplementation, surprisingly has not negatively affected production responses unless the frequency of supplementation is less than once every 3 d. In some cases, oscillation of dietary protein concentration has improved N retention compared with animals fed an equal amount of dietary protein on a daily basis. This may reflect benefits of Orn cycle adaptations and sustained recycling of urea to the gut. The microbial symbiosis of the ruminant is inherently adaptable to asynchronous N and energy supply. Recycling of urea to the gut buffers the effect of irregular dietary N supply such that intuitive benefits of rumen synchrony in terms of the efficiency of N utilization are typically not observed in practice.

Intracellular signalling pathways regulating the adaptation of skeletal muscle to exercise and nutritional changes

The focus of the present review is to assimilate current knowledge concerning the differing signalling transduction cascades that control muscle mass development and affect skeletal muscle phenotype following exercise or nutritional uptake. Effects of mechanical loading on protein synthesis are discussed. Muscle growth control is regulated by the interplay of growth promoting and growth suppressing factors, which act in concert. Much emphasis has been placed on understanding how increases in the rate of protein synthesis are induced in skeletal muscle during the adaptive process. One key point to emerge is that protein synthesis following resistance exercise or increased nutrient availability is mediated through changes in signal transduction involving the phosphorylation of mTOR and sequential activation of downstream targets. On the other hand, AMPK activation plays an important role in the inhibition of protein synthesis by suppressing the function of multiple translation regulators of the mTOR signalling pathway in response to cellular energy depletion and low metabolic conditions. The effects of exercise and/or nutritional uptake on the activation of signalling molecules that regulate protein synthesis are highlighted, providing a better understanding of the molecular changes in the cell.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

A system for studying epithelial-stromal interactions reveals distinct inductive abilities of stromal cells from benign prostatic hyperplasia and prostate cancer

The development of normal and abnormal glandular structures in the prostate is controlled at the endocrine and paracrine levels by reciprocal interactions between epithelium and stroma. To study these processes it is useful to have an efficient method of tissue acquisition for reproducible isolation of cells from defined histologies. Here we assessed the utility of a standardized system for acquisition and growth of prostatic cells from different regions of the prostate with different pathologies, and we compared the abilities of stromal cells from normal peripheral zone (PZ-S), benign prostatic hyperplasia (BPH-S), and cancer (CA-S) to induce the growth of a human prostatic epithelial cell line (BPH-1) in vivo. Using the tissue recombination method, we showed that grafting stromal cells (from any histology) alone, or BPH-1 epithelial cells alone produced no visible grafts. Recombining PZ-S with BPH-1 cells also produced no visible grafts (n = 15). Recombining BPH-S with BPH-1 cells generated small, well-organized and sharply demarcated grafts approximately 3-4 mm in diameter (n = 9), demonstrating a moderate inductive ability of BPH-S. Recombining CA-S with BPH-1 cells generated highly disorganized grafts that completely surrounded the host kidney and invaded into adjacent renal tissue, demonstrating induction of an aggressive phenotype. We conclude that acquisition of tissue from toluidine blue dye stained specimens is an efficient method to generate high quality epithelial and/or stromal cultures. Stromal cells derived by this method from areas of BPH and cancer induce epithelial cell growth in vivo which mimics the natural history of these diseases.

Influence of gut morphology on passive transport of freshwater bryozoans by waterfowl in Donana (southwestern Spain)

Waterbirds have been proposed as important vectors for the passive dispersal of those aquatic invertebrates and plants that lack a capacity for active dispersal between isolated water bodies. We analysed the frequency of internal transport of bryozoan propagules (statoblasts) by waterbirds in Donana, Spain, by examining their presence in the intestines and ceca of dead birds and analysing the role of different aspects of gut characteristics in explaining variation in the presence/absence and abundance of statoblasts. Of the 228 samples examined, 7.9% presented intact statoblasts of Plumatella fungosa (Pallas, 1768), Plumatella emarginata Allman, 1844, and two unidentified Plumatella species. For a given bird species, individuals with heavier gizzards and shorter ceca had a lower incidence and abundance of statoblasts in the lower gut. Grit mass and intestine length were unrelated to the presence or abundance of statoblasts. Our results suggest that waterbirds frequently transport bryozoans on a local scale, with lighter gizzards and longer ceca favouring such transport. Lighter gizzards are likely to destroy fewer propagules before they reach the lower gut. Species and individuals with longer ceca are particularly good candidates for long-distance dispersal of bryozoans, given the longer passage time of propagules that enter the ceca.

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the glucooligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.