Short communication: Effects of oral flavonoid supplementation on the metabolic and antioxidative status of newborn dairy calves.

Scientific proof for flavonoids as a health tool in calf nutrition is inconsistent. We investigated the effects of the most abundant flavonoid, quercetin, and of a green tea extract (GTE) containing various catechins on the metabolic and antioxidative traits in dairy calves to clarify their potential health-promoting effects. Male newborn German Holstein calves (n=7 per group) received either no flavonoid (control group), 10mg of quercetin equivalents as quercetin aglycone or as rutin/kg of body weight (BW) per day, or 10mg/kg of BW per day of a GTE from d 2 to 26 of life. The supplements were provided with the morning and evening feeding. The calves were fed colostrum and milk replacer, and BW, feed intake, and health status were evaluated daily. Blood samples were collected from a jugular vein on d 1, 5, 12, 19, and 26 before the morning feeding to investigate the metabolic and antioxidative status of the calves. The growth performance and health status remained unchanged, but the GTE-fed calves had fewer loose feces than the controls. The plasma concentrations of quercetin changed over time and were higher in the rutin-fed group than in the control group, whereas the catechins were below the detection limit. The plasma Trolox equivalent antioxidative capacity and ferric reducing ability of plasma were measured as markers for plasma antioxidative capacity. The concentrations of Trolox equivalent antioxidative capacity increased, whereas ferric reducing ability of plasma decreased after the first day of life in all the groups. The oxidative stress markers in the plasma were measured as thiobarbituric acid reactive substances and F2-isoprostanes, but these did not indicate treatment or time effects. The plasma concentrations of total protein, albumin, urea, lactate, glucose, and nonesterified fatty acids and of insulin and cortisol varied over time, but no group differences were caused by the flavonoid supplementation. In summary, orally administered quercetin and catechins at the dosages used in the present study resulted in weak effects on health and no effects on the metabolic and antioxidative status of newborn dairy calves.

Bioavailability of the flavonol quercetin in neonatal calves after oral administration of quercetin aglycone or rutin.

Polyphenols, such as flavonoids, are secondary plant metabolites with potentially health-promoting properties. In newborn calves flavonoids may improve health status, but little is known about the systemically availability of flavonoids in calves to exert biological effects. The aim of this study was to investigate the oral bioavailability of the flavonol quercetin, applied either as quercetin aglycone (QA) or as its glucorhamnoside rutin (RU), in newborn dairy calves. Twenty-one male newborn German Holstein calves were fed equal amounts of colostrum and milk replacer according to body weight. On d 2 and 29 of life, 9 mg of quercetin equivalents/kg of body weight, either fed as QA or as RU, or no quercetin (control group) were fed together with the morning meal. Blood samples were taken before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 12, 24, and 48 h after feed intake. Quercetin and quercetin metabolites with an intact flavonol structure (isorhamnetin, tamarixetin, and kaempferol) were analyzed in blood plasma after treatment with glucuronidase or sulfatase by HPLC with fluorescence detection. Maximum individual plasma concentration was depicted from the concentration-time-curve on d 2 and 29, respectively. Additional blood samples were taken to measure basal plasma concentrations of total protein, albumin, urea, and lactate as well as pre- and postprandial plasma concentrations of glucose, nonesterified fatty acids, insulin, and cortisol. Plasma concentrations of quercetin and its metabolites were significantly higher on d 2 than on d 29 of life, and administration of QA resulted in higher plasma concentrations of quercetin and its metabolites than RU. The relative bioavailability of total flavonols (sum of quercetin and its metabolites isorhamnetin, tamarixetin, and kaempferol) from RU was 72.5% on d 2 and 49.6% on d 29 when compared with QA (100%). Calves fed QA reached maximum plasma concentrations of total flavonols much earlier than did RU-fed calves. Plasma metabolites and hormones were barely affected by QA and RU feeding in this experiment. Taken together, orally administrated QA resulted in a greater bioavailability of quercetin than RU on d 2 and 29, respectively, and quercetin bioavailability of quercetin and its metabolites differed markedly between calves aged 2 and 29 d.

Ruminal degradation of quercetin and its influence on fermentation in ruminants.

The aim of the present study was to investigate the ruminal degradation of the flavonol quercetin and to determine its potential antimicrobial effects on ruminal fermentation in cows. Ruminal degradation of quercetin (0 or 100µmol/L, respectively) as well as its influence on ruminal gas production (0, 50, or 100µmol of quercetin equivalents/L, respectively, either applied as aglycone or as its glucorhamnoside rutin) using concentrate, grass hay, and straw as substrates were investigated in vitro using the Hohenheim gas test. Additionally, the influence of quercetin on ruminal concentrations of volatile fatty acids and their molar ratio in rumen-fistulated, nonlactating cows (n=5) after intraruminal application of quercetin as aglycone or as rutin (0, 10, or 50mg of quercetin equivalents/kg of BW, respectively) was evaluated. Quercetin was rapidly and extensively degraded, whereby the disappearance of quercetin was accompanied by the simultaneous appearance of 2metabolites 3,4-dihydroxyphenylacetic acid and 4-methylcatechol. In vitro total gas and methane production were not reduced by the addition of quercetin aglycone or rutin, respectively, using concentrate, grass hay, and straw as substrates. As expected, however, effects of the substrates used were detected on total gas and methane production. Highest gas production was found with concentrate, whereas values obtained with grass hay and straw were lower. Relative methane production was highest with grass hay compared with concentrate and straw (27.1 vs. 25.0 and 25.5%). After intraruminal application of the quercetin aglycone or rutin, respectively, neither total concentration nor the molar ratio of volatile fatty acids in the rumen fluid were influenced. Results of the present study show that quercetin underlies rapid ruminal degradation, whereby 3,4-dihydroxyphenylacetic acid and 4-methylcatechol are the main metabolites, whereas the latter one most likely is formed by dehydroxylation from 3,4-dihydroxyphenylacetic acid. Regarding antimicrobial effects of quercetin, results obtained indicate that fermentation processes in the forestomachs are not substantially influenced by quercetin or rutin, respectively. With regard to potential health-promoting effects of quercetin, its application in cows, especially in the form of the better available rutin, might not be accompanied by negative effects on ruminal fermentation.

Investigating the efficacy of purified tannin extracts from underutilized temperate forages in reducing enteric methane emissions in vitro.

The study investigated how the concentration and composition of purified tannin extracts, at various inclusion rates, affect the ruminal in vitro fermentation parameters. Tannin extracts were isolated from four different forage species: birdsfoot trefoil (Lotus corniculatus), sulla (Hedysarum coronarium), big trefoil (Lotus pedunculatus), and salad burnet (Sanguisorba minor). Plants extracts were purified by Sephadex LH-20 gel chromatography and analyzed by UPLC-ESI-MS/MS. The results showed a large variation among the extracts from different species in terms of tannin composition and structural features. The extracts from salad burnet were dominated by hydrolysable tannins, comprising mainly ellagitannins. The extracts derived from sulla and big trefoil contained predominantly proanthocyanidins (PA), primarily composed of prodelphinidins with high mean degree of polymerisation (mDP). Birdsfoot trefoil extracts comprised procyanidin-rich PAs with low mDP. To determine whether the combined presence of tannins and flavonoid together lead to synergistic or antagonistic effects, the tannin extracts were incubated both with or without rutin at concentrations of 10, 20, and 30 g/kg DM, using a base substrate of perennial ryegrass (Lolium perenne, control). In general, all the tannin extracts decreased methane (CH4) production compared to the control, while no significant effect of rutin was observed on both gas (GP) and CH4 production, neither pure, nor in the simultaneous presence of tannins. The highest CH4 reduction (15%, at 30 g/kg DM) was observed from sulla and big trefoil extracts compared to control, but this was also supplemented with a concomitant reduction in GP (11%) indicating a reduction in feed digestibility. The extracts from birdsfoot trefoil and salad burnet reduced CH4 by up to 12% without significantly reducing GP, indicating the importance of tannin composition on ruminal fermentation.

Bioavailability of quercetin from its aglycone and its glucorhamnoside rutin in lactating dairy cows after intraduodenal administration.

Because of their health-promoting properties, flavonoids are used in feed supplements for ruminants, although scientific evidence for their efficacy in vivo is limited. It has been shown recently that bioavailability of quercetin is low after ruminal administration in cows because of degradation by the ruminal microbiota. It is unknown whether quercetin could be absorbed from the small intestine in ruminants if degradation is prevented; therefore, we investigated the bioavailability of quercetin after duodenal administration in 6 German Holstein cows. On 88 ± 3 d in milk, each cow received equivalent doses of quercetin [9, 18, or 27 mg of quercetin equivalents (QE)/kg of body weight] either as quercetin aglycone (QA) or as its glucorhamnoside rutin (RU). In addition, 2 control studies with duodenal administration of NaCl solution (0.9%) were conducted per cow to examine concentrations of flavonoids in plasma during regular feeding. Blood samples were collected at defined time intervals over a period of 24h before and after administration of the test compounds. A washout period of 2d was applied between the runs to avoid possible carryover effects. Concentrations of plasma quercetin aglycone and its metabolites isorhamnetin, tamarixetin, and kaempferol were measured after treatment with glucuronidase/sulfatase by HPLC with fluorescence detection. After administration of RU, levels of plasma quercetin did not increase above baseline, irrespective of dose administered. After duodenal administration of QA, the plasma concentration of QA and its methylated metabolites clearly increased above baseline. The maximal plasma concentrations of total flavonols (about 2h after application) increased in a dose-dependent manner but showed high interindividual variability (range 368.8 to 983.3 nmol/L at 27 mg of QE/kg of body weight) but peak time did not differ. Preadministration baseline values of total flavonols were reached again 3 to 4h after QA administration. The bioavailability of quercetin and its metabolites, as measured by the area under the concentration-time curve, was affected by the quercetin source applied, whereby quercetin from RU was unavailable. Taken together, duodenal administration enhanced bioavailability of QA almost to values previously reported in pigs after oral administration of QA. In contrast to findings in monogastrics or after oral administration in cows, quercetin from RU seems to be unavailable when administered duodenally.

Influence of 4-week intraduodenal supplementation of quercetin on performance, glucose metabolism, and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows.

Quercetin has been shown to be a potent antioxidant, acts hepatoprotectively, and affects glucose and lipid metabolism in monogastrics. If this is also true in ruminants, quercetin could be beneficial in periparturient high-yielding dairy cows by ameliorating the negative effects of free radical formation and reducing the severity of liver lipidosis and ketosis. In a first attempt to evaluate effects of a long-term quercetin treatment, we intraduodenally administered twice daily 18 mg of quercetin (Q)/kg of body weight to 5 late-lactation (215d in milk) dairy cows over a period of 28 d. Frequent blood samples were taken before and during administration to determine plasma concentrations of flavonols and metabolites. Before and after 1 and 4 wk of Q administration, glycogen and fat content as well as mRNA expression of selected genes were measured in liver biopsies. Furthermore, euglycemic, hyperinsulinemic, and hyperglycemic clamp studies were conducted before and after 2 wk of Q administration. During the experiment, dry matter intake and most other zootechnical data remained unchanged. Milk protein content was increased in wk 2 and 4 of Q administration compared with basal values, whereas fat and lactose contents of milk remained unchanged. Plasma nonesterified fatty acids, γ-glutamyl transferase, cholesterol, glutamate dehydrogenase, triglyceride, and albumin concentrations, as well as liver fat and glycogen concentrations, were not affected by Q supplementation. Plasma glucose and ß-hydroxybutyrate concentrations in plasma decreased and increased, respectively, under the influence of quercetin. During hyperglycemic clamp conditions, the relative increase of plasma insulin was higher after 2 wk of Q administration, and a tendency for an increased rQUICKI (revised quantitative insulin sensitivity check index) was observed. The relative mRNA expression levels of selected genes related to glucose metabolism, fat metabolism, and antioxidative status were not altered after 1 or 4 wk of Q supplementation. In conclusion, the effects on insulin release and sensitivity support the assumption that administration of Q could have positive effects on the metabolic adaption of high-yielding cows to early lactation. The increase of milk protein content in response to Q supplementation needs to be verified.

Bioavailability of the flavonol quercetin in cows after intraruminal application of quercetin aglycone and rutin.

The bioavailability of quercetin has been intensively investigated in monogastric species, but knowledge about its bioavailability in ruminants does not exist. Thus, the aim of the present study was to determine the bioavailability of quercetin in nonlactating cows equipped with indwelling catheters placed in one jugular vein after intraruminal and additionally after i.v. application, respectively. Quercetin was administered intraruminally in equimolar amounts, either in the aglycone form or as its glucorhamnoside rutin, each at 2 dosages [10 and 50 mg of quercetin/kg of body weight (BW)]. In a second trial, 0.8 mg of quercetin aglycone/kg of BW was applied i.v. Blood samples were drawn 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after intraruminal application and every 5 min (first hour), every 10 min(second hour), and at 3 and 6h after i.v. bolus application, respectively. Quercetin and quercetin metabolites with an intact flavonol structure (isorhamnetin, tamarixetin, and kaempferol) in plasma samples were analyzed by HPLC with fluorescence detection. After intraruminal application of quercetin and rutin, respectively, quercetin and its methylated (isorhamnetin, tamarixetin) and dehydroxylated (kaempferol) derivatives were present in plasma mainly as conjugated forms, whereas free quercetin and its derivatives were scarcely detected. For rutin, the relative bioavailability of total flavonols (sum of conjugated and nonconjugated quercetin and its conjugated and nonconjugated derivatives after intake of 50 mg/kg of BW) was 767.3% compared with quercetin aglycone (100%). Absolute bioavailability of total flavonols was only 0.1 and 0.5% after quercetin aglycone and rutin applications, respectively. Our data demonstrate that bioavailability of quercetin from rutin is substantially higher compared with that from quercetin aglycone in cows after intraruminal (or oral) application, unlike in monogastric species.

Dietary flavonoids do not affect vitamin E status in growing rats.

This study aimed at investigating potential effects of the flavonoids genistein, quercetin and catechin and the role of co-ingested dietary fat on vitamin E concentrations in rats. In experiment 1, genistein, quercetin and catechin were fed to rats, incorporated into semisynthetic diets at concentrations of 2 g/kg, either as individual compounds or in combination to investigate their individual and possible synergistic actions towards alpha-tocopherol in plasma and selected tissues. For experiments 2 and 3, quercetin was selected as a representative model flavonoid to study the effects of the quantity (5% vs. 10%) and type of dietary fat (coconut fat plus corn oil vs. rapeseed oil; experiment 2) and the role of cholesterol (experiment 3) on potential flavonoid-vitamin E interactions. The concentrations of alpha-tocopherol and gamma-tocopherol in the plasma, liver, lung and cortex of flavonoid-fed rats were not significantly different from the concentrations measured in control rats in all three experiments. However, increasing the amount of coconut fat plus corn oil from 5 to 10% resulted in lower alpha-tocopherol concentrations in plasma and tissue. The alpha-tocopherol concentrations in the rats fed rapeseed oil were significantly higher than in rats fed coconut fat plus corn oil. The addition of 0.2% cholesterol to the diet did not influence the tocopherol concentrations in plasma and tissue in both quercetin-supplemented and control rats. Additionally, the mRNA levels of alpha-TTP, CYP3A4, CYP4F and Mdr2, which are integral proteins involved in vitamin E homeostasis were measured. Only genistein reduced the Mdr2 mRNA level, but none of the other transcripts. All other flavonoids were without effect. In conclusion, co-ingested dietary fat appears to influence vitamin E concentrations in rats, but does not seem to be an important determinant of flavonoid-vitamin E interactions.

Ochratoxin A impairs Nrf2-dependent gene expression in porcine kidney tubulus cells.

The mycotoxin, ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is frequently present in feedstuffs. Ochratoxin A exhibits a wide range of toxic activities including nephrotoxicity. However, the underlying molecular mechanisms of OTA-induced cellular nephrotoxicity have yet not been fully elucidated. Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of antioxidant and xenobiotic metabolizing enzymes in the kidney. Therefore, in the present study, the effects of OTA on the nuclear translocation and transactivation of the transcription factor Nrf2 as well as mRNA levels of Nrf2 target genes including glutathione-S-transferase and gamma-glutamylcysteinyl synthetase have been studied in cultured porcine kidney tubulus cells (LLC-PK1). Nrf2 was induced by sulforaphane, a well-known activator of this transcription factor. Ochratoxin A significantly decreased gamma-glutamylcysteinyl synthetase and glutathione-S-transferase mRNA levels in LLC-PK1 cells. Decreased mRNA levels of gamma-glutamylcysteinyl synthetase and glutathione-S-transferase were accompanied by a lowered nuclear translocation and transactivation of Nrf2. Furthermore, OTA also lowered Nrf2 mRNA levels. Current data indicate that OTA nephrotoxicity may be, at least partly, mediated by an Nrf2-dependent signal transduction pathway.

Effect of ochratoxin A on redox-regulated transcription factors, antioxidant enzymes and glutathione-S-transferase in cultured kidney tubulus cells.

Ochratoxin A (OTA), a mycotoxin mostly produced by Aspergillus ochraceus and Penicillium verrucosum, is a worldwide contaminant of food and feedstuff. OTA is nephrotoxic and a renal carcinogen in rodents. The underlying molecular and cellular mechanisms by which OTA exhibits its toxicity have yet not been fully clarified. In the present study the effects of ochratoxin A on the activity of redox-regulated transcription factors, antioxidant enzymes, as well as glutathione-S-transferase (GST) have been studied in cultured kidney tubulus cells (LLC-PK1). Confluent LLC-PK1 cells were incubated with increasing concentrations of OTA for 24h. OTA decreased SOD activity and enhanced intracellular levels of reactive oxygen species (ROS) as measured by flow cytometry. Furthermore OTA resulted in a down-regulation of GST mRNA and activity levels. Lower GST levels were accompanied by a decreased transactivation of activator protein-1 (AP-1) and NF-E2-related factor-2 (Nrf2), which mediate GST gene transcription. Present data indicate that enhanced ROS production and an impairment of GST activity, possibly due to an AP-1 and Nrf2 dependent signal transduction pathway, may be centrally involved in OTA induced nephrotoxicity.

Dietary green tea polyphenols do not affect vitamin E status, antioxidant capacity and meat quality of growing pigs.

Supplementation of pigs with vitamin E, the most important lipid-soluble antioxidant, has been shown to improve meat quality and animal health. Previous studies in cultured cells and laboratory animals indicate synergistic effects between polyphenols and vitamin E. The present feeding trial was undertaken to investigate the effects of dietary green tea polyphenols (GTP) on vitamin E status, antioxidative capacity and parameters of meat quality in growing pigs. Eighteen castrated, crossbred, male pigs received a flavonoid-poor diet based on corn starch, caseinate and rapeseed oil with a total vitamin E content of 17 IU/kg diet over a period of 5 weeks. This basal diet was supplemented with green tea extract to provide daily doses of 0 (control), 10 and 100 mg GTP/kg body weight. Dietary supplementation of growing pigs with GTP did not affect serum, liver, lung and muscle vitamin E (alpha- and gamma-tocopherol) concentrations, plasma antioxidant capacity (ferric reducing ability of plasma, trolox equivalent antioxidant capacity) or parameters of meat quality including meat temperature, pH, conductivity, colour and drip loss. In conclusion, supplementation of pig diets with green tea catechins is not associated with improved antioxidant status and meat quality under practice-oriented conditions.

Effects of a six-week intraduodenal supplementation with quercetin on liver lipid metabolism and oxidative stress in peripartal dairy cows.

The purpose of this study was to evaluate possible effects of quercetin (Q) on liver lipid metabolism and antioxidative status in periparturient dairy cows. The periparturient period is associated with enormous metabolic changes for dairy cows. Energy needs for incipient lactation are too high to be balanced by feed intake, leading to negative energy balance and body fat mobilization. It has been estimated that this leads to the development of fatty liver in about 50% of cows, which are at high risk for disease. Furthermore, the antioxidative status of these cows may be impaired. Quercetin is a plant flavonoid having hepatoprotective and antioxidative potential and the ability to reduce liver lipid accumulation in monogastric animals. Little information is available in regard to these effects in ruminants. To prevent microbial Q degradation in the rumen, Q was administered via a duodenal fistula to improve systemic availability. Five cows of the Q-treated group received, daily, 100 mg of quercetin dehydrate/kg BW in a 0.9% sodium chloride solution from d -20 until d 20 relative to calving, whereas 5 control (CTR) cows received only a sodium chloride solution. Blood samples were taken weekly and liver biopsies were performed in wk -4, -2, and 3 relative to calving. Cows treated with Q showed a tendency ( = 0.082) for lower liver fat content compared with CTR cows. Liver glycogen, glutathione concentrations, and relative mRNA abundance of genes related to hepatic lipid metabolism and antioxidative status as well as parameters of antioxidative status in plasma were not affected ( > 0.1) by Q supplementation. In conclusion, liver fat content in dairy cows tended to be reduced by Q supplementation, but potential underlying mechanisms remain unclear because analyzed parameters related to hepatic lipid metabolism and antioxidative defense were not altered by Q supplementation.

Bioavailability and metabolism of the flavonol quercetin in the pig.

During the last years, much data pointing to putative health-promoting effects of dietary plant-derived flavonoids (stemming mainly from epidemiological and in vitro studies) have been published. Our knowledge, however, concerning the systemic availability of these substances after ingestion with food is only sketchy. In the present study, we have investigated the bioavailability of the flavonol quercetin after intravenous and oral application in pigs equipped with a permanent jugular catheter. Each animal received a single intravenous dose of quercetin (0.4 mg/kg body weight) and one week later an oral dose of 50 mg/kg. A single animal additionally received an oral dose of 500 mg/kg one week after the lower oral dose. Blood samples were drawn at defined intervals over a total period of three days following the application of quercetin. Analysis of quercetin and some of its metabolites (isorhamnetin, tamarixetin, kaempferol) in plasma samples were performed by HPLC. The calculated apparent bioavailability of free, unchanged quercetin after intake of 50 mg quercetin/kg body weight was 0.54+/-0.19%. Bioavailability was, however, considerably increased to 8.6+/-3.8% after additionally taking into account conjugated quercetin and further increased to 17.0+/-7.1% by including quercetin's metabolites. Our results further indicate, that the conjugation of orally administered quercetin with glucuronic and sulfuric acid appears to preferentially occur in the intestinal wall.

Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans.

BACKGROUND: Quercetin, a flavonoid present in the human diet, which is found in high levels in onions, apples, tea and wine, has been shown previously to inhibit platelet aggregation and signaling in vitro. Consequently, it has been proposed that quercetin may contribute to the protective effects against cardiovascular disease of a diet rich in fruit and vegetables. OBJECTIVES: A pilot human dietary intervention study was designed to investigate the relationship between the ingestion of dietary quercetin and platelet function. METHODS: Human subjects ingested either 150 mg or 300 mg quercetin-4'-O-beta-D-glucoside supplement to determine the systemic availability of quercetin. Platelets were isolated from subjects to analyse collagen-stimulated cell signaling and aggregation. RESULTS: Plasma quercetin concentrations peaked at 4.66 microm (+/- 0.77) and 9.72 microm (+/- 1.38) 30 min after ingestion of 150-mg and 300-mg doses of quercetin-4'-O-beta-D-glucoside, respectively, demonstrating that quercetin was bioavailable, with plasma concentrations attained in the range known to affect platelet function in vitro. Platelet aggregation was inhibited 30 and 120 min after ingestion of both doses of quercetin-4'-O-beta-D-glucoside. Correspondingly, collagen-stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accompanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2, components of the platelet glycoprotein VI collagen receptor signaling pathway. CONCLUSIONS: This study provides new evidence of the relatively high systemic availability of quercetin in the form of quercetin-4'-O-beta-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

Interaction of quercetin glucosides with the intestinal sodium/glucose co-transporter (SGLT-1).

Recently it has been postulated that flavonoid glucosides may be absorbed by the small intestine via the SGLT-1. Therefore, we applied an in vitro mucosal uptake method to investigate mucosal uptake of the non-metabolisable glucose analogue methyl-alpha-D-glucopyranoside (MDG) as influenced by quercetin-3-glucoside (isoquercitrin) and quercetin-4'-glucoside (spiraeosid). We found that both glucosides significantly inhibited SGLT-1-mediated MDG uptake whereas the aglycon quercetin or the quercetin-3-rhamnoglucoside (rutin) were ineffective. Calculated apparent kinetic parameters (Km, Vmax) of initial Na+-dependent MDG uptake by the jejunal mucosa indicate a competitive type of inhibition.

Enzymatic cleavage of thymopoietin oligopeptides by pancreatic and intestinal brush-border enzymes.

The intestinal enzymatic degradation of the immunomodulating peptides thymotrinan (TP3), thymocartin (TP4), and thymopentin (TP5), three oligopeptides derived from the naturally occurring thymus hormone thymopoietin, was investigated to evaluate their potential for peroral drug delivery. In the presence of brush-border membrane vesicles, crude pancreas extract and everted rings from duodenum, jejunum, ileum, and colon, all peptides were shown to be degraded both by pancreatic enzymes and brush-border aminopeptidases. Degradation clearances (Cldeg) of TP3, TP4, and TP5 were calculated for a quantitative comparison of peptide stability. In the presence of crude pancreas extract, there was a rapid degradation of TP5 (Cldeg 17.9 ml/min) in comparison with TP3 and TP4 (Cldeg 0.95 and 0.56 ml/min, respectively, at 0.2 mM peptide concentration) caused by the cleavage of the C-terminal tyrosine by carboxypeptidase A, whereas TP3 and TP4 underwent hydrolysis by aminopeptidase N. In the presence of brush-border membrane vesicles, the degradation clearances were 3.9, 3.1, and 2.4 ml/min at 0.2 mM concentrations of TP4, TP5, and TP3, respectively. The clearance of all peptides was lowered with increasing peptide concentrations, indicating saturable degradation processes. The degradation of the thymopoietin oligopeptides in the presence of brush-border membrane enzymes was exclusively catalyzed by aminopeptidase N. The degradation of all peptides was highly dependent on the intestinal segment, with the lowest degradation clearance observed in the colon.

Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs.

Plasma samples of dogs given 5 mg kg-1 bodyweight of a broad spectrum antimicrobial with the active ingredient enrofloxacin were assayed by two different methods (bioassay: Escherichia coli 14 ICB 4004 on ISO sensitest agar; high performance liquid chromatography with a RP C18 column). At concentrations up to 1000 ng ml-1 a linear correlation between values obtained by the two assay methods was found. At concentrations above 1000 ng ml-1 no correlation could be determined. The main metabolite of enrofloxacin, ciprofloxacin, is an important determinant of overall antimicrobial activity and hence influences bioassay results.

Metabolism and excretion of ochratoxin A fed to sheep.

Hydrolysis of the mycotoxin ochratoxin A (OA) to ochratoxin alpha (Oalpha) by microorganisms within the gastrointestinal tract leads to the excretion of OA as the nontoxic alpha form. The Oalpha form is the principal means for the detoxification of OA. In the current experiment, three groups of four sheep were fed diets consisting of 70% concentrates and 30% hay (dry matter basis, energy to supply 1.1 times the requirement for maintenance) for 4 wk with three dietary concentrations of OA (0, 2, or 5 mg/kg of concentrate feed). The OA content did not affect feed intake or nutrient digestibility. In a preliminary experiment, an OA dose of 20 mg/kg of concentrate feed greatly reduced feed intake. After 1, 2, 3, and 4 wk of the trial, significant concentrations of OA were detected in the serum of the animals fed 2 or 5 mg of OA/kg feed. This suggested that even at a dosage of 2 mg of OA/kg of concentrate feed, considerable amounts of OA were not degraded by ruminal and intestinal microorganisms. The analysis of the feces and urine samples reflected these findings; OA and Oalpha were found in significant concentrations, escaping fermentation in the rumen and in the hindgut. The current experiment demonstrates that OA hydrolysis in the gastrointestinal tract of sheep is substantially less than previously described, especially if OA is ingested in combination with concentrate-rich diets.

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite.

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of 75Se-labeled selenite (10 mumol/L) was investigated in rat jejunum. For comparison, the effect of L-cysteine (1 mmol/L) on in vivo absorption of 75Se-labeled selenite was also studied. In the in vitro uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH. L-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlike L-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occurring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine.

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of 75Se from 75Se-labeled selenite (conc. 10 mumol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique. L-Cysteine (L-Cys) stimulated 75Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum. D-Cys was less effective in the jejunum and similarly effective in the cecum. L-Leucine (L-Leu) and L-glutamic acid significantly reduced the stimulatory effect of L-Cys on Se uptake in the distal jejunum, whereas the respective effect of D-Cys was not diminished by L-Leu. Cysteamine stimulated mucosal 75Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced 75Se uptake in the distal jejunum. The stimulatory effects of L-Cys, mercaptopyruvate, and thioglycolate were Na(+)-dependent, whereas the effect of cysteamine also occurred in the absence of Na+. Mercaptosuccinate, D-penicillamine, ergothioneine, and thiosulfate did not enhance mucosal 75Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na(+)-dependent and Na(+)-independent mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.