Analysis of time-related metabolic fluctuations induced by ethionine in the rat

The time-course of metabolic events following response to a model hepatotoxin ethionine (800 mg/kg) was investigated over a 7 day period in rats using high-resolution (1)H NMR spectroscopic analysis of urine and multivariate statistics. Complementary information was obtained by multivariate analysis of (1)H MAS NMR spectra of intact liver and by conventional histopathology and clinical chemistry of blood plasma. (1)H MAS NMR spectra of liver showed toxin-induced lipidosis 24 h postdose consistent with the steatosis observed by histopathology, while hypertaurinuria was suggestive of liver injury. Early biochemical changes in urine included elevation of guanidinoacetate, suggesting impaired methylation reactions. Urinary increases in 5-oxoproline and glycine suggested disruption of the gamma-glutamyl cycle. Signs of ATP depletion together with impairment of the energy metabolism were given from the decreased levels in tricarboxylic acid cycle intermediates, the appearance of ketone bodies in urine, the depletion of hepatic glucose and glycogen, and also hypoglycemia. The observed increase in nicotinuric acid in urine could be an indication of an increase in NAD catabolism, a possible consequence of ATP depletion. Effects on the gut microbiota were suggested by the observed urinary reductions in the microbial metabolites 3-/4-hydroxyphenyl propionic acid, dimethylamine, and tryptamine. At later stages of toxicity, there was evidence of kidney damage, as indicated by the tubular damage observed by histopathology, supported by increased urinary excretion of lactic acid, amino acids, and glucose. These studies have given new insights into mechanisms of ethionine-induced toxicity and show the value of multisystem level data integration in the understanding of experimental models of toxicity or disease.

Carbon and nitrogen isotopic ratios of urine and faeces as novel nutritional biomarkers of meat and fish intake

Purpose Meat and fish consumption are associated with changes in the risk of chronic diseases. Intake is mainly assessed using self-reporting, as no true quantitative nutritional biomarker is available. The measurement of plasma fatty acids, often used as an alternative, is expensive and time-consuming. As meat and fish differ in their stable isotope ratios, δ13C and δ15N have been proposed as biomarkers. However, they have never been investigated in controlled human dietary intervention studies. Objective In a short-term feeding study, we investigated the suitability of δ13C and δ15N in blood, urine and faeces as biomarkers of meat and fish intake. Methods The dietary intervention study (n = 14) followed a randomised cross-over design with three eight-day dietary periods (meat, fish and half-meat–half-fish). In addition, 4 participants completed a vegetarian control period. At the end of each period, 24-h urine, fasting venous blood and faeces were collected and their δ13C and δ15N analysed. Results There was a significant difference between diets in isotope ratios in faeces and urine samples, but not in blood samples (Kruskal–Wallis test, p < 0.0001). In pairwise comparisons, δ13C and δ15N were significantly higher in urine and faecal samples following a fish diet when compared with all other diets, and significantly lower following a vegetarian diet. There was no significant difference in isotope ratio between meat and half-meat–half-fish diets for blood, urine or faecal samples. Conclusions The results of this study show that urinary and faecal δ13C and δ15N are suitable candidate biomarkers for short-term meat and fish intake.

The type and quantity of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome ‘at-risk’ population syndrome.

An obese-type human microbiota with an increased Firmicutes:Bacteroidetes ratio has been described that may link the gut microbiome with obesity and metabolic syndrome (MetS) development. Dietary fat and carbohydrate are modifiable risk factors that may impact on MetS by altering the human microbiome composition. We determined the effect of the amount and type of dietary fat and carbohydrate on faecal bacteria and short chain fatty acid (SCFA) concentrations in people ‘at risk’ of MetS.

The impact of the catechol-O-methyltransferase genotype on vascular function and blood pressure after acute green tea ingestion

SCOPE: Evidence for the benefits of green tea catechins on vascular function is inconsistent, with genotype potentially contributing to the heterogeneity in response. Here, the impact of the catechol-O-methyltransferase (COMT) genotype on vascular function and blood pressure (BP) after green tea extract ingestion are reported. METHODS AND RESULTS: Fifty subjects (n = 25 of the proposed low-activity [AA] and of the high-activity [GG] COMT rs4680 genotype), completed a randomized, double-blind, crossover study. Peripheral arterial tonometry, digital volume pulse (DVP), and BP were assessed at baseline and 90 min after 1.06 g of green tea extract or placebo. A 5.5 h and subsequent 18.5 h urine collection was performed to assess green tea catechin excretion. A genotype × treatment interaction was observed for DVP reflection index (p = 0.014), with green tea extract in the AA COMT group attenuating the increase observed with placebo. A tendency for a greater increase in diastolic BP was evident at 90 min after the green tea extract compared to placebo (p = 0.07). A genotypic effect was observed for urinary methylated epigallocatechin during the first 5.5 h, with the GG COMT group demonstrating a greater concentration (p = 0.049). CONCLUSION: Differences in small vessel tone according to COMT genotype were evident after acute green tea extract.

Investigation of sainfoin (Onobrychis viciifolia) cultivar differences on nitrogen balance and fecal egg count in artificially infected lambs

Research in ruminant nutrition and helminth control with forages, which contain condensed tannins (CT), suggests that varying responses may depend not only on CT concentration but also on CT composition. An experiment was designed to test this by feeding 2 dried sainfoin cultivars (Visnovsky and Perly), which differed in CT properties, to lambs that were artificially infected with the abomasal blood-sucking nematode Haemonchus contortus. Twenty-four infected lambs received one of these 2 cultivars; the feeds were either untreated or treated with the CT-binding polyethylene glycol over 4 wk (n = 6). The 2 cultivars were also fed to 2 × 6 uninfected lambs. Nutrient digestibility, N balance, ADG, plasma urea together with indicators of infection [fecal egg count (FEC), abomasal worm count, per capita female fecundity, erythrocytic indices, and serum protein] were determined. The specific effects of sainfoin cultivar, CT, and infection were evaluated by contrast analysis. Digestibility of both NDF and ADF were lower (P < 0.001) with Perly compared to Visnovsky. The apparent nutrient digestibility was reduced (P < 0.001) by CT. However, no clear cultivar effects were evident on N excretion and retention. Condensed tannins reduced (P = 0.05) body N retention and shifted (P < 0.001) N excretion from urine to feces. Unlike cultivar and CT, infection decreased (P = 0.002) ADG. Plasma urea concentration was lower (P = 0.007) in Perly- compared to Visnovsky-fed lambs and was decreased (P < 0.001) by CT. Plasma concentrations of essential and semi-essential AA were increased (P < 0.001) by CT. The groups of infected lambs did not clearly differ in abomasal worm counts and erythrocytic indicators. In the last 2 to 3 wk of the experiment, FEC was lower (P ≤ 0.01) when feeding CT. The lack of substantial cultivar effects suggests that the differences in CT properties may have been too small to result in nutritional and anthelmintic effects. The present results indicate that sainfoin CT had a mitigating effect on FEC and, consequently, pasture infectivity. However, the reduction was too low to expect any significant benefits in an Haemonchus-dominated system. Therefore, the use of sainfoin for controlling H. contortus should only be one component within an integrated worm control system.

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

MALDI MS profiling of post-DRE urine samples highlights the potential of b-Microseminoprotein as a marker for prostatic diseases

BACKGROUND. To use spectra acquired by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) from pre- and post-digital rectal examination (DRE) urine samples to search for discriminating peaks that can adequately distinguish between benign and malignant prostate conditions, and identify the peaks’ underlying biomolecules. METHODS. Twenty-five participants with prostate cancer (PCa) and 27 participants with a variety of benign prostatic conditions as confirmed by a 10-core tissue biopsy were included. Pre- and post-DRE urine samples were prepared for MALDI MS profiling using an automated clean-up procedure. Following mass spectra collection and processing, peak mass and intensity were extracted and subjected to statistical analysis to identify peaks capable of distinguishing between benign and cancer. Logistic regression was used to combine markers to create a sensitive and specific test. RESULTS. A peak at m/z 10,760 was identified as b-microseminoprotein (b-MSMB) and found to be statistically lower in urine from PCa participants using the peak’s average areas. By combining serum prostate-specific antigen (PSA) levels with MALDI MS-measured b-MSMB levels, optimum threshold values obtained from Receiver Operator characteristics curves gave an increased sensitivity of 96% at a specificity of 26%. CONCLUSIONS. These results demonstrate that with a simple sample clean-up followed by MALDI MS profiling, significant differences of MSMB abundance were found in post-DRE urine samples. In combination with PSA serum levels, obtained from a classic clinical assay led to high classification accuracy for PCa in the studied sample set. Our results need to be validated in a larger multicenter prospective randomized clinical trial.

Effect of cattle urine addition on the surface emissions and subsurface concentrations of greenhouse gases in a UK peat grassland

Grazing systems represent a substantial percentage of the global anthropogenic flux of nitrous oxide (N2O) as a result of nitrogen addition to the soil. The pool of available carbon that is added to the soil from livestock excreta also provides substrate for the production of carbon dioxide (CO2) and methane (CH4) by soil microorganisms. A study into the production and emission of CO2, CH4 and N2O from cattle urine amended pasture was carried out on the Somerset Levels and Moors, UK over a three-month period. Urine-amended plots (50 g N m−2) were compared to control plots to which only water (12 mg N m−2) was applied. CO2 emission peaked at 5200 mg CO2 m−2 d−1 directly after application. CH4 flux decreased to −2000 μg CH4 m−2 d−1 two days after application; however, net CH4 flux was positive from urine treated plots and negative from control plots. N2O emission peaked at 88 mg N2O m−2 d−1 12 days after application. Subsurface CH4 and N2O concentrations were higher in the urine treated plots than the controls. There was no effect of treatment on subsurface CO2 concentrations. Subsurface N2O peaked at 500 ppm 12 days after and 1200 ppm 56 days after application. Subsurface NO3− concentration peaked at approximately 300 mg N kg dry soil−1 12 days after application. Results indicate that denitrification is the key driver for N2O release in peatlands and that this production is strongly related to rainfall events and water-table movement. N2O production at depth continued long after emissions were detected at the surface. Further understanding of the interaction between subsurface gas concentrations, surface emissions and soil hydrological conditions is required to successfully predict greenhouse gas production and emission.

Hydrophilic interaction chromatography–mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats

Hydrophilic interaction chromatography–mass spectrometry (HILIC–MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial–host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC–MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance (1H NMR) spectroscopy. Using CE–MS and 1H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC–MS and CE–MS. Overall, HILIC–MS appears to be highly complementary to CE–MS and 1H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

The effects of tannin-containing ground pine bark diet upon nutrient digestion, nitrogen balance, and mineral retention in meat goats

Background Pine bark is a rich source of phytochemical compounds including tannins, phenolic acids, anthocyanins, and fatty acids. These phytochemicals have potential to significantly impact on animal health and animal production. The goal of this work is to measure the effects of tannins in ground pine bark as a partial feed replacement on feed intake, dietary apparent digestibility, nitrogen balance, and mineral retention in meat goats. Results Eighteen Kiko cross goats (initial BW = 31.8 ± 1.49 kg) were randomly assigned to three treatment groups (n = 6). Dietary treatments were tested: control (0 % pine bark powder (PB) and 30 % wheat straw (WS)); 15 % PB and 15 % WS, and 30 % PB and 0 % WS. Although dry matter (DM) intake and digestibility were not affected (P > 0.10) by feeding PB, neutral detergent fiber (linear; P = 0.01), acid detergent fiber (linear; P = 0.001) and lignin digestibility (linear; P = 0.01) decreased, and crude protein (CP) digestibility tended to decrease (P = 0.09) as PB increased in the diet, apparent retention of Ca (P = 0.09), P (P = 0.03), Mg (P = 0.01), Mn (P = 0.01), Zn (P = 0.01) and Fe (P = 0.09) also increased linearly. Nitrogen intake and fecal N excretion were not affected (P > 0.05) by addition of PB in the diet, but N balance in the body was quadratically increased (P < 0.01) in the 15 % PB diet compared to other diets. This may be due to more rumen escape protein and less excreted N in the urine with the 15 % PB diet. The study showed that a moderate level of tannin-containing pine bark supplementation could improve gastrointestinal nitrogen balance with the aim of improving animal performance. Conclusion These results suggest that tannin-containing PB has negative impact on fiber, lignin, and protein digestibility, but positively impacted on N-balance.

Evaluating nitrogen utilization efficiency of nonpregnant dry cows offered solely fresh cut grass at maintenance levels

The present study aimed to identify key parameters influencing N utilization and develop prediction equations for manure N output (MN), feces N output (FN), and urine N output (UN). Data were obtained under a series of digestibility trials with nonpregnant dry cows fed fresh grass at maintenance level. Grass was cut from 8 different ryegrass swards measured from early to late maturity in 2007 and 2008 (2 primary growth, 3 first regrowth, and 3 second regrowth) and from 2 primary growth early maturity swards in 2009. Each grass was offered to a group of 4 cows and 2 groups were used in each of the 8 swards in 2007 and 2008 for daily measurements over 6 wk; the first group (first 3 wk) and the second group (last 3 wk) assessed early and late maturity grass, respectively. Average values of continuous 3-d data of N intake (NI) and output for individual cows ( = 464) and grass nutrient contents ( = 116) were used in the statistical analysis. Grass N content was positively related to GE and ME contents but negatively related to grass water-soluble carbohydrates (WSC), NDF, and ADF contents ( < 0.01), indicating that accounting for nutrient interrelations is a crucial aspect of N mitigation. Significantly greater ratios of UN:FN, UN:MN, and UN:NI were found with increased grass WSC contents and ratios of N:WSC, N:digestible OM in total DM (DOMD), and N:ME ( < 0.01). Greater NI, animal BW, and grass N contents and lower grass WSC, NDF, ADF, DOMD, and ME concentrations were significantly associated with greater MN, FN, and UN ( < 0.05). The present study highlighted that using grass lower in N and greater in fermentable energy in animals fed solely fresh grass at maintenance level can improve N utilization, reduce N outputs, and shift part of N excretion toward feces rather than urine. These outcomes are highly desirable in mitigation strategies to reduce nitrous oxide emissions from livestock. Equations predicting N output from BW and grass N content explained a similar amount of variability as using NI and grass chemical composition (excluding DOMD and ME), implying that parameters easily measurable in practice could be used for estimating N outputs. In a research environment, where grass DOMD and ME are likely to be available, their use to predict N outputs is highly recommended because they strongly improved of the equations in the current study.

Interactions between cocoa flavanols and inorganic nitrate: additive effects on endothelial function at achievable dietary amounts

Dietary intervention studies have shown that flavanols and inorganic nitrate can improve vascular function, suggesting that these two bioactives may be responsible for beneficial health effects of diets rich in fruits and vegetables. We aimed to study interactions between cocoa flavanols (CF) and nitrate, focusing on absorption, bioavailability, excretion, and efficacy to increase endothelial function. In a double-blind randomized, dose-response crossover study, flow-mediated dilation (FMD) was measured in 15 healthy subjects before and at 1, 2, 3, and 4 h after consumption of CF (1.4-10.9 mg/kg bw) or nitrate (0.1-10 mg/kg bw). To study flavanol-nitrate interactions, an additional intervention trial was performed with nitrate and CF taken in sequence at low and high amounts. FMD was measured before (0 h) and at 1h after ingestion of nitrate (3 or 8.5 mg/kg bw) or water. Then subjects received a CF drink (2.7 or 10.9 mg/kg bw) or a micro- and macronutrient-matched CF-free drink. FMD was measured at 1, 2, and 4 h thereafter. Blood and urine samples were collected and assessed for CF and nitric oxide (NO) metabolites with HPLC and gas-phase reductive chemiluminescence. Finally, intragastric formation of NO after CF and nitrate consumption was investigated. Both CF and nitrate induced similar intake-dependent increases in FMD. Maximal values were achieved at 1 h postingestion and gradually decreased to reach baseline values at 4 h. These effects were additive at low intake levels, whereas CF did not further increase FMD after high nitrate intake. Nitrate did not affect flavanol absorption, bioavailability, or excretion, but CF enhanced nitrate-related gastric NO formation and attenuated the increase in plasma nitrite after nitrate intake. Both flavanols and inorganic nitrate can improve endothelial function in healthy subjects at intake amounts that are achievable with a normal diet. Even low dietary intake of these bioactives may exert relevant effects on endothelial function when ingested together.