Establishment of an In Vitro Co-Culture Model of the Piglet Gut to Study Inflammatory Response and Barrier Integrity.

In intensive farming, piglets are exposed to various challenges that activate intestinal inflammatory processes, negatively affecting animal health and leading to economic losses. To study the role of the inflammatory response on epithelial barrier integrity, co-culture systems that mimic in vivo complexity are more and more preferred over cell monocultures. In this study, an in vitro gut co-culture model consisting of intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells was established. The model provides an appropriate tool to study the role of the inflammatory response on epithelial barrier integrity and to screen for feed and food components, exerting beneficial effects on gut health. In the established model, inflammation-like reactions and damage of the epithelial barrier, indicated by a decrease of transepithelial electrical resistance, were elicited by activation of peripheral blood mononuclear cells via one of 3 stimuli: lipopolysaccharide, lipoteichoic acid, or concanavalin A. Two phytogenic substances that are commonly used as feed additives, licorice extract and oregano oil, have been shown to counteract the drop in transepithelial electrical resistance values in the gut co-culture model. The established co-culture model provides a powerful in vitro tool to study the role of intestinal inflammation on epithelial barrier integrity. As it consists of porcine epithelial and porcine blood cells it perfectly mimics in vivo conditions and imitates the inter-organ communication of the piglet gut. The developed model is useful to screen for nutritional components or drugs, having the potential to balance intestinal inflammation and strengthen the epithelial barrier integrity in piglets.

Free Radical Scavenging Activity of Carbonyl-Amine Adducts Formed in Soybean Oil Fortified with Phosphatidylethanolamine.

Non-enzymatic browning reactions between lipid aldehydes and aminophospholipids might play an important role in the oxidative stability of cold-pressed vegetable oils. We, therefore, aimed to study the Maillard-type reaction between hexanal, a lipid oxidation product of linoleic acid, and phosphatidylethanolamine (PE (16:0/18:1)) at a ratio of 2:1 at conditions representative of the extraction of cold-pressed soybean oils (CPSBO) and determine the radical scavenging activity of the carbonyl-amine adducts with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The reaction product, 2-pentyl-3,5-dibutyl-dihydropyridine, could be identified by means of LC-ESI-QTOF-MS/MS. The formation of this nitrogen-containing heterocycle significantly increased with time and temperature (p < 0.05). The products formed during the carbonyl-amine reaction between PE (16:0/18:1) and hexanal at 60 °C showed a radical scavenging activity of approximately 20% (p < 0.05). The fraction, containing 2-pentyl-3,5-dibutyl-dihydropyridine, contributed to, but was not solely responsible for, the radical scavenging activity (p < 0.05). Incubation of CPSBO fortified with PE (16:0/18:1) at 60 °C for 60 min had the strongest radical scavenging activity of 85.1 ± 0.62%. Besides 2-pentyl-3,5-dibutyl-dihydropyridine, other carbonyl-amine adducts might impact the radical scavenging activity of CPSBO as well. The oxidative stability of CPSBO might be increased by promoting the formation of carbonyl-amine reaction products, such as 2-pentyl-3,5-dibutyl-dihydropyridine.

Simultaneous Analysis of Epoxidized and Hydroperoxidized Triacylglycerols in Canola Oil and Margarine by LC-MS.

The progress of lipid oxidation in foods is evaluated by measuring the peroxides and their scission products. However, hydrogen abstraction-independent pathways are not considered by commonly applied methods despite the known reactivity of epoxides toward biomolecules. Herein, a novel liquid chromatography tandem-mass spectrometry method was developed to detect hydroperoxidized and epoxidized triacylglycerols (TAGs) without derivatization or hydrolyzation of food samples. Epoxidized TAGs could be detected in refined canola oil at concentrations of 96.8 ± 2.08 µM, while only 5.77 ± 0.04 µM hydroperoxidized TAGs could be determined. In contrast to canola oil, margarine was more resistant to lipid oxidation since generation of epoxidized TAGs could only be marginally enhanced from 21.7 ± 0.48 to 28.8 ± 0.64 µM in margarine after treatment at 180 °C for 60 min, as also reflected by a peroxide value of 0.80 ± 0.00 mequiv O2/kg, which remained unchanged. The new method allows the assessment of food safety by the simultaneous measurement of hydroperoxidized and epoxidized TAGs without hydrolysis and laborious sample preparation.

Influence of phytogenics on recovery of the barrier function of intestinal porcine epithelial cells after a calcium switch.

BACKGROUND: The gut barrier is essential for animal health as it prevents the passage of potentially harmful foreign substances. The epithelial tight junctions support the intestinal barrier and can be disrupted by stress caused, for example, by pathogens or dietary or environmental factors, predisposing the host to disease. In animal husbandry, phytogenics (plant-derived feed additives) are used to support and maintain growth, feed efficiency and health. Therefore, several phytogenics were tested in vitro for their influence on the barrier function recovery of intestinal porcine epithelial cells (IPEC-J2) after disruption, particularly on the abundance of tight junction proteins. RESULTS: IPEC-J2 treated with 1,000 µg/ml liquorice root extract, 80 µg/ml plant powder mix, or 80 µg/ml angelica root powder showed significantly higher trans-epithelial electric resistance (TEER) 24 hr after tight junction disruption via a calcium switch assay than the control. In contrast, cells treated with 1,000 µg/ml oak bark extract showed a significantly lower TEER after 6 hr but not at later time points. The increased TEER caused by the liquorice root extract correlated with an increase in the abundance of the tight junction protein claudin-4. CONCLUSIONS: This study suggests potential beneficial effects of liquorice and angelica root extracts on the gut barrier function when used as feed additives for livestock. Further studies, especially in vivo, are necessary to confirm these findings.

Effects of phytogenic feed additives on cellular oxidative stress and inflammatory reactions in intestinal porcine epithelial cells1.

Due to increasing concerns about the use of antibiotic growth promoters (AGP) in livestock production and their complete ban in the European Union in 2006, suitable alternatives are urgently needed. Among others, anti-inflammatory activities of AGP are discussed as their putative mode of action. As numerous phytochemicals are known to modulate the cellular antioxidant capacity and immune response, we studied the antioxidative and anti-inflammatory properties of a phytogenic (plant-derived) feed additive (PFA) in intestinal porcine epithelial cells (IPEC-J2). The effects of the PFA were compared with those of selected phytogenic ingredients (grape seed extract [GRS], licorice extract [LIC], menthol [MENT], methyl salicylate [MES], oak bark extract [OAK], oregano essential oil [ORE], and a plant powder mix [PLA]), and with the effects of the AGP tylosin (TYL). Oxidative or inflammatory stress was induced by stimulating IPEC-J2 with hydrogen peroxide (H2O2; 0.5 mM) or tumor necrosis factor alpha (TNF-α; 10 ng/mL), respectively. The antioxidative effects of feed additives were assessed with a reactive oxygen species (ROS)-sensitive probe and by measuring the expression of 6 antioxidative target genes via quantitative real-time PCR (RT-qPCR). Anti-inflammatory potential was analyzed using a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter gene assay. Moreover, the expression levels of 6 NF-κB target genes were measured using RT-qPCR analysis, and the release of IL-6 was analyzed via ELISA. Significant decreases in cellular ROS upon H2O2 treatment were observed for the PFA (P < 0.001), LIC (P < 0.001), ORE (P < 0.05), and GRS (P < 0.01). No significant changes in the expression of antioxidative genes were found. NF-κB activation upon TNF-α treatment was significantly inhibited by the PFA (P < 0.05) and by ORE (P < 0.001). Moreover, the PFA and ORE significantly reduced the gene expression of IL-6 (P < 0.001), IL-8 (P < 0.001), and C-C motif chemokine ligand 2 (CCL2; P < 0.05), as well as the release of IL-6 (P < 0.05). The other phytogenic compounds as well as the AGP TYL did not significantly affect any of the inflammatory parameters. In summary, we revealed the antioxidative properties of the PFA, LIC, ORE, and GRS, as well as anti-inflammatory properties of the PFA and ORE in IPEC-J2, providing a better understanding of the mode of action of this PFA under our experimental conditions.

Folate deficiency and over-supplementation causes impaired folate metabolism: Regulation and adaptation mechanisms in Caenorhabditis elegans.

SCOPE: Impaired folate metabolism increases the risk of birth defects, neurodegenerative and cardiovascular disease, osteoporosis and cancer. We used Caenorhabditis elegans to investigate impaired folate metabolism by RNA interference of key enzymes in the methionine synthase (MS) and thymidylate synthase (TS) cycle and by folate deficiency and over-supplementation feeding studies. METHODS AND RESULTS: Folate status is influenced by genetic variations (polymorphisms), folate deficiency and supplementation. Single RNAi of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR) and MS revealed that gene regulation is largely affected in both folate cycles. Adaptation requires a close transcriptional connection between TS and MS cycle. Coupled DHFR and MS expression is required to balance both cycles, but seems to reduce the overall rate of folate conversion. Feeding studies showed that folate over-supplementation to functioning metabolism inactivates MS and MTHFR expression and enhances TS activity, which favors DNA synthesis over methylation reactions. Folate deficiency disrupted homeostasis by favoring TS cycle and led to malformation in C. elegans offspring. Embryos show aneuploidy and are nonviable lacking DNA repair during meiotic stage of diakinesis. CONCLUSION: Single gene silencing alters gene expression in both cycles and disrupts folate homeostasis. Folate over-supplementation and deficiency favors TS over MS cycle and causes prophase DNA damage.

Influence of intravenous thiamine supplementation on blood lactate concentration prior to cardiac surgery: A double-blinded, randomised controlled pilot study.

BACKGROUND: Stress related to surgery and critical illness depletes thiamine, essential in energy metabolism, and might result in high blood lactate concentrations and higher mortality. OBJECTIVES: We hypothesised that thiamine supplementation would increase blood concentration of thiamine and reduce blood lactate concentration postoperatively. Moreover, we aimed to identify the prevalence of, and risk factors for, high blood lactate concentrations. DESIGN: This was a double-blind, randomised controlled pilot study from February to July 2012 including 30 patients scheduled for cardiac surgery with cardiopulmonary bypass. INTERVENTIONS: Patients were assigned randomly to receive thiamine (300 mg in 0.9% Normal saline solution) or placebo (0.9% Normal saline) preoperatively. MAIN OUTCOME MEASURES: One arterial blood sample was taken preoperatively and another postoperatively to measure thiamine concentration, and multiple samples were taken during surgery and ICU stay to determine lactate concentrations. Twenty-four hour urine samples were collected to measure urinary thiamine concentration. Preoperatively, we assessed extracellular mass to body cell mass ratio (ECM/BCM). RESULTS: The mean (SD) age of the patients was 58 (12) years, 73% were overweight, 10% were malnourished and the prevalence of thiamine deficiency was 10%. Patients in the thiamine group had significantly higher blood thiamine concentrations 2 days postoperatively [805.2 ± 289.8 ng g(-1) haemoglobin (Hb)] than those in the placebo group (591.2 ± 100.7 ng g(-1) Hb, P < 0.01). The mean blood lactate concentration changed significantly over time, but did not differ significantly between the groups. Patients with ECM/BCM more than 1 had higher lactate concentrations on admission to ICU than those with ECM/BCM less than 1 (2.1 ± 0.7 vs. 1.7 ± 0.6, P = 0.09) and were at a significantly greater risk of having a higher lactate concentration on ICU admission [odds ratio (OR) 13.5, 95% confidence interval (95% CI) 1.0 to 179.4, P < 0.05]. On the basis of these results, a sample size calculation for a larger study has been facilitated. CONCLUSION: Thiamine supplementation caused normalisation of blood and urine concentrations postoperatively but without a significant reduction in lactate concentration or clinical outcome. Body composition played an important role in lactate formation. Further research focusing on preoperative screening and optimal treatment of high lactate concentrations in this specific population is warranted. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01524315.

Mental imagery interventions reduce subsequent food intake only when self-regulatory resources are available.

Research has shown that imagining food consumption leads to food-specific habituation effects. In the present research, we replicated these effects and further examined whether the depletion of self-regulatory resources would reduce the habituation effects of imagined food consumption. Since self-regulatory resources have been shown to reduce habituation effects during the perception of emotional stimuli, we expected a reduction in habituation effects from imagined food consumption when self-regulatory resources were depleted. In Study 1, we replicated habituation effects as a response to imagining gummy bear consumption with a high (36) and medium number (18) of repetitions in a camouflaged taste test. Participants imagining gummy bear intake showed decreased food intake compared with participants who imagined putting a coin into a laundry machine. The number of repetitions did not significantly moderate the observed habituation effect. In Study 2, we investigated whether self-regulatory depletion would impede habituation effects evoked by the imagination of walnut consumption. Participants in a depleted state did not show a reduction in food intake after imagining walnut intake compared with participants in a non-depleted state. We discuss directions for future research and processes that might underlie the observed moderating effect of self-regulatory resources.

Micronutrient deficiencies. Hohenheim Consensus Conference.

OBJECTIVE: The aim of this study was to consider the risk of micronutrient deficiencies and approaches for intervention, and to summarize existing knowledge and identify areas of ignorance. DESIGN: Experts from a range of relevant disciplines received and considered a series of questions related to aspects of the topic. INTERVENTION: The experts met and discussed the questions and arrived at a consensus. CONCLUSION: Though healthy balanced diet is available for the general European population, a few defined groups are at risk of micronutrient deficiencies. In addition, the intake of specific micronutrients such as iron, folic acid, vitamin D and vitamin B12 are often marginal. To overcome these deficiencies, either selected micronutrients or a mixture of different micronutrients might be recommended. However, to define and detect micronutrient deficiencies, specific biomarkers are only available for a few micronutrients (e. g. vitamin D, folic acid, vitamin C, iron). The definition of a risk group, based on scientific data, might be an appropriate way to justify intervention with supplements.