Torbjörn Lundh

Metabolism of Estrogenic Isoflavones in Domestic Animals

Abstract The metabolism of estrogenic isoflavones in cattle and sheep is reviewed. Results from in vitro and in vivo studies are discussed, mainly regarding whether differences in sensitivity to phytoestrogens between cattle and sheep depend on differences in metabolism, particularly in conjugative capacity. Results from a feeding experiment with pigs fed red clover meal are presented. Levels of phytoestrogens in plasma from the pig are compared with those found in plasma from ruminants fed red clover silage. Some aspects relating to the possibility of pigs being exposed to risks when fed with feed containing estrogenic isoflavones are briefly discussed.

Determination of free amino acids in pig plasma by precolumn derivatization with 6-N-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography

A high-performance liquid chromatographic method for measuring amino acids in pig plasma has been developed by using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent. With the method presented here it was possible to separate 24 amino acids in pig plasma within one single run in 45 min, while only 18 amino acids were properly separated with the original AccQ.Tag conditions used for analysis of hydrolysate samples. The recovery was above 90% for most amino acids, and the mean coefficient of variation of the retention times below 0.30%. The within- and between-assay reproducibility for the determination of plasma amino acids showed C.V.s below 2.2% and 4.5%, respectively, for most amino acids analyzed. In the present work, most of the plasma amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on very small amounts of sample.

Dietary flavonoids with a catechol structure increase α-tocopherol in rats and protect the vitamin from oxidation in vitro

To identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (−)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of α-tocopherol (α-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of α-T, the capacities of the flavonoids to i) protect α-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce α-tocopheroxyl radicals (α-T · ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected α-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of α-T · . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the α-T · by the flavonoids.

Inclusion of Chicory (Cichorium intybus L.) in Pigs' Diets Affects the Intestinal Microenvironment and the Gut Microbiota

ABSTRACT The content and composition of prebiotic plant fiber in the diet is important in promoting gut-related health. This study investigated the effects of the dietary inclusion of chicory forage and roots on the intestinal microenvironment of pigs. Thirty-seven-week-old pigs were fed 1 of 5 diets for 18 days, including a cereal-based control diet and 4 diets with the inclusion of 80 and 160 g kg−1 of body weight chicory forage (CF80 and CF160), 80 g kg−1 chicory root (CR80), and a mix of 80 g kg−1 forage and 80 g kg−1 chicory root (CFR). The animals maintained good performance and health irrespective of diet. Bacterial community structure and diversity in ileal and colonic samples was assessed using terminal restriction fragment length polymorphism (T-RFLP), combined with cloning and sequencing. Samples clustered perfectly according to gut segment with a higher bacterial diversity in colon than ileum. Distal ileum was dominated by lactic acid bacteria (LAB), and the relative amount of this group was increased by the CF160 and CFR diets. The colonic bacterial community was dominated by butyrate-producing bacteria and Prevotella. The increased relative abundance of butyrate-producing bacteria in the colon was positively correlated with the molar proportion of acetic acid and furthermore linked to the chicory forage diets (CF80 and CF160). Diets including chicory roots (CR80 and CFR) were correlated with a higher colonic abundance of Megasphaera elsdenii. The fermentation products and pH in digesta responded to diet type and were correlated with shifts in the microbiota, showing that chicory influences the intestinal microenvironment of pigs.

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47‐HY and Lactobacillus reuteri strain P43‐HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC‐J2 intestinal epithelial cell line model. The IPEC‐J2 cells polarized on a permeable filter exhibited villus‐like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC‐J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC‐J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC‐J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell–cell contact, reduced IPEC‐J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens‐1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens.

Dietary secoisolariciresinol diglucoside and its oligomers with 3-hydroxy-3-methyl glutaric acid decrease vitamin E levels in rats.

Secoisolariciresinol diglucoside (SDG) is an important dietary lignan that is found at very high levels in flaxseed (1-4 %, w/w). Flaxseed lignans have received much research interest in recent years because of reported phyto-oestrogenic, anticarcinogenic, and anti-atherogenic effects. Previously, flaxseed feeding has been shown to decrease vitamin E concentrations in rats despite the antioxidant potential of SDG in vitro. Sesamin, a sesame lignan, on the other hand has been shown to increase vitamin E concentrations in rats. The aim of the present study was to investigate the effect of dietary SDG and its oligomers on vitamin E and cholesterol concentrations in rats. SDG was extracted from defatted flaxseed flour with a dioxane-ethanol mixture and purified by silica column chromatography. The major oligomers with 3-hydroxy-3-methyl glutaric acid, containing a high ratio of SDG to p-coumaric and ferulic acid glucosides, were purified from the extracts by reversed-phase liquid chromatography. When fed to rats at 0.1 % in the diet for 27 d, both SDG and its oligomers had no effect on animal performance but caused an increase in liver cholesterol and a 2-fold reduction in the levels of alpha- and gamma-tocopherols in rat plasma and liver. It is notable that a phenolic antioxidant, such as SDG, causes a vitamin E-lowering effect in rats. This cannot be explained at present, but warrants further investigations with respect to the magnitude, mechanism, and significance of the observed effect for human nutrition.

Ileal amino acid digestibilities in pigs of barley-based diets with inclusion of lucerne (Medicago sativa), white clover (Trifolium repens), red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne).

Two experiments were performed with post-valve T-cannulated growing pigs, using five animals in each experiment in a change-over design to evaluate the effect of inclusion of four different dried forage meals on ileal crude protein (CP) and amino acid (AA) digestibilities. The control diets (C1 and C2) were barley-based and the experimental diets were formulated by replacing the barley with 100 or 200 g/kg of either lucerne (Medicago sativa) or white clover (Trifolium repens) meal in Expt 1 and red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne) meal in Expt 2. A decrease (P < 0.05) in the apparent ileal digestibility of CP and most of the essential and nonessential AA was found with the inclusion of luceme, white clover and perennial ryegrass meal in the barley-based diets. When red clover meal was included, only the apparent ileal digestibilities of CP, leucine, phenylalanine, tyrosine and glutamic acid were found to decrease (P < 0.05). The estimated apparent ileal digestibilities of most essential AA in the forage meals were lower than in the barley-based diets. The ileal flow of glucosamine and ornithine was found to increase (P < 0.05) with increasing proportion of fibre in the diet, suggesting an increase in endogenous N secretions and small-intestinal microbial activity. With the minor changes found for ileal essential AA digestibilities with forage meal inclusion in the diet the present data confirm the potential of forage meals as a source of protein in pig diets.

Thyroid gland function in ovariectomized ewes exposed to phytoestrogens

Phytoestrogens are by definition plant-derived substances that are able to activate the mammalian oestrogen receptors. We examined the possible effects of phytoestrogens on the secretion of thyroid hormones as well as on the immunoreactivity to oestrogen receptor alpha (ER alpha) in the thyroid glands of ovariectomized ewes. Eight ovariectomized ewes were fed 3.5 kg of 100% red clover silage for 14 days. Blood samples were collected before and on day 14 of exposure to phytoestrogens. After 5 months, four of the ewes were re-exposed to red clover silage as described above and the other four served as controls. Blood samples were collected as above. All ewes were slaughtered at the end of the experiment and the thyroid glands were weighed and examined for macroscopical changes. Tissue samples were taken for immunohistochemistry and image analysis. Ewes exposed to red clover silage had significantly higher plasma concentrations of total T(3) and free T(3) than ewes fed hay. The cross-section area of thyroid follicles tended to be larger in ewes fed red clover silage than in the control animals. ER alpha immunoreactivity was stronger in thyroid glands from ewes exposed to phytoestrogens than in ewes fed hay. In conclusion, daily ingestion of 81-95 mg phytoestrogens per kg body weight for 14 days stimulated secretion of thyroid hormones and tended to increase follicle size and ER alpha immunoreactivity of thyroid glands of ovariectomized ewes.

Activities of Enzymes Involved in Glutamine Metabolism in Connection with Energy Production in the Gastrointestinal Tract Epithelium of Newborn, Suckling and Weaned Piglets

Changes in the activity of enzymes involved in glutaminolysis and energy metabolism in the entire gastrointestinal (GI) tract of developing piglets are presented for the first time. The activities of glutaminase, glutamate dehydrogenase, oxoglutarate dehydrogenase, isocitrate dehydrogenase and alanine aminotransferase in the epithelium along the gastrointestinal tract from newborn, suckling (2–4 weeks old) and weaned (9 weeks old) piglets were investigated. The activity of glutaminase in the epithelium from the small intestine and colon was higher (p < 0.05) in weaned piglets than in newborn and suckling piglets. In addition, glutamate dehydrogenase and alanine aminotransferase activities in the small intestinal epithelium were higher (p < 0.05) for weaned piglets than for newborns. The activity of oxoglutarate dehydrogenase in the epithelium of the small intestine was significantly lower in newborn and suckling piglets compared with weaned individuals. The activity of isocitrate dehydrogenase in the epithelium along the gastrointestinal tract was higher (p < 0.05) for suckling and weaned piglets than for newborn piglets. The present data indicate that the utilization of substrates for energy production differs markedly between the stomach, small intestine and colon of growing piglets. Also, the capacity of enzymes in the epithelium of the GI tract to utilize acetyl-CoA as an energy substrate in the tricarboxylic acid cycle increased with piglet age. The epithelium of the GI tract of the newborn, suckling and weaned piglets showed a high capacity to metabolize α-ketoglutarate.

Heat Shock Proteins: Intestinal Gatekeepers that Are Influenced by Dietary Components and the Gut Microbiota.

Trillions of microorganisms that inhabit the intestinal tract form a diverse and intricate ecosystem with a deeply embedded symbiotic relationship with their hosts. As more detailed information on gut microbiota complexity and functional diversity accumulates, we are learning more about how diet-microbiota interactions can influence the immune system within and outside the gut and host health in general. Heat shock proteins are a set of highly conserved proteins that are present in all types of cells, from microbes to mammals. These proteins carry out crucial intracellular housekeeping functions and unexpected extracellular immuno-regulatory features in order to maintain the mucosal barrier integrity and gut homeostasis. It is becoming evident that the enteric microbiota is one of the major determinants of heat shock protein production in intestinal epithelial cells. This review will focus on the interactions between diet, gut microbiota and their role for regulating heat shock protein production and, furthermore, how these interactions influence the immune system and the integrity of the mucosal barrier.