Dietary Macromolecules Research Articles

Biofilm and invertebrate consumption by western sandpipers (Calidris mauri) and dunlin (Calidris alpina) during spring migratory stopover: insights from tissue and breath CO2 isotopic (δ 13C, δ 15N) analyses.

Shorebirds use key migratory stopover habitats in spring and fall where body proteins are replenished and lipids stored as fuel for the remaining journey. The Fraser River estuary, British Columbia, Canada, is a critical spring stopover site for hundreds of thousands of migrating western sandpiper, Calidris mauri, and dunlin, Calidris alpina. Intertidal biofilm in spring is an important nutritional source for western sandpiper, with previous isotopic research predicting 45-59% of total diet and 50% of total energy needs. However, these studies relied on isotopic mixing models that did not consider metabolic routing of key dietary macromolecules. Complexity arises due to the mixed macromolecular composition of biofilm that is difficult to characterize isotopically. We expanded on these earlier findings by considering a protein pathway from diet to the body protein pool represented by liver tissue, using a Bayesian mixing model based on δ 13C and δ 15N. We used δ 13C measurements of adipose tissue and breath CO2 to provide an estimate of the carbohydrate and protein δ 13C values of microphytobenthos and used these derived values to better inform the isotopic mixing models. Our results reinforce earlier estimates of the importance of biofilm to staging shorebirds in predicting that assimilated nutrients from biofilm contribute ~35% of the protein budgets for staging western sandpipers (n = 13) and dunlin (n = 11) and at least 41% of the energy budget of western sandpiper (n = 69). Dunlin's ingestion of biofilm appeared higher than anticipated given their expected reliance on invertebrate prey compared to western sandpiper, a biofilm specialist. Isotopic analyses of bulk tissues that consider metabolic routing and that make use of breath CO2 and adipose lipid assays can provide new insights into avian physiology. We advocate further isotopic research to better understand biofilm use by migratory shorebirds in general and as a critical requirement for more effective conservation.

Hydrogen isotope assimilation and discrimination in green turtles.

Although hydrogen isotopes (δ2H) are commonly used as tracers of animal movement, minimal research has investigated the use of δ2H as a proxy to quantify resource and habitat use. While carbon and nitrogen are ultimately derived from a single source (food), the proportion of hydrogen in consumer tissues originates from two distinct sources: body water and food. Before hydrogen isotopes can be effectively used as a resource and habitat tracer, we need estimates of (net) discrimination factors (Δ2HNet) that account for the physiologically mediated differences in the δ2H values of animal tissues relative to that of the food and water sources they use to synthesize tissues. Here, we estimated Δ2HNet in captive green turtles (Chelonia mydas) by measuring the δ2H values of tissues (epidermis and blood components) and dietary macromolecules collected in two controlled feeding experiments. Tissue δ2H and Δ2HNet values varied systematically among tissues, with epidermis having higher δ2H and Δ2HNet values than blood components, which mirrors patterns between keratinaceous tissues (feathers, hair) and blood in birds and mammals. Serum/plasma of adult female green turtles had significantly lower δ2H values compared with juveniles, likely due to increased lipid mobilization associated with reproduction. This is the first study to quantify Δ2HNet values in a marine ectotherm, and we anticipate that our results will further refine the use of δ2H analysis to better understand animal resource and habitat use in marine ecosystems, especially coastal areas fueled by a combination of marine (e.g. micro/macroalgae and seagrass) and terrestrial (e.g. mangroves) primary production.

Compound-specific δ2H analysis highlights the relationship between direct assimilation and de novo synthesis of amino acids from food and water in a terrestrial mammalian omnivore.

Hydrogen isotope (δ2H) analysis has been routinely used as an ecological tracer for animal movement and migration, yet a biochemical understanding of how animals incorporate this element in the synthesis of tissues is poorly resolved. Here, we apply a new analytical tool, amino acid (AA) δ2H analysis, in a controlled setting to trace the influence of drinking water and dietary macromolecules on the hydrogen in muscle tissue. We varied the δ2H of drinking water and the proportions of dietary protein and carbohydrates with distinct hydrogen and carbon isotope compositions fed to house mice among nine treatments. Our results show that hydrogen in the non-essential (AANESS) and essential (AAESS) AAs of mouse muscle is not readily exchanged with body water, but rather patterns among these compounds can be described through consideration of the major biochemical pathway(s) used by organisms to synthesize or route them from available sources. Dietary carbohydrates contributed more hydrogen than drinking water to the synthesis of AANESS in muscle. While neither drinking water nor dietary carbohydrates directly contributed to muscle AAESS, we did find that a minor but measurable proportion (10-30%) of the AAESS in muscle was synthesized by the gut microbiome using hydrogen and carbon from dietary carbohydrates. δ2H patterns among individual AAs in mice muscle are similar to those we previously reported for bacteria, which provides additional support that this approach may allow for the simultaneous analysis of different AAs that are more influenced by drinking water (AANESS) versus dietary (AAESS) sources of hydrogen.

Improved hydrolysis of α-tocopherol acetate emulsion and its bioaccessibility in the presence of polysaccharides and PEG2000

Dietary macromolecules are often used for food formulation. Most of the published literature usually focuses on a certain polysaccharide reducing lipid digestion, but the non-specific physical effects such as the excluded volume effect of the surrounding macromolecules, which will possibly enhance the hydrolysis of lipids, is rarely discussed. As the influence of polysaccharides on the gastrointestinal fate of α-tocopherol acetate (VE-A) encapsulated in emulsion have not been conducted so far, in this research, we study the effect of crowded medium created by polysaccharides (Ficoll400) on the hydrolysis of VE-A and its bioaccessibility. The results show that the presence of Ficoll400 increased the bioaccessibility and conversion of VE-A 1.6 times that of a dilute buffer. An enzyme-catalyzed kinetic reaction rate of VE-A hydrolysis is increased up to 3.5 times. Both the improved thermodynamic conversion yields and kinetic rates of the hydrolysis of the VE-A emulsion was further observed in a synthetic macromolecule PEG2000 medium. This research improves our understanding of the crowding effect of macromolecules on VE-A/lipid emulsion digestion.

Assimilation and isotopic discrimination of hydrogen in tilapia: implications for studying animal diet with δ2H

AbstractHydrogen isotope (δ2Η) analysis has become a valuable tool in the study of animal migration; however, the biochemical framework required for ecologists to confidently apply δ2Η to quantify resource use has yet to be adequately resolved. In contrast to carbon (δ13C) and nitrogen (δ15N) isotopes where food is the only source of these elements, there are two distinct sources of hydrogen available to consumers for tissue synthesis: food and water. To effectively use tissue δ2Η values as a tracer of food and water resources, two fundamental questions need to be examined in animals that inhabit different environments: (1) What proportion of hydrogen in tissues is derived from sources of food vs. water? and (2) What is the magnitude and degree of variation in total δ2Η discrimination (Δ2Hnet) between consumer tissues and these sources? We completed a 3 × 3 controlled feeding experiment on Nile tilapia (Oreochromis niloticus) in which we varied the δ2Η of tank water and dietary macromolecules to examine these questions in two tissues commonly analyzed by ecologists: muscle and liver. We found that the proportion of hydrogen in tilapia tissue derived from tank water was similar for muscle (~23%) and liver (~25%). We then used linear regression and an isotope mixing model based on accompanying δ13C data to estimate the proportion of hydrogen in muscle and liver tissue derived from dietary protein (34–44%), cornmeal (21–27%), corn syrup (4–5%), and lipids (≤1%). With this information and the δ2Η values of water, protein, carbohydrates, and lipids supplied to fish in each treatment, we calculated Δ2Hnet values of −47‰ ± 5‰ for muscle and −41‰ ± 5‰ for liver. Our experiment is the first to quantify the relative proportion of hydrogen from different dietary macromolecules used by an omnivore to synthesize its tissues. Such information is needed to further refine the use of δ2Η analysis as a dietary tracer for aquatic animals.

Variability in the routing of dietary proteins and lipids to consumer tissues influences tissue-specific isotopic discrimination.

The eco-physiological mechanisms that govern the incorporation and routing of macronutrients from dietary sources into consumer tissues determine the efficacy of stable isotope analysis (SIA) for studying animal foraging ecology. We document how changes in the relative amounts of dietary proteins and lipids affect the metabolic routing of these macronutrients and the consequent effects on tissue-specific discrimination factors in domestic mice using SIA. We also examine the effects of dietary macromolecular content on a commonly used methodological approach: lipid extraction of potential food sources. We used carbon ((13) C) and nitrogen ((15) N) isotopes to examine the routing of carbon from dietary proteins and lipids that were used by mice to biosynthesize hair, blood, muscle, and liver. Growing mice were fed one of four diet treatments in which the total dietary content of C4 -based lipids (δ(13) C = -14.5‰) and C(3) -based proteins (δ(13) C = -27‰) varied inversely between 5% and 40%. The δ(13) C values of mouse tissues increased by approximately 2-6‰ with increasing dietary lipid content. The difference in δ(13) C values between mouse tissues and bulk diet ranged from 0.1 ± 1.5‰ to 2.3 ± 0.6‰ for all diet treatments. The mean (±SD) difference between the δ(13) C values of mouse tissues and dietary protein varied systematically among tissues and ranged from 3.1 ± 0.1‰ to 4.5 ± 0.6‰ for low fat diets and from 5.4 ± 0.4‰ to 10.5 ± 7.3‰ for high fat diets. Mice used some fraction of their dietary lipid carbon to synthesize tissue proteins, suggesting flexibility in the routing of dietary macromolecules to consumer tissues based on dietary macromolecular availability. Consequently, all constituent dietary macromolecules, not just protein, should be considered when determining the relationship between diets and consumer tissues using SIA. In addition, in cases where animals consume diets with high lipid contents, non lipid-extracted prey samples should be analyzed to estimate diets using SIA.

Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

Amino acid δ13C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore.

Stable-isotope analysis (SIA) has revolutionized animal ecology by providing time-integrated estimates of the use of resources and/or habitats. SIA is based on the premise that the isotopic composition of a consumer's tissues originates from its food, but is offset by trophic-discrimination (enrichment) factors controlled by metabolic processes associated with the assimilation of nutrients and the biosynthesis of tissues. Laboratory preparation protocols dictate that tissues both of consumers and of their potential prey be lipid-extracted prior to analysis, because (1) lipids have carbon isotope (δ(13)C) values that are lower by approximately 3-8‰ than associated proteins and (2) amino acids in consumers' proteinaceous tissues are assumed to be completely routed from dietary protein. In contrast, models of stable-isotope mixing assume that dietary macromolecules are broken into their elemental constituents from which non-essential amino acids are resynthesized to build tissues. Here, we show that carbon from non-protein dietary macromolecules, namely lipids, was used to synthesize muscle tissue in an omnivorous rodent (Mus musculus). We traced the influence of dietary lipids on the synthesis of consumers' tissues by inversely varying the dietary proportion of C4-based lipids and C3-based protein while keeping carbohydrate content constant in four dietary treatments, and analyzing the δ(13)C values of amino acids in mouse muscle after 4 months of feeding. The influence of dietary lipids on non-essential amino acids varied as function of biosynthetic pathway. The source of carbon in ketogenic amino acids synthesized through the Krebs cycle was highly sensitive to dietary lipid content, with significant increases of approximately 2-4‰ in Glutamate and Aspartate δ(13)C values from the 5% to 15% dietary lipid treatment. Glucogenic amino acids (Glycine and Serine) were less sensitive to dietary lipid, but increased by approximately 3-4‰ from the 25% to 40% lipid diet. As expected, the δ(13)C values of essential amino acids did not vary significantly among diets. Although lipids provide a calorie-rich resource that fuels energy requirements, our results show that they also can be an important elemental source of carbon that contributes to the non-essential amino acids used to build structural tissue like muscle. As such, the calculation of trophic-discrimination factors for animals that consume a lipid-rich diet should consider lipid carbon as a building block for proteinaceous tissues. Careful consideration of the macromolecular composition in the diet of the consumer of interest will help to further refine the use of SIA to study animal ecology and physiology.

The Onset of Enhanced Intestinal Permeability and Food Sensitivity Triggered by Medication Used in Dental Procedures: A Case Report

Enhanced intestinal permeability and food sensitivity are two of the many proven causes of gastrointestinal disorders. This present report describes a woman with no previous gastrointestinal (GI) complaints, who underwent dental root canal, bone graft, and implant procedures. Postsurgery she experienced an allergic reaction to the combined medications. In the weeks that followed, she presented with multiple food intolerances. Four weeks after the final dental procedure, she was assessed serologically for mucosal immune function, salivary, and blood-gluten reactivity, intestinal permeability, and other food sensitivities. Compared to her test reports from two months prior to her first dental procedure, the patient's results showed high total secretory IgA (SIgA) and elevated salivary antibodies to alpha-gliadin, indicating abnormal mucosal immunity and loss of tolerance to gluten. Her serologic assessments revealed immunoglobulin G (IgG) and IgA antibodies to a range of wheat/gluten proteins and peptides, gut bacterial endotoxins and tight junction proteins. These test results indicate gut dysbiosis, enhanced intestinal permeability, systemic gluten-reactivity, and immune response to other dietary macromolecules. The present case suggests that patients who experience severe allergic or pseudoallergic reactions to medication should be assessed and monitored for gut dysfunction. If left untreated this could lead to autoimmune reactions to self tissues.

Isotopic fractionation and turnover in captive Garden Warblers (Sylvia borin): implications for delineating dietary and migratory associations in wild passerines

There is currently a great deal of interest in using stable-isotope methods to investigate diet and migratory connections in wild passerines. To apply these methods successfully, it is important to understand how stable isotopes discriminate or change between diet and the tissue of interest and what the element-turnover rates are in metabolically active tissues. Of particular use are studies that sample birds non-destructively through the use of blood and feathers. We investigated patterns of isotopic discrimination between diet and blood and feathers of Garden Warblers (Sylvia borin) raised on an isotopically homogeneous diet (48% C, 5% N) and then switched to one of two experimental diets, mealworms (56.8% C, 8.3% N) and elderberries, Sambucus niger (47.4% C, 1.5% N). We established that the discrimination factors between diet and blood appropriate for stable carbon (δ13C) and nitrogen (δ15N) isotopes are +1.7‰ and +2.4‰, respectively. For feathers, these values were +2.7‰ and +4‰, respectively. Turnover of elemental nitrogen in whole blood was best approximated by an exponential-decay model with a half-life of 11.0 ± 0.8 days (mean ± SD). Corresponding turnover of carbon was estimated to range from 5.0 ± 0.7 to 5.7 ± 0.8 days. We conclude that this decoupling of nitrogen- and carbon-turnover rates can be explained by differences in metabolic routing of dietary macromolecules. Our results suggest that tracking frugivory in migratory passerines that switch diets between insects and fruits may be complicated if only a trophic-level estimate is made using δ15N measurements.