Bioavailability of lysine in heat-treated foods and feedstuffs

During the processing of foodstuffs, lysine can react with other compounds present to form nutritionally unavailable derivatives, the most common example of which are Maillard products. Maillard products can cause serious problems when determining the available lysine content of processed foods or feedstuffs as they can revert to lysine during amino acid analysis. Several methods have been developed to determine the dietary lysine available for the metabolic processes of animals including animal growth-based assays, reactive lysine chemical methods and digestibility assays. However, growth-based assays are laborious, highly variable and tend to determine utilization rather than availability. Chemically reactive lysine assays do accurately determine the unmodified lysine in a food or feedstuff, but do not determine available lysine as they incorrectly assume that reactive lysine digestion and absorption is 100%. Ileal digestibility assays measure digestible total lysine rather than digestible reactive lysine (available lysine) and so are inaccurate, especially when applied to processed protein sources. This thesis describes the development of a true ileal digestible reactive lysine assay for determining dietary (bio)available lysine. This assay couples the guanidination reaction, for determining reactive lysine, with a true ileal digestibility assay. The resulting apparent digestibility estimate is corrected to a true digestibility value by accounting for the endogenous ileal lysine flow. Selected reaction conditions for the guanidination of lysine in a heated lactose/casein mixture and digesta of rats fed unheated casein and heated lactose/casein was examined. Overall, suitable reaction conditions were 0.6 M O-methylisourea for 7 d in a shaking waterbath at 21 ± 2 °C with an O-methylisourea to lysine ratio of 1000 and a reaction mixture pH of 10.6 for casein and heated lactose/casein and 11.0 for digesta. The accuracy of the guanidination method for determining reactive lysine in a range of “ready-to-eat ” cereal-based breakfast foods and selected feedstuffs was tested by comparison with the reactive lysine content of the same protein sources when determined using the fluorodinitrobenzene method. Overall, there was excellent agreement between the two methods. The accuracy of the newly developed bioassay for determining digestible reactive (available) lysine for predicting lysine deposition was also tested using a heated skim milk powder. The true ileal total and reactive lysine digestibilities were determined for the heated skim milk powder which was then fed to pigs, along with two control diets which were formulated based on either total lysine digestibility or reactive lysine digestibility. All diets were limiting in lysine. The pigs fed the heated skim milk powder deposited the same (P > 0.05) amount of lysine (9.1 g d-1) as the pigs fed the control diet that was formulated based on reactive lysine digestibility (9.1 g d-1) but deposited significantly (P The new assay demonstrated that for a range of milk protein-based foods, there was little difference between digestible total lysine and digestible reactive lysine for most of the milk products tested. In contrast, for a range of “ready-to-eat” cereal-based breakfast foods, available lysine was 5 – 50% lower than that determined using the traditional assay, which is of concern given that breakfast cereals are perceived to be “healthy” foods. Similarly, the available lysine content of a range of moist and dry commercial cat foods was significantly (P <br/

Cases of Conscience: The Supreme Court and Conscientious Objectors to Military Service During the Post World War II Era

This thesis examines the history of American conscientious objectors to military service during the aftermath of World War II. It describes why conscientious objectors were viewed with distrust and suspicion for their refusal to bear arms in defense of the nation and considers how groups such as the American Legion and the Veterans of Foreign Wars attempted to prevent COs from enjoying key benefits of U.S. citizenship by demanding that conscientious objectors be excluded from public employment and denied most forms of government assistance. This thesis focuses on decisions of the United States Supreme Court following World War II that defined and extended the rights of conscientious objectors. Some of those decisions reflected and continued a debate over the protection of speech and claims of conscience that developed among the Justices on the Supreme Court following the end of World War I. This paper explores and evaluates the connections between the World War I era cases and decisions that followed the end of World War II. Analysis of the post World War II decisions reveals how the Supreme Court moved away from ideological debates over the protection of conscience towards the imposition of procedural rules designed to insure that administrative and judicial hearings involving COs met due process standards. The contests over the rights of conscientious objectors that followed the end of World War II displayed the expanding role the Supreme Court assumed in protecting the civil liberties of all Americans. The Supreme Court cases concerning conscientious objectors discussed here also showed how judicial protection of claims of conscience were influenced by Cold War fears that the philosophy of COs might undermine the ability of the nation to defend itself

Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus

BACKGROUND: The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function. RESULTS: We collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members. CONCLUSION: We have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family

Empirical Evaluation of Bone Extraction Protocols

The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone

Gut luminal endogenous protein: Implications for the determination of ileal amino acid digestibility in humans

The true ileal digestibility assay provides the most informative measure of digestibility to assess bioavailability of amino acids in foods for humans. To determine ‘true’ estimates of ileal amino acid digestibility, requires that endogenous amino acids present in digesta at the terminal ileum be quantified. The amounts of endogenous amino acids in ileal digesta can be determined after feeding an animal or human a protein-free diet (traditional approach) or by various methods after giving a protein-containing diet. When the protein-free method has been applied with adult human subjects an overall mean value (three separate studies) for endogenous ileal nitrogen flow of 800 mg N/d has been reported. This value is considerably lower than a comparable value obtained after feeding protein of 1852 mg N/d (mean of four separate studies), and thus endogenous ileal N and amino acids should be measured under conditions of protein alimentation. There is some confusion concerning the terminology used to define digestibility, with the term “true” digestibility having different adopted meanings. Here, true amino acid digestibility is defined as apparent amino acid digestibility corrected for the basal amino acid losses determined after giving either a protein-free or a protein-containing diet. Basal losses should be determined at a defined dry-matter and protein intake. The protein-free diet approach to determining endogenous amino acids is considered unphysiological and basal losses refer to ileal endogenous amino acid flows associated with digesta dry-matter flow, and not including “specific” effects of dietary factors such as non starch polysaccharides and anti nutritional factors. Arguments are advanced that the enzyme hydrolysed protein/ultra filtration method may be suitable for routine application with a cannulated pig model, to obtain physiologically-valid basal estimates of ileal endogenous amino acids to allow calculation of true ileal amino acid digestibility in the pig, and then prediction (via statistical relationships) of true coefficients of amino acid digestibility in humans.falsePublishe

Amino Acid Analysis

Amino acid analysis is used to determine the amino acid content of amino acid–, peptide- and protein-containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of l-amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine