Protein is an important component of bone, but the role of dietary protein intake in osteoporosis remains controversial. The Utah picture-sort food frequency questionnaire was found to produce a useful estimation of usual dietary intake in the elderly. This method of dietary assessment was used in a population-based case-control study to examine the relationship between protein intake and risk of osteoporotic hip fracture in elderly Utah residents. Analyses of risk of hip fracture across increasing quartiles of protein intake were stratified by age-group. Higher protein intake was associated with a reduced risk of hip fracture in men and women aged 50-69 years but did not appear to increase or decrease risk of hip fracture in those aged 70-89 years. The relationship appeared to be modified by age. Modification of protein intake late in life may be a useful means to prevent hip fractures in the elderly.

Excess nitrogen (N) excretion from animal agriculture results in reduced air and water quality, and poses a risk to human health. Although the dairy industry utilizes milk urea N (MUN) to monitor protein feeding and N excretion, phenotypic diversity among cows may influence MUN and thus bias feed management. An initial study using data from 2 previously published research trials and a field trial, observed that cow had a significant effect on MUN variation. Regression models, utilized to predict MUN, corrected for dietary nutrients and some animal effects, and thus the observed effect of cow on MUN variation may reflect genetic selection decisions of animals with either poor or efficient urea transport. A second trial observed that MUN and PUN concentrations were positively correlated with gut urea clearance, providing evidence for differences in urea transport activity among cows. The presence of urea transport variation suggests that current protein recommendations may not estimate true requirements. A third trial observed that animals fed sub-NRC levels of RDP and RUP had reduced N intake and excretion of fecal N, urinary urea-N, and MUN. Animals maximized N efficiency and had no loss in milk production, suggesting a possible overestimation of RDP and RUP in the current NRC prediction model. The present project provides evidence for phenotypic variation among cows, which may be partially explained by differences in urea transport activity. Future work confirming genetic variation among urea transporters may provide an opportunity to improve feeding management if cow urea efficiency is known. / Master of Science

To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function. / Ph. D.

<p>Diet is the number one leading modifiable cause of poor health globally, with poor diets accounting for 10.9 million (22%) of all deaths among adults in 2017. In addition, one of our generation’s forthcoming challenges is the rapid expansion of the population aged 60 years and older. Although people are living longer, there is an associated increase in the prevalence of aged-related chronic diseases and functional impairment, such as cardiometabolic diseases and sarcopenia. As such, dietary components can play a role in positively or negatively influencing the prevention and treatment of chronic cardiometabolic diseases and sarcopenia. One such dietary component is dietary protein, which is essential throughout the life course, from gestation through old age. Evidence supports dietary protein playing an important role in reducing the risk of developing age-related chronic diseases such as sarcopenia and cardiometabolic diseases. </p> <p><em><strong>     Study 1, Chapter 2:</strong></em> The Dietary Guidelines for Americans (DGA) recommends consuming a variety of “Protein Foods” based on “ounce equivalent” (oz-eq) portions. In addition, the 2020-2030 Strategic Plan for NIH Nutrition Research includes to “define the role of nutrition across the lifespan” with an objective to “assess the role of nutrition in older adults to promote healthy aging.” However, there is a paucity of primary research that directly compares EAAs bioavailability between young and older adults consuming the same oz-eq portions of varied Protein Foods. No study has assessed the same oz-eq portions of animal- versus plant-based Protein Foods on essential amino acids (EAAs) bioavailability for protein anabolism in young and older adults. Therefore, we conducted two sequential randomized, investigator-blinded, crossover, acute feeding trials with the same study design; first in a cohort of young adults and second in a cohort of older adults. The primary objective of this project was to assess the effect of consuming two oz-eq portions of animal-based (unprocessed lean pork or whole eggs) vs. plant-based (black beans or sliced almonds) Protein Foods as part of a mixed whole foods meal on plasma EAAs bioavailability for protein anabolism. Consistent with our hypotheses, participant age did not affect postprandial EAAs bioavailability, and consuming a meal with two oz-eq of unprocessed lean pork or whole eggs resulted in greater postprandial EAAs bioavailability compared to a meal with two oz-eq of black beans or raw sliced almonds in 1) young adults; 2) older adults; and 3) young and older adults combined. These findings show on the same oz-eq basis, consuming these animal- vs. plant-based Protein Foods more effectively provide bioavailable EAAs for protein anabolism. </p> <p><em><strong>     Study 2, Chapter 3:</strong></em> Poultry meat is the most consumed type of meat worldwide and in the US. Poultry is generally considered to be a “healthy” meat as it is a high-quality protein source and provides other essential nutrients. However, research assessing poultry and its effects on and relations with chronic diseases in humans is sparse, and the forms of poultry typically consumed in the US, are not necessarily in line with recommendations provided by the DGA. Therefore, we conducted a scoping review to systematically search and chronicle scientific literature pertinent to poultry intake and human health. Main findings from this project were 1) historically, little research, especially randomized diet-controlled feeding trials, has been conducted to understand associations between and effects of consuming poultry products on human health; 2) the majority of research is from observational studies assessing relationships between poultry intake and risks of morbidity and mortality from various types of cancer; 3) a paucity of research exists to support chicken as a health-promoting food in children; and 4) research taking into account poultry product processing and cooking methods is needed. Science and health professionals, the poultry industry, and the public will benefit from new observational and experimental research to address cutting-edge scientific, public policy, and consumer topics pertinent to poultry intake and human health. </p> <p><em><strong>     Study 3, Chapter 4:</strong></em> Emerging research on whey protein supplementation suggests it may be a potential modifier of type 2 diabetes mellitus (T2DM) risk factors, including glucose control. As systematic reviews and/or meta-analyses of randomized controlled trials are gaining importance in nutrition literature, we conducted an umbrella systematic review to search for and chronicle published systematic reviews and/or meta-analyses of randomized controlled trials pertinent to whey protein supplementation and T2DM modifiable risk factors (study 3, Chapter 4). Among the 13 systematic reviews, including 12 meta-analyses critically assessed for this umbrella review, no reviews reported any adverse effects of whey protein on any reported T2DM-related risk factor. Collectively, a preponderance of evidence indicates whey protein supplementation improves multiple clinical indicators of glucose control in apparently healthy adults and those at increased risk for type 2 diabetes mellitus. </p>

A review of the literature revealed that diet plays an important role in serious human noninfectious large bowel diseases including cancer and inflammatory bowel diseases. Dietary protein (especially as red and processed meats) has been implicated as a positive risk factor for colorectal cancer while starch which is not digested in the small intestine (resistant starch, RS) appears to be protective. The series of experiments described in this thesis were aimed to determine the effects of dietary proteins and RS on indices of colon health in an animal model, the laboratory rat. Genetic damage is a prerequisite for carcinogenesis and this was assessed by a specific assay (the comet assay) which gives a measure of DNA strand breaks. Loss of mucus barrier function is thought to contribute to inflammatory bowel disease by permitting bacterial translocation and this was measured optically using a microscope micrometer. Other biomarkers were measured as described below. There were four major experiments. 1. Effects of dietary red meat and casein on colonic DNA damage and interaction with resistant starch Previous studies had shown that higher dietary protein (as casein) induced genetic damage in rat colonocytes and that RS (fed as a high amylose maize starch) was protective. This study was aimed at establishing whether a high protein diet fed as cooked red meat had similar effects and whether RS was protective. Rats were fed diets containing either 15 % or 25% casein or 25% barbecued lean red beef, each with or without 48% high amylose maize starch (as a source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic single-strand DNA breaks compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% more DNA damage than the high casein diet but reduced mucus thickness to a similar degree as casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. It is thought that RS promotes large bowel health through the SCFA produced by the large bowel bacteria. One acid in particular (butyrate) has been associated particularly with promotion of normal large bowel function and protection against disease. In keeping with this hypothesis, caecal and faecal short chain fatty acid pools (including those of butyrate) were increased by inclusion of RS in the diet. DNA damage is an early step in the initiation of cancer and these findings agree with the population data which suggest that total dietary protein and red meat promote risk of colorectal cancer. However, inclusion of resistant starch in the diet could significantly reduce that risk. 2. Differential effects of dietary whey, soy and casein on colonic DNA damage and interaction with resistant starch The preceding experiments showed that high levels of animal-derived proteins increased colonocyte genetic damage and loss of the mucus barrier in rats. This second experiment was designed to determine whether diets high in different types of dairy protein (casein or whey) or a plant protein isolate (soy) had similar adverse effects on colonic DNA and mucus barrier function and whether inclusion of RS in the diet was protective. Adult male Sprague Dawley rats were fed a diet containing 15 % or 25 % casein, whey or soy protein, each with or without 48 % high amylose maize starch for 4 weeks. In confirmation of the earlier studies, higher levels of dietary casein increased colonocyte DNA damage significantly. However, whey did not increase genetic damage. Colonic DNA damage was highest for soy when fed at both 15% and 25% protein in the absence of RS. Inclusion of RS in the diet attenuated colonocyte DNA damage due to higher dietary protein in all three groups. The colonic mucus barrier was thinner in rats fed higher dietary protein but the effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were low in rats fed the digestible starch and were higher in rats fed RS. However, there was no relationship between caecal or faecal SCFA and genetic damage or mucus thickness. Caecal and colonic tissue weight and colon length were higher in rats fed RS, consistent with greater SCFA supply. These data confirm that higher dietary protein of animal (casein) or plant (soy) origin increases genetic damage and loss of the mucus barrier indicating that this is an effect of protein and not its source. These findings accord with the epidemiological data which link dietary protein to greater risk of colorectal cancer and inflammatory bowel disease. However, the data show also that dietary proteins differ in their specific actions on genetic damage and mucus thickness. Further, the data from the feeding of whey suggest that not all proteins are equivalent in their capacity to provoke adverse changes in colonic integrity. While the data show that RS raised large bowel and faecal SCFA, they indicate their levels were not related directly to these biomarkers. 3. Dose response effects of resistant starch on protein induced colonic DNA damage The accumulated data linking greater protein intakes to adverse changes in the colon were obtained at dietary levels which were not unreasonable in terms of animal or human consumption. However, the dietary level of RS which were fed were relatively high (48% by weight) so this study was conducted to determine its effectiveness at lower levels of dietary inclusion. It was also important to ascertain whether there was a dose-response relationship between RS intake and the observed effects. One of the mechanisms proposed for the induction of colorectal cancer by high dietary protein intakes is oxidative damage to DNA. In this experiment, this was done by assaying with endonuclease III. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% high amylose maize starch for 4 weeks. As in the preceding studies comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% RS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by RS. There was no significant difference between dietary groups associated with oxidative DNA damage as measured by endonuclease III. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary RS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. The data show that RS prevents protein induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% high amylose maize starch was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools. Intakes of this order are not unreasonable in terms of human consumption of RS. 4. Dose response effects of red and white meat on colonic DNA damage and interaction with resistant starch The accumulated evidence from large prospective human studies links diet to colorectal cancer risk strongly. The evidence from the animal studies described in this thesis that dietary protein induces colonocyte genetic damage supports a role for high protein intakes in increasing risk. Recently, several large epidemiological studies and a meta-analysis of prospective studies have found that consumption of dietary red or processed meats, but not white (poultry) meat, is associated with increased risk of colorectal cancer. This is consistent with the data from the preceding studies that specific proteins affected colonic integrity differentially. A large prospective European study (European Prospective Investigation into Cancer and Nutrition) has reported that dietary fibre was protective. The findings reported in this thesis that RS opposes the effects of high dietary protein accord with that conclusion. This study aimed to compare the effects of cooked red (beef) or white (chicken) meat on DNA damage and mucus barrier thickness in rats. The study was designed to determine whether the relationship between the intakes of these meats was dose-dependent. Double-strand DNA breaks are thought to relate more closely to carcinogenesis than single-strand breaks so both were measured. Adult male Sprague-Dawley rats were fed a diet containing 15%, 25% or 35% cooked beef or cooked chicken each with or without 20% high amylose maize starch for four weeks. Both red and white meat increased colonic DNA damage dose-dependently. However, both single and double strand breaks were significantly greater when the rats were fed the red meat diets compared to those fed the white meat. Colonocyte DNA damage was reduced by the consumption of RS while large bowel SCFA were increased. The findings of this study are consistent with the epidemiological data which show that red meat consumption is associated with greater risk of colorectal cancer but that white meat is not. Summary The data reported in this thesis support the findings of prospective population studies that high dietary protein, red meat in particular, appears to be harmful to the health of the large bowel. However, the data demonstrate also that different protein types have differential effects on the integrity of the colonocyte DNA. Furthermore, the addition of RS to the diet protects against protein-induced colonic DNA damage and maintenance of the colonic mucus barrier, apparently through increased SCFA production by colonic fermentation. The results of these experiments indicate a strong potential for RS to be effective in maintenance of large bowel integrity in the face of high dietary protein. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292858 / Thesis(Ph.D.)-- School of Molecular and Biomedical Science, 2007.

A review of the literature revealed that diet plays an important role in serious human noninfectious large bowel diseases including cancer and inflammatory bowel diseases. Dietary protein (especially as red and processed meats) has been implicated as a positive risk factor for colorectal cancer while starch which is not digested in the small intestine (resistant starch, RS) appears to be protective. The series of experiments described in this thesis were aimed to determine the effects of dietary proteins and RS on indices of colon health in an animal model, the laboratory rat. Genetic damage is a prerequisite for carcinogenesis and this was assessed by a specific assay (the comet assay) which gives a measure of DNA strand breaks. Loss of mucus barrier function is thought to contribute to inflammatory bowel disease by permitting bacterial translocation and this was measured optically using a microscope micrometer. Other biomarkers were measured as described below. There were four major experiments. 1. Effects of dietary red meat and casein on colonic DNA damage and interaction with resistant starch Previous studies had shown that higher dietary protein (as casein) induced genetic damage in rat colonocytes and that RS (fed as a high amylose maize starch) was protective. This study was aimed at establishing whether a high protein diet fed as cooked red meat had similar effects and whether RS was protective. Rats were fed diets containing either 15 % or 25% casein or 25% barbecued lean red beef, each with or without 48% high amylose maize starch (as a source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic single-strand DNA breaks compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% more DNA damage than the high casein diet but reduced mucus thickness to a similar degree as casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. It is thought that RS promotes large bowel health through the SCFA produced by the large bowel bacteria. One acid in particular (butyrate) has been associated particularly with promotion of normal large bowel function and protection against disease. In keeping with this hypothesis, caecal and faecal short chain fatty acid pools (including those of butyrate) were increased by inclusion of RS in the diet. DNA damage is an early step in the initiation of cancer and these findings agree with the population data which suggest that total dietary protein and red meat promote risk of colorectal cancer. However, inclusion of resistant starch in the diet could significantly reduce that risk. 2. Differential effects of dietary whey, soy and casein on colonic DNA damage and interaction with resistant starch The preceding experiments showed that high levels of animal-derived proteins increased colonocyte genetic damage and loss of the mucus barrier in rats. This second experiment was designed to determine whether diets high in different types of dairy protein (casein or whey) or a plant protein isolate (soy) had similar adverse effects on colonic DNA and mucus barrier function and whether inclusion of RS in the diet was protective. Adult male Sprague Dawley rats were fed a diet containing 15 % or 25 % casein, whey or soy protein, each with or without 48 % high amylose maize starch for 4 weeks. In confirmation of the earlier studies, higher levels of dietary casein increased colonocyte DNA damage significantly. However, whey did not increase genetic damage. Colonic DNA damage was highest for soy when fed at both 15% and 25% protein in the absence of RS. Inclusion of RS in the diet attenuated colonocyte DNA damage due to higher dietary protein in all three groups. The colonic mucus barrier was thinner in rats fed higher dietary protein but the effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were low in rats fed the digestible starch and were higher in rats fed RS. However, there was no relationship between caecal or faecal SCFA and genetic damage or mucus thickness. Caecal and colonic tissue weight and colon length were higher in rats fed RS, consistent with greater SCFA supply. These data confirm that higher dietary protein of animal (casein) or plant (soy) origin increases genetic damage and loss of the mucus barrier indicating that this is an effect of protein and not its source. These findings accord with the epidemiological data which link dietary protein to greater risk of colorectal cancer and inflammatory bowel disease. However, the data show also that dietary proteins differ in their specific actions on genetic damage and mucus thickness. Further, the data from the feeding of whey suggest that not all proteins are equivalent in their capacity to provoke adverse changes in colonic integrity. While the data show that RS raised large bowel and faecal SCFA, they indicate their levels were not related directly to these biomarkers. 3. Dose response effects of resistant starch on protein induced colonic DNA damage The accumulated data linking greater protein intakes to adverse changes in the colon were obtained at dietary levels which were not unreasonable in terms of animal or human consumption. However, the dietary level of RS which were fed were relatively high (48% by weight) so this study was conducted to determine its effectiveness at lower levels of dietary inclusion. It was also important to ascertain whether there was a dose-response relationship between RS intake and the observed effects. One of the mechanisms proposed for the induction of colorectal cancer by high dietary protein intakes is oxidative damage to DNA. In this experiment, this was done by assaying with endonuclease III. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% high amylose maize starch for 4 weeks. As in the preceding studies comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% RS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by RS. There was no significant difference between dietary groups associated with oxidative DNA damage as measured by endonuclease III. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary RS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. The data show that RS prevents protein induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% high amylose maize starch was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools. Intakes of this order are not unreasonable in terms of human consumption of RS. 4. Dose response effects of red and white meat on colonic DNA damage and interaction with resistant starch The accumulated evidence from large prospective human studies links diet to colorectal cancer risk strongly. The evidence from the animal studies described in this thesis that dietary protein induces colonocyte genetic damage supports a role for high protein intakes in increasing risk. Recently, several large epidemiological studies and a meta-analysis of prospective studies have found that consumption of dietary red or processed meats, but not white (poultry) meat, is associated with increased risk of colorectal cancer. This is consistent with the data from the preceding studies that specific proteins affected colonic integrity differentially. A large prospective European study (European Prospective Investigation into Cancer and Nutrition) has reported that dietary fibre was protective. The findings reported in this thesis that RS opposes the effects of high dietary protein accord with that conclusion. This study aimed to compare the effects of cooked red (beef) or white (chicken) meat on DNA damage and mucus barrier thickness in rats. The study was designed to determine whether the relationship between the intakes of these meats was dose-dependent. Double-strand DNA breaks are thought to relate more closely to carcinogenesis than single-strand breaks so both were measured. Adult male Sprague-Dawley rats were fed a diet containing 15%, 25% or 35% cooked beef or cooked chicken each with or without 20% high amylose maize starch for four weeks. Both red and white meat increased colonic DNA damage dose-dependently. However, both single and double strand breaks were significantly greater when the rats were fed the red meat diets compared to those fed the white meat. Colonocyte DNA damage was reduced by the consumption of RS while large bowel SCFA were increased. The findings of this study are consistent with the epidemiological data which show that red meat consumption is associated with greater risk of colorectal cancer but that white meat is not. Summary The data reported in this thesis support the findings of prospective population studies that high dietary protein, red meat in particular, appears to be harmful to the health of the large bowel. However, the data demonstrate also that different protein types have differential effects on the integrity of the colonocyte DNA. Furthermore, the addition of RS to the diet protects against protein-induced colonic DNA damage and maintenance of the colonic mucus barrier, apparently through increased SCFA production by colonic fermentation. The results of these experiments indicate a strong potential for RS to be effective in maintenance of large bowel integrity in the face of high dietary protein. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292858 / Thesis(Ph.D.)-- School of Molecular and Biomedical Science, 2007.

BACKGROUND: After proteolysis, the majority of released amino acids from dietary protein are transported to the liver for gluconeogenesis or to peripheral tissues where they are used for protein synthesis and eventually catabolized, producing ammonia as a byproduct. High ammonia levels in the brain are a major contributor to the decreased neural function that occurs in several pathological conditions such as hepatic encephalopathy when liver urea cycle function is compromised. Therefore, it is important to gain a deeper understanding of human ammonia metabolism. The objective of this study was to predict changes in blood ammonia levels resulting from alterations in dietary protein intake, from liver disease, or from partial loss of urea cycle function. METHODS: A simple mathematical model was created using MATLAB SimBiology and data from published studies. Simulations were performed and results analyzed to determine steady state changes in ammonia levels resulting from varying dietary protein intake and varying liver enzyme activity levels to simulate liver disease. As a toxicity reference, viability was measured in SH-SY5Y neuroblastoma cells following differentiation and ammonium chloride treatment. RESULTS: Results from control simulations yielded steady state blood ammonia levels within normal physiological limits. Increasing dietary protein intake by 72% resulted in a 59% increase in blood ammonia levels. Simulations of liver cirrhosis increased blood ammonia levels by 41 to 130% depending upon the level of dietary protein intake. Simulations of heterozygous individuals carrying a loss of function allele of the urea cycle carbamoyl phosphate synthetase I (CPS1) gene resulted in more than a tripling of blood ammonia levels (from roughly 18 to 60 μM depending on dietary protein intake). The viability of differentiated SH-SY5Y cells was decreased by 14% by the addition of a slightly higher amount of ammonium chloride (90 μM). CONCLUSIONS: Data from the model suggest decreasing protein consumption may be one simple strategy to decrease blood ammonia levels and minimize the risk of developing hepatic encephalopathy for many liver disease patients. In addition, the model suggests subjects who are known carriers of disease-causing CPS1 alleles may benefit from monitoring blood ammonia levels and limiting the level of protein intake if ammonia levels are high.

A control diet (CN diet; 17.4% CP and 4.37% Ca) was compared with an experimental diet with a 0.4-percentage-unit reduction in protein content and a 35% replacement of limestone by a CaSO4-zeolite mixture [5.76% CaSO4 and 1.18% zeolite; reduced-emission diet (RE diet)] in laying hens to evaluate the effects on apparent N retention, egg production, egg composition, and excreta pH measured at excreta collection or after 7 d of storage. In previous studies, it was demonstrated that the RE diet reduced NH3 emissions by 48%. Laying hens (192 total; 48 replicate cages per diet, with 2 hens per cage) were fed experimental diets from 33 to 49 wk of age. Apparent N retention (48.2%), egg production (83.6%), and number of shell-less eggs (0.18%) were not affected by the diet. Eggs tended to be heavier (59.4 vs. 58.8 g/egg, P = 0.06), and yolk percentage (29.7 vs. 29.0%, P = 0.013) was greater with the RE diet. At 48 wk of age, the total solids content per egg was also greater from hens fed the RE diet (13.2 vs. 12.6 g/egg, P = 0.032). Other egg components were not influenced by diet. Thus, a slight reduction in dietary CP content and replacing a portion of the Ca from CaCO3 with CaSO4 did not affect egg production nor did it impair shell quality. At the end of the experiment, excreta were collected from all cages (excreta from 3 cages were mixed and pooled; 16 pools of excreta per diet). At collection, excreta of hens fed the RE diet had lower pH (5.89 vs. 6.54, P < 0.001) than those of hens fed the CN diet. After 7 d of storage, excreta pH of hens fed the RE diet continued to be lower (6.30 vs. 8.36, P < 0.001). The reduction of excreta pH, even after 7 d of storage, may control nitrogenous emissions from excreta by maintaining excreted N as NH4+.

To better understand the digestive and absorptive capacities of the chick intestine so that we may feed diets that better meet the nutritional needs of the chick, it is important to understand how expression of nutrient transporter genes changes in response to various factors. A series of feeding trials were conducted to evaluate the dietary and developmental regulation of nutrient transporter mRNA abundance in the small intestine of two lines of broilers selected on corn-based (Line A) or wheat-based (Line B) diets. Abundance of mRNA was quantified in all experiments using real time PCR and the absolute quantification method. The objective of the first study was to investigate intestinal nutrient transporter and enzyme mRNA in Line A and B broilers at embryo day 18 and 20, day of hatch, and d 1, 3, 7, and 14 posthatch. Genes evaluated included the peptide transporter, PepT1, 10 AA transporters (rBAT, bo,+AT, ATBo,+, CAT1, CAT2, LAT1, y+LAT1, y+LAT2, BoAT and EAAT3), four sugar transporters (SGLT1, SGLT5, GLUT5, and GLUT2), and a digestive enzyme, APN. For PepT1, Line B had greater quantities of mRNA compared with Line A (P = 0.001), suggesting a greater capacity for absorption of AA as peptides. Levels of PepT1 mRNA were greatest in the duodenum (P < 0.05), whereas the abundances of SGLT1, GLUT5 and GLUT2 mRNA were greatest in the jejunum (P < 0.05). Abundances of EAAT3, bo,+AT, rBAT, BoAT, LAT1, CAT2, SGLT5 and APN mRNA were greatest in the ileum (P < 0.05). Quantities of PepT1, EAAT3, BoAT, SGLT1, GLUT5, and GLUT2 mRNA increased linearly (P < 0.01), while CAT1, CAT2, y+LAT1, and LAT1 mRNA decreased linearly (P < 0.05) with age. The objective of the second study was to evaluate the effect of dietary protein quality on intestinal peptide, AA, and glucose transporter, and digestive enzyme mRNA abundance in Line A and B broilers. At day of hatch (doh), chicks from both lines were randomly assigned to corn-based diets containing 24% crude protein (CP) with either soybean meal (SBM) or corn gluten meal (CGM) as the supplemental protein source, ad libitum. Groups of chicks from both lines were also assigned to the SBM diet at a quantity restricted to that consumed by the CGM group (SBM-RT). Abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B (P < 0.03), while APN and SGLT1 were greater in Line A (P < 0.04). When feed intake was equal (CGM vs restricted SBM), a greater abundance of PepT1 and bo,+AT mRNA was associated with the higher quality SBM (P < 0.04), while a greater abundance of EAAT3 and GLUT2 mRNA was associated with the lower quality CGM (P < 0.01). When feed intake was restricted (SBM vs SBM-RT), a greater abundance of PepT1 mRNA was associated with the restricted intake (P < 0.04). The objective of the third study was to determine the effect of dietary protein composition on mRNA abundance of peptide and AA transporters, and a digestive enzyme. From day 8 to day 15 posthatch, Line A and B broilers were fed equal amounts of 1 of 3 diets (24% CP). Dietary protein sources included whey protein concentrate (whey), a partial whey hydrolysate (hydro), or a mixture of free amino acids (AA) similar to the composition of whey. Intestine was collected at days 8, 9, 11, 13, and 15. Expression of all genes except LAT1 was greater (P < 0.05) in Line B compared with A. Abundance of PepT1, EAAT3, y+LAT2, CAT1, bo,+AT, and APN mRNA varied little across diets in Line A but for CAT1 mRNA was greatest (P = 0.005) in Line A birds that consumed the AA diet. Expression of these genes was greatest (P < 0.006) in Line B birds consuming the hydro diet. A greater (P < 0.05) age response of bo,+AT, EAAT3, CAT1, and APN mRNA was observed in birds consuming the hydro or AA diets relative to the whey diet. Results from these studies collectively demonstrate that nutrient transporter gene expression is responsive to a variety of factors, including developmental stage, dietary manipulation, and genetic selection. Information from these studies can be used to improve dietary formulation so that nutrient utilization is enhanced, resulting in improved growth of the broiler. / Ph. D.

Em função de sua disponibilidade no mercado e qualidade nutricional, derivados da soja são importantes fontes de proteína na alimentação animal. Apresentam perfil de aminoácidos semelhante ao de fontes animais, o que estimula seu uso como potencial substituto da farinha de peixe nas rações para aquicultura. No entanto, são deficientes em aminoácidos sulfurados e apresentam fatores antinutricionais que prejudicam o crescimento de peixes. O processamento da soja permite obter produtos mais refinados e altamente digestíveis, como concentrados proteicos (CPS), que têm elevado teor proteico e baixa quantidade de fatores antinutricionais, extraídos no processo de fabricação. Neste contexto, foi determinado o Coeficiente de Digestibilidade Aparente (CDA) do CPS em dietas para juvenis de duas espécies de Characidae autóctones: pacu (Piaractus mesopotamicus) e dourado (Salminus brasiliensis). Também foram avaliados os efeitos do uso deste ingrediente em níveis crescentes de substituição da farinha de peixe na dieta (ensaios com seis tratamentos e quatro repetições), com o objetivo de determinar o nível seguro de substituição sem prejudicar o ganho de peso. O pacu apresentou CDA para proteína (95,33 %) e energia (84,29 %) mais altos que o dourado (CDAproteína = 82,6 %; CDAenergia = 66,6 %), indicando maior plasticidade da espécie para o aproveitamento de fontes vegetais. Baseado em ensaios de desempenho, nas dietas para pacu foi possível substituir até 78,05 % da farinha de peixe das dietas experimentais, enquanto que para dourados, até 37,2%. Acima destes níveis, houve redução no crescimento. A substituição crescente da farinha de peixe pelo CPS exerceu efeito regulatório sobre a atividade de enzimas pancreáticas nos primeiros segmentos do intestino das duas espécies, mensurada através da atividade da protease inespecífica, lipase inespecífica e &alpha;-amilase. Também promoveu redução na altura das dobras intestinais quando a substituição foi total, detectada através da morfometria da parede intestinal do intestino anterior. Estes efeitos podem ser consequência da ação conjunta de fatores antinutricionais da soja, principalmente inibidores de enzimas, potencializados quando a farinha de peixe foi totalmente substituída pelo CPS; e da habilidade com a qual cada espécie digere / absorve os nutrientes. Com base nos resultados obtidos, foi possível concluir que o CPS pode ser utilizado em substituições parciais da farinha de peixe em dietas para peixes onívoros e carnívoros. / Because of market availability and nutritional quality, soybean and its by-products are important sources of protein for the animal feed industry. The amino acid profile of soy products is similar to that of animal sources, encouraging their use as surrogate protein source to fishmeal (FM) in aquafeeds. However, soy products and by-products are deficient in sulfur amino acids and contain anti-nutritional factors that hamper fish growth. Secondary processing of soybean meals yield more refined and highly digestible protein ingredients such as the soy protein concentrate (SPC), which have higher protein contents and lower concentration of anti-nutritional factors, partially extracted or inactivated during the manufacturing process. In such a context, this work determined SPC\'s Apparent Digestibility Coefficient (ADC) in diets for two juveniles neotropical, native Characins species: pacu (Piaractus mesopotamicus), and dourado (Salminus brasiliensis), and also studied the effects of replacing graded levels of FM by SPC (six treatment levels and four repetions) in the aim to determine the optimum substitution level for better growth. Pacu presented higher ADC for protein (95,33 %) and energy (84,29 %) than dourado (ADCprotein = 82,6 %; ADCenergy = 66,6 %), indicating the plasticity of the specie to utilize plant sources. Based on growth performance, for pacu it was possible to substitute until 78,05% of FM, whereas for dourado, until 37,20%. Above these levels, there were reduction on growth performance. The increasing replacement of FM by SPC had regulatory effect on the activity of pancreatic enzymes in the first segment of the intestine of both species, measured through enzymatic assays for unspecific protease, unspecific lipase and &alpha;-amylase. It also caused a reduction on intestinal fold height of proximal intestine. All of these effects can be attributed to soybean anti-nutritional factors, especially enzyme inhibitors, whose action was enhanced when FM was completely replaced by the SPC; and the specific ability of each specie to digest / absorb plant nutrients. It is safe to infer that SPC can only be used as partial replacement of FM in diets for omnivore and carnivore fish.