PSX-A-17 Late-Breaking: Methionine and total sulfur amino acid requirements for pups (>14 wk to 9 months), adults, and senior Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Methionine and cystine are often considered limiting amino acids in canine diets but limited requirement studies have been conducted especially for different life stages. Eighteen Labrador Retrievers (6 pups (>14 wk-9 month), 6 adults, and 6 seniors [>8yr)] were utilized in feeding studies to evaluate the changing requirements of methionine (Met) and total sulfur amino acids (TSAA) as canines age. For this study, the indicator amino acid oxidation (IAAO) technique was utilized to determine the amino acid (AA) requirements in each of the three age groups. Dogs were subjected to diets ranging from deficient to excess, with indispensable amino acids formulated at 1.6x NRC values. To allow for adaptation, a control diet with same dietary ingredients, protein and amino acid levels was fed for two days prior to feeding the test diets on the third day. On test day, a baseline breath sample was collected for determining CO2 production using a respiration mask (Oxymax, Columbus Instruments). A priming dose of L-[1-13C] phenylalanine (Cambridge Isotope Laboratories, Inc.) based on body weight was utilized, followed by [1-13C] Phe doses every 30 minutes, spanning a four hour period. After each dose 13CO2 was collected, and enrichment was determined by isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using a piecewise model of best fit (JMP® Pro 16). A segmented line regression showed Met and TSAA mean and population requirements for pups (>14 wk-9 mo.) were 0.78 ± 0.16 and 1.53 ± 0.21 g/1000kcal (mean ± 2SD), respectively. Meanwhile, for adults, mean and population requirements for Met and TSAA were estimated to be 0.68 ± 0.19 and 1.4 ± 0.30 g/1000kcal (mean ± 2SD), respectively, and for seniors, Met and TSAA mean and population requirements were determined to be 0.62 ± 0.17 and 1.27 ± 0.23 g/1000kcal (mean ± 2SD), respectively.

PSX-A-5 Late-Breaking: Branched chain amino acid and threonine requirements for puppy (>14 wk-9mo) Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Branched chain amino acids are heavily involved in protein synthesis and turnover, emphasizing the need to establish requirement for growing animals. On the other hand, threonine is vital for supporting proteins necessary for gut health. Thus, it is very important to supply branched chain amino acids and threonine in appropriate amounts to growing animals. In this experiment, the indicator amino acid oxidation (IAAO) technique was utilized to determine valine, isoleucine, leucine, and threonine requirements in six puppy Labrador Retrievers (>14wk-9mo). Puppies were subjected to diets ranging from deficient to excess, with each of the indispensable amino acids formulated at 1.6x NRC values. The control diet was fed for two days of adaptation, followed by one experimental day in which the test diet was fed. On the test day, a breath sample was collected using a using a respiration mask (Oxymax, Columbus Instruments). A priming dose of L-[1-^13C]phenylalanine (Cambridge Isotope Laboratories, Inc.) based on body weight was supplied to each puppy, followed by [1-^13C]Phe doses every 30 minutes, for a four hour period. ^13CO[2] was collected after each dose and enrichment was determined by isotope ratio mass spectrometry (IRMS). Results from IRMS were converted to atom percent excess (APE) and analyzed using a segmented line model (JMP^® Pro 16). Each of the Four Rivers mean and population requirements were as follows: 1.72 ± 0.11 g/1000 kcal ME for valine; 1.43 ± 0.24 g/1000 kcal ME for isoleucine; 2.25 ± 0.15 g/1000 kcal ME for leucine; 1.74 ± 0.16 g/1000 kcal ME for threonine (mean ± 2SD). The knowledge gained from this study is highly useful as the lean mass deposited as a puppy influences the animal throughout their lifetime.

A pan-cancer transcriptomic study showing tumor specific alterations in central metabolism

Recently, there has been a resurgence of interest in metabolic rewiring of tumors to identify clinically relevant genes. However, most of these studies have had either focused on individual tumors, or are too general, providing a broad outlook on overall changes. In this study, we have first curated an extensive list of genes encoding metabolic enzymes and metabolite transporters relevant to carbohydrate, fatty acid and amino acid oxidation and biosynthesis. Next, we have used publicly available transcriptomic data for 20 different tumor types from The Cancer Genome Atlas Network (TCGA) and focused on differential expression of these genes between tumor and adjacent normal tissue. Our study revealed major transcriptional alterations in genes that are involved in central metabolism. Most tumors exhibit upregulation in carbohydrate and amino acid transporters, increased glycolysis and pentose phosphate pathway, and decreased fatty acid and amino acid oxidation. On the other hand, the expression of genes of the tricarboxylic acid cycle, anaplerotic reactions and electron transport chain differed between tumors. Although most transcriptomic alterations were conserved across many tumor types suggesting the initiation of common regulatory programs, expression changes unique to specific tumors were also identified, which can provide gene expression fingerprints as potential biomarkers or drug targets. Our study also emphasizes the value of transcriptomic data in the deeper understanding of metabolic changes in diseases.

A pan-cancer transcriptomic study showing tumor specific alterations in central metabolism

Recently, there has been a resurgence of interest in metabolic rewiring of tumors to identify clinically relevant genes. However, most of these studies have had either focused on individual tumors, or are too general, providing a broad outlook on overall changes. In this study, we have first curated an extensive list of genes encoding metabolic enzymes and metabolite transporters relevant to carbohydrate, fatty acid and amino acid oxidation and biosynthesis. Next, we have used publicly available transcriptomic data for 20 different tumor types from The Cancer Genome Atlas Network (TCGA) and focused on differential expression of these genes between tumor and adjacent normal tissue. Our study revealed major transcriptional alterations in genes that are involved in central metabolism. Most tumors exhibit upregulation in carbohydrate and amino acid transporters, increased glycolysis and pentose phosphate pathway, and decreased fatty acid and amino acid oxidation. On the other hand, the expression of genes of the tricarboxylic acid cycle, anaplerotic reactions and electron transport chain differed between tumors. Although most transcriptomic alterations were conserved across many tumor types suggesting the initiation of common regulatory programs, expression changes unique to specific tumors were also identified, which can provide gene expression fingerprints as potential biomarkers or drug targets. Our study also emphasizes the value of transcriptomic data in the deeper understanding of metabolic changes in diseases.

PSXI-31 Program Chair Poster Pick: Determination of the threonine requirement in adult and senior Labrador Retrievers using the indicator amino acid oxidation technique

Although nutrient profiles for canines have been developed in the past, the need to update amino acid (AA) requirements has gained importance as genetic selection changes the recommended nutrients. Correctly feeding AA to canines can have enormous effects, considering a deficiency or excess of such nutrient can lead to weight loss, disease, or in some cases, death. Amino acid requirements can be determined through the nitrogen balance or indicator amino acid oxidation (IAAO) methods. In this experiment, the IAAO technique was used to determine the threonine (Thr) requirement in Labrador retrievers. A total of six dogs (6 adult and 6 senior) were subjected to six diets with varying levels of Thr, ranging from deficient to excess. Diets were formulated to 1.6x NRC values for all indispensable amino acids. The control diet was fed for two days, followed by a day in which the test diet was fed, a tracer AA was supplied, and breath samples were collected. On test day, a priming dose of L-[1-13C]phenylalanine (Cambridge Isotope Laboratories, Inc.) based on the subject’s body weight was first supplied, followed by [1-13C]Phe doses every thirty minutes, spanning a four hour period. A respiration mask was placed on each subject every thirty minutes (Oxymax, Columbus Instruments), 13CO2 was collected, and enrichment was determined by isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using a piecewise model of best fit (JMP® Pro 15). The segmented line regression showed that the Thr mean and population requirements were determined to be 1.21 ± 0.24 and 0.92 ± 0.17 g/1000kcal (mean ± 2SD) for adult and senior dogs, respectively. As the pet food industry becomes more specialized in diets relating to aging, and diseased canines, updating the amino acid requirements related to such animals is increasingly important.

PSVI-30 Requirements of the sulfur amino acids in growing Labrador Retrievers using the indicator amino acid oxidation technique

Over time, the need to update amino acid (AA) requirements for canines becomes increasingly important to ensure animals are healthy and free of nutritional disease. Each of the AA plays a crucial role in the metabolism and health of mammals. Of the essential AA, the total sulfur amino acids (TSAA), namely methionine (Met) and cysteine (Cys), are involved in metabolic functions such as protein metabolism, intestinal health, and urinary function. Additionally, Met can function as a precursor in the requirement of Cys. The purpose of this experiment was to use the indicator amino acid oxidation technique to determine amino acid requirements on growing Labrador Retrievers. A total of 12 dogs were subjected to 12 diets with varying levels of Met and Cys, ranging from deficient to excess. Diets were formulated to 1.6x NRC values for all indispensable amino acids. The control diet was fed for two days, followed by a day in which the test diet was fed, a tracer amino acid was supplied, and breath samples were collected. On test day, a priming dose of L-[1-13C]phenylalanine (Cambridge Isotope Laboratories, Inc.) based on the subject’s body weight was first supplied, followed by [1-13C]Phe doses every thirty minutes, spanning a four hour period. A respiration mask was placed on each subject every thirty minutes (Oxymax, Columbus Instruments), 13CO2 was collected, and enrichment was determined by isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using a broken line model (JMP® Pro 15). Results showed that the Met and TSAA mean and population requirements were 0.78 ± 0.16 and 1.53 ± 0.21 g/1000kcal (mean ± 2SD), respectively. Knowledge gained from these studies is necessary as the petfood industry aims to deliver accurate diet formulations to the expansive canine population.

Metabolic Availability of Lysine in Milk and a Vegetarian Cereal–Legume Meal Determined by the Indicator Amino Acid Oxidation Method in Indian Men

ABSTRACT Background Lysine rich foods such as milk and legumes serve as important food additions to the lysine deficient cereal-based diets of vegetarian populations in low- and middle-income countries (LMICs) to alleviate the risk of quality corrected dietary protein inadequacy. Dietary protein quality can be determined by estimating the metabolic availability (MA) of lysine. Objectives The study aimed to estimate the MA of lysine in spray-dried cow milk powder (SMP), heat-treated spray-dried cow milk powder (HSMP), and a habitually consumed cereal-legume based vegetarian meal (VM), using the indicator amino acid oxidation (IAAO) slope-ratio method. Methods The MA of lysine in SMP, HSMP, and VM was estimated in 7 healthy young men aged 19–24 y with BMI of 21.5 ± 0.5 kg/m2 in a repeated measures design. The IAAO response slopes with 2 graded lysine intakes (10.5 and 15.0 mg·kg−1·d−1) from the SMP and VM were compared with the response slope generated with 3 graded crystalline lysine intakes (6.0, 10.5, and 15.0 mg·kg−1·d−1) at the subrequirement level. To produce HSMP, pasteurized cow milk was heat treated and spray dried. The MA of lysine in HSMP was tested at a single level of lysine intake (15 mg·kg−1·d−1). A total of 8 IAAO experiments were conducted on each participant in randomized order. The IAAO slopes were estimated using a linear mixed-effect regression model. Results The MA of lysine in SMP, HSMP, and VM was 91.9%, 69.9%, and 86.6% respectively. Conclusions Heat treatment reduced the MA of lysine by 22% in HSMP compared with SMP in healthy Indian adults. The lysine MA estimates can be used to optimize lysine limited cereal-based diets, with the addition of appropriately processed legumes and milk powder, to meet the protein requirement. This trial was registered at Clinical Trials Registry of India (http://ctri.nic.in) as CTRI/2019/08/020568.

Bioavailable Lysine, Assessed in Healthy Young Men Using Indicator Amino Acid Oxidation, is Greater when Cooked Millet and Stewed Canadian Lentils are Combined

ABSTRACT Background Pearl millet is the chief source of energy in the diet in some developing regions, but has a limited amount of indispensable amino acid lysine. Complementation with pulses like lentils can improve the protein quality of millet diets, but the knowledge of lysine bioavailability (BA) in millet and lentils is lacking. Objectives The study objectives were to determine the BA of lysine in millet and lentils separately and to assess the effect of complementation of millet and lentils in a mixed meal format. Methods We studied 9 healthy young men (≤30 y; BMI <25) in a repeated-measure design using the indicator amino acid oxidation (IAAO) method, with L-[1-13C] phenylalanine as the indicator. Each subject completed 7 or 8 experiments in random order. On the reference diet, subjects received 4 graded levels of L-lysine (5, 8, 12, and 15 mg·kg−1.d−1) from a crystalline amino acid mixture patterned after egg protein; on the test diets, they received 3 levels of lysine (10, 12, and 15 mg·kg−1.d−1) from either steamed millet or stewed lentils; and on the complementation diet, they received 1 level of lysine from a mixed meal of steamed millet and stewed lentils. The BA of lysine and the effect of complementation were assessed by comparing the IAAO responses to the test diets and the complementation diet with the IAAO response to L-lysine intakes in the reference protein, using the slope ratio method. Results The BA of lysine was 97% from millet and 80% from lentils. Complementation of steamed millet with stewed lentils decreased the oxidation of L-[1-13C] phenylalanine by 27% (P < 0.05), signifying improved quality of the combined millet and lentil protein. Conclusions Lysine has high BA but is still limiting in steamed pearl millet. Complementation with lentils in a 2:1 ratio is recommended to meet the lysine and protein requirements for adult men consuming a millet-based diet. This trial was registered at clinicaltrials.gov as NCT03674736 and NCT03339167.