Indicator Amino Acid Oxidation Technique Research Articles

PSIX-13 Methionine requirements across the life cycle of working Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine is a limiting amino acid in canine diets, but few requirement studies have been conducted across life stages in large-breed dogs. The objective of this study was to determine the minimum requirement (MR) for methionine (Met) in Labrador retrievers at 4 life stages using the indicator amino acid (AA) oxidation (IAAO) technique. In total, 24 dogs were used: 6 puppies aged 10 to 14 wk, 6 puppies aged 14 wk to 9 mo, 6 adults aged 2 to 5 yr, and 6 seniors aged over 8 yr in feeding studies to evaluate the changing requirements of methionine (Met) as canines age. After 2-d adaptation to Met-adequate basal diet (Met = 0.56% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of Met ranging from deficient to sufficient (final Met content in experimental diets was 0.1982, 0.2386, 0.279, 0.49, 0.525, 0.56% dry matter, with indispensable amino acids formulated at 1.6x NRC values. Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C]phenylalanine based on body weight was first given, followed by [1-13C]Phe doses every 30 min spanning a 4-h period), and breath samples were collected via respiration mask every 30 min. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for Met were estimated to be 0.84 ± 0.05 g/1000 kcal (mean ± 2SD) for puppies 10 to 14 wk, 0.78 ± 0.16 g/1,000kcal (mean ± 2SD) for puppies 14 wk to -9 mo, 0.68 ± 0.19 g/1,000kcal (mean ± 2SD) for adults 2 to 5 yr, and 0.62 ± 0.17 g/1,000kcal (mean ± 2SD) for seniors. This research provides important information to assist in accurate formulation of diets that contain appropriate methionine for large breed dogs at all life stages.

PSLBI-17 Total sulfur amino acid requirements across the canine life cycle in Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine and cysteine are the principal sulfur-containing amino acids that are incorporated into proteins, although homocysteine and taurine also have important physiological roles in canines. As limited studies have been conducted for total sulfur-containing amino acids (TSAA) requirements across the canine life cycle, our objective was to determine the minimum requirements (MR) for TSAA at 4 life stages using the indicator amino acid oxidation (IAAO) technique. Labrador Retrievers (n = 24) were used in feeding studies to evaluate the changing requirements of TSAA as dogs mature: 6 puppies aged 10 to 14 wk, 6 puppies >14 wk to 9 mos, 6 adults 2 to 5 yr (yr), and 6 seniors > 8yr. Following a 2-d adaptation to a TSAA-adequate basal diet (TSAA = 1.12% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of TSAA ranging from deficient to sufficient (final TSAA content in experimental diets were 0.41, 0.46, 0.51, 0.98, 1.05, and 1.12% dry matter, with indispensable amino acids formulated at 1.6x NRC values). The test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was included (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 spanning 4 h). Breath samples were collected via a respiration mask every 30 min. Total CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for TSAA were estimated to be 1.55 ± 0.069 g/1,000 kcal (mean ± 2 SD) for puppies 10 to 14 wk, 1.53 ± 0.21 g/1,000 kcal (mean ± 2 SD) for puppies 14 wk to 9 mo, 1.4 ± 0.30 g/1000 kcal (mean ± 2 SD) for adults 2 to 5 yr, and 1.27 ± 0.23 g/1,000 kcal (mean ± 2 SD) for seniors. These results may contribute to the formulation of diets that meet the age-appropriate requirements of TSAA for large-breed dogs at all life stages.

PSLBI-18 Isoleucine requirements of young adult and senior Labrador retrievers

Abstract Age-appropriate diets that meet the unique nutritional needs of aging are in greater demand as senior dogs represent an increasing percentage of the pet population. Studies targeting the specific amino acid requirements of the aging canine are important because they may inform dietary formulations for this specific life stage. In this study, the indicator amino acid oxidation (IAAO) technique was used to determine the minimum requirements for isoleucine (Iso) in young adult and senior Labrador retrievers. After 2-d adaptation to Iso-adequate basal diet (Iso = 0.608% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of isoleucine ranging from deficient to sufficient (final Iso content in experimental diets were 0.244, 0.285, 0.326, 0.532, 0.57 and 0.608% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 min spanning a 4 h period), and breath samples were collected via respiration mask every 30 min. CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for isoleucine were 898 ± 71 mg/1,000 kcal ME (mean ± 2 SD) for young adults and 885 ± 91 mg/1,000 kcal ME (mean ± 2 SD) in senior dogs, which is slightly greater than the National Research Council’s minimum requirement. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

Total sulfur amino acid requirements are higher during late gestation compared with early gestation in healthy Canadian pregnancies in a repeated-measures trial

BackgroundDietary Reference Intake (DRI) Recommendations for total sulfur amino acids (TSAAs; methionine + cysteine) during pregnancy are based on factorial calculations using data from adult males. To date, no data exist on TSAA requirements obtained directly during pregnancy. ObjectivesThe objective of this study was to examine whether TSAA requirements during early (11–20 wk) and late (31–40 wk) gestation in healthy females with singleton pregnancies are different than current recommendations, and different between early and late gestation using the indicator amino acid oxidation (IAAO) technique. MethodsTwenty-five females 20–40 y with a healthy singleton pregnancy were studied using the IAAO technique in a repeated measures design for a total of 70, 8-h d. On each study day a methionine test intake (range: 0–40 mg⋅kg−1⋅d−1) was provided in 8 hourly, isonitrogenous and isocaloric meals with cysteine excluded from the diet. Breath samples were collected at baseline and isotopic steady state of orally provided L-1-13C-Phenylalanine for measurement of phenylalanine oxidation. The requirement was determined using biphasic linear regression crossover analysis to identify a breakpoint in 13CO2 production, representing the estimated average requirement (EAR). ResultsThe TSAA requirement in healthy pregnant participants in early gestation was 11.1 mg⋅kg−1⋅d−1 {R2m = 0.79, R2c = 0.79; 95% confidence interval [CI] (8.9, 13.3 mg⋅kg−1⋅d−1)} and 15.0 mg⋅kg−1⋅d−1 (R2m = 0.72, R2c = 0.79; 95% CI [13.0, 17.0 mg⋅kg−1⋅d−1]) in late gestation. The difference between confidence intervals of the 2 breakpoints was = −3.9 ± 3.0, and statistically different. ConclusionsWe directly measured TSAA requirements in healthy pregnant mothers, and our findings suggest that requirements are lower than current DRI recommendations of 20 and 25 mg⋅kg−1⋅d−1, as the EAR, and Recommended Dietary Allowance, respectively. Late gestation TSAA needs are significantly different and increased 35% compared with early gestation. Recommendations for TSAA intake need to be tailored for gestational stage.This clinical trial was registered at clinicaltrials.gov as NCT04326322.

Beyond the Bowl: Understanding Amino Acid Requirements and Digestibility to Improve Protein Quality Metrics for Dog and Cat Foods.

The determination of amino acid (AA) requirements for mammals has traditionally been done through nitrogen (N) balance studies, but this technique underestimates AA requirements in adult animals. There has been a shift toward researchers using the indicator amino acid oxidation (IAAO) technique for the determination of AA requirements in humans, and recently in dogs. However, the determination of AA requirements specific to adult dogs and cats at maintenance is lacking and the current requirements outlined by the National Research Council are based on a dearth of data and are likely underreporting the requirements of indispensable AA (IAA) for the population. To ensure the physiological requirements of our cats and dogs are met, we need methods to accurately and precisely measure digestibility. In vivo methods, such as ileal cannulation, are most commonly used, however, due to ethical considerations, we are moving away from animal models and toward in vitro methods. Harmonized static digestion models have the potential to replace in vivo methods but work needs to be done to have these methods more accurately represent the gastrointestinal tract (GIT) of cats and dogs. The Digestible IAA Score (DIAAS) is one metric that can help define protein quality for individual ingredients or mixed diets that uses AA SID estimates and ideally those can be replaced with in vitro AA digestibility estimates. Finally, we need accurate and reliable laboratory AA analyses to measure the AA present in complete diets, especially those used to quantify methionine (Met) and cysteine (Cys), both often limiting AAs in cat and dog diets. Together, this will guide accurate feed formulation for our companion animals to satisfy requirements while avoiding over-supplying protein, which inevitably contributes to excess N excretion, affecting both the environment and feed sustainability.

Minimum methionine requirement in adult cats as determined by indicator amino acid oxidation.

There is a lack of empirical data on the dietary Met requirement, in the presence of Cys or cystine, in adult cats. Thus, the aim of this study was to determine the Met requirement, in the presence of excess Cys, in adult cats at maintenance using the indicator amino acid oxidation (IAAO) technique. Six adult neutered male cats were initially selected and started the study. Cats were adapted to the basal diet sufficient in Met (0.24% dry matter, DM) for 14 d prior to being randomly allocated to one of eight dietary levels of Met (0.10%, 0.13%, 0.17%, 0.22%, 0.27%, 0.33%, 0.38%, and 0.43% DM). Different dietary Met concentrations were achieved by supplementing the basal diet with Met solutions. Alanine was additionally included in the solutions to produce isonitrogenous and isoenergetic diets. Cats underwent a 2-d adaptation period to each experimental diet prior to each IAAO study day. On IAAO study days, 13 meals were offered corresponding to 75% of each cat's daily food allowance. The remaining 25% of their daily food intake was offered after each IAAO study. A bolus dose of NaH13CO3 (0.44mg kg-1) and l-[1-13C]-phenylalanine (13C-Phe; 4.8mg kg-1) were provided in fifth and sixth meals, respectively, followed by a constant dose of 13C-Phe (1.04mg kg-1) in the next meals. Breath samples were collected and total production of 13CO2 was measured every 25min through respiration calorimetry chambers. Steady state of 13CO2 achieved over at least three breath collections was used to calculate oxidation of 13C-Phe (F13CO2). Competing models were applied using the NLMIXED procedure in SAS to determine the effects of dietary Met on 13CO2. Two cats were removed from the study as they did not eat all meals, which is required to achieve isotopic steady. A breakpoint for the mean Met requirement, with excess of Cys, was identified at 0.24% DM (22.63mg kg-1) with an upper 95% confidence limit of 0.40% DM (37.71 mg·kg-1), on an energy density of 4,164 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. The estimated Met requirement, in the presence of excess of Cys, is higher than the current recommendations proposed by the National Research Council's Nutrient Requirement of Dogs and Cats, the Association of American Feed Control Officials, and the European Pet Food Industry Federation.

PSX-7 Determination of Tryptophan Requirements of Adult and Senior Labrador Retrievers Using the Indicator Amino Acid Oxidation Technique

Abstract Nutritional requirements vary across the life stages of canines, and the consumer demand for advanced diets that meet the age-appropriate needs of their pets has steadily increased. Availability of veterinary care and high-quality nutrition has led to a longer average lifespan in companion animals, and although senior dogs represent a significant proportion of the pet population, few studies have targeted the specific amino acid requirements of the aging canine. In this study, the indicator amino acid oxidation (IAAO) technique was used to compare the tryptophan (Trp) requirements of young adult and senior Labrador retrievers. After 2-day adaptation to Trp-adequate basal diet (Trp = 0.256% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of tryptophan ranging from deficient to sufficient (final Trp content in experimental diets was 0.05, 0.1, 0.15, 0.224, 0.25 and 0.256% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] Phe based on body weight was first given, followed by [1-13C] Phe doses every thirty minutes spanning a four-hour period), and breath samples were collected via respiration mask every 30 minutes. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for tryptophan were 377.7 ± 31.9 mg/1,000 kcal ME (mean ± 2SD) in the young adults and 362.9 ± 31.5 mg/1,000 kcal ME for senior dogs, which are greater than the currently recommended amounts by NRC and AAFCO. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

Applying the indicator amino acid oxidation technique in the domestic cat: results of a pilot study and development of a non-steady state prediction model.

The aim of this study was to evaluate whether the indicator amino acid oxidation (IAAO) method could be applied in the domestic cat. Six adult male cats were used in a replicated 3 × 3 Latin square design. Three semi-synthetic diets were developed: a methionine (Met) and total sulfur AA (TSAA) deficient diet (T-BASAL; 0.24% Met+Cys - DM basis) and two Met and TSAA-sufficient diets in which either dl-Met (T-DLM) or 2-hydroxy-4-(methylthio)-butanoic acid (T-MHA) were supplemented, respectively, on an equimolar basis to meet the TSAA requirement (0.34%). After a 2-d diet adaptation, IAAO studies were performed. Cats were offered 13 small meals. The sixth meal contained a priming dose (4.8 mg/kg-BW) of l-[1-13C]-Phe and the remaining meals a constant dose (1.04 mg/kg-BW). Breath samples were collected every 25 min to measure 13CO2 enrichment. The following morning, fasted blood samples were collected. Cats returned to the T-BASAL top dressed with a dl-Met solution for 4 d prior to being fed a new dietary treatment. Isotopic steady state was evaluated through visual inspection. Data were analyzed using PROC GLIMMIX procedure in SAS 9.4. While 13CO2 enrichment was successfully captured in breath samples, cats failed to reach 13CO2 steady state. Thus, a non-steady state isotope model was developed and coded in ACSLX (V3.1.4.2) individually for each cat on each study day to predict 13CO2 enrichment, and then, calculate oxidation of l-[1-13C]-Phe (F13CO2). A higher predicted F13CO2 was observed for cats fed T-BASAL compared to the others (P < 0.05), while no differences were observed between T-DLM and T-MHA (P > 0.05). Cats fed T-DLM tended to have higher plasma Met concentrations compared to those fed T-BASAL with cats fed T-MHA intermediate (P = 0.0867). Plasma homocysteine concentrations were higher in cats fed T-BASAL compared to the others (P < 0.05), while threonine concentrations tended to be higher in cats fed T-BASAL compared to those fed T-MHA (P = 0.0750). In conclusion, short-term provision of a semi-synthetic diet deficient in Met may elicit a metabolic response aiming to conserve Met. The successful quantification of 13CO2 enrichment in breath and the higher predicted F13CO2 in cats fed a Met deficient diet suggest that the IAAO technique may be used in cats. Adaptations in the isotope protocol should be made to achieve 13CO2 steady state in breath and avoid mathematical modeling to predict F13CO2.

106 Minimum Methionine Requirement in Adult Cats Using the Indicator Amino Acid Oxidation Technique

Abstract Despite the importance of Met for protein synthesis and a wide range of metabolic functions, little is known about its requirement in adult cats. Current recommendations for adult cats are based solely on studies using kittens and growth or nitrogen balance as the outcomes. Thus, this study aimed to determine the minimum Met requirement in mature cats using the indicator amino acid oxidation (IAAO) technique. Four adult neutered male cats (4.90 + 0.77 kg BW) were used in a Latin rectangle design. Cats were fed a semi-synthetic diet sufficient in Met for 2 weeks prior to being randomly allocated to one of the eight dietary Met treatments (0.12%, 0.17%, 0.21%, 0.28%, 0.35%, 0.42%, 0.50% and 0.57% dry-matter). Cys was provided in excess to spare the Met requirement and meet the Cys requirement. After 2d of adaptation, individual IAAO studies were performed. During each IAAO study day, cats were placed in indirect calorimetry chambers and fed 75% of their daily ration (12.38g/kg BW) split in 13 ½-hourly small meals. A priming dose of NaH13CO3 (0.44mg/kg) and L-[1-13C]-Phe (4.8mg/kg) were provided in the 4th and 5th meal, respectively, following by a constant dose (1.04mg/kg) of L-[1-13C]-Phe in the next meals. Breath samples were collected ½-hourly to measure 13CO2 enrichment to quantify 13CO2 and calculate F13CO2 during isotopic steady state (Phe oxidation). Competing models, broken-line linear (BLL) ascending model, and a broken-line quadratic (BLQ) ascending model, were fitted on F13CO2 to determine the minimum requirement of Met (breakpoint). The BLL yielded the best model with mean minimum Met requirement estimated at 0.305% (dry-matter), and the 95% confidence interval estimated at 0.521% (dry-matter) for adult cats. In conclusion, the estimated min Met requirement in this study is greater than those currently recommended by the NRC (2006; 0.17% dry-matter) and AAFCO (0.2% dry-matter).

PSXI-12 Determination of Protein Requirements for Labrador Retriever Pups (&amp;gt; 14 wk-9 Mo) Using the Indicator Amino Acid Oxidation (Iaao) Technique

Abstract Providing protein to growing puppies in accurate amounts ensures that animals have enough energy and nutrients for depositing muscle, growing bones, and maintaining joint health. Inclusion amounts must be provided at correct doses, considering a diet with excess protein can lead to harmful outcomes such as hyperkalemia or acidosis. To determine the protein requirements in growing Labrador Retrievers, the indicator amino acid oxidation (IAAO) technique was utilized in six puppies (&amp;gt;14 wk-9 mo). Dogs were supplied with constant dietary Phe across diets. 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 body weight of the subject was supplied, followed by [1-13C]Phe doses every 30 minutes, spanning a four-hour period. A respiration mask was placed on each subject every 30 minutes (Oxymax, Columbus Instruments), 13CO2 was collected, and enrichment was determined by isotope ratio mass spectrometry (IRMS). IRMS results were converted to atom percent excess (APE) and analyzed using a segmented model of best fit (JMP Pro 16). The outcome of this experiment determines that if essential amino acids and non-essential amino acid nitrogen requirements are met, Labrador Retriever puppies will have mean and population safe requirements (mean±2SD) of 47.5 ± 1.325 g/1,000 kcal.

Amino acid oxidation methods to determine amino acid requirements: do we require lengthy adaptation periods?

Determination of indispensable amino acid (IAA) requirements necessitates a range of intakes of the test IAA and monitoring of the physiological response. Short-term methods are the most feasible for studying multiple intake levels in the same individual. Carbon oxidation methods measure the excretion of 13CO2 in breath from a labelled amino acid (AA) in response to varying intakes of the test AA following a period of adaptation. However, the length of adaptation to each AA intake level has been a source of debate and disagreement among researchers. The assertion of the minimally invasive indicator amino acid oxidation (IAAO) technique is that IAA requirements can be estimated after only a few hours (8 h) of adaptation to each test AA intake, suggesting that adaptation occurs rapidly in response to dietary adjustments. On the contrary, the assertion of most other techniques is that 6-7 d of adaptation is required when determining IAA needs. It has even been argued that a minimum of two weeks is needed to achieve complete adaptation. This review explores evidence regarding AA oxidation methods and whether long periods of adaptation to test IAA levels are necessary when estimating IAA requirements. It was found that the consumption of experimental diets containing lower test IAA intake for greater than 7 d violates the terms of a successful adaptive response. While there is some evidence that short-term 8 h IAAO is not different among different test amino acid intakes up to 7 d, it is unclear whether it impacts assessment of IAA requirements.

O187 Development of a stable isotopic protocol to achieve steady-state during the indicator amino acid oxidation technique in the domestic cat

O187 Development of a stable isotopic protocol to achieve steady-state during the indicator amino acid oxidation technique in the domestic cat

Evaluation of threonine requirements in mature horses fed 1:1 ratio of forage to concentrate using the indicator amino acid oxidation technique

Threonine has been considered as the second limiting amino acid in equine diets following the first limiting amino acid, lysine, and its digestibility and bioavailability may be affected by dietary fiber content. Therefore, evaluation of threonine requirements in horses fed different diet compositions will improve diet formulation. The objective of this study was to evaluate threonine requirements in mature horses fed a 1:1 ratio of timothy hay and concentrate using the indicator amino acid oxidation (IAAO) technique. Six threonine intake levels [45, 56, 67, 79, 90, and 102 mg/kg body weight (BW)/day] were prepared by mixing two experimental concentrates containing different threonine concentrations (2.9 and 14.8 g of threonine/kg of concentrate). Each of six treatments was administrated to six Thoroughbred mares (4–16 years old; 580.1 ± 47.2 kg) in a randomly determined order. After 5 days of adaptation to each treatment, pre-feeding and 90 min post-feeding blood samples were collected on day 6 to evaluate plasma amino acid concentrations using high performance liquid chromatography. The IAAO procedure was conducted on day 7 of each experimental period. During the first two hour intravenous infusion of [13C]sodium bicarbonate, total CO2 production was evaluated from breath samples. From the subsequent four-hour oral administration of [1-13C]phenylalanine, blood and breath samples were collected to estimate phenylalanine oxidation to CO2. Plasma [13C]phenylalanine concentration and breath 13CO2 enrichment were evaluated using gas chromatography–mass spectrometry and an infrared isotope analyzer, respectively. Experimental data were analyzed using a one-way analysis of variance, including orthogonal linear and quadratic contrasts, in the mixed procedures of SAS version 9.3. Increased threonine intake resulted in higher plasma threonine concentration before and 90 min after feeding (P > 0.05), indicating higher bioavailability of threonine. However, phenylalanine oxidation rate was not affected by threonine consumption as evidenced by the lack of a breakpoint (P > 0.05). The results suggest that threonine requirements might be lower than 45 mg/kg BW/day in mature horses fed a 50% concentrate diet.

111 Validation of the Indicator Amino Acid Oxidation Technique in the Domestic Cat

Abstract There is a lack of data on the minimum Met or total sulfur AA (SAA) requirements for adult cats. Non-invasive and precise techniques, such as indicator AA oxidation (IAAO) can be used for these purposes. This pilot study aimed to validate the IAAO technique in cats and investigate the effect of dietary Met concentrations and sources on fasted plasma SAA concentrations. Six cats were fed three experimental diets in random order: Met-deficient diet (BASAL; 0.23% Met+Cys-DM) and two Met-sufficient diets in which either DL-Met (DLM) or Met-hydroxy-analogue (MHA) were supplemented, respectively, on an equimolar basis to meet the total SAA requirement (0.34%-DM). After 2d diet adaptation, IAAO studies were performed. Cats were offered 13 ½-hourly small meals. The 6th meal contained a priming dose (4.8 mg/kg-BW) of L-[1-13C]-Phe and the remaining meals the constant dose (1.04 mg/kg-BW). Breath samples were collected ½-hourly to measure 13CO2 enrichment. The following morning after an 16hr fast, blood samples were collected. Isotopic steady-state was evaluated through linear regression models. Plasma AA data were analyzed using PROC GLIMMIX procedure in SAS (Version 9.4). Met concentrations tended to be higher in cats fed DLM compared to BAS (P = 0.0877), but similar to those fed MHA (P &amp;gt; 0.05). Total cysteine plasma concentrations were similar between treatments (P &amp;gt; 0.05). Cats fed BAS had greater concentrations of plasma HCys compared to DLM and MHA (P = 0.0093). Cats did not reach isotopic steady-state in atom percent excess (P &amp;gt;0.05) regardless of the dietary treatment provided. Non-steady-state kinetics models are under development to predict 13CO2 flux. This study indicates that the short-term feeding of a Met-deficient diet in cats results in greater plasma Hcys and a tendency for lower plasma Met concentrations. A higher prime dose of L-[1-13C]-Phe is warranted to achieve isotopic steady-state and successfully apply the IAAO to estimate AA requirements.

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

Abstract 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 (&amp;gt;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.

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

Abstract 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 (&amp;gt;14 wk-9 month), 6 adults, and 6 seniors [&amp;gt;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 (&amp;gt;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.

Adequate protein intake in older adults in the context of frailty: cross-sectional results of the Nutrition and Health Survey in Taiwan 2014–2017

Emerging evidence suggests that a dietary protein intake higher than the current recommended dietary allowance of 0.8 g/kg body weight (BW)/d may be needed to maintain optimal muscle mass, strength, and function in older adults. However, defining optimal protein intake in this age group remains a challenge. In this study we sought to describe the dietary protein intake in frail, prefrail, and robust older Taiwanese adults. Data for 1920 older adults were collected from the Nutrition and Health Survey in Taiwan from 2014 to 2017. Dietary intake was assessed using the 24-h recall method. Frailty was determined using the modified Fried's criteria. Body composition was assessed using DXA. Sex-specific dietary protein intakes, measured as values/kg of BW, fat-free mass (FFM), and lean mass (LM), were estimated for the 3 age groups (65-69, 70-79, and ≥80y) and the 3 frailty levels. In both males (P for trend=0.034) and females (P for trend=0.015), there were significant downward trends for protein intake/kg of BW with the severity of frailty. The age-adjusted protein intake/kg of BW was still significant in males (P for trend=0.009), but no longer in females. This phenomenon was also seen for protein intake at lunch and dinner but not at breakfast. Age-adjusted trends for protein intake/kg FFM or LM were not significant in either sex. The median protein intake in robust older males and females was 1.21 and 1.19 g/kg BW/d, respectively, and the mean intakes were even higher. Median protein intake in robust Taiwanese older adults was approximately 1.2 g/kg BW/d, with higher mean values. The protein adequate intake in Taiwanese older adults was higher than the current recommended daily allowance (RDA) level but within the RDA range derived from the state-of art indicator amino acid oxidation technique.

Assessing the whole-body protein synthetic response to feeding in vivo in human subjects.

All tissues are in a constant state of turnover, with a tightly controlled regulation of protein synthesis and breakdown rates. Due to the relative ease of sampling skeletal muscle tissue, basal muscle protein synthesis rates and the protein synthetic responses to various anabolic stimuli have been well defined in human subjects. In contrast, only limited data are available on tissue protein synthesis rates in other organs. Several organs such as the brain, liver and pancreas, show substantially higher (basal) protein synthesis rates when compared to skeletal muscle tissue. Such data suggest that these tissues may also possess a high level of plasticity. It remains to be determined whether protein synthesis rates in these tissues can be modulated by external stimuli. Whole-body protein synthesis rates are highly responsive to protein intake. As the contribution of muscle protein synthesis rates to whole-body protein synthesis rates is relatively small considering the large amount of muscle mass, this suggests that other organ tissues may also be responsive to (protein) feeding. Whole-body protein synthesis rates in the fasted or fed state can be quantified by measuring plasma amino acid kinetics, although this requires the production of intrinsically labelled protein. Protein intake requirements to maximise whole-body protein synthesis may also be determined by the indicator amino acid oxidation technique, but the technique does not allow the assessment of actual protein synthesis and breakdown rates. Both approaches have several other methodological and inferential limitations that will be discussed in detail in this paper.

Dietary Aromatic Amino Acid Requirements During Early and Late Gestation in Healthy Pregnant Women

Phenylalanine and tyrosine (referred to as total aromatic amino acids; TAAs) are essential for protein synthesis, and are precursors for important catecholamines. Current estimated average requirement (EAR) recommendations for TAA during pregnancy are 36 mg·kg-1·d-1, and has not been experimentally determined. The aim was to determine TAA requirements (dietary phenylalanine in the absence of tyrosine) during early and late gestation using the indicator amino acid oxidation (IAAO, with L-[1-13C]leucine) technique. Nineteen healthy pregnant women (age 22-38 y) were studied at a range of phenylalanine intakes (5 to 100 mg·kg-1·d-1) in early (13-19 wk) and/or late (33-39 wk) pregnancy for a total of 51 study days. Graded test intakes were provided as 8 hourly isonitrogenous and isocaloric meals. Breath samples were collected for 13C enrichment analysis on an isotope ratio mass spectrometer. A plasma sample was collected and analyzed for phenylalanine and tyrosine concentrations on an amino acid analyzer. The TAA requirement in early and late pregnancy was calculated using 2-phase linear regression crossover analysis that identified breakpoints in 13CO2 production (the requirement) in response to phenylalanine intakes. TAA requirement during early pregnancy was 44 mg·kg-1·d-1 (95% CI: 28.3, 58.8) and during late pregnancy was 50 mg·kg-1·d-1 (95% CI: 36.1, 63.1). In early and late pregnancy, plasma phenylalanine and tyrosine concentrations rose linearly in response to graded phenylalanine intakes. Our results suggest that the current EAR of 36 mg·kg-1·d-1 for TAAs is underestimated. When compared with results previously determined in nonpregnant adults, early pregnancy requirements were similar (43 compared with 44 mg·kg-1·d-1, respectively). During late pregnancy, a 14% higher TAA requirement was observed when compared with early pregnancy. The results from this study have potential implications for creating gestation stage-specific TAA recommendations.

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

Abstract 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.