Chicken meal is not an appropriate reference protein for estimating protein quality of ingredients used in extruded diets intended for dogs.

The indicator amino acid oxidation (IAAO) method has been used to determine metabolic availability (MA) of amino acids in feedstuffs for pigs, humans, and preliminarily for cats. Peas are a commonly used protein source in grain-free extruded dog diets. However, peas have a poor sulfur amino acid (AA) ratio (methionine (Met):cysteine) with Met being the first limiting AA. Furthermore, little is known about the MA of Met in peas fed to dogs. Therefore, our objective was to compare the MA of Met in peas to chicken meal (CM), as a gold-standard reference protein. The study was done as a replicated 5 x 5 complete Latin square design. Ten neutered male mixed-breed dogs (1.5 years old; 26.0 kg ±2.4 kg body weight; BW) fed to maintain ideal BW received all dietary treatments: BAS: lamb-based diet (deboned lamb and lamb meal) providing Met at 50% of its requirement (0.27 g/100g DM), CHK: CM and lamb-based diet, and PEA: ground dried pea and lamb-based diet both providing Met at 68% of its requirement (0.35 and 0.37 g/100g DM, respectively). Two other treatments were created by blending BAS with PEA (BAP) and the BAS with CHK (BAC) to create diets with Met at 59% of requirement (0.32 and 0.31 g/100g DM, respectively). This resulted in three graded levels of Met for both CM and peas to allow for a slope-ratio assay approach to quantify MA with the BAS diet as the common first point. All other AAs were provided to meet at least 120% of the AAFCO recommendations for adult dogs. The BAS diet, with supplemental DL-Met, was fed for a 2-wk wash-in period. After 2 days of diet adaptation IAAO was performed. Dogs were fed 13 small meals where meal 6 contained a priming dose (9.4 mg/kg BW) of L-[1-13C]-phenylalanine (Phe; 99%) as well as a constant dose (2.4 mg/kg BW) in meals 6-13. Breath samples were collected and enrichment of 13CO2 was measured using isotope-ratio mass spectrometry to calculate the rate of Phe oxidation (F13CO2 umol/kg BW/h). Oxidation was analyzed via SAS using proc GLIMMIX with dog and period as random effects, and diet, %Met, and their interaction as fixed effects. Unexpectedly, the slope of Phe oxidation, in response to increasing Met intake, from CM was 31% of that of peas, indicating a lower MA for Met in CM as compared to peas. This finding may be due to damage of AAs during rendering. At this time, CM in extruded diets is not an acceptable reference protein to determine MA of AAs in dogs and the MA of Met from peas cannot be confidently assessed.

Leucine requirement determined in healthy young adult males using the indicator amino acid oxidation method.

BACKGROUND: Previous studies proposed varying leucine requirements for adults ranging from 25 to 40 mg⋅kg-1⋅d-1, but often these studies did not test intakes exceeding 40 mg⋅kg-1⋅d-1. Data using the indicator amino acid oxidation (IAAO) method suggest a higher requirement of 55 mg⋅kg-1⋅d-1 on the basis of the total branched-chain amino acids requirement, but not leucine independently. OBJECTIVES: The IAAO method was used to determine the leucine requirement in healthy young adult males. METHODS: Ten healthy adult males (26.9 ± 1.87 y, mean ± SEM) were studied at 7 leucine intakes; each studied over a 3-d period. Following 2-d of preadaptation to adequate protein intake (1.0 g⋅kg-1⋅d-1), subjects received experimental diets containing the randomly assigned test leucine intake (10, 20, 30, 40, 50, 65, and 75 mg⋅kg-1⋅d-1) for 8 h. The rate of the release of 13CO2 from the oxidation of L-[1-13C]phenylalanine (F13CO2) was measured on the third day, and the leucine requirement was determined using mixed-effect change-point regression and the F13CO2 data in R. The 95% confidence interval was calculated using parametric bootstrap. The effect of leucine intake on the concentration of plasma amino acids, insulin, and glucose were assessed using repeated measures analysis of variance and linear mixed effects. RESULTS: The mean leucine requirement was 33.6 mg⋅kg-1⋅d-1 with a lower and upper 95% confidence of 26.16, 41.04 mg⋅kg-1⋅d-1. Higher leucine intakes were associated with increased plasma leucine, and decreased valine, isoleucine, and serine concentrations. CONCLUSIONS: The leucine requirement of young adult males is ∼34 mg⋅kg-1⋅d-1, which aligns with previously published tracer balance experiments. This trial was registered at http://clinicaltrials.gov (https://clinicaltrials.gov/study/NCT05394155?term=leucine%20young%20adult&rank=1) as NCT05394155.

Metabolic availability of amino acids in humans.

Knowledge of amino acid bioavailability and the effect of combining complementary protein sources are required to determine how to best meet an individual's protein and indispensable amino acid needs. Traditionally, protein quality of foods has been assessed using digestibility data. Digestibility may overestimate bioavailability of some amino acids particularly those more susceptible to heat and processing. The indicator amino acid oxidation (IAAO) method has been validated and applied to determine amino acid bioavailability termed metabolic availability of the first limiting amino acid of a proteinaceous food. The metabolic availability of the limiting amino acid in the test protein is determined as a ratio of the indicator amino acid oxidation response to graded intakes of the test protein compared to the indicator response to a reference protein (crystalline amino acid patterned after egg protein). The IAAO method has also been applied to assess the effect of protein complementation directly in humans on the overall protein quality of the diet. The results demonstrate that protein complementation augments the limiting amino acid supply and increases protein synthesis.

Dietary Lysine Requirements of Older Adults Stratified by Age and Sex.

BACKGROUND: Current recommendation for lysine in older adults, 30 mg/kg/d, is based on young adult data. Evidence suggests that amino acid requirements may differ between young and old adults with both sex and age having an effect in the elderly. OBJECTIVES: This study aimed to define the lysine requirements in healthy older adults using the indicator amino acid oxidation (IAAO) method with L-[1-13C] phenylalanine as the indicator and to compare the derived estimates based on age: 60-69 y and >70 y. METHODS: Fourteen healthy males and 16 healthy females [>60 y, body mass index (BMI) = 26.3 kg/m2] were randomly assigned to receive 3-7 lysine intakes from 10 to 80 mg/kg/d. Subjects were adapted to a standard liquid diet providing 1.0 g/kg/d protein and adequate energy, for 2 d, with indicator oxidation measurements performed on day 3. The rate of release of 13CO2 from the oxidation of L-[1-13C] phenylalanine was measured in breath. A 2-phase linear mixed-effect model, and parametric bootstrap were used to determine mean lysine requirements and the 95% confidence intervals (CIs). The overlap of the 95% CI between the 2 age groups were used to compare the requirement estimates. The null hypothesis was accepted if the interval contained zero. RESULTS: The mean and upper 95% CI of the lysine requirement for females were 32.9 and 40.9 and 46.2 and 53.7 mg/kg/d for those aged 60-69 y and >70 y, respectively. The mean and upper 95% CI of the lysine requirement for the 2 groups of males were not different so was combined to yield a mean and 95% CI of 32.2 and 38.2 mg/kg/d. CONCLUSIONS: To our knowledge, this is the first study to report on the lysine requirement in adults aged >60 y. These results provide a basis from which the adequacy of diets to meet lysine needs of older adults can be assessed. The trial was registered at clinicaltrials.gov as NCT02008955 (https://clinicaltrials.gov/study/NCT02008955).

Quantification and interpretation of postprandial whole-body protein metabolism using stable isotope methodology: a narrative review.

Stable isotopes are routinely applied to determine the impact of factors such as aging, disease, exercise, and feeding on whole-body protein metabolism. The most common approaches to quantify whole-body protein synthesis, breakdown, and oxidation rates and net protein balance are based on the quantification of plasma amino acid kinetics. In the postabsorptive state, plasma amino acid kinetics can easily be assessed using a constant infusion of one or more stable isotope labeled amino acid tracers. In the postprandial state, there is an exogenous, dietary protein-derived amino acid flux that needs to be accounted for. To accurately quantify both endogenous as well as exogenous (protein-derived) amino acid release in the circulation, the continuous tracer infusion method should be accompanied by the ingestion of intrinsically labeled protein. However, the production of labeled protein is too expensive and labor intensive for use in more routine research studies. Alternative approaches have either assumed that 100% of exogenous amino acids are released in the circulation or applied an estimated percentage based on protein digestibility. However, such estimations can introduce large artifacts in the assessment of whole-body protein metabolism. The preferred estimation approach is based on the extrapolation of intrinsically labeled protein-derived plasma bioavailability data obtained in a similar experimental design setting. Here, we provide reference data on exogenous plasma amino acid release that can be applied to allow a more accurate routine assessment of postprandial protein metabolism. More work in this area is needed to provide a more extensive reference data set.

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.

[Changes in protein requirements of elderly people over 5 years assessed using the indicator amino acid oxidation method].

OBJECTIVE: To evaluate the changes in protein requirements of the elderly during the past five years. METHODS: Based on the previous study of protein requirements of 14 elderly in 2017, 4 of these elderly(70-80 y) were included as study participants and protein requirements were re-evaluated using the indicator amino acid oxidation method. There were seven protein levels: 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 g/(kg·d). Maintenance diets were given for the first two days of each protein level. A stable isotope study was conducted on the day 3, using L-~(13)C-phenylalanine as an indicator on the basis of an amino acid rationed diet, which was orally ingested into the body along with the amino acid rationed diet, and breath and urine samples were collected when the metabolism of L-~(13)C-phenylalanine reached steady state in the body. By measuring the kinetic parameters of labeled amino acids in the samples, a nonlinear mixed-effects model was constructed for the protein intake to be tested and the oxidation rate of labeled amino acids. The mean protein requirement of the study population was determined by the protein intake corresponding to the inflection point of the curve. RESULTS: Based on the production rate of ~(13)CO_2 in exhaled breath of four elderly people at different protein levels, the mean protein requirement was 1.05(95%CI 0.51-1.60) g/(kg·d). The protein recommended nutrient intake was 1.31(95%CI 0.64-2.00) g/(kg·d) was estimated by applying the coefficient of variation of the mean protein requirement to derive the recommended nutrient intake. CONCLUSION: Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Reevaluation of the protein requirement in Chinese elderly adults without sarcopenia with the indicator amino acid oxidation technique.

It is now generally believed that elderly may have slightly higher dietary protein requirements than those of the young-middle-aged adults. We have previously conducted related studies by the indicator amino acid oxidation (IAAO) technique, but more research data are needed to revise the protein requirements of the elderly. The main objective was to reevaluate the dietary protein requirements of healthy Chinese adults (65-80 years) without sarcopenia by using the IAAO technique. Nine healthy adult men and seven healthy adult women participated in the study, with protein intakes ranging from 0·1 to 1·8 g/(kg·d). Diets that delivered energy at a 1·5 resting energy expenditure were isocaloric. The amounts of phenylalanine and tyrosine needed to remain constant for each protein dosage. By applying a nonlinear mixed-effects model analysis on the F13CO2 data, which revealed a breakpoint in F13CO2 in response to graded protein intakes, the mean protein requirement was calculated. The mean estimated average requirement (EAR) for healthy elderly Chinese adults without sarcopenia was determined to be 0·94 g/(kg·d). The protein recommended nutrient intake (RNI) determined using various derivation approaches ranged from 1·13 to 1·36 g/(kg·d). The EAR for Chinese adults without sarcopenia aged 65-80 years in this study is 6·8 % higher than the current recommended EAR (0·88 g/(kg·d)). The RNI derived using various derivation approaches are all greater than the current RNI (0·98 g/(kg·d)). This trial was registered with the Chinese clinical trial registry as ChiCTR2200061382.

The Minimum Methionine Requirement for Adults Aged ≥60 Years Is the Same in Males and Females.

The minimum methionine requirement in the presence of excess dietary cysteine has not been determined in older adults. This study aimed to determine the minimum methionine requirement in healthy older adults using the indicator amino acid oxidation (IAAO) method. Fifteen healthy adults ≥ 60 years of age received seven methionine intakes (0 to 20 mg/kg/d) plus excess dietary cysteine (40 mg/kg/d). Oxidation of the indicator, L-[1-13C]phenylalanine (F13CO2), was used to estimate the mean minimum methionine requirement using a change-point mixed-effect model. There was no statistical difference between male and female requirement estimates, so the data were pooled to generate a mean of 5.1 mg/kg/d (Rm2 = 0.46, Rc2 = 0.77; p < 0.01; 95% CI: 3.67, 6.53 mg/kg/d). This is the first study to estimate the minimum methionine requirement in healthy older adults, which is the same between the sexes and as our lab's previous estimate in young adults. The findings are relevant considering current recommendations for increased consumption of plant foods, which will help to establish the appropriate balance of methionine and cysteine intake required to satisfy the sulphur amino acid requirements of older adults.

Evaluation of protein requirements using the indicator amino acid oxidation method: a scoping review.

BACKGROUND: The indicator amino acid oxidation (IAAO) method has been accepted as an approach to evaluate habitual protein requirements under free-living conditions. OBJECTIVES: This scoping review reports on literature that evaluated protein requirements in humans using the IAAO methods. METHODS: Three databases (PubMed/Medline, Web of Science, and ProQuest) were systematically searched to identify studies that evaluated protein requirements using the IAAO method published in English until 5 June, 2023. We evaluated the study quality using previously developed criteria. We extracted the characteristics of the study design and the results of protein requirements. Two reviewers conducted both reviews and quality assessments independently; any differences among them were resolved by consensus or agreement of all team members. RESULTS: We extracted 16 articles targeting children, young adults (including pregnant women, resistance training athletes, endurance-training athletes, and team sports), and older adults. In quality assessment, 14 studies were evaluated "strong," but the remaining 2 were "moderate." These studies were conducted in only 3 countries and did not include all sexes or life stages. The range of the estimated average protein requirements of each life stage was 1.30 g/kg body weight/d for children, 0.87 to 2.10 (0.87-0.93 for general young adults, 1.22-1.52 for pregnant women, 1.49-2.00 for resistance-trained athletes, 1.65-2.10 for endurance athletes, and 1.20-1.41 for team sports athletes) g/kg body weight/d for young adults, and 0.85 to 0.96 g/kg body weight/d for older adults. CONCLUSIONS: Protein requirements in 14 studies were higher than the current reference for each sex, life stage, and physical activity that are related to protein requirements. In the future, protein requirements of various populations including sex and life stage could be assessed using the IAAO methods worldwide.

The dietary requirement for total sulfur amino acids in adults aged ≥60 years appears to be higher in males than in females.

BACKGROUND: The total sulfur amino acid (TSAA) recommendation in older adults is based on data from young adults. Physiological evidence suggests that older adults have a higher requirement than young adults. OBJECTIVES: The objective of this study was to determine the TSAA requirement in healthy men and women aged ≥60 y. METHODS: The TSAA requirement was determined using the indicator amino acid oxidation method with L-[1-13C]phenylalanine as the indicator. At recruitment, 15 older adults (n = 7 men and n = 8 women; BMI < 30 kg/m2) were assigned to receive 7 methionine intakes (5, 10, 15, 19, 25, 35, and 40 mg/kg/d) without dietary cysteine. Intake levels were randomly assigned to each subject. Following enrollment, 2 subjects completed 2 intakes and 3 completed 3, while the remainder completed all 7. Mean TSAA requirement was determined from oxidation of L-[1-13C]phenylalanine using a mixed-effect change-point model. The 95% CI was calculated using parametric bootstrap. To test whether breakpoints were different between men and women, the overlap in the 95% CI was calculated. RESULTS: The mean TSAA requirement was 26.2 (Rm2 = 0.39, Rc2 = 0.89; P < 0.001) and 17.1 mg/kg/d (Rm2 = 0.22, Rc2 = 0.79; P < 0.001) for men and women, respectively. The requirement was significantly higher in men than in women (difference in CI: 9.1 ± 8.85). CONCLUSIONS: To our knowledge, this is the first study to determine the TSAA requirement in older adults. The requirement in older women is similar to current recommendations but is 75% higher in older men. These findings are important given recommendations for increased plant protein consumption. They will help in the assessment of diet quality and provide the basis of dietary guidelines for older adults consuming a plant-based diet. This trial was registered at clinicaltrials.gov as NCT04595188.

Length of Adaptation Has No Effect on the Threonine Requirement Determined in Healthy Young Adult Males Using the Indicator Amino Acid Oxidation Method.

BACKGROUND: The indicator amino acid oxidation (IAAO) method is minimally invasive; therefore, it is applicable to study the amino acid (AA) requirements of individuals in various age groups. However, the accuracy of this method has been criticized because of the 8 h (1 d) protocol, which has been suggested to be too short an adaptation time for estimating AA requirements. OBJECTIVES: The IAAO method was used to determine whether 3 or 7 d of adaptation to each threonine intake alters the threonine requirement in adult men compared to 1 d of adaptation. METHODS: Eleven healthy adult men (19-35 y, body mass index (BMI) 23.4 in kg⋅m-2) were studied at 6 threonine intakes; each intake was studied over a 9 d period. Following 2 d of pre-adaptation to adequate protein intake (1.0 g·kg-1⋅d-1), subjects received experimental diets containing the randomly assigned test threonine intake (5, 10, 15, 20, 25, or 35 mg·kg-1·d-1) for 7 d. IAAO studies were performed on days 1, 3, and 7 of adaptation to the experimental diet. The rate of release of 13CO2 from the oxidation of L-[1-13C]phenylalanine (F13CO2) was measured, and the threonine requirement was determined by applying mixed-effect change-point regression to the F13CO2 data in R version 4.0.5. The 95% confidence interval (CI) was calculated using parametric bootstrap, and the requirement estimates on days 1, 3, and 7 were compared using analysis of variance (ANOVA). RESULTS: The mean threonine requirements (upper, lower 95% CI) for days 1, 3, and 7 were 10.5 (5.7, 15.9), 10.6 (7.5, 13.7), and 12.1 (9.2, 15.0 mg·kg-1·d-1), respectively; and these requirements were not statistically different (P = 0.213). CONCLUSIONS: We demonstrated that the short, 8 h IAAO protocol results in a threonine requirement that is not statistically different from that obtained on days 3 or 7 of adaptation in healthy adult males. This trial was registered at www. CLINICALTRIALS: gov as NCT04585087.

Is It Time to Reconsider the U.S. Recommendations for Dietary Protein and Amino Acid Intake?

Since the U.S. Institute of Medicine's recommendations on protein and amino acid intake in 2005, new information supports the need to re-evaluate these recommendations. New lines of evidence include: (1) re-analysis/re-interpretation of nitrogen balance data; (2) results from indicator amino acid oxidation studies; (3) studies of positive functional outcomes associated with protein intakes higher than recommended; (4) dietary guidance and protein recommendations from some professional nutrition societies; and (5) recognition that the synthesis of certain dispensable amino acids may be insufficient to meet physiological requirements more often than previously understood. The empirical estimates, theoretical calculations and clinical functional outcomes converge on a similar theme, that recommendations for intake of protein and some amino acids may be too low in several populations, including for older adults (≥65 years), pregnant and lactating women, and healthy children older than 3 years. Additional influential factors that should be considered are protein quality that meets operational sufficiency (adequate intake to support healthy functional outcomes), interactions between protein and energy intake, and functional roles of amino acids which could impact the pool of available amino acids for use in protein synthesis. Going forward, the definition of "adequacy" as it pertains to protein and amino acid intake recommendations must take into consideration these critical factors.

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.

Evaluation of standardized ileal digestibility of amino acids and metabolic availability of methionine, using the indicator amino acid oxidation method, in black soldier fly larvae (Hermetia illucens) meal fed to growing pigs.

Standardized ileal digestibility (SID, %) of crude protein (CP) and amino acid (AA) and the metabolic availability (MA) of Met using the indicator amino acid oxidation (IAAO) method, in partially defatted black soldier fly larvae (PD-BSFL) meal were determined in growing pigs in 2 experiments. The Met SID value was then compared numerically with the Met MA to understand how different SID is compared with its MA value. In Exp. 1, 6 ileal-cannulated barrows (initial body weight [BW] = 18.03 ± 0.34 kg) were used in a 2-period switch back design and fed either a nitrogen-free diet (NFD) or test diet, with PD-BSFL meal as the sole source of AA, over two 11-d experimental periods, at a feeding level of 2.8 × estimated maintenance digestible energy requirement. Barrows were adapted for 9-d to the diet, followed by continuous 8-h ileal digesta collection on day 10 and 11. Digesta were pooled per pig within period. The SID of CP and Met of PD-BSFL meal were 76.1 ± 6.2% and 90.4 ± 3.9%, respectively. In Exp. 2, 7 barrows (initial BW = 18.77 ± 0.69 kg) were used in a 7 × 7 Latin square design with L-[1-13C]-Phe as the indicator AA. Each pig was randomly assigned to 1 of 7 dietary treatments over seven 3-d experimental periods. Two diet types were studied including reference (crystalline AA) and PD-BSFL test diets, each supplying graded intakes of Met at 55, 65, and 75% of the estimated SID requirement (NRC, 2012). The MA of Met was determined by comparing the IAAO response between the reference and PD-BSFL test diet using the slope-ratio method. Linear regression determined a negative slope of the best fit line for both the reference and test diets (P < 0.05). The MA of Met in PD-BSFL meal was 53.3%, which is as expected lower than the SID value. While it is generally appreciated that MA will be less than SID, the use of SID is more practical. In cases where SID cannot explain physiological outcomes of feeding a novel ingredient, IAAO may provide additional insight into whether MA should be explored.

The interest in black soldier fly larvae (BSFL) meal as a protein ingredient in swine feed has grown in the past years. As a novel protein ingredient, it is beneficial to evaluate the amino acid (AA) digestibility and metabolic availability (MA) of the limiting AA, Met, in pigs, in BSFL meal prior to incorporation in feed for a more precise formulation. Two different methodologies were used to determine the AA digestibility and MA, standardized ileal digestibility (SID) and indicator amino acid oxidation (IAAO) method, respectively. Based on the SID values, the AA digestibility of BSFL meal, for some, but not all AA, is comparable to other commonly used protein ingredients in commercial swine feed. When compared with the MA result of Met, the Met SID value is much lower. This indicates that not all digested Met is available for protein synthesis or other metabolic processes in the animal.

An Evaluation of the Protein Requirements of Japanese Young Men by Consuming Two Types of Dairy Proteins Using the Indicator Amino Acid Oxidation Method.

The indicator amino acid oxidation method is a relatively new method for determining protein requirements. Our hypothesis was that the protein requirement of the casein-whey protein mixture (70% casein and 30% whey protein) was lower than the protein requirement of plain casein, because casein and whey proteins compensate for the lack of the first-limiting amino acids. The optimal mixing ratio was determined based on the amino acid scoring pattern which is used to calculate the digestible indispensable amino acid score. In this study, digestibility was not considered to determine the optimal mixing ratio because dairy protein is a good source of digestible protein. This study aimed to evaluate the protein requirements of Japanese young men by consuming casein and casein-whey protein mixture. Ten healthy young men (22±0.2 y old) participated in 12 experiments according to a graded protein intake (0.5, 0.7, 0.9, 1.0, 1.2, 1.4 g/kg/d) of casein and casein-whey protein mixture. The mean protein requirement was calculated as the breakpoint of breath 13CO2 enrichment using change-point regression models. The mean protein requirements of Japanese young men by consuming casein and casein-whey protein mixture were estimated to be 1.00 g/kg/d and 0.90 g/kg/d, respectively. These estimated requirements were consistent with the protein quality expected from the amount of the first-limiting amino acids. The indicator amino acid oxidation method may be useful to evaluate protein quality.

Metabolic Availability of Methionine Assessed Using Indicator Amino Acid Oxidation Method, Is Greater when Cooked Lentils and Steamed Rice Are Combined in the Diet of Healthy Young Men.

BACKGROUND: Lentil is considered a high protein source. However, it is low in sulphur amino acids (SAA) and their metabolic availability (MA) is further affected by antinutritional factors in lentils. The combination of lentils with grains such as rice can enhance the protein quality of a lentil-based meal but the MA of SAA in lentils must first be known. OBJECTIVES: The objectives of the current study were to assess the MA of methionine in lentils and to test the effects of consumption of complementing lentils with rice in young adults. METHODS: Five healthy young men [age <30 y, BMI <25 (in kg/m2)] were each studied at 8 or 10 intake amounts of methionine in random order; 4 daily intake amounts of l-methionine: 0.5, 1, 2, and 3 mg.kg-1.d-1 (reference diet), 3 daily intake amounts of methionine from lentils, and 3 daily intake amounts of the mixed meal of lentils + rice (test diets). The MA of methionine and the effects of complementation were assessed by comparing the indicator amino acid oxidation (IAAO) response to varying intakes of methionine in cooked Canadian lentils, and in rice + lentils combined, compared with the IAAO response to l-methionine intakes in the reference protein (crystalline AA mixture patterned after egg protein) using the slope ratio method. l-[1-13C] phenylalanine was used as the indicator. Data were analyzed using the procedure "MIXED" with subject as a random variable, and oxidation day as repeated measure. RESULTS: The MA of methionine from lentils was 69%. Complementation of cooked lentils with rice decreased the oxidation of l-[1-13C] phenylalanine by up to 16% (P < 0.05). CONCLUSIONS: The content and MA of methionine are low in lentils. However, combination of lentils with rice in a 1:1 ratio can improve the protein quality of lentil-based diets, resulting in increased protein synthesis in young healthy adults. This trial was registered at www.clinical trials.gov as NCT03110913.

Evaluation of Protein Quality in Humans and Insights on Stable Isotope Approaches to Measure Digestibility - A Review.

The recent Food and Agricultural Organization/World Health Organization/United Nations University expert consultations on protein requirements and quality have emphasized the need for the new Digestible Indispensable Amino Acid Score (DIAAS), as a measure of protein quality. This requires human measurements of the true ileal digestibility of individual indispensable amino acids (IAAs) until the end of the small intestine. Digestibility is measured using standard oro-ileal balance methods, which can only be achieved by an invasive naso-ileal intubation in healthy participants or fistulation at the terminal ileum. Significant efforts have been made over the last 2 decades to develop noninvasive or minimally invasive methods to measure IAA digestibility in humans. The application of intrinsically labeled (with stable isotopes like 13C, 15N, and 2H) dietary proteins has helped in circumventing the invasive oro-ileal balance techniques and allowed the differentiation between endogenous and exogenous protein. The noninvasive indicator amino acid oxidation (IAAO) technique, which is routinely employed to measure IAA requirements, has been modified to estimate metabolic availability (a sum of digestibility and utilization) of IAA in foods, but provides an estimate for a single IAA at a time and is burdensome for participants. The recently developed minimally invasive dual isotope tracer method measures small intestinal digestibility of multiple amino acids at once and is suitable for use in vulnerable groups and disease conditions. However, it remains to be validated against standard oro-ileal balance techniques. This review critically evaluates and compares the currently available stable isotope-based protein quality evaluation methods with a focus on the digestibility and metabolic availability measurements in humans. In view of building a reliable DIAAS database of various protein sources and subsequently supporting protein content claims in food labeling, a re-evaluation and harmonization of the available methods are necessary.

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

BACKGROUND: 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. OBJECTIVE: In this study we sought to describe the dietary protein intake in frail, prefrail, and robust older Taiwanese adults. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Estimation of inter-individual variability of protein requirement by indicator amino acid oxidation method.

The indicator amino acid oxidation (IAAO) method is a recently developed method to determine the protein requirement and is particularly useful for analyzing human subjects because of its minimal invasiveness. IAAO study is performed using two-phase regression analysis, with the break-point between these phases being the estimated average requirement. However, this method requires that the break-point lie within a certain range in advance, which is in practice difficult. Recently, the change-point regression model (CPRM) has been proposed to be more effective for two-phase regression analysis. There is also a need to re-evaluate the value corresponding to the recommended dietary allowance. Calculation of the recommended dietary allowance requires data on the average requirement and the inter-individual variability of this requirement. However, no inter-individual variability values have been reported in the IAAO method. The aim of this study was thus to estimate the inter-individual variation in protein requirement using CPRM. From seven IAAO studies, the inter-individual variability was estimated as a coefficient of variation of about 20%. The coefficient of variation of the protein requirement determined by IAAO study was wider than the ordinary coefficient of variation obtained from the nitrogen balance test.