Indicator Amino Acid Oxidation Method Research Articles

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

BackgroundCurrent 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. ObjectivesThis 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. MethodsFourteen 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. ResultsThe 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. ConclusionsTo 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).

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

To evaluate the changes in protein requirements of the elderly during the past five years. 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. 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. Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Protein intake affects erythrocyte glutathione synthesis in healthy adults aged ≥60 years in a repeated-measures trial

BackgroundProtein recommendations for older adults are based on nitrogen balance data from young adults. Physiological studies using the indicator amino acid oxidation method suggest they need 30% to 50% more protein than current recommendations. We herein present glutathione (GSH) as a physiological estimate of protein adequacy in older adults. ObjectivesThe objective was to measure GSH kinetics in response to varying protein intakes in a repeated-measures design in healthy adults aged ≥60 y using the precursor-product method. MethodsSixteen healthy older adults (n = 8 male and n = 8 female; body mass index ≤30 kg/m2) were studied. Each received 4 of 6 protein intakes in random order (0.66, 0.8, 0.9, 1.1, 1.3 and 1.5 g⋅kg−1⋅d−1). At each intake level, participants underwent isotope infusion studies of 7 h duration following a 3-d adaptation to the test level of protein. On the fourth day, GSH fractional (FSR) and absolute synthesis (ASR) rates were quantified by measuring the incorporation of U-[13C2-15N]glycine into GSH at isotopic steady state. A mixed-effect change-point regression model was used to determine a breakpoint in FSR and ASR. Secondary outcomes included plasma concentrations of oxidative stress markers, homocysteine, 5-L-oxoproline (5-OP), and urinary sulfate. The effect of secondary outcomes on GSH kinetics was analyzed using a joint linear mixed-effect model and Tukey’s post hoc test. ResultsA protein intake of 1.08 g⋅kg−1⋅d−1 (95% confidence interval [CI]: 0.83, 1.32; Rm2 = 0.207; Rc2 = 0.671; P < 0.001) maximized GSH FSR. There was no effect of protein intake on concentrations of erythrocyte GSH, plasma homocysteine, oxidative stress markers, or 5-OP (P > 0.05). Protein intake had a positive effect on urinary sulfate excretion (P < 0.0001). ConclusionA protein intake of 1.08 g⋅kg−1⋅d−1 from a high-quality protein maximized GSH synthesis in adults ≥60 y. This lends support to data suggesting a requirement higher than the current recommendation.This study was registered at clinicaltrials.gov as NCT02971046.

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

BackgroundThe indicator amino acid oxidation (IAAO) method has been accepted as an approach to evaluate habitual protein requirements under free-living conditions. ObjectivesThis scoping review reports on literature that evaluated protein requirements in humans using the IAAO methods. MethodsThree 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. ResultsWe 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. ConclusionsProtein 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

BackgroundThe 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. ObjectivesThe objective of this study was to determine the TSAA requirement in healthy men and women aged ≥60 y. MethodsThe 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. ResultsThe 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). ConclusionsTo 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

BackgroundThe 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. ObjectivesThe 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. MethodsEleven 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). ResultsThe 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). ConclusionsWe 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.

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 ± 0.34kg) 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 x estimated maintenance digestible energy requirement. Barrows were adapted for 9-d to the diet, followed by continuous 8-h ileal digesta collection on days 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 ± 0.69kg) 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.

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.

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.

The Total Sulphur Amino Acid Requirement Is Higher in Older Men Than Women

ObjectivesThe primary aim of this study was to determine the total sulphur amino acid (TSAA) requirement of adults > 60 years, using the indicator amino acid oxidation (IAAO) method and to compare the requirement estimates between older males and females. The secondary aim was to compare the requirement estimates in older adults to the previously derived IAAO requirement in young males.MethodsFifteen healthy older adults (n = 7 male and n = 8 female participants), were randomized to receive 2 to 7 methionine intakes: 5, 10, 15, 19, 25, 35 and 40 mg. kg−1. d−1 without dietary cysteine. The TSAA requirement was determined using the IAAO method with L-[1–13C]phenylalanine as the indicator. The mean TSAA requirement was determined using two-phase linear mixed effect model, which identified a breakpoint in the F13CO2 in response to graded methionine intakes. The parametric bootstrap method was used to determine the 95% confidence interval (CI). To assess whether the breakpoints were different between older males and females or between older adults and young males the overlap in 95% CI was calculated. The null hypothesis was accepted if the interval contained zero.ResultsThe mean TSAA requirement for older males was 26.2 mg · kg−1· d−1 and 17.1 mg · kg−1· d−1 for older females. The TSAA requirement of older males is higher than older females (Difference in CI: 9.1 ± 8.85) and higher compared to young males based on our previous IAAO estimate of 12.6 mg · kg−1· d−1 (Difference in CI: 13.6 ± 5.98).ConclusionsTo our knowledge this is the first study to experimentally derive the TSAA requirement in older adults. The results can be used to assess whether the amino acid requirements of older adults can be met on a plant-based diet, especially given the current recommendations for increased plant protein consumption.Funding SourcesThis study was funded by CIHR. First author Alyssa Paoletti funded by Restracomp Scholarship provided by the Research Training Centre at SickKids Hospital.

Dietary Lysine Requirement of Women Over 60 Years Estimated Using Indicator Amino Acid Oxidation (IAAO) Technique, Is Not Different From Current Recommendations

ObjectivesThe goals of the current study were to 1) define the lysine requirement in female adults >60years using the indicator amino acid oxidation method (IAAO) method with L-[1–13C]phenylalanine as the indicator and to 2) compare the derived estimate to our previous estimates in young women done in both luteal and follicular phase of the mensural cycle. MethodsSixteen healthy women (>60 years) were randomly assigned to receive 3 to 7 lysine intakes from 10 to 80 mg. kg–1. d–1. Subjects were adapted to a standard liquid diet providing 1.0 g. kg–1.d–1protein and adequate energy, for 2 days, with indicator oxidation measurements on day 3. The rate of release of13CO2 from the oxidation of L-[1–13C]phenylalanine was measured in breath. A two-phase linear mixed effect model, and parametric bootstrap was used to determine the mean and 95% confidence interval (CI) of the lysine requirement. The overlap of the 95% CI between the older and younger women were used to compare the requirement estimates. The null hypothesis was accepted if the interval contained zero. ResultsThe mean and upper 95% CI of the lysine requirement of older women was 29 and 33 mg.kg–1.d–1respectively. This was not different from our previous estimate of 30.9 mg.kg–1.d–1for young women in the follicular phase of the mensural cycle. However, it was lower than our previous estimate of 37.7 mg.kg–1.d–1derived during the luteal phase of the mensural cycle. ConclusionsThis is the first study to report on the lysine requirement in older women >60 years. Although the estimate in older women is not different from current recommendations derived in young men, the results suggest that lysine requirement in young and older women may differ based on mensural cycle phase. Funding SourcesCIHR.

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

BackgroundLentil 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. ObjectivesThe 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. MethodsFive 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. ResultsThe 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). ConclusionsThe 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.

Bioavailable Lysine Assessed Using the Indicator Amino Acid Oxidation Method in Healthy Young Males is High when Sorghum is Cooked by a Moist Cooking Method

BackgroundSorghum is the fifth most consumed cereal grain but limiting in the indispensable amino acid lysine. Complementing sorghum with lentils can improve the quality of sorghum-based diets. However, knowledge of lysine bioavailability in sorghum is lacking. ObjectivesThe study objectives were to determine the bioavailability of lysine in sorghum and to assess the effect of complementation of sorghum and lentils in a mixed-meal format. MethodsWe studied 5 healthy young men (≤30 years; BMI <25 kg/m2) in a repeated-measure design using the indicator amino acid oxidation (IAAO) method, with L-[1–13C] phenylalanine as the indicator. Each subject participated in 8 determinations in random order. On the reference diet, subjects received 4 amounts 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 diet, they received 3 levels of lysine (8.2, 12.5, and 15.7 mg. kg–1. d–1) from sorghum, and on the complementation diet they received 1 level of lysine from a mixed meal of sorghum and lentils. The bioavailability of lysine in sorghum was estimated by comparing the IAAO response to the test diet with the IAAO response to the reference diet using the slope-ratio method. Effectiveness of complementation was assessed by comparing the IAAO response to the mixed meal to the IAAO response to the test protein. ResultsThe bioavailability of lysine from sorghum was 94%. Upon complementation with lentils, there was a decline in the oxidation of L-[1–13C] phenylalanine by 19% (P < 0.0495), reflecting an improvement in available lysine in the mixed meal due to increased lysine intake. ConclusionsAlthough the bioavailability of lysine in sorghum is high, its lysine content is limiting. Complementation with lentils in a 1:1 ratio is recommended to achieve the lysine requirement for adult men consuming a sorghum-based diet. This trial was registered at http://www.clinicaltrials.gov clinicaltrials.gov as NCT03411005.

Determining ideal balance among branched-chain amino acids in medical formula for Propionic Acidemia: A proof of concept study in healthy children

BackgroundPropionic Acidemia (PROP) is an inherited metabolic disorder, with defect in the enzyme propionyl-CoA carboxylase (PCC) which catalyzes catabolism of two of the branched-chain amino acids (BCAA), valine, isoleucine. Nutritional management in PROP depends on dietary protein restriction and consumption of medical formula depleted of the offending amino acids. Recently, concerns have been raised about medical formula due to imbalanced content of BCAA (high leucine – another BCAA, and no valine/isoleucine), which negatively impacts plasma concentrations of BCAA, and growth in children with PROP. Objectives and methodsTo determine an optimal BCAA ratio at which total body protein synthesis is optimized in healthy children using the indicator amino acid oxidation method (oxidation of L-13C-Phenylalanine to 13CO2). This was accomplished by reducing leucine intake gradually from the current high dose in medical formula, in order to compare protein synthesis, under different BCAA ratios. ResultsA total of 8 healthy children were studied, completing 42 study days. Significant differences in F13CO2 with different BCAA ratios were found. BCAA ratio (leucine: isoleucine: valine) 1:0:0 was associated with the highest F13CO2 (low protein synthesis) compared to other ratios. By reducing leucine intake, and isoleucine and valine at minimum PROP recommendations, BCAA ratio between1:0.26:0.28 to 1:0.35:0.4 was associated with optimal protein synthesis. ConclusionBCAA ratio of 1:0:0, present in medical formula limited total body protein synthesis. A balanced BCAA ratio was found between 1:0.26:0.28 and 1:0.35:0.4 (leucine:isoleucine:valine). Future research is needed to test this optimal BCAA ratio for optimizing protein synthesis in patients with PROP. SynopsisThe article describes a proof-of-concept study done on healthy school-aged children testing different ratios of branched chain amino acid (BCAA, leucine:isoleucine:valine), in order to determine an optimal ratio at which total body protein synthesis is improved and has implications for dietary management of children with Propionic Acidemia (PROP).

Glycine, a Dispensable Amino Acid, Is Conditionally Indispensable in Late Stages of Human Pregnancy

Recently, we showed that there are higher protein, lysine, and phenylalanine requirements in late stages of pregnancy compared with early stages. Animal studies have suggested an increased dietary need for specific dispensable amino acids in pregnancy; whether such a need exists in human pregnancies is unknown. The objective of the current study was to examine whether healthy pregnant women at midgestation (20-29 wk) and late gestation (30-40 wk) have a dietary demand for glycine, a dispensable amino acid, using the indicator amino acid oxidation method and measurement of plasma 5-oxoproline concentrations. Seventeen healthy women (aged 26-36 y) randomly received different test glycine intakes (range: 5-100 mg·kg-1·d-1) during each study day in midgestation (∼26 wk, n=17 observations in 9 women) and late gestation (∼35 wk, n=19 observations in 8 women). Diets were isocaloric with energy at 1.7 × resting energy expenditure. Protein was given as a crystalline amino acid mixture based on egg protein composition at current estimated average requirement (EAR; 0.88 g·kg-1·d-1). Breath samples were collected at baseline and isotopic steady state to measure oxidation of L-[1-13C]phenylalanine to 13CO2 (F13CO2). Plasma was collected at the sixth hour of the study day. Linear regression crossover analysis and simple linear regression were used to assess responses in F13CO2 and plasma 5-oxoproline concentrations to different glycine intakes. No statistically significant responses were observed in midgestation. However, in late gestation, lower glycine intakes resulted in higher rates of F13CO2 (suggesting low protein synthesis) with a breakpoint for phenylalanine oxidation at >37mgglycine·kg-1·d-1 and higher plasma 5-oxoproline (suggesting low glycine availability) with a breakpoint >27mg glycine·kg-1·d-1. The findings suggest that glycine should be considered a "conditionally" indispensable amino acid during late gestation, especially when protein intakes are at 0.88 g·kg-1·d-1, the current EAR. This trial was registered at clinicaltrials.gov as NCT02149953.

Dietary leucine requirement of older men and women is higher than current recommendations

Current national (34 mg . kg-1 . d-1) and international (39 mg kg-1 . d-1) recommendations for leucine in older adults are based on data from young adults. Evidence suggests that the leucine requirements of older adults are higher than those of young adults. The objective of the current study was to directly determine the leucine requirements in healthy older adult male and female study participants aged >60 y. Leucine requirement was determined using the indicator amino acid oxidation method (IAAO) with l-[1-13C]phenylalanine as the indicator. Sixteen older adults (n=7 male and n=9 female participants) were randomly assigned to receive 3 to 7 leucine intakes from 20 to 120 mg . kg-1 . d-1. The rate of release of 13CO2 from l-[1-13C]phenylalanine oxidation was measured, and breakpoint analysis was used to estimate the leucine requirement. The 95% CI was calculated using the parametric bootstrap method. The mean leucine requirement for male participants was 77.8 mg . kg-1 . d-1 (upper 95% CI:81.0) and for female participants, it was 78.2 mg . kg-1 . d-1 (upper 95% CI: 82.0) with no sex effect based on body weight. The data were therefore combined to yield a mean of 78.5 mg . kg-1 d-1 (upper 95% CI: 81.0 mg . kg-1 . d-1 ) for both sexes. On the basis of fat-free mass, the mean±SEM leucine requirements were 115±3.2 and 127±2.4 mg . kg-1 . d-1 for male and female participants, respectively (P<0.005). The estimated leucine requirement of older adults is more than double the amount in current recommendations. These data suggest that leucine could be a limiting amino acid in the diet of older adults consuming the current RDA for protein and those consuming a plant-based diet. In view of the functional and structural role of leucine, especially its importance in muscle protein synthesis, current leucine recommendations of older adults should be revised. This trial was registered at clinicaltrials.gov as NCT03506126.

Lysine Bioavailability in School-Age Children Consuming Rice Is Reduced by Starch Retrogradation

Rice is one of the most commonly consumed cereal grains and is part of staple diets in the majority of the world. However, it is regarded as an incomplete protein, with lysine being a limiting amino acid. Our objectives were to determine the bioavailability of lysine in school-age children consuming cooked white rice and to assess the effect of rice starch retrogradation. Bioavailability or metabolic availability (MA) of lysine was determined using the indicator amino acid oxidation (IAAO) method in a repeated-measures design. Six healthy school-age children (3 boys, 3 girls) with a mean±SD age of 6.8±0.98y randomly received 4 crystalline l-lysine intakes (2, 6, 10, 14 mg · kg-1 · d-1), and 5 rice intakes to provide lysine at 8, 11, or 14 mg · kg-1 · d-1. The 14 mg · kg-1 · d-1 intakes were measured twice as warm rice and once as cold rice (to assess the impact of starch retrogradation on MA). Diets provided protein at 1.5 g · kg-1 · d-1 and calories at 1.7 times the participant's measured resting energy requirement, and were isonitrogenous. Breath samples were collected at baseline and during an isotopic steady state for 13C enrichment measurement. The MA of lysine from rice was determined by comparing the IAAO response of rice with l-lysine using the slope-ratio and single intake methods. Starch retrogradation was characterized using differential scanning calorimetry. MA of lysine in warm rice measured in school-age children was 97.5% and was similar to a repeated rice study (97.1%) within the same study population. MA of lysine was reduced significantly (P<0.05) to 86.1% when the cooked rice was consumed cold, which corresponded to detectable starch retrogradation. To our knowledge, this is the first study to measure the MA of lysine from rice in school-age children. Although the bioavailability of lysine from rice is high, it can be reduced by retrogradation of its starch component.This trial was registered at clinicaltrials.gov as NCT04135040.

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

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. 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. 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±0.5kg/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. The MA of lysine in SMP, HSMP, and VM was 91.9%, 69.9%, and 86.6% respectively. 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.