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).

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

BACKGROUND: Protein 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. OBJECTIVES: The 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. METHODS: Sixteen 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. RESULTS: A 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). CONCLUSION: A 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.

Protein intake affects erythrocyte glutathione synthesis in young healthy adults in a repeated-measures trial.

BACKGROUND: In 2005, the Institute of Medicine advised using methods other than nitrogen balance (NB) for determining protein requirements. Since then, protein requirements using indicator amino acid oxidation (IAAO) have been published and are higher than NB. Glutathione (GSH), a tripeptide of cysteine, glutamate, and glycine, is a principal antioxidant that can be used as a functional indicator of protein adequacy. OBJECTIVES: The aim of this study was to measure changes in erythrocyte GSH kinetics [fractional synthesis rate (FSR) and absolute synthesis rate (ASR)] in healthy adults following a range of protein intakes at and above the current recommendations. METHODS: Sixteen healthy adults [8 males and 8 females, aged 25.6 ± 0.9 y (mean ± SEM)] were studied at 4 of 6 protein intakes ranging from 0.6 to 1.5 g⋅kg-1⋅d-1. Erythrocyte GSH kinetics were assessed during a 7-h infusion of [U-13C2-15N]glycine following 2 d of adaptation to each protein intake. Blood and urine tests were performed to measure oxidative stress markers, plasma homocysteine, triglycerides, plasma amino acid concentrations, 5-L-oxoproline (5-OP), and urinary sulfate. The protein intake that maximized GSH synthesis was determined using mixed-effect change-point regression in R. Primary and secondary outcomes were analyzed using linear mixed-effects and repeated-measures analysis of variance with Tukey's post hoc test. RESULTS: The protein intake that maximized GSH FSR at 78%⋅d-1 was 1.0 g⋅kg-1⋅d-1 (95% confidence interval: 0.63, 1.39). GSH ASR was significantly lower at 0.6 and 0.8 g⋅kg-1⋅d-1 than at 1.5 g⋅kg-1⋅d-1 (2.03 and 2.17, respectively, compared with 3.71 mmol⋅L-1⋅d-1). Increasing the protein intake led to increased urinary sulfate but did not affect erythrocyte GSH concentration, plasma oxidative stress markers, triglycerides, homocysteine, or 5-OP. CONCLUSIONS: A protein intake of 1.0 g⋅kg-1⋅d-1 maximized GSH synthesis, which is in agreement with earlier IAAO-derived protein requirements of 0.93 to 1.2 g⋅kg-1⋅d-1. These findings suggest that recommendations based on NB (0.66 g⋅kg-1⋅d-1) may underestimate protein needs for adequate health. This trial 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.

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.

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.

Methionine and cysteine oxidation are regulated in a dose dependent manner by dietary Cys intake in neonatal piglets receiving enteral nutrition.

Methionine (Met) is an indispensable amino acid (AA) in piglets. Met can synthesize cysteine (Cys), and Cys has the ability to reduce the Met requirement by 40% in piglets. However, whether this sparing effect on Met is facilitated by downregulation of Cys synthesis has not been shown. This study investigated the effects of graded levels of Cys on Met and Cys oxidation, and on plasma AA concentrations. Piglets (n = 32) received a complete elemental diet via gastric catheters prior to being randomly assigned to one of the eight dietary Cys levels (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.40, 0.50 g kg-1d-1) with an adequate Met concentration (0.25g kg-1d-1). Constant infusion of L-[1-14C]-Met and L-[1-14C]-Cys were performed for 6 h on d 6 and d 8 to determine Met and Cys oxidation, respectively. Met oxidation decreased as Cys intake increased (P<0.05). At higher Cys intakes (0.15 to 0.5g kg-1d-1), Met oxidation decreased (P<0.05) at a slower rate. Cys oxidation was similar (P>0.05) among dietary Cys intakes; however, a significant polynomial relationship was observed between Cys oxidation and intake (P<0.05, R2 = 0.12). Plasma Met concentrations increased (P<0.05) linearly with increasing levels of dietary Cys, while plasma Cys concentrations changed (P<0.05) in a cubic manner and the highest concentrations occurred at the highest intake levels. Increasing dietary levels of Cys resulted in a reduction in Met oxidation until the requirement for the total sulfur AA was met, indicating the sparing capacity by Cys of Met occurs through inhibition of the transsulfuration pathway in neonatal piglets.

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.

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.

BACKGROUND: Sorghum 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. OBJECTIVES: The 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Although 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 clinicaltrials.gov as NCT03411005.

A Human Milk-Based Protein Concentrate Developed for Preterm Infants Retains Bioactive Proteins and Supports Growth of Weanling Rats.

BACKGROUND: Bovine milk-based protein modulars are currently available to nutrient-enrich enteral feedings; however, they have limitations for use in very-low-birth-weight infants. OBJECTIVES: Our objectives were to develop a human milk-based protein (HMP) concentrate and to conduct a preclinical assessment of the HMP concentrate in weanling rats. METHODS: An HMP concentrate was produced from donor milk using pressure-driven membrane filtration processes and high hydrostatic pressure processing. Protein and lactoferrin concentrations and lysozyme activity were determined by Kjeldahl, HPLC, and turbidimetric assay, respectively. Male Sprague Dawley rats 24 d old (n = 30) were randomly assigned to 1 of 3 isocaloric AIN-93G diets for 4 wk containing 100% casein (control) or with 50% of the casein replaced with the HMP concentrate (treatment) or a bovine whey protein isolate (treatment). Body weight, food intake, fat mass, plasma amino acid profiles, and organ weights were measured. Data were analyzed using linear regression models. RESULTS: Raw donor milk contained (mean ± SD) 101 ± 6 g protein/kg and 5 ± 1 g lactoferrin/kg of milk solids. Postprocessing, protein and lactoferrin concentrations were 589 ± 3 g/kg and 29 ± 10 g/kg, respectively. Lysozyme activity was initially 209 ± 4 U/kg and increased to 959 ± 39 U/kg in the HMP concentrate. There were no statistically significant differences in body weight, food intake, fat mass, or plasma amino acid profiles between rats fed diets containing the HMP concentrate and the control diet. Full cecum weights were higher in rats fed the HMP concentrate than in those fed control diets (mean difference: 5.59 g; 95% CI: 4.50, 6.68 g; P < 0.0001), likely reflecting the concentration of human milk oligosaccharides. No differences were found for other organ weights. CONCLUSIONS: The HMP concentrate retained important bioactive proteins and supported normal rat growth in the preclinical assessment.

Intestinal resection affects whole-body arginine synthesis in neonatal piglets.

BACKGROUND: Previous studies in piglets show a direct relationship between intestinal mass and arginine (Arg) synthesis. We aimed to study the effects of 75% intestinal resection on whole-body Arg synthesis. METHODS: Piglets were allocated to sham or jejunocolic (JC) surgery and to enteral nutrition (EN) at 20% [sham (n = 8), JC (n = 10)], or 40% [sham (n = 4), JC (n = 5)]. A gastric tube was placed for EN and a venous catheter for parenteral nutrition and blood sampling. On day 6, a primed bolus and constant infusion of Arg m + 2 label and proline m + 1 label was delivered. In addition, 40% EN piglets received a citrulline (Cit) m + 3 tracer. Blood sampling was undertaken and whole-body Arg synthesis was calculated. On day 7, intestinal length was measured, and samples were collected for gene expression (PCR quantification) and histopathology. RESULTS: On Day 7, sham piglets showed intestinal lengthening compared to JC (p = 0.02). Whole-body Arg synthesis was similar between groups (p = 0.50). Adjusting for absolute small intestinal length, JC piglets had greater Arg synthesis (p = 0.01). Expression of arginosuccinase was upregulated in the jejunum of JC compared to sham on 20% EN (p = 0.03). CONCLUSION: This demonstrates for the first-time adaptive changes in intestinal Arg synthesis following intestinal resection. IMPACT: The intestine makes a critical contribution to whole-body arginine synthesis, particularly in neonates, a human population at risk for short bowel syndrome. Therefore, we studied intestinal arginine synthesis in a neonatal piglet model of short bowel syndrome and demonstrated adaptive changes in the intestine that may preserve whole-body arginine synthesis, despite loss of intestinal mass. This research adds new information to our understanding of the effects a massive intestinal resection has on amino acid metabolism during neonatal development.

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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 ± 0.98 y 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. RESULTS: 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. CONCLUSIONS: 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.

Dispensable Amino Acids, except Glutamine and Proline, Are Ideal Nitrogen Sources for Protein Synthesis in the Presence of Adequate Indispensable Amino Acids in Adult Men.

BACKGROUND: Nutritionally, there is a dietary requirement for indispensable amino acids (IAAs) but also a requirement for nitrogen (N) intake for the de novo synthesis of the dispensable amino acids (DAAs). It has been suggested that there might be a dietary requirement for specific DAAs. OBJECTIVES: Experiment 1 tested whether 9 of the DAAs (Ala, Arg, Asn, Asp, Gln, Glu, Gly, Pro, Ser) are ideal N sources using the indicator amino acid oxidation (IAAO) technique. Experiment 2 examined whether there is a dietary requirement for Glu in adult men. METHODS: Seven healthy men (aged 20-24 y) participated in 11 or 2 test diet intakes, in experiment 1 and 2, respectively, in a repeated measures design. In experiment 1, a base diet consisting of the IAA provided at the RDA was compared with test intakes with the base diet plus addition of individual DAAs to meet a 50:50 ratio of IAA:DAA on an N basis. In experiment 2, the diets corresponded to the amino acid pattern present in egg protein, in which all Glu and Gln was present as Glu, or removed, with Ser used to make the diets isonitrogenous. On each study day the IAAO protocol with l-[1-13C]phenylalanine was used to measure whole-body protein synthesis. RESULTS: In experiment 1, repeated measures ANOVA with post hoc multiple comparisons showed that 7 of the 9 DAAs (Ala, Arg, Asn, Asp, Glu, Gly, Ser) decreased IAAO significantly (P < 0.05) compared with base IAA diet, the exceptions being Gln and Pro. In experiment 2, a paired t test did not find significant (P > 0.05) differences in the IAAO in response to removal and replacement of Glu intake. CONCLUSIONS: The results suggest that in healthy men most DAAs are ideal N sources for protein synthesis, in the presence of adequate IAAs, and that endogenous synthesis of Glu is sufficient.Registered clinicaltrials.gov identifier: NCT02009917.

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

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

Bioavailable Methionine Assessed Using the Indicator Amino Acid Oxidation Method Is Greater When Cooked Chickpeas and Steamed Rice Are Combined in Healthy Young Men.

BACKGROUND: In general, pulse protein is limiting in the indispensable amino acid methionine, and antinutritional factors in pulses can affect methionine bioavailability. Complementation with grains such as rice can improve pulse protein quality, but knowledge of methionine bioavailability in pulses and grains is necessary to correct for available methionine when planning and assessing dietary protein intake. OBJECTIVES: The study objectives were to determine the bioavailability of methionine in rice and chickpeas separately and to assess the effect of complementation of chickpeas and rice. METHODS: Eleven healthy young men (<30 y, BMI <25 kg/m2) were studied in a repeated-measures design using the indicator amino acid oxidation (IAAO) method, with l-[1-13C]phenylalanine as the indicator. Each received 7 or 10 methionine intakes in random order: 4 intakes of l-methionine-0.5, 1, 2, and 3 mg⋅kg-1⋅d-1 (reference diet); 3 intakes of methionine from rice and from chickpeas; and 3 intakes from the mixed meal of chickpeas plus rice (test diets). The bioavailability of methionine and the effect of complementation were assessed by comparing the IAAO response to varying intakes of methionine in rice, in cooked Canadian chickpeas, and in rice plus chickpeas combined compared with the IAAO response to l-methionine intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) using the slope ratio method. RESULTS: The bioavailability of methionine from rice and from chickpeas was 100% and 63%, respectively. Complementation of cooked chickpeas with rice decreased the oxidation of l-[1-13C]phenylalanine by up to 14% (P < 0.05), suggesting an improved protein quality of the combined chickpeas plus rice protein. CONCLUSIONS: When chickpeas are the main protein source in the diet of young adult men, the combination of rice and chickpeas in a 3:1 ratio is recommended to improve dietary protein quality. This trial was registered at clinicaltrials.gov as NCT03339154 and NCT03674736.