Indicator Amino Acid Oxidation Technique Research Articles

Using the indicator amino acid oxidation technique to study threonine requirements in horses receiving a predominantly forage diet

Threonine has been reported to be the second limiting amino acid in typical equine diets, but its actual requirement has not been determined in horses. To evaluate amino acid metabolism and requirements, the indicator amino acid oxidation (IAAO) method has been successfully used in other species. The objective of this research was to estimate threonine requirements in mature horses fed timothy hay and concentrate in 4:1 ratio using the IAAO method. Six Thoroughbred mares (579.9±46.7kg) received each of 6 levels of threonine intake, 41, 51, 61, 70, 80 and 89mg/kg BW/day, in a randomly determined order. Each study period was 7-day long, and on day 6, blood samples were collected before and 90min after feeding to measure amino acid concentrations using HPLC. On day 7, horses underwent IAAO procedures, which included a 2-hr primed, constant intravenous infusion of [13 C]sodium bicarbonate to measure total CO2 production and a 4-hr primed, constant oral administration of [1-13 C]phenylalanine to estimate phenylalanine oxidation to CO2 . Blood and breath samples were collected to measure blood [13 C]phenylalanine, using GC-MS analysis and breath 13 CO2 enrichment, using an infrared isotope analyser. Increasing threonine intake levels did not affect plasma phenylalanine oxidation by the ANOVA test (p>0.05) but resulted in a linear decrease in phenylalanine oxidation (p=0.04) without a breakpoint by the orthogonal linear contrast. This study is the first attempt to evaluate threonine requirements in horses by the IAAO method; however, threonine requirements are still unknown in mature horses at this time.

Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African Cornmeal Assessed in Healthy Young Men Using the Indicator Amino Acid Oxidation Technique

Maize is a staple food in many regions of the world, particularly in Africa and Latin America. However, maize protein is limiting in the indispensable amino acids lysine and tryptophan, making its protein of poor quality. The main objective of this study was to determine the protein quality of white African cornmeal by determining the metabolic availability (MA) of lysine and tryptophan. To determine the MA of lysine, 4 amounts of l-lysine (10, 13, 16, and 18 mg · kg-1 · d-1 totaling 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement of 35 mg · kg-1 · d-1, respectively) were studied in 6 healthy young men in a repeated-measures design. To determine the MA of tryptophan, 4 amounts of l-tryptophan (0.5, 1, 1.5, and 2 mg · kg-1 · d-1 totaling 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement of 4 mg · kg-1 · d-1, respectively) were studied in 7 healthy young men in a repeated-measures design. The MAs of lysine and tryptophan were estimated by comparing the indicator amino acid oxidation (IAAO) response with varying intakes of lysine and tryptophan in cooked white cornmeal compared with the IAAO response to l-lysine and l-tryptophan intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) with the use of the slope ratio method. The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively. Our study provides a robust estimate of the availability of lysine and tryptophan in African white maize to healthy young men. This estimate provides a basis for postproduction fortification or supplementation of maize-based diets. This trial was registered at www.clinicaltrials.gov as NCT02402179.

Lysine Requirements of Healthy Pregnant Women are Higher During Late Stages of Gestation Compared to Early Gestation

Lysine is the first limiting amino acid in cereal proteins and is found mainly in animal-derived products. Current Dietary Reference Intake (DRI) recommendations extrapolate lysine requirements during pregnancy from nonpregnant adult data, and may underestimate true requirements. Our objective is to define a quantitative lysine requirement in healthy pregnant women and to determine whether requirements vary between 2 phases of gestation. Fourteen pregnant women in early (12-19 wk) and 19 women in late (33-39 wk) gestation were studied using the indicator amino acid oxidation technique. Individual lysine intakes (6-84 mg · kg-1 · d-1, deficient to excess) were tested on each study day as a crystalline amino acid mixture based on egg protein composition. Isonitrogenous diets maintained protein intake at 1.5 g · kg-1 · d-1 and calorie intake at 1.7 times resting energy expenditure during each study day. Phenylalanine and tyrosine intakes were held constant across all lysine intakes. Breath and urine samples were collected at baseline and isotopic steady state. Lysine requirements were determined by measuring the oxidation of L-[1-13C]-phenylalanine to 13CO2 (F13CO2). Biphase linear regression crossover analysis was used to determine a breakpoint (which represents the estimated average requirement, EAR) in F13CO2. The EAR for lysine during early gestation was determined to be 36.6 mg · kg-1 · d-1 (R2 = 0.484, upper 95% CI = 46.2 mg · kg-1 · d-1), similar to an earlier adult requirement of 36 mg · kg-1 · d-1. The EAR for lysine during late gestation was determined to be 50.3 mg · kg-1 · d-1 (R2 = 0.664, upper 95% CI = 60.4 mg · kg-1 · d-1), 23% higher than the current pregnancy DRI EAR recommendation of 41 mg · kg-1 · d-1. Our results suggest that lysine requirements are higher during late gestation compared to early gestation, and that current dietary lysine recommendations during late stages of pregnancy may be underestimated. The results have implications for populations consuming cereal-based diets as their primary source of protein. This trial was registered at clinicaltrials.gov as NCT01776931.

Indicator Amino Acid–Derived Estimate of Dietary Protein Requirement for Male Bodybuilders on a Nontraining Day Is Several-Fold Greater than the Current Recommended Dietary Allowance

Background: Despite a number of studies indicating increased dietary protein needs in bodybuilders with the use of the nitrogen balance technique, the Institute of Medicine (2005) has concluded, based in part on methodologic concerns, that "no additional dietary protein is suggested for healthy adults undertaking resistance or endurance exercise."Objective: The aim of the study was to assess the dietary protein requirement of healthy young male bodybuilders ( with ≥3 y training experience) on a nontraining day by measuring the oxidation of ingested l-[1-13C]phenylalanine to 13CO2 in response to graded intakes of protein [indicator amino acid oxidation (IAAO) technique].Methods: Eight men (means ± SDs: age, 22.5 ± 1.7 y; weight, 83.9 ± 11.6 kg; 13.0% ± 6.3% body fat) were studied at rest on a nontraining day, on several occasions (4-8 times) each with protein intakes ranging from 0.1 to 3.5 g · kg-1 · d-1, for a total of 42 experiments. The diets provided energy at 1.5 times each individual's measured resting energy expenditure and were isoenergetic across all treatments. Protein was fed as an amino acid mixture based on the protein pattern in egg, except for phenylalanine and tyrosine, which were maintained at constant amounts across all protein intakes. For 2 d before the study, all participants consumed 1.5 g protein · kg-1 · d-1 On the study day, the protein requirement was determined by identifying the breakpoint in the F13CO2 with graded amounts of dietary protein [mixed-effects change-point regression analysis of F13CO2 (labeled tracer oxidation in breath)].Results: The Estimated Average Requirement (EAR) of protein and the upper 95% CI RDA for these young male bodybuilders were 1.7 and 2.2 g · kg-1 · d-1, respectively.Conclusion: These IAAO data suggest that the protein EAR and recommended intake for male bodybuilders at rest on a nontraining day exceed the current recommendations of the Institute of Medicine by ∼2.6-fold. This trial was registered at clinicaltrials.gov as NCT02621294.

Investigation of the postruminal methionine requirement of growing lambs by using the indicator amino acid oxidation technique

Methionine was demonstrated to be the most limiting amino acid for growing sheep when microbial protein is the predominant source of absorbable AA, but its specific requirement was not well quantified. The indicator amino acid oxidation (IAAO) method is based on the concept that when the most limiting amino acid (LAA) is deficient for protein synthesis, then all other AAs will be oxidized. With increasing intakes of the LAA, IAAO will decrease until the requirement for the LAA is met, and there is no further change in the indicator oxidation afterwards. Therefore, the catabolism of 13C-Phe was used to indicate the effects of varying duodenal infusing levels of Met on the oxidation rate by 4-month-old Kazakh lambs (30.8±0.4kgBW). After a 6-d adaptation period, the IAAO test was conducted on d 7. Each lamb received a priming dose of 0.67mg/kg BW of 13C-Phe injected via the jugular vein, together with 0.18mg/kg BW of NaH13CO3 at time 0. Then, constant infusions [1.33mg 13C-Phe/(kgh)−1] were started. Breath samples were collected prior to and half-hourly between 90 and 210min after the start of the 13C-Phe infusions. An estimate of the mean Met requirement was determined by a breakpoint analysis of the rate of 13CO2 released by 13C-Phe oxidation, with a 2-phase linear regression model. The oxidation rate of 13C-Phe decreased (P<0.001) with increasing amounts of methionine infused in a dose-dependent fashion up to 1.65g/day, and it was not different from 1.65 to 2.15g/day (P>0.05). The mean Met requirement was estimated to be 1.28±0.11g/day of supplementation. The total postruminal minimum requirement of Met would be 2.02g/day when the basal passage of Met (0.74g/day) was summed.

Variable Intensity Exercise Increases Protein Requirements in Active Male and Female Adolescents as Determined by the Indicator Amino Acid Oxidation (IAAO) Technique

Adolescence is characterized by the greatest increase in linear growth and accrual of fat‐free mass (FFM) during the life cycle. This growth spurt is optimized by adequate energy and protein intake. Active adolescents also display a greater FFM than their sedentary peers with the sexual dimorphism of puberty leading to a greater total gain in males than females. Given current evidence which suggest that protein requirement is higher in active adults than their sedentary counterparts, we aimed to determine the protein requirements of active, growing adolescents by utilizing the minimally invasive Indicator Amino Acid Oxidation (IAAO) technique. Fourteen active male (n=7; 13±0.6 years; 171±2.8 cm; 57.3±4.2 kg; 48.4±5.0 kg FFM; 0.3±0.2 years from peak height velocity (PHV); means±95% CI) and female (n=7; 11±0.4 years; 159±3.3 cm; 51.6±8.1 kg; 41.5±6.7 kg FFM; 0.5±0.4 years from PHV; means±95% CI) participants each underwent 6 metabolic trials that included an acute bout of a variable intensity exercise (modified Loughborough Intermittent Shuttle Test) followed by the ingestion of 8 hourly meals providing a variable amount of protein (0.2 – 2.67 g·kg−1·d−1), 6 g·kg−1 of carbohydrate, and sufficient energy to match habitual intake. Protein was provided as crystalline amino acid mixtures modeled on the basis of egg protein, with the exception of tyrosine (40 mg·kg−1·d−1) and phenylalanine (30.5 mg·kg−1·d−1 of which 5.46 mg·kg−1 was L‐[13C] phenylalanine over the final 4 drinks). After the determination of breath 13CO2 enrichment by continuous‐flow isotope ratio mass spectrometry and CO2 production by indirect calorimetry, the estimated average requirement (EAR) was determined from the breakpoint of the 13CO2 excretion (F13CO2) after application of a bi‐phase linear regression. Preliminary analysis of F13CO2 revealed breakpoints of 1.53 g·kg−1·d−1 (r2 = 0.57) and 1.34 g·kg−1·d−1 (r2 = 0.69) for males and females respectively. When normalized to FFM, the breakpoints were 1.97 ± 0.33 g·kg FFM−1·d−1 (r2 = 0.68) and 1.45 ± 0.22 g·kg FFM−1·d−1 (r2 = 0.63) for males and females, respectively. The breakpoints derived herein exceed current estimated breakpoint values using the IAAO method for sedentary adults and children (0.93 g·kg−1·d−1, and 1.3 g·kg−1·d−1 respectively). Adjusting for the upper 95% CI, a recommended dietary allowance (RDA) was determined to be 1.83 g·kg−1·d−1 and 1.54 g·kg−1·d−1 for males and females respectively. Our estimate of the RDA in active adolescents exceeds the current protein RDA based on the factorial estimate of nitrogen balance for adolescents (0.9 g·kg−1·d−1), suggesting that variable intensity exercise increases daily protein requirements in active adolescents (and especially males) to potentially support greater FFM development and/or the replenishment of exercise‐induced oxidative losses.Support or Funding InformationSupported by the Ajinomoto Innovation Alliance Program.

Assessment of Protein Turnover in Health and Disease.

To identify protein requirements, it is necessary to be familiar with basic physiology, practical methods, and dose response of protein delivery. This review evaluates available methodology, with emphasis on the limitations of existing techniques most often related to the underlying assumption. Historically, nitrogen balance has been the dominating technique. It is still the gold standard, although there are considerable problems related to the underlying assumptions to have reliable readings. When minimal requirements needed to be defined, the indicator amino acid oxidation technique came into practice. In situations of longer term steady states, it serves a purpose. In situations of disease or in aging, it has proven to be more problematic. More recently, whole-body protein turnover measurements have shown to be useful in situations where the underlying assumptions for the other techniques are not possible to meet.

The Indicator Amino Acid Oxidation Method with the Use of L-[1-13C]Leucine Suggests a Higher than Currently Recommended Protein Requirement in Children with Phenylketonuria

BackgroundPhenylketonuria is characterized by mutations in the Phe hydroxylase gene that leads to the accumulation of Phe in plasma and the brain. The standard of care for phenylketonuria is nutritional management with dietary restriction of Phe and the provision of sufficient protein and energy for growth and health maintenance. The protein requirement in children with phenylketonuria is empirically determined based upon phenylketonuria nutritional guidelines that are adjusted individually in response to biochemical markers and growth. ObjectiveWe determined dietary protein requirements in children with phenylketonuria with the use of the indicator amino acid oxidation (IAAO) technique, with L-[1-13C]Leu as the indicator amino acid. MethodsFour children (2 males; 2 females) aged 9–18 y with phenylketonuria [mild hyperphenylalanemia (mHPA); 6–10 mg/dL (360–600 μmol/L)] were recruited to participate in ≥7 separate test protein intakes (range: 0.2–3.2 g ⋅ kg−1 ⋅ d−1) with the IAAO protocol with the use of L-[1-13C]Leu followed by the collection of breath and urine samples over 8 h. The diets were isocaloric and provided energy at 1.7 times the resting energy expenditure. Protein was provided as a crystalline amino acid mixture based on an egg protein pattern, except Phe and Leu, which were maintained at a constant across intakes. Protein requirement was determined with the use of a 2-phase linear-regression crossover analysis of the rate of L-[1-13C]Leu tracer oxidation. ResultsThe mean protein requirement was determined to be 1.85 g ⋅ kg−1 ⋅ d−1 (R2 = 0.66; 95% CI: 1.37, 2.33). This result is substantially higher than the 2014 phenylketonuria recommendations (1.14–1.33 g ⋅ kg−1 ⋅ d−1; based on 120–140% above the current RDA for age). ConclusionsTo our knowledge, this is the first study to directly define a quantitative requirement for protein intake in children with mHPA and indicates that current protein recommendations in children with phenylketonuria may be insufficient. This trial was registered at clinicaltrials.gov as NCT01965691.

Dietary Protein Requirement of Men >65 Years Old Determined by the Indicator Amino Acid Oxidation Technique Is Higher than the Current Estimated Average Requirement

Background: The current estimated average requirement (EAR) and RDA for protein of 0.66 and 0.8 g ⋅ kg−1 ⋅ d−1, respectively, for adults, including older men, are based on nitrogen balance data analyzed by monolinear regression. Recent studies in young men and older women that used the indicator amino acid oxidation (IAAO) technique suggest that those values may be too low. This observation is supported by 2-phase linear crossover analysis of the nitrogen balance data.Objective: The main objective of this study was to determine the protein requirement for older men by using the IAAO technique.Methods: Six men aged >65 y were studied; each individual was tested 7 times with protein intakes ranging from 0.2 to 2.0 g ⋅ kg−1 ⋅ d−1 in random order for a total of 42 studies. The diets provided energy at 1.5 times the resting energy expenditure and were isocaloric. Protein was consumed hourly for 8 h as an amino acid mixture with the composition of egg protein with L-[1-13C]phenylalanine as the indicator amino acid. The group mean protein requirement was determined by applying a mixed-effects change-point regression analysis to F13CO2 (label tracer oxidation in breath 13CO2), which identified a breakpoint in F13CO2 in response to graded intakes of protein.Results: The estimated protein requirement and RDA for older men were 0.94 and 1.24 g ⋅ kg−1 ⋅ d−1, respectively, which are not different from values we published using the same method in young men and older women.Conclusions: The current intake recommendations for older adults for dietary protein of 0.66 g ⋅ kg−1 ⋅ d−1 for the EAR and 0.8 g ⋅ kg−1 ⋅ d−1 for the RDA appear to be underestimated by ∼30%. Future longer-term studies should be conducted to validate these results. This trial was registered at http://clinicaltrials.gov as NCT01948492.

Lysine Requirements of Moderately Undernourished School-Aged Indian Children Are Reduced by Treatment for Intestinal Parasites as Measured by the Indicator Amino Acid Oxidation Technique ,

Lysine requirements of well-nourished children from developing regions have been found to be similar to those of children from developed regions (33.5 mg · kg⁻¹ · d⁻¹). However, intestinal parasites have been shown to increase lysine requirements in undernourished adults, and it is not known if a similar phenomenon occurs in undernourished children from poor and unsanitary environments. Our objective was to measure the lysine requirement of moderately undernourished school-aged Indian children by the indicator amino acid oxidation technique before and after successful treatment for intestinal parasites. Twenty-one undernourished school-aged children (∼8 y of age) with z scores between -2 SD and -3 SD for height-for-age or weight-for-age, who tested positive for intestinal parasites, were studied before and after successful antiparasite treatment. Children were fed any 2 of 7 levels of lysine intakes (5, 15, 25, 35, 50, 65, and 80 mg · kg⁻¹ · d⁻¹) in random order. The lysine requirement was determined by applying a 2-phase linear regression crossover analysis on the fractional oxidation rate of the tracer L-[1-¹³C] phenylalanine in response to the graded lysine intakes. The lysine requirement of undernourished children with intestinal parasite infestations was determined to be 42.8 mg · kg⁻¹ · d⁻¹ (95% CI: 32.6, 53.1 mg · kg⁻¹ · d⁻¹), and after successful antiparasitic treatment it was determined to be 35.5 mg · kg⁻¹ · d⁻¹ (95% CI: 25.5, 45.5 mg · kg⁻¹ · d⁻¹). The results were significantly different (P < 0.05), although the 95% CIs overlapped. The lysine requirement in undernourished children is similar to that of well-nourished children, and intestinal parasitic infestation increased the lysine requirement by ∼20%.

45 Using the indicator amino acid oxidation technique to study threonine requirements in horses

45 Using the indicator amino acid oxidation technique to study threonine requirements in horses

Nonessential Amino Acids as Nitrogen Sources in Adult Men Examined Using the Indicator Amino Acid Oxidation Technique

Nutritionally, there is a dietary requirement for the essential amino acids (EAA) but also a requirement for nitrogen (N) intake for the de novo synthesis of the nonessential amino acids (NEAA). It has been suggested that some NEAA may be more metabolically important than others. The goal of this study was to determine whether among the NEAA (Ala, Arg, Asn, Asp, Gln, Glu, Gly, Pro, Ser) there was an ideal N source using the indicator amino acid oxidation (IAAO) technique.Subjects were maintained on an adaptation diet for 2 days prior to each test day. Each subject participated in 10 test diet intakes, assigned randomly. One test intake was a base diet consisting of only the EAA provided at the recommended dietary allowance. All other test intakes involved the base diet with the addition of one NEAA to meet a 50:50 ratio of EAA: NEAA on a N basis. The diets were isocaloric and isonitrogenous, given as hourly meals. Each study day followed the IAAO protocol using 13C‐Phenylalanine as the indicator. Breath and urine samples were collected at baseline and isotopic steady state. Enrichments of 13C in breath were analyzed by continuous‐flow isotope ratio mass spectrometry to calculate F13CO2.Repeated measures ANOVA with post hoc multiple comparisons were done on the F13CO2 data. Preliminary results indicate that 7 of the 9 NEAA decrease IAAO significantly and suggest that in healthy males, most NEAA are good N sources, in the presence of adequate EAAs.(Support by Ajinomoto Co. Inc.)

Dietary Protein Requirement of Female Adults >65 Years Determined by the Indicator Amino Acid Oxidation Technique Is Higher Than Current Recommendations1–3

Studies on protein requirements in vulnerable groups such as older adults are few, and results are conflicting. The main objective of this study was to determine the protein requirements of free-living women >65 y by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO2 in response to graded intakes of protein. Twelve subjects participated in the study, with protein intakes ranging from 0.2 to 2.0 g · kg(-1) · d(-1) for a total of 82 studies. The diets provided energy at 1.5 times each subject's resting energy expenditure and were isocaloric. Protein was given as an amino acid mixture on the basis of the egg protein pattern, except for phenylalanine and tyrosine, which were maintained constant across the protein intake amounts. All subjects were adapted for 2 d before the study day to a protein intake of 1.0 g · kg(-1) · d(-1). The mean protein requirement was determined by applying a mixed-effects change-point regression analysis to F(13)CO2 (label tracer oxidation in (13)CO2 breath), which identified a breakpoint in the F(13)CO2 in response to graded amounts of protein. The mean estimated average requirement (EAR) and upper 95% CI (approximating the RDA) protein requirement of women >65 y were 0.96 and 1.29 g · kg(-1) · d(-1), respectively. These estimates of protein requirements for older women are higher than the current EAR and RDA based on nitrogen balance data, which are 0.66 and 0.80 g · kg(-1) · d(-1), respectively. This trial was registered at clinicaltrials.gov as NCT01604980.

Barley does not change threonine requirement in growing pigs fed a barley–casein-based diet compared to a casein-based diet, as determined by the indicator amino acid oxidation method

Ileal threonine (Thr) losses increase when young pigs are fed high fiber ingredients such as barley. Barley contains several anti-nutritive factors, e.g., insoluble fibers, which stimulate the secretion of intestinal mucin, a Thr-rich glycoprotein. We hypothesized that because the gut utilizes the majority of dietary Thr, primarily for mucin synthesis, an increase in mucin secretion and ileal losses of Thr will increase the whole body Thr requirement. The objective of this experiment was to compare the Thr requirement of pigs fed a barley–casein diet to a casein diet, using the indicator amino acid oxidation technique. Six barrows were fed diets containing 12% casein or 50% barley/6% casein at 50g/kg body weight/d. After a 7-d adaptation on the 0.35g Thr/kg body weight casein-based diet, pigs were fed casein diets with each of seven Thr levels (0.21–0.54g Thr/kg body weight in a random order) for 2-d with phenylalanine oxidation measured at the end of each period. After a 10-d adaptation to the barley-based diet, the same protocol was followed for barley-based diets with seven Thr levels in the same pigs. Using two-phase linear regression crossover analysis to determine breakpoints in individual pigs, the requirement on casein diet was 0.355±0.014g Thr/kg body weight/d, and 0.397±0.038g Thr/kg body weight/d on barley diets. Overall there was no difference (P>0.05) in Thr requirement between diets.

Assessment of protein requirement in octogenarian women with use of the indicator amino acid oxidation technique

Data on the protein requirements of elderly adults are limited, because it is impractical to conduct repeated nitrogen balance protocols in these vulnerable humans. This study was designed to determine the dietary protein requirement of elderly women by using the recently developed minimally invasive indicator amino acid oxidation (IAAO) technique. Six white women aged 80-87 y [mean ± SEM: 82 ± 1 y and body mass index (in kg/m²) 26 ± 2] completed a 3-d protocol 7 times. Each woman consumed an adaptation diet for 2 d and on day 3 consumed a complete test diet with a crystalline amino acid mixture containing 1 of 7 protein intakes (0.1, 0.3, 0.6, 0.9, 1.2, 1.5, or 1.8 g · kg⁻¹ · d⁻¹) tested randomly. A group-based protein requirement was assessed by using a nonlinear mixed model of protein intake and L-[1-¹³C]phenylalanine oxidation. The breakpoint, at which there was no further decline in the rate of appearance of ¹³C in the breath, was used as an index of the mean protein requirement. The mean protein requirement (95% CI) was 0.85 (0.60, 1.09) g · kg⁻¹ · d⁻¹. This requirement is 29% higher than the current Estimated Average Requirement (EAR) for adults of 0.66 g · kg⁻¹ · d⁻¹ based on the nitrogen balance technique, although the 95% CI includes the current EAR. The corresponding adequate protein allowance of 1.15 (0.77, 1.54) g · kg⁻¹ · d⁻¹ is 44% higher, although the 95% CI includes the Recommended Dietary Allowance (RDA) of 0.80 g · kg⁻¹ · d⁻¹. Notwithstanding uncertainty about the validity of the use of the IAAO technique to assess protein requirements, the results of this study with octogenarian women suggest that the current EAR and RDA for elderly women may be underestimated. The limitations of this short-term, noninvasive method underscore the need for new research that uses alternative experimental designs and measuring physiologic, morphologic, and health-related outcomes.

Examination of lysine requirement of healthy young male adults on a Chinese habitual diet by the modified indicator amino acid oxidation method

There is currently no reference for intake of lysine for Chinese people; therefore, the present study was conducted to determine the lysine requirement of Chinese young male adults on a habitual Chinese mixed diet based on the modified indicator amino acid oxidation method. Seven young men with a mean age of 23.7 ± 2.2 years that were healthy based on questionnaire, physical examinations and screening tests were evaluated. Subjects were evaluated over five consecutive 7 day periods, during which time they were administered decreasing amounts of lysine via the diet (65, 55, 45, 35, 25 mg·kg-1·d-1). Subjects were allowed to adapt from day 1 to 6 and the isotopes were measured on day 7 in each period. The subjects' body weights, body compositions and plasma proteins were also examined during the study. Amino acid kinetics were measured based on the indicator amino acid oxidation technique using the 13CO2 release rate and phenylalanine oxidation rate to estimate lysine requirements. Body weights, body compositions, and plasma proteins of subjects did not change significantly relative to those at baseline. The mean and the upper 95% CI of lysine requirements of Chinese habitual diets were determined to be 58.41 and 70.09 mg·kg-1·d-1, respectively, based on the 13CO2 release rate and 54.28 and 65.14 mg·kg-1·d-1, respectively, based on the phenylalanine oxidation rate.

Evaluation of the Protein Requirement in Chinese Young Adults Using the Indicator Amino Acid Oxidation Technique

ObjectiveTo accurately calculate the protein requirements in Chinese young adults using the indicator amino acid oxidation technique. MethodsNine women and ten men received a restricted daily level of protein intake (0.75, 0.82, 0.89, 0.97, and 1.05 g/kg), along with L-[1-13C]-leucine. Subjects' protein requirement was determined by a biphasic linear regression crossover analysis of F13CO2 data. In doing so, a breakpoint at the minimal rate of appearance of 13CO2 expiration specific to each level of dietary protein was identified. This trial was registered with the Chinese clinical trial registry as ChiCTR-ONC-11001407. ResultsThe Estimated Average Requirement (EAR) and the Recommended Nutrient Intake (RNI) of protein for healthy Chinese young adults were determined to be 0.87 and 0.98 g/(kg·d), respectively, based on the indicator amino acid oxidation technique. ConclusionThe EAR and RNI of mixed protein are 5% and 16% that are lower than the current proposed EAR and RNI (0.92 and 1.16 g/(kg·d), respectively), as determined by the nitrogen balance method. The respective EAR and RNI recommendations of 0.87 and 0.98 g/(kg·d) of mixed protein are estimated to be reasonable and suitable for Chinese young adults.

Dietary protein requirement of 65–75 year old adult males using indicator amino acid oxidation (IAAO) technique

Current dietary protein requirement for adult males aged 65–75 are based on nitrogen balance data and suggest no difference in requirement between the elderly and young adults. Recent studies in young adult males using IAAO technique suggest that nitrogen balance estimates are too low. The objective of this study was to determine the protein requirement of elderly men using the IAAO technique with L‐[1‐13C]‐phenylalanine as the indicator. Preliminary studies have been conducted in 3/7 (65–75 year old) men. Each received a minimum of 7 protein intakes (0.2 – 1.8 g.kg−1.d−1) in random order after a 2 day adaptation providing energy at 1.7 times measured resting energy expenditure and protein at 1.0 g.kg−1.d−1. Oxidation study day diets were amino acid based liquid diets patterned after egg protein. The mean protein requirement was determined by visual inspection of the preliminary F13CO2 obtained from the oxidation of L‐[1‐13C]‐phenylalanine in response to graded protein intakes. Preliminary estimates suggest a mean protein requirement of 0.8 g.kg−1.d−1. This preliminary data suggest that the mean protein requirement of elderly men aged 65–75 years is 20% higher than current recommendations.This study was supported by a grant from the Canadian Institute for Health Research (CIHR).

Dietary protein requirement of 65–75 year old females using indicator amino acid oxidation (IAAO) technique

Studies of dietary protein requirement in vulnerable groups, including elderly adults, are needed using methods other than nitrogen balance. The objective of this study was to determine the protein requirement in 8 healthy free‐living elderly females (65 – 75y old) by measuring the oxidation of L‐[1‐13C]‐phenylalanine to 13CO2 in response to graded intakes of protein. Subjects received a maximum of 7 protein intakes (0.2 to 2.0g.kg−1.d−1) for a total of 43 studies (preliminary data). The diets provided energy at 1.7 times measured resting energy expenditure and were isocaloric. Protein was given as an amino acid mixture patterned after egg protein. All subjects consumed controlled diets containing 1 g protein.kg−1.d−1 for 2 days prior to the study day. The mean protein requirement was determined by applying a 2‐phase linear regression crossover analysis on F13CO2 (label tracer oxidation in 13CO2), which identified a breakpoint in the F13CO2 in response to graded intakes of protein. Mean protein requirement was determined to be 1.1 g.kg−1.d−1. These preliminary results suggest the mean protein requirement of elderly women is higher than current recommendation derived from nitrogen balance studies. This study was supported by a CIHR grant.

Lysine from Cooked White Rice Consumed by Healthy Young Men Is Highly Metabolically Available When Assessed Using the Indicator Amino Acid Oxidation Technique

Cooked white rice (CWR) provides up to 71% of the dietary protein for many people worldwide. The protein digestibility-corrected amino acid (AA) score is the method adopted by FAO/WHO to evaluate protein quality. Our group has proposed the metabolic availability (MA) of AAs as another determinant of protein quality. It measures the percentage of an indispensable AA that is incorporated during protein synthesis. This study is the first to our knowledge to assess the MA of l-lysine (l-Lys) from CWR in humans using the indicator AA oxidation (IAAO) technique. Three amounts of l-Lys, 10, 15, and 19 mg · kg−1 · d−1 (= 28.5, 42.8, and 54.3% of the mean l-Lys requirement of 35 mg · kg−1 · d−1), were studied in 5 healthy young men in a repeated-measures design. To test the principle that the Maillard reaction has an effect on the MA of l-Lys, we also assessed the MA of l-Lys in oven-browned, cooked rice (n = 3) in the amount of 19 mg · kg−1 · d−1l-Lys. The MA of l-Lys was estimated by comparing the IAAO response with varying l-Lys intakes in rice compared with the IAAO response to varying l-Lys intakes in the reference protein (crystalline AA mixture patterned after egg protein) using the slope ratio method. The MA of l-Lys from CWR was high (97%), but the effect of the Maillard reaction reduced it to 70%. The results show that despite its relatively low content in rice, l-Lys has a high MA when the rice is cooked without being browned.