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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- 13 C] 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/m 2 ] 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 13 CO 2 from the oxidation of L-[1- 13 C] 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)., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

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., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Background: 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., Objective: 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., Methods: 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., Results: The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively., Conclusion: 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.

Background: 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., Objective: Our objective is to define a quantitative lysine requirement in healthy pregnant women and to determine whether requirements vary between 2 phases of gestation., Methods: 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., Results: 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., Conclusions: 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., (© 2018 American Society for Nutrition. All rights reserved.)

Background: 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., Objective: 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., Methods: 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., Results: 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., Conclusions: The lysine requirement in undernourished children is similar to that of well-nourished children, and intestinal parasitic infestation increased the lysine requirement by ∼20%., (© 2015 American Society for Nutrition.)

We previously showed that the splanchnic bed of neonates takes up a significantly higher amount (43%) of threonine in contrast to lysine (12%). We questioned whether this same pattern applied in adult humans and found no information in the literature. Therefore, our objective in the current study was to determine the difference in splanchnic first pass disappearance of threonine and lysine in adult humans during the fed state. During two 5-d study periods, 6 healthy men received l-[1-(13)C]threonine or l-[1-(13)C]lysine enterally or parenterally randomized to either study d 3 or 5, respectively. The diets were in the form of an amino acid mixture providing a protein intake of 1 g · kg(-1) · d(-1) and an energy intake of 1.5 times the resting energy expenditure. Blood and breath samples were collected at baseline and plateau for amino acid and 11CO(2) enrichment. Splanchnic disappearances in adult males were similar for threonine and lysine. The splanchnic disappearance of threonine as a percentage of intake was 17.9% (8.4 mg · kg(-1) · d(-1)) in the healthy men compared with 18.5% (11.2 mg · kg(-1) · d(-1)) for lysine. We conclude that the threonine requirement of the gut of healthy men consuming a liquid formula diet based on free amino acids is similar to the lysine requirement, which is in contrast to our previous findings for neonates.

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 LLys, we also assessed the MA of L-Lys in oven-browned, cooked rice (n = 3) in the amount of 19 mg · kg(-1) · d(-1) L-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.

Current AA recommendations for sows are to provide a fixed amount of AA intake throughout gestation; however, the demand for nutrients changes from maternal lean tissue in early gestation (EG) to fetal and mammary growth in late gestation (LG). The objective of this study was to determine the Lys requirement in EG (d 24 to 45) and LG (d 86 to 110) using the indicator AA oxidation method with simultaneous determination of heat production. Each of 7 Large White × Landrace sows received 6 diets in random order in both EG and LG. Three semisynthetic diets (14.0 MJ ME/kg) based on corn were formulated and mixed to produce a basal diet (60% of 1998 NRC Lys requirement) and high diets for EG and LG (150% and 185% of 1998 NRC Lys requirements, respectively). The 6 test diets provided Lys intakes of 7.5 to 19.3 g/d in EG and 8.1 to 23.7 g/d in LG. Sows were placed in respiration chambers, and expired air and blood were collected every 30 min for 5.5 h. The tracer AA, l-[1-(13)C]Phe, was given orally at a rate of 2 mg/(kg BW ⋅ h) over the last 4 h, divided into 8, 0.5-h meals. Expired air was measured for (13)CO(2) enrichment, and plasma was measured for l-[1-(13)C]Phe enrichment and free Lys concentration. Background (13)CO(2) was subtracted from plateau (13)CO(2) enrichment. Requirements were determined using a 2-phase nonlinear model. Mean maternal BW gain in gestation (43.7 kg; pooled SE, 1.2 kg), litter size (14.6 total born piglets; pooled SE, 0.8), and litter weight (19.4 kg; pooled SE, 0.9 kg) did not differ between parities. Sow weight gain and BW was greater (P = 0.001) in LG than EG. Lysine requirement was 9.4 and 17.4 g/d in EG and LG, respectively. Phenylalanine retention in LG was maximized at a Lys intake of 17.7 g/d. Heat production was more (P = 0.069) and energy retention less (P = 0.019) in LG than EG. Energy retention in LG was not different from 0. Quantitative Phe kinetics in EG were not affected by Lys intake. In LG, Phe retention increased with Lys intake (P = 0.004), whereas Phe oxidation decreased (P = 0.005). The Lys requirement was determined to be less than current recommendations in EG and more than current recommendations in LG. To meet the change in requirements, diets with increased lysine content are needed in LG. Increasing the feed allowance in LG is necessary to maintain a positive energy balance throughout gestation.

The activity of lysine α-ketoglutarate reductase (LKR), the initial enzyme in the principal pathway of lysine catabolism, is a primary determinant of whole-body lysine status. Past research indicated that LKR activity was predominantly hepatic; recent in vivo data suggest that other tissues can also catabolize lysine. The hypothesis of this investigation was that lysine catabolism takes place in extrahepatic tissues in pigs and that the enzymes involved may be subject to inhibition or activation. Using mitochondria from various tissues of market-age pigs, the activities of LKR and saccharopine dehydrogenase were measured. Liver mitochondria had the highest LKR activity, and the enzyme was subject to substrate inhibition. Mitochondria from the muscle, kidney, heart, and intestinal epithelial cells all had measurable LKR activity. The LKR activity was significantly inhibited by a variety of compounds including saccharopine, α-aminoadipate, α-ketoadipate, 5-hydroxy-l-lysine, and several metals. Oxidation of (14)C-lysine to (14)CO(2) was demonstrated in mitochondria isolated from the liver, muscle, and intestinal epithelial cells. Western blotting confirmed the presence of the α-aminoadipate δ-semialdehyde synthase protein in some extrahepatic tissues. These data show a significant capacity for lysine degradation in these extrahepatic tissues, most notably in cells of the intestine and muscle. These tissues should be considered important contributors to whole-body lysine catabolism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The lysine requirement of human neonates receiving parenteral nutrition (PN) has not been determined experimentally., Objective: The objective was to determine the parenteral lysine requirement for human neonates by using the minimally invasive indicator amino acid oxidation technique with l-[1-(13)C] phenylalanine as the indicator amino acid., Design: Eleven postsurgical neonates were randomly assigned to 15 lysine intakes ranging from 50 to 260 mg . kg(-1) . d(-1). Breath and urine samples were collected at baseline and at plateau for (13)CO(2) (F(13)CO(2)) and amino acid enrichment, respectively. The mean lysine requirement was determined by applying a 2-phase linear regression crossover analysis to the measured rates of F(13)CO(2) release and l-[1-(13)C]phenylalanine oxidation., Results: The mean parenteral lysine requirement determined by F(13)CO(2) release oxidation was 104.9 mg . kg(-1) . d(-1) (upper and lower CIs: 120.6 and 89.1 mg . kg(-1) . d(-1), respectively). The mean lysine parenteral requirement determined by phenylalanine oxidation was 117.6 mg . kg(-1) . d(-1) (upper and lower CIs: 157.5 and 77.6 mg . kg(-1) . d(-1), respectively). Graded intakes of lysine had no effect on phenylalanine flux., Conclusion: We recommend a mean lysine requirement for the postsurgical PN-fed neonate of 104.9 mg . kg(-1) . d(-1), which is 32-43% of the lysine concentration presently found in commercial PN solutions (246-330 mg . kg(-1) . d(-1)). This trial was registered at clinicaltrials.gov as NCT00779753.

We recently reported the lysine requirement of school-aged children living in Canada consuming a mixed diet to be 35 mg.kg(-1).d(-1). Because the majority of children in the world live on cereal-based diets in developing countries, we measured the daily lysine requirement in healthy children living in India and consuming a mostly cereal-based diet. Our objective in this study was to determine the lysine requirement in healthy, school-aged children in the developing world by using the indicator amino acid oxidation method with l-[1-(13)C] phenylalanine. Six healthy, school-aged children consumed 7 levels of lysine (5, 15, 25, 35, 50, 65, and 80 mg.kg(-1).d(-1)) each in a random order along with an amino acid mixture providing energy and protein intakes of 1.7 x resting energy expenditure and 1.5 g.kg(-1).d(-1), respectively. The mean lysine requirement was determined by applying a 2-phase linear regression crossover analysis on tracer oxidation (F(13)CO(2)) data, which identified a breakpoint (requirement) at minimal F(13)CO(2) in response to the graded lysine intakes. The mean lysine requirements with the upper 95% CI for children were determined to be 33.5 and 46.6 mg.kg(-1).d(-1), respectively, by breakpoint analysis of the F(13)CO(2) data. The mean lysine requirements of Indian children were almost identical to that of Canadian children (35 mg.kg(-1).d(-1)). There is no evidence for any adaptation in lysine requirements in children from developing countries such as India.

The number of days of adaptation to a specific amino acid intake required prior to the determination of amino acid requirements using the indicator amino acid oxidation method (IAAO) is still in debate. In this study, our objective was to determine whether adaptation for 8 h, 3 d, and 7 d to a wide range of lysine intakes had any effect on the oxidation of the indicator amino acid, l-[1-(13)C]phenylalanine, to (13)CO(2) (F(13)CO(2)). Five healthy young men randomly received each of 4 levels of lysine (5, 20, 35, and 70 mg x kg(-1) x d(-1)) along with an amino acid mixture to achieve a protein intake of 1.0 g x kg(-1) x d(-1) and energy intake of 1.5x resting energy expenditure during 4 separate 7-d study periods. IAAO studies were conducted on d 1, 3, and 7. During each study day, oral consumption of l-[1-(13)C]phenylalanine was followed by collection of breath for F(13)CO(2) and plasma for measurement of phenylalanine enrichment. F(13)CO(2) was affected by lysine intake but did not differ among adaptation periods of 8 h, 3 d, or 7 d. Phenylalanine flux was not significantly affected by period of adaptation. These results suggest that the minimally invasive IAAO model, where participants are adapted prior to protein intake for 2 d followed by study day adaptation to the test amino acid intake for 8 h, may be sufficient to estimate individual amino acid requirements in healthy young men.

Background: The current Dietary Reference Intake (DRI) recommendations for lysine requirements in children are based on a factorial estimate., Objective: The objective of the current study was to determine the lysine requirement in healthy school-age children by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2) (F(13)CO(2)) in response to graded intakes of lysine., Design: Five healthy school-age children randomly received each of 7 lysine intakes (5, 15, 25, 35 50, 65, and 80 mg x kg(-1) x d(-1)) along with an amino acid mixture to give a final calculated protein intake of 1.5 g x kg(-1) x d(-1) and an energy intake of 1.7 x resting energy expenditure (REE). The mean lysine requirement was determined by applying 2-phase linear regression crossover analysis on F(13)CO(2) data, which identified a breakpoint (requirement) at minimal F(13)CO(2) in response to graded lysine intakes., Results: The mean and population-safe (upper 95% CI) lysine requirements were determined to be 35 and 58 mg x kg(-1) x d(-1), respectively., Conclusions: The mean and population-safe lysine requirements for children are similar to those for adults (36 and 52 mg x kg(-1) x d(-1), respectively), which suggests that the findings from the current study reflect predominantly the maintenance lysine requirements in children and not all requirements for growth. Therefore, to ensure age-appropriate growth in school-age children, we propose the addition of the requirement of lysine for growth (approximately 6 mg x kg(-1) x d(-1)) to the mean estimate. The new mean and population-safe lysine requirements are 41 and 58 mg x kg(-1) x d(-1), respectively; these values are significantly higher than the current DRIs of 37 and 46 mg x kg(-1) x d(-1), respectively.

Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.

Although AA requirements for the mean in a population of growing pigs are well established, there are no direct estimates of their variability within the population. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs. The objective was to determine the Lys requirement in individual pigs to derive a first estimate of the population mean requirement and its variability. Nine individually housed barrows (15 to 18 kg) were surgically implanted with venous catheters for isotope infusion. Pigs were offered, in random order, isonitrogenous and isoenergetic diets with one of seven Lys concentrations (4.8 to 15.5 g of Lys/kg diet, as-fed basis). The pigs were fed twice daily, except for study days when they received one-half of the daily allowance in eight equal hourly meals. After a validated minimum adaptation period, indicator (Phe) oxidation was determined for each dietary Lys level during a 4-h primed, constant infusion of L-[1-(14C)]Phe at a rate of 464 kBq/h. The Lys requirement was calculated using a two-phase linear regression crossover analysis within individual pigs. For each pig, Phe oxidation decreased linearly (P < 0.02) as the dietary Lys concentration increased until the requirement was reached; thereafter, Phe oxidation was not different. The true ileal digestible Lys requirement ranged from 7.5 to 10.6 g/kg of diet (as-fed basis) for the nine animals. The mean requirement for all pigs was 9.1 g/d (CV, 11.6%) or 93.9% (CV, 9.8%) of the predicted (NRC, 1998) requirement based on each pig's mean BW and energy intake. The measured and predicted requirements did not differ. The indicator AA oxidation method gave values for Lys requirement similar to conventional methods. The short (< 3 wk) experimental period allows, for the first time, the estimate of population variability, which provides for more accurate calculation of the effect of altering Lys intake on herd performance and production economics. This method is suitable to use with all dietary indispensable AA.

The aim of this study was to investigate whether the phases of the menstrual cycle affect lysine requirement in healthy adult females, as determined by the indicator amino acid oxidation (IAAO) method. Five healthy females with regular menstrual cycles were studied at seven graded levels of lysine intake, in random order, with an oral [13C]phenylalanine tracer protocol in both the follicular and luteal phases. A total of 14 studies were conducted for each subject. Breath and plasma samples were collected according to the standard IAAO protocol. Serum 17beta-estradiol and progesterone concentrations were measured on each IAAO study day. The rate of release of 13CO2 from [13C]phenylalanine oxidation (F13CO2) was measured, and a two-phase linear regression crossover model was applied to determine lysine requirement. F13CO2 was higher during the luteal phase (P < 0.001) and was positively associated with serum concentrations of 17beta-estradiol and progesterone. The F13CO2 data were adjusted for subjects and sex hormones and used to define breakpoints for lysine requirements. The lysine requirement of healthy females in the luteal phase was 37.7 mg.kg(-1).day(-1) and higher (P = 0.025) than that of females in the follicular phase (35.0 mg.kg(-1).day(-1)). At all lysine intake levels, plasma amino acids were lower and phenylalanine oxidation was higher in the luteal relative to the follicular phase. Therefore, we reason that the higher lysine requirement observed in the luteal phase is probably due to higher amino acid catabolism.

Inevitable catabolism contributes to the inefficiency of using dietary lysine intake for body protein deposition (PD). This study was conducted to determine the effects of true ileal digestible (TID) lysine intake, body weight (BW), and growth potential on lysine catabolism in growing pigs. Starting at 15 kg BW, 16 female Yorkshire pigs were offered a purified diet providing all nutrients in excess of requirements for maximum protein deposition (PDmax). At approximately 25 kg BW, the pigs' PDmax was determined using the N-balance method. Thereafter, 4 pigs were allocated to each of 4 diets, first-limiting in lysine, providing lysine intakes corresponding to 60, 70, 80, and 90% of estimated requirements for PDmax. The pigs were surgically fitted with catheters in the jugular and femoral veins. Lysine catabolism was determined at 2 BW (40-45 kg, low; 70-75 kg, high) either directly (oxidation) using a primed, constant infusion of l-[1-(14)C]-lysine or indirectly (disappearance) using the N-balance method. There was no effect of BW on the rate (g/d) or fraction of TID lysine intake catabolized. Lysine catabolism decreased with increasing growth potential. Lysine disappearance and lysine oxidation (% of TID lysine intake) were independent of lysine intake, except for the lowest lysine intake level, where they were lower. When lysine catabolism was independent of intake, lysine oxidation based on plasma free lysine specific radioactivity (SRA) was lower (9.9% of TID intake) than lysine disappearance (17.4% of TID intake) or lysine oxidation based on liver free lysine SRA (13.4% of TID intake).

There is disagreement about the adaptation time required when using the indicator amino acid oxidation (IAAO) technique. Our objective was to establish the adaptation time required to obtain a plateau in indicator (L-[1-(14)C]-phenylalanine) oxidation in response to a test diet using growing and adult pigs. Four barrows (20 kg) and 4 sows (240 kg) were surgically implanted with venous catheters for isotope infusion. Growing Pigs: After 7 d of adaptation to an adequate lysine intake of 8.8 g/d, phenylalanine oxidation in growing pigs was 9.38 +/- 1.25% of the infused dose. At 2, 3, 4, or 6 d after reducing lysine intake to 3.8 g/d, and then increasing it back to 8.8 g/d, phenylalanine oxidation was 16.94 +/- 0.84% (P < 0.05) and 9.70 +/- 0.80% (P < 0.05), respectively, with no significant effect of days of adaptation to diet. Adult Pigs: After 14 d of adaptation to an intake of 200% of the amino acid maintenance requirement, phenylalanine oxidation in sows was 4.23 +/- 0.45% of dose. Changing the intake to 100 and 50% of the maintenance requirement, increased (P < 0.05) phenylalanine oxidation to 5.95 +/- 0.26 and 7.90 +/- 0.26%, respectively, with no significant effect of time (1, 2, 5, 6, 9, and 10 d) after diet change. The CV for repeated phenylalanine oxidation measurements within pigs and diets was 13.5% for growing and 8.8% for adult pigs. This demonstrates that the IAAO requires <2 d of adaptation regardless of age, dietary challenge (individual amino acid or total protein) or direction (increase or decrease) of change, and that the measured oxidation rate (% of dose) is highly repeatable.

Oxidation contributes to the inefficiency of lysine utilization for protein deposition. The influences of feeding frequency and type of isotope tracer on estimated lysine oxidation were studied in growing pigs fed lysine-limiting diets. Yorkshire gilts (n = 11) weighing 40-45 kg were fitted with venous catheters. They were fed, in 3 or 8 equal meals daily, a purified diet based on casein and cornstarch. Lysine intake limited the pigs' protein deposition to 70% of their potential. After a 5-d N-balance period, lysine oxidation was estimated by a primed, constant 26-h infusion of [1-14C]L-lysine and [6-3H]L-lysine. Feeding frequency and type of tracer did not affect lysine oxidation (P > 0.1). Increasing feeding frequency from 3 to 8 times daily reduced the variance and fluctuation of lysine oxidation by 46 and 30%, respectively. The mean lysine oxidation, as a fraction of the true ileal digestible lysine intake, was 9.2% based on the free lysine specific radioactivity (SRA) in plasma, 20.1% based on free lysine SRA in liver and 21.8% calculated from N-balance data. On the basis of liver free lysine SRA, tracer dilution methods and N-balance data give similar quantitative estimates of lysine oxidation (P > 0.10). Isotope tracer studies that cover one or more complete feeding cycles, i.e., feeding-to-feeding periods, can be used to obtain valid daily lysine oxidation values.

The indicator amino acid oxidation (IAAO) method allows the determination of amino acid requirements under conditions of low growth rate as found in pre-laying broiler breeder pullets. Cobb 500 breeder pullets (20 wk old; 2290 +/- 280 g, n = 4) were adapted (6 d) to a pelleted, purified control diet containing all nutrients at >or=110% of NRC recommendations. After recovery from surgery for implantation of a jugular catheter, each bird was fed, in random order, test diets containing one of nine levels of lysine (0.48, 0.96, 1.92, 2.88, 3.84, 4.80, 7.68, 9.60 and 14.40 g/kg of diet). Indicator oxidation was determined during 4-h primed (74 kBq/kg body), constant infusions (44 kBq x h(-1). kg body(-1)) of L-[1-(14)C]phenylalanine. Using the breakpoint of a one-slope broken-line model, the lysine requirement was determined to be 4.88 +/- 0.96 g/kg of diet or 366 +/- 72 mg x hen(-1) x d(-1) with an upper 95% CI of 6.40 g/kg of diet or 480 mg x hen(-1) x d(-1). IAAO allows determination of individual bird amino acid requirements for specific ages and types of birds over short periods of time and enables more accurate broiler breeder pullet diet formulation.

Dietary restriction of phenylalanine is the main treatment for phenylketonuria (PKU), and current estimates of requirements are based on plasma phenylalanine concentration and growth. The present study aimed to determine more precisely the phenylalanine requirements in patients with the disease by use of indicator amino acid oxidation, with L-[1-13C]lysine as the indicator. Breath 13CO2 production (F 13 CO2) was used as the end point. Finger-prick blood samples were also collected for measurement of phenylalanine to relate phenylalanine intake to blood phenylalanine levels. The mean phenylalanine requirement, estimated using a two-phase linear regression crossover analysis, was 14 mg. kg(-1). day(-1), and the safe population intake (upper 95% confidence interval of the mean) was found to be 19.5 mg. kg(-1). day(-1). A balance between phenylalanine intake and the difference between fed and fasted blood phenylalanine concentration was observed at an intake of 20 mg. kg(-1). day(-1). The similarity between these two values (19.5 and 20 mg. kg(-1). day(-1)) suggests that the maximal phenylalanine intake for children with PKU should be no higher than 20 mg. kg(-1). day(-1).

To investigate whether splanchnic uptake of the indicator amino acid ([1-(13)C] phenylalanine) during the fed state alters the estimate of lysine requirement, five healthy men were studied at graded levels of lysine intake, with either an oral or intravenous (IV) tracer protocol, in a randomized, crossover design. Splanchnic extraction of the oral tracer was expressed as the difference between the ratio of the enrichments in urinary phenylalanine between tracer protocols. The rate of release of (13)CO(2) from (13)C-phenylalanine oxidation (F(13)CO(2)) was measured and a two-phase linear regression crossover model was applied to determine the lysine requirement. Mean splanchnic extraction of the oral tracer was approximately 19%. Although actual F(13)CO(2) was higher during oral tracer infusion (P < 0.001), the breakpoint was not different from that determined with IV infusion (P = 0.98), with both yielding a mean lysine requirement of 36.6 mg/(kg. d). The upper 95% confidence intervals were 52.5 and 53.3 mg/(kg. d) for the oral and IV isotope infusions, respectively. These results demonstrate that routes of isotope administration using the indicator amino acid oxidation technique do not affect the estimated amino acid requirement. Therefore, the indicator amino acid oxidation method using the oral route, which is less invasive and allows for studies in vulnerable groups such as infants and children, should be the preferred method for studying amino acid requirements.

Tyrosine (Tyr) is an essential amino acid in phenylketonuria (PKU) because of the limited hydroxylation of phenylalanine (Phe) to Tyr. The recommended intakes for Tyr in PKU are at least five times the recommended phenylalanine intakes. This suggests that Phe and Tyr contribute approximately 20 and 80%, respectively, of the aromatic amino acid (AAA) requirement (REQ). In animals and normal humans, dietary Tyr was shown to spare 40-50% of the Phe requirement, proportions that reflect dietary and tissue protein composition. We tested the hypothesis that the Tyr REQ in PKU would account for 45% of the total AAA REQ by indicator amino acid oxidation (IAAO). Tyr REQ was determined in five children with PKU by examining the effect of varying dietary Tyr intake on lysine oxidation and the appearance of (13)CO(2) in breath (F(13)CO(2)) under dietary conditions of adequate energy, protein (1.5 g x kg(-1) x day(-1)), and phenylalanine (25 mg x kg(-1) x day(-1)). Lysine oxidation and F(13)CO(2) were determined using a primed 4-h oral equal-dose infusion of L-[1-(13)C]lysine. Lysine oxidation and F(13)CO(2) decreased linearly as Tyr intake increased, to a break point that was interpreted as the mean dietary Tyr requirement (16.3 and 19.2 mg x kg(-1) x day(-1), respectively). At Tyr intakes of >16.3 and 19.2 mg x kg(-1) x day(-1), lysine oxidation and F(13)CO(2), respectively, were low and constant. This represents 40.4 and 44.4%, respectively, of the total AAA intake. The current recommendations for Tyr intake in PKU patients appear to be overestimated by a factor of approximately 5. This study is the first application of the IAAO technique in a pediatric population and in humans with an inborn error of metabolism.

Urine sampling of the free amino acid pool serves to reflect plasma enrichment and is used as a noninvasive means to determine isotope enrichment in studies of amino acid metabolism. We determined the effect of D-[13C]phenylalanine and D-[13C]lysine content of tracers on urinary amino acid enrichment following oral infusion of L-[13C]phenylalanine in 18 preterm infants and L-[1-(13)C]lysine in seven healthy adult females. Urinary [13C]phenylalanine enrichment was higher (P < .0001) for L-[13C]phenylalanine containing 0.4% D-[13C]phenylalanine (28.6 +/- 7.1) versus L-[1-(13)C]phenylalanine that contained undetectable D-[13C]phenylalanine (10.2 +/- 1.5). D-[13C]phenylalanine, measured by chiral column gas chromatography-mass spectrometry (GC-MS), accounted for 10% to 30% (20.5% +/- 7%) of total phenylalanine in the urine of infants who received 0.4% D-[13C]phenylalanine, and was absent from the urine of infants receiving tracer with undetectable [13C]phenylalanine. Urinary L-[13C]phenylalanine enrichment did not differ between tracer groups (9.8 +/- 1.5 and 9.8 +/- 2.5). In adult females, the use of L-[1-(13)C]lysine (1.6% D-lysine) resulted in a higher (P < .02) urine total L,D-[13C]lysine enrichment compared with plasma enrichment (40.8 +/- 4.1 v 11.1 +/- 0.7). This study demonstrates the significant presence of D-[13C]amino acids in urine that originate as contaminants from commercially manufactured tracers, as a result of renal tubular discrimination of D-amino acids. A tracer containing detectable amounts of D-[13C]isomer cannot be recommended for any study in which urine will be used to reflect enrichment in the arterial plasma pool.

The primed, continuous intravenous infusion of amino acids labeled with 13C together with measurement of isotopic enrichment in plasma is commonly used to study amino acid metabolism. However, a less invasive, oral infusion that also produces an isotopic steady state in CO2 and urine would be useful, particularly for pediatric studies. We measured the 13C enrichments of expired CO2, plasma and urine free phenylalanine and lysine and estimated flux and oxidation rates in adult humans (n = 12) who received a 4-h oral, primed, equal dose infusion of either L-[1-13C]phenylalanine, L-[1-13C]lysine (D-lysine = 1.6%) or L-[1-13C]lysine (D-lysine

The indicator amino acid oxidation technique was adapted for use during total parenteral nutrition. Twenty-one male Yorkshire piglets (3 d of age) fitted with jugular and femoral vein catheters, received total parenteral nutrition for 6 d. Total parenteral nutrition included 15 g amino acids .kg-1.d-1 (1.25 g lysine.kg-1.d-1), lipid + glucose (1.1 MJ.kg-1.d-1), and micronutrients. Piglets were randomly assigned to lysine intakes of 0.40, 0.55, 0.70, 0.85, 1.00, 1.15, or 1.30 g.kg-1.d-1 and received a 4-h primed, constant infusion of L-[1-14C]phenylalanine. Phenylalanine oxidation (% of dose) decreased significantly (P < 0.05) from 18.6% at the lowest lysine intake to 3.0% at a lysine intake of 0.85 g.kg-1.d-1. Further increases in lysine intake had no effect on phenylalanine oxidation. Regression analysis yielded a mean lysine requirement of 0.79 g.kg-1.d-1 and a safe intake (upper 95% confidence limit) of 0.84 g.kg-1.d-1. Changes in the plasma lysine concentration and phenylalanine oxidation rates supported this estimate. The indicator amino acid oxidation technique can now be used to determine amino acid requirements during total parenteral nutrition. This means that the ideal amino acid pattern for neonates receiving total parenteral nutrition can now be defined by a sensitive and minimally invasive measurement of amino acid kinetics.

Recently, we reported the lysine requirement to be 41.2 mg.kg-1.d-1 using indicator amino acid oxidation (IAAO) in healthy adult males fed a protein intake of 1.0 g.kg-1.d-1. A lower protein intake has been hypothesized to significantly lower the requirement estimate. We tested this hypothesis using IAAO by estimating the lysine requirement at a protein intake of 0.8 g.kg-1.d-1 in five healthy adult males. Lysine requirement was determined from the rate of appearance of 13CO2 in breath (F13CO2) by using a primed 4-h continuous infusion of L-[1-(13)C]phenylalanine. Phenylalanine flux was not affected by graded increases in dietary lysine. Phenylalanine oxidation and F13CO2 decreased linearly (P < 0.05) as lysine intake increased to a breakpoint, after which the rates were not significantly different. Two-phase linear regression determined this breakpoint (mean lysine requirement) to occur at a dietary lysine intake of 45.0 mg.kg-1.d-1. We conclude that no reduction appears in the lysine requirement with a lower protein intake. Thus, lysine requirement appears not to be altered by protein intakes within the lower end of the protein requirement range.

Knowledge of amino acid availability in feedstuffs is central to accurate diet formulation. Dietary lysine oxidation was evaluated as a means of predicting dietary lysine availability. Growing pigs (30 kg) were offered control (1.43% lysine), free lysine-supplemented soybean meal-, or cottonseed meal-supplemented diets. Supplemented diets provided equivalent total lysine (approximately 24.2 g/d at 30 kg BW) but availabilities of lysine, determined by slope-ratio assay, in the free lysine, soybean meal, and cottonseed meal were 100, 90, and 30%, respectively. Feed was offered in eight equally spaced meals per day to achieve three times maintenance energy intake. Following a meal containing L-[U-14C]lysine (1 mu Ci/kg BW), lysine oxidation, as 14CO2 expired, was lower (P < .05) for the control diet but not different between the other three diets, contrary to the hypothesis. Lysine oxidation following an intravenous bolus dose was lowest (P < .05) for pigs fed the control, highest (P < .05) for pigs fed the cottonseed diet, and intermediate (P < .05) for pigs fed the free lysine-supplemented diet (27.1, 80.2, and 47.5, dpm/kg x 10(-2), respectively). Plasma lysine concentration was lower (P < .05) and lysine specific radioactivity tended to be higher (P < .10) following a meal containing cottonseed than following a meal containing free lysine, indicating that the lysine pool was smaller in pigs receiving the cottonseed-meal diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysine requirement was determined in seven adult males by examining the effect of varying dietary lysine intake on phenylalanine flux and oxidation under dietary conditions of adequate energy and phenylalanine (14 mg.kg-1 x day-1) and excess tyrosine (40 mg.kg-1 x day-1). Phenylalanine flux was determined from primed, constant intravenous infusions of L-[1-13C]phenylalanine (1.2 mg.kg-1 x day-1) and L-[ring-2H5]phenylalanine (0.5 mg.kg-1 x day-1) and measurement of isotopic enrichments of phenylalanine in plasma. Phenylalanine flux was not affected by graded increases in dietary lysine intake or by the isotope infused. Mean phenylalanine conversion to tyrosine was low (3.4%) and not significantly affected by lysine intake. Phenylalanine oxidation, estimated from the rate of 13CO2 released in expired air during the infusion of L-[1-13C]phenylalanine, decreased linearly as lysine intake increased to a break point that was interpreted as the mean dietary lysine requirement (37 mg.kg-1 x day-1). At lysine intakes of > 37 mg.kg-1 x day-1 phenylalanine oxidation was low and constant. Plasma lysine concentrations supported this estimate of requirement. These data show that: 1) indicator amino acid oxidation can be used as a new method to determine amino acid requirements of humans and 2) the lysine requirement of adult males is three times greater than the World Health Organization recommendation of 12 mg.kg-1 x day-1.

We examined leucine and lysine metabolism in adult humans (n = 8 and n = 5, respectively) to assess the current estimate of protein requirement. Each subject consumed a controlled isoenergetic liquid diet for three 10-d periods at three intakes of protein, 0.6, 0.8 and 1.0 g.kg-1.d-1, in random order. Measurements of flux and catabolism were determined in each subject in both the fed and postabsorptive states, with a primed, 4-h continuous infusion of L-[1-13C]leucine and L-[alpha-15N]lysine. Fluxes of leucine and lysine were not affected by feeding state or protein intake. Adaptation to different protein intakes did not affect the oxidation of leucine and lysine in the postabsorptive state, but leucine oxidation was significantly higher during the fed period compared with the postabsorptive period at a protein intake of 1.0 g.kg-1.d-1 (P less than 0.05) and tended to be greater (P = 0.06) at an intake of 0.8 g.kg-1.d-1. Nitrogen balance assessed over the last 72 h of each dietary period was positive for all subjects at protein intakes of 0.8 and 1.0 g.kg-1.d-1 of protein and, hence, was consistent with the leucine oxidation data. These data suggest that experiments conducted during the postabsorptive period are not appropriate for estimating protein requirements. Leucine oxidation data obtained during the fed state estimate a protein requirement for young men of between 0.6 and 0.8 g.kg-1.d-1.