The indicator amino acid oxidation technique defined a protein requirement that is comparable with that estimated by the application of a biphase linear regression model to nitrogen balance data in the literature. Our data and the reanalysis of the preexisting nitrogen balance data suggest that the current recommended protein requirements are too low and require reassessment.

Background Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Methods Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T-cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. Results Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27-year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343 ± 48μM (normal 30-119μM) to 854 ± 25μM (treatment goal ≤360μM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900 μM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. Conclusions Radiation preconditioning and serial rejection-risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure.

The indicator amino acid oxidation (IAAO) method is based on the concept that when 1 indispensable amino acid (IDAA) is deficient for protein synthesis, then all other IDAA, including the indicator amino acid, will be oxidized. With increasing intakes of the limiting amino acid, IAAO will decrease, reflecting increasing incorporation into protein. Once the requirement for the limiting amino acid is met, there will be no further change in the indicator oxidation. Originally, the IAAO method was designed to determine amino acid requirements in growing pigs. The minimally invasive IAAO method developed in humans has been systematically applied to determine IDAA requirements in adults. Due to its noninvasive nature, the IAAO method has also been used to determine requirements for amino acids in neonates and children, and in disease. The IAAO model has recently been applied to determine the metabolic availability (MA) of amino acids from dietary proteins and to determine total protein requirements. The IAAO method is robust, rapid, and reliable; it has been used to determine amino acid requirements in different species, across the life cycle, and in diseased populations. The recent application of IAAO to determine MA of amino acids and protein requirements is also very novel.

Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans.

No abstract

During the past 25 years a significant amount of research has been conducted to determine amino acid requirements in humans. This is primarily due to advancements in the application of stable isotopes to examine amino acid requirements. The indicator amino acid oxidation (IAAO) method has emerged as a robust and minimally invasive technique to identify requirements. The IAAO method is based on the concept that when one indispensable dietary amino acid (IDAA) is deficient for protein synthesis, then the excess of all other IDAA, including the indicator amino acid, will be oxidized. With increasing intakes of the limiting amino acid, IAAO will decrease, reflecting increasing incorporation into protein. Once the requirement for the limiting amino acid is met there will be no further change in the indicator oxidation. The IAAO method has been systematically applied to determine most IDAA requirements in adults. The estimates are comparable to the values obtained using the more elaborate 24h-indicator amino acid oxidation and balance (24h-IAAO/IAAB) model. Due to its non-invasive nature the IAAO method has also been used to determine requirements for amino acids in neonates, children and in disease. The IAAO model has recently been applied to determine total protein requirements in humans. The IAAO method is rapid, reliable and has been used to determine amino acid requirements in different species, across the life cycle and in disease. The recent application of IAAO to determine protein requirements in humans is novel and has significant implications for dietary protein intake recommendations globally.

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.

These estimates are considerably higher than the EAR of 0.88 g · kg(-1) · d(-1) currently recommended by the Dietary Reference Intakes. To our knowledge, this study is the first to directly estimate gestational stage-specific protein requirements in healthy pregnant women and suggests that current recommendations based on factorial calculations underestimate requirements. This trial was registered at clinicaltrials.gov as NCT01784198.