Rajavel Elango

Amino acid requirements in humans: with a special emphasis on the metabolic availability of amino acids

Due to advances made in the development of stable isotope based carbon oxidation methods, the determination of amino acid requirements in humans has been an active area of research for the past 2 decades. The indicator amino acid oxidation (IAAO) method developed in our laboratory for humans has been systematically applied to determine almost all indispensable amino acid requirements in adult humans. Nutritional application of experimentally derived amino acid requirement estimates depends upon the capacity of food proteins to meet the amino acid requirements in humans. Therefore, there is a need to know the proportion of dietary amino acids which are bioavailable, or metabolically available to the body for protein synthesis following digestion and absorption. Although this concept is widely applied in animal nutrition, it has not been applied to human nutrition due to lack of data. We developed a new in vivo method in growing pigs to identify the metabolic availability of amino acids in foods using the IAAO concept. This metabolic availability method has recently been adapted for use in humans. As this newly developed IAAO based method to determine metabolic availability of amino acids in foods is suitable for rapid and routine analysis in humans, it is a major step forward in defining the protein quality of food sources and integrating amino acid requirement data with dietary amino acid availability of foods.

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

BACKGROUND Data on the protein requirements of elderly adults are limited, because it is impractical to conduct repeated nitrogen balance protocols in these vulnerable humans. OBJECTIVE 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. DESIGN 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. RESULTS 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. CONCLUSIONS 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.

Protein requirement of healthy school-age children determined by the indicator amino acid oxidation method

BACKGROUND The current Dietary Reference Intake (DRI) recommendations for protein requirements in children are based on a factorial estimate and have not been directly determined. OBJECTIVE The objective of the current study was to determine the protein requirement in healthy, school-age children by measuring the oxidation of L-[1-(13)C]-phenylalanine to (13)CO(2) [label tracer oxidation (F(13)CO(2))] in response to graded intakes of protein. DESIGN Seven healthy children (6-11 y old) each randomly received a minimum of 7 protein intakes (range: 0.1-2.56 g · kg(-1) · d(-1)) for a total of 56 studies. The diets provided energy at 1.7 times the resting energy expenditure and were made isocaloric by using carbohydrate. Protein was given as an amino acid mixture on the basis of the egg-protein pattern, except for phenylalanine and tyrosine intakes, which were maintained constant across intakes. The mean protein requirement was determined by applying a 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 amounts of protein intake. RESULTS Mean and population-safe (upper 95% CI) protein requirements were determined to be 1.3 and 1.55 g · kg(-1) · d(-1), respectively. These results are significantly higher than the mean and population-safe protein requirements currently recommended by the DRI 2005 for macronutrients (0.76 and 0.95 g · kg(-1) · d(-1), respectively). CONCLUSION To our knowledge, this study was the first to directly estimate protein requirements in children by using stable isotopes and indicated that current recommendations are severely underestimated.

Evidence that protein requirements have been significantly underestimated.

Purpose of reviewThis review discusses recent evidence that suggests a significant underestimation of protein requirements in adult humans. Recent findingsTraditionally, total protein requirements for humans have been determined using nitrogen balance. The recent Dietary Reference Intake recommendations for mean and population-safe intakes of 0.66 and 0.8 g/kg/day, respectively, of high-quality protein in adult humans are based on a meta-analysis of nitrogen balance studies using single linear regression analysis. We reanalyzed existing nitrogen balance studies using two-phase linear regression analysis and obtained mean and safe protein requirements of 0.91 and 0.99 g/kg/day, respectively. The two-phase linear regression analysis is considered more appropriate for biological analysis of dose–response curves. Considering the inherent problems associated with the nitrogen balance method, we developed an alternative method, the indicator amino acid oxidation technique, to determine protein requirements The mean and population-safe requirements in adult men were determined to be 0.93 and 1.2 g/kg/day and are 41 and 50%, respectively, higher than the current Dietary Reference Intakes recommendations. SummaryThe indicator amino acid oxidation-based requirement values of 0.93 and 1.2 g protein/kg/day and the reanalysis of existing nitrogen balance studies are significantly higher than current recommendations. Therefore, there is an urgent need to reassess recommendations for protein intake in adult humans.

Recent advances in determining protein and amino acid requirements in humans

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.

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

BACKGROUND Studies on protein requirements in vulnerable groups such as older adults are few, and results are conflicting. OBJECTIVE 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. METHODS 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 subjects 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. RESULTS 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. CONCLUSION 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.

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimers disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Indicator Amino Acid Oxidation Is Not Affected by Period of Adaptation to a Wide Range of Lysine Intake in Healthy Young Men

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.

Individual amino acid requirements in humans: an update

Purpose of reviewTo discuss recent amino acid requirement studies in adult humans and school-age children, primarily determined using the indicator amino acid oxidation technique. Recent findingsUsing the minimally invasive indicator amino acid oxidation model, requirements for most indispensable amino acids have been defined in adult humans. The estimates are comparable to the values obtained using the more elaborate 24-h indicator amino acid oxidation and balance model. The less-invasive indicator amino acid oxidation model has also been successfully applied to define requirements in healthy school-age children and children with disease. A recent adaptation of the indicator amino acid oxidation method to determine protein requirements in adult humans resulted in mean and safe values of 0.93 and 1.2 g protein/kg/day, respectively. These estimates are 40–50% higher than current recommendations and suggest an urgent need to reassess recommendations for protein intake in humans. SummaryIn summary, indicator amino acid oxidation is a robust technique, and has resulted in the definition of amino acid and protein requirements in adult humans and children. A wider application of the technique in other vulnerable populations across life stages and in other diseases is now possible.

Protein Requirements of Healthy Pregnant Women during Early and Late Gestation Are Higher than Current Recommendations

BACKGROUND Adequate maternal dietary protein intake is necessary for healthy pregnancy. However, current protein intake recommendations for healthy pregnant women are based on factorial calculations of nitrogen balance data derived from nonpregnant adults. Thus, an estimate of protein requirements based on pregnancy-specific data is needed. OBJECTIVE The objective of this study was to determine protein requirements of healthy pregnant women at 11-20 (early) and 31-38 (late) wk of gestation through use of the indicator amino acid oxidation method. METHODS Twenty-nine healthy women (24-37 y) each randomly received a different test protein intake (range: 0.22-2.56 g · kg(-1) · d(-1)) during each study day in early (n = 35 observations in 17 women) and late (n = 43 observations in 19 women) gestation; 7 women participated in both early and late gestation studies. The diets were isocaloric and provided energy at 1.7 × resting energy expenditure. Protein was given as a crystalline amino acid mixture based on egg protein composition, except phenylalanine and tyrosine, which were maintained constant across intakes. Protein requirements were determined by measuring the oxidation rate of L-[1-(13)C]phenylalanine to (13)CO2 (F(13)CO2). Breath and urine samples were collected at baseline and isotopic steady state. Linear regression crossover analysis identified a breakpoint (requirement) at minimal F(13)CO2 in response to different protein intakes. RESULTS The estimated average requirement (EAR) for protein in early and late gestation was determined to be 1.22 (R(2) = 0.60; 95% CI: 0.79, 1.66 g · kg(-1) · d(-1)) and 1.52 g · kg(-1) · d(-1) (R(2) = 0.63; 95% CI: 1.28, 1.77 g · kg(-1) · d(-1)), respectively. CONCLUSIONS 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.