Ronald O. Ball

Proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal piglets

The indispensability of arginine has not been conclusively established in newborns. Because parenteral feeding bypasses the gut (where de novo synthesis of arginine occurs from proline), a dietary supply of arginine that is sufficient to maintain urea cycle function may be of greater importance during intravenous compared with enteral feeding. Two-day-old piglets ( n = 12) were fed nutritionally complete diets for 5 days via either a central vein catheter (IV pigs, n = 6) or a gastric catheter (IG pigs, n = 6). Subsequently, each piglet received three incomplete test diets [arginine free (-ARG/+PRO), proline free (-PRO/+ARG), or arginine and proline free (-ARG/-PRO)] in a randomized crossover design. Plasma ammonia was assayed every 30 min for 8 h or until hyperammonemia was observed. Ammonia increased rapidly in IV pigs receiving -ARG/+PRO and -ARG/-PRO (84 ± 36 and 74 ± 37 μmol ⋅ l-1 ⋅ h-1, respectively), requiring early diet cessation. A rapid increase was also exhibited by IG pigs receiving the -ARG/-PRO, but not the -ARG/+PRO diet (31 ± 15 vs. 11 ± 7 μmol ⋅ l-1 ⋅ h-1, respectively, P < 0.05). Plasma arginine and proline were indicative of deficiency (IG and IV groups) when deplete diets were infused. Arginine is indispensable in parenteral and enteral nutrition, independent of dietary proline.The indispensability of arginine has not been conclusively established in newborns. Because parenteral feeding bypasses the gut (where de novo synthesis of arginine occurs from proline), a dietary supply of arginine that is sufficient to maintain urea cycle function may be of greater importance during intravenous compared with enteral feeding. Two-day-old piglets (n = 12) were fed nutritionally complete diets for 5 days via either a central vein catheter (IV pigs, n = 6) or a gastric catheter (IG pigs, n = 6). Subsequently, each piglet received three incomplete test diets [arginine free (-ARG/+PRO), proline free (-PRO/+ARG), or arginine and proline free (-ARG/-PRO)] in a randomized crossover design. Plasma ammonia was assayed every 30 min for 8 h or until hyperammonemia was observed. Ammonia increased rapidly in IV pigs receiving -ARG/+PRO and -ARG/-PRO (84 +/- 36 and 74 +/- 37 micromol. l(-1). h(-1), respectively), requiring early diet cessation. A rapid increase was also exhibited by IG pigs receiving the -ARG/-PRO, but not the -ARG/+PRO diet (31 +/- 15 vs. 11 +/- 7 micromol. l(-1). h(-1), respectively, P < 0.05). Plasma arginine and proline were indicative of deficiency (IG and IV groups) when deplete diets were infused. Arginine is indispensable in parenteral and enteral nutrition, independent of dietary proline.

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.

The In Vivo Sparing of Methionine by Cysteine in Sulfur Amino Acid Requirements in Animal Models and Adult Humans

Sulfur amino acid metabolism has been receiving increased attention because of the link to chronic diseases such as cardiovascular disease, Alzheimers disease, and diabetes. In addition, the role of cysteine and optimal intakes for physiological substrates such as glutathione are currently of considerable interest in human health. Although the dietary indispensability of methionine is not in question, the ability of cysteine to substitute for a portion of its requirement has been the topic of much debate. Methionine is often the most limiting amino acid in the diets of the developing worlds population because of its low concentration in cereal grains. Therefore, the ability of cysteine to substitute for methionine requirement is not just biologically interesting; it is also of considerable economic and social importance. The primary goal of this review is to discuss the available evidence on the effect of cysteine substitution for methionine to meet the total sulfur amino acid requirement in adult humans, including an assessment of the methodological features of experiments with conflicting results. Assessment of the requirement experiments for amino acids with complex metabolism such as methionine and cysteine must begin with a careful definition of requirements and what substitution means. As a result of these definitions, a set of criteria for the intakes of methionine that will allow demonstration of the substitution effect have been developed. Some recent publications are assessed using these definitions and criteria, and a possible reason for the conflicting results in the literature is proposed. An approach to estimating tolerable upper intakes is also proposed. Research on in vivo sulfur amino acid metabolism in humans is tremendously difficult, and therefore, we do not wish to be overly critical of the high-quality work of the ambitious and highly intelligent men and women who have conducted various studies. Our goal is to objectively review the data for the reader in a logical and comprehensive manner and propose methods that may avoid difficulties in future studies.

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.

The Effect of Graded Intake of Glycyl-l-Tyrosine on Phenylalanine and Tyrosine Metabolism in Parenterally Fed Neonates with an Estimation of Tyrosine Requirement

Although tyrosine is considered indispensable during the neonatal period, its poor solubility has limited its inclusion in parenteral amino acid solutions to less than 1% of total amino acids. Dipeptides of tyrosine are highly soluble, have been shown to be well used and safe in animal models and humans, and, therefore, may be used as an effective means of providing tyrosine in the parenterally fed neonate. The goal of the present study was to determine the tyrosine requirement of the parenterally fed neonate receiving graded intakes of glycyl-l-tyrosine as a source of tyrosine. Thirteen infants receiving adequate energy (340 ± 38 kJ·kg-1·d-1) and protein (2.4 ± 0.4 g·kg-1·d-1) were randomized to receive parenteral nutrition with one of five graded levels of glycyl-l-tyrosine. The mean requirement and safe level of intake were estimated using a 1-13C-phenylalanine tracer and linear regression cross-over analysis that identified a break point in the response of label appearance in breath CO2 (F13CO2) and phenylalanine oxidation to graded tyrosine intake. Based on the mean estimates of whole-body phenylalanine oxidation, the tyrosine mean requirement and safe level of intake were found to be 74 mg·kg-1·d-1 and 94 mg·kg-1·d-1, respectively. This represents 3.1 and 3.9% of total amino acids, respectively, considerably higher than levels found in present commercially available pediatric amino acid solutions. These data raise concern regarding the adequacy of aromatic amino acid intake in the parenterally fed neonate.

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.

Current total parenteral nutrition solutions for the neonate are inadequate.

The amino acid requirements of the parenterally fed neonate are poorly defined. Newborn infants are at risk for amino acid deficiency and toxicity, due to lack of small intestinal metabolism and metabolic immaturity. We discuss recent evidence that identifies inadequacies of commercial amino acid solutions with respect to the balance and quantity of aromatic amino acids, and sulphur amino acids. We present data demonstrating that impaired small intestinal metabolism (or lack of first pass metabolism) alters the whole body requirement for methionine, threonine, and arginine, and discuss the potential adverse effects of excess or inadequate parenteral amino acid intake.

Determination of amino acid requirements by indicator amino acid oxidation: applications in health and disease.

Indispensable amino acid requirements for children, patients and pregnant women are largely unknown. We describe a new, non-invasive technique that can estimate requirements in vulnerable populations. Future applications will lead to optimal nutritional care for populations in which an appropriate balance of amino acids is essential for metabolic health and growth.

The design and validation of a diet for studies of amino acid metabolism in adult humans

Abstract A dietary protocol developed for use in studies of amino acid and protein metabolism is reported in detail. The diet consisted of a mixture of free amino acids, an amino nitrogen-free liquid formula and protein-free cookies. Energy requirement for a group of adult men (n=10) was determined by multiplying each individuals resting metabolic rate, measured by indirect calorimetry, by a factor of 1.7. Subjects maintained a constant weight while consuming the experimental diet and had few complaints regarding palatability during a nine day study period. No significant variation (P=0.69) was evident over a six hour period in measurements of 13 CO 2 enrichment in the breath of subjects (n=4) consuming small hourly meals of the diet. Dietary adaptation to a constant level of 13 C enrichment in breath CO 2 with this dietary protocol was accomplished in a short period of time as shown by the lack of change in the mean (±SD) 13 C enrichments of 0.02764±0.00084, 0.02818±0.00067 and 0.02812 ±0.00062 atoms percent enrichment in subjects (n=7) on days 3, 6 and 9 of the dietary period, respectively. The small within subject variation in repeated 13 CO 2 enrichment measurements indicates the reliability of the dietary protocol in studies where a steady isotope background is essential. This dietary protocol provides a constant and stable metabolic milieu for studies of amino acid metabolism, especially, for those involving the use of stable isotopes.