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Nutrient Requirements and Optimal Nutrition

The Indicator Amino Acid Oxidation Method Identified Limiting Amino Acids in Two Parenteral Nutrition Solutions in Neonatal Piglets^1,2

³ Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada, T6G 2P5; ⁴ Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada, A1B 3X9; and ⁵ Departments of Paediatrics and Nutritional Sciences, University of Toronto, Toronto, ON, Canada, M5G 1X8

^* To whom correspondence should be addressed. E-mail: jbrunton{at}mun.ca.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Recent studies using the indicator amino acid oxidation (IAAO) technique in TPN-fed piglets and infants have been instrumental in defining parenteral amino acid requirements. None of the commercial products in use are ideal when assessed against these new data. Our objectives were to determine whether the oxidation of an indicator amino acid would decline with the addition of amino acids that were limiting in the diets of TPN-fed piglets, and to use this technique to identify limiting amino acids in a new amino acid profile. Piglets (n = 26) were randomized to receive TPN with amino acids provided by Vaminolact (VM) or by a new profile (NP). After 5 d of TPN administration, lysine oxidation was measured using a constant infusion of L- [1-¹⁴C]-lysine. Immediately following the first IAAO study, the piglets were further randomized within diet group to receive either 1) supplemental aromatic amino acids (AAA), 2) sulfur amino acids (SAA) or 3) both (AAA+SAA) (n = 4–5 per treatment group). A second IAAO study was carried out 18 h later. In the first IAAO study, lysine oxidation was high for both groups (18 vs. 21% for VM and NP, respectively, P = 0.055). The addition of AAA to VM induced a 30% decline in lysine oxidation compared with baseline (P < 0.01). Similarly, SAA added to NP lowered lysine oxidation by 30% (P < 0.01). The application of the IAAO technique facilitates rapid evaluation of the amino acids that are limiting to protein synthesis in parenteral solutions.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The indicator amino acid oxidation (IAAO)⁶ technique has been used successfully to quantify amino acid requirements in the neonatal TPN-fed piglet model (6) and subsequently in infants (7). The technique is based on the theory that amino acids are not stored, such that if one amino acid is limiting in the diet, all others will be in excess and must be oxidized. The IAAO technique utilizes an isotopically labeled amino acid that is provided in a constant concentration (ratio of labeled to unlabeled) over a variety of dietary conditions. The lowest measured oxidation of the indicator amino acid identifies when that amino acid (and presumably all others) is being utilized to the greatest extent. We used this technique to identify limitations in the amino acid concentrations of a new parenteral amino acid profile (NP). To validate whether the IAAO technique was sensitive enough to detect small changes in the dietary concentration of the limiting amino acid, we conducted IAAO studies in piglets fed an amino acid solution, Vaminolact (VM). We have previously demonstrated that the concentration of aromatics in Vaminolact is low relative to the requirements of infants and piglets (7,8). Therefore, our objectives were 1) to determine whether the oxidation of an indicator amino acid would decline significantly with the addition of amino acids that are limiting in the diets of TPN-fed piglets, and 2) in our piglet model, to identify limiting amino acids in a proposed new amino acid profile being developed for use in neonates.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Twenty-six male piglets (Landrace/Large White) from a minimal disease herd (University of Alberta Swine Research Centre) were removed from the sow 24–48 h after birth. All experimental procedures were approved by The Faculty of Agriculture, Forestry and Home Economics Animal Policy and Welfare Committee at the University of Alberta. The experiment was conducted as 6 replicates of 4 pigs. Each replicate consisted of 2 pigs randomized to 1 of 2 parenteral diets, differing only in amino acid composition. One additional pair of piglets was required due to an unplanned mortality, resulting in a total of 25 piglets studied. The piglets were transported to the surgical suite of the metabolic research facility where they were anaesthetized with isofluorane (2%) mixed with oxygen (1.5 L/min). Subsequently, 2 catheters were surgically implanted under aseptic conditions. One catheter was introduced into the femoral vein and advanced to the inferior vena cava to enable blood sampling. The second catheter was introduced into the jugular vein for TPN and isotope infusion. Postoperatively, piglets received intramuscular injections of gentamicin sulfate (3.5 mg) (Garasol, Schering Plough Animal Health), trimethoprim and sulfadoxine (40 mg) (Borgal, Hoechst Roussel, Vet Canada) and buprenorphine hydrochloride (0.03 mg/kg) (Buprenex, Reckitt Benckiser Pharmaceuticals). A second dose of buprenorphine hydrochloride was given 12 h following the first dose. Parenteral nutrition was initiated immediately following surgery at 50% of maximal, and infusion rate was increased to provide the targeted intake by 24 h postoperatively. Piglets were housed individually in metabolic cages that allowed visual and aural contact. Each morning piglets were weighed on an electronic balance to calculate weight gain, blood was sampled, and the catheters were flushed with heparinized saline to maintain patency.

The diets were prepared under aseptic conditions with the amino acids supplied by the manufacturer (Fresenius Kabi AB) as either a 10% solution (New Profile, NP) or as VM 6.5%. The amino acid composition of the diets is presented in (Table 1). Both diets were designed to deliver 15 g protein · kg^–1 · d^–1, and were isoenergetic, providing 1.1 MJ · kg^–1 · d^–1. Carbohydrate and fat provided the nonprotein energy in a 1:1 ratio. The diets were not isonitrogenous, with VM providing 2.15 g nitrogen · kg^–1· d^–1 vs. 2.40 g · kg^–1 · d^–1 from the NP (Table 1). This was predominantly due to the high concentration of glutamine in the NP and accurately resembles the clinical scenario because TPN prescriptions are devised based on delivery of total amino acids as opposed to a calculated nitrogen intake. Immediately prior to infusion, the amino acid, dextrose, and electrolyte solutions were made complete by the addition of vitamins (Multi-12/K Pediatric, Rhone-Poulenc Rorer), trace minerals, and iron that were provided to exceed NRC recommended intakes for young swine (9). Lipid was provided as Intralipid 20% (Fresenius-Kabi AB). On the morning of d 5, the piglets underwent an indicator amino acid oxidation study (described in detail below). Following this IAAO study, the piglets were randomized within diet to receive 1 of 3 supplemental amino acid treatments. Piglets that received VM from d 0 to 5 were randomized to either 1) VM plus aromatic amino acids (AAA) (phenylalanine and tyrosine) (VM + AAA, n = 4); 2) VM plus sulfur amino acids (methionine and cysteine) (VM + SAA, n = 4); or 3) VM plus both aromatic and sulfur amino acids (VM + Both, n = 4). Piglets that received NP from d 0–5 were randomized to either 1) NP plus aromatic amino acids (NP + AAA, n = 4); 2) NP plus sulfur amino acids (NP + SAA, n = 4); or 3) NP plus both aromatic and sulfur amino acids (NP + Both, n = 5). A second IAAO study was carried out on d 6, after 18 h of feeding the supplemented diets.

    Sulfur amino acids supplement. Methionine was added to the VM and NP diets to increase the concentration from 2.0% of total amino acids to 2.7%. This provided methionine in excess of the requirement we previously determined for piglets receiving a diet with no cysteine (11). Free L-cysteine was utilized in both VM and NP diets to eliminate the question of bioavailability of cysteine sources and was added to a final concentration of 2.0% of total amino acids.

    Aromatic plus sulfur amino acid supplement. The combination of aromatic and sulfur amino acids was used because a greater response than for either supplemented group alone would indicate that both aromatic and sulfur amino acids were limiting in the diets. The supplemented VM and NP diets were isonitrogenous to the diets provided prior to supplementation (i.e., VM was isonitrogenous to VM+AAA, VM+SAA and VM+Both). Alanine concentration was reduced corresponding to the additional nitrogen concentration from the supplemental amino acids for both VM and NP groups.

    Blood sampling for amino acid analyses and clinical chemistry. Blood was sampled at the same time daily, and the plasma was stored at –70°C for subsequent analysis of amino acids, dipeptides (glycyl-tyrosine, alanyl-glutamine), N-acetylcysteine, and ammonia concentrations.

    Plasma amino acid analysis by HPLC. Plasma amino acids and dipeptides were measured using the method of Bidlingmeyer et al. (12) with modifications for biological samples according to House et al. (13), using HPLC.

    Nitrogen balance and plasma ammonia concentration. Between d 2 and 5 of the study period urine was collected on ice. Twenty-four-hour samples were pooled and an aliquot was stored at –20°C until analyzed. Total nitrogen concentration of the urine was determined by Kjeldahl method (14). Fecal losses in TPN-fed piglets were negligible during the 3-d collection period. Plasma ammonia concentration was determined daily. The spectrophotometric assay used in the analysis was based on the amination of 2-oxoglutarate to glutamate with simultaneous oxidation of NADPH (Sigma Procedure No. 171-UV, Sigma Diagnostics).

    Measurement of lysine oxidation via the indicator amino acid oxidation technique. Lysine was chosen as the indicator amino acid because its concentration in Vaminolact and NP was the same, and lysine was well in excess of the parenteral requirement for neonatal piglets (6). Lysine oxidation was determined using a primed (18.5 kBq/kg), constant intravenous infusion (5.55 kBq · kg^–1 · h^–1) of a tracer solution containing L- [1-¹⁴C]-lysine (9.25 MBq/L). The infusion continued over 4 h to reach a plateau in breath labeling. Details of the ¹⁴CO₂ and blood collection procedures have been previously described (6).

    Statistical analyses. The experiment was analyzed as a completely randomized design. A previous study from our laboratory comparing 2 TPN amino acid solutions in neonatal piglets demonstrated a 5% difference in nitrogen retention with a SD of 3.3% (n = 6) (10). Thus, with = 0.025, ß = 0.8, and SD = 3.3%, to detect a 5% difference required 10 piglets per group. We chose to use 12 piglets per group, which resulted in a 90% (ß = 0.9) chance of rejecting the null hypothesis with a 5% difference in nitrogen retention at = 0.025. Differences between the 2 treatments were determined by Student's t test. Differences within a group (before and after supplementation) were determined by paired Student's t test. Differences in the percentage of decline with the addition of supplemental amino acids was determined by 1-way ANOVA; if the overall P < 0.05 then a post-test (Bonferroni's Multiple Comparison Test) was conducted (GraphPad Prism, version 4.00 for Windows, GraphPad Software). For data in which there was repeated samples (plasma ammonia, plasma amino acids) data were analyzed using repeated measures ANOVA (SAS/STAT, version 8.1, SAS Institute) to determine whether there was an effect of sampling times from the initiation of the test diet on the dependent variable. If P < 0.05 for the F-value of the model, the slope of the response over time was considered to be significant. Values in the text and figures are means ± SD.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
There was one unplanned mortality in the VM group on d 5 and none in the NP group. Postmortem examination of the piglet by a veterinary pathologist was inconclusive. The piglet was subsequently replaced to achieve the desired sample size, but, in order not to cause stress due to isolation of a social animal, an additional piglet was included, resulting in n = 13 for the NP group. All other piglets remained active and interested in the environment and completed all outcome measures of the study.

    Body weight and water balance. There was no difference between groups in body weight at entry into study (both groups combined, 1.82 ± 0.23 kg) or on d 5 (2.73 ± 0.29 vs. 2.57 ± 0.29 kg for Vaminolact and NP, respectively). However, piglets receiving the VM-based TPN had a greater mean daily weight gain from d 2 to d 5 (0.224 ± 0.044 kg vs. 0.182 ± 0.033 kg, P = 0.02). Fluid balance was not different between groups (data not shown).

    Nitrogen balance, plasma ammonia, and indicator amino acid oxidation. Piglets fed NP had significantly greater nitrogen intake and retention, although there were no differences in nitrogen excretion or in percentage of intake retained (Table 2). Plasma ammonia concentrations did not differ between treatment groups at any time during the study (Fig. 1) or when compared as a mean of 5 d for each piglet (85.3 ± 43.0 vs. 95.4 ± 35.2 µmol/L for VM and NP groups, respectively, P > 0.05). On d 5, both treatment groups had mean plasma ammonia concentrations that were approximately twice that of sow-fed reference piglets (58 ± 32 µmol/L) (15). However, both groups exhibited great variability in plasma ammonia concentration, with a range of 21 to 377 µmol/L in the VM group, and 11 to 197 µmol/L in the NP group on d 5 of the study. The median value was similar for the groups, at 91 and 96 µmol/L for VM and NP groups, respectively.

View larger version (10K): [in this window] [in a new window]   FIGURE 1  Plasma ammonia concentrations for piglets fed VM or NP. Values are means ± SD, n = 12 per group. The dashed lines represent a reference mean ± SD for a group of sow-fed piglets (15).

View larger version (10K): [in this window] [in a new window]   FIGURE 2  Lysine oxidation in individual piglets on d 5 when piglets were fed the unsupplemented VM or NP, and on d 6 when diets were supplemented with amino acids. The dashed lines represent the group mean (± SD). VM + AAA (A), VM + Both (C), NP + SAA (E) and NP + Both (F) all declined significantly (P < 0.05) compared with d 5. VM + SAA (B) and NP + AAA (D) did not change from d 5 to d 6. All groups had n = 4 piglets except NP + Both, which had n = 5.

View larger version (14K): [in this window] [in a new window]   FIGURE 3  Plasma methionine (A), cystine (B), phenylalanine (C) and tyrosine (D) concentrations in piglets fed VM and NP. *Represents significant differences between diet groups on the days indicated, P < 0.05. +Represents a significant effect of time within that treatment group (P < 0.05).

View larger version (11K): [in this window] [in a new window]   FIGURE 4  Plasma arginine (A), ornithine (B), citrulline (C), glutamine (D), glutamate (E) and aspartate (F) concentrations in piglets fed VM and NP. *Represents significant differences between diet groups on the days indicated (P < 0.05). +Represents a significant effect of time within that treatment group (P < 0.05).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Vaminolact was included partly to validate our technique, because works from our laboratories have previously demonstrated that the phenylalanine + tyrosine concentration in Vaminolact is below that required for neonatal piglets (8) and infants (7). A 30% decline in oxidation of the lysine indicator strongly supports our hypothesis that the addition of aromatic amino acids, including tyrosine, would improve the efficiency of utilization of the dietary amino acids in this solution. The further decline with the addition of the sulfur amino acids suggests that they are the second limiting amino acids for protein synthesis in VM-fed piglets.

We applied the IAAO technique to evaluate a new amino acid profile (NP) that was formulated to address some of the nutritional shortcomings of the commercially available solutions. In NP, the aromatic amino acids were provided in a balance that was superior to any product currently on the market (64% phenylalanine and 36% tyrosine), but the total concentration (5.6%) was less than the safe intake (upper 95% CI) suggested by House et al. (8). The NP also had a total sulfur amino acid concentration that was low compared with most commercial solutions. The dietary concentration of methionine and cysteine just met the mean requirement determined for TPN-fed neonatal piglets (16), but was well below the estimated safe level of intake (upper 95% CI). Thus, we felt it was necessary to assess the effects of both additional sulfur amino acids and aromatic amino acids to the NP. Adding methionine and cysteine to the NP resulted in a 28% decline in lysine indicator oxidation. However, the addition of aromatics to NP did not induce a significant decrease, suggesting that 5.6% of total amino acids as a balance of phenylalanine and tyrosine was adequate for these piglets. The solubility of L-tyrosine is far below that required to provide the optimal balance of phenylalanine and tyrosine. In the NP, the high tyrosine content was achieved by the inclusion of the di-peptide glycyl-tyrosine. Higher plasma tyrosine concentration and the lack of response in lysine oxidation with aromatic amino acid supplementation suggests that this form of tyrosine was highly bioavailable. Studies investigating phenylalanine and tyrosine requirements in parenterally fed piglets and infants suggest that the hydroxylation of phenylalanine to form tyrosine is inadequate to meet tyrosine requirements (7,8). More recently we have shown that phenylalanine alone may not be enough to meet the total aromatic amino acid needs in enterally fed school aged children (17). Therefore, tyrosine must be considered an indispensable amino acid. Human milk has 4–6% of total amino acids as tyrosine, which is well beyond the concentration found in any commercial product. Clearly, the addition of glycyl-tyrosine in the NP is potentially a very important advancement in parenteral amino acid profiles.

Another novel aspect about the formulation of NP was the inclusion of cysteine as N-acetyl-cysteine (NAC). NAC has specific manufacturing advantages, because free L-cysteine will form cystine (cysteine-cysteine dimer) under conditions of heat processing and long-term storage. Cystine precipitates at low concentrations, whereas NAC is highly stable and soluble. NAC has proven to be highly bioavailable as source of cysteine. In TPN-fed neonatal piglets, we recently demonstrated that the retention of NAC was >80% (18). In that study, piglets received methionine at 50% of the total sulfur amino acid requirement plus graded concentrations of NAC. The TPN-fed piglets receiving methionine plus NAC had similar growth rates and nitrogen retention compared with controls fed an isomolar amount of L-cysteine. In the current study, the NP group had lower plasma methionine concentration on d 5 compared with VM despite the same dietary intake, which may be due to a greater conversion to cysteine. This is supported by the 30% reduction in lysine oxidation with the addition of cysteine to NP, demonstrating that the NAC concentration in NP should be increased further.

Glutamine comprised 15% of total amino acids in NP, supplied as the di-peptide alanyl-glutamine. The high concentration of glutamine in the formulation was balanced by the complete exclusion of glutamic and aspartic acids. NP contained no preformed glutamic acid but plasma concentrations remained similar to that of sow-fed reference piglets (10) and thus were physiologically normal. Plasma aspartate was low in NP-fed piglets, averaging only one-third the concentration measured in sow-fed piglets. The inclusion of glutamine in parenteral solutions has been under investigation for a number of years. In piglets, we showed that the addition of glutamine to parenteral nutrition solution in isonitrogeneously fed piglets did not improve growth or nitrogen balance (13). Recent studies, including a meta-analysis of parenteral and enteral feeding, have demonstrated no benefits in terms of reduced morbidity, mortality or time to reach full enteral feedings (19,20). This conclusion was challenged by recent data from Kalhan et al. (21) who used isotopic tracer techniques to determine whole body protein metabolism in preterm infants receiving glutamine supplemented TPN. Their results suggested that glutamine supplementation reduced whole body proteolysis, which could lead to enhanced protein accretion. It is important to note that the studies investigating the effects of glutamine-supplemented TPN for neonates used a commercial formulation (Trophamine) as the basis for the diet. Thus, to achieve isonitrogenous intakes with glutamine supplementation, the volume of the base solution was reduced, thereby reducing the delivery of all other amino acids by 20% (19,21). Therefore, the reduction of proteolysis observed by Kalhan et al. (21) could have been due to an overall deficiency in amino acids. The lack of beneficial effects observed by others could be due to perturbations in other amino acids rather than strictly a glutamine effect.

We used nitrogen balance as a classical technique to compare the efficiency of amino acid utilization between NP and VM groups, which did not identify one formulation as superior over the other. Whereas NP-fed pigs retained more nitrogen on an absolute basis, there was no difference in the percentage of nitrogen intake that was retained, and both groups had lower nitrogen retention than previous studies from our laboratory of TPN-fed piglets. Both VM and NP groups had nitrogen retentions of 76%, compared with 80% measured in piglets on similar TPN study protocols using aromatic amino acid supplemented TPN (13,22). This suggests that further improvements in the amino acid profile are possible.

It has been >15 y since a new pediatric parenteral amino acid solution was introduced commercially. During that time, there has been considerable progress toward understanding the amino acid requirements when parenteral feeding is necessary. Unfortunately, the cost of developing a new solution is substantial and the global market for such products is small, which may contribute to the lack of new product development. The IAAO technique facilitates rapid evaluation of small changes in an amino acid profile. A number of test diets can be assessed in one animal with only short-term adaptation to new dietary levels required prior to an indicator oxidation study. The IAAO technique is an ideal method to help elucidate the pattern of amino acids necessary to optimize amino acid utilization and protein synthesis in infants requiring parenteral feeding.

	FOOTNOTES

 
¹ Supported by joint funds from Fresenius-Kabi AB and the Canadian Institutes of Health Research University-Industry Program; Fresenius Kabi AB also donated the parenteral amino acid and lipid solutions used in this study.

² Author disclosures: J. A. Brunton, no conflicts of interest; A. K. Shoveller, no conflicts of interest; P. B. Pencharz, no conflicts of interest; R. O. Ball, no conflicts of interest.

⁶ Abbreviations used: AAA, aromatic amino acids; IAAO, indicator amino acid oxidation; NAC, N-acetyl-L-cysteine; NP, new profile; VM, Vaminolact.

Manuscript received 4 August 2006. Initial review completed 19 September 2006. Revision accepted 19 February 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

1. Duffy B, Gunn T, Collinge J, Pencharz P. The effect of varying protein quality and energy intake on the nitrogen metabolism of parenterally fed very low birthweight (less than 1600 g) infants. Pediatr Res. 1981;15:1040–4.[Medline]

2. Brunton JA, Ball RO, Pencharz PB. Current total parenteral nutrition solutions for the neonate are inadequate. Curr Opin Clin Nutr Metab Care. 2000;3:299–304.[Medline]

3. Bertolo RF, Brunton JA, Pencharz PB, Ball RO. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am J Physiol Endocrinol Metab. 2003;284:E915–22.[Abstract/Free Full Text]

4. Bertolo RF, Chen CZ, Law G, Pencharz PB, Ball RO. Threonine requirement of neonatal piglets receiving total parenteral nutrition is considerably lower than that of piglets receiving an identical diet intragastrically. J Nutr. 1998;128:1752–9.[Abstract/Free Full Text]

5. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J Nutr. 1998;128:606–14.[Abstract/Free Full Text]

6. House JD, Pencharz PB, Ball RO. Lysine requirement of neonatal piglets receiving total parenteral nutrition as determined by oxidation of the indicator amino acid L-[1–14C]phenylalanine. Am J Clin Nutr. 1998;67:67–73.[Abstract]

7. Roberts SA, Ball RO, Moore AM, Filler RM, Pencharz PB. The effect of graded intake of glycyl-L-tyrosine on phenylalanine and tyrosine metabolism in parenterally fed neonates with an estimation of tyrosine requirement. Pediatr Res. 2001;49:111–9.[Medline]

8. House JD, Pencharz PB, Ball RO. Tyrosine kinetics and requirements during total parenteral nutrition in the neonatal piglet: the effect of glycyl-L-tyrosine supplementation. Pediatr Res. 1997;41:575–83.[Medline]

9. National Research Council. Nutrient requirements for swine, 10th edition. Washington (DC): National Academies Press, 1998.

10. Wykes LJ, House JD, Ball RO, Pencharz PB. Amino acid profile and aromatic amino acid concentration in total parenteral nutrition: effect on growth, protein metabolism and aromatic amino acid metabolism in the neonatal piglet. Clin Sci (Lond). 1994;87:75–84.[Medline]

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12. Bidlingmeyer BA, Cohen SA, Tarven TL. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr. 1984;336:93–104.[Medline]

13. House JD, Pencharz PB, Ball RO. Glutamine supplementation to TPN promotes extracellular fluid expansion in piglets. J Nutr. 1994;124:396–405.[Abstract/Free Full Text]

14. Association of Official Analytical Chemists. Official methods and analysis. 10th edition. Washington, (DC): Association of Official Analytical Chemists; 1965. pp. 15–6.

15. Brunton JA, Bertolo RFP, Pencharz PB, Ball RO. Proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal piglets. Am J Physiol. 1999;277:E223–31.[Medline]

16. Shoveller AK, Brunton JA, House JD, Pencharz PB, Ball RO. Dietary cysteine reduces the methionine requirement by an equal proportion in both parenterally and enterally fed piglets. J Nutr. 2003;133:4215–24.[Abstract/Free Full Text]

17. Hsu JW, Ball RO, Pencharz PB. Evidence that phenylalanine may not provide the full needs for aromatic amino acid needs in children. Pediatr Res. 2007;61:361–65.[Medline]

18. Shoveller AK, Brunton JA, Brand O, Pencharz PB, Ball RO. N-acetylcysteine is a highly available precursor for cysteine in the neonatal piglet receiving total parenteral nutrition. JPEN J Parenter Enteral Nutr. 2006;30:133–42.[Abstract/Free Full Text]

19. Poindexter BB, Ehrenkranz RA, Stoll BJ, Wright LL, Poole WK, OH W, Bauer CR, Papile LA, Tyson JE, et al. National Institute of Child Health and Human Development Neonatal Research Network. Parenteral glutamine supplementation does not reduce the risk of mortality or late-onset sepsis in extremely low birth weight infants. Pediatrics. 2004;113:1209–15.[Abstract/Free Full Text]

20. Tubman TR, Thompson SW, McGuire W. Glutamine supplementation to prevent morbidity and mortality in preterm infants. Cochrane Database Syst Rev. 2005;CD001457.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brunton, J. A. Articles by Ball, R. O. Search for Related Content PubMed PubMed Citation Articles by Brunton, J. A. Articles by Ball, R. O.