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Background: Parenteral nutrition-associated liver disease (PNALD) continues to cause morbidity and mortality for neonates with intestinal failure. Lipid peroxidation is one potential etiological factor. This study was designed to test if supplementing vitamin E into conventional soy-based lipid would reduce the risk of PNALD., Methods: Sixteen piglets, aged 2-5 days and weighing 1.8-2.5 kg, were randomized to parenteral nutrition (PN) with soy lipid (SO, n = 8) or the same lipid plus α-tocopherol, the most bioactive form of vitamin E (SO+E, n = 8). After 17 days, bile flow, liver chemistry, gene expression associated with bile acid metabolism, and bile acid composition were assessed. C-reactive protein (CRP) and oxidative stress markers, including plasma 8-isoprostane, were measured. All results were compared with a sow-reared control group (CON)., Results: Comparing PN-treated groups, SO vs SO+E mean bile flow (5.91 vs 5.54 µL/g liver; P = .83), serum bile acid concentration (39.2 vs 26.6 µmol/L; P = .12), and total bilirubin (35.2 vs 26.9 µmol/L; P = .56) were not different. Gene expression related to bile acid metabolism and bile composition was not different between PN groups. There was no difference in CRP (41.8 vs 36.8 µg/mL; P = .22) or in plasma 8-isoprostane (27.9 vs 26.1 pg/mL; P = .77)., Conclusions: In term neonatal piglets, supplemental vitamin E did not prevent cholestasis. Additional vitamin E was not associated with reduced inflammation or oxidative stress. The benefit of supplementing vitamin E into conventional lipid, vs adding fish oil, to prevent early onset of PNALD requires further clarification.

Background: The optimal parenteral lipid emulsion for neonates should reduce the risk of intestinal failure-associated liver disease and inflammation, while supporting growth and development. This could be best achieved by balanced content of ω-6 and ω-3 polyunsaturated fatty acids (PUFAs). Using a neonatal piglet model of parenteral nutrition (PN), we compared a 100% soy oil-based emulsion (ω-6:ω-3 PUFA: 7:1) with a mixed lipid emulsion comprising 30% soy oil, 30% medium-chain triglycerides, 25% olive oil, and 15% fish oil (ω-6:ω-3 PUFA: approximately 2.5:1) with regard to liver disease, inflammation, and fatty acid content in plasma and brain., Method: Neonatal piglets, 3-6 days old, underwent jugular catheter insertion for isonitrogenous, isocaloric PN delivery over 14 days. The IL group (n = 8) was treated with Intralipid; the ML group (n = 10) was treated with the mixed lipid (SMOFlipid). Bile flow, liver chemistry, C-reactive protein (CRP), and PUFA content in plasma phospholipids and brain were compared., Results: Compared with the IL group, ML-treated piglets had increased bile flow (P = .008) and lower total bilirubin (P = .001) and CRP (P = .023) concentrations. The ω-6 long-chain PUFA content was lower in plasma and brain for the ML group. The key ω-3 long-chain PUFA for neonatal development, docosahexaenoic acid (DHA), was not different between groups., Conclusion: The mixed lipid, having less ω-6 PUFA and more ω-3 PUFA, was able to prevent liver disease and reduce systemic inflammation in PN-fed neonatal piglets. However, this lipid did not increase plasma or brain DHA status, which would be desirable for neonatal developmental outcomes., (© 2015 American Society for Parenteral and Enteral Nutrition.)

Background: Parenteral nutrition (PN)-associated liver disease (PNALD) remains a significant cause of morbidity and mortality for neonates dependent on PN. Total fat emulsion dose and composition, particularly the large amount of ω-6 long-chain polyunsaturated fatty acids in plant oils, have been proposed as risk factors for PNALD. We hypothesized restriction of the dose of emulsion would prevent PNALD, regardless of the composition, but growth could be compromised., Methods: Using a neonatal piglet model, we compared conventional soy oil emulsion (Intralipid), dosed high (SO10, n = 8: 10 g/kg/d) and low (SO5, n = 6: 5 g/kg/d), with fish oil (Omegaven), dosed low (FO5, n = 8: 5 g/kg/d). Piglets were given isonitrogenous PN for 14 days. The normal range for all parameters was determined by measurement in equivalent aged sow-reared piglets., Results: Bile flow was lower with high-dose Intralipid, outside the normal range, while higher for the other groups (SO10, 5.4 µg/g; SO5, 8.6 µg/g; FO5, 13.4 µg/g; P = .010; normal range, 6.5-12.2 µg/g). Total body weight was low in all treatment groups (SO10, 4.4 kg; SO5, 4.5 kg; FO5, 5.0 kg; P = .038; normal range, 5.2-7.3 kg). Brain weight was not different between groups (SO10, 40.3 g; SO5, 36.0 g; FO5, 36.6 g; P = .122; normal range, 41.8-51.4 g). Corrected for body weight, brain weight was lowest in the fish oil group (SO10, 9.3 g/kg; SO5, 8.0 g/kg; FO5, 7.3 g/kg; P < .001; normal range, 5.9-9.0 g/kg)., Conclusion: Low-dose fat emulsions reduce the risk of developing PNALD. Further investigation of the risk to brain development in neonates exposed to dose restriction, particularly with fish oil, is required., (© 2014 American Society for Parenteral and Enteral Nutrition.)

Background: Because gut absorption and liver sequestration are major mechanisms of mineral homeostasis, intestinal and hepatic bypass with parenteral feeding can lead to mineral toxicities. We employed a piglet model with multiple feeding routes to identify target organs susceptible to abnormal accumulation of minerals and to determine the importance of intestinal or hepatic metabolism., Methods: Fifteen 2- to 4-day-old piglets were fed identical complete elemental diets continuously for 8 days via central venous (intravenous [IV]), gastric (intragastric [IG]), or portal (intraportal [IP]) catheters. Concentrations of calcium, phosphorus, magnesium, zinc, copper, iron, manganese, sodium, potassium, and chloride were measured in small intestinal mucosa, liver, kidney, and femur., Results: Compared with IG-fed piglets, mineral sequestration patterns in IV- and IP-fed piglets can be attributed to bypass of intestinal absorption inefficiencies, with moderate accumulations of minerals in tissues involved in mineral homeostasis. However, differences between IP and IV feeding were notable for iron, manganese, and copper, suggesting first-pass hepatic metabolism is key to homeostasis of these minerals. Moreover, manganese and copper sequestration patterns reflected that for zinc, suggesting induction of metallothionein by parenteral zinc could interfere with metabolism of other minerals., Conclusions: We conclude that parenteral delivery of minerals to neonates needs to consider not only absorption efficiency but also mineral interactions and potentially toxic accumulation in target tissues, with particular concern for iron, copper, and zinc.

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.

Controversy exists as to whether the parenterally (PN) fed human neonate is capable of synthesizing adequate cysteine from methionine if the total dietary requirement for sulfur amino acid (SAA) is provided as methionine only. The goal of this study was to gather data on whether glutathione (GSH) synthesis is maximized at a methionine intake previously shown to be adequate for protein synthesis in the PN-fed human neonate. We measured GSH concentration, fractional, and absolute synthesis rate in five PN-fed human neonates. Each neonate underwent two isotope infusion studies of 7 h duration after a 2-d adaptation to the total SAA requirement (methionine only) and again after a further 2-d adaptation to the same methionine intake supplemented with cysteine at 10 mg x kg(-1) x d(-1). Cysteine supplementation did not significantly affect GSH synthesis. These data suggest that term infants are capable of synthesizing cysteine from methionine, not only for protein but also for GSH synthesis.

Background: The threonine requirement of human neonates who receive parenteral nutrition (PN) has not been determined experimentally., Objective: The objective was to determine the parenteral threonine 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: Nine postsurgical neonates were randomly assigned to 16 threonine intakes ranging from 10 to 100 mg . kg(-1) . d(-1). Breath and urine samples were collected at baseline and at plateau for (13)CO(2) and amino acid enrichment, respectively. The mean threonine requirement was determined by applying a 2-phase linear regression crossover analysis to the measured rates of (13)CO(2) release (F(13)CO(2)) and L-[1-(13)C]phenylalanine oxidation., Results: The mean threonine parenteral requirement determined by using phenylalanine oxidation was 37.6 mg . kg(-1) . d(-1) (upper and lower confidence limits, respectively: 29.9 and 45.2 mg . kg(-1) . d(-1)) and by using F(13)CO(2) oxidation was 32.8 mg . kg(-1) . d(-1) (upper and lower confidence limits, respectively: 29.7 and 35.9 mg . kg(-1) . d(-1)). Graded intakes of threonine had no effect on phenylalanine flux., Conclusion: This is the first study to report on the threonine requirement for human neonates receiving PN. We found that the threonine requirement for postsurgical PN-fed neonates is 22-32% of the content of threonine that is presently found in commercial PN solutions (111-165 mg . kg(-1) . d(-1)).

Background: Except for tyrosine, the amino acid requirements of human neonates receiving parenteral nutrition (PN) have not been experimentally derived., Objectives: The objectives were to determine the total sulfur amino acid (TSAA) requirement (methionine in the absence of cysteine) of postsurgical, PN-fed human neonates by using the indicator amino acid oxidation (IAAO) technique with L-[1-(13)C]phenylalanine as the indicator., Design: Fifteen postsurgical neonates were randomly assigned to receive 1 of 18 methionine intakes ranging from 10 to 120 mg x kg(-1) x d(-1), delivered in a customized, cysteine-free amino acid solution. Breath and urine samples were collected for the measurement of (13)CO(2) and amino acid enrichment. Blood samples were collected at baseline and after the test methionine infusion for the measurement of plasma methionine, homocysteine, cystathionine, and cysteine concentrations., Results: Breakpoint analysis determined the mean TSAA requirements to be 47.4 (95% CI: 38.7, 56.1) and 49.0 (95% CI: 39.9, 58.0) mg x kg(-1) x d(-1) with the use of oxidation and F(13)CO(2), respectively., Conclusions: This is the first study to report the TSAA requirement of postsurgical, PN-fed human neonates. The estimated methionine requirement expressed as a proportion of the methionine content of current commercial pediatric PN solutions was 90% (range: 48-90%) of that found in the lowest methionine-containing PN solution.

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-(14)C]-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 approximately 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.

Background: Cysteine (CYS) is accepted as an indispensable amino acid for infants receiving parenteral nutrition (PN), and CYS is unstable in solution. Thus, developing a method to supply CYS in PN for neonates is needed. N-acetyl-L-cysteine (NAC) is stable in solution and safe for use in humans; therefore, NAC may be a means of supplying parenteral CYS., Methods: We determined the bioavailability of NAC in intravenously (IV)-fed piglets randomized to 1 of 4 diet treatments, each supplying 0.3 g/kg/d methionine and either 0.2 g/kg/d CYS (CON), 0 NAC (zeroNAC), 0.13 NAC (lowNAC), or 0.27 g/kg/d NAC (highNAC). Piglets (2 days old; 1.8 kg, n = 20) were surgically implanted with femoral and jugular catheters. On day 3 postsurgery, test diets were initiated and continued until day 8. Piglets were weighed daily. Blood was sampled 6 hours before test diet initiation and at 0, 6, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, and 120 hours. Urine was collected on ice in 24-hour sample periods., Results: Total mean weight gain was not different between groups; however, average daily gain in the zeroNAC and lowNAC groups declined significantly (p < .05) over the 5-day treatment period. Nitrogen retention was similar between the CON and highNAC groups, both were higher than the lowNAC group, and the zeroNAC treatment produced the lowest nitrogen retention. NAC percent retention was not different between lowNAC and highNAC and was 85.4% and 82.6%, respectively. Plasma NAC was higher in highNAC than lowNAC (p < .05)., Conclusions: These data demonstrate that NAC is available as a precursor for CYS to support growth and protein (nitrogen) accretion in piglets administered a parenteral solution.

The requirements for total branched-chain amino acids (BCAA), isoleucine, leucine and valine, in neonatal piglets receiving parenteral and enteral nutrition was determined recently. The optimum ratio among BCAA during different routes of feeding is not yet known. In this study, the ratio of BCAA during parenteral and enteral feeding was tested using the indicator amino acid oxidation (IAAO) technique. Male Yorkshire piglets (n=24) received amino acid-based diets containing adequate nutrients for 5 d. Phenylalanine oxidation and kinetics were determined from a 4-h primed, constant infusion of L-[1-14C]-phenylalanine on d 6 and 8. On d 6, all piglets received a BCAA diet which met 75% of the total BCAA requirement, based on our previous research, with a ratio of 1:1.8:1.2 of isoleucine/leucine/valine. On d 8, the piglets were randomly assigned to receive one of the 3 test diets supplemented with isoleucine (+isoleucine), leucine (+leucine) or valine (+valine) to meet 100% of requirement, with the remaining two BCAA at 75% of requirement. The difference in phenylalanine oxidation (% of dose) between d 6 and 8 was used as an indicator of BCAA adequacy. In enterally fed piglets, the change in the percentage of the dose oxidized was minimal for all 3 test diets (mean=1.15%). In parenterally fed piglets, the difference in phenylalanine oxidation (% of dose) between d 6 and 8 was +isoleucine (12.6%), +leucine (2%) and +valine (6.6%). The ratio of 1:1.8:1.2 of isoleucine/leucine/valine is appropriate for enteral feeding, but during parenteral feeding, isoleucine was first limiting and valine was second limiting.

The sulfur amino acids (SAA), methionine and cysteine, are normally supplied in a 50:50 ratio in the oral diet of pigs. In contrast, cysteine is not included in any appreciable amounts in parenteral solutions due to its instability in solution. Cysteine can replace part of the methionine requirement, but is not required when methionine is supplied at a level that meets the entire SAA requirement. However, the role of the gut on cysteine sparing has not been investigated. In the present study, the enteral and parenteral methionine requirement was determined, with excess dietary cysteine, by using the indicator amino acid oxidation (IAAO) technique. Piglets [n = 28, 2 d, 1.65 +/- 0.014 kg (SE)] were fed elemental diets containing adequate energy, phenylalanine and excess tyrosine, with varied methionine concentrations and excess cysteine [0.55 g/(kg. d)]. Diets were infused continuously via intravenous (parenteral) or gastric (enteral) catheters. Phenylalanine oxidation was determined during a primed, constant infusion of L-[1-(14)C]-phenylalanine, by measuring expired (14)CO(2) and plasma specific radioactivity (SRA) of phenylalanine. For both the parenteral and enteral groups, phenylalanine oxidation (% of dose) decreased linearly (P < 0.01) as methionine intake increased and then became low and unchanging. Using breakpoint analysis, the methionine requirement was estimated to be 0.25 and 0.18 g/(kg. d) for enteral and parenteral feeding, respectively. These data show that the parenteral methionine requirement is approximately 70% of the enteral requirement when measured in the presence of excess dietary cysteine (P < 0.05). A comparison with our previous studies in which methionine was the only source of sulfur amino acids shows that the addition of dietary cysteine reduces the methionine requirement by approximately 40% in both enterally and parenterally fed neonatal piglets. Therefore, dietary cysteine is equally effective in sparing dietary methionine whether fed enterally or parenterally.

The requirements for the sulfur amino acids (SAA), methionine (Met) and cysteine (Cys), have seldom been determined in neonates and to our knowledge have not previously been determined directly in parenterally fed neonates. The sulfur amino acids are catabolized largely in the liver and kidney, and their metabolism by the gut has been studied less frequently. In the present research, the enteral and parenteral Met requirement was determined, without dietary Cys, using the indicator amino acid oxidation (IAAO) technique. Piglets [n = 32, 2 d old, 1.66 +/- 0.13 kg (SD)] received elemental diets containing adequate energy, phenylalanine (Phe) and excess tyrosine, with varied Met concentrations and no Cys. Diets were infused continuously via intravenous or intragastric catheters. Phenylalanine oxidation was determined during a primed, constant infusion of L-[1-(14)C]-Phe, by measuring expired (14)CO(2) and plasma specific radioactivity of Phe. For both parenteral and enteral groups, Phe oxidation (% of dose) decreased linearly (P < 0.01) as Met intake increased, then became low and unchanging. Using breakpoint analysis, the Met requirement was estimated to be 0.42 and 0.29 g/(kg. d) for enteral and parenteral feeding, respectively. Breakpoint analysis using absolute phenylalanine oxidation [ micro mol/(kg. h)] resulted in an estimation of the Met requirement of 0.44 and 0.26 g/(kg. d) for enteral and parenteral feeding, respectively. These data show that the parenteral Met requirement is approximately 69% of the enteral requirement and suggest that extraction of SAA by first-pass splanchnic metabolism may be responsible for this difference.

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-(13)C-phenylalanine tracer and linear regression cross-over analysis that identified a break point in the response of label appearance in breath CO(2) (F(13)CO(2)) 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.

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.

Background: The amino acid requirement profile for infants is different than that for adults and needs to be established; this profile also is different for infants receiving total parenteral nutrition. We used the neonatal piglet as a model for the infant to address (1) the metabolic and physiologic changes due to route of feeding and (2) the adequacy of the amino acid pattern in a pediatric elemental diet., Methods: Diets differed only in their amino acid pattern (modified human milk [MHM] and a commercial pediatric elemental diet [PED]) and were fed continuously for 8 days. Control piglets were fed MHM diet via gastric catheters (IG) and were compared with pigs fed MHM diet via venous catheters (IV) or to pigs IG-fed PED., Results: MHM-IV piglets experienced enlarged livers and gut atrophy, and lower nitrogen retention and body protein content. Higher glutamine (and lower glutamate) in PED-IG, compared with MHM-IG, produced no apparent advantage with respect to gut growth or histology. Proline, histidine, and lysine may have been limiting, and isoleucine and valine excessive, in the PED-IG diet as indicated by plasma concentrations, compared with sow-fed piglets; however, imbalances in the amino acid profile were not excessive because nitrogen retention was not different between MHM-IG or PED-IG pigs., Conclusions: Therefore, the amino acid profile of MHM needs to be modified to improve nitrogen retention during parenteral feeding and the profile of oral PED could be improved to normalize plasma amino acid concentrations.

Kinetics of three amino acids with different sites and characteristics of metabolic regulation were studied in low-birth-weight infants during enteral and parenteral feeding regimens typical of clinical practice. Primed constant infusions of [15N]glycine, L-[1-13C]leucine, and L-[1-13C]phenylalanine were administered simultaneously by the same route as the feeding, with isotope enrichment measured in urine over 12 h. The effect of feeding regimen was specific to each amino acid (mean +/- SD): glycine flux was lower during parenteral feeding (470 +/- 15 vs 561 +/- 69 mumol.kg-1.h-1, P less than 0.05), leucine flux was unaffected (360 +/- 77 vs 388 +/- 78 mumol.kg-1.h-1), and phenylalanine flux was higher (106 +/- 29 vs 56 +/- 6 mumol.kg-1.h-1, P less than 0.01). Kinetics were influenced by the interaction of several factors, including amino acid intake and routes of feeding and tracer administration. Glycine was most affected by route of feeding and phenylalanine was most affected by intake whereas leucine was little affected. Estimates of whole-body protein turnover calculated from leucine and phenylalanine were different; thus calculations of protein turnover from kinetics of a single amino acid should be interpreted with caution.

The validity of measuring isotopic enrichment of amino acids in urine was tested in neonates during different feeding regimens and tracer administration protocols, and with different amino acid tracers. Twenty infants, fed enterally or parenterally, were given primed, constant infusions of 15N-glycine, L-[1-13C]leucine, and L-[1-13C]phenylalanine. Urine was collected every 2 h for 16 h. Blood was sampled only when required clinically. At baseline, enrichment in plasma and urine did not differ for any amino acid. At isotopic steady state, leucine enrichment was similar in plasma and urine, while the agreement was slightly weaker for glycine and phenylalanine, probably because of problems inherent in drawing blood samples from neonates. Study protocol did not affect the agreement. Since urine collection involves minimal invasive procedures, this approach could facilitate more indepth tracer studies in infants and children.