Clinical Nutrition 33 (2014) 982e990Contents lists available at ScienceDirect Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu Randomized control trials Adaptive regulation of amino acid metabolism on early parenteral lipid and high-dose amino acid administration in VLBW infants e A randomized, controlled trial Hester Vlaardingerbroek a, Jorine A. Roelants a, Denise Rook a, Kristien Dorst a, Henk Schierbeek a, b, c, Andras Vermes d, Marijn J. Vermeulen a, Johannes B. van Goudoever a, b, c, *, Chris H.P. van den Akker a a Department of Pediatrics, Division of Neonatology, Erasmus MC e Sophia Children’s Hospital, c/o Room SP3433, P.O. Box 2060, 3000 CB Rotterdam, The Netherlands b Department of Pediatrics, Emma Children’s Hospital e AMC, c/o Room H7-282, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands c Department of Pediatrics, VU University Medical Center, c/o Room ZH 9D11, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands d Hospital Pharmacy, Erasmus MC, P.O. Box 2060, 3000 CB Rotterdam, The Netherlands a r t i c l e i n f o s u m m a r y Article history: Received 25 June 2013 Accepted 3 January 2014 Background & aims: An anabolic state can be achieved upon intravenous amino acid administration during the immediate postnatal phase despite a low energy intake. The optimal dosing of amino acid and energy intake has yet to be established. The aim was to quantify the efficacy of early initiation of parenteral lipids and increased amounts of amino acids on metabolism and protein accretion in very low birth weight infants. Methods: 28 very low birth weight infants were randomized to receive parenteral nutrition with glucose and either 2.4 g amino acids/(kg$d) (control group), 2.4 g amino acids/(kg$d) plus 2e3 g lipid/(kg$d) (AA þ lipid group), or 3.6 g amino acids/(kg$d) plus 2e3 g lipid/(kg$d) (high AA þ lipid group) from birth onward. On postnatal day 2, we performed a stable isotope study with [1-13C]phenylalanine, [ring-D4] tyrosine, [U-13C6,15N]leucine, and [methyl-D3]a-ketoisocaproic acid to quantify intermediate amino acid metabolism. Results: The addition of lipids only had no effect on phenylalanine metabolism, whereas the addition of both lipids and additional amino acids increased the amount of phenylalanine used for protein synthesis. In addition, high amino acid intake significantly increased the rate of hydroxylation of phenylalanine to tyrosine, increasing the availability of tyrosine for protein synthesis. However, it also increased urea concentrations. Increasing energy intake from 40 to 60 kcal/(kg$d) did not increase protein efficiency as measured by phenylalanine kinetics. The leucine data were difficult to interpret due to the wide range of results and inconsistency in the data between the phenylalanine and leucine models. Conclusions: High amino acid and energy intakes from birth onwards result in a more anabolic state in very low birth weight infants, but at the expense of higher urea concentrations, which reflects a higher amino acid oxidation. Long-term outcome data should reveal whether this policy deserves routine implementation. This trial was registered at www.trialregister.nl, trial number NTR1445, name Nutritional Intervention for Preterm Infants-2. Ó 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. Keywords: Metabolism Preterm infant Parenteral nutrition Growth Stable isotopes 1. Introduction * Corresponding author. Emma Children’s Hospital e AMC, c/o Room H7-282, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands. Tel.: þ31 20 5667729; fax: þ31 20 5669683. E-mail addresses:
[email protected],
[email protected] (J.B. van Goudoever). The neonatal period is characterized by protein accretion and rapid growth. In response to protein administration, adults1,2 and term infants3,4 decrease proteolysis and increase their endogenous glucose production rate.5 However, several studies have shown that fetuses and preterm infants increase protein synthesis rather than 0261-5614/$ e see front matter Ó 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. http://dx.doi.org/10.1016/j.clnu.2014.01.002 2. In addition. the infants received glucose (at least 4. The envelopes were created by a research pharmacist who was not involved in clinical care and were based on a computer-generated block randomization list with variable block sizes that was provided by a statistician. Rotterdam.3. Within 6 h after birth. lipids were started immediately after randomization (always within 6 h after birth) (starting dose 2 g/(kg$d). the lipid source was ignored in the final analyses. The three groups received the same amino acid solution: Primene 10% (Baxter. Leucine has a unique role in protein synthesis in skeletal muscle. At higher intakes. 2. all of the technicians were blinded for study group randomization throughout the study and the analyses. minimal enteral feeding was initiated on the day of birth and feeding progressed to full enteral nutrition in the following days according to local protocol. the Netherlands. Infants were excluded in cases of congenital anomalies (including chromosome defects). number of prenatal steroid doses (0.2. Whenever possible. or an early lipid administration plus high amino acid intake (high AA þ lipid) group.4 g/(kg$d) of amino acids (always on stock on the ward) as part of standard clinical care. the attending physician included infants in the study by opening a sealed opaque randomization envelope stratified by weight (<1000 g versus 1000e1499 g) and gender. The main sites of hydroxylation are the liver and kidneys. 2. birth weight < 1500 g) infants by means of stable isotope modeling using [1-13C]phenylalanine and [ring-D4]tyrosine and a model using [U-13C6. We hypothesized that additional amino acids in combination with the administration of lipids from birth onward would augment protein accretion further.H.3. or 2). can act independently as a nutrient signal. 2.1. The study was conducted at the neonatal intensive care unit of the Erasmus MC e Sophia Children’s Hospital. Materials and methods 983 present study were a subset of those included in our larger study10 to determine the safety and efficacy of early lipid initiation with or without additional amino acids from birth onward. the experimental parenteral nutrition was substituted for the infants in the AA þ lipid and high AA þ lipid groups.11 gestational age based on best obstetric measurement (ultrasound in early pregnancy or last menstrual period). leading to enhanced net protein accretion.15N] leucine and [methyl-D3]a-ketoisocaproic acid. birth weight and birth weight z-score. and stimulates protein synthesis via the activation of translational initiation factors. In this study. increased next day to 3 g/(kg$d)). The study protocol was approved by the institutional medical ethical review board. high-dose amino acids (3. Immediately after randomization to one of the three study groups.6 g/(kg$d) from birth onward) and lipids (starting dose 2 g/(kg$d) from birth onward. sufficient energy should be provided to optimize this process. Infants were randomized to either a control group (no lipid during the first two days). with the greater beneficial effect of energy on protein accretion occurring at intakes of <50e60 kcal/ (kg$d). Because the type of lipid had no effect on amino acid kinetics. however. on amino acid metabolism in very low birth weight (VLBW. early lipid administration may increase protein synthesis and/or decrease protein breakdown. oxidized to carbon dioxide. Germany). in combination with various levels of amino acid administration. 1. renal or hepatic disorders. Tyrosine can be synthesized into proteins.2. Subjects 2. 2. Study design and tracer protocol We performed a randomized controlled trial on the initiation of parenteral nutrition to preterm infants in the early postnatal phase from June 2009 to January 2012. leucine stimulates insulin release and tissue sensitivity.0 mg/(kg$min)) and 2. metabolic diseases. The relationship between energy supply and protein synthesis appears to be curvilinear. The infants in the control group received Intralipid 20%. Control group The infants in the control group continued to receive only glucose and amino acids (2.6e8 Because protein synthesis is an energy-demanding process. Because of the provision of additional energy. The subjects were inborn VLBW infants (birth weight < 1500 g) with a central venous and arterial catheter inserted for clinical purposes and written informed consent from the parents. / Clinical Nutrition 33 (2014) 982e990 Abbreviations AA CRIB ECF GC/MS GMP KIC Leu MPE Phe Q RP S TG TTR VLBW amino acid Critical Risk Index for Babies ethyl chloroformate gas chromatography mass spectrometer good manufacturing practice a-ketoisocaproic acid leucine mole percent excess phenylalanine flux rate of amino acid release from protein rate of utilization of amino acid for protein synthesis triacylglycerol tracer-tracee ratio very low birth weight suppress protein breakdown in response to protein administration. increased next day to 3 g/(kg$d)). an early lipid administration (AA þ lipid) group. or endocrine. however.1.3. or metabolized into several neurotransmitters and the skin pigment melanin. The study group randomization was open after inclusion for logistical reasons.2. in addition to glucose from birth onward. the amount of supplied amino acids is more highly correlated with anabolism. we analyzed the effect of the administration of lipids (as an energy source) from birth onward.4 g/(kg$d)) during the first two days of life. Vlaardingerbroek et al. High AA þ lipid group The infants in the high AA þ lipid group received. The infants included in the The parameters recorded at baseline were gender.9 The optimal glucose and lipid intakes that maximize protein accretion and growth in the early neonatal phase have not yet been determined. and severity of . 2.2. The major routes of disposal of phenylalanine are protein synthesis and hydroxylation to tyrosine. AA þ lipid group The infants in the AA þ lipid group received glucose and amino acids similarly to those in the control group (2. The Netherlands). 2. The infants in the intervention groups were randomized to receive Intralipid 20% or SMOFlipid 20% (both Fresenius Kabi. Nutritional intervention As soon as intravenous access was obtained after birth.4 g/(kg$d)). plasma urea concentration 10 mmol/L or . After 6. / Clinical Nutrition 33 (2014) 982e990 illness at entry in the study based on the Apgar score at 5 min and the Critical Risk Index for Babies (CRIB) score. the release of label from body protein is small in relation to the rate of isotope infusion.13 Briefly.984 H. Middelburg. and [methyl-D3]aketoisocaproic acid ([D3]KIC) (all 94% atom enriched and tested for sterility and pyrogenicity) were from Cambridge Isotope Laboratories (Andover. Immediately before infusion.9% saline by the hospital pharmacy. 0.12 The actual daily nutritional intake (parenteral and enteral) was recorded throughout the study. Oss. The total amount of blood sampled did not exceed 5% of the patient’s estimated total blood volume (75 mL/kg). and 8 h of infusion. L-[ring-D4]tyrosine. 2) a labeled molecule is not distinguished from an unlabeled molecule.2 mL of the supernatant was added to the new vial and evaporated using a SpeedVac (GeneVac miVac. and 0. 3500 g). The differences between groups were analyzed using the c2-test. we used the model developed by Clarke and Bier14 with the enhancement proposed by Thompson et al.5. i is the tracer infusion rate in mmol/(kg$h). Analytical methods Plasma amino acid enrichments were analyzed as follows. 2.2 mL of prewashed Dowex suspension (Ag 50W-X8 Hþ.2 mL of ammonia 6N. and sterile and pyrogen-free status of the product. or ManneWhitney U test. The samples were collected in EDTA-containing tubes and immediately placed on melting ice and centrifuged (10 min. Somers. The Netherlands) on a VF-17ms. thoroughly shaken. L-[U-13C6. Kruskale Wallis test.6. phenylenediamine. The significance level was set to p < 0. The supernatant was discarded and the pellet was washed twice with 1 mL of H2O. After centrifugation. To isolate the amino acids. 7. the [D3]KIC powder was dissolved in the amino acid tracer solution. and centrifuged at 4000 rpm for 1 min.V. and analyzed with an MSD 5975C Agilent gas chromatography mass spectrometer (GC/MS) (Agilent Technologies. Hydroxylation ¼ Qtyr * (E plasma tyr 13C/E plasma phe) * (Qphe/(i phe þ Q phe)) Non-oxidative phenylalanine disposal (reflecting synthesis) ¼ Q phe hydroxylation Phenylalanine release from protein (reflecting breakdown) ¼ Qphe phe intake Tyrosine release from protein (reflecting breakdown) ¼ Qtyr tyr intake hydroxylation Phe and tyr intake were calculated from the molar content of parenteral and enteral intake (the latter was only a very small amount).15N]leucine (12 mmol/(kg$h)). therefore. birth weight. 200e400 mesh) was added to the acidified samples.2 mm filter) and sterilized according to GMP guidelines.. including rates of hydroxylation to tyrosine. For additional power. 30 m 0.1. The major reasons were adjustments of nutrition according to the local protocol (i. 200 mL of the supernatant was transferred to a vial. [ringD4]tyrosine (2 mmol/(kg$h)). The infants received a primed continuous infusion of [1-13C]phenylalanine (6 mmol/(kg$h)). we included 7e12 infants per group. The priming doses were equivalent to an hourly dose. NY). redissolved in 50 mL of ethyl acetate. England). A 50- mL aliquot of plasma was acidified with 20 mL of hydrochloride (pH < 3). MA). KIC enrichments were measured as described previously..05 and power of 0. and birth weight z-score. Vlaardingerbroek et al. samples were derivatized with silylquinoxalinol. All statistical analyses were performed using SPSS Version 20. all in (mmol/(kg$h)): Qphe ¼ i$phe * ((E infusate phe/E plasma phe) 1) Qtyr ¼ i$tyr * ((E infusate tyr/E plasma tyr) 1) where Q indicates flux in mmol/(kg$h).e. given an expected decrease in leucine oxidation of 15 mmol/(kg$h) and a standard deviation of 10 mmol/(kg$h).0 (IBM SPSS Statistics. Spearman correlations were calculated between the two isotope models.25 mm ID capillary column (Varian Inc.80. 2. The original vial was rinsed with 0.. Results 3. Demographic and clinical characteristics We included 28 infants (7e12 infants per nutritional group) in the final analyses (Table 1). and the solution was filtered (0.15 The following calculations were used.4. Amstelveen. 3. Separate vials with a precisely weighed dry powder form of [D3]KIC were aseptically produced by the hospital pharmacy according to GMP guidelines. [U-13C6. the Netherlands). 400 mL of the supernatant was added to the first portion. the recycling of label is negligible in comparison with the total amount of unlabeled substrate entering the mixing pool. The samples were redissolved in 200 mL of H2O and derivatized with ethyl chloroformate (ECF) by the addition of 140 mL of ethanol/pyridine (4:1) and 20 mL of ECF. Not all of the infants were eligible for further analysis following the randomization to the three nutritional regimens. The amino acid isotopes were dissolved in 0.5 mL of ammonia 6N and transferred to a new vial. and [D3]KIC (6 mmol/(kg$h)) for 8 h using a Perfusor fm infusion pump (BjBraun Medical B. the amino acids were extracted from the Dowex pellet with 0. and E is the tracer enrichment. Phenylalanine balance ¼ non-oxidative phenylalanine disposal phenylalanine release from protein Leucine calculations are presented in the Addendum. Tests were performed to ensure the correct identity. Statistics A power calculation based on leucine oxidation showed that.5 mL) was sampled from the arterial catheter.05.. blood (0. Calculations For the calculation of whole body phenylalanine and tyrosine kinetics. GeneVac Ltd. concentration. The extraction step was repeated. The combined solutions were evaporated under an N2 stream at room temperature. Ipswich. Linear regression analyses were used to correct for the potential effects of gestational age. as appropriate. The plasma was stored at 80 C until analysis. The samples were left at room temperature for 5 min and then extracted with 400 mL of hexane/dichloromethane/ECF (50:50:1). Good manufacturing practice (GMP) tested L-[1-13C]phenylalanine. 21 infants (7 in each group) were needed to detect a significant difference with a of 0. We made the following assumptions regarding the tracer model: 1) during the course of the experiment. 2. and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and analyzed by GC/MS. The results are expressed as median (IQR). The Netherlands). the second time. After centrifugation.15N]leucine. S1 (supplemental data). A stable isotope study was performed on the second day of life. The CONSORT flow is depicted in Fig. weeks Birth weight.5 (2. and tyr release from protein (RPtyr).7e8. 3. Phenylalanine balance was significantly higher in the high AA þ lipid group (Fig. IQR in parenthesis.3 (4.8 (76. 4.6 (4.2)a 40. g/(kg$d) Parenteral þ Enteral amino acid and protein intake. N Parenteral glucose intake.2) 3.5th percentiles.0) 2. g Birth weight z-score (13) Prenatal steroids (% 0/1/2 doses) Apgar score at 5 min CRIB score (14) Control group AA þ lipid group High AA þ lipid group 7 (6/1) 29.4e27.8 (3. Discussion Our results demonstrated that a high amino acid intake in combination with a high energy intake (lipids) increased phenylalanine hydroxylation and the use of phenylalanine for protein synthesis in preterm infants.7)a 4.4e2. Phenylalanine efficiency was calculated as the net phenylalanine balance divided by the phenylalanine intake and was not Table 2 Actual nutritional intake on day two of life.7) 2.8e50.0) 980 (670e1140) 3. CRIB. amino acid. Per definition.0) 12 4.2 (2.3 (2.4 g amino acids/(kg$d)).2) 0/0/100 800 (669e918) 0. / Clinical Nutrition 33 (2014) 982e990 985 Table 1 Clinical characteristics.6e122.6)a.b 25.4) 2.0) 102 (117. ml/(kg$d) Parenteral þ Enteral non-protein energy intake.g.6)a (57.6 (3.8e3. mmol/(kg$h) Total tyrosine intake.4)a 5. Correction for gestational age and birth weight did not affect the differences in phenylalanine metabolism. phe release from protein (RPphe). fluid restriction.5e35. Regression analyses showed a significant effect of birth weight for gestational age z-score on phenylalanine hydroxylation to tyrosine. mmol/(kg$h) Total leucine intake.9 (25.8) 6.5th and 97. Phenylalanine and tyrosine kinetics Phenylalanine and tyrosine enrichments at steady state are presented in Table 3 (Supplementary data).6e6.7) 81.1 (76. whereas the release of phenylalanine from protein (reflecting proteolysis) did not differ among the groups. g/(kg$d) Enteral intake.7 (3.1e27.6e3. N (male/female) Gestational age. kcal/(kg$d) Control group AA þ lipid group High AA þ lipid group 7 5. and 2. On the other hand.3e11.6) 12 (7/5) 26. AA.0e0.1 (2. (B) phe balance. Vlaardingerbroek et al.3) 3..9 (25.5e3.7) 78.6 g amino acids/(kg$d)) compared to the control group and the AA þ lipid group (2.7 59. Tracer enrichments were analyzed in plasma by means of GC/MS.7)a. unless otherwise indicated.6e3.b 5. triacylglycerol (TG) concentration 3 mmol/L) before the start or during the isotope study and clinical contra-indications for the isotope study (e.9 (2. Fig. high AA þ lipids (67% (62e77%)) group. mg/(kg$min) Parenteral amino acid intake. Therefore.4 (4. 3. hydroxylation (Hydr).1 (4. The phenylalanine and tyrosine tracers indicate whole body metabolism as all are .4 (6.0e5. phenylalanine hydroxylation increased in the high amino acid group. IQR. AA þ lipid (68% (65e75%)).2e2.7) 9 (4/5) 26.5) 2. different between groups: control group (82% (56e82%)).2. One may speculate that infants in this study received sufficient energy to facilitate the energy cost of protein synthesis. Boxes and whiskers indicate the medians.5 to 0. Phenylalanine (phe) and tyrosine (tyr) kinetics on day two of life.7e75.4e29. phenylalanine hydroxylation to tyrosine and utilization of phenylalanine for protein synthesis (Fig. the hydroxylation rate was higher for infants with a higher z-score.4) 62.6)a Data are presented as median.4 (27.6) 39. The addition of only lipids to standard-dose amino acids did not affect metabolism. a Significantly different from control group (ManneWhitney U test). Leucine and a-KIC kinetics The leucine model resulted in a wide range of outcomes and these data were difficult to interpret due to various reasons (see Discussion).7 (4.5 to 1.3) 9. In the high AA þ lipid group. The phenylalanine model versus the leucine model In this complicated stable isotope study we tried to combine several aspects of intermediate metabolism.4e61.2e5. nor after early lipid administration.4 (2.3e28. Critical Risk Index for Babies.9e81.2 to 0.5) 0/33/67 8 (4e9) 4 (1e8) 9 (7e9) 4 (3e8) 8 (6e9) 5 (2e9) Data are presented as median. IQR in parenthesis. phe utilization for protein synthesis (Sphe).6 (3.1.5 (4.4e3.3 (39. AA.b 3.8) 0.5e3. amino acid. (A) Phe intake.3e12. the actual intake on day 2 of life was in accordance with the intended study intakes (Table 2).6) 2.3e2.3) 9 4.0 (25.6 (54. restriction of blood withdrawals).H.9) 27. This might be due to the relative low numbers.4) 3. Phenylalanine balances were highest in the high AA þ lipid group (3.7 (30.4 (4. 4.8e111. 1. The release of phenylalanine from proteins (reflecting proteolysis) did not change after higher amino acid intake.7 (25. 1(A)) were significantly higher than in the groups with the standard amino acid intake.1e40. 1(B)). the leucine and a-KIC kinetics and comparisons between the phenylalanine and leucine model are presented in the Addendum. Amino acid kinetics were measured by primed continuous infusion of L-[1-13C]phenylalanine and L-[ring-D4]tyrosine.0 (0. mmol/(kg$h) Parenteral lipid intake.3e28.9 (3. g/(kg$d) Total phenylalanine intake.5 (2.3.01) 0/22/78 755 (642e975) 1.8)a. b Significantly different from AA þ lipid group (ManneWhitney U test). Phenylalanine metabolism did not differ between the control group and the AA þ lipid group.4e8. concentrations are for large part influenced by hydration status and kidney function.2 an additional energy supply results in reduced proteolysis. The transition point from ‘preterm’ metabolism to ‘mature’ metabolism most likely lies closer to the term age. We anticipated that the majority of infants included in our study were ventilated with noninvasive methods. However. or most likely is different resulting in higher oxidation rates and thus higher urea synthesis rates at high protein intake. 4. physicians measured urea concentrations at day 1. such that collection of expired air without mixing with surrounding air was not possible. since many assumptions were made and this model has not been used in published studies ever since. the situation gets more complex by the increasing amount of enteral nutrition which can be human milk or formula with different amino acid intakes.22 and in second and third trimester human fetuses. the preset guideline to lower amino acid intake based on urea concentrations is rather arbitrary and besides. Vlaardingerbroek et al.2. This tracer study was primarily designed to quantify the adaptive mechanism in intermediate metabolism due to different nutritional intakes.0e3.21. This was comparable to the relative number of adjustments in the larger study. the urea thresholds set in our study were arbitrary and the validity of urea as a monitoring tool for amino acid tolerance is questionable and is discussed in more detail in our previous study. before urea concentrations were available. If adaptations in amino acid dosage were necessary based on urea concentration above 10 mmol/L. e. in 48% of infants in the control group.10 However. It has been suggested that high rates of proteolysis in combination with even higher rates of protein synthesis are required to support the high remodeling and growth rates in preterm infants8 and that the primary mechanism for protein accretion in parenterally fed preterm infants is the stimulation of protein synthesis rather than the suppression of proteolysis. rather than solely by amino acid oxidation. 4. Indeed. isotope studies were started early in the morning.6 g amino acids/kg per day.3. infants in the control groups received lipids as well. Of course. the plasma tyrosine concentrations in these infants10 were lower than the concentrations observed in healthy term breast-fed infants20 but were comparable to previously reported ranges in preterm infants7. the endogenous tyrosine synthesis rates (phenylalanine hydroxylation) were twice as high as tyrosine intake. In some infants.17. since nutritional differences between groups were largest at that day.6e8 our results demonstrate that preterm infants are resistant to the suppression of proteolysis in response to parenteral amino acids.23 Sufficient hydroxylation rates are important because parenteral amino acid solutions have a low tyrosine content due to low solubility.18 In older children and adults. we also must realize that results from a single amino acid do not necessarily pertain to another amino acid or to protein metabolism in general. aiming at similar intakes on day four of life.4 g amino acids/kg while the larger study gives the effects on overall outcomes of the adjusted intakes. the applicability of our results to the whole population of VLBW infants might be slightly hampered by the few hours-difference in postponement of adjustments to the amino acid dose. the incidence of hyperuremia was significantly higher in the high AA þ lipid group than in the control and AA þ lipid groups. Further studies should aim at measuring intermediate metabolism at later life.6 versus 2. in those infants metabolism might be different. From the third day of life onwards. which included a total of 144 infants (47e49 infants per group).10 On the second day of life.0 mmol/ (kg$h)). This is also illustrated by the wide range of results in the leucine model and the inconsistency in the data between the phenylalanine and leucine models. Tracer data versus nitrogen balance data This isotope study was part of a larger trial. this model was developed in an animal study and was performed using radioactive tracers instead of stable isotopes.19 Still. the total tyrosine availability (intake plus synthesis from hydroxylation) was approximately two-thirds of the molar phenylalanine tissue deposition (phenylalanine balance). and 7 in the high AA þ lipid group. the number of infants in whom amino acid intake was reduced after the isotope measurements were finished was 2 in the control group. it is of interest whether the differences observed on the second day of life expand to a longer period. There is no other way to dispose phenylalanine than through protein synthesis or hydroxylation to tyrosine. in practice an intake of 2. Hence. The final decision to postpone adaptations to amino acid dose was made by the attending physician.4. Using leucine as a tracer of amino acid metabolism can be more problematic as it is in need CO2 sampling which can be problematic. to determine the safety and efficacy of early lipid initiation with normal or high-dose amino acids from birth onward. However.3. the regression analyses in this study did not show an effect of gestational age on amino acid metabolism.8 These rates are also comparable to the hydroxylation rates in healthy human fetuses at term gestation. although the median gestational ages were somewhat lower in the intervention groups than in the control group. In addition. In the isotope study.4 mmol/(kg$h)) than in the AA þ lipid group (9. one needs to correct for CO2 retention in the body. we cannot exclude that the higher urea concentrations in the high AA þ lipid group may be . This value is equivalent to the molar ratio of 0. Therefore. However. / Clinical Nutrition 33 (2014) 982e990 measured in steady state. 4 and 6.g. The higher hydroxylation rates in response to higher amino acid intake are in agreement with those of previous studies. these infants were not included in this substudy. the isotope study gives the effects on metabolism of administration of exact 3. The validity might be questioned in our patient group. these adaptations were postponed several hours until the end of the tracer study.1. However. As stated before. In the larger trial. we used the open twopool leucine model of Helland et al. and to determine the rate of anabolism shortly following birth. if possible.16 However.1 mmol/(kg$h)) or the control group (5. Effects of nutrition on metabolism in preterm infants In agreement with the findings of several previous studies. Disposal of phenylalanine by hydroxylation was significantly higher in the high AA þ lipid group (13. Term infants generally increase protein synthesis rates in response to the administration of additional energy as well. In fact. we presented the leucine data in the Addendum and based the conclusions of our manuscript on the phenylalanine data only.7 Despite the difference in metabolic response between preterm infants and more mature infants. However. It remains to be determined whether hydroxylation rates are not increased further because of impaired enzyme activity or insufficient phenylalanine intake.64 extracted in tissue proteins of deceased fetuses. and 39% of infants in the AA þ lipid group.10 In our study urea concentrations were measured routinely on day 2. resulting in lowering the amino acid dose as per protocol in 81% of infants in the high AA þ lipid group.24 These findings suggest that hydroxylation rates are marginally sufficient to cope with neonatal demands for protein synthesis. Demographics at baseline were not different between infants in the isotope study and the larger study. In order to measure leucine oxidation and transamination without the use of breath samples or assumptions about CO2 fixation. occasionally resulting in lowering the amino acid dose before start of the isotope study.986 H. 1 in the high AA þ lipid group. Using phenylalanine as an indicator of protein metabolism has been validated numerous times. Furthermore. Infants were deliberately studied on postnatal day two. 1016/j. especially during stable nutritional intake. Future longterm follow-up studies should demonstrate if 3.6 g amino acids/ (kg$d) can be safely implemented in clinical practice. The discrepancy in results using the nitrogen balance method and the stable isotope study can be explained in three ways. CvdA designed the research. illustrating increased protein accretion available for tissue growth. The addition of another 1. Third. due to the small number of infants in the isotope study. resulting in an improved amino acid balance.27 In our model. feces. Potential effects of type of lipid Methods Infants in the early lipid groups were randomized to two different lipid types. Therefore. Our conclusion is that the tracer data are more reliable than the nitrogen balance data. That higher protein synthesis rates require higher amounts of other substrates. or a consequence of suboptimal amino acid composition of the currently available amino acid solutions. isotope studies are generally considered a more specific method to analyze the effects of amino acid administration on metabolism. anabolism is of great clinical importance in VLBW infants. Addendum.clnu. A single amino acid may not reflect accurately what is happening on whole body level. while nitrogen excretion is per definition underestimated. Type of lipid had no effect on amino acid kinetics. Nitrogen balances are noninvasive and can give an easy to obtain. and drafted the manuscript. The disadvantage of the tracer data is the necessity to extrapolate data from one single amino acid to whole protein metabolism. . in the isotope study. However.26 Our planned follow-up study at two years of age will reveal whether high amino acid and energy intake in the first few years has any benefits on the long run. Acknowledgments We would like to thank Aimon Niklasson for calculation of the growth SD scores. and drafted the manuscript.4. Conflict of interest None of the authors has a conflict of interest with regard to the manuscript. This study adds to the body of evidence that amino acid supplementation up to a level of 3. reflect a higher amino acid oxidation rate. we still recommend measurement of nitrogen balances as a basic measurement during nutritional intervention studies. shifts in body pools of N containing substrates as ammonia. this does not indicate that nitrogen balances are useless. it could be coincidence.2014. Nitrogen balance techniques are based on measurement of end product concentrations. Appendix A. These amino acid balances can be converted from molar rates to grams of protein and grams of tissue based on the assumptions that 1 g of protein contains 246 mmol phenylalanine24 (Fig. both enteral and parenteral nutritional intakes were kept stable during the 8 h study period. while the intake was changed in the majority of infants during the nitrogen balance study. Supplementary data). and fish oil. To adjust for the contribution of the available isotopomers. First. since specific N-losses (hair. The plasma enrichment of [U-13C6]KIC is therefore very close to the intracellular [U-13C6] leucine enrichment. possible effects might have been missed. All of the authors read and approved the final manuscript.10 the present tracer study demonstrates a beneficial effects of high amino acid administration in combination with early lipids on whole body metabolism. which reflects a higher amino acid oxidation. such as micronutrients and energy is underscored in the present study and in others. not enough studies are available to determine the upper safe and most effective level of amino acid and lipid administration in very low birth weight infants. Although the nitrogen balances did not differ between the AA þ lipid group and the high AA þ lipid group in our larger study. AV manufactured the stable isotope preparations. Leucine model 4.doi. a pure soybean oil emulsion or a multicomponent emulsion containing soybean oil. The search for an optimal composition of amino acid solutions specially designed for preterm infants is therefore of crucial importance.6 g/(kg$d) seems to result in a more anabolic state in this group of infants. JvG designed the research. olive oil. The higher urea concentrations observed in several studies. Second.01. JR conducted the research. which has not shown to be correlated with any detrimental outcome in this population. suggesting that higher-dose amino acid administration to VLBW infants results in a more anabolic state during the first postnatal phase.H. MV drafted the manuscript. The value of measuring urea concentrations in a routine fashion remains questionable. Furthermore. and drafted the manuscript. medium-chain triacylglycerol. However. stable isotope techniques require highly sophisticated machines and much more lab work compared to nitrogen balance techniques. and drafted the manuscript. although it is often difficult to achieve in the first days of life. S2.25 In conclusion. In addition. DR conducted the research. general indication of anabolism. The nitrogen balance is less precise than tracer data as intakes may quite easily be overestimated.002. which is usually considered as beneficial. Studies on the effect of lipid type on protein metabolism are lacking. Vlaardingerbroek et al.2 g amino acids/(kg$d) resulted in a protein balance that is approximately 1 g/(kg$d) higher. 987 comparing a 50% MCT-50% LCT lipid emulsion and a pure LCT emulsion. various leucine isotopomers were available in plasma. Source of funding Funding was not received for this project. To date. / Clinical Nutrition 33 (2014) 982e990 due to increased oxidation of amino acids. a-ketoisocaproic acid (KIC). skin. KD and HS analyzed the data. and drafted the manuscript. including our own. although it also resulted in higher urea concentrations. Statement of authorship HV designed and conducted the research. although in the larger study10 type of lipid had no effect on nitrogen balances and urea kinetics either. phlebotomy) are not taken into account. analyzed the data.org/10. except for a single study Calculations Leucine is reversibly transaminated within the cell to its ketoacid. Stable isotope techniques track the metabolic processes in amino acid metabolism and can give information about several rates of intermediate metabolism. Supplementary data Supplementary data related to this article can be found at http:// dx. amino acid levels are also not taken into account. the plasma enrichments of leucine were calculated utilizing all of the measured molecular forms of leucine. because of any supposed energy deficit to finance the cost of protein synthesis as discussed before. because the infants participating in the stable isotope study are a subset from those included in the larger study on nitrogen balance. we used two carbon- Oxidationðmmol=ðkg$hÞÞ ¼ Utilization of leucine for protein synthesis (Sleu. In the high AA þ lipid group. thereby omitting a non-existing flux. 15 Nleucine þ ½U 13 C6 leucine þ ½15 Nleucine þ ½D3 leucine by the following formula28:where [x]leucine can be each of the specific tracer enrichments. This model is a modified version of the model proposed by Shipley and Clark. / Clinical Nutrition 33 (2014) 982e990 ½xleucine enrichment ¼ ½xleucine ½U 12 C6 leucine þ ½U 13 C6 .30 Because these models use radioactively labeled isotopes instead of stable isotopes. these three values should be equal. mmol/(kg$h))¼ i½U13 C6 . A1(B)) did not differ among the groups. reamination.15 Nleucine TTR½U13 C6 mean Fluxes (Q) were calculated as follows: Q leu ðmmol=ðkg$hÞÞ ¼ RPleu þ reamination þ intake ¼ Sleu þ deamination Q KIC ðmmol=ðkg$hÞÞ ¼ deamination ¼ oxidation þ reamination Leucine balanceðmmol=ðkg$hÞÞ ¼ Sleu RPleu The leucine balance was converted from molar rates to grams of protein under the assumption that 1 g protein contains 590 mmol leucine. the utilization of leucine for protein synthesis (Sleu) was significantly higher than in the AA þ lipid group (Fig. and whole-body fluxes (Fig. deamination. To measure amino acid oxidation.15N]leucine plus [U-13C6]leucine. We therefore used the alternative open twopool model (dual-isotope model) of Helland et al. Vlaardingerbroek et al. but discrepancies are averaged to improve model and measurement accuracy. A1(A)).15 Nleucine TTR½D3 leucine TTR½U13 C6 mean TTR½D3 KIC TTR½D3 leucine Deaminationðmmol=ðkg$hÞÞ ¼ i½D3 KIC TTR½U13 C6 mean TTR½U13 C6 mean TTR½D3 KIC TTR½D3 leucine Reaminationðmmol=ðkg$hÞÞ ¼ i½U13 C6 . This calculation is based on the assumption that the only source of KIC is from the deamination of leucine within the twopool system. but its use was not significantly higher than in the control group.15 Nleucine TTR½D3 KIC i½D3 KIC TTR½U13 C6 mean TTR½U13 C6 mean TTR½D3 KIC TTR½D3 leucine i½D3 KIC TTR½U13 C6 mean i½U13 C6 .988 H. Most of the infants in our study were ventilated with non-invasive methods on day 2 such that the collection of expired air without mixture with surrounding air was not possible. reflecting proteolysis). oxidation. reflecting proteolysis. The leucine balance was significantly higher in the high . most models measure labeled carbon dioxide in expired air. Release of leucine from protein (RPleu.15 Nleucine TTR½D3 leucine TTR½U13 C6 mean TTR½D3 KIC TTR½D3 leucine labeled amino acids simultaneously. Results Leucine and a-KIC kinetics Leucine and a-KIC enrichments at steady state are presented in Table 3. However.28. As such. mmol/(kg$h))¼ i½U13 C6 ..16 which allowed us to measure leucine oxidation and transamination without the use of breath samples or assumptions about CO2 fixation. %) instead of mole percent excess.31 ! intakeleucine where i is the tracer infusion rate in mmol/(kg$h) and TTR½U13 C6 mean is the mean TTR of [U-13C6]KIC and the total sum of [U-13C6. enrichments in the following equations are expressed as tracer-tracee ratios (TTR. The release of leucine from protein (RPleu.29 adapted to leucine metabolism. 97(3): 746e54. Milne E. Liechty EA. The International Neonatal Network.162(1): 253e61. 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