Ligation of a patent ductus arteriosus under fentanyl anesthesia improves protein metabolism in premature neonates

Journal of Pediatric Surgery VOL 35, NO 9

SEPTEMBER 2000

Ligation of a Patent Ductus Arteriosus Under Fentanyl Anesthesia Improves Protein Metabolism in Premature Neonates By Stephen B. Shew, Tamir H. Keshen, Nancy L. Glass, Farook Jahoor, and Tom Jaksic Houston, Texas; Iowa City, Iowa; and Boston, Massachusetts

Background/Purpose: Although surgical ligation effectively reverses the cardiopulmonary failure associated with patent ductus arteriosus (PDA), previous findings have suggested that such surgery itself elicits a catabolic response in premature neonates. Therefore, the authors sought to quantitatively assess whether PDA ligation under fentanyl anesthesia aggravated or improved the protein metabolism of premature neonates. Methods: Seven ventilated, premature neonates (birth weight 815 ⫾ 69 g) underwent PDA ligation with standardized fentanyl anesthesia (15 ␮g/kg) on day-of-life 8.4 ⫾ 1.2 and were studied immediately pre- and 16 to 24 hours postoperatively while receiving continuous total parenteral nutrition (TPN). Whole-body protein kinetics were calculated using intravenous 1-[13C]leucine, and skeletal muscle protein breakdown was measured from the urinary 3-methylhistidine to creatinine ratio (MH:Cr).

I

T IS ESTABLISHED that surgery can be detrimental to the well being of premature neonates because they have a higher postoperative morbidity and mortality rate than full-term children.1 The metabolic response to surgery in neonates, as in adults, is characterized primarily by an increased loss of body protein.2 This catabolic response is seen in premature neonates as evidenced by their augmented plasma concentrations of the counterregulatory hormones and an elevation of a biochemical marker of skeletal muscle breakdown (3-methylhistidine) after surgical patent ductus arteriosus (PDA) ligation.3-5 Because these patients, especially those with very low birth weight (VLBW) weighing less than 1,500 g, have minimal metabolic reserves of protein,6-9 it has been proposed that surgery elicits a protein catabolic response that may overwhelm their metabolic capacities.3 However, PDA of the premature neonate engenders progressively worsening cardiopulmonary failure, which may, itself, also elicit a protein catabolic response. Because the loss of lean body mass in the premature

Results: Whole-body protein breakdown (10.9 ⫾ 1.2 v 8.9 ⫾ 0.8 g/kg/d, P ⬍ .05), turnover (17.4 ⫾ 1.2 v 15.4 ⫾ 0.8 g/kg/d, P ⬍ .05), and MH:Cr (1.95 ⫾ 0.20 v 1.71 ⫾ 0.16 ␮mol:mg, P ⬍ .05) decreased significantly after operation. This resulted in a 60% improvement in protein balance (1.6 ⫾ 0.8 v 2.6 ⫾ 0.6 g/kg/d, P ⫽ 0.08) postoperatively. Conclusions: Because of decreased whole-body protein breakdown, whole-body protein turnover, skeletal muscle protein breakdown, and increased protein accrual, surgical PDA ligation under fentanyl anesthesia promptly improves the protein metabolism of premature neonates enduring the stress of a PDA. J Pediatr Surg 35:1277-1281. Copyright © 2000 by W.B. Saunders Company. INDEX WORDS: Patent ductus arteriosus, protein, metabolism, surgery, prematurity, neonate.

neonate is directly associated with increased morbidity and mortality rates,10-11 it is important that clinical care

From the USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Departments of Surgery, Pediatrics, and Anesthesiology, Texas Children’s Hospital, Houston, TX; the Department of Surgery, University of Iowa, Iowa City, IA; and the Department of Surgery, Children’s Hospital, Harvard Medical School, Boston, MA. This work is a publication of the US Department of Agriculture (USDA)/Agricultural Research Service (ARS) Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX. Funding has been provided from the USDA/ARS under Cooperative Agreement #6250-5100-6001. The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. Address reprint requests to Tom Jaksic, MD, PhD, Children’s Hospital, Division of Pediatric Surgery, 300 Longwood Ave, Boston, MA 02115. Copyright © 2000 by W.B. Saunders Company 0022-3468/00/3509-0001$03.00/0 doi:10.1053/jpsu.2000.9293

Journal of Pediatric Surgery, Vol 35, No 9 (September), 2000: pp 1277-1281

1277

1278

SHEW ET AL

includes strategies to attenuate the magnitude of the protein catabolic response to surgery and illness in the premature neonate. A previous study has shown a decrease in the initial hormonal stress response in premature neonates administered fentanyl anesthesia for surgical PDA ligation.5 However, the resultant effect on whole-body protein metabolism is still unknown. Therefore, we performed a study designed to assess quantitatively whether surgical ligation of PDA using fentanyl anesthesia improved or aggravated the protein metabolism of premature neonates being fed total parenteral nutrition (TPN). Our aim was to measure whole-body protein kinetics and skeletal muscle protein breakdown rates in VLBW neonates before and after surgical PDA ligation utilizing stable isotope tracer techniques and urinary 3-methylhistidine to creatinine ratio. MATERIALS AND METHODS

Subjects Approval of the Baylor College of Medicine Institutional Review Board and parental consent were obtained to study 7 VLBW neonates with PDA. Their mean gestational age was 25.1 ⫾ 0.5 (SEM) weeks and mean birth weight was 815 ⫾ 69 g. Their birth weights were used to base all calculations in an effort to eliminate short-term fluctuation in fluid status as a confounding factor. All neonates were studied during the immediate preoperative period, on mean day of life 7.6 ⫾ 1.1, and 16 to 24 hours postoperatively. During both phases of the study they were ventilated mechanically and received continuous TPN. The TPN supplied 104 ⫾ 8.3 kcal/kg/d and 3.1 ⫾ 0.04 g/kg/d amino acid (2.2% Trophamine; McGaw, Irvine, CA) preoperatively and 100 ⫾ 4.7 kcal/kg/d and 3.1 ⫾ 0.06 g/kg/d amino acids postoperatively (2.2% Trophamine; McGaw). All subjects had a clinically significant PDA with a left-to-right shunt, which was confirmed by bedside echocardiography. Three patients failed to respond to a full course of intravenous indomethacin therapy (4 doses over 48 hours), and the other 4 patients had contraindications to indomethacin administration. Surgery was performed through a left thoracotomy incision, at bedside, in the neonatal intensive care unit under aseptic and euthermic conditions. Chest tubes were not used. All patients received standardized doses of fentanyl anesthesia (15 ␮g/kg) and pancuronium neuromuscular blockade (0.2 mg/kg). The mean operating time was 32 ⫾ 3 minutes, and mean anesthesia time was 66 ⫾ 3 minutes. There were no transfusions of blood products during either phase of the study. None of the neonates suffered from hemorrhage, hypothermia, or hypoxia within 72 hours of surgery, and there were no intraoperative or postoperative complications. Mean dopamine administration was 15.0 ⫾ 2.3 ␮g/kg/ min preoperatively and 15.6 ⫾ 2.1 ␮g/kg/min postoperatively in the 4 patients who were receiving pressor support. Exclusion criteria for this study were neonates of diabetic mothers and those neonates with hepatic failure, sepsis, renal failure, or insulin administration.

Study Protocol The pre- and postoperative phases of the study were identical in protocol (Fig 1). Thirty minutes before initiation of each study phase, a self-contained FT-IR Purge Gas Generator (Whatman Inc; Haverhill, MA) was used to provide CO2-free air (⬍ 1 ppm) through the ventilator to overcome the possible contamination of the endogenously produced CO2 from ambient CO2 in the air supply. After baseline blood and CO2

Fig 1. Infusion and sampling protocol used for both the preoperative and postoperative phases of the study. The arrows starting with the letter P show the time course of the primed continuous infusions of 1-[13C]leucine and NaH13CO3. The solid squares indicate the timing of the breath samples, and the solid circles show the timing of the blood samples. The 24-hour urinary nitrogen collections were coincident with the infusion protocol.

samples were obtained to document respective baseline ␣-ketoisocaproic acid (␣-KICA) and CO2 isotopic enrichments, each neonate was administered a primed (9 ␮mol/kg), 2-hour continuous intravenous infusion (6 ␮mol/kg/h) of NaH13CO3 (13C isotopic enrichment 99%: Cambridge Isotope Laboratory; Andover, MA) followed by a primed (12 ␮mol/kg), 4-hour continuous intravenous infusion (9 ␮mol/kg/h) of 1-[13C]leucine (13C isotopic enrichment 99%: Cambridge Isotope Laboratory) through an indwelling umbilical vein catheter. Four sets of duplicate CO2 samples were then collected in 15-minute intervals over the last 1 hour of the NaH13CO3 infusion during known isotopic steady state.11,12 Four additional sets of duplicate CO2 samples and 3 blood samples were obtained during the last 2 hours of the 1-[13C]leucine infusion to establish isotopic enrichment of CO2 and ␣-KICA, respectively, at steady state. Twenty-four– hour urine collections were obtained both pre- and postoperatively. The CO2 samples were collected into 20 mL evacuated containers (Vacutainer, Becton-Dickinson, McGaw, IL) from a gas flow resistor attached to the patient’s endotracheal tube by an Exosurf adapter (Respiratory Support Products Inc, Irvine, CA). The blood samples were drawn from an indwelling umbilical artery catheter, in place at least 2 days before the preoperative phase of the study, into prechilled tubes containing Na2EDTA and 10 ␮L of a protease inhibitor solution comprised of sodium azide, merthiolate, and soybean trypsin inhibitor. The blood samples were then centrifuged immediately at 3,000 rpm for 10 minutes and the plasma stored at ⫺70°C until analyzed. The total amount of blood drawn per study was 2.0 mL. Urine was collected quantitatively from an already indwelling Foley catheter or bag collection system, kept on ice, and stored in 6-hour aliquots at ⫺70°C until analysis. Stock infusate solutions were prepared aseptically and tested for sterility and pyrogenicity on a routine basis.

Analytic Methods Concentrations of the individual NaH13CO3 infusates were measured by acid-base titration, using 6 mmol/L HCl. Concentrations of the individual 1-[13C]leucine infusates were measured by high-pressure liquid chromatography on a Beckmann 7300 amino acid analyzer (Beckman Instruments, Fullerton, CA). Because ␣-KICA is the intracellular transamination product of leucine, plasma ␣-KICA was used for analysis of protein kinetics.13 The plasma ␣-KICA (pentafluorobenzyl derivative) and CO2 isotopic enrichments were determined by negative chemical ionization gas chromatography–mass spectrometry (Hewlett-Packard 5989A; Palo Alto, CA) and by use of a continuous flow isotope ratio mass spectrometer (Europa Scientific; Crewe, UK), respectively.14 Steady-state values were obtained by determining the average atom percent excess (APE) for CO2 and mole percent excess (MPE) for ␣-KICA after reaching plateau.15 The kinetic parameters of

PDA LIGATION IMPROVES PROTEIN METABOLISM

1279

were calculated by multiplying the above values of leucine kinetics with a conversion factor of 590 ␮mol leucine per 1 g of whole-body protein.

Statistical Analysis Data are expressed as the mean ⫾ SEM. Statistical analyses were performed by paired Student’s t test using Minitab version 11.1 (Minitab Inc, State College, PA) statistical software on a IBM-compatible personal computer.

RESULTS

Fig 2. Isotopic plateau of 13CO2 during the NaH13CO3 (60 to 105 minutes) and 1-[13C]leucine (240 to 330 minutes) infusions in 7 VLBW neonates before and after surgical PDA ligation. Values are expressed as mean ⴞ SEM of the APE over baseline enrichment. The coefficients of variation are 8.1% and 3.7%, respectively.

whole-body protein turnover (from total leucine flux), whole-body protein breakdown (from endogenous leucine flux), whole-body amino acid oxidation (from intracellular leucine oxidation), and whole-body protein balance (from leucine balance) were computed using previously described standard steady-state kinetic equations, which are outlined in detail in the calculation section.16 Urinary 3-methylhistidine concentration was measured by highpressure liquid chromatography on a Beckmann 7300 amino acid analyzer (Beckman Instruments), and urinary creatinine concentration was measured by the Jaffe reaction on a Cobas Fara II (Roche Diagnostics, Indianapolis, IN). The 3-methylhistidine to creatinine ratio was calculated by dividing the total 3-methylhistidine by the total creatinine excreted in the urine for each 24-hour collection period. Urinary nitrogen concentration was determined using the micro-Kjeldahl method after immediate exposure to 6 N HCl, and nitrogen balance was calculated from the difference between parenteral intake and urinary excretion over each 24-hour collection period.17

Calculations The whole-body protein kinetic equations used in this study may be described as follows. Leucine enters the free plasma amino acid pool from TPN (I) and from the breakdown of endogenous protein stores (B) and exits the pool by incorporation into body protein (S) or by leucine oxidation (O). At steady state, Q ⫽ I ⫹ B ⫽ S ⫹ O, where Q is the total leucine flux, or rate of leucine moving through the free plasma amino acid pool. Q is calculated from the isotopic enrichment of ␣-KICA, the transamination product of leucine; hence, it reflects intracellular leucine enrichment but can be measured in plasma by: Q (␮mol/kg/h) ⫽ inf ⫻ [(Ei/Ep) ⫺ 1], where inf is the L-[1-13C]leucine infusion rate (␮mol/ kg/h), Ei is the enrichment of the L-[1-13C]leucine infusate (mole % excess), and Ep is the mean enrichment of ␣-KICA (mole % excess) at plateau.16 Endogenous leucine flux was calculated by subtracting the known rate of exogenous leucine administration in TPN from Q such that B ⫽ Q ⫺ I (␮mol/kg). Leucine oxidation was calculated as follows: O ⫽ (ECO2 ⫻ RaCO2)/E␣-KICA (␮mol/kg/h), where ECO2 is the mean enrichment of 13CO2 during the leucine infusion, RaCO2 is the rate of CO2 production (obtained from the NaH13CO3 infusion), and E␣-KICA is the mean enrichment of intracellular leucine at plateau. Nonoxidative disposal of leucine (the amount of leucine going into whole-body protein synthesis) is the difference between total leucine flux and leucine oxidation rate: S ⫽ Q ⫺ O (␮mol/kg/h). Net leucine balance was calculated as the difference of leucine oxidation (O) from the rate of exogenously administered leucine (I). Whole-body protein kinetics

Each patient was studied as their own control for the preoperative versus postoperative analyses. Steady state for CO2 (Fig 2) and ␣-KICA enrichment (Fig 3) was achieved within 2 hours after the start of each NaH13CO2 and 1-[13C]leucine infusion, respectively. The coefficient of variation (CV) for the CO2 isotopic enrichment was 8.1% at plateau during the NaH13CO2 infusion and 3.7% during the 1-[13C]leucine infusion (Fig 2). The wholebody protein kinetics data are shown in Fig 4. Wholebody protein turnover (17.4 ⫾ 1.2 g/kg/d preoperative v 15.4 ⫾ 0.3 g/kg/d postoperative, P ⬍ .05) and breakdown rates (10.9 ⫾ 1.2 g/kg/d preoperative v 8.9 ⫾ 0.8 g/kg/d postoperative, P ⬍ .05) decreased significantly after surgical ligation of the PDA (Fig 4). Concomitantly, there was a trend toward improved protein balance postoperatively (1.6 ⫾ 0.8 g/kg/d preoperative v 2.6 ⫾ 0.6 g/kg/d, P ⫽ .08). Furthermore, the 3-methylhistidine to creatinine ratio was significantly lower in the postoperative period compared with the preoperative period (1.71 ⫾ 0.16 ␮mol:mg v 1.95 ⫾ 0.20 ␮mol:mg; P ⬍ .05). Protein synthesis (12.5 ⫾ 0.8 g/kg/d preoperative v 11.5 ⫾ 0.3 g/kg/d postoperative) and protein oxidation (4.9 ⫾ 0.8 g/kg/d preoperative v 3.9 ⫾ 0.6 g/kg/d postoperative) both decreased somewhat in the postoperative period; however, this was not statistically significant. Urinary nitrogen balance also was not changed in a significant manner (353 ⫾ 16 mg/kg/d preoperative v 350 ⫾ 36 mg/kg/d postoperative).

Fig 3. Isotopic plateau of ␣-KICA during the 1-[13C]leucine infusion (240 to 360 minutes) in 7 VLBW neonates before and after surgical PDA ligation. Values are expressed as mean ⴞ SEM of the MPE over baseline enrichment. The coefficient of variation is 8.9%.

1280

SHEW ET AL

Fig 4. Whole-body protein kinetics (protein turnover, protein breakdown, amino acid oxidation, and protein balance) in 7 VLBW neonates before and after surgical ligation of PDA. Values are expressed as mean ⴞ SEM. Statistical analyses were performed by paired Student’s t test and compared preoperative versus postoperative measurements.

DISCUSSION

Although most well premature neonates can grow at rates similar to those in utero,18 this task becomes more formidable when their tenuous physiologic homeostasis is breached by the stress of illness or surgery. The data reported here show that whole-body protein breakdown, protein turnover, and skeletal muscle protein breakdown rates significantly improve in VLBW neonates after operation with fentanyl anesthesia for hemodynamically significant PDAs. Therefore, the protein catabolic response to surgical PDA ligation appears to be eliminated when the surgery is performed using adequate anesthesia. Furthermore, because the protein metabolic parameters measured actually improve immediately after surgery, the illness from a PDA appears to be a greater stress for the VLBW neonate than that of the operation itself. Although the neonates, on average, were in net positive protein balance during both phases of the study, the significant postsurgical reduction in whole-body protein breakdown rate resulted in a 60% increase in protein balance. This mean postoperative protein balance (2.6 ⫾ 0.6 g/kg/d) is equivalent to the normal protein accretion rate of the human fetus at 28 weeks’ gestational age.19 These rates of net protein synthesis also are similar to those of other healthy premature neonates receiving equivalent amounts of energy and protein (106 kcal and 3 g protein/kg/d) by oral feedings.20 The improvement in protein balance is particularly relevant for VLBW neonates because they have inherently limited total protein stores of 90 g/kg body weight compared with those of full-term neonates with 130 g/kg body weight.6-9 By not achieving normal rates of net protein accrual, the VLBW neonates preoperatively experienced a relative loss of

protein while enduring the physiological stress of a PDA. Whereas after surgical PDA ligation with fentanyl anesthesia, the relative protein catabolism was corrected promptly, and the protein metabolism normalized. This study did not address whether PDA ligation with other anesthetics might have similar beneficial effects. Anand et al3-5 have reported previously that the addition of fentanyl (in lower doses than those in the current study) to an anesthetic regimen of d-tubocurarine and nitrous oxide given to premature neonates during surgical ligation of PDA significantly muted the increase in plasma concentrations of several counter-regulatory hormones. Also, their neonates receiving fentanyl showed only a modest increase in 3-methylhistidine to creatine excretion postoperatively, compared with a marked increase in excretion of up to 3 days after operation seen in the neonates who did not receive fentanyl.5 This finding suggested that the magnitude of the catabolic response to surgery was blunted by the addition of fentanyl when compared with the magnitude of the response in those infants who did not receive fentanyl. Hence, the stress of surgery can be lessened by blocking the nociceptive response with appropriate analgesia.5 Although the study by Anand et al5 showed that the addition of fentanyl to the anesthetic regimen attenuated the hormonal stress response to surgery in premature neonates, there has not been a direct assessment of whole body protein metabolism in these patients until the current study. In addition to anesthesia, other steps were used in our study that potentially lowered the metabolic cost of surgery. These included performing the surgery at bedside to minimize the stress associated with moving the patient21 and providing continuous parenteral nutrition to meet nutritional requirements. Early, continuous administration of parenteral amino acid solutions to VLBW neonates has been shown to improve their protein balance, primarily by increasing protein synthesis, and to support growth rates similar to those found normally in utero.22-24 The relative quantitative import of these additional measures was not addressed by our experiment. The findings of the current study indicate that PDA ligation in VLBW neonates, under the conditions of fentanyl anesthesia, parenteral nutrition, and bedside surgery, does not elicit a protein catabolic state. Actually, surgical PDA ligation, under adequate anesthesia, promptly and significantly decreases protein breakdown and tends to restore normal protein balance in these patients. ACKNOWLEDGMENTS The authors thank Margaret Frazer and Melanie Del Rosario for technical assistance and Leslie Loddeke for editorial assistance.

PDA LIGATION IMPROVES PROTEIN METABOLISM

1281

REFERENCES 1. Kiely E: Surgery in very low birth weight children. Arch Dis Child 59:707-708, 1984 2. Goldstein SA, Elwyn DH: The effects of injury and sepsis on fuel utilization. Annu Rev Nutr 9:445-473, 1989 3. Anand KJS: Neonatal stress responses to anesthesia and surgery. Clin Perinatol 17:207-214, 1990 4. Anand KJS, Hansen DD, Hickey PR: Hormonal-metabolic stress responses in neonates undergoing cardiac surgery. Anesthesiology 73:661-670, 1990 5. Anand KJS, Sippell WG, Aynsley-Green A: Randomized trial of fentanyl anaesthesia in preterm babies undergoing surgery: Effects on the stress response. Lancet 1:243-247, 1987 6. Widdowson EM: Changes in body composition during growth, in Davis JA, Dobbing J (eds): Scientific foundations of paediatrics. London, Heinemann, 1981, pp 330-342 7. Fomon SJ, Haschke F, Zeigler EE, et al: Body composition of reference children from birth to age 10 years. Am J Clin Nutr 35:11691175, 1982 8. Forbes GB, Bruining GJ: Urinary creatinine excretion and lean body mass. Am J Clin Nutr 29:1359-1366, 1976 9. Munro HN: Nutrition and muscle protein metabolism. Fed Proc 37:2281-2282, 1978 10. Ballard FJ, Tomas FM, Pope LM, et al: Muscle protein degradation in premature human infants. Clin Sci 57:535-544, 1979 11. Bresson JL, Mariotti A, Narcy P, et al: Recovery of [13C]bicarbonate as respiratory 13CO2 in parenterally fed infants. Eur J Clin Nutr 44:3-9, 1990 12. van Aerde JEE, Sauer PJJ, Pencharz PB, et al: The effect of energy intake and expenditure on the recovery of 13CO2 in the parenterally fed neonate during a 4 hour primed constant infusion of NaH13CO3. Pediatr Res 19:803-810, 1985 13. Schwenk WF, Beaufrere B, Haymond MW: Use of reciprocal pool specific activities to model leucine metabolism in humans. Am J Physiol 249:E646-E650, 1985 14. Hachey DL, Patterson BW, Reeds PJ, et al: Isotopic determina-

tion of organic keto acid pentafluorobenzyl esters in biological fluids by negative chemical ionization gas chromatography/mass spectrometry. Anal Chem 63:919-923, 1991 15. Jahoor F, Burrin DG, Reeds PJ, et al: Measurement of plasma protein synthesis rates in the pig: An investigation of alternative tracer approaches. Am J Physiol 267:R221-227, 1994 16. Matthews DE, Schwartz RD, Yang RD, et al: Relationship of plasma leucine and ␣-ketoisocaproate during a L-[1-13C]leucine infusion in man: A method for measuring human intracellular leucine tracer enrichment. Metab Clin Exp 31:1105-1112, 1982 17. Munro HM, Fleck A: Analysis of tissues and body fluids for nitrogenous constituents, in Munro HM (ed): Mammalian Protein Metabolism, Vol. 3. New York, NY: Academic Press, 1969, pp 423525 18. Zlotkin SH, Bryan MH, Anderson GH: Intravenous nitrogen and energy intakes required to duplicate in utero nitrogen accretion in prematurely born human children. J Pediatr 99:115-120, 1981 19. Denne SC, Karn CA, Liu YM, et al: Effect of enteral versus parenteral feeding on leucine kinetics and fuel utilization in premature newborns. Pediatr Res 36:429-435, 1994 20. van Toledo-Eppinga VL, Kalhan SC, Kulik W, et al: Relative kinetics of phenylalanine and leucine in low birth weight children during nutrient administration. Pediatr Res 40:41-46, 1996 21. Pokorny WJ, Adams JM, McGill CW, et al: Ligation of patent ductus arteriosus in the Neonatal intensive care unit. Mod Probl Paediatr 23:133-142, 1985 22. Ziegler EE, O’Donnell AM, Nelson SE, et al: Body composition of the reference fetus. Growth 40:329-341, 1976 23. Duffy B, Pencharz P: The effects of surgery on the nitrogen metabolism of parenterally fed human neonates. Pediatr Res 20:32-35, 1986 24. Rivera A Jr, Bell EF, Bier DM: Effect of intravenous amino acids on protein metabolism of preterm infants during the first three days of life. Pediatr Res 33:106-111, 1993