Hellstern, Gerald*; Kaempf-Rotzoll, Daisy*; Linderkamp, Otwin*; Langhans, Klaus-Dieter†; Rating, Dietz†
Premature infants usually receive parenteral nutrition in the first days of postnatal life. Parenteral nutrition without amino acids leads to an estimated net protein loss of 0.9 g · kg−1 · d−1 in premature infants (1). Adding amino acids to parenteral nutrition increases nitrogen retention in premature infants (2–6). Tracer kinetic studies suggest an increase in whole body protein synthesis as a possible mechanism for this increase in nitrogen retention (2,4,6–8). Whether early parenteral administration of amino acids improves albumin and muscle protein synthesis remains unknown.
The newborn piglet has been validated as a model for parenteral nutrition in human newborns (9). As in humans, protein accretion in pigs is most rapid during the neonatal phase (10). We chose premature minipigs as model animals for human premature newborns. In our experience and reports from other investigators (11), delivery of piglets more than 9 days before the expected delivery date leads to a high mortality rate. We delivered the animals at day 106 of gestation (mean expected delivery date, 111–113 days) to assure prematurity of birth with a still reasonable mortality rate. In our study, we measured the effects of parenteral amino acids on albumin and mixed muscle protein fractional synthetic rates in premature minipigs.
ANIMALS AND METHODS
From 5 pregnant Yucatan minipig sows (Charles River, Sulzfeld, Germany), a total of 17 piglets were delivered by cesarean section on day 106 of gestation (mean expected gestation, 111–113 days). Birth weight was 813 g ± 87 g (mean ± SD) with a range from 650 g to 950 g. Immediately after birth, animals were placed in an incubator at 35°C, and umbilical vein and artery catheters were placed. The venous catheter was used for administration of tracer and nutrients, and the arterial catheter was used for blood sampling. The experiment protocol was started within 30 minutes after birth. All animals were randomized to two groups: group 1 (n = 9, control group) received intravenous glucose at a rate of 19 g · kg−1 · d−1 for 6 hours; group 2 (n = 8, treatment group) received intravenous glucose (also at 19 g · kg−1 · d−1) and a commercially available amino acid solution (Primene 10%; Baxter) at a rate of 10 g · kg−1 · d−1 for 6 hours. Both groups received a primed continuous rate infusion of L-[1–13C]valine (prime dose, 4 mg/kg; continuous rate, 4 mg · kg−1 · h−1). One-milliliter heparinized blood samples were collected before the start of the experiment and at hourly intervals. Plasma was separated from erythrocytes by centrifugation and stored at −20°C until analysis. After 6 hours, animals were killed by injection of 250 mg pentobarbital. Gastrocnemius muscle biopsy samples (approximately 1 g) were obtained and immediately frozen at −20°C. Two animals (one in each group) were excluded from analysis because of death before termination of the experiment. The experiment protocol was approved by the state ethics committee for research in laboratory animals.
Isolation of Albumin
Albumin was extracted from plasma by differential solubility in absolute ethanol from trichloroacetic acid (12). Briefly, 0.5 mL heparin plasma was deproteinized with 0.5 mL 20% trichloroacetic acid. After centrifugation (10 minutes, 2,000 g, 4°C), the pellet was redissolved in 1 mL ethanol. The ethanolic protein was evaporated in a vacuum centrifuge at 40°C, and the residue was redissolved in 1 mL 0.3 mol/L NaOH at 37°C for 30 minutes. Albumin was reprecipitated with 1 mL 2 mol/L HClO4. Free amino acids were removed by washing the pellet three times with 1 mL 0.2 mol/L HClO4 before hydrolysis. The purity of the albumin preparation was confirmed by high-resolution polyacrilamide electrophoresis, resulting in a single protein band corresponding to porcine albumin.
Mixed Muscle Protein
Mixed muscle protein was isolated as previously described (13). Briefly, 30 mg to 50 mg of frozen tissue was homogenized in 2 mL of 0.2 N trichloroacetic acid. After centrifugation at 1,800 g for 20 minutes at 4°C, the pellet was washed three times with 0.2 N trichloroacetic acid to remove free amino acids. Muscle protein and albumin were hydrolyzed in 6 mol/L hydrochloric acid at 110°C for 24 hours, and the resulting amino acids were dried in vacuum at 40°C.
Plasma Free Amino Acids
Amino acids were isolated from protein hydrolysates or deproteinized plasma by passing the amino acid–containing solutions through a cation exchange column (Maxi-Clean IC-H, 0.5-mL bed volume; Alltech, Deerfield, IL, U.S.A.). Amino acids were eluted with 0.5 mL of 25% ammonia solution. The eluate was dried in vacuum at 40°C. The dried sample was kept at room temperature until derivatization.
Gas Chromatography/Mass Spectrometry
13C-valine plasma enrichments were measured by gas chromatography/mass spectrometry as previously described (13). The N(O,S)-methoxycarbonyl methyl esters of the isolated amino acids were formed according to the method of Husek (14). A standard curve was used for each series of analyses, prepared by dissolving weighed amounts of L-[1–13C]valine and commercially available L-valine in distilled water. Two standard curves were made, one with a range from 0 to 0.30 tracer/tracee molar ratio, and one with a range from 0 to 0.02 tracer/tracee molar ratio. Valine was separated from other amino acids by gas chromatography (Varian 3400; Varian, Darmstadt, Germany) using a ZB-1 column (30 m × 0.32 mm inner diameter, 0.25 μm film thickness; Phenomenex, Torrance, CA, U.S.A.). Isotopic enrichments between 0.03 and 0.40 tracer/tracee ratio were measured by mass spectrometry using a Magnum mass spectrometer (Finnigan MAT, Bremen, Germany). The column effluent was treated by chemical ionization with methane, and ion monitoring was performed at m/z 190 (unlabelled valine) and m/z 191 (13C-valine). Isotopic enrichments between 0 and 0.02 tracer/tracee ratio were measured by combustion/isotope ratio mass spectrometry as previously described (13) with a delta S isotope ratio mass spectrometer (Finnigan MAT).
Fractional synthetic rates were calculated according to Wolfe (15):EQUATION
where Zval:pr is the tracer/tracee molar ratio of L-[1–13C]valine to unlabeled valine in protein (mixed muscle protein or albumin) at the end of the experiment, and Zval:pl is the plateau tracer/tracee molar ratio in plasma at isotopic steady state.
Amino Acid Analysis
Plasma amino acid concentrations were measured by tandem mass spectrometry in our neonatal screening laboratory (16).
The significance of differences was assessed by Mann-Whitney rank sum test. Data are expressed as mean ± SD unless otherwise stated. When multiple comparisons were made, the Bonferroni correction factor was used to determine significance.
Stable 13C-valine plateau enrichments were reached in all piglets. Mean plateau 13C-valine values in plasma were lower in piglets receiving amino acids (7.8 ± 1.51 vs. 18.2 ± 2.43 tracer/tracee mole ratio;P < 0.0001). Plasma amino acid concentrations are shown in Table 1. Although concentrations did not change significantly during infusion of glucose, concentrations of several amino acids increased during infusion of amino acids.
Incorporation of 13C-valine into albumin was linear in both groups (Fig. 1). Mixed muscle protein 13C-valine enrichments and calculated mixed muscle and albumin fractional synthetic rates are shown in Table 2. Mixed muscle protein and albumin fractional synthetic rates were approximately doubled by infusion of amino acids when compared with control animals receiving glucose only. Individual values for mixed muscle and albumin fractional synthetic rates are shown in Figures 2 and 3.
We found that parenteral administration of amino acids increases albumin and mixed muscle fractional protein synthetic rates in premature newborn minipigs in the first hours after birth. Early parenteral administration of amino acids improves nitrogen balance in premature infants with very low birth weight (2–5,7), even at low caloric intakes of approximately 30 kcal · kg−1 · d−1 (2). Whole body tracer kinetic studies suggest increases in protein synthesis as the possible mechanism for the observed improvement in nitrogen balance (2,4,6–8). For obvious ethical reasons, the organs affected by the observed increase in whole body protein synthesis cannot be distinguished in human neonates.
We found that hindlimb muscle protein fractional synthetic rates increased by 112% when parenteral amino acids were administered in premature minipigs in the first hours of life. Similar increases have been reported in newborn minipigs after feeding with colostrum in the first 6 hours of life (17), suggesting that parenteral amino acids are an adequate alternative to enteral proteins for increasing skeletal muscle protein synthesis. The values reported in this study are comparable to ours, although a different method (flooding dose method) was used to measure protein synthesis. Much smaller increases (approximately 30%) in muscle fractional synthetic rate have been reported in adult pigs after administration of parenteral amino acids (18). Fractional synthetic rates were also much lower in these adult pigs (1.5% per 24 hours without amino acids vs. 2% per 24 hours with amino acids) than in our neonatal piglets (5.6% per 24 hours without amino acids vs. 11.9% per 24 hours with amino acids). These differences suggest that skeletal muscle fractional synthetic rates in premature neonatal piglets are higher and more dependent on exogenous amino acid supply than in adult pigs.
In our study, the fractional rate of albumin synthesis in piglets receiving amino acids (87% per 24 hours) was much higher than the 10% to 11% reported by Jahoor et al. in 11-week-old enterally fed piglets (19) and the 28% reported by Stoll et al. in enterally fed piglets at 4 weeks of age (20). This difference could be attributable to the higher age of the piglets used in the other studies, as fractional protein synthesis in several tissues declines with age in piglets (21), and to a lower protein intake (60 mg · kg−1 · h−1 in the study by Stoll et al. compared with 400 mg · kg−1 · h−1 in our study; protein intake was not stated in the study by Jahoor et al.).
Breakdown of albumin was not measured in this study. Therefore, it is not possible to determine whether the increase in albumin synthesis results in a rapid expansion of the albumin pool or instead reflects a high albumin turnover rate. Our data do suggest a unique capacity of newborn piglets on the first day of life to increase the rate of albumin synthesis when amino acids are administered.
Furthermore, these large differences also correspond well to values for albumin fractional synthetic rates in premature infants measured by incorporation of [15N]glycine (22), which were severalfold higher than in young adults (23).
When using amino acids labeled with stable isotope or radioactive tracers to measure protein synthesis, the problem of the precursor pool needs to be addressed. The “true” precursor pool is believed to be represented by the labeling of aminoacyl-tRNA as the immediate precursor for protein synthesis (18). However, aminoacyl-tRNA enrichments are rarely measured in isotope infusion experiments because tRNA concentration in tissues is very low, and isolation is technically very difficult. In the few studies published, aminoacyl-tRNA enrichment values in liver (24,25) and muscle (25) were intermediate between extracellular and intracellular amino acid enrichment in the fed and postabsorptive states. In addition, the relation between the labeling of the extracellular pool (venous 13C-leucine) and the intracellular pool (13C-ketoisocaproate) has been shown to be independent of feeding in premature infants (26). The precursor pool used in our study, arterial free amino acid, would be expected to have a higher isotopic enrichment than aminoacyl-tRNA, and the reported synthesis values would thus underestimate true values by approximately 15%. This would not change the conclusions drawn from the results.
In the treatment group, amino acid values were higher at the end of the experiment when compared with values at the beginning. This suggests a prompt increase in pool size after the start of amino acid administration, with rapid establishment of a new equilibrium, as indicated by a constant tracer/tracee ratio during the experiment and linear incorporation of label into albumin in both treatment and control groups.
Although nonprotein energy intake was the same in both groups, the group of piglets receiving amino acids had a higher energy intake because of the caloric content of the amino acids administered. The study design was chosen to reflect clinical studies in premature newborns (2–8), in which nonprotein calories are usually the same in both treatment and control groups, and total caloric intakes are higher in the amino acid–supplemented groups. Because tolerance of nonprotein calories is limited in premature newborns, protein calories are added in clinical studies. Whether an observed increase in protein synthesis is only caused by a direct effect of amino acids or also influenced by protein energy cannot be answered by this design.
The treatment group showed a higher variability both in albumin and muscle protein synthesis compared with the control group. It is possible that this varying response to amino acid administration is associated with prematurity of birth or other clinical variables not measured in the study.
In conclusion, we found that parenteral amino acids increase albumin and skeletal muscle fractional protein synthetic rates in premature newborn minipigs. Our data provide further evidence that protein synthetic rates are highest in the neonatal period and decline with age.
The authors thank A. Fichtner, S. Zacharevics, and M. Herrmann for help with the laboratory work, and Dr. Becker for assistance with the animal experiments.
1. Pencharz PB, Steffee WP, Cochran W, et al. Protein metabolism in human neonates: Nitrogen-balance studies, estimated obligatory losses of nitrogen and whole-body turnover of nitrogen. Clin Sci Molec Med 1977; 52:485–98.
2. Goudoever JB, Colen T, Wattimena JLD, et al. Immediate commencement of amino acid supplementation in preterm infants: Effect on serum amino acid concentrations and protein kinetics on the first day of life. J Pediatr 1995; 127:458–65.
3. Saini J, Macmahon P, Morgan JB, et al. Early parenteral feeding of amino acids. Arch Dis Child 1989; 64:1362–6.
4. van Lingen RA, Goudoever JB, Luijendijk IHT, et al. Effects of early amino acid administration during total parenteral nutrition on protein metabolism in preterm infants. Clin Sci 1992; 82:199–203.
5. Anderson TL, Muttart CR, Bieber MA, et al. A controlled trial of glucose versus glucose and amino acids in premature infants. J Pediatr 1979; 94:947–51.
6. Clark SE, Karn CA, Ahlrichs JA, et al. Acute changes in leucine and phenylalanine kinetics produced by parenteral nutrition in premature infants. Pediatr Res 1997; 41:568–74.
7. Rivera A, Bell EF, Bier DM. Effect of intravenous amino acids on protein metabolism of preterm infants during the first three days of life. Pediatr Res 1993; 33:106–11.
8. Denne SC, Karn CA, Ahlrichs JA, et al. Proteolysis and phenylalanine hydroxylation in response to parenteral nutrition in extremely premature and normal newborns. J Clin Invest 1996; 97:746–54.
9. Wykes LJ, Ball RO, Pencharz PB. The development and validation of a total parenteral nutrition model in the neonatal piglet. J Nutr 1994; 123:1248–59.
10. Seve B, Reeds PJ, Fuller MF, et al. Protein synthesis and retention in some tissues of the young pigs as influenced by dietary protein intake after early-weaning: Possible connection to the energy metabolism. Reprod Nutr Develop 1986; 26:849–61.
11. Flecknell PA, Wootton R, John M, et al. Steady state glucose kinetics and their relation to plasma glucose concentration in the premature and full term neonatal piglet. Can J Physiol Pharamacol 1981; 59:1069–72.
12. de Sain-van der Velden MG, Kaysen GA, de Meer K, et al. Proportionate increase of fibrinogen and albumin synthesis in nephrotic patients: Measurements with stable isotopes. Kidney Int 1998; 53:181–8.
13. Reijngoud DJ, Hellstern G, Elzinga H, et al. Determination of low isotopic enrichment of L-[1–13C]valine by gas chromatography/combustion/isotope ratio mass spectrometry: A robust method for measuring protein fractional synthetic rates in vivo. J Mass Spectrom 1998; 33:621–6.
14. Husek P. Rapid derivatization and gas chromatographic determination of amino acids. J Chromatogr 1991; 552:289–99.
15. Wolfe RR. Radioactive and stable isotope tracers in biomedicine: Principles and practice of kinetic analysis. New York: Wiley-Liss, 1992.
16. Chase DH, Millington DS, Terada N, et al. Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin Chem 1993; 39:66–71.
17. Davis TA, Fiorotto ML, Burrin DG, et al. Intrauterine growth restriction does not alter response of protein synthesis to feeding in newborn pigs. Am J Physiol 1997; 272:E877–84.
18. Watt PW, Corbett ME, Rennie MJ. Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability. Am J Physiol 1992; 263:E453–60.
19. Jahoor F, Bhattiprolu S, del Rosario M, et al. Chronic protein deficiency differentially affects the kinetics of plasma proteins in young pigs. J Nutr 1996; 126:1489–95.
20. Stoll B, Burrin DG, Henry J, et al. Dietary amino acids are the preferential source of hepatic protein synthesis in piglets. J Nutr 1998; 128:1517–24.
21. Davis TA, Burrin DG, Fiorotto ML, et al. Skeletal muscle and jejunal protein synthesis is more responsive to feeding in 7- than in 26-day-old pigs. Am J Physiol 1996; 270:E802–9.
22. Yudkoff M, Nissim I, McNellis W, et al. Albumin synthesis in premature infants: Determination of turnover with [15N]glycine. Pediatr Res 1987; 12:49–53.
23. Gersovitz M, Munro HN, Udall J, et al. Albumin synthesis in young and elderly subjects using a new stable isotope methodology: Response to level of protein intake. Metabolism 1980; 29:1075–86.
24. Vidrich A, Airhart J, Bruno MK, et al. Compartmentation of free amino acids for protein synthesis. Biochem J 1977; 162:257–66.
25. Watt PW, Lindsay Y, Scrimgeour CM, et al. Isolation of aminoacyl-tRNA and its labelling with stable-isotope tracers: Use in studies of human tissue protein synthesis. Proc Natl Acad Sci U S A 1991; 88:5892–6.
26. Denne SC, Rossi EM, Kalhan SC. Leucine kinetics during feeding in normal newborns. Pediatr Res 1991; 30:23–7.
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