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1 From the Department of Pediatrics, Division of Neonatology (SRDvdS and JBvG) and the Departments of Internal Medicine (DLW), Clinical Genetics (JH), and Clinical Pharmacy (AV), Erasmus MC–Sophia Children's Hospital, Erasmus University, Rotterdam, Netherlands
2 Supported by the Sophia Foundation of Scientific Research (Kröger Foundation) and the Ajinomoto Amino Acid Research Programme. 3 Reprints not available. Address correspondence to JB van Goudoever, Department of Pediatrics, Erasmus MC–Sophia Children's Hospital, Erasmus University, Dr Molewaterplein 60, 3015 GJ, Rotterdam, Netherlands. E-mail: j.vangoudoever{at}erasmusmc.nl.
ABSTRACT
Background:Threonine is an essential amino acid that is abundantly present in intestinally produced glycoproteins. Animal studies show that intestinal first-pass threonine metabolism is high, particularly during a restricted enteral protein intake.
Objective:The objective of the study was to quantify intestinal first-pass threonine metabolism in preterm infants during full enteral feeding and during restricted enteral intake.
Design:Eight preterm infants (
Conclusion:In preterm infants, the splanchnic tissues extract a very large amount of the dietary threonine intake, which indicates a high obligatory visceral need for threonine, presumably for the purposes of synthesis.
Key Words: Threonine • preterm infants • intestine • stable isotopes • nutrition • splanchnic metabolism
INTRODUCTION
During the first few weeks of life, preterm infants are faced with the challenge of doubling their body weight (1). The high growth rate of the newborn infant puts significant pressure on the intestine to efficiently digest and absorb nutrients. This occurs at a time when the neonatal intestine is adapting to the enteral route of nutrition after a prenatal period in which amino acids (AAs) were delivered via the umbilical route. Therefore, it may be speculated that a large quantity of AAs is needed for the growth and maintenance of the premature gut to enable its optimal function and integrity.
Given the key role of the gut in the maintenance of neonatal health, there has been considerable interest in the significance of first-pass intestinal metabolism of dietary AAs (2–4). Enterally absorbed AAs can be used for incorporation into mucosal cellular proteins, for energy production, or for conversion via transamination into other AAs, metabolic substrates, and biosynthetic intermediates. It is known that, in animals, <20% of intestinal AAs are used for constitutive gut growth by the intestinal mucosa (1), and, although some essential AAs (EAAs) are known to be catabolized (3–5), the catabolism of EAAs does not account for their high utilization rate. Therefore, the synthesis of secretory glycoproteins by the enterocytes appears to be a major metabolic fate for EAAs.
Of particular interest is threonine, which is the AA that, in neonatal piglets, is used to the greatest extent by the portal-drained viscera (PDV)—ie, the intestines, pancreas, spleen, and stomach. In neonatal pigs, the splanchnic extraction of threonine ranges from 60% to 80% of the dietary intake (2, 3, 6, 7). This high intestinal requirement for threonine may reflect the use of enterally absorbed threonine for the synthesis of secretory glycoproteins as the major metabolic fate. Indeed, Roberton et al (8) found that the protein cores of secretory mucins contain large amounts of threonine. In addition, Bertolo et al (9) showed that the whole-body threonine requirement in total parenteral nutrition (TPN)–fed piglets is 40% of that observed in enterally fed piglets, which indicates that enteral nutrition itself induces metabolic processes that demand threonine, probably within the intestine. One may postulate that the compromised gut barrier function associated in humans with parenteral nutrition is caused by a sparse threonine availability combined with diminished intestinal mucin production, as has been shown in rats (10).
In view of the central role of the gut in nutrient processing and metabolism, we considered it important to investigate the effect of the amount of enteral intake on splanchnic and whole-body threonine metabolism in preterm infants. Accordingly, we simultaneously used 2 stable isotope–labeled threonine tracers—[U-13C]threonine and [15N]threonine—and administered them via intravenous and intragastric routes to determine the quantitative aspects of threonine metabolism in preterm neonates under both parenteral and enteral feeding conditions. This technique enabled us to measure both the first-pass intestinal threonine uptake and the whole-body threonine kinetics. Previously, our group (7) found in neonatal pigs that the considerable first-pass threonine utilization was not significantly affected by a lower protein intake. Therefore, we hypothesized that the first-pass utilization of threonine by the splanchnic tissues would be substantial in preterm infants and would be independent of the dietary threonine intake. Hence, the present study explores the effect of the amount of enteral formula intake on various components of first-pass and whole-body threonine metabolism in neonates.
SUBJECTS AND METHODS
Subjects
Splanchnic and whole-body threonine kinetics were quantified in 8 preterm infants during 2 consecutive periods of different enteral and parenteral intakes. Patients eligible for this study were premature infants with a birth weight of 750 to 1250 g that was appropriate for gestational age according to the charts of Usher and McLean (11). Excluded from the study were infants who had major congenital anomalies or gastrointestinal or liver diseases. All infants' Clinical Risk Index for Babies (CRIB; 12) scores on the first day of life were <5. The maximum CRIB score is 23, and the minimum score is 0 (with increasing scores, there is increased morbidity and mortality). Selected relevant clinical variables for the infants studied are shown in Table 1. The infants received a standard nutrient regimen according to our feeding protocol: a combination of the mother's milk or formula (Nenatal; Nutricia, Zoetermeer, Netherlands; 0.024 g/mL protein) and parenteral nutrition containing glucose, AAs (Primene 10%; Clintec Benelux NV, Brussels, Belgium; 0.1 g/mL protein), and lipids (Intralipid 20%; Fresensius Kabi, Den Bosch, Netherlands). Formula feeding was given as the sole enteral nutrition 12 h before the start of the study and during the study days.
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TABLE 1. Subject characteristics1
Written informed consent was obtained from the parents of the infants. The study protocol was approved by the Institutional Review Board of the Erasmus Medical Center.
Protocol
The study design consisted of 2 periods of one study day (period A: study day 1; period B: study day 2). During period A, the infants received 40% enteral feeding and 60% parenteral feeding; during period B, they received full enteral feeding. A schematic outline of the tracer-infusion studies is shown in Figure 1. During period A, both an arterial and an intravenous catheter were implanted in the infants for the infusion of tracers and withdrawal of blood samples. These catheters were already installed for clinical purposes. During period B, a peripheral intravenous catheter was available for the infusion of tracers, and blood samples were collected by heelstick.
FIGURE 1.. Schematic overview of study periods A (study day 1) and B (study day 2). IG, intragastric; IV, intravenous.
To collect breath samples from these preterm infants, we employed the method described by Perman et al (13), which used a nasal tube. This method has been validated in preterm infants for the collection of expiratory carbon dioxide after the administration of 13C-labeled substrates (14, 15). Briefly, a 6-Fr gastric tube (6 Ch Argyle; Sherwood Medical, Tullamore, Ireland) was carefully placed 1.0 cm into the nasopharynx, and a 15-mL sample of end-tidal breath was slowly taken with a syringe. Duplicate aliquots of expired air from each sampling point were stored in evacuated tubes (Vacutainer; Becton Dickinson, Rutherford, NJ) for later analysis.
Three stable-isotope infusions were performed on each study day during both periods. First, a primed, continuous infusion [10.02 µmol/kg priming dose and 10.02 µmol/(kg · h) of [13C]sodium bicarbonate (99 mol% 13C; Cambridge Isotopes, Woburn, MA) dissolved in sterile saline was administered at a constant rate for 2 h. The 13C-labeled bicarbonate infusion was immediately followed by primed, continuous infusion [14.4 µmol/kg priming dose and 14.4 µmol/(kg · h) of [U-13C]threonine (97 mol% 13C; Cambridge Isotopes) given intravenously and a second primed, continuous infusion [14.7 µmol/kg priming dose and 14.7 µmol/(kg · h) of [15N]threonine (95 mol% 15N; Cambridge Isotopes) given enterally for 5 h. This process was designed to assess whole-body and splanchnic threonine kinetics. All isotopes were tested and found to be sterile and pyrogen-free before they were used in our studies. Baseline blood and breath samples were collected at time 0. During the last hour of each tracer infusion, breath samples were collected at 15-min intervals, and blood samples were obtained at 390 and 420 min. The total amount of blood drawn on a study day was 1.5 mL, which is <2% of the blood volume of a 1000-g infant. Blood was centrifuged immediately (2500 x g, 4 °C, 10 min) and stored at –70 °C for further analysis.
Analytic methods
Small aliquots of plasma (100 µL) were taken for the measurement of AA concentrations by using an analyzer (Amino Acid Analyser Biochrom 20; Biochrom Ltd, Cambridge, United Kingdom). Plasma threonine enrichments were determined by gas chromatography–mass spectrometry. Briefly, 50 µL plasma was deproteinized with 50 µL of 0.24 mol sulfosalicylic acid/L. After centrifugation for 8 min at 4 °C and 14 000 x g, the supernatant was passed through an H+ column (AG50W-X8; Biorad, Richmond, VA). The column was washed with 3 mL water, and the AAs were eluted with 1.5 mL of 3 mol NH4OH/L. The eluate was dried at 70 °C under a stream of nitrogen, and, finally, derivatives of the AAs were formed by adding 350 µL acetonitril and 10 µL N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (Pierce Omnilabo, Breda, Netherlands) to the dried AAs (16). Analyses were carried out on a Carlo Erba GC8000 gas chromatograph coupled to a Fisons MD800 mass spectrometer (both: Interscience BV, Breda, Netherlands) by injecting 1 µL with a split ratio of 50:1 on a 25-m x 0.22-mm fused silica capillary column, coated with 0.11 µm HT5 (SGE, Victoria, Australia). Natural threonine, [15N]-threonine, and [U-13C]-threonine were measured by selective ion monitoring of masses 404, 405, and 408, respectively (16). Breath samples were analyzed for enrichment of 13CO2 on an isotope ratio–mass spectrometer (ABCA; Europa Scientific, Van Loenen Instruments, Leiden, Netherlands) (17).
Calculations
The rate of threonine turnover was calculated by measuring the tracer dilution at steady state as modified for stable isotope tracers, as previously described (18, 19). Plasma enrichments of threonine were used to calculate the rate of threonine turnover.
The threonine flux (intravenous or intragastric) was calculated according to the following equation:
RESULTS
All infants were appropriate for gestational age (mean gestational age: 29 ± 2 wk; Table 1). Seven patients were studied during both periods; in 5 of those infants, whole-body threonine oxidation was determined. During both feeding periods, 1 infant underwent mechanical ventilation, 4 infants received supplemental oxygen via a nasal prong, and 3 infants received nasal continuous positive airway pressure (CPAP), which did not allow us to obtain expired air. All infants received caffeine and were clinically stable at the time of the study. All routine blood chemistry and hematology tests (ie, electrolytes, calcium, glucose, acid base, hematocrit, thrombocyte count, and white blood cell count) were within normal limits; there were no significant changes in these variables in the periods 24 h before and after the study. Intakes of threonine, protein, carbohydrate, fat, and energy are shown in Table 2. We aimed to keep the different macronutrient intakes very close together during both study periods, but this was impossible because different feeds (parenteral nutrition and preterm formula) were used. No differences were found in the intakes of glucose, fat, or energy. The threonine intake was significantly lower during period A, but, in absolute amounts, the difference was only 5 µmol/(kg/d) (ie, 8% less). The protein intake during period B was significantly lower than that during period A, which was inevitable, because we aimed at comparable total threonine intakes.
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TABLE 2. Intakes of threonine, protein, carbohydrate, fat, and energy during period A and period B1
Isotopic plateau
Threonine kinetics were calculated from the plateau enrichment values for plasma threonine, breath carbon dioxide, and rates of carbon dioxide production. Details of the isotopic enrichments of plasma threonine at baseline and plateau during periods A and B are given in Figure 2.
FIGURE 2.. Mean (±SD) isotopic enrichments of plasma threonine at baseline and plateau during period A (A) and period B (B). , [U-13C]threonine; , [15N] threonine.
The background (baseline) recovery of the [13C]label in expiratory air did not differ significantly between the 2 periods [period A: 1.0955 ± 0.0079 atom percent excess (APE); period B: 1.0923 ± 0.0032 APE]. The 13CO2 enrichment in breath during [13C]sodium bicarbonate infusion rose rapidly during the first hour of infusion in both periods to become constant in all infants by 120 min, with <5% variation of the plateau (CV: 1.4 ± 0.4% in period A and 3.3 ± 1.4% in period B). In Figure 3, the isotopic steady state of 13CO2 excretion in expiratory air is shown both during the [13C]sodium bicarbonate infusion and the [U-13C]threonine infusion. Although we took 2 blood samples after 4 h of tracer infusion, we are sure that isotopic steady state was reached during the [U-13C]threonine infusion because we found an isotopic plateau in carbon dioxide excretion. Before a plateau in breath is reached, a plateau has to be reached at the site of threonine oxidation—ie, intracellularly. The mean ± SD CVs of breath [13C]threonine enrichment above baseline at plateau were 3.4 ± 4.4% during period A and 8.5 ± 0.6% during period B. We considered this variation to be acceptable and to permit the detection of differences in rates of threonine oxidation between the 2 feeding periods.
FIGURE 3.. Mean (±SD) isotopic steady state in 13CO2 excretion in expiratory air during the [13C]sodium bicarbonate infusion (0–120 min) and the [U-13C]threonine infusion (120–420 min). A, period A; B, period B; APE, atom percent excess.
Threonine kinetics
The plasma concentrations of all AAs in both feeding periods are shown in Table 3. We did not find a statistically significant difference in the plasma threonine concentration between the 2 feeding periods (period A: 169 ± 111 µmol/L; period B: 201 ± 98 µmol/L), but the concentrations showed a large variation. The high first-pass threonine uptake during both periods is shown in Figure 4. The first-pass threonine uptake, expressed as a percentage of dietary intake, was significantly higher during low enteral intake. Yet, approximately three-quarters of the dietary intake was utilized by the splanchnic tissues during full enteral feeding. However, and in contrast to our hypothesis, the first-pass threonine uptake, in absolute amounts, was significantly (P < 0.0001) lower during restricted enteral threonine intake [period A: 24 ± 7 µmol/(kg/h); period B: 44 ± 4 µmol/(kg/h)].
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TABLE 3. Plasma amino acid concentrations during period A and period B with a reference range in term breastfed infants1
FIGURE 4.. First-pass threonine uptake in 8 preterm infants during periods A and B expressed in absolute amounts [µmol/(kg/h)] and as a fraction of dietary intake. APE, atom percent excess. *,**Significant difference between periods: *P < 0.01, **P < 0.0001.
The threonine kinetics are shown in Table 4. There was no significant difference between the 2 feeding periods in the amount of threonine oxidized by the whole body [period A: 15 ± 9 µmol/(kg/h); period B: 17 ± 13 µmol/(kg/h)]. During both intakes, whole-body threonine oxidation accounted for 6% of threonine flux. The nonoxidative threonine disposal and the threonine release of protein did not differ significantly between the 2 periods. Whole-body threonine balance, calculated from the difference between nonoxidative disposal and whole-body protein degradation, was positive during both feeding periods and did not differ significantly between the 2 periods.
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TABLE 4. Whole-body threonine kinetics quantified in 8 preterm infants during restricted enteral intake (period A) and full enteral intake (period B)1
DISCUSSION
This study describes the splanchnic threonine kinetics in preterm infants during early postnatal life. The splanchnic tissues play an essential role in the delivery of dietary AAs to peripheral tissues. The systemic appearance of a given AA is a result of adequate digestion, transport, and intracellular metabolism. Because threonine is an EAA, high splanchnic utilization may result in the depletion of the systemic plasma pool of threonine.
Therefore, the most striking observation to emerge from this study was the very high fractional first-pass threonine uptake by the intestine in the first week of life in preterm infants during a restricted enteral intake. This observation indicates a high obligatory visceral need for threonine in neonates. Although the gastrointestinal tissues represent only 5% of body weight, because of their high rates of metabolism, they account for 15%–35% of whole-body oxygen consumption and protein turnover (22–24). Of the many factors that affect neonatal gut growth and adaptation, probably the most physiologically significant stimulus is enteral nutrition (25, 26). Enteral feeding acts directly by supplying nutrients for the growth and mucosal metabolism of epithelial cells. In the present study, we show that up to 82% of specific nutrients are utilized in first-pass uptake. We hypothesize that this mainly represents intestinal tissue utilization. We measured first-pass splanchnic uptake, which includes hepatic uptake. However, neonatal animal studies suggest that the intestine is the major site of utilization.
The route of administration of nutrition is a major issue in the clinical care of preterm infants, because of these infants' intolerance of enteral feeding and the associated morbidity (27). To reduce the complications of TPN and to accelerate the adaptation to full enteral feeding, many neonatologists often provide small volumes of enteral nutrition—ie, minimal enteral feeding—in combination with TPN to preterm infants in the first weeks of life (28). Studies in neonatal piglets showed that an enteral intake of 20% is necessary to prevent gut protein loss, whereas an intake of 40% is needed to maintain normal growth (29, 30). In the present study, preterm infants were enterally fed 40% of their total nutrient intake during period A, and the results show the fractional first-pass threonine requirements were significantly higher (82% of dietary intake) than those of fully fed infants (70% of dietary intake). Because the first-pass uptake is upregulated, even at 40% of enteral intake, this finding indicates that the enteral requirement is not yet reached.
The high enteral threonine uptake observed during both partial and full enteral feeding reflects the use of absorbed threonine for the synthesis of secretory glycoproteins, for the synthesis of mucosal cellular proteins, or for oxidative purposes (2–4). In neonatal piglets, we did not find substantial first-pass threonine oxidation, which indicates that enterally absorbed threonine is mostly used for the other 2 metabolic pathways. Although the intestinal mucosa is highly secretory and proliferatory tissue, dietary threonine is not incorporated into constitutive mucosal proteins to a great extent (4). However, the intestinal mucosa is protected by a complex network of glycoproteins, and the core proteins of the highly glycosylated domains of intestinal mucins contain large amounts of threonine (31). It is likely that a significant proportion of the utilized threonine is channeled toward mucin production. These secretory mucins play a key role in the defense of the mucosa. In fact, there is evidence that mucin production is impaired in piglets fed threonine-deficient diets, and supplying threonine parenterally cannot restore normal mucin production (10). Moreover, recent studies suggested that the restriction of dietary threonine significantly and specifically impairs intestinal mucin synthesis and consequently reduces gut barrier function (32, 33). Especially in preterm infants who are vulnerable to infections during the first weeks of life, mucus would be an important aspect of defense.
A second aim of this study was to determine whole-body threonine kinetics in neonates under 2 different feeding circumstances. During both partial and full enteral feeding, whole-body threonine oxidation accounted for 6% of the threonine flux, which is comparable to the fractional oxidation rates found by Darling et al (34) in breastfed preterm infants. However, Parimi et al (35) recently reported a higher fractional oxidation rate of threonine in newborn infants, although the total threonine intake was substantially lower.
In conclusion, the present study showed that the splanchnic tissues of preterm infants use the dietary threonine intake to a substantial degree—ie, more than three-quarters—irrespective of the amount of enteral threonine delivery. Furthermore, our data show that <10% of the threonine flux is oxidized, and the route of feeding does not affect this whole-body threonine oxidation. Overall, we suggest that the major metabolic fate of intestinal utilized threonine is mucosal glycoprotein synthesis.
ACKNOWLEDGMENTS
We thank Professor Doctors Dick Tibboel and Hans A Büller of Erasmus-MC Sophia Children's Hospital for helpful comments and review of the manuscript and Peter Reeds (since deceased), who inspired us to undertake the mission. We especially thank the parents who provided consent for their infants to participate in the study.
The authors' responsibilities were as follows—SRDvdS: recruitment of the participants, blood and breath sample collection, preparation and analysis of the data, and writing of the manuscript; DLW and JH: data analysis; JBvG (principal investigator): study design and supervision; and AV: preparation of stable isotopes. None of the authors had a personal or financial conflict of interest.
REFERENCES