Protein Metabolism in the Extremely Low–Birth Weight Infant

~ ~ NUTRITION AND METABOLISM OF THE MICROI'REMIE zyxwvutsrqponmlkjihgfedcbaZYXW 0095-5108/00 $15.00 + .OO PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT Satish C. Kalhan, MBBS, and Sabine Iben, MD z A large body of literature has been published over the years describing protein and nitrogen metabolism in the neonate, both in relation to gestational age and to optimal nutritional management for protein and nitrogen accretion, and growth. Such data are also extremely difficult to interpret, however, because of the impact of a number of confounding variables. Such is the case with the micropremie (extremely low-birth weight [ELBW] infant), who at present constitutes a majority of the population of neonatal intensive care units. Although major improvements have been made in the immediate postnatal care of these infants with the use of assisted ventilation, surfactant, antepartum glucocorticoids and so forth, these neonates tend to remain in a catabolic state for a prolonged period after birth due to difficulties in the optimal administration of nutrients and due to other metabolic perturbations related to immaturity. By the time the micropremie reaches a corrected gestational age of 40 weeks, most growth parameters may remain subnormal. Thus, physiologic data obtained during this time period have to be evaluated carefully and the numerous confounding variables carefully considered. In addition, these infants because of their small size present a considerable challenge to the investigator performing nutrition studies in a meticulous fashion. These studies, by their very nature, are prolonged and require repeated sampling of blood and other biologic materials. The difficulties in obtaining accurately timed urine samples for nitrogen balance studies are well known. Other variables to be considered include multiple blood and other transfusions that are often given to these infants, the inability to discontinue glucose and electrolyte solutions in order to obtain The cited studies from the investigator's laboratory were supported by grants HD11089 and RROOO80 from the National Institutes of Health. From the Robert Schwartz, MD, Center for Metabolism & Nutrition, and Department of Pediatrics, MetroHealth Medical Center, Case Western Reserve University School of Medicine, (SCK); and the Division of Neonatology, Rainbow Babies & Children's Hospital (SI), Cleveland, Ohio zyxwvut CLINICS IN I'ERINATOLOGY ~ VOLUME 27 NUMBER 1 * MARCH 2000 23 24 zyxwvutsr zyxwvuts KALHAN & IBEN fasting data, and other interventions necessary for the management of such immature infants (e.g., ventilators, therapeutic agents for respiratory and metabolic acidosis, vasopressors, anteparturn and postpartum steroids, and other pharmacologic agents, medications that may have a direct or indirect as yet unrecognized effect on protein metabolism). In spite of these limitations, significant new data have been obtained in recent years. This article attempts to include only those studies where the possible confounding variables have been clearly described, and makes every attempt to separate the data of healthy neonates from those who require significant clinical support. Various factors that can potentially impact protein-nitrogen metabolism in the newborn are listed as follows: 1. Ontogeny 2. Adaptation to extrauterine environment 3. Energy intake: quantity, quality 4. Protein intake: quantity 5. Sickness or stress with or without infections 6. Growth zyx Ontogeny, or development, is associated with the expression of certain genes and hence of corresponding protein at specific stages, which effects the measurements of protein metabolism. As discussed later, the activity of enzymes related to urea synthesis is low early in gestation and their activity is signifi97, *07, lo* The process of cantly lower than adult levels, even at term ge~tation.4~. birth and adaptation to extrauterine life is associated with marked hormonal surges, initiation of respiration, temperature regulation, and other processes 95,, 99 Therefore, measurerelated to release of energy-generating s ~ b s t r a t e s .88,~ ~ ments performed during this transitional period are influenced by these adaptive responses. Because of the difficulty in administration of appropriate quantity of energy and nutrients, partly owing to logistical problems and partly owing to the immaturity of digestive and absorptive functions, the preterm infant often does not receive adequate energy. In the absence of adequate calories, the organism has to rely on endogenous sources including protein. In addition, as discussed later, even in the presence of adequate calorie intake but without adequate protein intake, the neonate remains in negative nitrogen balance. These factors need to be considered for the accurate interpretation of data. Stress or intercurrent illness with associated hormonal and cytokine responses have been shown to have profound effects on protein metabolism in different body compartments, including increased production of certain (acute phase) proteins and change in turnover of structural proteins.41,45, 140*149 Finally, growth and protein accretion are associated with unique changes in protein turnover. Interpretation of data in the neonate requires careful consideration of all these confounding variables. zyxw zyxwv zyxw ENDOGENOUS ENERGY STORES IN THE FETUS AND NEONATE Based upon available data on body composition of the fetus and neonate, an estimate can be made of the available sources and quantity of energy stored by the infant. Ziegler et and F o m ~ used n ~ ~the published data from chemical analyses of the human fetus and newborn to construct a reference fetus and neonate of representative body composition. Table 1 is adapted from their data. As shown, in the very low-birth weight (VLBW) neonate, a very large fraction of body weight is represented by water. Recent studies using tracer dilution zy z zyxwvuts PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 25 Table 1. AVAILABLE ENERGY STORES IN THE FETUS AND NEWBORN Weeks 24 26 28 30 32 34 36 40 2 mo Wt (9) Water* Nonprotein Protein Lipid Glycogen (9) Energy (kcal) 690 880 1160 1480 1830 2230 2690 3450 5450 88.6 86.8 84.6 82.6 80.7 79 77.3 74 63.7 8.8 9.2 9.6 10.1 10.6 11.1 11.4 12 11.4 0.1 1.5 3.3 4.9 6.3 7.5 8.7 11.2 22.4 3.5 4.5 5 6.5 8 8.5 11.5 15.3 25 19.5 123.6 326.2 606.2 954.3 1372.1 1918.2 3152.4 9866 (%I (%I zy *Based upon mean data on liver weight and assuming glycogen = 10% of liver weight. Adnptedfiom Fomon SJ: Body composition of the male reference infant during the first year of life. Pediatrics 40863, 1967, and Ziegler EE, ODonnell AM, Nelson SE, et al: Body composition of the reference fetus. Growth 40329, 1976; with permission. methods have confirmed these observation^.'^^ Because water mainly represents fat-free mass, it can be inferred that the low-birth weight infant has very little fat stores. As shown, the very premature infant has negligible quantities of readily available sources of energy (i.e., fat and glycogen), so that in the absence of available exogenous sources of nutrition, the premature infant has to rely upon protein as a possible energy source. Calculations made by a number of investigators based upon such data have shown that if the neonate consumes 50 to 60 kcal*kg-'day-' for energy expenditure, the nonprotein energy ,stores are enough for less than 1 day for the neonate born at 24 weeks gestation, whereas they may last for several (-15) days in the full-term infant.69 From the perspective of protein stores, a preterm neonate born at 24 weeks gestation has only 60 g of protein (8.8% of 690 g body weight, see Table 1).If one assumes an obligatory nitrogen loss of 5 mgN-kg-lh-', which is equivalent to 0.78 gkg-'d-' of protein or 0.54 g for a 690 g infant, such an obligatory nitrogen-loss represents approximately 1% of total body protein stores. Of course, such losses are increased in the absence of adequate nonprotein nitrogen intake and in the presence of intercurrent illnesses leading to catabolic states. QUANTIFICATION OF PROTEIN METABOLISM Several different methods have been used to quantify protein metabolism in vivo in children and adults as follows: Nitrogen balance 3-methylhistidine excretion (myofibrillar protein) Isotopic tracer kinetics Nitrogen turnover ([15N]glycineend product method) Individual essential amino acid kinetics (leucine, phenylalanine, and so forth) zy Their respective limitations have been discussed in several review^.^, 90, 141 Nitrogen balance technique, which requires careful and precise measurements of total nitrogen intake and loss from the body, does not provide any 26 zyxwvuts zyxwvutsr zyxwvuts KALHAN & IBEN insight into the mechanism(s) of the changes observed. Although the intake can be precisely determined, accurate determination of losses, particularly extraurinary losses via skin and breath (i.e., as ammonia), can be problematic. In addition quantitative collection of urine and stools in the infant can be difficult. Furthermore, as discussed under urea metabolism, not all of the urea synthesized in vivo may be excreted in the urine due to hydrolysis in the gut. 3-Methylhistidine (3-MH) is a constituent of actin and heavy chain of myosin in the white muscle and, therefore, skeletal muscle represents the largest pool of 3-MH in the body."', I5O It is released on protein breakdown, is not reutilized for protein synthesis, and is quantitatively excreted in urine. Because creatinine excretion is proportional to skeletal muscle mass, the 3-MH-creatinine ratio in the urine has been used as a measure of the fractional rate of degradation of myofibrillar protein. The limitations of this method have been discussed by Rennie and Millward."' Based upon direct studies of skeletal muscle protein turnover using isotopic tracers, these investigators have described large difference in actual rate of protein degradation measured by tracers and those estimated by 3-MH-creatinine ratio in the urine. They suggested this discrepancy was related to the contribution of 3-MH in urine from gastrointestinal tract. Long et al,79,80 however, have demonstrated the usefulness of this method based upon their studies in adults with short bowel syndrome. Isotopic tracers of amino acids are the most commonly used methods to study protein turnover in vivo. These methods either use nonessential amino acids, such as glycine, to quantify whole body nitrogen turnover or are based upon measurements of kinetics of a representative essential amino (e.g., leucine, . ~ techniques have been further modilysine, phenylalanine, and so f ~ r t h )These fied depending upon route of tracer administration (i.e., enteral versus parenteral); methods of administration (i.e., single dose versus continuous infusion); and measurement of isotopic tracer in the amino acids or in the end products, such as breath CO, urinary ammonia, or urea. A detailed discussion of these methods is beyond the scope of this presentation. Although the methods based upon measurements of kinetics of a representative essential amino acid should give similar measure of the dynamic aspect of protein turnover, such an assumption has not been validated across all age groups and during development. As shown by van Toledo-Eppinga et al,I3' the relative kinetics of phenylalanine and leucine in low-birth weight infants were not similar to those observed in adults, nor were they identical to the theoretically proposed ratio in mixed body proteins. In fact, the relative rate of turnover of phenylalanine and leucine in low-birth weight infants, expressed as the ratio of Ra phe/Ra leu (Ra = rate of appearance; phe = phenylalanine; leu = leucine) was lower than expected from reported whole body protein composition and from that reported in adults. These data suggest that there is a differential contribution of various body protein to whole body mixed protein turnover in low-birth weight infants compared with adults. These considerations are important in the measurement of whole body protein dynamics because estimates using single amino acid kinetics may not adequately represent the dynamic aspects of whole body protein metabolism. zyxwvut zyxw PROTEIN TURNOVER IN THE NEONATE Before presenting the limited data on protein turnover in the micropremie, it is important to consider the available data in the full-term and the premature infant. zy z zyxwvu PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT Full-Term Infants During Fasting 27 Because prolonged caloric deprivation of the neonate is considered clinically and ethically unacceptable, very few studies have examined the basal rates of protein turnover during fasting. We could not find any data in VLBW neonates during fasting. Only one study has examined the rates of leucine-protein turnover in full-term normal healthy newborns following 9 hours of nutrient deprivation.28Rates of turnover and oxidation of leucine were measured in 12 normal newborns during the first 3 days after birth using [l-13C]leucinetracer. Their data are displayed in Table 2 and are compared with 11 healthy adults studied after 17 hours of fasting. That both groups were in similar postabsorptive state was evident by the significant increase in plasma ketones (betahydroxybutyrate) in both groups. The plasma concentration of leucine in the neonates was significantly lower, whereas the rate of turnover (flux) of leucine quantified by tracer dilution was significantly higher than that in adults. Because leucine flux represents protein breakdown or turnover during steady state, these data show that healthy newborn infants have a high rate of protein turnover compared with adults. The newborn infants also had a higher rate of oxidation of leucine as well as a higher rate of basal energy expenditure (see Table 2). An interesting observation of this study was a significant positive correlation between the birth weight of the neonate and the rate of turnover of leucine normalized to body weight (Fig. 1).The exact significance of this association remains unclear. As discussed by the investigators, it may represent the impact of prenatal protein and calorie supply. Therefore, these data show that a full-term healthy neonate during fasting has a higher rate of leucine-protein flux and oxidation, and basal energy expenditure. zyxwv Effect of Nutrient Administration Several studies in full-term and preterm neonates have examined the impact of macronutrient, either individually or as mixtures, on whole body proteinamino acid kinetics in both term and preterm infants. Like other studies in the newborn infant, all of these data suffer from the same problems (i.e., lack of normalcy). The majority of these studies were done on infants who were in the intensive care unit for one reason or the other, and although clinically healthy, the infants were receiving antibiotics for presumed sepsis. In particular, the zyxwvuts zyxwvuts zyxwvuts Table 2. LEUCINE-PROTEIN KINETICS IN NORMAL NEWBORN INFANTS AND ADULTS Leucine Flux Leucine Oxidation pmol-kg-'h -l pmobkg -lh -l % of flux Infants (12) Adults (11) P 164 2 28 87215 <0.001 34 Ifr 11 1523 <0.001 2229 17?3 <0.05 Protein Turnover Basal Energy Expenditure gakg-ld - I kcal-kg-Id - l 6.721.1 3.5 +. 0.6 <0.001 45.1 2 4.6 23.6 2 3.8 <0.001 Values are mean? SD. From Denne SC, Kalhan SC: Leucine metabolism in human newborns. Am J Physiol253:E608, 1987; with permission. 28 zyxwvutsr zyxwvuts zyxwvut KALHAN & IBEN zyxw P 4.005 a I 2.5 1 I I 3.0 3.5 4.0 Birth Weight (kg) Figure 1. Relation between birth weight and whole-body leucine flux in full term healthy neonates. (Modified from Denne SC, Kalhan SC: Leucine metabolism in human newborns. Am J Physiol 253:E60&E615, 1987; with permission.) zyxwv preterm infants were receiving parenteral fluids containing glucose or were receiving ventilatory support. In addition, because the current feeding practice in the neonate involves frequent (i.e., 3 to 4 hourly) feedings and because of the various rates of gastric emptying, a neonate cannot be considered to be in a fasting state unless they are deprived of food for several hours. In the following, we have presented the studies that were conducted in what appears to be a truly healthy newborn population. Enteral Nutrition. Protein kinetics were measured in 11 normal healthy fullThe term infants during the first 3 days after birth using [l-'3C]leucine tra~er.3~ neonates were studied while they were receiving hourly oral feedings of a standard commercial formula (4 ml-kg-lh-'), corresponding to 1.44gkg-ld protein, 64 kcabkg-Id-' and 47 p,mol*kg-'h-' of leucine. Their energy expenditure calculated from the rate of oxygen consumption corresponded to 68% of energy intake. The data were compared with another group of full-term healthy neonates studied previously during fasting. As shown in Table 3, in response to administration of protein (i.e., leucine) plus other nutrients, there was an increase in the rate of appearance of leucine in plasma; however, the contribution of leucine released from protein breakdown to the total flux remained unchanged. Furthermore, the rate of oxidation of leucine in fed and fasted neonates was similar so that most of the additional leucine administered in the feed was disposed via the nonoxidative pathway, presumably for protein synthesis. These data suggest that normal healthy neonates respond to enteral nutrient administration by increasing protein synthesis. This is in contrast to data in adults where the primary response to nutrient administration consists of suppression of protein breakdown. As discussed by the investigators, the observed lack of decrease in proteolysis in response to formula feeding may be either a unique neonatal response in these infants or may be the consequence of (1) cross- zy z zyxwvuts 29 PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT zyxwvu zyxw zy Table 3. LEUCINE KINETICS IN FULL-TERM BABIES IN RESPONSE TO FEEDING Total Flux Release from Protein Breakdown Oxidation NOD 201 f 16*t 164f8 156f 16 164k8 31 f 4 3423 170f13' 129& 9 Fed (11) Fasted (12) "P <0.05 fed versus fasted. t pmol.kg-'h-'. NOD = nonoxidative disposal. From Denne SC, Rossi EM, Kalhan S C Leucine kinetics during feeding in normal newborns. Pediatr Res 3033, 1991; with permission. zyxwvu sectional nature of the study resulting in inability to detect a small change, if present, in proteolysis induced by feeding, or (2) because of problems related to tracer modeling, because the tracer was administered intravenously and some of the dietary leucine may have been taken up in the first pass through the splanchnic circulation.6,24, 89 Parenteral Nutrition Glucose. The impact of carbohydrate energy (i.e., glucose) on proteolysis and irreversible protein loss has been examined in adults. Studies performed in normal healthy adults on a raft diet show that provision of carbohydrate equivalent to approximately 20% of daily energy requirement can reduce the irreversible protein-nitrogen loss to a minimum.42No such data have been obtained in human neonates for ethical considerations. The question is important because of inability to provide total energy requirements, particularly in the period immediately after birth, due to intercurrent illnesses and difficulty in administering large amounts of fluids. The response of intravenous glucose administration at 5.5 mg-kg-lmin-' (7.9 g-kg-ld-l 30 kcal-kg-'d-') on whole body rate of proteolysis as measured by the rate of appearance of leucine (Ra) was examined in healthy full-term infants (n= 11)by Denne et al.32The infants were infused with glucose starting at 2 hours prior to tracer infusion and throughout the study for 5 hours, and their data were compared with another group of healthy infants (n=10) who were last fed approximately 7 to 8 hours prior to obtaining the tracer isotopic data. The rates of appearance of leucine measured by isotopic tracer dilution during steady state showed no effect of glucose infusion on proteolysis (fasting 243 2 41 p,mol*kg-'h-'; glucose 255 2 51 pmol-kg-'h-', mean k SD). The investigators did not measure the rate of irreversible protein (leucine C) loss in this study. Of interest, the plasma glucose and insulin concentrations were significantly higher in the glucose-infused group (Glucose: fasting 74 2 13 mg.dL-', glucose 89 2 13 mg.dL-'. Insulin: fasting 2.2 2 1 pU-mL-', glucose 6.2 2 3.7 p,U*mL-'). The lack of response in proteolysis may be due to (1) a requirement of high rate of protein turnover during periods of rapid growth; (2) a small effect may not be detected by the methodology used; (3) that the fasting infants were not in a true state of fasting, having received their last feed 7 to 8 hours before measurements. It is important to separate protein sparing, which is a decrease in irreversible nitrogen loss often equated with protein oxidation, from proteolysis, which is protein breakdown and release of amino acid but not oxidation. The former is probably more responsive to the availability of energy, whereas the latter, being an important component of tissue remodeling and protein accretion, may not be as responsive to nutrient administration. zyxwvutsrq - zyxwvu 30 zyxwvutsr zyxwvutsr zyxwvuts KALHAN & IBEN Lipids. Similar to the administration of intravenous glucose, administration of Intravenous lipid emulsion in equivalent amounts (-28 kcal-kg'd-' or 150 mg-kg-'h-') did not have any effect on rates of leucine turnover or protein breakdown in acute 5 hour study.32 Glucose Plus Lipids. Simultaneous administration of glucose (5.5 mgkg-'min-') and Intralipid (150 mg.kg-'h-') at a rate providing the entire basal energy requirement (-60 kcabkg-Id-') also did not have any impact on whole body rate of proteolysis (leucine Ra). In this study also, the plasma concentration of glucose (91 -t 15 mg-dL-') and insulin (3.3 ? 1.6 yU-mL-') was significantly higher than that in the fasting Mixed Nutrients. The response to mixed nutrients (glucose 6 mg.kg-'min-', lipid 165 mg-kg-'h-', amino acid 105 mg.kg-'h-' = 90 kcalskg-'d-') on leucine and phenylalanine kinetics were quantified by Denne et al.29Infants were studied 3 to 4 hours after a normal feed, fasting data were obtained between 120 and 180 minutes after the start of tracer infusion, and response to nutrients was examined during the 120 to 150 minutes of parenteral nutrition administration. The plasma glucose and insulin concentrations were significantly higher during parenteral nutrition (glucose: basal 74 f 13, parenteral nutrition 108 f 8 mg.dL-'; insulin basal 3 f 1, parenteral nutrition 24 5 yU*mL-']. These concentrations are much higher than those obtained in previous studies where glucose was administered alone or with lipids. The calculated rate of endogenous leucine and phenylalanine Ra (proteolysis) was significantly reduced in response to the parenteral administration of mixed nutrients. These data suggest that nutrients administered via the parenteral route with a high rate of energy intake lead to a significant decrease in the rate of proteolysis in healthy term newborn infants. Stochastic calculations of this tracer model lead one to conclude that nutrient administration was also associated with an increased rate of protein synthesis. Effect of Change in Carbohydrate:Fat Ratio. Jones et aP6 examined the impact of change in carbohydrate and fat ratio on protein metabolism in postoperative infants who were receiving total parenteral nutrition. Their study group consisted of near-term infants (gestational age -36 weeks) who had been operated for gastrointestinal tract anomalies. The neonates were recruited at least 4 days after surgery and were receiving 86 kcal-kg-'d-' and amino acid intake of 2.5 g'kg-Id-'. Protein metabolism was examined using [l-'3C]leucine tracer and nitrogen balance measurements. A change in g1ucose:fat ratio from 0.55 to 0.95 in the presence of isonitrogenous isocaloric intake had no impact on rates of protein turnover, synthesis, breakdown, oxidation, or nitrogen balance. These studies in neonates are in contrast to those in older infants,I5 which show higher rates of protein turnover and oxidation when glucose was given alone with a constant protein intake as compared with an isocaloric mixture of glucose and fat. Of interest, in these infants the rate of oxygen consumption was higher than previously reported, and all infants were recovering from gastrointestinal illness. Nevertheless, the study was strengthened by a crossover design, with every patient having received two randomly assigned 8-day periods of isocaloric and isonitrogenous infusions differing only by the source of calories. zyxw z + zyxwvu zyxwv Low-Birth Weight Infants Nitrogen Balance and Nitrogen Turnover Earlier studies of whole body nitrogen-protein turnover were performed using ['5N]glycine tracer according to the end product method described by PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT zy 31 Picou and Taylor-Robert~,'~~ wherein 15Nenrichment of end products of protein metabolism (i.e., urea and ammonia) are measured following the administration of [15N]glycine in order to quantify the dynamic aspects of protein turnover. Because the investigative protocol requires the administration of tracer by repetitive oral doses and collection of urine for the measurement of 15Nenrichment in urea or ammonia, the majority of these studies were performed in the fed state (i.e., during administration of nutrients in combination with measurements of 35, 36, 96, 97, lol, lo4 Because these studies were done in enterally nitrogen balan~e).'~, fed infants, all of them include newborns around 30 to 33 weeks gestation, birth weight less than 1600 but more than 900 g, and who were studied at variable times after birth and were gaining weight. These data show that protein turnover in growing preterm neonates measured by [I5N]glycineend product method is consistently high (range 8 to 16 gkg-ld-') compared with older children and adults. As shown by Catzeflis et all9 and by 0thers,3~, 70-72,97, Io4 a significant positive relationship exists between (1) nitrogen intake and nitrogen balance (Fig. 2), (2) whole body rate of protein synthesis and the rate of protein gain (Fig. 3), and ( 3 ) resting energy expenditure and protein gain (Fig. 4), so that for each gram of protein gain, there is simultaneous increase of approximately 10 kcal of energy expenditure. The intercept at zero protein gain indicates that about 40 kcabkg-Id-' are expended to maintain zero energy ba1an~e.I~ These data are significant in that they provide an important understanding of the relation between energy expenditure, protein turnover, and the energy cost of protein gain. To our knowledge, no such studies have been done in healthy fullterm infants and in the ELBW infant for the clinical reasons discussed previously. zyxwvu zyxw zyx zyxwvutsrqp zyxwvutsr Leucine Kinetics Several investigators have reported the rates of appearance of leucine and changes in protein turnover in response to enteral and parenteral nutrient N Intake (gokg-Id-1) zyx Figure 2. Relationship between nitrogen intake and nitrogen balance in preterm infants. (Modified from Catzeflis C, Schultz Y, Micheli J, et al: Whole body protein synthesis and energy expenditure in very low-birth weight infants. Pediatr Res 19:679-687, 1985; with permission.) 32 zyxwvuts zyxwvutsr zyxwv KALHAN & IBEN zyxwv zyxwvut 2o - 15 - 10 0 y = 1.7 t 5 . 2 ~ r = 0.68 P<0.05 - 0 I I I I I I 0.5 1.o 1.5 2.0 2.5 3.0 Protein Gain (g*kg-Id-1) Figure 3. Relationship between protein gain and protein synthesis. (Modified from Catzeflis C, Schultz Y, Micheli J, et al: Whole body protein synthesis and energy expenditure in very low-birth weight infants. Pediatr Res 19:679-687, 1985; with permission.) 80 70 - y = 38.9 t 1 0 . 3 ~ r = 0.58 P<O.Ol 60 50 40 30 0 0.5 1.o 1.5 2.0 2.5 3.0 Protein Gain (kcal*kg-ld-I) Figure 4. Relationship between resting energy expenditure and protein gain in preterm infants. (Modified from Catzeflis C, Schultz Y, Micheli J, et al: Whole body protein synthesis and energy expenditure in very low-birth weight infants. Pediatr Res 19:679-687, 1985; with permission.) zy zyxwvut zyxwvu PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 33 administration in preterm infants.5,7, 22, 27, 29-31. 52, 92, 137. 148 Most of these data have been obtained in infants at approximately 32 weeks gestation and weighing approximately 1600 g, under a variety of clinical circumstances (e.g., while on ventilatory and other support; varying combinations of nutrient administration [enteral versus parenteral]; under optimal and suboptimal calorie administration; postsurgical procedures; gaining or not gaining weight; and so forth. All of these have resulted in difficulty in comparison of the quantitative data. Some qualitative inferences can be made, however, and a few specific studies are presented. All of these reports consistently show that in response to protein and other nutrient administration, there is a corresponding increase in whole body rate of leucine turnover, and a small decrease in release of leucine (i.e., protein breakdown) from endogenous sources. In addition, these studies show that when administered in excessive amounts in relation to other amino acids for protein synthesis (eg., in parenteral amino acid mixtures) the metabolic fate of excessive leucine is via decarboxylation into oxidation or other metabolic pathway rather 22 than protein ~ynthesis.~, Whole body leucine kinetics and the role of splanchnic tissue in the metabolism of enterally administered leucine was examined by Beaufrere et a16 in 13 low-birth weight neonates (mean gestational age, 32.5 weeks; weight, 1341 g). At the time of the study, all infants were gaining weight (mean daily weight gain 16 gkg-'d-'), mean weight 1742 g, and their mean conceptional age was 36.3 weeks. A combination of enterally and parenterally administered leucine tracers was used in order to quantify the contribution of splanchnic tissues to the whole body leucine kinetics. As shown in Table 4, leucine from dietary proteins contributed significantly to the total leucine turnover (Ra). Of greater significance, almost half of the dietary leucine (range 25% to 69%) was extracted by the splanchnic tissue. As pointed out by the investigators, this may be a slight overestimate, because a fraction of dietary leucine would have entered the systemic circulation as its ketoanalogue (a-ketoisocaproic acid) and would not have been estimated in their experimental protocol. Nevertheless, these data show a high rate of splanchnic extraction of leucine compared with and may be related to the high rate of protein turnover in splanchnic organs (i.e., gut and liver) in these infants. Additionally, all the infants were in a positive leucine balance (leucine intake-leucine oxidized) and leucine intake was positively correlated with leucine balance (Fig. 5). Increasing leucine (protein) intake resulted in a lower rate of endogenous leucine Ra (protein breakdown), but had no significant impact on nonoxidative disposal of leucine, a measure of protein synthesis. Thus, protein intake appears to promote protein gain by inhibiting z Table 4. LEUCINE KINETICS AND SPLANCHNIC LEUCINE EXTRACTION IN PREMATURE INFANTS (n = 13) zyxw Leu Intake* Total Leu Ra* Dietary Leu Ra* Splanchnic Extractiont Leu Balance* 2.6321.09 3.452 0.57 1.35 2 0.73 48.5 & 15.6 1.922 0.97 zyxwvut Infants were receiving protein enriched human mik, protein intake 3.23 ?0.9 gkg-Id-'. *kmol.kg-'min-'. t%. Mean? SE. From Beaufrere 8,Putet G, Pachiaudi C, et al: Whole body protein turnover measured with 13C leucine and energy expenditure in preterm infants. Pediatr Res 28:147, 1990; with permission. 34 zyxwvuts zyxwvuts KALHAN & IBEN protein catabolism rather than by stimulating protein synthesis over a wide range of protein intake. Mitton and GarlickgZexamined the changes in leucine kinetics longitudinally in preterm infants using [l-13C]leucinewhile receiving two different types of amino acid mixtures parenterally. The infants were first studied while receiving only intravenous glucose and again on each of the next 4 days as the total parenteral nutrition was gradually increased to a maximum of 0.43 g nitrogen/ D D y = 3.16 - 0 . 2 4 ~ r = 0.41 zyxw zyxwvut zyxwvut / y = 3.49 - 0.60~ r = 0.88 P < 0.01 Y (Intake - Oxidation) y = 0.37+ 0.87~ r = 0.98 P4.001 y = -0.32 + -0.36~r = 0.68 P<0.02 0 I I I I 1 2 3 4 5 Leu Intake (pmol*kg-lmin-1) Figure 5. Relationship between leucine intake and leucine kinetics in preterm babies. (Modified from Beaufrere B, Fournier V, Salle B, et al: Leucine kinetics in fed low-birth weight infants: Importance of splanchnic tissues. Am J Physiol 263:E214-E220, 1992; with permission.) PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT zy 35 k g d (-2.6 g protein) and 90 nonprotein kcabkg-Id-'. With increasing proteincalorie intake, there was a corresponding increase in leucine turnover (i.e., both protein breakdown and synthesis) and in nitrogen retention, irrespective of the type of amino acid mixture used. Although these infants were studied while on ventilatory support, and so forth, nonetheless these data show that nitrogen accretion is associated with increase in both protein breakdown and synthesis. In this study, however, the magnitude of change in breakdown was much smaller than in protein synthesis. zyxw zyxw VLBW Infants Hertz et a15*and Denne et alZ9quantified leucine kinetics in VLBW infants (gestational age approximately 26 weeks; birthweight approximately 900 g) using [l-'3C]leucinetracer. The infants were between 1and 5 days old and were on low ventilatory support. Because such infants cannot be evaluated during fasting, the basal data were obtained while they were receiving intravenous glucose at approximately 6.5 mg/kgmin (37 kcal-kg-Id-'). The basal rate of leucine turn20 pmol*kg-'h-' (n=5).52Increasing the over under these conditions was 209 rate of glucose infusion to 9 mgkg-'min-' did not have any significant impact on the rate of leucine turnover (213 -t 20 prnol-kg-'h-'). Because leucine turnover or appearance is a measure of whole body proteolysis, these data show that provision of additional glucose with corresponding increase in insulin concentration did not have any impact on whole body protein breakdown in ELBW infants. It is also possible that because these infants could be studied during fasting that the prior glucose infusion at 6.5 mgkg-'min-' may have already lowered the rate of protein breakdown with no further change with higher rate of glucose infusion. Denne et alZ9further examined the response to parenteral mixed nutrient administration on leucine-protein turnover in VLBW infants. Again, the basal data were obtained while the babies were receiving intravenous glucose. The parenteral nutrition consisted of glucose, 8 mgkg-'min-', lipid, 165 mgkg-'h-', amino acid, 105 mgkg-'h-', 90 kcal*kg-'d-', and protein, 2.5 gkg-'d-'. As shown in Table 5, in contrast to full-term infants there was a significantly greater increase in plasma leucine concentration and the rate of appearance (Ra) of leucine in extremely premature infants in response to parenteral nutrient admin- * Table 5. LEUCINE KINETICS DURING PARENTERAL NUTRITION IN EXTREMELY PREMATURE INFANTS Plasma Leucine (pmo1.L-l) EP (8) Term (10) Leucine Ra Endo Leu Ra (pmobkg-lh-l) (pmobkg-W1) zyxwvu Basal PN 96f 11 76f5 191f 15*t 125f 14* Basal PN PN zyxwvu 184f 13 216 f 11 297 f 17*t 283 f 16" 202 f 17 187k 16 Leucine kinetics were measured using [l-'3C]leucine tracer and plasma enrichment of leucine. *P<O.Ol, basal versus parenteral nutrition. tP<0.05, extremely premature versus term. From Denne SC, Kam CA, Ahlrichs JA, et al: Proteolysis and phenylalanine hydroxylation in response to parenteral nutrition in extremely premature and normal newborns. J Clin Invest 97:746, 1996; with permission. 36 zyxwvuts zyxwvutsr zyxwvutsr z KALHAN & IBEN istration. In addition, during parenteral nutrition the calculated rate of appearance of leucine from endogenous sources (i.e., total leucine Ra-leucine exogenously administered) was significantly higher in VLBW neonates. Thus, the VLBW infants in response to nutrient administration, either glucose alone or glucose plus amino acids and lipids, continue to have a higher rate of proteolysis than term infants. Whether the inability to detect a change is the consequence of prior glucose administration, clinical state of these infants (i.e., ventilatory and other support), or is related to prematurity cannot be resolved from these data. Phenylalanine Kinetics Phenylalanine, an essential amino acid released primarily in vivo from the skeletal muscle protein breakdown, has been used in adults and children in order to quantify whole body rates of protein turnover.” 75 It is catabolized primarily in the liver, via hydroxylation at the 4 position of the phenyl ring to tyrosine, catalyzed by phenylalanine hydroxylase. Kidney and pancreas are the other minor sites of hydroxylation. No demonstrable reverse conversion of tyrosine to phenylalanine has been demonstrated, even in states of phenylalanine deficiency. Estimates of phenylalanine hydroxylation in the low-birth weight infant are of interest because of the previous data suggesting the decreased ability of the preterm infant to hydroxylate phenylalanine to tyrosine?’, 131 These studies suggest that tyrosine is a conditionally essential amino acid for the preterm infant. The use of phenylalanine tracer to quantify protein turnover in vivo assumes that free phenylalanine and tyrosine pools are homogeneous and well mixed, that phenylalanine enters the pool only from protein breakdown during fasting, and the only pathways of phenylalanine removal from the pool are (1) protein synthesis and (2) hydroxylation to tyrosine. The tracer isotope approach for the measurement of phenylalanine turnover and its hydroxylation was described by Clarke and Bierz2and further refined by Thompson et a1.Iz8Most studies have utilized L-[ring2H5] phenylalanine tracer to quantify kinetics. Krempf et a175compared the phenylalanine kinetics using different tracers and oral versus intragastric route of tracer administration. Their data show that phenylalanine fluxes were estimated to be higher when tracer was administered via the intragastric route when compared with the intravenous route, suggesting a first pass uptake through the splanchmc area. Also, higher fluxes were found for [ring-2H5]phenylalanineas compared with [15N] and L-[ l-13C]phenylalanine tracers. Zello et a P 1 evaluated the validity of urinary tracer enrichments to quantify whole body phenylalanine kinetics using different tracers with the goal of eliminating blood sampling, particularly in the low-birth weight baby. These investigators showed that [13C]phenylalaninetracer showed a high correlation between urine and plasma values. In contrast, [2H5]phenylalanineresulted in significantly higher enrichment in urine than in plasma. In relation to estimation of phenylalanine hydroxylation, [1-13C]phenylalaninetracer has been shown to give a higher estimate when compared with [2H,]-labeled tracer.83These methodologic considerations are important when data are compared from different studies. Whole body kinetics of phenylalanine, its hydroxylation, and the response to parenteral and enteral alimentation have been examined by a number of investigators.21,29, 56, 73, 137, 148 These data are summarized in Table 6. Denne et alZ9 studied 10 full-term infants during fasting, 5 to 6 hours after the last normal feed, and in response to a parenteral alimentation consisting of glucose (8 zyxwv zyxwvutsr zyxwvutsrqpon zyxwvutsrq zy Table 6. PHENYLALANINE KINETICS IN THE NEONATE zyxwvutsrqpo zyxwvutsrq zyxwvutsrq Study Parenteral 1 L 3 4 5 6 7 8 9 10 Enteral 11 12 13 n Gest Age Wk 39 32 29 26t 30t 30t 31t 32t 34t 32t Weight kg 3.2 f 1.53 t 1.1 f 0.80 2 1.4 f 1.4 f 1.19 f 2.21 f 2.46 t 1.94 f 0.1 0.07 0.1 0.1 0.4 0.4 0.02 0.6 0.8 0 1.27 f 0.2 1.42 t 0.2 1.70 f 0.2 Age d 2.2 6.6 3.2 2.8 2.6 5.7 6 37 37 20 iz 0.4 f 1.1 iz 0.9 f 0.5 t 0.8 f 1.5 ? 1 f 36 f 43 k 15 Phe Ra Basal* pmobkg- 'h-' 75 iz 3 80 f 3* 88 f 5* 71.6 t 19.8' - 28 f 5 19 t 15 40 f 16 During Alimentation pmobkg-lh- Phe Intake pmobkg-'h-' Protein Intake gkg-'d-' Author 27 2.5 2.5 1.5 2.5 134 f 22.2 127 k 22 158 ? 39 158 & 39 106 f 29 19.2 59 28 59 2.7 1.8 2.9 2.7 2.7 Denne et alZ9 Clark et a12' Kilani et a173 Denne et aPZ9 Kilani et a F Kilani et a P Van Toledo-Eppinga et all37 Roberts et all1' Roberts et a P 4 Wykes et all" 145 f 17 129 k 15 56 f 6 30 38 28 3.2 3 2.7 Van Toledo-Eppinga et all3' Van Toledo-Eppinga et all3' Wykes et all" 83 t 3 90 ? 4 112 f 17 104 2 5 - 27 27 - zyxwvutsrqp *Infants were receiving intravenous glucose during basal state. tInfants requiring ventilatory and other support. +Used YClphenylalanine tracer and measurements performed in urine samples §Fed premature formula. IlFed fortified human milk. W U 38 zyxwvutsr zyxwvuts KALHAN & IBEN mg.kg-'min-'), lipid (165 mg.kg-'h-'), and amino acids (105 mg.kg-'h-' or 2.5 g-kg-'d-') (see Study 1, Table 6). Such an infusion provides phenylalanine at 27 Fmobkg-lh-'. As shown, the basal rate of appearance of phenylalanine was 75 bmol*kg-'h-', which increased to 83 pmol*kg-'h-' during parenteral alimentation. The lesser increase in phenylalanine Ra (8 Frnol*kg-'h-') compared with the amount infused exogenously is interpreted as a decrease in release of phenylalanine from endogenous sources or protein breakdown. A quantitatively similar response was observed by Clark et alZ1in nine healthy infants at 32 weeks' gestation examined under a similar protocol as in term infant. Kilani et al,73in a group of healthy preterm infants at 29 weeks' gestation who were receiving a lower protein intake, showed a somewhat higher but qualitatively similar rate of turnover of phenylalanine during parenteral nutrition. These data show that the healthy preterm infant responds to exogenous infusion of amino acids including phenylalanine by suppressing the endogenous protein breakdown, a response similar to that seen in healthy full-term infants. The data in other studies of preterm neonates are at variance from the previous data, in part, confounded by other variables. All the infants in studies 4 to 10 in Table 6 were in oxygen and on ventilatory support. As shown, during parenteral alimentation their responses were quite variable, suggesting for the most part a lesser degree of suppression of proteolysis-a response that may have been determined by the intercurrent illness and other mediators. The only variation are the data of Wykes et al,'48 study 10, which may be related to the particular tracer isotope used and the site of tracer analysis-in this instance in urine samples. The response to enteral feedings is even more difficult to interpret because no basal data were obtained in these infants and the measured phenylalanine turnover had no relation to the actually administered dose (Studies 11-13, Table 6). The following can be inferred from these data: the healthy preterm infant of 30 to 32 weeks' gestation responds in a similar manner to the full-term infant by decreasing proteolysis during exogenously administered amino acids including phenylalanine administration. The variable response seen in other studies is likely to be related to the associated illness (respiratory distress syndrome, septicemia, and so forth) or the clinical support required. That the preterm as well as full-term neonates can hydroxylate phenylalanine to tyrosine had been demonstrated indirectly by measuring the biochemical products of their metabolism in the urine.138Recently, direct evidence for the hydroxylation of phenylalanine in both preterm and term neonates has been obtained by using stable isotope tracers3" 56, 73 These studies show that even preterm neonates as early as 26 weeks gestation can hydroxylate substantial amounts of phenylalanine to tyrosine. Under what circumstances tyrosine becomes a conditionally essential amino acid for the preterm infant needs to be carefully re-evaluated. zyxwvuz zyxw zyxwv Myofibrillar Protein Degradation As discussed previously, the rate of excretion of 3-MH can be used to estimate the rate of myofibrillar protein breakdown and to estimate the rate of muscle protein degradation. Apart from the criticism of the specificity of this method regarding muscle versus nonskeletal muscle source of 3-MH, particularly in adults, this method further requires an appropriate dietary preparation. zy z zyxwvuts zy zyxwvuts zyxw PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 39 Table 7. RELATIONSHIP BETWEEN NITROGEN RETENTION AND MUSCLE PROTEIN DEGRADATION IN PRETERM INFANTS Nitrogen Retention (mmobd- 'kg-') >0 >10 >15 >20 < 0 (3) < 10 (10) < 15 (8) < 20 (15) < 25 (11) > 25 (8) Muscle Protein Degradation (gd-'kg- ') 1.21 f 0.14 1.05 f 0.06 1.15 f 0.06 1.04 f 0.06 0.91 f 0.05 0.97 f 0.04 (% d) 6.48 5.51 4.74 4.55 4.40 3.83 f 1.16 f 0.49 f 0.38 f 0.23 f 0.23 +. 0.12 zyxwvuts zyxwvuts Values are mean 2 SEM with the number of measurements given in parenthesis for the first 55 balance studies in Table 1. From Ballard FJ, Tomas FM, Pope LM, et al: Muscle protein degradation in premature human infants. Clin Sci 57535, 1979; with permission. Because meat consumption in diets is a major source of 3-MH in adults, the study subjects were placed on a meat-free diet for an adequate length of time. In contrast, newborn infants do not have a dietary source of 3-MH while on formula feeding, and hence 3-MH can be used to estimate myofibrillar protein 16, loo,lZ9Ballard et al,3 several years ago, carefully examined the degradati~n.~, muscle protein degradation in 36 premature infants by measuring 3-MH excretion rate in combination with nitrogen balance and energy consumption. The important features of their data are displayed in Tables 7 and 8. The investigators calculated the rate of muscle protein degradation based upon measured concen,'~ tration of 3-MH in total muscle protein obtained from autopsy ~ t u d i e s . ~There was a negative relationship between nitrogen retention and the rate of muscle protein degradation, and with body weight changes. Interestingly, there was no relation between energy intake and muscle protein degradation. Pencharz et allo1 did not find any effect of age on 3-MH excretion rate in premature neonates weighing more than 1500 g or less than 1500 g. Similar observations have been reported by other investigators.16,57, 58, lZo Collectively, these data show that the rate of muscle protein degradation in preterm infants is increased while they are sick or stressed, probably in order to meet other metabolic demands, such as gluconeogenesis, and decreases during positive nitrogen balance and protein accretion, and that this effect is independent of quantity of calories administered. Table 8. RELATIONSHIP BETWEEN MUSCLE PROTEIN DEGRADATIONAND BODY WEIGHT CHANGES IN PRETERM BABIES Muscle Protein Degradation (gd- 'kg-') (% d) 1.24 t 0.04 1.02 f 0.04 0.95 f 0.04 6.17 f 0.41 4.81 f 0.25 4 f 0.08 ~ Losing weight (10) Weight stable (22) Gaining weight (23) From Ballard FJ, Tomas FM, Pope LM, et al: Muscle protein degradation in premature human infants. Clin Sci 57535, 1979; with permission. 40 zyxwvuts zyxwvutsr zyxwvuts zyxwvut KALHAN & IBEN UREA METABOLISM Urea is the final end product of oxidation of amino acids and proteins and represents the irreversible loss of nitrogen from the body. In healthy human adults, quantitatively urea represents 80% to 90% of the total nitrogen excreted in the urine. This contribution of urea to total nitrogen in urine is reported to be lower in the neonate. In humans, the liver is the site of urea synthesis. Urea is primarily eliminated via the kidneys, although a small amount is excreted in the sweat via the skin. Because the gut is very permeable to urea, the urea appearing in the gastrointestinal fluid can be hydrolyzed to C 0 2 and ammonia by the colonic microflora. The ammonia then released gets reabsorbed in the portal circulation and can be reutilized for the synthesis of nonessential amino acids or recycled back to urea. This recycling of urea nitrogen for nonessential amino acid synthesis, urea salvage, has been suggested to be significant, particularly during marginal protein intake.60,61 The quantity of urea hydrolyzed in the gut can be estimated from the difference between the total amount of urea synthesized measured by isotopic tracers and the amount of urea excreted in the urine. Quantitative estimation of urea synthesis (i.e., production) in adults and in neonates can be done either by isotopic tracer dilution methods using urea labeled with either I5N or l8O, or by measuring the rate of urinary urea excret i ~ n . ~63,139 ~ , The latter results in an underestimation of urea production because of extraurinary excretion of urea, and due to hydrolysis of an unmeasured quantity of urea in the gut. In addition, significant difficulties are encountered in accurate timed collection of urine, particularly in female infants, resulting in unreliable estimate. Several investigators, however, have made careful estimates of urea nitrogen production using this method.65,86, 87 Several different methods of isotopic tracer infusion have been used and validated to quantify urea synthesis. The tracer can be infused as a single bolus or as continuous infusion, either intravenously or by the enteral route. The advantages and disadvantages of the bolus versus constant rate infusion have been discussed.6z, 67, 139 Because urea is released in the blood compartment, administration of the tracer intravenously and measurement of tracer dilution in blood is the preferred and, perhaps, represents the most accurate method.62, 67 The use of intravenous tracers in infants and children, however, may not be acceptable in all circumstances. In addition, due to the long time interval necessary for the establishment of isotopic or equilibrium steady state, attempts have been made to administer tracer urea by the oral route in intermittent 62, lZ5 Such an approach certainly is not applicable in the presence of Helicobacter pylori infection in the stomach, a common infection, particularly in third world countries. H. pylori contains an active urease that rapidly hydrolyzes the administered tracer.lZ3Hibbert et a153compared the oral and intravenous dose regimen in normal healthy adults while they were receiving a liquid proprietary formula. The dilution of tracer was measured in urinary urea. The investigators suggested that the two methods gave virtually identical estimates of urea kinetics. The orally administered dose, however, did result in higher estimates of urea synthesis (270 104 oral versus 205 It_ 15 IV, mgN/kg.d), with a larger magnitude of variance. The capacity of the human fetus in utero to synthesize urea was demonstrated by the presence of significant activity of all five urea cycle enzymes as early as the third week of gestation.108,'09 With advancing gestation there is a zyxwv zyx zy zyxw zyxwvuts zyxwvu PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 41 gradual increase in enzyme activity, reaching approximately 90% of the adult activity by the 36th week of gestation2,20, 23, 47, Io9 This is in contrast to the data from the rat fetus, where very low urea-producing capacity is detectable in utero. Rates of Urea Synthesis The concentration difference for urea in the umbilical arterial and venous blood is extremely low or not measurable. The urea concentration in the fetal arterial blood, however, is higher than the simultaneously obtained maternal arterial blood. Gresham et a148estimated the rate of urea production by the human fetus at term gestation from the umbilical arterial-maternal arterial urea concentration difference and from the placental clearance of urea measured in the subhuman primate by using radioactive tracer urea. Their data show that the human fetus at term gestation has a high rate of urea synthesis (Table 9). This estimate is consistent with the observation that the total nitrogen transferred from the mother to the fetus is in excess of that necessary for protein synthesis. The rates of urea synthesis or protein oxidation in the neonate have been measured either from the rate of urinary urea excretion or by isotopic tracer dilution method. The data from some of these studies are presented in Table 9. In the period immediately after birth, as measured by intravenously administered ['5N2]ureatracer dilution, the rate of urea synthesis is almost one third of that of the fetus in ~ t e r o These . ~ ~ estimates are still higher than those obtained in term infants from the rates of urinary nitrogen excretion.65,85, 86 In this context the studies of Barlow and McCance4 and McCance and Widdowsons6,87 are of interest. These investigators measured the rate of urea excretion in healthy fullterm babies during the first 48 hours after birth while the infants did not receive any nutrients. They also observed a relatively low rate of urinary nitrogen excretion. The rate of nitrogen excreted increased twofold between day 1 and day 2 after birth. These data are in contrast to those of Steinbrecher et al,125 who studied full-term healthy infants between day 29 and 88 after birth using intermittent oral dose of [15N2]~rea. Of interest, their data show that healthy full term infants receiving approximately 81 kcal-kg-WI and 1.99 kg&' protein synthesized urea at 265 mgN*kg-'d-' or 11 mgN*kg-'h-', and excreted 3.6 mgN*kg-'h-' in urine. The observed high rates of urea synthesis in this study are similar to those reported by these investigators in other infants'@,145 and could be the consequence of the tracer methodology used (i.e., intermittent oral dose and measurements of tracer dilution in the urinary urea). In contrast to tracer measured rate of urea synthesis, their measurements of urinary urea excretion rate were of the same magnitude as other studies of formula-fed infants.99,lz6 zyx Premature Infants Although all the enzymes of ornithine-urea cycle are expressed early during fetal development and their activity increases as the gestation advances, at birth they remain 90% of adult levels. Thus, a preterm neonate, depending upon the gestational age, is born with a significantly lower capacity to synthesize urea in response to protein-nitrogen load.lo8It should be underscored, however, that at least in adult humans and animals these enzymes are rapidly inducible in response to nitrogen load.'39In a series of elegant studies, Boehm et have zyxwvutsrqpon zyxw zy zyxwvutsrqp zyxwvutsr zy Table 9. RATES OF PRODUCTION OF UREA AND OXIDATION OF PROTEIN IN HUMAN FETUS AND NEWBORN Study 1 2 3 4 5 6 n Age Fetus Term Term Term Term Preterm Term <6 h < 48 h 48-96 h 96-114 h Day 1 Day 2 Day 1-3 Day 1-3 [‘5N2]Ureatracer dilution: *intravenous; toral STotal nitrogen. (14) (11) (8) (18) (14) (56) (61 Urea Production MWkg-’h-’ Protein Oxidation 9.kg-ld-I 10.5 5.58$ 0.76 1.05 1.41 1.6* 3.3* 2.24 2.63 2651. (109-619) 1.58 0.84 0.11 0.16 0.21 0.24 0.48 0.34 0.39 3.97 Author Gresham et Kalhadj7 Barlow & McCance4 McCance and Widowsona6,87 Jones et aP5 Steinbrecher et allz5 zyx zy zy PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 43 examined the capacity of the VLBW infant (i.e., 1000 to 1500 g) to synthesize urea. The functional immaturity of the ornithine-urea cycle was demonstrated by the lack of increase in blood urea nitrogen concentration in response to a protein load in the presence of appropriate increase in serum alpha-amino nitrogen concentration" on the 8th day after birth. A linear correlation was observed between protein intake and serum alpha-amino nitrogen Concentration. In contrast, the more mature infants (birth weight 1510 to 1990 g, gestational age 31 to 33 weeks) responded to a high protein intake by increased serum urea concentration. By the 21st day after birth, all infants, VLBW as well as low birth weight, responded to the increased protein load by similar increase in urea as well as alpha-amino nitrogen concentration. In another studylo the same investigators examined the change in urinary excretion of urea and ammonia between day 10 and 42 of postnatal age in VLBW infants. Their data show that with advancing age there was a progressive increase in urea excretion, whereas there was a decrease in ammonia excretion during this time. Although the change in ammonia excretion could be the result of a number of other factors, such as acid-base balance, overall the data are suggestive of a decreased capacity for urea synthesis in the VLBW infant in response to protein load during the first 7 to 10 days after birth. A similar inference could be drawn from the lower incorporation of [I5N]from administered ammonia chloride in urinary urea in the preterm infantI4 and the inconsistent incorporation into urea of [I5N]from administered [15N]glycinereported by several other investigator^.'^ Estimates of urea synthesis or urea nitrogen excretion during fasting are impossible to obtain, particularly in the low-birth weight infant, due to clinical and ethical considerations. All data have been obtained during the fed state in the presence of variable protein intake. These studies show that there is a linear relationship between protein intake and serum and urinary urea concentration. In the presence of varying protein intake in the range of 1.5 to 4 gkg-Id-', a normally growing VLBW appropriate for gestational age infant excretes urea at a rate ranging from 1.8 to 5 mg N-kg-lh-l.lz, 13, 19, Io6 These rates are very similar to those observed in full-term infants. Thus, even in the presence of limited capacity to synthesize urea, the VLBW neonate, while receiving enteral nutrients and protein during the first 7 to 10 days after birth, synthesizes urea at a rate similar to that in more mature infants. Only one study has actually quantified rates of urea synthesis in preterm, healthy, growing infants using [15NZ]~rea tracer m e t h ~ d Their . ~ data show that preterm infants at a parenteral protein intake of 2.1 g-kg-'d-' and 61 kcal-kg-ld-' produce urea at the rate of 2.9 mg N-kg-'h-'. These data are consistent with the estimates made from the rates of urinary excretion of urea.116, lz4 Because all the studies were performed during either parenteral or enteral energy and protein intake, it is not possible to quantify the obligatory loss of nitrogen. Pencharz et aPoZestimated the obligatory urinary and total nitrogen losses in six preterm and one full-term infants by examining the relation between nitrogen intake and total nitrogen loss. The mean total obligatory loss of nitrogen was estimated to be 145 mg-kg-W' or approximately 6 mg N*kg-'h-'. It is important to note that all of the previous data on urea production and excretion have been obtained in infants with birth weights over 1000 g, and that there is an immediate need for careful studies in the ELBW neonate, the micropremie. zyxwvu Urea Nitrogen Recycling and Salvage The fact that a certain amount of urea nitrogen can be salvaged through the metabolic activity of gastrointestinal flora and incorporated into the body protein 44 zyxwvuts zyxwvuts zyxwvut zy KALHAN & IBEN has been known for some time.112,132, 139 The quantitative contribution of this nitrogen salvage to overall nitrogen economy in healthy adults and children on adequate protein-energy intake, however, remains controversial.61, 112 The estimates of urea salvage are based upon the incomplete excretion in urine of the total urea synthesized, as measured by isotopic tracer dilution, in the body. Thus, it becomes extremely important that both components of the equation (i.e., the total urea synthesis as well as the rate of urinary excretion) are measured precisely. In addition, other extraurinary losses of urea from skin should also be quantified accurately. A significant amount of nitrogen released from the hydrolysis of urea in the gut is also known to be recycled back into urea or excreted as ammonia in the breath.8l. 145 In normal healthy adults on an adequate protein intake, about 20% of synthesized urea is not excreted in the urine and an insignificant amount of urea nitrogen is incorporated into body protein.81,112 Jackson60, 61 has suggested that colonic salvage of urea nitrogen is an important component in the handling of urea nitrogen in the newborn due to high metabolic demands of the neonate, both for energy and protein relative to the energy and protein intake. Their conclusions are based upon their reported data on normal breast-fed infants 3 to 6 weeks of age and on other infants recovering from surgical 145 Careful evaluation of their data, however, shows that the high rate of urea salvage reported was, for the most part, due to a high rate of synthesis measured in these studies, whereas the rate of urinary excretion of urea in most of these infants was similar to that reported by other investigators. Whether the estimated high rate of urea production was due to the route of tracer administration (i.e., intermittent oral dose) requires further evaluation. Of significance, the magnitude of urea nitrogen recycling measured from the reincorporation of tracer nitrogen back into urea was only a small component of urea synthesis in these studies. Based upon the available data thus far, the evidence for the bioavailability and salvage of urea nitrogen in the neonate is not compelling. Urea nitrogen salvage may be important under conditions of marginal protein intake, such as in malnourished infants, or in certain animal species, such as ruminants, or during hibernation in bears.z5,93 STRATEGIES FOR INTERVENTION Several clinical strategies have been used to improve protein-nitrogen accretion in the low-birth weight infant, particularly in the immediate neonatal period, a time of most attenuation in growth. For the most part, they appear to be limited by the inability to deliver adequate calories and other nutrients. The following is a review of some of the interventions attempted to date. Early Nutrient Administration Saini et a P 6 examined the effect of early amino acid administration on nitrogen retention and balance in ventilator-dependent low-birth weight infants (gestational age 28 weeks, birthweight 1000 g). Parenteral amino acid administration started on the first day after birth resulted in greater nitrogen retention and positive nitrogen balance in the first 3 days. In contrast, all infants who did not receive any nitrogen during the first 3 days were in negative nitrogen balance (i.e., 100 to 150 mg N*kg-'d-'). This study is significant from the following perspectives. (1) The amino acids administered early were well tolerated, apart PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT zy 45 from an increase in plasma concentration of phenylalanine in two infants (2 of 10) in the early group and tyrosine concentration (5 of 10 in early and 4 of 11 in late) in both groups. Three infants in the early group had periods when they could not receive amino acid solution because of hyperglycemia, presumably because of sepsis. (2) The difference in nitrogen balance disappeared when the control group was also started on parenteral amino acids on day 4. (3)There was no distinct clinical advantage of early amino acid administration as measured by change in weight, crown-heel length, or head circumference by day 10. When administered early, parenteral amino acids are tolerated by the majority of low-birth weight infants; such interventions can place these infants in a positive nitrogen balance. Whether such interventions result in any long-term biologic benefit remains, however, to be established. The impact of early amino acid administration on whole body leucine and protein turnover was evaluated by Rivera et al,'13 using [l-13C]leucinetracer in combination with respiratory calorimetry. They administered 1.5 rkg-ld-' of Aminosyn PF (Abbott Laboratories, North Chicago, IL) within 24 hours of birth, along with glucose. Leucine kinetics were measured on day 3 (i.e., when the infants were receiving the parenteral nutrients at a constant rate). Their data (Table 10) confirm that early amino acid administration is well tolerated by preterm infants; allows greater delivery of calories; places them in positive nitrogen balance, and more when cysteine hydrochloride is added to the parenteral amino acid solution; and leads to a hgher rate of nonoxidative disposal of leucine (i.e., higher rate of protein synthesis). Van Goudoever et also measured leucine kinetics on the first day after birth while the infants were receiving amino acids at 1.15 gkg-ld-' and total energy at approximately 28 kcalekg -'day-'. Again, the amino acid solution was well tolerated. Although the amino acid group achieved a zero nitrogen balance in contrast to a negative nitrogen balance, no difference in parameters of leucine kinetics was observed. Their study also may be limited in part due to low protein and energy administration. Similar impact of amino acid administration on protein-nitrogen kinetics has been shown in other studies.134It should be noted that infants in all these studies have been over 28 weeks gestation and more than 1000 g birth weight. Whether a similar response or tolerance is evident in the micropremie has not been evaluated. zyxwv zyx z zyxwvutsrqpon zyx Insulin Insulin is an important anabolic hormone that has been shown to increase protein synthesis in isolated skeletal muscle preparation in ~ i t r 0 .Several l~ investigators have examined the effect of insulin, with and without amino acids, on whole body and skeletal muscle protein metabolism in normal healthy adults.', 33, 40, 43 These data show that at physiologic plasma concentrations insulin causes a decrease in skeletal muscle protein breakdown or proteolysis. Recently, Hillier et a154have shown that at very high concentration of insulin, a strong stimulatory effect of insulin on human forearm muscle protein synthesis can be observed. Because of these effects of insulin on muscle protein turnover, attempts have been made to use parenteral insulin infusion in order to stimulate nitrogen accretion and growth in low-birth weight babies. Poindexter et allo5examined the acute effect of insulin infusion on protein metabolism in four ELBW neonates of a gestational age of 26 weeks and birth weight of 894 t 44 g (mean ? SEM) at 3 days of age. Infusion of insulin raised plasma-insulin concentration to 79 f 13 &J*mL-' and caused a 20% decrease in the rate of proteolysis as measured zyxwvutsrqpon zy zyxwvutsrqp zyxwv zyxwvutsr zyxwvutsrq zyxwvu zyxwvutsrqp Table 10. IMPACT OF EARLY ADMINISTRATION OF AMINO ACIDS ON PROTEIN AND NITROGEN METABOLISM IN VLBW INFANTS Study Groups Glucose Glucose + AA Protein-NitrogenMetabolism n Glucose Glucose + AA (12) (14) n Birth Weight (kg) Gestational Age (wk) Energy Intake kcaUkgd (12) 1.09 + 0.24 1.05 2 0.25 28.5 t 1.8 28.5 t 1.8 35 t 12 54 f 11 (14) N intake mgkg- ‘d- BUN mmo//- N balance mgkg-’d- n Leucine Ra pmotkg- ‘h- Leucine 0 pmo\kg-7h-f zyxwvutsrqpon 0 250 f 8 4.6 t 1.4 6.1 ? 2.9 - 135 k 45 88 f 54 (7) (5) 164 t 25 241 k 38 40 & 17 71 f 22 Mean ? SD. Leucine Ra: Rate of appearance of leucine. Leucine 0 Rate of oxidation of leucine. From Rivera AJ, Bell EF, Bier D M Effect of intravenous amino acids on protein metabolism of preterm infants during the first three days of life. Pediatr Res 33:106, 1993; with permission. PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT zy 47 by the rate of appearance of leucine or phenylalanine in plasma. The estimated rate of protein synthesis was also decreased by a similar magnitude. Thus, infusion of insulin and glucose alone, without additional amino acid, caused a parallel decrease in both proteolysis and protein synthesis, and therefore no significant change in net protein balance. The lack of any effect on proteinnitrogen balance underscores the need to provide exogenous protein or amino acid. Additional findings of the study of Poindexter et allo5include an anticipated increase in the rate of use of glucose and an unexpected increase in the plasma lactate concentration and mild metabolic acidosis. As suggested by the investigators, based upon these findings it is prudent to perform a careful investigation of metabolic effects of insulin prior to its routine use for its growth-promoting effect. zyxwvu zyx zyxwz zyxw Growth Hormone Exogenously administered growth hormone has been shown to impact protein metabolism, improve nitrogen accretion, and promote growth, in growth hormone-deficient states, in normal adults and in neonatal pigs.'8, 117,142 The metabolic response to growth hormone administration has not been examined 136 examined the effects in healthy full-term neonates. Van Toledo-Eppinga et of recombinant human growth hormone (rhGH) treatment during the early postnatal period on growth, body composition, and energy expenditure in seven intrauterine growth-retarded neonates. Seven infants were studied as controls. In addition, they examined the effect of rhGH treatment on glucose and protein turnover using [U-13C]glucoseand [l-13C]leucinetracer, respectively. rhGH treatment (1 IU*kg-'d-') was started at 1 week postnatally and was discontinued when the infants reached 2000 g body weight. Both the treatment and control groups were receiving similar enteral caloric (-120 kcalekg -Id - I ) and protein (-3.3 g.kg-'d-') and other nutrients. rhGH treatment had no effect on energy expenditure measured by [2H2][180] method, daily weight, height gain, or the rate of increase in skinfold measurements. There was no effect on the rate of appearance and oxidation of glucose as measured by [13C]glucosetracer. The data on leucine-protein kinetics are displayed in Figure 6. As shown, rhGH treatment resulted in a small increase in leucine flux ( P = 0.08), in the endogenous rate of appearance of leucine (protein breakdown, P = 0.06). Because there was no change in the rate of decarboxylation of leucine (P=NS), the amount of leucine used for protein synthesis was significantly increased (402 2 72 versus 337 ? 36 Fmol*kg-'h-', mean 2 SD, P=0.04). Net leucine balance, however, was unchanged. These data suggest either (1) resistance to rhGH in this study group or (2) the results could be limited by the energy intake, because both groups received similar calorie intake. Other investigators have used rhGH to reduce the catabolic effects of dexamethasone, used for the treatment of bronchopulmonary dysplasia, in ELBW premature infants at varying times in the neonatal period.130 These data in general have not shown any benefits of administration of rhGH on the growth arrest and other catabolic effects of dexamethasone. Glutamine The amino acid glutamine is the most abundant amino acid in the muscle and plasma of humans. It plays an important role as the primary oxidative fuel 48 zyxwvutsr KALHAN & IBEN 600 zyxwvut zyxwvu zyxwvutsr zyxw zyxwvu 'i 400 l z r +0 a, 0 5 300 200 100 n rhGH Controls zyxwvutsr Figure 6. Effect of human growth hormone (rhGH) administration on leucine kinetics in low-birth weight infants. In each group: First bar = flux (F); second bar = intake (I); third bar = protein B; fourth bar = protein S; fifth bar = oxidation (0). F = leucine flux; I = leucine intake; B = leucine from protein breakdown; S = leucine for protein synthesis; 0 = leucine oxidation. (Adapted from van Toledo-Eppinga L, Houdijk EC, Dellemarre-Van de Waal HA, et al: Leucine and glucose kinetics during growth hormone treatment in intrauterine growth-retarded preterm infants. Am J Physiol 270:E451, 1996; with permission.) for the dividing cells, such as enterocytes and lymphocytes.95a, 146 In addition, glutamine is a key substrate for ammonia production in the kidney,44for nitrogen transfer between tissues, precursor for purine and pyrimidine synthesis, and is a regulator for protein 153, Glutamine constitutes a major component of nitrogen transferred from the mother to the fetus in ~ t e r o .78~Several ~, studies in humans and animals have proposed that glutamine becomes conditionally essential during catabolic illness. Because of these roles of glutamine and, in particular, in relation to the metabolism of the gastrointestinal several studies have been performed examining the advantages of glutamine supplementation in catabolic states in adults, such as following surgery, trauma, and so forth in order to induce nitrogen retention, infection resistance, gut growth and repair, and so forth.64,83, Is4 Glutamine supplementation via the enteral or parental route in VLBW infants has been evaluated by Neu et a194and by Lacey et aLZ6,76, Parenteral administration of glutamine at a dose of 15% to 25% of total amino acids administered resulted in an increase in plasma glutamine concentration in a dose-response manner.76Glutamine supplementation was well tolerated by these infants with no change in blood ammonia concentration, although the mean blood urea nitrogen concentration was slightly higher in the supplemented group. No statistically significant advantage was demonstrated in the entire group of infants. When the groups were further separated based upon their weight, however, infants less than 800 g (13 supplemented and 11 controls), the glutamine supplemented infants had a shorter length of stay in the neonatal intensive care unit, required fewer days on parenteral nutrition, had a shorter length of time to full feeds, and needed less time on the ventilator. Whether zy zyxw PROTEIN METABOLISM IN THE EXTREMELY LOW-BIRTH WEIGHT INFANT 49 these advantages were due to glutamine supplementation or to some other factors needs to be examined further. Enteral supplementation of glutamine was examined in VLBW infants (birth weight -950 g) by Neu et a1.26,94, 115 Glutamine was added to commercial special care infant formula and administered in a dose of 0.08 g-kg-'day-' and increasing to 0.31 g-kg-'day-'. glutamine supplementation had no impact on plasma amino acid c~ncentration,"~ nor on the tracer isotope measured flux of glutamine or leucine.26There was no difference in growth, serum ammonia, urea, prealbumin, or liver transaminase concentration or in the mean hospital stay. There was, however, a suggestion for better formula tolerance and possibly decreased odds of developing sepsis. Again, whether these advantages are related to glutamine supplementation or due to other unrecognized factors remains to be established. These data thus far presented do not support routine supplementation with glutamine in the nutritional management of VLBW infants. SUMMARY Although extensive data are available on the impact of nutrient and protein administration on growth, plasma amino acids, and nitrogen balance in the newborn and growing infants, relatively few studies have carefully examined the dynamic aspects of protein metabolism in vivo and particularly in the micropremie or ELBW infant. These studies show that the very preterm infants, either because of immaturity or because of the intercurrent illness, have high rates of protein turnover and protein breakdown. This high rate of proteolysis is not as responsive to nutrient administration. Intervention strategies aimed at promoting nitrogen accretion, such as insulin, human growth hormone, or glutamine, have not thus far resulted in enhanced protein accretion and growth. This may be, in part, due to limitations in delivery of adequate calorie and nitrogen. ACKNOWLEDGMENT The secretarial support of Mrs. Joyce Nolan is gratefully appreciated. References 1. 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Kalhan, MBBS Department of Pediatrics Robert Schwartz, MD, Center for Metabolism & Nutrition MetroHealth Medical Center 2500 MetroHealth Drive Cleveland, OH 44109-1998 e-mail: sck8po.cwru.edu