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Nutrient Metabolism

The Contribution of Body Protein to the Supply of Energy in Starved Newborn Piglets Is Not Preferentially Suppressed by Intravenous Provision of Glucose and Fat^1,2

Barbara D. Mickelson^*, Frank R. Greer^*,** and Norlin J. Benevenga^*,,3

Departments of ^* Nutritional Sciences Animal Sciences, and ^** Pediatrics, University of Wisconsin-Madison, WI 53706

³To whom correspondence should be addressed. E-mail: njbeneve{at}ansci.wisc.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Newborn piglets were used to study body protein preservation because it is critical to the survival of premature infants. Quantitative estimates of endogenous fuel use were obtained from 12 to 72 h of age in male piglets. Of the 40 piglets used (1300 ± 205 g, mean ± SD), 16 served as a 12-h-old body composition reference (R), 16 were starved (S) and received water only, and 8 received supplemental energy (E), obtaining 70% [210 kJ/(kg · d)] of their resting energy requirement as an i.v. mixture of glucose and Intralipid® (65:35 energy ratio). Urine was collected continuously from the bladder via an umbilical urachal catheter. Total body water, glycogen, lipid, ash, and Kjeldahl-N were determined on whole-pig homogenates. Comparative slaughter was used to estimate the disappearance of body constituents of S and E pigs from 12 to 72 h of age. Midpoint body weight was used in these calculations. Supplemental energy decreased use of all body energy sources as indicated by the decrease in body dry matter disappearance, 41.6 ± 8.8 vs. 25.5 ± 5.9 g/kg (P = 0.0021) and protein (urinary N excretion), 995 ± 508 vs. 329 ± 135 mg/kg (P = 0.0119) over 60 h. Supplemental energy did not preferentially spare the percentage of the resting energy expenditure supplied by endogenous body protein (protein 37.6% ± 9.6 vs. 41.7% ± 10.4; lipid 25.7% ± 5.2 vs. 20% ± 4.1; glycogen 36.8% ± 7.5 vs. 38.3% ± 9.9; S vs. E) because it made up 40% of the total in food-deprived and supplemented piglets.

KEY WORDS: • piglets • neonates • nitrogen excretion • body composition

Body protein preservation is critical to the survival of premature infants, particularly with reference to the maintenance of the integrity of the respiratory system in humans, which is immature and susceptible to damage by both mechanical ventilation and oxygen therapy (1). Exposure to a high concentration of oxygen resulted in lower survival of undernourished newborn rats (2); only 44% of undernourished rat pups survived 7 d in a 95% O₂ environment compared with 73% of their well-nourished counterparts. The combined negative effects of hyperoxia and undernutrition on weight gain, lung weight, and lung DNA content were more detrimental than either alone. In the view of some (3), undernutrition should be considered to be an etiologic factor in the development of bronchopulmonary dysplasia (BPD)⁴ in infants because poor nutritional support can negatively influence the response of the lung to all of the major factors that are believed to be involved in the overall pathogenesis of BPD (hyperoxia, barotrauma, infection, and pulmonary structural and biochemical immaturity). As in premature infants, body protein preservation is a concern also with regard to survival of newborn piglets kept under standard rearing conditions (4).

The immediate postpartum time period was chosen to study the pattern of endogenous fuel use. Although it is current clinical practice to supply amino acids and energy in the first 48 h of life, it may be a week or more before sufficient energy and amino acids are provided to support growth, especially in sick human premature infants <1500 g (5,6). Similar nutritional concerns are raised when newborn piglets fail to establish themselves in the competitive environment of a litter. In premature infants, i.v. glucose may provide the only source of exogenous energy for 1–3 d after birth, providing only 125–165 kJ/(kg · d) [30–40 kcal/(kg · d)], or 50–65% of the resting energy expenditure (REE). The neonatal period for human neonates has been described as one of acute semistarvation, followed by a prolonged period during which nutrient intakes are sufficient to cover ongoing losses, but are not adequate to support growth (7). Recent emphasis has been on providing amino acid and lipid-containing solutions earlier in life. Newborn piglets may be challenged with this same nutritional environment. Like human premature infants, piglets have limited body fat, i.e., 0.5–2% of total body weight (8,9), and liver glycogen is depleted within 12 h of birth (9,10). Heird et al. (11) estimated that a 1000-g premature infant would have only 460 kJ/kg [110 kcal/kg] of nonprotein energy available at the time of birth; metabolic processes would then be maintained primarily by the catabolism of protein in the absence of nutritional intervention. Similar arguments were made for piglets (9).

Because premature infants and newborn piglets have similar body compositions (12,13) and birth weights (1000–1500 g) (14), we selected the newborn piglet as a reasonable model with which to study the distribution of endogenous fuel use over a period of total or partial starvation immediately after birth. We established that the REE of food-deprived pigs in the first few days of life is 293 kJ/(kg · d) [70 kcal/(kg · d)]. This is based on expired CO₂ [367 µmol/(min · kg^0.75)] and an assumed respiratory quotient of 0.86 (15). Assuming that liver glycogen was depleted by 18–24 h of age (16,17) and fat was a very limited energy source, comprising at most 2% of body weight (17,18), protein would then be the primary source of energy for both premature infants and newborn piglets.

The literature values concerning the contribution of body protein to provide energy to support the metabolism of newborn animals are suspect because estimates of the contribution of protein to heat production based on urinary N excretion during 24 h of starvation in newborn lambs and pigs indicate that it is <10% (4,19,20). Similar rates of nitrogen excretion were reported in premature infants over the first few days of life (21–23). Based on the limited availability of fat and glycogen as fuels in premature infants and piglets, one would expect body protein to make up at least 50% of the contribution of organic body constituents to meet REE needs. The low value of 10% may be due to losses of body N that have not been measured. Davis et al. (24) reported in experiments estimating maximum rates of urea production in newborn piglets infused at 25, 50, or 75% of REE with L-alanine, recovery of only 50–75% of infused alanine-N in the expanded total body urea pool and urinary-N at the end of 36 h of experiments. We recently reported that we could not recover all of the ¹⁵N from alanine infused into a piglet over 60 h in the piglet, its urine, or in the expired air (25). Apparently, we cannot measure all of the N lost from newborn piglets. Nitrogen losses that have not been measured would lead to an underestimation of body N loss and hence, an underestimation of the contribution of body protein in meeting REE.

The present study was an attempt to quantify the rate of use of endogenous body protein, lipid, and glycogen in newborn piglets totally or partially (70% of energy provided i.v.) starved. A secondary goal was to compare body N (protein) loss as estimated from comparative slaughter to that estimated from urinary N because nitrogen balance methodology is often used to assess the protein status of neonates.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Chemicals. Liquid phenol (90%), 98% sulfuric acid, and methanol were obtained from Fisher Scientific. Tyrosine, glycogen, and enzymes were obtained from Sigma Chemical. Sodium hydroxide, glucose, ammonium sulfate, 30% hydrogen peroxide, and chloroform were obtained from Mallinckrodt. Sodium nitroprusside was from Eastman Organic Chemicals, and urea was obtained from Amend Drug & Chemical. Intralipid came from Kabi Pharmacia.

    Animal preparation. Newborn piglets (n = 40; Large White, Landrace and Duroc breeding), 1300 ± 205 g (mean ± SD), were obtained several hours after birth (extent of colostrum intake unknown). After transport to the laboratory, pigs were allocated by weight to 1 of 3 treatment groups to maintain similar weight distributions in the groups. Piglets serving as the body composition reference were killed at 12 h (R, n = 16), pigs starved (given water only) from 12 to 72 h of age (S, n = 16), and pigs given the glucose and Intralipid i.v. energy during this time (E, n = 8) were killed at 72 h. Within 12 h of birth, the S and E pigs had 2 umbilical arterial catheters (for sampling and infusion) and an umbilical urachal (for continuous urine collection) catheter inserted under aseptic conditions, as described previously (24). Pigs were then fitted with an orogastric catheter (12 Fr, Bard) for infusion of water (Sage pump, Orion). Surgical procedures took <1 h/pig after which they were placed in Plexiglass cylinders (45 cm long x 14 cm i.d.) with vents at each end for air flow. Radiant heaters were placed over the cylinders and adjusted to provide a neutral thermal environment based on piglet behavior. Piglets kept under these conditions sleep at least 90% of the time. All pigs were given water intragastrically (i.g.) until they were 12 h of age, at which time the body composition reference (R) pigs were killed. The i.g. water infusion (10 mL/h) continued for the S pigs and was discontinued for the E pigs because the i.v. glucose-Intralipid infusion (10 mL/h) (Table 1) was initiated using a 4-channel Rainin peristaltic pump. The trials (n = 4) were completed as follows. Trials 1 and 2: 4 pigs each in R and S groups. Trials 3 and 4: 4 pigs each in R, S, and E groups, with 2–3 litters represented in each trial. The experimental protocol was approved by the University of Wisconsin-Madison, College of Agricultural and Life Sciences Animal Care and Use Committee.

    Body composition. Pigs were killed while under anesthesia by injection of a bolus (10–12 mL) of saturated KCl into the deep catheter or via heart puncture if no catheter was present (R pigs). Individual pigs were placed in plastic bags and plunged into ice water to limit postmortem glycogen breakdown and then frozen intact. Frozen piglets were cut into -8 pieces with a hand saw, ground through a 9.6-mm plate with a meat grinder (Hobart) twice, and then twice again through a 4.8-mm plate and blended. Subsamples (3 x 100 g) were collected and frozen for subsequent analysis; 2 subsamples were pulverized in liquid nitrogen in a Waring blender. Recovery of the weight of the piglet as a homogenate was 95 ± 3%. Dry matter was determined before refreezing of the initial subsamples, and again on the liquid nitrogen–prepared subsamples to account for water loss during processing. Dry matter and ash were measured by standard methods using 5–6 g of homogenate. Kjeldahl-N, ash, dry matter, lipid, and glycogen were determined in duplicate on the liquid nitrogen ground subsamples. Nitrogen was determined on the pig homogenates using a macro-Kjeldahl procedure. Recoveries of nitrogen from tyrosine, albumin, ammonium sulfate, and tyrosine added to homogenate averaged 99.6, 99.3, 98.5, and 98%, respectively. Lipids were extracted from 2.5–3 g subsamples with a chloroform:methanol:water mixture (30). Because no internal standard was added, the method was tested on samples sizes ranging from 0.5 to 3 g and the total lipid extracted was a constant proportion of the sample weight. Glycogen was determined by enzymatic analysis (9); 1 g of homogenate was mixed with 0.6 mol/L perchloric acid and centrifuged for 15 min at 5000 x g. A neutralized aliquot of the supernatant was incubated with and without amyloglucosidase (EC 3.2.1.3) and assayed for glucose as above. Recovery of added glycogen to homogenate averaged 102.5%.

Statistics and calculations

    Body composition. The measured body composition of the pigs used as reference standards (R, n = 16, 4/trial) was used to generate regression equations to predict the body composition at the start of the experiment of the starved pigs (S) and pigs given i.v. energy (E). Prediction equations (Table 2) were obtained by regressing 12-h body components (g of N, glycogen, lipid or dry matter) on 12-h body weight (g). Information from the 4 trials was pooled to develop the regression equations using Minitab, release 7.2. The best fit (highest R² and lowest SEE that added significance to the single line model) was obtained when the same slope was used for the 4 groups, but each was allowed to have a different intercept. Biologically, this means that the relation between body weight and the component was the same for all groups of pigs, but they may start (intercept) at a different point, due to litter or development (postconception, i.e., time) effects. A single line was adequate to describe the relation between body weight and dry matter. To estimate energy expenditure in metabolizable energy units, absolute losses of protein (N x 6.25), lipid, and glycogen (12 h estimated – 72 h measured) were multiplied by 20.0, 39.7, and 17.2 kJ/g, respectively (4.8, 9.5, and 4.1 kcal/g). Two pigs (one S, one E) were not included in any of the analyses due to technical difficulties because 12-h prediction of body components was less than that measured after a 60-h period of full or partial food deprivation.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Blood urea N and glucose. BUN (Fig. 1) in starved pigs doubled from 0 to 60 h (10 vs. 21 mmol/L), whereas in pigs administered the supplemental i.v. glucose and fat it decreased from 6.6 to 4 mmol/L (0 vs. 36 h) and then rose to 5.5 mmol/L from 36 to 60 h. The whole-blood glucose concentration (data not shown) in pigs given i.v. supplemental glucose and fat rose from 3 mmol/L before supplement administration to 4 mmol/L by 6 h and then declined linearly to 2.5 mmol/L at 60 h. In starved pigs, blood glucose decreased linearly from an initial value of 3 at 0 h to 1.5 mmol/L at 60 h. The rate of decrease did not differ (P = 0.17) in starved and supplemented pigs. Repeated-measures analysis for urinary urea nitrogen (UUN; data not shown), BUN, and blood glucose indicated significant treatment (P = 0.003, 0.0257, and 0.032, respectively) effects. Additionally, trend differences between starved and supplemented pigs were evident for UUN and BUN (P = 0.0055 and 0.0001, respectively).

View larger version (17K): [in this window] [in a new window]   FIGURE 1 BUN concentrations of pigs starved (n = 15) or administered i.v. energy (n = 7) from 12 to 72 h after birth. Time "0" refers to the start of collection when pigs are 12 h old, and "60" is the time at which pigs are killed, 72 h of age. The symbols represent the mean ± SD of BUN concentration (mmol/L). Repeated-measures analysis indicated a significant effect of time (P = 0.0024), as well as treatment (P = 0.0257) and trend (P = 0.0001) differences.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In contrast to the 140 mg N/(kg · d) reported by others (4,20), N excretion in starved pigs over 60 h (Table 4) averaged almost 1 g/(kg · 60 h) or 398 mg/(kg · d). Nitrogen excretion in premature infants (body weight 1000–1500 g) reportedly ranges from 75 to 190 mg/(kg · d) with parenteral supplementation of 125–335 kJ/(kg · d) [30–80 kcal/(kg · d)] with or without the addition of amino acids (21,22,32,33). This compares favorably to the 130 mg/(kg · d) in the pigs administered i.v. supplemental energy as a glucose and fat mixture (Table 4). Urea-N excretion was 360–500 mg/(kg · d) during the first 3 d of life in 700-g premature infants (34) with administration of glucose (125 kJ/(kg · d) [30 kcal/(kg · d)] and small amounts of amino acids. This rate of excretion corresponds more closely to that of the starved pigs [300 mg urea N/(kg · d)], and is 2–4 times that reported in larger (1000 g+) premature infants, suggesting an even greater dependence on body protein for energy when lipid and glycogen reserves are limited as they are in more premature infants.

From Table 3, a comparison of the total urinary-N loss [995 ± 508 mg N/(kg · 60 h) or 398 mg N/(kg · 24 h)] with the calculated body protein loss of [15.3 ± 6.0 g/(kg · 60 h) or 6.12 g/(kg · 24 h)] revealed that urinary N loss accounted for only 41% of the total body protein loss based on comparative slaughter calculations. A similar comparison can be made with the information presented by Rivera et al. (22) who compared calculated body protein loss in preterm infants over the d 1–3 of life based on the difference between protein synthesis and degradation using [L-1-¹³C] leucine with N-balance. N-balance accounted for <50% of the body protein loss compared with body protein loss based on stochastic modeling. In the current study, using the energy values for protein, lipid, and glycogen listed in Table 4 and converting the body substance loss to a 24-h basis of 6.12 g protein, 2.04 g lipid, and 6.48 g glycogen yields 314 ± 63 kJ/(kg · d) or 75 kcal/(kg · d). This value of 75 kcal/(kg · d) compares favorably with the calculated heat production of 69 kcal/(kg · d) based on a measured CO₂ production of 367 µmol/(min · kg^0.75) from one of our earlier studies with similar piglets (15). This provides support for the use of the prediction equations we developed (Table 2) to estimate 12-h body composition and the subsequent comparative slaughter calculations.

An unanticipated finding of the present study is the failure of urinary N excretion over 60 h to approach within 10–15% of the estimated body N loss based on the values calculated from comparative slaughter. In general, only 20–50% of the N lost from the body was recovered in urine. Virtually all of the urine produced was collected via the urachal catheter, and most pigs had little or no stool output. Possible N losses in hair and saliva were not measured, whereas N loss due to blood sampling was estimated to be 34 mg. Clearly, these potential losses could not account for the missing 1.1–1.4 g N/kg body weight that was not recovered in urine. Body N was determined on the intact piglet; thus, N in the bladder and gastrointestinal tract was not lost. The discrepancy cannot be explained by a lack of recovery of homogenate (95% of the weight of the pig was recovered) or by methods because recoveries of standards were 95%.

Discrepancies in nitrogen retention based on carcass or body measurements and calculated N-balance were reported to be on the order of 10–80%, with N-balance always higher, in pigs (35) and rats (36). N balance appears to overestimate N retention in rats, pigs, preterm and term infants, and adult humans (24,35–40) because the calculated retention is not associated with body weight or composition changes. Overestimation of intake and underestimation of output are generally regarded as reasons for this incongruity. However, in the present study, these potential sources of error were minimized in that there was no N intake, and urine was collected by way of a catheter.

Calculations of body component disappearance such as those used in comparative slaughter are dependent on a predicted body composition for an initial measurement. Thus, a total of 16 animals with a weight range of 820-1550 g were included in the initial slaughter group, which included 4 pigs from each block and at least 8 litters, in an attempt to capture the variability present in the population. The potential error associated with the prediction of total body nitrogen at the start of the experiment based on a body weight of 1.2 kg (Table 3) was 10% (2 g) based on the regression in Table 2. This error may affect the estimated loss of body nitrogen (0.4–5 g over 60 h) and may contribute to the lack of agreement between the estimates of body protein loss based on urinary N excretion and body protein loss predicted from comparative slaughter. A resampling technique called bootstrapping (41,42) was used to assess whether the range of estimates we obtained from the comparative slaughter calculations would be expected. The central idea behind the bootstrapping approach is that the stability of the results based on the original data set can be assessed by studying their variability across a large number of bootstrap samples. For the 22 pigs remaining in the analysis (S and E combined), 42 ± 41% of body N loss calculated from comparative slaughter was recovered in urinary N. The 10,000 bootstrap simulations generated a grand mean of 39.6 ± 126%, showing reasonable agreement although greater variability. The percentage of body N loss recovered in urine is a ratio; thus, the frequency distribution is not expected to be normal. The median is less sensitive to extremes of data and may be a more acceptable way to describe the data set. In the bootstrap simulations, 10% of means fell below zero, and 10% above 100% whereas the range of 10,000 medians from the simulations ranged from 16 to 57% with a mean of 29% and SD of 4.8%, similar to the median of the actual data set (30%). We conclude from this exercise that either there is reasonable evidence of another very significant source of N loss that was not quantified, which we reported recently (25), or the discrepancy is due to the 12-h prediction of initial body composition, or both. That the prediction errors for the 12-h body composition were random is supported by the bootstrapping results, and the agreement between the heat production calculated from body component loss and measured CO₂ production lend credibility to the comparative slaughter results. This again raises questions about the quantitative accuracy of nitrogen loss.

Provision of exogenous energy did not affect the proportion of endogenous fuel loss originating from body protein. In animals starved for 60 h and in those receiving 70% of the REE as glucose and lipid, protein made up 40% of the energy derived from body components. The continual rise in BUN (Fig. 1) and in UUN excretion (data not shown) from 57 ± 47 to 94 ± 38 mg/(kg · 6 h) throughout the 60 h of starvation, the increase in BUN after 36 h (Fig. 1), and UUN excretion (data not shown) from 15 ± 12 mg to 26 mg/(kg · 6 h) after 36 h in piglets administered i.v. energy are consistent with the increase in protein and amino acid catabolism over time.

The observation that nonprotein energy alone did not preferentially spare body protein was also observed in protein turnover studies in preterm infants provided glucose or glucose and lipid without amino acids. Premature infants (26 wk, <1000 g, n = 5) were given 2 levels of glucose infusion, (6.2 and 9.5 mg/(kg · min) corresponding to 146 and 230 kJ/(kg · d) [35 and 55 kcal/(kg · d)] (43). Proteolysis, as determined by the rate of leucine appearance, did not differ at the 2 levels of glucose infusion. Similarly, rates of proteolysis did not differ during starvation, an i.v. glucose infusion [134 kJ (32 kcal)/(kg · d)], an i.v. lipid infusion [134 kJ/(kg · d)], or a combined glucose and lipid infusion [268 kJ (64 kcal)/(kg · d)] in 2-d-old term infants (44).

The administration of amino acids [1–2.5 g/(kg · d)] to premature infants as soon as possible after birth, even when energy intake is less than a maintenance level of 209 kJ (50 kcal)/(kg · d), was shown to increase protein synthesis and improve N balance compared with infants receiving only i.v. energy (22,45). Additionally, variable reductions in proteolysis (up to 15%) in preterm and term infants were seen with amino acid administration (46), even when energy intake was less than maintenance (47).

The lack of an effect of supplementation of nonprotein energy on the rate of body protein breakdown may be related to the support of the process of protein synthesis itself. For protein synthesis to be maintained, an amount and array of indispensable and dispensable amino acids in suitable concentrations and ratios must be maintained at the site of protein synthesis. The mixture of amino acids available for protein synthesis is derived from tissue protein turnover and from that added to the mixture from the diet. In cases in which dietary protein is limiting, or in starving animals, the source of amino acids for protein synthesis is totally dependent on turnover. In these situations, the rate of protein synthesis is diminished, possibly due to a limitation of one or more of the amino acids. In situations such as these, supplementation of the indispensable amino acids, methionine and threonine, results in enhanced retention of N, possibly by supporting reutilization of the amino acids derived from protein breakdown. In early work with dogs and rats, diets supplemented with methionine (in some cases with added Thr) diminished N loss when either low protein or protein free diets were used (48–52). This suggests that methionine is first limiting for protein synthesis and therefore has a unique ability to spare N under conditions in which major losses of N were anticipated. Direct support of this idea is found in a 1986 paper by Muramatsu et al. (53), in which the rate constant K_s for whole-body protein synthesis of chicks fed a protein-free diet increased from 11.0 to 13.5% when Met was added to the protein-free diet. These observations clearly suggest that amino acids in addition to energy are required to suppress body protein loss in compromised neonates.

	ACKNOWLEDGMENTS

 
The authors thank Linda Haas for her technical assistance and commitment to this study. They also thank fellow student Julie Davis for her help during these experiments and Rick Nordheim for assisting with statistical analysis. The piglets were donated by Pig Improvement Company (Spring Green, WI).

	FOOTNOTES

 
¹ Presented in abstract form at Experimental Biology ’96, April 1996, New Orleans, LA [Mickelson, B. D., Thomsen, J. A., Greer, F. R. and Benevenga, N. J. (1996) Nonprotein fuel decreases body dry matter (DM) loss but does not preferentially spare body protein. FASEB J. 10: A736 (abs.)].

² Supported by U.S. Department of Agriculture Grant 9137203 and USDA-CREES 92–38420-7373 Graduate Fellowship Grant and University of Wisconsin College of Agricultural and Life Sciences.

⁴ Abbreviations used: BPD, bronchopulmonary dysplasia; BUN, blood urea nitrogen; REE, resting energy expenditure; TUN, total urinary nitrogen; UUN, urinary urea nitrogen.

Manuscript received 4 March 2005. Initial review completed 15 April 2005. Revision accepted 12 August 2005.

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