During the early neonatal period, there is a several-fold increase in organ growth and accelerated development associated with the transition from intrauterine to extrauterine life (Widdowson et al. 1976, Widdowson and Crab 1976). Much of the growth stimulus for many organs in the newborn undoubtedly results from the increased demands associated with physiological functions such as pulmonary respiration, thermogenesis (Herpin and LeDividich 1995) and gluconeogenesis (Girard 1986). The diet ingested by the newborn, namely colostrum, contains a rich source of nutrients that not only support these metabolic needs, but are critical for the rapid rate of organ growth and development. We have previously shown that the rate of protein synthesis in a number of organs is increased markedly when newborn pigs ingest either colostrum or mature milk (Burrin et al. 1992). However, in some organs, the proportional stimulation of protein synthesis is greater when pigs are fed colostrum rather than mature milk. These differences in the protein anabolic stimulus between colostrum and mature milk may be due to the differences in the concentration of either nutrients or non-nutritive components such as growth factors. Several researchers have identified a number of peptide growth factors, including insulin, insulin-like growth factor-I (IGF-I)⁶ and epidermal growth factor (EGF ) that are present in higher concentrations in colostrum than in mature milk (Donovan et al. 1994, Jaeger et al. 1987, Simmen et al. 1988). Because of the mitogenic or trophic potential of these growth factors, it has been hypothesized that their ingestion via colostrum enhances tissue growth and development of the neonate.

In this report we present additional information derived from a previously reported experiment (Burrin et al. 1995) by determining the effects of colostrum ingestion on protein synthesis in vital organs, including brain, heart, lung, kidney and spleen of newborn pigs. We tested the hypothesis that components of colostrum other than macronutrients are, in part, responsible for the marked stimulation of vital organ protein synthesis in newborn pigs. To test this hypothesis, we measured the rates of brain, heart, lung, kidney and spleen protein synthesis in newborn pigs fed colostrum, mature milk or a fortified formula having a macronutrient composition similar to that of colostrum but devoid of growth factors.

METHODS

The specific radioactivity of ³H-phenylalanine was determined in tissue samples and in whole blood samples obtained 5, 15, and 30 min after infusion of the ³H-phenylalanine. The tissue samples were homogenized in 0.2 mol perchloric acid/L (PCA) as described previously (Burrin et al. 1991). The PCA-soluble homogenate supernatants containing the tissue free amino acid pools were separated from the PCA-insoluble precipitates by centrifugation. The PCA-insoluble precipitates were neutralized, washed and solubilized in 0.3 mol NaOH/L for 1 h at 37°C. Aliquots of the NaOH solutions were assayed for protein as described by Lowry et al. (1951). The remainder of the solutions were reacidified with 2 mol PCA/L, and after precipitation at 4°C and centrifugation at 3000 × g for 15 min, the acid-soluble supernatant fractions were assayed for total RNA by the method of Munro and Fleck (1969). The acid-insoluble pellets, containing protein, were hydrolyzed with 6 mol HCl/L for 24 h at 110°C and then vacuum-dried in a Speed-Vac concentrator (Jouan, Winchester, VA). The dried residues from the protein hydrolysates were solubilized with 4 mL of water and vacuum-dried twice to remove any residual HCl. The protein hydrolysates, homogenate supernatants and blood supernatants were vacuum-dried and resuspended in water for determination of phenylalanine specific activity. Phenylalanine was separated from the other amino acids using anion exchange chromatography (AminoPac column, Dionex, Sunnyvale, CA). The radioactivity associated with the phenylalanine peak was collected automatically (Model 202, Gilson Medical Electronics, Middleton, WI) and measured using liquid scintillation counting (LS5000TD, Beckman Instruments, Fullerton, CA).

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Because most of the RNA in tissues is ribosomal, the RNA-to-protein ratio is an estimation of the protein synthetic capacity (C_s) of the tissue. Protein synthetic efficiency (K_rna) was estimated by multiplying the fractional synthesis rate (K_s/d) by the ratio of protein to RNA to yield grams of protein synthesized per gram of total RNA per day.

RESULTS

Table 1. VitalorganproteinandRNAcontentsrelativetobodyweight(BW)inunfednewbornpigsandthosefedmaturemilk,colostrumorformulafor24h1,2 [View Table]

Brain protein content (relative to BW) was lower in the three feeding groups than in the unfed newborn group (Table 1). However, the kidney protein content in colostrum and formula-fed pigs was higher than in either the unfed newborns or those fed milk. Spleen protein content was also higher in colostrum-fed pigs than in unfed newborn pigs. The heart and lung RNA content (relative to BW) in pigs fed colostrum were higher than in either unfed newborns or milk-fed pigs (Table 1). The spleen RNA content in pigs fed colostrum and formula were higher than in either unfed newborns or milk-fed pigs.

The fractional protein synthesis rate in all vital organs was significantly increased by feeding in all groups (Figs. 1 and 2). Among the three feeding groups, the brain and heart protein synthesis rates of colostrum-fed pigs were greater than those of either formula- or milk-fed pigs (Fig. 1). Kidney and spleen protein synthesis rates in colostrum- and formula-fed pigs were not significantly different, but both were higher than in milk-fed pigs (Fig. 2). Lung protein synthesis rates did not differ among the three feeding groups (Fig. 2).

The protein synthetic capacity of the heart and spleen in pigs fed either colostrum or formula did not differ, but were greater than either the unfed newborn or milk-fed groups (Table 2). Protein synthetic capacity of the lung in pigs fed either colostrum or formula also did not differ, but was greater in pigs fed colostrum than either the unfed newborn or milk-fed groups. In contrast, the protein synthetic capacity of the kidney in pigs fed colostrum and milk did not differ from unfed newborn pigs, while that of formula-fed pigs was lower than unfed newborn pigs. Similarly, the protein synthetic efficiency of the heart, kidney and spleen were significantly greater in milk- and colostrum-fed pigs than in unfed newborn pigs (Table 2). The protein synthetic efficiency in the kidney of pigs fed formula was higher than either unfed newborn or milk-fed pigs.

Table 2. Vitalorganproteinsyntheticcapacities(Cs)andefficiencies(Krna)inunfednewbornpigsandthosefedmaturemilk,colostrumorformulafor24h1,2 [View Table]

DISCUSSION

The maximal stimulation of brain protein synthesis by colostrum was particularly interesting given the recent evidence that the quality of the diet fed to preterm infants can have beneficial effects on developmental outcomes (Lucas et al. 1990, 1992 and 1994). In particular, these studies have shown that preterm infants fed human milk rather than formula during the neonatal period scored significantly higher on intelligence quotient, psychomotor and mental development tests when measured at 18 mo and 8 y of age. It has been hypothesized that this apparent neurodevelopmental advantage of human milk for the preterm infant may be related to the presence of long-chain lipids, such as docosahexanoic acid (DHA), hormones and growth factors that are not found in infant formulas (Lucas et al. 1992). Other recent findings demonstrate that dietary cholesterol supplementation improves the abnormally low exploratory behavior observed in neonatal pigs genetically selected for low serum cholesterol (Schoknecht et al. 1994). Based on the composition of ingredients and limited analysis (Burrin et al. 1995), the formula we used was devoid of DHA and growth factors. Therefore, this advantage of colostrum feeding on brain protein synthesis cannot be attributed to macronutrient intake, particularly of protein and energy. Furthermore, it has been argued that some of the results of the human studies are confounded by inherent socio-behavioral differences between breast- and formula-feeding mothers. However, unlike these human studies, this controlled study with neonatal pigs is not confounded by any inherent maternal effects among feeding groups.

The kidney and spleen protein synthesis rates of colostrum- and formula-fed pigs did not differ from each other and were greater than those of milk-fed pigs, presumably reflecting the greater nutrient concentration of colostrum and formula. Thus, the dominant stimulus for kidney and spleen protein synthesis was macronutrient intake and is consistent with our previous findings for other visceral organs, particularly the liver (Burrin et al. 1995). The response to feeding milk, colostrum, or formula was associated with metabolic and endocrine changes reported previously (Burrin et al. 1995), which may explain the stimulation of vital organ protein synthesis. Insulin and amino acids are critical factors that mediate the acute stimulation of protein synthesis in response to feeding (Garlick et al. 1983; Garlick and Grant, 1988). In this study, circulating insulin and amino acid concentrations were increased with feeding in all three groups, but after 4 h of feeding, tended to be higher in colostrum- and formula-fed pigs than in milk-fed pigs (Burrin et al. 1995). This pattern of circulating insulin and amino acid concentrations likely reflects the higher protein intake of colostrum- and formula-fed versus milk-fed pigs. Therefore, the maximal rates of kidney and spleen protein synthesis in both colostrum- and formula-fed pigs compared to those of pigs fed milk may be largely attributed to higher protein intake and perhaps the resultant increase in circulating insulin and amino acid concentrations in these groups.

Our results suggest that the cellular mechanisms by which feeding stimulated protein synthesis varied among organs. Our estimates of translational efficiency and protein synthetic capacity are based on the assumption that our measurement represents largely ribosomal RNA, but does not preclude the posssibilty that specific mRNA's are either up- or down-regulated in response to diet. The increases in heart, lung and spleen protein synthesis resulted from increases in both translational efficiency and protein synthetic capacity, whereas kidney protein synthesis was mediated by increased translational efficiency. The increase in protein synthetic capacity that was evident in colostrum- and formula-fed pigs was paralleled by increased total RNA content, suggesting either enhanced ribosomal RNA synthesis or reduced degradation. This is supported by evidence from studies in rats suggesting that both amino acids and insulin increase ribosomal protein synthesis (Ashford and Pain 1986) and inhibit RNA degradation (Lardeux and Mortimore 1987).

These results, in conjunction with our earlier observations from this study, suggest that the response of protein metabolism to early feeding varies among organs. The organs associated primarily with the digestion and absorption (gastrointestinal tissues), metabolism (liver) and excretion (kidney) of macronutrients, appear to respond only to macronutrient intake itself. However, skeletal and cardiac muscle, and critically, the brain, apparently require some unidentified component of colostrum to achieve a maximum rate of protein synthesis and growth. Whether this factor is a trace nutrient (such as a specific fatty acid), a growth factor or reflects the activation of a specific growth-regulatory pathway remains unknown. Nevertheless, these findings support the critical role of colostrum ingestion in developmental regulation during the immediate neonatal period.

ACKNOWLEDGMENTS