Robert F. Bertolo

Creatine Synthesis Is a Major Metabolic Process in Neonatal Piglets and Has Important Implications for Amino Acid Metabolism and Methyl Balance

Our objectives in this study were as follows: 1) to determine the rate of creatine accretion by the neonatal piglet; 2) identify the sources of this creatine; 3) measure the activities of the enzymes of creatine synthesis; and 4) to estimate the burden that endogenous creatine synthesis places on the metabolism of the 3 amino acids required for this synthesis: glycine, arginine, and methionine. We found that piglets acquire 12.5 mmol of total creatine (creatine plus creatine phosphate) between 4 and 11 d of age. As much as one-quarter of creatine accretion in neonatal piglets may be provided by sow milk and three-quarters by de novo synthesis by piglets. This rate of creatine synthesis makes very large demands on arginine and methionine metabolism, although the magnitude of the demand depends on the rate of remethylation of homocysteine and of reamidination of ornithine. Of the 2 enzymes of creatine synthesis, we found high activity of l-arginine:glycine amidinotransferase in piglet kidneys and pancreas and of guanidinoacetate methyltransferase in piglet livers. Piglet livers also had appreciable activities of methionine adenosyltransferase, which synthesizes S-adenosylmethionine, and of betaine:homocysteine methyltransferase, methionine synthase, and methylene tetrahydrofolate reductase, which are required for the remethylation of homocysteine to methionine. Creatine synthesis is a quantitatively major metabolic process in piglets.

The nutritional burden of methylation reactions.

Purpose of reviewMethyl group metabolism is a metabolically demanding process that has significant nutritional implications. Methionine is required not only for protein synthesis but also as the primary source of methyl groups. However, demethylated methionine can be remethylated by methyl groups from methylneogenesis (via folate) and betaine (synthesized from choline). This review discusses the impact of methylation precursors and products on the methionine requirement. Recent findingsRecent evidence has clearly demonstrated that transmethylation reactions can consume a significant proportion of the flux of methionine. In particular, synthesis of creatine and phosphatidylcholine consume most methyl groups and their dietary provision could spare methionine. Importantly, methionine can become limiting for protein and phosphatidylcholine synthesis when creatine synthesis is upregulated. Other research has shown that betaine and choline seem to be more effective than folate at reducing hyperhomocysteinemia and impacting cardiovascular outcomes suggesting they may be limiting. SummaryIt appears that methyl groups can become limiting when dietary supply is inadequate or if transmethylation reactions are upregulated. These situations can impact methionine availability for protein synthesis, which can reduce growth. The methionine requirement can likely be spared by methyl donor and methylated product supplementation.

Low birth weight is associated with reduced nephron number and increased blood pressure in adulthood in a novel spontaneous intrauterine growth-restricted model in Yucatan miniature Swine.

Background: Impaired fetal growth and rapid postnatal growth are associated with programming of hypertension and metabolic syndrome in adulthood. Objectives: This study evaluated this phenomenon in a novel spontaneous intrauterine growth-restricted (IUGR) model in Yucatan miniature pigs. Methods: IUGR piglets (n = 6, 3 days old, 0.73 ± 0.11 kg) were paired with a normal weight (NW) same-sex littermate (n = 6, 1.11 ± 0.13 kg), fed milk replacer for 4 weeks followed by a standard diet ad libitum for 5 h/day. At 9 months of age, arterial blood pressure (BP) telemeters were implanted to assess BP before (0.5% NaCl) and after (4.5% NaCl) a 7-day salt-loading period. At 10 months of age, nephron numbers were determined. Results: Prior to sexual maturity, IUGR pigs showed greater (p < 0.05) relative feed intake and experienced significant catch-up growth. Adult IUGR pigs also had higher BP (diastolic BP: 93.8 ± 5.5 vs. 90.0 ± 8.7 mm Hg, p < 0.05) and 43% fewer nephrons per kidney (p < 0.05). Nephron number was positively associated with birth weight and negatively correlated with BP (p < 0.05). Acute salt loading increased BP in both groups (p < 0.05); however, the degree of salt sensitivity was similar between groups (p > 0.05). Conclusions: In conclusion, IUGR piglets have reduced nephron endowment associated with a modest BP increase in early adulthood. This new model can be used to conduct longitudinal mechanistic studies on the early programming phenomenon.

Enteral Feeding Induces Early Intestinal Adaptation in a Parenterally Fed Neonatal Piglet Model of Short Bowel Syndrome

BACKGROUND Successful small intestinal (SI) adaptation following surgical resection is essential for optimizing newborn growth and development, but the potential for adaptation is unknown. The authors developed an SI resection model in neonatal piglets supported by intravenous and enteral nutrition. METHODS Piglets (n = 33, 12-13 days old) were randomized to 80% SI resection with parenteral nutrition feeding (R-PN), 80% SI resection with PN + enteral feeding (R-EN), or sham SI transection with PN + enteral feeding (sham-EN). In resected pigs, the distal 100 cm of ileum (residual SI) and 30 cm of proximal SI were left intact. All pigs received parenteral nutrition postsurgery. Enteral nutrition piglets received continuous gastric infusion of elemental diet from day 3 (40:60 parenteral nutrition:enteral nutrition). Piglets were killed 4, 6, or 10 days postsurgery. RESULTS By 10 days, R-EN piglets had longer residual SI than R-PN and sham-EN pigs (P < .05). At days 6 and 10, R-EN piglets had greater weight per length of intact SI (P < .05) and isolated mucosa (P < .05) compared to other groups. Greater gut weight in R-EN piglets was facilitated by a greater cellular proliferation index (P < .01) by 4 days compared to other groups and greater overall ornithine decarboxylase activity vs R-PN piglets (P < .05). CONCLUSIONS This new model demonstrated profound SI adaptation, initiated early postsurgery by polyamine synthesis and crypt cell proliferation and only in response to enteral feeding. These changes translated to greater gut mass and length within days, likely improving functional capacity long term.

A Comparison of Parenteral and Enteral Feeding in Neonatal Piglets, Including an Assessment of the Utilization of a Glutamine-Rich, Pediatric Elemental Diet

BACKGROUND The amino acid requirement profile for infants is different than that for adults and needs to be established; this profile also is different for infants receiving total parenteral nutrition. We used the neonatal piglet as a model for the infant to address (1) the metabolic and physiologic changes due to route of feeding and (2) the adequacy of the amino acid pattern in a pediatric elemental diet. METHODS Diets differed only in their amino acid pattern (modified human milk [MHM] and a commercial pediatric elemental diet [PED]) and were fed continuously for 8 days. Control piglets were fed MHM diet via gastric catheters (IG) and were compared with pigs fed MHM diet via venous catheters (IV) or to pigs IG-fed PED. RESULTS MHM-IV piglets experienced enlarged livers and gut atrophy, and lower nitrogen retention and body protein content. Higher glutamine (and lower glutamate) in PED-IG, compared with MHM-IG, produced no apparent advantage with respect to gut growth or histology. Proline, histidine, and lysine may have been limiting, and isoleucine and valine excessive, in the PED-IG diet as indicated by plasma concentrations, compared with sow-fed piglets; however, imbalances in the amino acid profile were not excessive because nitrogen retention was not different between MHM-IG or PED-IG pigs. CONCLUSIONS Therefore, the amino acid profile of MHM needs to be modified to improve nitrogen retention during parenteral feeding and the profile of oral PED could be improved to normalize plasma amino acid concentrations.

Enteral Tryptophan Requirement Determined by Oxidation of Gastrically or Intravenously Infused Phenylalanine Is Not Different from the Parenteral Requirement in Neonatal Piglets

We have recently shown that the requirements of several amino acids differ substantially when neonates are fed parenterally as opposed to enterally. Our first objective was to determine whether the tryptophan requirement was different in parenterally fed (IVfed/IVdose) versus enterally fed (IGfed/IVdose) piglets. Because of the extensive extraction of amino acids by the gut, our other objective was to determine whether the route of isotope administration [i.e. intragastric (IGfed/IGdose) versus i.v. (IGfed/IVdose) dose] affects the estimate of tryptophan requirement in enterally fed piglets. We used the indicator amino acid oxidation technique in piglets (10 ± 0.5 d old, 2.79 ± 0.28 kg) receiving a complete elemental diet for 6 d either intragastrically or intravenously. Piglets were randomly assigned to receive test diets containing one of seven levels of tryptophan. All animals received a primed, constant infusion of l-[1-14C]phenylalanine either parenterally (IVfed/IVdose and IGfed/IVdose) or enterally (IGfed/IGdose). The mean tryptophan requirements for IVfed/IVdose (0.145 ± 0.023 g/kg/d), IGfed/IVdose (0.127 ± 0.022 g/kg/d), and IGfed/IGdose (0.113 ± 0.024 g/kg/d) were similar as were the safe intakes (upper 95% confidence interval) (0.185, 0.164, 0.154 g/kg/d, respectively). These data indicate that tryptophan is not extensively used by the gut, in contrast to all the other amino acids we have studied. Furthermore, in spite of a splanchnic extraction of 27% of the phenylalanine dose, the route of isotope infusion does not affect the tryptophan requirement as determined by indicator amino acid oxidation.

Reduced aluminum contamination decreases parenteral nutrition associated liver injury

PURPOSE Parenteral nutrition-associated cholestasis remains a significant problem, especially for the surgical neonates. Aluminum is a toxic element known to contaminate parenteral nutrition. We hypothesize that parenterally administered aluminum causes liver injury similar to that seen in parenteral nutrition-associated cholestasis. METHODS Twenty 3- to 6-day-old domestic pigs were divided into 5 equal groups. A control group received daily intravenous 0.9% NaCl. Each subject in experimental groups received intravenous aluminum chloride at 1500 μg kg(-1) d(-1) for 1, 2, 3, or 4 weeks. At the end of the study, blood was sampled for direct bilirubin and total bile acid levels. Liver, bile, and urine were sampled for aluminum content. Liver tissue was imaged by transmission electron microscopy for ultrastructural changes. RESULTS Transmission electron microscopy revealed marked blunting of bile canaliculi microvilli in all experimental subjects but not the controls. Serum total bile acids correlated with the duration of aluminum exposure. The hepatic aluminum concentration correlated with the duration of aluminum exposure. CONCLUSIONS Parenterally infused aluminum resulted in liver injury as demonstrated by elevated bile acids and by blunting of the bile canaliculi microvilli. These findings are similar to those reported in early parenteral nutrition-associated liver disease.

Guanidinoacetate Is More Effective than Creatine at Enhancing Tissue Creatine Stores while Consequently Limiting Methionine Availability in Yucatan Miniature Pigs

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAA- or Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1–3 hours post feeding and a bolus of [methyl-3H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-3H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.

Proline Supplementation to Parenteral Nutrition Results in Greater Rates of Protein Synthesis in the Muscle, Skin, and Small Intestine in Neonatal Yucatan Miniature Piglets

Proline and arginine are each indispensable during parenteral feeding due to limited interconversion by an atrophied gut. Commercial amino acid parenteral products designed for neonates contain proline concentrations that differ by almost 4-fold. To assess the adequacy of the lowest concentration of proline provided in commercial total parenteral nutrition (TPN) products, we compared rates of tissue-specific protein synthesis and nitrogen balance in neonatal piglets provided TPN at 2 different proline concentrations. Yucatan miniature piglets (9-11 d old, n = 12) were randomized to complete isonitrogenous TPN diets with low proline (LP; L-proline as 3% of amino acids) or proline supplemented (PS; 9%). After 7 d of receiving TPN, rates of protein synthesis in liver, gastrocnemius muscle, jejunal mucosa, and skin were determined by the flooding dose technique and tissue free amino acids were measured. Nitrogen balance was assessed during the last 3 d. The LP TPN resulted in lower free proline concentrations in plasma, muscle, and skin (P < 0.05) and lower rates of protein synthesis in the jejunum (by 25%; P = 0.02), muscle (by 45%; P = 0.015), and skin (by 60%; P = 0.01); there was no difference in liver. Nitrogen retention was 20% lower in the LP group (P = 0.01). In conclusion, muscle and skin protein synthesis was profoundly sensitive to parenteral proline supply and the reduced protein synthesis in the intestine could affect intestinal integrity. Low-proline TPN solutions that are currently in wide use in neonatal care may result in impaired tissue growth.

The dynamics of methionine supply and demand during early development.

Methionine is an indispensable amino acid that, when not incorporated into protein, is converted into the methyl donor S-adenosylmethionine as entry into the methionine cycle. Following transmethylation, homocysteine is either remethylated to reform methionine or irreversibly trans-sulfurated to form cysteine. Methionine flux to transmethylation and to protein synthesis are both high in the neonate and this review focuses on the dynamics of methionine supply and demand during early development, when growth requires expansion of pools of protein and transmethylation products such as creatine and phosphatidylcholine (PC). The nutrients folate and betaine (derived from choline) donate a methyl group during remethylation, providing an endogenous supply of methionine to meet the methionine demand. During early development, variability in the dietary supply of these methionine cycle-related nutrients can affect both the supply and the demand of methionine. For example, a greater need for creatine synthesis can limit methionine availability for protein and PC synthesis, whereas increased availability of remethylation nutrients can increase protein synthesis if dietary methionine is limiting. Moreover, changes to methyl group availability early in life can lead to permanent changes in epigenetic patterns of DNA methylation, which have been implicated in the early origins of adult disease phenomena. This review aims to summarize how changes in methyl supply and demand can affect the availability of methionine for various functions and highlights the importance of variability in methionine-related nutrients in the infant diet.