Photoprotection But Not N-acetylcysteine Improves Intestinal Blood Flow and Oxidation Status in Parenterally Fed Piglets.

OBJECTIVES: The purpose of the present study was to determine if protecting parenteral nutrition solutions from ambient light and supplementing with N-acetylcysteine (NAC) improves mesenteric blood flow, gut morphology, and oxidative status of parenterally fed neonates. METHODS: Neonatal Yucatan miniature piglets (n = 23, 7-11 days old) were surgically fitted with central venous catheters and an ultrasonic blood flow probe around the superior mesenteric artery. Piglets were fed continuously for 7 days either light-protected (LP) or light-exposed (LE) complete parenteral nutrition that was enriched with either NAC or alanine (ALA). RESULTS: There were no differences in body weight or overall gut morphology among groups after 7 days. Plasma concentrations of NAC were greater and total homocysteine lower in NAC- versus ALA-supplemented pigs on day 7 (N-acetylcysteine: 94 vs 7 µmol/L; P < 0.001; homocysteine: 14 versus 21 µmol/L; P < 0.005); plasma total glutathione was not affected. Hepatic lipid peroxidation was reduced by 25% in piglets that received LP parenteral nutrition (P < 0.05). The mesenteric artery blood flow decreased in all pigs between days 2 and 6 (P < 0.001) because of parenteral feeding. Photoprotection alone (LP-ALA) attenuated the decrease in mesenteric blood flow to 66% of baseline on day 6 compared with LE-ALA (37%; P < 0.05) and LP-NAC pigs (43%; P = 0.062); LE-NAC piglets had intermediate reductions in blood flow (55%). CONCLUSIONS: Photoprotection of parenteral nutrition solutions is a simple, effective method to attenuate decline in blood flow to the gut and hepatic lipid peroxidation, which are both commonly associated with parenteral feeding.

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates.

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4-8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [13C1]methionine and [2H3-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 µg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70-80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.

Dietary Methyl Donors Contribute to Whole-Body Protein Turnover and Protein Synthesis in Skeletal Muscle and the Jejunum in Neonatal Piglets.

BACKGROUND: The neonatal methionine requirement must consider not only the high demand for rapid tissue protein expansion but also the demands as the precursor for a suite of critical transmethylation reactions. However, methionine metabolism is inherently complex because upon transferring its methyl group during transmethylation, methionine can be reformed by the dietary methyl donors choline (via betaine) and folate. OBJECTIVE: We sought to determine whether dietary methyl donors contribute to methionine availability for protein synthesis in neonatal piglets. METHODS: Yucatan miniature piglets aged 4-8 d were fed a diet that provided 38 µg folate/(kg·d), 60 mg choline/(kg·d), and 238 mg betaine/(kg·d) [methyl-sufficient (MS); n = 8] or a diet devoid of these methyl precursors [methyl-deficient (MD); n = 8]. After 5 d, dietary methionine was reduced from 0.30 to 0.20 g/(kg·d) in both groups. On day 6, piglets received a constant [1-13C]phenylalanine infusion to measure whole-body protein kinetics, and on day 8 they received a constant [3H-methyl]methionine infusion to measure tissue-specific protein synthesis in skeletal muscle, the liver, and the jejunum. RESULTS: Whole-body phenylalanine flux, protein synthesis, and protein breakdown were 13%, 12%, and 22% lower, respectively, in the MD group than in the MS group (P < 0.05). Reduced whole-body protein synthesis in the MD piglets was attributed to 50% lower protein synthesis in skeletal muscle and the jejunum than in the MS piglets (P < 0.05). Furthermore, methionine availability in skeletal muscle was halved in piglets fed the MD diet (P < 0.05), and the specific radioactivity of methionine was doubled in the jejunum of MD piglets (P < 0.05), suggesting lower intestinal remethylation. Liver protein synthesis did not significantly differ between the groups, but secreted proteins were not measured. CONCLUSIONS: Dietary methyl donors can affect whole-body and tissue-specific protein synthesis in neonatal piglets and should be considered when determining the methionine requirement.

Dietary methyl donors affect in vivo methionine partitioning between transmethylation and protein synthesis in the neonatal piglet.

Methionine metabolism is critical during development with significant requirements for protein synthesis and transmethylation reactions. However, separate requirements of methionine for protein synthesis and transmethylation are difficult to define because after transmethylation, demethylated methionine is either irreversibly oxidized to cysteine during transsulfuration, or methionine is regenerated by the dietary methyl donors, choline (via betaine) or folate during remethylation. We hypothesized that remethylation contributes significantly to methionine availability and affects partitioning between protein and transmethylation. 4-8-day-old neonatal piglets were fed a diet devoid (MD-) (n = 8) or replete (MS+) (n = 8) of folate, choline and betaine to limit remethylation. After 5 days, dietary methionine was reduced to 80 % of requirement in both groups of piglets to ensure methionine availability was limited. On day 7, an intragastric infusion of [13C1]methionine and [2H3-methyl]methionine was administered to measure methionine cycle flux. In MD- piglets, in vivo remethylation was 60 % lower despite 23-fold greater conversion of choline to betaine (P < 0.05) and transmethylation was 56 % lower (P < 0.05), suggesting dietary methyl donors spared 425 µmol methyl/day for transmethylation. The priority of protein synthesis versus transmethylation was clear during MD- feeding (P < 0.05), as an additional 6 % of methionine flux was for protein synthesis in those piglets (P < 0.05). However, whole body transsulfuration was unaffected in vivo despite reduced in vitro cystathionine-ß-synthase capacity in MD- piglets (P < 0.05). Our data show that remethylation contributes significantly to methionine availability and that transmethylation is sacrificed to maintain protein synthesis when methionine is limiting in neonates, which should be considered when determining the methionine requirement.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet.

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4-8 days of age were fed a diet deficient (MD-) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [(3)H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD- feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD- feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD- feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

Cysteinyl-glycine reduces mucosal proinflammatory cytokine response to fMLP in a parenterally-fed piglet model.

BACKGROUND: PepT1 transports dietary and bacterial peptides in the gut. We hypothesized that cysteinyl-glycine would ameliorate the inflammatory effect of a bacterial peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), in both sow-fed and parenterally-fed piglets. METHODS: An intestinal perfusion experiment was performed in piglets (N = 12) that were sow-reared or provided with parenteral nutrition (PN) for 4 d. In each piglet, five segments of isolated intestine were perfused with five treatments including cysteine and glycine, cysteinyl-glycine, fMLP, free cysteine and glycine with fMLP, or cysteinyl-glycine with fMLP. Mucosal cytokine responses and intestinal morphology was assessed in each gut segment. RESULTS: PN piglets had lower mucosal IL-10 by approximately 20% (P < 0.01). Cysteinyl-glycine lowered TNF-α response to fMLP in PN-fed animals and IFN-γ response to fMLP in both groups (P < 0.05). The free cysteine and glycine treatment reduced TNF-α in sow-fed animals (P < 0.05). fMLP affected villus height in parenterally (P < 0.05), but not sow-fed animals. CONCLUSION: Parenteral feeding conferred a susceptibility to mucosal damage by fMLP. The dipeptide was more effective at attenuating the inflammatory response to a bacterial peptide than free amino acids. This may be due to competitive inhibition of fMLP transport or a greater efficiency of transport of dipeptides.

Betaine is as effective as folate at re-synthesizing methionine for protein synthesis during moderate methionine deficiency in piglets.

PURPOSE: Both folate and betaine (synthesized from choline) are nutrients used to methylate homocysteine to reform the amino acid methionine following donation of its methyl group; however, it is unclear whether both remethylation pathways are of equal importance during the neonatal period when remethylation rates are high. Methionine is an indispensable amino acid that is in high demand in neonates not only for protein synthesis, but is also particularly important for transmethylation reactions, such as creatine and phosphatidylcholine synthesis. The objective of this study was to determine whether supplementation with folate, betaine, or a combination of both can equally re-synthesize methionine for protein synthesis when dietary methionine is limiting. METHODS: Piglets were fed a low methionine diet devoid of folate, choline, and betaine, and on day 6, piglets were supplemented with either folate, betaine, or folate + betaine (n = 6 per treatment) until day 10. [1-13C]-phenylalanine oxidation was measured as an indicator of methionine availability for protein synthesis both before and after 2 days of supplementation. RESULTS: Prior to supplementation, piglets had lower concentrations of plasma folate, betaine, and choline compared to baseline with no change in homocysteine. Post-supplementation, phenylalanine oxidation levels were 20-46 % lower with any methyl donor supplementation (P = 0.006) with no difference among different supplementation groups. Furthermore, both methyl donors led to similarly lower concentrations of homocysteine following supplementation (P < 0.05). CONCLUSIONS: These data demonstrate an equal capacity for betaine and folate to remethylate methionine for protein synthesis, as indicated by lower phenylalanine oxidation.

Enterally delivered dipeptides improve small intestinal inflammatory status in a piglet model of intestinal resection.

UNLABELLED: PepT1, a di/tripeptide transporter, is preferentially preserved over free amino acid transporters in situations of gut stress. Therefore, our objective was to determine the impact of enterally delivered dipeptide-containing diets on indices of intestinal adaptation in neonatal piglets after intestinal resection. METHODS: Piglets (n = 25, 10 ± 1 d old) underwent an 80% jejuno-ileal resection and were provided 50% of nutritional support as TPN, and 50% as one of five, enteral test diets: 1) a control diet containing free amino acids, or the same diet but with equimolar amounts of free amino acids replaced by 2) alanyl-alanine, 3) alanyl-glutamine, 4) cysteinyl-glycine, or 5) both alanyl-alanine and cysteinyl-glycine. After 4 d of enteral feeding, indices of intestinal adaptation were assessed. Outcome measures included plasma and mucosal amino acid concentrations, morphological and histological parameters, protein synthesis, PepT1 mRNA and protein expression, and mucosal cytokine concentrations. RESULTS: Intestinal length, organ weight and protein synthesis rates were not different amongst groups. All of the dipeptide-containing diets reduced pro-inflammatory cytokine concentrations in the mucosa (TNF-α, IFN-γ). The cysteinyl-glycine diet supported greater villus height compared to all other dipeptides and greater crypt depth compared to alanyl-glutamine; however, none of the dipeptide diets altered intestinal morphology compared to the free amino acid control diet. CONCLUSIONS: This study showed that while there was no explicit morphological benefit of enteral dipeptides over their constituent free amino acids, there was the potential for the amelioration of intestinal inflammation by reducing pro-inflammatory cytokines. Enteral provision of dipeptides impacted intestinal adaptation, but the response was dipeptide-specific.

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.

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.

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.

N-acetylcysteine is a highly available precursor for cysteine in the neonatal piglet receiving parenteral nutrition.

BACKGROUND: Cysteine (CYS) is accepted as an indispensable amino acid for infants receiving parenteral nutrition (PN), and CYS is unstable in solution. Thus, developing a method to supply CYS in PN for neonates is needed. N-acetyl-L-cysteine (NAC) is stable in solution and safe for use in humans; therefore, NAC may be a means of supplying parenteral CYS. METHODS: We determined the bioavailability of NAC in intravenously (IV)-fed piglets randomized to 1 of 4 diet treatments, each supplying 0.3 g/kg/d methionine and either 0.2 g/kg/d CYS (CON), 0 NAC (zeroNAC), 0.13 NAC (lowNAC), or 0.27 g/kg/d NAC (highNAC). Piglets (2 days old; 1.8 kg, n = 20) were surgically implanted with femoral and jugular catheters. On day 3 postsurgery, test diets were initiated and continued until day 8. Piglets were weighed daily. Blood was sampled 6 hours before test diet initiation and at 0, 6, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, and 120 hours. Urine was collected on ice in 24-hour sample periods. RESULTS: Total mean weight gain was not different between groups; however, average daily gain in the zeroNAC and lowNAC groups declined significantly (p < .05) over the 5-day treatment period. Nitrogen retention was similar between the CON and highNAC groups, both were higher than the lowNAC group, and the zeroNAC treatment produced the lowest nitrogen retention. NAC percent retention was not different between lowNAC and highNAC and was 85.4% and 82.6%, respectively. Plasma NAC was higher in highNAC than lowNAC (p < .05). CONCLUSIONS: These data demonstrate that NAC is available as a precursor for CYS to support growth and protein (nitrogen) accretion in piglets administered a parenteral solution.

The balance of dietary sulfur amino acids and the route of feeding affect plasma homocysteine concentrations in neonatal piglets.

Plasma total homocysteine (tHcy) concentrations are associated with atherogenesis in adults and increased risk of stroke in infants and children. After a series of experiments to compare the methionine (Met) requirement and cysteine (Cys)-sparing capacity in piglets that were parenterally or enterally fed, we examined the effects of route of feeding and dietary Cys on plasma tHcy concentrations. Piglets (n = 60; 6-8 d old) were fed elemental diets, intragastrically (n = 28) or intravenously (n = 32), with 0.55 g. kg(-1). d(-1) dietary Cys (n = 28) or without dietary Cys (n = 32). Dietary Met ranged from deficient to excess. Increasing Met intake increased (P < 0.01) plasma tHcy in all treatment groups. Plasma tHcy concentrations were higher (P < 0.05) in the enterally fed piglets that did not receive dietary Cys than in all other groups, which did not differ from each other. Therefore, both route of feeding and dietary supply of Met and Cys significantly affected the concentrations of plasma tHcy. These dramatic and rapid alterations in plasma tHcy warrant further studies of sulfur amino acid metabolism in neonatal animals.

Dietary cysteine reduces the methionine requirement by an equal proportion in both parenterally and enterally fed piglets.

The sulfur amino acids (SAA), methionine and cysteine, are normally supplied in a 50:50 ratio in the oral diet of pigs. In contrast, cysteine is not included in any appreciable amounts in parenteral solutions due to its instability in solution. Cysteine can replace part of the methionine requirement, but is not required when methionine is supplied at a level that meets the entire SAA requirement. However, the role of the gut on cysteine sparing has not been investigated. In the present study, the enteral and parenteral methionine requirement was determined, with excess dietary cysteine, by using the indicator amino acid oxidation (IAAO) technique. Piglets [n = 28, 2 d, 1.65 +/- 0.014 kg (SE)] were fed elemental diets containing adequate energy, phenylalanine and excess tyrosine, with varied methionine concentrations and excess cysteine [0.55 g/(kg. d)]. Diets were infused continuously via intravenous (parenteral) or gastric (enteral) catheters. Phenylalanine oxidation was determined during a primed, constant infusion of L-[1-(14)C]-phenylalanine, by measuring expired (14)CO(2) and plasma specific radioactivity (SRA) of phenylalanine. For both the parenteral and enteral groups, phenylalanine oxidation (% of dose) decreased linearly (P < 0.01) as methionine intake increased and then became low and unchanging. Using breakpoint analysis, the methionine requirement was estimated to be 0.25 and 0.18 g/(kg. d) for enteral and parenteral feeding, respectively. These data show that the parenteral methionine requirement is approximately 70% of the enteral requirement when measured in the presence of excess dietary cysteine (P < 0.05). A comparison with our previous studies in which methionine was the only source of sulfur amino acids shows that the addition of dietary cysteine reduces the methionine requirement by approximately 40% in both enterally and parenterally fed neonatal piglets. Therefore, dietary cysteine is equally effective in sparing dietary methionine whether fed enterally or parenterally.

The methionine requirement is lower in neonatal piglets fed parenterally than in those fed enterally.

The requirements for the sulfur amino acids (SAA), methionine (Met) and cysteine (Cys), have seldom been determined in neonates and to our knowledge have not previously been determined directly in parenterally fed neonates. The sulfur amino acids are catabolized largely in the liver and kidney, and their metabolism by the gut has been studied less frequently. In the present research, the enteral and parenteral Met requirement was determined, without dietary Cys, using the indicator amino acid oxidation (IAAO) technique. Piglets [n = 32, 2 d old, 1.66 +/- 0.13 kg (SD)] received elemental diets containing adequate energy, phenylalanine (Phe) and excess tyrosine, with varied Met concentrations and no Cys. Diets were infused continuously via intravenous or intragastric catheters. Phenylalanine oxidation was determined during a primed, constant infusion of L-[1-(14)C]-Phe, by measuring expired (14)CO(2) and plasma specific radioactivity of Phe. For both parenteral and enteral groups, Phe oxidation (% of dose) decreased linearly (P < 0.01) as Met intake increased, then became low and unchanging. Using breakpoint analysis, the Met requirement was estimated to be 0.42 and 0.29 g/(kg. d) for enteral and parenteral feeding, respectively. Breakpoint analysis using absolute phenylalanine oxidation [ micro mol/(kg. h)] resulted in an estimation of the Met requirement of 0.44 and 0.26 g/(kg. d) for enteral and parenteral feeding, respectively. These data show that the parenteral Met requirement is approximately 69% of the enteral requirement and suggest that extraction of SAA by first-pass splanchnic metabolism may be responsible for this difference.