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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.

Recent studies using the indicator amino acid oxidation (IAAO) technique in TPN-fed piglets and infants have been instrumental in defining parenteral amino acid requirements. None of the commercial products in use are ideal when assessed against these new data. Our objectives were to determine whether the oxidation of an indicator amino acid would decline with the addition of amino acids that were limiting in the diets of TPN-fed piglets, and to use this technique to identify limiting amino acids in a new amino acid profile. Piglets (n = 26) were randomized to receive TPN with amino acids provided by Vaminolact (VM) or by a new profile (N P). After 5 d of TPN administration, lysine oxidation was measured using a constant infusion of L- [[1-.sup.14]C]-lysine. Immediately following the first IAAO study, the piglets were further randomized within diet group to receive either 1) supplemental aromatic amino acids (AAA), 2) sulfur amino acids (SAA) or 3) both (AAA+SAA) (n = 4-5 per treatment group). A second IAAO study was carried out 18 h later. In the first IAAO study, lysine oxidation was high for both groups (18 vs. 21% for VM and NP, respectively, P = 0.055). The addition of AAA to VM induced a 30% decline in lysine oxidation compared with baseline (P < 0.01). Similarly, SAA added to NP lowered lysine oxidation by -30% (P < 0.01). The application of the IAAO technique facilitates rapid evaluation of the amino acids that are limiting to protein synthesis in parenteral solutions.

Arginine is conditionally indispensable in the neonate, and its synthesis in the intestine is not sufficient to meet requirements. It is not known how neonatal endogenous arginine synthesis is regulated and the degree to which proline and glutamate are used as precursors. Primed, constant intraportal and intragastric infusions of L-[U[sup.14]C]proline and L-[3,4-[sup.3]H]glutamate, and intragastric L-[guanido-[sup.14]C]arginine were used to measure whole body and first-pass intestinal arginine synthesis in 10 neonatal piglets fed generous (1.80 g*[kg.sup.-1]*[day.sup.-1]) or deficient (0.20 g*[kg.sup.-1]*[day.sup.-1]) quantities of arginine for 5 days. Glutamate tracer was not detected in arginine, indicating a biologically insignificant conversion of < 1% of arginine flux. Endogenous arginine synthesis from proline had obligatory (0.36 g*[kg.sup.-1]*[day.sup.-1]) and maximal (0.68 g*[kg.sup.-1]*[day.sup.-1]) levels (P < 0.05, pooled SE 0.05). Although first-pass gut metabolism is responsible for 42-63% of whole body arginine synthesis, the gut is incapable of upregulating proline to arginine conversion during arginine deficiency, compared with a more than threefold increase without first-pass gut metabolism. These data suggest that upregulation of proline-to-arginine conversion occurs via increased arterial extraction of proline by the gut or in nonintestinal tissues. This study demonstrates that dietary arginine is an important regulator of endogenous arginine synthesis in the neonatal piglet and that proline, but not glutamate, is an important precursor for arginine synthesis in the neonate. proline; glutamate; arginine requirement; biosynthesis; amino acid kinetics; first-pass metabolism

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 x [kg.sup.-1] x [d.sup.-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. J. Nutr. 134: 609-612, 2004. KEY WORDS: * homocysteine * methionine * cysteine * total parenteral nutrition * enteral feeding

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 [+ or -] 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.sup.-14]C]phenylalanine, by measuring expired [sup.14]C[O.sub.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 ~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 ~40% in both enterally and parenterally fed neonatal piglets. Therefore, dietary cysteine is equally effective in sparing dietary methionine whether fed enterally or parenterally. KEY WORDS: * cysteine * cysteine sparing * indicator amino acid oxidation * methionine * total parenteral nutrition

We have previously shown that arginine deficiency is exacerbated by the removal of dietary proline in orally, but not parenterally, fed piglets. Therefore, we hypothesized that the net interconversions of proline, ornithine, and arginine primarily occur in the small intestine of neonatal piglets. Ten intragastrically fed piglets received either intraportal (IP) or intragastric (IG) primed, constant infusions of [guanido-[sup.14]C] arginine and [U-[sup.14]C] ornithine + [2,3-[sup.3]H]proline. By infusing amino acid isotopes via the stomach compared with the portal vein, we isolated small intestinal first-pass metabolism in vivo. During IP infusion, fractional net conversions (%) from proline to ornithine (0), ornithine to arginine (11 [+ or -] 6), and ornithine to proline (5 [+ or -] 1) were lower (P < 0.05) than during IG infusion (39 [+ or -] 8, 18 [+ or -] 6, and 42 [+ or -] 12, respectively); we speculate that these data are due to the localization of ornithine aminotransferase to the gut. The balance of these conversions indicated a large synthesis of arginine (70.0 [micro]mol*[kg.sup.-1]*[h.sup.-1]) by the gut, with a corresponding degradation of ornithine (70.8 [micro]mol* [kg.sup.-1]*[h.sup.-1]) and no change in proline balance. Gut synthesis of arginine from proline (48.1 [micro]mol*[kg.sup.-1]*[h.sup.-1]) was 50% of its requirement, whereas proline synthesis from arginine (33.0 [micro]mol*[kg.sup.-1]*[h.sup.-1]) amounted to 10% of its requirement. Overall, arginine synthesis is more dependent on the gut than proline synthesis. In situations in which gut metabolism is compromised, such as during parenteral nutrition or gastrointestinal disease, arginine and proline are individually indispensable because their biosyntheses are negligible. biosynthesis; intraportal infusion; ornithine aminotransferase; amino acid kinetics; first-pass metabolism

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 [+ or -] 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-[sup.14]C]-Phe, by measuring expired [sup.14]C[O.sub.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 ~69% of the enteral requirement and suggest that extraction of SAA by first-pass splanchnic metabolism may be responsible for this difference. KEY WORDS: * amino acid requirements * methionine * cysteine * total parenteral nutrition * indicator amino acid oxidation * piglets

Piglet models fed identical diets either intravenously or intragastrically were used to clearly demonstrate that arginine and proline are essential amino acids for both parenterally and enterally fed neonates. Moreover, it was shown that the gut functions as an important mediator of whole body arginine supply by employing proline as a major substrate for arginine synthesis. However, dietary proline or glutamate cannot totally spare the whole body requirements for arginine.