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.

Pattern of carbon dioxide production and retention is similar in adult pigs when fed hourly, but not when fed a single meal.

BACKGROUND: The understanding of bicarbonate kinetics and CO2 retention in the body is necessary to conduct amino acid tracer oxidation studies in both humans and laboratory animals. Significant metabolic activity is associated with eating which can affect bicarbonate steady state kinetics. A study was conducted to assess the impact of feeding regimen on the recovery of labelled bicarbonate and energy expenditure in adult female pigs (sows). Five catheterized sows (235 +/- 5 kg) were fed semi-synthetic diets as: a single meal 2 h into the infusion after an overnight fast, or in eight hourly meals starting 2 h before the infusion. Oxygen consumption, CO2 production and 14CO2 recovery (ie fraction not retained) were determined during primed, constant intravenous infusions of NaH14CO3. RESULTS: The 14CO2 recovery (%) after fasting (58.1 +/- 4.8) was lower than that after single meal feeding (78.8 +/- 5.9) or hourly meal feeding (81.0 +/- 2.6, P = 0.03). CO2 production correlated with 14CO2 recovery during hourly feeding (r = 0.40, P = 0.01); this relationship was not significant after single meal feeding (P = 0.30), probably due to physical activity-associated CO2 production. CONCLUSIONS: The correlation of CO2 retention factors with CO2 production during hourly feeding suggests that this regimen should be preferred for future amino acid kinetics studies.

Development of the indicator amino acid oxidation technique to determine the availability of amino acids from dietary protein in pigs.

Standardized ileal ("true") digestibility is currently the best estimate of amino acid digestibility, but it does not measure bioavailability. Growth assays to determine amino acid bioavailability are expensive and laborious; thus, a rapid method is needed. Applying the principle of slope-ratio assay to the indicator amino acid oxidation (IAAO) method, we hypothesized that the reduction in indicator oxidation per gram of lysine in feedstuffs relative to that per gram of free lysine represented the bioavailability of lysine, here termed "metabolic availability." Indicator oxidation in pigs was linear over increasing lysine intakes (r = 0.90, P = 0.001) when the dietary lysine contents were 2 SD below the mean lysine requirement of the pigs. Peas were treated (raw, heated to reduce lysine availability, or heated with added lysine) to test the responsiveness of the IAAO to differing lysine availability. Free lysine reduced indicator oxidation by 3.16% of dose oxidized per gram added lysine, whereas the addition of protein lysine as raw (-2.81%) and heated peas (-1.73%) reduced oxidation to a lesser degree. Adding free lysine to heated peas decreased indicator oxidation, evidence that heating had worsened the utilization of pea protein for protein synthesis by reducing the bioavailability of lysine alone. Pea diets differed only in the availability of lysine; therefore IAAO detected differences in lysine bioavailability. Because the IAAO technique responds to lysine available at the sites of protein synthesis, the metabolic availability covers all losses during digestion, absorption, and utilization of lysine. This method can determine the metabolic availability of amino acids of a feedstuff within 2 wk.

Indicator amino acid oxidation responds rapidly to changes in lysine or protein intake in growing and adult pigs.

There is disagreement about the adaptation time required when using the indicator amino acid oxidation (IAAO) technique. Our objective was to establish the adaptation time required to obtain a plateau in indicator (L-[1-(14)C]-phenylalanine) oxidation in response to a test diet using growing and adult pigs. Four barrows (20 kg) and 4 sows (240 kg) were surgically implanted with venous catheters for isotope infusion. Growing Pigs: After 7 d of adaptation to an adequate lysine intake of 8.8 g/d, phenylalanine oxidation in growing pigs was 9.38 +/- 1.25% of the infused dose. At 2, 3, 4, or 6 d after reducing lysine intake to 3.8 g/d, and then increasing it back to 8.8 g/d, phenylalanine oxidation was 16.94 +/- 0.84% (P < 0.05) and 9.70 +/- 0.80% (P < 0.05), respectively, with no significant effect of days of adaptation to diet. Adult Pigs: After 14 d of adaptation to an intake of 200% of the amino acid maintenance requirement, phenylalanine oxidation in sows was 4.23 +/- 0.45% of dose. Changing the intake to 100 and 50% of the maintenance requirement, increased (P < 0.05) phenylalanine oxidation to 5.95 +/- 0.26 and 7.90 +/- 0.26%, respectively, with no significant effect of time (1, 2, 5, 6, 9, and 10 d) after diet change. The CV for repeated phenylalanine oxidation measurements within pigs and diets was 13.5% for growing and 8.8% for adult pigs. This demonstrates that the IAAO requires <2 d of adaptation regardless of age, dietary challenge (individual amino acid or total protein) or direction (increase or decrease) of change, and that the measured oxidation rate (% of dose) is highly repeatable.

Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs.

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-(14)C]arginine and [U-(14)C]ornithine + [2,3-(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 +/- 6), and ornithine to proline (5 +/- 1) were lower (P < 0.05) than during IG infusion (39 +/- 8, 18 +/- 6, and 42 +/- 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 micromol. kg(-1). h(-1)) by the gut, with a corresponding degradation of ornithine (70.8 micromol. kg(-1). h(-1)) and no change in proline balance. Gut synthesis of arginine from proline (48.1 micromol. kg(-1). h(-1)) was 50% of its requirement, whereas proline synthesis from arginine (33.0 micromol. kg(-1). h(-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.

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 (IV(fed)/IV(dose)) versus enterally fed (IG(fed)/IV(dose)) 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 (IG(fed)/IG(dose)) versus i.v. (IG(fed)/IV(dose)) 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-(14)C]phenylalanine either parenterally (IV(fed)/IV(dose) and IG(fed)/IV(dose)) or enterally (IG(fed)/IG(dose)). The mean tryptophan requirements for IV(fed)/IV(dose) (0.145 +/- 0.023 g/kg/d), IG(fed)/IV(dose) (0.127 +/- 0.022 g/kg/d), and IG(fed)/IG(dose) (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.

Arginine synthesis is regulated by dietary arginine intake in the enterally fed neonatal piglet.

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-14C]proline and L-[3,4-3H]glutamate, and intragastric L-[guanido-14C]arginine were used to measure whole body and first-pass intestinal arginine synthesis in 10 neonatal piglets fed generous (1.80 g.kg(-1).day(-1)) or deficient (0.20 g.kg(-1).day(-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(-1).day(-1)) and maximal (0.68 g.kg(-1).day(-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.