Janet A. Brunton

Improvement in the Accuracy of Dual Energy X-ray Absorptiometry for Whole Body and Regional Analysis of Body Composition: Validation Using Piglets and Methodologic Considerations in Infants

Previously, we conducted dual energy x-ray absorptiometry (DXA) (Hologic QDR-1000/W) scans and carcass analysis of piglets to evaluate the Pediatric Whole Body software (PedWB) (V5.35) for use in infants. A software upgrade designed for infant whole body (InfWB) (V5.56) led to a reassessment of DXA by: 1) reanalysis of the original scans using InfWB software and 2) comparison of InfWB-estimates of bone mineral content (BMC) and lean and fat mass with chemical analysis. Other assessments included 1) methods of regional analysis and 2) artifacts and the Infant Table Pad in the scan field. The mean coefficients of variation for InfWB whole body measures in small piglets(n = 10, weight 1575 ± 73 g) and large piglets (n = 10, weight 5894 ± 208 g) were less than 2.6% except for fat mass which was higher (8.0% versus 6.3% and 6.6% versus 3.5%, respectively) compared with PedWB. In large piglets InfWB produced good estimates of BMC, lean and fat masses. In small piglets, fat mass by InfWB was correlated with chemical analysis, but not by PedWB. There was improvement in the estimation of BMC with InfWB, from 27 ± 2.2 g to 32 ± 2.3 g(carcass ash = 38 ± 3.3 g). Femur BMC analysis by InfWB was precise and was accurate when compared with chemical analysis. Artifacts in the DXA scan field (diapers and blankets) resulted in an increase of the DXA-estimated fat and lean masses. The Infant Table Pad increased the estimate of fat mass in a small piglet by 50%, thus further study is required before it is used routinely. Improvements of the DXA technology have resulted in a more accurate tool, if scanning procedures are carefully implemented.

Proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal piglets

The indispensability of arginine has not been conclusively established in newborns. Because parenteral feeding bypasses the gut (where de novo synthesis of arginine occurs from proline), a dietary supply of arginine that is sufficient to maintain urea cycle function may be of greater importance during intravenous compared with enteral feeding. Two-day-old piglets ( n = 12) were fed nutritionally complete diets for 5 days via either a central vein catheter (IV pigs, n = 6) or a gastric catheter (IG pigs, n = 6). Subsequently, each piglet received three incomplete test diets [arginine free (-ARG/+PRO), proline free (-PRO/+ARG), or arginine and proline free (-ARG/-PRO)] in a randomized crossover design. Plasma ammonia was assayed every 30 min for 8 h or until hyperammonemia was observed. Ammonia increased rapidly in IV pigs receiving -ARG/+PRO and -ARG/-PRO (84 ± 36 and 74 ± 37 μmol ⋅ l-1 ⋅ h-1, respectively), requiring early diet cessation. A rapid increase was also exhibited by IG pigs receiving the -ARG/-PRO, but not the -ARG/+PRO diet (31 ± 15 vs. 11 ± 7 μmol ⋅ l-1 ⋅ h-1, respectively, P < 0.05). Plasma arginine and proline were indicative of deficiency (IG and IV groups) when deplete diets were infused. Arginine is indispensable in parenteral and enteral nutrition, independent of dietary proline.The indispensability of arginine has not been conclusively established in newborns. Because parenteral feeding bypasses the gut (where de novo synthesis of arginine occurs from proline), a dietary supply of arginine that is sufficient to maintain urea cycle function may be of greater importance during intravenous compared with enteral feeding. Two-day-old piglets (n = 12) were fed nutritionally complete diets for 5 days via either a central vein catheter (IV pigs, n = 6) or a gastric catheter (IG pigs, n = 6). Subsequently, each piglet received three incomplete test diets [arginine free (-ARG/+PRO), proline free (-PRO/+ARG), or arginine and proline free (-ARG/-PRO)] in a randomized crossover design. Plasma ammonia was assayed every 30 min for 8 h or until hyperammonemia was observed. Ammonia increased rapidly in IV pigs receiving -ARG/+PRO and -ARG/-PRO (84 +/- 36 and 74 +/- 37 micromol. l(-1). h(-1), respectively), requiring early diet cessation. A rapid increase was also exhibited by IG pigs receiving the -ARG/-PRO, but not the -ARG/+PRO diet (31 +/- 15 vs. 11 +/- 7 micromol. l(-1). h(-1), respectively, P < 0.05). Plasma arginine and proline were indicative of deficiency (IG and IV groups) when deplete diets were infused. Arginine is indispensable in parenteral and enteral nutrition, independent of dietary proline.

Current total parenteral nutrition solutions for the neonate are inadequate.

The amino acid requirements of the parenterally fed neonate are poorly defined. Newborn infants are at risk for amino acid deficiency and toxicity, due to lack of small intestinal metabolism and metabolic immaturity. We discuss recent evidence that identifies inadequacies of commercial amino acid solutions with respect to the balance and quantity of aromatic amino acids, and sulphur amino acids. We present data demonstrating that impaired small intestinal metabolism (or lack of first pass metabolism) alters the whole body requirement for methionine, threonine, and arginine, and discuss the potential adverse effects of excess or inadequate parenteral amino acid intake.

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.

Determination of amino acid requirements by indicator amino acid oxidation: applications in health and disease.

Indispensable amino acid requirements for children, patients and pregnant women are largely unknown. We describe a new, non-invasive technique that can estimate requirements in vulnerable populations. Future applications will lead to optimal nutritional care for populations in which an appropriate balance of amino acids is essential for metabolic health and growth.

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.

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.