R. E. Austic

L‐Arginine‐Dependent Production of a Reactive Nitrogen Intermediate by Macrophages of a Uricotelic Species

L‐arginine‐dependent production of reactive nitrogen intermediates (RNIs: nitric oxide, nitrite, and nitrate) by mammalian macrophages has been proposed to occur via an L‐arginine oxidative deimination pathway and is known to be responsible for certain antineoplastic and antimicrobial effector functions. The present study represents the first examination of this pathway in a non‐mammalian vertebrate. Because chickens, unlike mammals, lack a urea cycle and are incapable of de novo synthesis of L‐arginine, the possible existence of an avian macrophage pathway for production of RNIs is questionable. We have defined conditions under which chicken macrophages are able to produce nitrite. Sephadex‐elicited chicken peritoneal macrophages required a bacterial Ii‐popolysaccharide (LPS from Escherichia coli) signal to produce nitrite during 24 hour cultures in the presence of L‐arginine. As little as 5 ng/ml LPS resulted in significant nitrite production in culture. The relationship of nitrite production to both LPS and L‐arginine levels was dose‐dependent. D‐arginine was unable to substitute for L‐arginine but also produced no inhibitory effect In contrast, L‐NG‐monomethyl arginine showed a significant inhibitory effect on nitrite production. A virus‐transformed chicken macrophage cell line, HD11, also produced nitrite in a dose‐dependent manner relative to both LPS and L‐arginine concentration. Concentrations as low as 5 ng/ml LPS and 0.1 mM L‐arginine resulted in significant nitrite production, while maximum levels of nitrite production were obtained using ≥0.5 μg/ml LPS and ≥0.4 mM L‐arginine. These results indicate that chicken macrophages can produce RNIs. This production is dependent upon activation and is influenced by local L‐arginine concentration. Moreover, because the chicken does not possess the ability to synthesize arginine and has an absolute nutritional requirement for this amino acid, the chicken represents a highly controllable system to examine the in vivo effects of L‐arginine on macrophage‐related immune functions.

YOU CAN'T JUDGE A PIGMENT BY ITS COLOR: CAROTENOID AND MELANIN CONTENT OF YELLOW AND BROWN FEATHERS IN SWALLOWS, BLUEBIRDS, PENGUINS, AND DOMESTIC CHICKENS

Abstract The two main pigment types in bird feathers are the red, orange, and yellow carotenoids and the black, gray, and brown melanins. Reports conflict, however, regarding the potential for melanins to produce yellow colors or for carotenoids to produce brown plumages. We used high-performance liquid chromatography to analyze carotenoids and melanins present in the yellow and brown feathers of five avian species: Eastern Bluebirds (Sialia sialis), Barn Swallows (Hirundo rustica), King Penguins (Aptenodytes patagonicus), Macaroni Penguins (Eudyptes chrysolophus), and neonatal chickens (Gallus domesticus). In none of these species did we detect carotenoid pigments in feathers. Although carotenoids are reportedly contained in the ventral plumage of European Barn Swallows (Hirundo rustica rustica), we instead found high concentrations of both eumelanins and phaeomelanins in North American Barn Swallows (H. r. erythrogaster). We believe we have detected a new form of plumage pigment that gives penguin and domestic- chick feathers their yellow appearance. No Puedes Juzgar un Pigmento por su Color: Contenido de Carotenoide y Melanina de Plumas Amarillas y Marrones en Golondrinas, Azulejos, Pingüinos y Gallinas Domésticas Resumen. Los dos tipos principales de pigmentos que las aves incorporan en sus plumas son carotenoides, para desarrollar plumajes rojo, naranja o amarillo, y melaninas, para adquirir coloración negra, marrón, gris o tonalidades color tierra. Sin embargo, existe información conflictiva sobre la potencial coloración de plumas amarillas basadas en melanina y la presencia de caroteniodes en el plumaje marrón de ciertas especies. En este estudio, usamos cromatografía líquida de alto rendimiento para analizar los tipos y cantidades de carotenoides y melaninas presentes en las plumas amarillas y marrones de cinco especies de aves: el azulejo Sialia sialis y la golondrina Hirundo rustica, los pingüinos Aptenodytes patagonicus y Eudyptes chrysolophus y el plumón natal amarillo de la gallina doméstica Gallus domesticus. En ninguna de estas especies detectamos pigmentos carotenoides en las plumas. A pesar de que los carotenoides han sido encontrados en el plumaje ventral de la golondrina Hirundo rustica rustica, nosotros en cambio encontramos altas concentraciones de eumelaninas y feomelaninas en H. r. erythrogaster y en azulejos que variaron entre individuos y regiones de plumaje. Creemos que hemos detectado una nueva forma de pigmento de plumaje que le da a las plumas de pingüinos y pollos domésticos su apariencia amarilla.

Effect of feeding level and dietary electrolytes on the arginine requirement of rainbow trout (Salmo gairdneri)

Abstract Experiments were conducted to determine the effect of level of feeding (restricted feeding and feeding to satiation) and dietary variations in the balance of monovalent minerals on the arginine requirement of rainbow trout fingerlings. Based on growth and efficiency of feed utilization for growth, the arginine requirement was lower (3.5% of protein) when fish were fed to satiation than when they were fed at a restricted level (4.2% of protein). When calculated on the basis of the amount of arginine consumed per day, however, the requirements were similar for fish subjected to the two methods of feeding. Three balances (acidic, neutral, and alkaline) of sodium, potassium and chloride were used in several experiments. The arginine requirement tended to be higher when fish were fed diets containing the alkaline as compared to the acidic balance of minerals. These studies indicate that the method of feeding has a significant effect on the dietary concentration of arginine needed to maximize growth rate and feed utilization in rainbow trout.

Changes in protein synthesis due to an inflammatory challenge.

Abstract Rates of protein synthesis in various chick tissues were examined 16 hr after an inflammatory challenge. Protein synthetic rates were calculated from the rate at which [14C]leucine was incorporated into protein and the specific activity of [14C]leucine in the precursor pool. An injection of either Escherichia coli or sheep red blood cells (SRBC) decreased the rate of protein synthesis in the gastrocnemius muscle, and increased the rate in liver, bursa, spleen, and thymus. E. coli, but not SRBC, decreased protein synthesis in the pectoralis muscle. E. coli significantly decreased the aggregation of pectoralis muscle polysomes and increased the aggregation of polysomes in the thymus, bursa, and spleen. E. coli increased the aggregation of free, but not bound, polysomes in liver, suggesting an increase in synthesis of export proteins. SRBC significantly increased polysomal aggregation in bursa and spleen only. A crude preparation of leukocyte endogenous mediator, isolated from peritoneal macrophages, decreased muscle-polysomal aggregation. These studies indicate that tissue-specific changes in protein synthesis occur after a noninfectious inflammatory challenge. These changes may be part of a homeostatic mechanism which supports the immune response.

Changes in Protein Degradation in Chickens Due to an Inflammatory Challenge

Abstract Tissue-specific changes in protein catabolism were examined in chicks 16 hr following an inflammatory challenge. It was determined that tyrosine was not catabolized or converted to phenylalanine in muscle, thymus, bursa, or spleen. Therefore, rates of tyrosine release from protein were used to estimate rates of protein catabolism in these tissues. Arginine was not catabolized to urea by chick liver; consequently, arginine release from liver protein was used to measure protein catabolism in this tissue. An injection of sheep red blood cells (SRBC) or Escherichia coli did not change rates of protein catabolism in liver or bursa as compared to saline-injected controls. SRBC significantly increased protein catabolism in muscle and spleen by 29 and 15%, respectively. E. coli resulted in significant increases in muscle, spleen, and thymus of 43, 30, and 34%, respectively. These changes in protein catabolism, together with known changes in protein synthesis, suggest that an inflammatory response to SRBC and E. coli result in increased protein accretion in the bursa and liver, and net protein loss from muscle.

The effect of sodium bicarbonate or potassium bicarbonate on acid-base status and protein and energy digestibility in swine

Abstract Three experiments were conducted to evaluate the effects of Na or K on acid-base status and nutrient utilization in young growing pigs. These studies were carried out to develop a better understanding of the growth response to alkaline salts of these minerals that have been observed in lysine-deficient swine. In experiment 1, a 2×2 factorial arrangement of 2.2 g/kg vs. 7.4 g/kg Na and ad libitum vs. meal feeding regimens was used to evaluated the effects of Na on blood acid-base variables. Na was supplemented as the alkaline salt, NaHCO 3 . For each treatment regimen, blood samples were collected via indwelling vena cava catheters every 2 hr for 28 hr. Sodium supplementation increased (P 3 − and base excess and tended (P 3 (1.3% or 2.6%) or KHCO 3 (3.0%) on apparent nutrient digestibility and nitrogen balance was investigated utilizing pigs fitted with simple T cannulas in the ileum near the ileocecal junction. A 4×4 Latin square design was used. KHCO 3 or 2.6% NaHCO 3 increased (P 3 had no effect (P 3 decreased (P 3 on gastrointestinal pH was studied. No effects (P>0.05) were observed in the stomach, duodenum, jejunum, ileum, cecum or colon. NaHCO 3 or KHCO 3 alter the acid-base balance of the pig. However, their growth-promoting effects in lysine-deficient pigs are not due to enhanced lysine or protein digestibility

Intestinal absorption and renal excretion of dietary methionine sources by the growing chicken

Abstract The biopotency of DL-methionine (MET) and 2-hydroxy-4-[methylthio]butanoic acid (DL-HMB) for broiler chicks was determined using a semipurified diet. Based on growth rate and efficiency of feed utilization, DL-HMB was 72% as potent as DL-MET, while the potencies of L-MET and DL-MET were not different. Studies were conducted to determine if differences in intestinal absorption or urinary excretion of these compounds account for differences in bioavailability. 14 C-labeled methionine sources were fed to 2- to 3-week-old broiler chicks and the extent of their absorption determined using Cr 2 O 3 as a marker of digestibility. All three sources were completely absorbed in the small intestine. Urinary excretion was studied in 4- to 6-week-old broiler chicks. Saline solutions containing inulin, and 20 mmol/L L-MET, 20 mmol/L D-MET, or 5 or 20 mmol/L DL-HMB were infused intravenously. Blood and urine samples were taken at regular intervals over a 50-min period. The plasma concentrations of L-MET, D-MET, and DL-HMB increased during the course of infusion to 0.5 mmol/L. Urinary excretion of L-MET and D-MET remained less than 1% and 2.2% of the filtered load, respectively. Excretion of HMB increased from about 6% to about 18% of the filtered load as plasma concentration increased to about 0.3 mmol/L, and increased abruptly at higher plasma concentrations. Based on these data and plasma HMB concentrations determined in chicks fed DL-HMB, urinary losses of HMB would be less than 1% of the daily intake. These experiments indicate that differences of biopotency between methionine sources are not due to variations in intestinal absorption or urinary excretion.

Interactions among dietary minerals, arginine and lysine in rainbow trout (Salmo gairdneri)

Studies were conducted to determine whether interactions occur among dietary lysine, arginine and monovalent minerals in rainbow trout. In one experiment, rainbow trout fingerlings were fed diets containing three levels of lysine (2.4, 3.1 and 3.8 g per 100 g diet), two levels of arginine (1.7 and 2.5 g per 100 g diet) and two mixtures of Na+ K+ and Cl in a 3×2×2 factorial design. The mixtures varied in the proportions of cations to anions such that Cl equalled the sum of Na+ and K+ (cations − anions = 0 mEq/kg diet) in one mixture and exceeded the sum of Na+ and K+ (cations − anions = −200 mEq/kg diet) in the second mixture. Growth and efficiency of feed conversion were not affected by dietary lysine and arginine in fish fed diets containing − 200 mEq/kg balance, but when fish were fed diets containing a 0 mEq/kg balance, 3.8% lysine and a combination of 3.1% lysine and 2.5% arginine depressed both measures of response. Trout receiving the 0 mEq/kg cation-anion balance had significantly higher free histidine concentrations and lower free lysine concentrations in muscle and higher hepatic arginase activity (P≤0.01) than those receiving −200 mEq/kg. In another experiment, trout were fed diets containing three levels of K+ (21, 191 and 360 mEq/kg), two levels of Na+ (21 and 191 mEq/kg) and two levels of Cl− (179 and 347 mEq/kg) in a 3×2×2 factorial design. Growth and efficiency of feed conversion were depressed and hepatosomatic index increased with higher levels of dietary K+ (P≤0.01), Na+ (P≤0.05) and Cl (P≤0.01), with significant K+ x Cl+ (P≤0.01) and K+ x Na+ x Cl (P≤0.05) interactions. Increasing dietary K+ resulted in increased levels of muscle free histidine and decreased levels of muscle free lysine and arginine (P≤0.01), while increasing dietary Cl increased muscle free lysine, the effect of which was dependent on dietary potassium (K+ x Cl−, P≤0.01). It is concluded that dietary levels of K+, Na+ and Cl−, irrespective of overall cation-anion balance of these minerals, affects growth rate, efficiency of feed utilization and the metabolism of basic amino acids in tissues of trout. Excess lysine causes depressed growth and efficiency of feed utilization. These effects were due to a lysine toxicity rather than a lysine-arginine antagonism, as they were not prevented by supplemental dietary arginine.

Dietary Arginine Influences Rous Sarcoma Growth in a Major Histocompatibility B Complex Progressor Genotype

Abstract L-Arginine (L-Arg) can serve as a substrate for the production of reactive nitrogen intermediates. One of these metabolites, nitric oxide, has been shown to possess significant antitumor properties in vitro. To investigate the importance of this system in vivo, we have examined the dietary L-Arg host tumor interaction in the chicken. Since chickens are incapable of de novo L-Arg synthesis, concentration of this amino acid is readily controlled by diet. Line UNH 105 New Hampshire chickens having the major histocompatibility complex genotype, B 24/B 24, were used to study in vivo effects of dietary L-Arg on Rous sarcoma growth. After 5 weeks on a standard diet, 119 chicks were fed either a basal (0.92% L-Arg) diet or a high arginine (2.40% L-Arg) diet. One week later, chicks were wing-web inoculated with subgroup A Rous sarcoma virus. Tumor growth was monitored weekly for 12 weeks after inoculation. Plasma L-Arg levels and body weights from birds on each dietary treatment were analyzed. Neither body weight gains nor latent period for tumor development was affected by diet. However, plasma L-Arg levels were significantly different between dietary treatments (basal, 0.245 ± 0.01 μmol/ml; high, 0.738 ± 0.03 μmol/ml). In addition, mean tumor size scores were significantly (P < 0.05) lower over time in chickens fed the high L-Arg diet. The results suggest that dietary L-Arg in excess of the amount required for growth reduces tumor load.

Changes in plasma, tissue, and urinary nitrogen metabolites due to an inflammatory challenge.

Abstract Studies were undertaken to define the changes in protein metabolism that result from stimulation of the immune system by noninfectious inflammatory agents. Chicks were injected with inflammatory agents and metabolite concentrations were determined between 4 and 48 hr postchallenge. Inflammatory agents resulted in a generalized decrease in the concentration of plasma nitrogen metabolites, including ammonia, uric acid, urea, and several amino acids. Escherichia coli and sheep red blood cell (SRBC) injections induced changes in the concentrations of tissue-free amino acids at 16 hr postchallenge. After E. coli injections, free amino acid concentrations were increased by 175% in muscle and decreased by approximately 25% in liver, spleen, and bursa. A SRBC challenge resulted in similar decreases in free amino acid concentrations in the spleen and bursa as did E. coli; however, muscle and liver free amino acid concentrations were mostly unchanged. Urinary ammonia was increased, urinary uric acid was decreased, and urinary amino acids were not affected by E. coli injection. These findings indicate that stimulation of the immune system by noninfectious inflammatory agents induces tissue-specific changes in nitrogen metabolism. Changes in amino acid pool sizes in various tissues suggest alterations in rates of protein synthesis or degradation.