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Metabolic Fates of Ammonia-N in Ruminal Epithelial and Duodenal Mucosal Cells Isolated from Growing Sheep

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Metabolic Fates of Ammonia-N in Ruminal Epithelial and Duodenal Mucosal Cells Isolated from Growing Sheep J. Dairy Sci. 88:3963–3970 American Dairy Science Association, 2005. Metabolic Fates of Ammonia-N in Ruminal Epithelial and Duodenal Mucosal Cells Isolated from Growing Sheep M. Oba,1 R. L. Baldwin, VI,2 S. L. Owens,3 and B. J. Bequette3 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5 2Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705 3Department of Animal and Avian Sciences, University of Maryland, College Park 20742 ABSTRACT (Key words: sheep, ruminal epithelial cells, duodenal mucosal cells, ammonia) The objective of this experiment was to determine the capability of ruminant gut tissues to detoxify am- Abbreviation key: AONCG = ONCG + aspartate, monia-N using short-term incubations of isolated cells DMC = duodenal mucosal cells, GC-MS = gas chroma- in vitro. Ruminal epithelial cells (REC) and duodenal tography/mass spectrometry, NCG = N-carbamoylglu- mucosal cells (DMC) were isolated from growing Texel- tamate, ONCG = NCG + ornithine, REC = ruminal Polypay ram lambs (n = 4) fed a pelleted forage:concen- epithelial cells, t-BDMS = t-butyldimethylsilyl. trate-based diet. Immediately after isolation, primary cells were incubated for 60 min with glucose (1mM), INTRODUCTION glutamate (1mM), [15N]ammonium chloride (5, 10, 20, Extensive dietary protein catabolism by rumen mi- or 40 mM), and 1 of 4 combinations of substrates (1 crobiota and subsequent ammonia absorption result mM each) that could support urea synthesis [control, in decreased efficiency of dietary protein use for pro- N-carbamoylglutamate (NCG); NCG + ornithine ductive purposes in ruminant animals. Enhancing ru- (ONCG); and ONCG + aspartate (AONCG)]. Treat- men microbial protein production, increasing urea re- ments were arranged in a 4 × 4 factorial design. Incor- cycling to the rumen, optimizing amino acid balance poration of ammonia-15N into alanine, citrulline, argi- for intestinal absorption, and decreasing first-pass me- nine, and urea was determined by gas chromatogra- tabolism of absorbed amino acids have been areas of phy-mass spectrometry. For both cell types, ammonia- research targeted to improve the net efficiency of N N transfer to alanine was lower when incubation me- usage in ruminants. Recent studies (Wu, 1995; Mouille dium contained NCG compared with control, whereas et al., 1999; Oba et al., 2004a) indicate that ammonia- use of ammonia-N for net alanine synthesis increased N detoxification pathways exist in gut tissues, thus quadratically with ammonia concentration regardless providing another target for nutritional or physiologi- of substrate treatment. For REC, ammonia-N was not cal approaches to reduce ammonia absorption and en- incorporated into citrulline, arginine, or urea, nor into hance net efficiency of N use in ruminants. arginine or urea by DMC. Ammonia-N use for net ci- Our previous study indicated that ruminant gut tis- trulline synthesis exhibited an inverse relationship sues are capable of synthesizing urea from arginine with ammonia concentration, decreasing linearly as or from ammonia when stimulated by N-carbamo- media ammonia concentration increased. Thus, ala- ylglutamate (NCG), a stable analog of N-acetylgluta- nine synthesis may be a significant metabolic pathway mate (Oba et al., 2004a). However, that study did not for ruminant gut tissues to detoxify ammonia-N when conclusively demonstrate that ammonia-N is assimi- it is presented luminally at high concentrations as lated into urea, but rather that urea is net-released compared with detoxification by the ornithine-urea cy- by ruminant gut cells. This could have occurred via cle. Furthermore, DMC do exhibit a metabolic capabil- complete function of the ornithine-urea cycle or by ity to incorporate ammonia-N into citrulline, but low action of arginase on arginine. Nonetheless, our re- or absent activity of downstream enzymes of the orni- sults agreed with work with pig intestinal (Wu, 1995) thine-urea cycle appears to limit ammonia-N transfers and rat colonic (Mouille et al., 1999) cells in which to urea. ammonia was detoxified to urea and citrulline, respec- tively. Another potential metabolic route for ammonia detoxification is via amination of keto-acids to form nonessential amino acids (e.g., alanine). Indeed, net Received June 6, 2005. Accepted August 3, 2005. absorption of alanine by the portal-drained viscera is Corresponding author: M. Oba; e-mail: [email protected]. greater than for other amino acids in sheep (Wolff et 3963 3964 OBA ET AL. al., 1972) and steer (Seal and Parker, 1996), poten- Table 1. Ingredients and nutrient composition of experimental diet (% of dietary DM except for DM). tially with N derived from absorbed ammonia. To date, however, the metabolic capability of ruminant gut tis- Ingredient sues to detoxify ammonia-N via synthesis of citrulline, Alfalfa hay 55.0 arginine, urea, or alanine has not been explored. Ground corn 40.0 The overall aim of the present study was to deter- Soybean meal 3.5 Ammonium chloride 0.5 mine metabolic fates of ammonia-N in ruminant gut Premix of salt and trace mineral1 0.5 tissues. Specific objectives were to confirm our previ- Dicalcium phosphate 0.45 2 ous observations that ruminant gut tissues possess Premix of vitamin A, D, and E 0.05 a complete ornithine-urea cycle pathway for de novo Chemical composition 15 DM 88.8 synthesis of urea from [ N]-ammonia, and to deter- NDF 34.5 mine whether ammonia-N is assimilated into alanine ADF 23.7 by ruminal epithelial (REC) and duodenal mucosal CP 15.8 Ether extracts 2.5 cells (DMC) of ruminant sheep. To establish the exis- Calcium 1.2 tence and activity of the pathways leading to urea Phosphorus 0.42 synthesis, combinations of substrates that contribute 1Premix of salt and trace mineral contains minimum of 92.0% NaCl; to the ornithine-urea cycle pathway (ammonia, NCG, 8000 ppm Zn; 5500 ppm Fe; 2400 ppm Mn; 670 ppm Cu; 67 ppm I; ornithine, aspartate) were provided to cells and the 67 ppm Co; and 1.6 ppm Se. 2 relative partition of [15N]-ammonia into alanine, ci- Premix of vitamins contains 5,291 kIU/kg of vitamin A, 1,322 kIU/ kg of vitamin D, and 11,023 IU/kg of vitamin E. trulline, ornithine, arginine, and urea determined by gas chromatography-mass spectrometry (GC-MS). pH 7.4. Incubations were initiated by addition of 0.5 MATERIALS AND METHODS mL of cell suspension (1 × 107 viable cells) to freshly gassed (20 s under 95:5 O2:CO2) media, and flasks were Animals and Cell Isolation placed into a reciprocal-action shaking water bath at All animal procedures were approved by the Belts- 37°C. After 60 or 90 min of incubation, 0.2 mL of con- ville Agricultural Research Center Institutional Ani- centrated HClO4 was injected into the flasks to termi- mal Care and Use Committee (protocol #02-008). Ru- nate the incubation, followed by addition of 0.3 mL of men epithelium cells (REC) and duodenal mucosal 5.8 M K2CO3 to neutralize the medium. cells (DMC) were isolated from 4 growing Texel-Pol- Experiment 1. Primary REC and DMC were incu- ypay crossbred ram lambs purchased from a commer- bated in triplicate for 90 min in the presence of 5 mM 15 14 cial sheep farm in Maryland. Lambs were housed in ammonium chloride ([ N] or [ N]) and 5 mM glucose. 14 individual pens at the USDA-ARS research facility The parallel incubations with unlabelled ( N) ammo- (Beltsville, MD), and fed ad libitum a pelleted diet nium chloride were used for determination of unla- composed of 55% forage and 45% concentrate (Table beled metabolite concentrations and release rates by 1) for at least 2 wk before slaughter. Daily DM intake an isotope dilution technique (Calder et al., 1999). For and gains, and BW at slaughter were 1.5 ± 0.1 kg/d, these unlabelled incubations, medium was clarified of 0.33 ± 0.11 kg/d, and 34.6 ± 2.9 kg, respectively. Gut cellular debris by centrifugation (2300 × g for 7 min), cells were isolated separately for each sheep following and to a known weight (2 g) of clarified medium was the procedures described by Baldwin and McLeod added a known weight (0.5 g) of a solution containing 15 (2000) and Oba et al. (2004b). Cell viability (trypan a mixture of tracer standards ([ N]glutamate, 15 15 blue dye exclusion) averaged 79.0% for REC and 81.6% [ N]aspartate, and [ N]alanine, each at 250 nmol). 15 for DMC. For incubations containing [ N]ammonium chloride, clarified medium was analyzed for 15N-containing end- products to determine the contribution of ammonia-N Incubations to the synthesis of glutamate, aspartate, and alanine. For all experiments, 2 flasks were prepared as time- Experiment 2. Primary REC and DMC were incu- zero controls to allow correction for endogenous metab- bated for 60 min in basal medium containing glucose olites and for determination of background abundance (1 mM), glutamate (1 mM), and [15N]ammonium chlo- of 15N. Ammonium chloride (99 atom % 15N) was pur- ride (5, 10, 20, or 40 mM), plus 1 of 4 combinations of chased from Cambridge Isotope Laboratories, Inc. (An- substrates to support urea synthesis via the ornithine- dover, MA). Incubation medium (2.5 mL; Krebs-Ringer urea cycle [control, NCG, NCG + ornithine (ONCG), plus 25 mM HEPES and 0.12 M sodium bicarbonate) and ONCG + aspartate (AONCG); 1 mM each]. Glu- was oxygenated with O2:CO2 (95:5) and adjusted to cose and glutamate were included in the basal medium Journal of Dairy Science Vol. 88, No. 11, 2005 AMMONIA-N UTILIZATION BY RUMINANT GUT CELLS 3965 to act as substrates for de novo synthesis of N-acetyl- because t-BDMS arginine yields the same ion frag- glutamate, ornithine, and aspartate.
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