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Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, TX 77843-2471
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
This study was conducted to determine whether endogenous synthesis of arginine plays a role in regulating arginine homeostasis in postweaning pigs. Pigs were fed a sorghum-based diet containing 0.98% arginine and were used for studies at 75 d of age (28.4 kg body weight). Mitochondria were prepared from the jejunum and other major tissues for measuring the activities of 
1-pyrroline-5-carboxylate (P5C) synthase and proline oxidase (enzymes catalyzing P5C synthesis from glutamate and proline, respectively) and of ornithine aminotransferase (OAT) (the enzyme catalyzing the interconversion of P5C into ornithine). For metabolic studies, jejunal enterocytes were incubated at 37°C for 30 min in Krebs-Henseleit bicarbonate buffer containing 2 mmol/L L-glutamine, 2 mmol/L L-[U-14C]proline, and 0-200 µmol/L gabaculine (an inhibitor of OAT). The activities of P5C synthase, proline oxidase and OAT were greatest in enterocytes among all of the tissues studied. Incubation of enterocytes with gabaculine resulted in decreases (P < 0.05) in the synthesis of ornithine and citrulline from glutamine and proline. When gabaculine was orally administered to pigs (0.83 mg/kg body weight) to inhibit intestinal synthesis of citrulline from glutamine and proline, plasma concentrations of citrulline (
26%) and arginine (22%) decreased (P < 0.05), whereas those of alanine (+21%), ornithine (+17%), proline (+107%), taurine (+56%) and branched-chain amino acids (+21-40%) increased (P < 0.05). On the basis of dietary arginine intake and estimated arginine utilization, the endogenous synthesis of arginine in the 28-kg pig provided 
50.2% of total daily arginine requirement. Taken together, our results suggest an important role for endogenous synthesis of arginine in regulating arginine homeostasis in postweaning growing pigs, as previously shown in neonatal pigs.
INTRODUCTION
Arginine is a basic amino acid and serves as an essential precursor for the synthesis of biologically important molecules such as protein, ornithine, proline, polyamines, creatinine, nitric oxide and agmatine (Barbul and Dawson 1994
, Cynober et al. 1995, Li et al. 1994). Nitric oxide is an endothelium-derived relaxing factor, a neurotransmitter, a mediator of immune response and a signalling molecule (Bredt and Snyder 1994). Agmatine is a novel noncatecholamine ligand at 
2-adrenergic receptors (Li et al. 1994) and an inhibitor of nitric oxide synthase (Galea et al. 1996). Arginine also plays an important role in the detoxification of ammonia via the urea cycle and is a potent stimulator of secretion of insulin and growth hormone, important regulators of nutrient metabolism (Mulloy et al. 1982, Visek 1986). Although arginine can be synthesized by most mammals (except for cats and ferrets), it is classified as a nutritionally essential amino acid for young mammals and for adults at times of stress and illness (Visek 1986, Yu et al. 1996). Thus, regulation of arginine homeostasis is of nutritional and physiologic importance.
Factors that regulate arginine homeostasis include dietary arginine intake, endogenous synthesis and degradation of arginine, as well as intracellular protein turnover. We have recently demonstrated that endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in neonatal pigs nursed by sows (Flynn and Wu 1996). This is of nutritional and physiologic importance for neonates because of a remarkable deficiency of arginine in the milk (Davis et al. 1994, Wu and Knabe 1994). In contrast, arginine homeostasis has been suggested to be regulated mainly by dietary arginine intake and arginine oxidation rather than by endogenous arginine synthesis in adult humans (Castillo et al. 1993). In both of these studies, piglets and humans were in the fed state. The contrasting findings between piglets and adult humans may result from differences in species, age and dietary availability of arginine.
The synthesis of arginine from glutamine requires glutaminase, 1-pyrroline-5-carboxylate (P5C)4 synthase, ornithine aminotransferase (OAT), carbamoylphosphate synthase I (CPS-I), ornithine carbamoyltransferase (OCT), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) (Wu and Knabe 1995) (Fig. 1). Glutaminase, OAT, ASS and ASL are widely distributed in mammalian tissues, but CPS-I and OCT are located exclusively in the liver and small intestine (Curthoys and Watford 1995, Wakabayashi 1995). Because P5C synthase is almost exclusively located in enterocytes, the small intestine is the major, if not exclusive organ for synthesis of P5C from arterial glutamine and dietary glutamate/glutamine (Flynn and Wu 1996, Wakabayashi 1995). Thus, there is enormous interest in intestinal metabolism of glutamine and arginine in health and disease (Burrin and Reeds 1997). Glutamine/glutamate has generally been considered to be the only source of P5C for citrulline synthesis in the small intestine (Wakabayashi 1995). However, we have recently demonstrated that proline is an important source of P5C (via proline oxidase) and citrulline in pig enterocytes (Wu 1997). Pyrroline-5-carboxylate is the common intermediate in pathways for the synthesis of citrulline from both glutamine and proline; it is interconverted into ornithine by OAT in enterocytes (Wu 1997). An inhibition of OAT will lead to decreased synthesis of citrulline and arginine from both glutamine and proline in enterocytes, thereby resulting in arginine deficiency, as in OAT-gene knockout mice (Wang et al. 1995).
1-L-pyrroline-5-carboxylate (P5C) synthase (a bifunctional enzyme) that exhibits 
-glutamyl kinase activity; 3) glutamate aminotransferase; 4) P5C synthase (a bifunctional enzyme) that exhibits -glutamylphosphate reductase activity; 5) spontaneous chemical reaction; 6) ornithine aminotransferase; 7) proline oxidase; 8) ornithine carbamoyltransferase; 9) carbamoylphosphate synthase-I; 10) argininosuccinate synthase; 11) argininosuccinate lyase. Reactions 1-9 occur in mitochondria, and reactions 10-11 take place in the cytosol. Reaction 3 also occurs in the cytosol. Abbreviations used: Asp, L-aspartate; CP, carbamoylphosphate; Glu, L-glutamate; -KG, -ketoglutarate; NAG, N-acetyl-glutamate; OAA, oxaloacetate.
The objective of this study was to determine whether endogenous synthesis of arginine plays a role in maintaining arginine homeostasis in postweaning pigs. Intestinal synthesis of citrulline and arginine from glutamine and proline was inhibited by gabaculine, a suicide inhibitor of OAT in animal cells (Jung and Seiler 1978), including pig enterocytes (Flynn and Wu 1996).
MATERIALS AND METHODS
-ketoglutarate, ATP and NADPH from Boehringer Mannheim (Indianapolis, IN). L-[U-14C]Proline and L-[U-14C]glutamate were purchased from American Radiolabeled Chemicals (St. Louis, MO) and Amersham (Arlington Heights, IL), respectively. HPLC-grade methanol and H2O were obtained from Fisher Scientific (Houston, TX).
Animals.
Pigs were offspring of Yorkshire × Landrance sows and Duroc × Hampshire boars, and were obtained from the Texas A&M University Swine Center. Piglets were freely nursed by sows until 28 d of age, when they were weaned to a standard sorghum-soybean meal-based diet containing 20% crude protein (Hansen et al. 1993). Beginning at 60 d of age, pigs were fed a 16% crude protein sorghum-based diet containing 0.98% arginine (Table 1) that met NRC nutrient requirements (NRC 1988). For analysis of amino acids (except tryptophan) in the diet, 1.0 g of the diet was hydrolyzed in 100 mL of 6 mol/L HCl at 110°C for 24 h under N2, and amino acids in acid hydrolysates were measured by HPLC (Wu and Knabe 1994). Tryptophan in the diet was determined as described by La Rue (1985). Briefly, 0.2 g of diet was hydrolyzed in a polyallomer tube containing 10 mL of 4.2 mmol/L NaOH and 0.1 mL of thiodiglycol (an antioxidant, 25% aqueous solution) at 110°C for 20 h, and tryptophan was analyzed by HPLC (Wu and Knabe 1994). Pigs had free access to water and feed, and were used for studies at 75 d of age (28.4 ± 0.8 kg body weight; means ± SEM, n = 22). This study was approved by Texas A&M University's Institutional Animal Care and Use Committee.
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Table 1. Composition of diet1 |
). Briefly, pigs were preanesthetized with an intramuscular injection of ketamine and acetylpromazine at 4.76 and 0.24 mg/kg body weight, respectively. Halothane was administered with a face mask to achieve a surgical plane of anesthesia. A mid-jejunal segment (50 cm) was removed, rinsed thoroughly with saline, filled with calcium-free oxygenated (95% O2-5% CO2) Krebs-Henseleit bicarbonate buffer (119 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4 and 25 mmol/L NaHCO3, pH 7.4) containing 20 mmol/L HEPES (pH 7.4), 5 mmol/L EDTA (disodium), and 5 mmol/L D-glucose (95% O2/5% CO2), and incubated for 50 min at 37°C in a shaking water bath (70 oscillations/min). At the end of the incubation, the jejunum was gently patted with finger tips for 1 min, and the lumen was drained into polystyrene tubes. Enterocytes were obtained by centrifugation (400 × g, 2 min). Cells (pellet) were washed three times with oxygenated Krebs-Henseleit bicarbonate buffer (95% O2-5% CO2) containing 2.5 mmol/L CaCl2 (no EDTA), 20 mmol/L HEPES (pH 7.4), and 5 mmol/L D-glucose. Enterocytes were viable for at least 30 min of incubation, as assessed by linear consumption of oxygen (Wu et al. 1994).
Distributions of OAT, P5C synthase and proline oxidase activities in pig tissues.
Pigs (75 d old) were anesthetized as described above. Adipose tissue, brain, colon, heart, jejunum, kidney, liver, lung, pancreas, skeletal muscle (semitendinosus muscle) and spleen were dissected (Flynn and Wu 1996). The jejunum was rinsed 3 times with saline to remove luminal digesta. Mucosa was separated from the jejunal serosal smooth muscle layer with the use of a glass slide. The serosal layer was rinsed three times with saline to remove residual mucosa.
). Briefly, the assay mixture (2 mL) consisted of 75 mmol/L potassium phosphate buffer (pH 7.5), 20 mmol/L ornithine, 0.45 mmol/L pyridoxal phosphate, 5 mmol/L o-aminobenzaldehyde, 0 or 3.75 mmol/L -ketoglutarate and cell extracts. The amount of cell extract protein in the OAT assay was 0.1, 0.5 and 5 mg for jejunal mucosa, intact jejunum and other tissues, respectively. The OAT assay was linear with time and amount of protein used.
), except that protease inhibitors were used in tissue homogenization medium. Briefly, tissues (0.5 g) were homogenized at 4°C in 6 mL of buffer [250 mmol/L sucrose, 1 mmol/L EDTA and 2.5 mmol/L dithiothreitol in 50 mmol/L potassium phosphate buffer (pH 7.2)] containing protease inhibitors (5 mg/L phenylmethylsulfonyl fluoride, 5 mg/L aprotinin, 5 mg/L chymostatin and 5 mg/L pepstatin A). The homogenates were centrifuged at 600 × g and 4°C for 10 min, and the supernatant fractions were centrifuged at 12,000 × g for 10 min at 4°C. The pellets (mitochondria) were suspended in 0.5 mL of 50 mmol/L potassium phosphate buffer (pH 7.5) and stored at 20°C for 24 h before use for proline oxidase assay. The assay mixture (1.0 mL), which consisted of 15 mmol/L L-proline, 20 µmol/L ferricytochrome C, mitochondrial pellets and 50 mmol/L potassium phosphate buffer (pH 7.5), was incubated at 37°C for 0 or 30 min. The reaction was terminated by addition of 0.5 mL of 10% trichloroacetic acid, followed by addition of 0.1 mL of 100 mmol/L o-aminobenzaldehyde. After a 30-min period of standing at room temperature, the mixture was centrifuged at 600 × g for 5 min, and the absorbance of the supernatant fraction was measured at 440 nm. Blanks (0 min incubation) were subtracted from sample values. The mitochondrial protein content in proline oxidase assay was 0.5 mg for jejunal mucosa, 2 mg for intact jejunum, liver and kidney, and 5 mg for other tissues. The enzyme assay was linear with both time and amount of protein used for the tissues containing proline oxidase activity.
). The assay mixture (1 mL) contained 0.1 mol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 1 mmol/L gabaculine, 1 mmol/L [U-14C]glutamate (200 Bq/nmol), 3 mmol/L ATP, 0.2 mmol/L NADPH, 15 mmol/L phosphocreatine, 10 U of creatine kinase, 0.25% Nonidet P-40 and mitochondria (1 mg protein). [14C]P5C was separated from [14C]glutamate by anion-exchange chromatography (Dowex AG 1-X8 resin, acetate form, 200-400 mesh) (Wu et al. 1994). The recovery of P5C was >99% as determined with the use of a purified P5C standard. The P5C synthase assay was linear with time and amounts of protein used.
and Wakabayashi et al. (1983) for the enzyme that catalyzes P5C synthesis from glutamate in mammalian cells. The P5C synthase assay, which was developed by Wakabayashi et al. (1983) and adapted in our studies (Wu and Knabe 1995), measured the conversion of L-glutamate into P5C. This pathway involves the following two reactions: 1) phosphorylation of L-glutamate by -glutamyl kinase (-GK) to form L-glutamyl--phosphate, and 2) reduction of L-glutamyl--phosphate by -glutamylphosphate reductase (-GPR) to form L-glutamyl--semialdehyde, with the latter spontaneously cyclizing to P5C (Fig. 1). In Escherichia coli, -GK and -GPR are two separate enzymes encoded by two distinct genes, pro B and pro A, respectively (Deutch et al. 1984), and are considered to form an enzyme complex necessary for P5C synthesis from glutamate (Hayzer and Moses 1978a and 1978b). In both plants (Hu et al. 1992) and mammalian cells (Aral et al. 1996), the conversion of L-glutamate to P5C is catalyzed by a bifunctional enzyme (P5C synthase, a single polypeptide) that exhibits both -GK and -GPR activities.
). Net production of amino acids was calculated on the basis of differences in their concentrations in cell extracts between 0- and 30-min incubation periods in the presence of 2 mmol/L glutamine.
Effect of gabaculine on synthesis of ornithine and citrulline from proline in enterocytes.
For the determination of amino acid synthesis from proline, enterocytes (25 × 106 cells/mL) were incubated at 37°C for 0 or 30 min in 2 mL of oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, saturated with 95% O2/5% CO2) containing 20 mmol/L HEPES, 1% BSA, 5 mmol/L glucose, 2.0 mmol/L L-glutamine, 2 mmol/L L-[U-14C]proline (150 Bq/nmol), and 0-200 µmol/L gabaculine. L-Glutamine was added to the incubation medium to provide the following: 1) ammonia for CPS-I, 2) glutamate for OAT and 3) aspartate for ASS. Incubations were initiated by addition of cells. After a 30-min incubation period, 0.5 mL of incubation medium containing cells was removed from the flask for determination of the intracellular specific activities of [14C]proline, [14C]ornithine and [14C]citrulline as described by Wu (1997). The remaining portion of medium plus cells (1.5 mL) was acidified with 0.2 mL of 1.5 mol/L HClO4 and neutralized with 0.1 mL of 2 mol/L K2CO3. Cell extracts were used for analysis by HPLC of amino acids and [14C]ornithine, [14C]citrulline and [14C]arginine (Wu 1997). The intracellular specific activities of [14C]proline, [14C]ornithine and [14C]citrulline were used to calculate production of [14C]ornithine, [14C]citrulline and [14C]arginine from proline, respectively.
Effect of oral administration of gabaculine on plasma concentrations of free amino acids, ammonia and urea.
Pigs (75 d old) were randomly assigned to one of two groups with eight animals each. One group of pigs received oral administration of gabaculine (0.83 mg/kg body weight) at 600, 1000 and 1400 h. The other group received an equivalent volume of vehicle solvent (double distilled deionized water) according to the same time schedule. Gabaculine was dissolved in 5 mL of water and then delivered orally via a 5-mL syringe. Blood samples (5 mL) were drawn from the jugular vein into heparinized tubes at 1600 h of the day before gabaculine administration (initial blood sampling) and again at 1600 h on the day of gabaculine administration (final blood sampling). Blood samples were placed in ice and centrifuged at 2000 × g for 15 min to obtain plasma (the supernatant fraction). Plasma (1 mL) was deproteinized with 1 ml of 1.5 mol/L HClO4, and neutralized with 0.5 mL of 2 mol/L K2CO3. Neutralized plasma was centrifuged at 600 × g for 10 min, and the supernatant fraction was used for analysis of ammonia and urea by colorimetric methods (Wu and Knabe 1994) and of amino acids by HPLC (Wu et al. 1994).
Amino acid analysis by HPLC.
The HPLC apparatus and precolumn derivatization of amino acids with o-phthaldialdehyde were as previously described (Wu 1993). Amino acids [except proline and cyst(e)ine] were separated on a Supelco 3-µm reversed-phase C18 column (4.6 × 150 mm, i.d.) guarded by a Supelco 40-µm reversed-phase C18 column (4.6 × 50 mm, i.d.; Supelco, Bellefonte, PA). The HPLC mobile phase consisted of solvent A (0.1 mol/L sodium acetate/0.5% tetrahydrofuran/9% methanol; pH 7.2) and solvent B (methanol), with a combined total flow rate of 1.1 mL/min. A gradient program with a total running time of 49 min (including the time for column regeneration) was developed for satisfactory separation of amino acids (0 min, 14% B; 15 min, 14% B; 20 min, 30% B; 24 min, 35% B; 26 min, 47% B; 34 min, 50% B; 38 min, 70% B; 40 min, 100% B; 42 min, 100% B; 42.1 min, 14% B; 48.5 min, 14% B). Proline was measured by an HPLC method involving oxidation of proline to 4-amino-1-butanol and precolumn derivatization with o-phthaldialdehyde (Wu 1993). Cysteine and cystine were analyzed by an HPLC method as previously described (Wu and Knabe 1994). Briefly, for cysteine analysis, a 100-µL sample was mixed with 50 µL of 50 mmol/L iodoacetic acid (an alkylating agent) for 5 min at room temperature to convert cysteine to S-carboxymethylcysteine. The latter then reacts with o-phthaldialdehyde to form a highly fluorescent derivative. For cystine analysis, a 100-µL sample was mixed with 100 µL of 28 mmol/L 2-mercaptoethanol (a reducing agent) for 5 min at room temperature to convert cystine to cysteine, and the latter was then analyzed as described above.
Protein determination.
Protein in enterocytes, mitochondria and cytosolic fluid was determined by a modified Lowry procedure, with BSA as a standard (Wu et al. 1994).
Statistical analysis.
Results were statistically analyzed as described by Steel and Torrie (1980). Data on enzyme activities and metabolism of glutamine and proline were analyzed by one-way ANOVA and the Student-Newman-Keuls test. Unpaired t test was used to analyze the differences in plasma metabolites between control and gabaculine-treated pigs. Paired t test was used to analyze the differences in plasma metabolites between the initial and final blood sampling periods for each group of pigs. Probabilities <0.05 were taken to indicate statistical significance.
RESULTS
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Table 2. Ornithine aminotransferase (OAT), proline oxidase and pyrroline-5-carboxylate (P5C) synthase in pig tissues1 |
), indicating that gabaculine had no effect on net proteolysis. No detectable amount of [14C]-labeled amino acids other than [14C]ornithine, [14C]citrulline and [14C]arginine was produced from [14C]proline in enterocytes incubated in the presence of 0-200 µmol/L gabaculine.
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Table 3. Net production of amino acids by pig enterocytes incubated in the presence or absence of gabaculine1,2 |
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Table 4. Effect of gabaculine on net production of [14C]ornithine, [14C]citrulline and [14C]arginine from [14C]proline in pig enterocytes1,2 |
Table 5.
Plasma concentrations of free amino acids, ammonia and urea in pigs1
22%) and citrulline (26%), 2) increased (P < 0.05) plasma concentrations of alanine (+21%), isoleucine (+28%), leucine (+21%), ornithine (+17%), proline (+107%), taurine (+56%), threonine (+48%) and valine (+40%), and 3) had no effect (P > 0.05) on other amino acids, ammonia or urea.
DISCUSSION
). Because hepatic OAT is absent from periportal hepatocytes and is restricted to perivenous hepatocytes with no CPS-I or OCT activity (Kuo et al. 1991), proline-derived P5C cannot be converted to citrulline in perivenous hepatocytes or to ornithine in periportal hepatocytes. As a result, there is no synthesis of citrulline from proline in the liver. Similarly, because the kidney contains no CPS-I or OCT activity (Edmonds et al. 1987, Morris 1992), there is no synthesis of citrulline from proline in this organ. Thus, as the only organ that contains all enzymes for synthesizing citrulline from glutamine and proline in animals (Wu 1997, Wu and Knabe 1995), the small intestine plays a major role in citrulline and arginine metabolism. In postnatal pig enterocytes, ~80-90% of utilized proline carbons were recovered in ornithine plus citrulline plus arginine (with CO2 being a minor product) (Wu 1997). Considering the relatively large mass of the small intestine compared with the liver and kidney in growing pigs (e.g., 398, 246 and 41 g in 6-wk-old pigs, respectively) (Schoknecht and Pond 1993), we suggest that the small intestine is a major organ for metabolism of ornithine and proline and for synthesis of citrulline from glutamine and proline in postweaning pigs. The intestine-derived citrulline is utilized for arginine synthesis in the kidneys of postweaning pigs, as in adult rats (Dhanakoti et al. 1990), which have high activities of ASS and ASL (Wu and Knabe 1995).
, Herzfeld et al. 1977). Second, proline oxidase activity was found to be greatest in the small intestine of neonatal and postweaning pigs (Table 2) (Samuels et al. 1989), but was reported to be negligible in or absent from the intestine of neonatal and adult rats (Herzfeld et al. 1977). Our findings with pigs are in contrast to the current view that proline oxidase is present primarily in the liver, kidney and brain of mammals (Phang et al. 1995).
). An inhibition of OAT is expected to inhibit synthesis of ornithine and citrulline from both glutamine and proline in enterocytes. This suggestion is supported by our findings that gabaculine [a potent inhibitor of pig enterocyte OAT; Ki = 19 µmol/L (Davis 1997)] markedly decreased the synthesis of ornithine and citrulline from glutamine and proline by pig enterocytes in a concentration-dependent manner (Tables 2 and 3). Because gabaculine does not interfere directly with the urea cycle and because a short-term treatment of gabaculine in pigs (Flynn and Wu 1996) and mice (Alonso and Rubio 1989) does not result in ammonia toxicity or any other adverse effects, oral administration of gabaculine was an attractive approach to decrease synthesis of citrulline and arginine from glutamine and proline in pigs and to determine the role for endogenous arginine synthesis in regulating arginine homeostasis in vivo.
Effect of OAT inhibition on plasma concentrations of citrulline and arginine.
This is the first study to determine the role for endogenous synthesis of arginine in maintaining arginine homeostasis in postweaning growing pigs. Oral administration of gabaculine resulted in decreased plasma concentrations of citrulline and arginine in postweaning 75-d-old pigs in the fed state (Table 5), as in neonatal pigs nursed by sows (Flynn and Wu 1996). Indeed, citrulline and arginine were the only two amino acids whose plasma concentrations were reduced by gabaculine treatment. Because gabaculine inhibits arginine degradation (Wu et al. 1996) and has no effect on dietary arginine intake, and because citrulline is absent from dietary and tissue proteins, we interpret the decreased plasma concentration of citrulline and arginine in gabaculine-treated pigs as likely resulting from decreased endogenous synthesis of these two amino acids. To substantiate this suggestion, studies involving stable or radioactive tracers are required to quantify rates of arginine synthesis in control and gabaculine-treated pigs.
). On the basis of arginine content in pig tissue protein (6.18 g arginine/100 g protein) (Wilson and Leibholz 1981), arginine requirement for net protein deposition is 7.91 g/d (6.18 × 128/100 = 7.91). On the basis of the oxidation of plasma arginine to CO2 [10.6 µmol/(kg·h)] and the conversion of plasma arginine to proline, glutamate, citrulline and ornithine [a total of 51.7 µmol/(kg·h)] in the young pig (Murch et al. 1996), the rate of catabolism of arginine to these products is 7.28 g/d. Thus a total requirement for arginine by the 28-kg pig is 15.19 g/d (7.91 + 7.28 = 15.19). The amount of dietary arginine entering the portal vein is estimated to be 7.56 g/d, on the basis of dietary arginine intake (14.02 g/d) and the following assumptions: 1) digestibility of arginine in feed is 90% (Knabe et al. 1988); and 2) 60% of luminal free arginine is absorbed intact by the small intestine (Windmueller and Spaeth 1976). Thus endogenous synthesis of arginine in the 28-kg pig is estimated to provide 7.63 g/d of arginine (15.19 7.56 = 7.63), or 50.2% of total daily arginine requirement, suggesting that endogenous arginine synthesis plays an important role in regulating arginine homeostasis in postweaning growing pigs, as previously shown in neonatal pigs (Flynn and Wu 1996). This suggestion is not consistent with previous findings that arginine homeostasis is regulated mainly by dietary arginine intake and arginine oxidation rather than by endogenous arginine synthesis in adult humans (Castillo et al. 1993). It is important to determine whether endogenous synthesis of arginine from glutamine/glutamate and proline plays an important role in regulating arginine homeostasis in growing children with net protein deposition in the body.
). Such a paradoxical effect of OAT inhibition has been reported in mice and humans, in that a deficiency of OAT decreases plasma concentration of ornithine in neonates but resulted in hyperornithinemia in adults (Wang et al. 1995, Valle and Simell 1995). In gabaculine-treated pigs, the source of elevated plasma ornithine is likely to be arginine, because gabaculine decreased ornithine synthesis from glutamine and proline (Tables 2 and 3). Both arginase and OAT are ubiquitous in animal tissues (Jenkinson et al. 1996, Valle and Simell 1995). Arginase I is located almost exclusively in hepatocytes, whereas arginase II is widely distributed in extrahepatic tissues (Jenkinson et al. 1996). In the liver, OAT is restricted to perivenous hepatocytes (Kuo et al. 1991), which are now known to contain arginase activity (O'Sullivan et al. 1996). Thus, arginase I and arginase II play an important role in generating ornithine from arginine in hepatic and extrahepatic tissues, respectively. Because of underdeveloped arginase activity in neonatal tissues including the small intestine (Wu et al. 1996), liver (Greengard et al. 1970) and kidney (Konarska and Tomaszewski 1986), arginine may quantitatively be a more important source of ornithine in postweaning animals and adults than in neonates. Thus, an inhibition of OAT may have a greater effect on plasma ornithine accumulation in postweaning pigs than in neonatal pigs. This may contribute to increased plasma ornithine concentrations in gabaculine-treated postweaning pigs (Table 5) and in OAT-deficient mice and humans (Valle and Simell 1995, Wang et al. 1995). Because extracellular ornithine is a poor precursor of citrulline and arginine in pig enterocytes due to preferential conversion of ornithine to proline (Wu et al. 1996), an increase in plasma concentrations of ornithine would not contribute to a significant increase in the synthesis of citrulline and arginine. This is consistent with the previous findings that increasing plasma concentrations of ornithine had little effect on those of citrulline and arginine in pigs (Edmonds et al. 1987) and humans (Castillo et al. 1995). Thus, the compartmentation of ornithine metabolism at cellular and systemic levels, as well as developmental changes of tissue arginase activities may explain why plasma citrulline and arginine were decreased despite an increase in plasma concentrations of ornithine in postweaning pigs (Table 5). This may also help to explain the paradox that an OAT deficiency results in hypo-ornithinemia in neonates of mice and humans but hyperornithinemia in adults (Valle and Simell 1995, Wang 1995).
Effects of OAT inhibition on plasma concentrations of glutamine, proline and amino acids.
Gabaculine is an inhibitor of OAT and other pyridoxal phosphate-dependent aminotransferases such as L-alanine transaminase, L-aspartate transaminase, and branched-chain amino acid aminotransferase (Soper and Manning 1982). These enzymes catalyze metabolism of alanine, aspartate and branched-chain amino acids (isoleucine, leucine and valine), in addition to ornithine. This may explain our in vivo observation that oral administration of gabaculine increased plasma concentrations of alanine, branched-chain amino acids and particularly proline (Table 5). This result further supports the view that OAT plays a major role in proline degradation (Valle and Simell 1995). Gabaculine treatment increased plasma concentrations of taurine in postweaning pigs (Table 5) as in neonatal pigs (Flynn and Wu 1996), suggesting that gabaculine inhibits taurine degradation whose catabolism requires pyridoxal phosphate-dependent taurine--ketoglutarate aminotransferase (Toyama et al. 1978). Finally, it is noteworthy that gabaculine treatment had no effect on plasma concentrations of glutamine in postweaning 75-d-old pigs (Table 5), in contrast to neonatal pigs (Flynn and Wu 1996). Because dietary glutamine does not enter the portal vein because of its extensive catabolism in the small intestine (Curthoys and Watford 1995), plasma concentrations of glutamine depend on the balance between glutamine synthesis and degradation in the body. It is likely that gabaculine decreases glutamine utilization by the small intestine to an extent similar to that by which gabaculine inhibits glutamine synthesis from branched-chain amino acids in the animal.
ACKNOWLEDGMENTS
FOOTNOTES
-GK, -glutamyl kinase; -GPR, -glutamylphosphate reductase; OAT, ornithine aminotransferase; OCT, ornithine carbamoyltransferase; P5C, 1-pyrroline-5-carboxylate.
Manuscript received 3 June 1997. Initial reviews completed 16 July 1997. Revision accepted 27 August 1997.
LITERATURE CITED
1-pyrroline- 5-carboxylate synthetase (P5CS) cDNA: a bifunctional enzyme catalyzing the first 2 steps in proline biosynthesis.
C. R. Acad. Sci. (Paris)
1996;
319:171-1781-pyrroline-5-carboxylate in rat tissues.
Biochem. J.
1977;
166:95-103
[Medline]
1-pyrroline-5- carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants.
Proc. Natl. Acad. Sci. U.S.A.
1992;
89:9354-9358
-ketoglutarate aminotransferase of Archromobacter superficialis.
Inactivation and reactivation of enzymes. Biochim. Biophys. Acta
1978;
523:75-81
-aminotransferase have paradoxical neonatal hypo-ornithinaemia and retinal degeneration.
Nature Genet.
1995;
11:185-190
[Medline]
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  W. S. Jobgen, S. P. Ford, S. C. Jobgen, C. P. Feng, B. W. Hess, P. W. Nathanielsz, P. Li, and G. Wu Baggs ewes adapt to maternal undernutrition and maintain conceptus growth by maintaining fetal plasma concentrations of amino acids J Anim Sci, April 1, 2008; 86(4): 820 - 826. [Abstract] [Full Text] [PDF]
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R. D. Mateo, G. Wu, H. K. Moon, J. A. Carroll, and S. W. Kim Effects of dietary arginine supplementation during gestation and lactation on the performance of lactating primiparous sows and nursing piglets J Anim Sci, April 1, 2008; 86(4): 827 - 835. [Abstract] [Full Text] [PDF]
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 R. O. Ball, K. L. Urschel, and P. B. Pencharz Nutritional Consequences of Interspecies Differences in Arginine and Lysine Metabolism J. Nutr., June 1, 2007; 137(6): 1626S - 1641S. [Abstract] [Full Text] [PDF]
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G. Wu, F. W. Bazer, T. A. Cudd, W. S. Jobgen, S. W. Kim, A. Lassala, P. Li, J. H. Matis, C. J. Meininger, and T. E. Spencer Pharmacokinetics and Safety of Arginine Supplementation in Animals J. Nutr., June 1, 2007; 137(6): 1673S - 1680S. [Abstract] [Full Text] [PDF]
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J. W. Frank, J. Escobar, H. V. Nguyen, S. C. Jobgen, W. S. Jobgen, T. A. Davis, and G. Wu Oral N-Carbamylglutamate Supplementation Increases Protein Synthesis in Skeletal Muscle of Piglets J. Nutr., February 1, 2007; 137(2): 315 - 319. [Abstract] [Full Text] [PDF]
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F. Guay and N. L. Trottier Muscle growth and plasma concentrations of amino acids, insulin-like growth factor-I, and insulin in growing pigs fed reduced-protein diets J Anim Sci, November 1, 2006; 84(11): 3010 - 3019. [Abstract] [Full Text] [PDF]
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 W. Qiang, X. Kuang, J. Liu, N. Liu, V. L. Scofield, A. J. Reid, Y. Jiang, G. Stoica, W. S. Lynn, and P. K. Y. Wong Astrocytes Survive Chronic Infection and Cytopathic Effects of the ts1 Mutant of the Retrovirus Moloney Murine Leukemia Virus by Upregulation of Antioxidant Defenses. J. Virol., April 1, 2006; 80(7): 3273 - 3284. [Abstract] [Full Text] [PDF]
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W. Qiang, J. M. Cahill, J. Liu, X. Kuang, N. Liu, V. L. Scofield, J. R. Voorhees, A. J. Reid, M. Yan, W. S. Lynn, et al. Activation of Transcription Factor Nrf-2 and Its Downstream Targets in Response to Moloney Murine Leukemia Virus ts1-Induced Thiol Depletion and Oxidative Stress in Astrocytes J. Virol., November 1, 2004; 78(21): 11926 - 11938. [Abstract] [Full Text] [PDF]
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 H. Kwon, S. P. Ford, F. W. Bazer, T. E. Spencer, P. W. Nathanielsz, M. J. Nijland, B. W. Hess, and G. Wu Maternal Nutrient Restriction Reduces Concentrations of Amino Acids and Polyamines in Ovine Maternal and Fetal Plasma and Fetal Fluids Biol Reprod, September 1, 2004; 71(3): 901 - 908. [Abstract] [Full Text] [PDF]
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J. T. Self, T. E. Spencer, G. A. Johnson, J. Hu, F. W. Bazer, and G. Wu Glutamine Synthesis in the Developing Porcine Placenta Biol Reprod, May 1, 2004; 70(5): 1444 - 1451. [Abstract] [Full Text] [PDF]
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R. Kohli, C. J. Meininger, T. E. Haynes, W. Yan, J. T. Self, and G. Wu Dietary L-Arginine Supplementation Enhances Endothelial Nitric Oxide Synthesis in Streptozotocin-Induced Diabetic Rats J. Nutr., March 1, 2004; 134(3): 600 - 608. [Abstract] [Full Text] [PDF]
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S. W. Kim, R. L. McPherson, and G. Wu Dietary Arginine Supplementation Enhances the Growth of Milk-Fed Young Pigs J. Nutr., March 1, 2004; 134(3): 625 - 630. [Abstract] [Full Text] [PDF]
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 X.-J. Zhang, D. L. Chinkes, and R. R. Wolfe Measurement of protein metabolism in epidermis and dermis Am J Physiol Endocrinol Metab, June 1, 2003; 284(6): E1191 - E1201. [Abstract] [Full Text] [PDF]
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R. F. P. Bertolo, J. A. Brunton, P. B. Pencharz, and R. O. Ball Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs Am J Physiol Endocrinol Metab, May 1, 2003; 284(5): E915 - E922. [Abstract] [Full Text] [PDF]
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