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,,4
* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5; Health Sciences and Laboratory Animal Services, University of Alberta, Edmonton AB, Canada T6G 2S2; ** The Research Institute, The Hospital for Sick Children, Toronto, and the Departments of Paediatrics and Nutritional Science, University of Toronto, Toronto, ON, Canada M5G 1X8
4 To whom correspondence should be addressed. E-mail: ron.ball{at}ualberta.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • arginine biosynthesis • citrulline • first-pass splanchnic metabolism
Arginine metabolism in neonatal piglets is an important area of current research, not only because of the various metabolic uses of arginine (1), but because arginine intake was shown to be a limiting factor for suckling piglet growth (2), and reduced circulating arginine concentrations and a lower whole-body arginine flux were associated with human neonatal pathologies such as necrotizing enterocolitis (3) and persistent pulmonary hypertension of the neonate (4), respectively. Despite the metabolic importance of arginine, arginine intake from sow's milk accounts for only 
40% of daily arginine use (5); therefore, endogenous arginine synthesis is critical for maintaining arginine homeostasis (6).
The arginine synthetic pathway involves the conversion of precursor amino acids, either proline, glutamine, or glutamate to pyrroline-5-carboxylate (P5C),5 ornithine, citrulline and finally arginine [see (1) for a detailed figure]. Sow's milk does contain substantial amounts of proline, glutamine, and glutamate (7,8), and trace amounts of ornithine and citrulline (8). We showed previously that proline must be the major dietary source of the 5-carbon backbone of arginine in neonatal piglets (9–11). However, piglets fed a proline-containing arginine-deficient diet still do not maintain optimum arginine status (11,12). These data indicate that although proline is the major precursor of arginine, proline supplementation alone cannot optimize arginine status in neonatal piglets. Therefore, there is an upper capacity for endogenous arginine synthesis in neonatal piglets, and some portion of the arginine synthetic pathway is limiting.
The first objective of the present study was to determine the most effective arginine precursor as a means to identify which step of the arginine synthetic pathway may be limiting in enterally fed, 1-wk-old piglets. Although previous studies in dogs (13), rats (14), cats (15), and growing pigs (16) showed that citrulline is a better arginine precursor than ornithine, there are many interspecies and developmental differences in arginine metabolism (5,17,18). To add to this data, we compared arginine precursors in 1-wk-old piglets and, in addition to studying the traditional indicators of whole-body arginine status, namely, plasma ammonia, urea, and amino acid concentrations, we also used isotopes to quantify differences in whole-body arginine flux and determined the extent to which each arginine precursor would spare the use of proline for arginine synthesis. Piglets were given an arginine-deficient diet (basal), or the basal diet supplemented with equimolar amounts of either proline (+Pro), ornithine (+Orn), citrulline (+Cit), or arginine (+Arg). We showed previously that the amount of arginine provided in the +Arg diet exceeded the piglet's daily arginine requirement (5,11). Therefore, if any proline, ornithine, or citrulline was converted to arginine efficiently, then the piglets fed the +Pro, +Orn, and +Cit diets, respectively, should have an arginine status similar to that of piglets fed the +Arg diet.
We estimated previously that 51–57% of arginine is extracted during first-pass splanchnic metabolism (12) in piglets fed arginine-deficient and generous diets. However, this estimate was based on data from two separate studies (11,12); thus, we could not analyze the results statistically to determine whether dietary treatment had an effect on dietary arginine extraction. Castillo et al. (19) found that 38% of dietary arginine was extracted during first-pass splanchnic metabolism in adult men fed an arginine-rich diet, but did not investigate the effect of dietary arginine intake on this extraction. Arginine is critical for numerous metabolic functions in both neonates and adult mammals (5,20); therefore, it is important to know how much dietary arginine enters the general circulation under different dietary circumstances. The second objective of this study was to determine whether arginine intake or whole-body arginine status had a significant effect on the first-pass splanchnic extraction of dietary arginine.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diets. A complete elemental diet, designed to meet the nutrient requirements of neonatal piglets (12), was continuously infused enterally via the gastric catheter throughout the experiment using pressure-sensitive infusion pumps (IVAC 560). The targeted nutrient intakes were 15 g amino acid/(kg·d), and 1.1 MJ metabolizable energy/(kg·d), with glucose and lipid (Intralipid 20%; Fresenius Kabi AB) each providing 50% of the nonprotein energy intake. The amino acid composition of the complete diet was based on that of an elemental total parenteral nutrition solution, which is based on human milk protein (Vaminolact; Fresenius Kabi); the amino acid composition of the base solution is provided in Table 1. The base solution was prepared and stored using procedures similar to those previously described (21). Just before diet infusion, mixtures of fat- and water-soluble vitamins (Multi-12/K1 Pediatric; Sabex), minerals (supplied at 200% of the NRC (22) requirement), iron dextran (Ferroforte; Bimeda-MTC), and lipid were added to the base solution. The ratio of lipid to base solution was 1:5.
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On the morning of d 3, piglets were randomly assigned to 1 of 5 test diets (Table 1): an arginine-deficient basal diet, or the basal diet supplemented with an equimolar amount of proline (+Pro), ornithine (+Orn), citrulline (+Cit), or arginine (+Arg). To ensure that the diets were isonitrogenous, the concentrations of alanine and glycine were adjusted from the base solution concentrations (Table 1).
Blood sampling. Beginning on the morning of d 3, before allocation to test diets, blood samples (2 mL) were collected every 24 h until the end of the trial on the evening of d 7. The daily blood samples were used for the determination of plasma ammonia and urea nitrogen concentrations, and the blood sample taken on the morning of d 7 was also used for the determination of plasma amino acid concentrations. As described below, additional blood samples were taken during tracer infusions on d 5, 6, and 7.
Constant tracer infusions. On the morning of d 5, arginine kinetics were determined by a primed [111 kBq (3 µCi)/kg], constant [185 kBq (5 µCi)/(kg·h)] infusion of L-[guanido-14C]arginine (2.04 GBq/mmol; American Radiolabeled Chemicals; and 2.11 GBq/mmol; Moravek Biochemicals). Half of the piglets in each dietary treatment were given an i.v. infusion of the isotope via the jugular vein catheter, and the other piglets received an i.g. infusion via the gastric catheter. On d 7, piglets were infused with L-[guanido-14C]arginine via the route of infusion that was not used on d 5. The isotope was infused over a 5.5-h period, and blood (1 mL) was sampled at 0, 60, 120, 180, 210, 240, 270, 300, and 330 min. On d 7, additional samples were taken 1 h (–60 min) and 30 min (–30 min) before the start of isotope infusion to correct for the background-specific radioactivity of arginine in the blood. Infusing the arginine isotope by both the i.v. and i.g. routes enabled us to calculate the first-pass splanchnic extraction of arginine.
On the morning of d 6, proline kinetics and the conversion of proline to arginine were determined by a primed [740 kBq (20 µCi)/kg], constant [370 kBq (10 µCi)/(kg·h)] infusion of L-[U-14C]proline (8.58 GBq/mmol; Amersham Biosciences). The isotope was infused i.g. for 8 h. Blood (1 mL) was sampled at –60, –30, 0, 60, 120, 180, 240, 300, 330, 360, 390, 420, 450, and 480 min. The infusion doses and periods for all infusions were based on previous experiments (11,12,23). Diets were infused continuously throughout all isotope infusions.
After the isotope infusion on d 7, piglets were anaesthetized with halothane and were killed by the injection of 1000 mg sodium pentobarbital into the jugular vein catheter.
Analytical procedures. Plasma amino acid concentrations and the specific activity (SA) of arginine and proline were measured by reverse-phase HPLC using phenylisothiocyanate derivatives as previously described (24,25). The internal standards norleucine and L-[U-14C]leucine (10.81 GBq/mmol; American Radiolabeled Chemicals) were added to each 300-µL plasma sample. Postcolumn radioactive derivatives were collected in 3-mL fractions; 14 mL of scintillant (Biodegradable Counting Scintillant; Amersham Canada) was added, and samples were counted on a scintillation counter (Tri-Carb 4000 series, Canberra Packard). For urea, the plasma concentration for each of the infusion samples was measured using a spectrophotometric assay (Sigma Procedure No. 640; Sigma Diagnostics), and the associated radioactivity of each sample was measured by collecting the underivatized urea peak during HPLC analysis (23). The urea peak elution time was verified using a radioactive urea standard.
Plasma ammonia (Reference 200-02; Diagnostic Chemical Limited) and urea nitrogen concentrations were determined every 24 h during test diet infusion (d 3–7) using spectrophotometric assays.
Calculations.
Plasma SAs of the postcolumn radioactive derivatives of both amino acids and urea were calculated as:
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The d 7 infusion SA values for arginine and urea were corrected for radioactive background by subtracting the average arginine or urea SA of the –60, –30, and 0 min samples.
Plateau SA values for each amino acid were verified as having a slope not different from zero. All plateaus had 
3 time points. The plateau SA values were used in the fractional net conversion and the whole-body flux calculations outlined below.
Fractional net conversion is the overall contribution that a precursor amino acid makes to the product amino acid flux. Fractional net conversions of the precursor (either proline or arginine) to product metabolite (either arginine or urea) were calculated by:
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Whole-body fluxes for the i.g. infused proline and the i.v. and i.g. infused arginine were calculated as:
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The calculated flux values included the amino acids entering the plasma pool through the diet, de novo synthesis, and protein breakdown, or leaving the pool through protein synthesis, oxidation, or conversion to other metabolites. The i.g. fluxes are influenced by both first-splanchnic metabolism and the metabolism by other peripheral tissues (such as muscle, kidney, intestinal metabolism of arterial substrates, lung), whereas i.v. fluxes include only the effects of metabolism by peripheral tissues.
The absolute conversion of proline to arginine (Qproline to arginine) was calculated using the i.g. arginine flux values using the following formula, and was used as a measure of arginine synthesis:
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First-pass splanchnic arginine to urea conversion was calculated within piglet by subtracting the i.v. from the i.g. value, and the first-pass splanchnic extraction of arginine was calculated within piglet using the approach of Castillo et al. (19) as follows:
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Statistical analyses. Unless otherwise stated, all data were analyzed using the mixed model of SAS Version 8.3 (SAS Institute), and differences were considered significant at P < 0.05.
The dependent variables plasma ammonia and plasma urea nitrogen were analyzed using repeated-measures analysis where the fixed effect was diet and the random variables were piglet nested in diet and day. The Kenward-Roger option was used to estimate the denominator degrees of freedom. The variance-covariance matrix was chosen for each analysis based on Schwarz's Bayesian Criterion. When the effects were significant (P < 0.05), preplanned comparisons of least-squares means were made using the pdiff test, which is the two-tailed pairwise comparison test used by the mixed procedure.
The results from the i.v. and i.g. arginine infusions were analyzed using a 5 x 2 factorial design with diet, route of infusion, and the interaction between diet and route of infusion as the fixed effects. Day of isotope infusion was tested as a covariate for the arginine infusion data and was included in the model only when the effect was significant (P < 0.05). For all other experimental variables, an ANOVA with piglet nested in diet as the random term was used. When the model P < 0.05, preplanned pair-wise comparisons of least-squares means were made using the pdiff option, and the two-tailed P-values were used to assess significance.
The relation between plasma arginine concentration and each of proline to arginine fractional net conversion and arginine synthesis from proline were studied using the correlation procedure of SAS.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The treatment groups did not differ in piglet weight at the initiation of test diet infusion (pooled mean = 1.94 kg, pooled SE = 0.09 kg), rate of weight gain during test diet administration [pooled mean = 93 g/(kg·d), pooled SE = 7 g/(kg·d)], and final piglet weight (pooled mean = 2.66 kg, pooled SE = 0.11 kg). Based on our previous results (12) and because of the short duration of the trial, we did not expect to see differences in body weight or weight gains.
Plasma amino acid concentration. With the exceptions of alanine, aspartate, glutamate, histidine, and lysine, the d 7 plasma concentrations of all amino acids measured were affected by diet (P < 0.05) (Table 2). In general, piglets fed the +Cit and +Arg diets had a similar plasma amino acid profile. The reasons for the differences in the plasma concentrations of the indispensable amino acids are unclear, and require further investigation, although they may be related to changes in protein synthesis. However, similar differences in plasma methionine and threonine concentrations were described previously in piglets fed generous and deficient arginine diets (11); therefore, it does appear that these 2 amino acids are particularly affected by whole-body arginine status. Previously, we found higher plasma asparagine and glutamine concentrations in piglets fed a deficient vs. generous arginine diet (11,12), presumably due to their roles as ammonia scavengers.
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Plasma ammonia and urea concentrations. Plasma ammonia concentration was used as a measurement of the ability of the urea cycle to convert ammonia, from amino acid catabolism, to urea for excretion. Higher plasma ammonia concentrations were an indication of impaired urea cycle function. Day (P = 0.0002), diet (P < 0.0001), and the interaction between diet and day (P = 0.04) all had a significant effect on plasma ammonia concentrations (Table 3). With the initiation of test diet infusion, there was a significant increase in plasma ammonia concentrations within the first 24 h in piglets fed the basal, +Pro, and +Orn diets, whereas there was no change (P > 0.05) in the plasma ammonia concentration in piglets fed the +Cit and +Arg diets for the duration of the test diet infusion. Therefore, piglets fed the basal, +Pro, and +Orn diets had higher plasma ammonia concentrations than those fed the +Cit and +Arg diets from the morning of d 4 onwards (Table 3).
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Arginine synthesis from proline. Piglets fed the +Pro diet had the highest rate of arginine synthesis from proline (P < 0.05) (Table 7), and piglets fed the +Arg diet had the lowest rate of arginine synthesis from proline (P < 0.05) (Table 7). Piglets fed the basal, +Orn, and +Cit diets had intermediate rates of arginine synthesis from proline (Table 7). There were significant negative correlations between plasma arginine concentrations and both the fractional net conversion of proline to arginine (r = –0.68) (P = 0.0002) and arginine synthesis from proline (r = –0.49) (P = 0.02). Therefore, as arginine status improved, proline use for arginine synthesis was spared.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Previous studies in puppies (13), kittens (15), adult rats (14), and 7- to 10-kg growing pigs (16) all concluded that citrulline is a more effective arginine precursor than ornithine. Suckling piglets given twice daily boluses of citrulline had higher plasma arginine concentrations than control suckling piglets (260 vs. 140 µmol/L) (27). Wu and colleagues (27) hypothesized that N-acetyl glutamate (NAG), a cofactor for carbamoylphosphate synthetase I (EC number 6.3.4.16) (1), concentrations are low in suckling piglets, and therefore suckling piglets have a low capacity for citrulline synthesis. Arginine is an allosteric activator of NAG synthase (EC number 2.3.1.1) (28); therefore, as arginine concentrations fall, the ability of piglets to synthesize arginine may also decrease due to a lower NAG synthase activity. The basal, +Pro, and +Orn piglets had plasma arginine concentrations below the sow-fed reference range (50–267 µmol/L) (29); therefore, these piglets may have had an even greater limitation for citrulline and subsequently arginine formation than suckling piglets.
The significantly higher plasma citrulline concentrations in the +Cit vs. +Arg piglets, in spite of similar plasma arginine concentrations and arginine flux, suggests that arginine synthesis from citrulline was occurring at a maximal rate, resulting in the accumulation of citrulline in the plasma. Plasma citrulline concentrations were well outside the sow-fed reference range (73–151 µmol/L) (29) in the +Cit piglets. Therefore, citrulline levels were likely much higher than the Km for argininosuccinate synthetase (EC number 6.3.4.5), which is 60 µmol/L for the human isoform (30). Unlike ornithine and proline, citrulline cannot be metabolized by any other metabolic pathways without first being converted to arginine (28); this may explain why citrulline was a more effective arginine precursor than either proline or ornithine.
Ornithine is an ineffective arginine precursor.
Ornithine can be metabolized by 3 different enzymes, i.e., ornithine transcarbamoylase (EC number 2.1.3.3), which catalyzes citrulline formation for use in the urea cycle or for arginine synthesis (28); ornithine amino transferase (OAT, EC number 2.6.1.13), which converts ornithine to P5C, which can subsequently be metabolized to 
-ketoglutarate or proline (31); and ornithine decarboxylase (EC number 4.1.1.17) for polyamine synthesis. Of these 3 enzymes, in vitro work using 1-wk-old piglet enterocytes showed that the activity of OAT is the highest, (32,33), suggesting that the conversion of ornithine to P5C is the most favored route of ornithine metabolism. Indeed, an in vivo study in neonatal piglets, fed no dietary ornithine, found that 25 and 36% of ornithine flux, for i.g. and intraportally infused ornithine, respectively, was oxidized to CO2 (23). Alternatively, it is also possible that due to the limitation in citrulline formation, as previously discussed, the supplemented ornithine was oxidized to prevent accumulation. There was no difference in plasma proline concentrations between the basal and +Orn piglets (Table 2), indicating that there was probably not a large conversion of the supplemented ornithine to proline. These results indicate that a large portion of ornithine may have been oxidized, preventing it from being used to support urea cycle function or as an arginine precursor.
The ineffectiveness of ornithine as an arginine precursor could also be due to a limitation in the activity of ORNT1, the mitochondrial membrane transporter. In neonatal mice, ORNT1 activity in both the small intestine and liver increases during the early postnatal period (34). To the authors' knowledge, there have been no reports of the development of ORNT1 in neonatal piglets; however, if ORNT1 activity is low piglets of the age of those in the present study, then there may have been a limitation in the ability of the supplemental ornithine to be transported to mitochondria for citrulline formation.
Proline is an ineffective arginine precursor. Although proline is the major dietary precursor for arginine in 1-wk-old piglets (9,11), the results from the +Pro and basal piglets show that it is not the optimal arginine precursor. We have not determined previously the amount of proline required by piglets to support the maximal rate of arginine synthesis, although the amount is likely equal to or less than the 1.25 g proline/(kg·d) provided in the basal diet. Dillon et al. (35) found that there is a substantial conversion of proline to P5C, but only a very small portion, 5–6%, of proline-derived P5C is converted to arginine in 2-wk-old piglet enterocytes in vitro. Compared with other piglets of other ages, 1-wk-old piglet enterocytes have the lowest amount of total proline to ornithine conversion (36). These in vitro findings strongly support our in vivo finding that there is a limit to proline use for arginine synthesis in 1-wk-old piglets.
Effective arginine precursors spare the use of proline for arginine synthesis. We observed previously that piglets fed a generous arginine diet have a lower rate of conversion of proline to arginine than piglets fed inadequate arginine diets (11,12); therefore, when arginine status is favorable, proline use for arginine synthesis is spared. Arginine and citrulline were both effective arginine precursors and therefore had the lowest rates of conversion of proline to arginine. Piglets fed the +Orn diet had a greater reliance on proline for arginine synthesis than the +Cit and +Arg piglets, because ornithine was not an effective arginine precursor. Proline to arginine conversion was highest in piglets fed the +Pro diet, even higher than the level observed for the basal diet, which may indicate that the basal diet did not contain enough proline to maximally support endogenous arginine synthesis. However, none of the other parameters studied that relate to whole-body arginine status (Table 2–4, Table 6) were improved by proline supplementation. The reasons for this contradiction in the effectiveness of supplemental proline as an arginine precursor are unknown. However, the rate of arginine synthesis from proline may have been overestimated in the +Pro piglets because the proline pool was likely substantially larger in the +Pro piglets compared with the other groups, as illustrated by higher plasma proline concentrations and proline flux. In summary, citrulline and arginine, but not proline or ornithine addition to an arginine-deficient diet spared the use of proline for arginine synthesis.
Splanchnic extraction of dietary arginine. We believe that our finding that 52% (Table 5) of dietary arginine is extracted during first-pass splanchnic metabolism is the first to show that arginine intake and status do not affect splanchnic arginine use. The reason why the splanchnic region, which includes the intestine and liver, is extracting a substantial portion of dietary arginine, especially in the cases in which piglets were in an arginine-deficient state, is unknown. Piglets in the +Cit and +Arg groups had substantial conversion of arginine to urea during first-pass splanchnic metabolism, which was not seen in piglets in the other treatment groups, and this may be a mechanism whereby piglets with a favorable arginine status can maintain their plasma arginine concentrations within a certain range. However, the formation of urea from arginine resulted in the removal of the radioactive label from arginine. Although the resulting ornithine may have been recycled back to arginine, this arginine was calculated as extracted because it no longer carried the label. Therefore, the splanchnic extraction of arginine in piglets fed the +Cit and +Arg diets may have been overestimated due to the production of urea. Other possible metabolic fates of the extracted arginine include the use for splanchnic nitric oxide, polyamine, and/or protein synthesis. Because of the importance of arginine in supporting piglet growth (2) and implications in preventing certain neonatal pathologies (37), a thorough understanding of the uses of dietary arginine during first-pass splanchnic metabolism is necessary.
Contribution of dietary arginine to whole-body arginine flux. Using the 52% calculated value for first-pass splanchnic extraction to calculate the amount of dietary arginine entering general circulation, in combination with the i.v. arginine fluxes (Table 6), we calculated the portion of flux derived from dietary arginine for each group. Dietary intake represented 19, 19, 18, 6, and 57% of arginine flux in piglets fed the basal, +Pro, +Orn, +Cit, and +Arg diets, respectively. This finding illustrates the importance of endogenous sources of arginine, which may include arginine from both protein breakdown or de novo arginine synthesis, for arginine flux, particularly when dietary arginine is limiting. Regardless of whole-body arginine status, the splanchnic region extracted a minimum of 25 µmol/(kg·h) and this value was much higher when there was a generous intake of arginine.
In conclusion, citrulline was the most effective arginine precursor, whereas there was a limit to the extent that either proline or ornithine could be used as an arginine precursor. The formation or availability of citrulline at the site of endogenous arginine synthesis may limit arginine synthesis in piglets fed an arginine-deficient diet. The percentage of dietary arginine extracted during first-pass splanchnic extraction is independent of whole-body arginine status and accounts for 52% of intake. Arginine is critical for the growth and health of neonates; therefore, complete knowledge of the mechanisms that control its synthesis are necessary to optimize diets for young pigs, human neonates, and other young mammals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Alberta Pork Producers Development Corporation.
3 Present address: Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada N1G 2W1.
5 Abbreviations used: +Arg, arginine supplemented diet; +Cit, citrulline supplemented diet; +Pro, proline supplemented diet; +Orn, ornithine supplemented diet; NAG, N-acetyl glutamate; ORNT1, mitochondrial membrane transporter; P5C, pyrroline-5-carboxylate; OAT, ornithine aminotransferase; SA, specific activity.
Manuscript received 7 December 2005. Initial review completed 16 January 2006. Revision accepted 10 April 2006.
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