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Ornithine metabolism, but not arginine synthesis, is affected by the addition of ornithine to an arginine-deficient diet in enterally-fed piglets
Livestock Science 108 (2007) 137 – 141 www.elsevier.com/locate/livsci
Ornithine metabolism, but not arginine synthesis, is affected by the addition of ornithine to an arginine-deficient diet in enterally-fed piglets ☆ K.L. Urschel a , P.B. Pencharz a,b,c , R.O. Ball a,b,⁎ a
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton AB, Canada T6G 2P5 b Department of Nutritional Science, University of Toronto, Toronto ON, Canada M5G 1X8 c Department of Paediatrics, University of Toronto, Toronto ON, Canada M5G 1X8
Abstract In a previous study, ornithine addition to an arginine-deficient diet did not improve whole-body arginine status in enterally-fed piglets; however, the metabolic fates of the supplemental ornithine were not studied. This experiment determined the metabolic fates of the supplemental ornithine and whether ornithine metabolism was affected by the addition of α-ketoglutarate. Male piglets (n = 20, 1.8 kg), fitted with gastric catheters for diet and isotope infusion, portal vein catheters for isotope infusion and femoral vein catheters for blood sampling (d 0), received 2 d of a complete diet, followed by 5 d of 1 of 4 test diets: the arginine-deficient diet (basal), or the basal diet with either α-ketoglutarate [ + α - KG; 4.6 mmol/(kg d)], ornithine [ + Orn; 9.2 mmol/(kg d)] or both [ + α - KG/ + Orn; 4.6 mmol/(kg d) α - ketoglutarate + 9.2 mmol/(kg d) ornithine]. Piglets received primed, constant infusions of [1-14C]ornithine infused intragastrically (either d 5 and d 7) to determine ornithine kinetics, and [guanido-14C]arginine intragastrically to measure arginine flux (d 6). Piglets receiving the ornithine-containing diets had a higher intragastric ornithine flux (P b 0.0001) and ornithine oxidation (P b 0.05). Ornithine supplementation did not increase arginine synthesis, although the ornithine supplemented piglets had a greater conversion of ornithine to proline (P b 0.0001). The fates of supplemental ornithine in piglets fed an argininedeficient diet appear to be oxidation and proline synthesis; this was not affected by the presence of α-ketoglutarate. © 2007 Elsevier B.V. All rights reserved. Keywords: Ornithine; Arginine; Ornithine alpha-ketoglutarate; Neonatal piglet
1. Introduction In a recent study in enterally-fed piglets receiving an arginine-deficient diet, we found that there was a ☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. Tel.: +1 780 492 7151; fax: +1 780 492 4265. E-mail address: [email protected] (R.O. Ball).
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.096
limitation in the extent to which enterally-administered ornithine could be used as an arginine precursor; however, the detailed metabolism of ornithine was not quantified (Urschel et al., 2006). Other metabolic fates of ornithine include proline, glutamate and glutamine, oxidation to CO2 via the citric acid cycle and polyamine synthesis (Fig. 1). Ornithine α-ketoglutarate (OKG) dissociates in solution into 2 moles of ornithine and 1 mole of α-ketoglutarate. In healthy adults receiving a bolus dose of either OKG, ornithine-HCl or calcium α-ketoglutarate, only OKG administration resulted in a significant increase in
138
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
Fig. 1. Pathways of ornithine metabolism. Enzymes responsible for the various reactions: 1. Aminotransferase enzymes; 2. Glutamine synthetase; 3. Glutaminase; 4. Pyrroline-5-carboxylate dehydrogenase; 5. Pyrroline-5-carboxylate synthase; 6. Proline oxidase; 7. Pyrroline-5-carboxylate reductase; 8. Ornithine aminotransferase; 9. Ornithine transcarbamoylase; 10. Argininosuccinate synthetase; 11. Argininosuccinate lyase; 12. Arginase; and 13. Ornithine decarboxylase.
plasma arginine and proline concentrations (Cynober et al., 1990). Therefore, unlike ornithine alone, OKG may be an effective arginine precursor. The proposed mechanism whereby OKG may be an effective arginine precursor is that by administering two components that are related by a series of reversible reactions (Fig. 1), ornithine metabolism may be shifted towards the production of proline and arginine, and away from glutamate and α-ketoglutarate production (Cynober et al., 1990). To our knowledge, this proposed mechanism has not been tested rigorously in vivo using isotopes. The primary objective of the present study was to examine ornithine metabolism, specifically the use of ornithine for arginine synthesis, in piglets receiving an arginine-deficient basal diet or the basal diet supplemented with either α-ketoglutarate (+α-KG), ornithine (+Orn), or OKG as α-ketoglutarate and ornithine (+αKG/+Orn). 2. Materials, methods and techniques 2.1. Animals and surgical procedures All procedures in this study were approved by the Faculty of Agriculture, Forestry and Home Economics Animal Policy and Welfare Committee at the University of Alberta. Twenty intact male Landrace/ Large White/ Duroc (∼ 1.8 kg) were obtained at 1– 2 days of age and immediately underwent surgical procedures to implant a gastric catheter for diet and
isotope infusion and a femoral vein catheter for blood sampling (Urschel et al., 2005). 2.2. Diets and treatment groups A complete elemental diet, designed to meet the nutrient requirements of neonatal piglets (Urschel et al., 2005), was continuously infused via the gastric catheter. The amino acid composition of the diet was recently described (Urschel et al., 2006). On the morning of d 3 piglets were randomly assigned to one of four test diets (n = 5/diet): an arginine-deficient basal diet (basal; 4 mmol/L arginine), or the basal diet with either α-ketoglutarate (+α-KG; 17 mmol/L α-ketoglutaric acid), ornithine (+Orn; 34 mmol/L ornithine-HCl), or both α-ketoglutarate and ornithine (α-KG/+Orn; 34 mmol/L α-ketoglutaric acid and 17 mmol/L ornithine-HCl). The ornithine content was based on the previously used + Orn diet (Urschel et al., 2006), and the α-ketoglutarate level was selected to provide a 2:1 molar ratio of ornithine: α-ketoglutarate in the +α-KG/+Orn diet. Diets were made isonitrogenous using alanine and glycine. 2.3. Constant tracer infusions On the morning of either d 5 or d 7, whole-body ornithine kinetics were determined by a primed [481 kBq/kg], constant [370 kBq/(kg h)] intragastric infusion of L-[1–14C]ornithine. 2–3 piglets in each dietary treatment group received this infusion on d 5,
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
139
with the remaining piglets receiving the infusion on d 7. Piglets received a different infusion on the day that they did not receive the ornithine infusion; however, these results are not included in the current paper. To enable 14CO2 collection, for the measurement of [l14C]ornithine oxidation, piglets were contained in plexi-glass boxes for the duration of the infusion, and air was drawn out of these boxes and through bottles of CO2 absorber. Blood was sampled every 30– 60 min during the infusions. Samples of the CO2 absorber were taken every 30 min for the determination 14CO2 production (House et al., 1997). On d 7, an additional hour of breath sampling was completed prior to the initiation of isotope infusion to correct for background 14CO2 production. On the morning of d 6, arginine kinetics were determined in all piglets by a primed [111 kBq/kg], constant [185 kBq/(kg h)] intragastric infusion of L[guanido-14C]arginine. Blood was sampled as described for the ornithine infusion.
2.5. Calculations
2.4. Analytical procedures
The d 7 concentrations of selected amino acids, ammonia and urea, ornithine and arginine flux, ornithine oxidation, and whole-body arginine and proline synthesis from ornithine are presented in Table 1. Whole-body arginine status was not affected by diet (P N 0.05), although plasma ornithine concentration, and ornithine flux, oxidation and conversion to
Plasma ammonia, urea and amino acid concentrations; the specific activity (SA) of arginine, ornithine and proline in the infusion samples; and breath sample radioactivity were determined as previously described (House et al., 1997; Urschel et al., 2005).
The formulas used to calculate plasma SAs of the post-column radioactive derivatives of the amino acids, whole-body arginine and ornithine fluxes, ornithine oxidation, ornithine conversion to product amino acids were as previously described (Bertolo et al., 2003; House et al., 1997; Urschel et al., 2006). 2.6. Statistical analyses Data were analyzed using an ANOVA in the mixed procedure of SAS Version 8.3, with diet as the fixed effect and piglet as the random effect. Data were considered statistically significant if P b 0.05. When a dietary effect was observed (P b 0.05 or a trend, 0.05 b P b 0.10), pre-planned comparisons, of least squares means were made using the pdiff option. 3. Results
Table 1 Plasma metabolite concentrations and indicators of ornithine metabolism in enterally-fed neonatal piglets 1 Diet Basal
+α-KG
+Orn
+α-KG/+Orn
Pooled SE
P-value
d 7 plasma concentrations Ammonia (μmol/L) Urea (mmol/L) Arginine (μmol/L) Ornithine (μmol/L) Proline (μmol/L)
118 3.0 25 46a 624ab
119 3.5 26 37a 478a
81 1.5 22 213b 886b
97 1.8 58 168b 785ab
13 0.6 14 18 112
NS 2 NS NS b0.0001 b0.10
Whole-body amino acid fluxes Ornithine flux (μmol/(kg h)) Arginine flux (μmol/(kg h))
234a 442
240a 399
473b 370
527b 303
32 71
b0.0001 NS
Whole-body ornithine metabolism Ornithine oxidation (% dose oxidized) Ornithine to arginine conversion (μmol/(kg h)) Ornithine to proline conversion 3 (μmol/(kg h))
20b 145 51a
15a 150 52a
30c 137 149b
34c 128 148b
1 29 6
b0.0001 NS b0.0001
Values are least square means, n = 5. Means in a row without a common superscript differ, P b 0.05. NS = not significant (P N 0.10). 3 Although the intragastric proline flux was not measured in the present study, it was previously determined in a similar study, using similar piglets receiving the identical basal and + Orn diets and identical methods (Urschel et al., 2006). The previously determined flux value of 511 μmol/(kg h) was used for piglets receiving the basal and +α-KG diets, and a flux value of 649 μmol/(kg h) was used for piglets receiving the +Orn and + α-KG/ +Orn diets. 1 2
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Fig. 2. A stochastic model of ornithine metabolism in enterally-fed piglets receiving an arginine-deficient diet supplemented with ornithine and/or α ketoglutarate.
proline were greater in piglets receiving supplemental ornithine (P b 0.05). 4. Discussion
4.2. Ornithine metabolism A stochastic model was used to provide a summary of ornithine metabolism in the piglets in the present study:
4.1. Ornithine α-ketoglutarate as an arginine precursor We have previously shown that the addition of an effective arginine precursor, citrulline, to the argininedeficient basal diet resulted in lower plasma ammonia and urea concentrations and a higher plasma arginine concentration and whole-body arginine flux (Urschel et al., 2006). These effects were not observed in the piglets receiving the +α-KG/+Orn diet relative to the +Orn diet; therefore, OKG was no more effective than ornithine as an arginine precursor. Because OKG infusion did not affect whole-body arginine status and synthesis, we could not verify the proposed mechanism of OKG as an effective arginine precursor (Cynober et al., 1990) in our enterallyfed neonatal piglet model.
Ornithine flux ¼ ornithine intake þde novo ornithine synthesis ¼ ornithine oxidation þornithine conversion to other metabolites
Piglets in the basal and +α-KG groups had a ∼ 1-fold greater rate of de novo whole-body (intragastric) ornithine synthesis than the piglets receiving dietary ornithine (Fig. 2), suggesting that, similar to arginine intake regulating arginine synthesis (Urschel et al., 2005; Wilkinson et al., 2004), ornithine intake regulates ornithine synthesis. Piglets receiving dietary ornithine had a ∼3-fold greater rate of ornithine oxidation than those piglets not
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
receiving any ornithine (Fig. 2). Ornithine oxidation in neonatal piglets appears to be regulated, at least in part, by ornithine intake. On a whole-body basis, the +Orn and +α-KG/+Orn piglets had a ~75% greater rate of ornithine conversion to other metabolites, compared to the basal and +α-KG piglets (Fig. 2). Proline formation was a major metabolic fate of the supplemental ornithine. In subjects receiving an OKG versus control solution, proline concentrations in the plasma (Cynober et al., 1990; Loi et al., 2005) and tissues (Loi et al., 2005) were higher in the subjects receiving OKG. 5. Conclusion The objective of the present study was to determine whether OKG was a more effective arginine precursor than ornithine alone. OKG was no better than ornithine alone as an arginine precursor. This finding supports the data of our previous study which found that arginine synthesis was limited by the rate of citrulline formation (Urschel et al., 2006). In general, α-ketoglutarate addition to the diet did not modify ornithine metabolism; however, ornithine intake had profound effects on ornithine metabolism. Piglets receiving dietary ornithine had a lower rate of ornithine synthesis, and greater rates of ornithine oxidation and conversion to proline, than piglets not receiving any ornithine. The primary metabolic fates of supplemental ornithine were therefore oxidation and proline synthesis.
141
References Bertolo, R.F., Brunton, J.A., Pencharz, P.B., Ball, R.O., 2003. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am. J. Physiol.: Endocrinol. Metab. 284, E915–E922. Cynober, L., Coudray-Lucas, C., De Bandt, J.P., Guechot, J., Aussel, C., Salvucci, M., Giboudeau, J., 1990. Action of ornithine alphaketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects. J. Am. Coll. Nutr. 9, 2–12. House, J.D., Pencharz, P.B., Ball, R.O., 1997. Phenylalanine requirements determined by using L-[1–14C]phenylalanine in neonatal piglets receiving total parenteral nutrition supplemented with tyrosine. Am. J. Clin. Nutr. 65, 984–993. Loi, C., Nakib, S., Neveux, N., Arnaud-Battandier, F., Cynober, L., 2005. Ornithine alpha-ketoglutarate metabolism in the healthy rat in the postabsorptive state. Metabolism 54, 1108–1114. Urschel, K.L., Shoveller, A.K., Pencharz, P.B., Ball, R.O., 2005. Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet. Am. J. Physiol.: Endocrinol. Metab. 288, E1244–E1251. Urschel, K.L., Shoveller, A.K., Uwiera, R.R., Pencharz, P.B., Ball, R.O., 2006. Citrulline is an effective arginine precursor in enterally-fed neonatal piglets. J. Nutr. 136, 1806–1813. Wilkinson, D.L., Bertolo, R.F., Brunton, J.A., Shoveller, A.K., Pencharz, P.B., Ball, R.O., 2004. Arginine synthesis is regulated by dietary arginine intake in the enterally fed neonatal piglet. Am. J. Physiol.: Endocrinol. Metab. 287, E454–E462.
Ornithine metabolism, but not arginine synthesis, is affected by the addition of ornithine to an arginine-deficient diet in enterally-fed piglets ☆ K.L. Urschel a , P.B. Pencharz a,b,c , R.O. Ball a,b,⁎ a
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton AB, Canada T6G 2P5 b Department of Nutritional Science, University of Toronto, Toronto ON, Canada M5G 1X8 c Department of Paediatrics, University of Toronto, Toronto ON, Canada M5G 1X8
Abstract In a previous study, ornithine addition to an arginine-deficient diet did not improve whole-body arginine status in enterally-fed piglets; however, the metabolic fates of the supplemental ornithine were not studied. This experiment determined the metabolic fates of the supplemental ornithine and whether ornithine metabolism was affected by the addition of α-ketoglutarate. Male piglets (n = 20, 1.8 kg), fitted with gastric catheters for diet and isotope infusion, portal vein catheters for isotope infusion and femoral vein catheters for blood sampling (d 0), received 2 d of a complete diet, followed by 5 d of 1 of 4 test diets: the arginine-deficient diet (basal), or the basal diet with either α-ketoglutarate [ + α - KG; 4.6 mmol/(kg d)], ornithine [ + Orn; 9.2 mmol/(kg d)] or both [ + α - KG/ + Orn; 4.6 mmol/(kg d) α - ketoglutarate + 9.2 mmol/(kg d) ornithine]. Piglets received primed, constant infusions of [1-14C]ornithine infused intragastrically (either d 5 and d 7) to determine ornithine kinetics, and [guanido-14C]arginine intragastrically to measure arginine flux (d 6). Piglets receiving the ornithine-containing diets had a higher intragastric ornithine flux (P b 0.0001) and ornithine oxidation (P b 0.05). Ornithine supplementation did not increase arginine synthesis, although the ornithine supplemented piglets had a greater conversion of ornithine to proline (P b 0.0001). The fates of supplemental ornithine in piglets fed an argininedeficient diet appear to be oxidation and proline synthesis; this was not affected by the presence of α-ketoglutarate. © 2007 Elsevier B.V. All rights reserved. Keywords: Ornithine; Arginine; Ornithine alpha-ketoglutarate; Neonatal piglet
1. Introduction In a recent study in enterally-fed piglets receiving an arginine-deficient diet, we found that there was a ☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. Tel.: +1 780 492 7151; fax: +1 780 492 4265. E-mail address: [email protected] (R.O. Ball).
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.096
limitation in the extent to which enterally-administered ornithine could be used as an arginine precursor; however, the detailed metabolism of ornithine was not quantified (Urschel et al., 2006). Other metabolic fates of ornithine include proline, glutamate and glutamine, oxidation to CO2 via the citric acid cycle and polyamine synthesis (Fig. 1). Ornithine α-ketoglutarate (OKG) dissociates in solution into 2 moles of ornithine and 1 mole of α-ketoglutarate. In healthy adults receiving a bolus dose of either OKG, ornithine-HCl or calcium α-ketoglutarate, only OKG administration resulted in a significant increase in
138
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
Fig. 1. Pathways of ornithine metabolism. Enzymes responsible for the various reactions: 1. Aminotransferase enzymes; 2. Glutamine synthetase; 3. Glutaminase; 4. Pyrroline-5-carboxylate dehydrogenase; 5. Pyrroline-5-carboxylate synthase; 6. Proline oxidase; 7. Pyrroline-5-carboxylate reductase; 8. Ornithine aminotransferase; 9. Ornithine transcarbamoylase; 10. Argininosuccinate synthetase; 11. Argininosuccinate lyase; 12. Arginase; and 13. Ornithine decarboxylase.
plasma arginine and proline concentrations (Cynober et al., 1990). Therefore, unlike ornithine alone, OKG may be an effective arginine precursor. The proposed mechanism whereby OKG may be an effective arginine precursor is that by administering two components that are related by a series of reversible reactions (Fig. 1), ornithine metabolism may be shifted towards the production of proline and arginine, and away from glutamate and α-ketoglutarate production (Cynober et al., 1990). To our knowledge, this proposed mechanism has not been tested rigorously in vivo using isotopes. The primary objective of the present study was to examine ornithine metabolism, specifically the use of ornithine for arginine synthesis, in piglets receiving an arginine-deficient basal diet or the basal diet supplemented with either α-ketoglutarate (+α-KG), ornithine (+Orn), or OKG as α-ketoglutarate and ornithine (+αKG/+Orn). 2. Materials, methods and techniques 2.1. Animals and surgical procedures All procedures in this study were approved by the Faculty of Agriculture, Forestry and Home Economics Animal Policy and Welfare Committee at the University of Alberta. Twenty intact male Landrace/ Large White/ Duroc (∼ 1.8 kg) were obtained at 1– 2 days of age and immediately underwent surgical procedures to implant a gastric catheter for diet and
isotope infusion and a femoral vein catheter for blood sampling (Urschel et al., 2005). 2.2. Diets and treatment groups A complete elemental diet, designed to meet the nutrient requirements of neonatal piglets (Urschel et al., 2005), was continuously infused via the gastric catheter. The amino acid composition of the diet was recently described (Urschel et al., 2006). On the morning of d 3 piglets were randomly assigned to one of four test diets (n = 5/diet): an arginine-deficient basal diet (basal; 4 mmol/L arginine), or the basal diet with either α-ketoglutarate (+α-KG; 17 mmol/L α-ketoglutaric acid), ornithine (+Orn; 34 mmol/L ornithine-HCl), or both α-ketoglutarate and ornithine (α-KG/+Orn; 34 mmol/L α-ketoglutaric acid and 17 mmol/L ornithine-HCl). The ornithine content was based on the previously used + Orn diet (Urschel et al., 2006), and the α-ketoglutarate level was selected to provide a 2:1 molar ratio of ornithine: α-ketoglutarate in the +α-KG/+Orn diet. Diets were made isonitrogenous using alanine and glycine. 2.3. Constant tracer infusions On the morning of either d 5 or d 7, whole-body ornithine kinetics were determined by a primed [481 kBq/kg], constant [370 kBq/(kg h)] intragastric infusion of L-[1–14C]ornithine. 2–3 piglets in each dietary treatment group received this infusion on d 5,
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
139
with the remaining piglets receiving the infusion on d 7. Piglets received a different infusion on the day that they did not receive the ornithine infusion; however, these results are not included in the current paper. To enable 14CO2 collection, for the measurement of [l14C]ornithine oxidation, piglets were contained in plexi-glass boxes for the duration of the infusion, and air was drawn out of these boxes and through bottles of CO2 absorber. Blood was sampled every 30– 60 min during the infusions. Samples of the CO2 absorber were taken every 30 min for the determination 14CO2 production (House et al., 1997). On d 7, an additional hour of breath sampling was completed prior to the initiation of isotope infusion to correct for background 14CO2 production. On the morning of d 6, arginine kinetics were determined in all piglets by a primed [111 kBq/kg], constant [185 kBq/(kg h)] intragastric infusion of L[guanido-14C]arginine. Blood was sampled as described for the ornithine infusion.
2.5. Calculations
2.4. Analytical procedures
The d 7 concentrations of selected amino acids, ammonia and urea, ornithine and arginine flux, ornithine oxidation, and whole-body arginine and proline synthesis from ornithine are presented in Table 1. Whole-body arginine status was not affected by diet (P N 0.05), although plasma ornithine concentration, and ornithine flux, oxidation and conversion to
Plasma ammonia, urea and amino acid concentrations; the specific activity (SA) of arginine, ornithine and proline in the infusion samples; and breath sample radioactivity were determined as previously described (House et al., 1997; Urschel et al., 2005).
The formulas used to calculate plasma SAs of the post-column radioactive derivatives of the amino acids, whole-body arginine and ornithine fluxes, ornithine oxidation, ornithine conversion to product amino acids were as previously described (Bertolo et al., 2003; House et al., 1997; Urschel et al., 2006). 2.6. Statistical analyses Data were analyzed using an ANOVA in the mixed procedure of SAS Version 8.3, with diet as the fixed effect and piglet as the random effect. Data were considered statistically significant if P b 0.05. When a dietary effect was observed (P b 0.05 or a trend, 0.05 b P b 0.10), pre-planned comparisons, of least squares means were made using the pdiff option. 3. Results
Table 1 Plasma metabolite concentrations and indicators of ornithine metabolism in enterally-fed neonatal piglets 1 Diet Basal
+α-KG
+Orn
+α-KG/+Orn
Pooled SE
P-value
d 7 plasma concentrations Ammonia (μmol/L) Urea (mmol/L) Arginine (μmol/L) Ornithine (μmol/L) Proline (μmol/L)
118 3.0 25 46a 624ab
119 3.5 26 37a 478a
81 1.5 22 213b 886b
97 1.8 58 168b 785ab
13 0.6 14 18 112
NS 2 NS NS b0.0001 b0.10
Whole-body amino acid fluxes Ornithine flux (μmol/(kg h)) Arginine flux (μmol/(kg h))
234a 442
240a 399
473b 370
527b 303
32 71
b0.0001 NS
Whole-body ornithine metabolism Ornithine oxidation (% dose oxidized) Ornithine to arginine conversion (μmol/(kg h)) Ornithine to proline conversion 3 (μmol/(kg h))
20b 145 51a
15a 150 52a
30c 137 149b
34c 128 148b
1 29 6
b0.0001 NS b0.0001
Values are least square means, n = 5. Means in a row without a common superscript differ, P b 0.05. NS = not significant (P N 0.10). 3 Although the intragastric proline flux was not measured in the present study, it was previously determined in a similar study, using similar piglets receiving the identical basal and + Orn diets and identical methods (Urschel et al., 2006). The previously determined flux value of 511 μmol/(kg h) was used for piglets receiving the basal and +α-KG diets, and a flux value of 649 μmol/(kg h) was used for piglets receiving the +Orn and + α-KG/ +Orn diets. 1 2
140
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
Fig. 2. A stochastic model of ornithine metabolism in enterally-fed piglets receiving an arginine-deficient diet supplemented with ornithine and/or α ketoglutarate.
proline were greater in piglets receiving supplemental ornithine (P b 0.05). 4. Discussion
4.2. Ornithine metabolism A stochastic model was used to provide a summary of ornithine metabolism in the piglets in the present study:
4.1. Ornithine α-ketoglutarate as an arginine precursor We have previously shown that the addition of an effective arginine precursor, citrulline, to the argininedeficient basal diet resulted in lower plasma ammonia and urea concentrations and a higher plasma arginine concentration and whole-body arginine flux (Urschel et al., 2006). These effects were not observed in the piglets receiving the +α-KG/+Orn diet relative to the +Orn diet; therefore, OKG was no more effective than ornithine as an arginine precursor. Because OKG infusion did not affect whole-body arginine status and synthesis, we could not verify the proposed mechanism of OKG as an effective arginine precursor (Cynober et al., 1990) in our enterallyfed neonatal piglet model.
Ornithine flux ¼ ornithine intake þde novo ornithine synthesis ¼ ornithine oxidation þornithine conversion to other metabolites
Piglets in the basal and +α-KG groups had a ∼ 1-fold greater rate of de novo whole-body (intragastric) ornithine synthesis than the piglets receiving dietary ornithine (Fig. 2), suggesting that, similar to arginine intake regulating arginine synthesis (Urschel et al., 2005; Wilkinson et al., 2004), ornithine intake regulates ornithine synthesis. Piglets receiving dietary ornithine had a ∼3-fold greater rate of ornithine oxidation than those piglets not
K.L. Urschel et al. / Livestock Science 108 (2007) 137–141
receiving any ornithine (Fig. 2). Ornithine oxidation in neonatal piglets appears to be regulated, at least in part, by ornithine intake. On a whole-body basis, the +Orn and +α-KG/+Orn piglets had a ~75% greater rate of ornithine conversion to other metabolites, compared to the basal and +α-KG piglets (Fig. 2). Proline formation was a major metabolic fate of the supplemental ornithine. In subjects receiving an OKG versus control solution, proline concentrations in the plasma (Cynober et al., 1990; Loi et al., 2005) and tissues (Loi et al., 2005) were higher in the subjects receiving OKG. 5. Conclusion The objective of the present study was to determine whether OKG was a more effective arginine precursor than ornithine alone. OKG was no better than ornithine alone as an arginine precursor. This finding supports the data of our previous study which found that arginine synthesis was limited by the rate of citrulline formation (Urschel et al., 2006). In general, α-ketoglutarate addition to the diet did not modify ornithine metabolism; however, ornithine intake had profound effects on ornithine metabolism. Piglets receiving dietary ornithine had a lower rate of ornithine synthesis, and greater rates of ornithine oxidation and conversion to proline, than piglets not receiving any ornithine. The primary metabolic fates of supplemental ornithine were therefore oxidation and proline synthesis.
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References Bertolo, R.F., Brunton, J.A., Pencharz, P.B., Ball, R.O., 2003. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am. J. Physiol.: Endocrinol. Metab. 284, E915–E922. Cynober, L., Coudray-Lucas, C., De Bandt, J.P., Guechot, J., Aussel, C., Salvucci, M., Giboudeau, J., 1990. Action of ornithine alphaketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects. J. Am. Coll. Nutr. 9, 2–12. House, J.D., Pencharz, P.B., Ball, R.O., 1997. Phenylalanine requirements determined by using L-[1–14C]phenylalanine in neonatal piglets receiving total parenteral nutrition supplemented with tyrosine. Am. J. Clin. Nutr. 65, 984–993. Loi, C., Nakib, S., Neveux, N., Arnaud-Battandier, F., Cynober, L., 2005. Ornithine alpha-ketoglutarate metabolism in the healthy rat in the postabsorptive state. Metabolism 54, 1108–1114. Urschel, K.L., Shoveller, A.K., Pencharz, P.B., Ball, R.O., 2005. Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet. Am. J. Physiol.: Endocrinol. Metab. 288, E1244–E1251. Urschel, K.L., Shoveller, A.K., Uwiera, R.R., Pencharz, P.B., Ball, R.O., 2006. Citrulline is an effective arginine precursor in enterally-fed neonatal piglets. J. Nutr. 136, 1806–1813. Wilkinson, D.L., Bertolo, R.F., Brunton, J.A., Shoveller, A.K., Pencharz, P.B., Ball, R.O., 2004. Arginine synthesis is regulated by dietary arginine intake in the enterally fed neonatal piglet. Am. J. Physiol.: Endocrinol. Metab. 287, E454–E462.