The effect of amino acids on the metabolic fate of 15NH4Cl in isolated sheep hepatocytes

Q J Luo, S A Maltby, Gerald Lobley, Alexander Graham Calder, Michael A. Lomax

Research output: Contribution to journalArticle

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Abstract

Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal-drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cytoplasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66-88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threonine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.
Original languageEnglish
Pages (from-to)912-7
Number of pages6
JournalEuropean Journal of Biochemistry
Volume228
Issue number3
Publication statusPublished - 15 Mar 1995

Fingerprint

Urea
Hepatocytes
Sheep
Amino Acids
Ammonia
Nitrogen
Aspartic Acid
Glutamine
Arginine
Glutamic Acid
Carbamyl Phosphate
Fluxes
Branched Chain Amino Acids
Atoms
Molecules
Ornithine
Viscera
Ruminants
Threonine
Amides

Keywords

  • Amino Acids
  • Ammonium Chloride
  • Animals
  • Cells, Cultured
  • Liver
  • Male
  • Nitrogen Isotopes
  • Sheep
  • Urea

Cite this

The effect of amino acids on the metabolic fate of 15NH4Cl in isolated sheep hepatocytes. / Luo, Q J; Maltby, S A; Lobley, Gerald; Calder, Alexander Graham; Lomax, Michael A.

In: European Journal of Biochemistry, Vol. 228, No. 3, 15.03.1995, p. 912-7.

Research output: Contribution to journalArticle

Luo, Q J ; Maltby, S A ; Lobley, Gerald ; Calder, Alexander Graham ; Lomax, Michael A. / The effect of amino acids on the metabolic fate of 15NH4Cl in isolated sheep hepatocytes. In: European Journal of Biochemistry. 1995 ; Vol. 228, No. 3. pp. 912-7.
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T1 - The effect of amino acids on the metabolic fate of 15NH4Cl in isolated sheep hepatocytes

AU - Luo, Q J

AU - Maltby, S A

AU - Lobley, Gerald

AU - Calder, Alexander Graham

AU - Lomax, Michael A.

PY - 1995/3/15

Y1 - 1995/3/15

N2 - Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal-drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cytoplasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66-88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threonine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.

AB - Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal-drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cytoplasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66-88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threonine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.

KW - Amino Acids

KW - Ammonium Chloride

KW - Animals

KW - Cells, Cultured

KW - Liver

KW - Male

KW - Nitrogen Isotopes

KW - Sheep

KW - Urea

M3 - Article

VL - 228

SP - 912

EP - 917

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 3

ER -