On the mechanism of urea formation

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Abstract
Citrulline, when added to liver slices, gives rise to urea in presence of ammonium lactate. The ratio NH3-N disappearance to urea-N formation was found to be approximately 1. Amino-N remained practically unchanged during the synthesis and was markedly increased when citrulline was replaced by arginine, showing that no ornithine was formed from citrulline. A marked urea formation was also obtained in presence of ammonium lactate but in the absence of citrulline or any other amino-acid. When ammonium lactate was replaced by [alpha]-ketoglutaric acid and NH4Cl the urea formed in absence of citrulline was accompanied by a rise in amino-N and of amide-N, interpreted as a synthesis of glutamine. The role of ketonic acids in the mechanism of urea synthesis was further shown by expts. in presence of glycine and alanine. Urea formation from ketoglutaric acid and NH4Cl was inhibited in presence of glycine and partially inhibited in presence of alanine, owing to the action of the amino-acids on [alpha]-ketoglutaric acid, during which amino-N disappeared. A small but distinct synthesis of urea was observed in presence of citrulline and a-ketoglutaric acid or pyruvic acid in absence of NH3. This result suggested an oxidation of citrulline by the ketonic acids leading to the formation of urea and glutamic acid. A scheme for this reaction was outlined. Glutamic acid and glutamine both gave rise to urea formation in presence of NH3, but not in its absence. A satisfactory N-balance sheet was set up. It revealed a marked formation of glutamine from glutamic acid and a disappearance of amide-N from glutamine. These observations led to the concept of a cyclic formation and disappearance of glutamine, catalysing urea synthesis. Ornithine, like citrulline, gave rise to urea formation in presence of a-ketoglutaric acid and NH4Cl, but not in absence of the latter. This was in agreement with the hypothesis of its oxidation to glutamine which was supported by the appearance of amide-N, ascribed to glutamine according to the methods applied. Urea synthesis from arginine in presence of ketoglutaric acid and NH4Cl was found to be 3 times as fast, and from citrulline 1.5 times as fast as from ornithine. Again, little change in amino-N was found with citrulline and none with ornithine whereas amino-N markedly increased with arginine. The disappearance of NH3 was of about the same order in all 3 cases. The role of CO2 or CO2 precursors as a source of carbon for urea formation was investigated. Na formate markedly increased urea formation in presence of NH4Cl from glutamic acid or glutamine. A scheme was outlined suggesting urea synthesis from ornithine, citrulline and also by hydrolysis from arginine, thus connecting the mechanism shown by Krebs [1933] with that observed by Leuthardt [1938].

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