Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid |
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| Authors: | Lynette D. Fairbanks, Elizabeth A. Carrey, Katarzyna Rü ckemann, Ramasamyiyer Swaminathan, Bernhard Kirschbaum,H. Anne Simmonds |
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| Affiliation: | a Purine Research Laboratory, 5th Floor Thomas Guy House, GKT Guy’s Hospital, London Bridge, London SE1 9RT, UK;b Department of Chemical Pathology, GKT Guy’s Hospital, London Bridge, London SE1 9RT, UK;c Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London NW3 2PF, UK;d Hoechst Marion Roussel, Frankfurt D-65926, Germany |
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| Abstract: | Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [14C]carbamoyl aspartate from [14C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations. |
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| Keywords: | Leflunomide Brequinar Dihydroorotic acid Carbamoyl aspartate Carbamoyl phosphate |
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