d‐DOPA IS UNIDIRECTIONALLY CONVERTED TO l‐DOPA BY d‐AMINO ACID OXIDASE, FOLLOWED BY DOPA TRANSAMINASE

SUMMARY Many studies have shown that administration of d ‐3, 4‐dihydroxyphenylalanine ( d ‐dopa) produces contralateral rotation in hemi‐parkinsonian animals comparable to l ‐dopa, with less potency and slower onset. It was postulated that d ‐dopa was converted to l ‐dopa to produce these effects. To investigate the postulated chiral inversion of d ‐dopa to l ‐dopa and the related mechanism, an enantiomeric separation method for d ‐ and l ‐dopa using HPLC was first established. Then, rat kidney homogenates containing d ‐dopa or l ‐dopa were incubated and subjected to HPLC to detect traces of respective enantiomer generation. The mechanism of the chiral inversion of d ‐dopa was explored by direct measurement of the production of l ‐dopa in kidney homogenates. d ‐Dopa incubations containing different concentrations of an inhibitor of d ‐amino acid oxidase (DAAO) and an inhibitor of dopa transaminase were measured for l ‐dopa generation using HPLC. The role of DAAO in the chiral inversion of d ‐dopa to l ‐dopa was further investigated by using purified DAAO and mutant ddY/DAAO − mouse kidney lacking DAAO activity. In rat kidney homogenate, d ‐dopa was, indeed, converted to l ‐dopa, whereas l ‐dopa was not converted to d ‐dopa. Sodium benzoate, a selective inhibitor of DAAO, blocked l ‐dopa generation in a concentration‐dependant manner. In contrast with kidney homogenates of wild‐type ddY/DAAO + mice, those of mutant ddY/DAAO − mice lacking DAAO activity did not convert d ‐dopa to l ‐dopa unless exogenous DAAO protein was added. Conversely, when carbidopa, an inhibitor of dopa transaminase, was added to the homogenates, significant inhibition of l ‐dopa production was noted. These results prove the proposal that d ‐dopa undergoes unidirectional chiral inversion and further suggest that d ‐dopa is first oxidatively deaminated by DAAO to its α‐keto acid and then transaminated by dopa transaminase to l ‐dopa.