Aldehyde oxidase-catalysed oxidation of methotrexate in the liver of guinea-pig, rabbit and man

Although 7-hydroxymethotrexate is a major metabolite of methotrexate during high-dose therapy, negligible methotrexate-oxidizing activity has been found in-vitro in the liver in man. The goals of this study were to determine the role of aldehyde oxidase in the metabolism of methotrexate to 7-hydroxymethotrexate in the liver and to study the effects of inhibitors and other substrates on the metabolism of methotrexate. Methotrexate, (+/-)-methotrexate and (-)-methotrexate were incubated with partially purified aldehyde oxidase from the liver of rabbit, guinea-pig and man and the products analysed by HPLC. Rabbit liver aldehyde oxidase: was used for purposes of comparison. In-vitro aldehyde oxidase from the liver of man catalyses the oxidation of methotrexate to 7-hydroxymethotrexate, but the turnover is low. However, formation of 7-hydroxymethotrexate from all forms of methotrexate by the liver in guinea-pig and man was significantly inhibited in the presence of 100 mu M menadione and chlorpromazine, potent inhibitors of aldehyde oxidase. Allopurinol (100 mu M) had a negligible inhibitory effect on liver aldehyde oxidase from guinea-pig and man. Allopurinol is a xanthine oxidase inhibitor. The production of 7-hydroxymethotrexate was enhanced in the presence of allopurinol. Although aldehyde oxidase is also responsible for some of this conversion, it is also possible that the closely related xanthine oxidase is responsible for the formation of 7-hydroxymethotrexate. By employing potent selective inhibitors of aldehyde oxidase, menadione and chlorpromazine, we have demonstrated for the first time that liver aldehyde oxidase from man is minimally involved in methotrexate oxidation.