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In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate...