Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu(1)) and MTX-polyglutamates (MTX-Glu( 2-5)) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether as corbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic ac id (THF) directly or by circumventing the MTX inhibition of dihydrofolate r eductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glu(n) in the absence or presence of ascorbate, was determined by anal ytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading e lectrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histid ine, pH 6.0, as a terminating electrolyte (T), MTX-Glu(n) derivatives inclu ding MTX-Glu(1) could be easily separated, whereas the quantitative estimat ion of THF was not possible. A quantitative characterization of the DHFR re action by measuring NADPH, NADP(+) and ascorbate was achieved with another system (L: 0.01 M HCl/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.2 7). Nanomolar concentrations of MTX-Glu(1-5) inhibited consumption of NADPH and production of NADP(+). Ascorbic acid was not able to reduce DHF, neith er directly nor after inhibition of DHFR by MTX. However, ascorbic acid see med to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.