Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin c

The tetrazolium salt 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium br omide (MTT) is reduced to formazan by the succinate dehydrogenase system of active mitochondria, and hence, specifically used to assay for the viable cells, such as measurement of cell proliferation, cytotoxicity, and cell nu mber. However, in the present study we have shown that some component speci fically present in M199 but not in RPM1 1640 media can reduce MTT to formaz an in the absence of a living system. Further study revealed that ascorbic acid reduced MTT to formazan, which was profoundly increased by a very smal l amount of retinol, whereas retinol alone had no effect. Oxidation of asco rbic acid by H2O2 destroyed its ability to reduce MTT. The rate of MTT redu ction was directly proportional to the concentration of MTT in the absence of retinol, but approached a zero-order state beyond a certain concentratio n of MTT in the presence of retinol. Furthermore, retinol remained unchange d after the completion of the reaction. Taken together, these results showe d that retinol acts as a reductase that catalyzes the reduction of MTT to f ormazan using ascorbic acid as the cosubstrate (electron donor). (C) 2000 W iley-Liss, Inc.