The tetrazolium salt 3(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (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 number. However, in the present study we have shown that some component specifically present in M199 but not in RPMI 1640 media can reduce MTT to formazan in the absence of a living system. Further study revealed that ascorbic acid reduced MTT to formazan, which was profoundly increased by a very small amount of retinol, whereas retinol alone had no effect. Oxidation of ascorbic acid by H(2)O(2) destroyed its ability to reduce MTT. The rate of MTT reduction was directly proportional to the concentration of MTT in the absence of retinol, but approached a zero-order state beyond a certain concentration of MTT in the presence of retinol. Furthermore, retinol remained unchanged after the completion of the reaction. Taken together, these results showed that retinol acts as a reductase that catalyzes the reduction of MTT to formazan using ascorbic acid as the cosubstrate (electron donor).
Copyright 2000 Wiley-Liss, Inc.