GROWTH HORMONE-RESPONSIVE DT-DIAPHORASE-MEDIATED BIOREDUCTION OF TETRAZOLIUM SALTS

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of t etrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms respo nsible for the formazan production are not known. MTAs currently provi de the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrastin g tetrazolium salts, namely 4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltet razolium bromide (MTT) and henyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfo phenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bi oassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electro n transfer pathway which is exclusively mediated by DT-diaphorase. (C) 1996 Academic Press, Inc.