INVITRO OXIDATION OF VITAMIN-D METABOLITES BY STEROID DEHYDROGENASES

Steroid dehydrogenases have been shown to react with hydroxylated derivatives of vitamin D in vitro. This suggests that sensitive enzyme assays for vitamin D derivatives could be developed. The reactivity of 3α,20β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase with cholecalciferol (D3), 1α-hydroxycholecalciferol (1α-OH D3), 25-hydroxycholecalciferol (25-OH D3), 24R and S,25-dihydroxycholecalciferol (24R and S,25-(OH)2D3) and 1α,25-dihydroxychole-calciferol (1,25-(OH)2D3) have been studied. 3β-hydroxysteroid dehydrogenase was unreactive with any of the hydroxylated cholecalciferol derivatives. 3α- and 3α,20β-hydroxysteroid dehydrogenases were respectively weakly reactive and unreactive with both D3 and 1α-OH D3 but both enzymes reacted well with 24R and S,25-(OH)2D3, somewhat less well with 25-OH D3 and worst with 1,25-(OH)2D3. The KM values with the two reactive enzymes were different for the same vitamin D derivatives and ranged from 0.7 to 7.4 μmol/l. Since most of the vitamin D metabolites are present in very low concentrations in plasma enzymatic cycling would be necessary to measure the NADH produced. The malate cycling product was produced at a final concentration 10,000 times greater than the NADH formed in the primary steroid dehydrogenase reaction. This permitted quantification of 24R,25-(OH)2D3 down to 0.12 pmol (50 pg). © 1979.