Comparative studies on the 25-hydroxylations of cholecalciferol and 1 alpha-hydroxycholecalfierol in perfused rat...

The 25-hydroxylations of [(3)H]cholecalciferol and 1alpha-hydroxy[(3)H]cholecalciferol in perfused rat liver were compared. Results showed that about twice as much 1alpha(OH)D(3) (1alpha-hydroxycholecalciferol) was incorporated into the liver as cholecalciferol. 25-Hydroxy[(3)H]cholecalciferol and 1alpha-25-dihydroxy[(3)H]cholecalciferol were not incorporated significantly. Livers isolated from vitamin D-deficient rats formed the 25-hydroxy derivatives of cholecalciferol and 1alpha(OH)D(3) respectively linearly with time for at least 120min. The rate of 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxycholecalciferol) production increased exactly 10-fold on successive 10-fold increases in the dose of 1alpha(OH)D(3), suggesting that hepatic 25-hydroxylation of 1alpha(OH)D(3) is not under metabolic control. On the other hand, the rate of conversion of cholecalciferol into 25(OH)D(3) (25-hydroxycholecalciferol) did not increase linearly with increase in the amount of cholecalciferol in the perfusate. The 25-hydroxylation of cholecalciferol seemed to proceed at a similar rate to that of 1alpha(OH)D(3) at doses of less than 1nmol, but with doses of more than 2.5nmol, the conversion of cholecalciferol into 25(OH)D(3) became much less efficient, though the linear relation between the amounts of substrate and product was maintained. A reciprocal plot of data on the 25-hydroxylation of cholecalciferol gave two K(m) values of about 5.6nm and 1.0mum, whereas that for the 25-hydroxylation of 1alpha(OH)D(3) gave a single K(m) value of about 2.0mum. These results suggest that there are two modes of 25-hydroxylation of cholecalciferol in the liver, which seem to be closely related to the mechanism of control of 25(OH)D(3) production by the liver.