Measurement of mammalian 25-hydroxyvitamin D3 24R-and 1 alpha-hydroxylase

An in vitro assay of mammalian 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases in kidney has been developed. It had been suggested that 25-hydroxyvitamin D binding protein present in mammalian blood and tissues inhibits the enzyme activities in cell-free preparations by binding the substrate 25-hydroxyvitamin D3 more strongly than the hydroxylases bind it. This inhibitory effect is overcome by the addition of substantial amounts of unlabeled 25-hydroxyvitamin D3 to saturate the binding sites of this protein. The resulting metabolites produced in vitro by rat kidney homogenates were isolated and firmly identified by ultraviolet absorption spectrometry and mass spectrometry as 1,25-dihydroxyvitamin D3 and (24R)-24,25-dihydroxyvitamin D3. Maximal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 could be demonstrated in kidney homogenates prepared from vitamin D-deficient rats. Thyroparathyroidectomy of these rats resulted in total suppression of the 1 alpha-hydroxylase. Homogenates of kidney from rats given vitamin D showed little or no 1 alpha-hydroxylase and substantial 24R-hydroxylase activity. Thyroparathyroidectomy of these rts markedly increased the 24R-hydroxylase activity.