C-25 hydroxylation of 1α,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1):: metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D-3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D-3 (1alpha,25(OH)(2)D-3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D-3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D-3 (1alpha,24(R)(OH)(2)D-3) is a non-25-hydroxylated synthetic vitamin D-3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)(2)D-3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)(2)D-3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)(2)D-3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)(3)D-3 and 1alpha,24(OH)(2)-23-oxo-D-3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)(2)D-3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)(2)D-3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)(2)D-3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)(2)D-3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)(2)D-3 at C-25. (C) 2004 Elsevier Inc. All rights reserved.