Rat cytochrome P450C24 (CYP24) does not metabolize 1′25-dihydroxyvitamin D2 to calcitroic acid

1alpha-Hydroxy-23carboxy-24,25,26,27-tetranorvitamin D-3 (calcitroic acid) is known to be the major water-soluble metabolite produced during the deactivation of 1,25-(OH)(2)D-3. This deactivation process is carried out exclusively by the multicatalytic enzyme CYP24 and involves a series of oxidation reactions at C-24 and C-23 leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1,25-(OH)(2)D-3, 1alpha,25-1,25-(OH)(2)D-2 is also known to undergo side-chain oxidation and side-chain cleavage to form calcitroic acid (Zimmerman et al. [2001]. 1,25-(OH)(2)D-2 differs from 1,25-(OH)(2)D-3 by the presence of a double bond at C-22 and a methyl group at C-24. To date, there have been no studies detailing the participation of CYP24 in the production of calcitroic acid from 1,25-(OH)(2)D-2. We, therefore, studied the metabolism of 1,25-(OH)(2)D-3 and 1,25-(OH)(2)D-2 using a purified rat CYP24 system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase high-pressure liquid chromatography (HPLC analysis. As expected, 1,23(OH)(2)-24,25,26,27-tetranor D and calcitroic acid were the major lipid and aqueous-soluble metabolites, respectively, when 1,25-(OH)(2)D-3 was used as substrate. However, when 1,25-(OH)(2)D-2 was used as substrate, 1,24(R),25-(OH)(3)D-2 was the major lipid-soluble metabolite with no evidence for the production of either 1,23(OH)(2)-24,25,26,27-tetranor D or calcitroic acid. Apparently, the CYP24 was able to 24-hydroxylate 1,25-(OH)(2)D-2, but was unable to effect further changes, which would result in side-chain cleavage. These data suggest that the presence of either the double bond at C-22 or the C-24 methyl group impedes the metabolism of 1,25-(OH)(2)D-2 to calcitroic acid by CYP24 and that enzymes other than CYP24 are required to effect this process. Published 2002 Wiley-Liss Inc.(dagger).