1-ALPHA,25-DIHYDROXYVITAMIN D-3-24-HYDROXYLASE (CYP24) HYDROXYLATES THE CARBON AT THE END OF THE SIDE-CHAIN (C-26) OF THE C-24-FLUORINATED ANALOG OF 1-ALPHA,25-DIHYDROXYVITAMIN D-3

The sequential oxidation and cleavage of the side chain of 1 alpha,25- dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3) initiated by the hydroxyla tion at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolis m of a synthetic analog of 1 alpha,25(OH)(2)D-3, 24,24-difluoro-1 alph a,25-dihydroxyvitamin D-3 (F-2-1 alpha,25(OH)(2)D-3), C-24 of which wa s designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1 alpha,25(OH)(2)D-3-supplemented rats were incubated wi th F-2-1 alpha,25(OH)(2)D-3, it was mainly converted to a more polar m etabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1 alpha,25,26-trihydroxyvitamin D-3 (F-2-1 alpha,25,26( OH)(3)D-3) by ultraviolet absorption spectrometry, frit-fast atom bomb ardment liquid chromatography/mass spectroscopy analysis, and direct c omparison with chemically synthesized F-2-1 alpha,25,26 (OH)(3)D-3. Me tabolism of F-2-1 alpha,25(OH)(3)D-3 into F-2-1 alpha,25,26(OH)(3)D-3 by kidney homogenates was induced by the prior administration of 1 alp ha,25(OH)(2)D-3 into rats. The C-24 oxidation of 1 alpha,25(OH)(2)D-3 in renal homogenates was inhibited by F-2-1 alpha,25(OH)(2)D-3 in a co ncentration-dependent manner. Moreover, F-2-1 alpha,25,26(OH)(3)D-3 wa s formed in ROS17/2.8 cells transfected with a plasmid expressing 1 al pha,25(OH)(2)D-3-24-hydroxylase (CYP24) but not in the cells transfect ed with that expressing vitamin D-3-25-hydroxylase (CYP27) or containi ng inverted CYP27 cDNA. These results show that CYP24 catalyzes not on ly hydroxylation at C-24 and C-23 of 1 alpha,25(OH)(2)D-3 but also at C-26 of F-2-1 alpha,25(OH)(2)D-3, indicating that this enzyme has a br oader substrate specificity of the hydroxylation sites than previously considered.