1alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of 1alpha,25-dihydroxyvitamin D3.
The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.
Miyamoto
Y
Y
Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.
Shinki
T
T
Yamamoto
K
K
Ohyama
Y
Y
Iwasaki
H
H
Hosotani
R
R
Kasama
T
T
Takayama
H
H
Yamada
S
S
Suda
T
T
D000818:Animals; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000378:metabolism; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000378:metabolism*; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
C023370:24,24-difluoro-1,25-dihydroxyvitamin D3; D002244:Carbon; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; D002112:Calcifediol