Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.
We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.
Shankar
V N
VN
Department of Biochemistry, Queen's University, K7L 3N6, Kingston, ON, Canada.
Byford
V
V
Prosser
D E
DE
Schroeder
N J
NJ
Makin
H L
HL
Wiesinger
H
H
Neef
G
G
Steinmeyer
A
A
Jones
G
G
D006801:Humans; D006900:Hydroxylation; D008745:Methylation; D008968:Molecular Conformation; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism*
C116441:2-methyl-1,25-dihydroxyvitamin D3; C423843:20-methyl-1,25-(OH)2D3; D014807:Vitamin D