In vivo metabolism of the vitamin D analog, dihydrotachysterol. Evidence for formation of 1 alpha,25- and 1 beta,25-dihydroxy-dihydrotachysterol metabolites and studies of their biological activity.
Dihydrotachysterol (DHT), a reduced vitamin D analog in which the A-ring has been rotated through 180 degrees is a biologically active molecule which can be used to study the structural requirements for the calcemic and cell differentiating properties of the vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), as well as to investigate the specificity of the enzyme systems that catalyze the formation of this hormone. In this study we showed that dihydrotachysterol was metabolized in vivo into a significant polar metabolite observed on straight-phase high performance liquid chromatography (HPLC) which subsequently split into two peaks on reverse-phase HPLC. These two metabolites were identified by HPLC and gas chromatography-mass spectrometry techniques as 1 alpha,25-(OH)2DHT and 1 beta,25-(OH)2DHT. This pair of metabolites was formed from either DHT2 or DHT3. Standard 1 alpha,25-(OH)2DHTs were generated in vitro from chemically synthesized 1-hydroxydihydrotachysterol precursors using a liver hepatoma cell system. Both 1 alpha,25-(OH)2D2 and 1 alpha,25-(OH)2DHT3 showed a binding affinity to the mammalian vitamin D receptor only 50-100 less than 1 alpha,25-(OH)2D3 whereas 1 beta,25-(OH)2DHTs showed poor binding. On the other hand 1 beta,25-(OH)2DHT3 bound to the rat vitamin D transport protein (DBP) with stronger affinity than did 1 alpha,25-(OH)2DHT3. When tested in a COS-1 cell transfection assay system using a rat osteocalcin vitamin D responsive element coupled to a growth hormone reporter gene, 1 alpha,25-(OH)2DHT3 showed a biological activity only 10 times lower than 1 alpha,25-(OH)2D3. It is therefore suggested that 1 alpha,25-(OH)2DHT probably represents the metabolite of DHT responsible for some of its in vivo effects although we cannot rule out in vivo effects of other metabolites identified. Our studies suggest that 1 alpha,25-dihydroxylated DHTs represent a promising novel group of vitamin D analogs worthy of study for cell differentiation as well as calcemic properties.
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
F
F
Qaw
M J
MJ
Calverley
N J
NJ
Schroeder
D J
DJ
Trafford
H L
HL
Makin
G
G
Jones
D000818:Animals; D001711:Biotransformation; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004097:Dihydrotachysterol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D051381:Rats; D013237:Stereoisomerism; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured; D014809:Vitamin D-Binding Protein / Q000378:metabolism*
D014809:Vitamin D-Binding Protein; D014316:Tritium; C074743:1,25-dihydroxydihydrotachysterol(3); D013006:Growth Hormone; D004097:Dihydrotachysterol