Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats...

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher T_max value than others. When 23S,25(OH)₂D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)₃D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)₃D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)₃D3 via 23,25(OH)₂D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)₃D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)₃D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.