Figure 4.

Metabolism of 25(OH)D₃ in COS-7 cells transfected with the expression plasmid for P4501α. Metabolism of 25(OH)D₃ by the cells transfected with 10 μg of pcDNA 1α (A) and the same amount of pcDNA empty vector (B). The retention times of 25(OH)D₃, 24,25(OH)₂D₃, 10-oxo-19-nor-25(OH)D₃, and 1α,25(OH)₂D₃ are indicated. 25(OH)D₃ was metabolized into 1α,25(OH)₂D₃ only in the cells transfected with pcDNA 1α. The fraction containing 1α,25(OH)₂D₃ was further applied to HPLC under the following two conditions: column, Finepak SIL 4.6 × 250 mm; mobile phase, 8% 2-propanol in hexane; flow rate, 1 ml/min; column, J′-sphere ODS-AM 4.6 × 150 mm (YMC Co., Kyoto, Japan); mobile phase, 10% water in methanol; flow rate, 1.0 ml/min (data not shown). In all cases the putative 1α,25(OH)₂D₃ comigrated with authentic 1α,25(OH)₂D₃. Similar results were obtained in three independent sets of experiments.