Fig. 8.

Identification of minor metabolites of vitamin D3. Products of P450scc action on vitamin D3 with retention times of 32, 26 and 26.7 min in Fig. 1 were purified, incorporated into phospholipid vesicles at a molar ratio to phospholipid of 0.025, incubated with P450scc (1 μM) for 1 h and the products were analyzed by HPLC with gradient elution. (A) Test reaction of the RT = 32 min monohydroxyvitamin D3 product shown in Fig. 1, identified in this experiment as 23-hydroxyvitamin D3; (B) zero-time control for this substrate where the reaction mixture was extracted at the end of the pre-incubation; (C) 20,23-dihydroxyvitamin D3 standard. (D) Test reaction of the RT = 26 min dihydroxyvitamin D3 product shown in Fig. 1, identified in this experiment as 17α,20-dihydroxyvitamin D3; (E) zero-time control for this substrate; (F) 17α,20,23-trihydroxyvitamin D3 standard. (G) Test reaction of the RT = 26.7 min monohydroxyvitamin D3 product shown in Fig. 1, identified in this experiment as 17α-hydroxyvitamin D3; (H) zero-time control for this substrate; (I) zero-time control spiked with 17α, 20-dihydroxyvitamin D3 standard. Note that the retention times for reactants and products here are different from those described in Fig. 1 due to the use of a new HPLC column.