The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism

Abstract

We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.

Fig. 1

Metabolism of vitamin D3 by purified bovine P450scc. Incubations were carried out in a reconstituted system comprising purified P450scc (3 μM), FDXR, FDX1 and phospholipid vesicles containing vitamin D3 at a molar ratio to phospholipid of 0.2. Reaction products were analyzed by TLC as described in Experimental Procedures. Control (incubation without NADPH (1); experimental incubation with NADPH (2); pregnenolone standard (3): products of vitamin D3 metabolism, P1, P2 and P3, are marked by arrows.

Fig. 2

NMR spectra of the vitamin D3 metabolite (P1) identified as 20(S)-hydroxyvitamin D3. (A) ¹H NMR; (B) COSY; (C) HMBC.

Fig. 3

¹H NMR spectra of metabolite P3, identified as 20(R),22-dihydroxyvitamin D3. (A) ¹H NMR. The two hydroxyl groups (3.96 p.p.m. assigned to 3-OH and 4.10 p.p.m. assigned to 22-OH) are marked by arrows. Finger print peaks for D3 are labeled with * in the 1D proton spectrum. (B) ¹H-¹H COSY. The line indicates the correlations of 22-OH proton to protons at C22, C23 and C24.

Fig. 4

Mass spectrometry of product P3. (A) LC/MS/MS of fragment with (M + 1)⁺ at m/z 399. (B) LC/MS/MS of fragment with (M + 1)⁺ at m/z 415.

Fig. 5

Proposed sequence of P450scc catalyzed transformation of vitamin D3 and structures of the first two reaction products.

Fig. 6

LC/MS and UV spectra of products of vitamin D3 metabolism by adrenal mitochondria. (A, C, E) Control (incubation without NADPH and isocitrate); (B, D, F) experimental incubation (with NADPH and isocitrate). The HPLC elution profile was monitored by absorbance at 265 nm (A, B). The selected ion monitoring (SIM) mode was used to detect ions with m/z = 383 (E, F) and m/z 401 (C, D). The peaks designated as 1–10 correspond to vitamin D3 metabolites. The peak designated as 11 corresponds to vitamin D3. Product #4 has a retention time and mass spectrometric characteristics identical to 25OH-vitamin D3 standard. Elution was carried out as described in Experimental procedures.

Fig. 7

Inhibition of vitamin D3 metabolism by DL-aminoglutethimide. (A) Control (incubation without NADPH and isocitrate); (B) Experimental incubation (with NADPH and isocitrate); (C) Experimental incubation (with NADPH and isocitrate) in the presence of DL-aminoglutethimide (100 μM). The mobile phases were slightly modified in comparison with Fig. 6 and consisted of 85% methanol and 0.1% acetic acid from 0 to 25 min, followed by linear gradient to 100% methanol and 0.1% acetic acid from 25 to 35 min; and 100% methanol and 0.1% acetic acid from 35 to 55 min. Note the marked disappearance of products 6, 8 and 3.