An alternative pathway of vitamin D metabolism. Cytochrome P450scc (CYP11A1)-mediated conversion to 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2

Abstract

We report an alternative, hydroxylating pathway for the metabolism of vitamin D2 in a cytochrome P450 side chain cleavage (P450scc; CYP11A1) reconstituted system. NMR analyses identified solely 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2 derivatives. 20-Hydroxyvitamin D2 was produced at a rate of 0.34 mol x min(-1) x mol(-1) P450scc, and 17,20-dihydroxyvitamin D2 was produced at a rate of 0.13 mol x min(-1) x mol(-1). In adrenal mitochondria, vitamin D2 was metabolized to six monohydroxy products. Nevertheless, aminoglutethimide (a P450scc inhibitor) inhibited this adrenal metabolite formation. Initial testing of metabolites for biological activity showed that, similar to vitamin D2, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2 inhibited DNA synthesis in human epidermal HaCaT keratinocytes, although to a greater degree. 17,20-Dihydroxyvitamin D2 stimulated transcriptional activity of the involucrin promoter, again to a significantly greater extent than vitamin D2, while the effect of 20-hydroxyvitamin D2 was statistically insignificant. Thus, P450scc can metabolize vitamin D2 to generate novel products, with intrinsic biological activity (at least in keratinocytes).

Fig. 1

Metabolism of vitamin D₂ by purified bovine cytochrome P450 side chain cleavage (P450scc). Incubations were carried out in a reconstituted system comprising purified P450scc (3 μM), adrenodoxin reductase, adrenodoxin and phospholipid vesicles containing vitamin D₂ at a molar ratio to phospholipid of 0.2. (A) Reaction products were analyzed by TLC and visualized by charring. Lane 1, Experimental incubation with NADPH; lane 2, control incubation without NADPH; lane 3, pregnenolone (P) and vitamin D₂ standards. Metabolite 1 (M1) and metabolite 2 (M2) are marked by arrows. (B) Electron impact MS of metabolite 1. (C) Electron impact MS of metabolite 2.

Fig. 2

NMR spectra of vitamin D₂ metabolite 1 identified as 20-hydroxyvitamin D₂. (A) Proton–proton COSY of vitamin D₂ standard. (B) COSY of vitamin D₂ metabolite 1. (C) Proton–carbon correlation spectroscopy (HSQC) of vitamin D₂ standard. (D) HSQC of vitamin D₂ metabolite 1. The separation of 22/23 proton signals in metabolite 1 and the lack of scalar coupling between 20-CH and 22-CH at 5.54 p.p.m. (circle in (B)) clearly indicates hydroxylation at 20-C. The doublet-to-singlet transition of proton NMR with concurrent downfield shift of the 21-methyl signal (1.01 p.p.m. and 21.2 p.p.m. to 1.3 p.p.m. and 29.5 p.p.m.) confirms hydroxylation at the 20 position. Impurities in the methyl regions are probably from the TLC purification process.

Fig. 3

NMR spectra of vitamin D₂ metabolite 2 identified as 17,20-dihydroxyvitamin D₂. (A) Proton spectra of metabolite 2. (B) Proton spectra of vitamin D₂. (C) COSY of metabolite 2. (D) Proton–carbon correlation spectroscopy (HSQC) of methyl regions of metabolite 2. Numbers in (B) indicate proton positions in the vitamin D₂ standard. In metabolite 2, the 20-hydroxyl is clearly present and there are no other changes in the side chain as indicated by COSY and HSQC. The large downfield shift of 14-CH from 1.99 p.p.m. to 2.68 p.p.m. with disappearance of the 17-CH signal at 1.32 p.p.m. indicates that hydroxylation has occurred at the 17-C position.

Fig. 4

Proposed sequence for the cytochrome P450 side chain cleavage (P450scc)-catalyzed transformation of vitamin D₂ with chemical structures of the reaction products.

Fig. 5

RP-HPLC separation of products of vitamin D₂ metabolism by adrenal mitochondria. (A) Incubation of mitochondria in the absence of NADPH and isocitrate. (B) Experimental incubation with NADPH and isocitrate. (C) Experimental incubation with 200 μM aminoglutethimide. The HPLC elution profile was monitored by absorbance at 265 nm. 1–6, novel vitamin D₂ metabolites; 7, vitamin D₂.

Fig. 6

Metabolites of vitamin D₂ inhibit DNA synthesis and stimulate differentiation in human HaCaT keratinocytes. (A) HaCaT keratinocytes were synchronized and incubated for 24 h in Ham’s F10 medium containing serum and vitamin D₂ or its metabolites, and [³H]-thymidine. (B) HaCaT keratinocytes were transfected with a construct containing the involucrin promoter (IVL-Luc) or with empty (promoter-free) construct, synchronized and incubated for 24 h in Ham’s F10 medium containing serum and vitamin D₂ or its metabolites. Data are shown as mean ± SEM (n = 3–8).