Lumisterol is metabolized by CYP11A1: discovery of a new pathway

Abstract

Lumisterol3 (L3) is produced by photochemical transformation of 7-dehydrocholesterol (7-DHC) during exposure to high doses of ultraviolet B radiation. It has been assumed that L3 is biologically inactive and is not metabolized in the body. However, some synthetic derivatives of L3 display biological activity. The aim of this study was to test the ability of CYP11A1 to metabolize L3. Incubation of L3 with bovine or human CYP11A1 resulted in the formation of three major and a number of minor products. The catalytic efficiency of bovine CYP11A1 for metabolism of L3 dissolved in 2-hydroxypropyl-β-cyclodextrin was approximately 20% of that reported for vitamin D3 and cholesterol. The structures of the three major products were identified as 24-hydroxy-L3, 22-hydroxy-L3 and 20,22-dihydroxy-L3 by NMR. 22-Hydroxy-L3 was further metabolized by bovine CYP11A1 to 20,22-dihydroxy-L3. Both 22-hydroxy-L3 and 20,22-dihydroxy-L3 gave rise to a minor metabolite identified from authentic standard and mass spectrometry as pregnalumisterol (pL) (product of C20-C22 side chain cleavage of L3) and two trihydroxy-L3 products. The capability of tissues expressing CYP11A1 to metabolize L3 was demonstrated using pig adrenal fragments where 20,22-dihydroxy-L3, 22-hydroxy-L3, 24-hydroxy-L3 and pL were detected by LC/MS. Thus, we have established that L3 is metabolized by CYP11A1 to 22- and 24-hydroxy-L3 and 20,22-dihydroxy-L3 as major products, as well as to pL and other minor products. The previously reported biological activity of pL and the presence of CYP11A1 in skin suggest that this pathway may serve to produce biologically active products from L3, emphasizing a novel role of CYP11A1 in sterol metabolism.

Keywords: CYP11A1; Cytochrome P450scc; Hydroxylation; Lumisterol; Vitamin D3.

Fig. 1

Photosynthesis of previtamin D3, tachysterol and lumisterol.

Fig. 2

Analysis of the products of L3 metabolism by CYP11A1. (A,B) HPLC analysis of L3 metabolites. L3 (20 µM) in 0.45% cyclodextrin was incubated with 1.0 µM CYP11A1 for 3 h at 37°C in a reconstituted system containing adrenodoxin reductase (0.4 µM), adrenodoxin (15 µM) and NADPH (50 µM). Products were extracted with dichloromethane and analyzed by HPLC using a methanol-water gradient (see Experimental Procedures). A, Zero-time control incubation; B, Test incubation. (C,D) Time courses for the metabolism of L3 by CYP11A1. L3 (50 µM) in 0.45% cyclodextrin was incubated with 1.0 µM CYP11A1 and products analyzed by HPLC. (C) major initial products; (D) minor products.

Fig. 3

NMR analysis of Product B (24(OH)L3) (A) 1D Proton; (B),(C) ¹H–¹³C HSQC; (D) ¹H–¹H TOCSY; (E) ¹H–¹³C HMBC

Fig. 4

NMR analysis of product C (22(OH)L3) (A) 1D Proton; (B),(C) ¹H–¹³C HSQC; (D) ¹H–¹H TOCSY; (E) ¹H–¹³C HMBC.

Fig. 5

NMR analysis of product A (20,22(OH)₂L3) (A) 1D Proton of 20,22(OH)₂L3; (B) 1D Proton of lumisterol; (C) ¹H–¹³C HMBC; (D) ¹H–¹³C HSQC; (E) ¹H–¹H TOCSY

Fig. 6

Further metabolism of L3 products by CYP11A1. 22(OH)L3, 24(OH)L3, 20,22(OH)₂L3 or Product G (20 µM in 0.45% cyclodextrin) were incubated with 2 µM CYP11A1 for 1 h, as in Fig. 2. Products were extracted with dichloromethane and analyzed by HPLC using a methanol-water gradient. Chromatograms show metabolites produced from (A) 24(OH)L3, product B; (B) monohydroxylumisterol, product G; (C) 22(OH)L3, product C; (D) 20,22(OH)₂L3, product A. (E) reaction mixture from 20,22(OH)₂L3 spiked with authentic pL.

Fig. 7

Metabolism of L3 by human CYP11A1. L3 (25 µM) in 0.45% cyclodextrin was incubated with 2.0 µM human CYP11A1 for 3 h at 37°C as for Fig. 2. Products were extracted with dichloromethane and analyzed by HPLC using a methanol-water gradient. (A) Control incubation with NADPH omitted; (B) Test incubation.

Fig. 8

Production of monohydroxy-L3 in by pig adrenal fragments. L3 was incubated with pig adrenal fragments for 20 h, the metabolites extracted and a preliminary separation carried out by HPLC using an acetonitrile in water gradient. The collected metabolites were then analyzed by LC-MS qTOF) as described in the Materials and Methods. The EIC (extracted ion chromatogram) was analyzed using m/z = 383.3 [M-H₂O+H]⁺ for 20(OH)L3 and 24(OH)L3, and m/z = 423.3 [M+Na]⁺ for 24(OH)L3. (A), (B) and (C) pig adrenals incubated with 500 µM L3; (D), (E) and (F) pig adrenals incubated with vehicle only. (A) and (D) 22(OH)L3; (B) and (E) 24(OH)L3; (C) and (F), CYP11A1 metabolite (OH)L3, product G.

Fig. 9

Production of dihydroxy-L3 and pL by pig adrenal fragments. L3 was incubated with adrenal fragments for 20 h, the metabolites extracted and a preliminary separation done by HPLC as in Fig. 10. Metabolites were then analyzed by LC-MS (qTOF) as described in the Materials and Methods. The EIC (extracted ion chromatogram) was analyzed using m/z = 439.3 [M+Na]⁺ for 20,22(OH)₂L3 and unknown dihydroxy-L3, and 297.2 [M-H₂O+H]⁺ for pL3. (A), (B) and (C) pig adrenals incubated with 500 µM L3; (D), (E) and (F) pig adrenals incubated with vehicle only. (A) and (D) 20,22(OH)₂L3; (B) and (E) CYP11A1 metabolite (OH)₂L3, product E; (C) and (F), pL.

Fig. 10

Summary of the pathways for metabolism of L3 by CYP11A1. The major pathways with products being identified by NMR are shown with bold arrows. Minor pathways are shown with thin arrows. U, V, W, X and Y are positions where the site of hydroxylation is unknown. Letters above metabolite names refer to the original product labels as defined in Fig. 2 and Fig. 6.