Detection of novel CYP11A1-derived secosteroids in the human epidermis and serum and pig adrenal gland

Abstract

To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterized in vitro, occur in vivo, we analyzed samples of human serum and epidermis, and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)2D3, 20,22(OH)2D3, 20,23(OH)2D3, 20,24(OH)2D3, 20,25(OH)2D3, 20,26(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3, plus 1,20(OH)2D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH)D3, respectively, and at levels above those required for biological activity as measured in vitro. We also detected 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormones in vivo.

Figure 1. Detection of 7DHC and 7DHP in the human epidermis, human serum and pig adrenals.

Extracted ion chromatograms (EIC) are shown for the human epidermis (A,D), human serum (B,E) and the pig adrenal (C,F) using m/z = 367.2 [M + H − H₂O]⁺ for 7DHC (A–C) and m/z = 337.2 [M + Na]⁺ (D), 279.3 [M + H-2H₂O]⁺ (E) or 315.2 = [M + H]⁺ (F) for 7DHP. Arrows indicate the retention times of the corresponding standards. Inserts for panels (A–C) show the mass spectra corresponding in retention time of 7DHC while inserts of panels (D–F) panels show the mass spectra corresponding to the retention time of 7DHP. Note that the UPLC conditions were different for (F) where a longer column was used compared to the other panels (see Materials and Methods).

Figure 2. Detection of 22(OH)7DHC and 20,22(OH)₂7DHC in the pig adrenal, human epidermis and human serum.

The LC-MS spectra were measured on fractions with retention times corresponding to either 22(OH)7DHC or 20,22(OH)₂7DHC that were pre-purified on a Waters C18 column (250 × 4.6 mm, 5 μm particle size) with a gradient of acetonitrile in water (40–100%) (see Materials and Methods). Extracted ion chromatograms are shown for human epidermis (A,D), serum (B,E) and the pig adrenal (C,F), and were measured using m/z = 383.3 [M + H − H₂O]⁺ (A) or 401.3 [M + H]⁺ (B,C) for 22(OH)7DHC, and m/z = 439.3 [M+Na]⁺ (D,E) or m/z = 417.3 [M + H]⁺ (F) for 20,22(OH)₂7DHC. Arrows indicate the retention times of the corresponding standards. Inserts show the mass spectra corresponding to the retention time of either 22(OH)7DHC (A–C) or 20,22(OH)₂7DHC (D–F).

Figure 3. Detection of 20(OH)D3, 22(OH)D3 and 25(OH)D3 in the human epidermis, human serum and the pig adrenal gland.

The LC-MS spectra were measured on fractions that were pre-purified on a Waters C18 column (250 × 4.6 mm, 5 μm particle size) with a gradient of acetonitrile in water (40–100%) as in Fig. 2. EICs are shown for epidermis (A), serum (B) and the pig adrenal gland (C) and were measured using m/z = 383.3 [M + H − H₂O]⁺ for (A,C) and 25(OH)D3 in (B) or 401.3 [M + H]⁺ for 20(OH)D3 and 22(OH)D3 in (B). The identified peaks had RTs corresponding to either the 20(OH)D3, 22(OH)D3 or 25(OH)D3 standards, indicated by arrows. The inserts show the mass spectra of samples corresponding to the retention times of the standards.

Figure 4. Novel dihydroxyvitamin D3 metabolites can be detected in the human epidermis, human serum and the pig adrenal gland.

The LC-MS spectra were measured on fractions that were pre-purified on Waters C18 column (250 × 4.6 mm, 5 μm particle size) as in Fig. 2. EICs are shown for epidermis (A), serum (B) and the pig adrenal (C) and were measured using m/z = 399.3 [M + H − H₂O]⁺ for 20,22(OH)₂D3 and 20,23(OH)₂D3 in A, and 1,25(OH)₂D3, 20,22OH)₂D3, 20,24(HO)₂D3 and 20,26(OH)₂D3 in C; 417.3 [M + H]⁺ for 1,25(OH)₂D3 in (A) and 20,23(OH)₂D3 and 20,25(HO)₂D3 in C; 439.3 [M + Na]⁺ for 20,24(OH)₂D3, 20,25(OH)₂D3 and 20,26(OH)₂D3 in (A,B). Arrows indicate the retention times of the corresponding standards. The mass spectra of samples corresponding to the retention time of each dihydroxyvitamin D3 standard are shown in the inserts.

Figure 5. Novel trihydroxy-vitamin D3 metabolites are also present in in the human epidermis, human serum and pig adrenal.

The LC-MS spectra were measured on fractions which were pre-purified on a Waters C18 column (250 × 4.6 mm, 5 μm particle size), as in Fig. 2. EICs are shown for the epidermis (A), serum (B), adrenal (C) and were measured using m/z = 415.3 [M + H − H₂O]⁺ except 17,20,23(OH)3D3 in (C) (455.3 [M + Na]⁺). Each standard is identified by arrow and the mass spectra of samples corresponding to the retention time of each trihydroxyvitamin D3 standard are shown in the inserts.