In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes, Caco-2 colon cells and the adrenal gland

Abstract

We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)₂D2, 1,20(OH)₂D2, 25(OH)D2 and 1,25(OH)₂D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)₂D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)₂D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity.

Keywords: 20-Hydroxyvitmin D2; Adrenals; CYP11A1; Keratinocytes; Placenta; Vitamin D.

Figure 1

Detection of novel vitamin D2 hydroxy-derivatives in the human placenta incubated ex utero with vitamin D2. (A) HPLC chromatogram with peaks detected at 265 nm. Left panel: test incubation; right upper panel: negative control comprising boiled placenta incubated with vitamin D2 substrate; right lower panel: control where placenta was incubated without the substrate. Numbers show retention times (RT) corresponding to the authentic standards: 1, 17,20(OH)₂D2; 2, 1,20(OH)₂D2; 3, 25(OH)D2; 4, 20(OH)D2. (B) Mass spectra of metabolites 1–4 collected after HPLC separation obtained using an API-3000 LC-MS/MS mass spectrometer. (C) LC-QTOF MS analysis of collected peaks 1–4 using EIC (extracted ion chromatogram) at m/z = 435.4 [M + Na]⁺ for 20(OH)D2 and 25(OH)D2 and m/z = 451.4 [M + Na]⁺ for 1,20(OH) ₂D2 and 17,20(OH)₂D2. The identified compounds with RT corresponding to the authentic standards are marked by arrowheads. Inset in A shows Waters UPLC-qTOF MS analysis of cell extracts with masses for dihydroxyvitamin D2 determined using m/z = 451.4 [M + Na]⁺ for EIC.

Figure 2

LC-MS analysis of 17α,20(OH)₂D2 (A–D) and 20(OH)D2 (E–H) produced by placental mitochondria. Mitochondria were incubated with 200 μM vitamin D₂ for 4 h and the major products separated by TLC prior to RP-HPLC analysis, as described in the Materials and Methods. A) Chromatogram of the 17α,20(OH)₂D2 TLC zone for the control reaction with NADP⁺ and isocitrate omitted. B) Test reaction showing the 17α,20(OH)₂D2 TLC-zone products. C) Test reaction sample spiked with authentic 17α,20(OH)₂D2. D) Analysis of the test reaction sample via LC-MS/MS in the MRM mode for the parent to product transition m/z 411.2 → 393.3 for 17α,20(OH)₂D2. E) HPLC chromatogram of the 20(OH)D2 TLC zone for the control reaction with NADP⁺ and isocitrate omitted. F) Test reaction Test reaction showing the 20(OH)₂D2 TLC-zone products. G) Test reaction sample spiked with authentic 20(OH)D2. H) Analysis of the test reaction sample by LC-MS/MS in the MRM mode for the parent to product transition m/z 413.2 → 71.1 for 20(OH)D2.

Figure 3

Placental mitochondria metabolise 20(OH)D₂ to 1α,20(OH)₂D2, with the expected transformation of 25(OH)D2 to 1α,25(OH)₂D2 serving as a positive control. Placental mitochondria were incubated with 50 μM 20(OH)D2 (A–D) or 50 μM 25(OH)D2 (E–H) for 4 h at 37° C, products partially purified by TLC and analyzed by RP-HPLC, as described in Materials and Methods. A) Chromatogram for the control reaction with NADP and isocitrate omitted. B) Test reaction showing the chromatogram of the 1α,20(OH)₂D₂ TLC-zone products. C) Test reaction sample spiked with authentic 1α,20(OH)₂D2. D) Analysis of the test reaction sample via LC-MS/MS in the MRM mode for the parent to product transition m/z 411.2 → 393.3 for 1α,20(OH)₂D2. E) Chromatogram for the control reaction with NADP⁺ and isocitrate omitted. F) Test reaction showing the chromatogram of the 1α,25(OH)₂D2 TLC-zone products. G) Test reaction sample spiked with authentic 1α,25(OH)₂D2. H) Analysis of the test reaction sample by LC-MS/MS in the MRM mode for the parent to product transition m/z 411.2 → 393.3 for 1α,25(OH)₂D2.

Figure 4

22R-hydroxycholesterol inhibits production of 17α,20(OH)₂D2 and 20(OH)D2 by placental mitochondria. Placental mitochondria were incubated with 200 μM vitamin D₂ for 4 h at 37° C, products partially purified by TLC and analyzed by RP-HPLC. A) Chromatogram for the test reaction showing the 17α,20(OH)₂D2 TLC-zone products. B) Test reaction carried out in the presence of 100 μM 22R-hydroxycholesterol showing the 17α,20(OH)₂D2 TLC-zone products. C) Test reaction showing the chromatogram of the 20(OH)D2 TLC-zone products. D) Test reaction carried out in the presence of 100 μM 22R-hydroxycholesterol showing the 20(OH)D2 TLC-zone products.

Figure 5

Placental mitochondria transform 20(OH)D2 to 17α,20(OH)₂D2 and 1α(OH)D2 to 1α,20(OH)₂D2. Placental mitochondria were incubated with 50 μM 20(OH)D2 (A–E) or 120 μM 1α(OH)D2 (F–J) for 4 h at 37°C, products partially purified by TLC and analyzed by RP-HPLC as described in the Materials and Methods. A, F) Chromatogram for control reactions with NADP⁺ and isocitrate omitted. B, G) Chromatogram for the test reaction with 20(OH)D2 (B) or 1α(OH)D2 (G). C, H) Test reactions carried out in the presence of 100 μM 22R-hydroxycholesterol. D, I) Sample of the test reaction spiked with authentic 17α,20(OH)₂D2 (D) or 1α,20(OH)₂D2 (I). E, J) Analysis of the test reaction samples by LC-MS/MS in the MRM mode for the parent to product transition m/z 411.2 → 393.3 for dihydroxyvitamin D2.

Figure 6

Docking of cholesterol (native substrate) and vitamin D2 derivatives into the active site of human CYP11A1 (PBD entry: 3N9Y). The docking score for each ligand is indicated in brackets. A, general view of the cholesterol binding pocket with a molecular surface limited to 3Å around cholesterol. B to D, close views of vitamin D2 and its hydroxyderivatives (brown thick tube) binding positions relative to the heme group (blue thin tube). Distances between ligand carbons and iron in the heme group were measured and labeled as colored dash lines (red: distance to C22; green: distance to C20; violet: distance to C17).

Figure 7

Production of vitamin D2 hydroxy-derivatives by human placental fragments after incubation for 16 h with different concentrations of vitamin D2. The analysis was performed using an API-3000 LC-MS/MS mass spectrometer equipped with ESI source in the SIM mode with m/z = 435.4 [M+Na]⁺ for mono-hydroxyvitamin D2 and 451.4 [M+Na]⁺ for di-hydroxyvitamin D2. The relative concentrations of products were calculated from LC-MS peaks areas in relation to standards curves generated using the corresponding authentic standards. The vitamin D2 concentrations used were 10, 100 and 500 μM and data are presented as means ± SEM (n=3).

Figure 8

LC-MS analysis of vitamin D2 hydroxy-derivatives produced by HaCaT epidermal keratinocytes (A–E) and Caco-2 colon cells (F–J) following incubation with 50 μM vitamin D2. (A–D) After RP-HPLC separation (see Materials and Methods) the peaks with RT corresponding to the standards were collected and further analyzed by an API-3000 LC-MS mass spectrometer equipped with an ESI source using the SIM mode to monitor m/z = 435.4 [M + Na]⁺ (A, B, F, G) and m/z = 451.4 [M + Na]⁺ (C, D, H, I) for respective detection of monoxydroxy- or dihydroxyvitamin D2 derivatives. (E, J) UPLC Waters Xevo^™ G2 QTOF MS analysis of cell extracts with masses for dihydroxyvitamin D2 determined using m/z = 451.4 [M + Na]⁺ for EIC. Arrows to 20(OH)D2, 25(OH)D2, 1,20(OH)₂D2, 1,25(OH)₂D2 and 17,20(OH)₂D2 show masses and RT corresponding to the authentic standards.

Figure 9

UPLC-qTOF MS identification of 20(OH)D2, 25(OH)D2 (A) and 1,20(OH)₂D2, 1,25(OH)₂D2 and 17,20(OH)₂D2 (B) produced by neonatal epidermal keratinocytes incubated for 16 h with 500 μM vitamin D2. The chromatograms show m/z = 435.4 [M + Na]⁺ for mono-hydroxyvitamin D2 and 451.4 [M + Na]⁺ for di-hydroxyvitamin D2 derivatives Arrows show the corresponding RT of 20(OH)D2, 25(OH)D2, 1,20(OH)₂D2, 1,25(OH)₂D2 and 17,20(OH)₂D2 authentic standards.

Figure 10

Pathway for the in vivo metabolism of vitamin D2 by CYP11A1