UVB-induced conversion of 7-dehydrocholesterol to (1 alpha,25-dihydroxyvitamin D3) (calcitriol) in the human keratinocyte line HaCaT

Photochemistry and Photobiology, Dec 2000 by Lehmann, Bodo, Knuschke, Peter, Meurer, Michael

UV irradiation. Samples were exposed to UVB emitted by (1) a tuneable high intensity monochromator (part of the DERMOLUM UM Fa. Miller Optik-Elektronik, Moosinning, Germany); or (2) a fluorescence tube Philips TL-01 (Fa. Philips, Eindhooven; The Netherlands): the so-called "narrow-band UVB lamp." The TL-01 lamp is characterized by a spectral output of 74% in the narrow waveband 311-313 nm. The spectral irradiance ET(X) of the TL-01 tube is depicted in Fig. 1. Using (1) the high intensity monochromator (full wavelength at half maximum: 5 nm) an irradiance Ee = 0.28 mW/ cm2 (inhomogeneity within the irradiation spot of o 15 turn; - 10%) was measured at the bottom of the culture dish. The irradiance was controlled by a thermopile TS 50-1 (Physikalisch-Technische Werkstatten, Jena, Germany), calibration with PTB (Braunschweig, Germany). The culture dish (i 30 mm) was continuously rotated round the axis. The UV-spot (i 15 mm) was positioned at the radius of the rotating dish. The UVB doses used were adapted to these experimental conditions (effective dose = Def = applied UVB dose X 0.25). The irradiance of (2) the TL-01 lamp at the bottom of the culture dish was measured as Ee = 0.86 mW/cmz by an adapted photometer UV-Meter (Fa. Waldmann, Villingen-Schwenningen, Germany).

HPLC analysis. NP-HPLC: Merck/Hitachi; column: LiChroCART 250-4, Superspher Si 60, 5 Rm; solvent system 1 (n-hexane: 2-propanol = 95:5 [vol/vol]; flow rate: 0.5 mL/min) for the determination of pre-VD3, VD3 and 7-DHC; solvent system 2 (n-hexane: 2-propanol:methanol = 87:10:3 [vol/vol/vol]; flow rate: 1.0 mL/min) for separation of calcitriol. The peaks of pre-VD3 and VD; (retention times: 8.22 and 10.57 min, respectively) in the former system were quantified by UV detection at 265 nm. The peak homogeneity was checked using a diode array detector (L-4500, Merck/Hitachi). Fractions containing calcitriol were collected and analyzed for calcitriol by a radioreceptor assay. The results were converted to picomoles of 1,25(OH)2D3 and normalized to 106 cells. Reversed phase (RP)HPLC: the calcitriol generated was identified by cochromatography of the 3H-labeled standard using a Hibar column, 250-4, LiChrospher IOORP-18, 5 wm, (Merck, Darmstadt, Germany); solvent system 3: methanol:water = 85:15 (vol/vol), flow rate: 1 mL/min. Calcitriol-containing fractions were analyzed for their content of calcitriol.

Gas chromatography-mass spectrometry. The dried fractions from HPLC were derivatized to the trimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS). The derivatized sample (1 wL) was directly and manually injected into a model 5890/II gas chromatograph equipped with a 25 m X 0.2 mm HP-1 capillary column (crosslinked methylsiloxane, 0.33 Itm) and interfaced with a model 5989A MS-Engine (Hewlett-Packard, Palo Alto, CA). GC conditions were the following: carrier gas, helium; column head pressure, 10 psi; injector temperature, 260*C; oven temperature gradient, maintained at 200*C for 1 min, increased to 260*C at 30C/min, then increased to 300'C at 20'C/min, held at 300*C for 10 min; interface temperature 300'C. Electron impact (EI) MS conditions were the following: source temperature, 250'C; analyzer temperature, 120*C; energy, 70 eV. All samples were run in triplicate under the control of the HP-ChemStation data system. For selective ion monitoring, the most abundant ions at m/z 452 and m/ z 501 were monitored for lI,25(OH)2D3.