740
steroids 7 1 ( 2 0 0 6 ) 736–744
adhered, cells were temporarily transfected with pGL3 Basic
containing three copies of the Vitamin D response element
(3 × VDRE), and the pRL SV40 internal control plasmid. After
6 h, various concentrations of the new metabolite and 1␣,25-
(OH)2D3 were added, and the cells were incubated for 40 h at
37 ◦C. Cells were lysed using a passive lysis buffer and analyzed
on a microplate luminometer (ARVO, Wallac).
(OH)2D3 were 7.8 and 32 mg/L, respectively. The new metabo-
lite was detected by HPLC (Fig. 2), and its amount represented
about 8% of that of 1␣,25-(OH)2D3, based on the HPLC peak
area.
3.2.
new Vitamin D3 metabolite
2.8.
Human promyeloid leukemia (HL-60)
A flow sheet of the procedures used in the isolation of the
metabolite is shown in Fig. 1. After removal of the substrate
VD3 and hydrophilic substance by Amberlite XAD-7 column,
the fraction containing the more polar Vitamin D metabo-
lites was purified on a CPC equipped with the upper phase
of the solvent mixture (AcOEt/n-hexane/MeOH/water (6:4:5:5))
as the stationary phase, the lower phase of same mixture as
the mobile phase. The peak containing a new metabolite was
eluted just before the elution of 1␣,25-(OH)2D3, which was
observed as a major peak in this condition (Fig. 3A). Peak I
was collected and was rechromatographed on a LiChrosorb
Si 60 column with 15% (v/v) 2-propanol in hexane (Fig. 3B).
By means of this procedure, the new metabolite of Vitamin
D3 has been isolated in pure form. The structure of this new
metabolite has been unequivocally identified as 2␣,25-(OH)2D3
by means of UV absorption, mass, and NMR spectrometry.
The UV spectrum of the new metabolite isolated in its pure
form is presented in Fig. 4. The spectrum displays a ꢀmax at
265 nm and a ꢀmin 228 nm, exhibiting the presence of the con-
jugated triene chromophore typical of VD3.
The low-resolution mass spectrum of the new metabo-
lite is presented in Fig. 5. The mass spectrum exhibited an
apparent molecular ion (M+) at m/z 416. High-resolution mass
spectrometry of the metabolite revealed a molecular weight
of 416.3289, and the elemental composition was determined
to be C27H44O3 (calcd for C27H44O3, 416.3290). The molecular
formula indicates the incorporation of two additional oxygen
atoms into the starting substrate VD3 (m/z 284, M+). Fragment
ions resulting from the cleavage at the C(7)–C(8) double bond
and the elimination of the side chain are highly diagnostic
for the structural assignments of the Vitamin D compound
[8,9]. Characteristic ions at m/z 152 and 134 arising from the
dissociation of the C(7)–C(8) bond and from the subsequent
elimination of water, respectively, confirm the presence of two
hydroxyl groups on the A-ring. It is interesting to note that in
contrast with 1␣,25-(OH)2D3 (m/z 134 as a base peak), the ion
at m/z 152 was observed as a base peak in the new metabolite.
This ion shows that the 3-hydroxyl group from the substrate
is present in the A-ring and the additional hydroxyl group is
either at the C(2) or the C(4) position. Fragment ions at m/z 287
resulting from the cleavage at the C(17)–C(20) bond suggests
the presence of the other oxygen atom on the side chain. The
location of the hydroxyl group on the side chain cannot be
deduced from the mass spectral data.
differentiation-inducing assay
HL-60 cells were grown in RPMI-1640 media supplemented
with 10% heat-inactivated FBS and 20 g/mL of gentam-
icin. Induction of differentiation was estimated from the
ability of the cell to generate superoxide anion. Vitamin
D-induced cells were obtained by seeding HL-60 cells at
1 × 105 mL−1 in growth media and culturing them for 4 days
in the presence of various concentrations of Vitamin D3
analogues. The cells were washed free of the compounds
and suspended in
a 1.5 mL reaction mixture containing
80 M of ferricytochrome C (Sigma) and 500 ng/mL phor-
bol myristate acetate (Sigma) in a 0.1% gelatin Hanks’ bal-
anced salt solution without phenol red. The mixture was
incubated at 37 ◦C for 60 min and centrifuged for 10 min
at 400 × g at 4 ◦C. The optical density of the supernatant
was determined with
a Hitachi U-3200 dual-wavelength
(540–550 nm) spectrophotometer. The amount of superoxide
anion generated was calculated with a molar extinction coef-
ficient of 19.1 × 103 cm−1. Deoxidization cytochrome C con-
centration (nmol/L) = the amount of superoxide anion gener-
ated = [(OD550 − OD540)/(19.1 × 103) × 1.5]/103 × 109.
2.9.
Keratinocyte growth inhibition assay
Normal human keratinocytes (Sanko Junyaku Co., Ltd.)
were grown in serum-free media (KGM-2, Sanko Junyaku
Co., Ltd.). The cells were plated in 96-well plates at
2 × 103 cells/175 L/well. The test substance and the control
substance were diluted with KGM-2 media and then 25 L of
each diluted solution was added to separate wells (test sub-
stance: n = 6, control substance: n = 12). After addition of the
drugs, the cells were cultured for 3 days. [Methyl-3H]thymidine
was diluted with KGM-2 media. Then, 25 L of this diluted
solution (7.4 kBq) was added to each well, and the cells were
cultured overnight. The cells were then detached by 0.05%
trypsin–EDTA in PBS (−). Next, the cells were adsorbed onto
a filter (Printed Filtermat A, Perkin Elmer Japan Co., Ltd.), with
a cell harvester (Harvester 96 Mach III M, Tomtec Inc.), and
the filter was dried in a microwave oven. The filter was then
embedded in a scintillator (MeltiLex A, Perkin Elmer Japan Co.,
Ltd.), and the radioactivity taken up by the cells was measured
with a liquid scintillation counter (MicroBeta 1450, Wallac Oy).
Fig. 6 illustrates the 1H NMR spectrum of the new metabo-
lite. The spectrum afforded definite evidence for the structure
of the metabolite. It showed the typical resonance for the
olefinic protons of the Vitamin D 5,6-cis triene chromophore:
two AB-type doublets at ı 6.27 and 5.99 ppm for the C(6) and
C(7) protons, and two multiplets assigned to the C(19) pro-
tons at ı 4.91 and 5.12 ppm, whose chemical shifts are almost
identical with those observed in the starting VD3 [10,11]. The
3.
Results and discussion
3.1.
Bioconversion of VD3
VD3 was mainly converted to 25-OHD3 and 1␣,25-(OH)2D3 after
reacting for 72 h. The concentrations of 25-OHD3 and 1␣,25-